Biomedical subjects

J W Simons

Publications and source records attributed to J W Simons.

At least 109 records · Page 6Linked to original sources

PubMed1988

Phenotypic heterogeneity within the first complementation group of UV-sensitive mutants of Chinese hamster cell lines.

A DNA-repair mutant was characterized that has the extraordinary and interesting properties of extreme sensitivity to UV killing combined with a high level of nucleotide excision repair. The mutant V-H1 isolated from the V79 Chinese hamster cell line appeared very stable, with a reversion frequency of about 3.5 X 10(-7). Genetic complementation analysis indicates that V-H1 belongs to the first complementation group of UV-sensitive Chinese hamster ovary (CHO) mutants described by Thompson et al. (1981). This corresponds with data on cross-sensitivity and mutation induction after UV irradiation published by this group. Surprisingly, the mutant V-H1 shows only slightly reduced (to approximately 70%) unscheduled DNA synthesis (UDS) after UV exposure, while the other two mutants of this complementation group are deficient in UDS after UV. In agreement with the high residual UDS, in V-H1 also the amount of repair replication in response to UV treatment is relatively high (approximately 50%). It has also been shown that the incision step of the nucleotide excision pathway takes place in V-H1 (with a lower rate than observed in wild-type cells), whereas another mutant (UV5) of the same complementation group is deficient in incision. This heterogeneity within the first complementation group indicates that the repair gene of this complementation group may have more than one functionally important domain or that the gene is not involved in the incision per se but is involved in e.g. preferential repair of active genes.

Animals↗

PubMed1988

Identification of a new seventh complementation group of UV-sensitive mutants in Chinese hamster cells.

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.

Animals↗

PubMed1988

Characterization of an X-ray-hypersensitive mutant of V79 Chinese hamster cells.

A V79 Chinese hamster cell line XR-V15B exhibiting hypersensitivity to X-ray has been isolated and characterized. Additionally to increased X-ray-sensitivity (approximately 8-fold, as judged by D10 values), cross-sensitivity to bleomycin (3-fold increase), 4NQO (3-fold), H2O2, EMS, MMS (2-fold) were observed also. No increased sensitivity to UV and MMC was found. Genetic complementation analysis indicates that XR-V15B belongs to the same complementation group as the X-ray-sensitive (xrs) mutants of Chinese hamster ovary (CHO) cells described by Jeggo (1985). Biochemical analysis of XR-V15B confirms this finding: the mutant showed a decreased ability to rejoin double-strand breaks induced by X-ray as measured by neutral elution. After 4 h of repair more than 50% of the double-strand breaks remain in comparison to 3% in V79 cells. No difference was observed between wild-type and XR-V15B cells in the initial number of single-strand breaks induced, in the kinetics of their rejoining and in the final level of unrejoined single-strand breaks. Treatment with 5-azacytidine did not have an effect on the reversion frequency of XR-V15B, contrary to the results obtained with the xrs mutants. XR-V15B has been grown in continuous culture for more than 3 months without evidence of reversion. The mutation induction by X-ray irradiation at the HPRT locus is not significantly increased in the mutant, but at doses giving the same degree of cell killing, XR-V15B cells are hypomutable.

Animals↗

PubMed1988

Epidermal growth factor enhances N-ethyl-N-nitrosourea-induced morphological transformation of Syrian hamster embryo cells.

To improve the usefulness of the Syrian hamster embryo (SHE) cell transformation system as a short-term test, it was investigated whether the variation in results due to serum variability could be reduced by the addition of epidermal growth factor (EGF). It was found that EGF-significantly (3-7-fold) enhanced the frequency of morphological transformation induced by N-ethyl-N-nitrosourea or benzo[a]pyrene if added to growth medium supplemented with a batch of serum which had a low ability to support transformation. Furthermore, addition of EGF to the assay medium enabled the demonstration of dose dependence of transformation with relatively small group sizes (up to 2000 colonies). Finally, it was observed that the transformed phenotype was easier to recognize in the presence of EGF. These data suggest that routine addition of EGF to the assay medium might reduce variability and enhance sensitivity of the SHE transformation assay.

Animals↗

PubMed1987

Mutagen-sensitive cell lines are obtained with a high frequency in V79 Chinese hamster cells.

