Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, al... more 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. ...
Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the ... more Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the generation of hybrids comprised of cells of the primary tumor and tumor-infiltrating leukocytes. Fusion hybrids were generated in vitro between weakly metastatic Cloudman S91 mouse melanoma cells and normal mouse or human macrophages. Hybrids were implanted s.c. in the tail and mice were monitored for metastases. Controls included parental S91 cells, autologous S91 x S91 hybrids, and B16F10 melanoma cells. Of 35 hybrids tested, most were more aggressive than the parental melanoma cells, producing metastases sooner and in more mice. A striking characteristic was heterogeneity amongst hybrids, with some lines producing no metastases and others producing metastases in up to 80% of mice. With few exceptions, hybrids with the highest metastatic potential also had the highest basal melanin content whereas those with the lowest metastatic potential were basally amelanotic, as were the parental ...
In schizophrenia, genetic predisposition has been linked to chromosome 22q11 and myelin-specific ... more In schizophrenia, genetic predisposition has been linked to chromosome 22q11 and myelin-specific genes are misexpressed in schizophrenia. Nogo-66 receptor 1 (NGR or RTN4R) has been considered to be a 22q11 candidate gene for schizophrenia susceptibility because it encodes an axonal protein that mediates myelin inhibition of axonal sprouting. Confirming previous studies, we found that variation at the NGR locus is associated with schizophrenia in a Caucasian case-control analysis, and this association is not attributed to population stratification. Within a limited set of schizophrenia-derived DNA samples, we identified several rare NGR nonconservative coding sequence variants. Neuronal cultures demonstrate that four different schizophrenia-derived NgR1 variants fail to transduce myelin signals into axon inhibition, and function as dominant negatives to disrupt endogenous NgR1. This provides the first evidence that certain disease-derived human NgR1 variants are dysfunctional proteins in vitro. Mice lacking NgR1 protein exhibit reduced working memory function, consistent with a potential endophenotype of schizophrenia. For a restricted subset of individuals diagnosed with schizophrenia, the expression of dysfunctional NGR variants may contribute to increased disease risk.
Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accu... more Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20-30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M "injury responses" of other cell types might be expected, consistent with their differentiated phenotypes.
Fusion of mouse peritoneal macrophages or human blood monocytes with weakly metastatic mouse Clou... more Fusion of mouse peritoneal macrophages or human blood monocytes with weakly metastatic mouse Cloudman S91 melanoma cells resulted in hybrids with enhanced metastatic potential (Rachkovsky et al., 1998. Clin. Exp. Metastasis, 16: 299-312). With few exceptions, such hybrids also showed increased basal- and MSH-induced pigmentation, at least in part through increased N-glycosylation of melanogenic proteins (Sodi et al., 1998. Pigment Cell Res., 11: 299-309). Here we report analyses regarding expression of the melanocyte-stimulating hormone (MSH) receptor (melanocortin-1 receptor, MC1-R) and the melanogenic proteins, tyrosinase (E.C. 1.14.18.1), tyrosinase-related protein 1 (TRP-1), and the tyrosinase-related protein 2 (TRP-2, E.C. 5.3.2.3), by a panel of cell lines consisting of parental Cloudman S91 melanoma cells, macrophages from DBA/2J mice, artificially derived macrophage x melanoma hybrids of high and low metastatic potential, and a naturally occurring highly metastatic hybrid between a Cloudman S91 tumor cell and a DBA/2J tumor-infiltrating cell. We show that incubation of cells with MSH/isobutylmethylxanthine (IBMX) resulted in strong melanogenic and morphologic responses in high metastatic hybrids compared to parental cells and the low metastatic hybrid, and that high metastatic hybrids exhibit increased mRNA expression for MC1-R accompanied by increased 125I-alphaMSH binding. Although tyrosinase activity and the protein level for tyrosinase and TRP-2, but not for TRP-1, were increased in the high metastatic hybrids versus the other cells, no significant changes in mRNA either for tyrosinase or for TRPs were observed in them. Furthermore, unlike tyrosinase, the abundance and gel mobility pattern of TRP-2 did not correlate with changes in activity in all hybrids and parental melanoma cells. The results suggest that although the activity MC1-R and tyrosinase correlate with enhanced basal as well as MSH-induced melanogenesis in metastatic/melanotic hybrids, their expression is differentially regulated, i.e., regulation of MC1-R while at transcriptional level, the TRPs are primarily regulated via post-transcriptional mechanisms in high metastatic hybrids.
... Such a prop-erty could have important implications since primary tumors are often heavily inf... more ... Such a prop-erty could have important implications since primary tumors are often heavily infiltrated with macrophages and lymphocytes (Evans, 1972; Gauci and Alexander ... 1:500) (Jimenez et al., 1991; l%ukamoto et al., 1992), and aPEP-13 (carboxy terminus of F'mel-l7/siZver ...
