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Abstract

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

Id proteins (inhibitors of DNA binding/differentiation) are negative regulators of basic helix-loop-helix (bHLH) type transcription factors, which promote the differentiation of various cell types. In addition to their “classical” ability to inhibit cell differentiation, they are able to stimulate cell cycle progression. These facts suggest that Id proteins play a role in keeping precursor cells immature and in expanding the cell population size during development. In vitro as well as in vivo analyses in the last several years have shown that Id proteins have more complex activities; they induce apoptosis or function as survival factors, depending on the cell context. Furthermore, dysregulated expression of Id proteins has been reported in several human tumors and seems to be related to the malignant character of tumors. Here, we summarize and discuss the biological activities of Id proteins from the standpoint of cell growth control. J. Cell. Physiol. 190: 21–28, 2002. © 2002 Wiley-Liss, Inc.

Transcription factors bearing the basic helix-loop-helix (bHLH) motif have been identified in various cell types and tissues (Weintraub et al., 1991; Massari and Murre, 2000). Gain-of- and loss-of- function experiments have explicitly demonstrated that these bHLH factors are critically involved in diverse types of tissue-specific differentiation and cellular function (Massari and Murre, 2000). Examples include myogenin in skeletal muscle development, neurogenin in neurogenesis, and SCL/tal-1 in hematopoiesis (Massari and Murre, 2000). In general, bHLH factors form dimers with ubiquitously expressed bHLH factors, so-called E proteins that include HEB, E2-2, and E2A gene products, E12 and E47, and regulate tissue-specific gene expression that promotes cell differentiation (Massari and Murre, 2000). The activity of bHLH factors as transcription factors is negatively regulated at the protein level by the structurally related Id proteins (inhibitors of DNA binding/differentiation) (Benezra et al., 1990; Norton et al., 1998; Norton, 2000). The mechanism underlying the inhibitory activity of Id proteins is quenching out E proteins, the dimerization partners of tissue-specific bHLH factors (Benezra et al., 1990; Norton et al., 1998; Norton, 2000). Id proteins possess the HLH domain, through which they form dimers mainly with E proteins. As a result, the E proteins are functionally sequestered and can not form functional heterodimers with tissue-specific bHLH factors, leading to inhibition of differentiation. In addition, the Id/E protein heterodimers can not bind DNA, because Id proteins lack a region rich in basic amino acids that is required for DNA binding. In mammals, four members of the Id family, Id1 through Id4, have been identified so far (Benezra et al., 1990; Christy et al., 1991; Sun et al., 1991; Riechmann et al., 1994; Norton et al., 1998; Norton, 2000). In Drosophila, the counterpart of Id is emc, which regulates the development of sensory hairs (Ellis et al., 1990; Garrell and Modolell, 1990) and sex determination (Younger-Shepherd et al., 1992).

Id proteins are involved not only in cell differentiation control but also in the regulation of cell proliferation. Forced overexpression of Id proteins induces an increased rate of proliferation in various differentiation model systems of cultured cells, e.g., erythroleukemia cells (Lister et al., 1995) and myoblast cells (Atherton et al., 1996), in addition to inhibiting cell differentiation. Furthermore, transgenic mice directed to express Id1 or Id2 in T cells show defective T cell maturation and develop lymphoma/leukemia (Kim et al., 1999; Morrow et al., 1999). The dual functions of Id proteins, inhibition of differentiation and stimulation of proliferation, might be interdependent, because cell differentiation is generally coupled to the exit from the cell cycle. However, the cell cycle stimulatory activity of Id proteins is not restricted to situations where cells undergo differentiation or maturation (Norton et al., 1998; Norton, 2000). It is well known that Id gene expression is rapidly induced in many cultured cells by the addition of growth factors or by serum stimulation (Norton et al., 1998; Israel et al., 1999; Norton, 2000). Furthermore, the involvement of Id proteins in the regulation of cyclin-dependent kinase inhibitors (CDKIs) has been demonstrated in relation to replicative senescence of cells (Alani et al., 2001; Ohtani et al., 2001) as well as in a context dependent on differentiation (Lyden et al., 1999). These observations suggest that Id proteins are not simply differentiation-dependent stimulators of proliferation but are also important positive regulators of the cell cycle.

