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.

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.

Melanoma x macrophage hybrids with enhanced metastatic potential.

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 melanoma cells. A spontaneous in vivo supermelanotic hybrid between an S91 tumor cell and DBA/2J host cell was one of the most metastatic lines. Hybrids with the highest metastatic potential also exhibited markedly higher chemotaxis to fibroblast-conditioned media. Histologically, the metastatic hybrids demonstrated vascular invasion and spread to distant organs similar to that of metastatic melanomas in mice and humans. Thus previous findings of enhanced metastasis in leukocyte x lymphoma hybrids can now be extended to include leukocyte x melanoma hybrids. Whether such hybridization is a natural cause of metastasis in vivo remains to be determined; however the fusion hybrids with genetically-matched parents described herein so closely resembled naturally-occurring metastatic melanoma cells that they could serve as useful new models for studies of this complex and deadly phenomenon.

Antitumour effects of genetically engineered Salmonella in combination with radiation.

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.

UV light and MSH receptors.

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 alpha 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.

Upregulation of mRNA for the melanocortin-1 receptor but not for melanogenic proteins in macrophage x melanoma fusion hybrids exhibiting increased melanogenic and metastatic potential.

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.

Enhancement of the depigmenting effect of hydroquinone by cystamine and buthionine sulfoximine.

Glutathione (GSH) performs several important biological functions, including quenching of reactive oxygen species, and protection of cells from toxic compounds such as quinones. The first step in the synthesis of GSH is catalysed by gamma-glutamylcysteine synthetase, an enzyme which is inhibited by cystamine and buthionine sulfoximine (BSO). In this study, we examined the possibility that the effect of hydroquinone (HQ) on pigmentation could be potentiated by inhibiting the production of GSH. In vitro studies using melanoma cell lines demonstrated that both cystamine and BSO could potentiate the inhibitory effects of HQ on tyrosinase activity and melanin content. A synergistic decrease in hair pigmentation was observed when a combination of HQ (2 or 4%) and BSO (5%) was applied to the dorsal skin of C57BL mice. In black hairless guinea-pigs, the application of HQ plus either BSO or cystamine resulted in a significant decrease in epidermal pigmentation when compared with any of the agents alone. The possibility exists that in the future a combination of HQ plus cystamine or BSO could be used to treat disorders such as melasma and post-inflammatory hyperpigmentation.

Altered N-glycosylation in macrophage x melanoma fusion hybrids.

It was recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential (Rachkovsky et al., 1998). With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content, enhanced chemotactic responses to fibroblast-conditioned media, and stronger responsiveness to MSH compared to parental cells. Analyses revealed that altered N-glycosylation in metastatic hybrids could explain the multiple phenotypic changes. Tyrosinase, TRP-2 and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. The incorporation of 3H-glucosamine, as a marker of N-glycosylation, into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis, and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.

Effects of ultraviolet irradiation on the cell cycle.

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.

Melanoma x macrophage fusion hybrids acquire increased melanogenesis and metastatic potential: altered N-glycosylation as an underlying mechanism.

We recently reported that a majority of hybrids generated in vitro between weakly metastatic mouse Cloudman S91 melanoma cells and human or mouse macrophages showed enhanced metastatic potential. With few exceptions, hybrids with enhanced metastatic potential also had elevated basal melanin content and increased responsiveness to MSH compared to parental cells. Here we investigated the hybrid melanotic phenotype in more detail, comparing the pigmentary systems of hybrids and parental Cloudman S91 cells by several techniques. Cells were studied by electron microscopy, cell lysates were analyzed for tyrosinase (E.C.1.14.18.1) activity, and melanosomal proteins were analyzed by gel electrophoresis and immunoblotting. Melanosomes in parental Cloudman melanoma cells were few in number and relatively amorphous, whereas those in the hybrids were numerous and heavily pigmented, containing highly organized lattice structures. Both basal and MSH-inducible tyrosinase activities were elevated several fold in hybrids compared to parental cells. Tyrosinase, TRP-2, and LAMP-1 from hybrids migrated more slowly on gels compared to the same proteins from parental melanoma cells, consistent with increased glycosylation. Migration of LAMP-1 from hybrids was similar to that from peritoneal macrophages, which also appeared to be more heavily glycosylated than LAMP-1 from Cloudman cells. By using 3H-glucosamine as a marker of N-glycosylation, its incorporation into tyrosinase and LAMP-1 was found to be elevated in hybrids, suppressed by N-glycosylation inhibitors, and stimulated by MSH to a greater degree in hybrids compared to parental cells. These results indicate N-glycosylation as an important regulatory pathway for MSH-induced melanogenesis and further suggest that altered N-linked glycosylation may be an underlying mechanism for regulation of both melanogenesis and metastasis in macrophage x melanoma hybrids.