Joyce F. Remsen

Excision of gamma-ray induced thymine lesions by preparations from ataxia telangiectasia fibroblasts

The capacity of whole cell sonicates of skin fibroblasts of normal individuals and patients with the autosomal recessive disease Ataxia telangiectasia (AT) to remove aerobic gamma-ray products of the 5,6-dihydroxydihydrothymine type (tgamma02) from exogenous DNA substrates was investigated. All four AT strains (AT CRL 1312, AT CRL 1343, AT GM 367, AT 4BI) possessed normal capabilities to excise tgamma02 from irradiated bacteriophage DNA and irradiated chromatin isolated from normal and AT-skin fibroblasts.

Uptake of [3h]cholesterol from low density lipoprotein by cultured human fibroblasts

The uptake of [3H]cholesterol from low density lipoprotein (LDL) was studied in LDL receptor-positive and receptor-negative human fibroblasts. In both cell lines the uptake depended upon temperature, time of incubation and the concentration of LDL in the medium. Although the incorporation of 125I-labeled LDL was minimal after 2 h of incubation in the receptor-negative (homozygous familial hypercholesterolemia, FH) cells, the uptake of [3H]cholesterol was only slightly less than that of the receptor-positive (WI-38) cells. With longer periods of incubation, a larger difference in labeled cholesterol incorporation was observed; this appeared to be due to a continued accumulation of the steroid in the WI-38 cells. After 8 and 24 h of incubation, some of the [3H]cholesterol was present as the ester in the WI-38 cells, but not the FH cells. Modified (reduced and methylated) LDL did not enter WI-38 cells by the receptor-mediated pathway during 2 h of incubation, as indicated by 125I uptake. [3H]Cholesterol uptake, however, was not significantly different from modified and unmodified LDL. While experiments indicated that significant amounts of cholesterol moved rapidly from LDL to cultured cells with a dependence on time and LDL concentration, no increase in total cell cholesterol was detected in either cell line. FH cells contained less total cholesterol and had a higher 3H specific activity than the WI-38 cells. These data suggest that there may be important mechanisms in addition to the LDL pathway for the movement of lipids into cells.

In vitro reaction of radioactive 7β,8α,-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo(A)pyrene and 7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo(A)pyrene with DNA☆

The in vitro reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [3H9, 3H10]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [3H9, 3H10]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N2-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N2-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.

Inhibition of DNA-repair and DNA-synthesis by harman in human alveolar tumor cells

Abstract The effect of the tryptophan pyrolysis product harman on colony forming ability, DNA-synthesis and DNA-repair was investigated in human alveolar tumor cells A549. Colony forming ability and overall DNA synthetic capacity decreased linearly with increasing harman concentration and reached values of 20 % and 29 % of untreated controls, respectively, at 200 μM. Harman also inhibited the repair of N-acetoxy-2-acetylaminofluorene induced DNA damage, as measured by the alkaline elution procedure. While incision of parental DNA occurred normally in the presence of harman the reconstitution of control molecular weights was strongly inhibited. These effects of harman may play a role in its co-mutagenic activity.

Ultraviolet inactivation and miscoding of irradiated R17-RNA invitro

Summary Ultraviolet irradiation of the single-stranded Coliphage R17 leads to inactivation of the in vitro messenger activity of its RNA. Uridine-photohydration is the major reaction occurring in the RNA under these conditions. At a residual activity of the synthetase gene of 37 per cent, there are 11.2 uridine-photohydrates present in the total R17 genome, while only 5.3 photohydrates are necessary to decrease the translation of the coat gene to the same value. De novo incorporation of histidine into the coat protein is observed with R17-RNA from ultraviolet-irradiated phage, indicating that uridine-photohydrates can miscode as cytidine in an in vitro translation system.

Repair of DNA damage induced by ionizing radiation and benzo[a]pyrene in mammalian cells.

The biological effects of DNA‐damaging agents are codetermined by the structural characteristics of the lesions, the quality and extent of the local distortion of DNA and chromatin structure, and the mode(s) of damage processing used by a given type of cell. Persistent damage (i.e., damage that is not removed before it is reached by DNA replication) may be mostly responsible for metagenesis and carcinogenesis. To understand the effects of environmental physical and chemical DNA‐damaging agents on human health, the mechanisms of damage processing used by human cells have to be elucidated. We report our studies of the excision of gamma‐ray products of the 5,6‐dihydroxydihydrothymine type (tγ o2) in normal human fibroblasts and in fibroblasts from patients with the hereditary diseases Fanconis anemia (FA) and ataxia telangiectasia (AT). Both diseases are characterized by chromosomal instability and increased susceptibility for the development of cancer. Decreased capacities of nuclear and whole cell sonicat...

Removal of benzo[a]pyrene from cells by various components of medium

Benzo[a]pyrene is removed from cells in culture by various additions to the medium. During post-treatment incubation, WI-38 fibroblasts were incubated with a low density, very low density and high density lipoproteins, delipidated or complete serum or plasma, or serum albumin. The time course of removal was followed. Increasing concentrations of lipoproteins resulted in increasing percentages of removal of benzo[a]pyrene from cell membranes. The most efficient addition was 10% complete human plasma. These results indicate that benzo[a]pyrene remains at or close to the plasma membrane for at least several hours and readily redistributes to medium components.

Uptake of [3H]Vitamin D3 from Low and High Density Lipoproteins by Cultured Human Fibroblasts

Abstract The plasma distribution and cellular uptake of [3H]vitamin D3 was studied in vitro using cultured human fibroblasts. Incubation of [3H]vitamin D3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2–4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotien (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [3H] 1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [3H]vitamin D3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D3 was time, concentration, and temperature dependent. At a concentration of 50 μg LDL/ml of medium, the uptake of [3H]vitamin D3 from LDL at 37°C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.