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G C Das

Publications and source records attributed to G C Das.

At least 19 recordsLinked to original sources
PubMed2001

Taxol-induced cell cycle arrest and apoptosis: dose-response relationship in lung cancer cells of different wild-type p53 status and under isogenic condition.

The effective dose, schedule, molecular basis of the cytotoxicity of taxol and their dependence on the genetic background in tumor cells are still not well understood. Here, we examined how the dose-response relationship for taxol varies in lung cancer cells with different p53 status and under isogenic conditions. DNA content analyses in A 549 (p53, +/+) and H 1299 (p53, -/-) cells, showed that taxol progressively induced G2/M arrest in both cell lines in a concentration-dependent manner, which was accompanied by a parallel decrease in the G1 population. G2/M arrest, however, occurred at a lower concentration in A 549 cell lines than in H 1299 cells. The S-phase population in A 549 cells was not significantly changed up to 0.025 microM, but dropped by six-fold at 1.0 microM taxol, in contrast to that in H 1299 cells. A sub-G1 apoptotic population was present at 24 h, even at 0.002 microM taxol, when G2/M arrest was not appreciably detected. In both cell lines, the maximum apoptosis of about 28% was achieved at 0.025 microM taxol, implicating that wild-type p53 does not modulate the level of taxol-induced apoptosis. When we examined the role of the wild-type p53 in isogenic cell lines developed in a H 1299 background, the maximum level of apoptosis was in the range of 28-34% at a drug concentration around 0.03 microM, not significantly different from that observed in parental H 1299 cells. We conclude that taxol is effective in inducing apoptosis at very low doses (0.020-0.035 microM), and that the presence or absence of the wild-type p53 does not make a statistically significant difference in the level of apoptotic cell death in these lung cancer cell lines, but the maximum is attained at a lower drug concentration in the presence of p53.

Antineoplastic Agents, Phytogenic↗
PubMed2000

Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma.

Aflatoxin B1 (AFB1) induced mutation of the p53 gene at codon 249 (p53mt249) is critical during the formation of hepatocellular carcinoma (HCC) following hepatitis B virus (HBV) infection. p53mt249 markedly increases insulin-like growth factor II (IGF-II) transcription largely from promoter 4, accumulating the fetal form of IGF-II. Modulation of the transcription factor binding to IGF-II P4 by wild-type p53 and p53mt249 was identified. Wild-type p53 inhibited binding of transcription factors Sp1 and TBP on the P4 promoter, while p53mt249 enhanced the formation of transcriptional complexes through enhanced DNA-protein (Sp1 or TBP) and protein-protein (Sp1 and TBP) interactions. p53mt249 stimulates transcription factor Sp1 phosphorylation which might be a cause of increased transcription factor binding on the P4 promoter while wild-type p53 does not. Transfection of hepatocytes with p53mt249 impaired induction of apoptosis by the HBV-X protein and TNF-alpha. Therefore, the blocking of apoptosis through enhanced production of IGF-II should provide a favorable opportunity for the selection of transformed hepatocytes. These results explain the molecular basis for the genesis of HCC by p53mt249 which was found to be induced by a potent mutagen, AFB1.

Aflatoxin B1↗
PubMed1998

The A enhancer of polyomavirus: protein-protein interactions for the differential early and late promoter function under nonreplicating conditions.

The A enhancer of the polyomavirus early promoter is a 110-bp domain located in the late region and it contains the major late RNA initiation site. The 'core' of this enhancer binds several cellular proteins, including proteins PEA1, PEA2 and PEA3. Another element, NF-D/YY1, is also located in this enhancer. The A enhancer is known to stimulate the early promoter, contains auxiliary elements for replication and serves as the initiator for late transcription. It may also be involved in early-to-late switch. We were interested in investigating how the A-core- and NF-D-binding proteins regulate early and late promoter activity under nonreplicating conditions and how the protein-protein interactions affect the function of the individual elements. By point mutational analysis, we show that, except for PEA1, all other proteins activate the early and late promoters differentially under nonreplicating conditions. All three core-binding proteins, and the protein bound to the NF-D site, are activators and have a combinatorial effect on early promoter activity. On late transcription, only PEA1 acts positively and inactivation of the NF-D site is without any effect. In contrast, PEA2 and PEA3 have a repressor-like activity under nonreplicating conditions, indicating that these two proteins might be involved in repressing late transcription, probably early in infection. By increasing the spacing between two consecutive elements, we further show that protein-protein interaction is important for enhancer function. Transactivation of the early promoter was affected by mutations in all four protein-binding sites. Responsiveness of these factors in regard to the late promoter was parallel to their intrinsic promoter strength. The effects of middle and large T antigens are parallel for both the early and the late promoter, suggesting that the pathway(s) for transactivation function of these oncoproteins may overlap downstream. This study with cloned viral promoter will be reflective of situations in vivo, at least partially, under nonreplicating conditions.