In this review, we focus on the cell cycle-related functions of Id proteins and discuss the role of these proteins in cell proliferation and survival, and their involvement in apoptosis and carcinogenesis. Advances made in the last several years have indicated that Id proteins exhibit more complex activity than previously thought. We hope that this review will help readers to understand the multivalent functions of Id proteins and will attract more researchers to the field of Id biology.

Regulation of Id gene expression

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

Id genes are expressed in a variety of undifferentiated and proliferating cells in vivo during development and their expression levels decrease in terminally differentiated cells, as expected from their ability to inhibit cell differentiation (Wang et al., 1992; Neuman et al., 1993; Ellmeier and Weith, 1995; Riechmann and Sablitzky, 1995; Jen et al., 1996, 1997). In general, this pattern holds true in many in vitro model systems of cell differentiation, such as muscle (Jen et al., 1992; Atherton et al., 1996; Melnikova and Christy, 1996) and blood cells (Shoji et al., 1994; Lister et al., 1995). The subject of this review is the roles of Id proteins in cell growth control, and we focus on the expression of Id genes in relation to proliferation, especially with respect to the cell cycle.

In human diploid fibroblasts, which are not differentiated, Id1 and Id2 show a biphasic expression pattern after serum stimulation: peaks of expression are found at 2–3 h and 12 h after growth stimulation (Hara et al., 1994). These two peaks correspond to the early G1 phase and the G1-S transition during the cell cycle progression, respectively. A similar biphasic pattern of expression is observed in several other cell types (Simonson et al., 1993; Biggs et al., 1995; Loveys et al., 1996; Cooper et al., 1997; Deed et al., 1997), suggesting the involvement of Id proteins in cell cycle control. In fact, Id3 was identified as a gene that is induced in the early phase after mitogen stimulation (Murphy and Norton, 1990; Christy et al., 1991). In hematopoietic cells, Id1 displays a similar biphasic expression pattern, while Id2 is expressed in a different manner, depending on the cell types (Cooper et al., 1997; Cooper and Newburger, 1998). The difference may reflect a role of Id2 in cell differentiation control, the classical activity of Id proteins, in the respective cell types.

Many factors have been shown to induce expression of Id genes, including bone morphogenic proteins, BMP-2/4/7 (Ogata et al., 1993; Hollnagel et al., 1999; Clement et al., 2000; Dorai and Sampath, 2001; Houldsworth et al., 2001; Nakashima et al., 2001), transforming growth factor β1 (TGFβ1) (Kee et al., 2001), platelet-derived growth factor (PDGF) (Christy et al., 1991; Barone et al., 1994), nerve growth factor (NGF) (Einarson and Chao, 1995), epidermal growth factor (EGF) (Le Jossic et al., 1994), insulin-like growth factor-I (IGF-I) (Belletti et al., 2001), and estrogen (Lin et al., 2000). Among these proteins, PDGF and EGF have been shown to activate the RAS-ERK-MAPK pathway (Goustin et al., 1986). This pathway is also activated by T cell receptor-mediated activation during T cell maturation, known as positive selection, and induces Id3 expression via the immediate early growth response gene 1, EGR1 (Bain et al., 2001). EGR1, which is rapidly induced by a variety of growth-stimulating factors without new protein synthesis (Sukhatme, 1990; Liu et al., 1998), has also been demonstrated to directly regulate the transcription of Id1 in C2C12 myoblasts and C3H10T1/2 fibroblasts (Tournay and Benezra, 1996). These observations are consistent with Id proteins playing a role in cell cycle stimulation.

Modification and degradation of Id proteins

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

Protein modification is important in the regulation of protein function. Id proteins possess potential phosphorylation sites for protein kinase A, protein kinase C, cdc2 kinase, and casein kinase II, with similar but distinct combinations of the sites among the Id family members (Nagata et al., 1995). These sites, except for the casein kinase II sites, have been reported to be phosphorylated in Id1, Id2, and Id3 in vitro (Nagata et al., 1995). Phosphorylation of protein kinase A sites found in Id1 and Id2, however, does not affect the ability of these proteins to heterodimerize with bHLH factors. In contrast, the serine residue at the fifth amino acid position of Id2, Id3, and Id4, but not present in Id1, can be phosphorylated by cdc2, cyclin E/cdk2 and cyclin A/cdk2, and the resulting phosphorylated forms of Id2 and Id3 have different dimerization affinity from the unphosphorylated forms (Deed et al., 1997; Hara et al., 1997).

