Formation of benzo[a]pyrene-DNA adducts in blood monocytes from lung cancer patients with a familial history of lung cancer.

The in vitro formation of benzo[a]pyrene-DNA adducts was determined in peripheral blood monocytes of 22 lung cancer patients with at least one first-degree relative with lung cancer and compared to results obtained in 30 healthy controls. In patients, the mean (SEM) adduct formation was 2.8 (0.3) fmol/micrograms DNA as compared to 2.1 (0.1) fmol/micrograms in controls (p less than 0.05), and it was independent of age and smoking habits. These findings support the hypothesis that carcinogen-DNA adduct formation may be one factor of a constitutionally enhanced lung cancer risk.

Clinical and biochemical characterization of syndromes associated with defects of the insulin receptor.

During 3 years of collaboration we have detected 13 patients with insulin resistance syndromes among the 800 patients referred to our genetic counseling center for diagnosis during that time. It represents about 1.5% of all diagnostic problems of our genetic counseling center. Thus it seems to us that insulin receptor disorders are not as rare as previously appreciated. We describe here the clinical and biochemical findings of four of them as well as characteristics of disorders of insulin receptors. On cellular level these four patients represents the full scale of insulin receptor defects and demonstrate genetic heterogeneity of these disorders.

Elevated frequencies of micronuclei in cultured fibroblasts after freezing and thawing.

Spontaneous micronuclei (MN) were determined in 69 fibroblast lines in the first subculture after cryoconservation. 45 cultures (65%) showed micronuclei in the normal range (less than 10 MN/500 cells), but 24 (35%) exhibited an elevation up to 60 MN/500 cells. In order to determine whether freezing and thawing is responsible for the enhanced MN level we studied the persistence of elevated MN in 10 cell cultures after freezing and thawing, and found that the MN levels returned to normal after 3 subcultures. In addition, the micronucleus formation before and after freezing was investigated in 2 newly established cell cultures obtained from probands whose cells had a particularly high number of MN. Both cultures had regular MN levels before freezing but a more than doubled number of MN when frozen samples were recultivated. This effect of the freezing procedure was confirmed with 5 separate biopsies obtained from the same donor. Since a freezing-related increase in MN was constantly observed in cells from some individuals but not in others, it might be possible that it is a constitutive trait which causes cultured fibroblasts of some individuals to be hypersensitive to the freezing and thawing procedure. It is also noteworthy that fibroblasts from 8 out of 12 probands with a familial malignant melanoma exhibited this phenomenon.

32P-postlabelling analysis of DNA adducts in monocytes of smokers and passive smokers.

In a controlled study, ten male volunteers were subjected to different smoking and passive smoking conditions. After 60 h of strictly controlled nonsmoking, five smokers were exposed to mainstream smoke only, while five nonsmokers were exposed to the gas phase of environmental tobacco smoke (ETS). In a second experiment smokers were mainstream and ETS exposed, while nonsmokers were exposed to complete ETS. Blood was drawn before and after smoking and DNA adducts were analysed from blood monocytes by the 32P-postlabelling assay, using the nuclease P1 enhancement method. We detected DNA adducts in monocytes of all probands. These adducts unrelated to smoking showed interindividual differences but only minor intraindividual changes in four samples of the same donor. After smoking interindividually variable additional adducts were visible in active smokers only. These smoking-related adducts had disappeared after 40 h of nonsmoking and reappeared again in three out of five smokers after the second smoking period. We conclude that smoking causes an interindividually variable pattern of DNA adducts in active smokers. These adducts disappear in less than 2 d, owing to the fast turnover of monocytes in the intravascular system. The effects described could not be observed in heavily exposed passive smokers.

[Chromosome instability in patients with malignant melanoma of the skin]. / Chromosomale Instabilität bei Patienten mit malignem Melanom der Haut.

The certainty of a strong genetic predisposition to malignant melanoma was first established over 35 years ago. Since it has been shown that constitutive chromosomal instability is significantly correlated with the familial occurrence of cancer, we have studied spontaneous micronucleus rates in fibroblast cultures from 44 melanoma patients, 44 healthy probands and 78 patients with bronchial carcinoma. Here we report a significantly (p = less than 0.0005) increased spontaneous chromosomal instability in patients with cutaneous malignant melanoma compared to healthy controls and other tumor patients (bronchial carcinoma).

A new familial syndrome with impaired function of three related peptide growth factors.

We describe a new familial syndrome in three siblings; it is biochemically characterized by a combined defect of the action of the three related peptides insulin, insulin-like growth factor I (IGF I) and epidermal growth factor (EGF). Clinically, the disease has features of Werner syndrome with lipodystrophy, scleroderma-like alterations of the skin, alterations of the skeleton and contractures of joints. In addition, one of the patients has an insulin-resistant diabetes mellitus. Studies with cultured fibroblasts obtained from skin biopsies show a markedly reduced stimulation of RNA synthesis by the three growth factors and a decreased insulin stimulation of 2-deoxy-D-glucose uptake as compared with normal controls. Receptor binding of the three peptides occurred with normal capacity and affinity. We conclude that the signal transfer of different growth factors has a common denominator at the postreceptor level.

Ultraviolet-induced formation of micronuclei and sister chromatid exchange in cultured fibroblasts of patients with cutaneous malignant melanoma.

