OBJECTIVETo investigate the methylation rate of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase inhibitor 2B (CDKN2B) in the 9P21 region in children with acute myeloid leukemia (AML) and the association of gene methylation with clinical features and outcomes.

METHODSThe clinical data of 58 children who were newly diagnosed with AML between January 2010 and December 2012 were retrospectively analyzed. Thirty-eight healthy children were recruited as the control group. Genomic DNA was extracted from bone marrow or peripheral blood of the 58 patients and 38 healthy children. The methylation status of CDKN2A and CDKN2B was analyzed by methylation-specific multiplex ligation-dependent probe amplification.

RESULTSGene methylation was not found in healthy children. Methylation probes of 44 patients were detected in 58 patients. The methylation of CDKN2A was detected with 136 bp and 237 bp methylation probes. The methylation of CDKN2B was detected with 130 bp, 210 bp, 220 bp, and 417 bp methylation probes. The methylation rate of CDKN2A was 5%, while the methylation rate of CDKN2B was 76%. The methylation detected by some probes was associated with sex, hemoglobin, and platelet count at the first visit.

CONCLUSIONSThe methylation of CDKN2B is a common event in children with AML, while the methylation of CDKN2A is relatively rare.

Adolescent ; Child ; Child, Preschool ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Female ; Humans ; Infant ; Leukemia, Myeloid, Acute ; genetics ; Male

OBJECTIVETo investigate the frequency of p16 and p15 gene methylation in multiple myeloma (MM), and its relationship with bone marrow cell apoptosis and clinical outcome.

METHODSTwenty-two patients with MM were studied to detect p16 and p15 gene methylation. Methylation-specific polymerase chain reaction (MSP) was used to detect gene methylation, and terminal transferase-mediated dUTP nick end-labeling (TUNEL) was used to detect cell apoptosis.

RESULTSp16 and/or p15 gene methylatoin was detected in 10 of 22 patients (45.4%). There were 3 patients with p16 gene methylation, 9 patients with p15 gene methylation, and 2 patients with both genes methylation. The incidence of methylation of p15 gene was higher than that of p16 gene (P < 0.05). The patients with p16 and/or p15 gene methylation had a delayed cell apoptosis, poor response to chemotherapy, and a short over-all survival (OS).

CONCLUSIONThe methylation of p16 and/or p15 gene plays a key role in MM apoptosis pathogenesis. The patients with both p16 and p15 gene methylation had a poor prognosis.

Apoptosis ; Cell Cycle Proteins ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; DNA, Neoplasm ; genetics ; Gene Silencing ; Genes, p16 ; Humans ; Multiple Myeloma ; genetics ; pathology ; Prognosis ; Transcription Factors ; genetics ; Tumor Suppressor Proteins

Gastric cancer is the result of multiple risk factors, including environmental factors, genetic factors and the interaction between them. The environmental factors mainly include dietary, Helicobacter pylori infection and family history of gastric cancer. Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes, tumor suppress genes, cell cycle regulators, DNA repair genes and signaling molecules. This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.
Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Oncogenes ; Stomach Neoplasms ; etiology ; genetics

OBJECTIVETo investigate the association of p15 and pl6 genes deletion and STKI5 gene overexpression in primary hepatocellular carcinoma (PHC).

METHODSThe carcinoma tissue and the adjacent normal tissue were taken from 30 PHC patients during operations who had had neither chemotherapy nor radiotherapy preoperatively. DNA was extracted from the tissues and PCR was used to determine the homozygous deletion of p15 exon2 (pl5E2) and pl6 exon 2 (pl6E2). RNA was extracted, cDNA was synthesized by RT-PCR, and the expression of STKI5 gene was tested by PCR. Beta-actin was used as an internal control. Average density value (ADV) of STK15 gene and that of beta-actin gene were determined in both carcinoma tissue and the adjacent normal tissue.

RESULTSThe rate of p15E2 deletion was 13.3% (4/30) and the rate of p16E2 deletion was 16.7% (5/30) in the carcinoma tissue. The p15E2 and pl6E2 co-deletion rate was 6.7% (2/30). In 19 of the 30 cases (63.3%) the expression of STK15 gene in carcinoma tissue was higher than that in the adjacent normal tissue. The ratio of ADV of STK15 gene to ADV of beta-actin gene (1.53+/-0.31) in the carcinoma tissue was significantly higher than that (0.91+/-0.25) in the paired adjacent normal tissue (t = 2.86).

