Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells.
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ABSTRACT:
SUBMITTER: Engstrom W
PROVIDER: S-EPMC1152305 | biostudies-other | 1983 Sep
REPOSITORIES: biostudies-other
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Project description:This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells. Quantitative comparative analyses of episomal and in situ chromosomal replication origin activity as well as chromatin immunoprecipitation (ChIP) assays, using specific antibodies targeting members of the pre-replication complex (pre-RC) as well as open/closed chromatin markers encompassing both episomal and chromosomal origins, revealed that episomal origins had similar levels of in vivo activity, nascent DNA abundance, pre-RC protein association, and elevated open chromatin structure at the origin in both cell types. In contrast, the chromosomal origins corresponding to 20mer1, 20mer2, and c-myc displayed a 2- to 3-fold higher activity and pre-RC protein abundance as well as higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited similar levels of activity, pre-RC protein abundance, and higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in both cell types. Nucleosomal positioning analysis, using an MNase-Southern blot assay, showed that all the origin regions examined were situated within regions of inconsistently positioned nucleosomes, with the nucleosomes being spaced farther apart from each other prior to the onset of S phase in both cell types. Overall, the results indicate that cellular transformation is associated with differential epigenetic regulation, whereby chromatin structure is more open, rendering replication origins more accessible to initiator proteins, thus allowing increased origin activity.
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Project description:Using libraries of replication origins generated previously, we identified three clones that supported the autonomous replication of their respective plasmids in transformed, but not in normal cells. Assessment of their in vivo replication activity by in situ chromosomal DNA replication assays revealed that the chromosomal loci corresponding to these clones coincided with chromosomal replication origins in all cell lines, which were more active by 2-3-fold in the transformed by comparison to the normal cells. Evaluation of pre-replication complex (pre-RC) protein abundance at these origins in transformed and normal cells by chromatin immunoprecipitation assays, using anti-ORC2, -cdc6 and -cdt1 antibodies, showed that they were bound by these pre-RC proteins in all cell lines, but a 2-3-fold higher abundance was observed in the transformed by comparison to the normal cells. Electrophoretic mobility shift assays (EMSAs) performed on the most efficiently replicating clone, using nuclear extracts from the transformed and normal cells, revealed the presence of a DNA replication complex in transformed cells, which was barely detectable in normal cells. Subsequent supershift EMSAs suggested the presence of transformation-specific complexes. Mass spectrometric analysis of these complexes revealed potential new protein players involved in DNA replication that appear to correlate with cellular transformation.
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Project description:Virulent Agrobacterium tumefaciens strains integrate their T-DNA into the plant genome where the encoded agrobacterial oncogenes are expressed and cause crown gall disease. Essential for crown gall development are IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) and Ipt (isopentenyl transferase), which encode enzymes for the biosynthesis of auxin (IaaH, IaaM) and cytokinin (Ipt). Although these oncogenes are well studied as the tumor-inducing principle, nothing is known about the regulation of oncogene expression in plant cells. Our studies show that the intergenic regions (IGRs) between the coding sequences (CDS) of the three oncogenes function as promoters in plant cells. These promoters possess a eukaryotic sequence organization and cis-regulatory elements for the binding of plant transcription factors. WRKY18, WRKY40, WRKY60 and ARF5 were identified as activators of the Ipt promoter whereas IaaH and IaaM is constitutively expressed and no transcription factor further activates their promoters. Consistent with these results, the wrky triple mutant plants in particular, develops smaller crown galls than wild-type and exhibits a reduced Ipt transcription, despite the presence of an intact ARF5 gene. WRKY40 and WRKY60 gene expression is induced by A. tumefaciens within a few hours whereas the ARF5 gene is transcribed later during crown gall development. The WRKY proteins interact with ARF5 in the plant nucleus, but only WRKY40 together with ARF5 synergistically boosts the activation of the Ipt promoter in an auxin-dependent manner. From our data, we propose that A. tumefaciens initially induces WRKY40 gene expression as a pathogen defense response of the host cell. The WRKY protein is recruited to induce Ipt expression, which initiates cytokinin-dependent host cell division. With increasing auxin levels triggered by ubiquitous expression of IaaH and IaaM, ARF5 is activated and interacts with WRKY40 to potentiate Ipt expression and balance cytokinin and auxin levels for further cell proliferation.
