SEARCH

SEARCH BY CITATION

Keywords:

  • signal transduction;
  • therapy;
  • translation;
  • tumour

Abstract

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

In the past years, the attention of scientists has focused mainly on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. All therapeutic strategies against cancer are, to date, directed at DNA either with cytotoxic drugs or gene therapy. Little or no interest has been aroused by protein synthesis mechanisms. However, an increasing body of data is emerging about the involvement of translational processes and factors in control of cell proliferation, indicating that protein synthesis can be an additional target for anticancer strategies. In this paper we review the novel insights on the biochemical and molecular events leading to protein biosynthesis and we describe their involvement in cell proliferation and tumorigenesis. A possible mechanistic explanation is given by the interactions that occur between protein synthesis machinery and the proliferative signal transduction pathways and that are therefore suitable targets for indirect modulation of protein synthesis. We briefly describe the molecular tools used to block protein synthesis and the attempts made at increasing their efficacy. Finally, we propose a new multimodal strategy against cancer based on the simultaneous intervention on protein synthesis and signal transduction.


Abbreviations
IF

initiation factor

eIF

eukaryotic IF

EF

elongation factor

RF

release factor

eRF

eukaryotic RF

PABP

polyadenylated binding protein

PHAS

phosphorylated heat and acid stable protein

dsRNA-PK

double-stranded RNA-dependent protein kinase

PKR

double-stranded RNA-activated kinase

PTI-1

prostate tumour-inducing gene 1

5′ UTR

5′ untranslated region

CaM kinase III

calmodulin-dependent protein kinase III

EGF

epidermal growth factor

S6K

ribosomal S6 kinase

ERK

extracellular signal regulated kinase

GM-CSF

granulocyte monocyte colony stimulating factor

PE

pseudomonas exotoxin A

DT

diphteria toxin

TAA

tumour associated antigen

PGF-R

peptide growth factor receptor

JNK-1

N-terminal Jun kinase-1

MAPKp38

p38 mitogen-activated protein kinase

Molecular events involved in protein synthesis

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

In the past, protein synthesis has not been considered to be fundamental in the control of cell proliferation. However, data are emerging on the involvement of this process in cell growth and tumorigenesis. Protein biosynthesis is a central process in all living cells. It is one of the last steps in the transmission of genetic information stored in DNA on the basis of which proteins are produced to maintain the specific biological function of a given cell. Protein synthesis takes place on ribosomal particles where the genetic information transcribed into mRNA is translated into protein. The process of protein synthesis on the ribosome consists of three phases: initiation, elongation and termination.

The initiation phase

In prokaryotic protein biosynthesis the initiation phase is controlled by a small number of IFs (IFs), IF-1, IF-2 and IF-3. Among these, IF-2 is the most likely to form a ternary complex with GTP and initiator inline image. All three factors are involved in assembling the initiation complex of IFs, initiator inline image, the ribosomal subunits and mRNA. Eukaryotic initiation is much more complex and involves a large number of eukaryotic IFs [1–3]. The 80 S ribosomes dissociate, and 40 S subunits are captured for initiation by binding eIF-1A and eIF-3; the size of the latter causes the particle to sediment at 43S. Initiator tRNA binds, in the form of a ternary complex with eIF-2 and GTP, to produce the 43S preinitiation complex [4,5]. The 43 S preinitiation complex binds to mRNA at the 5′ terminal mGTP cap structure, and then migrates along the mRNA towards the AUG initiation codon [6,7]. The initial binding involves the factors eIF-4E, eIF-4G and eIF-4A, which assemble at the 5′-end of mRNA, thus creating the conditions that allow the melting of intramolecular secondary structures within the mRNA that would otherwise prevent the binding of the 43 S preinitiation complex [1,3,8–10]. The term 48 S preinitiation complex is frequently used, and refers to the 43S–globin–mRNA complex formed in the reticulocyte lysate [11]. When the 43S preinitiation complex stops at the initiation codon, the GTP molecule introduced as part of the eIF-2 complex is hydrolysed to GDP, and this gives energy for the ejection of the IFs bound to the 40 S ribosomal subunit [12]. The IF eIF-5 is involved in this process which is likely to accelerate the hydrolysis of GTP [12]. The release of these factors allows the association of a native 60 S ribosomal subunit, to reconstitute an 80 S ribosome at the initiation codon positioned to commence the elongation stage of translation. The continuity of initiation events requires the recycling of IF molecules. eIF-2 is released as a binary complex with GDP and requires a guanine nucleotide exchange factor, eIF-2B, to catalyse the regeneration of the eIF-2*GTP complex [12,13].

The elongation phase

The elongation phase has been the most extensively studied phase in prokaryotes, both functionally and structurally, over the last 30 years [14]. This phase is controlled by three elongation factors (EFs). EF-Tu (EF-1 in eukaryotes) forms a ternary complex with GTP and amino-acylated tRNAs, it protects the amino-acid ester bond against hydrolysis and carries the amino-acylated tRNA to the ribosomal A-site for the decoding of mRNA by codon–anticodon interactions [15]. When correct codon–anticodon recognition occurs, GTP hydrolysis on EF-Tu is stimulated by the ribosome and EF-Tu:GDP is released [16,17]. However, when compared with other G-proteins EF-Tu has a very low intrinsic GTPase [18]. It works as a time-delayed molecular switch, and the GTPase activity is thus highly stimulated when the ternary complex interacts with ribosome. The nucleotide exchange factor EF-Ts (EF-1 in eukaryotes) converts EF-Tu:GDP into active EF-Tu:GTP. After a proofreading step, the amino-acylated-tRNA is brought into contact with the peptidyl-tRNA in the ribosomal P-site, where peptide bond formation is catalysed adding one amino acid to the growing peptide [19]. The last EF, EF-G (EF-2 in eukaryotes), together with GTP controls the translocation of tRNAs and mRNA on the ribosome [19]. It has been demonstrated that the EF-G*GTP complex mimics the ternary complex of EF-Tu*GTP with tRNA [20]. Therefore, it is likely to make an ‘imprint’ on the ribosome for the ternary complex [21,22]. In this way the site it leaves will become fit for the ternary complex. Translocation will then take place by a conformational change in EF-G so that its ‘tRNA mimicking part’ will occupy the anticodon region of the A-site [20]. Recent results [23] suggest that this conformational change and translocation are subsequent to the GTP hydrolysis. Another interesting finding is that the main contacts between the ribosome and EF-Tu are between domain 1 and the 50 S subunit and between domain 2 and the 30 S subunit. This is interesting, not only because these two domains are common between EF-Tu and EF-G, but also because similar domains are present in all the translational G-proteins [24]. The implication is that all of them will bind to the ribosome in the same way, and that the GTPase activity will be stimulated by the same mechanism. The latter could explain for the late onset of GTPase activity in the translational G-proteins.

The termination phase

Termination of protein synthesis occurs on the ribosomes and requires the presence of a termination (stop) codon in the mRNA at the ribosomal A-site and the polypeptide chain release factor (RF) of class 1 (reviewed in [25–30]). Termination is controlled by RFs which have been extensively studied in recent years [29–31]. In prokaryotes, the stop codons are specifically recognized by RF-1 (that recognizes the UAG and UAA stop signals) and RF-2 (that recognizes the UGA and UAA stop signals), while a third non codon-specific factor, RF-3, in complex with GTP stimulates the activities of the other two factors and the release of the fully synthesized protein from the ribosome [32]. Finally, RF-4 (ribosome release or recycling factor) displaces peptidyl-tRNA from the ribosome in a way related to its proposed function in removing deacylated tRNA during ribosome recycling [33]. The termination reaction leads to hydrolysis of the peptidyl-tRNA, which generates a free nascent polypeptide chain and a free (uncharged) tRNA followed by the release of these components from the ribosome. In eukaryotes, translational termination is governed by a single RF which recognizes all three termination codons [34]. When the structure of this factor had been established it was termed eRF-1 [35]. In contrast with earlier observations [36,37] where the termination reaction was accompanied by concomitant GTP hydrolysis, eRF-1 appeared to be able to catalyse in vitro peptidyl-tRNA hydrolysis at a high termination codon concentration in the absence of GTP and other factors [35–39]. This apparent controversy was resolved when a second RF, termed eRF-3, was identified and shown to be an eRF-1-dependent and ribosome-dependent GTPase [38,39]. It was shown that eRF-1 and eRF-3 bound to each other both in vitro and in vivo[39–42]. eRF-1*eRF-3 complex formation and eRF-3 GTPase activity were strongly interrelated as eRF-3 GTPase activity depended on the complex formation. eRF-3 alone, even capable of binding GTP, is virtually inactive in catalysing GTP hydrolysis on the ribosome [38]. Recently, the mammalian homolog of eRF-3, the GTP-binding protein GSPT (for G1 to S phase transition), has been cloned [43,44]. Its C-terminal binds EF-1A while its N-terminal interacts with the polyadenilate binding protein (PABP) [45]. GSPT inhibits the multimerization of PABP with poly(A) indicating that GSPT/eRF-3 may play an additional role in the regulation of mRNA stability [45].

The overall process of translation termination highly resembles the elongation process except for the fact that a stop codon, instead of a sense codon, is decoded at the A-site of the ribosome [31]. A molecular mimicry has been found between the EF-Tu ternary complex and the RF-1/2-RF-3-GTP complex [31]. The ribosome plays an active part during the translation of both sense and stop codons [26]. The ribosomal A-site decoding centre at the bottom of the intersubunit cavity is opened upon starvation for amino-acylated-tRNA [46]. Ribosomes that reach a stop codon probably undergo a similar conformational change to expose the A-site with the stop codon for RF interaction. Termination/readthrough at a stop codon is sensitive to the flanking codons [47]. The nonsense codon context effect suggests that the RF complex or the competing EF-Tu ternary complex with the near-cognate tRNA, or both, are sensitive to the neighbouring codon(s) either directly or indirectly [47].

Moreover, the mRNA sequence immediately downstream of the stop codon is important for termination efficiency. Conversely, an A as the first base downstream of the stop codon produces inefficient termination. Thus, termination is ruled by a signal of four bases instead of the three that constitute the termination codon itself [48]. In addition to the P-site tRNA that seems to interact with RF, the corresponding last amino acid in the nascent peptide also influences termination efficiency. Thus, a correlation can be found between a high propensity of this amino acid to participate in ordered structures (αL helix, β strand) and efficient termination at UGAA [49].

Recently, ribosome recycling factor or RF-4 has been described in prokaryotes. It unassembles the post-termination complex formed by ribosomes, mRNA and tRNA [34,50–52]. The biochemical processes of protein synthesis are described in Fig. 1.

image

Figure 1. The biochemical mechanisms of protein synthesis. The 40 S ribosomal subunit binds to eIF-1A and eIF-3 thus forming the 43 S complex. The latter allows the binding of eIF-2 (2)*GTP and the initiator t-RNA (⌋met), thus forming the preinitiation 43 S complex. The next step requires the melting of intramolecular secondary structure of mRNA performed by the complex eIF-4B (4B)*eIF-4A(4A)*eIF(4E). eIF-4E is sequestred by PHAS when protein synthesis is stopped. The phosphorylation of PHAS allows the release of eIF-4E and the formation of the complex that, after binding mRNA, allows the assembling of eIF-4F (4F) with the mRNA itself and with the ribosome. The resulting complex sediments at 48 S. GTP in eIF-2 is hydrolysed to GDP (a process to be accelerated by eIF-5) and eIF-2*GDP is ejected from the complex and recruited for novel initiation by the nucleotide exchange factor eIF-2B (2B) with the consequent formation eIF-2*GTP. This event marks the end of initiation and allows the binding of the 60 S ribosomal subunit and the ejection of the other IFs that leads to the formation of the 80 S complex. EF-1*GTP and amino acyl tRNA (aatRNA) binds to the A-site of the ribosome, GTP is again hydrolysed allowing the ejection of EF-1 (E1)*GDP (this process is repeated n times). EF-2 (E2)GTP binds to the ribosome and translocates the mRNA and the tRNA on the ribosome from the A-site to the P-site (this process is repeated n times). When termination codon is encountered eRF-1*GTP binds to the complex recalling an eRF-3 molecule that hydrolyses GTP to GDP in eRF-1 thus allowing the disassembly of the complex.

Download figure to PowerPoint

Protein synthesis and cell proliferation

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

There is presently a growing body of evidence which suggests the involvement of protein synthesis and translational factors in the regulation of cell proliferation and transformation.

The regulation and overexpression of polypeptide chain IFs for cell transformation and malignancy

Over the past few years, studies aimed at understanding the regulation and function of protein synthesis IFs. Initially, study of eIF-4E culminated in the unexpected finding that a moderate overexpression of this factor results in dramatic phenotypic changes, including rapid proliferation and malignant transformation [54–59]. Conversely, the tumorigenic properties of cancer cells can be strongly inhibited by antisense RNA against IF, or overexpression of the inhibitory proteins, such as 4E-BPs also called phosphorylated heat and acid stable proteins I–III (PHAS I–III) [54–60].

When the eIF-4E is overexpressed in mammalian cells in vitro oncogenic transformation occurs [54–56,58,59]. Furthermore, it has been reported that c-myc expression stimulates cell growth by increasing expression of eIF-4E [57] and overexpression of eIF-4G also caused malignant transformation in NIH3T3 cells [61].

