Molecular analysis of the INK4 family of genes in prostate carcinomas.

PURPOSE: A newly recognized class of INK4 family of cyclin-dependent kinase inhibitors CDKIs) include its prototype, p16 (INK4A/MTS1/CDKN2), and three others, p15 (INK4B/MTS2), p18 (INK4C), and p19 (INK4D). The putative tumor suppressor gene, p16 is frequently altered in certain neoplasms and many cell lines. The potential role of INK4 CDKIs in pathogenesis of prostate carcinoma was studied. MATERIALS AND METHODS: Thirty-two primary prostate cancer samples and two prostate cancer cell lines were examined for alterations of the p16, p15, p18, and p19 genes by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and Southern blot analysis. RESULTS: Alteration of the p16 gene was found in one of 32 primary prostate cancer samples by PCR-SSCP. DNA sequencing of the sample showed a 24-basepair insertion in exon 1 of the p16 gene at codon 11. No other mutations were found in p15, p18, or p19 genes by PCR-SSCP. Furthermore, none of the p16, p15, p18, or p19 genes had alterations by Southern blot analysis. CONCLUSIONS: These results indicate that structural abnormalities of the INK4 CDKIs is a rare event in prostate carcinoma, and the loss of function of INK4 CDKIs by other mechanisms, such as methylation should be further explored.

p53 transactivation through various p53-responsive elements.

p53 is a nuclear phosphoprotein whose function is classified as tumor suppression. Studies have shown that p53 functions by binding to p53 DNA recognition sequences and regulates transcription of growth-regulatory genes. Various p53 recognition sequences have recently been identified. pOST2 contained two copies of a palindromic high-affinity DNA-binding sequence for p53; the other p53 recognition sequences included p53-binding fragments found in the human ribosomal gene cluster (pRGC) region and in the murine muscle creatine kinase promoter (pMCK). The purpose of this study was to compare the abilities of various p53 recognition sequences to mediate transcription in the presence of endogenously produced wild-type (wt) or mutant p53. Three p53-responsive chloramphenicol acetyltransferase (CAT) reporter constructs (pOST2, pRGC, and pMCK) that contain one or two copies of p53 recognition sequences upstream of a herpes thymidine kinase (TK) promoter and CAT reporter cDNA were constructed. Either a p53-responsive gene or a control reporter gene was transfected into human carcinoma cell lines (having various p53 mutations) either with or without a wt or mutant p53 expression vector. CAT activity was assayed to measure transactivation through the various p53-responsive elements. We showed that pOST2 had a greater ability to mediate transactivation by p53 than either pRGC or pMCK. p53 with a mutation at either codon 175 or 248 was unable to transactivate a reporter gene with pOST2, pRGC, or pMCK. We found it interesting that pOST2, but not pRGC or pMCK, was able to mediate transactivation in cell lines that produce codon 273-mutant p53. These findings suggest that various sensitivities of the different p53-responsive elements to specific mutant and wt p53s may be an important factor in the role of p53 as a transcriptional activator both under normal physiological conditions and during carcinogenesis.