p16INK4/p15INK4B gene inactivation is a frequent event in malignant T-cell lines

Abstract: The cell cycle regulators p16^INK4 and p15^INK4B have been mapped to the minimal region of overlap for chromosome 9p21 deletions, observed in a number of malignancies, suggesting that they could be tumor suppressor genes (TSGs). In the case of pl6^INK4 this has been further substantiated by the finding of small intragenic mutations. In this study we have investigated the p16^INK4 and p15^INK4B genes in 16 malignant T-cell lines by means of Southern blot, PCR and sequence analysis. p16^INK4 allelic deletions occurred in 15 of 16 cell lines; 12 of which were homozygous and 3 hemizygous. In 1 cell line (DND 41) the remaining p16^INK4 allele carried a microdeletion of 29 bp of exon 2, supporting the concept that p16^INK4 is a target TSG for deletions on 9p21. Most p16^INK4 deletions also included the p15^INK4B gene. However, 4 of the cell lines deleted for p16^INK4 showed no evidence of p15^INK4B loss, indicating that p15^INK4B is not the target in these cell lines.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

p16(INK4a) and p15(INK4b) gene methylations in plasma cells from monoclonal gammopathy of undetermined significance.

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.     

Detection of hypermethylation of the p16(INK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus

BACKGROUND/AIM: Inactivation of the p16(INK4A) (p16) tumour suppressor gene by promoter region hypermethylation has been demonstrated not only in many types of tumours, including hepatocellular carcinoma (HCC), but also in early preneoplastic lesions in the lung, colon, oesophagus, and pancreas. The aim of this study was to examine the methylation status of the p16 promoter in pre- and/or non-neoplastic liver diseases. PATIENTS/SUBJECTS/METHODS: The methylation status of p16 was evaluated in 22 HCC, 17 cirrhosis, 17 chronic hepatitis, nine primary biliary cirrhosis (PBC), eight autoimmune hepatitis, seven drug induced liver disease, six fatty liver, and three normal liver tissues using methylation specific polymerase chain reaction (MSP). p16 protein expression was also examined by immunohistochemical staining. RESULTS: Methylation of the p16 promoter was detected in HCC (72.7%, 16/22) and also in cirrhosis (29.4%, 5/17) and chronic hepatitis (23.5%, 4/17), all of which were positive for hepatitis B or C virus infections. Methylation was not detected in any of the other samples. All methylation positive HCC, cirrhosis, and chronic hepatitis samples showed loss of p16 expression, and a significant correlation was found between methylation and loss of expression. Analysis of serial samples from individual patients with methylation positive HCC revealed that loss of p16 expression with promoter methylation occurred in 18 of 20 patients at the stage of chronic hepatitis without clinically detectable carcinoma. CONCLUSIONS: Our results suggest that methylation of the p16 promoter and the resulting loss of p16 protein expression are early events in a subset of hepatocarcinogenesis and that their detection is useful in the follow up of patients with a high risk of developing HCC, such as those with hepatitis B or C viral infections.     

The association of increased p14ARF/p16INK4a and p15INK4a gene expression with proliferative activity and the clinical course of multiple myeloma

p14/p16 and p15 gene expression was assessed by quantitative polymerase chain reaction in purified plasma cells (PC) from 52 patients with symptomatic multiple myeloma (MM) and seven with smoldering MM in order to clarify the impact of these genes on the proliferative activity of tumor cells and patients' outcome. p15 expression was lower in symptomatic MM than in smoldering SMM (-1.80 vs.1.51,p=0.026); similar results were observed for p14/p16. MM patients whose PC displayed high p15 and/or p14/p16 expression had a lower percentage of S-phase PC than the remaining cases (1.79%+/-1.35 vs. 3.04%+/-1.42, p=0.028), favorable prognostic factors and longer survival (100% vs. 49%at 2.5 years; p=0.007).     

Silencing of the p18INK4c gene by promoter hypermethylation in Reed-Sternberg cells in Hodgkin lymphomas

p18INK4c is a cyclin-dependent kinase (CDK) inhibitor that interferes with the Rb-kinase activity of CDK6/CDK4. Disruption of p18INK4c in mice impairs B-cell terminal differentiation and confers increased susceptibility to tumor development; however, alterations of p18INK4c in human tumors have rarely been described. We used a tissue-microarray approach to analyze p18INK4c expression in 316 Hodgkin lymphomas (HLs). Nearly half of the HL cases showed absence of p18INK4c protein expression by Reed-Sternberg (RS) cells, in contrast with the regular expression of p18INK4c in normal germinal center cells. To investigate the cause of p18INK4c repression in RS cells, the methylation status of the p18INK4c promoter was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing. Hypermethylation of the p18INK4c promoter was detected in 2 of 4 HL-derived cell lines, but in none of 7 non-Hodgkin lymphoma (NHL)-derived cell lines. We also detected p18INK4c hypermethylation, associated with absence of protein expression, in 5 of 26 HL tumors. The correlation of p18INK4c immunostaining with the follow-up of the patients showed shorter overall survival in negative cases, independent of the International Prognostic Score. These findings suggest that p18INK4c may function as a tumor suppressor gene in HL, and its inactivation may contribute to the cell cycle deregulation and defective terminal differentiation characteristic of the RS cells.     

