Cyclin review



Keywords: onc, oncogenes, cancer, apoptosis, tumor suppressor genes, tumor viruses, molecular oncology, cell cycle, growth factors, growth factor receptors, apoptosis, growth regulatory genes
Description: Oncogene is one of the world’s leading cancer journals. It is published weekly and covers all aspects of the structure and function of Oncogenes.

    1 Department of Cell Biology, University of Cincinnati, Cincinnati, OH, USA 2 University of Cincinnati Cancer Center, University of Cincinnati, Cincinnati, OH, USA 3 Center for Environmental Genetics, University of Cincinnati, Cincinnati, OH, USA 4 Abramson Family Cancer Research Institute, University of Pennsylvania Cancer Center, University of Pennsylvania, Philadelphia, PA, USA 5 Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

Correspondence: Dr E Knudsen, Department of Cell Biology, University of Cincinnati Cancer Center, Center for Environmental Genetics, University of Cincinnati, 3125 Eden Avenue, Cincinnati, OH 45267, USA. E-mail: erik.knudsen@uc.edu

cell cycle, cyclin-dependent kinase, alternative splicing, nuclear localization, retinoblastoma tumor suppressor

The cancer relevance of the cyclin D1 gene was apparent upon its identification in 1991; the cyclin D1 locus (also called PRAD1 or CCND1) was initially identified based on its involvement in a chromosomal rearrangement of benign parathyroid tumors (Motokura et al.. 1991 ). Cyclin D1 was simultaneously identified by two groups; the first identified cyclin D1 in a subtractive screen for growth factor inducible genes (Matsushime et al.. 1991 ) and the second group identified cyclin D1 through its ability to complement G1 cyclin loss in S. cerevisae (Xiong et al.. 1991 ). These initial findings suggested that cyclin D1 harbors the capacity to modulate cell cycle progression. From these beginnings, a wealth of studies systematically defined the manner in which cyclin D1 functions to regulate cell cycle and oncogenic transformation in mammalian cells (Sherr, 1995 ; Diehl, 2002 ; Gladden and Diehl, 2005 ). The cyclin D1 locus is known to be amplified in specific tumor types (Jiang et al.. 1992 ; Buckley et al.. 1993 ; Zhang et al.. 1993 ; Gillett et al.. 1994 ; Sherr, 1995 ; Palmero and Peters, 1996 ), and it is thought that this event renders a net increase in the proto-oncogenic functions of the cyclin D1 protein. Recent evidence indicates that the action of cyclin D1 in cancer may also be influenced by a common polymorphism that is believed to alter splicing of the cyclin D1 transcript and influence cancer risk and/or outcome. The present review is focused on the role of this polymorphism in cancer risk, the alternative splicing of the cyclin D1 RNA, and role of the resultant cyclin D1 variant protein in human cancer.

