1. Top of page
  2. Abstract

FHIT is a tumor suppressor gene that is frequently inactivated in human cancer. Although the Fhit protein is known to hydrolyze diadenosine triphosphate (Ap3A), this hydrolase activity is not required for Fhit-mediated oncosuppression. Indeed, the molecular mechanisms and the regulatory elements of Fhit oncosuppression are largely unknown. Here, we review physiological and pathological aspects of Fhit in the context of the ApnA family of signaling molecules, as well as the involvement of Fhit in apoptosis and the cell cycle in cancer models. We also discuss recent findings of novel Fhit interactions that may lead to new hypotheses about biochemical mechanisms underlying the oncosuppressor activity of this gene. J. Cell. Physiol. 208: 274–281, 2006. © 2006 Wiley-Liss, Inc.

The human tumor suppressor gene FHIT is located on chromosome 3p14.2. FHIT, which contains the most active common fragile site of the human genome, FRA3B, and the t(3;8) familial-kidney-cancer-associated breakpoint (Ohta et al., 1996), is often inactivated in the most common epithelial tumors and leukemia (reviewed in Pekarsky et al., 2002). Fhit belongs to the histidine triad (HIT) superfamily characterized by the canonical motif of three histidines (HØHØHØØ), and exerts a hydrolase activity on dinucleoside triphosphates. Other than the identification of its enzymatic activity, little is known about the physiological role of Fhit. Moreover, while recent studies have focused on Fhit's role in tumorigenesis, and a large body of data on Fhit suppressor activity and its inactivation in tumors has been obtained, the precise molecular mechanisms underlying its function in cancer cells remain unclear. In this review, we use the ApnA family, which includes the Fhit substrate Ap3A, as a starting point for further clues about Fhit's physiological and pathological functions and on recent data pointing to novel Fhit interactions and functions as a tumor suppressor.


  1. Top of page
  2. Abstract

Fhit is a branch 2 member of the HIT superfamily of nucleoside monophosphate hydrolases and transferases (Brenner, 2002), and represents the major diadenosine triphosphate (Ap3A) hydrolase found in animals and fungi. Crystallographic and biochemical studies revealed that Fhit exists in the cell as a homodimer, which binds two molecules of Ap3A that are first sequestered and then hydrolyzed by Fhit to AMP and ADP (Barnes et al., 1996; Draganescu et al., 2000). Fhit can also bind Ap4A but with lower affinity (Brenner et al., 1997; Pace et al., 1998). ApnA were shown to be hydrolyzed by a surface-located enzyme in an asymmetric manner (Guranowski, 2000; Vollmayer et al., 2003). Evidence for the crucial role of Fhit in the cellular metabolism of Ap3A came from studies on tumor and non-tumor cell lines, which demonstrated a striking relationship between Fhit protein expression levels and the concentration of Ap3A (McLennan, 2000). Consistent with those findings, experiments using the yeast homolog of Fhit, Hnt2, showed that disruption of the gene encoding Hnt2 caused an increase in Ap3N (N refers to all other residues) concentration (Chen et al., 1998).

The ApnA family

The diadenosine polyphosphates (ApnA) were discovered in the mid-1960s (Zamecnik et al., 1966). They are ubiquitous compounds present in a wide range of organisms, from bacteria to higher eukaryotes. The naturally occurring ApnAs consist of two adenosine moieties linked 5′–5′ by a polyphosphate bridge containing from 2 to 7 phosphate groups. Diadenosine 5′,5″′-P(1), P(4)-tetraphosphate (Ap4A) and diadenosine 5′,5″′-P(1), P(3)-triphosphate (Ap3A) are the most studied members of this family. The predominant source of intracellular ApnAs is believed to be the back reaction of an aminoacyladenylate with an acceptor nucleotide, catalyzed by various aminoacyl-tRNA synthetases (aaRS) (Goerlich et al., 1982; Plateau and Blanquet, 1982, 1994; Brevet et al., 1989; Kisselev and Wolfson, 1994). Tryptophanyl-tRNA synthetase (TrpRS) is unusual in being the only aminoacyl- tRNA synthetase that is only able to make Ap3A and not Ap4A (Merkulova et al., 1994).

Studies have shown that ApnA act as intracellular and extracellular signaling molecules (Kisselev et al., 1998). Extracellular ApnA can play a role as neurotransmitters or as extracellular signaling molecules after secretion by cells in response to stressful stimuli (Pintor et al., 2000). Other recent studies indicate that they can affect intraocular pressure (Pintor et al., 2002, 2003) and heart vascular tone, and that they exert vasosuppressor action in some tissues but vasodilator action in others (Stavrou et al., 2001; Steinmetz et al., 2002; Stavrou, 2003; Steinmetz et al., 2003). By contrast, the role of intracellular ApnA is still unclear, despite the large spectrum of physiological and pathological effects on cells that have been associated with altered ApnA relative levels (n from 2 to 6), and especially with the Ap3A/Ap4A ratio.

Effects of ApnA levels

Several studies have explored the relationship between intracellular ApnA levels and the proliferative state of cells or tissue. Increased Ap4A levels associated with stalled replication forks have been correlated with slowed replication during S-phase, necessary to permit repair of DNA lesions (reviewed in McLennan, 2000). Other evidence points to a correlation between increased ApnA levels and cell growth inhibition. For example, Claes et al. (2001) showed that hydrolysis of adenosine nucleotides to AMP and adenosine is the key mechanism for nucleotide-induced growth inhibition in glioma cells, concluding that the MAPK cascade was inactivated by adenosine. Indeed, adenosine has been reported to inhibit cell proliferation by inactivation of the MAPK pathway (Hirano et al., 1996) after adenosine activates nucleotide receptors on the plasma membrane (Jackson and Carlson, 1992) or, alternatively, by an adenosine-dependent pyrimidine starvation resulting from adenosine uptake by adenosine transporters (Lasso de la Vega et al., 1994). Sillero et al. (2002) showed that intracellular ApnA levels can influence poly(A)-polymerase activity, consistent with the relationship between dinucleoside polyphosphates and an enzyme catalyzing the synthesis and/or modification of DNA or RNA. By contrast, other studies using different cellular models found no correlation between diadenosine levels and cell growth (reviewed in McLennan, 2000), although differences in extraction methods may have played a significant role in these and other discrepancies in dinucleotide measurements.

