Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line.

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol-wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin-resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.

Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin.

The biosynthesis of melanin is initiated by the catalytic oxidation of tyrosine to dopa by tyrosinase in a reaction that requires dopa as a cofactor. Tyrosine then catalyzes the dehydrogenation of dopa to dopaquinone. The subsequent reactions can proceed spontaneously in vitro. Tyrosinase, purified from murine melanomas and the skins of brown mice, has now been shown to catalyze a third reaction in mammalian melanogenesis, namely the conversion of 5,6-dihydroxyindile to melanochrome. This reaction requires dopa as a cofactor and is inhibited by tyrosine. Conversely, 5,6-dihydroxyindole inhibits the oxidation of tyrosine to dopa, so that the relative concentrations of tyrosine and 5,6-dihydroxyindole within the mammalian pigment cell are capable of regulating melanogenesis in a previously unrecognized fashion. Tyrosinase has the unusual property of catalyzing three distinct reactions within a single biochemical pathway: the hydroxylation of a monophenol, the dehydrogenation of a catechol, and the dehydrogenation of a dihydroxyindole. The first and third of these reactions require dopa as a cofactor; in the second reaction, dopa is a substrate.

Outcomes of growing rod surgery for severe compared with moderate early-onset scoliosis: a matched comparative study.

Aims: The aim of this study was to compare the outcomes of surgery using growing rods in patients with severe versus moderate early-onset scoliosis (EOS). Patients and Methods: A review of a multicentre EOS database identified 107 children with severe EOS (major curve ≥ 90°) treated with growing rods before the age of ten years with a minimum follow-up of two years and three or more lengthening procedures. From the same database, 107 matched controls with moderate EOS were identified. Results: The mean preoperative major curve was 101° (90 to 139) in the severe group and 67° (33° to 88°) in the moderate group (p < 0.001), which was corrected at final follow-up to 57° (10° to 96°) in the severe group and 40° (3° to 85°) in the moderate group (p < 0.001). T1-S1 height increased by a mean of 54 mm (-8 to 131) in the severe group and 27 mm (-4 to 131) in the moderate group at the initial surgery (p < 0.001), and by 50 mm (-17 to 200) and 54 mm (-11 to 212), respectively, during distraction (p = 0.84). The mean number of complications per patient was 2.6 (0 to 14) in the severe group and 1.9 (0 to 10) in the moderate group (p = 0.040). Five patients (4.7%) in the severe group and three (2.8%) in the moderate group developed a neurological deficit postoperatively (p = 0.47). Conclusion: Severe EOS can be treated effectively using growing rods, but the risk of complications is high. Cite this article: Bone Joint J 2018;100-B:772-9.

Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo.

Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, although its use could be limited by the potential induction of tumor necrosis factor alpha (TNFalpha)-mediated septic shock stimulated by lipid A. Genetic modifications of tumor-targeting Salmonella that alter lipid A and increase safety must, however, retain the useful properties of this bacteria. We report here that disruption of the Salmonella msbB gene reduces TNFalpha induction and increases the LD50 of this pathogenic bacteria by 10,000-fold. Notwithstanding this enormous difference, Salmonella retains its tumor-targeting properties, exhibiting tumor accumulation ratios in excess of 1000:1 compared with normal tissues. Administration of this bacteria to mice bearing melanoma results in tumors that are less than 6% the size of tumors in untreated controls at day 18. Thus, the antitumor activity previously demonstrated using tumor-targeting Salmonella with normal lipid A is retained. Lipid modification of tumor-specific bacterial vectors provides a means for reducing septic shock and further suggests that the antitumor activity of these bacteria may be independent of TNFalpha.

Increase in melanin formation and promotion of cytotoxicity in cultured melanoma cells caused by phosphorylated isomers of L-dopa.

A new class of compounds, termed "dopa phosphates," is described. The compounds contain phosphate ester linkages at positions 3 and/or 4 of the phenylalanine ring. Dopa phosphates are highly soluble compounds which are stable over a wide range of pH values and are not hydrolyzed by boiling in concentrated acid. Synthetic yields of greater than 90% can be obtained using dopa as starting material. Exposure to alkaline phosphatase results in hydrolysis of the phosphate moieties and production of dopa. Dopa phosphates do not inhibit dopa oxidase (tyrosinase, EC 1.14.18.1) activity. Dopa oxidase does not catalyze the conversion of dopa phosphates into melanin unless the dopa phosphates are first treated with alkaline phosphatase. Dopa phosphates, when compared to L-dopa, are stable in the presence of O2 and are not oxidized by serum proteins. In the presence of cultured melanoma cells, dopa phosphates are readily converted into melanin, indicating that the cells are able to produce dopa from dopa phosphates. At high concentrations, dopa phosphates are cytotoxic toward melanoma cells in culture. The cytotoxicity is enhanced at least 3-fold by pretreatment of cells with melanotropin and is prevented by phenylthiourea, an inhibitor of dopa oxidase activity. These results, combined with studies on the uptake of radioactive forms of dopa phosphates (32P and 14C), indicate that phosphorylated isomers of dopa are efficiently taken up by Cloudman melanoma cells and are readily converted by the cells into a melanin precursor, presumably L-dopa.

