Fyn is a redox sensor involved in solar ultraviolet light-induced signal transduction in skin carcinogenesis.

Solar ultraviolet (UV) light is a major etiological factor in skin carcinogenesis, with solar UV-stimulated signal transduction inducing pathological changes and skin damage. The primary sensor of solar UV-induced cellular signaling has not been identified. We use an experimental system of solar simulated light (SSL) to mimic solar UV and we demonstrate that Fyn is a primary redox sensor involved in SSL-induced signal transduction. Reactive oxygen species (ROS) generated by SSL exposure directly oxidize Cys488 of Fyn, resulting in increased Fyn kinase activity. Fyn oxidation was increased in mouse skin after SSL exposure and Fyn-knockout mice formed larger and more tumors compared with Fyn wild-type mice when exposed to SSL for an extended period of time. Murine embryonic fibroblasts (MEFs) lacking Fyn and cells in which Fyn expression was knocked down were resistant to SSL-induced apoptosis. Furthermore, cells expressing mutant Fyn (C448A) were resistant to SSL-induced apoptosis. These findings suggest that Fyn acts as a regulatory nexus between solar UV, ROS and signal transduction during skin carcinogenesis.

Prospects for 3D, nanometer-resolution imaging by confocal STEM.

Confocal STEM is a new electron microscopy imaging mode. In a microscope with spherical aberration-corrected electron optics, it can produce three-dimensional (3D) images by optical sectioning. We have adapted the linear imaging theory of light confocal microscopy to confocal STEM and use it to suggest optimum imaging conditions for a confocal STEM limited by fifth-order spherical aberration. We predict that current or near-future microscopes will be able to produce 3D images with 1 nm vertical resolution and sub-Angstrom lateral resolution. Multislice simulations show that we will need to be cautious in interpreting these images, however, as they can be complicated by dynamical electron scattering.

Measurement of chemopreventive efficacy in skin biopsies.

OBJECTIVE: To explore methods suitable for quantitative assessment of the efficacy of chemopreventive intervention. STUDY DESIGN: High-resolution imagery of nuclei from the suprabasal and basal cell layers of sun-damaged skin were recorded. There were 10 cases. A shave biopsy was taken from an area of clearly evident solar keratosis before and after treatment with 2-difluoromethyl-dlornithine (DFMO) and from the colateral forearm, treated with a placebo. A number of karyometric variables were computed and combined to derive marker features that provided a numeric measure of the degree of nuclear deviation from normal. RESULTS: DFMO treatment was effective overall in reducing the degree of nuclear abnormality seen in the biopsies; in 8 of the 10 cases there was a significant improvement. The placebo-treated arm did not show a statistically different abnormality from the untreated arm. CONCLUSION: Karyometric analysis can provide numeric measures that allow documentation of statistically significant regression of actinic keratotic lesions following treatment with DFMO.

Ki-ras proto-oncogene mutations in sporadic colorectal adenomas: relationship to histologic and clinical characteristics.

BACKGROUND & AIMS: [corrected] The goal of this study was to examine the relationship between Ki-ras mutations in colorectal adenomas and characteristics of both the subject (age, gender, and family/personal history of colonic neoplasia) and the adenoma (multiplicity, size, location, and histologic features). METHODS: Ki-ras mutations were detected by direct sequencing in 738 adenomatous polyps removed at baseline from 639 participants in a nutritional trial of adenoma recurrence. RESULTS: Ki-ras mutations were detected in 17.2% of the adenomas. Ki-ras mutations were unrelated to gender, family, or personal history of colonic neoplasia, location within the colorectum, or adenoma multiplicity, but were more common in older subjects (P = 0.01 for trend), in larger adenomas (P < 0.0001 for trend), in adenomas with villous histology (odds ratio [OR], 3.2; 95% confidence interval [CI], 2.1-4.9 vs. tubular), and in adenomas with high-grade dysplasia (32.0% vs. 13.6%; OR, 3.0; 95% CI, 1.9-4.6 vs. low-grade dysplasia). Multivariate analysis showed Ki-ras mutations to be independently associated with subject age (P = 0.01 for trend), tubulovillous/villous histology (OR, 2.3; 95% CI, 1.5-3.7), and high-grade dysplasia (OR, 1.9; 95% CI, 1.2-3.1). Adenoma size was not independently related to Ki-ras mutation. CONCLUSIONS: Ki-ras mutations are associated with the histologic features of adenoma progression (villous histology and high-grade dysplasia) rather than with adenoma growth.

