Papers by Asok Mukhopadhyay
PDF file, 157K, Includes supplementary data regarding Viral transduction, Real-time PCR (includin... more PDF file, 157K, Includes supplementary data regarding Viral transduction, Real-time PCR (including primers), Flow cytometry, and additional experimental methods.
Oncogene, Jan 30, 2018
Epithelial ovarian carcinoma (EOC) patients often acquire resistance against common chemotherapeu... more Epithelial ovarian carcinoma (EOC) patients often acquire resistance against common chemotherapeutic drugs like paclitaxel and cisplatin. The mechanism responsible for the same is ambiguous. We have identified a putative drug-resistant tumour cell phenotype (EpCAM+CD45+) in the ascitic fluid of EOC patients, which appears to originate from the primary tumour. These cells represent the major tumour burden and are more drug resistant compared to EpCAM+ tumour cells due to the over-expression of SIRT1, ABCA1 and BCL2 genes. We have found that the entire EpCAM+CD45+ population is highly invasive with signature mesenchymal gene expression and also consists of subpopulations of ovarian cancer stem cells (CD133+ and CD117+CD44+). Additionally, we demonstrate that the EpCAM+CD45+ tumour cells over-express major histocompatibility complex class I antigen, which enable them to evade the natural killer cell-mediated immune surveillance. Preliminary evidence obtained in OVCAR-5 cells suggests t...
Vaccine, 1998
The growth of Mycobacterium w, a candidate strain for leprosy vaccine in submerged culture, was i... more The growth of Mycobacterium w, a candidate strain for leprosy vaccine in submerged culture, was inhibited by the presence of over 40% oxygen saturation in the medium. Intracellular levels of superoxide dismutase and catalase were very low in the beginning. However; under controlled oxygenation, these levels increased with time. The augmentations of these antioxidant enzymes were associated with the elevated oxygen consumption by the culture. By maintaining the oxygen level below 20% during @day culture, it was possible to grow Mycobacterium w in five production batches up to a cell density of 3.7 f 0.70 x lo9 bacilli ml-'. The shelf life of the vaccine produced in diRerent batches was more than 2 years, both at 4°C and at 26°C. This provides a costeflective, unit culture technology for the production of this candidate leprosy vaccine from a nonpathogenic organism, which will facilitate the widespread use of the vaccine.
Cytotherapy, 2016
Cell replacement therapy is considered a promising alternative in the treatment of degenerative d... more Cell replacement therapy is considered a promising alternative in the treatment of degenerative diseases, and in this context, mesenchymal stromal cells (MSCs) have been proposed for transplantation in Parkinson disease (PD). Thus far, the results of animal studies are found to be inconsistent and inconclusive regarding the therapeutic ability of the cells. This study investigated the efficacy of fetal liver (FL)-MSC-derived dopaminergic (DA) neuronal primed cells for correction of parkinsonian symptoms in mice. FL-MSCs were differentiated for 21 days in the presence of a combination of neurotropic factors. The extent of cellular reprogramming was analyzed by quantitative polymerase chain reaction for DA-specific neuronal gene expressions and protein expressions by immuno-cytochemistry. The functionality of the cells was determined by electrophysiology and dopamine release assays. Ten-day-primed neuron-like cells or unprimed MSCs were transplanted into the 6-hydroxydopamine (6-OHDA)...
Stem Cells, 2015
Liver fibrosis is strongly associated with chronic inflammation. As an alternative to conventiona... more Liver fibrosis is strongly associated with chronic inflammation. As an alternative to conventional treatments for fibrosis, mesenchymal stem cells (MSCs) therapy is found to be attractive due to its immunomodulatory functions. However, low survival rate and profibrogenic properties of MSCs remain the major concerns, leading to skepticism in many investigators. Here, we have asked the question whether bone marrow (BM)-derived CD45 cells is the better candidate than MSCs to treat fibrosis, if so, what are the molecular mechanisms that make such distinction. Using CCl4-induced liver fibrosis mouse model of a Metavir fibrosis score 3, we showed that BM-CD45 cells have better antifibrotic effect than adipose-derived (AD)-MSCs. In fact, our study revealed that antifibrotic potential of CD45 cells are compromised by the presence of MSCs. This difference was apparently due to significantly high level expressions of matrix metalloproteinases-9 and 13, and the suppression of hepatic stellate ...
