Search

Introduction of human chromosome 7 by microcell-mediated chromosome transfer suppresses indefinite division of SUSM-1, anin vitroestablished 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 β-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.

Studies on fibronectin, endothelin-1, and mortalin from our laboratory are reviewed here. Fibronectin expression has been analyzed as upregulated during in vitro serial passaging of human fetal lung and neonatal foreskin fibroblasts as well as umbilical vein endothelial cells. In vivo aging of skin fibroblasts, as well as aortic endothelial cells, are also accompanied by upregulation of fibronectin expression. Fibronectin promoter binding proteins from young and old cell nuclear extracts were further explored by gel retardation assay. Preliminary analyses have detected age-related differential binding activities with respect to AP-1, CRES, TFIID, CTF, and AP-2 regions, whereas Sp1 binding proteins remain unaltered. Endothelin-1 expression is also seen as upregulated during in vitro and in vivo aging of endothelial cells. This can contribute to the hypertension commonly observed in elderly patients. Mortalin, a novel member of hsp70 family of proteins, was initially identified by virtue of its association with a cellular mortal phenotype. Subsequently, normal cells and the ones with an immortal phenotype have been found to have differential subcellular localization of this protein. Antiproliferative activity of this protein in normal cells and the deregulation of expression in transformed cells is observed which suggests the association of mortalin in pathways that determine cellular divisional phenotype.

We examined the biological effects of low dose rate (LDR) of 60Co γ-ray irradiation (from 0.1–10 cGy/h) on the transcriptional activity of p53, using NIH/PG13Luc cells stably transfected with p53-dependent luciferase reporter plasmid. Microarray analysis revealed up-regulations of six P53-mediated genes (CDKN1A/p21, MDM2, SIP27, CCNG1/cyclin G1, EI24/PIG8 and Dinb/POLK) by exposure of cells for 72 h to LDR 60Co γ-ray irradiation. Using a real-time PCR, a significant elevation in the expression of CCNG1/cyclin G1, MDM2 and CDKN1A/p21 was observed at dose rates over 5 cGy/h. The dose rate dependency of these three p53-mediated genes was also observed. The expression of CCNG1/cyclin G1 at high dose rates (HDRs) γ-rays was higher than that for LDR. However, the expression of MDM2 for LDR γ-rays was higher than for HDR. Cells irradiated with γ-rays at 0.1 and 1 cGy/h appeared to show G1 phase arrest. Furthermore, G2 growth arrest was observed in cells irradiated with γ-rays at 5 and 10 cGy/h, being correlated with p53-mediated CCNG1/cyclin G1 up-regulation. These results imply that cellular response to radiation differs between the two LDRs used, 0.1–1 cGy/h and over 5 cGy/h.

A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with 32 P-labeled DNA probe in buffer in the presence of excess of nonspecific E. coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 m M ) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of the Drosophila clone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA–Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.

To determine the genetic relation between the pancytosolic (uniformly distributed in cytoplasm-p66 mot-1 ) and the perinuclear (p66 mot-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.

Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Conventional therapy of neuroblastoma includes the differentiation agents. Unlike chemo-radiotherapy, differentiation therapy shows minimal side effects on normal cells, because normal non-malignant cells are already differentiated. Keeping in view the limited toxicity of Withania somnifera (Ashwagandha), the current study was aimed to investigate the efficacy of Ashwagandha water extract (ASH-WEX) for anti-proliferative potential in neuroblastoma and its underlying signalling mechanisms. ASH-WEX significantly reduced cell proliferation and induced cell differentiation as indicated by morphological changes and NF200 expression in human IMR-32 neuroblastoma cells. The induction of differentiation was accompanied by HSP70 and mortalin induction as well as pancytoplasmic translocation of the mortalin in ASH-WEX treated cells. Furthermore, the ASH-WEX treatment lead to induction of neural cell adhesion molecule (NCAM) expression and reduction in its polysialylation, thus elucidating its anti-migratory potential, which was also supported by downregulation of MMP 2 and 9 activity. ASH-WEX treatment led to cell cycle arrest at G0/G1 phase and increase in early apoptotic population. Modulation of cell cycle marker Cyclin D1, anti-apoptotic marker bcl-xl and Akt-P provide evidence that ASH-WEX may prove to be a promising phytotherapeutic intervention in neuroblatoma related malignancies.

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.

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.

Mortalin, p66mol-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.