The involvement of eIF-4E in carcinogenesis has been demonstrated also in vivo. In fact, eIF-4E is overexpressed not only in all head and neck squamous cell cancers but also in some dysplastic lesions [54,62,63]. Moreover, strong expression of eIF-4E and 2α was found in the germinal centres of reactive follicles of non-Hodgkin's lymphomas [64] and in active growing lymphocytes [64]. eIF-4E expression is also high in colon tumours, and this increase is paralleled, in most but not all cases, by an increase in cyclin D1 levels [65]. These findings suggest that eIF-4E may represent a downstream target of the APC/β-catenin/Tcf-4 pathway, and it is likely to be involved in colon tumorigenesis [65]. Besides IF overexpression, another mechanism capable of inducing cell transformation is the disregulation of IF activity. In fact, mitogenic stimulation of protein synthesis is accompanied by an increase in eIF-4E phosphorylation that enhances its activity [66]. On the other hand, the effects induced on protein synthesis by differentiation are less known. When P19 embryonal carcinoma cells are treated with the differentiating agent retinoic acid, protein synthesis increased during the first hour [66]. However, the phosphorylation state, as well as the turnover of phosphate on eIF-4E, remained unchanged. Increased phosphorylation of the translational repressor protein 4E-BP1 was found in a Rous sarcoma virus-transformed cell line [67]. This was accompanied by its dissociation from the complex with IF eIF-4E [67]. More data seem to confirm the involvement of eIF-4E in the tumour invasiveness and angiogenic process. In fact, indirect evidence exists about the interaction between eIF-4E and the ras-dependent transduction pathway as ras-transformed rat fibroblast cells with reduced eIF-4E had delayed and reduced invasiveness and decreased experimental metastasis [68]. Reduced eIF-4E levels correlates with decreased expression of the metastasis-associated 92-kDa collagenase type IV and of the CD44 adhesion molecule and with decreased levels of the putative metastasis-suppressor protein nm23 [68]. Moreover, eIF-4E facilitates the synthesis of powerful tumour angiogenic factors by selectively enhancing their translation [62]. In vivo evidence has demonstrated a marked increase in eIF-4E in breast invasive ductal carcinoma and in islets of viable cells in the centre of poorly vascularized ductal carcinoma in situ[69]. Expression of eIF-4E was increased by hypoxia and, presumably, in hypoxic areas of these lesions. Clonal expansion of cancer cells, permanently over-expressing eIF-4E, is likely to give them a critical advantage to survive after hypoxia and marks the transition to the vascular phase of cancer progression [69]. In the last years other IFs have been correlated with tumorigenesis, such as eIF-2α and eIF-3-p40 [57,64,70,71]. Moreover, their involvement in tumour cell survival and protection from cytotoxic agents has been shown. In fact, eIF-4G is specifically degraded by caspases during apoptosis in lymphoma cell lines, suggesting its involvement in the protection of tumour cells from programmed cell death [72]. eIF-2α phosphorylation is induced in growth arrested cells by nitric oxide suggesting that nonspecific inhibition of protein synthesis may be a generalized response of cells exposed to high levels of nitric oxide and that inhibition of protein synthesis may contribute to many of the described cytostatic actions of nitric oxide [73].

The actions of the eIF-2 α-specific protein kinase PKR in the control of cell growth and apoptosis

A translational inhibitor was initially isolated from unstimulated Friend leukaemia cells. The inhibitor is a heat-labile, sulfhydryl reagent-insensitive protein [74]. It inhibits protein synthesis in the peptide chain initiation phase by preventing IF-dependent binding of methionyl-tRNA to 40 S ribosomal subunits [74]. The inhibitor contains protein kinase activity which phosphorylates the eIF-2α on Ser51 and inactivates it [74,75]. Later it became clear that eIF-2α kinase is the double-stranded RNA-dependent protein kinase (dsRNA-PK) thought to be a key mediator of the antiviral and antiproliferative effects of interferons (IFNs) and called INF-induced double-stranded RNA-activated kinase (PKR) [75,76]. PKR is a 68-kDa protein in human cells with serine/threonine kinase activity [76]. Thr446 and Thr451 in the PKR activation loop are required in vivo and in vitro for high level kinase activity [77]. In fact, mutation of either residue to Ala impaired translational control by PKR or autophosphorylation and eIF-2α phosphorylation by PKR in vitro[77]. Studies of the physiological function of the kinase suggest that it participates in cell growth and differentiation by regulating protein synthesis. Further data suggest that PKR has additional substrates, and that the kinase may also regulate gene transcription and signal transduction pathways [75]. In fact, PKR may have a tumour suppressor function under normal conditions [78,79]. Expression of a functionally defective mutant of human PKR in NIH3T3 cells resulted in malignant transformation, suggesting that PKR may function as a suppressor of cell proliferation and tumorigenesis through phosphorylation of eIF-2α[76]. NIH3T3 cells transformed by a dominant negative PKR mutant have a dramatically reduced eIF-2α phosphorylation. Furthermore, expression of a mutant form of eIF-2α, which cannot be phosphorylated on Ser51 also caused malignant transformation of NIH3T3 cells [76]. Moreover, studies of human malignancies and tumour cell lines suggest that, in general, patients bearing tumours with a higher PKR content have a more favourable prognosis [75]. In human breast carcinoma cells, dysregulation of PKR may be associated with the establishment or maintenance of the transformed state [80]. In fact, breast tumour cell lines express higher levels of active PKR as compared with nontransformed breast cells suggesting that cancer cell extracts contain a transdominant inhibitor of PKR [80]. A cellular inhibitor of PKR (referred to as p58) has been purified and characterized recently [80,81]. Subsequent cloning and sequencing have revealed that p58 is a member of the tetratricopeptide repeat family of proteins [81]. The stable transfection of p58 in NIH3T3 cells resulted in a transformed phenotype and reduced murine PKR activity and endogenous levels of eIF-2α phosphorylation. Therefore, p58 functions as an oncogene through the downregulation of PKR and subsequent deregulation of protein synthesis even if its expression in tumour cell lines is still to be investigated. The central role of eIF-2α phosphorylation in the regulation of protein synthesis and cell proliferation is proved by the discovery of the phosphorylative loop that involves the cellular glycoprotein p67 [82]. This protein protects eIF-2α from phosphorylation and is, in turn, regulated by an enzyme (p67 deglycosylase) that, when active, removes the carbohydrate moieties from p67, inactivates it and, subsequently, protein synthesis is inhibited. It has recently been found that the phosphorylation of eIF-2α increases at the G2/M phase of cell cycle, and p67 is deglycosylated at the same time. In addition, the level and the activity of p67 deglycosylase also increase at the G2/M boundary of cycling U2-OS cells [82].

EIF-5A and hypusine

Eukaryotic translation IF-5A (eIF-5A; 18 kDa) is highly conserved from yeast to mammalian cells [83,84]. The human cDNA that encodes the eIF-5A precursor has been cloned and sequenced [85]. eIF-5A precursor [(ec-eIF-5A(lys)] is the only cellular protein known to contain a specific lysine residue which is transformed into the unique amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)-lysine]. This amino acid is formed by a series of post-translational reactions starting with the transfer of the butylamine moiety from spermidine to the ε-amino group of one of the lysine residues in the eIF-5A precursor protein, thus forming peptide-bound deoxyhypusine [86]. This intermediate is not accumulated in cells but it is immediately hydroxylated at C-2 of the incoming 4-aminobutyl moiety to form hypusine [87–89]. Hypusine plays a key role in the regulation of eIF-5A function because eIF-5A precursors, which do not contain hypusine, have little, if any, activity [90]. Moreover, the Lys50[RIGHTWARDS ARROW]Arg variant is unable to stimulate methionyl-puromycin synthesis in vitro[90,91] and is inactive in vivo[85]. These data suggest that hypusine synthesis is required for the biological activity of the protein and for interaction with the ribosome.

More recently it has been found that eIF-5A undergoes a post-translational modification catalysed by transglutaminase [92].

eIF-5A promotes the formation of the first peptide bond during the initial stage of protein synthesis [1]. The actual in vivo function of eIF-5A, however, is still only partially known. A series of observations suggests that eIF-5A plays a role in cell growth and differentiation. In fact, the ec-eIF-5A(lys) modification is correlated with cell proliferation [93–96] and is vital for Saccharomyces cerevisiae growth [97]. Moreover, agents that block the lys/hyp transformation [84,98–100] inhibit the growth of mammalian cells [101] inducing reversible arrest at the G1/S boundary of the cell cycle [102–104]. In fact, d,l-α-difluoromethylornithine depresses spermidine and, as a consequence, hypusine formation and it produces a G1/S block in 9L brain tumour cells [102]. In hydralazine-treated cloned mouse T cells, growth arrest in late G1 and inhibition of deoxyhypusyl hydroxylation occur at the same time. In Chinese hamster ovary cells, 2-(4-hydroxy-toluene-3-yl)-4,5dihydro-5-carboxythiazole causes both inhibition of hypusine biosynthesis and reversible cell cycle arrest in late G1 and suppresses proliferation of human T lymphocytes in vitro at the G1/S boundary [103]. Hypusine synthesis increases after mitogen treatment of human peripheral blood lymphocytes [104]. Moreover, both polyamine and hypusine levels (together with the enzymes that regulate their metabolism) [88,89,95,97,105] are correlated to normal and malignant growth. More recently a correlation has been found between the polyamine-dependent modification of eIF-5A and the triggering of apoptosis in tumour cells [106]. In fact, excess putrescine accumulation in hepatoma tissue culture DH23A/b cells induces apoptosis and suppresses the formation of hypusine-containing eIF-5A [106]. Moreover, DH23A cells overexpressing ornithine decarboxylase accumulate putrescine with a consequent increase in apoptosis [107]. The latter effect induces an inhibition of the post-translational modification of eIF-5A that is also blocked by diaminohepatne, another strong inducer of apoptosis in DH23A cells [107]. eIF-5A is found in the cytoplasm in two pools: one free and another bound to endoplasmic reticulum (probably its proper site of action) [108]. However, it is also been recently reported that eIF-5A can accumulate at nuclear pore-associated intranuclear filaments in mammalian cells [109]. Moreover, the factor interacts with the general nuclear export receptor CRM1 and is transported from the nucleus to the cytoplasm [109]. These findings open a new scenario in which eIF-5A may also function as nucleocytoplasmic shuttle protein of mRNAs eventually correlated with cell proliferation.

The involvement of the EF-1 family in oncogenesis

Wide evidence suggests the involvement of the EFs at the onset of oncogenesis. Altered expression of translation EF-1α, a core component of protein synthesis, and closely related sequences have been linked to transformed phenotypes in several independent studies in diverse systems [110,111]. EF-1α is also a microtubule-severing protein [112] whose up-regulation by p53 may be a cause of cell death. A dominant acting oncogene, prostate tumour-inducing gene 1 (PTI-1) has provided further evidence of the link between EF-1α and cell transformation. PTI-1 appears to be a hybrid molecule with components derived from both prokaryotic and eukaryotic origins. The predicted protein coding moiety represents an EF-1α molecule, with a truncated N-terminal to amino-acid residue 68 and six additional point mutations. This coding sequence is linked to a 5′ untranslated region (UTR) that shows a very strong homology to ribosomal RNA derived from Mycoplasma hyopneumoniae[111]. Expression studies have confirmed the oncogenic nature of the molecule. A broad spectrum of tumour-derived cell lines, from varied tissue sources and blood samples from patients with prostate carcinoma, were positive for expression of PTI-1, while corresponding normal tissues or blood samples were negative. Moreover, blocking PTI-1 expression with antisense PTI-1 results in reversion of transformed phenotype in PTI-1-expressing cells. Therefore, it has been proposed that PTI-1 represents a new class of oncogene whose transforming capacity probably arises through mechanisms including: (a) protein translational infidelity, resulting in the synthesis of mutant polypeptides due to the loss of proofreading function during peptide chain elongation; (b) its association with and alteration of the cytoskeleton; (c) impinging on one particular or several different signal transduction pathways through its properties as a G protein [111]. Furthermore, overexpression of EF-1α mRNA has been correlated with increased metastatic potential in mammary adenocarcinoma probably due to its interaction with the actin cytoskeleton, an effector in metastasis [113]. It has been shown that EF-1α protein is overexpressed in metastatic compared with nonmetastatic cells and whole tumours. Similarly to other EF-1α, both types of tumour EF-1α bind to F-actin, but EF-1α from metastatic cells has a reduced affinity for actin. Following stimulation with epidermal growth factor (EGF), there is a parallel increase in the amount of F-actin and EF-1α associated with the cytoskeleton. Therefore, it has been proposed that a weakened association of EF-1α with actin may be related to the metastatic process via an altered organization of the actin cytoskeleton and the differential translation of mRNAs associated with the cytoskeleton [113]. Also EF-1γ is often overexpressed in gastrointestinal cancers, and a cDNA clone, covering part of the C-terminal domain of human EF-1δ, has been isolated from mammary cancer cells by subtractive hybridization [114–116].

The Ca2+/Calmodulin-EF-2 loop and the tumorigenesis

eEF-2 is the target for a very specific Ca2+/calmodulin-dependent eEF-2 kinase [117]. Phosphorylation of eEF-2 makes it inactive in translation, which suggests that protein synthesis can be regulated by Ca2+ through eEF-2 phosphorylation [117]. Recent data demonstrate that eEF-2 phosphorylation can be involved in cell cycle regulation and other processes where changes of intracellular Ca2+ concentration induce a new physiological state of a cell. The main role of eEF-2 phosphorylation in these processes is the temporary inhibition of the overall translation in response to a transient increase in the Ca2+ concentrations in the cytoplasm [117].

Calmodulin-dependent protein kinase III (CaM kinase III, EF-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family [118]. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases [118]. The activity of CaM kinase III is selectively activated in proliferating cells, while the inhibition of the kinase blocked cells in G0/G1–S and decreased viability [119–122].

The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than the one seen in a variety of cell lines with similar rates of proliferation [119–122]. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue [118]. Moreover, the combination of insulin-like growth factor I and EGF stimulated cell proliferation and activated CaM kinase III and the inhibition of the enzyme activity blocked cell proliferation induced by growth factors [122]. Moreover, CaM kinase III was increased in S-phase more than in other phases of the cell cycle. The activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer [118]. A basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III [121]. Serum addition to nonproliferating rat glial cells induces cell growth and restores CaM kinase III activity [120]. Conversely, 8-Br-cAMP, a well known differentiating agent, stimulates dephosphorylation of eEF-2, promoting protein synthesis that leads to neuronal differentiation [123].

In conclusion, the involvement of EFs in cell growth regulation can be explained with the following hypothesis. Temporary changes of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors and thus the activation of expression of new genes.

A description of the involvement of translational factors in cell proliferation and transformation is summarized in Table 1.

