Biomedical subjects

K U Kumar

Publications and source records attributed to K U Kumar.

14 recordsLinked to original sources

PubMed2000

BAG-1, a novel Bcl-2-interacting protein, activates expression of human JC virus.

Transcription of the human polyomavirus JC virus (JCV) genome is regulated by cellular proteins and the large tumour (T) antigen. Earlier studies led to the identification of nuclear factor-1 (NF-1)-binding sites in the JCV enhancer by DNase I protection assays of extracts from retinoic acid (RA)-differentiated P19 embryonal carcinoma (EC) cells. In this study, a cDNA clone that encodes a protein capable of binding to the JCV NF-1 sites was isolated from an RA-differentiated EC cell cDNA library. Sequence analysis revealed that the cDNA isolated was identical to the previously described Bcl-2-interacting protein BAG-1 (Bcl-2-associated athano gene-1). Results from RNA studies indicated that BAG-1 is expressed in several cell types. Co-transfection of a recombinant BAG-1 expression plasmid with JCV promoters indicated that BAG-1 stimulates transcription of the JCV(E) promoter and to a lesser extent the JCV(L) promoter. Mutations in the NF-1 sites in the JCV(E) promoter eliminated the activation by BAG-1. Thus, BAG-1 is a novel transcription factor that may play a role in JCV expression.

Amino Acid Sequence↗

PubMed1999

Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.

Animals↗

PubMed1998

Identification and requirement of three ribosome binding domains in dsRNA-dependent protein kinase (PKR).

The interferon-inducible, double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis initiation by phosphorylating the alpha-subunit of eukaryotic translation initiation factor 2 (eIF-2). The amino-terminal half of PKR contains two dsRNA binding domains, and the kinase domain resides in the carboxy-terminal half of the protein. PKR is a ribosomal-associated protein. In this report, we provide evidence that PKR contains three ribosome interaction sites, two that are localized in each of the dsRNA binding domains and one that is localized in the kinase domain. All three domains can associate with polysomes independently. The ribosome association of the dsRNA binding domains requires dsRNA binding activity. Ribosome interaction of either the individual or the combined dsRNA binding domains was disrupted by 0.1 M KCl. In contrast, the ribosome interaction of intact PKR and the isolated kinase domain was largely resistant to 0.5 M KCl. These results indicate that all three domains of PKR contribute to the high-affinity ribosomal association. After dissociation of polysomes with EDTA, both intact PKR and the isolated kinase domain were primarily associated with the 60S ribosomal subunit. Coexpression of the adenovirus VAI RNA, an RNA polymerase III gene product that binds and inactivates PKR, disrupted ribosomal association of intact PKR, but not of the isolated PKR kinase domain. The results support a model where VAI RNA induces a major conformational change in PKR to prohibit ribosome association of all interaction sites. In contrast, other inhibitors of PKR including vaccinia virus E3L and K3L gene products, and the HIV trans-activating response (TAR) element binding protein TRBP, did not disrupt ribosome association of PKR. The results suggest a novel mechanism by which viral RNAs may inactivate PKR through disrupting ribosome association.

Adenoviridae↗

PubMed1998

Phosphorylation of eukaryotic translation initiation factor 2 mediates apoptosis in response to activation of the double-stranded RNA-dependent protein kinase.

The interferon-inducible, double-stranded (ds) RNA-dependent serine/threonine protein kinase (PKR) plays a role in viral pathogenesis, cell growth, and differentiation and is implicated as a tumor suppressor gene. Expression of a trans-dominant negative, catalytically inactive mutant PKR protected NIH3T3 cells from apoptosis in response to either treatment with tumor necrosis factor alpha (TNF alpha), serum deprivation. In cells expressing mutant PKR, TNF alpha, but not dsRNA induced transcription from a nuclear factor kappa B-dependent promoter, demonstrating specificity for dsRNA in signaling through the PKR pathway. Serum or platelet-derived growth factor addition to serum-deprived mutant PKR-expressing cells induced transcription of the early response genes c-fos and c-jun, indicating that the immediate early response signaling was intact. Overexpression of wild-type PKR in a transient DNA transfection system was sufficient to induce apoptosis. TNF alpha-induced apoptosis correlated with increased phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the primary physiological substrate of the PKR. Furthermore, forced expression of a nonphosphorylatable S51A mutant eIF-2 alpha partially protected cells from TNF alpha-induced apoptosis, and expression of a S51D mutant eIF-2 alpha, a mutant that mimics phosphorylated eIF-2 alpha, was sufficient to induce apoptosis. Taken together, these studies identify a novel requirement for PKR in stress-induced apoptosis that is mediated through eIF-2 alpha phosphorylation.

3T3 Cells↗

PubMed1998

Identification and characterization of a JC virus pentanucleotide repeat element binding protein: cellular nucleic acid binding protein.

