[Diagnosis of hemophilia A carrier state using analysis of intergenic and intergenic polymorphism of factor VIII gene]. / Diagnostyka nosicielstwa hemofilii a na podstawie analizy wewnatrzgenowego i okologenowego polimorfizmu genu czynnika VIII.

Carrier state of haemophilia A was assessed in women at risk by using Bcl I polymorphism of factor VIII gene in intron 18, CA repeating sequence polymorphism in intron 13, and intergenic polymorphism at DXS52 locus. Appropriate fragments of DNA in intron 18 and 13 were amplified by PCR. Polymorphism at DXS52 locus was determined by the method of Southern after digesting DNA with endonuclease Taq I and employing F814 and ST14-1 probes. The diagnosis of carrier state of haemophilia A was made in 12 women from 9 families.

Modification by caffeine of acute cytotoxic response of cultured L5178Y cells to hydroxyurea treatment.

The effect of caffeine (CAF) on acute cytotoxic response of L5178Y lymphoblasts to hydroxyurea (HU) treatment was studied. The following events were examined: abnormal cell enlargement (giant cell formation), the rate of recovery of cell reproduction and DNA synthesis after releasing the cells from the HU blockage, parental DNA breakage and cell death. The presence of CAF at nontoxic concentration prevented giant cell formation, enhanced cell growth inhibition and cell killing. The effect of CAF was variable, dependent on the duration of exposure to HU and the time of exposure to CAF. To obtain maximal effect, the continuous presence of CAF during HU treatment and posttreatment time was necessary. Hydroxyapatite chromatography assay of single strand (ss) and double strand (ds) fractions in parental DNA and the measurement of the rate of post-treatment recovery of DNA synthesis indicated that CAF enhanced HU-induced DNA lesions. It is concluded that the results give further evidence that even short HU treatment can damage not only newly formed but also parental DNA. The lesions are normally, at least partly repaired and can be expressed under the conditions of DNA repair inhibition.

Mechanism of unbalanced growth-induced cell damage. II. A probable relationship between unbalanced growth, DNA breakage and cell death.

This study examines the relationship between unbalanced growth, DNase II activity, DNA breakage and cell survival during the exposure of L5178Y cells to hydroxyurea (HU), excess thymidine (dThR) or HU with excess of four deoxyribonucleosides (dNR). It has been found that in the cells arrested by HU or dThR, but still appearing viable with the trypan blue exclusion test, Protein/DNA imbalance and abnormal cell volume are correlated with enhancement of DNase II activity in the cells and in the medium and with moderate increase in parental DNA breakage. The incidence of DNA breaks was markedly potentiated in the presence of non-toxic concentration of caffeine (CAF), used to inhibit DNA repair. In HU+dNR arrested cells, in which unbalanced growth was abolished, enhancement of DNase II activity and of DNA breakage in the presence or absence of CAF was substantially prevented. Comparison of posttreatment cell survival in the presence or absence of CAF confirmed the differential effect of CAF: while in HU or dThR arrested cells the presence of CAF induced marked cell killing, in HU+dNR arrested cells the influence of CAF was negligible. Only a slight effect of CAF was observed in cells in which dThR-induced arrest and unbalanced growth were reversed by deoxycytidine (dCR) addition. It is suggested that the involvement of DNA nucleases in the unbalanced growth-induced overproduction of numerous hydrolytic enzymes, with their progressive leakage through the cell membranes, can lead to progressive DNA digestion. DNA breaks produced in this way are normally, at least partly, repaired. Concomitant exposure of such cells to DNA repair inhibitor can markedly enhance the level of breaks, leading to potentiation of unbalanced growth-induced cell killing.

Intragenomic distribution of 5-methylcytosine in various forms of human and murine leukemic cells.

The degree of 5-methylcytosine formation in DNA sequences differing in reassociation rate and susceptibility to DNase II digestion has been investigated in human chronic myelogenic leukemia and acute leukemia leukocytes, human PHA-stimulated lymphocytes and murine L5178Y lymphoblasts cultured in various phases of growth. The results indicate that in all forms of cells studied by us the general pattern of intragenomic 5-methylcytosine distribution is similar, with two preferentially methylated regions: the sequences fast reassociating and rendered Mg++-soluble after DNase II digestion of nuclei. The most variable fraction as regards the level of methylation seemed to be DNA of Mg++-soluble fraction of DNase II digest, which in acute leukemia leukocytes, PHA-stimulated lymphocytes and exponentially growing L5178Y cells revealed about twice greater relative proportion of methylated cytosines than in leukocytes of chronic myelogenic leukemia and L5178Y cells maintained at saturation density.

