Damodar Gupta

E2F4 regulates a stable G2 arrest response to genotoxic stress in prostate carcinoma

The retinoblastoma (pRB) family proteins regulate the E2F transcription factors; their complexes regulate critical transitions through the cell cycle. The function of these pRB family/E2F complexes, which includes p130/E2F4, in response to genotoxic agents, is not well understood. We investigated the role of E2F4 in the genotoxic stress response. Following radiation treatment, E2F4 colocalized with p130 in the nucleus during a radiation-induced stable G2-phase arrest. Arrested cells had significantly decreased expression of Cyclins A2 and B1 and decreased phosphorylation of mitotic protein monoclonal-2 (MPM-2) mitotic proteins. Small interference RNA (siRNA)-mediated knockdown of E2F4 sensitized cells to subsequent irradiation, resulting in enhanced cellular DNA damage and cell death, as determined by caspase activation and decreased clonogenic cell survival. Downstream E2F4 targets potentially involved in the progression from G2 into M phase were identified by oligonucleotide microarray expression profiling. Chromatin immunoprecipitation localized E2F4 at promoter regions of the Bub3 and Pttg1 mitotic genes following irradiation, which were among the downregulated genes identified by the microarray. These data suggest that in response to radiation, E2F4 becomes active in the nucleus, enforces a stable G2 arrest by target gene repression, and thus provides increased cell survival ability by minimizing propagation of cells that have irreparable DNA damage.

E2F4 Function in G2: Maintaining G2-Arrest to Prevent Mitotic Entry with Damaged DNA

Mammalian cells undergo cell cycle arrest in response to DNA damage through multiple checkpoint mechanisms. One such checkpoint pathway maintains genomic integrity by delaying mitotic progression in response to genotoxic stress. Transition though the G2 phase and entry into mitosis is considered to be regulated primarily by Cyclin B1 and its associated catalytically active partner Cdk1. While not necessary for its initiation, the p130 and Rb-dependent target genes have emerged as being important for stable maintenance of a G2 arrest. It was recently demonstrated that by interacting with p130, E2F4 is present in the nuclei and plays a key role in the maintenance of this stable G2 arrest. Increased E2F4 levels and its translocation to the nucleus following genotoxic stress results in down-regulation of many mitotic genes and as a result promote a G0-like state. Irradiation of E2F4-depleted cells leads to enhanced cellular DNA double-strand breaks that may be measured by comet assays. It also results in cell death that is characterized by caspase activation, sub-G1 and sub-G2 DNA content, and decreased clonogenic cell survival. Here we review these recent findings and discuss the mechanisms of G2 phase checkpoint activation and maintenance with a particular focus on E2F4.

Modification of radiation damage to mitochondrial system in vivo by Podophyllum hexandrum: Mechanistic aspects

The present study was undertaken to investigate whether RP-1 treatment protected mitochondrial system against radiation damage and also to unravel the mechanism associated with this process. Radioprotection of mitochondrial system by Podophyllum hexandrum (RP-1) was investigated to understand its mechanism of action. Levels of superoxide anion (O2−), reduced or oxidized glutathione (GSH or GSSG), thiobarbituric acid reactive substance (TBARS), protein carbonyl (PC), ATP, NADH-ubiquinone oxidoreductase (complex-I), NADH-cytochrome c oxidoreductase (complex I/II), succinate-cytochrome c oxidoreductase (complex II/III) and mitochondrial membrane potential (MMP) were studied in mitochondria isolated from liver of mice belonging to various treatment groups. Whole body γ-irradiation (10 Gy) significantly (p < 0.01) increased the formation of O2−, PC, and TBARS, upto 24 h as compared to untreated control. RP-1 treatment (200 mg/kg b.w.) to mice 2 h before irradiation reduced the radiation-induced O2− generation within 4 h and formation of TBARS and PC upto 24 h significantly (p < 0.01). Singularly irradiation or RP-1 treatment significantly (p < 0.01) increased the levels of glutathione within an hour, as compared to untreated control. Pre-irradiation administration of RP-1 enhanced levels of GSH induced increase in complex I (upto 16 h), complex I/III (4 h) complex II/III activity (upto 24 h;p < 0.01) and inhibited the radiation-induced decrease in MMP significantly (24 h;p < 0.01). The present study indicates that RP-1 itself modulates several mitichondrial perameters due to its influence on the biochemical milieu within and outside the cells. However, RP-1 treatment before irradiation modulates radiation induced perturbations such as the increase in electron transport chain enzyme activity, formation of O2−, TBARS and PC to offer radioprotection (Mol Cell Biochem 266: 65–77, 2004)

Protection of mitochondrial system by Hippophae rhamnoides L. against radiation-induced oxidative damage in mice

