Akshay Pandey - Academia.edu (original) (raw)

Papers by Akshay Pandey

Research paper thumbnail of Author Correction: Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

Research paper thumbnail of Abstract 1504: Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells

Molecular and Cellular Biology / Genetics, 2018

The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. ... more The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. However, the significance of its over-expression in cancers, including poorly differentiated hepatocellular carcinoma (HCC), is not known. Here we provide evidence for a causal role of the over-expressed FoxM1 in driving high-grade progression of HCC. Immunohistochemical staining of human HCC specimens revealed opposite expression patterns of FoxM1 and the liver differentiation genes FoxA1/A2. Moreover, using a transgenic mouse model for oncogenic Ras-driven HCC as well as cell-based studies, we provide in vivo genetic evidence for a direct repression of FoxA1/A2 by FoxM1. Interestingly, FoxM1 represses these differentiation genes mainly in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Moreover, repression of FoxA1/A2 in G1 phase is important, as these genes are capable of inhibiting expression of the pluripotency genes that are mainly expressed in S/G2 phases. Also, we show that FoxA1/A2 inhibit expression of FoxM1 by inhibiting binding of FoxM1 to its own promoter and thus blocking its auto-activation. Our observations identify a significant new mechanism in which FoxM1 and the FoxA genes play opposing roles that determine the differentiation state of the HCC cells. Citation Format: Vaibhav Chand, Akshay Pandey, Dragana Kopanja, Grace Guzman, Pradip Raychaudhuri. Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1504.

Research paper thumbnail of Opposing Roles of the Forkhead Box Factors FoxM1 and FoxA2 in Liver Cancer

Molecular Cancer Research, 2019

The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) devel... more The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) development, and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S–G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1 and inhibits FoxM1-induced tumorigenicity. Also, FoxA2 inhibits Ras-induced HCC progression that involves FoxM1. Implications: The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 ...

Research paper thumbnail of Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

Scientific reports, Jan 7, 2017

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor... more FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and...

Research paper thumbnail of Abstract B07: Ras-driven HCCs are addicted to FoxM1

Molecular Cancer Research, 2014

Hepatocellular carcinoma (HCC) is the fifth most fatal malignancy worldwide. In the US alone, acc... more Hepatocellular carcinoma (HCC) is the fifth most fatal malignancy worldwide. In the US alone, according to American Cancer Society, about 17,550 new cases and over 15,000 deaths are reported annually. The fork-head box gene FoxM1 is highly significant in that regard, as it is over-expressed in HCC, and its over-expression coincides with highly aggressive, poorly differentiated HCC. Moreover, over-expression of FoxM1 correlates with poorer overall survival after surgery. Previous work demonstrated that FoxM1 is essential for chemical carcinogen (DEN)-induced development of HCC. Since the Ras-pathway is frequently mutated in human liver cancers, we investigated the effects of FoxM1 deletion on the progression of oncogenic Ras-induced HCC. In the mouse model we used, HRas-V12 is expressed specifically in the adult liver under control of the Alb-promoter. In this mouse model, majority (80 to 90%) of the male mice develop HCC by 8/9 months of age. We crossed that transgenic line with Fox...

Research paper thumbnail of Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

International Journal of Radiation Biology, 2013

To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in ra... more To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Research paper thumbnail of An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

Cytotechnology, 2011

Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sampl... more Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sample processing, involvement of multiple steps and requirement of expensive reagents. The present study demonstrates a rapid, easy-to-perform cost-effective method for detection of apoptotic DNA fragments with considerable improvement in the sensitivity by avoiding loss of DNA fragments. It involves a few minutes of procedure involving direct lysis of cells with dimethyl sulphoxide (DMSO), brief vortexing, addition of 2% SDS-TE buffer, and a single step of centrifugation. This cost-and time-efficient method reduces the assay time considerably and can be used for a large number of samples with excellent sensitivity.