3T3 Cells↗
PubMed1996

Interaction of human polyomavirus BK with the tumor-suppressor protein p53.

We have been studying the interaction of the oncogenic human polyomavirus BK (BKV) with the tumor-suppressor protein p53 to understand the biology of this virus as well as to understand the basic mechanisms of p53 transactivation. We here demonstrate that p53 binds specifically to the viral promoter at two different sites, S-I (np 361-383) and S-II (np 314-336) in the late region. Site S-I is a 23 bp domain comprising an unique combination of a 10 bp consensus monomer binding site (Pu Pu Pu C (A/T) (T/A) G Py Py Py) which is contiguous with a GC-rich Sp-1 motif that binds p53 in the SV40 promoter. Site S-II also spans a 23 bp sequence containing two tandem consensus binding sites with three base pair mismatches in each and a one base pair deletion. A dimer of a 100 bp region spanning both the binding sites or the site S-I alone induced p53 responsiveness to a basal promoter when cloned upstream from the TATA box, but a similar construct using S-II did not. One tumor-derived mutant protein, p53-175 H, which is defective in DNA binding, also failed to transactivate the reporter gene. We further show that p53 binding-dependent transactivation is abrogated by BKV large T antigen, thereby suggesting an interaction between these two proteins in vivo. In contrast to the isolated p53 binding site, viral early promoter is repressed by p53 in H 1299 cells and the mutants are defective in this function to varying extent. This is suggestive of an involvement of cellular factors in modulating p53's function in the context of the whole promoter. p53 binding sites in BKV are flanked by the binding sites for transcription factors Sp-1 and NF-1 and we show that these transcription factors are present in the immunocomplex with purified p53, implicating modification of p53's transactivation function by protein-protein interaction. Thus, oncoprotein synthesis in this virus might be modulated by p53 in vivo by a complex mechanism other than simple DNA binding and sequestration of the TATA binding protein. Together with SV40 and polyomavirus, which also harbor p53 binding sites, this viral system will serve as a model to understand the role of p53 in viral infection.

Antigens, Viral, Tumor↗
PubMed1995

Cell-specific modulation of the papovavirus promoters by tumor-suppressor protein p53 in the absence of large T-antigen.

The oncoproteins from several DNA tumor viruses form a complex with p53 and inactivate its function. Wild-type p53 is a transcription factor and can regulate eukaryotic promoters both positively and negatively. To elucidate the basis of the opposing functions and to understand whether and how oncoprotein synthesis in papovaviruses is regulated by p53, we studied modulation of the early promoters of SV40, polyomavirus and BK virus in the absence of the interfering effect of viral large T antigens. We here show that murine p53 can regulate the viral promoters either positively or negatively depending on the cell type. A temperature-sensitive mutant p53, 135 Val, at 37 degrees C also showed a cell-specific effect. These results suggest that promoter activation by p53 is not solely determined by p53 binding site, but host factors modulate p53's transactivation function. A TATA-less polyomavirus late promoter was also repressed in HeLa cells and the level of repression was much less in the presence of active early promoter. As p53 and 135 Val were overexpressed to similar extent in different transfected cell lines, variation in transactivation function is not due to the difference in the level of expressed protein. Our observations thus suggest that p53 interactions with cellular factors in addition to the TATA binding protein (TBP) are important for activator and repressor functions of p53. Well-defined viral promoters could thus provide us with an important tool for the identification and characterization of cellular factors that modulate p53-binding dependent gene regulation in animal cells.

3T3 Cells↗
PubMed1995

Acute renal failure associated with freshwater fish toxin.

Clinical observations on toxicity after ingestion of gall bladder of Labeo rohita, a freshwater fish found commonly in India, were recorded from 22 patients between 1985 to 1990. The gall bladder in raw, cooked or desiccated form was swallowed as a traditional method of treatment for various chronic diseases. Patients generally presented with gastrointestinal symptoms such as cramping pain, nausea and vomiting within 12 hours (mean 4.6 +/- 3.7 hours) after ingestion. Subsequently renal failure was observed in all and hepatic dysfunction in some (36.3%) patients. The outcome in general is good. From the clinical course the role of toxic substance(s) is presumed as the causative factor(s), which needs further evaluation.

