Diploid Cell Strains Research Articles

Radiorésistance en relation avec l'activation d'oncogène

Despite considerable research over the past three decades, the factors that control the response of human tumors to radiation remain poorly understood. Normal human cell strains studied in vitro show a rather narrow range in radiosensitiv ity (1), whereas human tumor cells vary considerably in their sensitivity to the cytotoxic effects of radiation (2,3). Cells from some types of tumors such as soft tissue sarcomas are quite radiosensitive in vitro while a number of cell lines established from various human tumors including squamous cell carcinomas are unusually radioresistant, well beyond the range observed in normal cells (3). The reasons for this variation are not known. Mutations in a number of different oncogenes and tumor suppressor genes have been associated with different types of human cancer. Two observations have suggested a possible role for such genes in the sensitivity of cells to killing by ionizing radiation. First, certain human diploid fibroblast cell strains transformed by the SV-40 virus were found to be less radiosensitive than their non-transformed counterparts. Second, evidence has been presented that transfection of rodent cells with an activated ras oncogene may render these cells more resistant to killing by radiation (4,5). We have studied these two phenomena systematically in normal human diploid fibroblasts, as well as in two human tumor cell lines. Three human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or the SV-40 T-antigen (6). Multiple clones were isolated from each strain and examined for their sensitivity to x-irradiation in vitro, as well as for morphological alterations, immortalization and tumorigenicity in nude mice. Cells transfected with the activated ras oncogene alone showed no morphological alterations nor significant changes in radiosensitiv ity. Clones expressing SV-40 T-antigen, on the other hand, were significantly radioresistant as compared with their parental cells or clones tranfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV-40 T-antigen but not activated Ha-ras plays a significant role in the radiosensitivity of human diploid cells. The radioresistant phenotype was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalizati on itself.

Stimulation of human embryonic lung fibroblasts by TGF-beta and PDGF acting in synergism. The role of cell density.

The concerted action of TGF-beta and PDGF on a diploid human embryonic lung fibroblast cell strain (Flow 2002) grown in an homologous environment is investigated here. In sparse cultures, TGF-beta stimulates DNA synthesis over a broad concentration range (0.1-10 ng/ml). Furthermore, it acts in synergism with PDGF, a phenomenon which persists also during in vitro aging of the cells. Preincubation of TGF-beta with the fibroblasts up to 12 hours reduces the subsequent PDGF binding to the cells, while prolonged preincubation restores PDGF binding to control levels. Finally, in cultures of higher cell densities, TGF-beta ceases to stimulate DNA synthesis, whereas PDGF continues even at cell confluency, retains its stimulatory activity suggesting different roles for the two growth factors during the wound healing process.

Prolonged Cell Cycle Delay in Radioresistant Human Cell Lines Transfected with Activated ras Oncogene and/or Simian Virus 40 T-Antigen

Normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) and/or SV40 T-antigen. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant (D0 = 1.68-2.73 Gy) compared with their parent cells or clones transfected with the neo gene only (D0 = 1.20-1.35 Gy). This radioresistant phenotype persisted in postcrisis, immortalized cell lines. Cell cycle perturbations after X irradiations were studied in four immortalized, radioresistant cell lines transfected with EJ-ras plus SV40 T or SV40 T alone as well as in two nontransfected parental cell strains. Exponentially growing cells were exposed to various doses of X radiation and the distributions within the cell cycle were determined by flow cytometry. The time of onset and duration of division delay were also measured by cell counting. All cells underwent a dose-dependent G2 arrest; the duration of this division delay was proportional to radiation dose. The radioresistant cell lines had a longer arrest in G2 phase of the cell cycle compared to that of the parental cell strains. These data suggest that a prolonged cell cycle delay may be one of the factors involved in the radioresistance acquired by transfection of human diploid cells with SV40 T-antigen.

Differential Modulation of bFGF Receptors by TGF-β in Adult Skin, Scleroderma Skin, and Newborn Foreskin Fibroblasts

Effects of transforming growth factor beta (TGF-beta) on proliferative responses to basic fibroblast growth factor (bFGF) were studied in human diploid fibroblasts cell strains derived from three different sources: adult skin, scleroderma, and newborn foreskin. All three types of cell strains were similarly responsive to TGF-beta, whereas adult skin fibroblasts were significantly more responsive to bFGF. Incubation of cells with TGF-beta prior to bFGF addition substantially increased responsiveness of adult skin fibroblasts to this latter cytokine, slightly increased that of scleroderma fibroblasts, and decreased that of foreskin fibroblasts. Modulation of bFGF receptors by TGF-beta correlated positively with these mitogenic effects. Adult skin fibroblasts showed increases of both high- and low-affinity receptors and scleroderma fibroblasts showed small increases of high-affinity receptors only, whereas foreskin fibroblasts showed no changes. Heparitinase treatment of adult skin fibroblasts during TGF-beta pre-incubation resulted in reduced bFGF binding to low-affinity receptors and reduced mitogenic response to bFGF, suggesting that the TGF-beta-stimulated increase of low-affinity receptors in these cells contributes to the observed enhanced mitogenic effects of bFGF. Abnormal responses of scleroderma fibroblasts to TGF-beta/bFGF stimulation, particularly failure to synthesize low-affinity receptors in response to TGF-beta, adds a new characteristic to the fibrotic phenotype of scleroderma fibroblasts.

