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In JoVE (1)
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Articles by Calley Hirsch in JoVE
JoVE General
मानव प्रेरित pluripotent स्टेम सेल का चयन और अलग कालोनियों वयस्क Fibroblasts से reprogrammed किया
1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute
हम मानव प्रेरित pluripotent स्टेम कोशिकाओं के साथ (hiPSC) रेट्रोवायरल Oct3 / 4, Sox2, Klf4 और ग Myc (OSKM) एन्कोडिंग वैक्टर और जीना धुंधला द्वारा सही ढंग से reprogrammed किया hiPSC की पहचान का उपयोग कर में कुशल मानव दैहिक कोशिकाओं के reprogramming के लिए एक प्रोटोकॉल वर्तमान Tra 1-81 एंटीबॉडी.
Other articles by Calley Hirsch on PubMed
Histone Deacetylase Inhibitors Regulate P21WAF1 Gene Expression at the Post-transcriptional Level in HepG2 Cells
Histone deacetylase inhibitors (HDIs) are thought to act primarily at the level of transcription inducing cell cycle arrest, differentiation and/or apoptosis in many cancer cell types. Induction of the potent cdk/cyclin inhibitor p21WAF1 is a key feature of this HDI mediated transcriptional re-programming phenomenon. However, in the current study we report that HDIs are also capable of inducing p21WAF1 through purely post-transcriptional events, namely increased mRNA stability. These studies highlight our growing appreciation for the complexities of HDI mediated effects and challenge our preconceptions regarding the action of these promising anti-neoplastics.
5-Aza-2'-deoxycytidine (decitabine) Can Relieve P21WAF1 Repression in Human Acute Myeloid Leukemia by a Mechanism Involving Release of Histone Deacetylase 1 (HDAC1) Without Requiring P21WAF1 Promoter Demethylation
Decitabine is a potent demethylating agent that exhibits clinical activity against myeloid malignancies. Numerous genes silenced by hypermethylation are reactivated by decitabine through a mechanism involving promoter demethylation with subsequent release of histone deacetylases (HDACs) and accumulation of acetylated histones. Recent studies indicating that decitabine also induces regional chromatin remodeling of some unmethylated genes suggest additional mechanisms of action. Decitabine reactivates unmethylated p21WAF1 in some AML cell lines but the possible occurrence of p21WAF1 methylation in AML in vivo has not been studied in detail and decitabine effects on p21WAF1 chromatin remodeling have not been reported. We found that p21WAF1 mRNA was undetectable in 6 of 24 AML patient samples and 4 of 5 AML cell lines but there was no evidence of p21WAF1 promoter methylation. However, decitabine induced p21WAF1 in AML cell lines KG-1 and KG-1a in association with release of HDAC1 and increased acetylated histone H3 at the unmethylated p21WAF1 promoter. Decitabine effects on p21WAF1 histone acetylation and induction were enhanced by the HDAC inhibitor trichostatin A and were independent of wild type p53. Our findings indicate that decitabine can relieve p21WAF1 repression in AML by a mechanism that involves release of HDAC1 without requiring promoter demethylation. Furthermore, our study provides evidence that combined decitabine and HDAC inhibitor treatment can enhance chromatin remodeling and reactivation of an unmethylated tumor suppressor gene. This latter finding is of relevance to the clinical use of these agents in AML as we found the p21WAF1 promoter to be unmethylated in vivo.
Src Family Kinase Members Have a Common Response to Histone Deacetylase Inhibitors in Human Colon Cancer Cells
Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c-Src proto-oncogene expression in many cancer cell lines. Activation and/or overexpression of c-Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer-preventive agent. However, c-Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose- and time-dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c-Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.
Multiple Faces of the SAGA Complex
The SAGA complex provides a paradigm for multisubunit histone modifying complexes. Although first characterized as a histone acetyltransferase, because of the Gcn5 subunit, SAGA is now known to contain a second activity, a histone deubiquitinase, as well as subunits important for interactions with transcriptional activators and the general transcription machinery. The functions of SAGA in transcriptional activation are well-established in Saccharomyces cerevisiae. Recent studies in S. pombe, Drosophila, and mammalian systems reveal that SAGA also has important roles in transcript elongation, the regulation of protein stability, and telomere maintenance. These functions are essential for normal embryo development in flies and mice, and mutations or altered expression of SAGA subunits correlate with neurological disease and aggressive cancers in humans.
Histone Deacetylase Inhibitors Mediate Post-transcriptional Regulation of P21WAF1 Through Novel Cis-acting Elements in the 3' Untranslated Region
Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21(WAF1). However, HDACIs primarily increase p21(WAF1) expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis-acting elements in the 3' untranslated region (UTR) of p21(WAF1) are identified that control the ability of HDACIs to induce p21(WAF1) mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation.
Ubp8 and SAGA Regulate Snf1 AMP Kinase Activity
Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well-studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels.