Research paper thumbnail of FoxM1 in Liver Cancer

Research paper thumbnail of INVESTIGATION OF THE EFFECT OF CURRENT ON TENSILE STRENGTH AND NUGGET DIAMETER OF SPOT WELDS MADE ON AISI-1008 STEEL SHEETS

International Journal of Technical Research and Applications

The results of the investigation indicate the welding current to be the most significant paramete... more The results of the investigation indicate the welding current to be the most significant parameter controlling the weld tensile strength as well as the nugget diameter. The contribution of welding current holding time and pressure to tensile strength are 61%, 29%, 4% respectively and the contribution of these parameters to nugget diameter are 81%, 17%, 1.7% respectively. Relationship graph have been plotted between tensile strength and nugget diameter with parametric variations according to orthogonal array Keywords— resistance spot welding, taguchi method, tensile strength, nugget diameter, annova etc

Research paper thumbnail of Essential roles of FoxM1 in Ras-induced liver cancer progression and in cancer cells with stem cell features

Journal of hepatology, Jan 27, 2015

Over-expression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreov... more Over-expression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1. FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1-siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. Ras-driven tumors over-express FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1-deleted HCC cells. Moreover, FoxM1-deletion caused a disproportiona...

Research paper thumbnail of Predictive role of mitochondrial genome in the stress resistance of insects and nematodes

Bioinformation, 2010

Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as ... more Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as excellent experimental models to study the cellular stress signaling mechanisms since these organisms are far more stress-resistant as compared to mammalian system. Multiple factors have been implicated in this unusual response, including the oxidative stress response mechanisms. Radiation or chemical-induced mitochondrial oxidative stress occurs through damage caused to the components of electron transport chain (ETC) leading to leakage of electrons and generation of superoxide radicals. This may be countered through quick replacement of damaged mitochondrial proteins by upregulated expression. Since the ETC comprises of various proteins coded by mitochondrial DNA, variation in the composition, expressivity and regulation of mitochondrial genome could greatly influence mitochondrial role under oxidative stress conditions. Therefore, we carried out in silico analysis of mitochondrial DNA in these organisms and compared it with that of the stress-sensitive humans/mammals. Parameters such as mitochondrial genome organization, codon bias, gene expressivity and GC 3 content were studied. Gene arrangement and Shine-Dalgarno (SD) sequence patterns indicating translational regulation were distinct in insect and nematodes as compared to humans. A higher codon bias (ENC<35) and lower GC 3 content (<0.20) were observed in mitochondrial genes of insect and nematodes as compared to humans (ENC>42; GC3>0.20), coupled with low codon adaptation index among insects. These features indeed favour higher expressivity of mitochondrial proteins and might help maintain the mitochondrial physiology under stress conditions. Therefore, our study indicates that mitochondrial genome organization may influence stress-resistance of insects and nematodes. : SD sequences in the upstream region of protein coding genes (Bombyx mori, Manduca sexta, C. elegans and Homo sapiens). Rectangular boxes over the sequences mark the position of SD sequences in the respective upstream region.

Research paper thumbnail of An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

Research paper thumbnail of Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

International Journal of Radiation Biology, 2013

To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in ra... more To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Research paper thumbnail of Author Correction: Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

Research paper thumbnail of Abstract 1504: Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells

Molecular and Cellular Biology / Genetics, 2018

The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. ... more The pro-proliferation transcription factor FoxM1 is over-expressed mainly in high-grade cancers. However, the significance of its over-expression in cancers, including poorly differentiated hepatocellular carcinoma (HCC), is not known. Here we provide evidence for a causal role of the over-expressed FoxM1 in driving high-grade progression of HCC. Immunohistochemical staining of human HCC specimens revealed opposite expression patterns of FoxM1 and the liver differentiation genes FoxA1/A2. Moreover, using a transgenic mouse model for oncogenic Ras-driven HCC as well as cell-based studies, we provide in vivo genetic evidence for a direct repression of FoxA1/A2 by FoxM1. Interestingly, FoxM1 represses these differentiation genes mainly in G1 phase, a phase in the cell cycle in which cells can undergo differentiation. Moreover, repression of FoxA1/A2 in G1 phase is important, as these genes are capable of inhibiting expression of the pluripotency genes that are mainly expressed in S/G2 phases. Also, we show that FoxA1/A2 inhibit expression of FoxM1 by inhibiting binding of FoxM1 to its own promoter and thus blocking its auto-activation. Our observations identify a significant new mechanism in which FoxM1 and the FoxA genes play opposing roles that determine the differentiation state of the HCC cells. Citation Format: Vaibhav Chand, Akshay Pandey, Dragana Kopanja, Grace Guzman, Pradip Raychaudhuri. Opposing roles of FoxM1 and the hepatic specification genes FoxA1/A2 dictate differentiation state of liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1504.

