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R Wadhwa

Publications and source records attributed to R Wadhwa.

At least 19 recordsLinked to original sources
PubMed2000

Extramitochondrial localization of mortalin/mthsp70/PBP74/GRP75.

Subcellular fractionation and immunofluorescence microscopy were used to identify the specific sites of intracellular residence of mortalin, also called a mitochondrial homologue of the hsp70 family, in immortal human cell lines previously assigned to four distinct complementation groups (A-D) for indefinite cell division. In addition to the mitochondria it was seen in the endoplasmic reticulum (ER) fractions of all the cell lines analyzed. Interestingly, three of the group A cells lines (EJ, GM639, and HT1080), in addition to the mitochondria and ER, exhibited cytosolically (extra-organelle) localized pool of mortalin. These findings demonstrate that mortalin is not present exclusively in mitochondria. Its residence in different organelles may be the basis of differential distribution observed previously in different human cell lines.

Biomarkers↗
PubMed2000

Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan.

By immunoscreening with an antibody raised against a plasma membrane protein, we have cloned a growth suppressor gene, Gros1 and assigned it to short arm of human chromosome 1. Two alternatively spliced forms of the gene encoding 84- and 41-kDa (carboxy-terminus truncated) proteins were cloned. The two transcripts, 4.4 and 2.7 kb, were expressed weakly in most of the human tissues, with a high expression of the smaller transcript in placenta, ovary and testis. Normal human fibroblasts in culture showed two transcripts, with a higher level of expression of the 4.4 kb transcript. Transformed cells on the other hand showed predominant expression of the 2.7 kb transcript. Two Gros1 transcripts were also detected in most of the mouse tissues. Stable transfection of the mouse cDNA encoding the 85-kDa protein into NIH3T3 cells resulted in their slow growth and reduced colony-forming efficiency. Stable clones expressing antisense RNA on the other hand exhibited higher colony forming efficiency. While our data implied that Gros1 is a novel growth suppressor gene on human chromosome 1, an independent study has recently characterized its rat-homolog as a leucine proline-enriched novel basement membrane-associated proteoglycan leprecan. We describe here cloning, expression and biological activity analysis implying that this novel proteoglycan is a potential growth suppressor on chromosome 1p31, frequently altered in many malignancies.

3T3 Cells↗
PubMed2000

Structurally and functionally distinct mouse hsp70 family members Mot-1 and Mot-2 proteins are encoded by two alleles.

The mouse mortalin proteins Mot-1 and Mot-2 differ by two amino acids in their carboxy-terminus. These proteins are differentially localized in the cell cytoplasm and have contrasting biological activities. The genetic relationship between Mot-1 and Mot-2 was deciphered by mouse family analyses. Mot-1 and Mot-2 segregated in F1 and F2 progeny, providing direct evidence that the two proteins are encoded by two alleles.

Alleles↗
PubMed1999

Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin.

Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.

Amino Acid Sequence↗
PubMed1999

Identification of a 55-kDa ezrin-related protein that induces cytoskeletal changes and localizes to the nucleolus.

Normal and transformed human cells when stained for ezrin, an F-actin-binding ERM (ezrin/radixin/moesin) family protein, revealed a faint and intense immunofluorescence, respectively. Surprisingly, nuclear staining that was assigned to the nucleolus by confocal laser and immunoelectron microscopy was detected in both cell types and was more prominent in normal cells due to the absence of glistering cytoplasmic fluorescence. By Western analysis the nuclear fraction was seen to have a 55-kDa ezrin-reactive protein that did not react to the antibodies raised against the C-terminus of the protein, suggesting that it may correspond to an endogenously cleaved N-terminus of the protein. Transfections of cells with a cDNA encoding full-length ezrin tagged with green fluorescent protein (GFP) at its N-terminus indeed resulted in two GFP-tagged products corresponding to full-length and 55-kDa endogenously cleaved forms. Transfection with a cDNA encoding approximately 55 kDa of the ezrin N-terminus (N-ezrin) showed that it can translocate to the nucleus. N-ezrin transfected cells exhibited irregular cell edges and collapse of actin fibers. Similar changes were seen following microinjection of anti-p81/ezrin antibody, suggesting that N-ezrin may function as a dominant negative competitor of ezrin. These data demonstrate the existence of an N-terminal cleavage form of ezrin that localizes to the nucleolus and that its overexpression induces cytoskeletal changes.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a novel gene, striamin, that interacts with the tumor suppressor protein p53.

