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This work introduces, for the first time, simultaneous monitoring of fluorescence and absorbance using Bead Injection in a Lab-on-valve format. The aim of the paper is to show that when the target species, cells immobilized on a stationary phase, are exposed to reagents under well-controlled reaction conditions, dual monitoring yields valuable information. The applicability of this technique is demonstrated by the development of a Bead Injection method for automated measurement of cell density and intracellular hydrogen peroxide.

Background: Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%., Methods and Results: We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin-nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%., Conclusions: This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species-related cardiovascular diseases.

Rarely, dysplasia in Barrett's esophagus (BE) is composed of crypts lined by cuboidal-shaped cells that contain a centrally located nucleus, markedly increased nuclear/cytoplasmic ratio, but without nuclear stratification characteristic of conventional "adenomatous" dysplasia. The aim of this study was to evaluate the clinical and pathologic features, natural history, and DNA content flow cytometric abnormalities of BE patients with non-adenomatous dysplasia (NAD) in a cohort of BE patients enrolled in a prospective surveillance program. Eighteen patients with NAD identified over a 6 year period, in a cohort of 270 consecutive patients with BE and without esophageal adenocarcinoma (EA) at baseline, were evaluated for clinical and pathologic features, including association with conventional adenomatous dysplasia and EA, DNA content flow cytometric abnormalities (tetraploidy and aneuploidy) and outcome, over a mean follow-up period of 4.1 years. The findings in the 18 study patients were compared to those in the 252 remaining (control) patients without NAD. Control patients included 228 with metaplasia/indefinite for dysplasia, and 24 with conventional adenomatous dysplasia (13 low-grade, 11 high-grade). The prevalence rate of NAD in our BE cohort was 6.7% Of the 18 study patients, there were 17 were males and 1 female of mean age 66.7 years. The mean length of BE was 3.9 cm NAD foci were associated with goblet or non-goblet epithelium in 62% and 38% of cases, respectively. Ninety-four percent of patients with NAD (17/18) also had conventional adenomatous dysplasia (four with low-grade, 13 with high-grade) elsewhere in the esophagus at the same endoscopic procedure as the one that detected NAD. Patients with NAD had a significantly shorter length of BE compared to control patients with conventional adenomatous dysplasia (N=24) (p=0.03). Patients with NAD also showed a significantly higher rate of DNA content flow cytometric abnormalities compared to the entire cohort of control patients (38% vs. 11%, p=0.05). However, no significant differences regarding either flow cytometric abnormalities or progression to EA were found when the NAD patients were compared only to the 24 controls with conventional adenomatous dysplasia. NAD is a high grade histologic variant of intraepithelial neoplasia that is episodic in nature, and shows a high association with conventional adenomatous high-grade dysplasia.

No adequate means exist to identify the minority of ulcerative colitis (UC) patients destined to undergo neoplastic progression. Recognition of this subset would advance UC cancer surveillance by focusing the available management options onto the highest risk patients. Three different assays of genomic alterations in nondysplastic UC biopsies show promise for distinguishing patients with neoplasia (UC progressors) from those without (UC nonprogressors), including assays of telomere length, anaphase bridges, and chromosomal fluorescence in situ hybridization. Expanding the number of patients and testing of assays simultaneously in the same biopsy further validated their utility. A panel approach also improved testing outcome. A total of 14 UC progressors was readily separable from 15 UC nonprogressors and 6 normal controls. Chromosomal entropy (ie, the extent of alteration diversity) proved to be the most useful test. By receiver-operating characteristic analysis, mean chromosomal entropy in 28 patients over all four chromosomes yielded 100% sensitivity and 92% specificity for distinguishing progressors from nonprogressors with optimum choice of threshold. Moreover, separation was achieved using only nondysplastic and predominantly rectal (82.8%) biopsies that were remote from neoplasia, suggesting that full colonoscopy with extensive biopsies might be avoided for the majority of UC patients, the nonprogressors. These data further strengthen the concept that genomic biomarkers can distinguish UC progressors from nonprogressors and improve cancer surveillance in UC.

Purpose: Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barrett's esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation., Experimental Design: Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barrett's esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point., Results: Diploid S and 4N (G(2)/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G(1), or 4N fractions. High Ki67-positive and G(1)-phase fractions were not associated with the future development of esophageal adenocarcinoma (P=0.13 and P=0.15, respectively), whereas high diploid S-phase and 4N fractions were (P=0.03 and P<0.0001, respectively)., Conclusions: High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barrett's esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.

Background & Aims: Telomere shortening is implicated in cancer and aging and might link these 2 biologic events. We explored this hypothesis in ulcerative colitis (UC), a chronic inflammatory disease that predisposes to colorectal cancer and in which shorter telomeres have been associated with chromosomal instability and tumor progression., Methods: Telomere length was measured by quantitative polymerase chain reaction in colonocytes and leukocytes of 2 different sets of UC patients and compared with normal controls across a wide range of ages. For a subset of patients, telomere length was measured in epithelium and stroma of right and left colon biopsy specimens. A third set of biopsy specimens was analyzed for phosphorylation of histone H2AX (gammaH2AX), a DNA damage signal, by immunofluorescence and for telomere length by quantitative fluorescence in situ hybridization. Relationships between telomere length, gammaH2AX intensity, age, disease duration, and age of disease onset were explored., Results: Colonocyte telomeres shorten with age almost twice as rapidly in UC patients as in normal controls. This extensive shortening occurs within approximately 8 years of disease duration. Leukocyte telomeres are slightly shorter in UC patients than in controls, but telomeres of colon stromal cells are unaffected. gammaH2AX intensity is higher in colonocytes of UC patients than in controls and is not dependent on age or telomere length., Conclusions: Colonocytes of UC patients show premature shortening of telomeres, which might explain the increased and earlier risk of cancer in this disease. Shorter leukocyte telomeres and increased gammaH2AX in colonocytes might reflect oxidative damage secondary to inflammation.