Cyclin D1 expression is highly regulated, as would be expected based on its powerful role in proliferative control. Cyclin D1 expression is induced as a delayed early response to many mitogenic signals (Sherr et al.. 1992 ; Sherr, 1995 ), and is universally associated with the transition from quiescence into the proliferative cycle (Won et al.. 1992 ; Winston and Pledger, 1993 ; Lukas et al.. 1994b ; Ladha et al.. 1998 ; Diehl, 2002 ). Among the cyclins that regulate G1 progression, it is hypothesized that stimulation of cyclin D1 expression represents the point at which mitogenic signal transduction cascades are integrated to mediate engagement of the cell cycle machinery (Figure 1 ). After induction, both cyclin D1 mRNA and protein levels are under stringent regulation. First, cyclin D1 mRNA levels are dramatically increased following mitogenic stimulation. This occurs in part through direct induction of the cyclin D1 promoter (Herber et al.. 1994 ; Muller et al.. 1994 ), such as after estrogen stimulation in breast cancer cells (Sabbah et al.. 1999 ). Additionally, many oncogenes (e.g. Ras and -catenin) harbor the capacity to induce cyclin D1 promoter activity (Albanese et al.. 1995 ; Tetsu and McCormick, 1999 ). However, the cyclin D1 promoter is very complex and in general is only weakly induced in in vitro reporter assays after mitogen stimulation. This result suggests that much of the regulation of cyclin D1 action could occur either through intrinsic complexities associated with the transcription initiation/elongation, such as chromatin structure or long-range enhancer elements or via coordinate effects on mRNA stability. In fact, it has been shown that cyclin D1 mRNA regulation occurs through mitogen-induced stabilization of the mRNA transcript (Dufourny et al.. 2000 ; Lin et al.. 2000 ). Once produced, the cyclin D1 protein is intrinsically unstable, thus providing an additional level of regulation. Instability of the protein has been attributed to a C-terminal PEST sequence (Dragnev et al.. 2001 ) and the presence of a specific GSK-3B phosphorylation site (threonine 286) (Diehl and Sherr, 1997 ; Diehl et al.. 1997 ). Although highly invoked in the literature, there has not been a detailed analysis of the role for the PEST sequence in regulating cyclin D1 protein stability. In contrast, phosphorylation of the T286 residue has been shown to target the cyclin D1 protein for nuclear export and proteasomal degradation (Alt et al.. 2000 ). Indeed, phosphorylation-dependent destruction of cyclin D1 provides a critical barometer to prevent overexpression of cyclin D1 resulting from continuous mitogen-dependent cyclin D1 transcription. As such, mitogen-dependent induction of cyclin D1 and the resultant cell cycle progression reflects the induction of a threshold of cyclin D1 protein, which is determined by the rate of expression versus destruction. Lastly, cyclin D1 is marked for degradation after genomic insult (Agami and Bernards, 2000 ), thus preventing cell cycle progression in the presence of DNA damage. Combined, these mechanisms contribute to the accumulation of cyclin D1 protein and are believed to be critical for ensuring the progression into the cell cycle is restricted to the appropriate mitogenic context.

Schematic role for cyclin D1 in the cell cycle. Mitogenic and antiproliferative signals are integrated to dictate the levels of cyclin D1 in the cell. Cyclin D1 can associate with the CDK4 or CDK6 proteins to activate catalytic activity. This action of cyclin D1 is antagonized by p16ink4a, which binds to the CDK-moiety and disrupts the association with cyclin D1. Active CDK/cyclin D1 complexes initiate phosphorylation of the retinoblastoma tumor suppressor, which disrupts ability RB-mediated transcriptional repression of E2F and facilitates cell cycle progression.

The predominant action of cyclin D1 in cell cycle control is believed to be manifest through interactions with CDK4 and/or CDK6 (Matsushime et al.. 1992. 1994 ; Meyerson and Harlow, 1994 ; Sherr, 1995 ). CDK4 and CDK6 belong to a distinct subarm of the family of cyclin-dependent kinases, which specifically bind D-type cyclins. Association with cyclin D1 activates the CDK moiety and promotes phosphorylation of specific substrates (Figure 1 ). CDK/cyclin D1 complexes act on a limited repertoire of substrates (Matsushime et al.. 1992. 1994 ), relative to other promiscuous CDK/cyclin complexes. The first substrate defined for CDK4/cyclin D1 complexes was the retinoblastoma tumor suppressor protein, RB (Ewen et al.. 1993 ; Kato et al.. 1993 ; Matsushime et al.. 1992. 1994 ). In its active state, RB inhibits cell cycle progression through its ability to repress transactivation of genes required for DNA replication and G2/M progression (reviewed in Sherr and McCormick, 2002 ; Cam and Dynlacht, 2003 ; Cobrinik, 2005 ). Activated CDK4/cyclin D1 complexes initiate phosphorylation of RB in response to mitogens (Mittnacht, 1998 ; Harbour et al.. 1999 ). This phosphorylation event disrupts the ability of RB to mediate transcriptional repression and therefore nullifies its antiproliferative function (Hinds et al.. 1992 ; Dowdy et al.. 1993 ; Ewen et al.. 1993 ; Harbour et al.. 1999 ). Early evidence suggested that the ability of cyclin D1 to promote RB phosphorylation is crucial for its cell cycle function, as cancer cells that are defective in RB are refractory to cyclin D1 action (Lukas et al.. 1994a. 1995a ). This hypothesis is further supported by the finding that cyclin D1 expression is often attenuated in RB-deficient tumors (Bartkova et al.. 1994 ; Muller et al.. 1994 ), suggesting that the two events confer redundant growth advantages. The concept that RB is a critical downstream target of cyclin D1 was reinforced by study of the p16ink4a tumor suppressor. p16ink4a binds directly to CDK4 and CDK6 and displaces cyclin D1 from the complex (Serrano et al.. 1993 ; Koh et al.. 1995 ; Russo et al.. 1998 ; Jeffrey et al.. 2000 ). Through this ability to antagonize cyclin D1 function, p16ink4a blocks cell cycle progression, and the antiproliferative action of p16ink4a is dependent both on the presence of CDK4/6 activity and RB (Koh et al.. 1995 ; Lukas et al.. 1995b ). Clinical analyses support a model wherein p16ink4a, cyclin D1 and RB fall in a common pathway (Figure 1 ), as loss of p16ink4a, loss of RB, or excessive activation of CDK4/cyclin D1 are mutually exclusive events in human cancers (Otterson et al.. 1994 ; Kelley et al.. 1995 ; Palmero and Peters, 1996 ). Disruption of this 'p16-cyclin D1-RB' axis is known to occur in the majority of human tumors (Palmero and Peters, 1996 ; Sherr and McCormick, 2002 ), thus underscoring the importance of this pathway in growth regulation. Several additional substrates of CDK4/cyclin D1 have been identified, including Smad3 (Liu and Matsuura, 2005 ), CDT1 (Liu et al.. 2004 ), and the RB-related proteins p107 (Matsushime et al.. 1992 ; Leng et al.. 2002 ) and p130 (Canhoto et al.. 2000 ). Each putative substrate harbors an established role in cell cycle control and therefore may influence tumorigenesis. Identification of the comprehensive cohort of CDK/cyclin D1 substrates and assessing each specific contribution to cyclin D1 action will reveal the biological consequence of CDK-mediated cyclin D1 function.

