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PubMed2006

Antigenotoxic effects of three essential oils in diploid yeast (Saccharomyces cerevisiae) after treatments with UVC radiation, 8-MOP plus UVA and MMS.

Essential oils (EOs) extracted from medicinal plants such as Origanum compactum, Artemisia herba alba and Cinnamomum camphora are known for their beneficial effects in humans. The present study was undertaken to investigate their possible antigenotoxic effects in an eukaryotic cell system, the yeast Saccharomyces cerevisiae. The EOs alone showed some cytotoxicity and cytoplasmic petite mutations, i.e. mitochondrial damage, but they were unable to induce nuclear genetic events. In combination with exposures to nuclear mutagens such as 254-nm UVC radiation, 8-methoxypsoralen (8-MOP) plus UVA radiation and methylmethane sulfonate (MMS), treatments with these EOs produced a striking increase in the amount of cytoplasmic petite mutations but caused a significant reduction in revertants and mitotic gene convertants induced among survivors of the diploid tester strain D7. In a corresponding rho0 strain, the level of nuclear genetic events induced by the nuclear mutagens UVC and 8-MOP plus UVA resulted in the same reduced level as the combined treatments with the EOs. This clearly suggests a close relationship between the enhancement of cytoplasmic petites (mitochondrial damage) in the presence of the EOs and the reduction of nuclear genetic events induced by UVC or 8-MOP plus UVA. After MMS plus EO treatment, induction of these latter events was comparable at least per surviving fraction in wildtype and rho0 cells, and apparently less dependent on cytoplasmic petite induction. Combined treatments with MMS and EOs clearly triggered switching towards late apoptosis/necrosis indicating an involvement of this phenomenon in EO-induced cell killing and concomitant decreases in nuclear genetic events. After UVC and 8-MOP plus UVA plus EO treatments, little apoptosis and necrosis were observed. The antigenotoxic effects of the EOs appeared to be predominantly linked to the induction of mitochondrial dysfunction.

Apoptosis↗

PubMed2005

IVM media, oocyte diameter and donor genotype at RYR1 locus in relation to the incidence of porcine diploid oocytes after maturation in vitro.

In the present study three factors were investigated that may affect the process of the first polar body extrusion in pig oocytes matured in vitro: IVM medium, oocyte diameter and donor genotype at the ryanodine receptor (RYR1) locus. In the first experiment, COCs were collected by the aspiration of slaughterhouse ovaries. Oocytes were matured in vitro at 39 degrees C, in humidified 5% CO(2) atmosphere for 44 h using the following media: (1) TCM199+hCG+eCG+follicular fluid (FF), (2) TCM199+hCG+17beta-estradiol and (3) NCSU23+hCG+eCG+FF. According to cytogenetic analysis, 98.1% of cells reached the second metaphase stage (MII). No significant differences were observed among IVM groups in terms of diploidy level. In the second experiment, oocytes collected by the aspiration or slicing of individual ovaries were matured in vitro in groups reflecting their origin. One ovary was considered a donor. IVM was carried out under conditions described in experiment I, with the use of TCM199+hCG+17beta-estradiol. A total of 68 ovaries/donors were included in this study. Granulosa cells collected from each ovary were used as DNA source in molecular (RFLP) analysis. Genotype frequencies at the RYR1 locus were as follows: CC, 0.46; CT, 0.48 and TT, 0.06. After maturation the diameter of each denuded oocyte was determined with the use of a computer aided system. Five size categories were distinguished: <90, 90-100, 100.1-110, 110.1-120 and >120 microm. The average diameter of haploid oocytes at MII stage was 111.7 microm, whereas that of diploid cells was 110.4 microm. According to statistical analysis, diploidy was not related to the oocyte diameter. That trait, however, was influenced by the donor genotype at the RYR1 locus. The TT genotype was associated with a higher rate of diploidy.

Animals↗

PubMed2006

Effect of pyridoxylidene aminoguanidine on human diploid cells B-HEF-2: in vitro cytotoxicity test and cytogenetic analysis.

