Repair of chromosome damage induced by X-irradiation during G2 phase in a line of normal human fibroblasts and its malignant derivative.

A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G2 phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. On the assumption that each chromatid contains a single continuous DNA double helix, chromatid breaks would represent unrepaired DNA double-strand breaks; the gaps may represent single-strand breaks. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or beta-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G2 phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H2O2, or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G2 phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

Virion-associated and cellular RNA methylase activity in normal and neoplastic mammary tissue from mammary tumor virus-infected and -uninfected mice.

A comparison of cellular RNA methylase activities and patterns between normal and neoplastic mouse mammary tissue indicated the following. The rna methylases of mammary tumor tissue extracts are qualitatively different from those of normal lactating mammary tissue, based on differences in extent of methylation; the normal lactating tissue extracts have a greater capacity of methylate RNA than do the tumor extracts studied to date. There is no correlation between capacity and either the malignant state or the etiological agent. There is a qualitative effect on methylation patterns attributable to the presence of virus. Finally, both the etiological agent, mouse mammary tumor virus, and its putative nucleoprotein core, intracytoplasmic A particles, have a N-2-guanine RNA methyltransferase integrally associated with them. These conclusions are consistent with the aberrant nucleic acid methylation hypothesis, with the reservation that aberrant does not imply hypermethylation.

Repair of DNA-protein cross-links in an excision repair-deficient human cell line and its simian virus 40-transformed derivative.

DNA-protein cross-links are induced in mammalian cells by X-rays, ultraviolet light, fluorescent light, and numerous chemical carcinogens. Others have shown that these cross-links are repaired by normal cells but that excision repair-deficient xeroderma pigmentosum (XP) Group A cells, XP12BE, are deficient in repair of these bulky adducts. This paper compares the DNA-protein cross-link repair competency of another XP Group A strain, XP20S, with its more rapidly proliferating simian virus 40-transformed derivative line and with normal human skin fibroblasts. DNA-protein cross-links were induced with 20 microM transplatinum(II)diamminedichloride and assayed by the membrane alkaline elution procedure of Kohn. Treated and untreated cells are lysed on a polycarbonate membrane filter, and the coelution rates of the DNA at pH 12.2 are compared; DNA-protein cross-links retard elution of DNA. The repair competency of XP20S cells for trans-platinum(II)diamminedichloride-induced DNA-protein cross-links was similar to that of XP12BE cells, but the competency of the simian virus 40-transformed XP20S cells was nearly equal to that of normal human skin fibroblasts. These results suggest that either cell cycling compensates for the genetic deficiency present in the nucleotide excision process of XP Group A cells or that a process other than nucleotide excision can repair these lesions; this process requires cell cycling or activation by the virus.

Chromosomal radiosensitivity of human tumor cells during the G2 cell cycle period.

Thirteen cell lines derived from human tumors of diverse tissue origin and histopathology were compared with 12 lines of normal skin fibroblasts with respect to chromatid damage induced by 25, 50, or 100 R of X-irradiation during the G2 period of the cell cycle. Only cells in metaphase were examined, and these had been irradiated 1.5 hr before fixation. When irradiated under identical conditions, the tumor cells showed significantly more chromatid breaks and gaps than did the normal cells at all doses tested. The data suggest that the increased G2 chromosomal radiosensitivity of the tumor cells is associated with deficient DNA repair during the G2-prophase period of the cell cycle.

Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture.

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containing the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.

Effect of visible light on benzo(a)pyrene binding to DNA of cultured human skin epithelial cells.

The ubiquity of the photosensitive carcinogen benzo(a)pyrene (BP) and visible light in the environment suggests that their interaction might lead to photoproducts harmful to humans. To test the combined impact of these two agents on human epithelial cells, binding of BP to cellular DNA was assessed following treatment of cultures with BP and low-intensity (4.6 watts/sq m) intermittent (12 hr daily, 3 to 5 days) cool white fluorescent light. Light exposure reduced the formation of covalent BP adducts 20-fold (from 150 to 7 pmol BP per mg DNA) in cells treated with 1 microgram BP per ml and completely inhibited cytotoxicity; even with 10 microgram BP per ml, light exposure markedly inhibited cytotoxicity. However, at low BP dosage (0.1 microgram/ml), covalent adducts (2 pmol/mg DNA) to cellular DNA are produced and their formation is not influenced by light. These adducts persisted for at least 7 days following treatment; this observation suggests that chronic low-level exposure of human epithelium to BP may lead to an accumulation of DNA damage.

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.

Isopentenyl diphosphate isomerase deficiency in Synechocystis sp. strain PCC6803.

Isopentenyl diphosphate isomerase (IPP isomerase) in many organisms and in plastids is central to isoprenoid synthesis and involves the conversion between IPP and dimethylallyl diphosphate (DMAPP). It is shown that Synechocystis PCC6803 is deficient in IPP isomerase activity, consistent with the absence in its genome of an obvious homologue for the enzyme. Incorporation of [1-(14)C]IPP in cell extracts, primarily into C(20), occurs only upon priming with DMAPP in Synechocystis PCC6803 and in Synechococcus PCC7942. Isoprenoid synthesis in these cyanobacteria does not appear to involve interconversion of IPP and DMAPP, raising the possibility that they are not within the plastid evolutionary lineage.

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.

Factors affecting and significance of G2 chromatin radiosensitivity in predisposition to cancer.

The frequencies of chromatid breaks and gaps in metaphase cells fixed 2 h after G2 phase X-irradiation (1 Gy) were in almost all cases at least two- to three-fold higher in skin fibroblasts from individuals with genetic conditions predisposing to cancer than in comparable cells from clinically normal controls. Previously, we reported this response in all cancer-prone genetic disorders tested including ataxia telangiectasia, Bloom's syndrome, Fanconi's anemia, xeroderma pigmentosum (XP), familial polyposis, Gardner's syndrome, hereditary malignant melanoma, dysplastic nevus syndrome and cancer family members. One exception was XP-A. In this report we add information on skin fibroblasts from retinoblastoma, Wilms' tumor and XP-C patients, 13 clinically normal controls and six cell lines from fetal or infant cells. Factors affecting the response are identified and include pH, temperature, cell density, culture medium or serum, microbial contamination and visible light exposure (effective wavelength 405 nm). Because of experimental variability, known normal controls should be used in each group of assays. With adequate control of the above factors this response could provide the basis of a test for detecting individuals carrying genes that predispose to a high risk of cancer.

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells.

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.

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.