The use of ATP content as a measurement for cell growth was evaluated in the LNCaP prostatic cancer cell line. ATP content was found to correlate well with cell counts and was an easy and reliable method for following the effect of substances on cell growth. During cultivation for 9 days no effect on cell counts or ATP content could be seen when testosterone (10(-10) to 10(-6) M), estradiol-17 beta (10(-10) to 10(-5) M), 5 alpha-DHT (10(-9) to 10(-6) M), prolactin, vitamin A, or antiandrogen was added to the cell medium in different combinations. However, a weak positive effect was seen on the mitotic index when 10 or 100 nM 5 alpha-DHT was added to the cells, whereas 1 microM 5 alpha-DHT inhibited cell growth. Thus despite the fact that this LNCaP line contained 16 fmol androgen receptor/mg protein (Kd 0.6 nM), it is unresponsive to hormones and should be designated LNCaP-r (resistant). Chromosome analysis revealed that a shift in the modal chromosome number had occurred from the original LNCaP line, which could account for the lack of hormonal sensitivity.

The interaction of the antimitotic agent estramustine with bovine microtubule proteins and purified tubulin was investigated. Direct photoaffinity labeling of microtubule protein with [14C]estramustine resulted in the labeling of both alpha- and beta-tubulin, and this was inhibited with unlabeled estramustine in a dose-dependent manner. [14C]Estramustine was incorporated into both the soluble and polymerized forms of tubulin. The affinity constant for estramustine binding to tubulin was determined by equilibrium dialysis to be 23 +/- 5 mM. Estramustine did not affect [3H]vinblastine binding, and vinblastine had no effect on direct labeling with [14C]estramustine. Both rhizoxin and paclitaxel decreased the covalent labeling of tubulin with [14C]estramustine in a dose-dependent fashion and were noncompetitive inhibitors of the binding of estramustine to tubulin. The binding of colchicine to tubulin was not inhibited by estramustine as detected by fluorescence and DEAE filter assays. The estramustine binding site on tubulin is therefore distinct from that of colchicine and vinblastine and may at least partially overlap with the binding site for paclitaxel. In both bovine brain microtubules and cytoskeletal proteins from human prostatic carcinoma cells, the incorporation of [14C]estramustine into the beta III isotype of tubulin was found to occur with a reduced efficiency compared to that of the other beta-tubulin isotypes and alpha-tubulin. Since this isotype is overexpressed in estramustine resistant human prostate carcinoma cells, these results indicate that beta III-tubulin may play a role in the response to the effects of estramustine.

No abstract

Linomide, a quinoline-3-carboxamide, has growth-inhibitory effects against a series of Dunning R-3327 rat prostatic cancers in vivo [Ichikawa et al.: Cancer Res 52:3022-3028, 1992]. In addition, we have demonstrated that daily linomide treatment can inhibit angiogenic responses in nontumor-bearing rats and reduce tumor blood flow in tumor-bearing rats [Vukanovic et al.: Cancer Res 53:1833, 1993]. In the present study we have demonstrated that the reduced tumor blood flow is due to linomide's ability to inhibit tumor angiogenesis, as documented by decreased number of blood vessels in prostatic carcinomas growing in rats treated daily with linomide. Due to linomide's ability to inhibit tumor angiogenesis, and since tumor angiogenesis is required not only for the growth of the primary cancer but also for its ability to metastasize, the effect of linomide on metastasis was directly tested using a quantitation metastasis assay. These in vivo experiments demonstrated that daily linomide treatment decreased by 3-fold the extent of dissemination of cancer cells to the lungs. To test if this antimetastatic response is due to direct effects of linomide on the metastatic cells themselves as well as an induced effect upon inhibition of tumor angiogenesis, additional studies were performed. These studies demonstrated that linomide is not converted in vivo to metabolite(s) which are directly cytotoxic or cytostatic to the prostatic cancer cells themselves. These studies also demonstrated that linomide does not decrease the attachment, migration, or invasive abilities of metastatic cancer cells. These results suggest that the major mechanism for the antitumor and antimetastatic effects of linomide is via its inhibition of tumor angiogenesis. Additional studies have demonstrated that in vivo linomide treatment results in the apoptotic death of thymocytes. This cytotoxic effect is not required for linomide's antitumor effect, nor is it due to elevated plasma levels of glucocorticoid.