Syrian Hamster Melanoma Cells Research Articles

Steroid hormone receptor studies in melanoma model systems.

The transplantable B-16 melanotic melanoma carried in syngeneic C57Bl/6J female mice and the Syrian hamster melanoma cell line, RPMI 3460, were utilized to determine whether steroid-hormone receptors are present in animal melanomas. In the B-16 melanoma, a cytoplasmic-estrogen receptor is detectable, but there is no evidence for androgen or progestin receptors. Some tumors contain a glucocorticoid-binding macromolecule. Sucrose-density gradient centrifugation of cytosol after incubation with [3H]-estradiol revealed an 8S peak that was suppressed by excess radioinert diethylstilbesterol. Binding varied from 5-35 fmoles per mg cytosol protein. Scatchard analysis of [3H]-estradiol binding in cytosol yielded a single class of high-affinity binding sites; the dissociation constant is 6 x 10(-10) M. The receptor molecule is shown to be estrogen-specific by ligand competition assays. In contrast to B-16 melanoma, no estrogen, androgen, or progestin receptor can be found in the Syrian hamster melanoma cell line. However, a substantial level of specific binding is observed using [3H]-dexamethasone. Sucrose-gradient centrifugation of cytosol from this cell line after incubation with [3H]-dexamethasone revealed a 7S peak that was suppressed by excess radioinert dexamethasone. Scatchard analysis indicated a single class of high-affinity sites with a dissociation constant of 2 x 10(-9) M. Binding levels from 70-610 fmoles per mg cytosol protein were observed. The Syrian hamster melanoma cells also exhibit a biological response to glucocorticoids: Dexamethasone causes both an inhibition of growth and a decrease in final-cell density in these cells.

Resistance to bromodeoxyuridine mutagenesis and toxicity in mammalian cells selected for resistance to hydroxyurea.

Mutant cell lines resistant to hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase, were selected from a line of Syrian hamster melanoma cells. Mutant lines were selected for resistance to 0.3 mM HU, and from these lines, second-step mutants were selected for resistance to 1.9 mM HU. The HUr lines were tested ffor their responses to 5-bromodeoxyuridine (brdU), in terms of toxicity, mutagenesis, and incorporation of BrdU into DNA. All of the HUr lines showed increased resistance to the toxic effects of BrdU. in addition, the HUr lines all were resistant to BrdU mutagenesis. Overall, there was good correlation among the levels of resistance to HU toxicity, BrdU toxicity, and BrdU mutagenesis in the HUr lines. These tests were carried out under conditions such that the parental and HUr cells incorporated equal amounts of BrdU into nuclear DNA. Therefore, the resistance of the HUr cells to the effects of BrdU cannot be attributed to decreased incorporation of BrdU into DNA. These results suggest that the HUr cells have an lateration in ribonucleotide reductase activity that simultaneously confers resistance to HU and BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU. The properties of the HUr cells suggest that the perturbation of deoxcytidine metabolism by BrdU triphosphate inhibition of ribonucleotide reductase activity plays a key role in the toxic and mutagenic effects of BrdU in mammalian cells.

Altered thymidylate kinase substrate specificity in mammalian cells selected for resistance to iododeoxyuridine

A clone of Syrian hamster melanoma cells was selected for resistance to high levels of the thymidine (dT) analog 5-iododeoxyuridine (IdU). Unlike cell lines previously isolated as IdU resistant (IdU r), these IdU r lines had normal levels of thymidine kinase (EC 2.7.1.21) activity, grew in HAT medium, and readily incorporated exogenous dT and 5-bromodeoxyuridine (BrdU) into DNA. However, these IdU r cells were found to preferentially exclude IdU from their DNA. Analyses of the soluble nucleotide pools of the IdU r cells showed that they were able to take up and phosphorylate exogenous dT as well as the wild-type cells, and both mutant and wild-type cells accumulated dTTP as the major phosphorylated component. In contrast, while the wild-type cells in the presence of exogenous IdU accumulated significant levels of IdUTP (as well as IdUMP), the IdU r cells accumulated only IdUMP. Thus, the mutant cells appear to have a markedly decreased ability to phosphorylate IdU beyond the monophosphate level. Assays of thymidylate kinase (EC 2.7.4.9) activity in extracts of the IdU r cells indicated a marked preference for dTMP as substrate over IdUMP (in comparison to the wild-type enzyme activity). The cell lines described in this study represent a new phenotype arising from selection for resistance to a halogenated dT analog. The resistance appears to involve a change in the substrate specificity of thymidylate kinase, such that the enzyme in the IdU r cells has an enhanced ability to discriminate between very closely related compounds.

Bromodeoxyuridine mutagenesis in mammalian cells is stimulated by purine deoxyribonucleosides.