Tumor-targeted Salmonella as a novel anticancer vector.

There has been little investigation of bacteria as gene delivery vectors. Here, we demonstrate that genetically engineered Salmonella have many of the desirable properties of a delivery vector, including targeting of multiple tumors from a distant inoculation site, selective replication within tumors, tumor retardation, and the ability to express effector genes, such as the herpes simplex virus thymidine kinase (HSV TK). When wild-type Salmonella were introduced into melanoma-bearing mice, the bacteria were found within the tumor at levels exceeding 10(9) per g, although as pathogens, they caused the death of the mice. However, when attenuated, hyperinvasive auxotrophic mutants were used, the tumor-targeting and amplification phenomena were retained, whereas their pathogenicity was limited. With such attenuated strains, the tumor:liver ratios ranged between 250:1 and 9000:1. When these auxotrophs were inoculated i.p. into C57B6 mice bearing B16F10 melanomas, they suppressed tumor growth and prolonged average survival to as much as twice that of untreated mice. A plasmid containing the HSV TK gene with a beta-lactamase secretion signal was constructed that, when expressed, resulted in translocation to the periplasm and phosphorylation of the prodrug ganciclovir. Melanoma-bearing animals inoculated with HSV TK-expressing Salmonella showed ganciclovir-mediated, dose-dependent suppression of tumor growth and prolonged survival in addition to that seen with bacteria alone. The results demonstrate that attenuated Salmonella would be useful both for inherent antitumor activity and delivery of therapeutic proteins to cancer cells in vivo.

A spontaneous murine melanoma lung metastasis comprised of host x tumor hybrids.

Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail of a BALB/c nu/nu mouse, were comprised chiefly of host x tumor hybrids. These lung metastasis cells showed: (a) 30-40% increased DNA content; (b) resistance to 10(-4) M hypoxanthine, 4 x 10(-7) M aminopterin, and 1.6 x 10(-5) M thymidine (HAT) + G418; and (c) the presence in genomic DNA of genes for both wt and albino tyrosinase, reflecting the DBA/2J (Cloudman S91) and BALB/c mouse genotypes, respectively. Individual clones of lung metastasis cells expressed enhanced pigmentation, motility, and responsiveness to MSH/IBMX, a behavior similar to that recently reported for artificially generated melanoma x macrophage fusion hybrids. These similarities suggested that the host fusion partner generating the lung metastasis hybrids might have been a macrophage, although formal proof for this was not possible. The results provide the first direct evidence that host x tumor hybridization could serve as an initiating mechanism for melanoma metastasis.

Production and release of proopiomelanocortin (POMC) derived peptides by human melanocytes and keratinocytes in culture: regulation by ultraviolet B.

It is demonstrated that ultraviolet B (UVB) radiation stimulates increased expression of the proopiomelanocortin (POMC) gene which is accompanied by production and release of alpha-melanocyte stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH) by both normal and malignant human melanocytes and keratinocytes. The production and release of both peptides are also stimulated by dibutyryl cyclic adenosine monophosphate (dbcAMP) and interleukin 1 alpha (IL-1 alpha) but not by endothelin-1 (ET-1) or tumor necrosis factor-alpha (TNF-alpha). N-acetyl-cysteine (NAC), a precursor of glutathione (GSH), an intracellular free radical scavenger, abolishes the UVB-stimulated POMC peptide production and secretion. Conclusions are as follows: (1) Cultured human cells of cutaneous origin, namely keratinocytes and melanocytes, can produce and express POMC; (2) POMC expression is enhanced by exposure to UVB, possibly through a cyclic AMP-dependent pathway; and (3) The action of UVB on POMC production may involve a cellular response to oxidative stress.

Factors regulating growth and pigmentation of melanoma cells.

Growth and melanization are intimately related in melanoma cells. MSH, by promoting elevated cyclic AMP levels, causes increases in melanization, cessation of growth, and gross morphologic changes in Cloudman S-91 melanoma cells. Growth inhibition results from high levels of cyclic AMP while growth stimulation occurs with lower levels. During melanization, oxidation products of tyrosine are generated which are toxic to the cells. Genetic studies have revealed that some of these processes are related through common biochemical pathways. This article reviews work of recent years on such regulatory mechanisms in melanoma.

UVB-induced melanogenesis may be mediated through the MSH-receptor system.

