Raymond Gantt

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.

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.

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.

Serum-induced chromosome damage and neoplastic transformation of mouse cells in vitro

SummaryIn previous studies, mouse cells grown in medium supplemented with horse serum (HS) developed more chromosomal aberrations and underwent malignant transformation earlier than cells from the same pool grown with fetal bovine serum (FBS) supplement. In the present study cells derived from C3Hf/HeN mouse embryos were grown in medium NCTC-135 supplemented with various combinations of large- and small-molecule fractions of HS and FBS in an effort to determine the effective components. The results indicate that the large-molecule fraction of HS (mare or stallion) produces alterations in chromosome number and structure. HS is also shown to cause chromatid breaks and exchanges at or near the centromere in contrast to fluorescent-light-induced breaks which occur randomly along the chromatid. However, efforts to control completely chromosome stability and malignant transformation through the use of large-and small-molecule fractions of HS and FBS or combinations thereof were unsuccessful. In comnection with this study, diagnosis of malignant transformation in vitro was made by a direct sampling method based on cytologic criteria previously described and documented. With one exception, the diagnoses of 11 different cell lines were consistent with results of in vivo assays.

Effect of fractions of horse and fetal bovine serum on neoplastic conversion of C3H mouse cells in tissue culture

SummaryCultures derived from C3H/He mouse embryos were grown in medium NCTC 135 supplemented with horse serum, fetal bovine serum, or various combinations of large and small molecule fractions of horse and fetal bovine serum. Cultures in medium NCTC 135 alone or in medium 135 supplemented with the small molecule fraction of either horse or fetal bovine serum did not grow as continuous long term lines. The best growth was obtained when the cultures were in medium containing the large molecule fraction of fetal bovine serum either alone or in combination with a small molecule fraction. Cells grown in the presence of the low molecular weight fraction of horse serum invariably produced tumors on injection into syngeneic animals. Cells in the small molecular weight fraction of fetal bovine serum combined with the large molecular weight fraction of horse serum produced tumors after a prolonged period in vitro. *** DIRECT SUPPORT *** A00S8010 00003

Structural studies on avian myeloblastosis virus: Conditions for isolation and biochemical characteristics of the core component

Abstract The core component of avian myeloblastosis virus (AMV) was isolated in amounts sufficient for biochemical analysis. Core material which approached homogeneity by electron microscopy had a composition by weight of 86% protein, 8% RNA, and 6% phospholipid. This amount of phospholipid may indicate either contamination by non-core structures, or more probably the presence of phospholipid as an intrinsic constituent of AMV cores. The influence of various conditions of the isolation procedure on the yield of AMV cores from intact virus was also examined. The highest yield of core material was obtained by use of a sodium phosphate buffer with EDTA, a 10-min exposure of unfrozen Ficoll-purified AMV (protein concentration of 1 mg/ml) to Sterox SL (final concentration 1.5%, v/v), and velocity sedimentation through a layer of 48% (w/w) sucrose into a pellet.

Measurement of DNA-protein crosslinks in mammalian cells without X-irradiation

To study the mechanisms of formation and repair of DNA-protein crosslinks in mammalian cells, the best general method to assay these lesions is the Kohn membrane alkaline elution procedure. Use of this sensitive technique requires the introduction of random strand breaks in the DNA by X-irradiation to reduce the very high molecular weight so that it elutes off the filter at an appropriate rate. This report describes an alternative method for fragmenting the DNA in the absence of X-irradiation equipment. Convenient reproducible elution rates of DNA from various mouse and human cells in culture without X-irradiation result from elution through polyvinyl chloride filters with 75 mM sodium hydroxide (0.33 ml/min) instead of the standard 20 mM EDTA-tetrapropylammonium hydroxide, pH 12.2 (0.03 to 0.04 ml/min). Dose-dependent retardation of the DNA elution was observed over the range 0 to 30 microM trans-platinum(II)diamminedichloride, and proteinase K treatment during cell lysis restored the elution rate to that of the untreated control cell DNA. In the absence of X-irradiation, this elution method measures DNA-protein crosslinks with higher sensitivity and equivalent reproducibility as the air-burst procedure.

Methylation of satellite deoxyribonucleic acid in mouse neoplastic and non-neoplastic cell cultures.

SummaryComparisons of nucleic acid methylation between paired neoplastic and non-neoplastic mouse cell lines have shown a striking difference in the deoxyribonucleic acid (DNA) peak eluted from methylated albumin-kieselguhr columns (R. Gantt and V. J. Evans, 1969, Cancer Res. 29: 536–541). Since mouse satellite DNA is relatively highly methylated, its 5-methylcytosine content was compared with mainband DNA in these two paired cell lines to determine whether this might account for the observed differences. The cell DNA was labeled with methyl-labeled methionine and isolated from the cells by repeated neutral cesium chloride isopycnic centrifugation. The satellite DNA strands were then separated in an alkaline cesium chloride gradient. Both the 5-methylcytosine content and the relative amounts of satellite DNA were indistinguishable in the paired cell lines. Further, the results showed that both strands of satellite DNA had virtually equal amounts of 5-methylcytosine, although the heavy strand contains 1.5 times more cytosine than the light strand.

Comparison of methyl-labeled tRNA in a neoplastic cell line with a paired non-neoplastic control

Abstract A comparison has been made between methylation of tRNA in intact cells from a non-neoplastic mouse cell line and that from two related neoplastic mouse cell lines. By adding 3 H-methyl-methionine and 14 C-methyl-methionine to the growth medium, it was possible to separately label and then to simultaneously extract, isolate, and chromatograph the tRNA from neoplastic and non-neoplastic cells. Isotope ratios were calculated from reversed-phase chromatography profiles and a consistently reproducible variation in the ratio was seen through the region where the first tRNAs were eleuted, indicating chromatographic heterogeneity. Although this difference between tRNA methylation in non-neoplastic and that in neoplastic cells is small, the data are consistent with the view of Srinivasan and Borek that aberrant methylation may be a factor in neoplastic conversion.