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PubMed1987

Interrelationships among X-ray-induced anchorage independence, mutagenesis and chromosomal rearrangements in human diploid fibroblasts.

The changes in the frequencies of X-ray-induced mutations to 6-thioguanine resistance, chromosomal abnormalities and transformation to anchorage-independent growth were examined during confluent holding recovery in density-inhibited cultures of human diploid fibroblasts. Chromosomal abnormalities studied included non-stable aberrations, deletions, and reciprocal translocations measured up to 20 mean population doublings post-irradiation. Complex recovery kinetics were observed. A comparison of these kinetics suggests that reciprocal translocations are associated with the induction of both transformation and mutations. Mutagenesis correlated with deletions and translocations, whereas transformation correlated with translocations only.

Cell Adhesion↗

PubMed1993

DNA methylation in the Alu sequences of diploid and haploid primary human cells.

We have investigated DNA methylation in human Alu sequences, both in general and in specific Alu sequences associated with the genes for alpha 1 globin, tissue plasminogen activator (tPA), adrenocorticotropic hormone (ACTH) and angiogenin. We studied DNAs from lymphocytes, granulocytes, brain, heart muscle and sperm, and from the human HeLa and KB cell lines by using cleavage with methylation-sensitive restriction enzymes combined with Southern blot hybridization and by using genomic sequencing. The results can be summarized as follows. (i) In differentiated primary human cells, Alu elements are often highly methylated even when they are in very 5'-CG-3'-rich regions. This finding is not consistent with the notion that hypermethylation would be a sufficient condition in itself for 5'-CG-3' sequences to undergo loss of 5-methyl-deoxycytidine (5-mC) due to deamination and subsequent mutation. (ii) There are distinct differences in the levels of methylation in the specific Alu sequences. (iii) Alu elements in the DNA of haploid spermatozoa are much less methylated than in diploid cells. Preliminary data indicate that spermatozoa contain Alu-specific RNAs. (iv) The results of cell-free transcription experiments with Alu elements suggest that the in vitro transcription of Alu elements can be inhibited by 5'-CG-3' methylation. High levels of 5'-CG-3' methylation in Alu elements could contribute to their general transcriptional inactivity. (v) The patterns of methylation observed in the Alu elements and in the surrounding sequences are characterized by cell type specific interindividual concordance.

Adrenocorticotropic Hormone↗

PubMed1994

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits differentiation of normal diploid rat osteoblasts in vitro.

The influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue-like organization in primary cultures of normal diploid calvarial-derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post-proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone-cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post-confluent formation of multicellular nodules that develop bone tissue-like organization was dramatically suppressed. Consistent with TCDD-mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post-proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D-responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone control of differentiation.

Animals↗

PubMed2003

Putative role for EPC-1/PEDF in the G0 growth arrest of human diploid fibroblasts.

EPC-1/PEDF expression is closely associated with reversible growth arrest in normal human diploid fibroblast-like (HDF) cells and is diminished with proliferative senescence in vitro. EPC-1 expression in HDF cells is induced under conditions of density-dependent contact inhibition and growth factor deprivation. Antiserum generated against EPC-1 recognizes a secreted protein of approximately 50 kDa from medium conditioned by early passage HDF cells, but not from senescent cells. The addition of EPC-1 antiserum to early population doubling level (PDL) cultures near the plateau phase of growth significantly increases the number of cells entering DNA synthesis. Affinity purified EPC-1 antibodies alone enhance the ability of near plateau-phase early PDL WI-38 cells to synthesize DNA by as much as threefold. Further, the addition of recombinant EPC-1 (rEPC-1) to logarithmically growing cells resulted in a marked decrease in the ability of these cells to enter DNA synthesis. We also demonstrate the loss of EPC-1 expression in WI-38 and IMR-90 HDF cell lines with both senescence and simian virus 40 (SV40) transformation. The loss of EPC-1 expression with SV40 transformation occurs at the level of steady-state mRNA and protein accumulation with genomic EPC-1 sequences grossly intact. Taken together, these results suggest that EPC-1 may play a role in the entry of early passage fibroblasts into a G(0) state or the maintenance of such a state once reached.