Effect of subacute ibuprofen dosing on rectal mucosal prostaglandin E2 levels in healthy subjects with a history of resected polyps.

Nonsteroidal antiinflammatory drugs are among the most promising chemopreventive agents for colorectal cancer. Although the mechanism by which nonsteroidal antiinflammatory drugs exert such effects remains to be further characterized, their best known pharmacological effect is inhibition of prostaglandin synthetase, which leads to decreases in tissue prostaglandin levels. We conducted a randomized, double-blind, controlled study to examine the effect of daily ibuprofen treatment on the rectal mucosal prostaglandin E2 (PGE2) levels in healthy subjects with a history of resected polyps. Study participants (n = 27) completed a 2-week run-in period and were then randomized to take a single, daily dose of ibuprofen (300 or 600 mg) or of a placebo for 4 weeks. Rectal biopsy specimens were taken before and after the run-in period and at 2 and 4 weeks after the ibuprofen/placebo treatment. Notably large between- and within-subject variability in the rectal mucosal PGE2 content was seen. The changes in PGE2 levels after ibuprofen/placebo treatment correlated with the baseline PGE2 content. After adjustment of the baseline values, 2 weeks of 300 mg/day of ibuprofen treatment resulted in significantly more suppression of PGE2 levels than that observed after the placebo treatment (55% versus 22% suppression from baseline; P = 0.033). Although other ibuprofen treatment schedules and doses appeared to result in suppression in the PGE2 levels, the suppression was not statistically significant because of the large variability in this measurement. Because lower doses are associated with fewer adverse effects, a dose of 300 mg of ibuprofen/day should be considered for future Phase II chemoprevention studies. Stratifying study participants, based on their baseline PGE2 levels and inclusion of a larger number of study subjects, are recommended for future trials where the rectal mucosal PGE2 level is to be used as a surrogate end point biomarker.

Chemoprevention of human actinic keratoses by topical 2-(difluoromethyl)-dl-ornithine.

Alpha-2-(Difluoromethyl)-dl-ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, has been shown to suppress skin carcinogenesis in murine models after oral or topical administration. We designed a randomized, placebo-controlled study using a topical hydrophilic ointment formulation with or without 10% (w/w) DFMO. Forty-eight participants with moderate-severe actinic keratoses (AKs) on their forearms (i.e., at least 10 well-circumscribed lesions on the lateral surface) completed a 1-month run-in on placebo ointment. Before randomization, all lateral forearm AKs were circled, counted, photographed, and skin biopsies were obtained for DFMO and polyamine levels. Then participants were randomized to receive DFMO ointment on the right versus the left forearm and placebo hydrophilic ointment on the contralateral forearm twice daily for 6 months. DFMO was not detected in the blood of any subject, and there were no systemic toxicities. None of a subsample of 17 placebo forearms had measurable concentrations of DFMO, whereas 13 of the corresponding DFMO-treated forearms had high DFMO skin levels. As compared with placebo, the 6-month DFMO treatment caused a 23.5% reduction in the number of AKs (P = 0.001) as well as significant suppression of AK biopsy spermidine levels (26%; P = 0.04). Seven of the 48 (14.6%) participants experienced severe (2; 4.2%) or moderate (5; 10.4%) inflammatory reactions on their DFMO-treated arms which required dosing modification. Topical DFMO for 6 months can reduce the number of AK lesions and skin spermidine concentrations in high-risk participants and deserves additional study as a skin cancer chemopreventive agent.

Effects of piroxicam on prostaglandin E2 levels in rectal mucosa of adenomatous polyp patients: a randomized phase IIb trial.

