Computer repair st louis

ABSTRACT

Spatial positioning of pericentric chromosome regions in human lymphocyte cell nuclei was investigated during repair after H^sub 2^O^sub 2^/L-histidine treatment. Fifteen to three-hundred minutes after treatment, these regions of chromosomes 1, 15, and X were labeled by fluorescence in situ hybridization. The relative locus distances (LL-distances), the relative distances to the nuclear center (LC-distances), and the locus-nuclear center-locus angles (LCL-angles) were measured in ~5000 nuclei after two-dimensional microscopy. Experimental frequency histograms were compared to control data from untreated stimulated and quiescent (G^sub 0^) nuclei and to a theoretical two-dimensional projection from random points. Based on the frequency distributions of the LL-distances and the LCL-angles, an increase of closely associated labeled regions was found shortly after repair activation. For longer repair times this effect decreased. After 300 min the frequency distribution of the LL-distances was found to be compatible with the random distance distribution again. The LL-distance frequency histograms for quiescent nuclei did not significantly differ from the theoretical random distribution, although this was the case for the stimulated control of chromosomes 15 and X. It may be inferred that, concerning the distances, homologous pericentric regions appear not to be randomly distributed during S-phase, and are subjected to dynamic processes during replication and repair.

INTRODUCTION

The three-dimensional architecture of the cell nucleus is not randomly organized but has a functional meaning (T. Cremer et al., 1993, 2000; C. Cremer et al., 1996; Cremer and Cremer, 2001; Parada et al., 2004). It has been shown by means of whole chromosome painting that each chromosome occupies a distinct, mutually exclusive territory in the cell nucleus. The radial positioning of these territories and their subchromosomal elements are relatively fixed, depending upon the gene density or genetic activity, respectively. Gene-dense chromosomes are found in the interior; gene-poor chromosomes are found in the periphery of a nucleus (M. Cremer et al., 2001, 2003; Kozubek et al., 2002; Falk et al., 2002). Computer simulations support the concept of global positioning that is dependent upon DNA content and gene density of a chromosome territory (Kreth et al., 2004).

In contrast to the radial positioning, the angular positioning of chromosomes and subchromosomal elements has been found to be random (Kozubek et al., 2002; Luk??sov?? et al., 2002; Falk et al., 2002). Within chromosome territories the centromeres show a peripheral orientation whereas the telomeres are positioned toward the nuclear center (Amrichov?? et al., 2003; Weierich et al., 2003). In addition, the centromeres seem to play a significant role in the functional correlation of genome architecture and gene expression (Volpi et al., 2000; B??rtov?? et al., 2002; Taslerov?? et al., 2003). The functionally determined positioning of chromosome territories was evolutionarily conserved (Tanabe et al., 2002), and seems to follow similar rules for several types of normal cells, as in, for instance, lymphocytes, fibroblasts, or amniotic fluid cells (M. Cremer et al., 2001; Boyle et al., 2001).

During the cell cycle (G^sub 1^-S-G^sub 2^ phase) the chromosome territories undergo only very limited large-scale translational movements, whereas more extended positional changes were observed during the first few hours after mitosis (Parada and Misteli, 2002; Walter et al., 2003). Quantitative live cell imaging suggests that chromosome territories are only subjected to an independent Brownian-diffusionlike motion (Edelmann et al., 2001). A higher mobility with diffusion coefficients in the order of 10^sup -2^ ??m^sup 2^/s has been reported for Drosophila and yeast (Gasser, 2002; Marshall, 2002). More complex rearrangements of chromosome territories have been found during mitosis and it is controversially discussed, how and whether chromosome positioning is maintained in daughter cell nuclei (Walter et al., 2003, Gerlich et al., 2003).

