Inhibition of growth and induction of differentiation of metastatic melanoma cells in vitro by genistein: chemosensitivity is regulated by cellular p53

Abstract

Summary We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genistein's growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed.

Suppression of tumorigenic and metastatic potentials of human melanoma cell lines by mutated (143 Val-Ala) p53

“…The p53 expression plasmids contain wild-type or mutant (143 Val-Ala) p53 cDNA sequences under the control of cytomegalovirus (CMV) promoter-enhancer sequences. To isolate cells stably transfected with p53 expression plasmids or the expression vector, UISO-MEL-4 cells were co-transfected with plasmids pC53-SN3 or pC53-SCX3 and PIRVGalNeo as described previously (Rauth et al, 1997). Three days after transfection, the cells were subcultured in the presence of 1 mg ml G418 (Sigma Chemical).…”

“…p21 mRNA was analysed by reverse transcription and polymerase chain reaction (RT-PCR) using primers that give rise to 224-bp p21 cDNA fragment. P2-microglobulin mRNA, used as an internal control, was also analysed by RT-PCR using primers that yield a 120-bp product precipitation procedure, cells were transfected with wild-type or mutant (143 Val-Ala) p53 cDNA containing expression vector, as described previously (Rauth et al, 1997). The (143 Val-Ala) p53 mutant has been shown to retain DNA binding and some of the transactivation function of wild-type p53 Friedlander et al, 1996).…”

“…The selective media with G418 was replaced every 3 days, and colonies that appeared after 10-14 days were pooled and subcultured for further analysis. and 0.1% Nonidet p-40 as described previously (Rauth et al, 1997). Cell extracts containing equal amounts of protein (150 gg) were separated on a SDS 12.5% polyacrylamide gel.…”

“…p53 and p21 mRNAs in the transfected cells were analysed using RT-PCR of total cellular RNA as described previously (Rauth et al, 1993a;1997). To avoid the plateau effect, PCR reaction conditions were optimized and run for 25 cycles.…”

“…Here, we examined the effects of wild-type and mutated (143 Val-Ala) p53 on tumorigenic and metastatic potentials of two human melanoma cell lines. The cell line UISO-MEL-4 contains wild-type p53 and is tumorigenic, whereas UISO-MEL-6 lacks p53 and is metastatic upon s.c. injection into athymic mice (Rauth et al, 1993a(Rauth et al, , 1994(Rauth et al, , 1997. We report that UISO-MEL-4 melanoma cells, stably transfected with wild-type p53 expression vector, expressed high levels of p53 and p21 and produced s.c. tumours in vivo as the vectortransfected or parental cells.…”