Prostaglandin E2 (PGE2) has served as a surrogate end point biomarker in colorectal tumor progression. Colonic mucosa PGE2 levels of patients with colorectal adenomas or carcinomas have been shown to be higher than in control subjects. Our dose-finding study on piroxicam, a nonsteroidal anti-inflammatory drug with chemopreventive effects in preclinical colon carcinoma models, suggested that 7.5 mg/day was well tolerated and associated with significant depression of rectal mucosa PGE2 concentrations in comparison with baseline values. We therefore conducted a randomized Phase IIb cancer prevention clinical trial to investigate the chemopreventive properties of piroxicam in patients with a history of resected colorectal adenomatous polyps. After a 2-month run-in period, 47 participants were randomized to piroxicam at a dose of 7.5 mg/day, and 49 were randomized to a placebo. Rectal biopsy specimens were taken at the initial visit, at 2 months later during the run-in period, and at 6, 12, and 24 months after the start of the interventions. Mean PGE2 concentrations in the rectal mucosa of the piroxicam-treated patients differed significantly between visits (P < 0.001), and the values at the 6-month visit (P < 0.001) and 12-month visit (P = 0.005) differed significantly from the average baseline value. Unfortunately, we observed an incidence of adverse gastrointestinal side effects in patients treated with 7.5 mg/day of piroxicam similar to that seen for arthritis patients treated with 20 mg/day. Consequently, the gastrointestinal toxicities appear to override the potential benefit that piroxicam may offer as a long-term colon cancer chemopreventive agent.

Risk factors for Ki-ras protooncogene mutation in sporadic colorectal adenomas.

The Ki-ras protooncogene frequently is mutated in colorectal adenocarcinomas, but the etiology of this molecular event is uncertain. We investigated the association between variables known or suspected to be related to risk for colorectal cancer and the occurrence of Ki-ras mutations in colorectal adenomas. This study was conducted among 678 male and female participants, 40-80 years of age, enrolled in a phase III trial testing the effects of a wheat bran fiber supplement on adenoma recurrence. Exposure information on the risk factors of interest was assessed through self-administered questionnaires. Mutations in codons 12 and 13 of the Ki-ras protooncogene were analyzed in baseline adenomas 0.5 cm or larger by PCR amplification followed by direct sequencing. Eighteen percent (120 of 678) of the participants had one or more adenoma(s) with Ki-ras mutations. A higher risk of Ki-ras mutations was associated with increasing age and a lower intake of total folate. The odds ratio (OR) for Ki-ras mutations for individuals >72 years of age was 1.98 [95% confidence interval (CI) = 1.19-3.27; P for trend = 0.008] compared with those less than 65 years of age. Compared with individuals in the lower tertile of total folate, those in the upper tertile had an approximately 50% lower risk of having Ki-ras mutation-positive adenomas (OR = 0.52; 95% CI = 0.30-0.88; P for trend = 0.02). There was a suggestion of a stronger inverse association of total folate with G-->T transversions (OR = 0.41; 95% CI = 0.20-0.87) than G-->A transitions (OR = 0.61; 95% CI = 0.31-1.21), although the CIs for the associations overlap. The results of these analyses suggest that the protective effect of folate in colon cancer observed in published studies may be mediated through folate's effect on Ki-ras mutations.

Expression in normal human tissues of five nucleotide excision repair genes measured simultaneously by multiplex reverse transcription-polymerase chain reaction.

DNA repair is central to the integrity of the human genome. Reduced DNA repair capacity has been linked to genetic susceptibility to cancer. An adequate expression level of DNA repair genes is essential for normal DNA repair activities. Although there is tissue specificity in the expression, searching for a surrogate tissue is needed for molecular epidemiological studies. In this study, the relative expression levels of five selected human nucleotide excision repair (NER) genes (ERCC1, XPB/ERCC3, XPG/ERCC5, CSB/ERCC6, and XPC) in 20 different types of human normal tissue were simultaneously measured by a new multiplex reverse transcription (RT)-PCR assay using the expression level of the beta-actin gene as an internal control. Transcripts of each of the five NER genes were detectable, but the levels varied in these normal tissues. Both mitogen (phytohemagglutinin)-stimulated and unstimulated human peripheral lymphocytes showed similar expression patterns for the five NER genes. In general, the expression levels of stimulated lymphocytes were also similar to most of the rapidly proliferating tissues, such as the skin, breast, intestine, liver, testis, ovary, placenta, or prostate, but was relatively higher than that of the slowly proliferating or nonproliferating tissues such as adipose, brain, hippocampus, muscle, spleen, or lung. The data suggested that although the five NER genes were expressed at different levels in the normal tissues examined, PHA-stimulated peripheral lymphocytes may be used as a surrogate tissue for estimating expression levels of these genes in proliferating tissues. This new multiplex RT-PCR assay may help detect aberrant expression of these NER genes in both normal and tumor tissues.

