Specific tyrosinases associated with melanoma replicative senescence and melanogenesis.

Replicative senescence occurs in normal cells, in contrast to their malignant counterparts which are generally immortal in vitro. We now show that induction of melanogenesis in subconfluent B16 melanoma cells deprived of growth factors can lead to irreversible growth arrest but continued cell viability, concurrent with the expression of specific glycosylated high molecular weight tyrosinases. These tyrosinase activities identify withdrawal from the cell cycle since they were not detected in reversibly arrested quiescent melanocytes, serum-deprived melanoma, or apoptotic melanoma. Our data suggest that different tyrosinases can distinguish cycling and noncycling cells of melanocytic origin and also imply that replicative senescence can be restored in some tumor cells when induced to terminal differentiation in the absence of growth-promoting agents.

Accessibility to DNA in carcinoma chromatin is promoted by nanomolar okadaic acid: effect on AT-rich DNA binding proteins.

Differential accessibility to DNA in tumor cell chromatin is important to growth, differentiation apoptosis, and the targeting of DNA modifying drugs. We now show that endonuclease accessibility to DNA in the nuclei of A431 human carcinoma cells is increased within 90 min by nontoxic nanomolar levels of okadaic acid, known to inhibit protein phosphatase 2A. This genomic hypersensitivity was partly enhanced by joint treatment with epidermal growth factor and okadaic acid but did not appear without the latter. Nuclei with greater DNA susceptibility showed a decrease in M(r) 80,000 DNA binding protein doublet specific for dAT-rich sequences concurrent with the "apparent" hyperphosphorylation of a M(r) 70,000 nuclear matrix protein. We propose that some of the tumor-promoting effects of okadaic acid may be partly associated with its ability to promote genomic susceptibility.

Heterogeneity in cell-associated transformation-sensitive proteins antigenically related to fibronectin.

Antiserum to purified fibronectin has been used to investigate transformation-associated heterogeneity in surface components antigenically related to fibronectin. Examination of extracts from surface-labeled rat cells by gradient gel electrophoresis revealed in "normal" cells the presence of two species with approximate molecular weights of 250,000 and 230,000, which were decreased in wild-type transformed cells. Reaction with antifibronectin serum revealed the selective precipitation of the two transformation-sensitive surface components. A similar experiment with ts-NT3-KR that expresses a normal phenotype at 37 degrees and a transformed morphology at 33 degrees did not reveal a markedly altered surface labeling pattern at both temperatures. However, reaction with antifibronectin serum did show a weak but detectable recognition of a 230,000-dalton doublet in the cells grown at 37 degrees, and immune precipitation of components in the 100,000- and 60,000-dalton region in cells grown at 33 degrees. Experiments with normal mouse cells revealed a different ratio of two fibronectin-related external proteins with similar molecular weights to those seen in normal rat cells.

Effect of malignant transformation and arginine limitation on fibronectin and other cell surface macromolecules of liver epithelial cultures.

Surface labeling of "normal" nontumorigenic rat liver epithel cells permitted the detection of a 140,000-dalton species and of another 230,000-dalton molecule that comigrated with fibronectin, an external transformation-sensitive protein of fibroblasts. Reaction of surface-labeled normal liver epithelial cells with anti-fibronectin serum led to the selective recognition of the 230,000-dalton component, which revealed a peptide composition similar to that of the homologous fibroblasts fibronection. Similar experimental conditions with the tumorigenic liver epithelial cell counterpart revealed decreased labeling in the fibronectin region and preferential labeling in the 140,000- and 60,000-dalton regions. Arginine limitation was found to produce growth arrest in both normal and malignant liver epithelial cells, concurrent with a marked decrease in the labeling of the surface-associated fibronectin and different surface macromolecular alterations in normal and transformed cells. Mild proteolysis combined with brief neuraminidase treatment did not produce marked alterations in tumorigenic cell surface labeling but did lead to a decrease in the fibronectin of normal cells and to the increased expression of other transformation-associated changes in surface macromolecules different from fibronectin. Our results show the involvement of an arginine-regulated fibronectin and other cell surface macromolecules among the changes that occur during the conversion of normal liver epithelial cells to a malignant state.

