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PubMed1980

Ribonucleotide reduction in intact human diploid fibroblasts.

There have been very few studies on ribonucleotide reductase activity in human tissue. In this report we describe a rapid and convenient procedure for determining purine and pyrimidine ribonucleotide reduction in normal human diploid fibroblasts and use the method to examine some general properties of the activity in these cells. ADP and CDP reductase was characterized for its response to the positive effectors, ATP and dGTP, the negative effector dATP, and the reducing agent dithiothreitol. Apparent Km values for ADP and CDP were determined to be 0.1 mM and 0.04 mM respectively. THe antitumor agent hydroxyurea inhibited both purine and pyrimidine reductase in a noncompetitive fashion, giving Ki value of 0.40 mM and 0.41 mM for ADP and CDP respectively. These Ki estimates are about four to five times higher than those reported for some permanent cell lines. An examination of the cytotoxic effects of hydroxyurea indicated a close correlation between the concentration of drug which inhibited enzyme activity and decreased colony-forming ability. Clearly the ability to investigate ribonucleotide reduction in low numbers of normal human diploid cells will be useful for genetic and biochemical studies.

Adenosine Diphosphate↗

PubMed1980

Complement-dependent induction of DNA synthesis and proliferation of human diploid fibroblasts.

The effects of fresh human serum (FHS) and heat-inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1-10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of Cl, was shown by the inability of Clq-depleted FHS to support enhanced DNA synthesis whereas Clq-depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4 microgram/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled Cl complex to the fibroblast surface was required for FHS-mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement-dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20-fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody-independent role for complement in the activation of a subpopulation of human diploid fibroblasts.

Animals↗

PubMed1982

In vitro proliferation and lifespan of human diploid fibroblasts in serum-free BSA-containing medium.

We have developed two serum-free chemically defined media (RITC 78-6 and RITC 80-7) that support the growth in culture of human diploid fibroblasts to the same extent as Eagle's basal medium (BME) supplemented with 10% fetal bovine serum (FBS). These two media contain modified Eagle's minimum essential medium (MEM) supplemented with nonessential amino acids, various trace metals, organic compounds and growth factors [insulin, mouse epidermal growth factor (m-EGF), transferrin and triiodothyronine (T3)]. RITC 80-7 medium differs from RITC 78-6 in that it contains thymidine, hypoxanthine, and vitamin B12 and supports the long-term serial cultivation of human diploid cultures. The addition of commercial bovine serum albumin (BSA, 5 g/liter) to the medium enhances cell growth. This effect is not observed if BSA is first delipidized, but reconstitution of BSA with certain lipids restores its ability to promote growth. BSA has an inhibitory effect on cellular attachment but this is overcome when fibronectin (FN, 10 mg/liter is added to the medium.

Cell Adhesion↗

PubMed1984

Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S phase).

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1-10 microM), followed by irradiation with near-UV (5-10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival.

Bromodeoxyuridine↗

PubMed1986

Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene.

The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.

Cell Count↗

PubMed1991

Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts.

We examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA-binding and hsp 70 gene promoter activities during aging of the IMR-90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15-48 were heat shocked at temperatures of 39, 42, and 45 degrees C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE-binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE-binding activity. A substantial and rapid (within 30 min) increase in HSE-binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45 degrees C. However, PDL 35 cells did not respond to 39 degrees C, and PDL 48 cells responded slowly to heat shock at 45 degrees C, but not 39 or 42 degrees C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age-dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype.

Base Sequence↗

PubMed1994

Global change of gene expression at late G1/S boundary may occur in human IMR-90 diploid fibroblasts during senescence.