The whole extract of the fresh berries of Hippophae rhamnoides L. (RH−3), which has been reported to provide protection to whole mice, various tissues, cells and cell organelles against lethal irradiation, was further investigated for its effects on mitochondria isolated from mouse liver. Superoxide anion, reduced (GSH) and oxidized glutathione (GSSG) levels, NADH‐ubiquinone oxidoreductase (complex I), NADH‐cytochrome c oxidoreductase (complex I/II), succinate‐cytochrome c oxidoreductase (complex II/III), mitochondrial membrane potential (MMP), lipid peroxidation (LPx) and protein oxidation (PO) were determined for RH‐3‐mediated radioprotective manifestation. Pre‐irradiation treatment of mice with RH‐3 (30 mg kg−1, i.p.; single dose; −30 min) significantly inhibited the radiation‐induced increase in superoxide anions, GSSG, thiobarbituric acid reactive substances (TBARS), complex I, complex I/III activity and MMP maximally at 4 h (P < 0.05). This treatment inhibited the oxidation of proteins (P < 0.05) at all the time periods studied here. This study suggests that pre‐irradiation treatment of mice with RH‐3 protects the functional integrity of mitochondria from radiation‐induced oxidative stress.

Evaluation of silymarin as a promising radioprotector.

Silymarin, a purified extract of seeds of Silybum marianum L. and well known for its hepatoprotective abilities, has been evaluated for inherent utility as a radioprotective agent. A fraction (INM-7035) was authenticated by characterizing the percentage composition of silybin A and B (39.9% and 57.4%). Free radical scavenging activities of INM-7035 against superoxide radicals (>68%), hydroxyl radicals (>33.75%), DPPH (67.2%), and ABTS (32.4%) were also evaluated. The fraction chelated (>30%) ferrous ions, thereby able to restrict amplification. INM-7035 exhibited >50% peroxyl radical scavenging activity in the lipid phase along with dose-dependent (R2 = 0.990) reducing power in the aqueous phase. Radiation-induced free radical flux can lead to disruption of biomolecules like membrane lipids. INM-7035 completely inhibited lipid peroxidative stress in case of membranes against supralethal radiation stress in the liposomal system. The ability of INM-7035 to modulate the levels of NF-κB, indicated its inherent potential as a radioprotective bioactive constituent.

Radical scavenging and radiomodulatory effects of Psoralea corylifolia Linn. substantiated by in vitro assays and EPR spectroscopy.

The present study is the first report of the radiomodulatory effects of Psoralea corylifolia Linn. The extract (IBG-RA-26) prepared from P. corylifolia was chemically analysed by HPLC, LC-MS/MS and NMR. The total polyphenolic content of IBG-RA-26 was 0.287 mg/ ml of quercetin equivalents. IBG-RA-26 exhibited a dose-dependent increase in 2,2-diphenyl- 1-picrylhydrazyl (DPPH) radical scavenging activity. It exhibited comparable (> 50%) site-specific and non-site-specific hydroxyl radical scavenging activity in higher concentration ranges (500 - 1000 μg/ml), while at lower concentrations (5 - 50 μg/ml) it exhibited significantly (p < 0.05) higher non-site-specific scavenging ability compared to site-specific activity. Nitric oxide scavenging activity of IBG-RA-26 (5 - 1000 μg/ml) increased in a concentrationdependent manner, while maximum superoxide ion scavenging ability (79%) was observed at 50 μg/ml. The electron donation potential of IBG-RA-26 was found to be higher than that of ascorbic acid at lower concentrations (up to 5 μg/ml). Analysis of the ability of IBGRA- 26 to protect membranes against γ-radiation, utilizing an artificial membrane system (liposome), revealed a significant (p < 0.05) decrease in the formation of malondialdehyde (MDA) as a function of the concentration of IBG-RA-26. Radiation-induced lysis of human erythrocytes was monitored and efficacy of IBG-RA-26 was tested in the concentration range 25 - 1000 μg/ml, with significant protective efficacy observed in the range 25 - 50 μg/ml. IBG-RA-26 rendered significant (p < 0.05) protection against radiation (0.25 kGy)-induced DNA damage. EPR spectroscopy was used to investigate the DPPH radical scavenging capacity of IBG-RA-26. IBG-RA-26 exhibited a good DPPH radical scavenging capacity in a concentration-dependent manner. By direct EPR spectroscopy we have also demonstrated the possible formation of free radical species in a solution of IBG-RA-26. The wide spectrum of radioprotective and antioxidant properties exhibited by IBG-RA-26 indicate that P. corylifolia has potential as a radiomodulatory agent.

Development of Novel Anti-Cd20 Monoclonal Antibodies and Modulation in Cd20 Levels on Cell Surface: Looking to Improve Immunotherapy Response.

Rituximab has been revolutionized and validated CD20 targeting monoclonal antibody. Although, it is widely used for lymphoma therapy and many patients have been benefited. However significant numbers of patients are refractory or developed resistance to current therapies due to low level of CD20 expression and/or availability on cells surface. Thus development of novel anti-CD20 mAbs with great cell killing ability and enhance CD20 levels on cell surface can potentially exploit lymphoma therapy. In this scenario, we are summarizing the recently developed mAbs against CD20 and compounds that have ability to induce CD20 expression at significant level. We also are providing information regarding combination strategy for use of radiation and anti-CD20 mAbs in vitro. However, it will need to be determined by rigorous at pre-clinical and clinic testing. We hope this review will be beneficial for current research in the area of immunotherapy or radio-immunotherapy.

