Biomedical subjects

D Chakravarti

Publications and source records attributed to D Chakravarti.

At least 19 recordsLinked to original sources

PubMed2002

Initiation of cancer and other diseases by catechol ortho-quinones: a unifying mechanism.

Exposure to estrogens is a risk factor for breast and other human cancers. Initiation of breast, prostate and other cancers has been hypothesized to result from reaction of specific estrogen metabolites, catechol estrogen-3,4-quinones, with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine by 1,4-Michael addition. The catechol of the carcinogenic synthetic estrogen hexestrol, a hydrogenated derivative of diethylstilbestrol, is metabolized to its quinone, which reacts with DNA to form depurinating adducts at the N-7 of guanine and N-3 of adenine. The catecholamine dopamine and the metabolite catechol (1,2-dihydroxybenzene) of the leukemogen benzene can also be oxidized to their quinones, which react with DNA to form predominantly analogous depurinating adducts. Apurinic sites formed by depurinating adducts are converted into tumor-initiating mutations by error-prone repair. These mutations could initiate cancer by estrogens and benzene, and Parkinson's disease by the neurotransmitter dopamine. These data suggest a unifying molecular mechanism of initiation for many cancers and neurodegenerative diseases and lay the groundwork for designing strategies to assess risk and prevent these diseases.

Animals↗

PubMed2001

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

Animals↗

PubMed2001

Regulation of CLOCK and MOP4 by nuclear hormone receptors in the vasculature: a humoral mechanism to reset a peripheral clock.

Circadian clock genes are expressed in the suprachiasmatic nucleus and in peripheral tissues to regulate cyclically physiological processes. Synchronization of peripheral oscillators is thought to involve humoral signals, but the mechanisms by which these are mediated and integrated are poorly understood. We report a hormone-dependent interaction of the nuclear receptors, RAR alpha and RXR alpha, with CLOCK and MOP4. These interactions negatively regulate CLOCK/MOP4:BMAL1-mediated transcriptional activation of clock gene expression in vascular cells. MOP4 exhibits a robust rhythm in the vasculature, and retinoic acid can phase shift Per2 mRNA rhythmicity in vivo and in serum-induced smooth muscle cells in vitro, providing a molecular mechanism for hormonal control of clock gene expression. We propose that circadian or periodic availability of nuclear hormones may play a critical role in resetting a peripheral vascular clock.

3T3 Cells↗

PubMed2001

Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the set oncoprotein.

Acetylation of histones by p300/CBP and PCAF is considered to be a critical step in transcriptional regulation. In order to understand the role of cellular activities that modulate histone acetylation and transcription, we have purified and characterized a multiprotein cellular complex that potently inhibits the histone acetyltransferase activity of p300/CBP and PCAF. We have mapped a novel acetyltransferase-inhibitory domain of this INHAT (inhibitor of acetyltransferases) complex that binds to histones and masks them from being acetyltransferase substrates. Endogenous INHAT subunits, which include the Set/TAF-Ibeta oncoprotein, associate with chromatin in vivo and can block coactivatormediated transcription when transfected in cells. We propose that histone masking by INHAT plays a regulatory role in chromatin modification and serves as a novel mechanism of transcriptional regulation.

Acetylation↗

PubMed2000

Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia.

Initiation of skin tumors in mice is associated with the formation of oncogenic mutations in the H-ras gene. Mice treated on the dorsal skin with the potent polycyclic aromatic hydrocarbon (PAH) carcinogen dibenzo[a,l]pyrene (DB[a,l]P) form papillomas carrying the H-ras codon 61 (CAA to CTA) mutations. These mutations are induced in early preneoplastic skin within 1 day after DB[a,l]P treatment (Oncogene 16 (1998) 3203-3210) and appear to be related to DB[a,l]P-Ade-depurinating adducts (Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10422-10426). The rapid kinetics of mutation induction suggests that abasic sites generated from base depurination may undergo error-prone excision repair in pre-S-phase cells to induce these mutations. Analysis of mutations in the H-ras exon 1 and 2 region in DB[a,l]P-treated early preneoplastic skin indicated great changes in mutation spectra in the preneoplastic period. The initial spectra contained abundant A-->G mutations, which frequently occurred 3' to a putative conserved sequence (TGN-doublet). These mutations appeared to be induced initially as mismatched (G.T) heteroduplexes and then converted into double-stranded mutations by one round of replication. Unlike the A-->G mutations found in DB[a, l]P-treated skin (which forms 99% depurinating adducts), A-->G mutations found in anti-DB[a,l]P-diol epoxide-treated skin (forms 97% stable adducts) did not appear to be G.T heteroduplexes. These results, therefore, suggest that under these conditions, the repair errors occurred only from abasic sites but not from stable adducts. Initiated cells carrying specific oncogenic mutations, formed presumably by misrepair, underwent rapid clonal expansion and regression (transient clonoplasia). The multiplication of initiated stem cells during transient clonoplasia may be a factor determining the tumor-initiating potential of some PAH carcinogens.

Animals↗

PubMed1999

A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity.

Nucleosomal histone modification is believed to be a critical step in the activation of RNA polymerase II-dependent transcription. p300/CBP and PCAF histone acetyltransferases (HATs) are coactivators for several transcription factors, including nuclear hormone receptors, p53, and Stat1alpha, and participate in transcription by forming an activation complex and by promoting histone acetylation. The adenoviral E1A oncoprotein represses transcriptional signaling by binding to p300/CBP and displacing PCAF and p/CIP proteins from the complex. Here, we show that E1A directly represses the HAT activity of both p300/CBP and PCAF in vitro and p300-dependent transcription in vivo. Additionally, E1A inhibits nucleosomal histone modifications by the PCAF complex and blocks p53 acetylation. These results demonstrate the modulation of HAT activity as a novel mechanism of transcriptional regulation.