One study demonstrating increased intracellular concentrations of both Ap3A and Ap4A in pancreatic islet cells incubated with glucose at concentrations sufficient to induce insulin release suggests a role for Ap4A and Ap3A as second messengers in mediating the glucose-induced blockade of β-cell KATP channels (Martin et al., 1998). A similar role for these compounds in cardiac myocytes has also been proposed (Jovanovic et al., 1997). However, it should be kept in mind that both ATP and ADP, which are present at much higher concentrations than ApnAs, also affect KATP channel activity.

Effect of Ap3A/Ap4A ratio

In addition to ApnA levels, the Ap3A/Ap4A ratio may also act as a signal in regulating the status of a cell. How this ratio serves in signaling might be best understood by analogy to G proteins and their binding to the known signal transducers GTP or GDP, since binding of ApnA-binding proteins to either Ap4A or Ap3A is similar to that. In G proteins, the GTP/GDP ratio is regulated by GTPase activity of the G proteins. Whereas Ap3A and Ap4A are hydrolyzed by their specific hydrolase, they might also be regulated non-enzymatically depending on affinity of ApnA and Apn-1A for the same ApnA-binding proteins. A selective synthesis and/or degradation of a specific ApnA species may lead to a shift in the proportion of various ApnA derivatives, which may serve as a signal to the cell. Vartanian et al. (1997) demonstrated a modulation of the Ap3A/Ap4A ratio upon stimulation with interferons (IFNs). Human cultured cells treated with IFN-alpha or -gamma displayed a loss of proliferative potential, with increased Ap3A and decreased Ap4A concentrations, whereas cells induced to die by apoptosis showed decreased Ap3A but increased Ap4A concentrations. Ap3A is synthesized by mammalian interferon-inducible TrpRS, which, unlike most other aminoacyl RNA synthetases, cannot synthesize Ap4A. Moreover, the interferon-inducible 2′,5′-oligoadenylate synthetase (2′,5′-OAS) is known to catalyze the 2′-adenylation of various ApnA. Fhit, the predominant Ap3A hydrolase, does not recognize mono- or bis-adenylated (or mono- and bis-deoxyadenylated) Ap3A. On the contrary, the diadenosine tetraphosphate counterparts are substrates for the human (asymmetrical) Ap4A hydrolase (Guranowski et al., 2000). The difference in the recognition of the 2′-adenylated Ap3As versus the 2′-adenylated diadenosine tetraphosphates by the respective dinucleoside polyphosphate hydrolases provides a potential mechanism to regulate the ratio of the 2′-adenylated forms of the signaling molecules Ap3A and Ap4A, and the individual levels of Ap3A and Ap4A, in vivo (Fig. 1).

thumbnail image

Figure 1. Possible mechanisms regulating the Ap3A/Ap4A ratio. Increased of Ap3A/Ap4A ratio can be obtained by IFNγ via its receptor that leads to increased activity of tryptophanyl-tRNA synthetase (TrpRS) and, therefore, of Ap3A. IFNγ pathway activation also induced 2′,5′-oligoadenylate synthetase (2′,5′-OAS). Decrease of Ap3A/Ap4A is mediated mainly by Ap3A hydrolysis by Fhit.

Download figure to PowerPoint

An alternative approach to studying the biological relevance of these dinucleotides might involve the identification of specific Ap3A- or Ap4A-binding proteins. The structural dissimilarities among ApnA family members, due to different charge and conformational states of different length oligophosphate chains, may account for the different patterns of binding proteins for each ApnA molecule. Also, a single protein may bind several ApnA but with different affinity, so that concentrations of various ApnA become essential. Of particular interest will be the isolation of proteins with narrow specificity for ApnA derivatives, that is, proteins that bind Ap4A but not Ap3A and vice versa, since such ApnA binders promise to be very helpful in elucidating the signal transduction pathway from ApnA to target molecules (Fuge and Farr, 1993; Baxi et al., 1994; Chavan et al., 1994; Baxi and Vishwanatha, 1995; Barnes et al., 1996; Gasmi et al., 1996; Vartanian, 2003).

Further studies on all aspects of ApnA functions and interactions are needed, since the emerging data increasingly point to the crucial role of this family in the maintenance and regulation of vital cellular functions, and in a second messenger capacity.

Function of the Fhit–Ap3A complex

Fhit oncosuppressive activity was initially believed to be related to its Ap3A hydrolytic activity, although further studies disproved this theory (Siprashvili et al., 1997; Pace et al., 1998; Trapasso et al., 2003). Mutation of His96 of Fhit, which delays Fhit hydrolase activity by 7 orders of magnitude, did not affect its tumor suppressor activity (Siprashvili et al., 1997; Pace et al., 1998). Trapasso et al. (2003), using a series of alleles of Fhit designed to reduce substrate binding (increase in Km) and/or to decrease hydrolytic rates (decrease in kcat), found no correlation between kcat and biological activity, since a His-to-Asn change at position 96 (a nucleophilic mutation that specifically reduces kcat) did not affect the pro-apoptotic activity of Fhit. Furthermore, the authors showed that changes in Km could account for all the differences in pro-apoptotic activity between the alleles. This evidence was taken as clear proof that the pro-apoptotic function of Fhit depends on formation of an enzyme–substrate complex, which is limited by substrate binding but unrelated to substrate hydrolysis, and has led to the postulate that this complex represents the active form of Fhit.

The Fhit–substrate complex (Fhit dimers bound to two Ap3A molecules) presents to the cell an extensively phosphorylated surface plus two adenosine moieties in a deep cavity lined with histidines, arginines, and glutamines (Pace et al., 1998); this negatively supercharged surface might favor Fhit binding to secondary effector molecules and activate Fhit anti-proliferative or pro-apoptotic mechanisms. To date, it cannot be excluded that Fhit interacts directly with other domains, for example, the sequence surrounding Tyr114, which might allow binding through an SH2 domain.

A more complicated mechanism can also be envisioned that takes into account the fine regulation of ApnA levels and the Ap3A/Ap4A ratio. Because the Fhit–Ap3A complex has a short half-life, any mechanisms that serve to stabilize the complex might be critical in prolonging Fhit tumor suppressor function. No less important, however, is the effect of complex stabilization and, in general, the effect of any modulation of Fhit enzymatic activity on intracellular Ap3A concentrations. We also cannot exclude the possibility that variation in the levels of free Ap3A per se affects cell survival. Thus, complex formation might have indirect effects, such as decreasing the pool of free Ap3A by sequestration and/or by subsequent hydrolysis, thereby changing the Ap3A/Ap4A ratio. Other aminoacyl-tRNA synthetases might also form analogous pairs with Ap4A hydrolases, thus regulating the Ap4A level. As discussed more extensively in the last part of this review, interactions of Fhit with proteins such as Ubc9 (Shi et al., 2000; Golebiowski et al., 2004) and the cytoplasmic kinase Src (Pekarsky et al., 2004) might play a role in Fhit–substrate complex stabilization.


