| Introductionpi5 gene, mapped to the 9p21 region of human chromosome, is a tumor - suppressive gene and inhibitor of cyclin - dependent kinase 4/6 and can halt cell - cycle progression. p15 gene is silenced in many human tumors in ways of deletion or point mutation, but recent research has revealed that the hypermethylation of 5' CpG island on p!5 is the major mechanism of its inactivation in hematological malignancies such as leukemias and the frecjuency is very high. Methylation - specific PCR offers an effective methods to examine the hypermethylation of CpG island on a gene. In this study, we examined the methy-lation status of p15 gene in forty leukemias, eleven myelodysplastic syndromes , five chronic myeloid leukemias, thirteen non - Hodgkin' s lymphomas and seven plasma cell dyscrasias, and analyzed the clinical characteristics of ANLL and MDS and obtained some experimental data for further research and clinical practices.MethodsObjects in this study are forty cases of leukemias, eleven cases of myelodysplastic syndromes, five cases of chronic myeloid leukemia, thirteen cases of non - Hodgkin' s lymphoma and seven cases of multiple myelomas. Samples are obtained from these patients' peripheral blood. Twelve healthy volunteers are chosen as normal controls and positive control is Raji cell line which is previously reported to have p15 gene methylation. Mononuclear cells obtained from peripheral blood samples were isolated by Ficoll - Hypaque centifugation. TheDNA was obtained by proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. The DNA was treated with NaH-SO3: DNA(2.5ul) in a volume of 12.5ul was denatured by NaOH (0.4mmol/L,12. 5ul)for 10 minutes at 31℃. Fifteen microliters of 10mM hydroquinone and 260ul of 3M sodium bisulfite at pH5 were added, and samples were incubated under mineral oil at 50℃ for 16hr. After incubation, added 5mM NaAc at 4℃ for 4hr, 0. 5mM C6H6O2 at 4℃ for 4hr, 0. 5M NaAc 4℃ overnight. DNA was eluted into sterile water twice at 4℃ for 4hr and dissolved in sterile water at 4℃ , overnight. 0. 6mol/L NaOH 50 ul was added at room temperature for 5 minutes, add 6mol/L NH4Ac 50ul and ethanol l00ul, -20℃ overnight. Then they were centifugated at 12000r/min for 10 min, DNA was resuspended in water and stored at -20℃ for PCR. The PCR mixture contained 10 x PCR buffer( 16. 6mM ammonium sul-fate/67mM Tris, PH 8. 8/6. 7mM MgCl2/10mM 2 - mercaptoetha-nol) , dNTP(each at 2(ul) , primers(0. 5ul each per reaction) , bisulfite -modified DNA 4ul, Taq polymerase 0. 2ul. Amplification was carried out for 35 cycles: 5second at 96℃ , 30sec at 95℃ , 30sec at 60℃ , 30sec at 72℃ , followed by a final 7 - min extention at 72℃. Controls were performed for each set of PCRs. Each PCR was loaded onto 8% polyacrylamide gels, and directly visualized under UV illumination, taken pictures. Complete methylation is that sample can amplify with p15M but not p15U; incomplete methylation is that sample can amplify both with p15M and p15U; non - methylation is that sample can amplify with p15U but not p15M. We use t test and x2 test to check the statistics.ResultsIn patients of primary leukemia or relapse, those with positive MSP results were 62.2% (23 of 37) , ANLL were 77. 3% ( 17 of 22) , ALL were 40% (6 of 15) , and there was no difference between two types ( P > 0. 05, x2 =3- 80) . One patient in ANLL complete relieve phase showed methylated p15 gene. The percentage of p15 gene methylation is 36. 36% in MDS(4 of 11). Five chronic myeloid leukemias showed no evidence of p15 gene methylation. The percentage of p15 gene methylation is 30. 8% in non - Hodgkin' s lymphoma (4 of 13) and 42. 9% in multiple myelomas(3 of 7).Discussionp15 gene is frequently inactivated by deletion in many human tumors which include leukemias, and recent research has revealed that the hypermethylation of CpG island on promoter and transcriptional start of p15 gene is another important mechanism of its inactivation. Our study investigated the aberrant methylation status... |
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