Table 1. Evidence of protein synthesis involvement in tumorigenesis.
Experimental dataReference
 eIF-4ETransformation of cells in vitro and specific induction of c-myc
Overexpression in lymphomas, head and neck and colon carcinomas[54,62–65]
Involvement in invasiveness, metastatization and expression of VEGF and FGF-2[65,71]
 eIF-2ATransformation of cells in vitro and overexpression in colon carcinomas
 eIF-4GTransformation of cells in vitro
Degraded by caspases during apoptosis[72]
 4E-BP1 (PHAS)Hyperphosphorylation in transformed cells
 eIF-2APhosphorylation in NO-induced growth arrested cells
Its kinase (PKR) is a tumour suppressor gene[75,76,78,79]
Cancers with higher PKR have a more favourable prognosis[75]
The transdominant inhibitor of PKR (p58) functions as an oncogene[80,81]
p67 protects eIF-2A from phosphorylation and is deglycosylated and inactivated by an enzyme[82]
 involved in transformation 
 eIF-4EMitogens activate protein synthesis by eIF-4E phosphorylation
 Hypusine formation in eIF-5ABlockade of hypusine formation inhibits tumour cell proliferation
Inhibition of hypusine formation correlates with apoptosis[106,107]
 EF-1αOverexpression in transformed cells
Homology with the prostate oncogene PTI-1[111]
Overexpression in metastatic cells[113]
 EF-1γOverexpression in gastrointestinal cancers
 EF-1δOverexpression in cancer cells
 EF-2Its kinase, CaM Kinase III, is specifically activated in growing cells
EGF and IGF-I activate CaM Kinase III[118]
8-Br-cAMP, a differentiating agent, dephosphorylates EF-2[123]

Signal transduction pathways and protein synthesis

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

Interactions between signal transduction and protein synthesis components

Data are emerging on the interactions between the signal transduction pathways that control cell proliferation and the components of the protein synthesis machinery. In fact, the ribosomal S6 kinase (S6K) and several EFs and IFs are targets of signal transduction pathways [124]. The ribosomal S6 protein is phosphorylated at five Ser residues [125]. The major activity responsible for phosphorylation of ribosomal protein S6 is p70 S6K [125], which is activated by hierarchical phospholylation of seven Ser/Thr sites. It has been reported that multipotential S6K is activated and becomes membrane associated in response to insulin [126]. Moreover, p90 S6K is activated in response to growth factors by forming complexes with extracellular signal regulated kinase 1 and 2 (ERK-1 and -2) and then by undergoing phosphorylation [127]. Proteins homologous to p90 S6K are Mnk-1 and 2 and they were identified as binding partners and substrates of ERK-1 and -2 [128]. They are activated by phosphorylation at Thr197 and Thr202 both by a mitogen-activated pathway via ERK-1 or -2 and by a stress-activated pathway via p38 mitogen-activated protein kinase (MAPKp38) [129]. Mnk-1 and -2, in turn, phosphorylate the mRNA cap-binding protein at Ser209 in vivo[128–131], resulting in an increase of its affinity for caps [132]. Interestingly, Mnk-1 also binds the C-terminus of eIF-4G, creating a steric link between this factor and signal transduction pathway [129–133]. The effect of S6 phosphorylation on protein synthesis is unclear. However, there is a correlation between the activation of p70 S6K and the translation of mRNAs containing a 5′ TOP tract [134], and the treatment of 80 S ribosomes with multipotential S6 kinase doubles the elongation rate [126]. A recent study involves S6 phosphorylation in the interaction between 40 S subunits and mRNA [135]. However, the multipotential S6K also has components of protein synthesis different from S6 as substrates; the multipotential S6K is also able to phosphorylate, at unknown sites, eIF-4G as part of the eIF-4F complex [136]. The effect of the phosphorylation is the stimulation of in vitro protein synthesis by the complex eIF-4G–eIF-4F and of the binding of mRNA to the 43 S initiation complex. Another substrate of the multipotential S6K is eIF-1 that is further phosphorylated also by protein kinase C [136]. These phosphorylations result in a stimulation of elongation and GDP/GTP exchange, respectively. Also eIF-2α can be phosphorylated in response to normal signal transduction pathways, e.g. those regulated by amino acids [137] and IL-3 [138]. Phosphorylation of eIF-2α on Ser51 by several different kinases causes the formation of a stable complex with eIF-2B, thereby reducing the concentration of active eIF-2B. Growth factors and their signalling can interfere with the activity and phosphorylation status of eIF-4E. In fact, the treatment of leukemic cells with granulocyte monocyte colony stimulating factor (GM-CSF) stimulates significantly ERK-1/2 activity and the phosphorylation of both eIF-4E and PHAS [139]. Increased PHAS phosphorylation induces eIF-4E activation. These data suggest that GM-CSF exerts part of its growth-promoting effects through the activation of ERK-1/2 and enhancement of eIF-4E and PHAS phosphorylation and dissociation [139]. For a description of these interactions see also Fig. 2.

image

Figure 2. Interactions between protein synthesis and signal transduction: new modalities of intervention. Receptor tyrosine kinase (RTK) autophosphorylation recruits GRB-2*SOS complex on the inner side of the plasma membrane where SOS encounters RAS (attached to the membrane with a farnesyl residue) thus allowing the exchange of a GDP with a GTP and activating RAS. RAS, in turn, activates a kinase cascade that has as an upstream effector RAF-1. The latter stimulates MEK-1 that, finally, induces ERK-1/2 activity. The latter increases protein synthesis through p90S6K and Mnk that acts on eIF-4G. This pathway can be inhibited by tyrosine kinase inhibitors (TK inhibitors), antisense oligonucleotides for RTK (RTK oligo AS), dominant negative RAS (DNRAS), dominant negative RAF-1 (DNRAF-1), MEK-1 inhibitors (PD098059) and 8ClcAMP (that acts directly on ERK-1/2). RTKs can also activate phosphatidylinositol-3-kinase (PI3K) that leads to the formation on plasma membrane of phosphatidylinositol 3,4,5 phosphate [PtdIns (3,4,5)P3] which activates PDK that, in turn, stimulates protein kinase B (PKB) that phosphorylates mTOR. All three factors activate p70S6K. In addition, mTOR inhibits PHAS by phosphorylation thus activating protein synthesis. Finally, RTK can activate phospholipase Cγ (PLCγ) that lyses phosphatidylinositol (4,5) diphosphate [PtdIns (4,5)P2] in inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 induces an intracellular Ca2+ increase that activates calmodulin (CaM) and subsequently CaM kinase that inhibits EF-2 by phosphorylation. DAG activates protein kinase Cα (PKCα) that stimulates EF-1. PKCα can be inhibited by CGP 41251. Cytotoxic drugs, biological agents and INF-α can induce RTK upregulation and subsequent increase of IT binding that is internalized and inhibits EF-2 thus inducing apoptosis. We have demonstrated that INF-α is also able to inhibit hypusine synthesis on eIF-5A (thus inhibiting another step of protein synthesis). Moreover, INF-α induces apoptosis through JNK-1 and MAPKp38 activation. Strategies based on the concomitant administration of its INF-α and JNK-1 activators (drugs or wild-type JNK-1 plasmids) must be explored in order to enhance apoptosis in tumour cells.

Download figure to PowerPoint

Effects of the modification of translational factors on the species of translated mRNAs

On the basis of these data, one can assume that elevated protein synthesis may mark a critical transition in cancer progression. Establishing a greater protein synthesis output may be a necessary step for cancer cells in order to support their rapid proliferation [54,124]. However, analysis of cells transformed by several components of the translational process revealed that the synthesis of only a few proteins was greatly enhanced, while synthesis of most of them was slightly increased. One possible explanation for this is the following: translational factors cause these effects by increasing the translational efficiency of several specific oncogene transcripts, leading to the overexpression of their products. The feasibility of this hypothesis was confirmed experimentally by identifying the interaction between signalling and translational components and by finding several growth regulating proteins, such as growth factors and proto-oncogenes, that are specifically upregulated [124]. In fact, signalling pathways do not stimulate translation of all mRNAs equally and messenger RNAs differ widely in translational efficiency. Factors contributing to low efficiency of translation include a highly structured 5′ UTR, the presence of upstream AUGs, and poor sequence context for the initiating AUG [140]. Many proteins involved in cell growth and cell cycle progression are translated by mRNAs with low efficiency of translation [140]. These mRNAs are poorly translated in quiescent cells but preferably recruited to ribosomes after a mitogenic signal [141–143]. Overexpression of eIF-4E in cultured cells preferentially stimulates translation of a number of mRNAs with high 5′ UTR secondary structure that are involved in cell growth and division [140]. Expression of cell cycle-dependent proteins like c-myc [144] and cyclin D [145] requires mTOR, a Ser-Thr kinase effector of protein kinase B. The inhibition of mTOR by rapamycin prolongs the G1 phase in both T cells [146] and yeast [147]. These results suggest that pathways which activate the unwinding machinery, i.e. by phosphorylation of eIF-4E, eIF-4G, disproportionately stimulate translation of growth-regulated mRNAs with high 5′ UTR secondary structure predominantly involved in cell proliferation. 5′ TOP mRNAs are also differentially regulated in response to extracellular signals [148]. These encode many translational components, including ribosomal proteins, eEF-1α, eEF-2, and PABP. 5′ TOP mRNAs are recruited to polysomes in a growth-dependent fashion that is selectively inhibited by rapamycin [149,150]. These data support the hypothesis that protein synthesis machinery can be regulated by signal transduction effectors. However, this finding alone does not distinguish between signalling through PHAS and signalling through p70 S6K, because the pathway bifurcates downstream of mTOR (Fig. 2) [151]. However, ectopic expression of a dominant interfering p70 S6K blocks both activation of p70 S6K and 5′ TOP mRNA translation, indicating a direct role for 70 S6K [134]. Therefore, mitogen-induced protein synthesis is not only a general phenomenon involving the production and duplication of the components necessary for cell division. Growth factor-induced signalling also interacts specifically with translational factors in order to induce expression of proteins responsible for the progression in the cell cycle. In fact, the insulin-stimulated pathway that leads to general protein synthesis and to growth-regulated protein synthesis in 32D cells can be dissected with rapamycin [144]. Insulin-stimulated total protein synthesis is only 10% inhibited by rapamycin and actin synthesis is not affected at all, but insulin-stimulated c-myc synthesis is inhibited completely. Also, expression of constitutively active protein kinase Cz in the absence of insulin response substance-1, which bypasses the mTOR pathway, allows insulin-stimulated general protein synthesis but not c-myc synthesis [152]. This suggests that the insulin signal bifurcates at some point, one pathway stimulating general protein synthesis and the other stimulating growth-regulated protein synthesis, accounting for 10% of the total translational process, through eIF-4E and S6. This example demonstrates that signalling can differentially regulate the synthesis both of intracellular general proteins and of growth inducers specifically interacting with different translational factors that can represent useful targets for the inhibition of tumour cell proliferation.

Protein synthesis as a target of anticancer agents

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

Several biotechnological agents of natural origin can interfere and block protein synthesis. They are called toxins for their cytotoxic action on eukaryotic cells.

Bacterial and vegetal toxins

The most frequently used bacterial toxins are Pseudomonas exotoxin A (PE) [153,154] and diphteria toxin (DT) [155,156]. These toxins are synthesized as single polypeptide chains with domains devoted to binding, translocation and toxicity [157]. DT is processed after synthesis in its mature form, made up of two disulfide-bonded chains [155,156]. The B chain carries both a cell binding region (domain I that probably binds the galactose receptor) and a translocation region (domain II) that facilitates the insertion of the A chain (domain III) through the membrane of an acidic intracellular compartment in the cytosol [158–160]. In fact, the N-terminal region of the B fragment contains many hydrophobic residues, the presence of which suggests that the B fragment forms a pore which allows the translocation of the A fragment [161]. The A chain kills the cell by catalysing a modification of EF-2 that prevents its participation in protein synthesis. In detail, EF-2 is irreversibly ADP-ribosylated at a modified histidine residue, diphtamide, located at position 715 [162]. PE kills cells by a mechanism similar to that of DT except for the fact that PE remains as a single polypeptide chain after synthesis although proteolysis of the molecule to release the enzymatically active domain in the cytosol is necessary [162,163]. The residues at the carboxyl end of PE are Arg-Glu-Asp-Leu-Lys [164]. Deletion of Lys613 has no effect, but removal of Leu612 or additional amino acids greatly reduces cytotoxicity because the substitution makes the toxin unable to reach the cytosol [165]. As REDL resembles the sequence KDEL, which functions to retain newly synthesized proteins in the endoplasmic reticulum, PE ending in KDEL was made and found to be fully active (somewhat more active than PE itself) [166]. Therefore, after binding, PE enters the cell by endocytosis and is exposed to an acidic pH within an early endocytic vesicle (endosome) that unfolds the toxin and activates a cellular protease [167,168]. The protease cleaves PE between Arg279 and Gly280 [164]. The 37-kDa fragment that is produced by proteolysis and reduction of the disulfide bond (Cys265–287) is transferred by membrane fusion from endocytic vescicle to the trans-golgi and from there to a compartment that returns KDEL-tagged proteins to the endoplasmic reticulum [164]. Once in the endoplasmic reticulum, the 37-kDa fragment may use pre-existing protein transport complexes or pores to gain access to the cytosol. Domain Ia is not necessary for the translocation process.

The most frequently used plant toxins are ricin and abrin. Both are synthesized as single polypeptide chains and are processed post-translationally to yield the mature toxin, which is composed of two disulfide bonded subunits, A and B [169]. The B chain is a galactose-specific lectin that binds a ubiquitous animal cell receptor. The B subunit at the C-terminus has two galactose-binding domains composed of three ancient galactose-binding units, of which only one is active. After binding, the toxin enters the cell by endocytosis and the A chain is translocated across the membrane of an intracellular vescicle into the cytosol, a process facilitated by B chain [170,171]. The process of translocation of A subunit into the cytosol is poorly understood. The A chain is then released from the B chain by reduction and kills the cell by catalitically removing an adenine residue at position 4324 from the ribosomal 60 S subunit, which is necessary for the binding to EF-2 [172,173]. Proteins analogous to the A subunit of ricin and abrin are gelonin and saporin that are single chain ribosome inactivating proteins [174–176]. They are evolutionarily related to the A chain of ricin and inactivate ribosome in an identical fashion. They lack B chain and, consequently, the nonspecific binding.

The tumour associated antigens: peptide growth factor receptors

It is not feasible to use ‘pure’ toxins in anticancer approaches as they bind aspecifically to all eukaryotic cells. Therefore, it is necessary to gain specificity for neoplastic tissues. This has been achieved by targeting tumour surface molecules called tumour associated antigens (TAA) which are proteic, glycoproteic or sphingolypidic structures expressed at higher density on the surface of tumour cells. The bullets generally used to recognize TAAs are mAbs or their fragments. In order to reduce their immunogenicity in vivo, chimeric or humanized mAb have been designed, produced and used in clinical trials. Alternatively, ligands of peptide growth factor receptors (PGF-R) can be satisfactorily used to target TAA. In fact, PGF-Rs can really be considered TAAs. Alterations of growth factor signal transduction are a common finding in cancer cells. Moreover PGFs are involved in the control of tumour cell proliferation in vitro[177,178] and the study of PGF-R expression on tumour biopsies indicates that the levels of PGF-Rs are much higher on tumours than on the normal tissues from which they have been derived [179–182].

Biotechnological products and immunotoxins

Biotechnological products, called immunotoxins, have been constructed in order to link toxins and bullets. An immunotoxin is a cytotoxic agent that consists of a cell binding moiety and a toxic moiety linked via a chemical cross-linker, natural peptide, or disulfide bond. Genetically engineered immunotoxins are single polypeptides in which the toxin and the binding domain of an antitumour mAb are fused by DNA recombination. An additional genetic construct for the selective targeting of tumour cells is made by linking the toxin to a peptide growth factor, or a peptidic hormone that specifically binds receptors at the tumour cell surface. The toxic moiety can be an intact toxin, its toxic A chain, a naturally occurring single chain ribosome inactivating protein, or recombinant toxins [183].