Ultraviolet B radiation (UVB) elicits an increase in melanin production in mammalian skin. The mechanisms regulating this process are not understood, although it is well documented that there is an increase in the number of melanin-producing melanocytes. The melanotropins (MSH) are a family of peptides that increase the melanin content of melanocytes through an interaction with high affinity receptors. We have obtained evidence that the effects of UVB on melanogenesis may be mediated through an increase in MSH receptor activity on melanocytes. First, exposure of Cloudman S91 mouse melanoma cells to UVB resulted in increased binding of 125I-MSH to cells within 24 h. In five separate experiments, UVB-irradiated cultures displayed 2-10-fold increases in MSH binding capacity over that of unirradiated control cultures (optimum doses 10-20 mJ/cm2). Second, UVB and MSH potentiated one another in promoting cutaneous melanogenesis in both mice and guinea pigs. In the areas of guinea pig skin that received both UVB and MSH, there was a fivefold increase in active melanocytes/mm2 over the sum of active melanocytes/mm2 in areas receiving either MSH or UVB separately. Our results suggest that UVB light causes an increase in MSH receptor activity on cutaneous melanocytes, thus increasing cellular responsiveness to MSH. Implicit in this mechanism is a transduction of radiant energy into chemical energy during the process of UVB-induced melanogenesis.

Retinoic acid is a potent inhibitor of inducible pigmentation in murine and hamster melanoma cell lines.

Melanocyte-stimulating hormone (MSH) induces melanogenesis in Cloudman mouse melanoma cells. The activities of two enzymes in the melanogenesis pathway, tyrosinase and dopachrome conversion factor, are increased as part of the induction process. Trans retinoic acid (RA), at concentrations as low as 0.1 nM, inhibited the induction of tyrosinase, dopachrome conversion factor, and melanogenesis, but had no effect on the basal levels of either enzyme or of cellular melanin content. Half-maximal effects of RA occurred at a concentration of 10 nM; maximal effects were observed at 1 microM. The effects of RA on melanogenesis were independent of its effects on cellular growth since one Cloudman line tested was growth-inhibited by RA and another was growth-stimulated by RA, but the induction of melanogenesis by MSH in both lines was inhibited by RA. Mixing experiments with cell lysates failed to demonstrate the induction of a tyrosinase inhibitor by RA. The effects of RA were not limited to MSH or to Cloudman melanoma cells since RA blocked cholera toxin-inducible melanogenesis in Cloudman cells, as well as the induction of tyrosinase activity by L-tyrosine in Bomirski hamster melanoma cells. The effects of RA were specific to melanogenesis, however, since RA did not interfere with MSH-induced changes in cellular morphology and growth. Thus, RA appears to be a new and potent tool for understanding mechanisms regulating induction of the pigmentary system.

Ultraviolet B and melanocyte-stimulating hormone (MSH) stimulate mRNA production for alpha MSH receptors and proopiomelanocortin-derived peptides in mouse melanoma cells and transformed keratinocytes.

Cell lines of cutaneous origin, namely melanocytes and keratinocytes, were previously demonstrated to exhibit functional melanocyte-stimulating hormone (MSH) receptors that are up-regulated by ultraviolet (UV) radiation and by MSH itself. In this study, it is demonstrated that UVB irradiation, exposure to MSH, or exposure to N6,O2-dibutyryl cyclic adenosine monophosphate stimulates production of mRNAs for both alpha MSH receptors and proopiomelanocortin in cultured mouse Cloudman S91 melanoma cells, and that UVB stimulates production and release of MSH and adrenocorticotropin peptides in both melanoma cells and transformed PAM 212 mouse keratinocytes. The results add support to the hypothesis that the effects of UVB on cutaneous melanogenesis are mediated through a series of coordinated events in which MSH receptors and proopiomelanocortin-derived peptides play a central role.

New regulators of melanogenesis are associated with purified tyrosinase isozymes.

Three new regulatory factors in the melanogenesis pathway were recently described: dopachrome conversion factor accelerates the conversion of dopachrome to 5,6-dihydroxyindole; indole conversion factor accelerates the conversion of 5, 6-dihydroxyindole into melanin; and indole blocking factor retards the conversion of 5, 6-dihydroxyindole into melanin. Exposure of wild-type Cloudman melanoma cells in culture to melanotropin (MSH) removes blocking factor activity and increases indole conversion factor activity. The chemical nature of factors has not yet been determined. In this report we demonstrate that highly purified isozymes of tyrosinase from C57B1/6N murine hair bulbs and B16 murine melanoma are closely associated with conversion and blocking factor activities. The soluble isozymes. T1, T2, and T2 contain blocking factor activity, while isozyme T4, the major tyrosinase species found in melanosomes, contains activity that accelerates melanin formation from dopachrome. The results suggest that melanogenesis is regulated by the association of these different factors with the specific tyrosinase isozymes.

Assay of melanotropic peptides in an in vitro mammalian system.