The hallmark of cellular aging is the failure of senescent diploid cells to enter or to complete the S phase of the cell cycle. The cause for such failure may hold the key for our understanding of the molecular basis of cellular aging. We have previously shown that aging of IMR-90 human diploid fibroblasts in culture is accompanied by a five to sevenfold decrease in both thymidine kinase activity and thymidine kinase mRNA level (Chang and Chen, 1988, J. Biol. Chem., 263:11431-11435). To examine whether attenuation of gene expression at G1/S boundary is unique for thymidine kinase or it may involve most, if not all, of other G1/S genes, we compared the expressions of two classes of G1/S genes in young and in old IMR-90 cells following serum stimulation. We found that the expression of all these genes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), ribonucleotide reductase (PNR), proliferating cell nuclear antigen (PCNA), histone H1, histone H2A + 2B, histone H3, and histone H4, was induced to high levels in young IMR-90 cells but not in old IMR-90 cells. The mRNA levels of all G1/S genes in young cells were more than tenfold higher than that in old cells 12 hr after serum stimulation. The enzymes encoded by TS and DHFR genes and dUTPase also exhibited similar age-dependent attenuation in activities. In contrast, expression of growth-related genes such as eIF-5A, c-Ha-ras, and beta-actin did not show significant differences between young and old cells after serum stimulation. Computer analysis of the promoter region of these G1/S genes revealed an Sp-1 binding site as the most common cis-element. Taken together, our results suggest that the suppression of G1/S gene expressions during senescence may be a global phenomenon and that G1/S genes may be coordinately controlled.

Actins↗

PubMed1986

The histochemical identification of primordial germ cells in diploid parthenogenetic mouse embryos.

An attempt has been made to improve the early post-implantation development potential of diploid parthenogenetic mouse embryos by transferring parthenogenetic blastocysts to one uterine horn of a pseudopregnant recipient and a similar number of fertilized embryos to the contralateral horn. In control studies, diploid parthenogenetic embryos were transferred to both uterine horns of appropriate recipients. Unfortunately no obvious advantage appeared to be gained by carrying out the former manoeuvre. A significant improvement in the development potential of the parthenogenones could have indicated that their poor post-implantation survival might have been associated with a deficiency, possibly of hormonal origin, in the functioning of their decidual reaction. However, sufficient somite-containing parthenogenetic embryos were obtained in this study to allow a comparison to be made between them and fertilized embryos that were morphologically at a comparable stage of development. The parthenogenones were found to have a markedly smaller crown-rump length than their fertilized counterparts. A high proportion of both the parthenogenetic and fertilized embryos were subsequently fixed and appropriately stained in order to localize alkaline phosphatase activity. The analysis of this material clearly demonstrated that parthenogenetic mouse embryos are in fact capable of producing primordial germ cells. The latter were recognized by their morphology, histochemical staining appearance, and characteristic location, being found in the early 'turned' embryos within the dorsal mesentery in close proximity to the developing gut tube, and in the more advanced limb-bud stage embryos within the gonadal ridges.

Animals↗

PubMed1991

Cleavage rates of diploid and tetraploid mouse embryos during the preimplantation period.

Despite the fact that spontaneous tetraploidy is a rare phenomenon in mice, such embryos may be produced experimentally by a variety of means, though only a very limited degree of postimplantation development has been achieved. Despite this apparent limitation, much data on the rate of development of preimplantation tetraploid embryos has been published. However, the findings from these studies has often been conflicting. In the light of the recent successful achievement of advanced postimplantation tetraploid development in our laboratory, we decided it was an opportune time to re-evaluate the preimplantation development of these embryos in as near to optimal conditions as we could achieve. Three groups were studied, namely 1) control (diploid) embryos developing in vivo, 2) control (diploid) embryos that had been isolated at the 2-cell stage, briefly retained in culture, then transferred to the oviducts of pseudopregnant recipients, and 3) tetraploid embryos produced by electrofusion of blastomeres at the 2-cell stage, then transferred to the oviducts of pseudopregnant recipients. Embryos were isolated from females from each group at specific times after the HCG injection to induce ovulation. The total cell number of each embryo was established and the log mean values were plotted against time. From the gradients of the lines it was possible to establish that there was a significant difference between the cell doubling time of the transferred controls (group 2) compared to the in vivo controls (group 1) with cell doubling times of 15.86 +/- 1.45 h and 10.27 +/- 0.24 h, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Relationship between cell size and nuclear volume in nucleated red blood cells of developmentally matched diploid and tetraploid mouse embryos.

