Biomedical subjects
Publications and source records attributed to R Wadhwa.
We have undertaken the studies to investigate the presence of various activities of the leaf extract of Ashwagandha (Lash), a commonly used shrub in Indian traditional medicine, Ayurveda. In the present study, we studied the effect of Lash against MNNG-induced genotoxicity in onion root tip cells. We report that Lash offered substantial protection against the mutagenic effects of MNNG.
Withania somnifera (Ashwagandha) is used in Indian traditional medicine, Ayurveda and is believed to have a variety of health promoting effects. Molecular mechanisms and pathways underlying these effects have not been studied. We tried to characterize various activities of leaf extract of Ashwagandha (Lash) raised in the field and in the laboratory. We found that the Lash from field-raised plants has a significant anti-proliferative activity in human tumorigenic cells. However, it did not impart any protection against the oxidative damage caused by high glucose and hydrogen peroxide to human tumor cells suggesting that it can be used as an anti-tumor, but not as an anti-oxidant, substance.
Mouse and human cells have most frequently been used for studies that have led to the elucidation of various molecular pathways involved in senescence. The ARF-p53 pathway has been assigned as one of the major protagonists in these phenomena. ARF is an alternative reading frame protein encoded along with p16INK4A by the INK4a locus on human chromosome 9p21 and the corresponding locus on mouse chromosome 4. Whereas the mouse ARF (p19ARF) consists of 169 amino acids, the human ARF (p14ARF) consists of 132 amino acids, truncated at the C-terminus. Molecular studies on the regulation of ARF activity by its binding partners have revealed that mouse ARF protein, but not human ARF protein, interacts with a cytoplasmic protein, Pex19p. This interaction of mouse ARF with Pex19p results in its milder p53 activation function in mouse cells as compared to human cells and thus accounts, at least in part, for the weaker tumor surveillance and frequent immortalization of mouse cells.
Mortalin is a novel member of the hsp70 family of proteins that exhibits a different staining pattern in normal and immortal cells. It was also cloned as glucose regulated protein, GRP75 and peptidebinding protein, PBP74. It has been assigned multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation and tumorigenesis. The present article compiles and reviews information on multiple sites and functions of mortalin. In view of its upregulation in many tumors and transcriptional inactivation function of p53, its potential use in biotechnology and biomedicine is discussed.
A fusion between the promyelocytic leukemia (PML) protein and the retinoic acid receptor-alpha (RARalpha) results in the transforming protein of acute promyelocytic leukemia, PML-RARalpha. PML has growth-suppressive properties and is localized within distinct nuclear structures referred to as nuclear bodies. PML participates in numerous cellular functions, including transcriptional activation, apoptosis, and transcriptional repression, whereas PML-RARalpha blocks these functions. However, the role played by PML-RARalpha in leukemogenesis remains unclear. Here we report that PML is required for transcriptional repression mediated by the tumor suppressor Rb. Rb interacts with the histone decaetylase (HDAC) complex containing co-repressors and represses the transcription of the E2F target genes. Overexpression of PML enhanced Rb-mediated repression. The degree of Rb-mediated repression was weakened by injecting anti-PML antibodies and was lower in Pml-deficient mouse embryonic fibroblasts. PML-RARalpha inhibited Rb-mediated repression, and two co-repressor-interacting sites on the PML-RARalpha molecule were required for this activity. Furthermore, PML-RARalpha blocked the interaction between Rb and HDAC. Thus, aberrant binding of PML-RARalpha to co-repressor-HDAC complexes may inhibit their association with Rb, resulting in the abrogation of Rb activity. Thus, the disruption of Rb-mediated repression may be a contributory factor in leukemogenesis.
A heat-shock protein (hsp) 70 family member mortalin/glucose-regulated protein (GRP) 75/peptide-binding protein 74 (PBP74) has been localized to various cellular compartments including mitochondria, endoplasmic reticulum and cytoplasmic vesicles. Here we describe its interactions with an endoplasmic reticulum protein GRP94, a member of the hsp90 family of GRPs. Interactions were identified, confirmed and characterized by far-Western screening, in vivo reporter and co-immunoprecipitation assays. Interacting domains of the two proteins were also characterized by mutational analysis. Such interactions of these two GRPs may be important for function of either or both and therefore provide important information for further studies.
DNA methylation is essential for development in the mouse and plays an important role in inactivation of the X chromosome and genomic imprinting. MeCP2 is the founder member of a family of methyl-CpG-binding proteins. MeCP2 directly binds to the co-repressor mSin3, which interacts with class I histone deacetylase, recruiting them to methyl-CpG regions to suppress transcription. Here, we report that MeCP2 directly binds to two co-repressors, c-Ski and N-CoR, in addition to mSin3A, and that the c-Ski, which is encoded by the c-ski proto-onocogene, is required for MeCP2-mediated transcriptional repression. The two regions of c-Ski, including the C-terminal coiled-coil region, interact with the transcriptional repression domain in the center of the MeCP2 molecule. The immunostaining signals for c-Ski and MeCP2 overlap in the nuclear heterochromatin region, suggesting the co-localization of the two proteins. The degree of transcriptional repression mediated by a Gal4-MeCP2 fusion protein was abrogated by overexpression of the putative dominant negative form of c-Ski. Furthermore, injection of antibodies against c-Ski and Sno almost completely abolished the transcriptional repression mediated by the Gal4-MeCP2 fusion protein. These results suggest that the ski gene family is involved in methyl CpG-mediated transcriptional repression.
