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R Wadhwa

Publications and source records attributed to R Wadhwa.

At least 55 records · Page 3Linked to original sources
PubMed1996

Genetic differences between the pancytosolic and perinuclear forms of murine mortalin.

To determine the genetic relation between the pancytosolic (uniformly distributed in cytoplasm-p66mot-1) and the perinuclear (p66mot-2) mortalin proteins found in normal and immortal mouse cells, respectively, we initiated the present study using PCR cloning, sequencing, and single nucleotide primer extension analyses. The results indicate that the difference between mot-1 and mot-2 at 2 bp in the open reading frame does not arise by mutations in the immortalized cells. Intriguingly, both mot-1 and mot-2 sequences were detected in genomic DNA from normal (CMEF) and immortal (RS-4) cells derived from the CD1-ICR mouse strain. We extended the analyses to four other strains of mouse and detected both mot-1 and mot-2 sequences in genomic DNA from the Balb/c mouse also. Swiss and C57BL/6 mice exhibited only mot-2 and the C3H He mouse only mot-1. Southern analyses on a mouse strain that showed the presence of only mot-2 (Swiss) and one that showed both mot-1 and mot-2 (CD1-ICR) by PCR revealed that there are structural differences between mot-1 and mot-2 loci. Furthermore, there appears to be a correlation between frequency of spontaneous immortalization of cultured mouse fibroblasts and the mot loci present in the mouse strain from which they were derived.

Animals↗
PubMed1996

An effective elimination of false positives isolated from differential display of mRNAs.

A reverse Northern analysis that effectively eliminates the false positives isolated from differential display of mRNAs (DD) is demonstrated. Preparation of probe by one-step labeling in reverse transcription reaction is found to be more effective and specific as compared to the preparation of probe by random priming of reverse transcribed cDNA pool. Reverse Northern assay of DNA fragments isolated from DD prior to their cloning into plasmid, analysis of multiple fragments on single slot blot, and requirement of RNA only in small amounts as compared to conventional Northern makes the protocol quick, effective, and economic.

3T3 Cells↗
PubMed1995

Inhibitors of cGMP-dependent protein kinase block senescence induced by inactivation of T antigen in SV40-transformed immortal human fibroblasts.

Immortal human fibroblasts isolated following transfection with thermolabile simian virus 40 T antigen lost division potential upon shift up in temperature due to heat inactivation of the antigen. Such cells showed a concomitant change in the distribution of a mortality marker, mortalin, from a juxtanuclear cap like distribution of immortal cells to a uniform cytosolic distribution of mortal cells. We made an attempt to modulate the above inducible system of cellular senescence using various protein kinase inhibitors. Among the indolocarbazole type inhibitors tested, only KT5823, defined as a specific inhibitor of cGMP-dependent protein kinase, blocked the loss of division potential as determined by cell growth and colony forming ability. This inhibitor also prevented the above change in mortalin distribution due to temperature shift. In addition, the isoquinoline sulfonamide derivatives H8, H9, H88 and H89, all shown to inhibit cGMP-dependent protein kinase, suppressed the senescence. Inhibitors specific to other types of protein kinases, protein phosphatases or tyrosine kinases tested had no effect. Since there was no difference between the effective and non-effective inhibitors in their effects on cell cycle progression, cell cycle arrest by itself cannot account for the above phenomenon. These results suggest that a signaling pathway possibly mediated by cGMP-dependent protein kinase is involved in the induction of cellular senescence.

Alkaloids↗
PubMed1995

Mouse and human chromosomal assignments of mortalin, a novel member of the murine hsp70 family of proteins.

Mortalin has been shown to exhibit differential distributions in cells with mortal and immortal phenotypes. In the present study, we report mot-2 cDNA cloning from RS-4 cells--an immortal clone from CD1-ICR mouse embryonic fibroblasts--and the chromosomal assignments of mortalin related genes to mouse chromosomes 18 and X by fluorescence in situ hybridization. Similar analysis assigned the gene to chromosome 5q31.1 in human.

Animals↗
PubMed1995

Correlation between complementation group for immortality and the cellular distribution of mortalin.

