72–74 For instance, Rice et al. showed that over-replication of cellular DNA is induced by H/R, which is followed by amplification of the dihydrofolate reductase gene under methotrexate selection.73 Hypoxia followed by re-oxygenation also induces fragile sites that trigger DNA breakages and gene amplification.75 Fragile sites are chromosomal sites that show gaps and breaks after inhibition of DNA synthesis.76 They are usually associated with repetitive sequences with tri-, tetra- and dodeca-nucleotide repeats or with adenosine-thymidine (AT)-rich repeats. These repeats form DNA secondary structures. Based on these unique sequences in fragile

sites, Durkin and Glover proposed a molecular model for fragile site instability.77 LDK378 in vitro In this model, first, a dissociation of DNA-unwinding by the helicase/topoisomerase complex and DNA synthesis occurs when the action of DNA polymerase is inhibited. This creates XL765 order a long stretch of single-strand DNA around the fragile site. Second, AT-rich-repeats within a single strand of DNA form a hairpin

structure by self annealing. This structure further causes replication fork stalling. Although most of these structures will be detected and repaired by DNA repair machinery, some forks collapse, resulting in formation of single or double stand breaks, and present themselves as gaps or breaks on metaphase chromosomes Unoprostone at fragile sites.77 In support of this model, Pires et al. demonstrated that acute and severe hypoxia (<0.02% O2 for <8 h) blocks DNA synthesis of human cancers through inhibition of replication initiation and elongation. This blockage is due to the reduction of levels of the four dinucleotide triphosphate molecules that are required for DNA synthesis.54 A break at a hypoxia-induced fragile site may initiate gene amplification through the breakage-fusion-bridge mechanism.78 Another example of H/R-induced chromosomal alterations was reported by Rofstad et al.79 They examined the effects of severe hypoxia (<0.01% O2 for 24 h) on chromosome contents

of diploid as well as hyperdiploid human melanoma cell lines. They found that a subpopulation of diploid cells was arrested at the G2/M boundary during hypoxia exposure. During the first M phase after re-oxygenation, they observed a cell population which showed tetraploid chromosomes where homologous chromosomes were grouped in pairs (diplochromosomes), suggesting that severe H/R may disturb cell mitosis.79,80 Lee et al. placed phytohemagglutinin-stimulated normal human lymphocytes from 40 healthy donors under mild hypoxia (3% oxygen concentration) for 12 h or 24 h.81 After hypoxia exposure the cells were subjected to chromosomal analysis. They found that the frequency of sister chromatid exchange (SCE) (recombination between homologous sister chromatids) was higher in hypoxia treated cultures than normoxia cultures.