Search

Background: The histopathology of melanocytic tumors sometimes presents diagnostic problems. Applicable parameters other than routine pathology are needed., Objective: We assessed the feasibility of multiplex ligation-dependent probe amplification (MLPA), a novel PCR-based genome profiling method, in the classification of melanocytic tumors., Method: We extracted DNA from paraffin-embedded tissue sections of 24 primary melanomas, 14 Spitz nevi and 17 common melanocytic nevi. We analyzed the copy number gains or losses of a total of 76 genes spanning almost all chromosome arms using commercially available MLPA kits., Results: Although four melanocytic nevi and three Spitz nevi did not yield sufficient DNA for reliable analysis due to small tumor size, the MLPA analysis was feasible and applicable to the remaining 88% of samples. We found multiple genetic aberrations in primary melanomas. The total number of aberrations in each tumor ranged from 1 to 32 (average, 12.04). All but two melanomas showed aberrations at more than three genetic loci. Seventeen (70.8%) of the 24 melanomas showed a copy number loss of either the CDKN2A or CDKN2B gene on chromosome 9p21. All the Spitz nevi and 7 (50%) of 14 common melanocytic nevi had copy number changes at one or two gene loci (average, 1.04). The receiver operator characteristic curve analysis showed that the threshold value of copy number aberrations corresponding to 98% specificity for melanoma was 2.42 and the sensitivity using this threshold value was 92.5%., Conclusions: MLPA could be used as an adjunctive diagnostic tool for melanocytic tumors.

Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14(ARF) and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14(ARF) exon 1beta, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14(ARF) exon 1beta alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA.

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.

Decitabine (5-aza-2'-deoxycytidine) inhibits DNA methylation and has dual effects on neoplastic cells, including the reactivation of silenced genes and differentiation at low doses and cytotoxicity at high doses. We evaluated, in a phase 1 study, low-dose prolonged exposure schedules of decitabine in relapsed/refractory leukemias. Patient cohorts received decitabine at 5, 10, 15, or 20 mg/m2 intravenously over one hour daily, 5 days a week for 2 consecutive weeks, doses 5- to approximately 30-fold lower than the maximum tolerated dose (MTD). There were 2 groups that also received 15 mg/m2 daily for 15 or 20 days. A total of 50 patients were treated (44 with acute myelogenous leukemia [AML]/myelodysplasia [MDS], 5 with chronic myelogenous leukemia [CML], and 1 with acute lymphocytic leukemia [ALL]), and the drug was well tolerated at all dose levels, with myelosuppression being the major side effect. Responses were seen at all dose levels. However, the dose of 15 mg/m2 for 10 days appeared to induce the most responses (11 of 17 or 65%), with fewer responses seen when the dose was escalated or prolonged (2 of 19 or 11%). There was no correlation between P15 methylation at baseline or after therapy and response to decitabine. We conclude that decitabine is effective in myeloid malignancies, and low doses are as or more effective than higher doses.

5-fluorouracil (5-FU) has been widely used for chemotherapy of head and neck cancer, and is known to affect the cell cycle and induce apoptotic death of cancer cells. However, the molecular actions of 5-FU on the cell cycle regulatory mechanism have not been fully explained. Herein we analyzed the effects of 5-FU on the expression of G1/S-related cell cycle regulators in oral cancer cell lines. In vitro 5-FU treatment of oral cancer cells resulted in an increase in G1/S phase cells. p21 expression was augmented by 5-FU without any notable changes in p53 expression. A remarkable up-regulation of cyclin E and a concomitant down-regulation of cyclin D were observed after 24 h 5-FU treatment. Our results suggest that 5-FU-induced changes in cell cycle regulation of oral cancer cells might associate with an alteration of G1 cyclins expression. p21 was remarkably up-regulated, but it was speculated that its activity might be cancelled by an increased binding to CDK4.

To screen genes involved in P15(INK4b) regulation during cell cycle, differential display method was applied to compare mRNAs from G(1) synchronized cells of MLIK6, which overexpressed P15(INK4b) gene, and its control MLC2. By using this approach, 15 cDNA fragments that were preferentially expressed in MLIK6 cells, but not in MLC2 cells, were screened out. A novel gene named P15RS was identified with further analysis. Combining the sequence from DD-PCR, homology analysis against EST database and RACE, a 4,404 bp complete cDNA sequence of P15RS was generated. Sequence analysis revealed that P15RS cDNA encoded a 312-amino-acid peptide containing a RAR domain that is involved in regulation of nuclear pre-mRNA, which suggests that P15RS may be a nuclear regulation protein. Genomic sequence analysis demonstrated that human P15RS gene was localized on chromosome 18q12 with seven exons and six introns. Expressing antisense P15RS in MLIK6 cells can up-regulate the expression of cyclinD1 and cyclinE. These data indicate that P15RS may act as a negative regulator in G(1) phase.

p16 and p15, 2 inhibitors of cyclin-dependent kinases, are frequently hypermethylated in hematologic neoplasias. Decitabine, or 5-Aza-2'-deoxycytidine, reverts hypermethylation of these genes in vitro, and low-dose decitabine treatment improves cytopenias and blast excess in ~50% of patients with high-risk myelodysplastic syndrome (MDS). We examined p15 and p16 methylation status in bone marrow mononuclear cells from patients with high-risk MDS during treatment with decitabine, using a methylation-sensitive primer extension assay (Ms-SNuPE) to quantitate methylation, and denaturing gradient gel electrophoresis (DGGE) and bisulfite-DNA sequencing to distinguish individually methylated alleles. p15 expression was serially examined in bone marrow biopsies by immunohistochemistry. Hypermethylation in the 5' p15 gene region was detected in 15 of 23 patients (65%), whereas the 5' p16 region was unmethylated in all patients. Among 12 patients with hypermethylation sequentially analyzed after at least one course of decitabine treatment, a decrease in p15 methylation occurred in 9 and was associated with clinical response. DGGE and sequence analyses were indicative of hypomethylation induction at individual alleles. Immunohistochemical staining for p15 protein in bone marrow biopsies from 8 patients with p15 hypermethylation revealed low or absent expression in 4 patients, which was induced to normal levels during decitabine treatment. In conclusion, frequent, selective p15 hypermethylation was reversed in responding MDS patients following treatment with a methylation inhibitor. The emergence of partially demethylated epigenotypes and re-establishment of normal p15 protein expression following the initial decitabine courses implicate pharmacologic demethylation as a possible mechanism resulting in hematologic response in MDS.

