1030

CDKN2B

CDK4I|INK4B|MTS2|P15|TP15|p15INK4b;cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4);CDKN2B;cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4)

CDK inhibitory protein|CDK4B inhibitor|MTS-2|cyclin-dependent kinase 4 inhibitor B|cyclin-dependent kinases 4 and 6 binding protein|multiple tumor suppressor 2|p14-INK4b|p14_CDK inhibitor|p14_INK4B|p15 CDK inhibitor|p15-INK4b|p15_INK4B

9p21

protein-coding

Recent studies in our laboratory reported frequent loss of heterozygosity (LOH) on mouse chromosome 4 in T-cell lymphomas, identifying three candidate tumor suppressor regions (TLSR1-3). To determine the possible existence of other tumor suppressor gene loci on the proximal-mid part of chromosome 4 and to clarify whether the p16(INK4a) (alpha and beta) and p15(INK4b) genes are the inactivation targets of deletion at TLSR1, we have tested 73 gamma-radiation-induced T-cell lymphomas of F1 hybrid mice by LOH analysis. Frequent LOH was found at the INK4a and INK4b loci and the surrounding markers D4Mit77, D4Mit245 and D4Wsm1. In addition, we identified two distinct regions of significant allelic losses in the proximal-mid part of chromosome 4, defined by the markers D4Mit116 (TLSR4) and D4Mit21 (TLSR5). Taken together, this evidence and our previous data indicate the existence of at least five different candidate sites for tumor suppressor genes on chromosome 4, thus revealing a main role for this chromosome in the development of mouse T-cell lymphomas.

The INK4 (inhibitor of cyclin-dependent kinase 4) family consists of four tumor-suppressor proteins: p15(INK4B), p16(INK4A), p18(INK4C), and p19(INK4D). While their sequences and structures are highly homologous, they show appreciable differences in conformational flexibility, stability, and aggregation tendency. Here, p16 and p18 were first compared directly by NMR for line broadening and disappearance, then investigated by three different approaches in search of the causes of these differences. From denaturation experiments it was found that both proteins are marginally stable with low denaturation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear (1)H-(15)N nuclear Overhauser enhancement measurements revealed very limited conformational flexibility on the pico- to nanosecond time-scale for both p16 and p18. H/(2)H exchange of amide protons monitored by NMR on three proteins (p16, p18 as well as p15), however, revealed markedly different rates in the order p18

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the proteins conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of pl6INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 A for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.

Sulphation is an important detoxification pathway for numerous xenobiotics; however, it also plays an important role in the metabolism and bioactivation of many dietary and environmental mutagens, including heterocyclic amines implicated in the pathogenesis of colorectal and other cancers. A major sulphotransferase (SULT) enzyme in humans, SULT1A1, is polymorphic with the most common variant allele, SULT1A1*2, occurring at a frequency of about 32% in the Caucasian population. This allele codes for an allozyme with low enzyme activity and stability compared to the wild-type (SULT1A1*1) enzyme, and therefore SULT1A1 genotype may influence susceptibility to mutagenicity following exposure to heterocyclic amines and other environmental toxins. Previously, a significant association of SULT1A1*1 genotype with old age has been observed, suggesting a chemoprotective role for the high-activity phenotype. Here we have compared the frequencies of the most common SULT1A1 alleles in 226 colorectal cancer patients and 293 previously described control patients. We also assessed whether SULT1A1 genotype was related to various clinical parameters in the patient group, including Dukes classification, differentiation, site, nodal involvement and survival. There was no significant difference in allele frequency between the control and cancer patient populations, nor was there a significant association with any of the clinical parameters studied. However, when the age-related difference in allele frequency was considered, a significantly reduced risk of colorectal cancer (odds ratio = 0.47; 95% confidence interval = 0.27-0.83; P = 0.009), was associated with homozygosity for SULT1A1*1 in subjects under the age of 80 years. These results suggest that the high activity SULT1A1*1 allozyme protects against dietary and/or environmental chemicals involved in the pathogenesis of colorectal cancer.

