Radiosensitivity of murine and human melanoma cells: a comparative study with different models.

The in vitro radiosensitivity of one murine melanoma cell line (Cloudman S91 CCL 53.1) and three human melanoma cell strains (C8146C, C8161, and R83-4) were studied. Cells were irradiated by single dose X-rays and plated either in agar or on plastic. The survival curves were fitted by the single-hit multitarget, two-hit multitarget, and quadratic models. Multiple comparisons of the residual sum of squares suggested that the two-hit model was clearly inferior to the single-hit and quadratic models. No statistically significant difference was suggested for either the single-hit or quadratic models. Furthermore, on examination of the differences in correlations between the observed and predicted values, the residual plots (observed minus predicted over dose) failed to suggest a clear advantage of either the single-hit multitarget or the quadratic models. Either model could be recommended for analysis of in vitro radiation data.

Effect of single dose ionizing radiation on the cellular proliferation and apparent radiosensitivity of melanoma.

An important variable in determining in vitro cell survival is the time interval between treatment and assay. One manifestation of radiation damage is the introduction of a division delay in which the post-radiation division rate is less than the pre-radiation division rate. We have examined the influence of length of incubation on radiosensitivity of murine and human melanoma cells in a soft-agar assay. In general, the longer the incubation period the larger was the median colony diameter within control or experimental groups. Hence apparent radiosensitivity decreased with increased length of incubation.

Human recombinant alpha- and gamma-interferons enhance the cytotoxic properties of tumor necrosis factor on human melanoma.

Three short-term human melanoma cell lines were tested for sensitivity to human recombinant alpha-tumor necrosis factor (TNF) in a semisolid agar colony formation assay. Cells from three pigmented and one amelanotic strain displayed low sensitivity to TNF. The ID50 for the inhibition of melanoma colony formation ranged from 2,500 to 20,000 U/ml. We then tested the ability of human recombinant alpha-interferon (IFN-alpha) and gamma-interferon (IFN-gamma) to interact with TNF to inhibit melanoma colony formation. Analysis of the TNF-IFN mixtures using the median effect method demonstrated that both IFNs interacted synergistically with TNF to inhibit melanoma colony formation. On a unit basis, IFN-gamma was more active with TNF than IFN-alpha. The addition of the second interferon to the mixture enhanced the ability of TNF to promote the cytolysis of human melanoma cells. The enhanced killing effect seen with the combination of IFN-alpha, IFN-gamma, and TNF suggests an interesting strategy for the treatment of human melanoma.

Radiation survival of murine and human melanoma cells utilizing two assay systems: monolayer and soft agar.

The radiation response of murine and human melanoma cells assayed in bilayer soft agar and monolayer was examined. Cells from the murine melanoma Cloudman S91 CCL 53.1 cell line and three human melanoma cell strains (C8146C, C8161, and R83-4) developed in our laboratory were irradiated by single dose X-rays and plated either in agar or on plastic. D0 values were the same within 95% confidence intervals for cells from the human melanoma cell strains C8146C, C8161, and R83-4 but were dissimilar for the murine cell line CCL 53.1 Dq values were different for all cells studied. The shape of the survival curve for all four melanomas was not identical for cells assayed in soft agar versus cells grown on plastic. This would indicate that apparent radiosensitivity was influenced by the method of assay although there were no apparent consistent differences between the curves generated by monolayer or bilayer soft agar assays.

Effect of tumor colony definition on ionizing radiation survival curves of melanoma-colony forming cells.

Definition of survival and measurement of colony size in soft agar assays is important in establishing in vitro radiation survival curves. Conventionally, survival is assessed according to colony-forming ability. The distinction between small colonies that are abortive and those that are viable often involves a difficult and arbitrary choice for the investigator. We have examined the effect of different minimum colony sizes (greater than or equal to 25, greater than or equal to 50, greater than or equal to 75, and greater than or equal to 100 cells) on ionizing radiation survival curves for cells from established murine (CCL 53.1) and human (M1RW5) melanoma cell lines as well as from short-term human melanoma cell strains (C8146A, C8146C, C8161, C83-2C, C82-7A1, and C8442) and patient biopsy (83-4). Single cell suspensions were plated in the upper layer of the agar bilayer and cells were irradiated by single dose X rays. Giant cells did not form in colonies containing 50 or more cells. D0 values were highest (D0 values, from 390 to 100 cGy) for cells forming smaller colonies (greater than or equal to 25 cells, greater than or equal to 4-5 doublings) and lowest (D0 values, from 190 to 50 cGy) for cells forming larger colonies (greater than or equal to 100 cells, greater than or equal to 6-7 doublings). Therefore, apparent radiosensitivity was dependent on colony size selected for analysis. Precise measurement of colony size was important in establishing radiation survival curves because errors in determining the colony size will alter apparent radiosensitivity of cells. These results should help define the biological meaning of tumor colony growth in semisolid medium, and alter the interpretation of survival curves which measure sensitivity to agents using this assay.