The p16INK4a and p15INK4b 5' CpG island hypermethylation has been described as one of the most frequent mechanisms leading to inactivation of these tumor suppressor genes in hematological malignancies. The p16 and p15 promoter regions were studied using methylation-specific polymerase chain reaction in 53 CD30 non-Hodgkin's lymphomas (25 anaplastic large-cell, 13 peripheral T cell, and 15 anaplastic diffuse large B cell) and 26 Hodgkin's lymphomas, with the aim of comparing the methylation status of these tumor suppressor genes in anaplastic large-cell lymphomas and other related entities. p16 and p15 methylation was detected, respectively, in 28% and 60% of CD30 non-Hodgkin's lymphomas and in 38% and 42% of Hodgkin's neoplasms. This confirms the p16-methylated status in Hodgkin's cases described in a single previous study and adds information concerning the p15 gene that was also found to be methylated in this lymphoma subtype. Methylation incidence within cases at diagnosis and at relapse suggests that it is an early event in anaplastic large-cell lymphomas, being involved in tumor progression in Hodgkin's cases. Our results show that although p16 and/or p15 methylation is involved in non-Hodgkin's and Hodgkin's tumors that share morphological and phenotypic features, differences in incidence, pattern of methylation, and implication in tumor progression are observed.

This study aimed at determining the prevalence of INK4 deletions and their impact on outcome in 125 children with acute lymphoblastic leukemia (ALL) at first relapse using real-time quantitative polymerase chain reaction. Patients were enrolled into relapse trials ALL-REZ BFM (ALL-Relapse Berlin-Frankfurt-Münster) 90 and 96. The prevalence of p16(INK4a) and p15(INK4b) homozygous deletions was 35% (44 of 125) and 30% (38 of 125), respectively. A highly significant association of both gene deletions was found with the 2 major adverse prognostic factors known for relapsed childhood ALL: T-cell immunophenotype and first remission duration. There was no correlation between INK4 deletions and probability of event-free survival. These findings argue against an independent prognostic role of INK4 deletions in relapsed childhood ALL.

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.

There are few data on the molecular pathogenesis of cutaneous T cell lymphomas. A recent allelotyping study by our group identified frequent allelic loss on 9p, 10q, and 17p including losses on 9p21 in 16% of patients with mycosis fungoides and 46% with Sezary syndrome. The P15 and P16 genes are intricately linked on 9p21 and can be inactivated in melanoma and non-Hodgkin's lymphoma. We have therefore studied 76 patients with either mycosis fungoides or Sezary syndrome for abnormalities of these genes. DNA samples were analyzed for loss of heterozygosity, homozygous deletion, intragenic mutations, and promoter methylation. In addition P15 and P16 protein expression was assessed. Microsatellite analysis was informative in 73 of 76 cases: allelic loss on 9p21 was identified in 18 patients (25%), including 12 of 57 with mycosis fungoides (21%) and six of 16 with Sezary syndrome (37%). Single strand conformation polymorphism analysis of the entire coding regions of both genes did not identify any mutations, although two polymorphisms were identified including C613A, which has not previously been described. P15 and P16 gene promoter methylation was found in 45% and 29% of patients, respectively. Furthermore aberrant P15 protein expression was detected in 85% of patients analyzed with P15 gene abnormalities and abnormal P16 expression in 59% with P16 gene abnormalities. These abnormalities were not dependent on cutaneous stage of disease. This study suggests that abnormalities of the P15 and P16 genes are common in both early and advanced stages of mycosis fungoides and Sezary syndrome and that these genes may be inactivated by allelic loss and aberrant promoter methylation.

Germ-line events, such as paternal mutation or genomic imprinting, contribute to the early onset of childhood cancers such as retinoblastoma, Wilms tumors, and neuroblastoma. Given the high frequency of deletion involving chromosome 9p in childhood acute lymphoblastic leukemia (ALL), this study investigated whether 9p deletion might reflect preexisting germ-line gene inactivation. To do this the parental origin of deletion was determined in 10 cases of ALL with 9p21 loss of heterozygosity. Of these cases, 9 showed loss of the maternally derived allele, suggesting that a germ-line event involving a 9p gene may play a role in the onset of childhood ALL.

The p14(ARF), p15(INK4B) and p16(INK4A) genes were localized to 9p21, where genetic alterations have been reported frequently in various human tumors. We performed a molecular analysis of the mechanism of inactivation in cell lines and 32 oral squamous cell carcinoma (OSCC), using deletion screening, PCR-SSCP, methylation-specific-PCR and cycle sequencing. We detected homozygous deletion of p14(ARF)-1Ebeta in 9 (26.5%), of p15(INK4B) in one (3.1%), and of p16(INK4A) in 22 (56.3%) tumor samples. Three mutations were detected in the p16(INK4A) genes. We detected aberrant methylation of the p14(ARF) genes in 14 (43.8%), of the p15(INK4B) gene in 9 (28.1%), and of the p16(INK4A) gene in 16 (50.0%) tumor samples. Altogether, 87.5% of the samples harbored at least one of the alterations in the p14(ARF), p15(INK4B), and p16(INK4A) genes, indicating that the frequent inactivation of these genes may be an important mechanism during OSCC development.

We investigated 67 meningothelial tumors (20 benign meningiomas, 34 atypical meningiomas, and 13 anaplastic meningiomas) for losses of genetic information from chromosome arms 1p and 9p, as well as for deletion, mutation, and expression of the tumor suppressor genes CDKN2A (p16(INKa)/MTS1), p14(ARF), CDKN2B (p15(INK4b)/MTS2) (all located at 9p21) and CDKN2C (1p32). Comparative genomic hybridization and microsatellite analysis showed losses on 1p in 11 anaplastic meningiomas (85%), 23 atypical meningiomas (68%), and 5 benign meningiomas (25%). One atypical meningioma with loss of heterozygosity on 1p carried a somatic CDKN2C mutation (c.202C>T: R68X). Losses on 9p were found in five anaplastic meningiomas (38%), six atypical meningiomas (18%), and one benign meningioma (5%). Six anaplastic meningiomas (46%) and one atypical meningioma (3%) showed homozygous deletions of the CDKN2A, p14(ARF), and CDKN2B genes. Two anaplastic meningiomas carried somatic point mutations in CDKN2A (c.262G>T: E88X and c.262G>A: E88K) and p14(ARF) (c.305G>T: G102V and c.305G>A: G102E). One anaplastic meningioma, three atypical meningiomas, and one benign meningioma without a demonstrated homozygous deletion or mutation of CDKN2A, p14(ARF), or CDKN2B lacked detectable transcripts from at least one of these genes. Hypermethylation of CDKN2A, p14(ARF), and CDKN2B could be demonstrated in one of these cases. Taken together, our results indicate that CDKN2C is rarely altered in meningiomas. However, the majority of anaplastic meningiomas either show homozygous deletions of CDKN2A, p14(ARF), and CDKN2B, mutations in CDKN2A and p14(ARF), or lack of expression of one or more of these genes. Thus, inactivation of the G(1)/S-phase cell-cycle checkpoint is an important aberration in anaplastic meningiomas.