Antibodies↗

PubMed1977

Messenger RNA regulation in humam diploid fibroblasts.

In resting, non-growing human diploid fibroblasts the amount of rRNA is reduced 1.8-fold, cytoplasmic polysomes are disaggregated, and the level of poly-A RNA (mRNA) is reduced 1.8-fold in relation to growing cells. The distribution of poly-A RNA is altered in resting, non-growing cells so that an average of 64% of the total cytoplasmic poly-A RNA sediments along with particles lighter than 80S (prepolysomal) in sucrose density gradients. By camparison, in growing cells only 30% of the cytoplasmic poly-A RNA sediments in the prepolysomal region. In SDS sucrose gradients, the sedimentation profile of the prepolysomal poly-A RNA from resting cells resembles that of polysomal poly-A RNA from those cells. In contrast, the average size of prepolysomal poly-A RNA from growing cells is much smaller than that of the polysomal poly-A RNA from those cells. These data are compatible with the possibility that resting cell prepolysomal poly-A is untranslated mRNA. Also consistent with this interpretation are experiments which demonstrate that one-quarter to one-third of the prepolysomal poly-A RNA of resting cells is recruited into polysomes in the presence of of cyoloheximide.

Cell Division↗

PubMed1977

Characterization of the cell cycle of cultured human diploid cells: effects of aging and hydrocortisone.

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G1. The duration of S remained constant, while a small delay in G 2 was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G1 and possibly the G2 periods.

Cell Division↗

PubMed1977

Proliferative response of human diploid fibroblasts to intermittent light exposure.

Three- to four-hour exposure to fluorescent light, one to three times weekly, reproducibly enhanced the proliferation rate of human diploid fibroblasts. This enhancement was observed in WI-38 and a line from whole embryo mince at late population doubling level (PDL) as well as in a line from adult skin at early PDL. Single or multiple exposures of short duration stimulated proliferation, whereas exposures of long duration were cytotoxic. This proliferative response is reversible, and is mediated through the culture medium, Dulbecco Vogt's supplemented with 10% fetal bovine serum. Apparently light produces some mitogenic substance(s) in the culture medium that accumulates in the cells and is toxic or growth-stimulatory depending on its concentration per cell. Another possibility is that light produces in the medium both cytotoxic and growth-stimulatory substances.

Cell Division↗

PubMed1978

Regulation of protein synthesis in human diploid fibroblasts: reduced initiation efficiency in resting cultures.

The level of poly A+ RNA in growing cultures of human diploid fibroblasts is 1.8-fold times greater than in resting cultures. The level of functional ribosomes in growing cultures is 2.8 times that in resting cultures. Since transit times are similar in both types of cells, it can be concluded that the rate of protein synthesis in growing cultures is 2.8 times that in resting cultures. a reduced efficiency of mRNA translation at the level of initiation in resting cultures is proposed as a probable explanation for the fact that the decrease in protein synthesis rates is greater than the decrease in mRNA levels. This hypothesis is supported by the observations that: (a) poly A+ RNA is associated with smaller polysomes in resting than in growing cells, and (b) cycloheximide treatment of resting cells results in recruitment of nonpolysomal poly A+ RNA into polysomes and a shift of polysomal poly A+ RNA into larger polysomes.

Cell Cycle↗

PubMed1979

Superoxide dismutase specific activities in cultured human diploid cells of various donor ages.

It has been postulated that superoxide dismutase (SOD) protects cells from free radical-induced damage. In these experiments SOD specific activity was measured as established human diploid cell lines from various donor ages progressed through their in vitro lifespan. Significant elevations in activity occurred during the in vitro lifespans of cells from fetal and newborn donors, but no change in activity was detected during the lifespan of cells from an adult donor. In addition, a direct relationship between enzyme activity and donor age was detected with the following relative activities: adult greater than newborn greater than fetal. The possible relationship between these findings and the free radical theory of aging is discussed.