Lower cyclin H and cyclin-dependent kinase-activating kinase activity in cell cycle arrest induced by lack of adhesion to substratum.

Knowledge about adhesion checkpoints is important to counteract dissemination of cells from solid tumors. Lack of anchorage in adherent cells is associated with growth arrest and inhibition of cyclin-dependent kinases (cdks) required to drive cell cycle progression. Because cyclin-cdk complex activation requires CDK-activating kinase comprising cdk7 and cyclin H, we now investigated their relationship to decreased proliferation by lack of cell spreading. This report shows that either UV irradiation on an adhesive substrate or culture on a nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition of proliferation by UV primarily induced the cdk inhibitor p21WAF1 without a significant effect on cyclin H and cdk7. In contrast, lack of adhesion to substratum decreased cyclin H but not cdk7 with accumulation of a slower migrating, presumably unphosphorylated cdk4 isoform. These results were paralleled by decreased cdk7-mediated phosphorylation of GST-cdk2 and lower activation of a baculovirus-derived cdc2-cyclin B kinase complex. This is the first report showing that cyclin H-mediated down-regulation of cdk-activating kinase activity is involved in growth arrest induced by lack of anchorage.

Relationship of a Mr 140 fibronectin receptor and other adhesion-related glycoproteins to tumor cell-cell interaction.

Primary melanocytes attach poorly to collagen type IV and laminin, in contrast to their firm attachment to collagen type I/III and fibronectin [Gilchrest et al., In vitro (Rockville), 21: 114-120, 1985]. We have now found that metastatic B16 melanoma cells attach well to collagen type IV, laminin, vitronectin, and fibronectin but show a selective defect in attachment and cell aggregation on native collagen type I. Both flattened and aggregated melanoma cells revealed the presence of a Mr 120,000 surface-iodinated species with affinity for a matrix containing the hexapeptide (glycylarginylglycylaspartylserylproline) which includes the fibronectin cell attachment sequence, but only flattened cells showed significant exposure of a Mr 140,000 iodinated component with affinity for a large cell attachment-promoting fibronectin polypeptide. Decrease of the Mr 140,000 fibronectin-binding external protein in the collagen-cultured melanoma cells was also associated with an inability to respond to the cell attachment activity of fibronectin, laminin, or vitronectin added to the collagen gels. Metabolic labeling with [3H]glucosamine and electrophoretic analysis showed that lack of attachment and cell aggregation was associated with an increase in high molecular weight wheat germ agglutinin-binding glycoconjugates and an increase in Mr 55,000 concanavalin A-binding glycoprotein species. Our data suggest that: (a) melanoma cell attachment requires the expression of the Mr 140,000 fibronectin receptor which appears to be down regulated in cells exposed to poorly adhesive substrates; (b) expression of the Mr 120,000 iodinated species with affinity for the fibronectin attachment sequence (arginylglycylaspartic acid) may be necessary but not sufficient for firm cell-substratum interactions; (c) increased tumor cell-cell interaction may involve a decreased attachment to substrate and the expression of different glycoproteins which may modulate cell-cell association.

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

Tumor suppression without differentiation or apoptosis by antisense cyclin D1 gene transfer in K1735 melanoma involves induction of p53, p21WAF1 and superoxide dismutases.

In mammalian cells, terminal differentiation is mutually exclusive with proliferation. However, resistance to differentiation-inducing therapy requires alternative strategies to control poorly responsive tumors. We now show that retroviral transfer of the antisense cyclin D1 gene to differentiation-refractory K1735 melanoma leads to loss of in vivo tumorigenicity, shortened replicative ability, induction of the tumor suppressor p53 protein and of the cdk-inhibitor p21WAF1, increased beta-galactosidase pH 6.0 activity, and elevation in the ratio of superoxide dismutases to peroxidases, all properties associated with replicative senescence. However, pigmentation and tyrosinase expression, characteristic of differentiated melanocytic cells or apoptosis-associated PARP cleavage, were not increased by antisense cyclin D1 transduction. Our data suggests that targetting cyclin D1 inhibition suppresses melanoma tumorigenicity by promoting a cytostatic differentiation-independent pathway, mediated by activation of p53 and anti-oxidant functions.

