-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in severa... more -MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of -MSH (KPV / KP-DV) are the ...
The purpose of this study was to examine which intracellular signalling systems influence the att... more The purpose of this study was to examine which intracellular signalling systems influence the attachment of normal uveal melanocytes and uveal melanoma cells to extracellular matrix (ECM) proteins in vitro. Uveal melanocytes were found to attach strongly to fibronectin in preference to plastic, collagens type I, III or IV, or laminin. In contrast, uveal melanoma cells attached equally well to fibronectin and collagens I, III and IV in preference to plastic or laminin. Manipulation of intracellular cyclic AMP or protein kinase C had little, if any, effect on the attachment of either cell to fibronectin. In contrast, inhibition of calmodulin significantly inhibited the attachment of both normal and transformed cells, as did manipulating intracellular free calcium. We noted that the intracellular free calcium in melanoma cells was less than half that seen in melanocytes. Fibronectin, laminin and Arg-Gly-Asp (RGD) were all capable of acutely increasing the intracellular free calcium in both cells. ECM-induced increases in calcium were more apparent in low density than high density cells and appeared more sustained in melanocytes than in melanoma cells. We conclude that both normal and neoplastic uveal melanocytes require an intracellular signal or signals which involves calcium and calmodulin in the few minutes following cell binding to ECM proteins in order for successful cell attachment to occur. While the transformed cell does not differ significantly from the normal cell in this respect, this dependency on calcium and calmodulin may nevertheless offer an approach for pharmacological intervention in the prevention or arrest of metastatic spread and merits further investigation.
Several laboratories are pursuing the question of whether the expression of pigment genes can be ... more Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with alpha-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by alpha-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; alpha-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or alpha-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinase-related genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.
alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in s... more alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.
Tamoxifen plays a major role in the management of breast cancer in women and is currently used to... more Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti-estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen-receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.
BACKGROUND/AIMSPosterior uveal melanoma is the most common intraocular tumour in adults, responsi... more BACKGROUND/AIMSPosterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The β1 family of integrins, αvβ3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement
Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physio... more Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physiological regulation of melanocyte proliferation and differentiation is not yet fully defined and this study summarises several separate lines of evidence which suggest that, in vivo, some of the signals required for melanocyte proliferation and differentiation may derive from extracellular matrix (ECM) proteins adjacent to these cells. Culture of cutaneous and uveal melanocytes on cell-derived and individual ECM proteins was found to influence cell morphology with such effects being most noticeable in mitogen-deficient media. Similarly, cell-derived and individual ECM proteins increased tyrosinase activity in normal cutaneous melanocytes and effects of these ECM proteins were seen most consistently in mitogen-deficient media. Uveal melanocytes (as has been reported for cutaneous melanocytes) showed preferential attachment to fibronectin over other ECM substrates. This attachment was particularly sensitive to drugs which affected intracellular calcium or calmodulin activity. Acute addition of fibronectin to coverslips of uveal melanocytes loaded with Fura-2 produced an acute and transient increase in intracellular calcium which was more prevalent in low density than higher density cells. We conclude that ECM proteins in vitro are capable of influencing melanocyte morphology, tyrosinase activity, and proliferation and that an ECM-induced elevation in intracellular calcium may be part of the signalling system that transmits ECM information into the cell.
Following our recent finding that calmodulin antagonists can reduce cancer cell attachment to ext... more Following our recent finding that calmodulin antagonists can reduce cancer cell attachment to extracellular matrix proteins, we investigated the calmodulin antagonistic and anti-attachment properties of the non-steroidal anti-oestrogens tamoxifen and droloxifene. These drugs and four of their active metabolites were found to have calmodulin antagonist activity with IC50 values of 2-4 microM and to be capable of inhibiting attachment of murine B16 melanoma to extracellular matrix proteins in vitro. IC50 values for inhibition of attachment were 11 microM for tamoxifen and ranged from 5 to 40 microM for the other five compounds tested. (Poor reproducibility in drug potency between attachment experiments was almost certainly due to the low aqueous solubility of these drugs.) The effects of tamoxifen on cell/matrix adhesion were most evident between 15 min and 3 h of cell attachment. No effects of tamoxifen were evident in cells which had been allowed to attach for 6 h or more. Tamoxifen at concentrations between 0.1 and 30 microM was without effect on intracellular free calcium concentration. Tamoxifen also inhibited attachment of human ocular melanoma cells and human breast cancer (MCF7) cells to type I collagen. The concentration at which tamoxifen and its metabolites affect cell attachment in vitro (2-14 microM) is of the same order as the tissue concentrations of these drugs achieved clinically. The possibility exists that reduction of cell/matrix interactions may contribute to the clinical anti-metastatic efficacy of tamoxifen and some of its active metabolites.