Tetrazolium staining by optical scanning overestimates colony size and number of colonies counted.

We measured the effect that staining with 2-(P-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) had on the number and size distribution of tumor colonies counted using an optical image analyzer (FAS II). Staining increased the number of tumor colonies counted. By using opaque tumor cells or pigmented melanoma cells and measuring colony growth kinetics, we demonstrated that the use of INT staining to assist in counting tumor colonies artificially increased the size of viable tumor cell aggregates by adding a red precipitate to the outside surface of the cells. Laboratories that are using the INT method for drug screening are probably measuring colonies down to and below 42 microns in diameter. These small colonies could result from as few as one or two divisions. Thus, potentially useful drugs may be missed in the screen because of the presence of abortive colonies: i.e., lethally damaged cells completing only one or two divisions.

Median effect and long term recovery analysis of biological modifier interactions with difluoromethylornithine on the proliferation of human melanoma cells.

The ability of difluoromethylornithine (DFMO), a specific polyamine synthesis inhibitor, to interact with various biological modifiers to inhibit the colony-forming growth of human melanoma cells was determined by using the median effect principle to computer model the strength of two agent interactions. Either alpha- or gamma-IFN (interferon) in combination with DFMO resulted in a synergistic inhibition on human melanoma colony formation. For human melanoma cells which were not resistant to 13-cis RA (retinoic acid), an additive suppression on colony formation was obtained with the retinoid-DFMO combination. Dexamethasone (DEX) interacted with DFMO to yield a synergistic reduction in melanoma colony number on glucocorticoid sensitive cells and no growth enhancement with DFMO on glucocorticoid resistant melanoma lines. Human melanoma cells displayed differential long-term growth sensitivity to DFMO treatment. C8146C human melanoma cells were terminally growth-inhibited by a 96 h exposure to DFMO, in a manner which was concentration and time dependent. The proliferation of C82-7A melanoma cells was inhibited by 95% in presence of DFMO, but upon removal of DFMO the cells regained their ability to proliferate and form colonies. The simultaneous addition of DEX plus alpha-IFN plus 13-cis-RA with DFMO caused most of the human melanoma cells in these lines to become permanently growth arrested. Pre-treatment with DEX plus alpha-IFN plus 13-cis RA, but without DFMO, did not have any long term effect on the ability of melanoma cells to recover and proliferate in soft agar.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation-related growth factors and their receptors.

Cellular transformation may be accomplished in vitro and in vivo through the concerted action of growth factors and oncogenes. This association has demonstrated that malignant growth results from aberrations in growth factor-signal transduction pathways that normally operate to control proliferation. Activation of genes that code for growth factors and/or their receptors provides tumor cells with potential mechanisms to maintain their proliferative state. Tumor cells have been shown to produce endogenous substances that augment their growth (autocrine stimulation), as well as responding to exogenous substances (paracrine stimulation). With solid tumor cells these responses have been shown to involve aberrant expression of growth factor and/or receptor genes. The study of the interrelationship of these various growth regulatory molecules is important not only in the identification of gene products essential to cellular proliferation, but also in providing clues as to what forces are driving tumor cell growth.

Biological characterization of an acid-sensitive growth factor from human platelets: role in the proliferation of human melanoma and bovine endothelial cells.

An acid-sensitive growth factor for human melanoma has been partially purified from human platelets. TSK gel filtration HPLC provides a molecular weight estimation of 60,000 daltons. This factor is not only mitogenic for human melanoma, but also stimulates 3H-thymidine uptake and increases the number of bovine aortic endothelial cells, while fibroblasts are nonresponsive. Radioiodination of active HPLC fraction has been accomplished. The human melanoma cell line, M1RW5 demonstrates specific, time-dependent binding of the labeled protein. These studies suggest that the growth of human melanoma may in part be regulated by a newly described growth factor present in human platelets.

