S91 Melanoma Research Articles

An improved imaging agent for malignant melanoma, based on [Nle4,D-Phe7]alpha-melanocyte stimulating hormone.

Two compounds have been synthesized based on [Nle4,D-Phe7]alpha-melanocyte stimulating hormone (NDP-MSH) in which either one or two peptide sequences were covalently linked through their N'-termini to a single molecule of diethylenetriamine pentaacetic acid (DTPA). These two compounds (monoNDP-MSH-DTPA and bisNDP-MSH-DTPA, respectively) bound indium-111 (111In) stably and showed hormonal activity as great or greater than alpha-melanocyte stimulating hormone (alpha-MSH). Both compounds were able to target 111In to Cloudman S91 melanomas in DBA2 mice. MonoNDP-MSH-DTPA gave the highest tumour:blood and tumour:tissue ratios and showed least unspecific radioactivity in the liver and kidney. Radioscintigraphy of mice showed good tumour localization of 111In with both compounds, clear images being obtainable within 2 h of injection. Scans with monoNDP-MSH-DTPA showed some kidney and thyroid but no liver radioactivity, whereas bisNDP-MSH-DTPA gave extensive abdominal radioactivity, most of which was associated with the liver and kidneys. MonoNDP-MSH-DTPA was cleared from the tumour much less rapidly and gave more favourable tumour:blood ratios than other alpha-MSH derivatives previously investigated. It is concluded that monoNDP-MSH-DTPA offers promise as a melanoma imaging agent in man.

Growth and pigmentation in genetically related Cloudman S91 melanoma cell lines treated with 3‐isobutyl‐1‐methyl‐xanthine and β‐melanocyte‐stimulating hormone

4 clonal sublines of Cloudman S91 melanoma cells, S91/mel, S91/I3, S91/6 and S91/amel, were evaluated for changes in growth, pigment content and plating efficiency during and after treatment with a cyclic-AMP phosphodiesterase inhibitor-melanin-stimulating agent, 3-isobutyl-1-methyl-xanthine (IBMX) plus beta-melanocyte stimulating hormone (beta-MSH) or IBMX alone. After combined treatment, increases in melanin content on day 3 were 48, 27, 11, and 2 pg/cell in the four cell lines respectively. In each case IBMX alone was less effective than IBMX plus beta-MSH. Doubling time increased and plating efficiency decreased with increased melanization. The increases in doubling time and decreases in plating efficiency were cell line dependent. The greatest rate of increase in doubling time and decrease in plating efficiency as a function of melanin content were seen in S91/amel, which produced the least pigment. The lowest rates of increase/decrease were seen in S91/mel, which produced the most pigment. Melanin pigment induced in the cells was classified as eumelanin by EPR determination. The differential response to induction of pigmentation makes these cell lines suitable models for comparative studies on the role of melanin in pigment cell biology.

Evidence that Retinoid X Receptors Mediate Retinoid-dependent Transcriptional Activation of the Retinoic Acid Receptor β Gene in S91 Melanoma Cells

S91 melanoma cells are growth arrested and differentiate when treated with retinoids. These processes correlate with expression of the retinoic acid receptor (RAR) beta gene, which is induced through a retinoic acid response element (beta RARE). We wished to determine which endogenous retinoid receptors (RARs and retinoid X receptors, RXRs) mediate induction of the RAR beta gene. We show that RXR alpha and RXR beta are constitutively expressed. Electrophoretic mobility shift assays with nuclear extracts show specific binding to the beta RARE (Complex I) in untreated cells, which can be supershifted by antibodies against RXRs but not by anti-RAR antibodies. After 48 h of treatment with retinoic acid, Complex I is replaced by a faster migrating Complex II, which can be supershifted by anti-RAR beta and anti-RXR alpha antibodies. This suggests that induction of the RAR beta gene is largely mediated by RXRs only. Accordingly, we also find that 9-cis RA, which activates both RAR and RXR, is a more potent inducer of the RAR beta gene than RA, which only activates RAR. After 48 h, all RXRs appear to be titrated by the newly synthesized RAR beta into an RAR beta.RXR heterodimer complex. Thus, it appears that the beta RARE is sequentially occupied by RXR dimers and RAR-RXR heterodimers.

Cancer vaccines: the interleukin 2 dosage effect.

Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.

