Molecular mechanism of adenomatous polyposis coli-induced blockade of base excision repair pathway in colorectal carcinogenesis.

Colorectal cancer (CRC) is the third leading cause of death in both men and women in North America. Despite chemotherapeutic efforts, CRC is associated with a high degree of morbidity and mortality. Thus, to develop effective treatment strategies for CRC, one needs knowledge of the pathogenesis of cancer development and cancer resistance. It is suggested that colonic tumors or cell lines harbor truncated adenomatous polyposis coli (APC) without DNA repair inhibitory (DRI)-domain. It is also thought that the product of the APC gene can modulate base excision repair (BER) pathway through an interaction with DNA polymerase ß (Pol-ß) and flap endonuclease 1 (Fen-1) to mediate CRC cell apoptosis. The proposed therapy with temozolomide (TMZ) exploits this particular pathway; however, a high percentage of colorectal tumors continue to develop resistance to chemotherapy due to mismatch repair (MMR)-deficiency. In the present communication, we have comprehensively reviewed a critical issue that has not been addressed previously: a novel mechanism by which APC-induced blockage of single nucleotide (SN)- and long-patch (LP)-BER play role in DNA-alkylation damage-induced colorectal carcinogenesis.

DNA polymerase ß deficiency is linked to aggressive breast cancer: a comprehensive analysis of gene copy number, mRNA and protein expression in multiple cohorts.

Short arm of chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications in breast cancer. Although such genetic changes may have phenotypic consequences, the identity of candidate gene(s) remains to be clearly defined. Pol ß gene is localized to chromosome 8p12-p11 and encodes a key DNA base excision repair protein. Pol ß may be a tumour suppressor and involved in breast cancer pathogenesis. We conducted the first and the largest study to comprehensively evaluate pol ß in breast cancer. We investigated pol ß gene copy number changes in two cohorts (n = 128 &n = 1952), pol ß mRNA expression in two cohorts (n = 249 &n = 1952) and pol ß protein expression in two cohorts (n = 1406 &n = 252). Artificial neural network analysis for pol ß interacting genes was performed in 249 tumours. For mechanistic insights, pol ß gene copy number changes, mRNA and protein levels were investigated together in 128 tumours and validated in 1952 tumours. Low pol ß mRNA expression as well as low pol ß protein expression was associated high grade, lymph node positivity, pleomorphism, triple negative, basal-like phenotypes and poor survival (ps < 0.001). In oestrogen receptor (ER) positive sub-group that received tamoxifen, low pol ß protein remains associated with aggressive phenotype and poor survival (ps < 0.001). Artificial neural network analysis revealed ER as a top pol ß interacting gene. Mechanistically, there was strong positive correlation between pol ß gene copy number changes and pol ß mRNA expression (p < 0.0000001) and between pol ß mRNA and pol ß protein expression (p < 0.0000001). This is the first study to provide evidence that pol ß deficiency is linked to aggressive breast cancer and may have prognostic and predictive significance in patients.

Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome.

In myelodysplastic syndromes (MDS) increased chromosomal breaks point toward defects in DNA repair machinery including base excision repair (BER) pathway involved in handling of oxidative DNA damage. We investigated whether defects in this pathway can be found in MDS. Elevated levels of 8-oxoguanine (8-OG) were found in a significant proportion of MDS patients, indicating increased oxidative DNA damage or defective handling of oxidative load. In a distinct subgroup of patients, increased 8-OG content was associated with increased hOGG1 mRNA expression and activity. In some patients, increased numbers of abasic sites (AP sites) correlated with low levels of POLbeta. To further investigate the nature of this defect, we examined genetic lesions potentially explaining accumulation of 8-OG and AP sites. We genotyped a large cohort of MDS patients and found a correlation between increased oxidative damage and the presence of the hOGG1-Cys326 allele suggesting inadequate compensatory feedback. Overall, this hOGG1 variant was more frequent in MDS, particularly in advanced forms, as compared to controls. In summary, we demonstrated that BER dysfunction in some MDS patients may be responsible for the increased 8-OG incorporation and explains one aspect of the propensity to chromosomal breaks in MDS but other mechanisms may also be involved.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChip sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrate.

The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChip. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to alpha-cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50 mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase beta tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE).

Metaplastic potential of p53 down-regulation in ovarian surface epithelial cells affected by ovulation.

That ovulation is a predisposing factor in common (surface) epithelial ovarian cancer is widely recognized; however, the molecular events which underscore early-stage disease have not been elucidated. In vivo and in vitro studies using an ovine model system were designed to address a premise that oxidative distresses to DNA inflicted upon ovarian surface epithelial cells within a limited diffusion radius of the ovulatory site of follicular rupture, if gone uncorrected by p53-dependent cycle arrest/repair pathways, could yield a progenitor of tumorigenic potential. Immunofluorescence image analysis was used to quantitate the DNA damage marker 8-oxoguanine, the tumor suppressor p53, the base-excision repair polymerase beta, and apoptotic internucleosomal DNA fragmentation in ovarian surface epithelial cells isolated from the perimeter of ovulated follicles. Up-regulation of p53 coincided with accretion of 8-oxoguanine lesions. Oxidative disturbances to DNA were reconciled during the consequent luteal phase (before replicative repair of the ovarian rupture wound). Production of p53 was not related to apoptosis, but rather to induction of polymerase beta. Oxoguanine modifications persisted in cells affected by ovulation in which synthesis of p53 was negated in culture by an antisense oligonucleotide. Inhibition of p53 was associated with discordant cellular growth rates and expression of the cancer antigen CA-125 - a phenotype of metaplastic transformation. It is suggested that the integrity of DNA of ovarian surface epithelial cells is compromised by reactive oxidants and inflammatory mediators generated during the ovulatory process and that malfunction in a damage-recognition and(or) repair mechanism is a determinant in the etiology of ovarian metaplasia and carcinogenesis.

Rapid segmental and subdomain motions of DNA polymerase beta.

DNA polymerase (pol) beta is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between alpha-helix N and the nascent base pair. Kinetic and structural characterization of pol beta has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and alpha-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of alpha-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol beta were determined. The results revealed that the segmental motion of alpha-helix N was rapid ( approximately 1 ns) and far more rapid than the step that limits chemistry. Binding of Mg(2+) and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in alpha-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within alpha-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of alpha-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol beta deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors.

Ovulation-induced DNA damage in ovarian surface epithelial cells of ewes: prospective regulatory mechanisms of repair/survival and apoptosis.

Oxidative base (8-oxoguanine) damage, DNA fragmentation, and apoptosis occurred among ovarian surface epithelial cells within the formative site of ovulation in sheep. The incidence of 8-oxoguanine adducts in surviving antiapoptotic Bcl-2/base excision repair polymerase beta-positive cells at the margins of ruptured follicles (which avoid the focal point of the ovulatory assault) was intermediate between apoptotic and outlying healthy epithelium. Cells containing perturbations to DNA expressed the tumor suppressor p53. Localized reactions of DNA injury and programmed cellular death were averted by ovulation blockade with indomethacin. Progesterone enhanced the biosynthesis of polymerase beta in ovarian surface epithelial cells exposed in vitro to a sublethal concentration of H(2)O(2). Ovulation is a putative etiological factor in common epithelial ovarian cancer. A genetically altered progenitor cell, with unrepaired DNA, but not committed to death, could give rise to a transformed phenotype that is hence propagated upon healing of the ovulatory wound; it appears that this incongruity is normally reconciled by up-regulation of the base excision repair pathway during the ensuing luteal phase.