  1. Top of page
  2. Abstract

The tumor suppressor functions attributed to Fhit protein (cell cycle and apoptosis regulator) are often observed when its expression is restored in cells that have lost sensitivity to damage signals. In this respect, several lines of evidence point to a role for Fhit as a sensor that can restore the sensitivity of cancer cells to external and/or internal stimuli. It has been hypothesized that modulation of Fhit expression in normal cells does not produce gross phenotypic changes, but rather results in a different behavior under particular conditions (e.g., UV or mitomycin C treatment). Consistent with this hypothesis is the phenotype of Fhit −/− mice, which are viable and grow normally, but have a higher incidence of spontaneous and especially carcinogen-induced tumors compared to the normal littermates.

Insights from mouse models

Several studies have been conducted in mouse models, since the FHIT murine locus is highly similar to the human locus (Glover et al., 1998), encompasses the chromosomal fragile site, and is susceptible to DNA breaks after exposure to carcinogens that inactivate the FHIT gene in preneoplastic and neoplastic lesions.

In vivo models using mouse strains with one or both inactivated alleles have been established to investigate the events required for cancer development (Table 1). Fong et al. (2000) compared the susceptibility of Fhit +/+ and Fhit +/− mice to tumor formation induced by NMBA, a carcinogen that produces esophageal and forestomach tumors. The study showed that after eight doses of carcinogen, 100% of Fhit hemizygous mice developed tumors of the gastrointestinal tract as compared to only 25% of Fhit +/+ mice. In addition, 60% of Fhit +/− mice also developed sebaceous tumors with a phenotype similar to that of tumors in patients with Muir–Torre syndrome, suggesting that Fhit may be a target of damage in a fraction of mismatch repair-deficient cancers. Of interest, the development of NMBA-induced tumors in these mice could be prevented by administration of Fhit-expressing viral vectors (Dumon et al., 2001a).

Table 1. Tumors incidence in Fhit-deficient mice
GenotypeTumors incidence (%)
SpontaneousForestomach (NMBA eight doses)Forestomach (NMBA single dose)Bladder (BBN)Lung (cross with Vhl +/−)
Fujishita et al., 2004Zanesi et al., 2001Fong et al., 2000Zanesi et al., 2001Vecchione et al., 2004Zanesi et al., 2005
  • NMBA: N-nitrosomethylbenzylamine, BBN: N-butyl-N-(4-hydroxybutyl) nitrosamine, Vhl: Von Hippel–Lindau, MTS: Muir–Torre Syndrome, ND: not done.

  • *

    Preventable with administration of Fhit-expressing viral vectors (Dumon et al., 2001).

FHIT +/+30%8.3%25%7.7%8%None
FHIT +/−60%52.9%100%* (60% MTS-like)78.3%46%ND
FHIT −/−77%52.9%ND89.5%28%44%

A subsequent study in which mice with one, both, or neither intact FHIT allele were treated with a single dose of NMBA (Zanesi et al., 2001) found that more than 75% of mice Fhit +/− and Fhit −/− developed tumors compared to 8% of wild-type mice. Additionally, the authors found that more than 50% of heterozygous and nullizygous mice developed spontaneous tumors compared to only 8% of Fhit +/+ mice. Other authors have also demonstrated that untreated Fhit +/− and Fhit −/− knockout mice have a higher incidence of spontaneous tumors than do wild-type mice (Fujishita et al., 2004). These studies provided evidence that the loss of one Fhit allele had the same effect on tumor development as the loss of both alleles, suggesting that Fhit might be haplo-insufficient for tumor suppression.

The situation seems to be more complicated when other organs are considered. Recently, Vecchione et al. (2004)) examined the role of Fhit in the development of bladder cancer using FHIT knockout mice treated with BBN, a potent carcinogen that induces bladder tumors. The authors observed that although 76% of Fhit +/+ mice developed hyperplasia and mild dysplasia, only 8% of those mice showed the invasive carcinoma present in 46% of Fhit +/− and 28% of Fhit −/− mice. However, Fhit +/− mice appeared to be more susceptible to carcinogens compared to Fhit−/− mice, and the authors raise the possibility that Fhit plays a role in modulating unknown partners in bladder carcinogenesis.

Several studies have reported the loss of Fhit expression in lung preneoplastic and neoplastic lesions (Sozzi et al., 1998; Tseng et al., 1999), which frequently exhibit alterations on chromosome 3p, the site of FHIT and other tumor suppressor genes. To analyze the potential cooperation in tumor suppression by different genes on 3p, the incidence of spontaneous and induced lung tumors was evaluated in a cross between mice deficient for FHIT and Vhl, a 3p26-p25 gene frequently altered in lung cancer. The authors observed that 44% of Fhit −/− Vhl +/− mice developed spontaneous lung tumors by 2 years of age compared to none of the single Vhl +/− or Fhit −/− mice. In addition, double-deficient mice had a tumor incidence of 100% after carcinogen treatment. This incidence was higher than that observed for Fhit −/− mice (40%), suggesting that FHIT-deficient mice required further alteration for lung cancer progression. However, the number of mice analyzed in that study was relatively small, and the relationship between FHIT and Vhl requires further investigation (Zanesi et al., 2005).

Cell lines derived from the FHIT mouse model are also proving to be valuable tools in dissecting the molecular pathways involving Fhit. Ottey et al. (2004) observed reduced levels of apoptosis and enhanced clonogenic survival in Fhit −/− cells compared to Fhit +/+ cells after exposure to mitomycin C and UV-C treatment. In addition, Fhit −/− cells had a strong DNA checkpoint, regulated by an over-activation of the ATR/CHK1 pathway, which contributed to the radio-resistance and thus cell survival (Hu et al., 2005a). In particular, Fhit and Chk1 appear to have opposing roles in homologous recombination repair (Hu et al., 2005b), with loss of Fhit expression leading to enhanced repair activity and possibly to increased survival of cells with increased mutation burdens (Fig. 2).

thumbnail image

Figure 2. FHIT and damage activated pathways. Schematic representation of how loss of Fhit expression could contribute to carcinogenesis by providing survival advantage of damaged cells.

Download figure to PowerPoint

Both in vitro and animal model studies continue to provide valuable pieces of the puzzle of Fhit's role in tumorigenesis and are instrumental in developing novel therapeutic tools in cancer prevention and treatment (Ishii et al., 2004).

