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Sarma P.,Indian Institute of Chemical Technology | Ramaiah M.J.,Indian Institute of Chemical Technology | Kamal A.,Indian Institute of Chemical Technology | Bhadra U.,Functional Genomics and Gene Silencing Group | Pal Bhadra M.,Indian Institute of Chemical Technology
Cancer Biology and Therapy | Year: 2014

DNA damage response (DDR) that includes cell cycle check points, DNA repair, apoptosis, and senescence is intimately linked with cancer. It shields an organism against cancer development when genomic integrity fails. DNA repair pathways protect the cells from tumor progression caused as a result of DNA damage induced by irradiation or due to chemotherapeutic treatment. Many promising anticancer agents have been identified that target specific DNA repair pathways in response to DNA damage thereby leading to apoptosis. here we identified a novel bisindole-PBD conjugate that possess potent anticancer activity in breast cancer cells. Further studies aimed at understanding the mechanism of action of the molecule showed its role in DNA damage induced apoptosis via inhibition of DNA repair pathway. Trypan blue and BrdU assay exhibited a dose-dependent effect. single-stranded DNA damage was observed by COMET assay. In addition DNA damage induced ROS generation with simultaneous activation of ATM and ATR upon compound treatment was observed. Further downregulation of Bcl-XL and activation of Bax showed DNA damage induced apoptosis in MCF-7 and MDAMB-231 cells. In conclusion, it can be summarized that bisindole-PBD conjugate induces DNA damage in a dose dependent (2, 4, and 8 μM) manner by inhibiting the DNA repair genes. © 2014 Landes Bioscience.

Pushpavalli S.N.C.V.L.,Indian Institute of Chemical Technology | Sarkar A.,Indian Institute of Chemical Technology | Ramaiah M.J.,Indian Institute of Chemical Technology | Chowdhury D.R.,Functional Genomics and Gene Silencing Group | And 2 more authors.
BMC Molecular Biology | Year: 2013

Background: In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established.Results: Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk) is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks.Conclusion: mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using Drosophila as model system and carry out the interaction of MOF with the known components of the DNA damage pathway. © 2013 Pushpavalli et al.; licensee BioMed Central Ltd.

Shinde S.,Functional Genomics and Gene Silencing Group | Bhadra U.,Functional Genomics and Gene Silencing Group
BioMed Research International | Year: 2015

Small noncoding regulatory RNA exist in wide spectrum of organisms ranging from prokaryote bacteria to humans. In human, a systematic search for noncoding RNA is mainly limited to the nuclear and cytosolic compartments. To investigate whether endogenous small regulatory RNA are present in cell organelles, human mitochondrial genome was also explored for prediction of precursor microRNA (pre-miRNA) and mature miRNA (miRNA) sequences. Six novel miRNA were predicted from the organelle genome by bioinformatics analysis.The structures are conserved in other five mammals including chimp, orangutan, mouse, rat, and rhesus genome. Experimentally, six human miRNA are well accumulated or deposited in human mitochondria.Three of them are expressed less prominently inNorthern analysis. To ascertain their presence in human skeletalmuscles, total RNAwas extracted from enriched mitochondria by an immunomagnetic method. The expression of six novel pre-miRNA and miRNA was confirmed by Northern blot analysis; however, low level of remaining miRNA was found by sensitive Northern analysis. Their presence is further confirmed by real time RT-PCR.Thesix miRNA find theirmultiple targets throughout the human genome in three different types of software.The luciferase assay was used to confirm that MT-RNR2 gene was the potential target of hsa-miR-mit3 and hsamiR-mit4. Copyright © 2015 S. Shinde and U. Bhadra.

Pushpavalli S.N.C.V.L.,Functional Genomics and Gene Silencing Group | Bag I.,Indian Institute of Chemical Technology | Pal-Bhadra M.,Indian Institute of Chemical Technology | Bhadra U.,Functional Genomics and Gene Silencing Group
Chromosome Research | Year: 2012

Argonaute-1 (Ago-1) plays a crucial role in gene regulation and genome stability via biogenesis of small non-coding RNAs. Two "Argonaute" family genes, piwi and Ago-2 in Drosophila are involved in multiple silencing mechanisms in the nucleus, transgene cosuppression, long-distant chromosome interaction, nuclear organization and heterochromatin formation. To investigate whether Ago-1 also plays a similar role, we have generated a series of Ago-1 mutations by excising P element, inserted in the Ago-1 promoter (Ago-1 k08121). AGO-1 protein is distributed uniformly in the nucleus and cytosol in early embryos but accumulated predominantly in the cytoplasm during the gastrulation stage. Repeat induced silencing produced by the mini-white (mw) array and transcriptional cosuppression of non-homologous transgenes Adh-w/w- Adh was disrupted by Ago-1 mutation. These effects of Ago-1 are distict from its role in microRNA processing because Dicer-1, a critical enzyme for miRNA biogenesis, has no role on the above silencing. Reduction of AGO-1 protein dislodged the POLYCOMB, EZ (enhancer of zeste) and H3me3K27 binding at the cosuppressed Adh-w transgene insertion sites suggesting its role in Polycomb dependent cosuppression. An overall reduction of methylated histone H3me2K9 and H3me3K27 from the polytene nuclei precisely from the mw promoters was also found that leads to concomitant changes in the chromatin structure. These results suggest a prominent role of Ago-1 in chromatin organization and transgene silencing and demonstrate a critical link between transcriptional transgene cosuppression, heterochromatin formation and chromatin organization. We propose Drosophila Ago-1 as a multifunctional RNAi component that interconnects at least two unrelated events, chromatin organization in the nucleus and microRNA processing in the cytoplasm, which may be extended to the other systems. © 2012 Springer Science+Business Media B.V.

Kota S.K.,Functional Genomics and Gene Silencing Group | Roy Chowdhury D.,Functional Genomics and Gene Silencing Group | Rao L.K.,Functional Genomics and Gene Silencing Group | Padmalatha V.,Functional Genomics and Gene Silencing Group | And 2 more authors.
Chromosoma | Year: 2015

In mammals, X-inactivation process is achieved by the cis-spreading of long noncoding Xist RNA over one of the female X chromosomes. The Xist binding accumulates histones H3 methylation and H4 hypoacetylation required for X inactivation that leads to proper dosage compensation of the X-linked genes. Co-transcription of Tsix, an antisense copy of Xist, blocks the Xist coating on the Xi. In mice ES cells, an RNase III enzyme Dicer1 disrupts Xist binding and methylated H3K27me3 accumulation on the Xi. Later, multiple reports opposed these findings raising a question regarding the possible role of Dicer1 in murine X silencing. Here, we show that reduction of DICER1 in human female cells increases XIST transcripts without compromising the binding of the XIST and histone tail modifications on the Xi. Moreover, DICER1-depleted cells show differential upregulation of many human X-linked genes by binding different amounts of acetylated histone predominantly on their active promoter sites. Therefore, X-linked gene silencing, which is thought to be coupled with the accumulation of XIST and heterochromatin markers on Xi can be disrupted in DICER1 depleted human cells. These results suggest that DICER1 has no apparent effect on the recruitment of heterochromatic markers on the Xi but is required for inactivation of differentially regulated genes for the maintenance of proper dosage compensation in differentiated cells. © 2014, Springer-Verlag Berlin Heidelberg.

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