Physical activity, body mass index, and prostaglandin E2 levels in rectal mucosa.

BACKGROUND: Evidence suggests a relationship between prostaglandin levels in colonic mucosa and risk of colon cancer. Physical inactivity and a higher body mass index (BMI; weight in kilograms divided by [height in meters]2) have been consistently shown to increase risk of this cancer. We investigated whether higher levels of leisure-time physical activity or a lower BMI was associated with lower concentrations of prostaglandin E2 (PGE2) in rectal mucosa. METHODS: This study was conducted in 41 men and 22 women, 42-78 years of age, with a history of polyps, who participated in a randomized clinical trial testing the effects of piroxicam on rectal mucosal PGE2 levels. An [125I]PGE2 radioimmunoassay kit was used to determine PGE2 levels in samples of extracted rectal mucosa collected before randomization. Leisure-time physical activity was assessed through a self-administered questionnaire collected at baseline. The reported time spent at each activity per week was multiplied by its typical energy expenditure, expressed in metabolic equivalents (METs), to yield a MET-hours per week score. A repeated measures model was used to assess the effect of BMI and physical activity as predictors of PGE2 concentration. All statistical tests were two-sided. RESULTS: After adjustment for age, a higher BMI was associated with higher PGE2 levels (P = .001). A higher level of leisure-time physical activity was inversely associated with PGE2 concentration (P<.03). An increase in BMI from 24.2 to 28.8 kg/m2 was associated with a 27% increase in PGE2. An increase in activity level from 5.2 to 27.7 MET-hours per week was associated with a 28% decrease in PGE2. CONCLUSIONS: Physical activity and obesity may alter the risk of colon cancer through their effects on PGE2 synthesis.

Protective role of wheat bran fiber: data from marker trials.

The effects of wheat bran fiber on surrogate endpoint biomarkers for colon cancer risk have been studied in rats and humans. In both species, there is little evidence that wheat bran fiber significantly modifies epithelial cell proliferation. In rat studies, however, dietary supplementation with wheat bran fiber has decreased mucosal formation of aberrant crypt foci, an important marker currently used to estimate the efficacy of colon cancer chemoprevention agents. In humans, wheat bran fiber has been shown to consistently decrease fecal bile acid concentrations, mainly by reducing toxic secondary bile acids.

Relationship of p53 mutations to epidermal cell proliferation and apoptosis in human UV-induced skin carcinogenesis.

Human skin is continually subjected to UV-irradiation with the p53 gene playing a pivotal role in repair of UV-induced DNA damage and apoptosis. Consequently, p53 alterations are early events in human UV-induced skin carcinogenesis. We studied 13 squamous cell carcinomas (SCC), 16 actinic keratoses (AK), 13 samples adjacent to an AK (chronically sun-damaged), and 14 normal-appearing skin samples for p53 mutation, p53 immunostaining (IHC), apoptosis (in situ TUNEL and morphology), and proliferation (PCNA). The frequency of p53 mutation increased from 14% in normal skin, to 38.5% in sun-damaged skin, 63% in AK, and 54% in SCC. p53 IHC increased similarly. Apoptosis (TUNEL) increased from 0.06 +/- 0.02%, to 0.1 +/- 0.2, 0.3 +/- 0.3, and 0.4 +/- 0.3 in normal skin, sun-damaged skin, AK, and SCC, respectively. Apoptosis was strongly correlated with proliferation (i.e., TUNEL and PCNA, r = 0.7, P < 0.0001), and proliferation was significantly increased in the progression from normal skin to SCC. Bax was significantly increased in SCC compared to AK. These data imply that apoptosis in samples with a high frequency of p53 mutation may not necessarily be p53-dependent. We suggest that there is a mechanism for apoptosis in response to increased cellular proliferation that is p53-independent.

Expression of p53 protein in actinic keratosis, adjacent, normal-appearing, and non-sun-exposed human skin.