Id proteins are generally very short-lived, with half-lives ranging from 20 to 60 min depending on the cell types (Deed et al., 1996; Bounpheng et al., 1999), and are stabilized by formation of dimers with bHLH factors (Deed et al., 1996; Bounpheng et al., 1999). The rapid turnover is reminiscent of proteins involved in cell cycle regulation and suggests the importance of precise regulation of Id protein functions. The rapid degradation seems to be due to proteolysis through the ubiquitin-proteasome pathway (Bounpheng et al., 1999). Id1, Id2, and Id3 proteins are ubiquitinated and are degraded by the 26S proteasome. Ubiquitination of Id4 is also observed and is dependent on the E1 ubiquitin-activating enzyme, like ubiquitination of the other Id family members, but Id4 degradation appears to be insensitive to the 26S proteasome (Bounpheng et al., 1999). The biological significance of the difference is unclear. The amino acid sequences responsible for the degradation of Id proteins remain unknown. Although, loose consensus sequences for the destruction box are found in Id1, Id2, and Id4, as in A- and B-type cyclins, analyses with deletion mutants of Id1 failed to identify a domain required for the degradation (Bounpheng et al., 1999).

Mechanisms underlying growth-stimulatory function of Id proteins

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

As discussed above, the regulation of Id gene expression and of the stability of Id proteins is consistent with the notion, which was derived mainly from experiments with cultured cells, that Id proteins stimulate cell cycle progression. How do Id proteins carry out this function? A couple of mechanisms have been proposed based on in vivo (Lyden et al., 1999; Lasorella et al., 2000; Mori et al., 2000) as well as in vitro experiments (Iavarone et al., 1994; Lasorella et al., 1996; Prabhu et al., 1997; Norton et al., 1998; Norton, 2000; Pagliuca et al., 2000).

One mechanism involves the regulation of CDKIs, which inhibit complex formation between cyclins and cyclin-dependent kinases (CDKs) or suppress the complex activity (Fig. 1A) (Sherr and Roberts, 1999). Terminal differentiation of cells is generally associated with the exit from the cell cycle (Ferrari et al., 1992). In skeletal muscle cells, for example, elevated expression of a CDKI, p21, is associated with terminal differentiation and cell cycle arrest, and is induced by MyoD (Halevy et al., 1995; Parker et al., 1995). The promoter region of p21 contains E boxes, consensus binding sites for bHLH factors, that mediate transactivation of the gene by MyoD and/or E proteins (Prabhu et al., 1997; Pagliuca et al., 2000). In the presence of Id1, the transactivation of p21 gene expression by bHLH factors is suppressed and, probably, the activity of cyclin A or E/cdk2 complexes decreases, leading to cell cycle progression into the S phase (Prabhu et al., 1997). Because this mechanism relies on the common biological activity of Id proteins as negative regulators of bHLH factors, it seems plausible that the other Id family members would also function in a similar manner (Norton, 2000). The protein expression of p27, a member of the same protein family as p21, has been shown to be induced by neurogenic bHLH factors during neuronal differentiation of P19 mouse embryonal carcinoma cells (Farah et al., 2000). Additionally, Id1 has been shown to regulate the expression of p16, another CDKI that inhibits cyclin D/cdk4 or 6, through interactions with Ets proteins (Ohtani et al., 2001) and with bHLH factors (Alani et al., 2001). The regulation of p16 by Id proteins seems to be related particularly to replicative senescence (see below). In vivo, neuroblasts of the Id1−/−Id3−/− fetal brain exit prematurely from the cell cycle, and this is accompanied by elevated expression of p16 and p27 (Lyden et al., 1999). Furthermore, Id2−/− mammary epithelial cells are defective in proliferation during pregnancy, and exhibit elevated levels of p21 and p27 (Mori et al., 2000). Therefore, the negative regulation of CDKI expression by Id protein appears in fact to play a role in the control of the cell cycle.