In myelodysplastic syndrome (MDS), the expression of the cyclin-dependent kinase inhibitor p15(ink4B) (p15) is frequently decreased because of the aberrant methylation of the gene promoter; p15 is normally up-regulated during megakaryocytic differentiation. It was hypothesized that p15 methylation and deregulation of gene expression contribute to defective megakaryocytopoiesis in patients with MDS. Here it is shown that the increasing autocrine production of TGF-beta1 stimulates megakaryocytic differentiation in normal CD34(+) cells and that p15 mediates, at least in part, this effect. This TGF-beta1-dependent pathway is altered in MDS CD34(+) progenitors because of p15 methylation. The demethylating agent 2-deoxyAZAcytidin can restore the normal demethylated state of the p15 gene and increase its expression. Nevertheless, MDS CD34(+) cells only poorly differentiate to the megakaryocytic lineage. These findings suggest that p15 methylation occurs in a neoplastic clone with a profound defect of cell proliferation, survival, and differentiation that cannot be overcome by using a demethylating drug.

Saikosaponin a, a purified ingredient of Chinese herb with known antitumor activity, can inhibit cell growth and DNA synthesis of hepatoma cell line HepG2. Both mRNA and protein of the CDK inhibitor p-16(INK4a) and p-15(INK4b) in HepG2 were greatly induced by saikosaponin a while that of p-21(CIP), p-27(KIP) and other cell cycle related genes were not. In addition, reduced phosphorylation of RB protein is observed in saikosaponin a-treated HepG2. Staurosporin, one of the PKC inhibitors, significantly prevented the saikosaponin a induced growth inhibition suggesting PKC pathway be involved. On the other hand, the phorbol ester tumor promoter TPA (12-O-Tetredecanolyphorbol 13-acetate) also inhibited HepG2 growth and specifically induced p-16(INK4a) and p-15(INK4b) mRNA expression. The results suggest that both saikosaponin a and TPA-induced HepG2 growth inhibition are associated with p-15(INK4a) and p-16(INK4b) gene expression and might be mediated by PKC signaling pathway., (Copyright 2001 Academic Press.)

p15(INK4b) and p16(INK4a) proteins are cell cycle regulators involved in the inhibition of G1 phase progression. High frequency of methylation of both genes has been reported in multiple myeloma (MM), but it remains to be determined how and when these alterations contribute to tumorigenesis. Monoclonal gammopathy of undetermined significance (MGUS) represents an early disease stage in a fraction of MMs. Plasma cells from 33 patients with MGUS and 33 patients with MM were isolated and analyzed for p15(INK4b) and p16(INK4a) methylation by methylation-specific polymerase chain reaction. Selective methylation was found in 19% for p16(INK4a), 36% for p15(INK4b), and 6.5% for both genes in MGUS, and frequencies were similar in MM suggesting that methylation of these genes is an early event, not associated with transition from MGUS to MM. p15(INK4b) and p16(INK4a) gene methylation might contribute to immortalization of plasma cells rather than malignant transformation in the natural history of MM.

Aberrant hypermethylation of tumor suppressor genes plays an important role in the development of many tumors. Recently identified new DNA methyltransferase (DNMT) genes, DNMT3A and DNMT3B, code for de novo methyltransferases. To determine the roles of DNMT3A, DNMT3B, as well as DNMT1, in the development of leukemia, competitive polymerase chain reaction (PCR) assays were performed and the expression levels of DNMTs were measured in normal hematopoiesis, 33 cases of acute myelogenous leukemia (AML), and 17 cases of chronic myelogenous leukemia (CML). All genes were constitutively expressed, although at different levels, in T lymphocytes, monocytes, neutrophils, and normal bone marrow cells. Interestingly, DNMT3B was expressed at high levels in CD34(+) bone marrow cells but down-regulated in differentiated cells. In AML, 5.3-, 4.4-, and 11.7-fold mean increases were seen in the levels of DNMT1, 3A, and 3B, respectively, compared with the control bone marrow cells. Although CML cells in the chronic phase did not show significant changes, cells in the acute phase showed 3.2-, 4.5-, and 3.4-fold mean increases in the levels of DNMT1, 3A, and 3B, respectively. Using methylation-specific PCR, it was observed that the p15(INAK4B) gene, a cell cycle regulator, was methylated in 24 of 33 (72%) cases of AML. Furthermore, AML cells with methylated p15(INAK4B) tended to express higher levels of DNMT1 and 3B. In conclusion, DNMTs were substantially overexpressed in leukemia cells in a leukemia type- and stage-specific manner. Up-regulated DNMTs may contribute to the pathogenesis of leukemia by inducing aberrant regional hypermethylation. (Blood. 2001;97:1172-1179)

Alterations of p16 and p15 genes have been reported in cancer cell lines and in certain malignant neoplasm. These genes are designated as candidate tumor suppressor genes because they encode proteins that function as negative cell cycle regulators at G(1)-S checkpoint. One hundred and sixty eight tumor tissue, 20 schistosomal tissue, and 50 normal tissue samples were examined. The status of p16 and p15 genes in these tissues was determined by the polymerase chain reaction and by sequencing the DNA fragments produced during PCR. In addition, the expression of p16 and p15 proteins was examined by Western blot analysis. p16 and p15 genes were detected in all normal and schistosomal tissues. Deletion of both p16 and p15 genes was observed in 72 and 36 bladder tumors, respectively. Twenty eight of the 72 cases that exhibited p16 deletions also displayed deletions of p15. Only eight cases showed loss of the p15 gene while retaining p16 gene, and p16 deletion with apparently intact p15 gene was identified in 44 cases. The present analysis also reveals that deletion in the two genes are associated with low-stage, low grade bladder cancer, schistosomiasis-associated bladder cancer (SABC) and squamous cell carcinoma type (SCC). No point mutations were identified in either gene. The expression of p16 and p15 proteins was undetectable in 75 and 38 bladder tumors, respectively, by Western blot analysis. Alteration of the p16 and p15 genes appears to be an early event in bladder cancer which occurs more frequently in SABC and SCC, and may play an important role in the development of schistosomal bladder cancer.

The transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1-mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6-induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6-mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15(INK4B) and p16(INK4A), inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias. (Blood. 2000;96:475-482)

We used a sensitive, quantitative bisulfite PCR assay, methylation sensitive single nucleotide primer extension (Ms-SNuPE), to measure methylation of the 5' CpG islands of c-abl and p15 in chronic myelogenous leukemia (CML) patients during progression. We found that the Pa promoter of c-abl was methylated in 81% (17/21) of the white blood cells (WBCs) of CML patients, which correlates with previous reports. In contrast, WBCs from healthy donors, acute myelogenous leukemias, acute lymphocytic leukemias, and myelodysplastic syndromes were unmethylated at the c-abl Pa promoter locus. We also observed p15 hypermethylation in 24% (8/34) of CML cases. Methylation of the p15 but not c-abl Pa promoters was associated with CML progression (P = 0.047 vs 0.46), and the two events were independently acquired. We conclude that de novo methylation of c-abl and p15 both occur in CML, and analysis of DNA methylation changes using the bisulfite-based MS-SNuPE assay allows both a sensitive and quantitative assessment of these molecular events compared to other methods currently utilized. (Blood. 2000;95:2990-2992)

To test the hypothesis that cell cycle regulatory gene abnormalities are determinants of clinical outcome in adult acute lymphoblastic leukemia (ALL), we screened lymphoblasts from patients on a Southwest Oncology Group protocol for abnormalities of the genes, retinoblastoma (Rb), p53, p15(INK4B), and p16(INK4A). Aberrant expression occurred in 33 (85%) patients in the following frequencies: Rb, 51%; p16(INK4A), 41%; p53, 26%. Thirteen patients (33%) had abnormalities in 2 or more genes. Outcomes were compared in patients with 0 to 1 abnormality versus patients with multiple abnormalities. The 2 groups did not differ in a large number of clinical and laboratory characteristics. The CR rates for patients with 0 to 1 and multiple abnormalities were similar (69% and 54%, respectively). Patients with 0 to 1 abnormality had a median survival time of 25 months (n = 26; 95% CI, 13-46 months) versus 8 months (n = 13; 95% CI, 4-12 months) for those with multiple abnormalities (P <.01). Stem cells (CD34+lin-) were isolated from adult ALL bone marrows and tested for p16(INK4A) expression by immunocytochemistry. In 3 of 5 patients lymphoblasts and sorted stem cells lacked p16(INK4A) expression. In 2 other patients only 50% of sorted stem cells expressed p16(INK4A). By contrast, p16 expression was present in the CD34+ lin- compartment in 95% (median) of 9 patients whose lymphoblasts expressed p16(INK4A). Therefore, cell cycle regulatory gene abnormalities are frequently present in adult ALL lymphoblasts, and they may be important determinants of disease outcome. The presence of these abnormalities in the stem compartment suggests that they contribute to leukemogenesis. Eradication of the stem cell subset harboring these abnormalities may be important to achieve cure.

We prospectively analyzed p15 and p16 promoter methylation patterns using methylation-specific polymerase chain reaction (PCR) in patients with adult and childhood acute leukemias and studied the association of methylation patterns with chromosomal abnormalities and prognostic variables. In nearly all French-American-British leukemia subtypes, we found p15 methylation in bone marrow or peripheral blood cells from 58% (46/79) of patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), or acute biphenotypic leukemia (ABL). An identical alteration was detected in blood plasma from 11 of 12 of these patients (92%). We also demonstrated for the first time concomitant p16 and p15 methylation in 22% (8/37) of adults with AML or ALL, exclusively in those with M2, M4, or L2 subtypes. According to cytogenetic data from 35 patients with ALL, AML, or ABL, 82% (14/17) of those with unmethylated p15 alleles had normal karyotypes or hyperdiploidies associated with a favorable prognosis. Conversely, 44% (8/18) of patients with p15 methylation had chromosomal translocations, inversions, or deletions, suggesting an interplay of these abnormalities with p15 methylation. As a prognostic marker for disease monitoring, p15 methylation appears to be more widely applicable than BCR-ABL, AF4-MLL, and AML1-ETO transcripts, which were detectable in only 8% (4/48) of patients by reverse transcriptase-PCR. Thirty-nine of 43 blood samples (91%) sequentially collected from 12 patients with AML, ALL, or ABL showed p15 methylation status in excellent concordance with morphologic disease stage. Early detection of p15 methylation at apparent remission or its acquisition during follow-up may prove valuable for predicting relapse. Overall survival of patients with p15 methylation was notably shortened among 38 adults with AML and 12 adults with ALL. Aberrant p15 methylation may have important prognostic implications for clinical monitoring and risk assessment. (Blood. 2000;95:1942-1949)

The promoter region of the cyclin-dependent kinase inhibitor p15(INK4B) contains a CpG island that is hypermethylated in many hematologic malignancies. To explore the relationship between patterns of methylation and gene transcription, we used bisulfite genomic sequencing to obtain a detailed analysis of methylation in acute leukemia, leukemia cell lines, and normal lymphocytes. The entire CpG island region of p15 was largely devoid of methylation in normal lymphocytes, but methylation of varying density was found in primary acute leukemia. Methylation density was generally conserved between the alleles from each sample, but marked heterogeneity for the specific CpG sites methylated was observed. Patterns of methylation were compared and expression assessed with reverse-transcriptase polymerase chain reaction (RT-PCR). The density of methylation within the CpG island, and not any specific location, correlates best with transcriptional loss. Leukemias with methylation of approximately 40% of the CpG dinucleotides on each allele had complete gene silencing, with variable, but diminished expression with less dense CpG island methylation. Our results suggest that the transcriptional silencing of p15 in conjunction with aberrant hypermethylation is best understood as an evolutionary process that involves progressively increasing methylation of the entire p15 CpG island.