The dominance of cellular senescence over the immortal phenotype has been demonstrated by cell fusion experiments utilizing human and mouse cells. Mortalin, a novel 66-kDa member of the murine hsp70 family of proteins, has recently been identified as a marker of the mortal phenotype by virtue of its characteristic cytosolic distribution in mortal cells. Here we report the mortalin immunostaining observations on 21 human cell lines. These cell lines have previously been assigned by somatic cell hybridization analysis to one (18 lines) or more than one (3 lines) of the four complementation groups (A, B, C, and D) for immortalization. Four patterns of mortalin immunostaining were observed: granular-juxtanuclear cap, granular-gradient from nuclear to cell membrane, granular-juxtanuclear arch, and fibrous-perinuclear. In 17 of 18 cell lines assigned to a single complementation group, the mortalin staining corresponded with the complementation group. In two of the three cell lines previously assigned to multiple complementation groups, the mortalin staining corresponded to one of the assigned groups. Two cell lines, however, exhibited staining patterns which did not match to their assigned complementation groups. The basis of correlation between cellular distribution of mortalin and the complementation group remains unclear at present. However, the data (i) suggest that the intracellular distribution of mortalin can be used to distinguish mortal and immortal cells, confirming the association of mortalin with senescence; (ii) provide supportive evidence for the existence of at least four different pathways of immortalization in human cells; and (iii) indicate that mortalin is involved in processes that result in immortalization.

Animals↗
PubMed1994

Enhanced expression of multiple forms of VEGF is associated with spontaneous immortalization of murine fibroblasts.

The mechanism(s) involved in immortalization that constitute the first step during malignant transformation has been the subject of our interest. By the use of spontaneously immortalized mouse embryonic fibroblasts we have earlier identified two stages of immortalization which are characterized by growth characteristics of the cells, their conditioned medium and the protein markers such as p53, p81 and mortalin (Kaul et al. (1994) Biochim. Biophys. Acta, in press). The present study was planned to purify the mitogenic factors from the conditioned medium of stage II cells. Sequential purification by chromatography followed by peptide sequencing has characterized one of these as vascular endothelial growth factor (VEGF). Further analysis by RT-PCR suggests that the spontaneously immortalized stage II fibroblasts have enhanced synthesis and secretion of VEGF as compared to their mortal parent cells. Expression of a novel 304 bp long form of VEGF is identified in immortal fibroblasts in addition to the three known alternatively spliced forms. The study points to the involvement of VEGF function during spontaneous immortalization of mouse embryonic fibroblasts.

Alternative Splicing↗
PubMed1994

Identification of genetic events involved in early steps of immortalization of mouse fibroblasts.

The spontaneously immortalized early passaged fibroblasts from three different strains of mouse are observed to represent two distinct stages of immortalization. The cells at stage I are characterized by slow growth rate, contact inhibition and requisition of serum factors for their growth and proliferation. Stage II cells are marked by fast, multilayer growth that is independent of serum supplementation in growth medium and by the elevated levels of the two marker proteins, i.e., p53 and p81. The change from cytosolic distribution of mortalin, a senescence inducing protein (J. Biol. Chem. (1993) 268, 6615-6621; 22239-22242) to the perinuclear locale is detected as an early event during cellular immortalization. Furthermore, the distinct stages could be characterized by thermal analysis of intact cells, that to the best of our knowledge is employed for the first time for the analysis of cellular mortal and immortal phenotypes. The study characterizes at least two distinct end points in rodent transformation suggesting that there are multiple routes to immortalization.

Animals↗
PubMed1994

Sex- and tissue-specific Bkm(GATA)-binding protein in the germ cells of heterogametic sex.

The ZZ male/ZW female system of sex determination (female heterogamety) is found in snakes and birds whereas XY male/XX female system of sex determination (male heterogamety) operates in mammals including humans. The W and Y chromosomes are largely heterochromatic and undergo cycles of condensation and decondensation in the germ cells of ovary and testis, respectively, whereas they remain highly condensed and transcriptionally inactive in all somatic cells. Both chromosomes have enriched stretches of GATA repeats along their entire length (which is identified as banded krait minor satellite DNA and called Bkm) that are highly conserved through widely separated orders of eukaryotes. Here we report the existence of a factor, which specifically binds to Bkm, in the germ cells of the heterogametic sex (ovary in female heterogamety and testis in male heterogamety) where decondensation (activation) of the W and Y chromosomes, respectively, occurs; it has been purified as a polypeptide of 57.5 kDa from the rat snake ovary and designated as Bkm-binding protein (BBP) by virtue of its binding to GATA repeats of Bkm. Such a sex- and tissue-specific BBP is also present in the ovary of other species of snakes and in the testis of mouse and human where the Y chromosome is highly decondensed. We suggest that GATA repeats of Bkm brings about a coordinated decondensation of the W and Y sex chromosomes in the germ cells of the heterogametic sex in response to BBP which may serve as a "switch" for the activation of the genes present on the W and Y chromosomes.