The incidence of esophageal adenocarcinoma (EA) and its precursor condition, Barrett's esophagus, has risen rapidly in the United States for reasons that are not fully understood. Therefore, we evaluated the association between use of supplemental vitamins and minerals and risk of neoplastic progression of Barrett's esophagus and EA. The Seattle Barrett's Esophagus Program is a prospective study based on 339 men and women with histologically confirmed Barrett's esophagus. Participants underwent baseline and periodic follow-up exams, which included endoscopy and self-administered questionnaires on diet, supplement use, and lifestyle characteristics. Use of multivitamins and 4 individual supplements was calculated using time-weighted averages of reported use over the observational period. Cox proportional-hazards models were used to calculate hazard ratios (HR) for each endpoint: EA, tetraploidy, and aneuploidy. During a mean follow-up of 5 yr, there were 37 cases of EA, 42 cases of tetraploidy, and 34 cases of aneuploidy. After controlling for multiple covariates including diet, nonsteroidal anti-inflammatory drug use, obesity, and smoking, participants who took 1 or more multivitamin pills/day had a significantly decreased risk of tetraploidy [HR = 0.19; 95% confidence interval (CI) = 0.08-0.47) and EA (HR = 0.38; 95% CI = 0.15-0.99] compared to those not taking multivitamins. Significant inverse associations were also observed between risk of EA and supplemental vitamin C (> or = 250 mg vs. none: HR = 0.25; 95% CI = 0.11-0.58) and vitamin E (> or = 180 mg vs. none: HR = 0.25; 95% CI = 0.10-0.60). In this cohort study, use of multivitamins and single antioxidant supplements was associated with a significantly reduced risk of EA and markers of neoplastic progression among individuals with Barrett's esophagus.

Purpose: Leukocyte telomere length has gained attention as a marker of oxidative damage and age-related diseases, including cancer. We hypothesize that leukocyte telomere length might be able to predict future risk of cancer and examined this in a cohort of patients with Barrett's esophagus, who are at increased risk of esophageal adenocarcinoma and thus were enrolled in a long-term cancer surveillance program., Patients and Methods: In this prospective study, telomere length was measured by quantitative PCR in baseline blood samples in a cohort of 300 patients with Barrett's esophagus followed for a mean of 5.8 years. Leukocyte telomere length hazard ratios (HR) for risk of esophageal adenocarcinoma were calculated using multivariate Cox models., Results: Shorter telomeres were associated with increased esophageal adenocarcinoma risk (age-adjusted HR between top and bottom quartiles of telomere length, 3.45; 95% confidence interval, 1.35-8.78; P = 0.009). This association was still significant when individually or simultaneously adjusted for age, gender, nonsteroidal anti-inflammatory drug (NSAID) use, cigarette smoking, and waist-to-hip ratio (HR, 4.18; 95% confidence interval, 1.60-10.94; P = 0.004). The relationship between telomere length and cancer risk was particularly strong among NSAID nonusers, ever smokers, and patients with low waist-to-hip ratio., Conclusion: Leukocyte telomere length predicts risk of esophageal adenocarcinoma in patients with Barrett's esophagus independently of smoking, obesity, and NSAID use. These results show the ability of leukocyte telomere length to predict the risk of future cancer and suggest that it might also have predictive value in other cancers arising in a setting of chronic inflammation.

Background: Insulin-like growth factor-I (IGF-I) is a potent mitogen. IGF-I and its main binding protein, IGF binding protein-3 (IGFBP-3), and their polymorphisms have been investigated in relation to risk of many cancers, but not esophageal adenocarcinoma., Materials and Methods: We used data and specimens from a longitudinal study of persons with Barrett's esophagus (n=344; median, 5.4 years follow up) to determine whether baseline serum concentrations of IGF-I and IGFBP-3 and associated polymorphisms were related to the risk of developing esophageal adenocarcinoma or flow cytometric abnormalities., Results: Overall, circulating concentrations of IGF-I and IGBP-3 were not associated with risk of esophageal adenocarcinoma or flow cytometric abnormalities, with the exception of an approximate tripling of risk of aneuploidy among participants with higher IGFBP-3 levels [above median; adjusted hazard ratio (HR) comparing subjects with levels lower than median versus higher of equal to median, 2.7; 95% confidence interval (95% CI), 1.2-6.0; P=0.01]. Genotypic analyses revealed that persons with the IGF-I [cytosine-adenine (CA)](19) or the IGFBP-3 A-202C C allele were associated with lower circulating concentrations of IGF-I (P(trend)=0.01) and IGFBP-3 (P(trend)=0.002), respectively. Persons with two copies of the IGF-I receptors 2-bp deletion allele had a nonsignificant 2-fold increased risk of tetraploidy (HR, 2.3; 95% CI, 0.9-5.9; P(trend)=0.11). After adjustment for IGFBP-3 levels, participants carrying two IGFBP-3 C alleles had a significantly higher risk of developing aneuploidy (HR, 3.8; 95% CI, 1.0-14.0; P(trend)=0.04) than carriers of A alleles; whereas no associations were observed between the outcomes studied and the IGF-I receptors AGG trinucleotide repeat polymorphism at position 97., Conclusion: Our findings, although based on a relatively small number of outcomes and subject to several limitations, indicate a potential role of the complex IGF system in neoplastic progression among persons with Barrett's esophagus.

Chromosomal instability is regarded as an underlying mechanism of neoplastic progression, integral to the clonal selection and evolution that leads to cancer. We evaluated chromosomal instability in premalignant Barrett's esophagus tissue using high resolution Affymetrix mapping 100K SNP arrays as patients progressed through three molecular stages of disease-CDKN2A(LOH) only, CDKN2A(LOH)/TP53(LOH), and CDKN2A(LOH)/TP53(LOH) with aneuploidy. Within individuals over time, we observed increases in both numbers and sizes of regions of LOH or copy number change. In the earliest CDKN2A(LOH) only samples, we detected few regions with both copy change and LOH, whereas copy loss and LOH were highly correlated in more advanced samples. These data indicate that genomic instability increases in severity and changes character during neoplastic progression. In addition, distinct patterns of clonal evolution could be discerned within a segment of Barrett's esophagus. Overall, this study illustrates that pre-malignant disease can be associated with extensive instability and clonal dynamics that evolve from an initial stage characterized by small recombination-based alterations to one with larger copy change events likely associated with mitotic instability., ((c) 2007 Wiley-Liss, Inc.)