BACKGROUND: Pyridoxylidene aminoguanidine is an appropriate inhibitor of protein glycation, respectively formation of advanced glycation products, which are connected with mechanism of pathogenesis in chronic diabetic complications. Moreover, it was found that in comparison with aminoguanidine, pyridoxylidene aminoguanidine does not influence the level of vitamin B6 in liver and kidneys in vivo. The aim of this study was to test cytotoxic effect of pyridoxylidene aminoguanidine in vitro, in regard to its potential use as inhibitor of advance protein glycation in diabetic patients. METHODS: The potential genotoxic activity of pyridoxylidene aminoguanidine in vitro was assessed by the micronucleus test and the karyological analysis. The direct contact method using diploid human cell line B-HEF-2 was performed to evaluate cytotoxicity. The concentrations of 5 x 10(-3), 2.5 x 10(-3) and 1 x 10(-3) ml/l were used in all tests. RESULTS: Microscopic analysis did not proved any changes in morphology of exposed fibroblasts. The inhibitive effect of pyridoxylidene aminoguanidine was increased with rising concentration. The proliferative activity of exposed cells to concentrations of 1 x 10(-3), 2.5 x 10(-3), 5 x 10(-3) mol/l was inhibited approximately by 30%, 60% and 80%, respectively. The frequency of micronuclei and rate of numerical or structural aberrations was not increased. CONCLUSION: Obtained results confirmed that pyridoxylidene aminoguanidine in selected concentrations has an inhibitive effect on the proliferation activity of exposed cells, but did not develop any cytotoxic effect on B-HEF-2 cells.

Cell Line↗

PubMed2005

Rabies human diploid cell vaccine elicits cross-neutralising and cross-protecting immune responses against European and Australian bat lyssaviruses.

The ability of antibodies elicited against the rabies human diploid cell vaccine (HDCV) to neutralise European bat Lyssaviruses (EBLV types-1 and -2), Australian bat Lyssavirus and classical rabies virus (RABV) has been evaluated using modified fluorescent antibody virus neutralisation (mFAVN) assays. Ninety-six percent (48 of 50) of the human post-vaccinated sera tested cross-neutralised these viruses (>or=0.5 IU/ml). Cross-protection experiments using inbred mice (RIII, k/k haplotype) were also assessed. Mice were given HDCV (twice by the intra-peritoneal route) and challenged (intra-cranially or peripherally) with a lethal dose (25 MLD(50)) of the individual viruses. The vaccine conferred statistically significant protection in 80--100% of animals challenged via the peripheral route. Levels of protection were lower following intra-cranial (i.c.) challenge. Our data provides strong evidence for broad spectrum cross-neutralisation and cross-protection of phylogroup I lyssaviruses using rabies HDCV.

Animals↗

PubMed2004

Molecular pathways executing the "trophic sentinel" response in HPV-16 E7-expressing normal human diploid fibroblasts upon growth factor deprivation.

In response to oncogenic insults, normal human cells execute a defense response that culminates in cellular suicide, apoptosis. Normal human diploid fibroblasts expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein are predisposed to apoptosis when they are deprived of growth factors. Even though a dominant negative p53 mutant abrogates the cell death response, it is not accompanied by p53 phosphorylation, the DNA binding capacity of p53 remains unaltered, and no activation of common p53-dependent transcriptional targets is observed. Expression of two insulin-like growth factor-1 binding proteins, IGFBP-2 and -5, is increased presumably in response to enhanced NF-kappaB activity in HPV-16 E7-expressing serum-starved cells. Phosphorylation of AKT, an important modulator of IGF-1 survival signaling, is lower in serum-starved E7-expressing cells, and exogenously added IGF-1 can partially inhibit the cell death response. This suggests that IGFBP-2 and -5 may limit IGF-1 availability thus decreasing survival signaling. Caspase 3 but not caspase 8 is activated in serum-starved HPV-16 E7-expressing cells. Caspase inhibition affects nuclear DNA fragmentation, but cell death is not inhibited. Although mitochondria play important roles in caspase-dependent as well as -independent forms of cell death, there is no evidence for cytochrome c release and thus for mitochondrial permeabilization in growth factor deprived HPV-16 E7-expressing cells.

Apoptosis↗

PubMed2004

Inhibition of phosphatidylinostol 3-kinase uncouples H2O2-induced senescent phenotype and cell cycle arrest in normal human diploid fibroblasts.