Increase in cytotoxic effects of agents raised against protein synthesis machinery

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

Immunotoxins and related agents have already been used in preclinical and clinical studies, but their activity has been somehow limited [183]. The limitations of this approach are related to the vehicles used to deliver the toxin to the tumour cell surface and by the tumour cell biology itself (host immune response, moiety size, antigen density) [184]. In fact, it has been demonstrated that the efficiency of in vivo targeting of human tumour cells is correlated to the number of membrane antigen sites expressed by the cells [185,186]. Therefore, it was suggested that a more efficient tumour cell targeting could be accomplished by the use of agents able to increase the expression of antigens at the cancer cell surface [186–188]. Another reason of failure for such anticancer approaches is the tumour cell homeostasis linked to the cancer biology. In fact, several mechanisms of escape to immunotoxins can start in a tumour cell with the consequent activation of compensatory or alternative mechanisms of survival. All toxins are raised against EF-2, however EF-2 does not represent the only translational factor involved in the control of tumour cell proliferation. Therefore, the concomitant intervention on the other translational factors directly or through the modulation of the signalling components that control protein synthesis machinery, can constitute an additional therapeutic approach.

Several attempts have been made in order to increase the activity and efficacy of biotechnological products raised against protein synthesis machinery.

Combinatory approaches

As in the case of chemotherapy, the use of different agents prevents the onset of tumour cell resistance and allows the killing of the different clones that constitute the tumour cell population that is often differently sensitive to anticancer drugs. Therefore, immunotoxins have been used in combination with conventional drugs or biological agents, but with poor results [189–195]. An alternative approach is the use of immunotoxins raised against different antigens [196–198] or having different toxic moieties [199,200].

Increase in TAA expression

The increase in TAA expression can be a useful tool to overcome one of the major limitations of anticancer targeting as described above. INF-s have been shown to act as powerful modulators of antigenic expression on tumour cells and appear also capable of increasing the immunotargeting of tumour tissues in vivo[201–207]. In this light we have recently reported that INF-α upregulates the cell receptor for EGF and transferin (TRF) in human epidermoid cancer cells [96,208–210]. EGF-R expression is also increased by other antiproliferative agents such as retinoic acid, tumour necrosis factor-α, conventional anticancer drugs at a low concentration, cytosine arabinoside and 5-aza-2′deoxycytidine [211–220]. Although these studies showed that the binding of immunotoxins to target cells could be increased, they showed no inhibition of the onset of protective responses involving signalling and protein translation that can overcome the antiproliferative activity of the toxin.

Modalities of intervention on signalling and/or specific translational factors

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

As described above, the close interaction between protein synthesis and proliferative signal transduction pathways justifies the intervention on signalling in order to increase the inhibition of protein translation and vice versa.

Protein synthesis is one of the most complicated biochemical processes undertaken by the cell, requiring approximatley 150 different polypeptides and 70 different RNAs [124]. Yet seven polypeptides (eIF-2α, eIF-2Bε, eIF-4E, eIF-4G, S6, eEF-1, and eEF-2) have been identified as targets for regulatory pathways to date [124]. Multiple IFs and EFs were phosphorylated in response to a single extracellular signal [222,223]. This redundancy now seems more comprehensible as the modification of some factors affects the overall rate of translation whereas modification of others affects the spectrum of mRNAs translated. A better understanding of the pathways for the regulation of protein synthesis can open up novel approaches for cancer intervention [224], especially if pathways leading to growth-dependent protein synthesis can be selectively inhibited. In this respect, a widely studied signal transduction pathway is the ras-dependent mitogen activated protein kinase cascade which transduces signals from the growth factor receptor tyrosine kinases to the nucleus, thus initiating proliferation [225].

Growth factors activating signals throughout phosphorylation or other modifications regulate EFs and affect protein synthesis, as demonstrated before. Several attempts have been made to inhibit this pathway whose components are individually capable of inducing malignant transformation when aberrantly controlled [226]. The possibility to interfere at the level of growth factor receptor interaction, receptor activation or at protein target distal to the receptor such as ras and raf has been widely explored in preclinical study and interestingly, in recent years, in some clinical trials. For a description of the most common anticancer strategies raised against signal transduction pathways that, as described above, could represent a useful target for intervening on protein synthesis machinery see Table 2 and Fig. 2.

Table 2. Modalities of intervention on signal transduction pathways.
Molecular tools References
Inhibition of peptide growthAnti-sense oligonucleotides raised against PGF-R such as EGF-R, ErbB2, ErbB3 and ErbB4[215, 225–238]
 factor receptor (PGF-R)Blocking MAbs to specific receptors, e.g. EGF-R, erb-B2[239]
 expression and/or activityDrugs inhibiting receptor-associated tyrosin kinase activity (i.e. quinazolines, pyridopyrimidines[240–243]
 pyrrolpyrimidines) 
Inhibition of rasInhibition of farnesyl-transferase and geranyl-geranyl transferase[244–246]
Interfering genes for ras have been constructed such as RASN17[247,249,250,251]
Inhibition of raf-1Antisense oligonucleotide targeting c-Raf-1[248]
Pharmacological inhibition ofFlavonoids inhibitors of MEK-1 (i.e. PD098059) or blockers of ERK-1/2 activity (i.e. 8 CL-cAMP)[252,254–256]
 ERK-1/2 activation  
PKC inhibitionPKC inhibitors such as the staurosporine analogue CGP 41[253,257–259]

New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

The cross-talk existing between signal transduction and protein synthesis and the availability of different molecular tools interfering with both processes suggest the design of new multimodal therapeutic strategies. Our proposal is summarized in Fig. 2. We have demonstrated that INF-α induces apoptosis on KB cells, but the upregulation of the EGF-R caused by INF-α appeared to mediate an antiapoptotic signal. In fact, INF-α and EGF had opposite effects on apoptosis induction, expression of heat shock proteins and activity of the terminal enzymes of the stress signalling pathway. In more detail, we have found that the activity of N-terminal Jun Kinase-1 (JNK-1) is increased in INF-α-treated cells whereas EGF reduces the activity and the expression of the phosphorylated isoforms of JNK-1 in these cells. Furthermore, the transfection of KB cells with JNK-1wt itself induces apoptosis that was potentiated by INF-α and antagonized by EGF suggesting a direct involvement of JNK-1 in the death mechanisms of INF-α. On the other hand, EGF antagonized apoptosis induced by INF-α either in JNK-1wt-transfected or parental cells. The latter effect suggests that the protection from apoptosis induced by EGF is likely to target JNK-1 as a signalling molecule [260]. These effects are paralleled by modulation of hypusine synthesis. In fact, INF-α induces a strong reduction in hypusine synthesis that is restored when INF-α-treated KB cells are exposed to EGF for 12 h [96]. As post-translational modifications of eIF-5 A are essential for its activity, by reducing hypusine synthesis, INF-α is likely to inhibit the function of the translational factor. Therefore, we have hypothesized that the use of a fusion protein that targets EGF-R (that is also upregulated by INF-α) and inhibits protein synthesis through a downstream step (EF-2) could be useful in increasing the antiproliferative activity of INF-α. We have found that INF-α increases (by about 27-fold) the effect of TP40, formed by the fusion of transforming growth factor α (a ligand of EGF-R) and the modified toxin of Pseudomonas aeruginosa[96]. This effect could be attributed both to INF-α-induced EGF-R upregulation and to INF-α- and TP40-induced inhibition of multiple steps of protein synthesis (see Fig. 2). On the other hand, it has been reported that the cytotoxic effect induced by recombinant toxins could be, at least in part, due to the onset of programmed cell death [261]. Therefore, it can be hypothesized that the intervention on the stress-dependent JNK pathway evoked by INF-α and on the translational factors eIF-5A and EF-2 (induced by the cytokine itself and by TP40, respectively) can produce a synergistic effect on the induction of apoptosis in cancer cells (see Fig. 2). Moreover, the selection of pharmacological inhibitor/s of eIF-5A and/or the use of plasmid/s encoding for dominant negatives of other translational factors could be an additional approach to the selective inhibition of cell proliferation. Therefore, eIF-5A, on the basis of its intrinsic biochemical properties, could represent a useful target in combined approaches for the inhibition of different translational factors.

It was even demonstrated that the transfection of dominant negatives of other translational factors can inhibit cell proliferation (Table 1). Several efforts are being made by the gene therapy and clinical studies on the gene transfer through the use of viruses. It would be interesting to investigate if the concomitant inhibition of different protein synthesis factors could give an advantage in terms of cell growth inhibition. As it has been demonstrated that protein kinase C-, ras- and cAMP-dependent pathways regulate the activity of several elongation and IFs [124] the combined inhibition of these factors through the modulation of signal transduction pathways should be investigated (see Fig. 2). Moreover, the availability of the dominant negatives for the different translational factors could be useful not only for the design of new therapeutic approaches, but also for the identification of their role in the regulation of cell growth by the different pathways. The data obtained could again be useful in the design of new cancer treatments.

Conclusions

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References

As our understanding of mechanisms of malignant transformation and tumour proliferation improves, new molecular targets are continuously revealed, thus providing support to the hope of developing more effective and selective anticancer therapies. On this topic the academic as well as biotechnology company research programs have already produced several clinical grade products, some of which are being used in clinical studies. It is now reasonable to consider that protein synthesis targeting could be an important component of a multimodal approach which might involve cytotoxic compounds as well as immunostimulatory or antiproliferative cytokines. We think that the understanding of the specific interactions at the molecular level among these proliferative and translational pathways could readily increase the activity of combined antitumour approaches that represent the next generation in the fight against cancer.