Primary cutaneous B cell lymphomas represent a distinct group of lymphoproliferative disorders that can be distinguished from systemic lymphoma by their good response to local treatment and favorable prognosis. In systemic B cell lymphoma, inactivation of p15(INK4b) and p16(INK4a) is frequently observed and may be associated with a poor prognosis. There have been no comprehensive studies in primary cutaneous B cell lymphomas, however. Mechanisms of p15/p16 inactivation include loss of heterozygosity, homozygous deletion, promotor region hypermethylation, and point mutation. We analyzed DNA from 36 cases of primary cutaneous B cell lymphomas, four systemic B cell lymphomas, and six benign B cell lymphoproliferative infiltrates for abnormalities of p15 and p16 using microsatellite markers for 9p21, methylation specific polymerase chain reaction, and polymerase chain reaction/single stranded conformational polymorphism analysis with exon specific primers. expression of both p15 and p16 protein was assessed by immunohistochemistry. Loss of heterozygosity at 9p21 was identified in 2 out of 36 primary cutaneous B cell lymphomas. Hypermethylation of p15 and p16 promotor regions was identified in 8 of 35 (23%) and 15 of 35 (43%) cases, respectively. In two cases p16 hypermethylation was identified in recurrent disease but not in the initial tumor. No point mutations were identified. In seven patients, however, a polymorphism was observed in exon 3 of the p16 gene. In primary cutaneous B cell lymphomas with allelic loss or promotor hypermethylation of either p15 or p16, loss of expression in tumor cells was identified in 5 of 8 and 9 of 10 cases, respectively. Our findings suggest that p15(INK4b) and p16(INK4a) biallelic gene abnormalities are common in primary cutaneous B cell lymphomas, most frequently as a result of promotor hypermethylation. The presence of abnormalities in recurrent disease in some cases suggests that inactivation of p15 and p16 may be involved in disease progression.

PURPOSE: Malignant peripheral nerve sheath tumor (MPNST) can arise sporadically or in association with neurofibromatosis type 1. Deletions at the 9p21 locus have been reported in these tumors. To additionally characterize the status of this chromosomal region, in this study we performed a comprehensive, mostly PCR-based molecular analysis of the three tumor suppressor genes p15(INK4b), p14(ARF) and p16(INK4a) located at the 9p21 locus in 26 cryopreserved MPNSTs. EXPERIMENTAL DESIGN: Fourteen neurofibromatosis type 1-related and 12 sporadic cases were investigated for homozygous deletion coupled with fluorescent in situ hybridization, promoter methylation, and mutational analysis, as well as m-RNA expression. RESULTS: The results showed that an inactivation of one or more genes occurred in 77% of MPNSTs and was mainly achieved through homozygous deletion (46%), which, in turn, encompassed all of the three tandemly linked genes in 83% of the deleted cases. Promoter methylation was at a less extent involved in gene silencing (18%), and no mutations were found. Loss of function at DNA level strongly correlated with loss of mRNA expression accounting for 80% of the cases. Because of the close relationship between p14(ARF) and TP53 and between p15(INK4b)/p16(INK4a) and Rb, these results support a model of a coinactivation of TP53 and Rb pathways in 75% of MPNSTs, with functional consequences on cell growth control and apoptosis. CONCLUSIONS: The inactivation of the 9p21 locus is a frequent and peculiar hallmark of MPNST genetic profile leading also to an impaired apoptosis that could be taken into account in treatment planning of these tumors.

cancer is a multistep process resulting from an accumulation of several genetic changes. The determination of cooperating events in experimental models can help scientists decipher specific neoplastic pathways and place genes with similar functions in complementation groups. In leukemia models, retrovirus tagging is a powerful approach to determine genes that cooperate with oncogenic transgenes or tumor suppressors that have undergone targeted deletion. Experimental models for B and T cell leukemias involving transgenic c-myc were the first to show the utility of retroviral tagging. Here we review these experiments and present examples of new models of myeloid leukemia where retroviruses have collaborated with a transgene [Cbfbeta-MYH111 from Inv(16)] and with loss of a tumor suppressor (Ink4b) mice to induce disease.

