Efficient repair of DNA double-strand breaks in malignant cells with structural instability.

Aberrant repair of DNA double-strand breaks (DSBs) is thought to be important in the generation of gross chromosomal rearrangements (GCRs). To examine how DNA DSBs might lead to GCRs, we investigated the repair of a single DNA DSB in a structurally unstable cell line. An I-SceI recognition site was introduced into OVCAR-8 cells between a constitutive promoter (EF1alpha) and the Herpes simplex virus thymidine kinase (TK) gene, which confers sensitivity to gancyclovir (GCV). Expression of I-SceI in these cells caused a single DSB. Clones with aberrant repair could acquire resistance to GCV by separation of the EF1alpha promoter from the TK gene, or deletion of either the EF1alpha promoter or the TK gene. All mutations that we identified were interstitial deletions. Treatment of cells with etoposide or bleomycin, agents known to produce DNA DSBs following expression of I-SceI also did not generate GCRs. Because we identified solely interstitial deletions using the aforementioned negative selection system, we developed a positive selection system to produce GCR. A construct containing an I-SceI restriction site immediately followed by a hygromycin phosphotransferase cDNA, with no promoter, was stably integrated into OVCAR-8 cells. DNA DSBs were produced by an I-SceI expression vector. None of the hygromycin resistant clones recovered had linked the hygromycin phosphotransferase cDNA to an endogenous promoter, but had instead captured a portion of the I-SceI expression vector. These results indicate that even in a structurally unstable malignant cell line, the majority of DNA DSBs are repaired by religation of the two broken chromosome ends, without the introduction of a GCR.

Gene expression patterns distinguish colonoscopically isolated human aberrant crypt foci from normal colonic mucosa.

Aberrant crypt foci (ACF) are considered the earliest identifiable preneoplastic colonic lesions; thus, a greater understanding of the nature of genetic changes underlying the transformation of normal colonic mucosa (NM) into ACF may provide insight into the mechanisms of carcinogenesis. ACF were identified by indigo carmine spraying onto colonic mucosa during colonoscopy and isolated as standard pinch biopsies of the mucosal areas containing the ACF. RNAs isolated from ACF and matched NM biopsies from the ascending and descending colons of 13 patients were analyzed on arrays containing 9128 cDNAs. Thirty-four differentially expressed (P < 0.001) genes were found in a paired comparison of the ACF and NM samples, and 25 of 26 matched pairs of ACF and NM could be correctly classified in leave-one-out cross-validation. Differential expression for seven of eight genes was confirmed by real-time reverse transcription-PCR. Furthermore, ACF and NM samples, including six pairs of ACF and NM samples that had not previously been analyzed by array hybridization, can be correctly classified on the basis of the overexpression in ACF of three selected genes (REG4, SRPN-B5, and TRIM29) evaluated by real-time reverse transcription-PCR. In a separate analysis of 13 biopsy pairs from either ascending or descending colon, ACF and NM samples could also be correctly classified by the gene expression patterns. Analysis of gene expression differences in ACF from the ascending and descending colon versus NM samples indicates that ACF from these distinct colonic locations are converging toward similar gene expression profiles and losing differences in gene expression characteristic of NM from the ascending versus descending colon.

Celecoxib treatment alters the gene expression profile of normal colonic mucosa.