The methylation status of p15(INK4b) (MTS2), p16(INK4a) (MTS1) and p14(ARF) (p16beta) was analyzed in 56 lymphomas by restriction-enzyme related polymerase chain reaction (PCR) (REP), methylation-specific PCR (MSP), and bisulfite genomic sequencing (BGS). Methylation of the p15 and p16 genes was detected, respectively, in 64% and 32% of the B-cell lymphomas, in 44% and 22% of the T-cell lymphomas, and in none of the 5 reactive lymph nodes analyzed. Both p15 and p16 genes were methylated more often in the high-grade (78% and 50%, respectively) than in the low-grade B-cell lymphomas (55% and 21%, respectively). For 5 cases, mapping of the methylated CpGs of the p16 promoter region confirmed the results of REP and MSP. In addition, a large variation in the methylation patterns of p16 exon 1 was observed, not only from one lymphoma to another, but also within a given tumor. Methylation of p15 and p16 was associated with an absence of gene expression, as assessed by reverse transcription-PCR. The p14 gene was unmethylated and normally expressed in all 56 tumors. We found no mutations of p15, p16, or p14 in any of the 56 lymphomas. Our results suggest a role for p15 and p16 gene methylation during lymphomagenesis and a possible association between p15 and p16 inactivation and aggressive transformation in B-cell and T-cell lymphomas.

The INK4 cyclin dependent kinase inhibitors (CDKI), such as p15(INK4B) and p16(INK4A), block cell cycle progression from G to S phase. This is mediated by inhibition of phosphorylation of proteins, including the retinoblastoma susceptibility protein (Rb), by cyclin dependent kinases. Ectopic over-expression of the p16(INK4A) CDKI can inhibit growth of cell lines depending on Rb status. Cell lines lacking Rb, with few exceptions, are resistant to growth inhibition by p16(INK4A). The effects of ectopic over-expression of p15(INK4B) in cell lines with and without wild type Rb were examined by measuring cell recovery. Proliferation was inhibited in cells lacking Rb as well as in cells with wild type Rb expression. Experiments analyzing the effectiveness of chimeric p15(INK4B)/p16(INK4A) proteins indicated that the Rb independent growth inhibition required N-terminal residues of p15(INK4B). Linker insertion mutation of p15(INK4B) showed that the inhibition was dependent on intact ankyrin structures. Double staining flow cytometry found that the growth inhibition correlated with a decrease in cells in G2/M phases of the cell cycle. These findings are consistent with Rb independent inhibition of the progression from G1 to S caused by overexpression of p15(INK4B)., (Copyright 1999 Academic Press.)

Background: Angiotensin II has been reported to induce renal tubular hypertrophy, but the mechanisms of this hypertrophy are not well known. We evaluated the roles of cyclin-dependent kinase (CDK) inhibitors in renal tubular hypertrophy., Methods: To elucidate whether CDK inhibitors cause renal tubular hypertrophy, we produced adenovirus vectors containing coding sequences of the CDK inhibitors p27Kip1 (AxCAp27), p21CIP1 (AxCAp21), and p16INK4 (AxCAp16), and we investigated the effect of these gene transfers on epidermal growth factor (EGF)-induced proliferation in LLC-PK1 cells. We evaluated the cell cycle and hypertrophy by measurements of the [3H]-leucine and [3H]-thymidine incorporation, the protein:DNA ratio, flow cytometry, and CDK4 and CDK2 kinase assays., Results: AxCAp27 and AxCAp21 caused significant increases in [3H]-leucine incorporation and the protein:DNA ratio but did not change the [3H]-thymidine incorporation. Conversely, AxCAp16 inhibited EGF-stimulated [3H]-thymidine incorporation but did not change the [3H]-leucine incorporation. AxCAp27, AxCAp21, and AxCAp16 all inhibited EGF-stimulated CDK4 kinase activity (to 15.6, 14.1, and 21.9% of control, respectively). Forward light-scatter analysis demonstrated that AxCAp27 and AxCAp21 increased the cell size but that AxCAp16 effected no change in cell size., Conclusion: These findings suggest that p27Kip1 and p21CIP1 may play an important role in hypertrophy of renal tubule cells by reducing pRb phosphorylation. On the other hand, p16INK4 was not found to cause hypertrophic changes in EGF-treated LLC-PK1 cells.

Transforming growth factor beta (TGF-beta) can inhibit epithelial cell growth and induce extracellular matrix formation through signal transduction via its two receptors and its downstream intracellular Smad proteins. We recently reported a germline mutation, i.e., substitution of methionine for threonine at codon 315 in the kinase subdomain IV, of the TGF-beta type II receptor gene in a kindred of hereditary nonpolyposis colorectal cancer without microsatellite instability and found that the mutant receptor abolished the signal transduction for growth inhibition by TGF-beta. In this study, we performed further functional analysis of this mutant receptor. The results showed that, in contrast to its failure to mediate growth inhibition by TGF-beta, the mutant receptor still retained the ability to induce one of the extracellular matrix proteins, plasminogen activator inhibitor type 1, upon TGF-beta treatment. However, coincident with its failure to mediate growth inhibition by TGF-beta, the mutant receptor failed to transcriptionally upregulate one of the cyclin-dependent kinase inhibitors, p15(INK4B), in response to TGF-beta. These data suggest that threonine 315 of the TGF-beta type II receptor is dispensable for extracellular matrix protein production, but is essential for the growth inhibition by TGF-beta, and that the lack of growth inhibition due to the mutant receptor is possibly mediated through its failure to upregulate p15(INK4B)., (Copyright 1999 Academic Press.)

We have previously shown in transgenic mice that transforming growth factor (TGF)-alpha dramatically enhances c-myc-induced hepatocarcinogenesis by promoting proliferation and survival of hepatocellular carcinoma (HCC) cells. As transgenic livers display increased levels of mature TGF-beta1 from the early stages of hepatocarcinogenesis, we have now assessed whether impairment of TGF-beta1 signaling contributes to the deregulation of cell cycle progression and apoptosis observed during this process. Focal preneoplastic lesions lacking expression of TGF-beta receptor type II (TbetaRII) were detected in c-myc/TGF-alpha but not in c-myc livers. In c-myc/TGF-alpha mice, 40% (2/5) of adenomas and 90% (27/30) of HCCs showed down-regulation of TbetaRII expression in comparison with 11% (2/18) of adenomas and 47% (14/30) of HCCs in c-myc mice. Down-regulation of the TGF-beta1-inducible p15(INK4B) mRNA and reduced apoptotic rates in TbetaRII-negative HCCs further indicated the disruption of TGF-beta1 signaling. Furthermore, both TbetaRII-negative and -positive c-myc TGF-alpha HCCs, but not c-myc HCCs, were characterized by decreased levels of the cell cycle inhibitor p27. These results suggest 1) an inverse correlation of decreased p27 expression with the particularly strong expression of TGF-alpha in these lesions, consistent with the capacity of TGF-alpha signaling to post-transcriptionally regulate p27, and 2) the presence of alternative, downstream defects of TGF-beta1 signaling in c-myc/TGF-alpha HCCs that may impair the growth-inhibitory response to TGF-beta1. Thus, the accelerated neoplastic development in c-myc/TGF-alpha mice is associated with an early and frequent occurrence of TbetaRII-negative lesions and with reduced levels of p27 in HCC cells, indicating that disruption of TGF-beta1 responsiveness may play a crucial role in the enhancement of c-myc-induced hepatocarcinogenesis by TGF-alpha.