The JC virus (JCV) control region contains AGGGAAGGGA, the tandem pentanucleotide repeat element (Pnt2). Several proteins specifically interacted via Pnt2 to regulate the expression of JCV early promoter-enhancer (JCV(E)) or late promoter-enhancer (JCV(L)). In this study, a JCV Pnt2 oligonucleotide probe was used to screen a cDNA expression library from glial P19 mouse embryonal carcinoma cells. A cDNA clone was isolated by Southwestern blot assay and it produced a protein that reproducibly and specifically bound to Pnt2. This cDNA had 100% homology to one of three previously identified mouse cDNAs called cellular nucleic acid binding proteins (Cnbps). Cnbps are a highly homologous family of eukaryotic genes implicated in functional interactions with cytoplasmic RNA and regulatory DNA elements. An mRNA of 2.2 kb of Pnt2-interacting Cnbp (PCnbp) was seen in undifferentiated, muscle or glial P19 cells. When expressed from a cDNA expression vector as a fusion protein that also contained 115 kDa from beta-galactosidase, a Pnt2 binding protein (PCNBP) specifically bound to Pnt2 in Southwestern blots as a 30 kDa component of the 145 kDa fusion protein. Furthermore, JCV(E) expression was negatively regulated by PCnbp produced in vivo from the cDNA expression vector. Regulation of JCV(L) was unaffected. We suggest a novel role for CNBP as a PCNBP that interacts with Pnt2 in the negative transcriptional regulation of JCV(E).

5' Untranslated Regions↗

PubMed1997

Dual NF1-requiring effect of human neurotropic JC virus composite pentanucleotide repeat elements on early and late viral gene expression.

Human polyoma JC virus (JCV) is a largely brain cell-specific virus that is associated with AIDS. The neurotropism involves the various transcription factors interacting with their cognate sequences in the JCV early (JCVE) and late (JCVL) promoters-enhancer, but the mechanism is unclear. The JCV tandem AGGGA pentanucleotide double repeat element (Pnt2) and the abutting NF1 site form a composite Pnt2 (cPnt2). Here, we studied the roles of both sites of cPnt2 in the expression of JCVE and JCVL. JCVE activity was progressively increased in U-87 MG human glioblastoma cells following the site-specific mutation of Pnt2 in one and both 98-bp repeats. In contrast, the activity for JCVL was progressively reduced by mutating single and double Pnt2s. However, JCVE activity was unaffected by mutating both Pnt2s when both NF1 sites of cPnt2 were mutated. The activity of JCVL was also unaffected by double Pnt2 mutations after the NF1 sites were mutated. Differentiating P19 glial cells showed similar results. Next, Pnt2-interacting proteins were examined by two in vitro assays with a Pnt2 oligonucleotide. Mobility shift assays showed one Pnt2-protein complex for U-87 MG cell extracts and two for P19 glial cell extracts. However, similar results were obtained for glial, muscle, and undifferentiated P19 cells. In UV cross-linking assays, two Pnt2-binding proteins (Pnt2BPs) were detected. The results suggested that the effects on JCVE and JCVL expression of Pnt2BP and NF1 via cPnt2 are dual and require a glial cell-specific NF1 function.

Binding Sites↗

PubMed1996

Human JC virus cAMP response elements functional for enhanced glial cell expression in differentiating embryonal carcinoma cells.

Human JC virus (JCV) is glial cell-specific for growth and expression. This specificity is attributed to the cis-acting sequences of the two 98-bp tandem repeats in the JCV regulatory region. JCV causes brain lesions, especially in AIDS patients. To study the expression of JCV in glial cells, the role of both repeat region TGAGCTCA sequences, which are homologous to the classical TGAGCTCA cAMP response element (CRE), was examined. The effect of the CRE on expression of the JCV early promoter (JCVE) in response to cAMP was studied with undifferentiated, glial and muscle P19 embryonal carcinoma cells. The results showed a threefold increase in response to cAMP only in the glial cells in which JCV is efficiently expressed. The direct in vivo role of the JCV CRE was confirmed by site-directed mutagenesis. Additionally, a CRE oligonucleotide was induced by cAMP in vivo, and in in vitro transcription assays with glial cell extracts. The early promoter of human BK virus containing nonhomologous CRE sequences was previously shown not to be glial cell-specific and failed to respond to cAMP in glial P19 cells in this study. Mobility shift assays showed the cAMP-induced in vitro interaction of glial cell protein(s) with the CRE oligonucleotide. Southwestern blot and uv crosslinking experiments identified an approximately 43-kDa protein interacting with the JCV CRE oligonucleotide. The results indicate that the in vivo expression of JCVE is specifically increased in response to cAMP only in glial cells and JCV CRE in vitro protein complexes are only detected in response to cAMP for glial cell extracts.

