Up-regulation of expression and lack of 5' CpG island hypermethylation of <Emphasis Type="Italic">p16 INK4a</Emphasis>in HPV-positive cervical carcinomas

Background  

High risk type human papilloma viruses (HR-HPV) induce carcinomas of the uterine cervix by expressing viral oncogenes E6 and E7. Oncogene E7 of HR-HPV disrupts the pRb/E2F interaction, which negatively regulates the S phase entry. Expression of tumor suppressor p16^ink4a drastically increases in majority of HR-HPV associated carcinomas due to removal of pRb repression. The p16^ink4a overexpression is an indicator of an aberrant expression of viral oncogenes and may serve as a marker for early diagnostic of cervical cancer. On the other hand, in 25–57% of cervical carcinomas hypermethylation of the p16 INK4a promoter has been demonstrated using a methylation-specific PCR, MSP. To evaluate a potential usage of the p16 INK4a 5' CpG island hypermethylation as an indicator of tumor cell along with p16^ink4a overexpression, we analyzed the methylation status of p16 INK4a in cervical carcinomas     

Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas

Aberrant hypermethylation of CpG islands in the promoter region plays a causal role in the inactivation of various key genes involved in the cell cycle regulatory cascade, which could result in a loss of cell cycle control. The aim of the present study was to examine in more detail the prevalence and role of the promoter methylation of genes with a proven involvement in the cell cycle regulation of pituitary adenomas, since their tumorigenesis has not yet been clearly defined. We profiled the CpG island methylation status of a series of well-characterized cell cycle regulation genes: the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes, in 34 pituitary adenomas as determined by a methylation-specific polymerase chain reaction assay. Promoter hypermethylation of the RB1, p14 ^ARF , p15 ^INK4b , p16 ^INK4a , p21 ^Waf1/Cip1 , p27 ^Kip1 , and p73 genes was detected in 12 (35%), 2 (6%), 11 (32%), 20 (59%), 1 (3%), 0 (0%), and 4 (12%) of the adenomas, respectively. In total, 88% (30 of 34) of the adenomas displayed methylation of at least one of such cell cycle regulatory genes, especially methylation of the member genes of the RB1 pathway (29 of 34; 85%). Promoter hypermethylation of p15 ^INK4b coincided with RB1 and/or p16 ^INK4a methylation, whereas RB1 and p16 ^INK4a methylations tended to be mutually exclusive (p = 0.0048). Furthermore, promoter hypermethylations of p14 ^ARF , p21 ^Waf1/Cip1 , and p73 (not belonging to the member genes of the RB1 pathway) were also coincident with RB1 and/or p16 ^INK4a methylation except in one p73 methylated case. In contrast, none of the clinicopathological features, including the cell proliferation index, was significantly correlated with any particular methylation status. Our results suggested that aberrant hypermethylation of the key cell cycle regulatory genes occurs at a relatively high frequency in pituitary adenomas, especially in RB1 pathway genes with promoter hypermethylation of the p16 ^INK4a gene being the most common deregulation. We further obtained evidence to indicate that RB1 and p16 ^INK4a methylations tended to be mutually exclusive, but did occasionally coincide with other cell cycle regulation gene methylations.     

Methylation of the p16(Ink4a) tumor suppressor gene 5'-CpG island in breast cancer

Methylation of the p16(Ink4a) tumor suppressor gene 5'CpG island was analyzed in 104 primary breast cancer specimens using Southern blotting and methylation specific polymerase chain reaction (PCR) (MSP). Eight and four tumors, respectively, showed methylation, and all MSP positive tumors were detected by Southern blotting. To investigate possible methylation not detectable by these methods, bisulphite genomic sequencing was performed in 220 clones from 14 selected tumors. Absent methylation or methylation of single CpG dinucleotides prevailed in all tumors, but of the MSP positive tumors, three contained alleles with methylation of 31 or 32 of the 32 analyzed CpG dinucleotides in the island. Partially methylated alleles were also observed. In a group with low p16(Ink4a) expression determined by Western blotting, four randomly selected tumors contained several identical clones with methylation of 15 CpG dinucleotides by bisulphite genomic sequencing but with a methylation pattern that did not support detection by either Southern blotting or MSP, increasing the potential significance of p16(Ink4a) methylation in breast cancer.     