The heat shock chaperones mortalin/mitochondrial heat shock protein 70 (mtHsp70) and Hsp60 are found in multiple subcellular sites and function in the folding and intracellular trafficking of many proteins. The chaperoning activity of these 2 proteins involves different structural and functional mechanisms. In spite of providing an excellent model for an evolutionarily conserved molecular “brotherhood,” their individual functions, although overlapping, are nonredundant. As they travel to various locations, both chaperones acquire different binding partners and exert a more divergent involvement in tumorigenesis, cellular senescence, and immunology. An understanding of their functional biology may lead to novel designing and development of therapeutic strategies for cancer and aging.

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.

The median and maximum life spans of Zaprionus paravittiger at 26 +/- 2 degrees C were 35.45 +/- 0.39 and 70.0 +/- 1.00 days, respectively, in males and 39.93 +/- 0.40 and 77.67 +/- 0.58 days in females. Feeding of an antioxidant-sodium hypophoshite (SHP), 1, 10 1 X 10(2) and 1 X 10(3) microM-resulted in increased survival of both sexes. Further increase in SHP concentration accelerated the mortality rate. Secondly, the lipid peroxide level as measured by malondialdehyde content showed an age-related increase in normal as well as SHP-fed cohorts. However, the rate of increase was much slower in the latter. It is suggested that SHP, by virtue of its free-radical scavenging property, prolongs the life span of Z. paravittiger.

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.

The median and maximum life spans of Zaprionus paravittiger are 43 and 79 days, respectively, for males and 52 and 91 days for females at 24 +/- 2 degrees C. Sodium selenite (SS), an antioxidant, feeding prolongs the median as well as maximum life span of both the sexes. Antioxidative enzymes, catalase and peroxidase showed greater activity during the developmental stages. Females, at most of the age intervals, exhibited greater enzymatic activities as compared to their age-matched males. Both the sexes showed a statistically significant increase in the baseline activity of these enzymes as consequent upon SS feeding. It is suggested that SS acts by strengthening the antioxidative enzymes, namely catalase and peroxidase.

The median and maximum life spans of the drosophilid insect Zaprionus paravittiger reared on corn meal agar medium at 26±2°C were 35 and 70 days for males and 40 and 77 days for females, respectively. The addition of different concentrations of an antioxidant, sodium hypophosphite (SHP), in the diet resulted in increased median and maximum life spans of both the sexes: maximum by 21.7% and 34.2% in males and by 27.5% and 27.2% in females, respectively. DNA and RNA contents were found to decrease in normal as well as SHP fed insects, but the rate of decline was significantly lower in the latter. The results suggest that the damaging effect of free radicals is diminished by the antioxidizing property of SHP, thus supporting the free radical theory of aging.

Addition of an antioxidant, sodium hypophosphite (SHP; 1 m M ), to the diet of Zaprionus paravittiger (Diptera) results in depressed ATPase activity in both the sex

Sodium hypophosphite (1 x 10(3) microM) supplementation in the diet of Zaprionus paravittiger resulted in adaptive responses in the quantitative as well as qualitative activity of peroxidase. The changes in peroxidase were in compliance with the earlier reported sodium hypophosphite (SHP)-induced prolonged life span of flies (Wadhwa, R., Arch. Gerontol. Geriatr., 6 (1987) 141-147) and support the concept that antioxidative enzymes may serve as important longevity determinants.

The median and the maximum life spans of Drosophila bipectinata increased after feeding with various concentrations (10 microM, 10(2) microM and 10(3) microM) of butylated hydroxytoluene (BHT). Insects fed on the diet supplemented with 10(3) microM BHT had decreased rate of lipid peroxidation (measured by thiobarbituric acid test) with respect to the controls. It is suggested that the antioxidant BHT, which scavenges free radicals, prolongs the life span of Drosophila.

Zaprionus paravittiger fed with an antioxidant (sodium hypophosphite, 1 × 103μM) supplemented diet exhibited adaptive compensatory responses in catalase activity (quantitative as well as qualitative). The longevity and catalase activity were found to be positively linked. The study denotes that free radical formation and antioxygenic defenses are closely associated and are the possible determinants of life span and ageing.

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.

The mean life span of male and female Zaprionus paravittiger reared on corn meal agar medium at 26±2°C was found to be 35 and 40 days, respectively. The DNA content showed a perpetual decline in both sexes. The RNA content also showed a continuous decline in males while in females a significant leap is found during the first 17 days followed by a steady decline until the 41st day. The study supports the concept that protein synthesis (RNA-DNA) and thus the biological expression during different phases of life, are controlled at the DNA-RNA level.

