Lysyl oxidase catalyzes the final extracellular enzymatic step required for cross-linking collagen and elastin in the formation of a mature and functional extracellular matrix. Lysyl oxidase also has tumor suppressor activity in ras-transformed cells. The role of lysyl oxidase enzyme activity in tumor suppression has not been previously investigated. The hypothesis of this proposal is that lysyl oxidase gene expression is required for its tumor suppressor activity, but extracellular lysyl oxidase enzyme activity is not required. Preliminary studies in our laboratory show that phenotypic reversion of c-H-ras-transformed NIH3T3 cells (RS485 cells) by the anti-cancer drug suramin is accompanied by dramatically increased lysyl oxidase mRNA levels, but no increase in enzyme activity. Surprisingly, we found that suramin appears to be a potent inhibitor of lysyl oxidase enzyme activity.
Three specific aims are proposed.
Aim 1 will establish that lysyl oxidase gene expression is specifically required for phenotypic reversion of RS485 cells by suramin. RS485 cells will be stably transfected with an antisense lysyl oxidase expression vector, and the phenotype of transfected clones grown in the presence and absence of suramin will be determined. The hypothesis predicts that antisense- transfected RS485 cells grown in the presence of suramin will contain low lysyl oxidase mRNA levels and will resist phenotypic reversion. Cell phenotype will be characterized by cell morphology changes, growth rate changes, altered anchorage independent growth, and changes in cell cycle kinetics determined by flow cytometry. In addition, selected transformed oral human tumor cell lines, and selected murine tumor cell lines will be screened for suramin-induced phenotypic reversion, and the specific role of lysyl oxidase mRNA will be established using the same antisense approaches.
Aim 2 will rigorously establish that suramin inhibits lysyl oxidase enzyme activity, thus supporting the hypothesis of this proposal. Lysyl oxidase will be purified and suramin inhibition of lysyl oxidase will be characterized as follows: the I50, the reversibility of inhibition, the K1 and kinetics type of inhibition, and whether substrates protect against suramin inhibition.
Aim 3 will investigate the molecular mechanisms of tumor suppression by lysyl oxidase by determining whether inhibitors of pro-lysyl oxidase processing and activation prevent suramin-dependent phenotypic reversion of RS485 cells. The notion being tested is that proteolytic fragments of prolysyl oxidase such as the released propeptide, for example, may cause phenotypic changes by a feed-back mechanism. These studies will provide the novel approaches and new insights into mechanisms of tumor suppression relevant to oral cancer, will establish two separate activities for the lysyl oxidase gene, and will define a new class of lysyl oxidase inhibitor.
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