The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with ... more The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with X-rays in mice bearing B16F10 or Cloudman S91 melanomas. Each treatment alone slowed tumour growth and prolonged survival, and the combined treatments produced supra-additive antitumour effects. That is, in dose-response studies with single doses of Salmonella and increasing doses of radiation, the two agents together caused suppression of tumour growth that was greater than that calculated for additivity. The results suggest that the combination of these genetically engineered Salmonella with radiotherapy could be a new and beneficial treatment for solid tumours.
Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growt... more Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growth suppression of tumors in mice. We examined the effects of mutations on this anticancer phenotype in two Salmonella virulence gene clusters. Salmonella pathogenicity island (SPI)-1 genes promote systemic invasion from the intestine, whereas SPI-2 genes support systemic survival within macrophages and other cells. Disabling SPI-1 (prgH−) strongly reduced
Ultraviolet B (UVB) radiation in the skin induces pigmentation that protects cells from further U... more Ultraviolet B (UVB) radiation in the skin induces pigmentation that protects cells from further UVB damage and reduces photocarcinogenesis. Although the mechanisms are not well understood, our laboratory has shown that UVB radiation causes increased MSH receptor activity by redistributing MSH receptors from internal pools to the external surface, with a resultant increase in cellular responsiveness to MSH. By this means, UVB and MSH act synergistically to increase melanin content in the skin of mice and guinea pigs. In humans, MSH causes increased skin pigmentation, predominantly in sun-exposed areas. We have shown recently that UVB irradiation and exposure to MSH or to dbcAMP, stimulates production of mRNAs for both ␣ MSH receptors and POMC in human melanocytes and keratinocytes. This indicates that at least one action of UVB on the pigmentary system is mediated through increased MSH receptor production, as well as through the production of the signal peptides, MSH and ACTH, that can further activate MSH receptors. 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 POMC-derived peptides play a central role.
Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail... more 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...
Systemically administered tumor-targeted Salmonella has been developed as an anticancer agent, al... more 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. ...
Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the ... more Studies were conducted on the hypothesis that melanoma metastasis might be initiated through the generation of hybrids comprised of cells of the primary tumor and tumor-infiltrating leukocytes. Fusion hybrids were generated in vitro between weakly metastatic Cloudman S91 mouse melanoma cells and normal mouse or human macrophages. Hybrids were implanted s.c. in the tail and mice were monitored for metastases. Controls included parental S91 cells, autologous S91 x S91 hybrids, and B16F10 melanoma cells. Of 35 hybrids tested, most were more aggressive than the parental melanoma cells, producing metastases sooner and in more mice. A striking characteristic was heterogeneity amongst hybrids, with some lines producing no metastases and others producing metastases in up to 80% of mice. With few exceptions, hybrids with the highest metastatic potential also had the highest basal melanin content whereas those with the lowest metastatic potential were basally amelanotic, as were the parental ...
In schizophrenia, genetic predisposition has been linked to chromosome 22q11 and myelin-specific ... more In schizophrenia, genetic predisposition has been linked to chromosome 22q11 and myelin-specific genes are misexpressed in schizophrenia. Nogo-66 receptor 1 (NGR or RTN4R) has been considered to be a 22q11 candidate gene for schizophrenia susceptibility because it encodes an axonal protein that mediates myelin inhibition of axonal sprouting. Confirming previous studies, we found that variation at the NGR locus is associated with schizophrenia in a Caucasian case-control analysis, and this association is not attributed to population stratification. Within a limited set of schizophrenia-derived DNA samples, we identified several rare NGR nonconservative coding sequence variants. Neuronal cultures demonstrate that four different schizophrenia-derived NgR1 variants fail to transduce myelin signals into axon inhibition, and function as dominant negatives to disrupt endogenous NgR1. This provides the first evidence that certain disease-derived human NgR1 variants are dysfunctional proteins in vitro. Mice lacking NgR1 protein exhibit reduced working memory function, consistent with a potential endophenotype of schizophrenia. For a restricted subset of individuals diagnosed with schizophrenia, the expression of dysfunctional NGR variants may contribute to increased disease risk.
Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accu... more Cultured mouse Cloudman melanoma cells, EMT6 breast carcinoma cells, and 3T3 fibroblasts all accumulated in the G2/M phase of the cell cycle when exposed to UVB radiation. The effects of UVB were maximal at 20-30 mJ/cm2 for all three cell lines, and could be observed by flow cytometry as early as 12 hr post irradiation. It has been known since the mid-1970s that MSH receptor binding activity is highest on Cloudman melanoma cells when they are in the G2/M phase of their cycle. Here we show that either UVB irradiation or synchronization of Cloudman cells with colchicine results in a stimulation of MSH binding within 24 hr following treatment, a time when both treatments have resulted in accumulation of cells in the G2/M phase of the cycle. Furthermore, the two treatments performed together on the melanoma cells stimulated MSH receptor activity to the same extent as either treatment performed separately, suggesting that each may be influencing MSH receptor activity solely through a G2/M accumulation of cells. Together, these results raise the possibility that an increase in the number of cells in the G2 phase of the cell cycle is a generalized cellular response to injury, such as UV irradiation. However, in the case of pigment cells this response includes a mechanism for increasing melanin formation, i.e., increased MSH receptor activity. Should this be the case, similar G2/M "injury responses" of other cell types might be expected, consistent with their differentiated phenotypes.