The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pi... more The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F(2alpha) (PGF(2alpha)) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF(2alpha) receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25-30%) in dopa oxidase activity in response to 10(-7)-10(-5)m LT and PGF(2alpha). Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF(2alpha). FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF(2alpha) increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.
Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions includin... more Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of alpha-, beta- and gamma-MSH correlated clinically with malignant melanoma development, but other studies suggest alpha-MSH acts to retard invasion. In the present study, we investigated the action of alpha-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. alpha-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kappaB transcription factor. However, A375-SM and C8161 cells did not respond to alpha-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three li...
Epidemiological studies show female survival benefit in advanced metastatic melanoma. In investig... more Epidemiological studies show female survival benefit in advanced metastatic melanoma. In investigating a possible mechanism for this female survival benefit, we have previously reported that the female steroid 17beta-oestradiol significantly reduces invasion of a human melanoma cell line (A375-SM cells) and ocular melanoma cells through fibronectin. Neither cell type was found to possess oestrogen receptor-alpha. The aim of the current study was to obtain further information on the extent to which progression of cutaneous melanoma might be sex steroid sensitive by (a) examining the relationship between circulating sex steroids, sex hormone binding globulin and disease progression; (b) examining the relationship between sex steroid structure and the ability of steroids to reduce invasion of a melanoma cell line in vitro; and (c) examining the effects of sex steroids on proliferation of these cells in vitro. We report a significant reduction in circulating oestrone with disease progre...
The aim of this study was to investigate whether the presence of pigment affects the sensitivity ... more The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.
The aim of this study was to investigate whether the presence of pigment affects the sensitivity ... more The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.
The involvement of signal transduction systems in the initial attachment of two murine B16 melano... more The involvement of signal transduction systems in the initial attachment of two murine B16 melanoma clones of differing metastatic potential to extracellular matrix components was examined to learn more of the early events in cell-matrix interaction. Clones of high and low metastatic capacity attached similarly in the absence of any stimulators, exhibiting a two phase time course of attachment with 100% attachment by 60 min. A slight difference in attachment characteristics between the clones was seen in response to phorbol ester stimulation, which significantly inhibited attachment of the low metastatic clone but which had no effect on the highly metastatic clone. Total protein kinase C activity and distribution was similar for both clones. Attachment of both clones was severely reduced, however, if intracellular calcium was elevated or intracellular calmodulin inhibited. This study suggests that signal transduction mechanisms are involved in melanoma cell attachment to matrix proteins and offers an approach to pharmacological manipulation of these cell-matrix interactions which may be relevant to reducing metastatic spread.
-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in severa... more -MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of -MSH (KPV / KP-DV) are the ...
The purpose of this study was to examine which intracellular signalling systems influence the att... more The purpose of this study was to examine which intracellular signalling systems influence the attachment of normal uveal melanocytes and uveal melanoma cells to extracellular matrix (ECM) proteins in vitro. Uveal melanocytes were found to attach strongly to fibronectin in preference to plastic, collagens type I, III or IV, or laminin. In contrast, uveal melanoma cells attached equally well to fibronectin and collagens I, III and IV in preference to plastic or laminin. Manipulation of intracellular cyclic AMP or protein kinase C had little, if any, effect on the attachment of either cell to fibronectin. In contrast, inhibition of calmodulin significantly inhibited the attachment of both normal and transformed cells, as did manipulating intracellular free calcium. We noted that the intracellular free calcium in melanoma cells was less than half that seen in melanocytes. Fibronectin, laminin and Arg-Gly-Asp (RGD) were all capable of acutely increasing the intracellular free calcium in both cells. ECM-induced increases in calcium were more apparent in low density than high density cells and appeared more sustained in melanocytes than in melanoma cells. We conclude that both normal and neoplastic uveal melanocytes require an intracellular signal or signals which involves calcium and calmodulin in the few minutes following cell binding to ECM proteins in order for successful cell attachment to occur. While the transformed cell does not differ significantly from the normal cell in this respect, this dependency on calcium and calmodulin may nevertheless offer an approach for pharmacological intervention in the prevention or arrest of metastatic spread and merits further investigation.
