Biomedical subjects

Renu Wadhwa

Publications and source records attributed to Renu Wadhwa.

At least 19 recordsLinked to original sources

PubMed2005

Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60.

Mortalin/mtHsp70 (mitochondrial Hsp70) and HSP60 (heat-shock protein 60) are heat-shock proteins that reside in multiple subcellular compartments, with mitochondria being the predominant one. In the present study, we demonstrate that the two proteins interact both in vivo and in vitro, and that the N-terminal region of mortalin is involved in these interactions. Suppression of HSP60 expression by shRNA (short hairpin RNA) plasmids caused the growth arrest of cancer cells similar to that obtained by suppression of mortalin expression by ribozymes. An overexpression of mortalin, but not of HSP60, extended the in vitro lifespan of normal fibroblasts (TIG-1). Taken together, this study for the first time delineates: (i) molecular interactions of HSP60 with mortalin; (ii) their co- and exclusive localizations in vivo; (iii) their involvement in tumorigenesis; and (iv) their functional distinction in pathways involved in senescence.

Cell Division↗

PubMed2005

Quantum dots in bio-imaging: Revolution by the small.

Visual analysis of biomolecules is an integral avenue of basic and applied biological research. It has been widely carried out by tagging of nucleotides and proteins with traditional fluorophores that are limited in their application by features such as photobleaching, spectral overlaps, and operational difficulties. Quantum dots (QDs) are emerging as a superior alternative and are poised to change the world of bio-imaging and further its applications in basic and applied biology. The interdisciplinary field of nanobiotechnology is experiencing a revolution and QDs as an enabling technology have become a harbinger of this hybrid field. Within a decade, research on QDs has evolved from being a pure science subject to the one with high-end commercial applications.

Animals↗

PubMed2005

Mimotope-hormesis and mortalin/grp75/mthsp70: a new hypothesis on how infectious disease-associated epitope mimicry may explain low cancer burden in developing nations.

It is generally observed that countries with heavy infectious burden show lower cancer incidence as compared to more affluent nations. With the emerging paradigm on microbial heat shock proteins (hsps) as molecular link between infections and autoimmune diseases, we posit a new hypothesis, the "mimotope-hormesis", on the immunologic impact of infections on regional cancer prevention. According to this, assaults of infection during early adulthood could fortify the immune system to evoke more potent defenses against late-onset diseases, such as cancer, via autoimmunity. Interestingly, both experimental and clinical data support the beneficial role of autoimmunity in long-term cancer survivors. We illustrate this by a comprehensive in silico mimotope (epitope mimicry) analysis of human infectious pathogens against mortalin (mthsp70/PB74/GRP75), a type of hsp70 protein involved in control of cell proliferation, immortalization and tumorigenesis.

Communicable Diseases↗

PubMed2004

Imminent approaches towards molecular interventions in ageing.

Ageing is an innate feature of living organisms. Sensational progress in its molecular understanding in the last decade has culminated into a highly complex picture. Emerging from this complexity are the distinctive roles of some of the tumor suppressor pathways including p53 and pRB in maintenance of senescence phenotype, and telomere maintaining pathways in its escape. We discuss here the current scenario of molecular ageing and the use of modern approaches for its intervention in culture system, at least. Many of the tools we describe here are the newly emergent functional RNA tools that are proved to be fruitful in decoding the human genome. These post-genomic technologies will help us in the discovery of gene targets for interventions aiming to improve the quality at later years of life beyond their mere algebraic extension.

Aging↗

PubMed2004

Use of a randomized hybrid ribozyme library for identification of genes involved in muscle differentiation.

We have employed the hybrid hammerhead ribozyme-based gene discovery system for identification of genes functionally involved in muscle differentiation using in vitro myoblast differentiation assay. The major muscle regulatory genes (MyoD1, Mylk, myosin, myogenin, and Myf5) were identified endorsing the validity of this method. Other gene targets included tumor suppressors and cell cycle regulators (p19ARF and p21WAF1), FGFR-4, fibronectin, Prkg2, Pdk4, fem, and six novel proteins. Functional involvement of three of the identified targets in myoblast differentiation was confirmed by their specific knockdown using ribozymes and siRNA. Besides demonstrating a simple and an effective method of isolation of gene functions involved in muscle differentiation, we report for the first time that overexpression of Fem, a member of the sex-determining family of proteins, caused accelerated myotube formation, and its targeting deferred myoblast differentiation. This functional gene screening is not only helpful in understanding the molecular pathways of muscle differentiation but also to design molecular strategies for myopathologic therapies.

Animals↗

PubMed2004

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.

Animals↗

PubMed2004

Alternative reading frame protein (ARF)-independent function of CARF (collaborator of ARF) involves its interactions with p53: evidence for a novel p53-activation pathway and its negative feedback control.