Sensitization to DNA damage by okadaic acid or bromodeoxyuridine involves unequal effects on melanoma cell adhesion and differentiation.

Because melanoma tumors originate partly from excessive UV exposure but become relatively resistant to radiation, we have now compared the effects of okadaic acid, a phosphatase inhibitor, with that of the thymidine analog bromodeoxyuridine as sensitizers of DNA damage in B16 melanoma. We now show that 25 nM okadaic acid promotes DNA fragmentation in B16 melanoma, increasing cell detachment as well as pigmentation, a characteristic of melanocytic cell differentiation. At lower levels, okadaic acid synergizes with UV exposure to increase DNA fragmentation. Although bromodeoxyuridine also caused DNA damage, it did not increase pigmentation and it suppressed cell detachment. Okadaic acid was also more effective in promoting DNA laddering in growing versus quiescent melanocytes. Because DNA damaging effects of okadaic acid are mediated by different pathways from those used by nucleoside analogs, like bromodeoxyuridine, we propose their concurrent effect with radiation as sensitizers to DNA damage.

Cyclin-dependent kinase 2 and cyclin A interaction with E2F are targets for tyrosine induction of B16 melanoma terminal differentiation.

L-Tyrosine promotes a dramatic increase in melanogenesis and an apparent replicative senescence in B16 melanoma (M. Strasberg-Rieber and M. Rieber, Cancer Res., 53:2469-2471, 1993). Since cyclins are implicated in controlling cell proliferation and differentiation, we have now investigated their relationship to melanocytic growth arrest and pigmentation. In B16 melanoma cells enriched in G1 by serum starvation or synchronized in late G1/early S phase by exposure to hydroxyurea, L-tyrosine overrides mitogenic signals and induces terminal differentiation without cytotoxicity. This correlates with a decrease in cyclin A and cyclin E-dependent kinase 2 activity and with an altered interaction of cyclin A with the transcription factor E2F. This activity involves a lower level of the catalytic cdK2 kinase protein without a concomitant decrease in cyclin A or cyclin E. Upon addition of serum or removal of hydroxyurea, cells resume cell cycle progression and the ability to form tumors in vivo, but these properties are irreversibly inhibited in tyrosine-treated cells. Our data suggest that targeted inactivation of cdK2 with specific inducers of differentiation favors reacquisition of tumor growth control.

Differential genomic susceptibility in malignancy correlates with changes in ATATAT DNA-binding proteins.

Accesibility to DNA in the nucleus is important for the regulation of gene expression and for the effect of DNA-modifying drugs. We have now studied differential genome susceptibility in normal melanocytes and the corresponding malignant melanoma. DNA hypersensitivity assays revealed a markedly lesser degradation in melanoma nuclei compared to that in melanocytes. Cross-linking of DNA to nuclear proteins by ultraviolet light showed a cell-type dependent inverse correlation of genomic susceptibility with binding of (dA.dT) (dA.dT) sequences, compared to that shown with (dG.dC) (dG.dC), regardless of methylation in cytosines. Exposure to cholera toxin partly reversed genomic susceptibility and increased DNA/protein cross-linking in melanocytes. In contrast, melanoma cells showed decreased DNA/protein interactions and greater genome susceptibility after exposure to cholera toxin or okadaic acid. Our data suggest that a molecular mechanism for differential genome exposure in cancer cells involves a modified expression of sequence-specific DNA-binding proteins.

Early inhibition of protein phosphatases preferentially blocks phorbol ester-stimulated mitogenic signalling in melanocytes: increase in specific tyrosine phosphoproteins.