Analysis of control diploid and polyploid amphibia indicated that cell and nuclear volumes were closely related to their ploidy, so that an increase in ploidy was generally associated with an increase in cell size. This relationship is also believed to occur in mammalian polyploids. However, since the latter are only rarely encountered spontaneously, or only occasionally following experimental manipulation, no detailed morphometric studies have been carried out to date to confirm whether such a relationship exists. In this study, the cellular and nuclear volume of primitive red blood cells was analyzed in carefully developmentally matched control diploid mouse embryos and tetraploid embryos produced by the technique of electrofusion. All of the cells and/or nuclei studied had a characteristic spherical shape which greatly simplified the morphometric analysis. A defined and predictable relationship between ploidy and cellular and/or nuclear volume was observed in the red blood cells between 8.25 and 14.5 days of gestation. During this period the primitive red blood cells are gradually replaced by the definitive erythrocytes. The ratio of control values to tetraploid values was found to be close to the theoretical value of 1:2 when comparable cells and/or their nuclei were analyzed in carefully developmentally matched material.

Animals↗

PubMed1989

Cotransfection of plasmids with ras and myc oncogenes to diploid cells derived from rodent fetuses: alteration of neoplastic transformation frequency depending on the gestation period.

To analyze the mechanism of neoplastic transformation of rodent diploid cells by ras and myc oncogenes, human EJ c-Ha-ras and mouse c-myc second and third exons promoted by SV40 promoter were connected to pSV2neo and pSV2gpt, respectively. Mouse and rat primary fetal cells cotransfected with both genes formed transformed and nontransformed colonies in a medium containing G418 and mycophenolic acid (MPA). The proportion of transformed colonies in the total G418/MPA-resistant colonies decreased dependent on the stage of the gestation period of rat fetuses from which primary cells had been obtained. Analysis of randomly isolated colonies showed that the transformed colonies had a high copy number and high amount of expression of the introduced genes, were anchorage independent, and were tumorigenic in nude mice. On the other hand, the nontransformed colonies had a low copy number, low amount of expression, and no tumorigenicity. This contrast indicated not only that the activated Ha-ras and myc oncogenes had been integrated, but also that the amplification or overexpression (or both) of these genes was required for the rodent diploid cells to be transformed. We conclude that early-stage rat fetal cells might have endogenous factors that promote cell transformation. Alternatively, late-stage cells might have factors that suppress cell transformation by activated Ha-ras and myc oncogenes.

Animals↗

PubMed1994

Prohibitin expression during cellular senescence of human diploid fibroblasts.

Prohibitin is an evolutionarily conserved gene postulated to possess tumor suppressor activity and to contribute to the limited lifespan of human diploid fibroblast-like cells. Prohibitin mRNA and protein expression and its ability to become post-translationally modified were determined in human diploid fibroblast-like cells of different in vitro ages. The expression of prohibitin mRNA and protein changes little with increasing in vitro age; however, its protein product is post-synthetically modified in younger but not older cells. These results suggest that prohibitin is similar to the retinoblastoma gene product whose anti-proliferative activity remains active in older cells because it is not post-synthetically modified.

Cell Cycle↗

PubMed1994

ACPR, a STE12 homologue from Candida albicans, is a strong inducer of pseudohyphae in Saccharomyces cerevisiae haploids and diploids.

ACPR from Candida albicans encodes a protein antigenically related to the secretory acid proteinase of this yeast. Its amino terminal domain is highly similar to the amino terminal, DNA-binding domain of STE12 of Saccharomyces cerevisiae. STE12 is involved in mating of haploids and in pseudohyphae formation in diploids. ACPR, or its DNA-binding domain swapped into STE12, can support pseudohyphae formation in S. cerevisiae diploids. However, unlike STE12, these constructs affect the budding pattern and induce pseudohyphae formation in S. cerevisiae haploids as well, and this induction is independent of the nitrogen status of the medium. ACPR appears to be a stronger inducer of pseudohyphae than STE12 and is likely to be involved in the formation of pseudohyphae and hyphae in C. albicans.

Aspartic Acid Endopeptidases↗

PubMed2001

Analysis of p53-dependent mechanisms in the maintenance of genetic stability in diploid tumourigenic line SK-UT-1B of human uterine leiomyosarcoma.