Frequent but borderline methylation of<Emphasis Type="Italic"> p16</Emphasis><Superscript>INK4a</Superscript> and<Emphasis Type="Italic"> TIMP3</Emphasis> in medulloblastoma and sPNET revealed by quantitative analyses

Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 ^INK4A, p14 ^ARF, TIMP3, CDH1, p15 ^INK4B and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 ^INK4A (17/43), p14 ^ARF (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 ^INK4A and TIMP3 were methylated consistently in medulloblastomas (p16 ^INK4A 14%, TIMP3 11%) and p16 ^INK4A also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1–5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.     

Selective variegated methylation of the p15 CpG island in acute myeloid leukemia

Both p15and p16are tumor suppressor genes that have 5′ CpG islands; aberrant cytosine methylation of these islands has been associated with silencing of their expression. Deoxycytidine kinase (dCK) converts prodrugs to their cytotoxic form, has a 5′ CpG island and is a candidate gene for inactivation by hypermethylation. In our study, we used sodium bisulfite sequencing to generate high resolution maps of 5-methylcytosine in the CpG islands associated with p15, p16 and dCK in normal human bone marrow (BM), peripheral blood lymphocytes (PBL) and cytosine arabinoside (ara-C)-resistant acute myeloid leukemia (AML) patients, and established human hematopoietic tumor cell lines. In normal cells the p15, p16and dCK CpG islands were largely unmethylated. The p16 and dCK CpG islands were also unmethylated in the 8 AML specimens. In contrast, the p15CpG island was aberrantly methylated in 6 of the 8 AML specimens. Furthermore, bisulfite sequencing revealed that the p15CpG island is heterogeneously methylated in AML, with large intra-individual and inter-individual variability.Int. J. Cancer 78:561–567, 1998. © 1998 Wiley-Liss, Inc.     

Promoter hypermethylation in Indian primary oral squamous cell carcinoma

We evaluated promoter hypermethylation of a panel of tumor suppressor genes as a means to detect epigenetic alterations in oral squamous cell carcinomas (OSCC) of Indian‐origin and compare with North‐American head and neck squamous cell carcinomas (HNSCC). Quantitative‐methylation‐specific PCR was used to investigate the promoter methylation status of DCC, EDNRB, p16^INK4a and KIF1A in 92 OSCC, and compared to 48 paired normal tissues and 30 saliva and sera samples from healthy control subjects. Aberrant methylation of at‐least one of these genes was detected in 74/92 (80.4%) OSCC; 72.8% at EDNRB, 71.7% at KIF1A, 47.8% at p16^INK4a and 58.7% at DCC; and in 5 of 48 (10.4%) normal oral tissues. None of the saliva and sera samples from controls exhibited DNA methylation in these four target genes. Thirty‐two of 72 node positive cases harbored p16^INK4a and DCC hypermethylation (p = 0.005). Thus, promoter hypermethylation in genes analyzed herein is a common event in Indian OSCC and may represent promising markers for the molecular staging of OSCC patients. We found higher frequency of p16^INK4a methylation (47.8%) in this Indian cohort in comparison with a North‐American cohort (37.5%). In conclusion, aberrant methylation of EDNRB, KIF1A, DCC and p16^INK4a genes is a common event in Indian OSCC, suggesting that epigenetic alterations of these genes warrant validation in larger studies for their potential use as biomarkers.     