A reproducible and sensitive assay for melanotropic agents is described employing mouse melanoma cells in culture and measuring tyrosinase activity in terms of production of tritiated water from L-(ring-3,5-3H)-tyrosine. Molar concentrations of peptides inducing one-half maximal stimulation of tyrosinase activity were: beta-MSH, 1 +/- 2 x 10(-9); alpha-MSH and Beta h-LPH, 1 +/- 2 x 10(-8); ACTHp, 1 +/- 2 x 10(-7). Beta p 9-18-MSH and melanotropin potentiating factor, beta s 88-91-LPH exhibited no activity at concentrations as high as 10(-5)M.

Enhanced expression of melanocortin-1 receptor (MC1-R) in normal human keratinocytes during differentiation: evidence for increased expression of POMC peptides near suprabasal layer of epidermis.

Immunohistochemical staining of human skin specimen showed the stronger localization of proopiomelanocortin peptides near the suprabasal layer of the epidermis, where keratinocytes are mostly differentiated. To test the possibilities of whether the production of proopiomelanocortin peptides or their receptor-binding activity or both is increased during differentiation of keratinocytes, we treated the cells in culture with Ca2+ to induce their differentiation. The production of proopiomelanocortin peptides and its gene expression were not induced significantly, but the binding ability of melanocortin receptor, as well as its gene expression were stimulated by Ca2+. Ultraviolet B irradiation, an inducer of differentiation, stimulated both proopiomelanocortin production and melanocortin receptor expression. These data show that normal human keratinocytes express melanocortin receptor similar to melanocytes, and that it is induced during differentiation.

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.

Human monocyte x mouse melanoma fusion hybrids express human gene.

Artificial fusion of human monocyte with Cloudman S91 mouse melanoma cells resulted in hybrids that showed increased motility in vitro, enhanced metastatic potential in vivo, and also tended to be super melanotic (Rachkovsky et al., Clin. Exp. Metastasis 16 (1998) 299). However, no gene derived from monocytes has been shown to be expressed in these hybrids until now. Similar observations have also been noted in hybrids originating from mouse macrophage and mouse melanoma cells. Having the advantage of species differences in mouse x human hybrids, we are able, this time, to show by RT-PCR that some genes specific to the human genome are expressed in these hybrids, indicating that not only is the genomic DNA from parental monocytes integrated in the hybrids but also some genes are being expressed. This observation may lead us to find contributory genes from monocyte and/or macrophage that are responsible for modulating the genotypes and hence the phenotypes in the hybrids.

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 microM 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 analysis 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.

Evidence that dopachrome tautomerase is a ferrous iron-binding glycoprotein.

Dopachrome tautomerase (DT) (EC 5.3.2.3) is a melanocyte-specific, membrane-associated, heat-labile, non-dialyzable, protease-sensitive factor which catalyzes the isomeric rearrangement of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), apparently through a tautomerization reaction. Metal ions such as Cu, Ni, Co, Zn, Mn, Ca, Al, and Fe can also catalyze the dopachrome/DHICA isomerization. How is the reaction regulated in vivo? An attractive possibility would be that DT is a metalloenzyme. Here we present evidence that this may indeed be the case. Purified preparations of DT and tyrosinase, obtained from Cloudman S91 mouse melanoma cells, were assayed in the presence of a variety of metal chelators including EDTA (predominantly Ca and Mg), EGTA (predominantly Ca), phenylthiourea (PTU) (predominantly Cu), 2,2'-dipyridyl (predominantly Fe); 1,10-phenanthroline (predominantly Fe), and 2,3-dihydroxybenzoic acid (predominantly Fe). In addition, DT activity was assayed in the presence of two non-chelating structural analogs of 1,10-phenanthroline. Results were as follows: (i) iron chelators inhibited DT activity with no effects on tyrosinase activity; (ii) inhibition by the chelators was reversible with the addition of ferrous iron; (iii) 1,10-phenanthroline pre-complexed to ferrous iron was not inhibitory to DT; (iv) non-chelating analogs of phenanthroline were not inhibitory to DT; (v) PTU was inhibitory to tyrosinase but not DT; (vi) Ca2+ and Mg2+ chelators had little effect on either enzyme activity. Finally, studies with glycosylation inhibitors, glycosylase enzymes, and immobilized lectins, indicated that DT is a glycoprotein. The results suggest that DT is a metal-containing glycosylated enzyme, possibly with ferrous iron at its catalytic center.

Ultraviolet B stimulates production of corticotropin releasing factor (CRF) by human melanocytes.

Here we show that human melanocytes express the corticotropin releasing factor (CRF) gene and produce CRF peptide. The CRF production and secretion is markedly stimulated by ultraviolet B (UVB) radiation. This is the first demonstration that cutaneous melanocytes respond to environmental stress (UVB) through the production of CRF.