Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the Nterminal transactivation domain (DNp73) was described in mice. In this study, we report the cloning and characterisation of the human DNp73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human DNp73 lacks the transactivation domain and starts with an alternative exon (exon 3'). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, DNp73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the DNp73 is strongly upregulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of DNp73 is exerted through a p53 responsive element located on the DN promoter. Expression of DNp73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of DNp73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of DNp73 in primary tumours would clarify this issue. Cell Death and Differentiation (2001) 8, 1213 ± 1223.

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.

We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1␣ and the new isoforms, hDMP1␤ and hDMP1␥. The hDMP1␣, -␤, and -␥ splice variants have unique expression patterns in normal hematopoietic cells; hDMP1␤ mRNA transcripts are strongly expressed in quiescent CD34؉ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1␣. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1␤ transcripts, and hDMP1␥ is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19 ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1␣ but not hDMP1␤ and -␥, is a transcriptional activator. Furthermore, hDMP1␤ was found to inhibit the CD13/ APN promoter transactivation ability of hDMP1␣. Stable, ectopic expression of hDMP1␤ and, to a lesser extent hDMP1␥, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1␤ altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1␤ antagonizes hDMP1␣ activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels.

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...

Caspase-3 activity has been described to be essential for druginduced apoptosis. Recent results suggest that in addition to its downstream executor function, caspase-3 is also involved in the processing of upstream caspase-8 and -9. To test the absolute requirement for caspase-3, we examined mitomycin C (MMC)-induced apoptosis in the caspase-3 deficient human breast cancer cell line MCF-7. MMC was used as anticancer drug since this agent was preferentially active compared to chemotherapeutic compounds with differing mechanisms of action such as cisplatin, docetaxel, or lovastatin. MMC treatment led to pronounced caspase-8, -9, and -7 processing and early morphological features of apoptosis within 48 h. This could be inhibited by the broad-spectrum caspase inhibitor z-VAD.fmk and to a lesser extent by z-IETD.fmk and z-LEHD.fmk, which have a certain preference for inhibiting caspase-8 and -9, respectively. MMC induced apoptosis in MCF-7 cells was not mediated by the death receptor pathway as demonstrated by experiments using the inhibiting anti-Fas antibody ZB4 and transfections with CrmA, a viral serpin inhibitor of caspase-8, and the dominant negative Fasassociated death domain (FADD-DN). Stable expression with Bcl-2 significantly prevented the processing of caspase-9 but also of caspase-8 and blocked the induction of apoptosis. Thus, we provide evidence that caspase-3 activity is dispensable for MMC-induced apoptosis and for caspase-8 and -9 processing in MCF-7 cells.