Background:
D-type cyclins, in association with the cyclin-dependent kinases cdk4 and cdk6, promote progression through the g1 phase of the cell cycle. cdk activity is modulated by inhibitors such as p15ink4b and p16ink4a. loss of function of p15ink4b and p16ink4a (multiple tumor suppressor-i and cdk4 inhibitor) determines impairment in the control of the cell cycle and contributes to the transformation of several cell types.
Methods:
The authors examined 20 thyroid neoplasms (12 papillary carcinomas and 8 follicular adenomas) and 4 human thyroid carcinoma cell lines for gene mutations and epigenetic modifications of the p15ink4b and p16ink4a genes by southern blot analysis, single strand conformation polymorphism, and a polymerase chain reaction-based methylation assay.
Results:
Abnormalities of p16 were found in the four cell lines studied. in follicular carcinoma (wro) cells, both the p15 and p16 genes were homozygously deleted. undifferentiated carcinoma (fro) cells had a nonsense point mutation at codon 72 (cga-tga, arg-stop) of p16, whereas the poorly differentiated papillary carcinoma (npa) line harbored a point mutation at the exon 1-intron 1 boundary that altered the donor splicing site and caused an aberrantly spliced form of p16ink4a. furthermore, p16 allelic loss was evident in the dna of both fro and npa cells. finally, p16 expression was absent in the aro cell line, likely due to a de novo methylation of exon 1 of p16ink4a. regarding the primary thyroid tumors, a missense point mutation at codon 91 was found in 1 of 12 papillary thyroid carcinomas (gcc-gtc, ala-val). no mutations were found in follicular adenomas. however, in 6 of 20 primary tumors there was hypermethylation at exon 1 of p16.
Conclusions:
The high prevalence of p15 and p16 mutations in the cell lines described suggests involvement of these genes in immortalization in vitro. the p16 defects may have preexisted in a small subclone of the primary tumor that were selected for in vitro. alternatively, p16 mutations may have arisen de novo during cell culture. mutations of p15ink4b and p16ink4a do not appear to be critical events in the development of follicular adenomas or papillary carcinomas. however, de novo methylation of the 5' cpg island of p16 is common in primary tumors, indicating that the function of this gene may be lost as an epigenetic event during disease progression.