Although cyclin D1 acts through CDK4 to regulate cell cycle progression, a growing body of evidence demonstrates that cyclin D1 harbors CDK-independent activities (reviewed in Coqueret, 2002 ; Ewen and Lamb, 2004 ). Evidence for CDK-independent functions was first observed in clinical samples, wherein breast cancers overexpressing cyclin D1 failed to show enhanced proliferation rates, and control of the RB pathway was retained (Oyama et al.. 1998 ; Lamb et al.. 2003 ). In addition, B-cell lymphomas overexpressing cyclin D1 retained RB in its activated state (Zukerberg et al.. 1996 ). Thus, it was suspected that cyclin D1 likely harbors activities outside the cell cycle. Subsequent studies strikingly demonstrated that cyclin D1 is a modifier of gene transcription, and that this function of cyclin D1 has important implications in tumor biology. Specifically, cyclin D1 has been shown to regulate a number of sequence-specific transcription factors, including C/EBP (Lamb et al.. 2003 ), STAT3 (Bienvenu et al.. 2001 ), DMP1 (Inoue and Sherr, 1998 ), and BETA2/NeuroD (Ratineau et al.. 2002 ). The largest class of transcription factors regulated by cyclin D1 belong to the nuclear receptor superfamily, and include the estrogen receptor (Zwijsen et al.. 1998 ; Lamb et al.. 2000 ), androgen receptor (Knudsen et al.. 1999 ; Reutens et al.. 2001 ; Petre et al.. 2002 ; Burd et al.. 2005 ), thyroid hormone receptor (Pibiri et al.. 2001 ), and peroxisome proliferator activated receptor- (Qin et al.. 2003 ). In many cases cyclin D1 was shown to directly associate with the transcription factors, independent of CDK4 association, and modify transcription factor action through cell-cycle independent mechanisms. In this capacity, cyclin D1 can enhance transcriptional activation (e.g. through recruitment of coactivators such as P/CAF), or induce transcriptional repression (e.g. through association with histone deacetylases such as HDAC3). Each of these activities have been mapped to specific regions of the cyclin D1 protein. Specifically, transcriptional activation is associated with an LxxLL motif that is important for coactivator recruitment (Zwijsen et al.. 1998 ; McMahon et al.. 1999 ), whereas transcriptional repression is associated with the presence of a repressor domain for selected transcription factors (Petre-Draviam et al.. 2005 ). The biological significance of the cyclin D1 transcriptional regulatory function was solidified in a large scale, comprehensive analysis of genes deregulated in breast cancer specimens (Lamb et al.. 2003 ). This study unexpectedly revealed that cyclin D1 overexpression does not disrupt the RB function in breast cancer, and the cellular changes associated with cyclin D1 overexpression were not attributed to alterations in cell cycle. Rather, the principal action of cyclin D1 in this tissue type was shown to occur via direct modulation of C/EBP action. Thus, emerging evidence demonstrates that the ability of cyclin D1 to regulate transcription may strongly influence its function in cancer.

