The Preliminary Study On The Relationship Between P16 Gene Expression,Deletion,Mutation,Methylation Status And Clinico-Pathological Features In Human Sporadic Pituitary Adenomas

Objective: The multiple tumor suppressor gene P16 encode the P16 protein, which competitively binds to cyclin-dependent kinase 4 (CDK4) and inhibits its ability to interact with cyclinDl and phosphorylate the KB protein then to control the Gl/S check point of cell cycle as well. P16 gene has rapidly come under intense investigation as a spot-light tumor suppressor gene as it has been found to be inactivated in a wide variety of primary human tumors. The most common mechanism of P16 inactivation involves deletions, mutations and methylation. Because little is known about the clinical and pathological features of pituitary adenomas correlated with P16 gene down-regulation. We analyzed P16 gene status at genomic and protein levels in a series of 70 pituitary adenomas and compared these data with immunohistochemical tumor type, tumor size and invasiveness.Methods: A total of 70 human pituitary adenomas undergoingsurgery and 10 normal brain tissues as control were collected for study. Clinico-pathological features had been prospectively collected in a data including patient age and sex, endocrino-logical adenoma type (non-function, acromegaly, prolactinoma, Cushing's disease, gonadotroph and plurihormonal adenoma), tumor size and invasiveness as revealed during or after surgery were also collected. Of these tumors, there were 20 cases of invasive adenomas and 8 cases of recurrent tumors. The P16 mRNA and protein expression levels were examined by using reverse transcription polymerase chain reaction (RT-PCR) and Western Blot analysis in 70 pituitary adenomas and 10 normal brain tissues respectively. Methylation specific polymerase chain reaction (MSP-PCR) was used to assess the 5'CpG island methylation status in the promotor region of P16 gene in pituitary adenomas and normal brain tissues. The deletion and mutation of P16 gene exon 2 were examined by polymerase chain reaction (PCR) and polymerase chain reaction single-strand conformation polymorphism (PCR- SSCP) analysis. Moreover, all pituitary adenomaswere further examined for Gl/S check point regulators of cell cycle and their mutual relationship by using immunohistochemistry with Envision method, in the meanwhile, the P16 gene status and expression correlated with the clinico-pathological features of tumors were also analyzed.Results: In our group, 74.3%(52/70) pituitary adenomas had a negative or markedly reduced P16 expression. The deletion of P16 geneexon 2 was identified in 5 cases of 70 pituitary adenomas. The mutation of PI6 gene exon 2 was discovered with only 1 case having mobility shifts by PCR-SSCP analysis. But the methylation rate was 72.9%(51/70) in total tumors, whereas it was absent in normal brain tissues .The P16 gene methylation was found in most null cell, lactotroph, gonadotroph, plurihormonal adenomas (90%, 45/50), but it was rare in somatotroph (33.3%, 4/12) and corticotroph (37.5%, 3/8) adenomas. The 5'CpG island methylation in the promoter region of PI6 gene in pituitary adenomas were highly associated with P16 inactivation. Although the invasive and recurrent adenomas had a markedly decreased expression and higher methylation rate than that of noninvasive and nonrecurrent group respectively, the difference was not significant (p>0.05). Moreover, P16 expression deleted adenomas were obviously larger than pi6 - positive tumors (P<0.01), tumors carrying PI6 methylation were also larger than tumors without methylation (P<0.01). The deletion of P16 expression and overexpression of CyclinDl and CDK4 were common in pituitary tumors,but the RB deletion was not revealed by using immunohistochemistry with Envision method in most adenomas in our group. There was a negative relationship between PI6 and CyclinDl, CDK4, RB protein expression (P<0.01) and a positive relationship between CyclinDl and CDK4 expression (P<0.05). None of the above gene expression had an association with the pathological factors and invasiveness of pituitaryadenomas.Conclusions: P16 gene down-regulation is not only common in pitui...