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Figure 1. Schematic representations of Id protein function in cell cycle progression. A: A bHLH factor-dependent mechanism. The promoter regions of CDKI genes, p16 and p21, contain E-boxes that are consensus binding sites for bHLH factors (tissue-specific bHLH protein/E protein heterodimers or E protein hetero- or homodimers). Expression of CDKI genes are enhanced by bHLH factors, resulting in cell cycle arrest in the G0/G1 phase of the cell cycle. Id proteins prevent the formation of bHLH factor dimers and thereby inhibit the transactivation of CDKI genes. This leads to the activation of CDK activity and stimulation of the G1-S transition of the cell cycle. The expression of p15 gene, another CDKI, is also regulated similarly (Pagliuca et al., 2000). B: A bHLH factor-independent mechanism. Unphosphorylated Rb protein (pRb) binds and inactivates E2F, which is a stimulator of the G1-S transition. Phosphorylation of pRb by cyclin D/CDK4 and cyclin E/CDK2 complexes progresses at the early and late G1 phases of the cell cycle sequentially. E2F dissociates from the phosphorylated form of pRb (ppRb) and activates genes involved in cell cycle progression. Among the four members of the Id family, Id2 binds and antagonizes unphosphorylated forms of the Rb family proteins, which causes release of E2F from pRb. This mechanism is similar to those of viral oncoproteins such as the adenovirus E1A protein and the SV40 large T antigen. Note that neither Id1 nor Id3 has the ability to bind Rb family proteins. Id4 seems to target pRb.

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The other proposed mechanism involves Id protein interaction with the tumor suppressor retinoblastoma protein (pRb) (Fig. 1B). In the G1 phase of the cell cycle, pRb associates with and inactivates E2F proteins, which regulate a variety of genes required for cell cycle progression, resulting in suppression of the transition from G1 to S phase (Sherr and Roberts, 1999). Sequential phosphorylation of pRb by cyclinD/cdk4 and cyclin A or E/cdk2 releases E2F from pRb at the G1-S transition, and the cell cycle then progresses to the S phase (Sherr and Roberts, 1999). The interaction of pRb with E2F in the G1 phase occurs via the so-called pocket domain of pRb (Helin et al., 1992; Kaelin et al., 1992; Shan et al., 1992). Id2 has been shown to bind the unphosphorylated pRb through interaction between the HLH region and the pocket domain of the respective proteins, resulting in the release of E2F (Iavarone et al., 1994), as seen in the cases of viral oncoproteins such as E1A (Dyson et al., 1989; Weinberg, 1989), which eventually leads to the stimulation of cell cycle progression (Iavarone et al., 1994). Rb family proteins p107 and p130 are also antagonized by Id2 (Lasorella et al., 1996). Consistent with the antagonization of Rb proteins by Id2, apoptosis and differentiation arrest of neural cell progenitors derived from the cerebral cortex are induced by overexpression of Id2 and prevented by coexpression of Rb (Toma et al., 2000). Furthermore, defective phenotypes in Rb−/− mice are partially rescued in compound mutant Rb−/−Id2−/− mice (Lasorella et al., 2000). However, although Id4 also seems to antagonize the Rb protein family members (Norton, 2000), Id1 and Id3 lack the activity (Iavarone et al., 1994), despite the fact that all members of the Id protein family have similar biological activity (Norton et al., 1998; Norton, 2000). The differences among Id proteins with respect to the interaction with Rb proteins remain to be clarified.

Additionally, it has been demonstrated that Id2 is a direct target of myc and that the cell cycle stimulatory activity of Myc is highly dependent on the presence of Id2 (Lasorella et al., 2000). Thus, Id2 seems to be a transducing molecule of Myc activity to the cell cycle machinery that functions through interaction with the Rb protein family. This explains the correlation between the expression of N-myc and Id2 in neuroblastoma-derived cells (Lasorella et al., 2000).