References

  1. Top of page
  2. Abstract
  3. Molecular events involved in protein synthesis
  4. Protein synthesis and cell proliferation
  5. Signal transduction pathways and protein synthesis
  6. Protein synthesis as a target of anticancer agents
  7. Increase in cytotoxic effects of agents raised against protein synthesis machinery
  8. Modalities of intervention on signalling and/or specific translational factors
  9. New multimodal therapeutic strategies based on protein synthesis inhibition via signal transduction
  10. Conclusions
  11. Acknowledgements
  12. References
  • 1
    Hershey, J.W. (1991) Translational control in mammalian cells. Annu. Rev. Biochem. 60, 717 755.
  • 2
    Merrick, W.C. (1992) Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291 315.
  • 3
    Pain, V.M. (1996) Initiation of protein synthesis in eukaryotic cells. Eur. J. Biochem. 236, 747 771.
  • 4
    Gaspar, N.J., Kinzy, T.G., Scherer, B.J., Humbelin, M., Hershey, J.W.B., Merrick, W.C. (1994) Translation initiation factor eIF-2. Cloning and expression of the human cDNA encoding the gamma-subunit. J. Biol. Chem. 269, 3415 3422.
  • 5
    Hannig, E.M., Cigan, A.M., Freeman. B.A., Kinzy, T.G. (1993) GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 506 520.
  • 6
    Kozak, M. (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J. Biol. Chem. 266, 19867 19870.
  • 7
    Kozak, M. (1989) The scanning model for translation: an update. J. Cell Biol. 108, 229 241.
  • 8
    Rhoads, R.E. (1993) Regulation of eukaryotic protein synthesis by initiation factors. J. Biol. Chem. 268, 3017 3020.
  • 9
    Merrick, W.C. (1994) Eukaryotic protein synthesis: an in vitro analysis. Biochimie 76, 822 830.
  • 10
    Rhoads, R.E., Joshi, B., Minich, W.B. (1994) Participation of initiation factors in the recruitment of mRNA to ribosomes. Biochimie 76, 831 838.
  • 11
    Bommer, U.A., Lutsch, G., Stahl, J., Bielka, H. (1991) Eukaryotic initiation factors eIF-2 and eIF-3: interactions, structure and localization in ribosomal initiation complexes. Biochimie 73, 1007 1019.
  • 12
    Price, N. & Proud, C. (1994) The guanine nucleotide-exchange factor, eIF-2B. Biochimie 76, 748 760.
  • 13
    Dholakia, J.N., Francis, B.R., Haley, B.E., Wahba, A.J. (1989) Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains. J. Biol. Chem. 264, 20638 20642.
  • 14
    Krab, I.M. & Parmeggiani, A. (1998) EF-Tu, a GTPase odyssey. Biochim. Biophys. Acta 1443, 1 22.
  • 15
    Kaziro, Y. (1978) The role of guanosine 5′-triphosphate in polypeptide chain elongation. Biochim. Biophys. Acta 505, 95 127.
  • 16
    Berchtold, H., Reshetnikova, L., Reiser, C.O.A., Schirmer, N.K., Sprinzl, M., Hilgenfeld, R. (1993) Crystal structure of active elongation factor Tu reveals major domain rearrangements. Nature 365, 126 132.
  • 17
    Kjeldgaard, M., Nissen, P., Thirup, S., Nyborg, J. (1993) The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure 1, 35 50.
  • 18
    Wolf, H., Chinali, G., Parmeggiani, A. (1977) Mechanism of the inhibition of protein synthesis by kirromycin. Role of elongation factor Tu and ribosomes. Eur. J. Biochem. 75, 67 75.
  • 19
    Clark, B.F.C., Thirup, S., Kjeldgaard, M., Nyborg, J. (1999) Structural information for explaining the molecular mechanism of protein biosynthesis. FEBS Lett. 452, 41 46.DOI: 10.1016/s0014-5793(99)00562-1
  • 20
    Nyborg, J. & Liljas, A. (1998) Protein biosynthesis: structural studies of the elongation cycle. FEBS Lett. 430, 95 99.DOI: 10.1016/s0014-5793(98)00624-3
  • 21
    Nyborg, J., Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Clark, B.F.C., Reshetnikova, L. (1996) Structure of the ternary complex of EF-Tu: macromolecular mimicry in translation. Trends Biochem. Sci. 21, 81 82.DOI: 10.1016/0968-0004(96)30008-x
  • 22
    Liljas, A. (1996) Imprinting through molecular mimicry. Protein synthesis. Curr. Biol. 6, 247 249.
  • 23
    Rodnina, M.V., Savelsbergh, A., Katunin, V.I., Wintermeyer, W. (1997) Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome. Nature 385, 37 41.
  • 24
    Övarsson, A., Brazhnikov, E., Garber, M., Zheltonosova, J., Chirgadze, Y., Al-Karadaghi, S., Svensson, L.A., Liljas, A. (1994) Three-dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. EMBO J. 13, 3669 3677.
  • 25
    Craigen, W.J., Lee, C.C., Caskey, C.T. (1990) Recent advances in peptide chain termination. Mol. Microbiol. 4, 861 865.
  • 26
    Tate, W. & Brown, C.M. (1992) Translational termination: ‘stop’ for protein synthesis or ‘pause’ for regulation of gene expression. Biochemistry 31, 2443 2450.
  • 27
    Kisselev, L.L. & Frolova, L. & Yu. (1995) Termination of translation in eukaryotes. Biochem. Cell Biol. 73, 1079 1086.
  • 28
    Tate, W.P., Poole, E.S., Mannering, S.A. (1996) Hidden infidelities of the translational stop signal. Prog. Nucleic Acids Res. Mol. Biol. 52, 293 335.
  • 29
    Buckingham, R.H., Grentzmann, G., Kisselev, L. (1997) Polypeptide chain release factors. Mol. Microbiol. 24, 449 456.
  • 30
    Nakamura, Y. & Ito, K. (1998) How protein reads the stop codon and terminates translation. Genes Cells 3, 265 278.
  • 31
    Nakamura, Y., Ito, K., Isaksson, L.A. (1996) Emerging understanding of translation termination. Cell 87, 147 150.
  • 32
    Buckingham, R.H., Grentzmann, G., Kisselev, L. (1997) Polypeptide chain release factors. Mol. Microbiol. 24, 449 456.
  • 33
    Heurgue-Hamard, V., Karimi, R., Mora, L., MacDougall, J., Leboeuf, C., Grentzmann, G., Ehremberg, M., Buckingham, R.H. (1998) Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome. EMBO J. 17, 808 816.
  • 34
    Konecki, D.S., Aune, K.S., Tate, W.P., Caskey, C.T. (1977) Characterization of reticulocyte release factor. J. Biol. Chem. 252, 4514 4520.
  • 35
    Frolova, L., Le Go¡, X., Rasmussen, H.H., Cheperegin, S., Drugeon, G., Kress, M., Arman, I., Haenni, A.-L., Celis, J.E., Philippe, M., Justesen, J., Kisselev, L. (1994) A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor. Nature 372, 701 703.
  • 36
    Goldstein, J.L., Beaudet, A.L., Caskey, C.T. (1970) Peptide chain termination with mammalian release factor. Proc. Natl Acad. Sci. USA 67, 99 106.
  • 37
    Beaudet, A.L. & Caskey, C.T. (1971) Mammalian peptide chain termination. II. Codon specificity and GTPase activity of release factor. Proc. Natl Acad. Sci. USA 68, 618 624.
  • 38
    Frolova, L., Le Goj, X., Zhouravleva, G., Davydova, E., Philippe, M., Kisselev, L. (1996) Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase. RNA 2, 334 341.
  • 39
    Zhouravleva, G., Frolova, L., Le Goj, X., Le Guellec, R., Inge Vechtomov, S., Kisselev, L., Philippe, M. (1995) Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14, 4065 4072.
  • 40
    Stans¿eld, I., Jones, K.M., Kushnirov, V.V., Dagkesamanskaya, A.R., Poznyakovski, A.I., Paushkin, S.V., Nierras, C.R., Cox, B.S., Ter-Avanesyan, M.D., Tuite, M.F. (1995) The products of the SUP45 (eRF1) and SUP35 genes interact to mediate translation termination in Saccharomyces cerevisiae. EMBO J. 14, 4365 4373.
  • 41
    Paushkin, S.V., Kushnirov, V.V., Smirnov, V.N., Ter-Avanesyan, M.D. (1997) Interaction between yeast Sup45p (eRF1) and Sup35p (eRF3) polypeptide chain release factors: implications for prion-dependent regulation. Mol. Cell. Biol. 17, 2798 2805.
  • 42
    Frolova, L.Yu, Simonsen, J.L., Merkulova, T.I., Litvinov, D.Y., Martensen, P.M., Rechinsky, V.O., Camonis, J., Kisselev, L.L., Justesen, J. (1998) Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors. Eur. J. Biochem. 256, 36 44.
  • 43
    Merkulova, T.I., Frolova, L.Y., Lazar, M., Camonis, J., Kisselev, L.L. (1999) C-terminal domains of human translation termination factors eRF1 and eRF3 mediate their in vivo interaction. FEBS Lett. 443, 41 47.DOI: 10.1016/s0014-5793(98)01669-x
  • 44
    Hoshino, S., Imai, M., Mizutani, M., Kikuchi, Y., Hanaoka, F., Ui, M., Katada, T. (1998) Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing factors (eRF). Its identification as eRF3 interacting with eRF1. J. Biol. Chem. 273, 22254 22259.
  • 45
    Hoshino, S., Imai, M., Kobayashi, T., Uchida, N., Katada, T. (1999) The eukaryotic polypeptide chain releasing factor (eRF3/GSPT) carrying the translation termination signal to the 3′-Poly (A) tail of mRNA. Direct association of erf3/GSPT with polyadenylate-binding protein. J. Biol. Chem. 274, 16677 16680.
  • 46
    Öfverstedt, L.G., Zhang, K., Tapio, S., Skoglund, U., Isaksson, L.A. (1994) Starvation in vivo for aminoacyl-tRNA increases the spatial separation between the two ribosomal subunits. Cell 79, 629 638.
  • 47
    Tate, W.P. & Mannering, S.A. (1996) Three, four or more: the translational stop signal at length. Mol. Microbiol. 21, 213 219.
  • 48
    Poole, E.S., Brown, C.M., Tate, W.P. (1995) The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J. 14, 151 158.
  • 49
    Björnsson, A., Mottagui-Tabar, S., Isaksson, L.A. (1996) Structure of the C-terminal end of the nascent peptide influences translation termination. EMBO J. 15, 1696 1704.
  • 50
    Janosi, L., Mottagui-Tabar, S., Isaksson, L.A., Sekine, Y., Ohtsubo, E., Zhang, S., Goon, S., Nelken, S., Shuda, M., Kaji, A. (1998) Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis. EMBO J. 16, 1141 1151.
  • 51
    Ryoji, M., Berland, R., Kaji, A. (1981) Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor. Proc. Natl Acad. Sci. USA 78, 5973 5977.
  • 52
    Janosi, L., Ricker, R., Kaji, A. (1996) Dual functions of ribosome recycling factor in protein biosynthesis: disassembling the termination complex and preventing translational errors. Biochimie 78, 959 969.DOI: 10.1016/s0300-9084(97)86718-1
  • 53
    Kaji, A., Teyssier, E., Hirokawa, G. (1998) Disassembly of the post-termination complex and reduction of translational error by ribosome recycling factor (RRF) − a possible new target for antibacterial agents. Biochem. Biophys. Res. Commun. 250, 1 4.DOI: 10.1006/bbrc.1998.9168
  • 54
    De Benedetti, A. & Harris, A.L. (1999) eIF4E expression in tumors: its possible role in progression of malignancies. Int. J. Biochem. Cell. Biol. 31, 59 72.DOI: 10.1016/s1357-2725(98)00132-0
  • 55
    Shantz, L.M. & Pegg, A.E. (1994) Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation. Cancer Res. 54, 2313 2316.
  • 56
    Raught, B., Gingras, A.C., James, A., Medina, D., Sonenberg, N., Rosen, J.M. (1996) Expression of a translationally regulated, dominant-negative CCAAT/enhancer-binding protein β isoform and up-regulation of the eukaryotic translation initiation factor 2alpha are correlated with neoplastic transformation of mammary epithelial cells. Cancer Res. 56, 4382 4386.
  • 57
    Rosenwald, I.B. (1996) Upregulated expression of the genes encoding translation initiation factors eIF-4E and eIF-2alpha in transformed cells. Cancer Lett. 102, 113 123.DOI: 10.1016/0304-3835(96)04171-7
  • 58
    Anthony, B., Carter, P., De Benedetti, A. (1996) Overexpression of the proto-oncogene/translation factor 4E in breast-carcinoma cell lines. Int. J. Cancer 65, 858 863.DOI: 10.1002/(sici)1097-0215(19960315)65:6<858::aid-ijc25>3.0.co;2-z
  • 59
    West, M.J., Sullivan, N.F., Willis, A.E. (1995) Translational upregulation of the c-myc oncogene in Bloom's syndrome cell lines. Oncogene 11, 2515 2524.
  • 60
    Shantz, L.M., Coleman, C.S., Pegg, A.E. (1996) Expression of an ornithine decarboxylase dominant-negative mutant reverses eukaryotic initiation factor 4E-induced cell transformation. Cancer Res. 22, 5136 5140.
  • 61
    Fukuchi-Shimogori, T., Ishii, I., Kashiwagi, K., Mashiba, H., Ekimoto, H., Igarashi, K. (1997) Malignant transformation by overproduction of translation initiation factor eIF4G. Cancer Res. 57, 5041 5044.
  • 62
    Nathan, C.A., Franklin, S., Abreo, F.W., Nassar, R., de Benedetti, A., Williams, J., Stucker, F.J. (1999) Expression of eIF4E during head and neck tumorigenesis: possible role in angiogenesis. Laryngoscope 109, 1253 1258.
  • 63
    Sorrells, D.L. Jr, Ghali, G.E., De Benedetti, A., Nathan, C.A., Li, B.D. (1999) Progressive amplification and overexpression of the eukaryotic initiation factor 4E gene in different zones of head and neck cancers. J. Oral Maxillofac. Surg. 57, 294 299.
  • 64
    Wang, S., Rosenwald, I.B., Hutzler, M.J., Pihan, G.A., Savas, L., Chen, J.J., Woda, B.A. (1999) Expression of the eukaryotic translation initiation factors 4E and 2alpha in non-Hodgkin's lymphomas. Am. J. Pathol. 155, 247 255.
  • 65
    Rosenwald, I.B., Chen, J.J., Wang, S., Savas, L., London, I.M., Pullman, J. (1999) Upregulation of protein synthesis initiation factor eIF-4E is an early event during colon carcinogenesis Oncogene 18, 2507 2517.
  • 66
    Kleijn, M., Voorma, H.O., Thomas, A.A. (1995) Phosphorylation of eIF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells. J. Cell Biochem. 59, 443 452.
  • 67
    Tuhackova, Z., Sovova, V., Sloncova, E., Proud, C.G. (1999) Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts. Int. J. Cancer 81, 963 969.DOI: 10.1002/(sici)1097-0215(19990611)81:6<963::aid-ijc20>3.0.co;2-c
  • 68
    Graff, J.R., Boghaert, E.R., De Benedetti, A., Tudor, D.L., Zimmer, C.C., Chan, S.K., Zimmer, S.G. (1995) Reduction of translation initiation factor 4E decreases the malignancy of ras-transformed cloned rat embryo fibroblasts. Int. J. Cancer 60, 255 263.
  • 69
    DeFatta, R.J., Turbat-Herrera, E.A., Li, B.D., Anderson, W., De Benedetti, A. (1999) Elevated expression of eIF4E in confined early breast cancer lesions: possible role of hypoxia. Int. J. Cancer 80, 516 522.DOI: 10.1002/(sici)1097-0215(19990209)80:4<516::aid-ijc6>3.0.co;2-7
