p16(INK4a) induces differentiation and apoptosis in erythroid lineage cells

OBJECTIVE: Hematopoiesis is regulated by proliferation, differentiation, and death. p16(INK4a) has been reported to regulate apoptosis and differentiation of diverse cells, as well as arresting the cell cycle at G1 phase. The aim of this study is to explore the properties of p16 in apoptosis and differentiation of erythroid cells. METHODS: We transfected the INK4a gene to K562 cells, which defect the INK4a gene, and compared the effect of enforced expression of p16(INK4a) with that of various additives, topoisomerase I inhibitor (SN 38), interferon-alpha, phosphatidyl-inositol-3 kinase inhibitor (LY294002), and serum deprivation, which arrest cell cycle at different phases. We also investigated the role of p16(INK4a) in normal day-6 human erythroid colony-forming cells by transfecting the INK4a gene. RESULTS: p16(INK4a) induced cell cycle arrest at the G0/G1 phase, and promoted erythroid differentiation in viable K562 cells, but induced apoptosis in K562 cells with incomplete differentiation. The apoptosis induced by p16 was accompanied with downregulation of bcl-x and nuclear NF-kappaB. These findings were not observed in K562 cells treated with various additives. p16(INK4a) decreased the cell viability and promoted apoptosis in day-9 ECFC. CONCLUSION: We propose that p16(INK4a) plays a role in maintaining homeostasis during erythroid differentiation, and that the mechanisms for this effect are not confined to those inducing cell cycle arrest.     

p16INK4A gene alterations are not a prognostic indicator for survival in patients with hepatocellular carcinoma undergoing curative hepatectomy

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is a common malignancy worldwide that is highly associated with chronic hepatitis B or C infection and cirrhosis. The tumor suppressor gene p16INK4A is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. The present study was performed to determine genetic and epigenetic alterations in the p16INK4A tumor suppressor gene and the effect of these on HCC progression. METHODS: The status of p16INK4A was evaluated in 117 HCC tumoral nodules and 110 corresponding peritumoral tissues by loss of heterozigosity (LOH) at the 9p21-22 region, homozygous deletions, single-strand conformation polymorphism-polymerase chain reaction (PCR) mutational analysis and methylation specific PCR. RESULTS: The most frequent inactivation mechanism was hypermethylation of the promoter region, which was found in 63.2% of the tumor samples and in 28.2% of the peritumoral samples. Loss of heterozygosity at the 9p21 region was detected in 27.3% and 10% of tumor and peritumoral tissues, respectively. Homozygous deletions and mutations were less common events in hepatocarcinogenesis. The authors found 5.9% of the tumor cases with exon 2 homozygous deletions and 8.6% with mutations. Two polymorphisms were detected, one at codon 148 (GCG --> ACG, Ala --> Thr) in three cases and the other in exon 3 at 540 bp (34.2% of the samples). No association was found between inactivation of p16INK4A and clinicopathological characteristics or prognosis. CONCLUSION: p16INK4A is altered frequently and early in HCC, being the predominant mechanism of inactivation promoter hypermethylation. The present results suggest that the p16INK4A gene plays an important role in the pathogenesis of HCC.     

High levels of p53 protein expression do not correlate with p53 mutations in hepatocellular carcinoma

To determine the relationship between p53 altered expression and p53 mutations in hepatocellular carcinoma (HCC), we analysed p53 protein immunohistochemically and assessed the presence of mutations in exons 4-8 of the p53 gene using SSCP assay in 117 HCCs corresponding to 78 patients. We also determined the relationship of p53 expression with cellular proliferation by immunostaining with monoclonal antibodies to Ki-67. We found significant levels of p53 protein expression in 23.1% of the 117 cases studied, but identified mutations in only 12 cases (10.3%). Only four of the p53-positive cases had mutations in the regions analysed. Six of the cases that displayed mutations at p53 gene were negative for immunohistochemical analysis (IHC) and two cases showed positive immunoreactivity in the cytoplasm of the cell. In conclusion, strong IHC reactivity for p53 protein is not an indicator of the presence of p53 gene mutations at exons 4-8 in HCC. Thus, p53 loss of function in HCC should be evaluated both by p53 mutation analysis and p53 protein expression, as both give complementary information about p53 status.     

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.     

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.     

血吸虫病患者肝癌细胞p53、p16蛋白表达的相关性研究

目的:探讨血吸虫病合并原发性肝细胞癌(hepatocellular carcinoma,HCC)与p53、p16蛋白的基因之间的相关性及其作用机制。方法:58例HCC患者分为两组:1组(HCC合并血吸虫病组)23例和2组(HCC不合并血吸虫病组)35例。采用免疫组织化学方法检测所有患者的p53、p16蛋白表达。结果:p53蛋白阳性表达率在1组中和2组中分别为73.9%(17/23)和31.4%(11/35),两组比较差异有显著性意义(P<0.01);而p16蛋白阳性率在1组和2组中分别为34.8%(8/23)和28.6%(10/35),两组比较差异无显著性意义(χ2=0.25,P>0.05)。p53蛋白及p16蛋白同时阳性仅7例,阳性率为12.1%(7/28);其中1组为13.0%(3/23),2组为11.4%(4/35),两组比较差异无显著性意义(χ2=0.052,P>0.05)。结论:患有日本血吸虫的肝细胞癌患者的癌组织中有较高的p53基因突变率,说明血吸虫感染对肝细胞癌组织细胞中p53突变蛋白的过量表达有促进作用;同时,血吸虫患者肝细胞癌患者肝组织细胞中p16肿瘤抑制基因-p16基因的丢失普遍。p16蛋白表达缺失,其与p53突变蛋白的过量表达同时存在,两者共同促进肿瘤的增长,可能是HCC恶性增殖的原因之一。     

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.     