We isolated a 33-kDa protein, Pex19p/HK33/HsPXF, as a p19ARF-binding protein in a yeast two-hybrid screen. We demonstrate here that Pex19p interacts with p19ARF in the cell cytoplasm and excludes p19ARF from the nucleus, leading to a concurrent inactivation of p53 function. Down-regulation of Pex19p by its antisense expression resulted in increased levels of p19ARF, increased p53 function, and a p53/p21WAF1-mediated senescence-like cell cycle arrest. The data demonstrated a novel mechanism of down-regulation of the p19ARF-p53 pathway.
We previously reported that endogenously expressed, intracellularly localized fibroblast growth factor (FGF)-1 interacts with mortalin. Here we report that FGF-1 added to the culture medium of quiescent BALB/c3T3 cells is taken up by the cells and interacts with mortalin in the cells in a regulated manner. Although both the internalized FGF-1 and mortalin were present at high levels throughout the FGF-1-initiated cell cycle, their interaction became apparent only in late G1 phase. Interestingly, mortalin was preferentially tyrosine phosphorylated at the same time, and when its normally weak phosphorylation in early G1 phase was augmented by treating the cells with vanadate, a strong interaction between mortalin and FGF-1 was established. Conversely, when phosphorylated mortalin was treated with tyrosine phosphatase, its interaction with FGF-1 was abrogated. These results indicate that FGF-1 taken up by cells preferentially interacts with mortalin in late G1 phase of the cell cycle, and that tyrosine phosphorylation of mortalin regulates this interaction.
We report here the cloning and characterization of a novel KRAB zinc finger gene, ZFQR, which has eight tandemly repeated zinc fingers, a complete KRAB box at the N-terminal region, and a unique C-terminal sequence. It is expressed in a variety of human tissues, and mRNA levels are upregulated in nondividing senescent and quiescent human fibroblasts. Overexpression of the protein in quiescent cells stimulated with serum growth factors results in inhibition of entry into the cell cycle. The latter activity is lost when the N-terminal KRAB domain is deleted. The KRAB domain is also required for the transcriptional repression activity of ZFQR and in maintaining association of the protein with the nuclear matrix. The gene has been mapped to human chromosome 19q13.4. The association of ZFQR with the nuclear matrix, transcriptional repression activity, increased expression in senescent and quiescent cells, and the ability to inhibit quiescent cells stimulated with growth factors from entering the cell cycle suggests a role for ZFQR in the maintenance of the nondividing state of normal human cells.
Fusion of the promyelocytic leukemia (PML) protein to the retinoic acid receptor-alpha (RARalpha) generates the transforming protein of acute promyelocytic leukemias. PML appears to be involved in multiple functions, including apoptosis and transcriptional activation by RAR, whereas PML-RARalpha blocks these functions of PML. However, the mechanisms of leukemogenesis by PML-RARalpha remain elusive. Here we show that PML interacts with multiple corepressors (c-Ski, N-CoR, and mSin3A) and histone deacetylase 1, and that this interaction is required for transcriptional repression mediated by the tumor suppressor Mad. PML-RARalpha has the two corepressor-interacting sites and inhibits Mad-mediated repression, suggesting that aberrant binding of PML-RARalpha to the corepressor complexes may lead to abrogation of the corepressor function. These mechanisms may contribute to events leading to leukemogenesis.
Mot-2 protein is shown to interact with p53 and inhibit its transcriptional activation function. Mot-2 overexpressing stable clones of NIH 3T3 cells were malignantly transformed, however, they had a high level of expression of a p53 downstream gene, p21WAF1. The present study was undertaken to elucidate possible molecular mechanism(s) of such upregulation. An increased level of p21WAF1 expression was detected in stable transfectants although an exogenous reporter gene driven by p21WAF1 promoter exhibited lower activity in these cells suggesting that some post-transcriptional mechanism contributes to upregulation. Western analyses of transient and stable clones revealed that upregulation of p21WAF1 in stable NIH 3T3/mot-2 cells may be mediated by cyclin D1 and cdk-2.
Mouse mortalin proteins, mot-1 and mot-2, differ by only two amino acid residues in their C-terminus. In previous studies we showed that they differ in their subcellular distributions and interactions with the tumor suppressor protein, p53. By using mot-1 deletion mutants and amino acid substitution constructs, we report here that inability of mot-1 to affect p53 activity in vivo is dependent on the presence of both of the unique mot-1 amino acids and all three of the predicted hsp70, EF hand, and leucine zipper motif regions. The two proteins and their single amino acid mutants showed different mobilities on SDS-polyacrylamide gel presenting an evidence for their different secondary structures. Taken together, the data suggest that each of the two differing amino acids between mot-1 and mot-2 is an important determinant of their secondary structures and in vivo activities.
MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.
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.
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.
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.
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