CONCLUSIONThe homozygous deletion of p15E2 and p16E2 and overexpression of STKI5 gene may play a role in the oncogenesis and malignant progression of PHC.

Aurora Kinase A ; Aurora Kinases ; Carcinoma, Hepatocellular ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Female ; Gene Deletion ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; Male ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics

Good progress has been made in the researches on correlating genes of breast cancer in recent years. Quite a few kinds of genes such as susceptibility gene, oncogene and tumor suppressor genes have been found with implications for diagnosis, therapy and prognosis. Abnormality of breast cancer susceptibility gene (BRCA) is of great significance, especially in the development of breast cancer.
BRCA1 Protein ; genetics ; BRCA2 Protein ; genetics ; Breast Neoplasms ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; Female ; Humans ; Mutation ; Neoplasm Proteins ; genetics ; Proto-Oncogene Proteins ; genetics ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo investigate the expression and alteration (including homozygous deletion and mutation) of MTS1 gene in precancerous lesions and squamous cell carcinomas (SCC) of oral mucosa, and to analyse the function of MTS1 gene alteration in oral mucosal carcinogenesis.

METHODSThe expression of p16 protein produced by MTS1 gene was examined with immunohistochemical SP method in 10 normal oral mucosas, 30 precancerous lesions (10 mild, 10 moderate and 10 severe dysplasia respectively) and 45 squamous cell carcinomas (SCCI18, SCCII 19, SCCIII 8). The deletion and mutation of exon1 and exon2 of MTS1 gene were examined with methods of PCR and SSCP in these same samples.

RESULTSAll the precancerous lesions had p16 protein expression and no alteration of MTS1 gene. In SCC, the positive rate of p16 protein was 60.0% with 72.2% in SCCI, 57.9% in SCCII, 37.5% in SCC III, and there were no significant difference among the three groups by chi2 test (P>0.05). Gene homozygous deletion of exon1 and/or exon2 was detected in 10 cases, and gene mutation in 4 cases. The whole rate of gene alteration was 31.1% (14/45). The MTS1 gene alteration rate was 27.8% in SCCI, 31.6% in SCCII, 37.5% in SCC III and there was also no significant difference among the three groups by chi2 test (P>0.05). In SCC with local lymph nodes metastasis, MTS1 alteration rate was 57.1%, while in SCC with no lymph nodes metastasis was 8.3%, and there was significant difference by chi2 test (P<0.05).

CONCLUSIONSMTS1 gene alteration is not an early event in the carcinogenesis of oral mucosa and can not be used as a biology mark to examine oral precancerous lesions. MTS1 gene may play a certain role in the progression of oral squamous cell carcinomas.

Carcinoma, Squamous Cell ; chemistry ; genetics ; pathology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Genes, p16 ; Humans ; Lymphatic Metastasis ; Mouth Neoplasms ; chemistry ; genetics ; pathology ; Mutation ; Precancerous Conditions ; genetics

This study was aimed to investigate the reversing effect of arsenic trioxide (As2O3) on methylation status and the regulatory effect on transcription of malignant lymphoma cell line CA46 p16 gene as well as their possibe mechanisms. The hypermethylated malignant lymphoma cell line CA46 was used as a subject of experiment for studying relation of gene methylation with expression. The effect of As2O3 on the proliferation and viability of CA46 was detected by SRB method, the change of p16 methylation status after exposure to As2O3 was determined by nMSP, the expressions of p16, DNMT1, DNMT3A, DNMT3B mRNA were assayed by RT-PCR, the influence of As2O3 on CA46 cell cycle was analyzed by flow cytometry using analytical method for DNA ploidy. The results showed that the methylation level of p16 gene was obviously reduced after treatment with As2O3 for 72 hours and the hypermethylation of p16 gene was successfully reversed; the expression of p16 gene in untreated (control) group was low while it was enhanced in treated groups; the gray scale ratios of p16 gene to beta-actin in groups treated with As2O3 of concentration 0.5, 1.0 and 2.0 micromol/L were 0.33+/-0.10, 0.57+/-0.11 and 0.67+/-0.09 respectively, exhibiting a significant difference in comparison with 0.73+/-0.13 of positive control (p<0.01); as compared with the untreated group, the expression of DNMT3A and DNMT3B in treated groups was obviously down-regulated in a concentration-dependent manner, while expression of DNMT1 was nearly unchanged; as compared with control, all the 3 different concentrations of As2O3 could inhibit the proliferation of CA46 cells and increase the cell number in G0/G1 phase. It is concluded that the As2O3 may up-regulate the expression of p16 gene, recover the activity of p16 gene, thereby promote the regulatory function on cell cycle resul-ting in arrest of cells in G0/G1 phase and inhibit growth of tumor cells through depressing the expression of DNMT3A and DNMT3B and/or directly reversing the methylation status of p16 gene.
Arsenicals ; pharmacology ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; DNA Methylation ; drug effects ; Genes, p16 ; Humans ; Lymphoma ; genetics ; Oxides ; pharmacology ; Transcriptional Activation