Heterogeneity of DNA methylation in murine L5178Y lymphoblasts.

Comparison of the extent of methylation in mouse DNA fragments rendered MgCl2 soluble after mild DNase II digestion of nuclei, with different reassociation rate and nucleoli-bound, revealed the existence of 3 regions of the genome particularly 5-methylcytosine-rich: the sequences considered to be related to the transcriptionally active chromatin with the highest content of this base and fast reassociating, as well as nucleolar DNA with somewhat lower proportion of the methylated cytosines.

DNA methylation in various growth phases of cultured L5178Y murine lymphoblasts.

The extent of methylation of DNA during the transition of cells from exponential phase of growth to the saturation density has been studied in cultured L5178Y mouse lymphoma cells by the comparison of the degree of 5-methylcytosine formation and the stability of this preformed base in various periods of their growth. It has been found that while the degree of 5-methylcytosine formation gradually fell down, the level of this base preformed during the exponential growth was stable during the subsequent periods until the saturation density was reached. At that state some small and variable loss of the base was found.

Differential cell-cycle-dependent inhibition of DNA synthesis by hydroxyurea and cytosine arabinoside in cultured L5178Y mouse lymphoma cells.

The effect of various concentrations of hydroxyurea and cytosine arabinoside on DNA synthesis, measured as 3H-thymidine incorporation, in exponentially growing and synchronized L5178Y cells in culture was investigated. The experiments revealed that each compound causes differential inhibition of DNA synthesis at various times of the S-phase. Maximum sensitivity to hydroxyurea occurred in cells in the late S, and maximum sensitivity to cytosine arabinoside in the early S-phase. When they were given simultaneously in low concentrations an additive effect was observed.

Fractionation of mouse DNA by precipitation with F1 histone intro fragments differing in their base composition.

Several fractions of mouse DNA were obtained by gradual precipitation with histone F1. The analysis of their base compositon revealed that histone interacted selectively with sequence of DNA rich in adenine plus thymine, regardless of the type of DNA molecules present in the DNA solution to be fractionated.

[Differential inhibition of synthesis and methylation of DNA fragments by hydroxyurea in L5178Y lymphoblasts in vitro]. / Róznicowe hamowanie syntezy i metylacji fragmentów dna przez hydroksymocznik w limfoblastach L5178Y in vitro.

In cultures of mouse L5178Y lymphoblasts treated with low concentrations of hydroxyurea during the logarithmic phase of growth differential inhibition of synthesis and methylation of various DNA fragments was observed. Some evidence is presented that the effect may be due to different sensitivity of cells to the action of hydroxyurea at various times of the S-phase.

Comparison of hydroxyurea and excess thymidine as synchronizing agents in cultures of L5178Y mouse lymphoma cells.

Comparison of double hydroxyurea and hydroxyurea-excess thymidine blockades as synchronizing procedures in cultured L5178Y cells revealed that hydroxyurea mimics the effect of thymidine as regards the degree of the resulting synchrony evaluated by the mitotic index and 3H-thymidine incorporating activity. However, the peaks of the mitotic and 3H-incorporating activity appear later after thymidine than after hydroxyurea blockade and the initial maximum incorporation of 3H-deoxycytidine into DNA was about twice higher in thymidine- than in hydroxyurea-treated cells. On the other hand, the timing of 14C-formate incorporation into purine DNA bases was closely similar in both cell populations.

Regulation of growth and DNA synthesis in mouse lymphoma L5178Y cells under conditions of nutrient depletion.

The effect of cultivation of L5178Y lymphoblasts to high density and in two kinds of nutrient-depleted medium on their growth was observed. It was found that in cell population grown to stationary phase the mitotic index and labeled thymidine incorporation dropped about 3 times in comparison with exponential control. Upon dilution with fresh medium the cells resume traverse the life cycle in partially synchronized manner. Cultivation of the cells in medium with 0-5% serum induced very rapid loss of their viability. Cultivation of the cells in medium without asparagine and with dialyzed serum caused significant slowing of their growth-rate and upon dilution with complete medium the cells entered the exponential rate of division in an asynchronous manner.