Given its role in promoting cellular proliferation and modulating transcription, it is not surprising that cyclin D1 is deregulated in cancer. Interestingly, cyclin D1 alterations are tumor-type specific, and occur through chromosomal translocation, gene amplification, or excessive accumulation of the protein. Chromosomal translocation of cyclin D1 is relatively rare in most cancers, but occurs in greater than 90% of Mantle Cell lymphoma, a mature B-cell malignancy (Bosch et al.. 1994 ; De Wolf-Peeters and Pittaluga, 1994 ; Bigoni et al.. 1996 ). This same translocation (11;14)(q13;q32) is also observed at a lower frequency mutliple myeloma and specific leukemias (Bosch et al.. 1994 ; Bergsagel and Kuehl, 2005 ). By contrast, cyclin D1 gene amplification or overexpression occurs with significant frequency in other tumor types, such as breast cancer and esophageal carcinoma, wherein 40–50% of primary carcinomas overexpress cyclin D1 (Jiang et al.. 1992. 1993 ; Buckley et al.. 1993 ; Gillett et al.. 1994 ). In addition to these mechanisms, it is tempting to speculate that activating mutations of cyclin D1 may also be selected for in human cancers, which may enhance association with CDK4 or alter the ability of cyclin D1 to modulate gene transcription. This hypothesis is not supported by the current literature, wherein surprisingly few tumor-derived mutations of cyclin D1 have been reported (Hosokawa et al.. 1995 ). However, recent evidence has indicated that a polymorphism of cyclin D1 (G/A 870) may influence cancer risk and outcome.

Over 100 single nucleotide polymorphisms have been identified spanning the cyclin D1 locus and catalogued in public single nucleotide polymorphism databases (dbSNP: www.ncbi.nlm.nih.gov/SNP/ ; HapMap: www.hapmap.org ; or GeneSNPs: www.genome.utah.edu/genesnps/ ). No common missense polymorphisms have been identified through these discovery efforts. However, whether this gene contains rare missense polymorphisms (1% frequency) will require deeper resequencing in large numbers of subjects with and without cancer phenotypes. Of the polymorphisms identified, the cyclin D1 G/A870 polymorphism has received the most investigation. The polymorphism frequency in the Caucasion population is approximately 44% A and 56% G, depending on the study (Simpson et al.. 2001 ; Sanyal et al.. 2004 ), but large variances between racial and ethnic groups have been reported. Owing to the significance of cyclin D1 in human cancer, a large number of epidemiological studies have challenged the influence of this particular polymorphism in cancer susceptibility and disease outcome. These studies generally compare the allelic frequency of G/A870 in disease affected or unaffected individuals, and assess correlations with clinical parameters (e.g. stage at diagnosis or overall survival). As summarized in Table 1. studies have shown specific genotypes harbor increased cancer risk or poor outcome for a large number of tumor types.

The majority of studies link the A-allele to increased cancer risk and poor disease outcome, with the largest associations observed with the A/A genotype. In these studies, relative risks were significant but typically modest, with many studies reporting less than a twofold effect. Such a result is in part to be expected for a common allele (i.e. A-870) that contributes to a complex phenotype. However, results have been inconsistent and some studies have implicated the G-allele with increased cancer risk, and others have ascribed no significant value to any allele of the G/A870 polymorphism. Combined, these results indicate that individual alleles may harbor differential effects in distinct tumor types. However, even within a specific tumor type (e.g. colorectal cancer) there have been disparate conclusions regarding the significance of the polymorphism. In part these disparities could be reflective of the patient population under study and the possible involvement of external factors (e.g. smoking or obesity) that have been suggested to cooperate with the polymorphism in specific studies (Buch et al.. 2005 ; Shu et al.. 2005 ). Thus, although the majority of observations are indicative of a role for the polymorphism in cancer risk, analysis of larger patient cohorts such as the NCI's Breast and Prostate Cancer Cohort Consortium will be required to resolve inconsistencies in the published literature.

