The costs of publication of this article were defrayed in part by the payment of page charges.Serumfree DMEM with mgliter unlabeled methionine was added to each plate in the presence or absence of M PKI.Pronase E, washed again, and filtered through a m nylon mesh. C, immunoblots of lysates from LAPC xenograft tumors harvested hafter the fifth daily dose of PKI. Mean tumor volumes at Revefenacin treatment begin was equal between groups.Statistical analyses comparing fold increases between groups were performed on the natural logarithms of the tumor volumes corrected for baseline volumes.Studentsttest was used for comparison of two groups.Mice bearing LAPC or LAPC xenograft tumors were treated daily for weeks with mgkg PKI versus vehicle. Data are expressed as fold tumor volume compared with day; bars, SE.B, top panel, testosterone rescues growth inhibition by PKI in castrated animals.Castrated male mice bearing androgenindependent xenografts were randomized to four treatment groups. Castrated male mice bearing LAPC xenograft tumors were treated with PKI testosterone for days, and EGF were harvested min later.Displayed are immunoblots for PY, ERK, and total ERK.Bottom panel, testosterone does not affect PKI bioavailability in mice.PKI Thujone levels in plasma were determined by reversedphase highperformance liquid chromatography in castrated mice after days of treatment with PKI testosterone.Blood and tumors were collected hafter the last dose of PKI and displayed as mean values. C, AW augments inhibitory effect of PKI on LAPC xenografts in intact male mice.Surgical castration was performed on the same day as PKI treatment was started.Data are expressed as fold tumor volume compared with day. Similarly sized bands have been observed previously after treatment of A cells with the lysosomal inhibitor methylamine and presumably represent an intermediate step in receptor degradation.SCID mice bearing tumors from the human prostate cancer xenografts LAPC or from the A cell line were treated for days with, and mgkg of PKI, and tumor tissue was harvested hafter the last dose was administered.Similar results were obtained in mice bearing LAPC or A xenografts. Androgenindependent sublines of the prostate cancer xenografts grown in castrated host mice were consistently more sensitive to growth inhibition by PKI than androgendependent sublines of the same xenograft growing in intact male. This observation was confirmed in multiple experiments and noted in both LAPC and LAPC xenografts. In both LAPC and LAPC xenografts, androgen addback partially rescued the growth inhibitory effects of PKI. To test this hypothesis, we randomized intact male SCID mice bearing the LAPC xenograft to four treatment groups.Compared with vehicletreated mice, AW by surgical castration slowed the growth of LAPC tumors. Growth inhibition by PKI given at mgkg daily did not reach statistical significance.However, the combination of PKI with AW resulted in nearly complete growth suppression. The difference between the combined treatment group and any of the other three treatment groups was highly statistically significant. The LAPC and LAPC xenografts have been passaged in mice over multiple generations, and various androgendependent and androgenindependent subclones derived from the original parental lines have been maintained independently.In the course of these studies we noted that subclones derived from the same parental line occasionally displayed differences in their sensitivity to PKI.

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