Acetyltransferases↗

PubMed1998

Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Animals↗

PubMed1998

Linear amplification mapping of polycyclic aromatic hydrocarbon-reactive sequences in H-ras gene.

Linear amplification, or primer directed single-strand DNA synthesis, is commonly used in applications such as cycle sequencing and mapping replication block sites in DNA. Although linear amplification reactions would be expected to synthesize full-length single-stranded DNA, the synthesis is often prematurely terminated. We describe the optimization of a linear amplification protocol for synthesizing a full-length (985-nt) single-stranded pBR322 segment. The enzyme activities of five DNA polymerases commonly used in PCR amplification, namely, AmpliTaq, Stoffel fragment, Tth, Pfu, and Vent, were tested either singly or in combination. The results indicate that the additive action of small amounts of proofreading DNA polymerases to a nick-translating polymerase is optimum for linear amplification. From these results, a linear amplification protocol was developed to map DNA synthesis-blocking sites generated by the reaction of (+/-) anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, or anti- or syn-dibenzo[a,l]pyrene-9,10-diol-11,12-epoxide with H-ras DNA surrounding the oncogenic codon 61 region. The results indicate that the central A of H-ras codon 61 (CAA) reacts with these polycyclic aromatic hydrocarbons.

Base Sequence↗

PubMed1997

Nuclear receptor coactivator ACTR is a novel histone acetyltransferase and forms a multimeric activation complex with P/CAF and CBP/p300.

We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.

Acetyltransferases↗

PubMed1997

Nuclear receptor repression mediated by a complex containing SMRT, mSin3A, and histone deacetylase.

The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. Here we show that SMRT and N-CoR directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. In addition, we demonstrate that the recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit repressor complex. Consistent with this model, we find that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and differentiation of myeloid leukemia (HL-60) cells. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests this type of regulation may be more widely conserved than previously suspected.

Antineoplastic Agents↗

PubMed1996

Two contact regions between Stat1 and CBP/p300 in interferon gamma signaling.

Interferon gamma (IFN-gamma) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-gamma-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP/p300 and the carboxyl-terminal region of Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-gamma-induced transcription. Because CBP/p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-gamma is based at least in part on direct competition by nuclear Stat1 with E1A for CBP/p300 binding.

Adenovirus E1A Proteins↗

PubMed1996

Role of CBP/P300 in nuclear receptor signalling.

The nuclear receptor superfamily includes receptors for steroids, retinoids, thyroid hormone and vitamin D, as well as many related proteins. An important feature of the action of the lipophilic hormones and vitamins is that the maintenance of homeostatic function requires both intrinsic positive and negative regulation. Here we provide in vitro and in vivo evidence that identifies the CREB-binding protein (CBP) and its homologue P300 (refs 6,7) as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signalling pathways.

CREB-Binding Protein↗

PubMed1996

Novel retinoic acid receptor ligands in Xenopus embryos.

Retinoids are a large family of natural and synthetic compounds related to vitamin A that have pleiotropic effects on body physiology, reproduction, immunity, and embryonic development. The diverse activities of retinoids are primarily mediated by two families of nuclear retinoic acid receptors, the RARs and RXRs. Retinoic acids are thought to be the only natural ligands for these receptors and are widely assumed to be the active principle of vitamin A. However, during an unbiased, bioactivity-guided fractionation of Xenopus embryos, we were unable to detect significant levels of all-trans or 9-cis retinoic acids. Instead, we found that the major bioactive retinoid in the Xenopus egg and early embryo is 4-oxoretinaldehyde, which is capable of binding to and transactivating RARs. In addition to its inherent activity, 4-oxoretinaldehyde appears to be a metabolic precursor of two other RAR ligands, 4-oxoretinoic acid and 4-oxoretinol. The remarkable increase in activity of retinaldehyde and retinol as a consequence of 4-oxo derivatization suggests that this metabolic step could serve a critical regulatory function during embryogenesis.

Animals↗

PubMed1995

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Animals↗

PubMed1995

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.

DNA-Binding Proteins↗

PubMed1993

Eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. Cloning, characterization, and expression of the gene encoding the 45,346-Da protein.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes hydrolysis of GTP bound to a 40 S ribosomal initiation complex with the subsequent joining of a 60 S ribosomal subunit to form an 80 S initiation complex. The yeast gene that encodes eIF-5, designated TIF5, has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. TIF5 is a single-copy gene that maps on yeast chromosome XVI and is essential for cell viability. The gene contains an intron-free open reading frame that encodes a protein of calculated M(r) 45,346 in close agreement with the apparent molecular weight of eIF-5 isolated from yeast cells. Sequence analysis of the gene reveals several interesting features. First, the presence of two in-frame translational start sites located 51 base pairs apart suggests the possibility that two proteins, differing by an amino-terminal extension of 17 amino acids, could be generated from the TIF5 gene via differential translational starts. This would explain the presence, in yeast cell lysates, of two forms of eIF-5 differing in molecular weight by about 2,000. Second, the predicted amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.

Amino Acid Sequence↗

PubMed1993

Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates. The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.

Animals↗

PubMed1992

Promoter-containing ribosomal DNA fragments function as X-Y meiotic pairing sites in D. melanogaster males.

The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X-Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X-Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X-Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X-Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P-element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X-Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter-containing rDNA fragments are autonomously capable of being transcribed and of functioning as X-Y pairing sites, but not of forming a mini-nucleolus.

Animals↗