Sir Peter MacCallum Department of Oncology - Research Publications
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ItemNo Preview AvailableNormal p53 function in primary cells deficient for Siah genes(AMER SOC MICROBIOLOGY, 2002-12)Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G(1) phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.
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ItemNo Preview AvailableThe yeast inositol polyphosphate 5-phosphatases Inp52p and Inp53p translocate to actin patches following hyperosmotic stress: Mechanism for regulating phosphatidylinositol 4,5-bisphosphate at plasma membrane invaginations(AMER SOC MICROBIOLOGY, 2000-12)The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] and PtdIns(3, 5)P(2). Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P(2) 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P(2) and PtdIns(4,5)P(2) at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.
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ItemNo Preview AvailableGeneration and analysis of Siah2 mutant mice(AMER SOC MICROBIOLOGY, 2003-12)Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.
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ItemNo Preview AvailableThe ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice(AMER SOC MICROBIOLOGY, 2002-04)The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.
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ItemNo Preview AvailablemTOR-Dependent regulation of ribosomal gene transcription requires S6K1 and is mediated by phosphorylation of the carboxy-terminal activation domain of the nucleolar transcription factor UBF(AMER SOC MICROBIOLOGY, 2003-12)Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.
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ItemNo Preview AvailableVascular endothelial growth factor d is dispensable for development of the lymphatic system(AMER SOC MICROBIOLOGY, 2005-03)Vascular endothelial growth factor receptor 3 (Vegfr-3) is a tyrosine kinase that is expressed on the lymphatic endothelium and that signals for the growth of the lymphatic vessels (lymphangiogenesis). Vegf-d, a secreted glycoprotein, is one of two known activating ligands for Vegfr-3, the other being Vegf-c. Vegf-d stimulates lymphangiogenesis in tissues and tumors; however, its role in embryonic development was previously unknown. Here we report the generation and analysis of mutant mice deficient for Vegf-d. Vegf-d-deficient mice were healthy and fertile, had normal body mass, and displayed no pathologic changes consistent with a defect in lymphatic function. The lungs, sites of strong Vegf-d gene expression during embryogenesis in wild-type mice, were normal in Vegf-d-deficient mice with respect to tissue mass and morphology, except that the abundance of the lymphatics adjacent to bronchioles was slightly reduced. Dye uptake experiments indicated that large lymphatics under the skin were present in normal locations and were functional. Smaller dermal lymphatics were similar in number, location, and function to those in wild-type controls. The lack of a profound lymphatic phenotype in Vegf-d-deficient mice suggests that Vegf-d does not play a major role in lymphatic development or that Vegf-c or another, as-yet-unknown activating Vegfr-3 ligand can compensate for Vegf-d during development.
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ItemTargeting mTOR-dependent with specific inhibitors: a model for personalized medicine based on molecular diagnoses(MULTIMED INC, 2009-02)Cancer cells are characterized by aberrant growth arising from deregulated signalling pathways. The mammalian target of rapamycin (mTOR) pathway integrates multiple growth signals coming from both intracellular and extracellular cues. In this short review, we summarize what is known about the efficacy of targeting the mTOR pathway to treat cancer patients, and we explain the rationale behind promising new inhibitors that could show more potent tumour growth inhibition than did the first generation of these drugs.
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ItemDifferential expression of selected histone modifier genes in human solid cancers(BMC, 2006-04-25)BACKGROUND: Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Here we report characteristic patterns of expression of 12 members of 3 classes of chromatin modifier genes in 6 different cancer types: histone acetyltransferases (HATs)- EP300, CREBBP, and PCAF; histone deacetylases (HDACs)- HDAC1, HDAC2, HDAC4, HDAC5, HDAC7A, and SIRT1; and histone methyltransferases (HMTs)- SUV39H1and SUV39H2. Expression of each gene in 225 samples (135 primary tumours, 47 cancer cell lines, and 43 normal tissues) was analysedby QRT-PCR, normalized with 8 housekeeping genes, and given as a ratio by comparison with a universal reference RNA. RESULTS: This involved a total of 13,000 PCR assays allowing for rigorous analysis by fitting a linear regression model to the data. Mutation analysis of HDAC1, HDAC2, SUV39H1, and SUV39H2 revealed only two out of 181 cancer samples (both cell lines) with significant coding-sequence alterations. Supervised analysis and Independent Component Analysis showed that expression of many of these genes was able to discriminate tumour samples from their normal counterparts. Clustering based on the normalized expression ratios of the 12 genes also showed that most samples were grouped according to tissue type. Using a linear discriminant classifier and internal cross-validation revealed that with as few as 5 of the 12 genes, SIRT1, CREBBP, HDAC7A, HDAC5 and PCAF, most samples were correctly assigned. CONCLUSION: The expression patterns of HATs, HDACs, and HMTs suggest these genes are important in neoplastic transformation and have characteristic patterns of expression depending on tissue of origin, with implications for potential clinical application.
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ItemA mouse with a loss-of-function mutation in the c-Cbl TKB domain shows perturbed thymocyte signaling without enhancing the activity of the ZAP-70 tyrosine kinase(ROCKEFELLER UNIV PRESS, 2003-02-17)The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl-mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4(+)CD8(+) double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice.
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ItemCbl associates with Pyk2 and Src to regulate Src kinase activity, αvβ3 integrin-mediated signaling, cell adhesion, and osteoclast motility(ROCKEFELLER UNIV PRESS, 2001-01-08)The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.