OBJECTIVETo detect the methylation pattern of the p15(INK4B) gene and to explore its significance in the pathogenesis of acute leukemia (AL) and leukemic transformation of myelodysplastic syndromes (MDS).

METHODSA total of 49 AL cases and 22 MDS cases were analyzed by methylation specific polymerase chain reaction (MSP) for methylation patterns in CpG islands of the p15(INK4B) gene.

RESULTSHypermethylation of the p15(INK4B) gene was found in 90% (26/29) of newly diagnosed AL, including 46% with complete methylation and 54% with partial methylation. All 3 evolved AL from MDS and 9 relapsed AL showed a methylated p15(INK4B) gene and the proportion of complete methylation was 67% and 56% respectively. Only 5 of 11 (45%) AL in remission, including 2 in complete remission (CR) and 3 in partial remission (PR), were partially methylated. The frequency of p15(INK4B) gene methylation in newly diagnosed or relapsed AL was significantly higher than that in AL in the remission stage (P = 0.002) p15(INK4B) gene methylation was found in 5 of 13 (38%) low-risk MDS (RA/RAS) patients and 80% of them showed only partial methylation. However, p15(INK4B) gene methylation was found in all 9 cases in the high-risk group (RAEB/RAEB-T), including complete methylation in 56%, significantly different from the low-risk MDS group (P = 0.002).

CONCLUSIONSHypermethylation of the p15(INK4B) gene occurs frequently in leukemia and high-risk MDS. It is possible that hypermethylation of this gene is related to the pathogenesis and development of AL and MDS. It may be used as a gene marker to detect minimal residual disease, relapse of AL and leukemic transformation in MDS.

Cell Cycle Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Myelodysplastic Syndromes ; genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Tumor Suppressor Proteins

Among tumor suppressor genes, p15(INK4b) gene is gaining more attention for its important role in the progression of myelodyplastic syndrome (MDS). Serial studies demonstrated that highly frequent hypermethylation of p15(INK4b) gene, which is located at the 5'CpG island in the promoter region of exon 1 and is the main reason of inactivation of p15(INK4b) gene, occurs during the development of MDS towards AML. The assay of methylation-specific PCR (MSP) is sensitive to this pattern of methylation which is restricted to the MDS clone. Apoptosis mediated by cytokines such as Fas antigen and TGF-beta, and bHLH proteins is inhibited by the inactivation of p15(INK4b) gene. This may result in the evolution of MDS clone to AML. In as much as the close relationship between p15(INK4b) gene methylation and MDS, modulation of the methylation status of p15(INK4b) gene may be considered as a noval treatment modality for MDS.
Cell Cycle Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Gene Deletion ; Genes, Tumor Suppressor ; Humans ; Mutation ; Myelodysplastic Syndromes ; etiology ; genetics ; therapy ; Tumor Suppressor Proteins

OBJECTIVETo explore the proliferation and invasive effects of inhibitors of kinase 4(INK4)(P15(ink4b) and P16(ink4a)/CDKN2) gene protein activation on RKO human colorectal cell in vivo and in vitro.

METHODSRKO human colorectal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo.

RESULTSINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression of RKO human colorectal cells after exposure to 1×10(-7), 5×10(-7) and 1×10(-6) mol/L 5-Aza-CdR concentrations(A, B, C groups) were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1(positive control group) to 32.4±7.2, 21.3±5.4 and 19.5±6.4 (3 experiment groups, all P<0.05) respectively. Transwell chamber assay showed that migrated cell number in positive control group(67.4±7.2) was significantly higher than those of 3 experimental groups(35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively). The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group (P>0.05). There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively(all P<0.01).

CONCLUSIONINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; Neoplasm Transplantation ; Transcriptional Activation