Important issues that remain to be resolved are whether the G/A870 polymorphism is the specific causal variant and whether there are other polymorphisms at this locus that are biologically relevant. It is formally possible that G/A870 is a proxy that is in linkage disequilibrium with the actual functional variant that is modulating cancer risk. Such a possibility may in part explain some of the discrepancies associated with the role of G/A870 in cancer in different study populations and emphasizes the importance of conducting linkage disequilibrium or haplotype-based investigations of common genetic variation across the entire locus. One group has coordinately analysed both the G/A870 and the G/C1722 polymorphism of cyclin D1 (Holley et al.. 2001 ). This study indicated that the two polymorphisms are in linkage disequilibrium, such that individuals harboring the A870-allele are most likely also carrying the C1722-allele. In spite of this observation, each polymorphism had a distinct influence on disease (Holley et al.. 2001 ), suggesting that other variants in addition to the G/A870 variant in cyclin D1 may be important. Therefore, although more exhaustive studies will be required to conclusively determine the involvement of the G/A870 polymorphism in cancer, there is significant evidence that it alters risk, thus necessitating study of the functional significance of the polymorphism.

The cyclin D1 gene is comprised of five distinct exons and until recently, splicing of cyclin D1 was not considered a significant regulatory factor. Two transcripts of cyclin D1 can be detected in normal cells (Howe and Lynas, 2001 ), although regulation of splicing appears to be most relevant in cancer. Numerous groups have detected a cyclin D1 message that derives from alternative splicing (Betticher et al.. 1995 ; Hosokawa et al.. 1997. 1999 ; Bala and Peltomaki, 2001 ). This isoform was initially defined following the identification of the G/A870 polymorphism, that occurs at the intron 4/exon 5 boundary. As shown in Figure 2. the G870 allele creates an optimal splice donor site, resulting in the well-described transcript for cyclin D1 ('transcript a'). By contrast, the A870 allele is predicted to hinder the splicing event, thus allowing for read-through into intron 4 and production of a variant splice product of cyclin D1, termed 'transcript b'. Consistent with this hypothesis, Betticher et al. (1995). Holley et al. (2001). and Howe and Lynas (2001) demonstrated that the A-allele of cyclin D1 was preferentially associated with transcript b production. However, individuals with the A/A genotype can still produce transcript a (Bala and Peltomaki, 2001 ; Holley et al.. 2001 ), thus indicating that the 870-A allele is not completely penetrant for transcript b production, and individuals with the G/G genotype can produce transcript b. It should be noted that the presence of the A-nucleotide at the splice donor site does represent a change from the consensus (Figure 2 ), wherein the G-Allele represents 91% consensus using a weighted splice site identification matrix and the A-Allele is 85% (Shapiro and Senapathy, 1987 ). Importantly, both sites would be predicted to be functional and mutations in splice donor sequence analgous to the A-allele are appropriately spliced (Wieringa et al.. 1983 ). Thus, modifiers in addition to the polymorphic 870-allele must influence the splicing event. In principle these could be additional polymorphisms within the cyclin D1 gene or trans -acting factors, which have yet to be defined.

Cyclin D1 polymorphism and splicing of exon 4 and exon 5. Depicted is a consensus splicing site and the sequences surrounding the exon/intron junctions of cyclin D1. The program splice site was utilized to determine the score for the splice donor and acceptor sites relative to the consensus sequence.

Interpretation of epidemiological studies comparing the G/A870 polymorphism to cancer risk may be confounded by the dysjunction between the genotype and transcript b production. To date, no study has directly examined the relationship of the G/A870 polymorphism to transcript b production using quantitative/mechanistic methodologies. However, individual assessment of the prognostic values of the polymorphism and transcript b production in colon cancers revealed that while the polymorphism held no predictive value, alterations in transcript b production were of significance (Bala and Peltomaki, 2001 ). Therefore, possible sources of the heterogeneous influence of the cyclin D1 polymorphism in cancer are factors which influence transcript 'a' versus 'b' splicing independently of the polymorphic nucleotide. Thus, there is an urgent need to solidify the relationship of the polymorphism to transcript b production, and to correspondingly assess the impact of transcript b in human disease.