Thymidine kinase in malignant melanoma

Thymidine kinase (EC 2.7.1.21) is an enzyme supporting DNA synthesis under conditions of increased cell proliferation. Although it has proved to be a useful marker for various malignant diseases, it has not been tested in malignant melanoma. Thymidine kinase activity was measured by means of a radioenzymic assay in two classical animal models of melanoma disease--B16 and Cloudman S91 melanoma-bearing mice. Tumour cell proliferation was assessed histochemically by measuring the expression of proliferating cell nuclear antigen (PCNA). Tumour cytosolic specific thymidine kinase activity was found to be higher in less pigmented Cloudman S91 melanoma than in differentiated, ie pigmented B16 melanoma, relative to the proliferative activity of the two tumours. Serum thymidine kinase levels were increased in melanoma-bearing animals of both types compared with healthy mice; this also reflected the efficacy of the therapy: cyclophosphamide-treated B16 melanoma-bearing mice in which the tumour development was slowed down had significantly lower serum enzyme levels in comparison with the non-treated group and the same levels compared with control, healthy mice. Our results suggest that serum thymidine kinase levels might be used as a marker to follow the effect of melanoma therapy.

Ultraviolet radiation-induced melanogenesis in human melanocytes. Effects of modulating protein kinase C.

The mechanism by which ultraviolet radiation induces melanogenesis in epidermal melanocytes is unknown. Previous observations that in cultured human melanocytes 1-oleoyl-2-acetylglycerol augmented both basal and ultraviolet radiation-induced melanogenesis, suggested that the responses were mediated via protein kinase C. However, paradoxically the phorbol ester TPA was without effect. Therefore, the present study has examined the involvement of protein kinase C in melanogenesis. Analysis of the isozyme profile of human melanocytes revealed the presence of protein kinase C alpha, beta I, epsilon and zeta but not the isozyme eta. Following exposure to 500 nM TPA for 24 hours, isozymes alpha, beta I and epsilon were downregulated, but zeta was unaffected. Similar isozyme profiles were observed in S91 and SKMEL3 melanoma cells. The melanogenic responses to 1-oleoyl-2-acetylglycerol and ultraviolet radiation were unaffected by inhibition of protein kinase C with Ro31-8220, or ablation by downregulation with 500 nM TPA, in human melanocytes and melanoma cells. 1-Oleoyl-2-acetylglycerol had no effect on protein kinase C activity in human melanocytes, as measured by rapid phosphorylation of the 80 kDa protein myristoylated alanine-rich C kinase substrate (MARCKS). Ultraviolet radiation induced a small increase in MARCKS protein phosphorylation but this effect was inhibited by pretreatment for 24 hours with 500 nM TPA, which had no effect on ultraviolet-induced melanogenesis. Overall, these findings indicate that 1-oleoyl-2-acetylglycerol and ultraviolet radiation activate melanogenesis via protein kinase C-independent pathways.

High-Molecular-Weight Forms of Tyrosinase and the Tyrosinase-Related Proteins: Evidence for a Melanogenic Complex

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.

Treatment of Human Melanocytes and S91 Melanoma Cells with the DNA Repair Enzyme T4 Endonuclease V Enhances Melanogenesis After Ultraviolet Irradiation

Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum. The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes. The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown. Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model. We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes. T4N5 treatment also increases cell yields following UV irradiation. These data suggest that tanning can be stimulated through enhanced DNA repair.

Partial characterization of IR-α-MSH peptides found in melanoma tumors

Our previous work indicated that IR-α-MSH (immunoreactive α-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-α-MSH. Further identification of the material in tumor extracts has been carried out in this study, and the results presented here show that the immunoreactivity is associated with a major fraction of about 16 kDa and another of 5–9 kDa. Significant amounts of the immunoreactive material were also found in human melanoma cells but not in culture supernatants. The presence of this material may be related to the melanogenic status of the tumor cells. We have estimated the intracellular IR-α-MSH to be within a 0.4 to 2.3 n M range in melanoma tumor cells. We have investigated the melanogenic effect of the IR-α-MSH material and its relationship to α-MSH. Purified extracts both from metastases and cultured cells were found to promote frog skin darkening as well as tyrosinase activity in Cloudman S91 melanoma cells. The IR material could also displace labeled α-MSH from its binding sites in human melanoma cells. Our data clearly indicate that melanoma cells engage in an autocrine production of α-MSH-like bioactive peptides by melanoma cells, of larger mol.wt., which are able to bind to MSH receptors. These peptides may be involved in the regulation of melanogenesis and possibly in the growth and proliferation of melanoma cells by an autocrine/paracrine mechanism.

Synthesis and biological activities of fatty acid conjugates of a cyclic lactam .alpha.-melanotropin

Four fatty acid conjugates of a cyclic lactam-bridged alpha-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than alpha-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse S91 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged activity.