Alterations in the p16INK4a/CDKN2A tumor suppressor gene in gastrinomas

The p16INK4a/CDKN2A gene (p16INK4a) is frequently altered by homozygous deletion, mutation, or methylation in many nonendocrine tumors, and these alterations may be predictive of recurrence, tumor growth, or aggressiveness. Whether this is true of neuroendocrine tumors such as gastrinomas is unclear. To address this question we analyzed the gastrinomas from 44 patients for p16INK4a gene mutations and correlated the results to the tumor's biological behavior, growth pattern, and aggressiveness. No gastrinomas had mutations of exon 1 or exon 2 of the p16INK4a gene, although polymorphisms were found in 54%. No homozygous deletions were found. In 52% of the gastrinomas, hypermethylation of a 5'-CpG island of the p16INK4a gene promoter was found. To assess the growth behavior of the gastrinomas, all patients were assessed yearly with at least three conventional imaging studies (computed tomography scan, magnetic resonance imaging, and ultrasound), and since 1994 have been assessed with radionuclide scanning using [111In-diethylenetriamine pentaacetic acid,DPhe1]octreotide. The mean follow-up was 5.1+/-0.4 yr (range, 1.2-11.7). The presence or absence of methylation of the p16INK4a gene did not correlate with clinical characteristics of the gastrinoma, biological behavior (gastrin release and basal or maximal acid output), the presence or absence of known prognostic factors (tumor size, gastrinoma location, lymph node metastases, liver metastases, and curability), or growth pattern of the gastrinoma postresection. These results indicate that methylation of the p16INK4a gene is the most common gene alteration described to date in gastrinomas. Furthermore, because it is independent of disease stage it is probably an early event in the pathogenesis and because it is independent of the primary gastrinoma location, which is now thought to have different origins, methylation of the p16INK4a gene is probably a central process in the molecular pathogenesis of these tumors.     

Expression of thymidylate synthase in human gastric and colorectal adenocarcinomas is upregulated by p16/INK4

BACKGROUND/AIMS: We observed the relationship between the expression of thymidylate synthase protein (pTS) and cell cycle regulators in gastric and colorectal adenocarcinoma tissues. METHODOLOGY: This study included 80 gastric and 50 colorectal adenocarcinomas. Immunohistochemical staining was performed using a polyclonal antibody to recombinant human pTS, and monoclonal antibodies to p53, p21/WAF1CIP1, p16/INK4, cyclin D1 and pRB. Each staining was quantified using computerized image analysis on a CAS 200 system. We selected the mean expression values as the cutoff values to distinguish between high and low expression of these substances. RESULTS: There was no relationship between pTS expression and p21/WAF1CIP1, cyclin D1, or pRB expression in gastric and colorectal carcinomas. In both gastric and colorectal carcinomas, the pTS expression was significantly low in the high p16/INK4 expression subgroup compared with the low p16/INK4 expression subgroup (P < 0.05). Further, the pTS expression was significantly high in the high p53 expression subgroup compared with the low p53 expression subgroup in colorectal adenocarcinomas (P < 0.05). CONCLUSIONS: pTS expression regulation in human gastric and colorectal adenocarcinomas in complex, and upregulated by p16/INK4.     

Frequent deletion of p16INK4a/MTS1 and p15INK4b/MTS2 in pediatric acute lymphoblastic leukemia