Aging↗

PubMed1980

Senescence of cultured human diploid fibroblasts. Are mutations responsible?

Two predictions of the error/mutation hypothesis of cellular senescence (Orgel, '73) namely,a) exponential accumulation of somatic mutations during the replicative lifespan and b) shortening of culture lifespan upon treatment with mutagens have been examined experimentally in a strain of cultured human diploid fibroblasts. Our studies show that as cells traverse the replicative lifespan (from 10 to 75 mean population doublings (MPD); total lifespan congruent to 95 MPD), no rapid and exponential increase occurs in the accumulation of mutations measured by the frequencies of Thgr(thioguanine resistance) and Dipr (diphtheria toxin resistance) mutants. Furthermore, repeated cycles of treatment (from 1- to 14-times) of human fibroblasts with two mutagens, ethyl methane sulfonate (EMS) and N-methyl-N' nitro-nitrosoguanidine, which led to a marked increase in the mutation frequency for the Dipr marker (congruent to 100-fold), failed to shorten the lifespan of cultured fibroblasts. On the contrary, repeated mutagen treatment (12 times with EMS) prolonged the lifespan of one replicative culture (110 MPD versus 94--98 MPD). These results strongly indicate that mutations are unlikely to be the primary event in cellular senescence and suggest instead that senescence is probably controlled by one or more (specific) gene(s) whose expression can be modified by mutations.

Cell Line↗

PubMed1980

Formation of sulfhydryl groups in the culture medium by human diploid fibroblasts.

When human diploid fibroblasts IMR-90 are cultured in routinely used medium (Eagle's basal medium supplemented with 10% fetal calf serum), sulfhydryl compounds appear in the medium. The major component of these sulfhydryl compounds is cysteine, and it is shown that a part of medium cystine is converted into cysteine by the cells. It is also shown that the sulfhydryl groups of serum albumin, which are masked and barely detectable before the culture, are restored. Probably cysteine formed by the cells reacts with serum albumin to give rise to the protein sulfhydryl groups via sulfhydryl-disulfide exchange reactions. Total sulfhydryl concentrations in the medium are maintained in a considerable level throughout the culture, and a possible physiological function of these sulfhydryl groups is discussed.

Cell Line↗

PubMed1980

Synergistic effects of epidermal growth factor and thrombin on the growth stimulation of diploid Chinese hamster fibroblasts.

The serum supplement used in the culture of a variety of mammalian cells can be replaced by known growth factors. Diploid Chinese hamster fibroblasts (CHEF/18) will grow for several days in a medium (4F) supplemented with four growth factors: epidermal growth factor (EGF), insulin, fibroblast growth factor (FGF), and transferrin. The growth rate is only about 50% as fast as when fetal calf serum is added. This difference is eliminated by thrombin (10--100 ng/ml; 0.3--3 nM). The CHEF/18 cell line is unique in that no other cell line responds to thrombin in this concentration range. Thrombin acts synergistically with other growth factors to stimulate CHEF/18 cell growth. By itself, thrombin is only mitogenic at elevated concentrations. Thrombin can largely compensate for the absence of EGF and partly for the absence of insulin in serum-free media. Chemically and "spontaneously" transformed cell lines related to CHEF/18 have lost requirements for both EGF and thrombin, and have retained requirements for insulin and transferrin expressed by CHEF/18. No CHEF cells in this work required FGF. These results suggest that the mechanisms by which EGF and thrombin stimulate cells to grow are related.

Animals↗

PubMed1981

Effect of glucose on aspartate and glutamate synthesis by human diploid fibroblasts.

