DNA microarrays can be used to formulate a molecular predictor of survival after chemotherapy for diffuse large-B-cell lymphoma.

Comparative genomic hybridization was applied to 5 breast cancer cell lHes and 33 primary tumors to discover and map regions of the genome with increased DNAsequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copynumber affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression.Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.Increased expression of specific genes plays an important role in the pathogenesis of solid tumors (1-3). Gene amplification, characterized by distinct cytogenetic structures, such as homogeneously stained regions, double-minute chromosomes (1-7), is commonly found in tumor cells and is considered an important mechanism by which tumor cells gain increased levels of expression of critical genes. Increased copy-numbers also occur as a result of extensive chromosomal rearrangements, such as duplications, isochromosomes, extra marker chromosomes, and acentric chromosomal fragments that may affect the gene dosage of numerous genes simultaneously. In breast cancer, cytogenetic evidence of increased DNA-sequence copy-number is common (4-7). For example, homogeneously stained regions have been found in 60% of primary breast carcinomas (7). Although genetic analysis has found amplification of oncogenes, such as ERBB2 (17q12), MYC (8q24), PRADII CYCLIN D (11q13), FLG (8p12), BEK (10q24), and IGFR-1/FES (15q24-q25) (8-12), in most cases these do not explain the presence of large homogeneously stained regions (13). Thus, amplification of currently unknown genes may often occur in breast cancer.We have recently developed a method, comparative genomic hybridization (CGH), for surveying entire genomes for DNA-sequence copy-number variation (14, 15). In CGH, the relative intensities of tumor DNA (detected using green fluorescence) and normal reference DNA (detected with red fluorescence) after hybridization to normal metaphase chromosomes is used to reveal and map regions of increased DNA-sequence copy number (14-16). These loci are visualized as chromosomal region(s) with predominantly green fluorescence ( Fig. 1) and quantified by digital image analysis as an increased green-to-red fluorescence intensity ratio (Fig. 2). As no specific probes or previous knowledge of aberrations is required, CGH is especially suitable for identification and mapping of previously unknown DNA copy-number changes that may highlight locations of important genes. In the present study, we have used CGH to identify and map increases in DNA-sequence copy number in 15 breast cancer cell lines and 33 uncultured primary breast tumors. MATERIALS AND ME...

We describe here the development of a new approach to the analysis of Escherichia coli replication control. Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with Gl, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division. Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass. The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9%o. Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation. In dnaA(Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that forwild-type cells. In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony. The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.Normally, cells growing under steady-state conditions duplicate their chromosomal complement once and only once between each cell division. In any given steady-state culture of Escherichia coli cells, initiation of DNA replication at the chromosomal origin, oriC, occurs at a specific time in the cell cycle and at a specific cell mass. Two questions regarding the relationship between cell growth and initiation are of particular importance.First, how is the timing of initiation coupled to cell growth? An early hypothesis suggested that the initiation process responds to cell mass per se (1). According to this model, initiation always occurs at a fixed ratio of mass to origins-i.e., the initiation mass is a constant which is independent of the growth conditions. More recent evidence suggests, however, that the situation is more complicated, since the experimentally determined initiation mass varies significantly with growth rate (2, 3).Second, how variable is the mass at the time of initiation, when the cells grow under steady-state conditions? The extent of variation indicates the tightness of coupling between initiation and cell physiology, of which cell mass is an indicator: the less the variation, the tighter the coupling.Appropriately controlled replication initiation requires the assembly of the replication machinery at oriC. A central component of this assembly is the initiator protein DnaA.

SummaryComparative genomic hybridization (CGH) is used to detect amplified and/or deleted chromosomal regions in tumours by mapping their locations on normal metaphase chromosomes. Forty-five sporadic colorectal carcinomas were screened for chromosomal aberrations using direct CGH. The median number of chromosomal aberrations per tumour was 7.0 (range 0-19). Gains of 20q (67%) and losses of 18q (49%) were the most frequent aberrations. Other recurrent gains of 5p, 6p, 7, 8q, 13q, 17q, 19, X and losses of 1p, 3p, 4, 5q, 6q, 8p, 9p, 10, 15q, 17p were found in > 10% of colorectal tumours. High-level gains (ratio > 1.5) were seen only on 8q, 13q, 20 and X, and only in DNA aneuploid tumours. DNA aneuploid tumours had significantly more chromosomal aberrations (median number per tumour of 9.0) compared to diploid tumours (median of 1.0) (P < 0.0001). The median numbers of aberrations seen in DNA hyperdiploid and highly aneuploid tumours were not significantly different (8.5 and 11.0 respectively; P = 0.58). Four tumours had no detectable chromosomal aberrations and these were DNA diploid. A higher percentage of tumours from male patients showed Xq gain and 18q loss compared to tumours from female patients (P = 0.05 and 0.01 respectively). High tumour S phase fractions were associated with gain of 20q13 (P = 0.03), and low tumour apoptotic indices were associated with loss of 4q (P = 0.05). Tumours with TP53 mutations had more aberrations (median of 9.0 per tumour) compared to those without (median of 2.0) (P = 0.002), and gain of 8q23-24 and loss of 18qcen-21 were significantly associated with TP53 mutations (P = 0.04 and 0.02 respectively). Dukes' C/D stage tumours tended to have a higher number of aberrations per tumour (median of 10.0) compared to Dukes' B tumours (median of 3.0) (P = 0.06). The low number of aberrations observed in DNA diploid tumours compared to aneuploid tumours suggests that genomic instability and possible growth advantages in diploid tumours do not result from acquisition of gross chromosomal aberrations but rather from selection for other types of mutations. Our study is consistent with the idea that these two groups of tumours evolve along separate genetic pathways and that gross genomic instability is associated with TP53 gene aberrations.

It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We

In wild‐type Escherichia coli cells, initiation of DNA replication is tightly coupled to cell growth. In slowly growing dnaA204 (Ts) mutant cells, the cell mass at initiation and its variability is increased two‐ to threefold relative to wild type. Here, we show that the DnaA protein concentration was two‐ to threefold lower in the dnaA204 mutant compared with the wild‐type strain. The reason for the DnaA protein deficiency was found to be a rapid degradation of the mutant protein. Absence of SeqA protein stabilized the DnaA204 protein, increased the DnaA protein concentration and normalized the initiation mass in the dnaA204 mutant cells. During rapid growth, the dnaA204 mutant displayed cell cycle parameters similar to wild‐type cells as well as a normal DnaA protein concentration, even though the DnaA204 protein was highly unstable. Apparently, the increased DnaA protein synthesis compensated for the protein degradation under these growth conditions, in which the doubling time was of the same order of magnitude as the half‐life of the protein. Our results suggest that the DnaA204 protein has essentially wild‐type activity at permissive temperature but, as a result of instability, the protein is present at lower concentration under certain growth conditions. The basis for the stabilization in the absence of SeqA is not known. We suggest that the formation of stable DnaA–DNA complexes is enhanced in the absence of SeqA, thereby protecting the DnaA protein from degradation.

Summary The mode of cell death induced by photodynamic treatment (PDT)

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O2), 7% O2 induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O2 induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O2 also extended the G0/G1 stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44bright cells. Correspondingly, the sorted CD44bright cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44dim cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44bright cells under normoxia formed significantly more colonies and spheres compared with the CD44dim cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.