A replica-plating technique has been adopted for the isolation of mutagen-sensitive mutants of Chinese hamster V79 and CHO cell lines. After the mutagenic treatment (ENU) clones derived from these cell lines were replica plated into micro wells and replicas were treated with UV (254 nm), X-ray, MMC, EMC or MMS. Clonal cell lines which demonstrated mutagen sensitivity were retested by the determination of survival. Only one UV-sensitive line was obtained in 1500 clonal lines derived from CHO cells. This mutant appeared also sensitive to 4NQO and MMC. The sensitivity to UV and MMC was 2-3-fold enhanced, while the increase in sensitivity to 4NQO was 4-5-fold. In V79 cells 9 mutagen-sensitive lines were found after screening of 500 clonal lines; six of them showed increased sensitivity towards UV, two towards MMC, and one cell line was found to be X-ray sensitive. A considerable cross-sensitivity for the various agents was found among the isolated mutants. When a 2-fold increase is taken as a minimum to indicate mutagen sensitivity 6 mutants were sensitive to UV, 8 mutants were sensitive to MMC, 6 mutants were sensitive to 4NQO and 4 mutants were sensitive to X-rays. The difference in sensitivity to UV versus 4NQO makes it unlikely that 4NQO can be considered as a UV-mimetic agent. The sensitivity to MMC appears to fall into 2 classes: a class with moderate sensitivity (2-8-fold) and a class with high sensitivity (30-100-fold). The presence of similar classes is indicated for UV. Except for the two lines V-E5, V-B7 and the two lines V-H11, V-H4 all obtained mutants have a different spectrum of mutagen sensitivities which suggests that different genetic alterations underly these effects. The observed high frequency of mutagen-sensitive mutants in V79 cells, although unexpected and substantially higher than those published for CHO cells and L5178Y cells, can still be explained by the presence of functionally hemizygous loci.

Animals↗

PubMed1987

Biological and biochemical consequences of the human ERCC-1 repair gene after transfection into a repair-deficient CHO cell line.

The consequences of the presence of the human gene ERCC1 in repair-deficient 43-3B cells were examined. The gene restores the sensitivity of this mutant not only to UV but also to 4NQO, N-Ac-AAF and alkylating agents to the normal level. Also, the frequency of mutation induction by UV at the Na+/K+-ATPase locus returns to the level of CHO wild-type cells. Additionally, the rate of cyclobutane pyrimidine dimer removal approaches that in wild-type CHO cells. The results obtained indicate that the human gene ERCC-1 restores the impaired functions in 43-3B, and that the gene is probably functionally homologous to the defective one in the 43-3B cell line. Some evidence was found for a difference between the human gene product and its rodent counterpart, as the restoration of normal sensitivity to 4NQO, ENU and N-Ac-AAF was complete whereas it was not for UV.

4-Nitroquinoline-1-oxide↗

PubMed1986

Analysis of repair processes by the determination of the induction of cell killing and mutations in two repair-deficient Chinese hamster ovary cell lines.

Two UV sensitive DNA-repair-deficient mutants of Chinese hamster ovary cells (43-3B and 27-1) have been characterized. The sensitivity of these mutants to a broad spectrum of DNA-damaging agents: UV254nm, 4-nitroquinoline-1-oxide (4NQO), X-rays, bleomycin, ethylnitrosourea (ENU), ethyl methanesulphonate (EMS), methyl methanesulphonate (MMS) and mitomycin C (MMC) has been determined. Both mutants were not sensitive to X-rays and bleomycin. 43-3B was found to be sensitive to 4NQO, MMC and slightly sensitive to alkylating agents. 27-1 was sensitive only to alkylating agents. The results suggest the existence of two repair pathways for UV-induced cytotoxicity: one pathway which is also used for the removal of 4NQO and MMC adducts and a second pathway which is also used for the removal of alkyl adducts. Parallel to the toxicity, the induction of mutations at the HPRT and Na+/K+-ATPase loci was determined. The increased cytotoxicity to UV, MMC and 4NQO in 43-3B cells and the increased cytotoxicity to UV in 27-1 cells correlated with increased mutability. It was observed that the increase in mutation induction at the HPRT locus was higher than that at the Na+/K+-ATPase locus. As only point mutations give rise to viable mutants at the Na+/K+-ATPase locus the lower mutability at this locus suggests that defective excision repair increases the chance for deletions. Despite an increased cytotoxicity to ENU in 27-1 cells the mutation induction by ENU was the same in 27-1 and wild-type cells at both loci, which suggests that the mutations are mainly induced by directly miscoding adducts (e.g. O-6 alkylguanine), which cannot be removed by CHO cells. As EMS and MMS treatment of 27-1 cells caused an increase in mutation induction at the HPRT locus and a decrease at the Na+/K+-ATPase locus it indicates that these agents induce a substantial fraction of other mutagenic lesions, which can be repaired by wild-type cells. This suggests that O-6 alkylation is not the only mutagenic lesion after treatment with alkylating agents.