Fhit regulation of the cell cycle and apoptosis

Evidence obtained mainly through analyses of cells isolated from the Fhit knockout mouse and through transduction experiments in cancer cell lines supports the involvement of the Fhit protein in cell cycle and apoptosis regulation. Although Fhit −/− and Fhit +/+ cells isolated from mouse kidneys do not differ in their proliferation properties, the Fhit-negative cells reportedly show an increase in the S-phase of the cell cycle (+ 10% of cells) and a decrease in the G1-phase (− 10% of cells) compared to the normal counterpart, suggesting that the S and G2 checkpoints might be over-activated (Ottey et al., 2004). Consistent with this notion is the apparent contribution of a strong S-phase checkpoint in Fhit-negative cells to the UV resistance of these cells, slowing damaged cells before an apoptotic response is triggered. It seems, therefore, that in normal cells Fhit could participate in the regulation of the S/G2/M transition, possibly by facilitating the entrance into apoptosis of cells with genomic alterations.

One of the first effects observed in transduction experiments using Fhit-negative lung cancer cells was a transient accumulation of cells in G2 phase, before the onset of an apoptotic effect resulting in the appearance of the sub-G0 peak and accumulation of the remaining cells in G1 and S phases (Roz et al., 2002).

In general, restoration of Fhit expression in cancer cells reduces the proliferation rate (Ishii et al., 2001a), although not all lines tested respond to Fhit restoration treatment (Werner et al., 2000). This non-responsiveness might rest in tissue-specific differences, in additional underlying genetic alterations, or in factors related to expression levels of either the transduced or the residual endogenous protein. In fact, an accurate analysis performed using an inducible system showed that the anti-proliferative effect of Fhit is exquisitely dose-dependent (Cavazzoni et al., 2004).

At the molecular level, the growth suppressive properties of Fhit have been linked to upmodulation of the cell cycle regulator p21waf1, a potent and tightly binding inhibitor of cyclin-dependent kinases. This link has been demonstrated in different experimental models at both the RNA and protein levels (Sard et al., 1999), and has been shown to be p53-independent (Cavazzoni et al., 2004). The relationship between Fhit and p53 pathways has also been investigated extensively in light of the high frequency of alterations of both genes in human cancers, and has revealed a synergistic oncosuppressor activity (Nishizaki et al., 2004). Although some reports suggest that Fhit does not require a functional p53 for its activity, since even p53-negative cells showed reduced growth and evidence of apoptosis after Fhit transduction (Ji et al., 1999; Roz et al., 2002), the coordinated expression of the two proteins has a very potent anti-tumor effect. While this synergistic effect might be due to stabilization of p53 related to Fhit-mediated downregulation of MDM2 (Nishizaki et al., 2004), it more likely rests in the convergence of the two pathways on a common mediator such as p21waf1 (Cavazzoni et al., unpublished data).

Other clues to the effects of Fhit on the cell cycle come from microarray experiments where the majority of regulated transcripts after FHIT restoration were represented by genes involved in mitotic control; coordinated downregulation of kinesin family members (KNSL1, KNSL6), DNA replication factors (MCM2, MCM5), and proteins involved in spindle assembly checkpoints (BUB1, KNTC2) has been observed (Roz et al., 2003).

The onset of apoptosis after Fhit expression restoration has been consistently reported in many studies (Ishii et al., 2001a). Three main characteristics appear to typify Fhit-induced apoptosis: (i) slow onset, requiring long-lasting and sustained Fhit expression; (ii) significant enhancement by other apoptotic stimuli; and (iii) general absence in cells with normal Fhit expression. Thus, a likely scenario is that Fhit acts to lower the apoptotic threshold of damaged (cancer) cells. A putative sensor role of Fhit would explain the time- and dose-dependence of the response, the synergy with other stimuli, and the absence of any perturbation in normal cells by overexpression of this protein.

Several mediators have been implicated in Fhit-mediated apoptosis, including activation of different caspases (-8, -9, -3, and -2), cleavage of multiple substrates (PARP, β-catenin, Bid), and loss of mitochondrial potential (Dumon et al., 2001b; Ishii et al., 2001b; Roz et al., 2002). Consistent with these data, caspase inhibitors have been shown to prevent apoptosis after Fhit restoration (Ishii et al., 2001b).

Two main apoptotic pathways have been described, the “extrinsic” (cytoplasmic) pathway, which is strongly linked to signals from membrane receptors and headed by caspase-8, and the “intrinsic” (mitochondrial) pathway, regulated mainly by Bcl-2 family members at the mitochondrial level and reliant on caspase-9 activation. To understand how Fhit relates to apoptosis, the connection of the two pathways at the cellular level by several mediators must be considered. Caspase-8-mediated activation of the mitochondrial pro-apoptotic factor Bid represents one of the best-known links. In cells where the two pathways are strongly linked, observable involvement of mediators of both pathways in the late stages of the apoptotic response is expected. However, lung cancer cells with a blocked mitochondrial apoptotic program due to Bcl-2 or Bcl-X(L) overexpression still exhibit apoptosis, despite the absence of cytochrome-c release from the mitochondria (Roz et al., 2004), indicating that this amplification step is not necessary for Fhit activity. This is consistent with the reported activation of caspase-8 in the early phases of Fhit-induced apoptosis (Roz et al., 2002) and suggests that Fhit exerts its apoptosis-facilitating function mainly at the cytoplasmic level.

These observations have potential clinical and therapeutic implications since knowledge of the apoptotic pathways triggered by different agents can provide valuable information about chemo-resistance mechanisms and the possible synergy or antagonism among different treatments. In particular, not only are Fhit −/− cells reportedly mitomycin C- and UV-C-resistant (Ottey et al., 2004), but also Fhit was recently shown to modulate sensitivity to cisplatin in lung cancer cells (Andriani et al., 2005).


  1. Top of page
  2. Abstract

Whereas a considerable body of evidence has been obtained for the role of Fhit in apoptosis and cell cycle regulation, there is still very little information on Fhit-interacting molecules despite the use of various biochemical approaches and two-hybrid systems. The discovery that the FHIT gene in D. melanogaster and C. elegans presents an additional amino-terminal domain, which is homologous to plant and bacteria nitrilase Nit, and that the two enzymes are then encoded as a separate genes in mammals (FHIT and Nit1) suggested a strict cooperation of the two enzymes in the same pathway (Pekarsky et al., 1998). Nevertheless, no physical interaction or reciprocal influence between the two enzymes has yet been reported.

