Biomedical subjects

Sunil C Kaul

Publications and source records attributed to Sunil C Kaul.

34 records · Page 2Linked to original sources

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

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

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↗

PubMed2003

A novel putative collaborator of p19ARF.

We performed a yeast interactive screen in search of p19(ARF)-binding partners and have isolated a novel serine rich protein that is assigned to human chromosome 4q35 and mouse chromosome 8. The human and mouse proteins showed 84.2% homology. It is named CARF for its role as a putative Collaborator of ARF. CARF binds to both mouse and human ARF proteins. Its expression was detected in a variety of human tissues. The cDNA was expressed in bacteria and mammalian cells as a soluble and predominantly nucleoplasmic protein, respectively. CARF is a novel binding partner of ARF and might be involved in its p53-dependent or -independent tumor suppressive functions.

Amino Acid Sequence↗

PubMed2003

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates.

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their i) broad absorption spectra allowing visualization with single light source, ii) exceptional photo-stability allowing long term studies and iii) narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.

Antibodies, Monoclonal↗

PubMed2003

Targeting mortalin using conventional and RNA-helicase-coupled hammerhead ribozymes.

Mortalin, also known as mot2/mthsp70/GRP75/PBP74, is a member of the heat-shock protein 70 family that is heat-uninducible. It is differentially distributed in cells that have normal and immortal phenotypes, has been localized to various subcellular sites, and has several binding partners and functions. Here, we describe the construction and use of mortalin-specific conventional and hybrid ribozymes to elucidate its crucial role in cell proliferation. Whereas conventional hammerhead ribozymes did not cause any repression of endogenous mortalin expression, RNA-helicase-linked hybrid ribozymes successfully suppressed the expression of mortalin, which resulted in the growth arrest of transformed human cells. We show that, first, RNA helicase-coupled hybrid ribozymes that have a linked unwinding activity can be used to target genes for which conventional hammerhead ribozymes are ineffective; second, the targeting of mortalin by RNA-helicase-coupled hybrid ribozymes causes growth suppression of transformed human cells and could be used as a treatment for cancer.

Animals↗

PubMed2002

Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3.

Transcription factor Glioblastoma-3 (Gli3) is cleaved in the anterior region of the limb bud to generate its repressor form. In contrast, Sonic hedgehog (Shh) signaling from the posterior zone of polarizing activity blocks Gli3 processing and then induces the expression of Gli3 target genes, including Gli1. Here we report that the Ski corepressor binds to Gli3 and recruits the histone deacetylase complex. The Gli3-mediated repression was impaired by anti-Ski antibody and in Ski-deficient fibroblasts, and Shh-induced Gli1 gene transcription mediated by full-length Gli3 was inhibited by Ski. Furthermore, a Ski mutation enhanced the digit abnormalities caused by the Gli3 gene mutation. Thus, Ski plays an important role in pattern formation.

Acyltransferases↗

PubMed2002

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53.

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.

3T3 Cells↗

PubMed2002

Rhodacyanine dye MKT-077 inhibits in vitro telomerase assay but has no detectable effects on telomerase activity in vivo.

MKT-077, a cationic rhodacyanine dye analogue, causes selective toxicity to cancer cells. Its cellular targets elucidated thus far include oncogenic Ras, F-actin, mortalin (hmot-2)/mthsp70, and telomerase. Here we report that MKT-077 causes growth arrest of cancer cells in culture independent of their Ras, p53, or telomerase status. Telomerase activity is inhibited in vitro by MKT-077 in the telomerase assay used. However, the in vivo toxicity seen in telomerase-positive cancer cells was not accompanied by inhibition of telomerase activity or telomere shortening. Furthermore, cells with an alternative mechanism for lengthening of telomeres were also sensitive to MKT-077 and showed enhanced formation of alternative mechanism for lengthening of telomeres-associated PML bodies in their nuclei. The data suggested that inhibition of telomerase activity is unlikely to be a prime cause of MKT-077-induced toxicity in cancer cells.

Cell Division↗

PubMed2002

A major functional difference between the mouse and human ARF tumor suppressor proteins.

Suppression of tumorigenesis is considerably more stringent in the human than in the much shorter lived mouse species, and the reasons for this difference are poorly understood. We investigated functional differences in the control of the ARF (alternative reading frame) protein that acts upstream of p53 and is encoded along with p16(INK4a) at a major tumor suppressor locus in both the human and mouse genomes. The mouse and human ARF proteins are substantially divergent at their carboxyl termini. We have shown that the mouse ARF protein (p19ARF) interacts with Pex19p in the cell cytoplasm leading to its nuclear exclusion and repression of its p53 activation function. The human ARF protein (p14ARF) is substantially smaller than its mouse counterpart and is not subject to this functional inactivation by Pex19p. In an identical cellular background, ribozymes directed against Pex19p enhanced p19ARF- but not p14ARF-activated p53 function. This is the first demonstration of a functional difference between the mouse and human ARF proteins. In view of the major role of ARF in tumor suppression, this distinction may contribute to the different levels of tumor proneness of these species.

3T3 Cells↗

PubMed2002

Hsp70 family member, mot-2/mthsp70/GRP75, binds to the cytoplasmic sequestration domain of the p53 protein.

Hsp70 family member mot-2/mthsp70/GRP75/PBP74 was shown to bind to the tumor suppressor protein p53. In this study, by in vivo coimmunoprecipitation of mot-2 with p53 and its deletion mutants, the mot-2 binding site of p53 was mapped to its C-terminal amino acid residues 312-352, a region of p53 that includes its cytoplasmic sequestration domain. These data demonstrate that cytoplasmic sequestration and inactivation of p53 by mot-2 occurs by its binding to the cytoplasmic sequestration domain. Therefore, perturbation of mot-p53 interactions can be employed to abrogate cytoplasmic retention of wild-type p53 in tumors.

3T3 Cells↗

PubMed2002

An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where?

Mortalin/mthsp70/PBP74/Grp75 (called mortalin hereafter), a member of the Hsp70 family of chaperones, was shown to have different subcellular localizations in normal and immortal cells. It has been assigned to multiple subcellular sites and implicated in 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 the multiple sites and functions of mortalin in different organisms. The relevance of its differential distributions and functions in normal and immortal cell phenotypes is discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Mortalin: present and prospective.

Mortalin, also known as mthsp70/PBP74/GRP75, resides in multiple subcellular sites including mitochondria, ER, plasma membrane, cytoplasmic vesicles and cytosol. It is differentially distributed in normal and cancerous cells; the latter, when reverted back to normal phenotype, also show change in mortalin staining pattern similar to normal cells. Depending on its different subcellular niche and binding partner therein, mortalin is expected to perform multiple functions relevant to cell survival, control of proliferation and stress response.

Animals↗