Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase α inhibitor

The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase α, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells.     

Protein kinase C β inhibition by enzastaurin leads to mitotic missegregation and preferential cytotoxicity toward colorectal cancer cells with chromosomal instability (CIN)

Enzastaurin is a selective inhibitor of protein kinase C β and a potent inhibitor of tumor angiogenesis. In addition, enzastaurin shows direct cytotoxic activity toward a subset of tumor cells including colorectal cancer cells (CRC). In spite of promising results in animal models, the clinical activity of enzastaurin in CRC patients has been disappointing although a subset of patients seems to derive benefit. In the present study we investigated the biological and cytotoxic activities of enzastaurin toward a panel of well-characterized CRC cell lines in order to clarify the mechanistic basis for the cytotoxic activity. Our results show that enzastaurin is significantly more cytotoxic toward CRC cells with chromosome instability (CIN) compared to cells with microsatellite instability (MSI). Since CIN is usually attributed to mitotic dysfunction, the influence of enzastaurin on cell cycle progression and mitotic transit was characterized for representative CIN and MSI cell lines. Enzastaurin exposure was accompanied by prolonged metaphase arrest in CIN cells followed by the appearance of tetraploid and micronuclei-containing cells as well as by increased apoptosis, whereas no detectable mitotic dysfunctions were observed in MSI cells exposed to isotoxic doses of enzastaurin. Our study identifies enzastaurin as a new, context dependent member of a heterogeneous group of anticancer compounds that induce “mitotic catastrophe," that is mitotic dysfunction accompanied by cell death. These data provide novel insight into the mechanism of action of enzastaurin and may allow the identification of biomarkers useful to identify CRC patients particularly likely, or not, to benefit from treatment with enzastaurin.     

Altered replication in human cells promotes DMPK (CTG)(n) · (CAG)(n) repeat instability

Myotonic dystrophy type 1 (DM1) is associated with expansion of (CTG)(n) · (CAG)(n) trinucleotide repeats (TNRs) in the 3' untranslated region (UTR) of the DMPK gene. Replication origins are cis-acting elements that potentiate TNR instability; therefore, we mapped replication initiation sites and prereplication complex protein binding within the ~10-kb DMPK/SIX5 locus in non-DM1 and DM1 cells. Two origins, IS(DMPK) and IS(SIX5), flanked the (CTG)(n) · (CAG)(n) TNRs in control cells and in DM1 cells. Orc2 and Mcm4 bound near each of the replication initiation sites, but a dramatic change in (CTG)(n) · (CAG)(n) replication polarity was not correlated with TNR expansion. To test whether (CTG)(n) · (CAG)(n) TNRs are cis-acting elements of instability in human cells, model cell lines were created by integration of cassettes containing the c-myc replication origin and (CTG)(n) · (CAG)(n) TNRs in HeLa cells. Replication forks were slowed by (CTG)(n) · (CAG)(n) TNRs in a length-dependent manner independent of replication polarity, implying that expanded (CTG)(n) · (CAG)(n) TNRs lead to replication stress. Consistent with this prediction, TNR instability increased in the HeLa model cells and DM1 cells upon small interfering RNA (siRNA) knockdown of the fork stabilization protein Claspin, Timeless, or Tipin. These results suggest that aberrant DNA replication and TNR instability are linked in DM1 cells.     

Knockdown of αII spectrin in normal human cells by siRNA leads to chromosomal instability and decreased DNA interstrand cross-link repair

Nonerythroid α-spectrin (αIISp) is a structural protein involved in repair of DNA interstrand cross-links and is deficient in cells from patients with Fanconi anemia (FA), which are defective in ability to repair cross-links. In order to further demonstrate the importance of the role that αIISp plays in normal human cells and in the repair defect in FA, αIISp was knocked down in normal cells using siRNA. Depletion of αIISp in normal cells by siRNA resulted in chromosomal instability and cellular hypersensitivity to DNA interstrand cross-linking agents. An increased number of chromosomal aberrations were observed and, following treatment with a DNA interstrand cross-linking agent, mitomycin C, cells showed decreased cell growth and survival and decreased formation of damage-induced αIISp and XPF nuclear foci. Thus depletion of αIISp in normal cells leads to a number of defects observed in FA cells, such as chromosome instability and a deficiency in cross-link repair.     

Is increased chromosomal diversity in house mice from Lombardy (Italy) congruent with genic divergence?