The cells of tumourigenic line SK-UT-1B combine features characteristic both of normal (diploid karyotype, a low level of polyploid cells, absence of chromosomal marker) and tumour cells (high level of chromosomal instability, high malignancy). We suggest that maintenance of diploid karyotype in this line is controlled via the p53/p21 pathway. We demonstrate that the amount of p53 increases following gamma-irradiation and accumulated p53 protein seems to be functional as p53-luc and p21/Waf-luc reporter plasmids were found to be activated. However, gamma-irradiation-induced increase of p53 was not accompanied by increase of p21/Waf on the protein level. Apparently this is one of the reasons for G1/S and G2/M checkpoint control disruption. The absence of these checkpoints could not prevent the proliferation of cells with intrachromosomal rearrangements. The only effective checkpoint in SK-UT-1B is the p53-dependent M checkpoint, which directed the cells with changed chromosome numbers to apoptosis and therefore strictly guarded the diploidy of the cell population. This indicates that p53 can control the preservation of genetic stability at different levels via different pathways.

Apoptosis↗

PubMed1995

Error propagation in reproduction of diploid organisms. A case study on single peaked landscapes.

Two versions of the diploid selection mutation equation as adapted to sequence space are studied. Focussing on diploid generalizations of the well-established single peaked landscape, quantitative effects of dominance on error thresholds in infinite populations are found, as well as unexpected qualitative features like multiple equilibria. Analogues of these phenomena are also recovered in stochastic versions for finite populations.

Animals↗

PubMed1999

Intramitotic and intraclonal variation in proliferative potential of human diploid cells: explained by telomere shortening.

Normal human diploid cells can only divide for a limited number of times (known as the Hayflick limit). They manifest two unique features during in vitro senescence. The division capability of individual cells in a clone, though all derived from a same ancestor, is heterogeneous with a distinct bimodal distribution. Two sister cells derived from a same parent cell can have a large difference in their doubling potentials. These two unique features have not been properly explained by any known physiological process since their observation in 1980. Here I represent a telomere-shortening model based on recent experimental measurement of telomere deletion in human cells. Using computer simulation, I show that the model satisfactorily explains the intraclonal and intramitotic variation in division capability of human diploid cells. Moreover, the simulations predict that human cells may only monitor the shortening of a few, most likely two, telomeres to regulate their proliferative potential.

Clone Cells↗

PubMed1998

Adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine.

A virulent bovine coronavirus isolate (newborn calf diarrheal) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). The same cells have been used to produce modified live porcine rotavirus and coronavirus vaccines that are federally licensed and sold worldwide. Growth of the bovine coronavirus resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Virus yields were relatively high in the ST cells and active replication was confirmed by immune electron microscopy and immunofluorescence. Adaptation of bovine coronavirus to a diploid swine cell line has not been previously reported. Different cell culture passage levels of bovine coronavirus were evaluated by oral inoculation of clean-catch, colostrum-deprived calves. A passage level of bovine coronavirus was identified that multiplied in the calf without the clinical signs of disease associated with virulent passages. The modified live bovine coronavirus vaccine remained safe and efficacious even after 5-backpassages in calves. Further efficacy studies have shown that the modified live bovine coronavirus vaccine significantly protected calves from highly virulent challenges with either winter dysentery or newborn calf diarrheal coronavirus isolates.

Adaptation, Biological↗

PubMed1975

Extensive segments of the Escherichia coli K12 chromosome in Proteus mirabilis diploids.

Various Escherichia coli K12 Hfr donors transfer at low frequency portions of the E. coli genome to Proteus mirabilis. By remating such Proteus hybrids with the same or a different E. coli Hfr strain, other genetic characters could be added to yield diploid Proteus hybrids which contained more than 30 percent of the E. coli genome. The extent of the E. coli genetic material in these unstable Proteus diploid hybrids included segments with the following selected markers: gal, lac, ara, mel, mtl, and malA. Unselected markers known to map throughout this region of the chromosome were also detected in these hybrids. Among the markers expressed in Proteus hybrids with the E. coli malA region was the receptor site for coliphage lambda. Although plaques were not seen, lambda was adsorbed by the Proteus hybrids. Examination of DNA from the various Proteus hybrids by CsCl density gradient centrifugation showed a satellite component of E. coli DNA with a size that corresponded to the extent of the E. coli genome present as determined by genetic analysis.

Centrifugation, Density Gradient↗