CDKN2B (INK4B), which encodes the cyclin-dependent kinase inhibitor p15(INK4b), is up-regulated by many cytokines found in hematopoietic environments in vivo. In human acute myeloid leukemias (AMLs), it is inactivated with high frequency. To gain insight into the regulatory pathways leading to the normal activation of p15(Ink4b) expression, we examined interferon beta (IFNbeta)-induced transcription. Using reporter gene assays in murine myeloid cells M1, we determined that a 328-bp fragment, located 117 to 443 bp upstream of the translation initiation site, was sufficient to activate transcription. Both the interferon consensus sequence-binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) and PU.1 were able to increase transcription from this region. It was determined that both ICSBP and PU.1 must bind to DNA to form a stable PU.1/ICSBP binding complex. Interestingly, introduction of the ICSBP into ICSBP-null Tot2 cells led to a significant increase in p15(Ink4b) RNA expression. This regulation of the Ink4b promoter is apparently myeloid specific because both ICSBP and PU.1 are myeloid commitment factors. Importantly, this provides a mechanism to explain in part the tumor suppressor activity of ICSBP, since ICSBP-deficient mice develop a chronic myelogenous leukemia (CML)-like disease and a high percentage of human AML and CML lack ICSBP transcripts.

OBJECTIVE: The tumor suppressor p15Ink4b (Ink4b) is a cell-cycle inhibitor that is inactivated in a high percentage of acute myeloid leukemia and myeloid dysplasia syndrome cases. Despite this, the role of Ink4b in hematopoiesis remains unclear. Here we examined the role of Ink4b in blood cell formation using Ink4b-deficient (Ink4b(-/-)) mice. METHODS: We compared the bone marrow (BM) of Ink4b(-/-) and wild-type mice using flow cytometric, colony-forming unit and competitive repopulating assays (CRA). The proliferation, differentiation, self-renewal, and apoptosis of progenitor cells were further compared by in vitro and in vivo methods. RESULTS: BM from Ink4b(-/-) mice contained increased numbers of granulocyte-monocyte progenitors and Gr-1(+) cells and showed a competitive advantage over wild-type cells in myeloid cell formation by CRA. Ink4b(-/-) progenitors did not demonstrate increased proliferation, self-renewing potential, or reduced apoptosis. Instead, Ink4b(-/-) common myeloid progenitors (CMPs) showed increased myeloid progenitor formation concomitant with reduced erythroid potential. CONCLUSIONS: This work establishes a role for Ink4b in regulating the differentiation of CMPs and indicates that loss of Ink4b enhances the formation of myeloid progenitors.

The inbred BDII rat is a valuable experimental model for the genetic analysis of hormone-dependent endometrial adenocarcinoma (EAC). One common aberration detected previously by comparative genomic hybridization in rat EAC is loss affecting mostly the middle part of rat chromosome 5 (RNO5). First, we applied an RNO5-specific painting probe and four region-specific gene probes onto tumor cell metaphases from 21 EACs, and found that rearrangements involving RNO5 were common. The copy numbers of loci situated on RNO5 were found to be reduced, particularly for the CDKN2A/2B locus. Second, polymerase chain reaction analysis was performed with 22 genes and markers and homozygous deletions of the CDKN2A exon 1beta and CDKN2B genes were detected in 13 EACs (62%) and of CDKN2A exon 1alpha in 12 EACs (57%) Third, the occurrence of allelic imbalance in RNO5 was analyzed using 39 microsatellite markers covering the entire chromosome and frequent loss of heterozygosity was detected. Even more intriguing was the repeated finding of allele switching in a narrow region of 7 Mb across the CDKN2A/2B locus. We conclude that genetic events affecting the middle part of RNO5 (including bands 5q31 approximately q33 and the CDKN2A locus) contribute to the development of EAC in rat, with the CDKN2A locus having a primary role.