Quiescence and proliferative response of normal human embryonic fibroblasts in homologous environment: Effect of aging

A cell culture system for the study of human serum growth factors is described. It is based on a diploid human embryonic lung fibroblast cell strain (Flow 2002) with a finite life span (60±3 CPDS). When maintained in homologous (human) plasma-derived serum (PDS) at concentrations as low as 0.05%, the cells attain quiescence. Under these conditions, they remain viable for at least 14 days and they readily respond when stimulated to proliferate by human serum or human PDS, as well as by growth factors, such as PDGF, EGF, FGF. During in vitro aging, the cells retain their responsiveness to these growth stimuli, although the net proliferative effect decreases as they approach senescence.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Permanent hexose transport upregulation in a respiration-deficient human fibroblast cell strain

The regulation of hexose transport was studied in a human diploid fibroblast respiration-deficient cell strain (WG750). Transport of 2-deoxy-D-glucose (2-DG) was greater than sixfold higher compared with an in vivo age-matched normal cell strain (MCH55). In addition, 3-O-methylglucose transport and 14CO2 production were elevated in the mutant cell strain. Kinetic analysis revealed that the increased sugar transport in mutant cells was due to an average 5.7-fold increase in the 2-DG maximal transport rate, with no observed differences in the transport Michaelis constant for both normal and mutant cells. Also, the inhibitor constants for D-glucose inhibition of 2-DG transport were nearly identical for both cell types. Glucose deprivation led to a similar time-dependent increase in hexose transport in both cell strains. Serum refeeding of glucose-fed serum-deprived cultures led to a progressive increase in 2-DG transport in normal cells, whereas mutant cells displayed a time-delayed increase in 2-DG transport. Exposure to 67 and 670 nM insulin stimulated 2-DG transport on average 1.99 +/- 0.25- and 2.33 +/- 0.26-fold, respectively, over basal transport in the normal cells, whereas the mutant cells were significantly less sensitive to the stimulatory effects of the hormone. Insulin binding and amino acid transport (i.e., alpha-aminoisobutyric acid uptake) in the normal and mutant cells were not different. Data obtained using Western blot analysis showed that WG750 (mutant) cells expressed an increase (approximately 4-fold) in total cellular HepG2 (erythroid-brain) transporter protein compared with normal cells, thus reflecting the changes seen in hexose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of an infinite life span diploid human fibroblast cell strain and a near-diploid strain arising from a clone of cells expressing a transfectedv- myc oncogene

Diploid human fibroblasts were transfected with a plasmid carrying av- myconcogene linked to the neo gene or with a vector control carrying a neo gene. Drug-resistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed thev- myc oncogene. After several months, these cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This cell strain was designated MSU-1.1. Similar analysis showed that cells from an earlier passage were diploid. These cells were designated MSU-1.0. Both strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both strains express thev- mycprotein and have the same integration site for the transfectedv- mycand neo genes. The MSU-1.0 cells cannot grow without exogenously added growth factors. The MSU-1.1 cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 cells. Since the chance of human cells acquiring an infinite life span in culture is very rare, the data suggest that MSU-1.1 cells are derived from MSU-1.0 cells. The expression ofv- myc is probably required for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression ofv- myc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 cells expressed this oncogene, but the vast majority of them senesced.

Advantages of multiple cell culture systems for detection of mixed-virus infections

Simultaneous infections by two or more viruses occur frequently, especially in immunosuppressed patients. In order to detect more than one viral agent in a single specimen, multiple cell systems have been employed in our laboratory. Specimens are routinely inoculated into four different cell cultures, namely: MRC-5, a human diploid lung fibroblast cell strain; A549, a human continuous cell line; primary guinea pig embryo (GPE) cell culture, and primary rhesus monkey kidney (RhMK) cell culture. For rapid detection of cytomegalovirus (CMV) antigen, MRC-5 cells grown in shell vials containing coverslips are also inoculated with the same specimens followed by centrifugation. During 1989, nine cases of multiple-virus isolations were obtained in this laboratory. In all nine patients, CMV was detected in MRC-5 cells. Five of the nine cases were co-infected with HSV-1, three were co-infected with adenovirus, and one was co-infected with both HSV-1 and adenovirus. All four adenovirus isolates were obtained in A549 cells. Of the six HSV-1 isolates, one was detected in all three cell cultures, e.g. MRC-5, A549 and GPE; one was detected in both MRC-5 and A549 cells, and four were isolated in a single-cell type only. For nine CMV-positive cases, five were obtained by both conventional and centrifugation cultures, two each were detected by centrifugation or conventional culture only. Thus for a maximum detection of viruses present in a single specimen, it is suggested that multiple-cell-culture systems, together with more than one technique, should be employed.