Inhibition of human melanoma growth by prostaglandin A, D, and J analogues.

The relative inhibitory potency of prostaglandin A (PGA) and prostaglandin J2 (PGJ2) analogues compared to prostaglandin A1 (PGA1) was determined in a clonogenic assay system. Three human melanoma cell strains (C8146A, C8146C, and C8161), a human melanoma cell line (M1RW5) and a human neuroblastoma cell line (IMR-32) were used. Prostaglandin analogues were screened in the clonogenic assay system and the dose effect curves were analyzed by linear regression utilizing the median effect relationship. The computer-generated 50% and 95% inhibitory doses showed that 15-deoxy-16-hydroxyl-16-vinyl-prostaglandin A2 (DHV-PGA2) was from two- to three-fold more active than PGA1 in inhibiting the clonogenic growth of human melanoma cells. Based on the 50% inhibitory dose, PGJ2 and its analogues were from two to five times more potent than PGA1. The delta 12- and delta 12,14-PGJ2 were the most potent of the prostaglandins tested. However, the 95% inhibitory dose for prostaglandin D2 (PGD2), PGJ2 and its analogues against neuroblastoma did not show any enhancement in activity in comparison to PGA1, suggesting that some tumor specificity in the activity of these analogues may be signified by the neuroblastoma data. Prostaglandins which contained a fluoride substitution at position 11 were also tested for activity. As we previously observed with other analogues which did not contain an alpha, beta-unsaturated carbonyl group in the cyclopentane ring, 9 beta, 15 alpha-dihydroxy-11 beta-fluoroprosta-5-cis-13-trans-dienoic acid and 9 alpha, 15 alpha-dihydroxy-11 beta-fluoroprosta-5-cis-13-trans-dienoic acid did not inhibit the clonogenic growth of human melanoma cells. Administration s.c. to established human melanoma tumors growing in athymic nude mice caused a significant growth inhibition. The treatment schedules ranged from 1 to 8 days. Injection s.c. of PGA1 at a dose of 40 mg/kg/day resulted in a 20% suppression in tumor growth. Higher doses (100 and 200 mg/kg/day) effected an 80% reduction in tumor growth. The higher doses were associated with reversible toxicities, diarrhea and skin inflammation. Administration of DHV-PGA2 at a dose of 20 mg/kg/day resulted in 40% reduction in tumor growth. The increased in vivo potency of DHV-PGA2 corresponds to the results obtained in the clonogenic assay system.

Actions of melanotropins on mouse melanoma cell growth in vitro.

Melanotropins induce melanogenesis in mouse Cloudman S91 melanoma cells by stimulating the activity of tyrosinase. In monolayer culture, alpha-melanocyte-stimulating hormone and the superpotent analogue 4-norleucine, 7-D-phenylalanine-alpha-melanocyte, which had prolonged effects on tyrosinase activity, did not inhibit the proliferation of melanoma cells even at concentrations that elicited maximal tyrosinase stimulation. In soft agar the melanotropins stimulated the formation of melanized colonies and increased the cloning and proliferative potentials of melanoma cells. Both melanotropins increased the number of small (42-104 microns in diameter) colonies at initial plating densities ranging from 625 to 7,500 cells/dish. The number of larger (greater than 104 microns in diameter) colonies was also increased except at densities 5,000 cells or more/dish, wherein the proliferative capacity was inhibited; yet the cloning efficiency was still increased. Therefore, in bilayer soft agar cultures, melanotropins stimulate the growth of the clonogenic S91 melanoma cell population under conditions that allow for optimal expression of the cloning and proliferative potentials of these cells.

Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent.

Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to alpha-melanotropin or its potent analog [Nle4, D-Phe7]-alpha-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with alpha-MSH or [Nle4, D-Phe7]-alpha-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2 X 10(6) cells/flask, and exposed for 24 h to 10(-7) M alpha-MSH, only the cultures seeded at low densities (0.2 and 0.4 X 10(6) cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10(-7) M alpha-MSH or [Nle4, D-Phe7]-alpha-MSH for 24 h, followed by removal of the melanotropins from the culture medium. The magnitude and duration of the residual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.

Difluoromethylornithine enhances inhibition of melanoma cell growth in soft agar by dexamethasone, clone A interferon and retinoic acid.