Acute Kidney Injury↗
PubMed1994

Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function.

The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.

3T3 Cells↗
PubMed1993

Identification of proteins binding to the F441 locus of polyomavirus B enhancer that are required for its activity in embryonic carcinoma cells.

A point mutation at nucleotide 5233 of the polyomavirus (A2 strain) enables it to overcome growth restriction in undifferentiated embryonal carcinoma cells. We analysed the binding of nuclear proteins from F9 cells to a 38 bp region that spans this site of mutation and encompasses two copies of the bovine papillomavirus core sequence, CCACCC, and characterized this domain by mutational analysis. Our results showed that the F441 mutation creates a sequence motif which binds TEF-1 or a TEF-1-like protein from F9 cells more strongly than its wild-type counterpart and increases its activity by about 10-fold. Another protein identified as CP1 binds with increased affinity in the presence of the F441 mutation to the CAT box-like sequences which is contiguous with the downstream CCACCC box. Point mutations within these two motifs that abolished binding in vitro also impaired the activity of the F441 locus in vivo. As neither the wild-type sequence without the F441 mutation, nor the F441 template without the CAT box has appreciable activity in vivo, interaction between these two elements is required for function. At a higher level of organization, this interaction is probably extended to factors bound to other domains in the A and B enhancer.

Animals↗
PubMed1991

Proteins of the nuclear factor-1 family act as an activator of the late promoter in human polyomavirus BK in vitro.

The cis-acting elements for the early and late promoters, as well as the enhancer in the prototype strains of human polyomavirus BK (BKV) are located within a 500 bp intergenic region. We previously studied the specificity of protein binding in this region in vitro and showed that the interaction of proteins of the nuclear factor-1 (NF-1) family is crucial for early promoter activity. We have now extended our study to the BKV late promoter. We show that the late promoter activity in HeLa cell extracts is poor compared to the activity of the early promoter. Using a high template to protein ratio, multiple start sites were detected by primer extension analysis. DNase I protection experiments revealed the presence of three NF-1 binding sites in the late side, in addition to those identified previously in the 68 bp repeats and C element. Competition transcription assays using binding sites for NF-1, AP-1, Sp-1 and a complete 68 bp repeat indicated that only the 68 bp repeat and the NF-1 binding site competed significantly with the late promoter activity. A point mutation in the NF-1 binding site, which destroys the ability of the oligonucleotide to bind NF-1, also impaired its capacity to compete with the late promoter. The ability of NF-1 to activate both the early and late promoters suggests that the proteins of this family act as a bidirectional transcriptional activator in this virus.

BK Virus↗
PubMed1991

Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

Expression of the chicken beta B1-crystallin gene was examined. Northern (RNA) blot and primer extension analyses showed that while abundant in the lens, the beta B1 mRNA is absent from the liver, brain, heart, skeletal muscle, and fibroblasts of the chicken embryo, suggesting lens specificity. Promoter fragments ranging from 434 to 126 bp of 5'-flanking sequence (plus 30 bp of exon 1) of the beta B1 gene fused to the bacterial chloramphenicol acetyltransferase gene functioned much more efficiently in transfected embryonic chicken lens epithelial cells than in transfected primary muscle fibroblasts or HeLa cells. Transient expression of recombinant plasmids in cultured lens cells, DNase I footprinting, in vitro transcription in a HeLa cell extract, and gel mobility shift assays were used to identify putative functional promoter elements of the beta B1-crystallin gene. Sequence analysis revealed a number of potential regulatory elements between positions -126 and -53 of the beta B1 promoter, including two Sp1 sites, two octamer binding sequence-like sites (OL-1 and OL-2), and two polyomavirus enhancer-like sites (PL-1 and PL-2). Deletion and site-specific mutation experiments established the functional importance of PL-1 (-116 to -102), PL-2 (-90 to -76), and OL-2 (-75 to -68). DNase I footprinting using a lens or a HeLa cell nuclear extract and gel mobility shifts using a lens nuclear extract indicated the presence of putative lens transcription factors binding to these DNA sequences. Competition experiments provided evidence that PL-1 and PL-2 recognize the same or very similar factors, while OL-2 recognizes a different factor. Our data suggest that the same or closely related transcription factors found in many tissues are used for expression of the chicken beta B1-crystallin gene in the lens.

