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PubMed1989

Note on the dispersion of generations among cells in senescing diploid fibroblast populations.

The cells in a cultured diploid fibroblast population have heterogeneous intermitotic times--even cells derived from the same mitosis may divide at different ages. This heterogeneity of inter-mitotic times results in asynchronous population growth and a dispersion of generations among members of the cell population. Because the appearance of non-dividing cells in middle-age populations has been attributed to the presence of lineages with more generations than average, we estimated the magnitude of the dispersion of cell generations as functions of the population doubling level and of the coefficient of variation of inter-mitotic times. For some data, such as that of Macieira-Coelho and Azzarone (Exp. Cell Res., 141 (1982) 325, the rate at which such non-proliferating lineages appear could be explained by a reasonable coefficient of variation of inter-mitotic times (25%). Most other data, however, would be fit only if the coefficient of variation of inter-mitotic times were 50% or greater, a variability that exceeds what has been observed in microcinematography experiments.

Cell Cycle↗

PubMed1990

Early kinase C dependent events in aging human diploid fibroblasts.

Two early kinase C dependent events, the induction of c-fos transcription and the phosphorylation of an 87 kDa protein, were investigated in aging IMR 90 human diploid fibroblasts. C-fos induction and 87-kDa protein phosphorylation were similarly regulated in aging cells and their young counterparts. The data suggests that G0 exit or the competence phenomenon is intact in aging cells and that the proliferative block occurs later in G0/S.

Aging↗

PubMed1991

Regulation of the retinoblastoma anti-oncogene in aging human diploid fibroblasts.

Cell cycle expression of the retinoblastoma (RB) gene was investigated in young and aging IMR 90 human diploid fibroblasts and in the immortalized but untransformed A31 BALB/c 3T3 mouse fibroblasts. RB RNA levels increased about 75% during late G1/S in both the young and old IMR 90 cells. Young and old cells had similar normalized amounts of RB RNA per cell. Kinase C activation did not stimulate RB transcription. The A31 cells showed a constant RB RNA level throughout G0/S. The data is consistent with the hypothesis that RB activity is regulated primarily post-transcriptionally and indicates that a change in the regulation of RB transcription is not part of the senescence phenomenon.

Animals↗

PubMed1992

Age related decline in the expression of proliferating cell nuclear antigen in human diploid fibroblasts.

Proliferating cell nuclear antigen mRNA and protein levels were determined in human diploid fibroblasts of different in vitro ages as they progressed through the cell cycle. Cells were analyzed at G0; at various stages of G1, including the G1/S interface; and during S. At all in vitro ages, PCNA message levels were low to undetectable at G0, were evident 8 to 12 h following entrance into G1, peaked at G1/S and declined during S phase. Message levels were 2-3-fold lower in older populations at all stages of the cell cycle tested. PCNA protein increased from G0 through S phase in both age groups with 2-3-fold less being found in older cells. The decline in PCNA mRNA in older populations was not the result of changes in mRNA turnover or transcription. The results suggest that the reduction in PCNA expression is due to an age related alteration in a post-transcriptional regulatory function. The decline in the expression of the PCNA gene would contribute to the inability of older cells to initiate replicative DNA synthesis.

Cells, Cultured↗

PubMed1994

Cell aging of human diploid fibroblasts is associated with changes in responsiveness to epidermal growth factor and changes in HER-2 expression.

The limited replicative life span of diploid human cells in vitro (cellular senescence) serves as a cellular model of aging. We examined the proliferative response of 2BS cells of different population doubling levels to epidermal growth factor (EGF). DNA synthesis was measured by thymidine incorporation. As the cells aged, there was a significant decrease both in the baseline level of DNA synthesis and in the stimulation of DNA synthesis by EGF addition. The effective concentration of EGF and the latent period prior to DNA synthesis did not change. EGF receptor mRNA expression also remained unchanged as the cells aged, in the absence or presence of EGF, suggesting that the defect in old cells lies downstream in the EGF signaling pathway. As the cells reached 100% of their life span, however, there was a 70% decrease in EGF receptor mRNA. Expression of the EGF receptor homologue HER-2 was also examined. The HER-2 mRNA level was significantly reduced in old cells. Moreover, HER-2 expression was stimulated by EGF addition in young cells but not in old cells. The results suggest that cell aging is associated with a progressive loss in the ability of cells to respond to growth factors.