Advances in Biochemical Engineering/Biotechnology, 2003
Advances in Biochemical Engineering/Biotechnology, 1999
The glycosylation machinery in eukaryotic cells is available to all proteins that enter the secre... more The glycosylation machinery in eukaryotic cells is available to all proteins that enter the secretory pathway. There is a growing interest in diseases caused by defective glycosylation, and in therapeutic glycoproteins produced through recombinant DNA technology route. The choice of a bioprocess for commercial production of recombinant glycoprotein is determined by a variety of factors, such as intrinsic biological properties of the protein being expressed and the purpose for which it is intended, and also the economic target. This review summarizes recent development and understanding related to synthesis of glycans, their functions, diseases, and various expression systems and characterization of glycans. The second section covers processing of N- and O-glycans and the factors that regulate protein glycosylation. The third section deals with in vivo functions of protein glycosylation, which includes protein folding and stability, receptor functioning, cell adhesion and signal transduction. Malfunctioning of glycosylation machinery and the resultant diseases are the subject of the fourth section. The next section covers the various expression systems exploited for the glycoproteins: it includes yeasts, mammalian cells, insect cells, plants and an amoeboid organism. Biopharmaceutical properties of therapeutic proteins are discussed in the sixth section. In vitro protein glycosylation and the characterization of glycan structures are the subject matters for the last two sections, respectively.
Stem Cells and Development, 2004
Cytotoxic drugs or irradiation are generally administered before bone marrow (BM) transplantation... more Cytotoxic drugs or irradiation are generally administered before bone marrow (BM) transplantation because of the idea that host bone marrow 'niches' become available to the donor cells for engraftment. How BM stromal cells respond to the radiation, which ultimately modulates grafting of donor cells, is poorly understood. In this study, we examined homing and marrow retention of PKH26+ donor cells in BM of age-matched C57BL/6J mice conditioned at different doses of irradiation. When we injected donor cells into mice that received 900 cGy, the percent homing was highest (15.8 +/- 1.5%) as compared to the lower doses of radiation. Despite the highest levels of homing of donor cells in these mice, about 70% (p < 0.005) homed cells were detached from the marrow within 72 h of transplantation. In contrast, a 2- to 2.5-fold (p < 0.03) multiplication of homed PKH-26+ Sca-1+ cells was observed in sublethally irradiated mice. While determining that CD45- CD106+ cells in BM of the mice received 900 cGy, we found that more than 80% of cells were depleted. It was also revealed from this investigation that grafted cells conferred partial protection to the endogenous myeloid colony-forming cells from radiation injury. Collectively, the present study implicates radiation-induced degeneration of stroma as a cause of poor retention of donor cells in BM of lethally irradiated mice. These results may have important clinical implications in designing conditioning regimens for BM transplantation.
Journal of Hematotherapy & Stem Cell Research, 2003
Homing and engraftment of hematopoietic stem cells (HSCs) to bone marrow (BM) is a complex proces... more Homing and engraftment of hematopoietic stem cells (HSCs) to bone marrow (BM) is a complex process that primarily depends on the cell-surface expression of adhesion molecules on stem and stromal cells. Here we report an in vitro model for homing of stem cells on pre-established stromal layer; the stroma-adhered cells were found to engraft, multiply, and differentiate in BM of agematched mice. In vitro study revealed that initially the adhesion of BM cells on irradiated stroma was increased with time, and it attained a peak at 2 h of contacts. During that time, 44.1 6 6.5% (n 5 8) cells were adhered, and this value was maintained up to 6 3 10 6 cells. The adhered cell fraction was enriched by 3.9-, 2.5-, and 1.7-fold Sca-1, colony forming cell (CFC), and cobblestone area forming cells (CAFC), respectively, as compared to the fresh BM cells. These adhered cells homed to BM with an engraftment efficiency of 11.8 6 2.5% (n 5 6). The homed cells reconstituted BM of myeloablative mice by self-renewing and differentiating into myeloid cells. Overall, a simple in vitro model system has been described to study homing and grafting of HSCs that can be deployed to any possible experimental conditions to investigate the interactions between stromal and stem cells.
Molecular Carcinogenesis, 2005
The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kapp... more The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF-kappaB distinctively, while modulating the pathways of JNK and AP-1 similarly. These differences may influence cellular processes such as proliferation, differentiation, and apoptosis.
