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Abstract
Dermal fibroblasts cultured from members of a family presenting multiple polyps and sarcomas were compared with fibroblast strains from unrelated healthy donors for sensitivity to killing by four genotoxic agents. Cells from the sister of the male proband (strain 3437T), mother (strain 3703T), two of his paternal aunts (3701T and 3704T) and one paternal uncle (3702T) displayed marked resistance (1.8 to 4.3 times greater than the normal mean) to 4-nitroquinoline 1-oxide (4NQO), a procarcinogen whose DNA-damaging properties encompass those of both far (254 nm) ultraviolet (UV) light and ionising radiation. These same 4NQO-resistant cells, however, responded normally to reproductive inactivation by UV light, 60Co gamma radiation or the alkylating agent methylnitrosourea, signifying that the abnormal resistance of these cells to 4NQO is not associated with aberrant DNA metabolism. In keeping with this conclusion, exposure to a given dose of 4NQO produced decreased amounts of DNA damage and stimulated lower levels of repair DNA synthesis in all five 4NQO-resistant strains than in normal controls. Moreover, exogenous radiolabelled 4NQO accumulated to a lesser extent in the 4NQO-resistant than in the normal fibroblasts. Cell sonicates of strains 3437T, 3701T and 3702T exhibited reduced capacities (40-60% of normal) to catalise the conversion of 4NQO to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide. However, the 4NQO-resistant strains 3703T and 3704T carried out 4NQO bioreduction at normal rates. Our data therefore indicate that enhanced resistance to 4NQO cytotoxicity in 3437T, 3701T and 3702T is a consequence of anomalies in both intracellular accumulation and enzymatic reduction of 4NQO, whereas 4NQO resistance in 3703T and 3704T appears to result solely from reduced intracellular drug accumulation.
Highlights
To explore the basis of the unusual carcinogen-resistance phenotype displayed by fibroblast strains from available informative members of the cancer-prone family under study, we have compared these strains with normal controls with respect to: (i) initial yield of DNA damage formed upon exposure to a given concentration of 4-nitroquinoline 1-oxide (4NQO); (ii) capacity to perform DNA repair following 4NQO treatment; and (iii) kinetics of intracellular accumulation and rate of bioreduction of the compound
This study demonstrated that skin fibroblast strains derived from five members of a polyposis/sarcoma family (Figure 1; Table I) exhibit abnormal resistance to reproductive inactivation by 4NQO (Table II), a partially UV-mimetic and radiomimetic carcinogen (Regan & Setlow, 1974; Hanawalt et al, 1979; Smith & Paterson, 1980)
This unusual cytotoxic response for noncancerous cell types correlated with the introduction of an abnormally low amount of genomic DNA damage on exposure to a given concentration of 4NQO, as a result of decreased accumulation of the chemical in resistant compared to normal fibroblasts (Table III)
Summary
The cells were cultured at 37°C in Ham's F12 medium supplemented with 10% (v/v) foetal bovine serum (Bockneck Laboratories Inc., Toronto, ON), 1 mM glutamine and antibiotics (100 IU ml-' penicillin G and 100 l.g ml-' streptomycin sulphate; GIBCO Laboratories, Grand Island, NY) in a humidified atmosphere of 5% CO2 in air. All strains were used between passages 10 and 18 (1:3-split passage). For alkaline sucrose sedimentation analysis, cellular DNA was labelled by incubating exponentially growing cultures for 24 h in thymidine (dThd)-free medium containing 2.4 x 101" Bq mmol-' [methyl-3H]dThd at 1.8 x l04 Bq ml-' or 2.0 x I0O Bq mmol-' [methyl-14C]dThd at 3.7 x 104Bq ml-' (New England Nuclear Canada, Lachine, PQ)
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