Animals↗

PubMed1996

Glial and muscle embryonal carcinoma cell-specific independent regulation of expression of human JC virus early promoter by cyclic AMP response elements and adjacent nuclear factor 1 binding sites.

The human polyoma JC virus (JCV) is a glial cell-specific virus and is the etiological agent for the terminal AIDS-associated brain disease, progressive multifocal leukoencephalopathy (PML). JCV contains several binding sites for transcriptional factors that are important for activity in glial cells, including cyclic AMP (cAMP) response elements (CREs) which are four nucleotides from nuclear factor 1 (NF1) sites within the two 98 bp repeat regions. We studied the combined role of cAMP and NF1 in regulating the expression of the JCV early promoter-enhancer (JCVE) in differentiating glial and muscle P19 embryonal carcinoma cells. JCVE expression remained several-fold higher in the presence of cAMP in glial cells, irrespective of whether the relatively strong activity of JCVE was greatly reduced by NF1 site mutations. In contrast, cAMP had no effect in muscle cells, independent of whether the modest activity of JCVE was two-fold higher due to NF1 site mutations. The in vivo effects were confirmed with in vitro transcription assays using glial cell extracts, competitors of CRE, and the NF1 site, and single repeat JCVE region with mutations in the NF1 II/ III binding sites as templates. The in vitro results also indicated that the effects were due to the CREs of JCV, rather than to the indirect effects of cAMP. Overall, the results indicated that NF1 and cAMP have independent, different, tissue-specific, and direct effects in the regulation of JCVE. These effects may contribute the neurotropic PML-inducing pattern of expression of JCVE.

Binding Sites↗

PubMed1996

Evidence for a mechanism of demyelination by human JC virus: negative transcriptional regulation of RNA and protein levels from myelin basic protein gene by large tumor antigen in human glioblastoma cells.

Human JC virus (JCV) is a neurotropic human polyomavirus that was found in the plaques and oligodendroglial cells of the brains of patients with the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Transgenic mice expressing JCV large tumor (T)-antigen from integrated DNA showed dysmyelination in the central nervous system. However, the role of T-antigen from episomal DNA in the demyelination in PML remains unclear. In this report, we examined the effect of episomally expressed JCV T-antigen on the expression of myelin basic protein (MBP) in U-87 MG human glioblastoma cells to study the mechanism of demyelination. Expression assays of the MBP promoter in U-87 MG detected a 2.5-fold reduction in cells expressing intact T-antigen. Next, U-87 MG expressing T-antigen were examined by RNase protection assays for mRNA accumulation from the endogenous MBP promoter. Also, the expression of the MBP promoter plasmid was determined using in vitro transcription assays with extracts from T-antigen expressing cells. Both assays found a similar down-regulation of the MBP promoter by T-antigen, confirming that negative regulation occurred at the transcriptional level for the endogenous and exogenous MBP promoters. Furthermore, in situ immunofluorescence assays and quantitative Western blot analysis provided convincing evidence of a similar reduction in the level of MBP produced from the functional endogenous gene in U-87 MG glioblastoma cells expressing T-antigen. Thus, we provide evidence for the role of T-antigen in a transcriptional control mechanism for the demyelination that is caused by JCV in PML patients.

Antigens, Polyomavirus Transforming↗

PubMed1996

Up-regulation of hormone response of human papillomavirus type 16 expression and increased DNA-protein binding by consensus mutations of viral glucocorticoid response elements.

Human papillomaviruses (HPVs) and steroid hormones are linked to the development of cervical cancer. Studies from our laboratory and others showed that the steroid glucocorticoid and progesterone hormones activated the expression of HPV type 16. This activation was attributed to the specific interaction of the glucocorticoid receptor (GR) with the three glucocorticoid response elements (GREs) in the HPV16 regulatory region. In the present study, we first examined the glucocorticoid response mediated through the GREs, using GRE consensus (GREc) mutations and expression assays from a heterologous basal promoter. Both single and triple HPV16 GREc constructs increased expression in the presence of the dexamethasone glucocorticoid in HeLa cervical carcinoma cells and primary baby rat kidney epithelial cells, in comparison with the triple wild-type GREs. Further, the hormone increased significantly the expression of the viral E6-E7 oncogene mRNA from intact HPV in primary human ectocervical cells in in situ hybridization assays. Three in vitro assays of DNA-protein interaction with oligonucleotides and HeLa cell extracts showed a higher binding of protein to two of the HPV16 GREcs than to the wild-type GREs. This applied especially to the GRE containing an overlapping NF1 half site, that also had a greater differential induction by dexamethasone of expression in vivo. The NF1 site was mutated in the GREc that also was bound by unique, lower-mobility complexes in electrophoretic mobility shift assays. UV-crosslinking assays confirmed the increased binding and showed binding by a 96-kDa protein, probably the GR. Our results show an important role of glucocorticoids in HPV16 expression. The direct action through the HPV16 GREs is suggested to be mediated by the hormone-activated GR in association with other factors.