4-Nitroquinoline-1-oxide↗

PubMed1986

Cyclosporine A, an in vitro calmodulin antagonist, induces nuclear lobulations in human T cell lymphocytes and monocytes.

Cyclosporine A is a noncytotoxic, natural, 11 amino acid cyclic peptide used clinically as an immunosuppressant to prevent organ rejection after transplantation. Cyclosporine A is an in vitro calmodulin antagonist. At the low concentrations required to inhibit calmodulin-dependent phosphodiesterase in vitro, cyclosporine A causes a dramatic alteration in the nuclear morphology of 23% of human peripheral blood mononuclear leukocytes in vitro without loss of viability. The shape of the nucleus changes from ovoid to a distinctive, radially splayed lobulated structure. The changes occur in a dose-dependent manner in 60 min at 37 degrees C. Specific monoclonal antibodies to human leukocytes identify the cells susceptible to nuclear lobulation by cyclosporine A as OKT4 antigen-positive T cell lymphocytes and monocytes. The lobulated nuclei are 2N as determined by flow cytometric measurement of ethidium bromide fluorescence of DNA. The cyclosporine A-induced lobulation of T cell nuclei requires both physiologic temperature and metabolic energy. Although structurally different than cyclosporine A, the calmodulin antagonists R24571 and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide] also produce T cell nuclear lobulations that are indistinguishable from the nuclear lobulations caused by cyclosporine A. These data indicate that nonmitotic structural elements that govern normal nuclear morphology in a subset of mononuclear leukocytes appear to require a calmodulin-mediated process. Cyclosporine A may be a useful noncytotoxic inhibitor of calmodulin-dependent systems that influence nuclear structure and function.

Antigens, Surface↗

PubMed1986

The wavelength dependence of u.v.-induced pyrimidine dimer formation, cell killing and mutation induction in human diploid skin fibroblasts.

In this study, we determined the wavelength dependence of u.v.-induced pyrimidine dimer formation, cell killing and mutation induction in human diploid skin fibroblasts. Pyrimidine dimers were quantified using the T4 endonuclease V assay, cell killing was measured as loss of colony forming ability and mutation induction was detected at the HPRT locus. U.v. irradiation was performed with monochromatic light of four different wavelengths (254, 297, 302 and 365 nm) and with polychromatic light of a Philips TL-01 lamp (predominantly 312 nm). The relative wavelength dependence for cell killing and mutation induction did not correlate with that for dimer formation. Toxicity and mutagenicity per equivalent initial dimer load increase with increasing wavelength. The relative wavelength dependence for cell killing and mutation induction is essentially the same, except at 365 nm.

Cell Survival↗

PubMed1986

Transformation of C3H10T1/2 cells by N-ethyl-N-nitrosourea occurs as a single, low frequency, mutation-like event.

C3H10T1/2 cells have been found to be relatively insensitive to transformation by N-ethyl-N-nitrosourea (ENU) and some other strongly carcinogenic, direct-acting, monofunctional alkylating agents. Because these agents are strong inducers of gene mutations we have considered the possibility that they transform 10T1/2 cells through a gene mutation. Such an event could have been missed in the standard assay, in which relatively low numbers of cells are treated. We found that the yield of ENU-induced transformed foci could be increased drastically by increasing the seeding density (up to 10(4) viable cells/cm2). The transformation frequency (foci/survivor) after ENU treatment was lower but much less dependent on cell density than after 3-methylcholanthrene. To investigate the mechanism of transformation by ENU in more detail, we used a modified transformation protocol which involved treatment of the cells at high density, followed by a period of exponential growth and reseeding at high density for focus selection. We found that ENU-induced transformation frequency is independent of the length of the growth period, thus independent of the total number of posttreatment cell divisions. This means that the focus-forming ability is completely fixed within maximally four generations. The transformation frequency does, however, depend on the number of divisions between the final reseeding and confluence. This suggests that the phenotypic expression of at least a part of the ENU-induced transformants is influenced by their clone size at confluence. Finally, we observed that the dose dependency of transformation was qualitatively and quantitatively similar to that of mutation at the Na-K-ATPase locus. Together, these experiments indicate that transformation of C3H10T1/2 cells by ENU is the result of a single, low frequency event, probably a gene mutation.