Animals↗
PubMed1994

Cellular mortality to immortalization: mortalin.

The roots of cellular mortality-limited capacity of normal cells to divide, and immortalization-unabated proliferation of cancerous cells remain undefined so far. Out of a variety of experimental strategies employed, the cell fusion approach has been proven to be significantly informative. The present article reviews some of the more important recent results and describes the use of natural and conditional aging systems obtained by the fusion of mortal and immortal mouse fibroblasts to identify putative senescence-determining and/or senescence-escaping genes. The strategy has led to the isolation of a novel 66-kDa protein, mortalin- a unique member of the mouse heat shock protein 70 (hsp 70) family. The intracellular distributions of mortalin, i.e., cytosolic and perinuclear, distinguish the mortal phenotype from the immortal one, respectively. Consistently, the cytosolic mortalin is seen to have a senescence-inducing function in contrast to the perinuclear mortalin which has no detectable effect on cellular phenotype. It is suggested that mortalin can be exploited to unravel some aspects of cellular mortality and immortality and also for the early detection of cancerous cells.

Animals↗
PubMed1993

Spontaneous immortalization of mouse fibroblasts involves structural changes in senescence inducing protein, mortalin.

Mortalin, a novel member of mouse heat shock protein 70 (hsp70) family, is seen to distinguish the cellular mortal and immortal phenotypes by virtue of its cytosolic and perinuclear distribution, respectively. We report here that the cytosolic and perinuclear forms of mortalin from CD1-ICR mouse embryonic fibroblasts and NIH 3T3 cells, respectively, differ by two amino acids, can be distinguished on two-dimensional SDS-polyacrylamide gel. The perinuclear mortalins from RS-4 and Balb/c 3T3 cells harbor the same two amino acids as that of NIH 3T3 cells. However, these when analyzed with C-MEF mortalin did not exhibit the mobility shift equivalent to C-MEF and NIH 3T3 mortalins. The data indicate that the perinuclear mortalin from different immortal fibroblasts are not identical and implicate the possibility of additional structural changes in mortalin during immortalization. Such differences may also contribute to the differential in vitro growth characteristics of these immortal cells.

3T3 Cells↗
PubMed1993

Induction of cellular senescence by transfection of cytosolic mortalin cDNA in NIH 3T3 cells.

We have recently identified a novel member of hsp70 family (mortalin) as a mortality marker (Wadhwa, R., Kaul, S. C., Ikawa, Y., and Sugimoto, Y. (1993) J. Biol. Chem. 268, 6615-6621). It has distinct intracellular distribution in mortal and immortal fibroblasts. Here, we report that the cytosolic (mot-1) and the perinuclear (mot-2) forms of mortalin cDNA cloned from mortal and immortal cells, respectively, differ by only two bases in the open reading frame, resulting in two amino acid changes. The induced expression of the cytosolic form by transfection of mot-1 cDNA (isolate from CD1-ICR mouse embryonic fibroblasts) to NIH 3T3 cells induced cellular senescence. However, the perinuclear form expressed by mot-2 cDNA (isolate from NIH 3T3 cells) did not yield an equivalent effect. The data suggest the senescence-inductive function of cytosolic mortalin and implicitly point to a genetic event involved in immortalization.

3T3 Cells↗
PubMed1993

On the cytosolic and perinuclear mortalin: an insight by heat shock.

We have identified, cloned and characterized a 66-kD protein from cytosolic fractions of mouse embryonic fibroblasts and named it mortalin (Wadhwa et al., J. Biol. Chem., 268, in press, 1993). Immortal fibroblasts were seen to harbor the same or very similar protein, however, localized in the perinuclear locale. The present report is on the differentially localized forms of p66 protein which are biochemically and structurally found to be widely the same. In fact, heat shock treatment could translocate the cytosolic form to the perinuclear position without any detectable biochemical modification. The observed phenomenon adds to the unique identity of mortalin in hsp70 family. Besides, it confers that only the minor differences in the protein probably enroute its differential cellular distribution and the associated function.