Inhibition of protein phosphatases has been suggested as an alternative mechanism of tumor promotion (H. Fujiki, Mol. Carcinog. 5:91, 1992). We have now used early melanocyte passages dependent on phorbol esters and serum for growth and later passages with partial phorbol ester independence, to investigate the role of protein phosphatases on melanocyte DNA synthesis. Neither okadaic acid, an inhibitor of ser/thr protein phosphatases, nor vanadate, an inhibitor of tyrosine phosphatases, can stimulate basal or serum-stimulated mitogenesis in contrast to phorbol esters. Moreover, both phosphatase inhibitors are able to suppress serum and phorbol ester-stimulated mitogenesis, if added within 4 hours of growth activation. Inhibition of mitogenesis by either inhibitor correlated with an early increase in a common set of tyrosine phosphoproteins, which included a major 33 Kd species. Our data suggest that protein phosphatase inhibitors are growth suppressors and antagonize phorbol ester effects in cells of melanocytic origin, implying an early requirement for protein phosphatase activity during mitogenic signalling in these cells.

Epidermal growth factor promoted changes in the triton-insoluble cytoskeletal matrix from human epidermal carcinoma cells: effect of bromodeoxyuridine.

We have used monolayers of bromodeoxyuridine (BrdU)-grown and control human epidermoid carcinoma (A431) cells to investigate the polypeptide changes resulting when cells are rounded by epidermal growth factor (EGF). Whereas no significant change was detected in Triton-soluble components, the urea-solubilized matrix fraction revealed greater levels of a 20 kd species in cells exposed to EGF, compared with the same cells not exposed to the growth factor. The corresponding urea fraction from BrdU-grown cells showed decreased levels of the 20 kd species as a result of exposure to EGF. Further evidence for a differential effect of EGF resulting from growth of the cells with the pyrimidine analog was observed in the matrix fraction soluble in SDS, which revealed a decrease in a 20 kd species resulting from exposure of control cells to EGF, with no comparable effect in BrdU-grown cells. Our results suggest that EGF induces a change in the properties of matrix-associated components of low molecular weight in an effect which appears to be modified by prior growth of cells with BrdU.

Transformed cells exhibit altered response to db cycle amp-mediated modulation of protein phosphorylation and different endogenous phosphoprotein acceptors.

Endogenous protein phosphorylation has been studied in extracts from rat cells transformed by temperature-sensitive derivatives of Rous sarcoma virus which can reversibly express a transformed behavior at 33 degrees C and reacquire "normal" properties at 39 degrees C. The expression of transformation appeared associated with marked alterations in the type of phosphoprotein acceptors and with an increased protein kinase activity, particularly in detergent solubilized fractions. A comparison of the effect of dibutyryl cyclic AMP on protein phosphorylation revealed a transformation-dependent response, as well as a different effect of the cycle nucleotide in cytoplasmic and detergent solubilized fractions. Our studies suggest that the processes leading to malignant transformation are accompanied by altered response of the phosphorylating system to cyclic nucleotide-mediated modulation and by marked alteration in phosphoprotein acceptors.

Differential susceptibility of fibroblastic and non-fibroblastic surface fibronectins to periodate-borohydride labelling and enzymic treatment.

Surface labelling of liver epithelioid cells permits the detection of a 230,000 dalton component reactive with antifibronectin serum, when tritiated borohydride labelling is preceded by oxidation of sugar residues with galactose oxidase but not with sodium periodate. Similar experiments with liver fibroblasts lead to the labelling of a similar 230,000 dalton component both when sugar oxidation is carried out with galactose oxidase or periodate. Neuraminidase treatment prior to surface labelling lead to an increased labelling in both cell types, although a decreased labelling in the fibronectin region was apparent in the epithelioid cells.

UV radiation induces DNA fragmentation and cell death in B16 melanoma sensitized by bromodeoxyuridine: impaired c-jun induction and defective tyrosine phosphorylation signalling.