Animals↗

PubMed1985

Increase in clonal variation in Chinese hamster ovary cells after treatment with mutagens.

Clonal variation has been studied in CHO cells. The variant phenotype was an altered morphology of clones in agar: the parental CHO cells give rise to solid clumps of cells (wild-type colonies); occasionally, dispersed colonies arise, and the cells display an invasive growth in agar (INGA-type colonies). The frequency of this altered phenotype can be enhanced by treatment with a variety of mutagens (EMS, ENU, 4NQO, N-Ac-AAF, ultraviolet light, and X-irradiation). Enhancement was not due to a selective killing of wild-type cells or to a side-effect of cytotoxicity, which suggests that DNA damage is the cause of the altered phenotype. The INGA-trait breeds true, but most of the isolated clones have an inherent instability.

4-Nitroquinoline-1-oxide↗

PubMed1985

Mutations induced by X-rays at the HPRT locus in cultured Chinese hamster cells are mostly large deletions.

We investigated the molecular basis of 19 X-ray-induced HPRT-deficient mutants of V79 Chinese hamster cells with Southern hybridisation techniques. 12 of those mutants suffer from a big deletion (greater than 10 kb) of HPRT DNA sequences. Cytological studies of chromosome preparations of those 12 deletion mutants showed that in at least 3 of these mutants part of the long arm of the X-chromosome was lost. After correction for spontaneous arising mutations we estimate that at least 70-80% of X-ray-induced mutations are caused by large deletions.

Animals↗

PubMed1985

Genetic and molecular mechanisms of the in vitro transformation of Syrian hamster embryo cells by the carcinogen N-ethyl-N-nitrosourea. I. Correlation of morphological transformation and enhanced fibrinolytic activity to gene mutation, chromosomal alterations and lethality.

The role of chromosomal alterations, as opposed to gene mutations, in the origin of early stages of the in vitro transformation of Syrian hamster embryo (SHE) cells was investigated. For that purpose we compared the rates at which SHE cells recover from potential tumorigenic, mutagenic, clastogenic and cytotoxic damage if they are held in confluence in low serum medium for 3 or 6 days following a single treatment of N-ethyl-N-nitrosourea (ENU) before the cells are allowed to divide and to express this damage. The results show: (i) that frequencies of gene mutations remain constant; (ii) frequencies of sister chromatid exchange (SCE) and cytotoxicity decrease with very similar kinetics; and (iii) frequencies of chromatid aberrations and micronuclei decrease rapidly in the first 3 days, but slowly or not at all between days 3 and 6. Thus, all the mutational damage and a small fraction of the clastogenic damage still persist after 6 days confluent holding. From the two early stages of in vitro transformation studied, morphological transformation and enhanced fibrinolytic activity, the former shows similar kinetic behaviour as chromatid aberrations and micronuclei, whereas the kinetics of the latter correspond with those of gene mutations. Neither is correlated to SCE or cytotoxicity. Our results suggest that chromosomal alterations can play a major role in induction of morphological transformation of SHE cells. Insofar as enhanced fibrinolytic activity is due to a genetic change, gene mutations can be responsible. Our observations further indicate that different types of ENU-induced DNA lesions are involved in gene mutations, SCE and cytotoxicity, and clastogenic damage. We have reported the results of experiments analysing these relationships in another paper.

Animals↗

PubMed1985

Genetic and molecular mechanisms of the in vitro transformation of Syrian hamster embryo cells by the carcinogen N-ethyl-N-nitrosourea II. Correlation of morphological transformation, enhanced fibrinolytic activity, gene mutations, chromosomal alterations and lethality to specific carcinogen-induced DNA lesions.

The stability of N-ethyl-N-nitrosourea (ENU)-induced DNA damage in Syrian hamster embryo (SHE) cells was determined to study correlations with ENU-induced mutation and transformation. Confluent cultures were treated with ENU and after 0, 3 or 6 days holding in low serum medium to inhibit cell proliferation, the extent of ethylation at different sites in the DNA was determined with h.p.l.c. The amounts of dTp(Et)dT-triester, and O4- and O2-EtThy remained constant during the 6-day period. O6-EtGua slowly decreased (t1/2: 14 days); the initial level was lower than expected from in vitro data, suggesting that Syrian hamster embryo cells contain alkyl transferase. Evidence for active removal was also obtained in the case of 7-EtGua (t1/2: 59 h), O2-EtCyt (t1/2: 96 h) and possibly 3-EtGua (t1/2: 102 h). As expected, the promutagenic O6-EtGua was found to correlate with gene mutations. In addition, however, we have found that the likewise promutagenic O4- and O2-EtThy also correlate with gene mutations. Furthermore, our data suggest that sister chromatid exchange (SCE) and cytotoxicity (clonal survival) have a similar molecular basis. Both correlate with O2-EtCyt and 3-EtGua which are located in the narrow groove and are therefore expected to block DNA replication. Chromatid aberrations and micronuclei could not be correlated to specific DNA lesions, but were found to correlate mainly to N-ethylations. The same holds for morphological transformation, but in this case there is also a small contribution of DNA O-ethylation. In contrast, enhanced fibrinolytic activity did correlate only with stable O-ethylations, including O6-EtGua.