Cell Division↗

PubMed1984

Induction of plasminogen activator in a human diploid fibroblast cell line (MRC-5) by conditions which cause induction of interferon: the role of calcium ions.

The human diploid fibroblast cell line, MRC-5, derived from embryo lung tissue produced only small quantities of plasminogen activator (PA) when harvested using a standard nutrient medium (Eagle's Minimal Essential Medium, MEM). Use of a schedule designed to induce high concentrations of fibroblast interferon in these cells also resulted in production of considerably enhanced levels of PA. The kinetics of PA production differed from those of interferon production; specifically, PA was produced for at least 6 days following induction despite the toxicity of the inducers whereas interferon synthesis continued for only 1 day. Further investigation of the induction conditions for PA revealed that double-stranded RNA which was absolutely required for interferon production was not required for induction of PA. Indeed, the stimulus for enhancement of PA production appeared to be solely an elevated concentration of calcium ions in the extracellular medium. The possible physiological relevance of this induction of PA by elevated concentrations of calcium ions is discussed.

Calcium↗

PubMed1986

A comparison of the reactivity and immunogenicity of RA 27/3 strain rubella vaccine prepared in WI-38 or MRC5 human diploid cells.

The immunogenicity and clinical reactivity of rubella vaccine derived from WI-38 or MRC5 human diploid cells was compared in 125 seronegative adolescent females. Seroconversion rates, assessed by single radial haemolysis testing of paired pre- and post-vaccination samples exceeded 98% (56/57 and 68/68 vaccinees, respectively) for both vaccines. Quantitative assessment of rubella-specific antibodies in 53 post-vaccination sera by an ELISA technique also failed to reveal any difference in immunogenicity between the vaccines. Assessable calendar records documenting the occurrence of local and systemic signs and symptoms in the four weeks following vaccination were returned by 106 subjects. No important statistically significant difference in parameters of clinical reactivity between the vaccine groups was observed although the incidence of pain at the injection site was found to be significantly higher for vaccinees receiving WI-38 derived vaccine.

Adult↗

PubMed1975

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.

Cell Division↗

PubMed1980

Loss of reiterated DNA sequences during serial passage of human diploid fibroblasts.

A specific family of tandemly repeated DNA sequences was found to diminish in the human genome after serial passage of three strains of diploid fibroblasts. Eco RI restriction fragments of 340 and 680 bp were significantly reduced in quantity at late passage as determined by autoradiography of 14C-DNA and also by ethidium bromide fluorescence. The reduction in these closely related DNA sequences was confirmed by saturation hybridization to excess 14H-RNA transcribed from a homogeneous restriction fragment recleaved from the 340 bp DNA. The maximal fraction of DNA hybridizing to the 3H-RNA probe declined by 33-50% over 21-41 population doublings. Divergence and/or methylation of such sequences could not account for these results since the thermal stability of cRNA:DNA duplexes actually increased by 0.3 degrees C at late passage. Total highly repetitive sequences assayed by reassociation kinetics were also substantially reduced at late passage, implying that depletion may be common to many repeat families in DNA. The denaturation temperature for such rapidly reassociated duplexes again increased slightly at late passage, possibly reflecting the minor decreases in DNA methylation which were detected in two of the cell strains. Karyotype analyses demonstrated that over 95% euploidy was maintained, with no specific chromosome loss and no visible deletions at late passage. The depletion of reiterated sequences during repeated cell division is thus attributed to numerous small DNA deletions, which may arise from unequal recombination coupled with selection or from a nonreciprocal mechanism such as excision.

Cell Division↗

PubMed1982

Establishment, characterization and differentiation induction of a new human diploid myelomonocytic cell line (HL-92) derived from a patient with acute myelomonocytic leukemia.