As depicted in Figure 3. transcript b gives rise to a cyclin D1 protein product with a completely divergent C-terminal domain, termed 'cyclin D1b'. The divergent C-terminal domain is defective in several motifs that typically regulate cyclin D1 turnover, including both the PEST motif and the residue that controls nuclear export and protein stability (threonine 286). Thus, it would be predicted that cyclin D1b is a more stable, constitutively nuclear protein, harboring increased capability to regulate CDK activity and cell cycle control. Several groups have recently assessed the action of cyclin D1b on these parameters. The expectation that cyclin D1b would exhibit increased protein stability (as compared to cyclin D1a) was not significantly observed (Lu et al.. 2003 ; Solomon et al.. 2003 ). Rather, the half-lives and relative accumulation of cyclin D1 and cyclin D1b were indistinguishable in most model systems (Lu et al.. 2003 ; Solomon et al.. 2003 ). By contrast, these same studies validated the prediction that cyclin D1b is constitutively nuclear in localization (Lu et al.. 2003 ; Solomon et al.. 2003 ). This was a critical observation, as constitutive nuclear localization of cyclin D1 (as achieved through mutation of T286) is known to promote oncogenic transformation (Alt et al.. 2000 ). Strikingly, it was demonstrated that cyclin D1b harbors increased transforming capability (as compared to cyclin D1) (Lu et al.. 2003 ; Solomon et al.. 2003 ), thus indicating that cyclin D1b may serve as a more potent oncogene in human cancers.

Domain structure of cyclin D1 and D1b. The predominant domains and their localization within the primary sequence of cyclin D1 and D1b are provided.

In light of this enhanced oncogenic action, substantial effort has been directed at delineating the mechanisms underlying cyclin D1b function. Cyclin D1b maintains its capacity to associate with CDK4 (Lu et al.. 2003 ; Solomon et al.. 2003 ), yet may have an altered capacity to drive CDK4 activity. In one set of reported experiments, cyclin D1b/CDK4 complexes immunopurified from cells phosphorylated recombinant retinoblastoma substrate as efficiently as cyclin D1/CDK4 complexes (Lu et al.. 2003 ). Conversely, in a different approach, cyclin D1b was relatively inefficient in its capacity to drive RB inactivation in cells as determined by assessing accumulation of phospho-RB in cells (Solomon et al.. 2003 ). As there is no detailed structural data on the CDK4-cyclin D1 kinase, it is hard to predict why alterations in the extreme C-terminus would effect kinase activity. Based on the crystal structure of other cyclin-CDK complexes, the C-terminus of the cyclin is not predicted to affect interactions with the CDK subunit (Russo et al.. 1998 ; Jeffrey et al.. 2000 ). Thus, one intriguing possibility is that the C-terminal alterations influence substrate recognition and thus substrate specificity. If so, one might anticipate disparate biological properties of the two molecules. Indeed, it was also observed that cyclin D1 but not cyclin D1b can efficiently advance cell cycle progression under conditions of RB-mediated cell cycle arrest or low serum (Solomon et al.. 2003 ; Holley et al.. 2005a ). Thus, while cyclin D1b has enhanced transforming properties, this function of cyclin D1b may be attributed to novel oncogenic functions. In addition, it will be important to determine if the cyclin oncogenic function of cyclin D1b is limited (or related) to tissue types wherein the A870-allele increases cancer risk.

In addition to functions on classical cell cycle control, it would be predicted that the loss of the LxxLL-motif and changes in nuclear localization may alter the transcriptional activity of cyclin D1b. Consistent with this idea, recent studies have demonstrated that in specific tumor types, the ability of cyclin D1b to regulate transcription factor action may be significantly altered and contribute to proliferative control (Burd CJ and Knudsen KE, unpublished data). Future studies to delineate the mechanisms of cyclin D1b function and the contribution of the novel C-terminus to cyclin D1b action will be critical to elucidate its contribution to cancer development and progression.

