Katherine K. Sanford

G2 chromosomal radiosensitivity of ataxia-telangiectasia heterozygotes

Five lines of skin fibroblasts from individuals heterozygous for ataxia-telangiectasia (A-T), compared with six cell lines from age-matched normal controls, show a much higher frequency of chromatid breaks and gaps following x-irradiation during the G2 phase of the cell cycle. The magnitude of this difference suggests that G2 chromatid radiosensitivity could provide the basis for an assay to detect A-T heterozygotes. Though clinically normal, A-T heterozygotes share a high risk of cancer with A-T homozygotes and constitute approximately 1% of the human population. Further, we propose that G2 chromosomal radiosensitivity, which appears to result from a DNA repair deficiency, may be associated with a genetic predisposition to cancer.

Influence of oxygen and pH on plating efficiency and colony development of WI-38 and Vero cells

Abstract Clonal growth of human (WI-38) and adult monkey (Vero) cells was enhanced by lowering the O2 content of the gas phase from atmospheric (18–20%) to 1%. The O2 sensitivity of the low-passage diploid cells was significantly greater than that of the long-term heteroploid monkey line. Several culture media and serum sources consistently promoted superior colony development at 1% O2, whereas variable growth and reduced plating efficiencies were observed in replicate cultures gassed with 20% O2.

Fluorescent light-induced chromosome damage in human IMR-90 fibroblasts role of hydrogen peroxide and related free radicals

Exposure of human fibroblasts (IMR-90) to cool-white fluorescent light causes chromatid breaks and exchanges. This chromatid damage is caused largely by the production of hydrogen peroxide (H2O2) since it can be prevented almost completely by the addition of catalase. In support of this conclusion, exogenous H2O2 is shown to induce chromatid breaks. The clastogenic amounts of H2O2 generated during light exposure are formed within the cell since cells illuminated in saline showed the same extent of damage as cells in culture medium. Addition of selenite to the cultures during light exposure significantly decreases the chromatid damage in a dose-related manner and may be necessary to maintain sufficient activity of glutathione peroxidase. The free hydroxyl radical, . OH, appears to be partially responsible for the light-induced chromatid damage. Of the free-radical scavengers tested, i.e., mannitol, vitamin E, and dimethyl sulfoxide, only mannitol, which scavenges . OH, significantly decreases the light-induced chromatid damage. Thus, both . OH and H2O2 formed within the cell during light exposure are agents that directly or indirectly cause chromatid damage.

Radiation-induced chromatid breaks and deficient DNA repair in cancer predisposition

Deficient repair of DNA double-strand breaks, resulting in an abnormally high frequency of chromatid breaks after G(2) exposure of cells to radiation, appears to be associated with cancer predisposition. Unrepaired DNA strand breaks contribute to genomic instability. Unrepaired chromatid breaks representing DNA strand breaks can result in chromosome deletions, translocations and gene amplifications seen in human cancers. This cytogenetic response of cells to radiation may be useful as a marker of cancer susceptibility and in identifying individuals at risk of developing cancer in cancer families.

Cytogenetic response to G2-phase X irradiation in relation to DNA repair and radiosensitivity in a cancer-prone family with Li-Fraumeni syndrome

Noncancerous skin fibroblasts from six family members with Li-Fraumeni syndrome, five with cancer of diverse tissue origin and one with a premalignant neoplasm, showed a high frequency of chromatid aberrations, 94 to 119 breaks and 58 to 95 gaps per 100 metaphase cells arrested with colcemid 0.5 to 1.5 h after X irradiation (1.75 x 10(-2) C/kg). This response results from deficient repair of the radiation-induced DNA damage. In contrast, skin fibroblasts from two unrelated normal controls and a spouse showed 19 breaks and 17 to 19 gaps per 100 cells. Whereas all six members of the cancer-prone family had a radioresistant phenotype, only four had an inherited p53 mutation. Fibroblasts from a radioresistant family member showed the same extent of chromatid damage directly (0 to 0.5 h) after G2-phase X irradiation as those from the radiosensitive control spouse. We conclude, therefore, that radiosensitivity, as determined by cell killing in asynchronous populations of skin fibroblasts, is unrelated to chromosomal sensitivity to G2-phase X irradiation. However, the persistence of a high frequency of chromatid breaks and gaps at 0.5 to 1.5 h after G2-phase X irradiation, a manifestation of deficient DNA repair, is associated with proneness to cancer in this family.

Visible light-induced DNA crosslinks in cultured mouse and human cells.

Cool-white fluorescent light induces crosslinks in DNA when proliferating cells are exposed at 37 degrees C for 20 h to 4.6 J/m2/s in culture medium supplemented with fetal bovine serum. Using the Kohn alkaline elution technique, we now find that: 1. Increased light intensity increases DNA crosslinks. 2. The crosslinking is medium-mediated. 3. Oxygen enhances the crosslinking. 4. The extent of crosslinking is decreased at high cell density. 5. The crosslinks can be removed by digestion with proteinase K (0.02 to 0.50 mg/ml). 6. Human cell lines including those derived from adult prostate, fetal lung (IMR-90) and mixed fetal tissues are susceptible to light-induced crosslinks. 7. Crosslinkage is not decreased by addition of catalase to the medium and the effective wavelength is probably between 450 nm and 490 nm. From these results we conclude that the mechanism of light-induced crosslinks differs from that of light-induced chromatid breaks and that the major lesion observed is protein-DNA cross-linkage rather than DNA strand breaks.