Journal of Cellular Biochemistry, 2002
Tumor necrosis factor (TNF) is one of the most potent activators of nuclear transcription factor ... more Tumor necrosis factor (TNF) is one of the most potent activators of nuclear transcription factor NF-kappaB, c-Jun N-terminal protein kinase (JNK), and apoptosis in a wide variety of cells. The biological effects of TNF are mediated through sequential interactions of various cytoplasmic proteins with intracellular domains of TNF receptors. Whether signal transducer and activator of transcription-1 (STAT1), which mediates interferon (IFN) signaling, also plays any role in the TNF-mediated activation of NF-kappaB, JNK, and apoptosis has not been established. Here, we report our investigation of the role of STAT1 in TNF signaling using STAT1-deficient U3A and STAT1-stably transfected U3A-PSG91 cells. IFNalpha inhibited the proliferation of STAT1-expressing U3A-PSG91 cells but had no effect on STAT1-negative U3A cells. TNF alone, even up to 10 nM, had no effect on the proliferation of either U3A-PSG91 or U3A cells. Irrespective of STAT1 status, TNF induced cytotoxic effects in the presence of cycloheximide (CHX) in both cell types. Additionally, TNF-induced caspase-3 and caspase-8 activation and TNF-induced PARP cleavage were unaffected by the presence or absence of STAT1. TNF activated NF-kappaB, consisting of p50 and p65, in both U3A and U3A-pSG91 cells in a dose- and time-dependent manner, but the degree and rate of activation were slightly lower in U3A cells, as were IkappaBalpha degradation and NF-kappaB-dependent reporter gene expression. STAT1 was, however, required for IFNalpha-mediated downregulation of TNF-induced NF-kappaB activation. TNF activated JNK in both cell types, but dose and time of exposure required for optimum activation differed slightly. Thus, overall our results indicate that STAT1 plays a minimal role in TNF-mediated cellular responses.
Journal of Biotechnology, 1994
European Journal of Haematology, 2012
In mitochondria, oxidative metabolism is associated with the generation of reactive oxygen specie... more In mitochondria, oxidative metabolism is associated with the generation of reactive oxygen species (ROS) that normally diffuse out in the cytosol. Owing to some unusual circumstances, especially exposure to high oxygen level, considerable amount of ROS are accumulated in the mitochondria, causing oxidative damage to the cells (1, 2). Long exposure to ROS may promote the onset of premature proliferative senescence, which results in detrimental effect on expansion potential of the cells (3-5). Dissolved O 2 was found to influence the growth of murine embryonic stem cells (6), muscle satellite cells (7), and neural precursor cells (8). In all cases, the cellular proliferation and survival were higher in the physiologic O 2 level (6% or less) than in non-physiologic (21%) culture conditions. Bone marrow (BM) microenvironment is hypoxic. Dissolved O 2 concentration in BM may vary from 0.1 to 4%, depending on the radial position of the marrow (9,
Biotechnology and Bioengineering, 1996
Synthesis of the p-subunit of human chorionic gonadotropin (PhCG) in Vero cells by the recombinan... more Synthesis of the p-subunit of human chorionic gonadotropin (PhCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of phCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of phCG, deduced from the pulse-chase experiments were "zero order." At 30 h postinfection, the relative values of net synthesis and secretion rates were 4.0 AU ' mm2 phCG/106 cells. h and 1.55 AU. mm2 phCGilO6 cells. h, respectively. The time required to secrete 50% of intracellular phCG was 210 min. Pulse-chase data also showed that 24% of PhCG was degraded intracellularly within 10 h, of which 17% was detected in the autoradiograrn. Along with 30 kD phCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia-derived phCG was 13 kD more that its nonglycosylated form, indicating extensive glycosylation in Vero cells. The mRNA levels in infected Vero cells at different postinfection times were quantified by excess DNA dot-blot hybridization. It appears that the Vero cell possesses some host cellassociated factor(s), which prevented the transcription of early phCG-mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half-life of phCG-mRNA, as determined by follow-up of decay after blocking transcription initiation, was found t o be 6.4 h. The synthesized phCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native ahCG t o form heterodimer hCG, which cornpeted with '251-hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells.