Besides such open questions of chromosome rearrangements during the normal cell cycle, chromosome organization, under certain functional conditions, has been reported that can be the result of large-scale chromosome movement-as in, for instance, homologous chromosome pairing in meiosis. The meiotic homolog pairing allows the exchange of DNA between homologous chromosomes that is known as genetic recombination (Scherthan, 2001). Similarly, homologous chromosome association has been observed in somatic cells. Homologous chromosome pairing during interphase has, for instance, been known for many decades for Drosophila (Metz, 1916). Somatic association of homologous chromosomes and/or subchromosomal regions has also been described for different mammalian cell types in different pathophysiological states (Arnoldus et al., 1989; Marschio et al., 1992; Lewis et al., 1993; La Salle and Lalande, 1996; Stout et al., 1999). In radiation biology, the rearrangement of chromosomes and subchromosomal elements like centromeres has been studied in human cell lines after ?¡À-irradiation (Aten et al., 2004) and after ??-irradiation of a few Gy (Dolling et al., 1997; B??rtov?? et al., 1999; Jirsov?? et al., 2001). Two and five hours, respectively, after irradiation, the homologous chromosomes or centromeres are located closer to each other than in the not-irradiated control. Recent findings described association processes after 4-Gy irradiation during the first hour of repair for heterochromatic regions, but not euchromatic regions (Abdel-Halim et al., 2004). These results, in principle, indicated a tendency toward homolog association during repair.

The work presented here is addressed to study whether somatic homolog association is a general feature of human peripheral blood lymphocytes after repair activation induced by strand breaks. In >5000 cell nuclei, homologous locus distances (LL-distances), locus-to-nuclear center distances (LC-distances), and the locus-nuclear center-locus angles of pericentric regions have been determined interactively for different chromosomes and different repair times after H^sub 2^O^sub 2^/L-histidine treatment, which is known to induce DNA single- and double-strand breaks (Szumiel et al., 1995), thus activating DNA repair. Assuming that centromere association is a basic effect for the association of whole chromosome territories, homolog association may be an essential effect to correlate two homologous DNA matrices for replication involved in repair activities and recombination.

MATERIALS AND METHODS

Cell preparation

Whole peripheral blood samples from two healthy female donors were taken into 0.5% heparin solution. The lymphocytes were stimulated and cultivated in tissue flasks with chromosome growth medium B, which included phytohemaglutinin (Biochrom, Berlin, Germany) for 78 h at 37?¡ãC. To enrich cells in G^sub 1^/S-phase, they were treated with thymidine (Sigma, St. Louis, MO) at a concentration of 300 ??g/ml and incubated for another 16 h. To identify S-phase cells, the cultures were pulse-labeled with bromodeoxyuriiline (BrdU). The cells were then centrifuged for 10 min at 200 g and washed once with fresh chromosome growth medium B, and the cells were incubated for 30 min in the presence of BrdU at a concentration 10 ??g/ml (Sigma). BrdU incorporation was stopped by centrifugation of the cell suspension at 200 g for 10 min and resuspending the pellet in a hypotonic solution containing 75 mM KCl (Carl Roth, Karlsruhe, Germany). After incubation at 37?¡ãC for 30 min and centrifugation at 200 g for 10 min, the cells were fixed either with cold (-20?¡ãC) methanol/acetic acid (3:1) according to standard procedures or with formaldehyde (see next paragraph).

H^sub 2^O^sub 2^/L-histidine treatment

The cells were treated with H^sup 2^O^sub 2^, which is known to induce single-strand DNA breaks (Szumiel et al., 1995). In combination with L-histidine, double-strand breaks are also induced (Sestiti et al., 1995; Hausmann et al., 1998). After BrdU incorporation, the cells were washed with fresh chromosome growth medium B and centrifuged at 200 g for 10 min. Ten milliliters of prewarmed (37?¡ãC) PSA (Gibco BRL, Invitrogen, Gaithersburg, MD) was carefully added and mixed into the cell pellet. After centrifugation (200 g for 10 min), the cell pellet was resuspended in 25 ml H^sub 2^O^sub 2^ mixture (100 ml PBS + 100 ??l H^sub 2^O^sub 2^ + 0.0209 g L-histidine resulting in 10 ??M H^sub 2^O^sub 2^ and 1 mM L-histidine final concentration in PBS; all chemicals from Sigma) for 30 min at 37?¡ãC. The pellet was resuspended in 25 ml fresh chromosome growth medium B after centrifugation at 200 g for 10 min and incubated for 15, 30, 60, 120, and 300 min at 37?¡ãC. Then the interphase nuclei were isolated as described above.