Nonmelanoma skin cancer, including both squamous cell carcinoma and basal cell carcinoma, is a significant and increasing health problem in the United States. The precursor lesion of cutaneous squamous cell carcinoma, actinic keratosis (AK), is a major risk factor for nonmelanoma skin cancer, and it is also a marker of long-term sun exposure. AKs themselves can serve as biomarkers in chemopreventive studies, but in addition, they may contain phenotypic and genetic alterations that are related to the process of UV-induced skin carcinogenesis. One of these alterations, the tumor suppressor gene p53, is altered early in UV-induced skin carcinogenesis in humans. p53 protein expression was measured by immunohistochemistry in biopsies from AKs, tissue immediately adjacent to AKs (AK-adjacent), normal-appearing upper medial arm skin, and non-sun-exposed skin from 19 subjects. There was a significant difference and a progressively increasing mean p53 labeling index in total epidermis (basal and suprabasal layers) between upper medial arm skin (0.9 +/- 1.8%) and AK-adjacent (12.1 +/- 14.4%; P = 0.0004) and between AK (27.7 +/- 21.3%) and AK-adjacent skin (P = 0.04), whereas upper medial arm skin was marginally different from non-sun-exposed skin (0.1 +/- 0.2; P = 0.05). This pattern of p53 expression was also seen when epidermis was separated into basal and suprabasal layers. We conclude that epidermal p53 protein expression is associated with histological evidence of chronic sun damage.

The effect of wheat bran fiber and calcium supplementation on rectal mucosal proliferation rates in patients with resected adenomatous colorectal polyps.

Colorectal cancers continue as the second most common cause of death from cancer in the United States. Only a few prospective, randomized clinical trials have been performed to evaluate the potential preventive effects of dietary fiber or calcium in patients with an increased risk for the development or recurrence of colorectal cancer. We designed and conducted a double-blinded, placebo-controlled randomized trial involving supplementation of fiber and calcium intake and measurements of [3H]thymidine labeling index (LI) percentages in rectal mucosal biopsies obtained from patients with resected colorectal adenomas to examine the potential mechanisms by which dietary interventions might reduce colorectal cancer risk. We performed a randomized, double-blinded, Phase II study, using a factorial design to measure the effects of supplemental dietary wheat bran fiber (2.0 or 13.5 g/day) and calcium carbonate (250 or 1500 mg/day elemental calcium) supplementation on [3H]thymidine LI percentages in rectal mucosal crypts and 24-h in vitro outgrowth cultures. Measurements were made at baseline randomization (i.e., after a 3-month placebo run-in period using 2.0 g of wheat bran fiber plus 250 mg of calcium carbonate) and after 3 and 9 months on treatment in 100 randomized participants who had a history of colon adenoma resection. Neither the wheat bran fiber nor the calcium carbonate supplements significantly reduced [3H]thymidine LI percentages in rectal mucosal crypts (total or compartmental analysis) or 24-h in vitro outgrowth cultures at either 3 or 9 months of daily supplementation in the 93 evaluable participants. We conclude that 9 months of high-dose wheat bran fiber and calcium carbonate supplementation in study participants with a history of recently resected colorectal adenomas does not have a significant effect on cellular proliferation rates in rectal mucosal biopsies, comparing 3- and 9-month results to baseline results. Ultimately, there is great need for the evaluation of these two different nutrient interventions in the setting of Phase III studies wherein adenomatous polyp recurrence, rather than a rectal mucosal biomarker, serves as the primary end point.

Surrogate end-point biomarkers as measures of colon cancer risk and their use in cancer chemoprevention trials.

Surrogate end-point biomarkers (SEBs) have become widely used in short-term cancer chemoprevention trials in place of cancer end points. This paper discusses criteria relevant to the selection and validation of SEBs for colon cancer risk and the use of SEBs in colon cancer chemoprevention trials. As with a number of other cancers, colon carcinogenesis is the result of a multistep process in which an increasing number of alterations, including specific gene mutations, occur as cells progress from normal to precancerous states of increasing size and dysplasia to cancer and finally to metastatic disease. Ideally, a SEB would show differential expression between the various phases of colon carcinogenesis (i.e., normal, premalignant, and malignant tissues) and be associated with risk of colon cancer. Some SEBs that do not meet these criteria may still be useful for demonstrating the effect of a particular agent. It is also necessary that a SEB be measured in tissues (or other sample material) accessible for multiple and sequential sampling and allow for development of appropriate quality control procedures. Some SEBs must have the potential for modulation by chemopreventive agents. Validation of SEBs for use in chemoprevention studies requires that a relationship between the marker and subsequent risk of cancer be established. Also, the assay reliability and accuracy for each SEB must be determined adequately in well-designed prospective studies.

