Defining the Role of CtBP2 in p53-Independent Tumor Suppressor Function of ARF: A Dissertation

ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF

Typing of bluetongue virus serotype 1 and 23 by RT-PCR

373-377Two primer pairs were evaluated for amplification of unique regions on the serotype-specific segment 2 (VP2 gene) of bluetongue virus (BTV) by reverse transcription-polymerase chain reaction (RT-PCR). A new set of primer pair [forward primer (FP): 1240-1271 bp and reverse primer (RP): 1844-1813 bp] specific to VP2 gene of BTV serotype 1 (BTV-1) was designed. This primer pair successfully amplified the cell-culture-grown six BTV-1 Indian isolates from different geographical regions, yielding an expected PCR product of 604 bp. However, the primer pair failed to amplify Indian isolates of BTV-18 and BTV-23. Similarly, the primer pair for BTV-23 serotype (FP: 604-623 bp; RP: 1262-1243 bp) amplified 658 bp amplicon with Indian isolates of only BTV-23 and not with the isolates of BTV-1 and BTV-18 serotypes. These VP2 gene based serotype specific primers when aligned with other BTV serotype sequences available in the GenBank revealed no significant sequence identity except with the serotypes for which they were designed. This strongly suggests that the serotype specific primers have potential application for detection of BTV-1 and BTV-23 by using rapid, reliable and specific RT-PCR assay

Restriction analysis of conserved and variable regions of VP2 gene of Indian isolates of bluetongue virus serotype 1

272-276Bluetongue virus (BTV) is a member of Orbivirus genus in family Reoviridae. The virus genome is composed of 10 double-stranded RNA segments. The RNA segment L2 encodes an outer capsid viral protein, VP2, which is the main determinant of neutralization and serotype-specific immune response. BTV serotype 1 (BTV-1) specific novel primer pair was designed using VP2 gene sequences available in GenBank to amplify 1240-1844 bp region because two hypervariable and three conserved regions have been reported within these 604 nucleotides. This primer pair successfully amplified cell culture adapted six Indian isolates of BTV-1 from different geographical regions of the country. The 604 bp PCR product of VP2 gene of all six BTV-1 yielded two fragments of 273 and 331 bp when digested with Taq I restriction enzyme. This indicated that there is only one TaqI site at 1513 bp (within 1240-1844 bp region) of VP2 gene of BTV-1 Indian isolates. The in silico restriction analysis revealed that in BTV-1 South African isolate (BTV-1 SA) there is no TaqI site while in BTV-1 Australian isolates (BTV-1 AUS), there are two TaqI sites (at 1513 and 1567 bp) within 1240-1844 bp region of VP2 gene. The earlier reported VP2 gene based primer pair for BTV-1 was used in the present study to amplify 2242-2933 bp region of six BTV-1 Indian isolates as three conserved regions have been reported within these 691 nucleotides. The digestion of 691 bp PCR products with XmnI yielded three fragments of 364, 173 and 154 bp with all the six Indian isolates of BTV-1 suggesting that there are two XmnI sites within 2242-2933 bp region of VP2 gene. A single XmnI site was observed in silica in BTV-1 AUS and BTV-1SA isolates at different positions within this region. The in vitro and in silico restriction profile analyses of partial VP2 gene sequences using TaqI and XmnI restriction enzymes indicated a close relationship of Indian isolates of BTV-1 with BTV-1 AUS isolates but not with BTYI BTV-1 SA isolate. &nbsp

The alternative reading frame tumor suppressor antagonizes hypoxia-induced cancer cell migration via interaction with the COOH-terminal binding protein corepressor

The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent activities critical to the prevention of cancer in mice and humans. Recent evidence from mouse models suggests that when p53 is absent, further loss of ARF can widen the tumor spectrum, and potentiate invasion and metastasis. A major target of the p53-independent activity of ARF is the COOH-terminal binding protein (CtBP) family of metabolically regulated transcriptional corepressors, which are degraded upon acute exposure to the ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, to repress epithelial and proapoptotic genes, and can mediate hypoxia-induced migration of cancer cells. The possibility that ARF could suppress tumor cell migration as part of its p53-independent activities was thus explored. Small-interfering RNA (siRNA)-mediated knockdown of ARF in human lung carcinoma cells led to increased cell migration, especially during hypoxia, and this effect was blocked by concomitant treatment with CtBP2 siRNA. Introduction of ARF into p53 and ARF-null human colon cancer cells inhibited hypoxia-induced migration. Furthermore, overexpression of CtBP2 in ARF-expressing cells enhanced cell migration, and an ARF mutant defective in CtBP-family binding was impaired in its ability to inhibit cell migration induced by CtBP2. ARF depletion or CtBP2 overexpression was associated with decreased PTEN expression and activation of the phosphatidylinositol 3-kinase pathway, and a phosphatidylinositol 3-kinase inhibitor blocked CtBP2-mediated cell migration. Thus, ARF can suppress cell migration by antagonizing CtBP2 and the phosphatidylinositol 3-kinase pathway, and these data may explain the increased aggressiveness of ARF-null tumors in mouse models

Targeting of C-Terminal Binding Protein (CtBP) by ARF Results in p53-Independent Apoptosis

ARF encodes a potent tumor suppressor that antagonizes MDM2, a negative regulator of p53. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF or p53 singly null mice, results in a broadened tumor spectrum and decreased tumor latency. To investigate the mechanism of p53-independent tumor suppression by ARF, potential interacting proteins were identified by yeast two-hybrid screen. The antiapoptotic transcriptional corepressor C-terminal binding protein 2 (CtBP2) was identified, and ARF interactions with both CtBP1 and CtBP2 were confirmed in vitro and in vivo. Interaction with ARF resulted in proteasome-dependent CtBP degradation. Both ARF-induced CtBP degradation and CtBP small interfering RNA led to p53-independent apoptosis in colon cancer cells. ARF induction of apoptosis was dependent on its ability to interact with CtBP, and reversal of ARF-induced CtBP depletion by CtBP overexpression abrogated ARF-induced apoptosis. CtBP proteins represent putative targets for p53-independent tumor suppression by ARF

Spontaneous expression of embryonic factors and p53 point mutations in aged mesenchymal stem cells: a model of age-related tumorigenesis in mice

Aging is the single most common risk factor for cancer. Peripheral and marrow-derived stem cells are long lived and are candidate cells for the cancer-initiating cell. Repeated rounds of replication are likely required for accumulation of the necessary genetic mutations. Based on the facts that mesenchymal stem cells (MSC) transform with higher frequency than other cell types, and tumors in aged C57BL/6 mice are frequently fibrosarcomas, we used a genetically tagged bone marrow (BM) transplant model to show that aged mice develop MSC-derived fibrosarcomas. We further show that, with aging, MSCs spontaneously transform in culture and, when placed into our mouse model, recapitulated the naturally occurring fibrosarcomas of the aged mice with gene expression changes and p53 mutation similar to the in vivo model. Spontaneously transformed MSCs contribute directly to the tumor, tumor vasculature, and tumor adipose tissue, recruit additional host BM-derived cells (BMDC) to the area, and fuse with the host BMDC. Unfused transformed MSCs act as the cancer stem cell and are able to form tumors in successive mice, whereas fusion restores a nonmalignant phenotype. These data suggest that MSCs may play a key role in age-related tumors, and fusion with host cells restores a nonmalignant phenotype, thereby providing a mechanism for regulating tumor cell activity