The composition of single MCF-7 breast cancer cells is characterized using 2-D CE. Individual MCF-7 cells were aspirated into a 30 mum inner diameter fused-silica capillary and lysed by contact with an SDS-containing buffer. Proteins and other primary amines were fluorescently labeled on-column using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled components were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by MEKC. Analytes were detected in a sheath-flow cuvette using LIF. The expression profiles for MCF-7 cellular homogenate and a single MCF-7 cell are compared. As a proof-of-principle investigation, variation in expression was also compared within and between G1 and G2/M cell cycle phases for MCF-7 cells. Following their treatment with the viable nuclear stain Hoechst 33342, MCF-7 cells were sorted by flow cytometry on the basis of their ploidy. Sorted cells were then analyzed by 2-D CE. The degree of variability was >2.5 times larger between cells of different phases than between cells of the same phase. In typical 1 h 2-D CE separations using MCF-7 cells, over 100 components are resolved.

Background: Barrett's esophagus, a risk factor for esophageal adenocarcinoma, is associated with reflux disease. The aim of this study was to assess the expression of bile acid receptors in the esophagus (normal, esophagitis, Barrett's esophagus and adenocarcinoma) and to investigate their possible function., Results: the expression of the bile acid receptors FXR and VDR in esophageal biopsies from patients with a normal mucosa, esophagitis, Barrett's esophagus or adenocarcinoma (n = 6 per group) and in cell lines derived from Barrett's esophagus and esophageal adenocarcinoma, was assessed by real time Q-PCR and immunohistochemistry. The effect of guggulsterone, an antagonist of bile acid receptors, on apoptosis of Barrett's esophagus-derived cells was assessed morphologically, by flow cytometry and by measuring caspase 3 activity. The expression of FXR was increased in esophagitis, Barrett's esophagus and adenocarcinoma compared to normal mucosa by a mean of 44, 84 and 16, respectively. Immunohistochemistry showed a weak expression in normal esophagus, a strong focal reactivity in Barrett's esophagus, and was negative in adenocarcinoma. VDR expression did not significantly differ between groups. In cell cultures, the expression of FXR was high in Barrett's esophagus-derived cells and almost undetectable in adenocarcinoma-derived cells, whereas VDR expression in these cell lines was not significantly different. In vitro treatment with guggulsterone was associated with a significant increase in the percentage of apoptotic cells and of the caspase 3 activity., Conclusion: the bile acid receptor FXR is significantly overexpressed in Barrett's esophagus compared to normal mucosa, esophagitis and esophageal adenocarcinoma. The induction of apoptosis by guggulsterone in a Barrett's esophagus-derived cell line suggests that FXR may contribute to the regulation of apoptosis.

Background: Defects in DNA damage recognition and repair have been associated with a wide variety of cancers. We conducted a prospective study to determine whether mutagen sensitivity, as determined by an in vitro assay, was associated with the future development of cancer in patients with Barrett's esophagus, which is associated with increased risk of progression to esophageal adenocarcinoma., Methods: We measured sensitivity to bleomycin in peripheral blood lymphocytes in a cohort of 220 patients with Barrett's esophagus. We followed these patients for 1,230 person-years (range, 3 months to 10.1 years; median, 6.4 years), using development of cancer and aneuploidy as end points. A subset of these patients was evaluated for inactivation of tumor-suppressor genes CDKN2A/p16 and TP53 [by mutation and loss of heterozygosity (LOH)] in their Barrett's segments at the time of, or before, the bleomycin test, and the patients were stratified by CDKN2A/p16 and TP53 status in an analysis of mutagen sensitivity and progression., Results: Bleomycin-sensitive patients were found to be at significantly greater risk of developing aneuploidy (adjusted hazard ratio, 3.71; 95% confidence interval, 1.44-9.53) and nonsignificantly greater risk of cancer (adjusted hazard ratio, 1.63; 95% confidence interval, 0.71-3.75). Among patients with detectable LOH at the TP53 locus (on chromosome 17p), increasing bleomycin sensitivity was associated with increased risk of developing cancer (P(trend) < 0.001) and aneuploidy (P(trend) = 0.005)., Conclusions: This study supports the hypothesis that sensitivity to mutagens increases the risk of neoplastic progression in persons with Barrett's esophagus, particularly those with 17p LOH including TP53.

Barrett's esophagus is a useful model for the study of carcinogenesis, as the metaplastic columnar epithelium that replaces squamous esophageal epithelium is at elevated risk for development of adenocarcinoma. We examined telomere length and chromosomal instability (CIN) in Barrett's esophagus biopsies using fluorescence in situ hybridization. To study CIN, we selected centromere and locus-specific arm probes to chromosomes 17/17p (p53), 11/11q (cyclin D1), and 9/9p (p16 INK4A), loci reported to be involved in early stages of Barrett's esophagus neoplasia. Telomere shortening was observed in Barrett's esophagus epithelium at all histologic grades, whereas CIN was highest in biopsies with dysplastic changes; there was, however, considerable heterogeneity between patients in each variable. Alterations on chromosome 17 were strongly correlated with telomere length (r = 0.55; P < 0.0001) and loss of the 17p arm signal was the most common event. CIN on chromosome 11 was also associated with telomere shortening (r =0.3; P = 0.05), although 11q arm gains were most common. On chromosome 9p, arm losses were the most common finding, but chromosome 9 CIN was not strongly correlated with telomere length. We conclude that CIN is related to telomere shortening in Barrett's esophagus but varies by chromosome. Whether instability is manifested as loss or gain seems to be influenced by the chromosomal loci involved. Because telomere shortening and CIN are early events in Barrett's esophagus neoplastic progression and are highly variable among patients, it will be important to determine whether they identify a subset of patients that is at risk for more rapid neoplastic evolution.