The effects of purine deoxyribonucleosides on bromodeoxyurdine (BrdU) mutagenesis in Syrian hamster melanoma cells were determined. Both deoxyguanosine (dG) and deoxyadenosine (dA) were found to stimulate mutagenesis without changing the amount of BrdU in DNA. In addition, the stimulation of mutagenesis by dG and dA was suppressed by the addition of deoxycytidine (dC). These results suggest that BrdU mutagenesis involves the perturbation of dC metabolism, which perturbation is enhanced by dGTP and dATP. The mutagenic activity of dG in the absence of BrdU was tested, as was that of thymidine (dT), which we had shown previously to stimulate BrdU mutageneis. With dG alone, no increase above the spontaneous mutation frequency was detected. However, at extremely high concentration, dT in the absence of BrdU was slightly mutagenic, and the mutagenesis by dT was enhanced by dG and suppressed by dC.

Isolation and properties of amelanotic variants of a hamster melanoma

Stable amelanotic variants, isolated from a line of pigmented Syrian hamster melanoma cells, show a constant association of (1) dopa oxidase deficiency, (2) flattened epithelial or fibroblast morphology, (3) anchorage dependence, and (4) a long latent period in tumor formation, each of these properties distinguishing them from the dopa-oxidase-positive, anchorage-independent, highly malignant pigmented cells of origin. One of the variants is shown to have the properties of a pleiotropic conditional “mutant” since its cells show the simultaneous but transitory restoration of pigmentation and melanoma-like morphology when cloned at alkaline pH. Cells of this variant have been hybridized with pigmented cells, derived both from the line of origin and from a mouse melanoma. Hybrids from the first cross show all of the properties characteristic of the pigmented parental cells, but produce segregants reexpressing the ensemble of properties of the amelanotic variant, while those of the second cross show extinction (followed by reexpression) of pigmentation. It is therefore concluded that cells of the amelanotic variant produce a factor capable of extinguishing dopa oxidase activity, as revealed directly by the properties of the hamster-mouse hybrids and indirectly by the amelanotic segregants of the hamster-hamster hybrids.

Bromodeoxyuridine mutagenesis in mammalian cells: mutagenesis is independent of the amount of bromouracil in DNA.

Studies were undertaken to determine how a line of mutant Syrian hamster melanoma cells (HAB-2E) that displays unlimited growth potential when all of the thymine residues in nuclear DNA are replaced by bromouracil (BrUra) could avoid the deleterious effects of bromodeoxyuridine (BrdUrd) mutagenicity. It was found that BrdUrd could be mutagenic to these cells. However, there was a nonlinear relationship between mutagenicity and the amount of BrUra in the DNA of the HAB-2E cells. With these cells, mutagenicity apparently is determined by the concentration of BrdUrd to which the cells are exposed rather than the amount of BrUra in DNA. These results were obtained with both the induction of ouabain resistance and thioguanine resistance as markers for mutagenesis. The dependence of BrdUrd mutagenicity on BrdUrd concentration was also observed for the parental melanoma cells.

Biological and biochemical effects of bromodeoxyuridine and deoxycytidine on Syrian hamster melanoma cells.

The addition of deoxycytidine (dCyd) to the growth medium of cultured Syrian hamster melanoma cells causes a reversal of the toxic effects of 5-bromodeoxyuridine (BrdU) and a decrease in the extent of incorporation of BrdU into nuclear DNA. These effects of dCyd can be accounted for, in part, by the intracellular conversion of the exogenously supplied dCyd to thymidine (dThd) nucleotides which can compete with BrdU nucleotides for incorporation into DNA. To some extent, the conversion of dCyd to dThd nucleotides can be inhibited by increasing the concentration of BrdU in the growth medium. The conversion of dCyd to dThd nucleotides is inhibited completely by aminopterin (Apt), and Apt also prevents dCyd from reversing BrdU toxicity and from decreasing the level of BrdU incorporation into nuclear DNA. In a clone of Syrian hamster melanoma cells, increasing the concentration of dCyd in the growth medium from 1 micron to 1000 micron resulted in a progressive increase in the percentage of dThd residues in nuclear DNA being derived from the exogenous dCyd, until more than 90% of the dThd residues came from the exogenous dCyd. However, despite the increasing amount of dThd derived from exogenous dCyd, there was a plateau in the decrease in BrdU incorporation into nuclear DNA at concentrations of dCyd above 8 micron.

Dissociation of plasminogen activator from the transformed phenotype in a 5-bromodeoxyuridine dependent mutant of Syrian hamster melanoma cells.

