| pi6 gene, mapped to the 9p21 region, which is also called MTS ( Mutiple Tumor Suppressor) , is an inhibitor of cyclin - dependent ki-nase 4/6 and can halt cell - cycle progression. p!6 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 p16 is the other mechanism of its inactivation in hematological malig-nancies, especially leukemias. Methylation - specific PCR offers an effective methods to examine the hypermethylation of CpG island on a. gene. The aberrant methylation of p16 has gained widespread impor-tance in hematological malignancies. In this study, we demonstrated the relationship between the methylation of p16 and leukemias, inves-tigated the relationship between the methylation of pl6 and other he-matological diseases, such as lymphomas, myelodysplastic syndromes and plasma cell dyscrasias, and obtained some experimental datas for further research and clinical practices.MethodsObjects in this study are forty cases of leukemias, five cases of chronic myeloid leukemia, thirteen cases of non - Hodgkin's lympho-ma, eleven cases of myelodysplastic syndromes, seven cases of multi-ple myelomas. Samples are obtained from these patients' peripheral blood, twelve healthy volunteers are chosen as controls. The DNA wasobtained from peripheral blood by proteinase K digestion, phenol/ chloroform extraction and ethanol precipitation. The DNA was treated with NaHS03: DNA(2. 5μl) in a volume of 12.5μl was denatured by NaOH(0.4mmol/L,12.5μl)for 10 minutes at 37癈. Fifteen microli-ters of 10 mM hydroquinone and 260 (xl of 3M sodium bisulfite at pH5 were added, and samples were incubated under mineral oil at 50T1 for 16hr. After incubation, added 5mM NaAc at 4T1 for 4hr, 0. 5mM C6H602 at 4癈 for 4hr, 0. 5M NaAc 4癈 overnight. DNA was eluted into sterile water twice at 4Tl for 4hr and dissolved in sterile water at 4癈 , overnight. 0. 6mol/L NaOH 50μl was added at room temperature for 5 minutes, add 6mol/L NH4Ac 50μl and ethanol l00μJ, -20癈 overnight. Then they were centifugated at 12000r/min for 10 min, DNA was resuspended in water and stored at - 20癈 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 2μJ) , primers(0. 5μ1 each per reaction) , bisulfite -modified DNA 4μd, Taq polymerase 0. 2μl0 Amplification was carried out for 35 cycles; 5second at 96癈, 30sec at 95癈 , 30sec at 60癈 , 30sec at 72癈 , followed by a final 7 - min extention at 72癈. Controls were performed for each set of PCRs. Each PCR was loaded onto 8% polyacrylamide gels, and directly visualized under UV illumination, taken pictures.ResultsIn 37 patients of primary leukemia or relapse, those with positive MSP results were 64. 9% (24 of 37) , ANLL were 59. 1% (13 of 22) , ALL were 73.3%(11 of 15) , and there was no difference between twotypes ( P > 0. 05, x2= 0. 2919). Three AL in complete relieve phase (2 ANLL, 1 ALL) showed negative. Five chronic myeloid leu-kemia showed no evidence of methylation, four cases were in the chro-nic - phase , one case was in the acute - phase. The percentage of p!6 methylation is 53. 8% in non - Hodgkin's lymphoma (7 of 13) , none in MDS or multiple myelomas.DiscussionThe p!6 gene blocks progression through the cell cycle by compe-ting with cyclinDl and binding to either cyclin - dependent kinase (CDK)4 or 6 and inhibiting the action of CDK4/6. p!6 is frequently inactivated by deletion in many human cancers which include leukemi-as, and recent research has revealed that the hypermethylation of CpG island on transcriptional start of pi6 is another important mechanism of its inactivation. Herman et al conclude the Methylation ?specific PCR, based on; after bisulfite treatment of DNA all cytosines are con-verted to uracils, but those are methylated ( 5 - methylcytosine ) are resistant to this modification and remain as cytosines. We can distin-guish methylated from unmethy... |
PDF Full Text Request