Evaluation of proliferating cell nuclear antigen as a surrogate end point biomarker in actinic keratosis and adjacent, normal-appearing, and non-sun-exposed human skin samples.

The incidence of nonmelanoma skin cancer, including both squamous cell carcinoma and basal cell carcinoma, is a significant health problem in the United States. Actinic keratosis (AK), the precursor of cutaneous squamous cell carcinoma, is a major risk factor for nonmelanoma skin cancer. In addition, AKs are tissue targets for the identification of biomarkers for use in chemopreventive studies. The biomarker addressed in this study is epidermal cell proliferation, as quantitated by proliferating cell nuclear antigen (PCNA). Shave biopsies were obtained from AKs, tissue immediately adjacent to AKs, normal-appearing, upper-medial arm skin, and non-sun-exposed skin from 19 subjects. When any degree of PCNA staining was considered positive (semiquantitative 1-4 scale), there was a significant difference and a progressively increasing mean PCNA labeling index (LI) in the total epidermis (basal and suprabasal layers), beginning with non-sun-exposed buttock skin, with the lowest LI (2.5 + or - 1.6%), followed by upper-medial arm skin (12.3 + or - 7.4%; P = 0.0015), skin adjacent to AKs (19.2 + or - 12.2%; P = 0.0218), and finally, AKs with the highest LI (34.6 + or - 20.1%; P = 0.0017). This same pattern was observed when the epidermis was separated into basal and suprabasal layers, with the exception of a nonsignificant result for upper-medial arm skin compared with adjacent skin in the basal layer (P = 0.3981). PCNA LIs were also analyzed separately by staining intensity (i.e., scores of 1-4). The PCNA LI in skin with varying degrees of sun damage and/or histological atypia is a candidate surrogate end point biomarker for skin cancer chemoprevention studies.

Reproducibility of the measurement of colonic proliferation using bromodeoxyuridine across laboratories.

Although measures of colonic cell proliferation are being used as potential intermediate markers in chemoprevention studies, measurement standardization is still ongoing. This study was designed to assess the reproducibility of the labeling index quantification, as measured by bromodeoxyuridine, across four laboratories experienced in its use. Each institution submitted 10 slides, with one circled area of each slide to be scored. Each site followed its standard procedures for scoring colonic crypts; no attempts to standardize these procedures were made. There was high concordance among the laboratories on whether scorable crypts were present on a particular slide, but only two pairs of laboratories demonstrated agreement statistically greater than that predicted by chance. The overall difference among the sites on the number of scorable crypts was marginally significant (P = 0.083), and there was a highly significant overall difference in the magnitude of the labeling index (P < 0.0001). Sites 1 and 2 tended to have similar results, as did sites 3 and 4, most likely due to common training. Even with these discrepancies, high correlation (r > 0.75) was observed among the reported labeling index values for each pair of laboratories. Without standardized training, these laboratories may differ in the crypts considered appropriate for counting and in whether cells are counted as labeled or unlabeled. These results suggest that standardized training in scoring across all sites performing labeling index determinations is required to assure reproducibility across sites or studies. These results may also help explain discrepancies in the average values of the labeling index reported in the literature.

Comparison of proliferating cell nuclear antigen versus the more standard measures of rectal mucosal proliferation rates in subjects with a history of colorectal cancer and normal age-matched controls.