锤头状核酶对肝癌突变基因p53的抑制作用

目的 探讨p53核酶对肝癌细胞突变型抑癌基因p53的抑制作用。方法 应用计算机设计并合成针对突变型p53(249位密码子AGG→AGT)的锤头状核酶RZ，构建其体外转录和真核表达载体，检测核酶对突变型p53(mtp53)的体外切割作用，并在Lipofect AMINE^TM2000的介导下转染肝癌细胞MHCC97，应用逆转录聚合酶联反应(RT—PCR)检测核酶对肝癌细胞突变型p53的抑制作用。结果 测序证实核酶基因被正确克隆人体外转录载体pBSKU6和真核载体pEGFPC1中。体外切割效率为42％，而野生型p53(wtp53)没有被切割。在Lipofect AMINETM2000的介导下成功转染肝癌细胞MHCC97，RT—PCR检测证实突变型p53的mRNA水平明显下降，细胞内的切割效率为69％。结论 p53核酶可成功抑制肝癌细胞中突变型p53的表达，为肝癌的基因治疗提供了一个新的选择。     

P53 mutation but not p16/MTS1 mutation occurs in intraductal papillary mucinous tumors of the pancreas

BACKGROUND/AIMS: Intraductal papillary mucinous tumors of the pancreas are rare lesions, which typically show a benign clinical course. However, some of these tumors have a malignant nature and grow in an invasive manner. The purpose of the study was to determine the prevalence of p53-, p16/MTS1- and K-ras mutations in benign and malignant intraductal papillary mucinous tumors with intent to value their importance for tumor progression. METHODOLOGY: Thirteen different archival tumor specimens were obtained at the Department of Pathology, University of Ulm. Three cases showed an invasive component of the tumor. Genomic DNA was extracted after laser capture microdissection of tumor cells from paraffin-embedded tissue sections. The corresponding sequences of p53 (exon 5, 6, 7, 8) and p16/MTS1 (exon 2) were amplified by polymerase chain reaction and subjected to single strand conformation polymorphism analysis. Codon 12 of K-ras was analyzed by the enrichment polymerase chain reaction-restriction fragment length polymorphism method. Positive samples were further investigated by sequencing. RESULTS: K-ras mutations occurred in benign and malignant intraductal papillary mucinous tumors (4/13), whereas an alteration of the coding p53 gene sequence could only be detected in the intraductal and invasive component of one malignant tumor. None of the tissue specimens revealed mutations in exon 2 of p16/MTS1. CONCLUSIONS: In contrast to K-ras mutations, alterations in the p53 gene may characterize ductal papillary mucinous carcinomas, which could be of major interest for their early diagnosis. The lack of mutations in the p16/MTS1 gene suggests that other genes may be involved in the formation of intraductal papillary mucinous neoplasias.     

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.     

Effects of p16 gene on biological behaviours in hepatocellular carcinoma cells

AIM: To investigate the effects of p16 gene on biological behaviours in hepatocellular carcinoma cells. METHODS: HCC cell lines SNU-449 and HepG2.2.15 were infected respectively by a replication defective, recombinant retrovirus capable of producing a high level of p16 protein expression (pCLXSN-p16). G418 resistant stable P16 protein expression cell lines were selected. And the biological behaviours of the p16 gene transfected HCC cells were observed. RESULTS: Initial in vitro experiments in HCC cell line SNU-449 with loss of p16 protein expression demonstrated the pCLXSN-p16 treatment significantly inhibited cell growth. But there was no treatment effect when the pCLXSN-p16 was used in another HCC cell line HepG2.2.15 which has positive p16 protein expression. Subsequent study in a nude mouse model demonstrated that the p16 gene transfected SNU-449 had a lower succeeding rate in the first time establishment of tumors and grew more slowly in the nude mice when compared with non-transfected SNU-449. Moreover, the nude mice inoculated with transfected SNU-449 had a longer surviving time than those inoculated with non-transfected SNU-449. CONCLUSION: Our results show that the p16INK4a gene transfer can inhibit the proliferation and reduce the invasion ability of hepatocellular carcinoma.     

P16 reactivation induces anoikis and exhibits antitumour potency by downregulating Akt/survivin signalling in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is one of the most malignant tumours with high rate of recurrence and metastasis. In HCC, deficiency of the P16/CDK4/Rb pathway is a frequent molecular event, and transferring the P16 gene into cancer cells can induce cell cycle arrest and apoptosis, suggesting that the P16 gene is a good target in cancer gene therapy. The previous study demonstrated that P16 re-expression mediated by adenovirus within cancer cells can induce cell apoptosis and exert potent antitumour efficacy in cancer xenografts in nude mice. However, the molecular mechanism of P16-induced apoptosis in cancer cells is not clear yet. In this resulting study, we found that P16 re-expression can downregulate survivin expression in HCC cells. As a member of the inhibitors of the apoptotic gene family, survivin has been reported to be overexpressed in most common human cancers and present multiple physiological and pathological functions including cell cycle control, inhibition of cell apoptosis, regulation of cell division and induction of angiogenesis, etc. Further investigation found that P16 reactivation led to a decrease of phosphorylated Akt on Thr308 and phosphorylated survivin on Thr34, then downregulated survivin expression. The P16-mediated decrease of nuclear survivin in cancer cells limited CDK4 import into nuclei, which restrained CDK4 functions of promoting cell proliferation, then exhibited the effect of cell cycle arrest and induction of detachment-induced apoptosis (anoikis). The antitumor potency of P16 by downregulating the Akt/survivin signalling was also demonstrated in HCC xenograft models in nude mice. This new insight into P16 function would help in designing better strategies for cancer gene therapy.