To date, the number of human cancers that may be influenced by cyclin D1b activity have been identified largely through association with the G/A870 polymorphism. Given the unreliable relationship between the genotype and cyclin D1b expression, there is a current emphasis on developing tools to screen for cyclin D1b expression. Although the transcript is relatively easy to detect by PCR based methods (Bala and Peltomaki, 2001 ; Howe and Lynas, 2001 ), detection of cyclin D1b protein is subject to the limitation that most available cyclin D1 antibodies were either raised against or preferentially recognize the C-terminus of cyclin D1a and therefore do not detect cyclin D1b. However, Hosokawa et al. (1999) detected cyclin D1b protein in B-lymphoid cell lines employing the DCS-6 monoclonal antibody, which is directed against a domain conserved within both cyclin D1 and D1b. Antibodies have also been developed against the unique sequences of cyclin D1b, so as to distinctly assess its expression levels in human specimens (Lu et al.. 2003 ; Solomon et al.. 2003 ). This approach proved successful in at least one study, which specifically detected the cyclin D1b protein in esophageal tumors (Lu et al.. 2003 ). Analysis of cyclin D1b in alternate tumor types is essential to ascertain its putative impact in tumorigenesis.

The prediction that alternative splicing might contribute to cancer-specific alterations in cyclin expression is not without precedent. Alternative splicing of cyclin E has been documented in both cancer-derived cell lines and primary cancer tissue (Mumberg et al.. 1997 ). In addition to regulated splicing, novel cyclin E isoforms that appear to be generated via proteolytic processing in cancer tissue have also been documented (Porter and Keyomarsi, 2000 ). These novel cyclin E isoforms contribute to a functionally active CDK2 kinase with altered substrate specificity (Porter and Keyomarsi, 2000 ; Porter et al.. 2001 ) and expression of these isoforms correlates with poor prognosis in breast cancer (Keyomarsi et al.. 2002 ). What remains unique with regard to cyclin D1 is the association between alternative splicing, the polymorphic 870 site and the unique expression of the variant D1b isoform in cancer. Still, the question as to whether alternative cyclin D1 splicing is a cause or an effect of cancer initiation and/or progression is of critical importance.

Cyclin D1 is a critical regulator of the cell cycle and transcriptional processes which is overexpressed at high frequency in human cancer. A large number of studies have implicated the cyclin D1 G/A870 polymorphism as a modulator of cancer risk and/or poor prognosis in human disease, and it has been suggested that specific polymorphic alleles may facilitate production of the cyclin D1b oncogene. However, significant questions remain regarding the influence of this polymorphism in human disease. First, large multiethnic studies are needed to assist in delineating the precise impact of the polymorphic alleles on tissue-specific cancers. Given the disparity in alleleic distribution across populations and the possibility that the G/A870 polymorphism may be a marker of another variant in the cyclin D1 gene, it may be necessary to analyse each group separately to designate prognostic value to the polymorphism. Second, the relationship between the polymorphism and cyclin D1b production should be clarified. Although it is apparent that specific polymorphic alleles may facilitate generation of transcript b, it is clear that additional factors influence the splicing event. When possible, concurrent analysis of genotype and cyclin D1b expression would clarify the relationship between the two parameters and assign their relative prognostic values in individual tumor types. Third, it will be of significant benefit to identify the mechanisms underlying the enhanced oncogenic action of cyclin D1b. The present data demonstrate that cyclin D1b incorporates novel mechanisms to regulate cellular transformation and transcriptional control, with the contribution of these functions to cancer development and progression as the focus of future studies. Finally, it will be essential to determine whether expression of the alternatively spliced cyclin D1b influences therapeutic efficacy. Although numerous small molecule inhibitors have been developed with a more pointed interest in targeting the CDK2 kinase, recent data suggest that the cyclin D1/CDK4 kinase may represent a good therapeutic target. CDK4/CDK6-specific small molecule inhibitors have been developed and one such molecule, PD 0332991, exhibits an IC50 in the nanomolar range, with excellent target specificity (Fry et al.. 2004 ; Toogood et al.. 2005 ). In addition, the cytostatic property of PD 0332991 is restricted to tumor-derived cell lines, which retain wild-type Rb and depend upon the cyclin D1/CDK kinase for proliferation (Fry et al.. 2004 ). However, if the cyclin D1b/CDK complexes exhibit alternative substrate specificities that reflect altered structural properties, expression of cyclin D1b will have a significant impact on therapeutic outcome. The current data support a model wherein cyclin D1b displays potentially novel mechanisms to regulate cellular proliferation, transcription factor action, and cellular transformation. Assessing the contribution of each function to cancer development and therapeutic intervention will contribute to our ability to develop cancer therapies and utilize these therapies in an appropriately targeted manner.






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