| Objective The essential attribute of tumor is the loss of control in the cell cycle regulation, and the cells proliferate unlimitedly. p16 protein, an inhibitor of CDK4 (cyclin-dependent kinase,CDK),plays an important role in the cellular cycle regulation. The inactivation of p16 gene and its product is relevant to the various tumorigenesis. Till now, the studies on relationship between p16 and leukemia have often been conducted in leukemia cell lines or focused on the the childhood acute leukemia, and little is known in the adult leukemia. We detected the homozygous deletion and methylation of p16 gene El and E2 in 75 patients with adult acute leukemia, respectively. Meanwhile, we also detected p16 mRNA and p16 protein levels in these samples. The aim of our study is to analyze if the inactivation of p16 corresponds to the adult acute leukemia and if p16 inactivation results from the regulatory abnormality of transcription and translation, and to explore the regulation mechanism in the p16 gene expression in the adult acute leukemia.Methods 75patients with adult acute leukemia were selected.32patients were acute lymphoblastic leukemia(ALL), 43 patients were acute myeloid leukemia(AML). All cases were diagnosed by MIC standard worked out by FAB in 1985. 20 people were selected as normal controls. 2 ml fresh marrow was obtained. 20g/L EDTA was taken as theanticoagulant. The genome DNA was extracted .Multiple comparative PCR was used to amplify the specific segments of exon 1 and 2 of p16 gene. Methylation sensitive restriction enzymes-PCR method was used to detect the methylation of p16 exon 1 and exon 2. At the same time, the cell suspensions were bloted onto the pretreated slides. We used hybridization in situ method and immunochemistry method to detect p16 mRNA and p16 protein expression, respectively. Applying software SPSS 10.0 to analyze the results.Results 1. p16 gene homozygous deletion Homozygous deletion of p16 gene was only found in 2 patients with ALL, not in patients with AML or in normal samples. 2. Abnormal mythelation of p16 gene Methylation were found in 5 ALL and 9 AML cases without homozygous deletion of p16. The p16 gene mythelation hi exon 1 has been found in 2 cases, and the p16 gene mythelation hi exon 2 has been found hi 2 cases, and the p16 gene mythelation in both exon 1 and exon 2 has been found in 10 cases. None of 20 normal samples were mythelated . By Fisher's exact test, the adult acute leukemia group and normal controls had significant differences (p=0.035). 3. Detection of p16 mRNA hi 73 patients without homozygous deletion and methylation of p16, p16 mRNA was present in 56 cases, and was absent in 19 cases. None of the normal samples was negative. By Fisher's exact test, the adult acute leukemia group and normal group had significant differences (p=0.010). 4.pl6 gene protein expression p16 protein was detectable in 54patients,and 19 were absent. None of normal samples was absent of p16 protein. By Fisher's exact test, the adult acute leukemia group and the normal group had significant differences (p=0.005). 5. Relationship of p16 gene structure,p16mRNA and p16 protein (1) p16 mRNA in 14 patients with p16 mythelation were not dectable. Maybe p16 gene methylation is associated with p16 inactivation. (2)Of 59 patients without p16 homozygous deletion and methylation, p16 mRNA expression in 3 cases were negative, 17 cases positive and 39 cases strongly positive. That indicates that p16gene structure does not conincide with p1 6 mRNA. Maybe the abnormality of transcription regulation is relevant to p16 inactivation in the adult acute leukemia. (3) Of 56 patients without p16 mRNA loss, p16 protein expression hi 2 cases were negative, 3 cases weak positive, 18 cases positive and 33 cases strongly positive. Data was analyzed by matched t test, the level of p16mRNA and p16 protein in patients had significant differences(p=0.004). p16 mRNA was not unanimous with p16 protein. Maybe the abnormality of translation regulation is associated with p16 inactivation in the ad... |
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