Apoptosis and Id

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

It has generally been believed that an inappropriately strong signal for proliferation induces apoptosis (White, 1996). Cytotoxicity of Id proteins has been demonstrated by overexpression of Id proteins in cultured cells established from myeloid progenitors (Florio et al., 1998), osteosarcoma (Florio et al., 1998), myoblasts (Jen et al., 1992), fibroblasts (Nakajima et al., 1998) as well as in primary cultures of embryonic fibroblasts (Norton and Atherton, 1998), neonatal cardiac myocytes (Tanaka et al., 1998), astrocytes (Andres-Barquin et al., 1999), and cortical neural progenitors (Toma et al., 2000). Consistent with these findings, it is difficult to obtain cells overproducing Id proteins by exogenous introduction of the respective genes in certain cell types (Mori and Yokota, unpublished observations). Depending on the cell line, overexpression of Id proteins results in either apoptosis (Florio et al., 1998; Nakajima et al., 1998; Norton and Atherton, 1998; Tanaka et al., 1998; Andres-Barquin et al., 1999) or cell proliferation (Iavarone et al., 1994; Desprez et al., 1995; Lister et al., 1995; Atherton et al., 1996). Id-induced apoptosis appears to be independent of p53 (Norton and Atherton, 1998). In apparent contrast to the general pattern, mammary epithelial cells of pregnant Id2−/− mice show enhanced apoptosis (Mori et al., 2000). These observations suggest that the precise regulation of the level of Id proteins is important for cell survival, depending on the cell context. Although, the precise molecular mechanisms are unclear, they may involve antagonization of the Rb proteins (Kaelin, 1999; Harbour and Dean, 2000) by Id2 (Iavarone et al., 1994) or Id4 (Norton, 2000), at least in part. On the other hand, Myc proteins have the ability to induce apoptosis (Askew et al., 1991; Evan et al., 1992) and this may involve the activation of Id2 expression, since the Id2 is a direct target of Myc proteins, as discussed above (Lasorella et al., 2000).

The N-terminal region of Id proteins, outside of the HLH region that is important for the interaction with other proteins including bHLH factors (Deed et al., 1997; Hara et al., 1997), has been shown to be sufficient for inducing apoptosis (Florio et al., 1998). Interestingly, the N-terminal region of Id3 lacks this activity. Although, the underlying mechanism is unclear, it is associated with the enhanced expression of proapoptotic BAX (Florio et al., 1998). This observation raises the interesting possibility that the N-terminal region of Id2 can activate a route for apoptosis.

During the development of the mouse, three members of the Id gene family, Id1, Id2, and Id3, are highly expressed in the interdigital web (Jen et al., 1996). This region is known to disappear via apoptosis of cells in the region (Hammar and Mottet, 1971). This suggests the intriguing possibility that Id proteins are able to function as components of the apoptosis pathway under the physiological conditions that occur during normal development.

Id proteins as survival factors

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

In contrast to their involvement in apoptosis, Id proteins appear to be required as survival factors for some cell types. In Id2−/− mice, Sertoli cells undergo apoptosis, which results in impaired spermatogenesis and infertility (Sablitzky, personal communication). Sertoli cells are unique somatic cells constituting the epithelial lining of the seminiferous tubules of the testis, and are supporting cells for germ cells. Their proliferation stops at puberty and they survive in adulthood without dividing (Russell et al., 1990). The requirement for Id2 in Sertoli cells in the adult strongly indicates that Id2 is a survival factor in these cells. Similarly, in the late phase of pregnancy, mammary epithelial cells of Id2−/− mice display increased apoptosis, which is associated with the activation of the p53-BAX pathway (Mori et al., 2000). Although, the detailed mechanisms are not yet known, these findings obtained from gene-deficient mice may provide clues about novel functional aspects of Id proteins.

Senescence and Id

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

Primary cells cultured from organs or tissues show a decrease of proliferation ability as they undergo increasing divisions in culture and finally stop dividing (Hayflick, 1965; Stanulis-Praeger, 1987; Goldstein, 1990). These are called aging and senescence, respectively (Stanulis-Praeger, 1987; Goldstein, 1990). The expression levels of Id1 and Id2 decrease during aging (Hara et al., 1994), suggesting a correlation between expression of Id proteins and proliferation ability. In accordance with this notion, human diploid fibroblasts can escape from aging by overexpression of Id2 together with the SV40 large T antigen (Hara et al., 1996b). Furthermore, overexpression of Id1 induces immortalization (Alani et al., 1999) or at least a delay in aging of human keratinocytes (Nickoloff et al., 2000). These observations demonstrate that there is a relationship between Id expression and the aging of cells.

