P14 Promoter Hypermethylation Research Articles

High frequency of p16 and p14 promoter hypermethylation and marked telomere instability in salivary gland tumors

Objectivesto investigate p16INK4a and p14ARF tumor suppressor gene methylation status, determine telomere length and assess the importance of these epigenetic and genetic parameters in the development of pleomorphic adenoma and carcinoma ex pleomorphic adenoma of the parotid salivary glands. Materials and MethodsGenomic DNA from paraffin-embedded samples of 50 pleomorphic adenomas and 10 carcinomas ex pleomorphic adenoma was subjected to methylation specific polymerase chain reaction for hypermethylation analyses and real time polymerase chain reaction for the relative telomere length calculations. ResultsPromoter hypermethylation of the two genes was a very frequent event in both neoplasms – between 60% and 90% of samples were hypermethylated – but without significant difference between the groups. The mean relative telomere length in the pleomorphic adenoma group was significantly increased in comparison to the control group (P=0.00), and significantly decreased in comparison to the carcinoma group (P=0.05). Telomeres were also longer in myxoid and cellular histological subtypes of adenomas than in the classic type (P=0.044 and P=0.018, respectively). Longer telomeres were more frequent in tumors with hypermethylated p14ARF alleles (P=0.013). ConclusionPromoter hypermethylations seems to be an important mechanism of p16INK4a and p14ARF inactivation in parotid gland tumors. Telomeric lengthening appears to be involved in the pathogenesis of both benign and malignant tumors of the parotid glands.

Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia and Lymphomas of Germinal Center Origin

INTRODUCTION. Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. This occurs as a response to stimulation by various antigens originating, sometimes, follicular hyperplasia (FH). GC have been known as a major source of B-cell lymphomas including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCL. Much less is known about the role of methylation in FH. We determined the methylation status of 6 tumor-suppressor genes in 43 patients with FH, 18 patients with FL and 49 patients with DLCL in order to see the differential implication of this epigenetic mechanism in the pathological and the physiological development of GC.MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 43 FH, 18 FL and 49 DLCL after being treated with EZ DNA-Methylation Kit (Zymo Research) with the manufacturer's instructions, were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14, SHP1, MGMT and PDRM1. All samples were obtained mostly from lymph nodes and tonsils. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin.RESULTS: DAP-k promoter methylation occurred with higher frequency in FL(89%) than in DLCL(78%) and FH(40%). SHP1 was methylated in 61% of FL, 58% of FH and 23% of DLCL. RARb was methylated in 67% of FL patients, 30% of DLCL and only 12% of FH. Eight (44%) FL, seventeen (35%) DLCL and four (10%) BFH patients showed MGMT methylation. Promoter hypermethylation of p14 was detected only in 5 (12%) FH, 2 (4%) DLCL and none FL patients. Methylation of PRMD1 was present only in 1 (6%) FL, 2 (6%) DLCL and 1 (4%) FH samples.CONCLUSIONS. Inactivacion of DAP-K and SHP1 is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a GC hyperplastic response. Inactivation of the retinoic acid response through the methylation of Rarâ is significantly more frequent in lymphomas than in FH. As reported in other tumors methylation of MGMT is more frequent in lymphomas than in FH. With our data methylation of Cyclin dependent kinase inhibitors p14 is not a differential pathogenic event in lymphomas of GC origin, in fact it is more frequent in FH. Promoter Methylation of PDRM1 is not the mechanism involved in lymphomagenesis in FL and DLCL, the two FH positive deserve further follow-up to determine its significance.

Promoter Methylation of Multiple Genes in Diffuse Large Cell Lymphoma (DLCL), Follicular Lymphoma (FL) and Bening Follicular Hyperplasia (BFH).

Transcriptional silencing of tumor suppressor genes, associated with DNA methylation, is a common epigenetic event in leukemias and myelodisplastic syndromes. Much less is known about the specific methylation changes that occur in DCLC, FL and their normal counterpart BFH. Methylation of cyclin dependent kinase inhibitors is more common in high grade lymphomas and may be an important step in the progression and transformation of follicular lymphoma. We determined the methylation status of 7 tumor suppressor genes in 31 patients with B-cell malignancies. Seven patients with benign follicular hyperplasia were used as normal controls. The target genes chosen are potentially involved in B-cells malignancies and encoding proteins implicated in apoptosis regulation (death associated protein kinase, DAP-K), Jak/STAT3 signalling pathway (SHP1), hormonal response (RARb), DNA repair (O6-methilguanine-DNA methyltranferase, MGMT), cell cycle control (p14ARF and p15INK4b) and detoxification of environmental xenobiotics such as doxorubicin (glutathione S-transferase P1, GSTP1). Genomic DNA extracted from paraffin-embedded samples of thirty one B-cell malignancies (twenty six DLCL and five FL) were analyzed by methylation-specific polymerase chain reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, MGMT, p14, p15 and GSTP1. Samples were obtained mostly from lymph nodes; spleen, stomach, skin, and parathyroid gland biopsies were also included. Specimens were collected at diagnosis before specific therapy. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin. DAP-k promoter methylation occurred with similar frequency in DLCL (100%), FL (100%) and BFH (86%). SHP1 was methylated in 75% of DLCL, all FL and the 7 patients with BFH. RARb was methylated in all DLCL and FL patients and 85% of the BFH. Sixteen (50%) DLCL, three (60%) FL and none BFH patients showed MGMT methylation. Promoter hypermethylation of p14 and p15 was detected in 11 (42%) and 13 (50%) DLCL, 2 (40%) and 3(60%) FL, 3(42%) and 5(71%) BFH patients respectively. Methylation of GSTP1 was absent in all samples. Inactivacion of DAP-K, SHP1 and Rarβ is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a BFH response. With our data methylation of Cyclin dependent kinase inhibitors such as p14 and p15 is not a differential pathogenic event in DLCL with respect to FL or BFH. Finally, the absence of GSTP1 methylation in DLCL and FL questions its relevance in B-cell neoplasia development.