Exposure of WI38 human diploid fibroblasts (HDFs) to hydrogen peroxide (H2O2) induced premature senescence. The senescent HDFs were permanently arrested and exhibited a senescent phenotype including enlarged and flattened cell morphology and increased senescence-associated beta-galactosidase (SA-beta-gal) activity. The induction of HDF senescence was associated with an activation of p53, increased expression of p21Cip1/WAF1, and hypophosphorylation of retinoblastoma protein (Rb), while no changes in the expression of p16Ink4a, p27Kip1, and p14Arf were observed. Exposure of WI38 cells to H2O2 also selectively activated phosphatidylinostol 3-kinase (PI3 kinase) and mitogen-activated protein kinase (MAPK) kinase (MEK), while no changes in p38 MAPK and Jun kinase (JNK) activities were observed. Selective inhibition of PI3 kinase activity with LY294002 abrogated H2O2-induced cell enlargement and flattened morphology and significantly attenuated the increase in SA-beta-gal activity, but did not affect H2O2-induced cell cycle arrest. In contrast, selective inhibition of MEK and p38 MAPK with PD98059 and SB203580, respectively, produced no significant effect on H2O2-induced senescent phenotype and cell cycle arrest. These findings demonstrate that expression of the senescent phenotype can be uncoupled from cell cycle arrest in prematurely senescent cells induced by H2O2 and does not contribute to the maintenance of permanent cell cycle arrest.

Cell Cycle↗

PubMed2002

Up-regulation of PDCD4 in senescent human diploid fibroblasts.

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.

Animals↗

PubMed1989

Suppression of TNF-stimulated proliferation of diploid fibroblasts and TNF-induced cytotoxicity against transformed fibroblasts by TGF-beta.

Human transforming growth factor-beta (TGF-beta) dose-dependently inhibited proliferation of WI-38 cells, normal human diploid fibroblasts, stimulated by tumor necrosis factor (TNF). Inhibition occurred at 1 ng/ml concentration of TGF-beta. Also, TGF-beta dose-dependently suppressed cytotoxicity of TNF against L-929 cells, murine transformed fibroblasts. The concentration of TNF required for 50% cytolysis of L-929 cells was changed from 30 ng/ml to 350 ng/ml by 10 ng/ml TGF-beta. This suppression was abolished when L-929 cells were treated with actinomycin D or cycloheximide, suggesting that TGF-beta might inhibit the action of TNF via de novo protein synthesis. This response was not due to down regulation of TNF receptors nor to alteration of the affinity of TNF for its receptor.

Animals↗

PubMed1979

An effect of ultraviolet light on RNA and protein synthesis in nondividing human diploid fibroblasts.

Nondividing human diploid fibroblasts maintained in medium containing 0.5% calf serum do not survive when exposed to low doses of UV (254 nm). The extent of killing is dose and strain dependent. DNA excision repair-proficient cells are more resistant than excision repair-deficient cells. Results of measurements of the effect of UV on RNA and protein synthesis in repair-proficient and -deficient (XP12BE) cells are reported. UV causes an immediate and equal depression of the RNA synthesis rate in both kinds of cells. A recovery to control rates was observed only at low (5 J/m2) doses in repair-deficient cells and at higher doses (20 J/m2) in repair-proficient cells. No recovery was observed at doses that cause substantial reductions in survival (greater than 5 J/m2 for XP12BE; greater than 40 J/m2 for repair-proficient populations). No initial effect on rate of protein synthesis was detected at doses less than 20 J/m2. However, in XP12BE populations, a decreased rate first evident at 15-30 h post-UV and before any cell degeneration and loss was observed for doses as low at 7 J/m2. This delayed effect was not observed in repair-proficient populations. The results are consistent with the hypothesis that the lethal action of UV in nondividing cells is one on DNA that leads to an inhibition of required protein synthesis by preventing RNA transcription.

Cell Division↗

PubMed1980

Repair of radiation-induced DNA damage in nondividing populations of human diploid fibroblasts.