Biochemical evidence for deficient DNA repair leading to enhanced G2 chromatid radiosensitivity and susceptibility to cancer

Human tumor cells and cells from cancer-prone individuals, compared with those from normal individuals, show a significantly higher incidence of chromatid breaks and gaps seen in metaphase cells immediately after G2 X irradiation. Previous studies with DNA repair-deficient mutants and DNA repair inhibitors strongly indicate that the enhancement results from a G2 deficiency(ies) in DNA repair. We report here biochemical evidence for a DNA repair deficiency that correlates with the cytogenetic studies. In the alkaline elution technique, after a pulse label with radioactive thymidine in the presence of 3-acetylaminobenzamide (a G2-phase blocker) and X irradiation, DNA from tumor or cancer-prone cells elutes more rapidly during the postirradiation period than that from normal cells. These results indicate that the DNA of tumor and cancer-prone cells either repairs more slowly or acquires more breaks than that of normal cells; breaks can accumulate during incomplete or deficient repair processes. The kinetic difference between normal and tumor or cancer-prone cells in DNA strand-break repair reaches a maximum within 2 h, and this maximum corresponds to the kinetic difference in chromatid aberration incidence following X irradiation reported previously. These findings support the concept that cells showing enhanced G2 chromatid radiosensitivity are deficient in DNA repair. The findings could also lead to a biochemical assay for cancer susceptibility.

BIOTIN, B12, AND OTHER VITAMIN REQUIREMENTS OF A STRAIN OF MAMMALIAN CELLS GROWN IN CHEMICALLY DEFINED MEDIUM.

Abstract A strain of mouse cells designated NCTC2071 has been cultured for years in chemically defined medium NCTC 109 free of antibiotics or any undefined supplements. This strain, derived from clone NCTC 929-L, and free of pleuropneumonia-like organisms, was examined with respect to vitamin requirements. The effects of the 18 vitamins in medium 109 on cell survival, rates of proliferation, and morphology were determined in long-term studies. Inter-relationships between the vitamins and other components of the medium such as the nucleic acid derivatives were explored. In a coenzyme-free modification of medium 109, the cells required 5 vitamins for survival, namely, niacinamide (or niacin), thiamine, riboflavin, pantothenate, and choline chloride. In addition, pyridoxal (or pyridoxine) prevented destruction of cells when subjected to mechanical agitation, and folic acid and biotin slightly increased rates of cell proliferation but were not required for continuous cell propagation. Eight vitamins appeared to have no significant effects on the cells, namely, vitamins A, D, K, E, C, p -aminobenzoic acid, B 12 , and i -inositol. When cells were cultured in a medium lacking all the nucleic acid derivatives except deoxycytidine, a requirement for folic acid was demonstrated. Neither thymidine nor vitamin B 12 completely substituted for this requirement. In a medium containing the seven vitamins and only two of the five nucleic acid derivatives, the cells required biotin for survival, a requirement that was not detected when cells were grown in the complete medium. The lots and/or levels of cysteine in medium 109 were found to be toxic for single well-isolated cells. A toxicity of glutathione was counteracted by vitamin C. However, all three reducing agents could be eliminated without detectable reduction in cell growth. A stimulatory effect of vitamin B 12 on cell proliferation was observed under one set of conditions. Cells that had been cultured for a long period in a medium lacking all nucleic acid derivatives exhibited a marked stimulatory response to the vitamin. However, cells cultured for months in an identical medium except that it contained thymidine and deoxycytidine failed to show any response to the vitamin. Interpretations of these results are discussed. The influence of the type of chemically defined medium on the transplantability and/or malignancy of the cells was tested. Several new formulations of chemically defined media with evaluations of their action on these cells are presented.

A new culture medium for human skin epithelial cells

SummaryA new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1∶1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell outgrowth from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell outgrowths. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of fibroblasts after only 19 to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial growth factor, hydrocortisone and insulin at 5 ng per ml, 4 μg per ml and 5 μg per ml, respectively did not appreciably enhance the growth of the epithelial cells.

Oxygen and light effects on chromosomal aberrations in mouse cells in vitro.

Abstract Decreasing the oxygen concentration in the gas phase from 18% (atmospheric) to 1% decreased the frequency of chromosomal aberrations in mass cultures of cells from adult lung and embryos of two inbred mouse strains. Both the rate of shift from the diploid number and the incidence of abnormal chromosomes were decreased at the lower oxygen level. Similarly, shielding mouse cells from room lights (cool white, fluorescent) during routine fluid renewals reduced the incidence of abnormal chromosomes, particularly minutes and metacentrics. The increased incidence of chromosomal abnormalities on exposure of cells to light and high oxygen presumably results from a photodynamic reaction affecting the DNA or associated proteins of the chromatin fibers.