A Radiation Resistance Factor in Cultured Cloudman S91 Mouse Melanoma Cells

The gamma-ray survival of a radiation-sensitive amelanotic subclone of Cloudman S91 mouse melanoma, S91/amel, is increased by the presence in the tissue culture dishes of heavily irradiated cells from the same cell line (amel-HRCells) and from clonally related melanotic S91/I3 radioresistant cells (I3-HRCells). The D0 of the target S91/amel cells increases from 1.25 to 2.08 Gy in the presence of 60,000 heavily irradiated S91/amel cells per dish. The presence of I3-HRCells in dishes of target S91/I3 cells does not increase their radioresistance. Comparable numbers of I3-HRCells are more effective than amel-HRCells at increasing survival of target S91/amel cells irradiated with 3 Gy of gamma rays. Conditioned medium from the S91 melanoma cells also increases the radioresistance of S91/amel, but is not as effective as the HRCells. Unirradiated cells can condition the medium as effectively as irradiated cells. It is concluded that the radiosensitive mouse melanoma cell line is made significantly more resistant by a diffusible cellular factor(s) elaborated more proficiently by radiation-resistant cells.

Stimulation of the receptor for melanocyte-stimulating hormone by retinoic acid

Treatment of Cloudman S91 melanoma cells with retinoic acid (RA) inhibits MSH-induced tyrosinase activity and melanin formation [(1990) J. Invest. Dermatol. 94, 461-464]. We report here, however, that in spite of inhibiting MSH-induced pigmentation, RA treatment caused a marked increase in MSH binding capacity for both cell surface and internal MSH binding sites. The stimulation was dose- and time-dependent and reversible, with half-maximal effects seen at 2 μM RA. Stimulation of MSH binding was seen as early as 3 h after exposure of cells to RA. Cell surface and internal binding activity increased in concert. Scatchard analyses indicated that increased MSH binding resulted from a 3–4-fold increase in the number of sites with no significant difference in their affinity for MSH. It appears that in suppressing MSH-induced melanogenesis, RA elicited a compensatory up-regulation of the MSH receptor system.

Inhibition of induced melanogenesis in cloudman melanoma cells by four phenotypic modifiers

Retinoic acid, hexamethylene bisacetamide, sodium butyrate, and dimethylsulfoxide, four compounds which modulate phenotypic expression in a variety of neoplastic cell lines, all inhibited the induction of tyrosinase activity and melanogenesis by the combination of melanocyte-stimulating hormone and isobutylmethylxanthine in Cloudman S91 melanoma cells. Results were the same in assays of whole cells or in extracts made from them. Only retinoic acid, however, was effective at inhibiting the activation of dopachrome isomerase, another regulatory enzyme in melanogenesis. Despite inhibiting the effects of melanocyte-stimulating hormone (MSH) and isobutylmethylxanthine on tyrosinase activity, all of the agents tested increased the binding of MSH to intact cells. Ultrastructural analysis of treated cells following DOPA cytochemistry revealed that both retinoic acid and hexamethylene bisacetamide arrested melanosomal maturation at stage I–II. Retinoic acid resulted in a derangement of melanosomal structure. The specificity of these agents for preventing the induction of melanogenesis makes them powerful tools for the dissection of this complex cellular process.

A chelating derivative of α-melanocyte stimulating hormone as a potential imaging agent for malignant melanoma

A chelating derivative of alpha-melanocyte stimulating hormone (MSH) has been synthesised, in which two molecules of the hormone are cross-linked by diethylenetriamine pentaacetic acid (DTPA). This compound, bisMSH-DTPA, was equipotent with MSH in an in vitro tyrosinase assay with Cloudman S91 melanoma cells. When DBA/2 mice bearing the same tumour were injected with bisMSH-DTPA labelled with the gamma-emitting isotope indium-111 (111In), the radioactivity became rapidly associated with the melanoma tissue. By 24 h post-injection, radioactivity in tumour tissue was significantly higher (P less than 0.001) than in spleen, lung, brain, eye and skin. Uptake of radioactivity by the tumours was inhibited by a 200-fold molar excess of MSH, whereas uptake by liver, kidney, spleen, lung, brain, eye and skin was unaffected. We conclude that bisMSH-DTPA may offer an alternative to antibody targeting in the imaging of malignant melanoma.

The biological activity of retinoids in melanoma cells: Induction of expression of retinoic acid receptor-β by retinoic acid in S91 melanoma cells

The expression of mRNA for retinoic acid receptor beta (RAR-β was induced by all trans-retinoic acid in murine S91 melanoma cells. The induction of RAR-β was dose-dependent, rapid and insensitive to cycloheximide. Both 13-cis-retinoic acid and 3,4-didehydro-all trans-retinoic acid also induced expression of RAR-β but were only effective at concentrations 100-fold greater than all trans-retinoic acid. The expression of RAR-α and RAR-γ was unaffected by retinoic acid.