The occurrence of DNA repair in UV- (254 nm) and X-irradiated normal human diploid fibroblasts maintained in a quiescent, nondividing state using low serum (0.5%) medium was ascertained. Techniques that detect different steps of the excision repair process were used so that the extent of completion of repair at single sites could be determined. These included measuring the disappearance of pyrimidine dimers by chromatography, detecting repair synthesis by density-gradient and autoradiographic methods and detecting the rejoining of repaired regions and repair of x-ray-induced single-strand DNA breaks using alkaline sucrose gradients. Results show that dimer excision occurs and the subsequent steps of repair synthesis and ligation are completed. About 50% of the dimers formed by exposure to 20 J/m2 is excised in the initial 24-h post-UV period. DNA repair (unscheduled DNA synthesis) can be detected through a 5-d post-UV period. The fraction of damaged sites eventually repaired is not known. X-ray-induced single-strand DNA breaks are repaired rapidly.

Cell Line↗

PubMedDate not supplied

Metal-induced DNA damage and repair in human diploid fibroblasts and Chinese hamster ovary cells.

Cloning efficiency and DNA strand breaks induction were compared in human diploid fibroblasts (HSBP) and chinese hamster ovary (CHO) cells treated with various metal salts. Cadmium (Cd2+), nickel (Ni2+) and chromate (Cr2O7) reduced the cloning efficiency of HSBP cells more than that of CHO cells whereas the reverse was true after treatment with mercury (Hg2+), manganese (Mn2+) and cobalt (Co2+). The effects on cloning efficiency did not consistently correlate with DNA strand breaking activity as all metals except Cr(VI) were more effective at producing DNA strand breaks in CHO cells than in human cells. The differential responses of the two cell types was shown to be only partially due to differences in cellular uptake of metals. DNA breaks induced in human cells by Hg2+ and Cr2O7 were shown most likely to be alkaline labile sites rather than true strand breaks since no damage was detected in a nick translation assay which measures the amount of free 3'-OH terminals. Damage induced by Mn2+ and Co2+, however, appeared to be comprised at least in part by true DNA strand breaks. DNA damage was also induced in HSBP cells following treatment with selenium but only in the presence of reduced glutathione. These studies indicate that DNA damage is not as major a consequence following some metal treatments in human cells as it appears to be in rodent cells. This suggests that rodent models for risk estimation of metal-induced tumorigenesis may not always be appropriate for extrapolation to humans.

Animals↗

PubMed1999

Effects of three epoxides--ethylene oxide, propylene oxide and epichlorohydrin--on cell cycle progression and cell death in human diploid fibroblasts.

Ethylene oxide (EtO), propylene oxide (PO), and epichlorohydrin (ECH) strongly influenced the G1/S progression in human diploid fibroblasts, VH-10. However, these epoxides did not affect substantially the G2/M progression. It was found that G1 arrest is induced by these epoxides 6-18 h after the treatment at doses above 5, 3, and 0.5 mMh for EtO, PO, and ECH, respectively. An inhibitory effect on DNA synthesis was also demonstrated at the same doses within the same time interval. On the contrary, the epoxides transiently stimulated DNA synthesis 3-18 h after the treatment with the lower doses (below 5, 3, and 0.5 mMh for EtO, PO, and ECH, respectively). This effect was manifested both as an elevated rate of DNA synthesis and as an increase in the number of cells in S-phase. Among the three studied epoxides EtO was the most effective one: the increases of the rate of DNA synthesis and of cells in S-phase were 35 and 55%, respectively. All the epoxides tested induced significant decrease of intracellular level of reduced glutathione (GSH) shortly after cell exposure. While low and moderate doses induced a transient decrease in GSH level the high doses induced its irreversible depletion. The extensive GSH depletion was related to cell death. Morphological examination of cell nuclei indicated that epoxide-treated cells die via necrosis. This conclusion is supported by the lack of such features of the apoptosis as chromatin condensation and the occurrence of so called 'apoptotic bodies'. The absence of nucleosomal fragmentation of DNA and an increase of the permeability of the plasma membrane after the epoxide treatment also indicated a necrotic form of cell death. ECH is about ten times more toxic than the two other epoxides, and it causes almost 100% necrosis at dose of 3.0 mMh.

Cell Cycle↗

PubMed2003

Cellular aging-dependent decrease in cholesterol in membrane microdomains of human diploid fibroblasts.