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the presence of differentiation-inhibited pro- and pre-T-cell blasts. The p16INK4a tumor suppressor gene has been shown to be frequently deleted in human T-ALL cases. Deletion of p16INK4a may be associated with poor prognosis and relapse of the disease. Radiation-induced murine T-ALL in C57B1/6 mice shares pathogenetic and molecular characteristics with the human disease. We used the murine disease as a model to study the status of the INK4/ARF gene locus and to examine the effect of p16INK4a-re-expression in T-ALL cells on their leukemic potential in vivo. In 9 of 17 radiation-induced murine T-ALL cell lines, the p16INK4a protein was not expressed as determined by immunoblotting. Southern blot analysis revealed homozygous deletions of the p16INK4a gene locus in three of the nine lines, along with the genes encoding p15INK4b and p19ARF. Transduction of p16INK4a-negative T-ALL lines with retrovirus encoding p16INK4a significantly inhibited their in vitro proliferation by inducing G1-arrest. Importantly, re-expression of p16INK4a in p16INK4a-negative T-ALL cells obliterated the induction of lethal disseminated leukemia in syngeneic mice. This is the first demonstration that re-establishment of p16INK4a expression is critical for in vivo growth regulation of T-ALL cells.

Although numerous somatic mutations that contribute to the pathogenesis of childhood acute lymphoblastic leukemia (ALL) have been identified, no specific cytogenetic or molecular abnormalities are known to be consistently associated with relapse. The p16(INK4A) (p16), which encodes for both p16(INK4A) and p19(ARF) proteins, and p15(INK4B) (p15) genes are inactivated by homozygous deletion and/or p15 promoter hypermethylation in a significant proportion of cases of childhood ALL at the time of initial diagnosis. To determine whether alterations in these genes play a role in disease progression, we analyzed a panel of 18 matched specimen pairs collected from children with ALL at the time of initial diagnosis and first bone marrow relapse for homozygous p16 and/or p15 deletions or p15 promoter hypermethylation. Four sample pairs contained homozygous p16 and p15 deletions at both diagnosis and relapse. Among the 14 pairs that were p16/p15 germline at diagnosis, three ALLs developed homozygous deletions of both p16 and p15, and two developed homozygous p16 deletions and retained p15 germline status at relapse. In two patients, p15 promoter hypermethylation developed in the interval between initial diagnosis and relapse. In total, homozygous p16 deletions were present in nine of 18 cases, homozygous p15 deletions in seven of 18 cases, and p15 promoter hypermethylation in two of eight cases at relapse. These findings indicate that loss of function of proteins encoded by p16 and/or p15 plays an important role in the biology of relapsed childhood ALL, and is associated with disease progression in a subset of cases.

9p21 chromosomal region contains p15INK4b and p16INK4a genes which regulate G1 phase of the cell cycle by inhibition of cyclin-cyclin dependent kinases. The p19ARF protein is translated from an alternative transcript of the p16INK4a gene and regulates G1 and G2 phase of the cell cycle by degradation of the MDM2 protein. p16INK4a and p15INK4b gene homozygous deletions occur mostly in acute lymphoblastic leukemia, ATL secondary to HTLV1 infection, and some lymphoma. Point mutations of p16INK4a or p15INK4b gene seem to be extremely rare, however selective methylations of the p15INK4b or p16INK4a promoters are frequently found in myelodysplastic syndromes and acute leukemias, or lymphomas and myelomas respectively. These data demonstrate that inactivation of gene of the 9p21 region is currently the main molecular event found in hematological malignancies.

Low-grade follicle center lymphoma (LGFCL) is characterized genetically by the t(14;18) translocation and an indolent clinical course. Histologic progression from LGFCL to an aggressive diffuse large B-cell lymphoma (DLCL) occurs in 60% to 80% of cases, and this transformation is associated with the accumulation of secondary genetic alterations. Using 10 polymorphic microsatellite markers spanning the chromosome 9p21 region harboring the p15 (p15(INK4B)/MTS-2/CDKN2B) and p16 (p16(INK4A)/MTS-1/CDKN2) tumor-suppressor gene loci, we analyzed 11 matched pairs of LGFCL and their corresponding progressed DLCL biopsies for loss of heterozygosity and homozygous deletions at 9p21. A comparative multiplex polymerase chain reaction assay was also used for the detection of homozygous deletions. Deletions were identified in 8 of the 11 cases studied (73%): 6 homozygous (54%) and 2 hemizygous (18%). The deletions were identified exclusively in the progressed DLCL biopsies. Immunohistochemical studies showed an excellent correlation with the results from the genetic analyses. Of the 9 matched pairs of LGFCL and progressed DLCL with interpretable immunohistochemical staining, 9 of 9 (100%) of the LGFCL showed diffuse reactivity for p16. Four of the 9 (44%) immunohistochemically evaluable cases of progressed DLCL showed loss of or, in 1 case, markedly diminished p16 expression. All 4 of these cases correspondingly showed homozygous deletions at 9p21. Five of the 9 progressed DLCL cases showed p16 expression and demonstrated retention of one or both 9p21 alleles by genetic analysis. This is the first longitudinal series examining sequential biopsy specimens of low-grade and progressed FCL for genetic loss at 9p21 encompassing the p16 and p15 loci. The high frequency and exclusive occurrence of deletions involving p16 in the progressed DLCLs suggests that genetic loss at 9p21 targeting p16 and/or p15 is an important secondary genetic event in the histologic progression of FCL.