Recent studies in metacentric (MC) populations of the house mouse, Mus musculus domesticus, singled out underdominance more so than recombination suppression as the foremost barrier to gene flow. Here, MC populations from Lombardy (Italy) were sampled to identify the nature and strength of the barriers to gene flow. The chromosomal analysis recovered the three major MC populations (abbreviated to IBIN, IGAL, both with 2n = 24 and ICRE, 2n = 22), but revealed the existence of a new one (IONE, 2n = 24) which likely derived from IGAL through a single WART (Whole‐Arm Reciprocal Translocation). This, once again, highlights the paramount role of WARTs in the chromosomal diversification of this subspecies. Contacts between MC and standard populations coincided with rivers confirming these hybrid zones as tension zones. Divergence between populations was estimated using available allozyme data. Although the overall low genetic structure globally agreed with the chromosome structure, a large variation in divergence levels was retrieved that only partially matched the underdominance degree. This disparity from expectations highlighted the additional contribution of physical barriers and geographic isolation to the differential rate of evolution of the MC populations of the house mouse.     

Partial structure of the human α2(IV) collagen chain and chromosomal localization of the gene (COL4A2)

Summary We have isolated a 2.1-kb cDNA clone from a human placental library encoding part of the 2 chain of collagen IV, a major structural protein of basement membranes. The DNA sequence encodes 446 amino acids in the triplehelical domain plus the 227 amino acids of the carboxy-terminal globular domain. The latter structure is composed of two homologous subdomains and is highly conserved between the 1 and 2 chains. The triple-helical domain contained seven interruptions of the Gly-X-Y repeat and these interruptions were in general larger than their counterparts in the 1 chain. DNA from human rodent hybrid cell lines was analyzed under conditions in which there was no cross-hybridization of the 2(IV) cDNA probe with the gene for the 1(IV) collagen chain. An Eco RI fragment characteristic of the 2 chain had a concordance of 0.97 with chromosome 13. This result was confirmed and extended with in situ localization of the gene at 13q34. Since the 1(IV) gene has previously been localized to 13q34, the two type IV collagen genes reside in the same chromosome region (13q34), possibly in a gene cluster. The presence of the genes for type IV collagen chains on chromosome 13 excludes a primary role for these genes in adult polycystic kidney disease and X-linked forms of hereditary nephritis.     

Epstein–Barr Virus DNase (BGLF5) induces genomic instability in human epithelial cells

Epstein–Barr Virus (EBV) DNase (BGLF5) is an alkaline nuclease and has been suggested to be important in the viral life cycle. However, its effect on host cells remains unknown. Serological and histopathological studies implied that EBV DNase seems to be correlated with carcinogenesis. Therefore, we investigate the effect of EBV DNase on epithelial cells. Here, we report that expression of EBV DNase induces increased formation of micronucleus, an indicator of genomic instability, in human epithelial cells. We also demonstrate, using γH2AX formation and comet assay, that EBV DNase induces DNA damage. Furthermore, using host cell reactivation assay, we find that EBV DNase expression repressed damaged DNA repair in various epithelial cells. Western blot and quantitative PCR analyses reveal that expression of repair-related genes is reduced significantly in cells expressing EBV DNase. Host shut-off mutants eliminate shut-off expression of repair genes and repress damaged DNA repair, suggesting that shut-off function of BGLF5 contributes to repression of DNA repair. In addition, EBV DNase caused chromosomal aberrations and increased the microsatellite instability (MSI) and frequency of genetic mutation in human epithelial cells. Together, we propose that EBV DNase induces genomic instability in epithelial cells, which may be through induction of DNA damage and also repression of DNA repair, subsequently increases MSI and genetic mutations, and may contribute consequently to the carcinogenesis of human epithelial cells.     

Cloning and chromosomal assignment of the bovine interleukin-2 receptor alpha (IL-2Rα) gene

The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number U24226.     

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30–50 keV) and neutrons (14 MeV)

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).     

