The eventual long term scientific goal of this research proposal is to achieve an understanding of the molecular mechanisms which are important for vesicle transport and digestive enzyme secretion leading to invasive activities of prostatic tumor cells. We are searching for ways to destroy tumors more effectively by identifying critical cellular targets (ATPases) and more specifically studying drugs which inhibit the ATPases critical for cell motility events related to tumor invasion. Estramustine, a synthetic product of nor nitrogen mustard and estradiol represents a class of anti-tumor drugs we have worked with whose effectiveness in inhibiting a specific class of ATPases, vesicle transport, enzyme secretion and invasive activities of prostatic tumor cells will be assessed. We have found that a novel microtubule associated cytoplasmic ATPase, termed kinesin powers secretory vesicle streaming along microtubules isolated from DU 145 cells. The motion is inhibited in vivo and in vitro with micromolar levels of estramustine (Piazza and Stearns, Nature, in preparation), and agents (vanadate) which are known to inhibit dynein and kinesin ATPase activity. Because of these preliminary findings, in depth biochemical studies of estramustine and kinesin are planned. Specifically, these include, (a) purification and characterization of prostatic cell kinesin from a variety of tumor cell lines; (b) immunofluorescent and immunogold localization studies of kinesin(s) and assessment of the relevance of such localizations to secretory vesicle motility along microtubules, (c) drug combination studies with estramustine and several analogues in an attempt to establish if ATPase activity and vesicle motility are inhibited and more importantly, to determine if drug combination studies inhibit collagenase secretion by invasive DU 145 cells or primary tumor cell lines established from human prostate biopsies. The results from these studies should establish if different tumor cell lines contain kinesin or novel kinesin(s) important for critical cellular functions during tumor invasion. The effect of estramustine dosages to cell performance will be determined with respect to its affinity for kinesin and inhibitory effects on kinesin ATPase activity and related motility functions in tumor invasion including vesicle transport and secretion.