p15(INK4b) gene is an inhibitor of cyclin-dependent kinase (CDK) 4 and CDK6 whose expression is induced by transforming growth factor (TGF)beta. Recent reports suggest frequent methylation of the p15(INK4b) gene promoter in leukemias, and it has been proposed that this methylation could be necessary for leukemic cells to escape TGF beta regulation. We investigated the methylation status of p15(INK4b) gene in 53 myelodysplastic syndromes (MDS) cases, including nine that had progressed to acute myeloid leukemia (AML), using a recently described sensitive method where polymerase chain reaction (PCR) is preceded by bisulfite modification of DNA (methylation specific PCR). p15(INK4b) methylation was observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients with greater than 10% bone marrow blasts had p15(INK4b) methylation (including all nine patients who had progressed to AML) as compared with none of MDS patients with <10% bone marrow blasts. No correlation between karyotypic abnormalities and methylation status was found. Patients with p15(INK4b) methylation had a worse prognosis, but the prognostic significance of p15(INK4b) methylation was no more found by multivariate analysis, due to its strong correlation to the percentage of marrow blasts. In 10 MDS cases, sequential DNA samples were available. In five of them, methylation of the p15(INK4b) gene was detected at leukemic transformation, but not at diagnosis. Our results showed that methylation of the p15(INK4b) gene in MDS is correlated with blastic bone marrow involvement and increases with disease evolution toward AML. It suggests that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle, and possibly of TGF beta inhibitory effect.

The fact that the p16/INK4a and p15/INK4b genes are frequently inactivated in human malignancies and that p16/INK4a null mice spontaneously develop B-cell lymphomas prompted us to examine the status of both genes in Burkitt's Lymphoma (BL). We found a low frequency of p16/INK4a and p15/INK4b deletions and mutations in BL cell lines and biopsies. However, p16/INK4a exon 1 was methylated in 17 out of 19 BL lines (89.5%) and in 8 out of 19 BL biopsies (42%) analyzed. p15/INK4b Exon 1 was also methylated, although at a lower frequency. p16/INK4a mRNA was readily detected in BL lines carrying unmethylated p16/INK4a, but not in those carrying methylated p16/INK4a. No p16/INK4a protein was detected in any of the BL lines and biopsies examined. In contrast, only one out of seven lymphoblastoid cell lines (LCLs) examined was methylated in p16/INK4a exon 1, and three out of the six LCLs with unmethylated p16/INK4a expressed detectable levels of p16/INK4a protein. Thus, the frequent p16/INK4a methylation in BL lines correlates with downregulation of p16/INK4a expression, suggesting that exon 1 methylation is responsible for silencing the p16/INK4a gene in BL.

Neoplastic cells simultaneously harbor widespread genomic hypomethylation, more regional areas of hypermethylation, and increased DNA-methyltransferase (DNA-MTase) activity. Each component of this "methylation imbalance" may fundamentally contribute to tumor progression. The precise role of the hypomethylation is unclear, but this change may well be involved in the widespread chromosomal alterations in tumor cells. A main target of the regional hypermethylation are normally unmethylated CpG islands located in gene promoter regions. This hypermethylation correlates with transcriptional repression that can serve as an alternative to coding region mutations for inactivation of tumor suppressor genes, including p16, p15, VHL, and E-cad. Each gene can be partially reactivated by demethylation, and the selective advantage for loss of gene function is identical to that seen for loss by classic mutations. How abnormal methylation, in general, and hypermethylation, in particular, evolve during tumorigenesis are just beginning to be defined. Normally, unmethylated CpG islands appear protected from dense methylation affecting immediate flanking regions. In neoplastic cells, this protection is lost, possibly by chronic exposure to increased DNA-MTase activity and/or disruption of local protective mechanisms. Hypermethylation of some genes appears to occur only after onset of neoplastic evolution, whereas others, including the estrogen receptor, become hypermethylated in normal cells during aging. This latter change may predispose to neoplasia because tumors frequently are hypermethylated for these same genes. A model is proposed wherein tumor progression results from episodic clonal expansion of heterogeneous cell populations driven by continuous interaction between these methylation abnormalities and classic genetic changes.

Previous studies have shown that the cyclin-dependent kinase inhibitor (CDKI) genes p15INK4B and p16INK4A are frequently inactivated by genetic alterations in many malignant tumors and that they are candidate tumor-suppressor genes. Although genetic alterations in these genes may be limited to lymphoid malignancies, it has been reported that their inactivation by aberrant methylation of 5' CpG islands may be involved in various hematologic malignancies. In this study, we investigated the p15INK4B and p16INK4A genes to clarify their roles in the pathogenesis of myelodysplastic syndrome (MDS). Southern blotting analysis showed no gross genetic alterations in either of these genes. However, hypermethylation of the 5' CpG island of the p15INK4B gene occurred frequently in patients with MDS (16/32 [50%]). Interestingly, the p15INK4B gene was frequently methylated in patients with high-risk MDS (refractory anemia with excess blasts [RAEB], RAEB in transformation [RAEB-t], and overt leukemia evolved from MDS; 14/18 [78%]) compared with patients with low-risk MDS (refractory anemia [RA] and refractory anemia with ring sideroblast [RARS]; 1/12 [8%]). Furthermore, methylation status of the p15INK4B gene was progressed with the development of MDS in most patients examined. In contrast, none of the MDS patients showed apparent hypermethylation of the p16INK4A gene. These results suggest that hypermethylation of the p15INK4B gene is involved in the pathogenesis of MDS and is one of the important late events during the development of MDS.

Both p16 and p15, encoded by the genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases (CDK4/6) and the upstream regulators of Rb function. In hematopoietic malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid, and more particularly from B-lineage malignancies except multiple myeloma (MM). To investigate whether these genes are inactivated by deletions, mutations, and hypermethylation of the 5' CpG islands, we examined 12 MM patients by Southern hybridization and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. No deletions nor mutations of the p16 and p15 genes were found. However, hypermethylation was observed in 75% for p16 and 67% for p15 in our group of MM patients. Such high frequencies of involvement of these genes in MM make them hitherto the most common genetic abnormalities in this disease. Concomitant hypermethylation, uncommon thus far in the literature of the study of these genes, is a rather common phenomenon, occurring in 67% of our patient group. Moreover, hypermethylation of p16/p15 was associated with blastic disease and concomitant hypermethylation of both genes may be pathogenetically related to plasmacytoma development. These results indicate that these genes are important in MM pathogenesis. Here we report, for the first time in the literature, the high incidences of p16 and p15 alterations in MM, not by homozygous deletions or mutations, but solely by hypermethylation of the 5' CpG islands, which may be a specific mechanism in this disease.

