Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism

Aim： To explore the effect of androgen receptor （AR） on the expression of the cell cycle-related genes, such as CDKNIA and BTG1, in prostate cancer cell line LNCaP. Methods： After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction （RT- PCR） tests were carried out to confirm the results of the chips. Results：After AR antagonist flutamide treatment, three hundred and twenty-six genes （3.93 %） expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDCIO, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKNIA and BTG2. The CDKNIA and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. Conclusion： Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

Relationships between cell cycle pathway gene polymorphisms and risk of hepatocellular carcinoma

AIM: To investigate the associations between the polymorphisms of cell cycle pathway genes and the risk of hepatocellular carcinoma(HCC). METHODS: We enrolled 1127 cases newly diagnosed with HCC from the Tumor Hospital of Guangxi Medical University and 1200 non-tumor patients from the First Affiliated Hospital of Guangxi Medical University. General demographic characteristics, behavioral information, and hematological indices were collected by unified questionnaires. Genomic DNA was isolatedfrom peripheral venous blood using Phenol-Chloroform. The genotyping was performed using the Sequenom Mass ARRAY i PLEX genotyping method. The association between genetic polymorphisms and risk of HCC was shown by P-value and the odd ratio(OR) with 95% confidence interval(CI) using the unconditional logistic regression after adjusting for age, sex, nationality, smoking, drinking, family history of HCC, and hepatitis B virus(HBV) infection. Moreover, stratified analysis was conducted on the basis of the status of HBV infection, smoking, and alcohol drinking.RESULTS: The HCC risk was lower in patients with the MCM4 rs2305952 CC(OR = 0.22, 95%CI: 0.08-0.63, P = 0.01) and with the CHEK1 rs515255 TC, TT, TC/TT(OR = 0.73, 95%CI: 0.56-0.96, P = 0.02; OR = 0.67, 95%CI: 0.46-0.97, P = 0.04; OR = 0.72, 95%CI: 0.56-0.92, P = 0.01, respectively). Conversely, the HCC risk was higher in patients with the KAT2 B rs17006625 GG(OR = 1.64, 95%CI: 1.01-2.64, P = 0.04). In addition, the risk was markedly lower for those who were carriers of MCM4 rs2305952 CC and were also HBs Ag-positive and non-drinking and nonsmoking(P < 0.05, respectively) and for those who were carriers of CHEK1 rs515255 TC, TT, TC/TT and were also HBs Ag-negative and non-drinking(P < 0.05, respectively). Moreover, the risk was higher for those who were carriers of KAT2 B rs17006625 GG and were also HBs Ag-negative(P < 0.05).CONCLUSION: Of 12 cell cycle pathway genes, MCM4, CHEK1 and KAT2 B polymorphisms may be associated with the risk of HCC.

5-aza-2'-deoxycitydine induces demethylation and up-regulates transcription of p16^(INK4A) gene in human gastric cancer cell lines

Background To investigate the effects of DNA methylation on the expression of tumor-associated genes and the cell cycle in human gastric cancer cells. Methods Two gastric cancer cell lines (MKN-45 and HGC-27) were treated with DNA methyltransferase (DNMT) inhibitor,5-aza-2’-deoxycytidine (5-aza-dC). The expressions of p16 INK4A,p21 WAF1,p53 , p73 ,c-Ha-ras and c-myc genes mRNA were detected by using reverse transcription PCR (RT-PCR). DNA methylation status of p16 INK4A gene promoter was assayed by bisulfite modification and sequencing. The cell cycle was analyzed by using flow cytometry (FCM). Results 5-aza-dC induced the demethylation of p16 INK4A gene promoter. The expression of p16 INK4A mRNA was obviously up-regulated by treatment with 10 μmol/L (MKN-45 cells) or 5 μmol/L (HGC-27 cells) of 5-aza-dC for 24 hours. However,5-aza-dC treatment failed to regulate the expressions of p21 WAF1,p53 , p73 ,c-Ha-ras and c-myc genes in MKN-45 and HGC-27 cells. Furthermore,5-aza-dC induced the cell cycle arrest in G1 phase in HGC-27 cell,but not in MKN-45 cell. Conclusions DNA methylation regulates the transcription of p16 INK4A but not p21 WAF1 and proto-oncogenes in human gastric cancer cell lines MKN-45 and HGC-27.