Alkylation↗

PubMed1985

Genetic analysis of the susceptibility of mouse cytomegalovirus to acyclovir.

Eight independently derived mouse cytomegalovirus (MCMV) mutants resistant to acyclovir (ACV) were obtained by the sequential plating of wild-type virus in increasing concentrations of ACV. Results of complementation studies among these eight mutants suggest that all had mutations within the same or closely associated genes. A ninth MCMV mutant resistant to phosphonoacetate (PAA) derived by plating wild-type virus in the presence of 100 micrograms of PAA per ml displayed coresistance to ACV and was unable to complement any of the ACV-derived mutants. Recombination experiments among all combinations of the nine MCMV mutants were performed and supported the complementation data in that no recombination could be detected. Seven of the eight ACV-resistant mutants demonstrated cross-resistance to PAA and hypersensitivity to aphidicolin. The one mutant not coresistant to PAA was more susceptible to PAA than was the parent virus. Only a few mutants demonstrated coresistance when the mutants were tested against 9-beta-D-arabinofuranosyladenine (ara-A). The ACV mutant that demonstrated increased susceptibility to PAA was 30-fold more susceptible to ara-A but remained unchanged in susceptibility to aphidicolin. Two of the parent-mutant combinations were selected for DNA synthesis analysis in the presence of ACV (5 microM). A significant decrease in DNA synthesis was demonstrated for both parent viruses, and there was little effect on mutant virus DNA synthesis at the same drug concentration. These results suggest that susceptibility of MCMV to ACV is confined to a product of a single gene and that a mutation of this gene can lead to an altered phenotype when compared with parent virus in susceptibility of DNA synthesis to PAA, ara-A, and aphidicolin, drugs that are known to inhibit DNA polymerase activity.

Acyclovir↗

PubMed1984

Relationship between cell killing, chromosomal aberrations, sister-chromatid exchanges and point mutations induced by monofunctional alkylating agents in Chinese hamster cells. A correlation with different ethylation products in DNA.

Several monofunctional alkylating agents (AA) were compared for their ability to induce chromosomal aberrations, cell killing, sister-chromatid exchanges (SCE) and point mutations in Chinese hamster cells (CHO and V79 cells). The AAs chosen varied in their reaction kinetics as well as their affinity to nucleophilic sites (different s values). AAs with low s values were more mutagenic in comparison to those with high s values, whereas the reverse was true for induction of cytotoxic effects. Neither SCEs nor chromosomal aberrations correlated with the induction of point mutations, indicating that different primary DNA lesions and repair pathways are involved in these biological processes. Molecular dosimetric studies indicate that O6 alkylation of guanine is the most probable cause of lesions in DNA leading to point mutations following treatment with ethyl methanesulphonate and ethyl nitrosourea.

Alkylating Agents↗

PubMed1984

Interaction of far- and near-ultraviolet radiation. The occurrence of photo-augmentation and photo-recovery in cultured mammalian cells.

Guided by the phenomena of photo-augmentation and photo-recovery, which have been described with respect to the induction of erythema in human skin, experiments were undertaken with cultured mammalian cells to study whether irradiation with far- and near-ultraviolet radiation results in an interaction at the cellular level with respect to cell survival and induction of mutations. Evidence was found for both photo-augmentation and photo-recovery. Photo-augmentation (more than an additive effect) was observed for cell survival when the long-wave ultraviolet irradiation (UVA) preceded the short-wave ultraviolet irradiation (UVB). Photo-recovery (less than an additive effect) was observed for cell survival if the UVA was given after or simultaneously with the UVB. The latter effect, however, was strongly influenced by dose: doses of UVA higher than 20 000 J/m2 no longer lead to photo-recovery in cell survival. For mutation induction, reduction in mutant frequency appears indicated for both combinations of UVA and UVB and for high and low doses of UVA.

Animals↗

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↗