""""""""Basic and translational research has investigated the role of nm23 in the regulation of tumor metastasis. Five transfection studies have documented that overexpression of nm23 cDNA in either breast carcinoma or melanoma cell lines results in a 50-90% decrease in tumor metastatic potential in vivo. The biochemical mechanism whereby Nm23 suppresses metastatic potential is under investigation. We have transfected site directed mutants of nm23-H1 into breast carcinoma cell lines and examined their in vitro motility to correlate Nm23 structure and biological function. Two mutations abrogated the tumor cell motility suppressive capacity of Nm23: proline 96, the killer of prune mutation in the Drosophila nm23 homolog, which can cause aberrant differentiation; serine 120, a site of mutation in human Stage IV neuroblastomas, and phosphorylation. In vitro assays of purified wild-type and mutant Nm23-H1 proteins found that the proline 96 and serine 120 mutant proteins were uniquely deficient in aspects of histidine-dependent protein phosphotransferase pathways. Histidine protein kinases are poorly described in mammalian cells, but form the """"""""""""""""two-component"""""""""""""""" or histidyl-aspartyl phosphorelay signal transduction system in prokaryotes. We have recently identified a 43 kDa protein from a bovine brain lysate which is a substrate of Nm23-H1 histidine protein kinase and, like two-component systems, exhibits an aspartate phosphorylation. Moreover, neither the Proline 96 nor serine 120 mutants of Nm23-H1 were capable of completing this reaction. These data suggest that (1) a histidine-dependent protein phosphotransferase activity may underlie Nm23-H1's suppression of tumor metastasis, and (2) two-component-like phosphorelay pathways may exist in mammalian cells, and may be germane to developmental or metastatic processes. We have identifed three proteins with homology to plant two component response receivers, and have preliminary data that Nm23-H1 co-immunoprecipiates with one of them. Two translational projects are underway: First, we have identified the promoter for nm23- H1, and the portion which determines whether breast carcinoma cell lines of varying metastatic potentials express high or low Nm23 levels. No mutations were detected in this region. Two regulatory mechanism are operative: A 250 bp region of the promoter which contains a set of mammary specific transcription factors appears to be differentially utilized. Second, differences in the methylation status of two CpG islands were detected in some breast carcinoma cell lines and,in these lines, incubation of cells with deazadeoxycytidine increased Nm23-H1 expression. Second, using the COMPARE computer program in collaboration with DTP, we have used Nm23 as a marker to identify novel compounds with preferential in vitro inhibitory activity against the most aggressive human breast and melanoma cell lines. One of these compounds, NSC 680718, exhibited systemic in vivo activity in the hollow fiber assay against human tumor cell lines traditionally difficult to treat by conventional chemotherapy, including metastatic breast carcinomas, non-small cell lung carcinoma, colon carcinoma and melanoma. In limited xenograft assays NSC 680718 showed anti-tumor activity against metastatic breast carcinoma and melanoma, but its optimal activity at the lowest dose tested suggested poor solubilization. The compound received Decision Network IIA approval for continued experimentation into solubility, formulation and toxicity.""""""""
Showing the most recent 10 out of 19 publications