Purpose of Review: Patients with chronic inflammatory bowel disease, such as ulcerative colitis and Crohn's disease, have an increased risk of colorectal cancer. Life-long colonoscopy surveillance is performed to detect the presence of dysplasia, but this approach is expensive and time-consuming. Thus, there is intensive research to identify molecular factors with prognostic value. This review summarizes recent research, with a special emphasis on the mechanisms underlying these molecular alterations., Recent Findings: The role of chromosomal instability in the progression to inflammatory bowel disease-associated colorectal cancer is clear and likely relates to chronic cycles of injury, inflammation, repair and telomere shortening. The role of microsatellite instability has been a subject of discussion, and data suggest that microsatellite instability in inflammatory bowel disease might be different from microsatellite instability in sporadic colorectal cancer. Methylation, as a mechanism of gene silencing, also plays a role in ulcerative colitis tumorigenesis. Chronic inflammation has been linked to p53 activation and oxidative stress, contributing to the extensive genomic DNA damage observed in ulcerative colitis., Summary: Improved understanding of the molecular biology of cancer progression in inflammatory bowel disease will hopefully lead to the identification of useful prognostic biomarkers. Efforts are needed to prove the clinical utility of the most promising markers now identified.

Little is known regarding the significance of esophageal biopsies that show dysplasia-like atypia limited to the bases of the crypts, without involvement of the surface epithelium in Barrett's esophagus (BE). The aim of this study was to evaluate the clinical, pathologic, immunohistochemical, and molecular characteristics of basal crypt dysplasia-like atypia (BCDA) with surface maturation in surveillance endoscopic mucosal biopsies to gain insight into its biologic significance. The Seattle Barrett's Esophagus Project is a prospective cohort study in which patients and their biopsies have been evaluated prospectively for clinical, pathologic, and molecular markers. As part of continued surveillance of the cohort, 206 consecutive BE patients were evaluated prospectively for BCDA between July 1, 2001 and August 13, 2003; 15 patients had BCDA (prevalence rate = 7.3%). These 15 patients were evaluated for clinical, pathologic, and immunohistochemical (p53 and MIB-1) features during the study period (2001-2003) as well as associations with clinical, pathologic, and molecular markers [17p(TP53) loss of heterozygosity (LOH), 9p(p16) LOH, tetraploidy, and aneuploidy] that were detected previously in the same patients in the cohort study (1983-2001). All BE patients with BCDA (male-to female ratio, 12:3; mean age, 72 years; mean length of BE, 7.0 cm; mean duration of BE, 95.1 months), except 2 (87%), had dysplasia or adenocarcinoma detected in biopsies either prior to or concurrent to the one that contained BCDA. In contrast, only 112 of 191 (59%) controls had neoplasia during the same time period (59%, P = 0.05). The difference between BCDA and controls was particularly significant with regard to the association with high-grade dysplasia (P = 0.004). Compared with adjacent nonatypical and nondysplastic (metaplastic) BE, areas of BCDA showed a significantly elevated prevalence rate of p53 positivity (60% vs. 13%, P<0.02) and a significantly elevated total crypt and basal crypt MIB-1 proliferation rate (P<0.001). Indeed, the MIB-1 proliferation rate in the basal portion of the crypts in BCDA was similar to that detected in conventional low- or high-grade dysplasia. Patients with BCDA showed a significantly increased rate of 17p(TP53) LOH (P = 0.016), aneuploidy (P = 0.004), and a trend in increased 9p(p16) LOH (P = 0.08), compared with control patients without BCDA. The clinical, pathologic, immunohistochemical, and molecular abnormalities were similar in BCDA cases that were considered low-grade versus those considered high-grade by histologic evaluation, except that high-grade cases tended to be older (79 years vs. 68 years, P = 0.06). BCDA with surface maturation, in mucosal biopsies from patients with BE, is an uncommon but significant pathologic change that shows a variety of proliferative and molecular abnormalities and has a high association with conventional dysplasia and/or adenocarcinoma. Based on these findings, BCDA warrants further investigation as a possible subtype of true dysplasia despite the morphologic appearance of surface maturation.

Background and Aims: 17p (TP53) loss of heterozygosity (LOH) has been reported to be predictive of progression from Barrett's esophagus to esophageal adenocarcinoma, but the mechanism by which TP53 LOH develops is unknown. It could be (a) DNA deletion, (b) LOH without copy number change, or (c) tetraploidy followed by genetic loss. If an alternative biomarker assay, such as fluorescence in situ hybridization (FISH), provided equivalent results, then translation to the clinic might be accelerated, because LOH genotyping is presently limited to research centers., Methods: We evaluated mechanisms of TP53 LOH to determine if FISH and TP53 LOH provided equivalent results on the same flow-sorted samples (n = 43) representing established stages of clonal progression (diploid, diploid with TP53 LOH, aneuploid) in 19 esophagectomy specimens., Results: LOH developed by all three mechanisms: 32% had DNA deletions, 32% had no copy number change, and 37% had FISH patterns consistent with a tetraploid intermediate followed by genetic loss. Thus, FISH and LOH are not equivalent (P < 0.000001)., Conclusions: LOH develops by multiple chromosome mechanisms in Barrett's esophagus, all of which can be detected by genotyping. FISH cannot detect LOH without copy number change, and dual-probe FISH is required to detect the complex genetic changes associated with a tetraploid intermediate. Alternative biomarker assay development should be guided by appreciation and evaluation of the biological mechanisms generating the biomarker abnormality to detect potential sources of discordance. FISH will require validation in adequately powered longitudinal studies before implementation as a clinical diagnostic for esophageal adenocarcinoma risk prediction.

Whereas free radical damage has been proposed as a key component in the tissue degeneration associated with aging, there has been little evidence that free radical damage limits life span in mammals. The current research shows that overexpression of the antioxidant enzyme catalase in mitochondria can extend mouse life span. These results highlight the importance of mitochondrial damage in aging and suggest that when targeted appropriately, boosting antioxidant defenses can increase mammalian life span.

Telomeres shorten with age, which may be linked to genomic instability and an increased risk of cancer. To explore this association, we analyzed telomere length in normal colorectal tissue of individuals at different ages using quantitative-fluorescence in situ hybridization (Q-FISH) and quantitative-PCR (Q-PCR). Using Q-FISH, we also examined the histologically normal epithelium adjacent to, or distant from, colon adenomas and cancers, in addition to the neoplasms. Q-FISH and Q-PCR showed that telomere length was inversely associated with age until approximately ages 60 to 70; surprisingly, beyond this age, telomere length was positively associated with age. This association was found exclusively in epithelial, and not in stromal, cells. Peripheral blood lymphocytes showed an inverse association between telomere length and age, but without any apparent increase in telomere length in the oldest individuals. Telomere length in larger adenoma lesions (>2 cm) was significantly shorter than in normal adjacent (P = 0.004) or normal distant (P = 0.05) tissue from the same individuals. However, telomere length in histologically normal epithelium adjacent to cancers or in adenomas <2 cm was not statistically different from that of the normal distant mucosa or from normal controls, evidence that a telomere-shortening field effect was not present. We suggest that the positive association between telomere length and age in the oldest patients is a consequence of selective survival of elderly patients with long colonocyte telomeres.