  • 70
    Eberle, J., Krasagakis, K., Orfanos, C.E. (1997) Translation initiation factor eIF-4A1 mRNA is consistently overexpressed in human melanoma cells in vitro. Int. J. Cancer 71, 396 401.DOI: 10.1002/(sici)1097-0215(19970502)71:3<396::aid-ijc16>3.0.co;2-e
  • 71
    Nupponen, N.N., Porkka, K., Kakkola, L., Tanner, M., Persson, K., Borg, A., Isola, J., Visakorpi, T. (1999) Amplification and overexpression of p40 subunit of eukaryotic translation initiation factor 3 in breast and prostate cancer. Am. J. Pathol. 154, 1777 1783.
  • 72
    Clemens, M.J., Bushell, M., Morley, S.J. (1998) Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines. Oncogene 17, 2921 2931.
  • 73
    Kim, Y.M., Son, K., Hong, S.J., Green, A., Chen, J.J., Tzeng, E., Hierholzer, C., Billiar, T.R. (1998) Inhibition of protein synthesis by nitric oxide correlates with cytostatic activity: nitric oxide induces phosphorylation of initiation factor eIF-2 alpha. Mol. Med. 4, 179 190.
  • 74
    Pinphanichakarn, P., Kramer, G., Hardesty, B. (1977) Partial purification and characterization of a translational inhibitor from Friend leukemia cells. J. Biol. Chem. 252, 2106 2112.
  • 75
    Jagus, R., Joshi, B., Barber, G.N. (1999) PKR, apoptosis and cancer. Int. J. Biochem. Cell. Biol. 31, 123 138.DOI: 10.1016/s1357-2725(98)00136-8
  • 76
    Donze, O., Jagus, R., Koromilas, A.E., Hershey, J.W., Sonenberg, N. (1995) Abrogation of translation initiation factor eIF-2 phosphorylation causes malignant transformation of NIH 3T3 cells. EMBO J. 14, 3828 3834.
  • 77
    Romano, P.R., Garcia-Barrio, M.T., Zhang, X., Wang, Q., Taylor, D.R., Zhang, F., Herring, C., Mathews, M.B., Qin, J., Hinnebusch, A.G. (1998) Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2. Mol. Cell. Biol. 18, 2282 2297.
  • 78
    Koromilas, A.E., Roy, S., Barber, G.N., Katze, M.G., Sonenberg, N. (1992) Malignant transformation by a mutant of the INF-inducible dsRNA-dependent protein kinase. Science 257, 1685 1689.
  • 79
    Meurs, E.F., Galabru, J., Barber, G.N., Katze, M.G., Hovanessian, A.G. (1993) Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase. Proc. Natl Acad. Sci. USA 90, 232 236.
  • 80
    Savinova, O., Joshi, B., Jagus, R. (1999) Abnormal levels and minimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells. Int. J. Biochem. Cell Biol. 31, 175 189.DOI: 10.1016/s1357-2725(98)00140-x
  • 81
    Barber, G.N., Thompson, S., Lee, T.G., Strom, T., Jagus, R., Darveau, A., Katze, M.G. (1994) The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties. Proc. Natl Acad. Sci. USA 91, 4278 4282.
  • 82
    Datta, B., Datta, R., Mukherjee, S., Zhang, Z. (1999) Increased phosphorylation of eukaryotic initiation factor 2alpha at the G2/M boundary in human osteosarcoma cells correlates with deglycosylation of p67 and a decreased rate of protein synthesis. Exp. Cell. Res. 250, 223 230.DOI: 10.1006/excr.1999.4508
  • 83
    Gordon, E.D., Mora, R., Meredith, S.C., Lee, C., Lindquist, S.L. (1987) Eukaryotic initiation factor 4D, the hypusine-containing protein, is conserved among eukaryotes. J. Biol. Chem. 262, 16585 16589.
  • 84
    Park, M.H., Chung, S.I., Cooper, H.L., Folk, J.E. (1984) The mammalian hypusine-containing protein, eukaryotic initiation factor 4D. Structural homology of this protein from several species. J. Biol. Chem. 259, 4563 4565.
  • 85
    Smit-McBride, Z., Dever, T.E., Hershey, J.W.B., Merrick, W.C. (1989) Sequence determination and cDNA cloning of eukaryotic initiation factor 4D, the hypusine-containing protein. J. Biol. Chem. 264, 1578 1583.
  • 86
    Park, M.H., Cooper, H.L., Folk, J.E. (1982) The biosynthesis of protein-bound hypusine (N epsilon-(4-amino-2-hydroxybutyl) lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N epsilon-(4-aminobutyl) lysine). J. Biol. Chem. 257, 7219 7222.
  • 87
    Abbruzzese, A., Park, M.H., Folk, J.E. (1985) Deoxyhypusinehydroxylase: distribution and partial purification from rat testis. Fed. Proc. 44, 1487.
  • 88
    Abbruzzese, A., Park, M.H., Folk, J.E. (1986) Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization. J. Biol. Chem. 261, 3085 3089.
  • 89
    Abbruzzese, A., Liguori, V., Park, M.H. (1988) Deoxyhypusine hydroxylase. Adv. Exp. Med. Biol. 250, 459 466.
  • 90
    Park, M.H., Wolff, E.C., Smith-McBride, Z., Hershey, J.W.B., Folk, J.E. (1991) Comparison of the activities of variant forms of eIF-4D. The requirement for hypusine or deoxyhypusine. J. Biol. Chem. 266, 7988 7994.
  • 91
    Hershey, J.W.B., Smit-McBride, Z., Schnier, J. (1990) The role of mammalian initiation factor eIF-4D and its hypusine modification in translation. Biochim. Biophys. Acta 1050, 160 162.
  • 92
    Beninati, S., Nicolini, L., Jakus, J., Passeggio, A., Abbruzzese, A. (1995) Identification of a substrate site for transglutaminases on the human protein synthesis initiation factor 5A. Biochem. J. 305, 725 728.
  • 93
    Abbruzzese, A. (1988) Developmental pattern for deoxyhypusine hydroxylase in rat brain. J. Neurochem. 50, 695 699.
  • 94
    Abbruzzese, A., Isernia, T., Liguori, V., Beninati, S. (1988) Polyamine-dependent post-translational modification of protein and cell proliferation. In Perspectives in Polyamine Research(Perin, A., Scalabrino, G., Sessa, A., Ferrioli, M.E., eds), pp. 79 84. Wichtig, Milan, Italy.
  • 95
    Beninati, S., Abbruzzese, A., Folk, J.E. (1990) High-performance liquid chromatographic method for determination of hypusine and deoxyhypusine. Annal. Biochem. 184, 16 20.
  • 96
    Caraglia, M., Passeggio, A., Beninati, S., Leardi, A., Nicolini, L., Improta, S., Pinto, A., Bianco, A.R., Tagliaferri, P., Abbruzzese, A. (1997) Interferon α2 recombinant and epidermal growth factors modulate proliferation and hypusine synthesis in human epidermal cancer KB cells. Biochem. J. 324, 737 741.
  • 97
    Schnier, J., Schwelbeerger, H.G., Smit-McBride, Z., Kang, H.A., Hershey, J.W.B. (1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 11, 3105 3114.
  • 98
    Abbruzzese, A., Hanauske-Abel, H.M., Park, M.H., Henke, S., Folk, J.E. (1991) The active site of deoxyhypusyl hydroxylase: use of catecholpeptides and their component chelator and peptide moieties as molecular probes. Biochem. Biophys. Acta 1077, 159 166.
  • 99
    Abbruzzese, A., Park, M.H., Beninati, S., Folk, J.E. (1989) Inhibition of deoxyhypusine hydroxylase by polyamines and by a deoxyhypusine peptide. Biochem. Biophys. Acta 997, 248 255.
  • 100
    Jakus, J., Wolff, E.C., Park, M.H., Folk, J.E. (1993) Features of the spermine-binding site of deoxyhypasine synthase as derived from inhibition studies. Effective inhibition by bis- and mono-guanylated diamines and polyamines. J. Biol. Chem. 268, 13151 13159.
  • 101
    Park, M.H., Wolff, E.C., Folk J.E. (1993) Is hypusine essential for eukaryotic cell proliferation? Trends Biochem. Sci. 18, 475 479.
  • 102
    Park, M.H. (1987) Regulation of biosynthesis of hypusine in Chinese hamster ovary cells. Evidence for eIF-4D precursor polypeptides. J. Biol. Chem. 262, 12730 12734.
  • 103
    Lalande, M. & Hanausske-Abel, H.M. (1990) A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary. Exp. Cell. Res. 188, 117 121.
  • 104
    Park, M.H., Cooper, H.L., Folk, J.E. (1981) Identification of hypusine, an unusual amino acid, in a protein from human lymphocytes and of spermidine as its biosynthetic precursor. Proc. Natl Acad. Sci. USA 78, 2869 2873.
  • 105
    Beninati, S., Abbruzzese, A., Cardinale, M. (1993) Differences in the post-translational modification of proteins by polyamines between weakly and highly metastatic B16 melanoma cells. Int. J. Cancer 53, 792 797.
  • 106
    Tome, M.E. & Gerner, E.W. (1997) Cellular eukaryotic initiation factor 5A content as a mediator of polyamine effects on growth and apoptosis. Biol. Signals 6, 150 156.
  • 107
    Tome, M.E., Fiser, S.M., Payne, C.M., Gerner, E.W. (1997) Excess putrescine accumulation inhibits the formation of modified eukaryotic initiation factor 5A (eIF-5A) and induces apoptosis. Biochem. J. 328, 847 854.
  • 108
    Shi, X.P., Yin, K.C., Zimolo, Z.A., Stern, A.M., Waxman, L. (1996) The subcellular distribution of eukaryotic translation initiation factor, eIF-5A, in cultured cells. Exp. Cell. Res. 225, 348 356.DOI: 10.1006/excr.1996.0185
  • 109
    Rosorius, O., Reichart, B., Kratzer, F., Heger, P., Dabauvalle, M.C., Hauber, J. (1999) Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1. J. Cell. Sci. 112, 2369 2380.
  • 110
    Wang, J.F., Engelsberg, B.N., Johnson, S.W., Witmer, C., Merrick, W.C., Rozmiarek, H., Billings, P.C. (1997) DNA binding activity of the mammalian translation elongation complex: recognition of chromium- and transplatin-damaged DNA. Arch. Toxicol. 71, 450 454.DOI: 10.1007/s002040050410
  • 111
    Gopalkrishnan, R.V., Su, Z.Z., Goldstein, N.I., Fisher, P.B. (1999) Translational infidelity and human cancer: role of the PTI-1 oncogene. Int. J. Biochem. Cell. Biol. 31, 151 162.DOI: 10.1016/s1357-2725(98)00138-1
  • 112
    Kato, M.V. (1999) The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria. Leuk. Lymphoma 33, 181 186.
  • 113
    Edmonds, B.T., Wyckoff, J., Yeung, Y.G., Wang, Y., Stanley, E.R., Jones, J., Segall, J., Condeelis, J. (1996) Elongation factor-1 alpha is an overexpressed actin binding protein in metastatic rat mammary adenocarcinoma. J. Cell. Sci. 109, 2705 2714.
  • 114
    Frazier, M.L., Inamdar, N., Alvula, S., Wu, E., Kim, Y.H. (1998) Few point mutations in elongation factor-1gamma gene in gastrointestinal carcinoma. Mol. Carcinog. 22, 9 15.DOI: 10.1002/(sici)1098-2744(199805)22:1<9::aid-mc2>3.0.co;2-j
  • 115
    Mathur, S., Cleary, K.R., Inamdar, N., Kim, Y.H., Steck, P., Frazier, M.L. (1998) Overexpression of elongation factor-1gamma protein in colorectal carcinoma. Cancer 82, 816 821.DOI: 10.1002/(sici)1097-0142(19980301)82:5<816::aid-cncr3>3.3.co;2-4
  • 116
    Kolettas, E., Lymboura, M., Khazaie, K., Luqmani, Y. (1998) Modulation of elongation factor-1 delta (EF-1 delta) expression by oncogenes in human epithelial cells. Anticancer Res. 18, 385 392.
  • 117
    Ryazanov, A.G. & Spirin, A.S. (1990) Phosphorylation of elongation factor 2: a key mechanism regulating gene expression in vertebrates. New Biol. 2, 843 850.
  • 118
    Parmer, T.G., Ward, M.D., Yurkow, E.J., Vyas, V.H., Kearney, T.J., Hait, W.N. (1999) Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. Br. J. Cancer 79, 59 64.
  • 119
    Cheng, E.H., Gorelick, F.S., Czernik, A.J., Bagaglio, D.M., Hait, W.N. (1995) Calmodulin-dependent protein kinases in rat glioblastoma. Cell Growth Differ. 6, 615 621.
  • 120
    Bagaglio, D.M. & Hait, W.N. (1994) Role of calmodulin-dependent phosphorylation of elongation factor 2 in the proliferation of rat glial cells. Cell Growth Differ. 5, 1403 1408.
  • 121
    Bagaglio, D.M., Cheng, E.H., Gorelick, F.S., Mitsui, K., Nairn, A.C., Hait, W.N. (1993) Phosphorylation of elongation factor 2 in normal and malignant rat glial cells. Cancer Res. 53, 2260 2264.
  • 122
    Calberg, U., Nilsson, A., Skog, S., Palmquist, K., Nygard, O. (1991) Increased activity of the eEF-2 specific, Ca2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells. Biochem. Biophys. Res. Commun. 180, 1372 1376.
  • 123
    Li, H., Chen, H.C., Huang, F.L. (1995) Identification of a rapidly dephosphorylating 95-kDa protein as elongation factor 2 during 8-Br.-cAMP treatment of N1E115 neuroblastoma cells. Biochem. Biophys. Res. Commun. 217, 131 137.DOI: 10.1006/bbrc.1995.2754
  • 124
    Rhoads, R.E. (1999) Signal transduction pathways that regulate eukaryotic protein synthesis. J. Biol. Chem. 274, 30337 30340.
  • 125
    Proud, C.G. (1996) p70, S6 kinase: an enigma with variations. Trends Biochem. Sci. 21, 181 185.DOI: 10.1016/0968-0004(96)10016-5
  • 126
    Chang, Y.W. & Traugh, J.A. (1997) Phosphorylation of elongation factor 1 and ribosomal protein S6 by multipotential S6 kinase and insulin stimulation of translational elongation. J. Biol. Chem. 272, 28252 28257.
  • 127
    Smith, J.A., Poteet-Smith, C.E., Malarkey, K., Sturgill, T.W. (1999) Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo. J. Biol. Chem. 274, 2893 2898.
  • 128
    Waskiewicz, A.J., Flynn, A., Proud, C.G., Cooper, J.A. (1997) Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2. EMBO J. 16, 1909 1920.
  • 129
    Waskiewicz, A.J., Johnson, J.C., Penn, B., Mahalingam, M., Kimball, S.R., Cooper, J.A. (1999) Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo. Mol. Cell. Biol. 19, 1871 1880.
  • 130
    Joshi, B., Cai, A.L., Keiper, B.D., Minich, W.B., Mendez, R., Beach, C.M., Stepinski, J., Stolarski, R., Darzynkiewicz, E., Rhoads, R.E. (1995) Phosphorylation of eukaryotic protein synthesis initiation factor 4E at Ser-209. J. Biol. Chem. 270, 14597 14603.
  • 131
    Flynn, A. & Proud, C.G. (1995) Serine 209, not serine 53, is the major site of phosphorylation in initiation factor eIF-4E in serum-treated Chinese hamster ovary cells. J. Biol. Chem. 270, 21684 21688.
  • 132
    Minich, W.B., Balasta, M.L., Goss, D.J., Rhoads, R.E. (1994) Chromatographic resolution of in vivo phosphorylated and nonphosphorylated eukaryotic translation initiation factor eIF-4E: increased cap affinity of the phosphorylated form. Proc. Natl Acad. Sci. USA 91, 7668 7672.
  • 133
    Pyronnet, S., Imataka, H., Gingras, A.C., Fukunaga, R., Hunter, T., Sonenberg, N. (1999) Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E. EMBO J. 18, 270 279.
  • 134
