Recent clinicopathologic studies have shown that many prostatic adenonomas express focal neuroendocrine differentiation and that neuroendocrine differentiation is most apparent in advanced anaplastic tumors. While studying growth-regulatory signal nsduction events in human prostate carcinoma cell lines, we found that in two of four cell lines, the androgen-sensitive line LNCaP and the highly metastatic androgen-independent line PC-3-M, elevation of cAMP through addition of cAMP analogues or phosphodiesterase inhibitors induced a markediy neuronal morphology. Also in LNCaP cells ultrtructural analysis showed that cAMP induced the appearance of neurosecretory cell-like dense-core granules. Phenotypic analysis of untreated LNCaP and PC-3-M cells showed that both cell lines express markers of the neural crest incuding S-100, chromogranin A, pp60 , and neuron-specific enolase as well as the epithelial marker KS1/4 and stage-specific embryonic antigen 4. In PC-3-M cells, cAMP markedly elevated neuron-specific enolase protein and caused an increase in the specific activity of the neuroendocrine marker ppWw, and in both cell lines expression ofKS1/4 and stage-specific embryonic antigen 4 was down-regulated. In addiion to effects on lineage markers, cAMP treatment induced GI synchronization, growth arrest, and loss of clonogenicity, indicating terminal differentiation. Our data provide direct evidence of plastic in the lineag commitment of adenocarcinoma of the prostate. We have shown that cellpermeant cAMP analogue can induce terminal differentiation, suggesting that hydrolysis-resistant cyclic nucleotides may present an additional approach to the treatment of advanced prostate cancer.In an effort to develop new anticancer drugs directed at unique aspects of prostate cancer biology, we have been studying the signal transduction pathways regulating the growth of human prostate adenocarcinoma cells in vitro. As reported previously, we found that addition of dibutyryl (db) cAMP to the androgen-independent prostate carcinoma cell line PC-3 causes induction of type (32 transforming growth factor (TGF-g32) mRNA, production of bioactive TGF-P2, and growth arrest (1).We have subsequently studied the effect of cAMP derivatives and phosphodiesterase inhibitors on the other two commonly available prostate carcinoma cell lines, DU 145 and LNCaP, as well as the highly metastatic variant of PC-3, PC-3-M, and found that all lines were growth inhibited by elevation of intracellular cAMP (data not shown). Data presented here demonstrate that in two ofthese lines, LNCaP and PC-3-M, elevation of intracellular cAMP induces permanent conversion from an epithelial to a neuronal morphology and that these cells express markers of the neuroendocrine phenotype. These data suggest that these cell lines, which are derived from metastatic adenocarcinoma of the prostate, contain or consist of multipotent cells capable of both neuroendocrine and epithelial differentiation.

Progression through the cell cycle is controlled by the induction of cyclins and the activation of cognate cyclin-dependent kinases. The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor lovastatin induces growth arrest and cell death in certain cancer cell types. We have pursued the mechanism of growth arrest in PC-3-M cells, a p53-null human prostate carcinoma cell line. Lovastatin treatment increased protein and mRNA levels of the cyclin-dependent kinase inhibitor p21 WAF1/CIP1, increased binding of p21 with Cdk2, markedly inhibited cyclin E-and Cdk2-associated phosphorylation of histone H1 or GST-retinoblastoma protein, enhanced binding of the retinoblastoma protein to the transcription factor E2F-1 in vivo, and induced the activation of a p21 promoter reporter construct. By using p21 promoter deletion constructs, the lovastatin-responsive element was mapped to a region between ؊93 and ؊64 relative to the transcription start site. Promoter mutation analysis indicated that the lovastatin-responsive site coincided with the previously identified transforming growth factor-␤-responsive element. These data indicate that in human prostate carcinoma cells an inhibitor of the HMG-CoA reductase pathway can circumvent the loss of wild-type p53 function and induce critical downstream regulatory events leading to transcriptional activation of p21.Cell cycle progression is controlled by the orderly activation of cyclin-dependent kinases (Cdks) 1 (1-4). Cdk activation is regulated at multiple levels including the induction and degradation of cyclin protein, Cdk phosphorylation by CAK, the cyclin-activating kinase, and the synthesis of Cdk-inhibitory proteins (5). Cdk inhibitors can negatively regulate cell cycle progression in response to a variety of antiproliferative signals including DNA damage, differentiation, contact inhibition, and senescence (6 -8). p21 WAF1/CIP1 , the first Cdk inhibitor to be identified, is required for p53-induced growth arrest (9). The p21 protein negatively regulates cell cycle progression by inhibiting Cdk catalytic activity and by inhibiting proliferating cell nuclear antigen, which is required for DNA synthesis (10). By inhibiting the activity of G 1 cyclin-Cdk complexes, p21 blocks phosphorylation of the retinoblastoma protein (pRB). This leaves pRB in its active, hypophosphorylated form, in which it is able to induce G 1 arrest through negative regulation of the heterodimeric transcription factor E2F/DP (11-13).We have found that human prostate carcinoma cells are very sensitive to the induction of growth arrest and cell death by the HMG-CoA reductase inhibitor lovastatin (14).2 Lovastatin blocks the rate-limiting step in the cholesterol-synthesis pathway, the formation of mevalonic acid from HMG-CoA (15-17). The study presented here investigates the mechanism by which lovastatin induces cell cycle arrest in the androgen-independent prostate carcinoma line PC-3-M, which is lacking both p53 alleles (18). The data show that lovastatin treatment caused a marked shift of pR...

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The signalling pathway leading to an activation of mitogen-activated protein (MAP) kinase subtypes Erk-1 and -2 upon stimulation of muscarinic receptor with carbachol in human neuroblastoma SK-N-BE2(C) cells was investigated. Carbachol activated Erk-1/-2 by stimulating M3 muscarinic receptor, as determined by specific antagonists for individual muscarinic receptors. The activation of Erk-1/-2 by carbachol was blocked by the inhibition or down-regulation of protein kinase C (PKC). Among the multiple PKC isoforms expressed in SK-N-BE2(C) cells, only PKCepsilon was activated by the treatment of carbachol, and selective down-regulation of PKCepsilon was sufficient to block Erk-1/-2 activation. Carbachol treatment induced activation of the serine/threonine protein kinase Raf, and an inhibition of Raf blocked Erk-1/-2 activation. Ectopic expression of inhibitory small GTPase Ras, RasN17, blocked the carbachol-induced Raf activation without affecting the activation of PKCepsilon, while the inhibition of PKC blocked the Raf activation. Thus, these results suggest that carbachol-induced activation of PKCepsilon mediates Erk-1/-2 activation by a sequential activation of Ras, Raf and MAP kinase kinase.

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