The relationship between plasminogen activator levels and the expression of the transformed phenotype was studied in a 5-bromodeoxyuridine (BrdUrd) dependent mutant of Syrian hamster melanoma cells. In terms of cell morphology and cellular interactions, the BrdUrd dependent cells resemble transformed cells when grown in the presence of BrdUrd but resemble untransformed cells when grown in the absence of BrdUrd. It was found that the BrdUrd dependent cells release significant levels of plasminogen activator only when cultured in the absence of BrdUrd. In the presence of BrdUrd, the release of plasminogen activator by the dependent cells is suppressed, and the decreased level of plasminogen activator released in the presence of BrdUrd seems to be due to the decreased production of active enzyme. Growth tests revealed that the BrdUrd dependent cells, when attached to a substrate, required BrdUrd in order to attain high densities. Furthermore, the cells are able to grow well in soft agar only in the presence of BrdUrd. These results suggest that the production and release of high levels of plasminogen activator are not related (either as cause or effect) to the expression of the transformed phenotype in the BrdUrd dependent cells. The effect of dog serum (as a plasminogen source) on the BrdUrd dependent cells also was tested. It was found that cells cultured in medium containing dog serum exhibit a morphological alteration, but only in the absence of brdUrd. The morphological response of the cells to dog serum resembles that previously observed with virus-transformed cells. In the BrdUrd dependent cells, the morphological response to dog serum appears correlated with the release of plasminogen activator but separated from other transformed characteristics.

Deoxycytidine reverses the suppression of pigmentation caused by 5-BrdUrd without changing the amount of 5-BrdUrd in DNA

5-Bromodeoxyuridine (BrdUrd) suppresses pigmentation in Syrian hamster melanoma cells in vitro, and this suppression is reversed by the addition of deoxycytidine (dCyd) to the culture medium. Exogenous dCyd also decreases the incorporation of BrdUrd into nuclear DNA. Over a certain range of dCyd concentrations, however, increasing the concentration of dCyd does not lead to any significant further decrease in the level of BrdUrd incorporation. With 0.5 μM BrdUrd in the medium, increasing the dCyd concentration from 0.4 mM to 1.0 mM produces essentially no detectable change in the amount of BrdUrd incorporated. This is the range of dCyd concentrations, however, in which the addition of dCyd has the greatest effect in reversing the suppression of pigmentation caused by BrdUrd. The effects of dCyd on pigmentation are prevented by aminopterin, which blocks the conversion of dCyd to thymidine nucleotides. The results suggest that dCyd reverses the suppression of pigmentation caused by BrdUrd through a mechanism that does not involve changes in the amount of BrdUrd in nuclear DNA, and that the effect of dCyd on pigmentation depends upon the conversion of the exogenously supplied dCyd to thymidine nucleotides.

Effects of thymidine analogs on Syrian hamster melanoma cells: phenotypes arising from selection for analog resistance.

In order to compare the biological effects of different thymidine (dT) analogs, two unusual cell lines (B-4 and HAB) previously isolated from a Syrian hamster melanoma line by selection with 5-bromodeoxyuridine (BrdU) were analyzed for their response to other analogs. B-4 cells require high concentrations of BrdU for optimal growth, and it was seen that the requirement for BrdU could be satisfied partially by 5-chlorodeoxyuridine (CldU) but not by the other dT analogs tested. HAB cells are able to grow with all the dT residues in nuclear DNA replaced by BrdU, and it was found that they could also grow with essentially all the dT residues in nuclear DNA replaced by CldU but not by other analogs. New cell lines resistant to 100 micrometer concentrations of CldU, 5-iododeoxyuridine (IdU), and 5-hydroxymethyldeoxyuridine (HMdU) were isolated from the melanoma line and tested for cross-resistance to the other dT analogs. A high level of cross-resistance was observed only with BrdU and CldU. The ability of the cell lines resistant to BrdU, CldU, and IdU to incorporate these analogs into nuclear DNA also was determined. BrdU and CldU were incorporated efficiently by all of the lines tested, but the IdU-resistant cells seemed to preferentially exclude IdU.

Inhibition of biological effects of bromodeoxyuridine by deoxycytidine: Correlation with decreased incorporation of bromodeoxyuridine into DNA

The effects of 5-bromodeoxyuridine (BrdU) on pigmentation, contact inhibition, cell morphology, and tumorogenicity of Syrian hamster melanoma cells are inhibited in the presence of deoxycytidine (dC). The inhibition of these biological effects of BrdU by dC is correlated with a decrease in the incorporation of BrdU into nuclear DNA. The results suggest that the intracellular changes resulting from the addition of dC to cells in the presence of BrdU are comparable to those resulting from a decrease in the concentration of BrdU in the medium.

Gene dosage dependence of pigment synthesis in melanoma x fibroblast hybrids (hamster cells-mouse fibroblast-DOPA-oxidase-irradiation).

Hybrids between Syrian hamster melanoma cells and mouse fibroblasts, containing one genome (1s) of each parent, produce neither melanin nor DOPA-oxidase ("extinction"). Attempts to induce loss of the fibroblast chromosomes by irradiation of the fibroblasts before fusion with melanoma cells resulted in the formation of colonies comprising pigmented hybrid cells, which contained 2s melanoma and 1s fibroblast chromosome-complements suggesting that extinction or re-expression of melanogenesis is a function of genic balance. This interpretation was confirmed by crosses between 2s melanoma cells with unirradiated 1s fibroblasts, which produced both pigmented and unpigmented hybrids. No correlation has thus far been established between karyotype and phenotype of the hybrid cells, but analysis of the karyological data suggests that the fibroblast chromosomes responsible for extinction cannot be numerous.