3T3 Cells↗
PubMed1993

Identification of a novel member of mouse hsp70 family. Its association with cellular mortal phenotype.

A novel 66-kDa protein, named p66mot-1, is identified to be associated with cellular mortality by virtue of its presence in cytosolic fractions of serially passaged mouse embryonic fibroblasts (MEF) and the mortal hybrids obtained by the fusion of mortal (MEF) and immortal (MN48-1, derivative of NIH 3T3) cells. Immortal cells lack this protein in their cytosolic fractions. cDNA cloning and homology search placed it in the heat shock protein 70 (hsp70) family. Microinjection of anti-p66 antibody to senescent MEF could transiently stimulate their cell division, which supports the cellular mortality-related function of p66mot-1.

3T3 Cells↗
PubMed1993

Identification and differential expression of yeast SEC23-related gene (Msec23) in mouse tissues.

We have isolated a yeast SEC23-related clone (Msec23) from mouse fibroblast cDNA library. It has an open reading frame of 1721 bp (64% homologous to SEC23-2.3 kb) which can potentially encode a 64.7 kDa protein (61% homologous to 85.4 kDa product of SEC23). The deduced Msec23 protein (Msec23p) sequence contains three successive Ig-like domains at the N-terminus followed by amphipathic alpha-helical regions, suggesting the potential of Msec23p to interact with other protein components. Further, Msec23 is differentially expressed in mouse tissues with its high level in brain and fibroblasts.

Amino Acid Sequence↗
PubMed1993

Differential subcellular distribution of mortalin in mortal and immortal mouse and human fibroblasts.

Mortalin, p66mot-1, from the cytosolic fractions of mouse embryonic fibroblasts (MEF) has been identified. We have cloned and characterized it as a novel member of mouse hsp70 family [R. Wadhwa, S. C. Kaul, Y. Ikawa, and Y. Sugimoto (1993) J. Biol. Chem. 268, 6615-6621]. We report that immortal clones from MEF such as NIH 3T3, RS4, and Balb/c 3T3 harbor the same or a very similar protein but in the perinuclear locale. Biochemical analysis revealed that the proteins from mortal (MEF) and immortal (NIH 3T3) fibroblasts do not show gross differences. However, the phenomenon of differential distribution of p66 protein in mortal and immortal fibroblasts is conserved in three different strains of mouse and, interestingly, in human fibroblasts, too. The data suggest that at least one common step is involved in immortalization of mouse and human fibroblasts and also point to the practical implication(s) of such easily detectable differences.

Animals↗
PubMed1991

Natural and conditional ageing of mouse fibroblasts: genetic vs. epigenetic control.

Mouse embryonic fibroblasts were fused with a spontaneously immortalized mouse fibroblast cell line and a large number of heterogeneous hybrid populations were obtained. These showed variations with respect to their chromosomal number as well as in vitro life span but none acquired immortal phenotype. Apart from demonstrating the dominant nature of senescence over immortalization in mouse system, we also provide the first report on the analysis of genomic DNA methylation during in vitro passaging of parental and hybrid cell populations representing the normal and conditional ageing, respectively. Since no random decline in DNA methylation could be detected in any of the cases, our results suggest that it is unlikely that mortality of cells in culture is the outcome of random loss of epigenetic control imposed by 5-methyldeoxycytidine at CpG sites in the genome.

Animals↗
PubMed1989

X chromosome restriction fragment length polymorphisms in five racial groups: rare variant detected with the RC8 (DXS9) probe in the Marathi population, India.

Restriction fragment length polymorphisms were investigated in five racial groups using the X chromosome probes DXS9 and DXS7. The allele frequencies of these polymorphisms showed significant differences and both DNA fragments were found to be highly polymorphic in the populations of south and southeast Asia. In the Marathi population of India, a rare allele B*3 (3 kilobases; kb) and an altered 7-kb fragment instead of the 6.6-kb constant band were found with DXS9. This is the first time that the rare B*3 allele is found in a non-European population.

Asian People↗