Aging and replicative senescence are correlated with the transcriptional activation of p16 (Alcorta et al., 1996; Hara et al., 1996a; Loughran et al., 1996; Reznikoff et al., 1996). Recently, Ets1 and Ets2, which are negatively regulated by Id1, were shown to be able to directly transactivate p16 expression by binding its promoter region (Ohtani et al., 2001). Consistent with a report demonstrating that Id proteins possess antagonistic activity against Elk-1, which is one of the Ets family proteins (Yates et al., 1999), Id1 can bind and antagonize Ets1 and Ets2. In proliferating human diploid fibroblasts, Id1 is expressed abundantly but the level of p16 is low. In contrast, the expression profiles are reversed in human diploid fibroblasts at replicative senescence, and Ets1 expression is correlated with that of p16. These observations suggest that replicative senescence is under the regulation of Ets proteins and that Id proteins are involved in this process through the regulation of Ets protein function. Id proteins may inhibit the execution of the senescence program by antagonizing Ets proteins, although it is unclear what determines Id gene expression during the process.

Consistent with the findings described above, Id1−/− primary mouse embryonic fibroblasts undergo premature senescence, showing increased expression of p16, but not p19, and decreased CDK activity (Alani et al., 2001). The up-regulation of p16 expression is also detected in the ventral telencephalon of Id1−/− mouse embryo at embryonic day 11.5 (Alani et al., 2001). In addition to the regulation by Ets proteins (Ohtani et al., 2001), p16 gene expression is also induced by bHLH factors via E-boxes present in the promoter and inhibited by Id1 (Alani et al., 2001).

Oncogenesis and Id

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

The cell cycle stimulatory activity of Id proteins suggests that they may be oncogenic or at least oncogenesis-related factors. In cell culture systems, overexpression of Id3 induces morphological changes in mouse NIH3T3 fibroblasts (Deed et al., 1993) and Id1 expression leads to activation of telomerase activity and immortalization of primary human keratinocytes (Alani et al., 1999). In mice, intestinal epithelia expressing Id1 form adenomas (Wice and Gordon, 1998) and overexpression of Id1 or Id2 in T cells results in T cell lymphoma/leukemia (Kim et al., 1999; Morrow et al., 1999). Recently, various reports have shown that increased expression of Id proteins is observed in primary human tumors as well as cultured tumor cells (Zhu et al., 1995; Andres-Barquin et al., 1997; Kleeff et al., 1998; Sablitzky et al., 1998; Israel et al., 1999; Maruyama et al., 1999; Kebebew et al., 2000; Langlands et al., 2000; Lasorella et al., 2000; Lin et al., 2000; Norton, 2000; Takai et al., 2001), although there is no direct evidence for the linkage of Id gene loci with chromosomal aberrations in cancers. Id2 is abundantly expressed in cultured cells derived from astrocytic tumors (Zhu et al., 1995; Andres-Barquin et al., 1997), pancreatic carcinomas (Kleeff et al., 1998; Maruyama et al., 1999), and neuroblastomas (Lasorella et al., 2000), and all Id proteins are abundantly expressed in seminomas (Sablitzky et al., 1998). In human clinical specimens, Id proteins have been found to be overexpressed in pancreatic cancers (Id1 and Id2; Kleeff et al., 1998; Maruyama et al., 1999), breast cancers (Id1; Lin et al., 2000), skin cancers (Id1, Id2, and Id3; Langlands et al., 2000), thyroid medullary carcinomas (Id1; Kebebew et al., 2000), and endometrial carcinomas (Id1; Takai et al., 2001).