With very few exceptions, it has not been possible to grow human myeloid cells for long periods in culture. We have recently developed techniques enabling the long-term in vitro propagation of normal immature myeloid cells from fresh foetal cord blood and monocytes from normal adult peripheral blood, and have utilized these procedures to initiate cultures of fresh peripheral blood leukocytes from leukemic donors. In four of 26 leukemic samples tested, leukocyte replication beyond that obtained in control cultures was observed, and in one of these HL-92, derived from the peripheral blood of a patient with acute myelomonocytic leukemia, the culture has continued to replicate slowly for over 2 years under the special growth conditions. Morphological, cytochemical, immunological and functional studies show that the culture consists predominantly of immature myeloid cells (myeloblasts through to myelocytes) but also contains some mature neutrophils and monocytes. At least a portion of HL-92 cells express Fc and complement receptors, contain histacompatibility locus antigens, including HLA-DR, and release GM-CSA, low levels of PGE and lysozyme. HL-92 cells can be induced with DMSO or RA to differentiate into mature neutrophils (an increase from 20 to 70% of the cell population) as determined by morphology, by an increase in phagocytic cells, and superoxide anion production. Fresh leukocytes from the patient's bone marrow appeared to have a diploid karyotype. However a consistent chromosomal abnormality observed in HL-92 was a deletion in the long arm of chromosome 11 [del(11)(q23)]. This is consistent with recent observations in monocytic leukemia. Since the few other established human myeloid cell lines have various chromosomal abnormalities, and some respond to differentiation inducers, while others do not, there appears to be no detectable common chromosome change required either for in vitro growth of myeloid cells or their response to inducers of differentiation. These cell lines and the application of the techniques described here for the growth of myeloid cells from other leukemic or normal sources should be helpful in the study of normal and leukemic myeloid cell growth and differentiation.

Cell Line↗

PubMed1982

[Production of rabies vaccine in animal diploid cells].

Modalities for production of inactivated rabies vaccine derived from diploid hamster cell cultures are reported. The inactivated concentrated virus, purified by zonal centrifugation, is utilised for the preparation of vaccines destinated to carnivores, either in the form of monovalent vaccine or associated with distemper and canine contagious hepatitis vaccines. The inactivated concentrated virus is utilised for the preparation of bovine vaccine. The procedure is compatible with industrial production. The results concerning safety and potency tests of the experimental lots are presented.

Animals↗

PubMed1987

The effect of cellular age on zinc levels in untreated and zinc-treated human diploid fibroblasts.

Cellular aging is accompanied by increased cellular permeability to zinc(II). The intrinsic zinc content of human diploid fibroblast cells increases with cell age, so that it quadruples from early to late passage, on a Zn(II) per cell or per cell volume basis, but it remains constant on a Zn(II) per protein basis. When the cells are challenged with toxic concentrations (0.2 mM) of Zn(II), both the rate of zinc incorporation into the cells and the amount of zinc incorporated at equilibrium increases considerably with age (unless measured as zinc per protein). In terms of growth inhibition, Zn(II) is more toxic to the cell than Cu(II), Mn(II), or Mg(II).

Cations, Divalent↗

PubMed1977

Comparative mutagenicity of ICR-191 to S. typhimurium and diploid human lymphoblasts.

Concentration-dependent mutagenicity of ICR-191 has been measured in Salmonella typhimurium strain TA98 and in a diploid human cell line. In both cell systems, approximately equigenerational exposure produced mutation linearly related to concentration in the lower range of ICR-191 concentrations tested. Saturation behavior was observed in the human cell assay but not in the bacterial assay. However, a 25-fold greater concentration of ICR-191 was required to induce a significant rise in the mutant fraction in the S. typhimurium assay than in the human cell assay. These differences may be linked to the differences in the biochemical events required for mutation or in the time of exposure to ICR-191.

Acridines↗

PubMed1978

Determination of radiation equivalence of the chemical furylfuramide (AF-2) for the induction of gene conversion in diploid yeast and estimation of genetic risk to the Japanese population.