CARF, a collaborator of ARF (alternative reading frame protein), was cloned as a novel ARF-binding protein from a yeast-interaction screen. It potentiated ARF-mediated p53 function, and also caused a moderate increase in p53 activity in the absence of ARF. We herein report the molecular mechanism of ARF-independent function of CARF. By employing a variety of approaches, including overexpression of CARF, its suppression by small interfering RNA and use of protease inhibitors, we demonstrate that: (i) CARF directly interacts with wild-type p53, causing its stabilization and functional activation; and (ii) CARF and p53 levels show an inverse relationship that is instigated by a negative-feedback control via a proteasome-mediated degradation pathway.

Alternative Splicing↗

PubMed2004

LIM kinase-2 targeting as a possible anti-metastasis therapy.

BACKGROUND: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. METHODS: We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. RESULTS: Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. CONCLUSIONS: Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells.

Base Sequence↗

PubMed2004

Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells.

BACKGROUND: Human normal cells have active p53 and pRB tumor suppressor pathways and undergo telomere shortening at each cell division. When immortalized, these exhibit functional inactivation of one or both tumor suppressor pathways and activation of telomere-maintaining mechanisms. Regulation of immortalization promoting molecular pathways by other genes is poorly understood and is an essential component of cancer therapeutics. In the present study, we have immortalized human normal cells by functional inactivation of p53 and pRB tumor suppressor proteins and simultaneous activation of telomerase function. We demonstrate that when the expression of mortalin (a member of hsp70 family proteins) was suppressed in such genetically defined immortal cells they underwent a permanent growth arrest. METHODS: WI-38 cells were transfected with expression plasmids encoding papilloma virus E6 and E7 proteins and a catalytic subunit of human telomerase enzyme. The derivative cells were compromised for p53 and pRB senescence pathways, showed telomerase activity and were immortalized. These cells were transfected with antisense expression plasmid for mortalin. The derivative clones were analyzed for mortalin expression, proliferation and telomerase activity. RESULTS: WI-38 cells were immortalized with E6, E7 and the catalytic subunit of human telomerase. The derivative-immortalized cells when suppressed for mortalin expression underwent senescence-like growth arrest. CONCLUSIONS: The data demonstrate that suppression of mortalin is sufficient to induce growth arrest in human immortalized cells that have compromised p53 and pRB functions and possess telomerase activity.

Antisense Elements (Genetics)↗

PubMed2004

Cell migration and metastasis as targets of small RNA-based molecular genetic analyses.

Metastatic tumor cells can migrate from one place to another in the body. This involves their adherence to host cell layers and subsequent transcellular movements by a complex process, molecular basis of which are yet to be clarified. Elucidation of genes functionally involved in metastasis may lead to deeper understanding of the mechanism of cell migration, and identification and designing of metastasis-modulating strategies for cancer therapeutics. We review here cell migration in tumor metastasis and the use of small RNA-based approaches to identify functional genes. We then describe our promising novel approach that uses randomized ribozyme libraries for identification of genes involved in cell migration, a consistent feature of metastatic cells.

Animals↗

PubMed2004

Know-how of RNA interference and its applications in research and therapy.

Double stranded RNA (dsRNA) mediates gene silencing in a sequence specific manner. Originally recognized in plants and lower organisms, it was recently extended to higher eukaryotes and established as an important evolutionary conserved phenomenon. It has been established that the double stranded short interfering RNAs (siRNAs) originate by the activity of a dsRNA-specific endonuclease, Dicer. siRNA in conjunction with a multiple enzyme complex called RNA-induced silencing complex (RISC) locates to the specific sites on mRNA and degrades it by endonuclease and exonuclease activities. In addition to gene silencing at transcript level (degradation of messenger RNA), siRNA was also shown to reduce the expression of protein by silencing of gene promoters via de novo methylation. By virtue of their specific gene silencing activity and owing to the recent discoveries on their plasmid and virus driven expression, small dsRNAs are being widely adopted in research and therapeutics. They are rapidly replacing the conventional gene knock-out technologies. siRNA libraries are also being recruited as a new tool in genome wide functional screenings. There is no doubt that further progress in understanding the mechanism of their action as well as strategies to achieve their tightly regulated and tissue specific expression will revolutionize basic and applied biomedical research.

Animals↗

PubMed2004

Emerging technologies: trendy RNA tools for aging research.

Aging is an inevitable biological phenomenon. Attempts to understand its mechanisms and, consequently, to therapeutically decelerate or even reverse the process are limited by its daunting complexity. Rapid and robust functional genomic tools suited to a wide array of experimental model systems are needed to dissect the interplay of individual genes during aging. In this article, we review principles that transcend the view of RNA, from a molecule merely mediating the flow of genetic information, into a unique molecular tool. In the form of catalytic molecular scissors (ribozymes), antibody-like antagonists (aptamers) and gene silencers (interfering RNAs, RNAi) can be effectively used to dissect biofunctions conserved throughout the evolution. In this review, application of recent RNA tools in aging research is discussed.

Aging↗

PubMed2004

World of small RNAs: from ribozymes to siRNA and miRNA.

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.

Animals↗

PubMed2004

Dose and dose-rate effects of low-dose ionizing radiation on activation of Trp53 in immortalized murine cells.