Risk factors for esophageal adenocarcinoma include obesity, high fat intake, and low consumption of fruits and vegetables. This trial tested whether an intervention to reduce these risk factors in patients with Barrett esophagus, a preneoplastic condition for esophageal adenocarcinoma, could reduce biomarkers of cellular proliferation and, by inference, the risk of neoplastic progression. Eighty-seven men and women with Barrett esophagus were randomized to an intensive dietary intervention or control group. At baseline, 18 and 36 months after intervention, biopsies were obtained at 2-cm intervals throughout the length of the Barrett segment. Ki67/DNA content flow cytometry was used to assess (a) % Ki67-positive proliferating diploid G(1) cells, (b) % total Ki67-positive proliferating cells, (c) presence of aneuploidy, and (d) presence of >6% of cells in the 4N (G(2)/tetraploid) fraction of the cell cycle. We also assessed re-epithelialization and length of the Barrett segment, reflux symptoms, and medication use. The intervention effects for energy, fat, fruits and vegetables, and weight were, respectively, -314 kcal, -12.2% energy, 1.8 servings/d, and -4.0 kg at 18 months (all P < 0.005) and were smaller but remained significant at 36 months. There were no significant effects of the intervention on any biomarker of cellular proliferation. The intervention effects +/- SE for mean %G(1) Ki67+ cells were 0.98 +/- 1.58 at 18 months and 1.79 +/- 1.31 at 36 months; the relative risks (95% confidence interval) for developing >6% of cells in 4N were 0.5 (0.1-2.6) at 18 months and 0.75 (0.2-3.1) at 36 months. A single control participant developed aneuploidy. There were no significant effects on re-epithelialization, segment length, or reflux medication use. We conclude that substantial dietary change has no short-term effects on biomarkers of cellular proliferation in Barrett esophagus or on clinical observations of the Barrrett segment.

Chronic inflammation predisposes to cancer. We used an inflammation-induced human model of tumorigenesis to explore how populations of mutated cells expand and initiate the earliest stages of cancer. Ulcerative colitis (UC) is a chronic inflammatory disease of the colon associated with an increased risk of colorectal cancer mediated through a process of genomic instability. In order to characterize the process of clonal expansion, arbitrary primed (AR) and inter-simple sequence repeat (ISSR) PCR DNA fingerprint mutation profiles of single crypts were compared with the mutational profiles from clusters of crypts and whole biopsies within the same individual. To provide information at the earliest steps of neoplastic progression, we examined histologically negative crypts, as well as dysplastic crypts. Crypts from UC dysplasia/cancer show alterations in 10-20% of DNA fingerprint sites, regardless of (i) whether the crypts were dysplastic or non-dysplastic and (ii) whether the DNA came from one crypt or thousands of crypts. Of the mutational changes in single crypts, almost half are clonally expanded to adjacent crypts and/or to the thousands of crypts in a single biopsy. Using fluorescent in-situ hybridization to examine p53 alterations in individual crypt cells, we demonstrate that the mechanism of clonal expansion can occur through crypt fission. DNA alterations are initiated in colonic crypts and expand to adjacent crypts through crypt fission. Our data suggest that a continuous process of DNA mutations, clonal expansion through crypt fission and clonal succession initiates the development of inflammatory-associated colon cancer; this mutational process is moderated by crypt cell turn-over and cell death. This paradigm may apply to other inflammatory-induced cancers.

Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end-to-end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ-based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence-conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin-fixed paraffin-embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar-embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image-analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.

Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G2/M phase, the least in G1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIalpha expression as the extent of SB did not differ between cell cycle compartments in topoIIalpha-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus response to topoII-targeting drugs.

Objective: This study tests the feasibility of using on-line analysis of tissue during surgical resection of brain tumors to provide biologically relevant information in a clinically relevant time frame to augment surgical decision making. For the purposes of establishing feasibility, we used measurement of deoxyribonucleic acid (DNA) content as the end point for analysis., Methods: We investigated the feasibility of interfacing an ultrasonic aspiration (USA) system with a flow cytometer (FC) capable of analyzing DNA content (DNA-FC). The sampling system design, tissue preparation requirements, and time requirements for each step of the on-line analysis system were determined using fresh beef brain tissue samples. We also compared DNA-FC measurements in 28 nonneoplastic human brain samples with DNA-FC measurements in specimens of 11 glioma patients obtained from central tumor regions and surgical margins after macroscopically gross total tumor removal to estimate the potential for analysis of a biological marker to influence surgical decision making., Results: With minimal modification, modern FC systems are fully capable of real-time, intraoperative analysis of USA specimens. The total time required for on-line analysis of USA specimens varies between 36 and 63 seconds; this time includes delivery from the tip of the USA to complete analysis of the specimen. Approximately 60% of this time is required for equilibration of the DNA stain. When compared with values for nonneoplastic human brain samples, 50% of samples (10 of 20) from macroscopically normal glioma surgical margins contained DNA-FC abnormalities potentially indicating residual tumor., Conclusion: With an interface of existing technologies, DNA content of brain tissue samples can be analyzed in a meaningful time frame that has the potential to provide real-time information for surgical guidance. The identification of DNA content abnormalities in macroscopically normal tumor resection margins by DNA-FC supports the practical potential for on-line analysis of a tumor marker to guide surgical resections. The development of such a device would provide neurosurgeons with an objective method for intraoperative analysis of a clinically relevant biological parameter that can be measured in real time.

