Dlk/ZIP kinase-induced apoptosis in human medulloblastoma cells: requirement of the mitochondrial apoptosis pathway

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours. © 2001 Cancer Research Campaign http://www.bjcancer.co

Dissecting the role of p53 phosphorylation in homologous recombination provides new clues for gain-of-function mutants

Regulation of homologous recombination (HR) represents the best-characterized DNA repair function of p53. The role of p53 phosphorylation in DNA repair is largely unknown. Here, we show that wild-type p53 repressed repair of DNA double-strand breaks (DSBs) by HR in a manner partially requiring the ATM/ATR phosphorylation site, serine 15. Cdk-mediated phosphorylation of serine 315 was dispensable for this anti-recombinogenic effect. However, without targeted cleavage of the HR substrate, serine 315 phosphorylation was necessary for the activation of topoisomerase I-dependent HR by p53. Moreover, overexpression of cyclin A1, which mimics the situation in tumors, inappropriately stimulated DSB-induced HR in the presence of oncogenic p53 mutants (not Wtp53). This effect required cyclin A1/cdk-mediated phosphorylation for stable complex formation with topoisomerase I. We conclude that p53 mutants have lost the balance between activation and repression of HR, which results in a net increase of potentially mutagenic DNA rearrangements. Our data provide new insight into the mechanism underlying gain-of-function of mutant p53 in genomic instability

Impaired CK1 Delta Activity Attenuates SV40-Induced Cellular Transformation In Vitro and Mouse Mammary Carcinogenesis In Vivo

Simian virus 40 (SV40) is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP) gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T) mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA repair. Our data show that inactivating mutations in CK1δ impair SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo

Transcriptional Regulation of PP2A-Aα Is Mediated by Multiple Factors Including AP-2α, CREB, ETS-1, and SP-1

Protein phosphatases-2A (PP-2A) is a major serine/threonine phosphatase and accounts for more than 50% serine/threonine phosphatase activity in eukaryotes. The holoenzyme of PP-2A consists of the scaffold A subunit, the catalytic C subunit and the regulatory B subunit. The scaffold subunits, PP2A-Aα/β, provide a platform for both C and B subunits to bind, thus playing a crucial role in providing specific PP-2A activity. Mutation of the two genes encoding PP2A-Aα/β leads to carcinogenesis and likely other human diseases. Regulation of these genes by various factors, both extracellular and intracellular, remains largely unknown. In the present study, we have conducted functional dissection of the promoter of the mouse PP2A-Aα gene. Our results demonstrate that the proximal promoter of the mouse PP2A-Aα gene contains numerous cis-elements for the binding of CREB, ETS-1, AP-2α, SP-1 besides the putative TFIIB binding site (BRE) and the downstream promoter element (DPE). Gel mobility shifting assays revealed that CREB, ETS-1, AP-2α, and SP-1 all bind to PP2A-Aα gene promoter. In vitro mutagenesis and reporter gene activity assays reveal that while SP-1 displays negative regulation, CREB, ETS-1 and AP-2Aα all positively regulate the promoter of the PP2A-Aα gene. ChIP assays further confirm that all the above transcription factors participate the regulation of PP2A-Aα gene promoter. Together, our results reveal that multiple transcription factors regulate the PP2A-Aα gene