As a step toward elucidating the biological function of FHIT gene, attempts have been made to clarify the exact intracellular localization of the Fhit protein. Analysis of Fhit protein expression in subcellular fractions of normal rat tissue suggested that the protein is localized at the plasma membrane and also in the nucleus (Golebiowski et al., 2001). Based on the reported in vitro physical interaction of both wild-type Fhit and FhitH96N with tubulin, leading to an increase in microtubule mass (Chaudhuri et al., 1999), Golebiowski et al. (2001) suggested that this interaction was compatible with Fhit localization at the plasma membrane. Nevertheless, a Fhit–tubulin interaction has not been confirmed in any in vivo system, and the significance of this interaction, if any, for the tumor suppressor role of Fhit remains unknown. Subcellular fractionation experiments in our laboratory using human cancer cell lines suggest the localization of Fhit in other cellular compartments in addition to the cytoplasm (Campiglio et al., unpublished data). Of interest, particular conditions in both immunohistochemical and biochemical analyses are required to unmask the Fhit molecule in non-cytoplasmic compartments, suggesting that Fhit is tightly complexed to other molecules and thus not easily revealed. Shi et al. (2000) using Chinese hamster ovary (CHO) cells first reported the binding, both in vitro and in vivo, of the C-terminal portion of Fhit to ubiquitin-conjugating enzyme 9 (Ubc9) and showed that this interaction occurred only when Fhit was complexed with Ap3A but not with free Fhit (Fig. 3). A subsequent study (Golebiowski et al., 2004) suggested that this interaction suppresses the enzymatic activity of Fhit, possibly leading to a prolonged half-life of the Fhit–Ap3A complex that represents the native signaling form of Fhit for its action as a tumor suppressor in the cell (Pace et al., 1998; Trapasso et al., 2003). Of interest is the involvement of Ubc9 in sumoylation pathways, which affect post-translational modifications of proteins able to regulate several processes, such as nuclear translocation of proteins or enhancement of protein stability (Muller et al., 2001; Seeler and Dejean, 2003). Thus, it has been hypothesized that Ubc9 binding to the Fhit–Ap3A complex not only increases Fhit's antitumor activity by means of prolonging the complex lifetime, but can also lead to sumoylation of Fhit and to its translocation to the nucleus. The latter phenomenon can also be influenced and controlled by Ap3A intracellular levels. Many questions remain about the Ubc9–Fhit–Ap3A interaction regarding its biological function and regulation in both normal and pathological conditions.

thumbnail image

Figure 3. Representation of known (equation image) and suggested (equation image) interactions of Fhit protein.

Download figure to PowerPoint

Recent data have highlighted another interesting regulation of the Fhit protein. Pekarsky et al. (2004) demonstrated that Fhit tyrosine 114 undergoes phosphorylation by activated Src in vitro and in vivo in normal human tissue and in human embryonic kidney cells. A subsequent study by the same authors (Garrison et al., 2005) provided evidence that the Fhit dimer can exist in three different phosphorylation states, that is, non-phosphorylated, mono-phosphorylated, or phosphorylated on both Fhit monomers. The mono- and di-phosphorylated Fhit state was correlated with a decrease in Km and kcat that results in the increased half-life of the Fhit–Ap3A complex. The sequence surrounding Fhit tyrosine 114 is compatible with binding by an SH2 domain. To interpret this Fhit modification by Src, it is important to consider that the cytoplasmic tyrosine kinase Src is involved in the regulation of a variety of normal and oncogenic processes, and is commonly activated by growth factors (Maa et al., 1995). Moreover, Src is a known downstream molecule of several tyrosine kinase receptors that are often abnormally expressed and activated, such as the EGFR family (EGFR, HER2, HER3, and HER4), whose members are overexpressed in 25% of human breast and ovarian carcinomas (Slamon et al., 1989). Our studies of breast pathology have revealed a decrease or absence of Fhit protein in almost 70% of breast tumors (Campiglio et al., 1999), and our recent analysis of 400 breast carcinomas indicating that Fhit-negative tumors are more frequently HER2-positive has raised the possibility of cooperation between the Fhit and HER2 pathways. Using a mouse model obtained by crossing heterozygous FHIT mice with transgenic HER2/neu mice, we were able to demonstrate a clear protection by the FHIT gene in the development of mammary tumor growth driven by transgenic HER2/neu (Campiglio et al., 2003). To further investigate this cooperation, we recently analyzed whether mitogenic stimulation via HER2 influences the steady-state level of the Fhit protein; indeed, the activation of overexpressed EGFR and/or HER2 led to decreased Fhit protein levels both in breast and ovarian cancer cell lines and properly transfected models, and phosphorylation of Fhit tyrosine-114 by Src appeared to be required for EGF-dependent Fhit downmodulation (Campiglio et al. unpublished data). Fhit protein downregulation can be attributed to post-translational machinery such as degradation by a proteasome system activated as a consequence of phosphorylation. The biological function of Fhit protein modifications by Src, together with a possible strict cooperation between Fhit and HER2, require further exploration since such studies may provide insight into a new biochemical pathway involving Fhit.


  1. Top of page
  2. Abstract

While hundreds of studies have been published on the FHIT gene, with extensive examination of its inactivation in several cancer histotypes and its role as a tumor suppressor gene, Fhit enzymatic activity and the functional role of its substrate in physiological and pathological conditions have received less attention. After rigorous confirmation that the hydrolase activity of Fhit on Ap3A is unrelated to its tumor suppressor function (Siprashvili et al., 1997; Trapasso et al., 2003), and after acceptance of the view that the Fhit–Ap3A complex exerts the apoptotic function of Fhit, the intracellular regulation of Ap3A has lost the attention of Fhit researchers. Today, the ApnA family can be considered important signaling molecules, a new perspective that warrants renewed focus on the role of the Fhit substrate in regulating Fhit activity. Recent findings on the interaction of the Fhit–Ap3A complex with Ubc9, and on the phosphorylation of Fhit by Src that regulates Fhit–Ap3A half-life and, in turn, the amount of the active signaling form of Fhit, underscore the importance of ApnA as modulators of Fhit activities both in normal and cancer cells. Further examination of the participants in Fhit pathways thus far identified and those players suggested by microarray analysis, as well as the new interactions that remain only partially explored, will increase our understanding of the physiological and pathological roles of Fhit. Dissection of these new pathways promises to shed light on the fine regulation exerted by Fhit on the cell cycle and apoptosis and thus its oncosuppressor activity. Clinical and therapeutic implications for cancer are likely to arise from this knowledge, especially with respect to chemo-resistance mechanisms and synergy/antagonism of different treatments.