Reciprocal relationship between methylation status and loss of heterozygosity at the p14(ARF) locus in Australian and South African hepatocellular carcinomas.

Chromosome 9p21, a locus comprising the tumor suppressor genes (TSG) p16(INK4a) and p14(ARF), is a common region of loss of heterozygosity (LOH) in hepatocellular carcinoma (HCC). p14(ARF) shares exon 2 with p16 in a different reading frame. p14 binds to MDM2 resulting in a stabilization of functional p53. This study examined the roles of p14, p16 and p53 in hepatocarcinogenesis, in 37 Australian and 24 South African patients. LOH at 9p21 and 17p13.1, p14 and p16 mutation analysis, p14 and p16 promoter methylation and p14, p16 and p53 protein expression was examined. LOH at 9p21 was detected more frequently in South African HCC (P = 0.04). Comparable rates of p53 LOH were observed in Australian and South African HCC (10/22, 45%vs 13/22, 59%, respectively). Hypermethylation of the p14 promoter was more prevalent in Australian HCC than in South African HCC (17/37, 46%vs 7/24, 29%, respectively). In Australian HCC the prevalence of p14 methylation increased with age (P = 0.03). p16 promoter methylation was observed in 12/37 (32%) and 6/24 (25%) in Australian and South African HCC, respectively. Loss of p16 protein expression was detected in 14/36 Australian HCC whereas p53 protein expression was detected in 9/36. Significantly, a reciprocal relationship between 9p21 LOH and p14 promoter hypermethylation was observed (P < or = 0.05). No significant association between p14 and p53 was seen in this study. The reciprocal relationship identified indicates different pathways of tumorigenesis and likely reflects different etiologies of HCC in the two countries.

Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines.

The tumor-suppressor genes p14(ARF), p16(INK4a) and Tp53 are commonly inactivated in many tumors. We investigated their role in the pathogenesis of 9 bile tract cancer cell lines and 21 primary sporadic extrahepatic bile duct carcinomas. p53 and p16 protein expression was examined by Western blot analysis and immunohistochemistry. Mutation screening of p53 was done by SSCP and direct sequencing. Inactivating mechanisms of p14 and p16 were addressed by screening for mutations, homozygous deletions, chromosomal loss of 9p21 (loss of heterozygosity [LOH] analysis) and promoter hypermethylation of the p14/p16 genes. p53 overexpression could be detected in 7 of 9 cell lines and 7 of 21 primary tumors, but mutations were found in 3 cell lines only. p16 expression was absent in all cell lines, due to homozygous deletion of the gene in 8 of 9 cell lines and hypermethylation of the p16 promoter in one cell line (CC-LP-1). p14 exon 1beta was homozygously deleted in 6 of 9 cell lines, while retained in CC-LP-1 and 2 additional lines. No p14 promoter hypermethylation could be detected. p16 expression was lost in 11 of 21 primary tumors. p16 promoter hypermethylation was present in 9 of 21 primary tumors, all with lost p16 expression. Allelic loss at 9p21 was detected in 13 of 21 primary tumors, 10 of 11 with lost p16 expression and 8 of 9 with methylated p16 promoter. No p14 promoter hypermethylation or p14/p16 mutations could be detected. Neither Tp53 nor p16 alterations showed obvious association with histopathologic or clinical characteristics. In conclusion, inactivation of the p16 gene is a frequent event in primary sporadic extrahepatic bile duct cancers, 9p21 LOH and promoter hypermethylation being the principal inactivating mechanisms. Therefore, p16, but not p14, seems to be the primary target of inactivation at the INK4a locus in bile duct cancers. Other mechanisms than Tp53 mutations seems to be predominantly responsible for stabilization of nuclear p53 protein in bile duct cancers.