The relevance of tyrosine phosphorylation and c-jun protooncogene expression to radiation sensitization was investigated in B16 melanoma. These cells are sensitized by bromodeoxyuridine (BrdU), a thymidine analog, showing extensive DNA fragmentation reminiscent of apoptosis, after UV radiation. UV-irradiated unsensitized cells did not reveal DNA fragmentation but showed increased expression of c-jun and greater protein tyrosine phosphorylation in response to sodium vanadate, an inhibitor of tyrosine phosphatases. However, these responses were inhibited in UV-irradiated BrdU-treated cells. Our data suggest that the bromodeoxyuridine-induced sensitization to radiation can lead to DNA fragmentation and cell death, partly because of a defective tyrosine kinase signalling and an impaired c-jun expression, both of which appear important for cell survival in response to UV radiation.

Improved detection of labile cell-surface components with zinc chloride-aprotinin: demonstration of glycoprotein differences in K-1735 metastatic melanoma variants.

Metabolic labelling of K-1735 melanoma variants with 3H-glucosamine and cell harvesting with the commonly used protease inhibitor phenylmethylsulfonylfluoride revealed a Triton-insoluble fibronection-like 230 kd component in poorly metastatic cells. This component was not evident in highly metastatic cells. Significantly improved surface labelling and detection of the 230 kd glycoprotein in the highly metastatic variant was achieved by zinc chloride-aprotinin treatment of cells prior to harvesting. This procedure also revealed an increase in a trypsin-sensitive glycoprotein of higher molecular weight in the Triton-insoluble fraction of the highly metastatic cell variant. Glycoprotein labelling in this fraction showed an electrophoretic pattern strongly resembling that reported by others for the high-molecular-weight human melanoma-associated glycoprotein complex. The differential detection of the high-molecular-weight glycoprotein species in melanoma variants with differing metastatic abilities in an animal model provides a means of studying their possible relevance to metastatic melanoma. Our data also suggest that zinc chloride-aprotinin can be used to improve the detection of labile cell-surface components.

Tumor hypersensitive DNA is enriched in c-myc sequences and reacts differentially with normal and malignant genomic DNA.

We now show that exposure of B16 melanoma cells to bromodeoxyuridine increases cell-substratum interactions concurrent with an increase in genome susceptibility to nucleases. Hypersensitive DNA was isolated after mild nicking of nuclei with DNase I followed by repair with DNA polymerase I in the presence of biotin-19-SS-dUTP and affinity chromatography on streptavidin-agarose. Dot blot studies showed that the hypersensitive DNA is enriched in c-myc sequences compared to total tumor genomic DNA, and hybridizes preferentially to the latter, compared to normal genomic DNA, particularly when prepared from BrdU-treated cells. Since hypersensitive DNA can hybridize with multiple Alu sequences in the genome, we postulate that one of the mechanisms for its differential reactivity may be by recognition of an unequal number of Alu repeats in normal and tumor genomic DNA.

Substrate-dependent effect of epidermal growth factor on intercellular adhesion and synthesis of triton-insoluble proteins in human carcinoma A431 cells.

Human epidermoid carcinoma A431 cells attach more rapidly to collagen type-I and -IV substrates than to surfaces coated with laminin or fibronectin. The diminished intercellular interaction and rounding up manifested when these cells are exposed to epidermal growth factor (EGF) in tissue culture plastic or collagen films is not shown when the assay is performed on 3-dimensional collagen. In the latter substrate, cells exposed to EGF reveal greater cell cohesion with interdigitations and desmosomal junctions, compared to the limited intercellular interaction detected in similarly treated cells assayed in tissue culture substrate. Although the protein synthesis inhibitor, cycloheximide, did not prevent these EGF-mediated changes, metabolic labelling indicated a substrate-dependent effect of EGF on the synthesis of proteins associated with the Triton-insoluble cytoskeletal matrix. The involvement of cytoskeleton components in the EGF effects on intercellular adhesion was shown by its susceptibility to cytochalasin B, known to disorganize actin-containing microfilaments. Some of the mechanisms of epithelial morphogenesis and the influence of extracellular matrix components on cell-receptor/growth-factor interactions may now be suitably analyzed by examining the EGF effects on A431 cells grown on different substrata.