Several laboratories are pursuing the question of whether the expression of pigment genes can be ... more Several laboratories are pursuing the question of whether the expression of pigment genes can be used as a useful marker for tumour progression. However, many melanoma tumours are amelanotic in vivo. The purpose of this study was to examine the relationship between the expression of tyrosinase-related genes [tyrosinase, tyrosinase-related protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2)] and pigmentation of melanoma cells. Fourteen cutaneous melanoma cell lines were examined for visible pigment, melanin content, and dopa oxidase activity and findings were related to the previously determined expression of the three tyrosinase-related genes in these cells in culture. Four of the cell lines were also stimulated with alpha-MSH, isobutylmethylxanthine, and forskolin to examine the relationship between induced pigmentation and upregulation of pigmentation genes. There was no simple correlation between pigmentation gene expression and dopa oxidase activity or total melanin content of the 14 melanoma cell lines in culture. In the majority of cells, there was no appreciable pigment, whereas, in contrast, half of the cells showed significant dopa oxidase activity. Upregulation of dopa oxidase activity was achieved by alpha-MSH in two out of four cell lines examined in detail and with IBMX in three out of four of these cell lines. IBMX increased tyrosinase gene expression in all four cell lines; alpha-MSH was without effect; and TRP-1 and TRP-2 expression were largely unaffected by IBMX or alpha-MSH. Modest changes in morphology were noted in response to IBMX. Overall, however, human melanoma cell lines were, with two exceptions, amelanotic in culture despite the fact that 10 out of the 14 lines expressed tyrosinase-related genes. We conclude that measurable pigmentation is not a necessary consequence of the expression of pigmentation genes. An implication of this work is that amelanotic tumours in vivo may nevertheless be positive for tyrosinase-related genes.
alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in s... more alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.
Tamoxifen plays a major role in the management of breast cancer in women and is currently used to... more Tamoxifen plays a major role in the management of breast cancer in women and is currently used to a lesser extent in other neoplasias. Many of the pharmacological properties of tamoxifen are consistent with anti-estrogen activity, but it also has significant, although lesser, benefit in patients whose tumours are estrogen-receptor negative. We recently reported that murine B16 melanoma cell attachment to extracellular matrix proteins can be inhibited by calmodulin antagonists. In seeking a calmodulin antagonist that could be used clinically, we investigated tamoxifen, which is known to have calmodulin antagonist activity in vitro, and confirmed that it will inhibit murine melanoma cell attachment in vitro. In the current study, we examined the effect of tamoxifen on the attachment of human ocular melanoma cell lines to a range of extracellular matrix substrates to evaluate the potential relevance of calmodulin antagonists, including tamoxifen, to reducing metastatic spread of these tumours. We report that six ocular melanoma cell lines established from choroidal melanoma tumours showed rapid attachment to a range of substrates and that this attachment can be significantly reduced by an experimental calmodulin antagonist (J8) and by tamoxifen. In summary, we conclude that the ability of calmodulin antagonists, including tamoxifen, to inhibit ocular melanoma cell attachment to matrix proteins in vitro merits further investigation as it may offer another approach to reducing metastatic spread of these tumours.
BACKGROUND/AIMSPosterior uveal melanoma is the most common intraocular tumour in adults, responsi... more BACKGROUND/AIMSPosterior uveal melanoma is the most common intraocular tumour in adults, responsible for the death of approximately 35% of patients. Hepatic metastases are most frequent, and once diagnosed survival is usually less than 1 year. The β1 family of integrins, αvβ3 and MMP-2 and MMP-9 have been implicated in the metastasis of several types of tumour. To study their involvement
Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physio... more Cutaneous and ocular melanocytes are routinely cultured in complex mitogen-rich media. The physiological regulation of melanocyte proliferation and differentiation is not yet fully defined and this study summarises several separate lines of evidence which suggest that, in vivo, some of the signals required for melanocyte proliferation and differentiation may derive from extracellular matrix (ECM) proteins adjacent to these cells. Culture of cutaneous and uveal melanocytes on cell-derived and individual ECM proteins was found to influence cell morphology with such effects being most noticeable in mitogen-deficient media. Similarly, cell-derived and individual ECM proteins increased tyrosinase activity in normal cutaneous melanocytes and effects of these ECM proteins were seen most consistently in mitogen-deficient media. Uveal melanocytes (as has been reported for cutaneous melanocytes) showed preferential attachment to fibronectin over other ECM substrates. This attachment was particularly sensitive to drugs which affected intracellular calcium or calmodulin activity. Acute addition of fibronectin to coverslips of uveal melanocytes loaded with Fura-2 produced an acute and transient increase in intracellular calcium which was more prevalent in low density than higher density cells. We conclude that ECM proteins in vitro are capable of influencing melanocyte morphology, tyrosinase activity, and proliferation and that an ECM-induced elevation in intracellular calcium may be part of the signalling system that transmits ECM information into the cell.