Research paper thumbnail of Opposing Roles of the Forkhead Box Factors FoxM1 and FoxA2 in Liver Cancer

Molecular Cancer Research, 2019

The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) devel... more The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) development, and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S–G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1 and inhibits FoxM1-induced tumorigenicity. Also, FoxA2 inhibits Ras-induced HCC progression that involves FoxM1. Implications: The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 ...

Research paper thumbnail of Plk1 Regulates the Repressor Function of FoxM1b by inhibiting its Interaction with the Retinoblastoma Protein

Scientific reports, Jan 7, 2017

FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor... more FoxM1b is a cell cycle-regulated transcription factor, whose over-expression is a marker for poor outcome in cancers. Its transcriptional activation function requires phosphorylation by Cdk1 or Cdk2 that primes FoxM1b for phosphorylation by Plk1, which triggers association with the co-activator CBP. FoxM1b also possesses transcriptional repression function. It represses the mammary differentiation gene GATA3 involving DNMT3b and Rb. We investigated what determines the two distinct functions of FoxM1b: activation and repression. We show that Rb binds to the C-terminal activation domain of FoxM1b. Analyses with phospho-defective and phospho-mimetic mutants of FoxM1b identified a critical role of the Plk1 phosphorylation sites in regulating the binding of FoxM1b to Rb and DNMT3b. That is opposite of what was seen for the interaction of FoxM1b with CBP. We show that, in addition to GATA3, FoxM1b also represses the mammary luminal differentiation marker FoxA1 by promoter-methylation, and...

Research paper thumbnail of Abstract B07: Ras-driven HCCs are addicted to FoxM1

Molecular Cancer Research, 2014

Hepatocellular carcinoma (HCC) is the fifth most fatal malignancy worldwide. In the US alone, acc... more Hepatocellular carcinoma (HCC) is the fifth most fatal malignancy worldwide. In the US alone, according to American Cancer Society, about 17,550 new cases and over 15,000 deaths are reported annually. The fork-head box gene FoxM1 is highly significant in that regard, as it is over-expressed in HCC, and its over-expression coincides with highly aggressive, poorly differentiated HCC. Moreover, over-expression of FoxM1 correlates with poorer overall survival after surgery. Previous work demonstrated that FoxM1 is essential for chemical carcinogen (DEN)-induced development of HCC. Since the Ras-pathway is frequently mutated in human liver cancers, we investigated the effects of FoxM1 deletion on the progression of oncogenic Ras-induced HCC. In the mouse model we used, HRas-V12 is expressed specifically in the adult liver under control of the Alb-promoter. In this mouse model, majority (80 to 90%) of the male mice develop HCC by 8/9 months of age. We crossed that transgenic line with Fox...

Research paper thumbnail of Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

International Journal of Radiation Biology, 2013

To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in ra... more To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.

Research paper thumbnail of An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

Cytotechnology, 2011

Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sampl... more Conventional DNA ladder assay has certain shortcomings such as loss of DNA fragments during sample processing, involvement of multiple steps and requirement of expensive reagents. The present study demonstrates a rapid, easy-to-perform cost-effective method for detection of apoptotic DNA fragments with considerable improvement in the sensitivity by avoiding loss of DNA fragments. It involves a few minutes of procedure involving direct lysis of cells with dimethyl sulphoxide (DMSO), brief vortexing, addition of 2% SDS-TE buffer, and a single step of centrifugation. This cost-and time-efficient method reduces the assay time considerably and can be used for a large number of samples with excellent sensitivity.

Research paper thumbnail of FoxM1 in Liver Cancer

Research paper thumbnail of INVESTIGATION OF THE EFFECT OF CURRENT ON TENSILE STRENGTH AND NUGGET DIAMETER OF SPOT WELDS MADE ON AISI-1008 STEEL SHEETS

International Journal of Technical Research and Applications

The results of the investigation indicate the welding current to be the most significant paramete... more The results of the investigation indicate the welding current to be the most significant parameter controlling the weld tensile strength as well as the nugget diameter. The contribution of welding current holding time and pressure to tensile strength are 61%, 29%, 4% respectively and the contribution of these parameters to nugget diameter are 81%, 17%, 1.7% respectively. Relationship graph have been plotted between tensile strength and nugget diameter with parametric variations according to orthogonal array Keywords— resistance spot welding, taguchi method, tensile strength, nugget diameter, annova etc

Research paper thumbnail of Essential roles of FoxM1 in Ras-induced liver cancer progression and in cancer cells with stem cell features

Journal of hepatology, Jan 27, 2015

Over-expression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreov... more Over-expression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1. FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1-siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. Ras-driven tumors over-express FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1-deleted HCC cells. Moreover, FoxM1-deletion caused a disproportiona...