    Jefferies, B.J., Fumagalli, S., Dennis, P.B., Reinhard, C., Pearson, R.B., Thomas, G. (1997) Rapamycin suppresses 5′TOP mRNA translation through inhibition of p70s6k. EMBO J. 16, 3693 3704.
  • 135
    Chiaberge, S., Cassarino, E., Mangiarotti, G. (1998) The phosphorylation of protein S6 modulates the interaction of the 40S ribosomal subunit with the 5′-untranslated region of a dictyostelium pre-spore-specific mRNA and controls its stability. J. Biol. Chem. 273, 27070 27075.
  • 136
    Morley, S.J., Dever, T.E., Etchison, D., Traugh, J.A. (1991) Phosphorylation of eIF-4F by protein kinase C or multipotential S6 kinase stimulates protein synthesis at initiation. J. Biol. Chem. 266, 4669 4672.
  • 137
    Hinnebusch, A.G. (1997) Translational regulation of yeast GCN4. A window on factors that control initiator-tRNA binding to the ribosome. J. Biol. Chem. 272, 21661 21664.
  • 138
    Ito, T., Jagus, R., May, W.S. (1994) Interleukin 3 stimulates protein synthesis by regulating double-stranded RNA-dependent protein kinase. Proc. Natl Acad. Sci. USA 91, 7455 7459.
  • 139
    Aronica, S.M., Gingras, A.C., Sonenberg, N., Cooper, S., Hague, N., Broxmeyer, H.E. (1997) Macrophage inflammatory protein-1alpha and interferon-inducible protein 10 inhibit synergistically induced growth factor stimulation of MAP kinase activity and suppress phosphorylation of eukaryotic initiation factor 4E and 4E binding protein 1. Blood 89, 3582 3595.
  • 140
    De Benedetti, A. & Harris, A.L. (1999) eIF4E expression in tumors: its possible role in progression of malignancies. Int. J. Biochem. Cell Biol. 31, 59 72.DOI: 10.1016/s1357-2725(98)00132-0
  • 141
    Darveau, A., Pelletier, J., Sonenberg, N. (1985) Differential efficiencies of in vitro translation of mouse c-myc transcripts differing in the 5′ untranslated region. Proc. Natl Acad. Sci. USA 82, 2315 2319.
  • 142
    Manzella, J.M., Rychlik, W., Rhoads, R.E., Hershey, J.W.B., Blackshear, P.J. (1991) Insulin induction of ornithine decarboxylase. Importance of mRNA secondary structure and phosphorylation of eucaryotic initiation factors eIF-4B and eIF-4E. J. Biol. Chem. 266, 2383 2389.
  • 143
    Nielsen, F.C., Ostergaard, L., Nielsen, J., Christiansen, J. (1995) Growth-dependent translation of IGF-II mRNA by a rapamycin-sensitive pathway. Nature 377, 358 362.
  • 144
    Mendez, R., Myers, M.G., White, M.F., Rhoads, R.E. (1996) Stimulation of protein synthesis, eukaryotic translation initiation factor 4E phosphorylation, and PHAS-I phosphorylation by insulin requires insulin receptor substrate 1 and phosphatidylinositol 3-kinase. Mol. Cell. Biol. 16, 2857 2864.
  • 145
    Muise-Helmericks, R.C., Grimes, H.L., Bellacosa, A., Malstrom, S.E., Tsichlis, P.N., Rosen, N. (1998) Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway. J. Biol. Chem. 273, 29864 29872.
  • 146
    Terada, N., Takase, K.P., Abstract, P., Nairn, A.C., Gelfand, E.W. (1995) Rapamycin inhibits ribosomal protein synthesis and induces G1 prolongation in mitogen-activated T lymphocytes. J. Immunol. 155, 3418 3426.
  • 147
    Barbet, N.C., Schneider, U., Helliwell, S.B., Stansfield, I., Tuite, M.F., Hall, M.N. (1996) TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell 7, 25 42.
  • 148
    Hornstein, E., Git, A., Braunstein, I., Avnil, D., Meyuhas, O. (1999) The expression of poly (A)-binding protein gene is translationally regulated in a growth-dependent fashion through a 5′-terminal oligopyrimidine tract motif. J. Biol. Chem. 274, 1708 1714.
  • 149
    Redpath, N.T., Foulstone, E.J., Proud, C.G. (1996) Regulation of translation elongation factor-2 by insulin via a rapamycin-sensitive signalling pathway. EMBO J. 15, 2291 2297.
  • 150
    Jefferies, H.B.J., Reinhard, C., Kozma, S.C., Thomas, G. (1994) Rapamycin selectively represses translation of the ‘polypyrimidine tract’ mRNA family. Proc. Natl Acad. Sci. USA 91, 4441 4445.
  • 151
    von Manteuffel, S., Dennis, P.B., Pullen, N., Gingras, A., Sonenberg, N., Thomas, G. (1997) The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k. Mol. Cell. Biol. 17, 5426 5436.
  • 152
    Mendez, R., Kollmorgen, G., White, M.F., Rhoads, R.E. (1997) Requirement of protein kinase C zeta for stimulation of protein synthesis by insulin. Mol. Cell. Biol. 17, 5184 5192.
  • 153
    Iglewski, B.H. & Sadoff, J.C. (1979) Toxin inhibitors of protein synthesis: production, purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 60, 780 793.
  • 154
    Chung, D.W. & Collier, R.J. (1977) Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect. Immunol. 16, 832 841.
  • 155
    Gill, D.M. & Dinius, L.L. (1971) Observations on the structure of diphtheria toxin. J. Biol. Chem. 246, 1485 1491.
  • 156
    Collier, R.J. (1975) Diphtheria toxin: mode of action and structure. Bacteriol. Rev. 39, 54 85.
  • 157
    Allured, V.S., Collier, R.J., Carrol, S.F., McCay, D.B. (1986) Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc. Natl Acad. Sci. USA 83, 1320 1324.
  • 158
    Kagan, B.L., Filkestein, A., Columbini, M. (1981) Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc. Natl Acad. Sci. USA 78, 4950 4954.
  • 159
    Donovan, J.J., Simon, M.I., Draper, R.K., Montal, M. (1981) Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc. Natl Acad. Sci. USA 78, 172 176.
  • 160
    Sandvig, K. & Olsnes, S. (1980) Diphtheria toxin entry into cells is facilitated by low pH. J. Cell Biol. 87, 828 832.
  • 161
    Johnson, V.G., Wilson, D., Greenfield, L., Youle, R.J. (1988) The role of the diphtheria toxin receptor in cytosol translocation. J. Biol. Chem. 263, 1295 1300.
  • 162
    Carroll, S.F. & Collier, R.J. (1984) NAD binding site of diphtheria toxin: identification of a residue within the nicotinamide subsite by photochemical modification with NAD. Proc. Natl Acad. Sci. USA 81, 3307 3311.
  • 163
    Carroll, S.F. & Collier, R.J. (1988) Amino acid sequence homology between the enzymic domains of diphtheria toxin and Pseudomonas aeruginosa exotoxin A. Mol. Microbiol. 2, 293 296.
  • 164
    Pastan, I., Chaudhary, V., Fitzgerald, D.J. (1992) Recombinant toxins as novel therapeutic agents. Annu. Rev. Biochem. 61, 331 354.
  • 165
    Chaudhary, V.K., Jinno, Y., Fitzgerald, D.J., Pastan, I. (1990) Pseudomonas exotoxin contains a specific sequence at the carboxyl terminus that is required for cytotoxicity. Proc. Natl Acad. Sci. USA 87, 308 312.
  • 166
    Munro, S. & Pelham, H.R. (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48, 899 907.
  • 167
    Jiang, J.X. & London, E. (1990) Involvement of denaturation-like changes in Pseudomonas exotoxin a hydrophobicity and membrane penetration determined by characterization of pH and thermal transitions. Roles of two distinct conformationally altered states. J. Biol. Chem. 265, 8636 8641.
  • 168
    Idziorek, T., Fitzgerald, D., Pastan, I. (1990) Low pH-induced changes in Pseudomonas exotoxin and its domains: increased binding of Triton X-114. Infect. Immunol. 58, 1415 1420.
  • 169
    Olsnes, S., Sandvig, K., Petersen, O.W., van Deurs, B. (1989) Immunotoxins – entry into cells and mechanisms of action. Immunol. Today 10, 291 295.
  • 170
    McIntosh, D.P., Edwards, D.C., Cumber, A.J., Parnell, G.D., Dean, C.J., Ross, W.C., Forrester, J.A. (1983) Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent. FEBS Lett. 164, 17 20.
  • 171
    Youle, R.J. & Neville, D.M. Jr. (1982) Kinetics of protein synthesis inactivation by ricin-anti-Thy 1.1 monoclonal antibody hybrids. Role of the ricin B subunit demonstrated by reconstitution. J. Biol. Chem. 257, 1598 1601.
  • 172
    Endo, Y. & Tsurugi, K. (1987) RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128 8130.
  • 173
    Endo, Y., Mitsui, K., Motizuki, M., Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J. Biol. Chem. 262, 5908 5912.
  • 174
    Stirpe, F. & Barbieri, L. (1986) Ribosome-inactivating proteins up to date. FEBS Lett. 195, 1 8.
  • 175
    Stirpe, F., Olsnes, S., Pihl, A. (1980) Gelonin, a new inhibitor of protein synthesis, nontoxic to intact cells. Isolation, characterization, and preparation of cytotoxic complexes with concanavalin A. J. Biol. Chem. 255, 6947 6953.
  • 176
    Stirpe, F., Gasperi Campani, A., Barbieri, L., Falasca, A., Abbondanza, A., Stevens, W.A. (1983) Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree). Biochem. J. 216, 617 625.
  • 177
    Cross, M. & Dexter, T.M. (1991) Growth factors in development, transformation, and tumorigenesis. Cell 64, 271 280.
  • 178
    Stoscheck, C.M. & King, L.E. (1986) Role of epidermal growth factor in carcinogenesis. Cancer Res. 46, 1030 1037.
  • 179
    Long, M.W. (1992) Blood cell cytoadhesion molecules. Exp. Hematol. 20, 288 301.
  • 180
    Keating, M.T. & Williams, L.T. (1988) Autocrine stimulation of intracellular PDGF receptors in v-sis-transformed cells. Science 239, 914 916.
  • 181
    Neal, D.E., Sharples, L., Smith, K., Fennelly, J., Hall, R.R., Harris, A.L. (1990) The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer 65, 1619 1625.
  • 182
    Yano, H., Shizaki, H., Kobayashi, K., Yano, T., Tahara, H., Tamura, S., Mori, T. (1991) Immunohistologic detection of the epidermal growth factor receptor in human esophageal squamous cell carcinoma. Cancer 67, 91 98.
  • 183
    Vitetta, E.S. & Thorpe, P.E. (1993)Immunotoxins. In Biological Approaches to Cancer Treatment(Mitchell, M.S., ed.), pp.482 495. McGraw-Hill, New York, USA.
  • 184
    Tagliaferri, P., Caraglia, M., Muraro, R., Pinto, A., Budillon, A., Zagonel, V., Bianco, A.R. (1994) Pharmacological modulation of peptide growth factor receptor expression on tumor cells as a basis for cancer therapy. Anti-Cancer Drugs 5, 379 393.
  • 185
    Goldenberg, A., Masui, H., Divgi, C., Kamrath, H., Pentlow, K., Mendelsohn, J. (1989) Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody. J. Natl Cancer Inst. 81, 1616 1625.
  • 186
    Sung, C., Shockley, T.R., Morrison, P.F., Dvorak, H.F., Yarmush, M.L., Dedrick, R.L. (1992) Predicted and observed effects of antibody affinity and antigen density on monoclonal antibody uptake in solid tumors. Cancer Res. 52, 377 383.
  • 187
    Boerman, O., Massuger, L., Makkink, K., Thomas, C., Kenemans, P., Poels, L. (1990) Comparative in vitro binding characteristics and biodistribution in tumor-bearing athymic mice of anti-ovarian carcinoma monoclonal antibodies. Anticancer Res. 10, 1289 1295.
  • 188
    Matzku, S., Bruggen, J., Brocker, E.B., Sorg, G. (1987) Criteria for selecting monoclonal antibodies with respect to accumulation in melanoma tissue. Cancer Immunol. Imunother. 24, 151 157.
  • 189
    Scadden, D.T., Schenkein, D.P., Bernstein, Z., Luskey, B., Doweiko, J., Tulpule, A., Levine, A.M. (1998) Immunotoxin combined with chemotherapy for patients with AIDS-related non-Hodgkin's lymphoma. Cancer 83, 2580 2587.DOI: 10.1002/(sici)1097-0142(19981215)83:12<2580::aid-cncr25>3.0.co;2-c
  • 190
    O'Connor, R., Liu, C., Ferris, C.A., Guild, B.C., Teicher, B.A., Corvi, C., Liu, Y., Arceci, R.J., Goldmacher, V.S., Lambert, J.M. (1995) Anti-B4-blocked ricin synergizes with doxorubicin and etoposide on multidrug-resistant and drug-sensitive tumors. Blood 86, 4286 4294.
  • 191
    Lidor, Y.J., O'Briant, K.C., Xu, F.J., Hamilton, T.C., Ozols, R.F., Bast, R.C. Jr (1993) Alkylating agents and immunotoxins exert synergistic cytotoxic activity against ovarian cancer cells. Mechanism of action. J. Clin. Invest. 92, 2440 2447.
  • 192
    Francisco, J.A., Kiener, P.A., Moran-Davis, P., Ledbetter, J.A., Siegall, C.B. (1996) Cytokine activation sensitizes human monocytic and endothelial cells to the cytotoxic effects of an anti-CD40 immunotoxin. J. Immunol. 157, 1652 1658.
  • 193
    Gelber, E.E. & Vitetta, E.S. (1998) Effect of immunosuppressive agents on the immunogenicity and efficacy of an immunotoxin in mice. Clin. Cancer Res. 4, 1297 1304.
  • 194
    Clinchy, B. & Vitetta, E.S. (1998) The use of an anti-CD3 immunotoxin to prevent the development of lymphoproliferative disease in SCID/PBL mice. J. Immunol. Methods 1, 141 153.
  • 195
    French, R.R., Bell, A.J., Hamblin, T.J., Tutt, A.L., Glennie, M.J. (1996) Response of B-cell lymphoma to a combination of bispecific antibodies and saporin. Leuk. Res. 20, 607 617.DOI: 10.1016/0145-2126(96)00007-0
  • 196
    Dean, G.S., Pusztai, L., Xu, F.J., O'Briant, K., DeSombre, K., Conaway, M., Boyer, C.M., Mendelsohn, J., Bast, R.C. Jr (1998) Cell surface density of p185 (c-erbB-2) determines susceptibility to anti-p185 (c-erbB-2) -ricin A chain (RTA) immunotoxin therapy alone and in combination with anti-p170 (EGFR)-RTA in ovarian cancer cells. Clin. Cancer Res. 4, 2545 2550.
  • 197
    Engert, A., Gottstein, C., Bohlen, H., Winkler, U., Schon, G., Manske, O., Schnell, R., Diehl, V., Thorpe, P. (1995) Cocktails of ricin A-chain immunotoxins against different antigens on Hodgkin and Sternberg–Reed cells have superior anti-tumor effects against H-RS cells in vitro and solid Hodgkin tumors in mice. Int. J. Cancer 63, 304 309.
  • 198
    Flavell, D.J., Boehm, D.A., Emery, L., Noss, A., Ramsay, A., Flavell, S.U. (1995) Therapy of human B-cell lymphoma bearing SCID mice is more effective with anti-CD19- and anti-CD38-saporin immunotoxins used in combination than with either immunotoxin used alone. Int. J. Cancer 62, 337 344.
  • 199
    Bolognesi, A., Tazzari, P.L., Olivieri, F., Polito, L., Lemoli, R., Terenzi, A., Pasqualucci, L., Falini, B., Stirpe, F. (1998) Evaluation of immunotoxins containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OM124): in vitro and in vivo studies. Br. J. Haematol. 101, 179 188.
  • 200