  1. Top of page
  2. Abstract
  • Andriani F, Perego P, Carenini N, Sozzi G, Roz L. 2005. Increased sensitivity to cisplatin in non-small lung cancer cell lines after FHIT gene transfer. Neoplasia 8 [Epub ahead of print].
  • Barnes LD, Garrison PN, Siprashvili Z, Guranowski A, Robinson AK, Ingram SW, Croce CM, Ohta M, Huebner K. 1996. Fhit, a putative tumor suppressor in humans, is a dinucleoside 5′, 5′′′-P1, P3-triphosphate hydrolase. Biochemistry 35: 1152911535.
  • Baxi MD, Vishwanatha JK. 1995. Uracil DNA-glycosylase/glyceraldehyde-3-phosphate dehydrogenase is an Ap4A binding protein. Biochemistry 34: 97009707.
  • Baxi MD, McLennan AG, Vishwanatha JK. 1994. Characterization of the HeLa cell DNA polymerase alpha-associated Ap4A binding protein by photoaffinity labeling. Biochemistry 33: 1460114607.
  • Brenner C. 2002. Hint, Fhit, and GalT: Function, structure, evolution, and mechanism of three branches of the histidine triad superfamily of nucleotide hydrolases and transferases. Biochemistry 41: 90039014.
  • Brenner C, Pace HC, Garrison PN, Robinson AK, Rosler A, Liu XH, Blackburn GM, Croce CM, Huebner K, Barnes LD. 1997. Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein. Protein Eng 10: 14611463.
  • Brevet A, Chen J, Leveque F, Plateau P, Blanquet S. 1989. In vivo synthesis of adenylylated bis(5′-nucleosidyl) tetraphosphates (Ap4N) by Escherichia coli aminoacyl-tRNA synthetases. Proc Natl Acad Sci USA 86: 82758279.
  • Campiglio M, Pekarsky Y, Ménard S, Tagliabue E, Pilotti S, Croce CM. 1999. FHIT loss of function in human primary breast cancer correlates with an advantages stage of the disease. Cancer Res 59: 38663869.
  • Campiglio M, Olgiati C, Fumagalli M, Fidanza V, Croce CM, Ménard S. 2003. Tumor growth protection by Fhit gene in HER2-transgenic mice. 94th Annual Meeting—Proc AACR 44: 961.
  • Cavazzoni A, Petronini PG, Galetti M, Roz L, Andriani F, Carbognani P, Rusca M, Fumarola C, Alfieri R, Sozzi G. 2004. Dose-dependent effect of FHIT-inducible expression in Calu-1 lung cancer cell line. Oncogene 23: 84398446.
  • Chaudhuri AR, Khan IA, Prasad V, Robinson AK, Luduena RF, Barnes LD. 1999. The tumor suppressor protein Fhit. A novel interaction with tubulin. J Biol Chem 274: 2437824382.
  • Chavan AJ, Haley BE, Volkin DB, Marfia KE, Verticelli AM, Bruner MW, Draper JP, Burke CJ, Middaugh CR. 1994. Interaction of nucleotides with acidic fibroblast growth factor (FGF-1). Biochemistry 33: 71937202.
  • Chen J, Brevet A, Blanquet S, Plateau P. 1998. Control of 5′,5′-dinucleoside triphosphate catabolism by APH1, a Saccharomyces cerevisiae analog of human FHIT. J Bacteriol 180: 23452349.
  • Claes P, Grobben B, Van Kolen K, Roymans D, Slegers H. 2001. P2Y(AC)(−)-receptor agonists enhance the proliferation of rat C6 glioma cells through activation of the p42/44 mitogen-activated protein kinase. Br J Pharmacol 134: 402408.
  • Draganescu A, Hodawadekar SC, Gee KR, Brenner C. 2000. Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates. J Biol Chem 275: 45554560.
  • Dumon KR, Ishii H, Fong LY, Zanesi N, Fidanza V, Mancini R, Vecchione A, Baffa R, Trapasso F, During MJ, Huebner K, Croce CM. 2001a. FHIT gene therapy prevents tumor development in Fhit-deficient mice. Proc Natl Acad Sci USA 98: 33463351.
  • Dumon KR, Ishii H, Vecchione A, Trapasso F, Baldassarre G, Chakrani F, Druck T, Rosato EF, Williams NN, Baffa R, During MJ, Huebner K, Croce CM. 2001b. Fragile histidine triad expression delays tumor development and induces apoptosis in human pancreatic cancer. Cancer Res 61: 48274836.
  • Fong LY, Fidanza V, Zanesi N, Lock LF, Siracusa LD, Mancini R, Siprashvili Z, Ottey M, Martin SE, Druck T, McCue PA, Croce CM, Huebner K. 2000. Muir–Torre-like syndrome in Fhit-deficient mice. Proc Natl Acad Sci USA 97: 47424747.
  • Fuge EK, Farr SB. 1993. AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB. J Bacteriol 175: 23212326.
  • Fujishita T, Doi Y, Sonoshita M, Hiai H, Oshima M, Huebner K, Croce CM, Taketo MM. 2004. Development of spontaneous tumours and intestinal lesions in Fhit gene knockout mice. Br J Cancer 91: 15711574.
  • Garrison PN, Robinson AK, Pekarsky Y, Croce CM, Barnes LD. 2005. Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms. Biochemistry 44: 62866292.
  • Gasmi L, McLennan AG, Edwards SW. 1996. The diadenosine polyphosphates Ap3A and Ap4A and adenosine triphosphate interact with granulocyte-macrophage colony-stimulating factor to delay neutrophil apoptosis: Implications for neutrophil: Platelet interactions during inflammation. Blood 87: 34423449.
  • Glover TW, Hoge AW, Miller DE, Ascara-Wilke JE, Adam AN, Dagenais SL, Wilke CM, Dierick HA, Beer DG. 1998. The murine Fhit gene is highly similar to its human orthologue and maps to a common fragile site region. Cancer Res 58: 34093414.
  • Goerlich O, Foeckler R, Holler E. 1982. Mechanism of synthesis of adenosine(5′)tetraphospho(5′)adenosine (AppppA) by aminoacyl-tRNA synthetases. Eur J Biochem 126: 135142.
  • Golebiowski F, Kowara R, Pawelczyk T. 2001. Distribution of Fhit protein in rat tissues and its intracellular localization. Mol Cell Biochem 226: 4955.
  • Golebiowski F, Szulc A, Szutowicz A, Pawelczyk T. 2004. Ubc9-induced inhibition of diadenosine triphosphate hydrolase activity of the putative tumor suppressor protein Fhit. Arch Biochem Biophys 428: 160164.
