Biomedical subjects

O Popanda

Publications and source records attributed to O Popanda.

24 records · Page 2Linked to original sources

PubMed1989

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains.

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.

Adenosine Triphosphate↗

PubMed1988

Comparison of DNA-incising capacities in fibroblast strains from the Mannheim XP collection after treatment with N-acetoxy-2-acetylaminofluorene and UV light.

The DNA-incising capacity was determined in 8 normal and 23 XP fibroblast strains of the Mannheim XP collection using the alkaline elution technique after treatment with both UV light and the "UV-like" carcinogen (Ac)2ONFln. Experimental conditions were chosen to allow for selective monitoring of repair-specific enzyme-catalyzed breaks. In order to compare DNA-incising capacities of the various cell strains after UV irradiation with those after treatment with (Ac)2ONFln, dose-response experiments including up to 8 dose levels were performed. The elution curves were analyzed by linear regression analysis. Elution velocities (in terms of DNA single-strand breaks per 10(6) nucleotides) were plotted against the square root of the doses. The slope of the resulting regression line yielded a characteristic term, designated EO, for the DNA-incising capacity of each cell strain. In contrast to normal fibroblasts, EO was found to be reduced in all XP cell strains belonging to the complementation groups A, C, D, E, F (or G) and I investigated, after treatment with both UV light or (Ac)2ONFln. Surprisingly, XP variant strains also exhibited lower EO values. A comparison of post-UV with post-(Ac)2ONFln DNA-incising capacities revealed that reduction in the EO values was very similar in all XP cell strains tested. These data suggest that the sensitivity of XP cells towards UV light or (Ac)2ONFln is due to the same enzymatic defect, namely impaired incision of DNA containing pyrimidine dimers or (Ac)2ONFln-DNA adducts.

2-Acetylaminofluorene↗

PubMed1985

Xeroderma pigmentosum patients from the Federal Republic of Germany: decrease in post-UV colony-forming ability in 30 xeroderma pigmentosum fibroblast strains is quantitatively correlated with a decrease in DNA-incising capacity.

A total of 16 normal and 46 XP fibroblast strains from the Mannheim Collection were investigated for colony-forming ability following exposure to both UV light and the "UV-like" carcinogen (Ac)2ONFln. The dose-response experiments included up to 13 dose levels. The exponential segments of the curves were analysed by linear regression and the negative reciprocal of the regression coefficient (D0) was calculated for each cell strain. For quantitating the DNA-incising capacity, DNA elution curves were determined at several UV dose levels. Plotting the initial velocities of the elution curves versus the UV dose yielded a regression line, the slope of which was used to obtain the characteristic value E0. Comparing D0 with E0 values showed that cell strains in which colony-forming ability was reduced suffered a reduction of DNA-incising capacity of the same magnitude. There were only 3 exceptional strains in which reduction of DNA-incising capacity was less pronounced than reduction of colony-forming ability. We have previously shown (Fischer et al. 1982) that D0 values from 27 XP strains of the Mannheim Collection were correlated with clinical symptoms. This correlation is now being extended by relating colony-forming ability to the magnitude of the DNA incision defect. From our data we conclude that the best quantitative biochemical denominator to explain the sun sensitivity of XP is that of a defective incision of UV-damaged DNA. A considerable similarity in sensitivity towards both UV light and (Ac)2ONFln was found in 16 normal and 46 XP strains. This seems to indicate that UV- and (Ac)2ONFln-induced DNA damage are removed to a large extent by the same pathways in human fibroblasts.

Acetoxyacetylaminofluorene↗

PubMed1985

A ninth complementation group in xeroderma pigmentosum, XP I.

A new complementation group of excision-deficient xeroderma pigmentosum (XP) is described in 2 patients living in the F.R.G. Dermatological, ophthalmological and neurological symptoms of XP are presented together with DNA repair characteristics such as unscheduled DNA synthesis, colony-forming ability and alkaline elution studied in cultured fibroblasts. The results are compared to normal controls.

Adolescent↗

PubMed1983

Spontaneous in vitro malignant transformation in a xeroderma pigmentosum fibroblast line.

This paper deals with a spontaneous malignant transformation in one of our XP fibroblast lines. This cell line, designated XP29MA, was derived from a 14-year-old boy who did not show skin tumors or precancerous alterations either at the time of clinical examination or when the biopsy was taken. We have compared the following features in both the malignant and the benign cell line from which the malignant line developed: tumor formation in nude mice, repair capacity, cytogenetic status, light and electron microscopic characteristics. The benign cell line XP29MA had a doubling time of 4.3 d, did not form tumors in nude mice, showed a very low repair capacity (as determined by colony-forming ability, unscheduled DNA synthesis and alkaline elution) but exhibited a normal cytogenetic and ultrastructural status. In contrast, the transformed cell line XP29MAmal grew three times faster, formed colonies in methyl cellulose, gave rise to fibrosarcomas in nude mice, showed a drastically higher repair capacity, and was characterized by an extreme genetic imbalance, resulting from numerical and structural chromosome alterations of Nos. 1, 3, 4, 8, 12, 16, 17, 18, 20 and 21. Ultrastructural examination revealed fusiform and polygonal cells, the latter exhibiting large indented nuclei, vesicular dilatations of the endoplasmatic reticulum and numerous lysosomes. The higher repair capacity in XP29MAmal cells is tentatively explained in terms of reversion, enhancement of post-replication repair and/or expression of SOS-type functions.

Adolescent↗

PubMed1982

XP patients from Germany: correlation of colony-forming ability, unscheduled DNA synthesis and single-strand breaks after UV damage in xeroderma pigmentosum fibroblasts.

DNA repair capacity was investigated in 25 normal and XP fibroblast lines after UV damage was induced, using the following methods: colony-forming ability, unscheduled DNA synthesis, and alkaline elution (which can serve as a measure of repair-specific DNA incision). The majority of the XP fibroblast lines was derived from biopsies of patients who are at present under clinical observation by Dr. E.G. Jung (Dept. of Dermatology, Mannheim Medical School). Colony-forming ability was determined at 12 different UV dose levels and expressed in terms of D0. Unscheduled DNA synthesis was measured autoradiographically. Dose-response curves (grains per nucleus versus UV dose) were established and analyzed by linear regression. The characteristic value of G0, defined as the linear increase in the mean number of grains per nucleus when the UV dose is multiplied by the factor e (i.e., 2.72), was derived from the slope of the regression lines. For quantitating DNA-incising activity of a cell line, DNA elution curves were determined at several UV dose levels. Plotting of the initial velocities of the elution curves versus the UV doses yielded a regression line, the slope of which was used to obtain the characteristic elution value, E0. A descriptive correlation of all three characteristic values, D0, G0 and E0, showed that in all cell lines in which colony-forming ability and unscheduled DNA synthesis were diminished, a reduction of DNA-incising activity occurred. We conclude that this reduction accounts for both the decreased colony-forming ability and unscheduled DNA synthesis.

Autoradiography↗