A derivative of immortalized murine NIH/PG13Luc cells stably transfected with a Trp53-dependent luciferase reporter plasmid was used to study the transcriptional activity of Trp53 in response to radiation. The cell line was sensitive enough to detect the response of Trp53 to 0.2 cGy of (60)Co gamma radiation. To examine the biological effects of low-dose-rate (60)Co gamma radiation (from 0.1-10 cGy/h), we have analyzed the cell cycle, Trp53 transcriptional activity, and gene expression profiles of control and treated cells. Microarray analysis revealed up-regulation of six Trp53-mediated genes (Cdkn1a/ p21, Mdm2, Sip27, Ccng1/cyclin G1, Ei24/Pig8 and Dinb/ Polk) after exposure of cells to low-dose-rate radiation for 72 h. Using real-time PCR, a significant elevation in the expression of Ccng1/cyclin G1, Mdm2 and Cdkn1A/p21 was observed with low-dose-rate irradiation at dose rates over 5 cGy/ h. A dose-rate dependence was also observed for these three Trp53-mediated genes. The expression of Ccng1/cyclin G1 at high dose rates of gamma rays was higher than that for low dose rate. However, the expression of Mdm2 for low-dose-rate gamma rays was higher than for the high dose rate. Cells irradiated at low dose rates of 0.1 cGy/h and 1 cGy/h underwent G(1)-phase arrest. Furthermore, G(2)-phase growth arrest was observed in cells irradiated at the low dose rates of 5 cGy/h and 10 cGy/h, which correlated with Trp53-mediated Ccng1/cyclin G1 up-regulation. These results show that cellular response to radiation depended on the dose rate used; i.e., the responses seen at dose rates from 0.1-1 cGy/h were different from those observed at dose rates over 5 cGy/h.

Animals↗

PubMed2004

Vectors for RNA interference.

Introduction of double-stranded RNA into cells causes gene silencing in a sequence-specific manner, involving the coordinated activity of enzymes such as Dicer and RNA-induced silencing complex. Several groups have recently demonstrated that this phenomenon of RNA interference (RNAi) occurs in mammalian cells when small interfering (si)RNAs are used, and have developed vector-based siRNA expression systems that can induce RNAi in living cells. These vector systems use polymerase III promoters, such as U6 or H1, and are classified into two groups based on the form of expressed RNA, tandem or hairpin. This review describes the basis for, and methodology of siRNA expression vectors for mammalian cells.

Animals↗

PubMed2003

Limited capacity of the nuclear matrix to bind telomere repeat binding factor TRF1 may restrict the proliferation of mortal human fibroblasts.

The maintenance of telomere integrity is essential for prolonged cell proliferation, and failure in this mechanism is a most consistent manifestation of cellular senescence. In this study, we investigated the role of telomere repeat binding factor (TRF1) in the proliferation of human fibroblasts. TRF1 expression is upregulated in a large variety of immortal human cells and supports de novo telomere formation in a dose-dependent manner. These observations suggest that the suppression of TRF1 might limit telomere maintenance and thus the life span of mortal cells. However, primary fibroblasts ectopically overexpressing TRF1 were unable to avoid senescence. On the other hand, exogenously expressed TRF1 in primary fibroblasts neither supported de novo telomere formation nor bound to the nuclear matrix as tightly as observed in immortal cells that show upregulated TRF1 expression. We present evidence suggesting that mortal human cells lack specific ligand(s) that anchor TRF1 to the nuclear matrix and that this contributes to their limited lifespan.

Cell Division↗

PubMed2003

Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts.

The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.

Cell Division↗

PubMed2003

Mortalin-MPD (mevalonate pyrophosphate decarboxylase) interactions and their role in control of cellular proliferation.

Mortalin (mot-2/GRP75/PBP74/mthsp70) is a member of the hsp70 family of proteins and is differentially distributed in normal and immortal cells. It was shown to be involved in pathways to cell senescence and immortalization. To elucidate its functional aspects, a yeast interactive screen for mortalin (mot-2) binding proteins was performed. Mevalonate pyrophosphate decarboxylase (MPD) was identified as one of the mortalin binding partners. The interactions were confirmed in mammalian cells by two-hybrid assay and in vivo coimmunoprecipitation. MPD is known to furnish prenyl groups required for prenylation, protein modification that is essential for the activity of many proteins including p21(Ras) (Ras). We have examined the effect of MPD-mot-2 interactions on the level and activity of p21(Ras) and its downstream effectors, p44 and p42 MAP kinases (ERK1/ERK2), in Ras-Raf pathway. An overexpression of mot-2 resulted in reduced level of Ras and phosphorylated ERK2. These were rescued by co-expression of MPD from an exogenous promoter demonstrating a functional link between mot-2, MPD, and Ras. Ras and its oncogenic forms act as key players in controlling proliferation of normal and cancerous cells. Assigning mot-2 upstream of p21(Ras) offers an important mechanism for influence over cell proliferation.

Animals↗