Modulation by Retinoids of mRNA Levels for Nuclear Retinoic Acid Receptors in Murine Melanoma Cells

Retinoids inhibit the growth and enhance the differentiation of murine S91-C2 melanoma cells. Specific alterations in gene expression are a plausible mechanism for these effects. Since nuclear retinoic acid receptors (RAR) are likely mediators of retinoid-induced changes in gene expression, we used Northern blotting to analyze the expression of RAR alpha, RAR beta, and RAR gamma in S91-C2 cells. mRNA for both RAR alpha and RAR gamma was detected in these cells, but no RAR beta mRNA could be found. Treatment with 10(-7) and 10(-6) M beta-all-trans-retinoic acid (RA) for 24 h caused a 1.5- to 2-fold increase in RAR alpha and RAR gamma mRNA, whereas lower concentrations of RA were ineffective. RAR beta mRNA, which was undetectable in untreated cells, was detected after 24 h of treatment with a RA concentration as low as 10(-9) M, and its level increased with up to 10(-6) M RA. At the latter dose, RAR beta mRNA induction occurred by 4 h and increased progressively, reaching a plateau after 24 h of treatment. RAR beta mRNA induction at 4 h was not inhibited by cycloheximide at a concentration that suppressed protein synthesis by more than 90%. Several retinoids and related synthetic compounds, including 13-cis RA, TTNPB, Ch55, Am80, and the trifluoromethyl nonyloxyphenyl analog of RA, also induced RAR beta mRNA, whereas a 24-h treatment with 10(-6) M retinol, TTNP (a decarboxylated analog of TTNPB), or the phenyl analog of RA failed to induce RAR beta mRNA. With the exception of retinol and the trifluoromethyl nonyloxyphenyl analog of RA, the ability of the retinoids to induce RAR beta mRNA and their growth inhibitory effect were correlated. However, S91-C154, a RA-resistant mutant subclone derived from S91-C2 cells, showed mRNA levels of RAR alpha and RAR gamma and induction of RAR beta by RA similar to those detected in the sensitive S91-C2 cells. Like the S91 melanoma cells, two other mouse melanoma cell lines, K-1735P and B16-F1, constitutively expressed RAR alpha and RAR gamma mRNAs. The level of RAR beta mRNA was increased by RA only in B16-F1 cells, although the growth of both was inhibited by RA. These results demonstrate that RA can, directly and rapidly, induce the expression of mRNA for a high affinity nuclear receptor in some murine melanoma cells and that this induction is not sufficient to inhibit growth.

Targeting of a chelating derivative of a short-chain analogue of α-melanocyte stimulating hormone to Cloudman S91 melanomas

Conference Article| October 01 1990 Targeting of a chelating derivative of a short-chain analogue of α-melanocyte stimulating hormone to Cloudman S91 melanomas DAVID R. BARD; DAVID R. BARD 1Strangeways Research Laboratory, Worts' Causeway, CB1 4RN, Cambridge U.K. Search for other works by this author on: This Site PubMed Google Scholar C. GRAHAM KNIGHT; C. GRAHAM KNIGHT 1Strangeways Research Laboratory, Worts' Causeway, CB1 4RN, Cambridge U.K. Search for other works by this author on: This Site PubMed Google Scholar D. PETER PAGE-THOMAS D. PETER PAGE-THOMAS 1Strangeways Research Laboratory, Worts' Causeway, CB1 4RN, Cambridge U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1990) 18 (5): 882–883. https://doi.org/10.1042/bst0180882 Article history Received: February 28 1990 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation DAVID R. BARD, C. GRAHAM KNIGHT, D. PETER PAGE-THOMAS; Targeting of a chelating derivative of a short-chain analogue of α-melanocyte stimulating hormone to Cloudman S91 melanomas. Biochem Soc Trans 1 October 1990; 18 (5): 882–883. doi: https://doi.org/10.1042/bst0180882 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: MSH, α-melanocyte stimulating hormone, DTPA, diethylenetriamine penta-acetic acid This content is only available as a PDF. © 1990 Biochemical Society1990 Article PDF first page preview Close Modal You do not currently have access to this content.