Animals↗
PubMed1990

Purification and biochemical characterization of hepatocyte nuclear factor 2 involved in liver-specific transcription of the human alpha 1-antitrypsin gene.

The upstream region of the human alpha 1-antitrypsin gene binds liver-specific proteins. Two of these proteins, hepatocyte nuclear factors 1 and 2, are essential for liver-specific transcription of this gene. We report for the first time the purification of hepatocyte nuclear factor 2 from rat liver nuclei. This protein, purified to apparent homogeneity by DNA sequence-specific affinity chromatography, appeared on sodium dodecyl sulfate-polyacrylamide gels as a single polypeptide band of molecular mass of 68 kDa. The polypeptide after extraction from the gel matrix and subsequent renaturation bound specifically to the recognition sequences (-88 to -125) and protected the same region of the promoter against DNase I digestion.

Animals↗
PubMed1989

Identification of HeLa cell nuclear factors that bind to and activate the early promoter of human polyomavirus BK in vitro.

Human polyomavirus BK (BKV), an oncogenic DNA virus, differs from other papovaviruses in the organization of the regulatory region and in tissue tropism for kidney cells. The noncoding regulatory region of the viral genome in prototype strains includes three 68-base-pair (bp) repeats, each containing a number of potential regulatory elements. Some of these signals are unique to human papovaviruses, and others are homologous to those identified in many viral and cellular genes. We evaluated the contribution of individual 68-bp repeats to the initiation of transcription from the early promoter in a HeLa cell extract and identified cis-acting elements to which human cellular factors bind to activate transcription. The early promoter with only one copy of the 68-bp repeat could accurately initiate transcription in vitro, but additional copies were required for its stimulation. DNA-binding assays and DNase I protection experiments identified six domains in the regulatory region protected by human cellular factors. Two of these footprints were located within the proximal and distal 68-bp repeats, and one was located at the late side of the repeats. These footprints were centered over a TGGA(N)5-6GCCA core and were produced by a protein of the nuclear factor 1 (NF-1) family. This protein is either identical or similar to that which binds to the high-affinity site at the origin of adenovirus DNA replication. Three other domains, two at the junctions of the 68-bp repeats and one in the late side of the repeats, were partially protected by proteins with AP-1- and Sp-1-like activities. Transcription initiation from the early promoter was drastically reduced when a complete 68-bp repeat or the NF-1 binding site was used as a competitor in the in vitro assay. However, a point mutation within the NF-1 binding site, which reduced NF-1 binding in vitro to a level comparable to that of nonspecific DNA, also eliminated its ability to compete with early transcription. The murine homolog of the AP-1 binding site had a modest effect on in vitro transcription. Our results suggest that, among the multiple HeLa cell nuclear factors, NF-1 acts as a major activator of the early promoter in vitro.

BK Virus↗
PubMed1988

Promoter activity of the two chicken delta-crystallin genes in a Hela cell extract.

The in vitro transcriptional activity of the two delta-crystallin genes (5'-delta 1-delta 2-3') of the chicken was studied in a whole Hela cell extract. Both the delta 1 and delta 2 promoters were recognized by RNA polymerase II in this heterologous system. The major RNA initiation site from the delta 1 promoter was the same in vitro as that which occurs in vivo, as judged by mapping with S1-nuclease, although other minor initiation sites upstream and downstream of the major initiation site were noted. A primer extension experiment showed that the longest RNA synthesized in vitro from a delta 2 template initiated near the beginning of the first exon. The delta 1 promoter was several-fold stronger than that of delta 2 under the present in vitro conditions. Transcription from the delta 1 promoter was abolished by a competitor fragment (c'-II; includes -328 to -63) purified from the delta 2 promoter, indicating that one or more common transcription factors binding upstream from the TATA box are required for in vitro function of the two delta-crystallin promoters. Thus, in the Hela cell extract both delta-crystallin genes contain a functional promoter. We consider the possibility that the single 5'CCAAT3' sequence present in the delta 1 promoter (but lacking in the delta 2 promoter) may contribute to its greater core activity under our conditions. The greater promoter activity of the delta 1-crystallin gene in the Hela cell extract was not sufficient to account for the large ratio of delta 1 to delta 2 mRNA (approximately 50 to 100) in the embryonic chicken lens.

Animals↗