Fusion of mouse peritoneal macrophages or human blood monocytes with weakly metastatic mouse Clou... more Fusion of mouse peritoneal macrophages or human blood monocytes with weakly metastatic mouse Cloudman S91 melanoma cells resulted in hybrids with enhanced metastatic potential (Rachkovsky et al., 1998. Clin. Exp. Metastasis, 16: 299-312). With few exceptions, such hybrids also showed increased basal- and MSH-induced pigmentation, at least in part through increased N-glycosylation of melanogenic proteins (Sodi et al., 1998. Pigment Cell Res., 11: 299-309). Here we report analyses regarding expression of the melanocyte-stimulating hormone (MSH) receptor (melanocortin-1 receptor, MC1-R) and the melanogenic proteins, tyrosinase (E.C. 1.14.18.1), tyrosinase-related protein 1 (TRP-1), and the tyrosinase-related protein 2 (TRP-2, E.C. 5.3.2.3), by a panel of cell lines consisting of parental Cloudman S91 melanoma cells, macrophages from DBA/2J mice, artificially derived macrophage x melanoma hybrids of high and low metastatic potential, and a naturally occurring highly metastatic hybrid between a Cloudman S91 tumor cell and a DBA/2J tumor-infiltrating cell. We show that incubation of cells with MSH/isobutylmethylxanthine (IBMX) resulted in strong melanogenic and morphologic responses in high metastatic hybrids compared to parental cells and the low metastatic hybrid, and that high metastatic hybrids exhibit increased mRNA expression for MC1-R accompanied by increased 125I-alphaMSH binding. Although tyrosinase activity and the protein level for tyrosinase and TRP-2, but not for TRP-1, were increased in the high metastatic hybrids versus the other cells, no significant changes in mRNA either for tyrosinase or for TRPs were observed in them. Furthermore, unlike tyrosinase, the abundance and gel mobility pattern of TRP-2 did not correlate with changes in activity in all hybrids and parental melanoma cells. The results suggest that although the activity MC1-R and tyrosinase correlate with enhanced basal as well as MSH-induced melanogenesis in metastatic/melanotic hybrids, their expression is differentially regulated, i.e., regulation of MC1-R while at transcriptional level, the TRPs are primarily regulated via post-transcriptional mechanisms in high metastatic hybrids.
... Such a prop-erty could have important implications since primary tumors are often heavily inf... more ... Such a prop-erty could have important implications since primary tumors are often heavily infiltrated with macrophages and lymphocytes (Evans, 1972; Gauci and Alexander ... 1:500) (Jimenez et al., 1991; l%ukamoto et al., 1992), and aPEP-13 (carboxy terminus of F'mel-l7/siZver ...
The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with ... more The antitumour efficacy of lipid A mutant Salmonella was evaluated alone and in combination with X-rays in mice bearing B16F10 or Cloudman S91 melanomas. Each treatment alone slowed tumour growth and prolonged survival, and the combined treatments produced supra-additive antitumour effects. That is, in dose-response studies with single doses of Salmonella and increasing doses of radiation, the two agents together caused suppression of tumour growth that was greater than that calculated for additivity. The results suggest that the combination of these genetically engineered Salmonella with radiotherapy could be a new and beneficial treatment for solid tumours.
Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growt... more Salmonella enterica servovar Typhimurium is capable of targeting, colonizing, and eliciting growth suppression of tumors in mice. We examined the effects of mutations on this anticancer phenotype in two Salmonella virulence gene clusters. Salmonella pathogenicity island (SPI)-1 genes promote systemic invasion from the intestine, whereas SPI-2 genes support systemic survival within macrophages and other cells. Disabling SPI-1 (prgH−) strongly reduced
Ultraviolet B (UVB) radiation in the skin induces pigmentation that protects cells from further U... more Ultraviolet B (UVB) radiation in the skin induces pigmentation that protects cells from further UVB damage and reduces photocarcinogenesis. Although the mechanisms are not well understood, our laboratory has shown that UVB radiation causes increased MSH receptor activity by redistributing MSH receptors from internal pools to the external surface, with a resultant increase in cellular responsiveness to MSH. By this means, UVB and MSH act synergistically to increase melanin content in the skin of mice and guinea pigs. In humans, MSH causes increased skin pigmentation, predominantly in sun-exposed areas. We have shown recently that UVB irradiation and exposure to MSH or to dbcAMP, stimulates production of mRNAs for both ␣ MSH receptors and POMC in human melanocytes and keratinocytes. This indicates that at least one action of UVB on the pigmentary system is mediated through increased MSH receptor production, as well as through the production of the signal peptides, MSH and ACTH, that can further activate MSH receptors. 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 POMC-derived peptides play a central role.
Cells from a lung metastasis, arising from Cloudman S91 melanoma cells implanted s.c. in the tail... more 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...
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Papers by Stefano Sodi