Five human melanoma cell lines (C8146C, C8161, C82-7A, C83-2CY and MIRW5) were shown to contain a significant number of melanoma colony-forming units resistant to single-agent treatment by dexamethasone, alpha-interferon and trans-retinoic acid. These biological modifiers were combined with difluoromethylornithine into a low-dose combination using concentrations below pharmacologically achievable levels. The suppression of melanoma colony formation induced by this combination was consistent and significantly higher than that seen with any single agent, colony formation being reduced by an average of 90%. Leaving either DEX or DFMO out of the 4-agent combination resulted in a significant decrease in the observed inhibition. This was also verified by the addition of putrescine which inhibited only the DFMO activity. Median effect analysis of the DFMO + IFN inhibition of C8161 cells demonstrated that the 2 agents interacted synergistically over the entire dose-response curve. Of the high-dose combination-treated melanoma colony-forming units, 97% did not form small growth units; most remained as arrested single cells, but the cells and small growth units could still metabolize tetrazolium stain after the experiment, suggesting that the high-dose combination arrested the growth of the melanoma colony-forming units via a non-cytotoxic mechanism.

Stimulation of human metastatic melanoma colony-forming cells by an acid-sensitive factor in human platelet sonicate.

A human platelet sonicate was evaluated for its effects on the growth of human metastatic melanoma colony-forming cells in soft agar from cells in culture and from biopsies. The addition of platelet sonicate increased both cloning efficiency and proliferative capacity in that more and larger colonies were formed. In more detailed studies under growth-limiting conditions, melanoma cellular responses to known growth factors were compared to the activity found in the platelet sonicate. None of the growth factors tested either alone or in combination, including platelet-derived growth factor, epidermal growth factor, alpha-type transforming growth factor, and beta-type transforming growth factor, were capable of inducing melanoma colony formation to the 12-fold stimulation observed with the platelet sonicate. Treatment of platelet sonicate with dithiothreitol, trypsin, or acid resulted in loss of activity for human melanoma. Our results suggest that human platelets contain an acid-sensitive protein which can support the expression of the transformed phenotype of human melanoma, and this factor is distinct from acid-stable activities previously characterized from human platelets.

Anchorage-independent growth of murine melanoma in serum-less media is dependent on insulin or melanocyte-stimulating hormone.

alpha-Melanocyte-stimulating hormone (MSH) is known to stimulate melanogenesis in murine melanoma, particularly in Cloudman S-91 melanoma cells. The effects of MSH and insulin on the proliferation of S91 murine melanoma cells have aroused controversy; in various reports, both hormones have been reported to either stimulate or inhibit murine melanoma growth. In our studies both MSH and insulin stimulated the colony-forming ability and the proliferative capacity of S-91 murine melanoma cells grown in soft agar with either serum-supplemented or serum-less medium. Unless insulin and/or MSH were present, Cloudman S-91 melanoma cells failed to clone in soft agar. The insulin effect was greater than that of MSH, and was more pronounced in serum-less than in serum-supplemented medium. The concurrent treatment of S91 melanoma cells with both MSH and insulin resulted in a greater increase in the total number of colonies formed than caused by treatment with either hormone alone. The combined MSH-insulin stimulation of anchorage-independent growth was specific, since the effect could not be mimicked by epidermal growth factor (EGF), gonadotropin-releasing hormone (GRH), luteinizing hormone (LH), nerve growth factor (NGF) or platelet-derived growth factor (PDGF). Therefore, MSH and insulin may be specific growth factors for murine melanoma cells.

Dexamethasone and zinc in combination inhibit the anchorage-independent growth of S-91 Cloudman murine melanoma.

Zinc inhibited the colony formation of Cloudman S-91 murine melanoma cells in a dose dependent manner with an ID50 of 3.4 ug/ml. Total inhibition of the melanoma colony-forming units occurred at a zinc concentration of 4.42 ug/ml. In the presence of dexamethasone the ID50 for zinc inhibition was reduced by 49% and total inhibition of anchorage-independent growth occurred at the achievable in vivo zinc concentration of 3.0 ug/ml. Dexamethasone and zinc in combination effected a greater than additive inhibition of the murine melanoma colony-forming units. Statistical evaluation of these results showed that zinc and dexamethasone interacted synergistically to inhibit the formation of murine melanoma colonies.