    Li, B.Y. & Ramakrishnan, S.J. (1994) Recombinant hybrid toxin with dual enzymatic activities. Potential use in preparing highly effective immunotoxins. J. Biol. Chem. 269, 2652 2658.
  • 201
    Greiner, J.W., Horan Hand, P., Noguchi, P., Fisher, P.B., Pestka, S., Schlom, J. (1984) Selective interferon-induced enhancement of tumor-associated antigens on a spectrum of freshly isolated human adenocarcinoma cells. Cancer Res. 44, 3208 3214.
  • 202
    Guadagni, F., Schlom, J., Johnston, W.W., Szpak, C.A., Goldstein, D., Smalley, R., Simpson, J.F., Borden, E.C. (1989) Selective interferon-induced enhancement of tumor-associated antigens on a spectrum of freshly isolated human adenocarcinoma cells. J. Natl Cancer Inst 81, 502 512.
  • 203
    Greiner, J.W., Guadagni, F., Noguchi, P., Pestka, S., Colcher, D., Fisher, B.P., Schlom, J. (1987) Recombinant interferon enhances monoclonal antibody-targeting of carcinoma lesions in vivo. Science 235, 895 898.
  • 204
    Rosenblum, M.G., Lamki, L.M., Murray, J.L., Carlo, D.J., Gutterman, J.U. (1988) Interferon-induced changes in pharmacokinetics and tumor uptake of 111In-labeled antimelanoma antibody 96.5 in melanoma patients. J. Natl Cancer Inst. 80, 160 165.
  • 205
    Khun, J.A., Wong, J.Y.C., Beatty, B.G., Esteban, J.M., Williams, L.E., Beatty, J.D. (1992) Gamma-interferon enhancement of carcinoembryonic antigen expression in human colon carcinoma xenografts. J. Immunother. 11, 257 266.
  • 206
    Bernstein, W., Zou, Z.-Q., Black, R.J., Pirollo, K.F., Chang, E.H. (1988) Association of interferon-gamma induced growth inhibition and modulation of epidermal growth factor receptor gene expression in squamous cell carcinoma cell lines. J. Biol. Regul. Homeost. Agents 2, 186 192.
  • 207
    Chang, E.H., Ridge, J., Black, R. et al. (1987) Interferon-gamma induces altered oncogene expression and terminal differentiation in A431 cells. Proc. Soc. Exp. Biol. Med. 206, 319 326.
  • 208
    Budillon, A., Tagliaferri, P., Caraglia, M., Torrisi, M.R., Normanno, N., Iacobelli, S., Palmieri, G., Stoppelli, M.P., Frati, L., Bianco, A.R. (1991) Upregulation of epidermal growth factor receptor induced by alpha-interferon in human epidermoid cancer cells. Cancer Res. 51, 1294 1299.
  • 209
    Caraglia, M., Leardi, A., Corradino, S., Ciardiello, F., Budillon, A., Guarrasi, R., Bianco, A.R., Tagliaferri, P. (1995) alpha-Interferon potentiates epidermal growth factor receptor-mediated effects on human epidermoid carcinoma KB cells. Int. J. Cancer 61, 342 347.
  • 210
    Caraglia, M., Libroia, A.M., Corradino, S., Coppola, V., Guarrasi, R., Barile, C., Genua, G., Bianco, A.R., Tagliaferri, P. (1994) Alpha-interferon induces depletion of intracellular iron content and upregulation of functional transferrin receptors on human epidermoid cancer KB cells. Biochem. Biophys. Res. Commun. 203, 281 288.DOI: 10.1006/bbrc.1994.2179
  • 211
    Jetten, A.M. (1980) Retinoids specifically enhance the number of epidermal growth factor receptors. Nature 284, 626 629.
  • 212
    Komura, H., Wakimoto, H., Chen, C.F., Terakawa, N., Aono, T., Tanizawa, O., Matsumoto, K. (1986) Retinoic acid enhances cell responses to epidermal growth factor in mouse mammary gland in culture. Endocrinology 118, 1530 1136
  • 213
    Adachi, K., Belser, P., Bender, H., Li, D., Rodeck, U., Beneviste, E.N., Wood, D., Schmiegel, W.H., Herlyn, D. (1992) Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor alpha. Cancer Immunol. Immunother. 34, 370 376.
  • 214
    Zuckier, G. & Tritton, T.R. (1983) Adriamycin causes up regulation of epidermal growth factor receptors in actively growing cells. Exp. Cell Res. 148, 155 161.
  • 215
    Baselga, J., Norton, L., Masui, H., Pandiella, A., Coplan, K., Miller, W.H. Jr, Mendelsohn, J. (1993) Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J. Natl Cancer Inst. 85, 1327 1333.
  • 216
    Caraglia, M., Tagliaferri, P., Correale, P., Genua, G., Pepe, S., Pinto, A., Del Vecchio, S., Esposito, G., Bianco, A.R. (1993) Cytosine arabinoside increases the binding of 125I-labelled epidermal growth factor and 125I-transferrin and enhances the in vitro targeting of human tumour cells with anti-(growth factor receptor) mAb. Cancer Immunol. Immunother. 37, 150 156.
  • 217
    Caraglia, M., Pinto, A., Correale, P., Zagonel, V., Genua, G., Leardi, A., Pepe, S., Bianco, A.R., Tagliaferri, P. (1994) 5-Aza-2′-deoxycytidine induces growth inhibition and upregulation of epidermal growth factor receptor on human epithelial cancer cells. Ann. Oncol. 5, 269 276.
  • 218
    Caraglia, M., Leardi, A., de Ricciardi, B., Laurentiis, M., Lentini Graziano, M.L., Matano, E., De Placido, S., Bianco, A.R., Tagliaferri, P. (1996) α-Interferon potentiates the growth inhibitory effects of anti-transferrin receptor monoclonal antibodies. Int. J. Oncol. 8, 847 850.
  • 219
    Caraglia, M., Leardi, A., Improta, S., Perin, V., Ricciardi, B., Arra, C., Ferraro, P., Fabbrocini, A., Pinto, A., Bianco, A.R., Tagliaferri, P. (1997) Transient exposure to cytarabine increases peptide growth factor receptor expression and tumorigenicity of melanoma cells. Anticancer Res. 17, 2369 2376.
  • 220
    Leardi, A., Caraglia, M., Selleri, C., Pepe, S., Pizzi, C., Notaro, R., De Fabbrocini, A., Lorenzo, S., Musicò, M., Abbruzzese, A., Bianco, A.R., Tagliaferri, P. (1998) Desferioxamine increases iron depletion and apoptosis induced by ara-C of human myeloid leukaemic cells. Br. J. Haematol. 102, 746 752.
  • 221
    Vaickus, L., Biddle, W., Cemerlic, D., Foon, K.A. (1990) Interferon gamma augments Lym-1-dependent, granulocyte-mediated tumor cell lysis. Blood 75, 2408 2416.
  • 222
    Duncan, R. & Hershey, J.W.B. (1985) Regulation of initiation factors during translational repression caused by serum depletion. Covalent modification. J. Biol. Chem. 260, 5493 5497.
  • 223
    Morley, S.J. & Traugh, J.A. (1989) Phorbol esters stimulate phosphorylation of eukaryotic initiation factors 3, 4B, and 4F. J. Biol. Chem. 264, 2401 2404.
  • 224
    Aktas, H., Fluckiger, R., Acosta, J.A., Savage, J.M., Palakurthi, S.S., Halperin, J.A. (1998) Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole. Proc. Natl Acad. Sci. USA 95, 8280 8285.
  • 225
    Denhardt, D.T. (1996) Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling. Biochem. J. 318, 729 747.
  • 226
    Huang, P.S. & Heimbrook, D.C. (1997) Oncogene products as therapeutic targets for cancer. Curr. Opin. Oncol. 9, 94 100.
  • 227
    Wagner, R.W. (1994) Gene inhibition using antisense oligodeoxynucleotides. Nature 372, 332 335.
  • 228
    Normanno, N., Bianco, C., Damiano, V., de Angelis, E., Selvam, M.P., Grassi, M., Magliulo, G., Tortora, G., Bianco, A.R., Mendelsohn, J., Salomon, D.S., Ciardiello, F. (1996) Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides. Clin. Cancer Res. 2, 601 609.
  • 229
    Kumar, R., Shepard, H.M., Mendelsohn, J. (1991) Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells. Mol. Cell Biol. 11, 979 986.
  • 230
    Fan, Z., Baselga, J., Masui, H., Mendelsohn, J. (1993) Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res. 53, 4637 4642.
  • 231
    Fan, Z., Masui, H., Atlas, I., Mendelsohn, J. (1993) Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res. 53, 4322 4328.
  • 232
    Fan, Z., Lu, Y., Wu, X., DeBlasio, A., Mendelsohn, J. (1995) Prolonged induction of p21Cip1/WAF1/CDK2/PCNA complex by epidermal growth factor receptor activation mediates ligand-induced A431 cell growth inhibition. J. Cell Biol. 131, 235 242.
  • 233
    Wu, X., Fan, Z., Masui, H., Rosen, N., Mendelsohn, J. (1995) Apoptosis induced by an anti-epidermal growth factor receptor monoclonal antibody in a human colorectal carcinoma cell line and its delay by insulin. J. Clin. Invest. 95, 1897 1905.
  • 234
    Baselga, J., Tripathy, D., Mendelsohn, J., Baughman, S., Benz, C.C., Dantis, L., Sklarin, N.T., Seidman, A.D., Hudis, C.A., Moore, J., Rosen, P.P., Twaddell, T.H.I.C., Norton, L. (1996) Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J. Clin. Oncol. 14, 737 744.
  • 235
    Wu, X., Rubin, M., Fan, Z., DeBlasio, T., Soos, T., Koff, A., Mendelsohn, J. (1996) Involvement of p27KIP1 in G1 arrest mediated by an anti-epidermal growth factor receptor monoclonal antibody. Oncogene 12, 1397 1403.
  • 236
    Peng, D., Fan, Z., Lu, Y., DeBlasio, T., Scher, H., Mendelsohn, J. (1996) Anti-epidermal growth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1 arrest in prostatic cancer cell line DU145. Cancer Res. 56, 3666 3669.
  • 237
    Mendelsohn, J. (1997) Epidermal growth factor receptor inhibition by a monoclonal antibody as anticancer therapy. Clin. Cancer Res. 3, 2703 2707.
  • 238
    Baselga, J., Norton, L., Albanell, J., Kim, Y.M., Mendelsohn, J. (1998) Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res. 58, 2825 2831.
  • 239
    Cobleigh, M.A., Vogel, C.L., Tripathy, D., Robert, N.J., Scholl, S., Fehrenbacher, L., Wolter, J.M., Paton, V., Shak, S., Lieberman, G., Slamon, D.J. (1999) Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J. Clin. Oncol. 17, 2639 2648.
  • 240
    Levitzki, A. (1994) Signal-transduction therapy. A novel approach to disease management. Eur. J. Biochem. 226, 1 13.
  • 241
    Levitzki, A. (1999) Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol. Ther. 82, 231 239.DOI: 10.1016/s0163-7258(98)00066-7
  • 242
    Shawver, L.K., Schwartz, D.P., Mann, E., Chen, H., Tsai, J., Chu, L., Taylorson, L., Longhi, M., Meredith, S., Germain, L., Jacobs, J.S., Tang, C., Ullrich, A., Berens, M.E., Hersh, E., McMahon, G., Hirth, K.P., Powell, T.J. (1997) Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide. Clin. Cancer Res. 3, 1167 1177.
  • 243
    Eckhardt, S.G., Rizzo, J., Sweeney, K.R., Cropp, G., Baker, S.D., Kraynakdagger, M.A., Kuhn, J.G., Villalona-Calero, M.A., Hammond, L., Weiss, G., Thurman, A., Smith, L., Drengler, R., Eckardt, J.R., Moczygemba, J., Hannah, A.L., Von Hoff, D.D., Rowinsky, E.K. (1999) Phase I and pharmacologic study of the tyrosine kinase inhibitor SU101 in patients with advanced solid tumors. J. Clin. Oncol. 17, 1095 1104.
  • 244
    Lowy, D.R. & Willumsen, B.M. (1993) Function and regulation of ras. Annu. Rev. Biochem. 62, 851 891.
  • 245
    Kohl, N.E., Oliff, A., Gibbs, J.B. (1994) Farnesyltransferase inhibitors: Ras research yields a potential cancer therapeutic. Cell 77, 175 178.
  • 246
    Boyartchuk, V.L., Ashby, M.N., Rine, J. (1997) Modulation of Ras and a-factor function by carboxyl-terminal proteolysis. Science 275, 1796 1800.
  • 247
    Scherr, M., Grez, M., Ganser, A., Engels, J.W. (1997) Specific hammerhead ribozyme-mediated cleavage of mutant N-ras mRNA in vitro and ex vivo. Oligoribonucleotides as therapeutic agents. J. Biol. Chem. 272, 14304 14313.
  • 248
    Monia, B.P., Johnston, J.F., Geiger, T., Muller, M., Fabbro, D. (1996) Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase. Nature Med. 2, 688 675.
  • 249
    Stacey, D.W., Feig, L.A., Gibbs, J.B. (1991) Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras. Mol. Cell. Biol. 11, 4053 4064.
  • 250
    Cai, H., Szeberenyi, J., Cooper, G.M. (1990) Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol. Cell. Biol. 10, 5314 5323.
  • 251
    Shu, J., Lee, J.H., Harwalkar, J.A., Oh-Siskovic, S., Stacey, D.W., Golubic, M. (1999) Adenovirus-mediated gene transfer of dominant negative Ha-Ras inhibits proliferation of primary meningioma cells. Neurosurgery 44, 579 578.
  • 252
    Crews, C.M. & Erikson, R.L. (1993) Extracellular signals and reversible protein phosphorylation: what to Mek of it all. Cell 74, 215 217.
  • 253
    Marshall, C.J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 80, 179 185.
  • 254
    Alessi, D.R., Cuenda, A., Cohen, P., Dudley, D.T., Saltiel, A.R. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J. Biol. Chem. 270, 27489 27494.
  • 255
    Caraglia, M., Di Gennaro, E., Barbarulo, D., Marra, M., Tagliaferri, P., Abbruzzese, A.&., Budillon, A. (1999) Up-regulated EGF receptors undergo to rapid internalization and ubiquitin-dependent degradation in human cancer cells exposed to 8-Cl-cAMP. FEBS Lett. 447, 203 209.DOI: 10.1016/s0014-5793(99)00292-6
  • 256
    Budillon, A., Di Gennaro, E., Caraglia, M., Barbarulo, D., Abbruzzese, A., Tagliaferri, P. (1999) 8-Cl-cAMP antagonizes mitogen-activated protein kinase activation and cell growth stimulation induced by epidermal growth factor. Br. J. Cancer 81, 1134 1141.
  • 257
    Ahmad, S., Mineta, T., Martuza, R.L., Glazer, R.I. (1994) Antisense expression of protein kinase C alpha inhibits the growth and tumorigenicity of human glioblastoma cells. Neurosurgery 35, 904 908.
  • 258
    Yazaki, T., Ahmad, S., Chahlavi, A., Zylber-Katz, E., Dean, N.M., Rabkin, S.D., Martuza, R.L., Glazer, R.I. (1996) Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide. Mol. Pharmacol. 50, 236 242.
  • 259
    Glazer, R.I. (1998) The protein kinase ABC's of signal transduction as targets for drug development. Curr. Pharm. Des. 4, 277 290.
  • 260
    Caraglia, M., Abbruzzese, A., Leardi, A., Pepe, S., Budillon, A., Baldassarre, G., De Selleri, C., Lorenzo, S., Fabbrocini, A., Giuberti, G., Vitale, G., Lupoli, G., Bianco, A.R., Tagliaferri, P. (1999) Interferon-alpha induces apoptosis in human KB cells through a stress-dependent mitogen activated protein kinase pathway that is antagonized by epidermal growth factor. Cell Death Differ. 6, 773 780.
  • 261
    Keppler-Hafkemeyer, A., Brinkmann, U., Pastan, I. (1998) Role of caspases in immunotoxin-induced apoptosis of cancer cells. Biochemistry 37, 16934 16942.