Among the cancers with the increased expression of Id, neuroblastomas are of interest. N-myc overexpression is detected in many clinical specimens of neuroblastoma and established neuroblastoma cell lines (Kohl et al., 1983, 1984; Schwab et al., 1984a,b) and is correlated with the stage of the disease (Brodeur et al., 1984), while elevated Id2 expression is observed in cultured neuroblastoma cell lines showing N-myc overexpression (Lasorella et al., 2000). The fact that Id2 is a direct target of Myc (Lasorella et al., 2000) prompts us to think that Id2 may also be correlated with the clinical grade of the neuroblastoma. In support of this notion, Id1 is more abundantly expressed in invasive breast cancer than in ductal carcinoma in situ (Lin et al., 2000) and stronger expression of Id proteins is detected in dysplastic regions than in pathologically milder regions in several cancers (Kleeff et al., 1998; Maruyama et al., 1999; Langlands et al., 2000; Takai et al., 2001). In addition, mammary epithelial cells engineered to express Id1 exhibit increased proliferation and invade the basement membrane (Desprez et al., 1998). Taken together, these observations suggest that Id proteins are related to the clinical grade of cancers.

Tumor cells form a mass and require a blood supply for their survival and proliferation. Tumor angiogenesis, which involves remodeling of pre-existing blood vessels in tissues and enables a sufficient blood supply to tumor cells, is critical for tumor growth. Id proteins are profoundly important in this process (Lyden et al., 1999). In Id1+/−Id3−/− mice, intradermally injected tumor cells do not grow and the mice can survive, whereas control mice treated similarly suffer from the growth of tumor masses and die. Even mice bearing a single copy deletion of Id1, Id1+/−Id3+/+, demonstrate partial resistance against the challenge of tumor transplantation. Id proteins seem to play a role in tumor angiogenesis through control of the expression of an adhesion molecule, αvβ3-integrin, and its associated metalloproteinase, MMP2 (Lyden et al., 1999). These observations demonstrate the involvement of Id proteins in controlling tumor growth as well as the levels of external factors. In contrast to the abundant expression of Id1, Id2, and Id3 in endothelial cells of blood vessels in peripheral tissues during development, their expression levels are very low in the corresponding normal adult tissues (Jen et al., 1996; Lyden et al., 1999). When endothelial cells are stimulated to proliferate, Id proteins may be expressed at elevated levels and take part in the regulation of adhesion molecules and tumor-associated proteases as well as in promoting cell cycle progression of endothelial cells. In this regard, inhibitors of Id proteins have been proposed to be useful as anti-angiogenic drugs to treat human cancers (Lyden et al., 1999).

Perspectives

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

Id proteins, which are involved in the control of differentiation and the cell cycle, are valuable tools for investigating those processes and elucidating the connections between them. Evidence that Id proteins function in controlling the cell cycle has been rapidly accumulated by analyzing a wide variety of systems in vitro and in vivo. However, important aspects still remain to be elucidated, although great advances have been made. None of the proposed mechanisms can consistently explain all aspects of the growth control by Id proteins. In addition, it is still unclear which effector molecules function under the control of Id proteins in apoptosis. Furthermore, recent reports have demonstrated that Id proteins can interact with and modulate the functions of many proteins that are devoid of a bHLH motif. Examples include Rb family proteins (Iavarone et al., 1994; Lasorella et al., 1996), Ets family proteins (Yates et al., 1999; Ohtani et al., 2001), Pax proteins (Roberts et al., 2001), and MIDA1 (Shoji et al., 1995; Inoue et al., 1999). Id proteins appear to carry out even more complicated functions in vivo. Thus, further investigations will clearly be required to fully clarify how Id proteins function in cell cycle control. Gene-inactivated mice for each Id protein are now available (Yan et al., 1997; Lyden et al., 1999; Pan et al., 1999; Yokota et al., 1999; Rivera et al., 2000, Sablizky, personal communication) and studies of these mice may reveal unexpected roles of Id proteins in cell cycle regulation that no one has yet imagined. Verification of known in vitro activities of Id proteins using Id-deficient mice will also be important for understanding the real functions of Id proteins. We are still on the way to answer “What are Id proteins?” but the way is full of spectacular findings.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED

We are grateful to Fred Sablitzky for sharing unpublished observations and to all members of the Yokota lab at Fukui Medical University for helpful discussions and comments on the manuscript. We also thank Y. Matsui for secretarial assistance.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. Regulation of Id gene expression
  4. Modification and degradation of Id proteins
  5. Mechanisms underlying growth-stimulatory function of Id proteins
  6. Apoptosis and Id
  7. Id proteins as survival factors
  8. Senescence and Id
  9. Oncogenesis and Id
  10. Perspectives
  11. Acknowledgements
  12. LITERATURE CITED
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