Differentiation Of Human Melanoma Cells Research Articles

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

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.

Transcriptional upregulation of TGF-alpha by phenylacetate and phenylbutyrate is associated with differentiation of human melanoma cells.

The aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB) induce tumour cell differentiation in experimental models and both are currently in clinical trials. The purpose of this study was to determine the effect of these antitumour agents on the expression of transforming growth factor-alpha (TGF-alpha) in neoplastic cells. Treatment of human melanoma 1011 cultures with either PA or PB caused over 40-fold increase in TGF-alpha biosynthesis and secretion into the media. Whereas elevation in TGF-alpha mRNA steady-state levels became evident within 6-12 h and reached peak quantities the following day, the amounts of its coded protein increased gradually over a period of 5 days of treatment. Further molecular analysis revealed that regulation of TGF-alpha expression occurred at the transcriptional level. In contrast to TGF-alpha, expression of its receptor remained below detectable levels, indicating that an autocrine loop involving this growth factor is unlikely. Interestingly, the increase in TGF-alpha production paralleled drug-induced cytostasis and differentiation defined by morphological changes and increased melanogenesis. Like PA and PB, other differentiation inducers such as all-trans-retinoic acid, dimethyl sulfoxide, and 5-aza-2'-deoxycytidine, all induced TGF-alpha expression in the melanoma cells. The close association between enhanced TGF-alpha production and melanoma cell differentiation suggests that this growth factor, often linked to mitogenesis, may play a novel role in tumour differentiation by PA and PB.

Modification of invasion and differentiation in human melanoma cell clones.

In an attempt to define the role of plasminogen activator in invasiveness and differentiation of human melanoma cells, the modulation of these parameters was studied in two melanoma clones characterized by marked differences in their basal features, using 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and retinoic acid, two differentiation inducers, and doxorubicin, a cytotoxic agent. TPA induced only slight reductions, whereas retinoic acid and doxorubicin caused an increase in invasiveness, enzymatic activity and differentiation in the clone showing low invasivity, low urokinase-type plasminogen activator levels and high differentiation. In contrast, in the clone showing high invasivity, high urokinase-type plasminogen activator levels and low differentiation it was found that: TPA was ineffective; retinoic acid induced a reduction of plasminogen activator but no modifications of invasiveness and differentiation; doxorubicin caused a decrease in invasiveness and plasminogen activator activity but no modification of morphological features. The different behaviour of the two clones thus could be related to the basal features of the clones. The results reported here indicate that in the presence of these drugs the associations between invasiveness and urokinase-type plasminogen activator activity and between invasiveness and differentiation are lost. Drug treatment therefore significantly affected the features of the clone characterized by low biological aggressiveness (high differentiation, low invasiveness), whereas the highly aggressive clone did not show a consistent response to drug treatment.

Spontaneous and induced differentiation of human melanoma cells.

Malignant melanoma cells can differentiate spontaneously in vivo and in vitro into cells with a finite lifespan. Analysis of differentiating cells from primary melanomas in culture revealed a flat, fibroblast-like morphology and expression of the fibroblast-associated marker leucine aminopeptidase (LAP). Differentiation was also observed in a minor sub-population of permanent cell lines derived from metastatic lesions. An experimental model of melanoma cell differentiation was then developed, using the pyrimidine analog bromodeoxyuridine (BUdR). BUdR-treated cells had a flat morphology, were contact-inhibited, had up to 20-fold increased surface area, expressed LAP, no longer proliferated anchorage-independently in soft agar, and 3 out of 4 cell lines were non-tumorigenic in athymic nude mice. Our results show that models of differentiation of melanoma cells can be established that help to define pathways of differentiation.

151. Multistep model of terminal differentiation in human melanoma cells treated with the combination of recombinant human fibroblast Interferon (IFN-β) and mezerein (MEZ)

151. Multistep model of terminal differentiation in human melanoma cells treated with the combination of recombinant human fibroblast Interferon (IFN-β) and mezerein (MEZ)

Genistein-Induced Cell Differentiation and Protein-Linked DNA Strand Breakage in Human Melanoma Cells

Genistein, an in vitro inhibitor of topoisomerase II and tyrosine kinases, elicited an inhibition of growth and increased melanin content in five human melanoma cell lines, after six days of treatment at a concentration of 45 microM. In two lines examined more thoroughly, HO and SK-MEL-131, treatment with genistein also increased other markers of differentiation, including tyrosinase activity, reactivity with CF21 monoclonal antibody, and dendrite-like structure formation. The genistein-evoked increases in melanin content and tyrosinase activity were concentration- and time-dependent. Treatment of HO and SK-MEL-131 cells with 45 microM genistein for 24 hr or 60-600 microM genistein for only 1 hr resulted in an increase in protein-linked DNA strand breaks. Our results suggest an association between the genistein-evoked, protein-linked, DNA strand breaks and the genistein-induced differentiation of human melanoma cells.