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Project description:Histone deacetylase inhibitors (HDACi) developed as anti-cancer agents have a high degree of selectivity for killing cancer cells. HDACi induce acetylation of histones and nonhistone proteins, which affect gene expression, cell cycle progression, cell migration, and cell death. The mechanism of the tumor selective action of HDACi is unclear. Here, we show that the HDACi, vorinostat (Suberoylanilide hydroxamic acid, SAHA), induces DNA double-strand breaks (DSBs) in normal (HFS) and cancer (LNCaP, A549) cells. Normal cells in contrast to cancer cells repair the DSBs despite continued culture with vorinostat. In transformed cells, phosphorylated H2AX (gammaH2AX), a marker of DNA DSBs, levels increased with continued culture with vorinostat, whereas in normal cells, this marker decreased with time. Vorinostat induced the accumulation of acetylated histones within 30 min, which could alter chromatin structure-exposing DNA to damage. After a 24-h culture of cells with vorinostat, and reculture without the HDACi, gammaH2AX was undetectable by 2 h in normal cells, while persisting in transformed cells for the duration of culture. Further, we found that vorinostat suppressed DNA DSB repair proteins, e.g., RAD50, MRE11, in cancer but not normal cells. Thus, the HDACi, vorinostat, induces DNA damage which normal but not cancer cells can repair. This DNA damage is associated with cancer cell death. These findings can explain, in part, the selectivity of vorinostat in causing cancer cell death at concentrations that cause little or no normal cell death.
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Project description:IntroductionCholine is an essential human nutrient that is particular important for proliferating cells, and altered choline metabolism has been associated with cancer transformation. Yet, the various metabolic fates of choline in proliferating cells have not been investigated systematically.ObjectivesThis study aims to map the metabolic products of choline in normal and cancerous proliferating cells.MethodsWe performed 13C-choline tracing followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of metabolic products in normal and in vitro-transformed (tumor-forming) epithelial cells, and also in tumor-derived cancer cell lines. Selected metabolites were quantified by internal standards.ResultsUntargeted analysis revealed 121 LCMS peaks that were 13C-labeled from choline, including various phospholipid species, but also previously unknown products such as monomethyl- and dimethyl-ethanolamines. Interestingly, we observed formation of betaine from choline specifically in tumor-derived cells. Expression of choline dehydrogenase (CHDH), which catalyzes the first step of betaine synthesis, correlated with betaine synthesis across the cell lines studied. RNAi silencing of CHDH did not affect cell proliferation, although we observed an increased fraction of G2M phase cells with some RNAi sequences, suggesting that CHDH and its product betaine may play a role in cell cycle progression. Betaine cell concentration was around 10 µM, arguing against an osmotic function, and was not used as a methyl donor. The function of betaine in these tumor-derived cells is presently unknown.ConclusionThis study identifies novel metabolites of choline in cancer and normal cell lines, and reveals altered choline metabolism in cancer cells.
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Project description:Transplanted adult bone marrow-derived cells (BMDCs) have been shown to adopt the phenotype and function of several nonhematopoietic cell lineages and promote tumorigenesis. Beyond its cancer enhancing potential, cell fusion has recently emerged as an explanation of how BMDCs regenerate diseased heptocytes, contribute to Purkinje neurons and skeletal and cardiac muscle cells, and participate in skin and heart regeneration. Although bone marrow-derived epithelial cells also have been observed in the intestine, fusion as a mechanism has not been investigated. Here, we show that transplanted BMDCs fuse with both normal and neoplastic intestinal epithelium. Long-term repopulation by donor-derived cells was detected in all principal intestinal epithelial lineages including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells, suggesting that the fusion partners of the BMDCs are long-lived intestinal progenitors or stem cells. Fusion of BMDCs with neoplastic epithelium did not result in tumor initiation. Our findings suggest an unexpected role for BMDCs in both regeneration and tumorigenesis of the intestine.
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