Conditional immortalization of primary adipocyte precursor cells.
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ABSTRACT:
SUBMITTER: Church C
PROVIDER: S-EPMC4496986 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells, and thus represent attractive drug targets for treating cancer.
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Project description:To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.
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Project description:Cardiac tissue engineering will remain only a prospect unless large numbers of therapeutic cells can be provided, either from small samples of cardiac cells or from stem cell sources. In contrast to most adult cells, cardiomyocytes are terminally differentiated and cannot be expanded in culture. We explored the feasibility of enabling the in vitro expansion of primary neonatal rat cardiomyocytes by lentivector-mediated cell immortalization, and then reverting the phenotype of the expanded cells back to the cardiomyocyte state. Primary rat cardiomyocytes were transduced with simian virus 40 large T antigen (TAg), or with Bmi-1 followed by the human telomerase reverse transcriptase (hTERT) gene; the cells were expanded; and the transduced genes were removed by adenoviral vector expressing Cre recombinase. The TAg gene was more efficient in cell transduction than the Bmi-1/hTERT gene, based on the rate of cell proliferation. Immortalized cells exhibited the morphological features of dedifferentiation (increased vimentin expression, and reduced expression of troponin I and Nkx2.5) along with the continued expression of cardiac markers (alpha-actin, connexin-43, and calcium transients). After the immortalization was reversed, cells returned to their differentiated state. This strategy for controlled expansion of primary cardiomyocytes by gene transfer has potential for providing large amounts of a patient's own cardiomyocytes for cell therapy, and the cardiomyocytes derived by this method could be a useful cellular model by which to study cardiogenesis.
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Project description:ObjectiveExpression of zinc finger protein 423 (ZFP423), a key proadipogenic transcription factor in adipocyte precursor cells, is regulated by interaction of the proadipogenic early B-cell factor 1 (EBF1) and antiadipogenic ZFP521. The ubiquitin ligase seven-in-absentia homolog 2 (SIAH2) targets ZFP521 for degradation. This study asked whether SIAH2 is expressed in adipocyte precursor cells and whether SIAH2 interacts with ZFP521 and EBF1 to regulate ZFP521 protein levels during adipogenesis.MethodsSIAH2 expression in precursor cells was assessed in primary cells and tissues from wild-type and SIAH2 null mice fed a control or high-fat diet. Primary cells, 3T3-L1 preadipocytes, and HEK293T cells were used to analyze Siah2, Ebf1, and Zfp521 expression and SIAH2-mediated changes in ZFP521 and EBF1 protein levels.ResultsSiah2 is expressed in platelet-derived growth factor receptor α (PDGFRα)+ and stem cell antigen-1 (SCA1)+ adipocyte precursor cells. SIAH2 depletion reduces Ebf1 gene expression and increases EBF1 protein levels in early but not late adipogenesis. In early adipogenesis, SIAH2 forms a protein complex with EBF1 and ZFP521 to enhance SIAH2-mediated ubiquitylation and degradation of ZFP521 while increasing EBF1 protein levels.ConclusionsSiah2 is expressed in PDGFRα+ adipocyte precursor cells and is linked to precursor cell commitment to adipogenesis by interacting with EBF1 and ZFP521 proteins to target the antiadipogenic ZFP521 for degradation.
| S-EPMC7902405 | biostudies-literature