Dose--effect relationships for the induction of gene conversion by AF-2 in diploid yeast have been established. On the basis of these, the rec value for AF-2 for induction of gene conversion has been calculated to be 0.085 microgram/ml.h. This compares well with the rec values obtained with other test systems and other genetic end-points. The genetic burden to the Japanese population due to consumption of AF-2 is estimated to be equivalent to 55 millirec. However, uncertainties in the consumption of AF-2 per person and its metabolic detoxication tend to lower this value to, probably, a small fraction of that due to natural background ionizing radiation.

Alleles↗

PubMed1978

A quick method for testing recessive lethal damage with a diploid strain of Aspergillus nidulans.

A simple method capable of detecting recessive lethal damage in a diploid strain of Aspergillus nidulans is described. The method scores the recessive lethals on the 1st, the 3rd and the 5th chromosomes, which represent about 40% of the total map of A. nidulans. Two examples of induced lethals, with ultraviolet irradiation and methyl methanesulfonate are shown. The frequency of lethals may reach 36% of the total population with UV irradiation.

Aspergillus nidulans↗

PubMed1979

Induction of gene conversion in diploid yeast by chemicals: correlation with mutagenic action and its relevance in genotoxicity screening.

More than 200 chemicals, comprising drugs, pesticides, herbicides, industrial chemicals, food additives, etc. tested by various workers for their ability to induce gene conversion in diploid yeast, have been reviewed. This review shows that a strong correlation exists between the convertogenic and mutagenic abilities of these chemicals. This conclusion confirms the earlier one by Zimmermann which was based on fewer mutagenic chemicals. Published data have further been analysed to confirm quantitatively Zimmermann's other important conclusion that gene conversion is not mutagen-specific. This is a positive advantage in using gene conversion in preliminary screening of chemicals because it reduces the chances of false negatives.

Alleles↗

PubMed1984

Use of low temperature for growth arrest and synchronization of human diploid fibroblasts.

The growth kinetics of human diploid fibroblasts at two different temperatures were followed. Proliferation of exponentially growing cells is reduced and eventually stops upon incubation at low temperature (i.e. 30 degrees C). The cells which are in S phase at the time of switching to low temperature complete their DNA synthesis and become arrested in the G1 phase of the cell cycle. The arrested cells can be stimulated to proliferate by restoration of the optimal growth temperature (37 degrees C). The kinetics of entry into S phase were investigated by measuring [3H]thymidine incorporation into TCA-precipitable material, by autoradiography and by flow cytofluorimetry. The synchronized cells initiate DNA synthesis at approximately 8 h and DNA synthesis peaks at 20.4 +/- 0.7 h after stimulation. In addition, the rates of UV-induced excision repair at 30 degrees C and 37 degrees C were compared. The results indicate that at 30 degrees C the excision-repair process is operative but at a slightly reduced rate in comparison with repair at 37 degrees C. This method will be useful for the study of S-phase-dependent processes, as well as for repair studies in the absence of cell division.

Cell Cycle↗

PubMed1989

Fixation and repair of radiation-induced potentially mutagenic damage sensitive to hypertonic treatment in human diploid fibroblasts.

The effect of hypertonic salt treatment on the repair of potentially lethal damage and potentially mutagenic damage in X-irradiated asynchronous and synchronous human diploid fibroblasts (IMR91) have been studied. Resistance to 6-thioguanine was used for the mutagenic end point. When cells in late-S-phase were treated with hypertonic salt solution immediately after X-irradiation, both cell killing and mutation induction were enhanced, as compared to X-irradiation alone. This suggests that X-irradiation of cells in late S phase induces both potentially lethal damage and potentially mutagenic damage and that both are sensitive to hypertonic salt solution. When cells were allowed 2 h for repair after exposure to X-rays, both types of damage were completely repaired. Almost the same results were obtained with asynchronous cells. These results are discussed in terms of the relationship between radiation damage leading to cell lethality and mutagenesis.

Aphidicolin↗