Research paper thumbnail of Predictive role of mitochondrial genome in the stress resistance of insects and nematodes

Bioinformation, 2010

Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as ... more Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as excellent experimental models to study the cellular stress signaling mechanisms since these organisms are far more stress-resistant as compared to mammalian system. Multiple factors have been implicated in this unusual response, including the oxidative stress response mechanisms. Radiation or chemical-induced mitochondrial oxidative stress occurs through damage caused to the components of electron transport chain (ETC) leading to leakage of electrons and generation of superoxide radicals. This may be countered through quick replacement of damaged mitochondrial proteins by upregulated expression. Since the ETC comprises of various proteins coded by mitochondrial DNA, variation in the composition, expressivity and regulation of mitochondrial genome could greatly influence mitochondrial role under oxidative stress conditions. Therefore, we carried out in silico analysis of mitochondrial DNA in these organisms and compared it with that of the stress-sensitive humans/mammals. Parameters such as mitochondrial genome organization, codon bias, gene expressivity and GC 3 content were studied. Gene arrangement and Shine-Dalgarno (SD) sequence patterns indicating translational regulation were distinct in insect and nematodes as compared to humans. A higher codon bias (ENC<35) and lower GC 3 content (<0.20) were observed in mitochondrial genes of insect and nematodes as compared to humans (ENC>42; GC3>0.20), coupled with low codon adaptation index among insects. These features indeed favour higher expressivity of mitochondrial proteins and might help maintain the mitochondrial physiology under stress conditions. Therefore, our study indicates that mitochondrial genome organization may influence stress-resistance of insects and nematodes. : SD sequences in the upstream region of protein coding genes (Bombyx mori, Manduca sexta, C. elegans and Homo sapiens). Rectangular boxes over the sequences mark the position of SD sequences in the respective upstream region.

Research paper thumbnail of An improved non-enzymatic “DNA ladder assay” for more sensitive and early detection of apoptosis

Research paper thumbnail of Radioresistant Sf9 insect cells undergo an atypical form of Bax-dependent apoptosis at very high doses of γ-radiation

International Journal of Radiation Biology, 2013

To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in ra... more To investigate the underlying mechanisms of cell-death at extremely high doses of radiation in radioresistant Spodoptera frugiperda-9 (Sf9) insect cells. Morphology, cell proliferation and DNA-fragmentation analysis was performed at 500-2000 Gy. Changes in intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cardiolipin oxidation and Annexin-V externalization were studied using flow-cytometry. Cytochrome-c release was measured using immunofluorescence microscopy. Inhibitors of apoptosis, i.e., Bongkrekic acid (BKA), Caspase-9 inhibitor (C9i), 5-(4-fluorosulfonylbenzoyl) adenosine hydrochloride (FSBA) and Cyclosporin-A (CsA) were used to dissect apoptotic mechanism at many classical steps. Caspase-3 activity was measured using a caspase-activity assay kit. A dose-dependent induction of typical apoptosis was observed at extremely high doses, marked by extensive apoptotic body formation. However, certain atypical responses such as cellular hypertrophy and the lack of phosphatidylserine-externalization were observed during the initial hours after radiation. Loss of mitochondrial membrane potential observed at 48 h following a 2000 Gy dose was accompanied by an increase in ROS that caused significant cardiolipin oxidation leading to cytochrome-c release, caspase activation and internucleosomal DNA fragmentation. Inhibitors of B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax)-mediated cytochrome-c release, apoptosome formation and caspase-9 effectively prevented radiation-induced apoptosis, strongly suggesting the role of Bax-dependent cell death mechanism. Our study demonstrates that the Sf9 insect cells display good homology with human cells in the mitochondria-dependent events during radiation-induced apoptosis, although doses eliciting similar responses were 50-200 times higher than human cells. Factors upstream to mitochondrial damage remain pertinent for a thorough understanding of this extreme radioresistance displayed by lepidopteran cells.