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Author(s): Vassilis G. Gorgoulis [1, 8]; Leandros-Vassilios F. Vassiliou [1, 8]; Panagiotis Karakaidos [1]; Panayotis Zacharatos [1]; Athanassios Kotsinas [1]; Triantafillos Liloglou [2]; Monica Venere [3, 4]; Richard A. DiTullio, [...]

Author(s): Yentram Huyen [1, 2]; Omar Zgheib [1, 2]; Richard A. DiTullio Jr [1, 2]; Vassilis G. Gorgoulis [1, 4]; Panayotis Zacharatos [1, 4]; Tom J. Petty [1, 2]; Emily [...]

Aim: Crohn's disease (CD) and ulcerative colitis (UC) are multifactorial diseases with a significant genetic background. Apart from CARD15/NOD2 gene, evidence is accumulating that molecules related to the innate immune response such as CD14 or Toll-like receptor 4 (TLR4), are involved in their pathogenesis. In further exploring the genetic background of these diseases, we investigated the variations in the CARD15/NOD2 gene (Arg702Trp, Gly908Arg and Leu1007fsinsC), and polymorphisms in the TLR4 gene (Asp299Gly and Thr399Ile) as well as in the promoter of the CD14 gene (T/C at position -159) in Greek patients with CD and UC., Methods: DNA was obtained from 120 patients with CD, 85 with UC and 100 healthy individuals. Genotyping was performed by allele specific PCR or by PCR-RFLP analysis., Results: The 299Gly allele frequency of the TLR4 gene and the T allele and TT genotype frequencies of the CD14 promoter were significantly higher in CD patients only compared to healthy individuals (P = 0.026<0.05; P = 0.0048<0.01 and P = 0.047<0.05 respectively). Concerning the NOD2/CARD15 mutations the overall presence in CD patients was significantly higher than that in UC patients or in controls. Additionally, 51.67% of the CD patients were carriers of a TLR4 and/or CD14 polymorphic allele and at least one variant of the NOD2/CARD15, compared to 27% of the UC patients. It should be pointed out that both frequencies significantly increased as compared with the 10% frequency of multiple carriers found in healthy controls. A possible interaction of the NOD2/CARD15 with TLR4 and especially CD14, increased the risk of developing inflammatory bowel disease (IBD)., Conclusion: Our results indicate that co-existence of a mutation in either the TLR4 or CD14 gene, and in NOD2/CARD15 is associated with an increased susceptibility to developing CD compared to UC, and to developing either CD or UC compared to healthy individuals.

E2F-1 is an intriguing transcription factor that accumulates the integrated signal of the G1-S transition regulators. Its role in cell fate, as depicted from in vivo models and a few studies on human tissues, is a matter of debate, since it confers a tissue-specific oncogenic or tumor suppressor behavior. In the present work, in an attempt to shed light on the role of E2F-1 in colon cancer, we examined E2F-1 expression in a series of 45 colon carcinomas and we further correlated it with tumor kinetics. E2F-1 expression and proliferation were evaluated by immunohistochemistry as the percentage of E2F-1 (E2F-1 index: EI) and Ki-67 (Proliferation index: PI)-positive cells, respectively; whereas apoptosis was estimated as the percentage of positive, by TUNEL assay, cells (Apoptotic index: AI). The relationship between E2F-1 expression and tumor kinetics was assessed by microscopical evaluation in semi-serial tissue sections and statistical analysis. Our results demonstrated that E2F-1 expression was inversely correlated with tumor growth (GI=PI/AI) (p=0.002). Specifically, the histological observations showed that E2F-1 was expressed in lesions with high apoptotic incidence and low proliferation. These results also supported the statistical findings showing that EI was inversely correlated with PI (p < 0.001) and positively associated with AI (p = 0.013). In conclusion, we suggest a tumor-suppressive behavior of E2F-1 in colon carcinomas.

A hallmark of cancer is inactivation of cell cycle checkpoints. However, very few mutations targeting mitotic checkpoint genes have been described, and in those instances, a wild-type copy of the gene was retained. chfr is a mitotic checkpoint gene that functions in early prophase delaying chromosome condensation in response to microtubule poisons. In a panel of 53 lung carcinomas for which matched normal tissue was available, we identified three missense mutations in the chfr gene, at least one of which was associated with loss of heterozygosity. In tissue culture checkpoint assays, the tumor-associated missense mutants had reduced activity or were inactive. Together with recent data suggesting that the chfr gene is frequently silenced in various tumors because of methylation of its promoter, these findings suggest that chfr is inactivated by multiple mechanisms in human cancer.

beta-catenin (beta-cat) is a versatile component of homotypic cell adhesion and signaling. Its subcellular localization and cytoplasmic levels are tightly regulated by the adenomatous polyposis coli (APC) protein. Mutations in beta-cat (exon 3) or APC (MCR) result in beta-cat aberrant overexpression that is associated with its nuclear accumulation and improper gene activation. Data from experimental models have shown that beta-cat overexpression has a multitude of effects on cell-cycle behavior. In many of these aspects its function depends on major G(1) phase regulators. To the best of our knowledge, most of these issues have never been addressed concurrently in tumors. For this reason we investigated in a panel of 92 non-small-cell lung carcinomas, beta-cat and APC expression, and their relationship with cell-cycle kinetics (PI and AI) and ploidy status. Moreover, the above correlations were examined in relation to the main G(1)/S-phase checkpoint regulators. Four beta-cat immunohistochemical expression patterns [membranous (11.1%), membranous-cytoplasmic (54.3%), cytoplasmic (9.9%), cytoplasmic-nuclear (24.7%)] and three APC immunohistochemical expression patterns [cytoplasmic (37.7%), cytoplasmic-nuclear (58%), nuclear (4.3%)] were observed, which were further confirmed by Western blot analysis on subcellular fractions in representative samples. The frequent presence of beta-cat in the cytoplasm is an indication of aberrant expression, whereas membranous and nuclear localization were inversely related. Absence of mutations in beta-cat (exon 3) and APC (MCR) suggest that beta-cat destruction mechanisms may be functional. However, expression analysis revealed attenuated levels for APC, indicating a residual ability to degrade beta-cat. Decreased levels were associated with loss of heterozygosity at the APC region in 24% of the cases suggesting that additional silencing mechanisms may be involved. Interestingly, the 90-kd APC isoform associated with apoptosis, was found to be the predominant isoform in normal and cancerous lung tissues. The most important finding in our study, was the correlation of nuclear beta-cat immunohistochemical localization with increased proliferation, overexpression of E2F1 and MDM2, aberrant p53, and low expression of p27(KIP), providing for the first time in vivo evidence that beta-cat-associated proliferation correlates with release of E2F1 activity and loss of p53- and p27(KIP)-dependent cell-cycle checkpoints. Loss of these checkpoints is accompanied by low levels of APC, which possibly reflects a diminished ability to degrade beta-cat. Taken together our data indicate that cases with nuclear beta-cat immunohistochemical expression represent a subset of non-small-cell lung carcinomas that have gained an increased proliferation advantage in contrast to the other beta-cat immunohistochemical expression profiles.