The quantity of aspartate, but not of glutamate, synthesized by human diploid fibroblasts (HDF) was inversely proportional to the glucose concentration. Forty hours after confluent cells were refed with media containing 1.5 mM or 55 microM glucose, the aspartate concentration was 8 microM in medium high in glucose and 28 microM in medium low in glucose; glutamate was 350 microM at both glucose levels. The label incorporation from [U-14C] glutamine (0.8 C/ml) into aspartate after 40 hours in 1.5 mM glucose medium was less then 1000 DPM/ml and into glutamate 540,000 DPM/ml. The respective label incorporation into aspartate in 55 microM glucose medium was 118,000 DPM/ml and into glutamate 450,000 DPM/ml. In media with 1.5 mM glucose, label incorporation into both aspartate and glutamate was observed from [6-14C] glucose, [3-14C] pyruvate, and [1-14C] pyruvate. Since [1-14C] pyruvate labeled aspartate in 1.5 mM glucose medium, whereas [U-14C] glutamine did not, the observations support a cystolic pathway of aspartate synthesis from glycolytic intermediates in the presence of glucose. However, when the glucose concentration is decreased, glutamine appears to be the primary carbon source for aspartate synthesis. Therefore, both the quantity and the pathway of aspartate synthesis in HDF are functions of the glucose concentration. It is postulated that increased accumulation of aspartate in the media of HDF at confluency may be explained by decreased availability of acetyl CoA or of NADH.

Aspartic Acid↗

PubMed1982

Effect of growth stage on histone H1 metabolism in human diploid fibroblasts.

Metabolism of histone H1 at different stages of cell growth was investigated in order to get a better understanding of the role of histone H1 in the cell growth of human diploid fibroblasts. Histone H1 content exhibited some fluctuation during the culture stage of cell growth. When cells entered confluent phase, the ratio of histone H1 to total histones decreased significantly. Histone H1 had a turnover half-life of 80 hours whereas nucleosomal histones did not significantly turn-over regardless of the growth stage. DNA synthesis was drastically diminished with increased cell density whereas histone synthesis was less sensitive to contact-inhibition. The gradual decline of histone H1 content with increased cell density suggests that its degradation is slightly superior to its residual synthesis. When the confluent cells were seeded at low density, cell proliferation resumed and histone H1 was synthesized and deposited to chromatin in a greater amount than nucleosomal histones, thus resulting in an abrupt increase of histone H1 content. The possible role of histone H1 metabolism in normal cells is discussed.

Cell Division↗

PubMed1982

Regulation of growth of human diploid fibroblasts by factors elaborated by activated lymphoid cells.

Fibroblasts growth synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80 degrees C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (alpha-LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.

Cell Cycle↗

PubMed1982

Transport of cystine and cysteine and cell growth in cultured human diploid fibroblasts: effect of glutamate and homocysteate.

Human diploid fibroblasts take up cystine in the culture medium and the cystine is immediately reduced to cysteine in the cells. It is found that cysteine thus formed is rapidly released from the cells into the medium and accumulates there. The system transporting cysteine is convincingly similar to the ASC system described by Christensen et al. (1967). Since cysteine in the medium is sensitive to autoxidation and readily changes back to cystine, the uptake of cystine seems crucial to the cells. Inhibitors of cystine uptake, such as glutamate and homocysteate, potently reduce the intracellular and extracellular levels of cysteine. These inhibitors modify the cell growth depending upon the cystine concentration is physiological. An excessive concentration of cystine is in itself inhibitory action is antagonized by glutamate or homocysteate.

Amino Acids↗

PubMed1983

Regulation of intracellular protein degradation in IMR-90 human diploid fibroblasts.

Human diploid fibroblasts (IMR-90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half-lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long-lived proteins without affecting degradation of short-lived proteins. Fetal calf serum reduces degradative rates of long-lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long-lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10(-5) M). Amino acid deprivation increases degradative rates of long-lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short-lived proteins. Although cells incubated at 27 degrees C have reduced rates of degradation for both short-lived and long-lived proteins compared to cells at 37 degrees C, lowered temperature reduces catabolism of long-lived proteins to a greater extent.

Amino Acids↗

PubMed1985

Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts.

When randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.

Animals↗