A clinical trial was recently conducted to evaluate the safety and efficacy of a selective inhibitor of cyclooxygenase-2 (celecoxib) in hereditary nonpolyposis colon cancer patients. In a randomized, placebo-controlled phase I/II multicenter trial, hereditary nonpolyposis colon cancer patients and gene carriers received either celecoxib at one of two doses or placebo. The goal was to evaluate the effects of these treatment arms on a number of endoscopic and tissue-based biomarker end points after 12 months of treatment. As part of this trial, we analyzed gene expression by cDNA array technology in normal descending (rectal) colonic mucosa of patients before and after treatment with celecoxib or placebo. We found that treatment of patients with celecoxib at recommended clinical doses (200 and 400 mg p.o. bid), in contrast to treatment with placebo, leads to changes in expression of >1,400 genes in the healthy colon, although in general, the magnitude of changes is <2-fold. Twenty-three of 25 pairs of colon biopsies taken before and after celecoxib treatment can be classified correctly by the pattern of gene expression in a leave-one-out cross-validation. Immune response, particularly T- and B-lymphocyte activation and early steps of inflammatory reaction, cell signaling and cell adhesion, response to stress, transforming growth factor-beta signaling, and regulation of apoptosis, are the main biological processes targeted by celecoxib as shown by overrepresentation analysis of the distribution of celecoxib-affected genes across Gene Ontology categories. Analysis of possible cumulative effects of celecoxib-induced changes in gene expression indicates that in healthy colon, celecoxib may suppress the immune response and early steps of inflammation, inhibit formation of focal contacts, and stimulate transforming growth factor-beta signaling.

Distinguishing right from left colon by the pattern of gene expression.

Distinct epidemiological and clinicopathological characteristics of colorectal carcinomas (CRCs) based on their anatomical location suggest different risk factors and pathways of transformation associated with proximal and distal colon carcinogenesis. These differences may reflect distinct biological characteristics of proximal and distal colonic mucosa, acquired in embryonic or postnatal development, that determine a differential response to uniformly distributed environmental factors. Alternatively, the differences in the epidemiology of proximal and distal CRCs could result from the presence of different procarcinogenic factors in the ascending versus descending colon, acting on cells with either similar or distinct biological characteristics. We applied cDNA microarray technology to explore the possibility that mucosal epithelium from adult proximal and distal colon can be distinguished by their pattern of gene expression. In addition, gene expression was studied in fetal (17-24 weeks gestation) proximal and distal colon. More than 1000 genes were expressed differentially in adult ascending versus descending colon, with 165 genes showing >2-fold and 49 genes showing >3-fold differences in expression. With almost complete concordance, biopsies of adult colonic epithelium can be correctly classified as proximal or distal by gene expression profile. Only 87 genes were expressed differently in ascending and descending fetal colon, indicating that, although anatomically relevant differences are already established in embryonic colon, additional changes in gene expression occur in postnatal development.

Genetic counseling and testing in families with hereditary nonpolyposis colorectal cancer.

BACKGROUND: Genetic testing to refine cancer risk is available. However, little is known about factors affecting the uptake of testing for the most common hereditary colon cancer, hereditary nonpolyposis colorectal cancer. This study investigated attitudes, intentions, and uptake of genetic testing within newly identified families with hereditary nonpolyposis colorectal cancer. METHODS: Cohort study conducted at the National Institutes of Health between April 15, 1996, and November 20, 1999. Data were collected through questionnaires before semistructured education sessions, individual counseling sessions, and the offer of genetic testing. RESULTS: Of the 111 eligible first-degree relatives, 51% chose to participate in education and individual counseling sessions. Participation was associated with greater numbers of first-degree relatives with cancer; no association was found between participation and personal history of cancer. Before education and individual counseling sessions, 64% of participants had heard little about genetic testing for cancers; however, most (97%) stated intentions to pursue testing. Fifty-one percent identified learning about their children's risks as the most important reason to consider testing. Thirty-nine percent identified the potential effect on their health insurance as the most important reason to not undergo testing. Of the 111 eligible first-degree relatives, 51% chose to undergo genetic testing. Participants' intentions to pursue genetic testing were significantly affected by concerns regarding the ability to handle the emotional aspects of testing and the psychosocial effect on family members. CONCLUSIONS: Genetic counseling and testing offers the potential to focus cancer screening resources in individuals truly at increased risk, thereby reducing mortality and morbidity. Fears of discrimination and concerns about psychological and psychosocial issues may present barriers to the use of current cancer prevention strategies, including genetic counseling and testing.