The tandemly linked p16INK4aMTS1 and p15INK4b/MTS2 genes on chromosome 9, band p21 encode proteins that function as specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. This locus undergoes frequent bi-allelic deletion in human cancer cell lines, suggesting that the encoded proteins may function as tumor suppressors. However, more recent analysis of primary tumor samples has shown a much lower frequency of abnormalities affecting this region, raising doubt over the importance of these proteins in human malignancies. Hemizygous deletions and rearrangements of chromosome 9, band p21, are among the most frequent cytogenetic abnormalities detected in pediatric acute lymphoblastic leukemia (ALL), occurring in approximately 10% of cases. To determine if the p16INK4a/p15INK4b locus might be the target of these chromosomal lesions, we analyzed both genes in primary clinical samples from 43 pediatric ALL patients using interphase fluorescence in situ hybridization, Southern blot analysis, and the polymerase chain reaction. Deletions of p16INK4a/p15INK4b were identified in 18 of 20 cases with cytogenetically observed abnormalities of 9p and 5 of 23 with apparently normal chromosomes 9p, with the majority containing bi- allelic deletions (16 homozygous/7 hemizygous). Although most homozygous deletions involved both genes, Southern blot analysis showed an interstitial deletion in a single case that was confined to p16INK4a, suggesting that p15INK4b was not the critical target gene in this case. Sequence analysis of both p16INK4a and p15INK4b in all seven cases with hemizygous deletions failed to show mutations within the coding regions of the retained alleles. In this select group of patients, deletion of p16INK4a/p15INK4b was associated with T-cell phenotype, nonhyperdiploid karyotype (< 50 chromosomes), and poor event- free survival. These findings indicate that deletion of the p16INK4a/p15INK4b locus is one of the most common genetic abnormalities so far detected in pediatric ALL, and that loss of one or more of these cell cycle kinase inhibitors is important in leukemogenesis.     

Methylation of the p16INK4A promoter is associated with malignant behavior in abdominal extra-adrenal paragangliomas but not pheochromocytomas

Pheochromocytomas and abdominal extra-adrenal paragangliomas are related to endocrine tumors of the sympathetic nervous system. Studies in animal models have shown that inactivation of the products of the cyclin dependent kinase inhibitor 2A (CDKN2A) gene locus, p16INK4A and p14ARF, promotes the development of pheochromocytoma, especially in malignant form. The present study evaluated the involvement of CDKN2A in human pheochromocytomas and abdominal extra-adrenal paragangliomas from 55 patients. Promoter methylation was assessed using quantitative Pyrosequencing and methylation-specific PCR, and mRNA expression was measured by quantitative real-time PCR. For p16, western blot analysis and sequencing were also performed. succinate dehydrogenase complex subunit B (SDHB) sequencing analysis included extra-adrenal paragangliomas, all tumors classified as malignant, and cases diagnosed at 30 years or younger. The p16INK4A promoter was heavily methylated in a subset of paragangliomas, and this was significantly associated with malignancy (P<0.0043) and SDHB mutation (P<0.002). p16INK4A mRNA expression showed moderate suppression in malignant cases (P<0.05). In contrast, very little p14ARF promoter methylation was seen and there was no significant difference in p14ARF expression between tumors and normal samples. The p16 protein expression was reduced in 16 tumors, and sequence variations were observed in four tumors including the missense mutation A57V and the single nucleotide polymorphism (SNP) A148T. The results suggest that p16INK4A, and not p14ARF, is a subject of frequent involvement in these tumors. Importantly, hypermethylation of the p16INK4A promoter was significantly associated with malignancy and metastasis, and SDHB gene mutations. This finding suggests an etiological link and could provide a clinical utility for diagnostic purposes.     

The hypermethylation and protein expression of p16 INK4A and DNA repair gene O6-methylguanine-DNA methyltransferase in various uterine cervical lesions

Purpose This study is aimed at investigating the significance of gene promoter methylation status and protein expression of p16 ^INK4A and O⁶-methylguanine-DNA methyltransferase (MGMT) in the various uterine cervical lesions.Materials and methods Methylation status by using methylation-specific polymerase chain reaction (MS-PCR) and protein expression by using immunohistochemistry for p16 ^INK4A and MGMT genes were performed in cervical squamous intraepithelial neoplasms (CIN), invasive squamous cell carcinomas (SCC), adenocarcinomas and non-neoplastic cervices.Results None of 20 non-neoplastic cervices showed p16 ^INK4A and MGMT gene hypermethylation, whereas at least one of these genes was hypermethylated with 50.0% (5/10) of CIN I, 65.0% (13/20) of CIN II–III, 70.2% (33/47) of SCC and 85.0% (17/20) of adenocarcinoma. p16 ^INK4A protein was totally negative in non-neoplastic cervices, but positive with 90.0% of CIN I, 100% of CIN II–III and adenocarcinoma, and 78.7% of SCC. MGMT protein was expressed in 10% of non-neoplastic cervices, but significantly increased in SCC (42.5%) and adenocarcinoma (70.0%). The protein expression of p16 ^INK4A and MGMT was not related to their gene promoter methylation status.Conclusions The hypermethylation of p16 ^INK4A and MGMT genes in the uterine cervix may indicate the presence of malignant cells, and p16 ^INK4A immunostaining is useful in grading CIN and diagnosing invasive SCC and adenocarcinoma.