Genetically enhanced sensitivity to ultraviolet (UV) radiation may play an important role in the development of cutaneous malignant melanoma (CMM). This was studied in cultured fibroblasts of 26 CMM patients and controls by micronucleus (MN) test and sister chromatid exchange (SCE) after UV irradiation (375 J/m2). Sister chromatid exchange and MN formation were used as parameters to detect the UV-induced genotoxic damage in the individual cell strains. We found that the UV-induced level of MN was significantly increased in CMM patients (p = 0.0005), being most pronounced in the familial cases (p = 0.0001). Ultraviolet-induced SCE was also elevated in CMM patients (p = 0.001), but there was no difference between familial and nonfamilial cases. The present findings indicate that genetic predisposition contributes to the development of CMM in a subset of CMM patients and may be due to an enhanced susceptibility to UV light.

Formation of DNA adducts and water-soluble metabolites of benzo[a]pyrene in human monocytes is genetically controlled.

Formation of DNA adducts and of water-soluble metabolites was studied in monocytes of 86 first-degree relatives of 15 families. Tests were performed with blood monocytes using (G-3H)-benzo[a]pyrene as a model pro-carcinogen. Variance analysis revealed significantly higher inter-familial than intra-familial variations. From these data we conclude that the formation of DNA adducts is genetically controlled. Therefore the enhanced formation of benzo[a]pyrene DNA adducts in lung cancer patients found in earlier studies may reflect a genetic predisposition for lung cancer in some patients.

Impaired insulin-induced RNA synthesis secondary to a genetically defective insulin receptor.

The stimulation of glucose uptake and RNA synthesis by insulin was studied in cultured fibroblasts from patients with an inherited affinity defect of the insulin receptor. Additional cell cultures were set up of two patients with Alstrøm syndrome, a genetic disease with insulin resistance but normal insulin receptor, and of eight healthy individuals. In receptor-defective patients we found a corresponding defect of the insulin-mediated stimulation of RNA synthesis but a normal stimulation of glucose uptake. We conclude that both postreceptor effects are controlled by different insulin-directed mechanisms.

Heterozygous carriers for Bloom syndrome exhibit a spontaneously increased micronucleus formation in cultured fibroblasts.

Cultured fibroblasts of homozygotes and heterozygotes for Bloom syndrome exhibit an enhanced formation of micronuclei. The number of spontaneously occurring micronuclei permit clear separation of heterozygotes from either normal controls or homozygous patients without overlap between these groups. The observed differences could not be enhanced further by the addition of various mutagens. We conclude from the increased chromosomal damage that heterozygotes for Bloom syndrome may have a higher risk for malignant diseases.

Functional deficiency of fibroblasts heterozygous for Bloom syndrome as specific manifestation of the primary defect.

The effect on the rate of sister chromatid exchanges (SCEs) in Bloom syndrome fibroblasts by cocultivation with Fanconi anemia and xeroderma pigmentosum fibroblasts and with Bloom syndrome heterozygotes was studied. Cells of Fanconi anemia and xeroderma origin reduced the rate of SCEs in Bloom cells by about 45%-50%, just as control cells do. In contrast, heterozygous Bloom cells reduced the rate of SCEs by only 16%-28%. In absolute figures, Fanconi cells reduced the mean rate of SCE in Bloom cells from 55.7 +/- 5.50- to 27.7 +/- 6.44, xeroderma cells to 30.5 +/- 5.73, and control cells to 28.3 +/- 5.35. Three different cell strains from Bloom syndrome heterozygotes reduced the rate to 40.1 +/- 8.81, 47.0 +/= 6.94, and 47.5 +/- 8.32. There was no effect on any of these cell strains by Bloom syndrome fibroblasts. We interpret the functional deficiency of heterozygous Bloom syndrome fibroblasts as a gene dosis effect. It probably represents a specific manifestation of the yet unknown primary defect, because it suggests the existence of a "corrective factor" that is inactive or absent in homozygous Bloom cells and reduced in heterozygotes. It may be identical with or closely related to the normal gene product at the Bloom locus.

Mutagen-induced sister chromatid exchange rate in Bloom syndrome remains unaltered in the presence of Bloom corrective factor.

Fibroblasts of a patient with Bloom syndrome (GM-1492) were cultured in the presence of either mitomycin C, ethylmethanesulfonate, or 4-nitroquinoline-1-oxide, (4-NQ1-O) and sister chromatid exchange was determined. The mutagens enhanced the sister chromatid exchange rate to different degrees, 4-NQ1-O being the most potent substance. Bloom corrective factor, which is present in normal cell-conditioned culture medium, reduced the spontaneously increased SCE in Bloom syndrome cells by about 20 SCE per metaphase but failed to reduce the additional mutagen-induced SCE increase. These findings indicate that only spontaneously, but not mutagen-induced, SCE in Bloom syndrome fibroblasts can be decreased by the Bloom corrective factor.

Decreased rate of benzo(a)pyrene-induced sister chromatid exchange in fibroblast cultures from patients with lung cancer.

Fibroblast cultures from 29 lung cancer patients including eight familial cases and from 25 healthy controls were investigated. We studied the benzprene-induced sister chromatid exchange (SCE) following incubation in the presence of 150 nM benzo(a)pyrene and found spontaneously increased SCE values in the patients' cells but a reduced response to benzprene as compared to the control cultures. The benzpyrene-induced SCE was positively correlated with both benzpyrene metabolism and DNA adducts in normal control cells but not in the patients' cells. The ratio of benzypyrene DNA adducts and benzpyrene-induced rate of SCE calculated for the individual cell strains exhibited a bimodal distribution with patients' cells, but not with control cells. We speculate that the described differences between patients' and control fibroblasts might reflect a genetic predisposition to the development of lung cancer in about one half of the patients tested.