Polyamine depletion in human melanoma cells leads to G(1) arrest associated with induction of p21(,)(WAF1/CIP1/SDI1) changes in the expression of p21-regulated genes, and a senescence-like phenotype

The cell cycle regulatory events that interface with polyamine requirements for cell growth have not yet been clearly identified. Here we use specific inhibitors of polyamine biosynthetic enzymes to investigate the effect of polyamine pool depletion on cell cycle regulation. Treatment or MALME-3M ce lls with either the ornithine decarboxylase inhibitor alpha -difluoromethyl ornithine or the S-adenosylmethionine decarboxylase inhibitor MIDL-73811 lo wered specific polyamine pools and slowed cell growth but did not induce ce ll cycle arrest. By contrast, treatment with the combination of inhibitors halted cell growth and caused a distinct G(1) arrest. The latter was associ ated with marked reduction of all three polyamine pools, a strong increase in p21(WAF1/CIP1/SDI1) (p21), and hypophosphorylation of retinoblastoma pro tein. All effects were fully prevented by exogenous polyamines. p21 inducti on preceded p53 stabilization in MALME-3M cells and also occurred in a poly amine-depleted, p53-nonfunctional melanoma cell line, indicating that p21 i s induced at least in part through p53-independent mechanisms. Conditional overexpression of p21 in a fibrosarcoma cell line was shown previously to i nhibit the expression of multiple proliferation-associated genes and to ind uce the expression of genes associated with various aspects of cell senesce nce and organism aging. Polyamine depletion in MALME-3M cells was associate d with inhibition of seven of seven tested p21-inhibited genes and with ind uction of 13 of 14 tested p21-induced genes. p21 expression is also known t o induce a senescence-like phenotype, and phenotypic features of senescence were observed in polyamine-depleted MALME-3M cells. Cells increased in siz e, appeared more granular, and expressed senescence-associated beta -galact osidase. Cells released from the polyamine inhibition lost the ability to f orm colonies, failed to replicate their DNA, and similar to 25% became bi- or multinucleated. These events parallel the outcome of prolonged p21 induc tion in fibrosarcoma cells. The results of this study indicate that polyami ne pool depletion achieved by specific biosynthetic enzyme inhibitors cause s p21-mediated G(1) cell cycle arrest followed by p21-mediated changes in g ene expression, development of a senescence-like phenotype, and loss of cel lular proliferative capacity.