Expression analysis of a novel cDNA isolated from immortal murine fibroblasts revealed a single transcript of 3.0 kilobase pairs that was highly expressed in mouse and human striated muscle and in mouse heart. The gene has therefore been named striamin. Its expression was confined to skeletal muscle types with a fast glycolytic (2B) contractile phenotype. It was also detected in C2C12 mouse myoblasts and was down-regulated during in vitro myogenesis. The cDNA has a single open reading frame encoding a predicted 16.8-kDa protein of 149 amino acids with no homology to known proteins. Microinjection and transfection of green fluorescence protein-tagged striamin demonstrated that it localizes to the nucleus. Coimmunoprecipitations revealed that it can interact with p53 (a positive marker for myoblast differentiation) in vivo and in vitro. Furthermore, it repressed p53 activity in p53-mediated reporter assays. Fluorescence in situ hybridization with a mouse P1 genomic clone localized the gene to chromosome 12C3, which is syntenic to human chromosome 14q21-22.

Amino Acid Sequence↗
PubMed1999

A novel testis-specific metallothionein-like protein, tesmin, is an early marker of male germ cell differentiation.

We have cloned a novel cDNA encoding testis-specific metallothionein-like protein, tesmin, by randomized RT-PCR on RNA from mouse tissues. Two tesmin-related transcripts (2.2 and 1.8 kb) in mouse and one (2.1 kb) in human were detected and cloned. These encode a cysteine-rich 32-kDa protein that contained a metallothionein-like motif. In situ hybridization analysis in adult mouse testis showed that tesmin is specifically expressed in spermatocytes. Quantitative RT-PCR at different stages of mouse postnatal development (days 4, 8, 12, 18, and 42) revealed that tesmin is expressed as early as day 8 and coincides with the entry of germ cells into meiosis. Furthermore, adult W/Wv sterile mice that harbor the c-kit mutation lacked tesmin expression. The gene is assigned to mouse chromosome 19B, which has been reported to translocate (11;19) in male sterile mice.

Amino Acid Sequence↗
PubMed1999

Viral ski inhibits retinoblastoma protein (Rb)-mediated transcriptional repression in a dominant negative fashion.

The mechanism by which the viral oncogene ski (v-ski) transforms chicken embryo fibroblasts is currently unknown. Recently, the c-ski gene product (c-Ski) was found to bind to N-CoR (nuclear hormone receptor co-repressor), an element implicated in transcriptional repression mediated by multiple transcriptional repressors including the nuclear hormone receptors and Mad. c-Ski is required for transcriptional repression mediated by Mad involved in negative regulation of cellular proliferation. v-Ski abrogates Mad-induced transcriptional repression in a dominant negative fashion. Here we report that v-Ski also inhibits transcriptional repression mediated by Rb, another tumor suppressor gene product. Rb forms a complex with c-Ski, Sin3A, and histone deacetylase (HDAC) via direct binding to c-Ski and HDAC. c-Ski is required for the transcriptional repression mediated by Rb. These results suggest that inhibition of Rb activity contributes, at least partly, to transformation by v-Ski.

Animals↗
PubMed1999

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor.

The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis.

Animals↗
PubMed1999

NIH 3T3 cells malignantly transformed by mot-2 show inactivation and cytoplasmic sequestration of the p53 protein.

In previous studies we have reported that a high level of expression of mot-2 protein results in malignant transformation of NIH 3T3 cells as analyzed by anchorage independent growth and nude mice assays [Kaul et al., Oncogene, 17, 907-11, 1998]. Mot-2 was found to interact with tumor suppressor protein p53. The transient overexpression of mot-2 was inhibitory to transcriptional activation function of p53 [Wadhwa et al., J. Biol. Chem., 273, 29586-91, 1998]. We demonstrate here that mot-2 transfected stable clone of NIH 3T3 that showed malignant properties indeed show inactivation of p53 function as assayed by exogenous p53 dependent reporter. The expression level of p53 in response to UV-irradiation was lower in NIH 3T3/mot-2 as compared to NIH 3T3 cells and also exhibited delay in reaching peak. Furthermore, upon serum starvation p53 was seen to translocate to the nucleus in NIH 3T3, but not in its mot-2 derivative. The data suggests that mot-2 mediated cytoplasmic sequestration and inactivation of p53 may operate, at least in part, for malignant phenotype of NIH 3T3/mot-2 cells. NIH 3T3/mot-2 cells show inactivation of p53 protein.