α-MSH causes a small rise in cAMP but has no effect on basal or ultraviolet-stimulated melanogenesis in human melanocytes

The effects of alpha-melanocyte stimulating hormone (alpha-MSH) were studied on levels of cyclic adenosine 3',5'-monophosphate (cAMP), melanin content and response to ultraviolet radiation (UVR) in cultured human melanocytes (HuMC). Foreskin HuMC were cultured in a hormone-supplemented system not dependent on the presence of phorbol esters. Following addition of alpha-MSH (10(-6) M) there was a rise in cAMP levels maximal between 5 and 15 min to 9.4 +/- 3.2 pM/10(5) cells, while control levels were 3.6 +/- 0.7 pM/10(5) cells. After 7 days' culture in the presence of alpha-MSH (10(-8) -10(-6) M) the melanin content increased by only 35%, whereas Forskolin (10(-5) M) induced a 9.5-fold rise in cAMP after 5 min and a 10.9-fold rise in melanin content after 7 days. When HuMC were irradiated daily for 6 days with UVR (Helarium fluorescent lamps emitting 20% UVB, 80% UVA) melanin content rose 2.7-fold (SE 0.3). This was unchanged or slightly reduced in the presence of alpha-MSH (10(-8)-10(-6) M). Parallel observations on Cloudman S91 melanoma cells showed that alpha-MSH caused only an 80% increase in melanin content after 4 days. The rise in melanin content induced by three daily UV-irradiations (2.4-fold, SE 0.5) was unchanged by alpha-MSH (10(-8)-10(-6) M). Although alpha-MSH induces a small rise in cAMP in HuMC this does not result in melanogenesis, and the response to UVR is not affected by alpha-MSH in either HuMC or S91 cells.

Mast cell tumor destruction by deionized water

SUMMARY In a controlled study, malignant murine P815 mastocytoma cells exposed in vitro to distilled and deionized water died as a result of progressive swelling, degranulation, and membrane rupture. A 90% mean cell death occurred when cells obtained directly from culture were exposed to deionized water for 2 minutes. Of 6 cryopreserved malignant murine cell lines, which included Cloudman S91 melanoma, CMT-93 rectum carcinoma, MMT-06052 mammary carcinoma, and S-180 Sarcoma, only P815 mastocytoma and YAC-1 lymphoma were significantly (P < 0.05) affected by hypotonic shock; Cloudman S91 melanoma cells were the most resistant. Mastocytoma cells were selectively killed by hypotonic solution, and lymphoma cells were also killed by isotonic saline solution. Local mast cell tumor (mct) recurrence and percentage survival were evaluated in 12 cats (21 mct) and 54 dogs (85 mct) subjected to surgery alone or local infiltration of deionized water as an adjunct to surgery. Of all 16 incompletely excised mct in cats, there was no local recurrence following injection. Four mast cell tumors (2 cats) regressed after being injected in situ. In dogs with clinical stage-I mct, local recurrence was detected in 50% (5/10), but with injection after incomplete excision, local mct recurrence was significantly (P < 0.05) less (6.6%, 1/15). Percentage recurrence was significantly (P < 0.05) less and survival significantly greater when incompletely excised grade-II mct were injected. Mean follow-up period after surgery in cats and dogs was 35 and 23.4 months, respectively.

Synthesis and Actions of a Melanotropin Conjugate, Ac-[Nle4, Glu(gamma-4′-hydroxyanilide)5, D-Phe7]α-MSH4–10-NH2, on Melanocytes and Melanoma Cells In Vitro

L-Glutamic acid (γ-4′-hydroxyanilide) (GHB) is oxidized by tyrosinase to a quinone which inhibits DNA polymerase, RNA polymerase, and mitochondrial energy production within mushrooms. It was previously shown that GHB can kill B16 melanoma cells in culture, but lacks cytotoxicity for nontyrosinase-containing cells. We have conjugated this drug to a superpotent melanotropic peptide and examined the bioactivity of this conjugate to melanoma cells. 4′-Hydroxyaniline was attached to glutamic acid at position 5 in the superpotent melanotropin fragment analogue, Ac-[Nle4, d-Phe7]α-MSH4–10-NH2. The melanotropin:anilide conjugate, Ac-(Nle4, Glu(γ-4′-hydroxyanilide)5, d-Phe7]α-MSH4–10-NH2, was not cytotoxic to B16 or Cloudman S91 mouse melanoma cells in culture, as determined by cell counts and protein assays. Interestingly, we also found that GHB stimulated melanoma cell tyrosinase above control levels in both melanoma cell lines. In our study, GHB itself also was found not to be cytotoxic to B16 or S91 melanoma cells in culture. In the frog skin bioassay, the melanotropin conjugate was more potent than α-MSH or Ac-[Nle4, d-Phe7]α-MSH4–10 in stimulating melanosome dispersion. These results demonstrate that putative chemotherapeutic ligands can be incorporated into active-site fragment analogues of MSH without loss of biological activity.