Background: Induced sputum, in contrast to bronchoscopic biopsies and lavages, is an easily obtained source of biological specimens. However, obtaining abnormal exfoliated cells for detailed molecular studies is limited because respiratory epithelial cells comprise only about 1% of sputum cell populations., Methods: We developed a multiparameter flow sorting strategy to purify epithelial cells from nonepithelial sputum cells, using anti-cytokeratin antibody AE1/AE3 to recognize human epithelial cells and DAPI to stain DNA. We excluded cells with a high degree of side-scatter, which were composed predominantly of squamous cells and contaminating macrophages. The remaining cytokeratin-positive respiratory epithelial cells were then sorted based on anti-cytokeratin (PE) vs DNA (DAPI) parameters., Results: In this proof of principle study, the AE1AE3 cytokeratin/DNA flow sorting strategy enriched rare diploid respiratory epithelial cells from an average of 1.1% of cells in unsorted induced sputum samples to average purities of 42%. Thus, AE1AE3 flow-sorting results in a 38-fold enrichment of these cells., Conclusions: We report a multiparameter flow cytometric assay to detect and enrich rare respiratory epithelial cells from induced sputum samples to average purities of 42%. With further development, this methodology may be useful as part of a molecular screening approach of populations at high risk for lung cancer., (Copyright 2004 Wiley-Liss, Inc.)

The Werner syndrome (WS) protein (WRN), a DNA helicase/exonuclease, is required for genomic stability and avoidance of cancer. Current evidence suggests that WRN is involved in the resolution of stalled and/or collapsed replication forks. This function is indicated, in part, by replication defects in WS cells and by hypersensitivity to agents causing major structural aberrations in DNA that block replication. We show here that antisense suppression of WRN in two human glioma cell lines reproduces hallmarks of the drug cytotoxicity profile of WS cells, namely, hypersensitivity to 4-nitroquinoline 1-oxide, camptothecin and hydroxyurea. We also show that antisense-treated cells are hypersensitive to methyl-lexitropsin, a site-specific alkylating agent that produces mainly N3-methyladenine, a cytotoxic and replication-blocking lesion. Antisense-treated cells are hypersensitive to O(6)-methylguanine adducts as well, but only when repair by O(6)-methylguanine-DNA methyltransferase is lacking. Our results illustrate the drug sensitivity caused by deficiency of WRN in a uniform genetic background. They extend the WRN DNA damage sensitivity spectrum to methyl base adducts that can result in blocked replication, and suggest that WRN may be required for resumption of processive replication when incomplete repair of DNA damage leaves blocking lesions at forks. The evidence that highly disparate lesions fall within the purview of WRN, and that abrogating DNA repair can reveal dependence on WRN, suggests that WRN may protect the genome from the lethal, mutagenic and carcinogenic effects of widely diverse DNA damage arising from endogenous processes and environmental agents.

Background: Telomeres are tandem repeated DNA sequences at the ends of every chromosome, which cap, stabilize, and prevent chromosome fusions and instability. Telomere regulation is an important mechanism in cellular proliferation and senescence in normal diploid and neoplastic cells. Quantitative methods to assess telomere lengths are essential to understanding how telomere dynamics play a role in these processes., Methods: Telomere lengths have been conventionally measured using terminal restriction fragment (TRF), quantitative fluorescence in situ hybridization (QFISH), and flow FISH. In this study, we have applied QFISH to measure average telomere lengths in cultured cells and human tissues of the GI tract. Importantly, this method can be used to analyze telomere lengths in sections using confocal microscopy. We describe and compare three image analysis algorithms: a simple pixel histogram calculation of background corrected fluorescence, a telomere spot-finding method, and a background curve subtraction algorithm., Results: Using normal human diploid fibroblasts (NHDF) either dropped on slides or sectioned after agar embedding, similar telomere length shortening is evident with increasing population doubling levels (PDLs), using peptide nucleic acid (PNA) and an N3'-P5'-phosphoamidate (PA) oligonucleotide probe for all three methods. Validation of these in situ telomere quantification methods showed excellent agreement with the commonly used telomere repeat fragment-Southern blot method. Telomere length reductions can also be demonstrated in tissue sections from histologically normal mucosa from patients with chronic ulcerative colitis (with dysplasia or cancer elsewhere in the colon), in colon adenomas, and in mucosal biopsies from patients with Barrett's esophagus. Both on slides and in tissue sections, the telomere spot-finding method has the greatest variability, while intra- and inter-biopsy variability in telomere length assessment using the other methods is relatively low., Conclusions: Accurate and reproducible telomere length measurements can be made in tissue sections using QFISH and confocal microscopy. The simplest methods proved the most reliable and make these methods readily accessible to many laboratories. The use of these methods will enhance the ability to measure telomere lengths in tissue samples and aid in the understanding of the role of telomere length in aging and disease., (Copyright 2004 Wiley-Liss, Inc.)

Elevated 4N (G(2)-tetraploid) cell populations are unstable intermediates in the development of many human cancers. However, 4N cell populations are intermixed with larger diploid fractions in vivo, limiting investigation of these key intermediates of neoplastic progression. Therefore, to study elevated 4N cell populations in human neoplasia, we used flow cytometry to purify populations of spontaneously arising TP53(wt) and TP53(mut) 4N cells from cell strains derived from premalignant Barrett's esophagus biopsies. Using oligonucleotide arrays, we identified 625 genes differentially expressed in at least one replicate 2N/4N comparison in each strain and in hTERT-immortalized cultures of the TP53(mut) strains. Strikingly, when hierarchically clustered, these data contained a large node of 124 genes that were up-regulated in 4N TP53(mut) cells in the absence of condensed chromosomes. Most of these genes function in G(2)-M to mediate processes such as chromosome condensation and segregation. These results describe the molecular phenotype of dysregulated G(2)-M functions and cell cycle checkpoints in a key intermediate of human neoplastic progression.

