Results from our laboratory and others over the past decade have indicated that the defect of altered methionine metabolism and unbalanced transmethylation is a candidate for a general biochemical alteration in cancer. The alteration is manifested frequently as methionine dependence which is the inability of cancer cells to proliferate when methionine is replaced by its immediate precursor homocysteine in the growth medium (Met- Hcy+medium). Related to methionine dependence is the elevated rate of overall transmethylation resulting in depleted pools of free-methionine and S-adenosylmethionine and an excess of S- adenosylhomocysteine in cancer cells incubated Met-Hcy+- medium. The defects, manifested in one or more of the above forms, occur in all types of cancer cells tested at 100% frequency as shown in our laboratory. Important new data from our laboratory has shown that lung, colon, breast and other tumor types are very frequently methionine dependent when cultured directly from surgery or biopsy in our newly developed three- dimensional, gel-supported, primary culture system. These data indicate that methionine dependence may be an important component of clinical cancer. We propose here to greatly expand our survey of methionine dependence on all the major human cancer types in primary culture including those of the lung, breast, colon, ovary, melanoma and other important human cancer types, all of which are able to be cultured at high frequency in the above-mentioned system. We have also recently developed a procedure whereby methionine dependence can be exploited to enhance selectivity of cell-cycle-specific chemotherapeutic drugs. The fact that the methionine dependency results in a reversible cell-cycle arrest in late-S/G2 in cancer cells when they are incubated in Met-Hcy+ medium is the basis of methionine- dependent chemotherapy. In methionine-dependent chemotherapy the use of cell-cycle specific drugs has been highly successful in eliminating methionine-dependent human tumor cell lines from co-cultures with normal cells while allowing the normal cells to flourish. We propose here to determine if tumors taken directly from surgery in primary culture are also susceptible to methionine-dependent chemotherapy with the idea of eventually using this approach as a new rational selective mans of therapy of human solid tumors. To understand the genetic basis of methionine dependence, cDNAs from normal cell types and methionine independent revertants we have isolated will be cloned in expression vectors. The recombinant plasmids will be transfected into methionine-dependent human cancer cells in Met-Hcy+ medium to induce methionine-independence in order to select genes involved in regulating methionine dependence. This experiment will serve as a means to understand the molecular basis of this important aspect of oncogenic transformation.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Pathology B Study Section (PTHB)
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University of California San Diego
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