Dexamethasone, prostaglandin A, and retinoic acid modulation of murine and human melanoma cells grown in soft agar.

The cloning efficiencies of a murine melanoma cell line (S91 CCL 53.1) and a human melanoma cell strain (C8146c) were inhibited by dexamethasone (DEX), prostaglandin A1 (PGA1), and beta-all-trans-retinoic acid (RA) in a dose-dependent manner. Murine melanoma tumor colony-forming units (MTCFU) were inhibited more than 99% by DEX (1 X 10(-7) M) and RA (1 X 10(-7) M) with a concentration needed to produce a 50% reduction in colony formation for both hormones of 5 X 10(-9) M. Combinations of DEX and RA effected a synergistic inhibition on colony formation, which was reflected by a 11/2 log reduction in the hormone concentration needed to produce a greater than 99% inhibition of colony formation. When PGA1 was added to DEX and RA, a greater than additive reduction in colony formation was observed. Human MTCFU from cell strain C8146c were inhibited more than 85% at an RA concentration of 1 X 10(-7) M, but they were reduced only to 40% of control at a DEX concentration of 1 X 10(-6) M. DEX-RA produced an additive inhibition of colony formation. Addition of submaximal amounts of PGA1 to DEX-RA combinations or to either hormone alone resulted in synergistic reduction of human MTCFU. These results demonstrated that the proliferative potential of human and murine melanomas can be simultaneously regulated by DEX, PGA1, and RA.

In vitro modulation of human and murine melanoma growth by prostanoid analogues.

The inhibitory effect of various prostaglandin analogues on the anchorage independent growth of murine and human melanoma cells was measured. PGA analogues (which were modified at C-16 and C-18) did not demonstrate any major improvement in activity over PGA alone. These included 16,16-dimethyl PGA1, 16,16-dimethyl-PGA2, 16,16-dimethyl-18-oxa-PGA2 and trans-delta-2-15-alpha acetoxy-16,16-dimethyl-18-oxa-11-deoxy-PGE1-methylester. The thromboxane synthetase inhibitor, U51605, demonstrated weak anti-proliferative activity. PGD2 (with a ketone at C-11 versus C-9 for PGA and PGE) was the most potent prostaglandin tested. Cells from melanoma lines displayed species differences in their sensitivities. PGA1 and PGE1 were the most potent inhibitors of the anchorage independent growth of murine melanoma cells. On human melanoma cells PGD2 was the most active prostaglandin, 2-3 times more potent than PGA1; PGE1 was a very weak inhibitor.

Inhibition of human malignant melanoma colony-forming cells in vitro by prostaglandin A1.

The direct effect of continuous exposure to prostaglandins on the cloning efficiency and proliferative capacity of human malignant melanoma colony-forming cells in soft agar was evaluated. Prostaglandin A1 (PGA1) and prostaglandin E1 (PGE1) effected a dose-dependent inhibition of colony formation and proliferative capacity. PGA1 at a concentration of 5 microgram/ml reduced colony formation of cells from human melanoma cell strains C8054, C8130, and C822 by at least 85%. PGA1 also inhibited colony formation of cells obtained directly from biopsies of melanoma tissues from eight patients by greater than 70% at a concentration of 5 microgram/ml. A steep dose-response curve was evident by the little effect of PGA1 on colony formation at a concentration of 0.5 microgram/ml. The mean 50% inhibitory doses for PGA1 and PGE1 were 1.25 and 4.25 microgram/ml, respectively. Prostaglandin A2 was much less effective than PGA1 in inhibiting melanoma colony formation. The related prostaglandins (prostaglandin B1, prostaglandin F1 alpha, and prostaglandin E2 alpha) had little or no effect on colony formation. Overall, these results suggested that the presence of a carbonyl group at position 9 of the cyclopentane ring may be required for inhibitory activity as prostaglandins of the A and E series inhibited human melanoma cell growth. PGA1 and PGE1 did not effect a rise in cyclic adenosine 3':5'-monophosphate levels in C8054 and C8130 cells. However, while alpha-melanocyte-stimulating hormone and prostaglandin F2 alpha did generate a rise in adenosine 3':5'-monophosphate levels in C8054 cells, these hormones had no effect on colony formation. These results are consistent with the notion that the PGA1 and PGE1 inhibition of melanoma colony-forming cells occurs via a noncyclic nucleotide mechanism.