IDENTIFICATION OF HUMAN TUMOR-SUPPRESSOR GENES BY MONOCHROMOSOME TRANSFER - RAPID GROWTH-ARREST RESPONSE MAPPED TO 9P21 IS MEDIATED SOLELY BY THE CYCLIN-D-DEPENDENT KINASE INHIBITOR GENE, CDKN2A (P16(INK4A))

Microcell transfer of intact normal human chromosomes into immortal mo use and hamster fibroblast cell lines has revealed growth suppressive activity associated with a small sub-set of the human complement, Here , we describe the results of a detailed study aimed at identifying the gene or genes responsible for the rapid growth-arrest response obtain ed with human chromosome-9, Initially, STS-PCR deletion mapping of seg regants arising in monochromosome transfer experiments was used succes sfully to localize the active sub-chromosomal region to 9p21, Subseque nt finestructure deletion mapping of previously uninformative hybrid s egregants, employing additional markers between D9S162 and D9S171, pro vided strong evidence that the cyclin-dependent kinase (cdk) inhibitor gene CDKN2A (p16(INK4A)) was solely responsible for the chromosome-9 effect; 9p21 microdeletions in a significant proportion of segregant c lones were restricted to a single CDKN2A exon, Transfection experiment s with CDKN2A and CDKN2B cDNA expression vectors, using mouse A9 cells and three human malignant melanoma cell lines as recipients, provided further evidence in support of this hypothesis, Collectively, our res ults indicate that expression of human CDKN2A (controlled either by it s natural regulatory elements, or by a cytomegalovirus promoter) is in compatible with in vitro proliferation in immortalized rodent cells an d in human melanoma cell lines, The rapidity of the growth inhibitory effects of CDKN2A was inconsistent with a mode of action involving ind uction of replicative cell senescence via telomerase repression, but w as consistent with a mechanism based on cell cycle arrest through cdk inhibition, The study described here has generated a panel of microdel eted monochromosome-9 donor hybrids which may prove valuable in functi onal investigations aimed at identifying other important tumour suppre ssor genes located on human chromosome-9.