DNA damage by chemically generated singlet oxygen.

A naphthalenic endoperoxide was used as a non-photochemical source of singlet oxygen (1O2) to examine some interactions between this reactive oxygen species and DNA. High molecular weight DNA (ca. 10(8) daltons) was exposed to 120 mol m-3 1O2 (cumulative concentration) and analyzed for interstrand crosslinkage by hydroxyl apatite chromatography following formamide denaturation. No evidence for 1O2-induced interstrand crosslinking was obtained. The capacity of 1O2 to generate strand breaks in single-stranded (ss) and double-stranded (ds) DNA was investigated by sucrose gradient centrifugation analysis of bacteriophage phi X174 DNA. No direct strand breaks could be detected at neutral pH, whereas extensive strand breakage was observed after treatment with alkali. Possible biological consequences of 1O2-exposure were assessed by examining the plaque-forming capacity of ss and ds phi X174 DNA molecules using wildtype Escherichia coli spheroplasts as recipients. Without any further treatment with heat or alkali, exposure to the endoperoxide resulted in a time- and dose-dependent inactivation, ss DNA being considerably more sensitive than ds DNA. From the present results and those reported earlier (Nieuwint et al.,) we infer that 1O2-induced inactivation of phi X174 DNA is not due to DNA backbone breakage nor to interstrand crosslinking, but rather to some form of damage to the base or sugar moiety of the DNA, the exact nature of which remains to be elucidated.

Assignment of human transcobalamin II (TC2) to chromosome 22 using somatic cell hybrids and monosomic meningioma cells.

Human transcobalamin II (TC2), a vitamin B12 binding serum protein, is synthesized and secreted into the medium by cells growing in vitro. Mouse-man somatic cell hybrids were analyzed in order to map the locus of TC2. The presence of human TC2 in the culture media was correlated with the results of genetic marker and chromosome analysis of the hybrid cells. Chromosome 22 showed 100% concordancy. However, chromosome 6 (90% concordancy) and chromosome 7 (96% concordancy) were not completely excluded. Meningioma cells obtained from patients heterozygous for TC2 showed a concomitant loss of one chromosome 22 and one of the TC2 alleles, strongly supporting the assignment to chromosome 22.

Effects of mitomycin C on the rate of DNA synthesis in normal and Fanconi anaemia cells.

The effect of low doses mitomycin C (MMC) on DNA synthesis of fibroblast cell lines derived from normal individuals or patients with Fanconi anaemia (FA) was studied. Using low doses of MMC (12 ng/ml), little or no effect was observed on DNA synthesis of normal cells, whereas DNA synthesis of FA cells was greatly inhibited 24 and 48 h after treatment. This effect was due to a decrease in the number of DNA-synthesizing cells, while the amount of radioactivity incorporated per cell (as measured with grain counting in autoradiograms) remained the same. These findings indicate that the inhibition of semiconservative DNA synthesis induced by MMC in FA cells is not due to an inhibitory effect of unrepaired lesions on the rate of DNA synthesis but rather to a block in cell cycle progression.

Pepsinogen synthesis in monolayer culture of human and rabbit gastric mucosal cells.

We have established monolayer cultures of human and rabbit gastric mucosal cells and of isolated rabbit gastric chief cells. These cultures were capable of de novo pepsinogen synthesis and secretion, demonstrated by electrophoresis and subsequent autoradiography of cell lysates and growth medium after culture in the presence of 14C-labelled amino acids. Cultures could be maintained for 1 week without overgrowth by fibroblasts.

Inability of chemically generated singlet oxygen to break the DNA backbone.

The capacity of a photodynamic and a chemical source of singlet molecular oxygen to cause DNA strand breakage at pH 7.8 was compared in the following systems: (1) dissolved rose bengal plus light (400-660 nm), (2) a novel water-soluble naphthalene-derived endoperoxide showing temperature-dependent singlet oxygen release, in the absence of light. Covalently closed circular DNA was efficiently converted to the open (relaxed) form upon exposure to dissolved rose bengal plus light in a time-dependent reaction, showing that this system was capable of causing DNA strand breakage at pH 7.8. The reaction was greatly reduced under hypoxic conditions (less than 5 p.p.m. O2), was stimulated when using D2O instead of H2O as a solvent and was not inhibitable by superoxide dismutase, indicating that singlet oxygen was a critical intermediate. However, comparatively large fluxes of singlet oxygen generated by the endoperoxide completely failed to produce DNA strand breaks. We conclude that, although singlet oxygen seems to play a role in DNA strand breakage by rose bengal plus light, singlet oxygen per se is very inefficient if not completely incapable of causing DNA strand breakage.

Fanconi anaemia cells are not uniformly deficient in unhooking of DNA interstrand crosslinks, induced by mitomycin C or 8-methoxypsoralen plus UVA.

Fanconi anaemia (FA) cells are extremely sensitive to crosslinking agents, e.g. mitomycin C, but only moderately sensitive to trimethylpsoralen plus UVA. Evidence has been reported suggesting that there is a deficient DNA crosslink repair mechanism in FA cells, but others failed to confirm this conclusion using other methods and other crosslinking agents. We reinvestigated the mitomycin C and 8-methoxypsoralen crosslink repair in FA cells with a high sensitivity to mitomycin C. Although an essentially similar methodology was used to that previously described, no difference between the control and FA cell strains was observed, neither for mitomycin C- nor for 8-methoxypsoralen-induced crosslinks.