Hypermethylation of promoter 5＇CpG island of p16 gene in glioma tissue and plasma

ObjectiveTo explore the clinical significance of methylation status of promoter CpG island of p16 gene in glioma tissue and plasma.MethodsMethylation specific polymerase chain reaction (MSP) was used to determine the methylation status of the promoter for p16 gene within glioma tissue and plasma.Immunohistochemicel method (SP) was used to analyze the expressions of p16 and Ki-67 proteins.ResultsHypermethylation was found in 17/40 (42.5%) of brain gliomas,in comparison with 11/40 (27.5%) plasma specimens (x2 = 1.9780,P = 0.1596).Loss of p16 expression was associated (P = 0.0229) with hypermethylation of CpG island of promoter regions.Hypermethylation of p16 gene CpG island was significantly related to the increase of malignant grade of brain glioma (TissueX2 = 11.4288,P = 0.0007;PlasmaX2 = 8.9439,P = 0.0028).The Ki-67 index increased significantly (P＜0.05) in brain gliomas methylated in contrast to those unmethylated.ConclusionP16 hypermethylation may be one of the major mechanisms of tumorigenesis of gliomas.Methylated tumor-specific DNA may be as a plasma biomarker for prognosis in patients with glioma.     

Human papillomavirus and p16 expression in inverted papillomas of the urinary bladder

Human Papillomaviruses (HPVs) have been found in association with benign and malignant growth of epithelia. The cell cycle inhibitor p16^Ink4a has been shown to be overexpressed in HPV-positive cervical pre-malignant and malignant lesions, probably as a result of pRB targeting by the viral E7 protein. Inverted papillomas of the urinary bladder are epithelial tumors considered to be of benign nature. In this report we analyze the expression of p16^Ink4a and the presence of HPV sequences in inverted papillomas and in non-tumoral bladder controls. Our results show no association of HPV infection and inverted papillomas. Further, no correlation between p16 overexpression and HPV positivity was found. We conclude that HPV does not play an indispensable role in the development of urinary bladder inverted papillomas and that overexpression of p16^Ink4a does not correlate with HPV infection in these tumors.     

Alterations of the p53 and pRB pathways in human astrocytoma

Human astrocytomas are characterized by a number of molecular changes affecting two critical tumor suppressor pathways: the pRB and the p53 pathways. Genetic alterations functionally eliminate pRB and p53 themselves or upstream and/or downstream molecules such as products of theInk4a/ARF locus, p16^Ink4a and p14^ARF. As a result, malignant cells are defective in critical cell cycle and apoptosis regulatory elements contributing to unrelenting tumour growth and invasion. Current research aims to discover effective means of reconstituting p53 and pRB pathway components in an effort to attenuate the aggressive phenotype of astrocytoma.     

Role of Helicobacter pylori infection in aberrant DNA methylation along multistep gastric carcinogenesis

CpG island hypermethylation is frequently found during gastric carcinogenesis. We investigated methylation profiles of p16, LOX, HAND1, THBD, p41ARC, and APC along multistep gastric carcinogenesis and determined their association with Helicobacter pylori infection. Methylation levels in these six genes were evaluated in noncancerous gastric biopsy specimens using quantitative methylation‐specific PCR in 459 patients with gastric cancer (GC), 137 with dysplasia, and 248 controls. Controls were divided into four subgroups sorted by current H. pylori infection status (active vs past or negative infection) and the presence of intestinal metaplasia (IM). In controls, active H. pylori infection significantly increased methylation levels in THBD, LOX, and HAND1 (all P < 0.001), and hypermethylation of THBD, HAND1, and APC was associated with IM. Aberrant DNA hypermethylation was correlated well with activity of H. pylori‐associated gastritis. However, methylation levels in LOX, HAND1, THBD, and p41ARC remained increased in cases with past H. pylori infection compared to those that were H. pylori negative (all P < 0.05). Hypermethylation of THBD, and possibly p16, was significantly associated with GC, regardless of the status of current H. pylori infection (all P < 0.05). These results suggest that aberrant DNA hypermethylation caused by H. pylori‐associated gastritis occurs in a gene‐specific manner along gastric carcinogenesis, which can be persistent even after the disappearance of H. pylori. Aberrant methylation of THBD might provide a link between H. pylori infection and development of GC. (Cancer Sci 2010)     