3T3 Cells↗
PubMed1998

Inactivation of tumor suppressor p53 by mot-2, a hsp70 family member.

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21(WAF-1) and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent protein-tagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.

3T3 Cells↗
PubMed1998

Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein.

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.

3T3 Cells↗
PubMed1998

A novel alternatively spliced form of murine vascular endothelial growth factor, VEGF 115.

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

Alternative Splicing↗
PubMed1997

Elevated levels of mortalin expression in human brain tumors.

We have performed immunohistochemical studies of mortalin in normal and tumor human brain sections. In normal brain sections, the expression was seen mainly as being confined to neurons. Normal astrocytes showed undetectable expression of this unique member of the heat shock 70 protein family. Three grades of astrocyte tumors (low-grade astrocytoma, anaplastic astrocytoma, and glioblastoma), however, showed an increasing number of mortalin-positive cells. Other types of brain tumors, such as meningiomas, neurinomas, pituitary adenomas, and metastases, also showed elevated levels of mortalin expression compared to those in the normal brain. Mortalin has earlier been reported to have differential intracellular distribution in normal and transformed cells in vitro. Therefore, we substantiated the present study with immunofluorescence localization of the protein in normal and glioblastoma cells. The observations indicated that the tumors might be expressing a nonpancytosolic mortalin. An increase in number of mortalin-positive cells with malignant progression of brain tumors and its correlation with Ki-67 (a cell proliferation marker)-positive cells further suggested an involvement of nonpancytosolic mortalin(s) in malignant transformation of cells in vivo.

Animals↗
PubMed1997

Decrease in amplified telomeric sequences and induction of senescence markers by introduction of human chromosome 7 or its segments in SUSM-1.

Introduction of human chromosome 7 by microcell-mediated chromosome transfer suppresses indefinite division of SUSM-1, an in vitro established human fibroblast line. This cell line has unusually long telomeric sequences although it lacks detectable telomerase activity. Thus, we examined whether such telomeric sequences change upon introduction of chromosome 7 or its segments. In the microcell hybrids that stopped dividing by introduction of chromosome 7, the telomeric sequences were found to be lost or markedly diminished. Introduction of various fragments (2-40 Mb) of chromosome 7 contained in radiation hybrids gave similar results. On the other hand, the telomeric sequences were not altered significantly in the unsuppressed hybrids, a revertant of one suppressed clone, or subclones of SUSM-1 used as controls. In the suppressed microcell hybrids, the distribution of a mortality marker, mortalin, was changed to the cytosolic type of mortal cells from the immortal type of perinuclear fibres. Also, senescence-associated beta-galactosidase was induced to a level similar to that of normally senesced diploid fibroblasts. These results suggest that human chromosome 7 induces senescence in SUSM-1 by suppressing its telomere maintenance mechanism, which does not depend on telomerase.

Animals↗
PubMed1997

Expression analysis of mortalin, a unique member of the Hsp70 family of proteins, in rat tissues.

We have investigated the expression of mortalin in rat tissues by Northern analysis, RNA in situ hybridization, and immunohistochemical studies. By Northern assay, the highest level of expression was detected in brain, heart, and skeletal muscle followed by lung, liver, and kidney, and the least level of expression was detected in testis and spleen. RNA in situ and immunohistochemical studies showed that mortalin expression is significantly higher in upper nondividing layers than in the lower basal layers of skin, in neurons and nerve fibers than in surrounding glial cells in brain, in cardiomyocytes than in nonmyocytes in heart, and in interstitial secretory tissue than in germinating follicles in ovary. Such tissue- and cell-specific expression patterns of mortalin coordinates with its earlier reported antiproliferative function in normal cells. However, a deregulation of the expression is observed in rat brain tumor along with the detection of nonpancytosolic mortalin in rat glioma cell line C6. The study suggests that mortalin is involved in pathways that regulate division capacities of cells in vivo.

Animals↗