The ZNF217 oncoprotein is a constituent of a core transcriptional complex that includes CoREST, histone deacetylase 1/2, lysine demethylase 1, and the C-terminal binding protein 1/2. We have combined genome-wide expression profiling and chromatin immunoprecipitation with directed selection and ligation (ChIP-DSL) to identify a subset of genes directly regulated by ZNF217. Our results establish p15(ink4b) as a direct target of the ZNF217 complex. Downregulation of ZNF217 in MCF-7 breast cancer cells resulted in a dramatic increase in p15(ink4b) expression and coincided with increases in dimethylation of H3-K4 and, surprisingly, a decrease in K9/K14-H3 acetylation. Stimulation of HaCaT cells with transforming growth factor beta (TGF-beta) resulted in a release of ZNF217 and a concomitant binding of SMAD2 to the proximal promoter, which preceded increases in ink4b protein expression. Furthermore, the changes in chromatin marks at the p15(ink4b) promoter following TGF-beta stimulation were similar to those observed following ZNF217 downregulation. Collectively, these results establish the ZNF217 complex as a novel negative regulator of the p15(ink4b) gene and may constitute an important link between amplification of ZNF217 and the loss of TGF-beta responsiveness in breast cancer.

Aberrant DNA methylation is considered an important epigenetic mechanism for gene inactivation. Monoclonal gammopathy of undetermined significance (MGUS) is believed to be a precursor of multiple myeloma (MM). We have analyzed methylation status of p15 INK4B , p16 INK4A , ARF, SOCS-1, p27 KIP1 , RASSF1A, and TP73 genes in bone marrow DNA samples from 21 MGUS and 44 MM patients, in order to determine the role of aberrant promoter methylation as one of the steps involved in the progression of MGUS to MM. Methylation specific polymerase chain reaction assay followed by DNA sequencing of the resulting product was performed. SOCS-1 gene methylation was significantly more frequent in MM (52%) than in MGUS (14%; p=0,006). Methylation frequencies of TP73, ARF, p15 INK4B , p16 INK4A , and RASSF1A were comparable in MGUS: 33%, 29%, 29%, 5%, and 0%, to that observed in MM: 45%, 29%, 32%, 7%, and 2%. All patients lacked methylation at p27 KIP1 gene. In both entities, a concurrent methylation of p15 INK4B and TP73 was observed. The mean methylation index of MGUS was lower (0.16) than that of MM (0.24; p<0.05). Correlations with clinicopathologic characteristics showed a higher mean age in MGUS patients with SOCS-1 methylated (p<0.001); meanwhile in MM, methylation of p15 INK4B was more frequent in males (p=0.009) and IgG isotype (p=0.038). Our findings suggest methylation of TP73, ARF, p15 INK4B , and p16 INK4A as early events in the pathogenesis and development of plasma cell disorders; meanwhile, SOCS-1 methylation would be an important step in the clonal evolution from MGUS to MM.

Inactivation of p15INK4b, an inhibitor of cyclin-dependent kinases, through DNA methylation is one of the most common epigenetic abnormalities in myeloid leukemia. Although this suggests a key role for this protein in myeloid disease suppression, experimental evidence to support this has not been reported. To address whether this event is critical for premalignant myeloid disorders and leukemia development, mice were generated that have loss of p15Ink4b specifically in myeloid cells. The p15Ink4b(fl/fl)-LysMcre mice develop nonreactive monocytosis in the peripheral blood accompanied by increased numbers of myeloid and monocytic cells in the bone marrow resembling the myeloproliferative form of chronic myelomonocytic leukemia. Spontaneous progression from chronic disease to acute leukemia was not observed. Nevertheless, MOL4070LTR retrovirus integrations provided cooperative genetic mutations resulting in a high frequency of myeloid leukemia in knockout mice. Two common retrovirus insertion sites near c-myb and Sox4 genes were identified, and their transcript up-regulated in leukemia, suggesting a collaborative role of their protein products with p15Ink4b-deficiency in promoting malignant disease. This new animal model demonstrates experimentally that p15Ink4b is a tumor suppressor for myeloid leukemia, and its loss may play an active role in the establishment of preleukemic conditions.