The DNA methylation system in proliferating and differentiated cells

The human melanoma cell line M21 can be induced to differentiate into oligodendrocyte-like cells with concommitant cessation of cell division. Cytosine-arabinoside, 5-aza-2'-deoxycytidine, hydroxyurea, aphidicolin, and phorbol-12-myristate-13-acetate were found to be potent differentiation inducers. We have analyzed the changes of methylation of DNA cytosines that occur after treatment of M21 cells with these compounds. Although DNA methylation levels remain unchanged in the presence of aphidicolin and phorbol ester, 5-aza-2'-deoxycytidine-induced differentiation of these cells results in a 40% DNA demethylation. On the other hand, hydroxyurea and cytosine-arabinoside treatment causes DNA hypermethylation, which, in the case of the cytidine analogue is of only transient nature. These results show that the differentiation of human melanoma cells can be accompanied by variable changes of DNA methylation levels. In another set of experiments, the DNA methylation levels have been analyzed during cytosine-arabinoside-induced differentiation of human K562 erythroleukemia cells. In this system, a transient DNA demethylation precedes the establishment of the differentiated phenotype. Since DNA replication is inhibited, this demethylation cannot be explained by inhibition of the maintenance activity of DNA methyltransferase, but is more likely caused by an active excision of 5-methylcytosine from DNA.

Induction of Differentiation in Human Melanoma Cells by the Combination of Different Classes of Interferons or Interferon Plus Mezereina

Induction of Differentiation in Human Melanoma Cells by the Combination of Different Classes of Interferons or Interferon Plus Mezerein^a

Variable DNA methylation changes during differentiation of human melanoma cells

The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2′-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase α inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

Effects of interferon on differentiation of normal and tumor cells

As described in this review, both partially purified and recombinant interferons are potent modulators of differentiation in diverse cell culture systems (Table 2). Depending on the target cell, interferon exerts either an inhibitory or an inductive effect on cell differentiation. In certain myeloid leukemic cells, such as HL-60, interferon by itself is growth suppressive but does not induce cell maturation, whereas in combination with inducers of differentiation, such as DMSO, TPA or retinoic acid, interferon potentiates their ability to stimulate differentiation in both sensitive and resistant cell populations (Grant et al., 1982, 1983; Tomida et al., 1982). Interferon also interacts synergistically with phorbol ester tumor promoters in inhibiting melanogenesis in murine B-16 cells (Fisher et al., 1981a, 1984a) and adipocyte formation in 3T3 cells (Cioe et al., 1980), whereas the combination is synergistic in inducing differentiation in human melanoma cells (Fisher et al., 1984b,c). In contrast, interferon and TPA display antagonistic effects on differentiation in human skeletal muscle cultures, i.e. interferon induces and TPA inhibits myogenesis (Fisher et al., 1982, 1983). Recent studies have demonstrated the presence of high affinity saturable cell membrane receptors for mouse and human interferons (Aguet, 1980; Branca and Baglioni, 1981, 1982; Mogensen et al., 1981; Branca et al., 1982; Anderson et al., 1982; Joshi et al., 1982; Faltynek et al., 1983; Yonehara et al., 1983; Langer and Pestka, in preparation). Similarly, specific membrane receptors have been identified for phorbol esters and mezerein (Driedger and Blumberg, 1980; Shoyab and Todaro, 1980; Horowitz et al., 1981; Fisher et al., 1981b). These findings suggest that the plasma membrane may be a primary target for mediating the biochemical effects induced by both interferon and phorbol esters. Although the mechanism by which interferon and phorbol esters transmit the necessary membrane signal(s) required for altering differentiation are not known, a possible component of this transmembrane signaling process may involve changes in the physical dynamics of the plasma membrane. It is therefore of interest that both interferon and TPA induce early changes in the fluidity of the plasma membrane (Fisher et al., 1979, 1981b, 1984d; Castagna et al., 1979; Kuhry et al., 1983).(ABSTRACT TRUNCATED AT 400 WORDS)