ACCELERATED PAPER: Identification of human tumour 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)

Abstract

Microcell transfer of intact normal human chromosomes into immortal mouse 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 obtained with human chromosome-9. Initially, STS-PCR deletion mapping of segregants arising in monochromosome transfer experiments was used successfully to localize the active sub-chromosomal region to 9p21. Subsequent fine-structure deletion mapping of previously uniformative hybrid segregants, employing additional markers between D9S162 and D9S171, provided strong evidence that the cyclin-dependent kinase (cdk) inhibitor gene CDKN2A (p16INK4A) was solely responsible for the chromosome-9 effect; 9p21 microdeletions in a significant proportion of segregant clones were restricted to a single CDKN2A exon. Transfection experiments 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 results indicate that expression of human CDKN2A (controlled either by its natural regulatory elements, or by a cytomegalovirus promoter) is incompatible with in vitro proliferation in immortalized rodent cells and in human melanoma cell lines. The rapidity of the growth inhibitory effects of CDKN2A was inconsistent with a mode of action involving induction of replicative cell senescence via telomerase repression, but was consistent with a mechanism based on cell cycle arrest through cdk inhibition. The study described here has generated a panel of microdeleted monochromosome-9 donor hybrids which may prove valuable in functional investigations aimed at identifying other important tumour suppressor genes located on human chromosome-9.

Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status

“…On the other hand, p16 has many of the attributes expected of a senescence mediator and it has recently been reported that ®broblasts from p16 nullizygous mice immortalize at a very high frequency, presumably indicative of a failure to senesce (Serrano et al, 1996). Moreover, the higher frequency of p16 deletions and mutations observed in human tumour cell lines as opposed to primary tumours likely re¯ects selection in culture, as a means of escaping senescence, and several studies have provided good evidence for a correlation between loss of p16 function and immortalization (Loughran et al, 1994(Loughran et al, , 1996Rogan et al, 1995;England et al, 1996;Noble et al, 1996;Rezniko et al, 1996). Although the current data imply that this selective pressure may be less critical for mouse cells, there is evidence that the mouse p16 gene also acts as a tumour suppressor.…”

Functional evidence for a squamous cell carcinoma mortality gene(s) on human chromosome 4

“…The repeated loss of exogenous, wild type, chromosome alleles in immortal segregant clones arising from MMCT experiments can indicate that there is a suppressor of cancer cell proliferation (England et al, 1996) or viability (Robertson et al, 1998) in that chromosome region and can be used to map the position of the genes. This approach has been successfully used to map the gene responsible for Leigh Syndrome (Zhu et al, 1998).…”

Molecular biology of prostate cancer

“…The TSG is either introduced into tumour cells as a cloned cDNA in an appropriate expression vector 104 or as an individual chromosome by microcell-mediated transfer. 105 Introduction of a retroviral vector containing normal wild type RB cDNA into the PC cell line DU145 resulted in an approximately 15-fold reduction in the tumorigenicity of the transfectants in nude mice.…”