Measurement of proliferation rates by the more standard in vitro uptake techniques of [3H]thymidine and 5'-bromo-2'-deoxyuridine (BrdUrd) labeling indices (LIs) were compared to proliferating cell nuclear antigen (PCNA) in rectal mucosal biopsies from 16 subjects with resected colorectal cancer and 14 normal age-matched controls. Correlation coefficients for BrdUrd versus PCNA, [3H]thymidine versus PCNA, and BrdUrd versus [3H]thymidine were 0.691, 0.876, and 0.770, respectively. No significant differences (P > 0.05) were detected in total mean LIs between the LI methods for the normal group. In contrast, total PCNA LIs were found to be significantly different in the resected cancer patients when compared to either BrdUrd (P = 0.005) or [3H]thymidine (P < 0.001). A significant difference (P = 0.010) in total PCNA LI but not in total [3H]thymidine or BrdUrd LIs was also observed between normal controls and resected colorectal cancer subjects. Compartmental analysis of the cancer group versus the normals showed a significant difference in compartments 1 and 3 for PCNA LIs only. The reproducibility of two PCNA LI counts was excellent (r = 0.9). In addition, the reliability of mean LIs were > 0.8 with the exception of [3H]thymidine in the normal group (0.7). These study results demonstrate that PCNA LIs in human rectal mucosal biopsies are correlated highly with other more commonly used cellular proliferation measurements; however, PCNA LIs were found to be significantly higher than the other two methods in the resected colorectal cancer subjects.

Measurement of prostaglandin E2 in rectal mucosa in human subjects: a method study.

It has been demonstrated and confirmed that certain nonsteroidal anti-inflammatory drugs which inhibit cyclooxygenase and the synthesis of prostaglandins and other eicosanoids, can reduce the formation of both colon polyps and cancers in experimental animals given known carcinogens. Additionally, the results of several epidemiologic studies have suggested that nonsteroidal anti-inflammatory drugs may reduce the risk of colon polyp occurrence and/or colon cancer mortality. We have carried out a study to evaluate the methodology of the measurement of prostaglandin E2 (PGE2) in human colonic mucosa because its concentration may serve as a valuable intermediate marker of the pharmacological activity in Phase II studies of nonsteroidal anti-inflammatory drugs as colon cancer preventive agents. We studied all aspects of the actual measurement of PGE2 including the extraction efficiency of the PGE2 from the mucosa, the precision of the assay and calculation of the PGE2 content in terms of milligrams of protein in the sample, the inhibition of PGE2 by indomethacin over time, the reproducibility of the measurement within one homogenate, the rate of PGE2 production over time, the effect of adding indomethacin versus snap freezing on PGE2 production, the stability of PGE2 in tissues over time stored in liquid nitrogen, and the variability of the measurement of PGE2 in separate biopsies from one individual. Our studies indicated that the most reliable method for accurate and consistent measurements of PGE2 was to add the mucosal tissue instantly after biopsy to an indomethacin buffer that effectively inhibited the in vitro formation of PGE2.

Do NSAIDs exert their colon cancer chemoprevention activities through the inhibition of mucosal prostaglandin synthetase?

Nonsteroidal antiinflammatory drugs (NSAIDs) have considerable potential as chemopreventive agents for colorectal cancer. Recent case-control drug surveillance and large cohort studies found that patients with regular aspirin use had a reduced incidence of colorectal cancer and/or decreased death rate from this disease. Several different NSAIDs reduce formation of both colon adenomatous polyps (the precursor lesion of colon cancer) and cancers in experimental animals given known carcinogens. Perhaps most convincing are reports that the NSAID sulindac promotes regression and inhibits recurrence of adenomatous colon polyps in patients with adenomatous polyposis coli. The best characterized pharmacologic effect of the NSAIDs is their reduction of prostaglandin synthesis by inhibiting prostaglandin synthetase PGE2, which catalyzes the formation of prostaglandin precursors from arachidonic acid. Several lines of evidence are contrary to the concept that inhibition of prostaglandin synthesis is central to the NSAIDs' chemopreventive effects. Relatively high levels of prostaglandins have been reported to inhibit tumor cell growth both in vivo and in vitro, and to inhibit differentiation in some tumor cell lines. We evaluated comparative chemopreventive effects on colon tumor formation in an azoxymethane (AOM)-induced colon carcinogenesis rat model using the NSAIDs piroxicam, sulindac, and sulindac sulfone, a metabolite of sulindac which lacks the anti-prostaglandin synthetase activity typically associated with NSAID-induced gastrointestinal toxicities. The results demonstrate that sulindac sulfone, a compound lacking anti-prostaglandin synthetase activity, inhibits AOM-induced colon cancer in rats. Substantial dose-dependent reductions in both tumor burden and tumor multiplicity were observed in the sulindac sulfone-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)