Surface levels of CD20 determine anti-CD20 antibodies mediated cell death in vitro

Background The sensitivity of human Burkitts lymphoma cells to rituximab (Rtx) and tositumomab (Tst) was assessed on cells expressing different levels of CD20 on surface. Cells that harbor low CD20 levels may resists against therapeutics response to CD20-specific antibodies. We postulated that, radiation-induced modulation of CD20 surface levels may play a crucial and central role in determining the relative efficacy of rituximab and tositumomab in treating Burkitts lymphoma disease. Here, we examined the γ-radiation-induced CD20 expression in the Burkitt lymphoma cell line ‘Daudi’ and the relation of differential levels of CD20 with anti-CD20 mAbs mediated cell death. Methodology In this study we examined kinetics of CD20 expression following sub lethal doses ofγ-radiation to Daudi cells and thereafter anti-CD20 mAbs (rituximab and tositumomab) were added in cell suspensions. The correlation of kinetics of CD20 expression and cells treated with anti-CD20 mAbs/or corresponding isotype Abs with special reference to changes in mitochondrial membrane potential and reactive oxygen species generation was also examined. Further, we also investigated the efficacy of anti-CD20 mAbs and possible induction of cell death in relation to levels of CD20 cell surface expression. Conclusion This report provides evidence that CD20 expression can be induced by exposure of cells to γ-radiation. In addition, these findings demonstrated that the efficacy of anti-CD20 mAbs is dependent on the surface levels of CD20. Based on these findings, we hypothesized (i) irradiation just prior to immunotherapy may provide new treatment options even in aggressive B cell tumors, which are resistant to current therapies in vivo (ii) The efficacy of induction of apoptosis varies with type of monoclonal antibodies in vitro.

NF-κB as a key player in regulation of cellular radiation responses and identification of radiation countermeasures

Nuclear factor (NF)-κB is a transcription factor that plays significant role in immunity, cellular survival and inhibition of apoptosis, through the induction of genetic networks. Depending on the stimulus and the cell type, the members of NF-κB related family (RelA, c-Rel, RelB, p50, and p52), forms different combinations of homo and hetero-dimers. The activated complexes (Es) translocate into the nucleus and bind to the 10bp κB site of promoter region of target genes in stimulus specific manner. In response to radiation, NF-κB is known to reduce cell death by promoting the expression of anti-apoptotic proteins and activation of cellular antioxidant defense system. Constitutive activation of NF-κB associated genes in tumour cells are known to enhance radiation resistance, whereas deletion in mice results in hypersensitivity to IR-induced GI damage. NF-κB is also known to regulate the production of a wide variety of cytokines and chemokines, which contribute in enhancing cell proliferation and tissue regeneration in various organs, such as the GI crypts stem cells, bone marrow etc., following exposure to IR. Several other cytokines are also known to exert potent pro-inflammatory effects that may contribute to the increase of tissue damage following exposure to ionizing radiation. Till date there are a series of molecules or group of compounds that have been evaluated for their radio-protective potential, and very few have reached clinical trials. The failure or less success of identified agents in humans could be due to their reduced radiation protection efficacy. In this review we have considered activation of NF-κB as a potential marker in screening of radiation countermeasure agents (RCAs) and cellular radiation responses. Moreover, we have also focused on associated mechanisms of activation of NF-κB signaling and their specified family member activation with respect to stimuli. Furthermore, we have categorized their regulated gene expressions and their function in radiation response or modulation. In addition, we have discussed some recently developed radiation countermeasures in relation to NF-κB activation

Differential Action of Anti-CD20 Monoclonal Antibodies: Role in Induction of Cell Death

Purpose: To study and assess to the sensitivity of Burkitt’s lymphoma cells harboring differential levels of cell surface CD20 for anti-CD20 monoclonal Antibodies. Material and methods: Burkitt’s lymphoma cell line ‘Daudi’ was used during present study. Cells were exposed to single dose of γ-radiation (0.5 Gy) and thereafter incubated with rituximab (Rtx) or tositumomab (Tst) (0.5 μg/ ml concentration each). The changes in expression of RelA, Akt and Bax/Bcl-2 ratio was measured to assess sensitivity of cells in term of cell death. Results: During the present investigation, we found that cells incubated with anti-CD20 mAbs at +20 hrs post radiation exposure showed higher levels of cell death in term of Bax/Bcl-2 ratio. In addition, we also found the significant changes in expression of pro-survival signaling proteins such as RelA and Akt pathways. Moreover, we found that tositumomab is a potent inducer of apoptotic cell death. Conclusion: These findings suggested that the efficacy of anti-CD20 mAbs depends on the number of CD20 molecules expressed on cell surface and type of antibody used. It may provide new treatment options for selection of anti-CD20 mAbs even in aggressive B-cell lymphoma, which harbors low levels of CD20 or even resistant to current therapies in vivo.