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

Pancreatic adenocarcinoma is a disease with high mortality for which chronic pancreatitis confers a markedly increased risk. We hypothesize that chromosome instability and genomic damage occur in pre-neoplastic pancreatic ductal epithelium, and that this damage may be related to oxidative stress. We used dual-color fluorescence in situ hybridization with centromere probes and locus-specific arm probes for chromosome arms 11q, 17p, and 18q to identify genomic instability in cultures of normal-appearing human pancreatic ductal epithelium from normal organ donor controls compared to patients with chronic pancreatitis or pancreatic adenocarcinoma. To examine early pancreatic tumorigenesis, we studied only normal-appearing pancreatic ductal cells adjacent to pancreatitis or carcinoma. We found that, compared to the finding in normal controls, chromosomal abnormalities are present in normal-appearing human pancreatic ductal epithelia obtained from patients with chronic pancreatitis or pancreatic adenocarcinoma. Furthermore, these chromosomal abnormalities could be induced in cultured pancreatic ductal epithelium from normal organ donors by chronic exposure to dilute hydrogen peroxide, suggesting that oxidative stress may contribute to the development of chromosomal instability in the pancreas. These results elucidate a potential mechanism linking chronic pancreatitis to pancreatic cancer and suggest that chromosomal instability may be an early event in the pathogenesis of pancreatic cancer., (Copyright 2003 Wiley-Liss, Inc.)

This article provides a framework for clinicians who are attempting the difficult task of interpreting the Barrett's biomarker literature with the goal of improving care for their patients. Although many articles. including more that 60 proposed biomarkers, have been published on this subject, only a few describe phase 3 and 4 studies that are of interest to the clinical gastroenterologist (Table 1). For year, dysplasia grade has been the sole means of risk stratification for patients with BE, and it likely will continue to be used in the foreseeable future. The current authors believe that dysplasia classification can be valuable using the team management approach and quality controls described previously. Significant problems, however, have emerged in phase 2 through 4 studies of dysplasia that make it imperative for the Barrett's field to incorporate additional biomarkers as they are validated. These problems include poor reproducibility of dysplasia interpretations, poor predictive value for negative, indefinite, and low-grade dysplasia, and inconsistent results for HGD in different centers, all of which makes it virtually impossible to develop national guidelines for surveillance. Some studies have even suggested that endoscopic biopsy surveillance using dysplasia may not be worthwhile. Currently, flow cytometric tetraploidy and aneuploidy have progressed furthest in biomarker validation (see Table 1). With proper handling, endoscopic biopsy specimens can be shipped to reference laboratories that have the instruments, computer analytic methods, and expertise to reproducibly detect tetraploidy and aneuploidy. The results of phase 4 studies indicate that flow cytometry appears to be useful in detecting a subset of patients who do not have HGD and yet have an increased risk of progression to cancer that cannot be identified by dysplasia grade. For many reasons, the authors anticipate that the number of validated biomarkers will increase substantially in the future. Biopsy repositories are now readily available for phase 3 studies that can evaluate and compare biomarkers. There are initiatives for multi-institutional Barrett's Centers of Excellence that could provide rapid progress in biomarker evaluation. In addition to new candidate biomarkers, the human genome project has provided high-throughput methodologies and methods for computer analysis of data, which can provide the volume and quality control required for clinically useful biomarkers. Currently, 17p (p53) LOH has progressed the furthest among molecular biomarkers. The authors do not recommend its routine clinical use at the present time, however. Finally, it is likely that clinicians will want to follow the results of clinical treatment-response studies and epidemiologic studies that evaluate relationship between clinical interventions or environmental risk and protective factors and surrogate endpoints, especially if the endpoints are progessing well along the phases of biomarker validation. These studies are likely to be of clinical interest because they may becoming the basis for randomized clinical trials to prevent cancer in BE.

Patients with extensive ulcerative colitis (UC) of longer than 8 years duration are at high risk for the development of colorectal cancer. The cancers in these patients appear to develop in a stepwise manner with progressive histological changes from negative for dysplasia --> indefinite for dysplasia --> dysplasia --> cancer. The aim of this study was to determine the timing and extent of genomic instability in the progression of UC dysplasia and cancer. Using two polymerase chain reaction (PCR)-based DNA fingerprinting methods, arbitrarily primed PCR and intersimple sequence repeat PCR, we assessed DNA sequence variation in biopsies across the spectrum of cancerous, dysplastic, and nondysplastic mucosa. UC patients with dysplasia/cancer had substantial genomic instability in both their dysplastic and nondysplastic colonic mucosa, whereas instability was not present in the majority of UC patients without dysplasia/cancer. The degree of instability in nondysplastic tissue was similar to that of dysplastic/cancerous mucosa from the same patient, suggesting that this instability was widespread and reached the maximum level early in neoplastic progression. These results suggest that UC patients who develop dysplasia or cancer have an underlying process of genomic instability in their colonic mucosa whereas UC patients who are dysplasia-free do not.

A dramatic increase in the incidence of esophageal adenocarcinoma has occurred among men in the United States over the last two decades. The underlying reasons remain largely unknown, although the increasing prevalence of obesity likely plays a role. Most adenocarcinomas arise in a metaplastic epithelium termed Barrett's esophagus (BE) that develops in approximately 10% of persons who have chronic gastroesophageal reflux. Persons with BE are at high risk (0.5-1.0%/year) of progressing to cancer. In a cross-sectional study of 429 persons with BE, we evaluated the associations between increased body mass index, anthropometric measures, cigarette smoking, use of nonsteroidal anti-inflammatory drugs (NSAIDs) and markers of increased risk, including aneuploidy, increased 4N fraction, loss of heterozygosity (LOH) of 17p and 9p alleles, and high-grade dysplasia (HGD). In logistic regression models adjusting for age, gender, NSAID use, and cigarette smoking, increasing waist:hip ratio was related to increasing risk of aneuploidy (trend P = 0.01), 17p LOH (trend P = 0.005), and 9p LOH (trend P = 0.007). The odds ratios comparing highest to lowest quartiles were 4.3 [95% confidence interval (CI), 1.2-15.6] for aneuploidy, 3.9 (95% CI, 1.3-11.4) for 17p LOH, and 2.7 (95% CI, 1.2-6.3) for 9p LOH. A nonsignificant trend was also observed for increased 4N fraction, whereas little association was found for HGD. Similar patterns of risk were noted for other anthropometric measures such as waist:thigh and abdomen:thigh ratios. There was no evidence that elevated body mass index increased risk of any of the biomarkers. Suggestive evidence also was found for a protective effect of NSAID use. The odds ratios for current users, compared with those who never used NSAIDs regularly, were 0.6 (95% CI, 0.3-1.4) for increased 4N, 0.6 (95% CI, 0.3-1.3) for aneuploidy, 0.3 (95% CI, 0.1-0.7) for 17p LOH, and 0.7 (95% CI, 0.4-1.2) for HGD. There was no association between NSAID use and risk of 9p LOH. We conclude that an abdominal distribution of body fat, which is more common in men and is termed male-pattern obesity, may be a strong predictor of risk of neoplastic progression among persons with BE and may account in part for the male predominance of BE and esophageal adenocarcinoma. We also conclude that NSAID use may reduce the risk of progression to cancer in this population. Prospective studies are needed to confirm these results.

