Estramustine is an effective drug in the treatment of prostatic cancer, although preclinical and clinical pharmacology has shown that its cytotoxic properties are distinct from its constituent nitrogen mustard/estradiol moieties. In fact, estramustine, as an intact molecule, has an entirely novel mechanism of action, inducing antimicrotubule effects in a diverse range of cells, including prostatic tumor cells, through binding to microtubule associated proteins (MAPs). The long term goal of this continua- tion is to understand which MAPs may be tumor specific in human tissues and to determine how tumor cells in culture acquire resistance to estramustine. Specifically, the following approaches will be adopted: (1) Following surgery, human biopsies of normal and malignant prostate and other tissues will be analyzed by immunohistochemical and immunocytochemical techniques to determine the presence of MAP-lA and a 210 KDa MAP. The former is a known target for estramustine whereas the latter is not and there are indications that differential expression of these MAPs may occur in tumor and normal tissue. (2) By using an existing cDNA probe for MAP-lA and constructing one for the 210 KDa MAP, the effect of estramustine on mRNA levels for each protein will be determined in a DU 145 prostatic carcinoma cell line. (3) Initially in the DU 145 cells and subsequently in other prostatic carcinoma cell lines, estramustine resistance will be induced through sequential selection by drug dose escalation. Established resistant lines will be characterized with respect to karyotype, drug uptake, glutathione S-transferase isoenzymes, estramustine binding protein content, collateral resistance/sensitivity to other antimicrotubule agents and estramustine analogues and quantitative/qualitative MAP content. (4) A number of estramustine analogues will be used in existing experimental approaches in order to determine which part(s) of the estramustine molecule are most critical to the drugs MAP-binding potential. These approaches will assist in determining whether clinical efficacy and/or selectivity for estramustine is based upon the drug degrees MAP binding properties. Of further significance, the drug is already proving to be a valuable tool for probing the role of MAPs in cytoskeletal structure. Continued progress will consolidate and expand the utility of the drug as an aid to cell biologists.