Recently it has been shown that cholesterol plays indispensable roles in the function of cholesterol-rich microdomains (rafts), such as in ligand-mediated signal transduction. Using a perfringolysin O derivative (BCtheta) that binds selectively to cholesterol in rafts without causing membrane damage (Proc. Natl. Acad. Sci. USA 98 (2001) 4926), we have investigated the effect of in vitro replicative aging of human diploid fibroblasts, TIG-1, on the distribution of plasma membrane cholesterol. The amount of BCtheta-labeled membrane cholesterol decreased during replicative aging of TIG-1 cells, whereas total cholesterol increased somewhat. The relationship was confirmed by double staining with BCtheta and senescence-associated-beta-galactosidase, a biomarker of senescent cells. Cell fractionation experiments revealed decreases in both cholesterol in rafts and a raft marker, flotillin, during replicative aging. In addition, hydroxyurea-induced prematurely senescent cells also showed a lower level of BCtheta-labeled cholesterol than untreated cells, despite maintaining the total amount of cholesterol. When TIG-1 cells were cultured in cholesterol-deficient medium, BCtheta labeling was first diminished and then premature senescence was induced. Taken together with the reduced signaling capacity of aged cells, these results suggest that plasma membrane cholesterol in raft microdomains is more sensitive to senescence than total cholesterol and is a primary target in aging.

Apoptosis↗

PubMed1983

Inhibition of DNA synthesis in young cycling human diploid fibroblast-like cells upon fusion to enucleate cytoplasts from senescent cells.

Previous studies have demonstrated that upon fusion with actively cycling human diploid fibroblast-like (HDFL) cells, senescent HDFL are capable of inhibiting the young nucleus from entry into DNA synthesis (Norwood et al. (1974) [10]; Rabinovitch et al. (1980) [1]). These studies have been interpreted as evidence in favor of the existence of cell cycle inhibitors in senescent cells. Enucleate cytoplasts derived from senescent HDFL cells are also capable of inhibiting the young nucleus from entry into DNA synthesis upon fusion with whole cycling HDFL cells. In contrast, no inhibition was observed in fusions between enucleate young cells and whole cycling cells. These results provide evidence for transmissible cell cycle-inhibitory factors in the cytoplasm of senescent cells.

Cell Cycle↗

PubMed2000

Rad24 is essential for proliferation of diploid cells in fission yeast.

The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein. We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells.

Cell Cycle Proteins↗

PubMed2001

Growth kinetics rather than stress accelerate telomere shortening in cultures of human diploid fibroblasts in oxidative stress-induced premature senescence.

WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 microM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 microM H2O2 stress or five repeated 30 microM t-BHP stresses along the same PD, respectively a 322 +/- 55 and 380 +/- 129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress.

Cell Division↗

PubMed2001

Clusterin/apolipoprotein J is a novel biomarker of cellular senescence that does not affect the proliferative capacity of human diploid fibroblasts.

Normal human fibroblasts have a limited replicative potential in culture and eventually reach a state of irreversible growth arrest, termed senescence. In a previous study aiming to identify genes that are differentially regulated during cellular senescence we have cloned clusterin/apolipoprotein J (Apo J), a 80 kDa secreted glycoprotein. In the current report we pursue our studies and show that senescence of human diploid fibroblasts is accompanied by up-regulation of both Apo J mRNA and protein levels, but with no altered biogenesis, binding partner profile or intracellular distribution of the two Apo J forms detected. To analyze the causal relationship between senescence and Apo J protein accumulation, we stably overexpressed the Apo J gene in primary as well as in SV40 T antigen-immortalized human fibroblasts and we showed no alteration of the proliferative capacity of the transduced cells. Despite previous reports on tumor-derived cell lines, overexpression of Apo J in human fibroblasts did not provide protection against apoptosis or growth arrest induced by hydrogen peroxide. Overall, our results suggest that Apo J overexpression does not induce senescence but it is rather a secondary consequence of the senescence phenotype. To our knowledge this is the first report that provides a functional analysis of human Apo J during replicative senescence.

Antigens, Differentiation↗

PubMed1998

Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts.

The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers. It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture. To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF). To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein. This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells. Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein. Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells. These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

Antennapedia Homeodomain Protein↗