  • Guranowski A. 2000. Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates. Pharmacol Ther 87: 117139.
  • Guranowski A, Galbas M, Hartmann R, Justesen J. 2000. Selective degradation of 2′-adenylated diadenosine tri- and tetraphosphates, Ap(3)A and Ap(4)A, by two specific human dinucleoside polyphosphate hydrolases. Arch Biochem Biophys 373: 218224.
  • Hirano D, Aoki Y, Ogasawara H, Kodama H, Waga I, Sakanaka C, Shimizu T, Nakamura M. 1996. Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells. Biochem J 316: 8186.
  • Hu B, Han SY, Wang X, Ottey M, Potoczek MB, Dicker A, Huebner K, Wang Y. 2005a. Involvement of the Fhit gene in the ionizing radiation-activated ATR/CHK1 pathway. J Cell Physiol 202: 518523.
  • Hu B, Wang H, Wang X, Lu HR, Huang C, Powell SN, Huebner K, Wang Y. 2005b. Fhit and CHK1 have opposing effects on homologous recombination repair. Cancer Res 65: 86138616.
  • Ishii H, Dumon KR, Vecchione A, Fong LY, Baffa R, Huebner K, Croce CM. 2001a. Potential cancer therapy with the fragile histidine triad gene: Review of the preclinical studies. JAMA 286: 24412449.
  • Ishii H, Dumon KR, Vecchione A, Trapasso F, Mimori K, Alder H, Mori M, Sozzi G, Baffa R, Huebner K, Croce CM. 2001b. Effect of adenoviral transduction of the fragile histidine triad gene into esophageal cancer cells. Cancer Res 61: 15781584.
  • Ishii H, Vecchione A, Fong LY, Zanesi N, Trapasso F, Furukawa Y, Baffa R, Huebner K, Croce CM. 2004. Cancer prevention and therapy in a preclinical mouse model: Impact of FHIT viruses. Curr Gene Ther 4: 5363.
  • Jackson JA, Carlson EC. 1992. Inhibition of bovine retinal microvascular pericyte proliferation in vitro by adenosine. Am J Physiol 263: H634H640.
  • Ji L, Fang B, Yen N, Fong K, Min JD, Roth JA. 1999. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res 59: 33333339.
  • Jovanovic A, Alekseev AE, Terzic A. 1997. Intracellular diadenosine polyphosphates: A novel family of inhibitory ligands of the ATP-sensitive K+ channel. Biochem Pharmacol 54: 219225.
  • Kisselev LL, Wolfson AD. 1994. Aminoacyl-tRNA synthetases from higher eukaryotes. Prog Nucleic Acid Res Mol Biol 48: 83142.
  • Kisselev LL, Justesen J, Wolfson AD, Frolova LY. 1998. Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules? FEBS Lett 427: 157163.
  • Lasso de la Vega MC, Terradez P, Obrador E, Navarro J, Pellicer JA, Estrela JM. 1994. Inhibition of cancer growth and selective glutathione depletion in Ehrlich tumour cells in vivo by extracellular ATP. Biochem J 298: 99105.
  • Maa M-C, Leu T-H, McCarley DJ, Schatzman RC, Parsons SJ. 1995. Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: Implications for the etiology of multiple human cancers. Proc Natl Acad Sci USA 92: 69816985.
  • Martin F, Pintor J, Rovira JM, Ripoll C, Miras-Portugal MT, Soria B. 1998. Intracellular diadenosine polyphosphates: A novel second messenger in stimulus-secretion coupling. FASEB J 12: 14991506.
  • McLennan AG. 2000. Dinucleoside polyphosphates-friend or foe? Pharmacol Ther 87: 7389.
  • Merkulova T, Kovaleva G, Kisselev L. 1994. P1,P3-bis(5′-adenosyl)triphosphate (Ap3A) as a substrate and a product of mammalian tryptophanyl-tRNA synthetase. FEBS Lett 350: 287290.
  • Muller S, Hoege C, Pyrowolakis G, Jentsch S. 2001. SUMO, ubiquitin's mysterious cousin. Nat Rev Mol Cell Biol 2: 202210.
  • Nishizaki M, Sasaki J, Fang B, Atkinson EN, Minna JD, Roth JA, Ji L. 2004. Synergistic tumor suppression by coexpression of FHIT and p53 coincides with FHIT-mediated MDM2 inactivation and p53 stabilization in human non-small cell lung cancer cells. Cancer Res 64: 57455752.
  • Ohta M, Inoue H, Cotticelli MG, Kastury K, Baffa R, Palazzo J, Siprashvili Z, Mori M, Mccue P, Druck T, Croce CM, Huebner K. 1996. The FHIT gene, spanning the chromosome 3p14.2 fragile site acid renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers. Cell 84: 587597.
  • Ottey M, Han SY, Druck T, Barnoski BL, McCorkell KA, Croce CM, Raventos-Suarez C, Fairchild CR, Wang Y, Huebner K. 2004. Fhit-deficient normal and cancer cells are mitomycin C and UVC resistant. Br J Cancer 91: 16691677.
  • Pace HC, Garrison PN, Robinson AK, Barnes LD, Draganescu A, Rosler A, Blackburn GM, Siprashvili Z, Croce CM, Huebner K, Brenner C. 1998. Genetic, biochemical, and crystallographic characterization of Fhit-substrate complexes as the active signaling form of Fhit. Proc Natl Acad Sci USA 95: 54845489.
  • Pekarsky Y, Campiglio M, Siprashvili Z, Druck T, Sedkov Y, Tillib S, Draganescu A, Wermuth P, Rothman JH, Huebner K, Buchberg AM, Mazo A, Brenner C, Croce CM. 1998. Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans. Proc Natl Acad Sci USA 95: 87448749.
  • Pekarsky Y, Zanesi N, Palamarchuk A, Huebner K, Croce CM. 2002. FHIT: From gene discovery to cancer treatment and prevention. Lancet Oncol 3: 748754.
  • Pekarsky Y, Garrison PN, Palamarchuk A, Zanesi N, Aqeilan RI, Huebner K, Barnes LD, Croce CM. 2004. Fhit is a physiological target of the protein kinase Src. Proc Natl Acad Sci USA 101: 37753779.
  • Pintor J, Diaz-Hernandez M, Gualix J, Gomez-Villafuertes R, Hernando F, Miras-Portugal MT. 2000. Diadenosine polyphosphate receptors. From rat and guinea-pig brain to human nervous system. Pharmacol Ther 87: 103115.
  • Pintor J, Carracedo G, Alonso MC, Bautista A, Peral A. 2002. Presence of diadenosine polyphosphates in human tears. Pflugers Arch 443: 432436.