Flow cytometry can measure a variety of functional parameters of increasing interest to cell biologists and clinicians. The distinguishing feature of these assays is that the information of interest is the functional behavior of the cell itself, rather than the phenotype. This unit discusses cell viability, oxidative metabolism, membrane potential, intracellular ionized calcium, intracellular pH, intracellular organelles, and gene reporter assays.

Background: Drug sensitivity is commonly determined by assays that utilize colony formation to discriminate between surviving and lethally treated cells. These assays require cells with high plating efficiency that form discernible colonies, are time-consuming and laborious, and require manual counting of large numbers of colonies. To overcome these drawbacks, we developed a flow cytometric technique that assays survival of proliferative capacity in cultured cells., Methods: Labeling with bromodeoxyuridine for 72 h followed by bivariate Hoechst 33258/ethidium bromide flow cytometry allows discrimination of nonproliferating cells from those that have undergone one to three divisions. Addition of an internal standard, chicken erythrocyte nuclei, permits determination of total cell number. To validate our assay, we used flow and colony-forming assays to determine the sensitivity of cell lines derived from Werner syndrome patients and unaffected individuals to 4-nitroquinoline-1-oxide (4NQO) and camptothecin., Results: The flow and colony-forming assays yielded comparable sensitivity for each drug and essentially identical increases in drug sensitivity exhibited by Werner syndrome cells., Conclusion: Our results indicate that the flow assay is a less laborious surrogate for colony-forming assays. The flow technique will also facilitate the analysis of drug sensitivity in cells that are not amenable to colony-forming assays., (Copyright 2002 Wiley-Liss, Inc.)

The clinical phenotype of Werner Syndrome (WRN) includes features reminiscent of accelerated aging and an increased incidence of sarcomas and other tumors of mesenchymal origin. This syndrome results from mutations in the WRN DNA helicase/exonuclease gene. We found that WRN deficient primary fibroblasts, as well as lymphoblastoid cell lines (LCLs), show reduced proliferative survival in response to 4-nitroquinoline-N-oxide (4NQO) and 8-methoxypsoralen (8MOP), compared with WRN-proficient cells. This is the first demonstration of drug hypersensitivity in primary cells of mesenchymal origin from WRN patients. Notably, 8MOP-induced DNA interstrand crosslinks, but not 8MOP mono-adducts, produced S-phase apoptosis in WRN-deficient LCLs. In contrast, 8MOP did not induce S-phase apoptosis in WRN-deficient diploid fibroblasts, in which drug hypersensitivity was entirely due to reduced cell proliferation. Such reduced proliferation of damaged mesenchymal cells in WRN patients may lead to earlier proliferative senescence. In addition, failure of WRN-deficient mesenchymal cells to undergo apoptosis in response to DNA damage in S-phase may promote genomic instability and could help clarify the increased risk of sarcoma in WRN patients. Because interstrand crosslinks are believed to be repaired through homologous recombination, these results suggest an important role for WRN in recombinational resolution of stalled replication forks.

We have optimized a flow cytometric DNA alkaline unwinding assay to increase the sensitivity in detecting low levels of DNA damage (strand breaks and alkali-labile sites) and to permit the measurement of the extent of DNA damage within each cell cycle compartment. The lowest gamma radiation dose that induced detectable DNA damage in each cell cycle phase of HeLa and CEM cells was 10 cGy. The lowest H(2)O(2) concentration that induced detectable DNA damage in each cell cycle phase was 0.5 microM in HeLa cells, and 1-2.5 TmicroM in CEM cells. For both HeLa cells and CEM cells, DNA damage in each cell cycle compartment increased approximately linearly with increasing doses of gamma radiation and H(2)O(2). Although untreated HeLa and CEM cells in S phase consistently exhibited greater DNA unwinding than did G(1) or G(2) cells (presumably due to DNA strand breaks associated with replication forks), there was no difference between the susceptibility of G(0)/G(1), S and G(2)/M phase cells to DNA damage induced by gamma radiation or H(2)O(2), or in the rate of repair of this damage. In each cell cycle phase, the susceptibility to gamma radiation-induced DNA damage was greater in CEM cells than in HeLa cells. In contrast to the lack of cell cycle phase-specific DNA damage induced by exposure to gamma radiation or H(2)O(2), the cancer chemotherapeutic drug doxorubicin (adriamycin) predominantly induced DNA damage in G(2) phase cells.

Analysis of allelic loss in archival tumor specimens is constrained by quality and quantity of tissue and by technical limitations on the number of chromosomal sites that can be efficiently evaluated in conventional analyses using polymorphic microsatellite markers. Newly developed array-based assays have the potential to yield genome-wide data from small amounts of tissue but have not been validated for use with routinely processed specimens. We used the Affymetrix HuSNP assay, composed of 1494 single nucleotide polymorphism sites, to compare allelic loss results obtained from both formalin-fixed and frozen breast tissue samples. Tumor cells were separated from normal epithelia and nonepithelial cells by dissection and bivariate cytokeratin/DNA flow sorting; normal breast cells from the same patient served as constitutive normal. Allele results from the HuSNP array averaged 96% reproducibility between duplicates and were concordant between the fixed and frozen normal samples. We also analyzed DNA from the same samples after whole-genome amplification (primer extension preamplification). Although overall signal intensities were lower, the genotype data from the primer extension preamplification material was concordant with genomic DNA data from the same samples. Results from genomic normal tissue DNA averaged informative single nucleotide polymorphism at 379 (25%) loci genome-wide. Although data points were clustered and some segments of chromosomes were not informative, our data indicated that the Affymetrix HuSNP assay could provide an efficient and valid genome-wide analysis of allelic imbalance in routinely processed and whole genome-amplified pathology specimens.