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J J McCormick

Publications and source records attributed to J J McCormick.

At least 127 records · Page 7Linked to original sources
PubMed1981

Induction of anchorage-independent growth in human fibroblasts by propane sultone.

We have demonstrated a dose-dependent increase in the frequency of diploid human cells capable of anchorage-independent (AI) growth after treatment with the carcinogen propane sultone, followed by exponential growth to allow full expression of this phenotype (8 to 13 population doublings). Exposure to these same concentrations of propane sultone also resulted in a dose-dependent increase in the frequency of 6-thioguanine-resistant cells in the population. Procedures such as synchronization of cells and treatment just after the onset of DNA synthesis or the use of special selective medium were not essential for this induction. A very low frequency of cells with the AI phenotype was found in the control population (background). Cells which exhibited the AI phenotype spontaneously or after carcinogen treatment retained the characteristic over as many generations as tested (greater than 13). The data suggest that AI growth is the result of a mutational event.

Anthralin↗
PubMed1980

A comparison of the DNA binding, cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens.

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G0 state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.

Acetoxyacetylaminofluorene↗
PubMed1980

Error-free excision of the cytotoxic,mutagenic N2-deoxyguanosine DNA adduct formed in human fibroblasts by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The ability of normal diploid human fibroblasts and excision repair-deficient xeroderma pigmentosum cells (XP12BE, complementation group A) to excise potentially cytotoxic or mutagenic lesions induced in DNA by (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BzaP-diol epoxide) was determined. Large populations of cells were prevented from replicating by being grown to confluence; after 3 days they were exposed to tritiated BzaP-diol epoxide for 2 hr. One set of cultures was immediately released and assayed for the number of residues covalently bound to DNA, percent survival of colony-forming ability, and frequency of induced mutations. After various periods of time in confluence, other sets were similarly released and assayed. The normal cells exhibited a gradual increase in survival with time held in confluence (recovery from potentially cytotoxic lesions) which was directly correlated with a gradual loss of radioactivity from their DNA and a gradual decrease in the frequency of induced mutations. In contrast, no loss of radioactively labeled carcinogen from the DNA of the XP12BE cells could be detected during a 6-day period and their percent survival and frequency of induced mutations did not change. DNA from normal cells harvested immediately after treatment or after 2, 4, or 8 days in confluence was enzymatically hydrolyzed and analyzed by high-pressure liquid chromatograhy. Only a single peak was detected that cochromatographed with a standard prepared from deoxyguanosine treated with BzaP-diol epoxide. The kinetics of decrease of tritium label in this specific peak corresponded to the decrease in radioactivity of the total DNA with time and with the kinetics of recovery of the cells from the potentially cytotoxic and mutagenic effects of BzaP-diol epoxide. These results suggest that the N2-deoxyguanosine adduct is responsible for these biological effects and indicate that excision repair of this lesion by the normal human cells is "error free."

Benzopyrenes↗
PubMed1980

Human cell-mediated benzo(a)pyrene cytotoxicity and mutagenicity in human diploid fibroblasts.

A human epithelial cell-mediated cytotoxicity and mutagenicity assay system for benzo(a)pyrene [B(a)P] has been developed with human fibroblasts as the target cells. Lethally X-irradiated human kidney carcinoma-derived epithelial cells, which had constant levels of B(a)P-metabolizing activity, were cocultivated with target human skin fibroblasts (XP12BE), which lack excision repair capability for B(a)P-DNA adducts. The optimal conditions determined for the cell-mediated cytotoxicity assay were a 48-hr exposure to B(a)P concentrations ranging from 0.1 to 1 microM at a metabolizing cell:target cell ratio of at least 1:1. Under these conditions, the frequency of mutations to 6-thioguanine resistance induced in the target XP12BE cells by B(a)P as well as the binding of tritium-labeled B(a)P to DNA was also shown to be concentration dependent. High-pressure liquid chromatography analysis of enzymatically degraded B(a)P-DNA adducts revealed two peaks: a major peak (82%) which cochromatographed with the guanosine adduct-standard synthesized from anti-isomeric-7,8-dihydrodiol-9,10-epoxide of B(a)P; and a minor peak (18%) which cochromatographed with the guanosine adduct-standard synthesized from syn-isomeric-7,8-diol-9,10-epoxide of B(a)P. Human liver carcinoma- and lung carcinoma-derived cell lines, capable of metabolizing B(a)P, proved equal to or better than the kidney carcinoma-derived cell line in producing cytotoxic B(a)P metabolites in the cell-mediated cytotoxicity assay with target XP12BE cells.

Benzo(a)pyrene↗
PubMed1979

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation.

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.

Cell Line↗
PubMed1979

Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.

The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.

Cell Cycle↗
PubMed1978

An improved acriflavine-Feulgen reagent for quantitative DNA cytofluorometry.

We have modified the acriflavine-Feulgen histochemical method for the quantitative determination of DNA by performing the staining of hydrolyzed cells affixed to cover slips with acriflavine dissolved in 90% ethanol. Compared to conventional techniques, in which the acriflavine is dissolved in aqueous HCl plus potassium metabisulfite, this method not only decreased non-Feulgen (background) staining in both the nucleus and the cytoplasm, but also increased the fluorescent intensity of Feulgen stained nuclei more than four-fold. Cells stained by our modified method exhibited a fluorescence maximum of 507 nm, which is similar to the 500 nm fluorescence maximum obtained with acriflavine bound to apurinic acid solution by a modification of the acriflavine-Feulgen method. These fluorescence maxima are in contrast to the 510 nm to 625 nm fluorescence maxima which are obtained when cells are stained according to conventional protocols. The fluorescence intensities of nuclei of synchronized cells stained by the modified method were proportional to DNA content. Thus, by the criteria of staining specificity, in situ and in solution fluorescence spectra agreement and quantitative staining, we conclude that our modified acriflavine-Feulgen method is more satisfactory for quantitatively measuring DNA in situ by cytofluorometry than the usual acriflavine-Feulgen method.

Acriflavine↗
PubMed1977

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts.

The cytotoxicity of the "K-region" epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several "K-region" epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ratio of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions. To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by "K-region" epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the "K-region" epoxide of benzo(a)pyrene (BP), 7,12-dimethyl-benz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.

Azaguanine↗
PubMed1977

Purification of commercial acriflavine by Sephadex LH-20 column chromatography.

Acriflavine supplies commercially was purified by removing contaminating proflavine by chromatography on a column of Sephadex LH-20 eluded with 65% methanol. The purified acriflavine, recovered in a yield that was 52% of the theoretical maximum, had a visible absorption maximum of 452 nm with a band width of 47 nm. These spectral properties agreed with those of acriflavine purified by precipitation, repeated recrystallization and chromatography on aluminum oxide, which results in a yield of 13% at best.

Acridines↗
PubMed1977

An improved acriflavine-Feulgen method.

The acriflavine-Feulgen method for the histochemical demonstration of deoxyribonucleic acid was modified by staining hydrolyzed cells with 0.01% acriflavine dissolved in 90% ethanol. This method offered the following advantages: (a) it simplified the preparation of the acriflavine-Feulgen reagent; (b) it left the cytoplasm essentially unstained while staining the nuclei bright green in hydrolyzed cells and left the cytoplasm and nuclei essentially unstained in unhydrolyzed cells; (c) it eliminated poorly defined reagents from the staining solutions. Because of these staining properties, this technique may be especially useful in the quantitative determination of deoxyribonucleic acid by cytofluorometry.

Acridines↗
PubMed1977

A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.

Acridines↗