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vacine production and reduce costs.

Changes in the cell surface of human diploid fibroblasts during cellular aging

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.

A new human male diploid cell strain, TIG-7: its age-related changes and comparison with a matched female TIG-1 cell strain

A new human diploid cell strain, TIG-7, which has the male karyotype, was established and characterized. Isozyme and histocompatibility typing of the cell strain was performed. The average in vitro life span of the cells is 73 population doublings. Changes in cell volume, doubling time, saturation density, the efficiency of cell attachment, plating efficiency, and relative DNA content were examined during in vitro cellular aging. Hydrocortisone slightly prolongs the life span of the cell strain when the hormone is administered to the cultures during middle passages. The age-related changes in the parameters of TIG-7 are not appreciably different from those of the previously established TIG-1 cell strain. These results show that this cell strain is useful for research on cellular aging; further profit is anticipated from research using a combination of these two sexually different cell strains.

Isozyme polymorphisms in human diploid cell strains for research on cellular aging

Six human lung diploid cell strains established for the study of in vitro cellular aging (TIG-1, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, and HeLa cells as a control) were studied by cellulose acetate membrane electrophoresis for allozymic differences at 18 enzyme loci. Eight enzyme loci (G6PD, PGM1, PGM3, PepA, PGD, ADA, GLO1, and ME), proved to be informative in establishing unique allozyme genetic signatures for all of the cell strains established from the same species and from the same organ. Changes in the allozyme genetic signatures were not observed throughout the life span of TIG-1 and MRC-9 cells. The allozyme genetic signatures can be used as a quick monitor of cell identification and intraspecific cell contamination.

Sensitivity of Human Diploid Fibroblast Cell Strains from Various Genetic Disorders to Acute and Protracted Radiation Exposure

The cytotoxic effect of acute X irradiation was studied by a colony formation assay in 114 human skin fibroblast cell strains from 31 apparently normal individuals and 83 patients with a variety of genetic disorders possibly associated with in vitro hypersensitivity to ionizing radiation. The effect of protracted exposure to beta radiation from tritiated water (HTO) was examined in parallel experiments in 65 of these strains. The disorders included neurological diseases and syndromes characterized by an increased susceptibility to spontaneous and radiation-induced cancer. Homozygous ataxia telangiectasia and Nijmegen break syndrome cells were highly sensitive to both types of radiation. However, the response of cells from the other genetic disorders fell within the broad range characteristic of normal cell strains. While HTO may be useful as a quantitative method for determining the cytotoxic response of human diploid cells to ionizing radiation, the present results indicate that it does not offer a more sensitive assay than acute X irradiation for detecting minor degrees of hypersensitivity.

Post Exposure Studies with Human Diploid Cell Rabies Vaccine and Purified Chick Embryo Cell Vaccine: Comparative Serological Responses in Man

Rabies-neutralising antibody responses to human diploid cell strain rabies vaccine (HDCSV) and purified chick embryo cell vaccine (PCECV) were studied in 125 patients previously exposed to rabid animals having received 3, 5 and 6 doses on days 0, 3, 7, 14, 30 and 90. Antibody response was significantly higher (P < 0.05) with HDCSV than PCECV in all subjects irrespective of their sex and age group. Three doses on day 0, 3, 7 given for post-exposure rabies prophylaxis to class I patients with a negligible risk elicited antibody titres significantly higher than the minimum protective level required (0.5 I.U./ml); the mean response was>15 I.U./ml in the case of PCECV and>32 I.U./ml in the case of HDCSV. The use of PCECV is cost-effective and suggested for use in developing countries.

Homologous Plasmid Recombination Is Elevated in Immortally Transformed Cells

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.

A rapid micro method for counting cells "in situ" using a fluorogenic alkaline phosphatase enzyme assay.

A new method has been developed to count cells "in situ", based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10(4) to 5 X 10(5) for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays.

Pre-exposure studies with purified chick embryo cell culture rabies vaccine and human diploid cell vaccine: serological and clinical responses in man

Clinical reactions and neutralizing antibody responses to six pre-exposure regimens of purified chick embryo cell culture rabies vaccine (PCECV) and human diploid cell strain rabies vaccine (HDCSV) were studied in 177 volunteers. Antibody kinetics, height of the response and persistence of antibody over two years were virtually identical after PCECV and HDCSV. An antibody response was detected in all subjects on day 14 when the highest titres were found after two intramuscular (i.m.) 1.0 ml doses of a schedule of immunization on days 0, 7 and 21. In comparison, a schedule of immunization on days 0, 28 and 56 ultimately evoked the highest titres 21 days after the final injection, but antibody persisted equally well over two years with either schedule. Neutralizing antibody titres were lower after intradermal (i.d.) vaccination with 0.1 ml compared to 1.0 ml i.m. on days 0, 7 and 21, but when given on days 0, 28 and 56 the responses were comparable. Three subjects with a personal or family history of atopy developed urticarial lesions after PCECV. Both vaccines were otherwise well tolerated.