RESUMEN
Recapitulating radioresistant cell features in pertinent cell line models is essential for deciphering fundamental cellular mechanisms. The limited understanding of passage and cell cycle phases on radioresistant cells revived post-cryopreservation led us to investigate the effect of sub-culturing in parental and radioresistant MCF-7 cells. In this study, the radioresistant cells showed high-intensity nucleic acid and cytochrome bands, which are potentially a radiation-induced spectral marker. Raman spectroscopy data showed dynamic biochemical alterations in revived radioresistant G2/M synchronized cells at early cell passages 1 and 3 with stabilization at a latter cell passage, 5. The study highlights the importance of cell passaging and cell cycle phases in potentially changing the biochemical parameters during in vitro experiments after the revival of radioresistant cells post-cryopreservation.
RESUMEN
Histone Post-Translational Modifications (PTMs) play fundamental roles in mediating DNA-related processes such as transcription, replication and repair. The histone mark H3K36me3 and its associated methyltransferase SETD2 (Set2 in yeast) are archetypical in this regard, performing critical roles in each of these DNA transactions. Here, we present an overview of H3K36me3 regulation and the roles of its writers, readers and erasers in maintaining genome stability through facilitating DNA double-strand break (DSB) repair, checkpoint signalling and replication stress responses. Further, we consider how loss of SETD2 and H3K36me3, frequently observed in a number of different cancer types, can be specifically targeted in the clinic through exploiting loss of particular genome stability functions.
Asunto(s)
Inestabilidad Genómica , Histonas , ADN , Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/metabolismo , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Poor-responsiveness of tumors to radiotherapy is a major clinical problem. Owing to the dynamic nature of the epigenome, the identification and targeting of potential epigenetic modifiers may be helpful to curb radio-resistance. This requires a detailed exploration of the epigenetic changes that occur during the acquirement of radio-resistance. Such an understanding can be applied for effective utilization of treatment adjuncts to enhance the efficacy of radiotherapy and reduce the incidence of tumor recurrence. RESULTS: This study explored the epigenetic alterations that occur during the acquirement of radio-resistance. Sequential irradiation of MCF7 breast cancer cell line up to 20 Gy generated a radio-resistant model. Micrococcal nuclease digestion demonstrated the presence of compact chromatin architecture coupled with decreased levels of histone PTMs H3K9ac, H3K27 ac, and H3S10pK14ac in the G0/G1 and mitotic cell cycle phases of the radio-resistant cells. Further investigation revealed that the radio-resistant population possessed high HDAC and low HAT activity, thus making them suitable candidates for HDAC inhibitor-based radio-sensitization. Treatment of radio-resistant cells with HDAC inhibitor valproic acid led to the retention of γH2AX and decreased H3S10p after irradiation. Additionally, an analysis of 38 human patient samples obtained from 8 different tumor types showed variable tumor HDAC activity, thus demonstrating inter-tumoral epigenetic heterogeneity in a patient population. CONCLUSION: The study revealed that an imbalance of HAT and HDAC activities led to the loss of site-specific histone acetylation and chromatin compaction as breast cancer cells acquired radio-resistance. Due to variation in the tumor HDAC activity among patients, our report suggests performing a prior assessment of the tumor epigenome to maximize the benefit of HDAC inhibitor-based radio-sensitization.
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Neoplasias de la Mama/radioterapia , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Ácido Valproico/farmacología , Acetilación/efectos de la radiación , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral/efectos de la radiación , Cromatina/efectos de la radiación , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/genética , Epigénesis Genética/efectos de la radiación , Femenino , Inhibidores de Histona Desacetilasas/metabolismo , Histonas/efectos de la radiación , Humanos , Incidencia , Recurrencia Local de Neoplasia/epidemiología , Fenotipo , Radioterapia/efectos adversos , Ácido Valproico/metabolismoRESUMEN
BACKGROUND: At present, 'pharmaco-epigenomics' constitutes the hope in cancer treatment owing to epigenetic deregulation- a reversible process and playing a role in malignancy. OBJECTIVE: Chemotherapy has many limitations like host-tissue toxicity, drug resistance. Hence, it is imperative to unearth targets to better treat cancer. Here, we intend to repurpose a set of our previously synthesized difluorinated Propanediones (PR) as Histone lysine Methyltransferase inhibitors (HMTi). METHODS: The cell lines of leukemic origin viz. histiocytic lymphoma (U937) and acute T-cell leukemia (JURKAT) were treated with PR-1 to 7 after docking studies with active pocket of HMT. The cell cycle analysis, in vitro methylation and cell proliferation assays were carried out to delineate their physiological role. RESULTS: A small molecule PR-4, at 1 and 10µM, has shown to alter the methylation of histone H3 and H4 in both cell lines. Also, treatment shows an increase in G2/M population and a subsequent decrease in the G0/G1 population in U937. In JURKAT, an increase in both G2/M and S phase population was observed. The sub-G1 population showed a steady rise with increase in dose and prolonged time intervals in U937 and JURKAT cell lines. In SRB assay, the PR showed a cell growth of 42.6 and 53.4% comparable to adriamycin; 44.5 and 53.2% in U937 and JURKAT, respectively. The study suggests that PR-4 could emerge as a potential HMT inhibitor. CONCLUSION: The molecule PR-4 could be a lead in developing more histone lysine methyltransferases inhibitors with potential to be pro-apoptotic agents.
Asunto(s)
Antineoplásicos/farmacología , Reposicionamiento de Medicamentos , Inhibidores Enzimáticos/farmacología , Histona Metiltransferasas/antagonistas & inhibidores , Hidrocarburos Fluorados/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Halogenación , Histona Metiltransferasas/metabolismo , Humanos , Hidrocarburos Fluorados/síntesis química , Hidrocarburos Fluorados/química , Conformación Molecular , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Células Tumorales Cultivadas , Células U937RESUMEN
Epigenetics have witnessed a renewed interest over the past decade and assays with recombinant histones has become an important tool for uncovering various aspects of histone biology. However, at times absence of recombinant histone accumulation in bacteria is encountered which is also commonly observed for many eukaryotic proteins in general. In this study, we have investigated the effect of multiple parameters on heterologous expression of proteins. We show that there is marked variability in the accumulation of H2A.2, H2B.1, H3.2 and H4 in the recombinant host, possibly owing to translational variability and degradation by the host proteases. We found that the variability could be overcome by incorporation of the commonly used purification tags, like GST or MBP, of appropriate size and position. Our results provide compelling evidence that transcript parameters like rare codon and GC content, mRNA secondary structure etc. together modulate translation kinetics and govern recombinant protein accumulation.
RESUMEN
Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.