Animals↗

PubMed1996

Human JC virus nuclear factor 1 binding motifs and large tumor antigen region rquired for transactivation of late promoter.

The nuclear factor 1 (NF-1) motifs, NF-1 II/III, In the two 98-bp repeats of the transcription-regulatory region of JC virus (JCV), have a critical role in brain-specific transcription from the JCV early promoter-enhancer. In this study, the role of these motifs in transactivation of the JCV late promoter-enhancer (JCVL) was examined in differentiating glial P19 embryonal carcinoma cells. The expression of papovaviral large tumor antigen (T-Ag) in the glial cells was shown by double immunofluorescence assays. By using site-directed mutagenesis and in vivo assays, the two wild-type NF-1 II/III sites, but not the third site, were found to be essential for the transactivation of JCVL by JCV T-Ag. In vitro transcription assays confirmed this specific transactivation and the transactivation was abolished by T-Ag antibody. In electrophoretic mobility shift assays, expression of JCV T-Ag increased the binding of a factor(s) to the 98-bp repeat. T-Ag antibody abolished the increase of binding. Binding assays with oligonucleotides of NF-1 11/111 motifs showed that the increased binding specifically required the wild-type NF-1 II/III sequences and confirmed the requirement of T-Ag. To determine the region of T-Ag necessary for transactivation Of JCVL, the coding sequences were mutated. The amino-terminal region of JCV Ag in amino acids 1-437 was essentially required for efficient transactivation. These results indicated that transactivation of JCVL and increased binding require a factor(s) found specifically in glial cells, the JCV NF-1 II/III sites, and the T-Ag amino-terminal region.

Animals↗

PubMed1996

Estimation of fetal age by histological study of kidney.

An attempt has been made to correlate the gestational age of a fetus with a histological study of the sequential development of nephrons in the kidneys. One hundred and eighteen dead fetuses aged between 14 and 40 weeks of gestation were examined. In each case, paraffin sections from the kidneys were studied microscopically and the number of glomerular generations present was counted and correlated with the reported period of gestation based on obstetric calculations. The results were analysed and statistically confirmed a high degree of correlation, indicating that this could be a reliable method of estimating fetal age in forensic situations, such as infanticide and criminal abortion.

Abortion, Illegal↗

PubMed1994

Differential regulation by c-jun and c-fos protooncogenes of hormone response from composite glucocorticoid response element in human papilloma virus type 16 regulatory region.

Glucocorticoid hormones positively regulate human papilloma virus (HPV) type 16 gene expression, and we have previously shown that this regulation is through three glucocorticoid response elements (GREs). The GRE at nucleotide 7640 is a composite GRE (cGRE) containing an overlapping activator protein-1 (AP-1) motif for the c-jun homodimer and c-jun/c-fos heterodimer. This report examined the effects of c-jun and/or c-fos AP-1 protooncogenes and the glucocorticoid hormone dexamethasone on expression of the HPV 16 cGRE in AP-1-deficient P19 embryonal carcinoma cells. The activity of the full-length HPV 16 enhancer was progressively increased with increasing levels of c-jun. The hormone induced an additional response. For the c-jun/c-fos heterodimer, the response to hormone was progressively diminished. Site-specific mutations of the cGRE revealed that the regulation by AP-1 and hormone required both GRE and the AP-1 motif. An enhancer fragment containing the cGRE and excluding the two simple GREs gave similar results. Two disruption mutations of the AP-1 site confirmed the requirement of this site for hormone response. A cGRE oligonucleotide construct substantiated the effect of c-jun for response to hormone. For heterodimer, activity and hormone response were both also progressively increased. The results reveal a unique cross-talk between the distinct AP-1- and hormone-signaling pathways, suggesting the involvement of a complex interaction of c-jun and c-fos and glucocorticoid hormone receptor with the HPV 16 cGRE, resulting in novel control patterns for regulating viral expression.

Animals↗

PubMed1993

Human JC virus perfect palindromic nuclear factor 1-binding sequences important for glial cell-specific expression in differentiating embryonal carcinoma cells.

The brain cell specificity of the human papovavirus JC virus was examined by site-directed mutagenesis of the nuclear factor 1 (NF1) motifs within the viral regulatory region. The NF1 motif sites, located within the 98-bp tandem repeats that contain 6-bp perfect inverted palindromic sequences, were important for glial cell-specific expression of JC virus in differentiated embryonal carcinoma cells in vivo. The NF1 site on the late side of the repeats was not important, a fact confirmed by in vitro transcription studies. These observations were correlated with in vitro DNase I footprinting and mobility shift assays, which demonstrated specific interactions of factors in glial cell nuclear extracts with NF1 sites.

Base Sequence↗