DNA methylation in small lung adenocarcinoma with bronchioloalveolar carcinoma components

We examined the methylation status in 100 specimens of lung adenocarcinomas measuring 2 cm or less and with bronchioloalveolar carcinoma (BAC) components (Noguchi types A–C) and then compared the methylation status between noninvasive tumors (Noguchi type A or B) and invasive tumors (Noguchi type C). Methylation-specific PCR was used to determine the methylation statuses of p16^INK4a, RASSF1A, CDH13, RARβ, and Cyclin D2. The methylation index that was regarded as representing the degree of methylation was calculated. We also determined the mutational statuses of EGFR exons 19 and 21 using a PCR-based method. A multivariate analysis showed that the aberrant methylation of p16^INK4a, RASSF1A, and CDH13 was significantly more frequent in invasive tumors than in noninvasive tumors [p16^INK4a, 36.5% versus (vs.) 8.3%, P = 0.0023; RASSF1A, 46.2% vs. 14.6%, P = 0.0012; CDH13, 42.3% vs. 10.4%, P = 0.0006]. The methylation index was significantly higher in invasive tumors than in noninvasive tumors (P = 0.004). The methylation of p16^INK4a was significantly more frequent in EGFR wild-type tumors than in EGFR mutant tumors (P = 0.021). Our results indicate the involvement of epigenetic alterations in the progression of adenocarcinoma with BAC components.     

Effect of <Emphasis Type="Italic">cis</Emphasis>-9, <Emphasis Type="Italic">trans</Emphasis>-11-conjugated linoleic acid on cell cycle of mammary adenocarcinoma cells(MCF-7)

Objective: To determine the effect of cis-9, trans-1 1-conjugated linoleic acid on the cell cycle of mammary cancer cells (MCF-7) and the possible mechanism of the inhibitory effect of c9,t11-CLA. Methods: Using cell culture and immunocytochemical techniques, we examined the cell growth, DNA synthesis, expression of PCNA, cyclin A, B₁, D₁, p16^ink4a and p21^cip/waf1 of MCF-7 cells at various c9,t11-CLA concentrations (25μM, 50μM, 100μM and 200μM), at 24h and 48h. 96% ethand was used as negative control. Results: The cell growth and DNA synthesis of MCF-7 cells were inhibited by c9,t11-CLA. After treatment with various doses of c9,t11-CLA mentioned above for 8 days, the inhibition frequency was 27.18%, 35.43%, 91.05%, and 92.86%, respectively. Inhibitory effect of c9,t11-CLA on DNA synthesis (except for 25μM, 24h) was demonstrated by significantly less incorporation of ³H-TdR than the negative control (P<0.05 and P<0.01). To further investigate the influence of the cell cycle progression, we found that c9,t11-CLA may arrest the cell cycle of MCF-7 cells. Immunocytochemical staining demonstrated that incubation with different concentration of c9,t11-CLA at various times significantly decreased the expression of PCNA, Cyclin A, B₁, D₁ in MCF-7 cells compared to the negative control (P<0.01), whereas the expression of p16^ink4a and p21^cip/waf1, cyclin-dependent kinases inhibitors (CDKI), were increased. Conclusions: The cell growth and proliferation of MCF-7 cells is inhibited by c9,t11-CLA via blocking cell cycle, accompanying reduced expression of cyclin A, B₁, D₁ and enhanced expression of CDKI (p16^ink4a and p21^cip/waf1). Foundation item: This work was supported by the National Natural Science Foundation of China(No.39870661). Biography: Liu Jia-ren(1968–), PhD, assistant professor, Public Health College, Harbin Medical University, majors in health toxicology.