  • Pintor J, Peral A, Pelaez T, Martin S, Hoyle CH. 2003. Presence of diadenosine polyphosphates in the aqueous humor: Their effect on intraocular pressure. J Pharmacol Exp Ther 304: 342348.
  • Plateau P, Blanquet S. 1982. Zinc-dependent synthesis of various dinucleoside 5′,5′ ′ ′-P1,P3-Tri- or 5′′,5′ ′ ′-P1,P4-tetraphosphates by Escherichia coli lysyl-tRNA synthetase. Biochemistry 21: 52735279.
  • Plateau P, Blanquet S. 1994. Dinucleoside oligophosphates in micro-organisms. Adv Microb Physiol 36: 81109.
  • Roz L, Gramegna M, Ishii H, Croce CM, Sozzi G. 2002. Restoration of fragile histidine triad (FHIT) expression induces apoptosis and suppresses tumorigenicity in lung and cervical cancer cell lines. Proc Natl Acad Sci USA 19: 36153620.
  • Roz L, Andriani F, Ferrario C, Gariboldi M, Alcalay M, Minardi SP, Delia D, Pierotti MA, Sozzi G. 2003. Analysis of FHIT-regulated gene expression patterns using oligonucleotide arrays. 94th Annual Meeting—Proc.AACR, 1206. Ref Type: Abstract.
  • Roz L, Andriani F, Ferreira CG, Giaccone G, Sozzi G. 2004. The apoptotic pathway triggered by the Fhit protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression. Oncogene 23: 91029110.
  • Sard L, Accornero P, Tornielli S, Delia D, Bunone G, Campiglio M, Colombo PM, Gramegna M, Croce CM, Pierotti MA, Sozzi G. 1999. The tumor suppressor gene FHIT is involved in the regulation of apoptosis and in the cell cycle. Proc Natl Acad Sci USA 96: 84898492.
  • Seeler JS, Dejean A. 2003. Nuclear and unclear functions of SUMO. Nat Rev Mol Cell Biol 4: 690699.
  • Shi Y, Zou M, Farid NR, Paterson MC. 2000. Association of FHIT (fragile histidine triad), a candidate tumour suppressor gene, with the ubiquitin-conjugating enzyme hUBC9. Biochem J 352: 443448.
  • Sillero MA, De Diego A, Osorio H, Sillero A. 2002. Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase. Eur J Biochem 269: 53235329.
  • Siprashvili Z, Sozzi G, Barnes LD, Mccue P, Robinson AK, Eryomin V, Sard L, Tagliabue E, Greco A, Fusetti L, Schwartz G, Pierotti MA, Croce CM, Huebner K. 1997. Replacement of Fhit in cancer cells suppresses tumorigenicity. Proc Natl Acad Sci USA 94: 1377113776.
  • Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SC, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich A, Press MF. 1989. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707712.
  • Sozzi G, Pastorino U, Moiraghi L, Tagliabue E, Pezzella F, Ghirelli C, Tornielli S, Sard L, Huebner K, Pierotti MA, Croce CM, Pilotti S. 1998. Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res 58: 50325037.
  • Stavrou BM. 2003. Diadenosine polyphosphates: Postulated mechanisms mediating the cardiac effects. Curr Med Chem Cardiovasc Hematol Agents 1: 151169.
  • Stavrou BM, Sheridan DJ, Flores NA. 2001. Contribution of nitric oxide and prostanoids to the cardiac electrophysiological and coronary vasomotor effects of diadenosine polyphosphates. J Pharmacol Exp Ther 298: 531538.
  • Steinmetz M, Janssen AK, Pelster F, Rahn KH, Schlatter E. 2002. Vasoactivity of diadenosine polyphosphates in human small mesenteric resistance arteries. J Pharmacol Exp Ther 302: 787794.
  • Steinmetz M, Gabriels G, Le TV, Piechota HJ, Rahn KH, Schlatter E. 2003. Vasoactivity of diadenosine polyphosphates in human small renal resistance arteries. Nephrol Dial Transplant 18: 24962504.
  • Trapasso F, Krakowiak A, Cesari R, Arkles J, Yendamuri S, Ishii H, Vecchione A, Kuroki T, Bieganowski P, Pace HC, Huebner K, Croce CM, Brenner C. 2003. Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding. Proc Natl Acad Sci USA 100: 15921597.
  • Tseng JE, Khuri F, Kemp B, Liu D, Hong WK, Mao LMD. 1999. Loss of pFhit expression is frequent but not associated with prognosis in stage-I non-small cell lung cancer (NSCLC). AACR Proceedings, 184. Ref Type: Abstract.
  • Vartanian AA. 2003. Gelsolin and plasminogen activator inhibitor-1 are Ap3A-binding proteins. Ital J Biochem 52: 916.
  • Vartanian A, Prudovsky I, Suzuki H, Dal PI, Kisselev L. 1997. Opposite effects of cell differentiation and apoptosis on Ap3A/Ap4A ratio in human cell cultures. FEBS Lett 415: 160162.
  • Vecchione A, Sevignani C, Giarnieri E, Zanesi N, Ishii H, Cesari R, Fong LY, Gomella LG, Croce CM, Baffa R. 2004. Inactivation of the FHIT gene favors bladder cancer development. Clin Cancer Res 10: 76077612.
  • Vollmayer P, Clair T, Goding JW, Sano K, Servos J, Zimmermann H. 2003. Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases. Eur J Biochem 270: 29712978.
  • Werner NS, Siprashvili Z, Fong LY, Marquitan G, Schroder JK, Bardenheuer W, Seeber S, Huebner K, Schutte J, Opalka B. 2000. Differential susceptibility of renal carcinoma cell lines to tumor suppression by exogenous Fhit expression. Cancer Res 60: 27802785.
  • Zamecnik PC, Stephenson ML, Janeway CM, Randerath K. 1966. Enzymatic synthesis of diadenosine tetraphosphate and diadenosine triphosphate with a purified lysyl-sRNA synthetase. Biochem Biophys Res Commun 24: 9197.
  • Zanesi N, Fidanza V, Fong LY, Mancini R, Druck T, Valtieri M, Rudiger T, McCue PA, Croce CM, Huebner K. 2001. The tumor spectrum in FHIT-deficient mice. Proc Natl Acad Sci USA 98: 1025010255.
  • Zanesi N, Mancini R, Sevignani C, Vecchione A, Kaou M, Valtieri M, Calin GA, Pekarsky Y, Gnarra JR, Croce CM, Huebner K. 2005. Lung cancer susceptibility in Fhit-deficient mice is increased by Vhl haploinsufficiency. Cancer Res 65: 65766582.