DNA content decreases during first 20 and 25 days of survival in male and female flies of Drosophila bipectinata respectively. RNA as well as RNA/DNA content show a similar trend in males while in females both rise significantly during the first 5 to 10 days of survival followed by a steady decline till day 25. The study suggests that decreasing nucleic acid content of aging D. bipectinata is accompanied by the decline in protein synthesizing capacity.

An antioxidant, sodium hypophosphite (SHP — 1 × 10 3 μ M), when added to the diet of Zaprionus paravittiger , induces various shifts in the age-related biochemical parameters. It maintains the viability of protein synthesis and normal energy-yielding processes through longer intervals besides decelerating the rate of loss of nucleic acids (Sharma and Wadhwa, 1986). Reduced body weight which indicates the low level of food restriction, and adaptive changes in amino acids and proteins, could be the important factors for the favourable effect of this antioxidant on life span.

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.

We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 μg and 60 μg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxyribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and -independent pathways.

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.

Aging in the beetle Caryedon serratus was accompanied by a decline in total DNA content (9.28% in males and 9.79% in females). Mitochondrial DNA remained constant until the 11th day of age and then decreased gradually with advancing age in both sexes. RNA content and the RNA/DNA ratio showed similar changes in whole body as well as mitochondrial fractions. The two parameters decreased continuously and significantly (p less than 0.001) in males whereas in females an initial rise during the reproductive period was followed by decline until death. The observations suggest that there is a loss of mitochondria in the senescent period and a decline in the total nucleic acid content and protein synthetic capacity of C. serratus.

Mortalin is a member of Hsp70 family of stress chaperones. It was first identified as a protein involved in the senescence of mouse cells. Genetic studies revealed that there are two mouse mortalin alleles coding for two proteins (mot-1 and mot-2) that differ in only two amino acids in the carboxy-terminus, but have contrasting activities. Whereas mot-1 accelerated senescence, mot-2 extended the lifespan of mouse cells in culture. In human cells, only one kind of mortalin protein has been identified so far and is shown to be functionally equivalent to mouse mot-2. Whereas mortalin is enriched in cancer cells and contributes to carcinogenesis, the old age brain disorders show its deficiency. As we demystify its deux de machina, accumulating evidence reveal that mortalin may be "druggable" bidirectionally to either treat cancer or neuro-degenerative disorders.

Background Embelin, a quinone derivative, is found in the fruits of Embelia ribes Burm (Myrsinaceae). It has been shown to have a variety of therapeutic potentials including anthelmintic, anti-tumor, anti-diabetic, anti-bacterial and anti-inflammation. Inflammation is an immunological response to external harmful stimuli and is regulated by an endogenous pyrogen and pleiotropic pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α production has been implicated in a variety of other human pathologies including neurodegeneration and cancer. Several studies have shown that the anti-inflammatory activity of embelin is mediated by reduction in TNF-α. The latter is synthesized as a membrane anchored protein (pro-TNF-α); the soluble component of pro-TNF-α is then released into the extracellular space by the action of a protease called TNF-α converting enzyme (TACE). TACE, hence, has been proposed as a therapeutic target for inflammation and cancer. Methods We used molecular docking and experimental approaches to investigate the docking potential and molecular effects of embelin to TACE and human cancer cell characteristics, respectively. Results We demonstrate that embelin is a potential inhibitor of TACE. Furthermore, in vitro studies revealed that it inhibits malignant properties of cancer cells through inactivation of metastatic signaling molecules including MMPs, VEGF and hnRNP-K in breast cancer cells. Conclusion Based on the molecular dynamics and experimental data, embelin is proposed as a natural anti-inflammatory and anticancer drug.

Fixed mortality of normal somatic cells is a well-estblished fact though the mechanism underlying this universal phenomenon remains unknown. Use of immortal cells in conjunction with their normal mortal counterparts has delineated the dominant genetic nature of the senescent phenotype over immortalization. Although the involvement of proteins in determining the entry/exit/arrest of cells in the cell cycle is evident from the literature, none of them has been confirmed for its role in senescence-associated irreversible cell cycle exit/arrest. The identification of true mortality markers might be possible by selecting a system of natural and conditional aging achieved by the fusion of mortal and spontaneously immoralized cells of the same origin. We report here a few such protein markers which might serve as useful handles to tease out the molecular events determining mortality/immortality of cultured cells.