Faculty Bibliography

Similarly to p53, p73alpha and p73beta induce growth arrest and/or apoptosis in response to DNA damage or when exogenously expressed. However, how they trigger apoptosis remains unresolved. After stable transduction of either p73alpha or p73beta, a greater apoptotic response was observed for p73beta in both primary and tumor cells. Consistently, blocking ectopic and endogenous p73beta expression by specific shRNA significantly decreased apoptotic levels after DNA damage. We found that p73beta targets the apoptotic program at multiple levels: (i) facilitating caspase activation through p53-dependent signals and (ii) inducing p57KIP2, while down-regulating c-IPA1 and IEX1 through a p53-independent mechanism. p73beta-mediated apoptosis was considerably reduced after inhibition of p57(KIP2) by small interfering RNA, IEX-1 overexpression, and in mouse embryo fibroblasts derived from p57-/- mice. Data from this study offer evidence for the apoptotic activity exclusive of p73beta. In the clinical context, these results might have potential therapeutic implications, because p73beta could induce alternative apoptotic responses in tumors harboring p53 mutations

Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers. These mutations promote tumour development by deregulating the E2F family of transcription factors leading to uncontrolled cell cycle progression. We show that the mitotic checkpoint protein Mad2 is a direct E2F target and, as a consequence, is aberrantly expressed in cells with Rb pathway defects. Concordantly, Mad2 is overexpressed in several tumour types, where it correlates with high E2F activity and poor patient prognosis. Generation of Rb pathway lesions in normal and transformed cells produces aberrant Mad2 expression and mitotic defects leading to aneuploidy, such that elevated Mad2 contributes directly to these defects. These results demonstrate how chromosome instability can arise as a by-product of defects in cell cycle control that compromise the accuracy of mitosis, and suggest a new model to explain the frequent appearance of aneuploidy in human cancer

MAD2 inhibits the anaphase-promoting complex when chromosomes are unattached to the mitotic spindle. It acts as a tumor suppressor gene because MAD2+/-cells enter anaphase early and display chromosome instability, leading to the formation of lung tumors in mice. Complete MAD2 inactivation has not been identified in human tumors, although partial defects are prevalent. By employing RNA interference in human somatic cells, we found that severe reduction of MAD2 protein levels results in mitotic failure and extensive cell death arising from defective spindle formation, incomplete chromosome condensation, and premature mitotic exit leading to multinucleation. Cyclin B is degraded prematurely in the MAD2 short interfering RNA-treated cells but not in MAD2+/- cells, suggesting an explanation for the spindle failure and mitotic catastrophe in the MAD2 knockdown cells. Thus, anaphase-promoting complex substrates exhibit distinct sensitivities in the presence of different MAD2 doses, which in turn determine MAD2's role as either a tumor suppressor or an essential gene

The Mdm2 oncoprotein physically associates with p53 and antagonizes its tumor suppressor functions. Previous studies indicate that some tumors express alternatively or aberrantly spliced Mdm2 variants that are unable to bind p53, but whether these actively contribute to carcinogenesis or are a byproduct of cancer progression has been unclear. In this study, we examined the ability of full-length Mdm2 and several tumor-derived splice variants to modulate tumor development in E micro -myc transgenic mice. We report that several tumor-derived Mdm2 splice variants promote tumorigenesis in a manner that is comparable with full-length Mdm2. Our results imply that the current paradigm for understanding Mdm2 action during oncogenesis is incomplete, and its splice variants contribute to human cancer

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo

BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle

During the metaphase-anaphase transition, the spindle checkpoint prevents segregation of chromosomes if the spindle assembly is perturbed. Critical components of this checkpoint are the MAD and BUB families of proteins, which prevent the proteolysis of Pds1 and B cyclins, producing mitotic arrest. In the present study, we first intended to resolve the role of the hsMAD2 gene in human cancer by determining the potential presence of hsMAD2 mutations in 44 primary bladder tumors, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas. The entire coding region of the hsMAD2 gene was analyzed using PCR-SSCP and sequencing. One of the bladder tumor samples showed a point mutation consisting of a transition, ATC-->GTC (Ile-->Val) in codon 190 of hsMAD2. However, no differences were found in the mitotic arrest between cells transfected with mutant and wild-type MAD2 cDNA. We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder tumors, 3 soft-tissue sarcomas and 1 hepatocellular carcinoma, consistent with a polymorphism at codon 143, CCA-->CCG (Pro-->Pro). Another polymorphism was identified in a hepatocellular carcinoma case at codon 22, GAG-->GAA (Glu-->Glu). In addition, a subgroup of 67 primary tumors was analyzed by Southern blot hybridization. No deletion or visible re-arrangements were detected by comparing tumor and normal DNA band signals. Two other important components of the spindle mitotic checkpoint, hBUB1 and hBUB3, were also screened for mutations: hBUB1 in 43 bladder tumors and 9 bladder cell lines and hBUB3 only in the cell lines. Two polymorphisms were found in hBUB1 at positions 144, CAG-->CAA (Gln-->Gln) in 1 primary tumor and 1 bladder cell line, and 913 (ATC-->ATT, Ile-->Ile) in 1 primary tumor. We did not find sequence alterations in hBUB3. These results suggest that mutations of the hsMAD2, hBUB1 and hBUB3 genes are very rare in bladder tumors and that hsMAD2 alterations are also infrequent in soft-tissue sarcomas and hepatocellular carcinomas

The VP-2 major capsid protein of the prototype strain of the parvovirus minute virus of mice (MVMp) was expressed, using a baculovirus vector, in Sf9 insect cells. Immunogold electron microscopy of infected Sf9 cells showed VP-2 localized in the nucleus and cytoplasm as is observed in mammalian cells during natural infections. The VP-2 subunits self-assembled into empty parvovirus-like particles (VLPs), which appeared morphologically similar to and immunogenically indistinguishable from native empty MVMp particles, which also contain the minor capsid protein, VP1. Incubations under different pH and temperature conditions showed that the MVMp VLPs and native empty MVMp capsids share comparable stability. Once heated the particles can be similarly and specifically cleaved by trypsin at the VP-2 N-terminal domain. This process mimics the further maturation of the 'rat-like' parvovirus virions, following viral DNA encapsidation, indicating that biologically relevant features of the MVMp capsid are maintained in the VLPs. Crystals have been obtained for the MVMp VLPs which were isomorphous to those used for the high-resolution structure determination of virions and native empty particles of the immunosuppressive strain of MVM (MVMi). The VLP crystals diffracted X rays to beyond 3-A resolution and are in space group C2 (a = 448.7, b = 416.6, c = 306.1 A, and beta = 95.9 degrees ). This is the first report of crystals from parvoviral particles produced in a heterologous system diffracting X rays to high resolution, indicating that VP-2 of some parvovirus capsids can self-assemble into ordered T = 1 icosahedral capsids in the absence of other viral and host cell functions