Biochemical Pharmacology, 1999
Beta-lapachone, the product of a tree from South America, is known to exhibit various pharmacolog... more Beta-lapachone, the product of a tree from South America, is known to exhibit various pharmacologic properties, the mechanisms of which are poorly understood. In the present report, we examined the effect of beta-lapachone on the tumor necrosis factor (TNF)-induced activation of the nuclear transcription factors NF-kappaB and activator protein-1 (AP-1) in human myeloid U937 cells. TNF-induced NF-kappaB activation, p65 translocation, IkappaBalpha degradation, and NF-kappaB-dependent reporter gene expression were inhibited in cells pretreated with beta-lapachone. Direct treatment of the p50-p65 heterodimer of NF-kappaB with beta-lapachone had no effect on its ability to bind to the DNA. Besides myeloid cells, beta-lapachone was also inhibitory in T-cells and epithelial cells. Beta-lapachone also suppressed the activation of NF-kappaB by lipopolysaccharide, okadaic acid, and ceramide but had no significant effect on activation by H2O2 or phorbol myristate acetate, indicating that its action is selective. Beta-lapachone also abolished TNF-induced activation of AP-1, c-Jun N-terminal kinase, and mitogen-activated protein kinase kinase (MAPKK or MEK). TNF-induced cytotoxicity and activation of caspase-3 were also abolished by beta-lapachone. Because reducing agents (dithiothreitol and N-acetylcysteine) reversed the effect of beta-lapachone, it suggests the role of a critical sulfhydryl group. Overall, our results identify NF-kappaB, AP-1, and apoptosis as novel targets for beta-lapachone, and this may explain some of its pharmacologic effects.
Biochemical and Biophysical Research Communications, 2005
Primitive hematopoietic stem cells are responsible for long-term engraftment in irradiated host. ... more Primitive hematopoietic stem cells are responsible for long-term engraftment in irradiated host. Here, we report that multi-drug resistance 1 (mdr1) gene expressing primitive hematopoietic cells were multiplied in ex vivo culture, with the support of extracellular matrix components and cytokines. About 20-fold expansion of total nucleated cells was achieved in a 10-day culture. Lin À Sca-1 + and long-term culture-initiating cells were increased by 54-and 26-fold, respectively. Expanded cells were long-term multi-lineage engraftible in sub-lethally irradiated mice. Donor-derived peripheral blood chimerism was significantly higher (73.2 ± 9.1%, p < 0.01) in expanded cells than in normal and 5-flurouracil-treated bone marrow cells. Most interestingly, the expression of mdr1 gene was significantly enhanced in cultured cells than in other two sources of donor cells. The mdr1 gene was functional since expanded cells effluxed Hoechst 33342 and Rh123 dyes. These results suggest that primitive engraftible stem cells can be expanded in the presence of suitable microenvironments.
Biotechnology and Bioengineering, 1995
ABSTRACT
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 1999
Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-... more Silymarin is a polyphenolic flavonoid derived from milk thistle (Silybum marianum) that has anti-inflammatory, cytoprotective, and anticarcinogenic effects. How silymarin produces these effects is not understood, but it may involve suppression of NF-kappa B, a nuclear transcription factor, which regulates the expression of various genes involved in inflammation, cytoprotection, and carcinogenesis. In this report, we investigated the effect of silymarin on NF-kappa B activation induced by various inflammatory agents. Silymarin blocked TNF-induced activation of NF-kappa B in a dose- and time-dependent manner. This effect was mediated through inhibition of phosphorylation and degradation of Iota kappa B alpha, an inhibitor of NF-kappa B. Silymarin blocked the translocation of p65 to the nucleus without affecting its ability to bind to the DNA. NF-kappa B-dependent reporter gene transcription was also suppressed by silymarin. Silymarin also blocked NF-kappa B activation induced by phorb...
Vaccine, 2000
Active immunization of women against human chorionic gonadotropin (hCG) has been considered as a ... more Active immunization of women against human chorionic gonadotropin (hCG) has been considered as a promising option for contraception. However, prototype hCG vaccines based on natural sources of antigen are expected to be costlier for use by common people. In the present report, a functionally active, cost-eective antigen of bacterial origin has been described. Sulfonation of thiol groups of the protein, anion-exchange puri®cation, refolding with concomitant formation of disul®de bonds in the presence of cysteamine±cystamine redox buer, and slow removal of denaturant resulted in 95% homogenous, monomeric form of the antigen. The recombinant processed antigen [CGb(p)] obtained this way was highly immunopotent. Cellular DNA and endotoxin contaminants were appreciably low in the ®nal product. The immunogenic response was drastically reduced with the unprocessed antigen. This ®nding envisages better prospect of a cost-eective hCG vaccine for birth control.
Oncogene, 2001
While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, ... more While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kB (NF-kB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-kB and AP-1 in the survival of human androgen-independent (DU145) and-dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-kB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative eects of tumor necrosis factor (TNF). TNF induced NF-kB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH 2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-kB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-kB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms. Oncogene (2001) 20, 7597 ± 7609
Uploads
Papers by Asok Mukhopadhyay