The aim of this review is to provide insight into the molecular mechanisms by which transforming growth factor-beta (TGF-beta) modulates cell cycle progression in different cell types. Particular attention is focused on the differences between these mechanisms in cells of epithelial origin and in mesenchymally derived cells. This is important because many transformed epithelial cells lose responsiveness to the growth-inhibitory effects of TGF-beta, thus generating a more fibroblast-like phenotype. Loss of negative growth control, including a lack of response to growth-inhibitory factors, is a common feature of many tumor cells. G1 phase cyclin-dependent kinases (cdks) and their inhibitors (ckis) are central to the pathways that regulate commitment to cellular division in response to positive as well as negative growth effectors. Many checkpoints are deregulated in oncogenesis, and this is often due to alterations in cyclin-cdk complexes. The loss of R-point regulation, in particular, can allow cell growth and division to proceed autonomously of external signals. This may occur due to either the aberrant expression of positive regulators, such as the cyclins and cdks, or the loss of negative regulators, such as the ckis. Beginning with a survey of the role of the cdks in the mammalian cell cycle, the review examines how cdk activity is modulated by cyclin binding, phosphorylation, and ckis, including the Ink4 proteins and the closely related inhibitors p21Cip1 and p27Kip1. Particular attention is paid to the role of p27Kip1 and p21Cip1 in the mechanisms of TGF-beta-induced suppression or stimulation of the cell cycle and how these mechanisms contrast between epithelial cells and cells of mesenchymal origin. Other aspects of TGF-beta signal transduction are discussed, including its effects on cyclin and cdk expression in various cell types, and the downstream targets of cdks and their modulation by TGF-beta and other growth factors are also discussed. These include proteins of the retinoblastoma family, and the related modulation of the transcriptional activity of the E2F family members. Finally, the role of cell cycle regulatory proteins in oncogenesis is review in view of the findings described here.

It is now evident that the cell cycle machinery has a variety of elements negatively regulating cell cycle progression. However, among these negative regulators in cell cycle control, only 4 have been shown to be consistently involved in the development of human cancers as tumor suppressors: Rb (Retinoblastoma susceptibility protein), p53, and two recently identified cyclin-dependent kinase inhibitors, p16INK4A/MTS1 and p15INK4B/MTS2. Because there are functional interrelations among these negative regulators in the cell cycle machinery, it is particularly interesting to investigate the multiplicity of inactivations of these tumor suppressors in human cancers, including leukemias/lymphomas. To address this point, we examined inactivations of these four genes in primary lymphoid malignancies by Southern blot and polymerase chain reaction-single-strand conformation polymorphism analyses. We also analyzed Rb protein expression by Western blot analysis. The p16INK4A and p15INK4B genes were homozygously deleted in 45 and 42 of 230 lymphoid tumor specimens, respectively. Inactivations of the Rb and p53 genes were 27 of 91 and 9 of 173 specimens, respectively. Forty-one (45.1%) of 91 samples examined for inactivations of all four tumor suppressors had one or more abnormalities of these four tumor-suppressor genes, indicating that dysregulation of cell cycle control is important for tumor development. Statistical analysis of interrelations among impairments of these four genes indicated that inactivations of the individual tumor-suppressor genes might occur almost independently. In some patients, disruptions of multiple tumor-suppressor genes occurred; 4 cases with p16INK4A, p15INK4B, and Rb inactivations; 2 cases with p16INK4A, p15INK4B, and p53 inactivations; and 1 case with Rb and p53 inactivations. It is suggested that disruptions of multiple tumor suppressors in a tumor cell confer an additional growth advantage on the tumor.

Melanoma is an important human cancer, the etiology of which has been the subject of much study. Recently a gene for familial melanoma, MLM, has been mapped and isolated. This gene encodes the cell-cycle regulator p16 and is mutated in a variety of sporadic human cancers in addition to melanoma. The isolation of MLM answers some questions in the area of melanoma biology, but raises others. Identification of p16 and other genes that contribute to melanoma development may be viewed as one step in the attempt to understand, diagnose, and treat this malignant disease.

Recent developments in the study of the cell cycle have shed much light on the origins of human cancer. We summarize these developments with an emphasis on the molecular characterization and the functional role of the cyclin-dependent kinase family of protein kinases (CDK) and their associated regulatory subunits. The Rb tumor suppressor in the progression from the G1 to S phase of the cell cycle and in tumor development is used as a paradigm for illustrating the importance of understanding the molecular regulatory events in the etiology of cancer. Recent developments with cyclin-dependent kinase inhibitors, most notably, p16 (CDKN2), indicate that these molecules represent new tumor suppressors in both skin and lung cancers. Insights from these cell cycle studies can provide avenues for the diagnosis, prognosis, and potential gene and chemotherapies for many cancers, including non-small cell lung cancer.

No constant genetic alteration has yet been unravelled in T-cell acute lymphoblastic leukemia (T-ALL), and, to date, the most frequent alteration, the SIL-TAL1 deletion, is found in approximately 20% of cases. Recently, two genes have been identified, the multiple tumor-suppressor gene 1 (MTS1) and multiple tumor-suppressor gene 2 (MTS2), whose products inhibit cell cycle progression. A characterization of the MTS locus organization allowed to determine the incidence of MTS1 and MTS2 inactivation in T-ALL. MTS1 and MTS2 configurations were determined by Southern blotting using 8 probes in 59 patients with T-ALL (40 children and 19 adults). Biallelic MTS1 inactivation by deletions and/or rearrangements was observed in 45 cases (76%). Monoallelic alterations were found in 6 cases (10%). The second MTS1 allele was studied in the 4 cases with available material. A point mutation was found in 2 cases. The lack of MTS1 mRNA expression was observed by Northern blot analysis in a third case. A normal single-strand conformation polymorphism pattern of MTS1 exons 1alpha and 2 was found and MTS1 RNA was detected in the fourth case, but a rearrangement occurring 5' to MTS1 exon 1 alpha deleting MTS1 exon 1Beta was documented. One case presented a complex rearrangement. Germline configuration for MTS1 and MTS2 was found in only 7 cases. The localization of the 17 breakpoints occurring in the MTS locus were determined. Ten of them (59%) are clustered in a 6-kb region located 5 kb downstream to the newly identified MTS1 exon 1Beta. No rearrangement disrupting MTS2 was detected and more rearrangements spared MTS2 than MTS1 (P<.01). MTS1 but not MTS2 RNA was detected by Northern blotting in the human thymus. These data strongly suggest that MTS1 is the functional target of rearrangements in T-ALL. MTS1 inactivation, observed in at least 80% of T-ALL, is the most consistent genetic defect found in this disease to date.