Following our recent finding that calmodulin antagonists can reduce cancer cell attachment to ext... more Following our recent finding that calmodulin antagonists can reduce cancer cell attachment to extracellular matrix proteins, we investigated the calmodulin antagonistic and anti-attachment properties of the non-steroidal anti-oestrogens tamoxifen and droloxifene. These drugs and four of their active metabolites were found to have calmodulin antagonist activity with IC50 values of 2-4 microM and to be capable of inhibiting attachment of murine B16 melanoma to extracellular matrix proteins in vitro. IC50 values for inhibition of attachment were 11 microM for tamoxifen and ranged from 5 to 40 microM for the other five compounds tested. (Poor reproducibility in drug potency between attachment experiments was almost certainly due to the low aqueous solubility of these drugs.) The effects of tamoxifen on cell/matrix adhesion were most evident between 15 min and 3 h of cell attachment. No effects of tamoxifen were evident in cells which had been allowed to attach for 6 h or more. Tamoxifen at concentrations between 0.1 and 30 microM was without effect on intracellular free calcium concentration. Tamoxifen also inhibited attachment of human ocular melanoma cells and human breast cancer (MCF7) cells to type I collagen. The concentration at which tamoxifen and its metabolites affect cell attachment in vitro (2-14 microM) is of the same order as the tissue concentrations of these drugs achieved clinically. The possibility exists that reduction of cell/matrix interactions may contribute to the clinical anti-metastatic efficacy of tamoxifen and some of its active metabolites.
The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pi... more The prostaglandin F2alpha derivative, latanoprost (LT), used in glaucoma treatment, can induce pigmentation in irises of patients with hazel or heterochromatic eye colour. The mechanism by which LT induces pigmentation in the iris is not yet established, although it does not appear to induce proliferation of iridial melanocytes. The purpose of this study was to develop an in vitro model in which to investigate this mechanism. The pigmentary responses to LT and prostaglandin F(2alpha) (PGF(2alpha)) were examined in human iridial melanocytes alone or in co-culture with epithelial cells (non-ocular human epidermal keratinocytes and iris pigment epithelial cells) or mesenchymal cells (non-ocular dermal fibroblasts or iridial fibroblasts). Melanogenesis was assessed after 4 days culture with prostanoids, using dopa oxidase activity. Prostaglandin FP expression on human iridial fibroblasts and melanocytes was investigated using an immunofluorescent technique employing antibody to PGF(2alpha) receptor and RT-PCR. Iridial melanocytes did not show a convincing increase in dopa oxidase when cultured alone but in the presence of fibroblasts (ocular or non-ocular) there was a significant increase (25-30%) in dopa oxidase activity in response to 10(-7)-10(-5)m LT and PGF(2alpha). Co-culture of melanocytes with epithelial cells, while leading to increased dopa oxidase activity, did not lead to any melanogenic response to LT or PGF(2alpha). FP receptor expression was detected on fibroblasts but not iridial melanocytes by immunocytochemistry and RT-PCR. The melanocyte/fibroblast co-culture model developed in this study also showed that LT and PGF(2alpha) increased dopa oxidase activity in melanocytes from donors with brown but not blue eyes. These results suggest that LT may be inducing pigmentation in the human iris indirectly through the FP receptor on adjacent fibroblasts.
Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions includin... more Alpha-melanocyte stimulating hormone (alpha-MSH) is known to have pleiotrophic functions including pigmentary, anti-inflammatory, antipyretic and immunoregulatory roles in the mammalian body. It is also reported to influence melanoma invasion with levels of alpha-, beta- and gamma-MSH correlated clinically with malignant melanoma development, but other studies suggest alpha-MSH acts to retard invasion. In the present study, we investigated the action of alpha-MSH on three human melanoma cell lines (HBL, A375-SM and C8161) differing in metastatic potential. alpha-melanocyte-simulating hormone reduced invasion through fibronectin and also through a human reconstructed skin composite model for the HBL line, and inhibited proinflammatory cytokine-stimulated activation of the NF-kappaB transcription factor. However, A375-SM and C8161 cells did not respond to alpha-MSH. Immunofluorescent microscopy and Western blotting identified melanocortin-1 receptor (MC-1R) expression for all three li...
Epidemiological studies show female survival benefit in advanced metastatic melanoma. In investig... more Epidemiological studies show female survival benefit in advanced metastatic melanoma. In investigating a possible mechanism for this female survival benefit, we have previously reported that the female steroid 17beta-oestradiol significantly reduces invasion of a human melanoma cell line (A375-SM cells) and ocular melanoma cells through fibronectin. Neither cell type was found to possess oestrogen receptor-alpha. The aim of the current study was to obtain further information on the extent to which progression of cutaneous melanoma might be sex steroid sensitive by (a) examining the relationship between circulating sex steroids, sex hormone binding globulin and disease progression; (b) examining the relationship between sex steroid structure and the ability of steroids to reduce invasion of a melanoma cell line in vitro; and (c) examining the effects of sex steroids on proliferation of these cells in vitro. We report a significant reduction in circulating oestrone with disease progre...
The aim of this study was to investigate whether the presence of pigment affects the sensitivity ... more The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.
The aim of this study was to investigate whether the presence of pigment affects the sensitivity ... more The aim of this study was to investigate whether the presence of pigment affects the sensitivity of pigmented cells of the eye, retinal pigment epithelium (RPE) and choroidal melanocytes (CMs) to the cytotoxic effects of xenobiotic drugs. Two approaches were used to compare pigmented versus unpigmented cells: RPE cells were repigmented by phagocytosis of synthetic melanin; UVB irradiation was used to induce an increase in pigment in both RPE and CMs. Three drugs known to induce toxicity in the eye, tamoxifen, chloroquine and thioridazine, were used to assess the sensitivity of cells to xenobiotic drugs. RPE cells were more resistant than CMs to the cytotoxic effects of all three drugs by a factor of 5-fold for tamoxifen, 7-fold for thioridazine and 30-fold for chloroquine. When RPE cells were repigmented using synthetic melanin, their sensitivity to tamoxifen was unchanged, they showed a slightly improved response to thioridazine (after 3 days of incubation with this drug), but they showed greatly increased toxicity to chloroquine (after 1 and 3 days of exposure to the drug), suggesting accumulation of this latter drug on the synthetic melanin. UVB irradiation was used to achieve an increase in the pigment content of both RPE and CMs. CMs were much more sensitive to UVB than RPE cells. CMs appeared to synthesise pigment via DOPA oxidase activity; RPE cells showed an increase in fluorescent material independent of any detectable DOPA oxidase activity. Irrespective of the nature of the pigment that UVB induced in melanocytes and RPE cells, their subsequent response to thioridazine and chloroquine was unchanged by the presence of this pigment.
The involvement of signal transduction systems in the initial attachment of two murine B16 melano... more The involvement of signal transduction systems in the initial attachment of two murine B16 melanoma clones of differing metastatic potential to extracellular matrix components was examined to learn more of the early events in cell-matrix interaction. Clones of high and low metastatic capacity attached similarly in the absence of any stimulators, exhibiting a two phase time course of attachment with 100% attachment by 60 min. A slight difference in attachment characteristics between the clones was seen in response to phorbol ester stimulation, which significantly inhibited attachment of the low metastatic clone but which had no effect on the highly metastatic clone. Total protein kinase C activity and distribution was similar for both clones. Attachment of both clones was severely reduced, however, if intracellular calcium was elevated or intracellular calmodulin inhibited. This study suggests that signal transduction mechanisms are involved in melanoma cell attachment to matrix proteins and offers an approach to pharmacological manipulation of these cell-matrix interactions which may be relevant to reducing metastatic spread.
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Papers by Mark Wagner