This study evaluates the incidence and prognostic impact of aberrant methylation of 25 tumor suppressor genes in 40 patients with RARS, a MDS subtype, by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) assay. Methylation of at least one gene was detected in 18 patients (45%). The genes methylated were CDKN2B (20%), RASSF1 (18%), RARB (10%), CDH13 (7.5%) and FHIT (5%). Patients with at least one methylated gene had a significantly shorter OS than patients without methylated genes. Aberrant methylation is a frequent event in patients with RARS as in patients with high-risk MDS appears to confer a worse prognosis.

A 42 kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16(INK4A), p14(ARF) and p15(INK4B), and is altered in an estimated 30-40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15(INK4B) and p16(INK4A), but not p14(ARF), inhibits the expression of ANRIL (antisense non-coding RNA in the INK4 locus), a 3.8 kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15(INK4B) locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15(INK4B) locus, increases the expression of p15(INK4B), but not p16(INK4A) or p14(ARF), and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15(INK4B) locus.

Four INK4 proteins can prevent cell proliferation by specifically inhibiting cyclin D-dependent kinases. Both p18INK4c and p19INK4d were widely expressed during mouse embryogenesis, but p16INK4a and p15INK4b were not readily detected prenatally. Although p15INK4b, p18INK4c and p19INK4d were demonstrated in many tissues by 4 weeks after birth, p16INK4a protein expression was restricted to the lung and spleen of older mice, with increased, more widespread mRNA expression during aging. Transcripts encoding the INK4a alternative reading frame product p19ARF were not detected before birth but were ubiquitous postnatally. expression of p16INK4a and p15INK4b was induced when mouse embryos were disrupted and cultured as mouse embryo fibroblasts (MEFs). The levels of p16INK4a and p18INK4c, but not p15INK4b or p19INK4d, further increased as MEFs approached senescence. Following crisis and establishment, three of four independently-derived cell lines became polyploid and expressed higher levels of functional p16INK4a. In contrast, one MEF line that sustained bi-allelic deletions of INK4a initially remained diploid. Therefore, loss of p16INK4a and other events predisposing to polyploidy may represent alternative processes contributing to cell immortalization. Whereas p18INK4c and p19INK4d may regulate pre- and postnatal development, p16INK4a more likely plays a checkpoint function during cell senescence that underscores its selective role as a tumor suppressor.

The cyclin-dependent kinase inhibitors p15INK4b and p16INK4a are involved in the development of a wide range of human and murine tumors. These tumor suppressor genes are inactivated by deletions frequently associated to point mutations in the coding regions or hypermethylation of their promoters. In this work, we describe a simple-sequence length polymorphism located in mouse Chromosome (Chr) 4, between the Cdkn2B (p15INK4b) and Cdkn2A (p16INK4a) genes, only 700 bp downstream of the Cdkn2B locus. This DNA region was analyzed in different inbred strains showing a variable AC-repetitive DNA sequence. We used this microsatellite to detect loss of heterozygosity of the Cdkn2A and Cdkn2B loci in gamma-irradiation-induced thymic lymphomas of C57BL/6J x RF/J F1 hybrids. Using this specific marker, we were able to locate additional allelic losses not detected by other microsatellites. Since the allelic losses can be detected by a simple PCR amplification, this AC-repetitive sequence is specially useful as a genetic marker for these Cdkn2 genes and specifically for the p15INK4b cell cycle inhibitor.