Is the Guadeloupean racoon (Procyon minor) really an endemic species? New insights from molecular and chromosomal analyses

The Guadeloupean racoon has been chosen as the emblematic species of the Parc National de Guadeloupe because of its supposed endemic specificity. However, its taxonomic position is not clearly defined. At what period did this animal colonize Guadeloupe and how it reached the island remain unsolved questions. To clarify these points, insular and continental individuals (Procyon lotor), coming from five localities and belonging to three subspecies, were compared using molecular (sequencing of a 294-bp of the left domain of the mtDNA control region) and chromosomal (C, G and R karyotypes) analyses. Genetic distance between the Guadeloupean racoon and lotor subspecies from Virginia and Maryland was very short (mean sequence divergence between haplotypes equals to 1.46%). A four times higher distance was found between P. l. lotor and P. l. pallidus, a continental subspecies inhabiting Arizona than between P. l. lotor and Procyon minor. A neighbour-joining phenogram is proposed; the Guadeloupean haplotype is on a branch close to the one clustering P. l. lotor from the East coast whereas the haplotype from Arizona diverged earlier. These results strongly suggest that the Guadeloupean racoon is included in the P. lotor species. Moreover, no differences were found between P. lotor and P. minor karyotypes, again suggesting a high genetic homogeneity between the two taxa. Furthermore, comparison of the control region variability within Procyon with intra- and interspecific variability observed in other Carnivores, clearly strengthens the hypothesis that P. minor is conspecific with P. lotor. These results imply that the taxonomic status of the Guadeloupean racoon should be revised. In a biogeographic perspective, it is suggested that the racoon has been recently introduced in Guadeloupe by man with individuals coming from the east coast of the USA. The consequences of these results for conservation of the racoon in Guadeloupe, and more generally, the definition and the use of the subspecies as a unit of conservation are discussed.     

How does DNA break during chromosomal translocations?

Chromosomal translocations are one of the most common types of genetic rearrangements and are molecular signatures for many types of cancers. They are considered as primary causes for cancers, especially lymphoma and leukemia. Although many translocations have been reported in the last four decades, the mechanism by which chromosomes break during a translocation remains largely unknown. In this review, we summarize recent advances made in understanding the molecular mechanism of chromosomal translocations.     

The sequence of the chromosomal mouse β-globin major gene: Homologies in capping,splicing and poly(A) sites

We have determined the entire nucleotide sequence of a cloned β-globin^maj gene derived from the BALB/c mouse. This sequence is 1567 bases long and includes the 5′ cap region as well as the presumptive poly(A) addition site of β-globin mRNA. The sequence establishes the fact that the gene is encoded in three discontinuous segments of DNA interrupted by two intervening sequences and precisely locates each. The smaller intervening sequence, 116 bases long, occurs between Arg and Leu codons at codon positions 30 and 31. The larger intervening sequence of 646 bases also occurs between Arg and Leu codons, but at codon positions 104 and 105. There is striking homology between the borders of the two intervening sequences, but no extensive dyad symmetry. Furthermore, the DNA region that just precedes and overlaps the 5′ cap structure of the mRNA shows homology to corresponding regions in other eucaryotic genes including the late adenovirus promoter. The 3′ untranslated sequence is closely homologous to that of the rabbit β-globin mRNA. The sequence thus allows us to identify several noncoding regions of potential importance for the expression and processing of genetic information. It also provides a basis for future comparison with other sequenced genes and a defined substrate for the development of direct tests of gene function.     

Recombination-suppression: how many mechanisms for chromosomal speciation?

Over the past decade several theoretical and empirical studies have revived interest in the role of chromosomes in speciation. The resulting models do not suffer from the problems experienced by previously proposed mechanisms of chromosomal speciation, because they invoke suppression of recombination rather than a reduction in the fitness of heterokaryotypes as their core process. However, they are not free from difficulties. The evidence for recombination-suppression models is discussed here. The general conclusion is that a consensus opinion on which models best describe the real-world situation is currently unlikely because of an inability of the available empirical evidence to fully distinguish between them, which may be due in part to a lack of exclusivity. I argue that future work should take this lack of exclusivity into account. Resolving the biogeography of speciation is also suggested in order to tell the various models apart. Further study is needed which focuses on confirming the operation of individual elements of the various models, rather than attempting to validate any single mechanism as a whole.     

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2(-•) and H2O2

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.     

Rare chromosomal aberrations in the Shereshevski?-Turner syndrome

Among 106 females with the Turner syndrome phenotype, two displayed rare chromosomal anomalies. In one patient, in addition to X-chromosome monosomy, among cultured lymphocytes cells with two isochromosomes made by long arms of X-chromosome were detected. Their frequency was 25%, and this value was the same in cultures obtained repeatedly after 3 and 5 months, which may suggest a certain stability of this clone. The other patient had a combination of two aberrations never reported before: the combination of isochromosome Xq and the Robertsonian translocation 13; 14. By their phenotype, these two women did not change from other patients with isochromosome Xq.