Effect of folate supplementation on mucosal cell proliferation in high risk patients for colon cancer.

AIMS: Intracellular folate deficiency has been implicated in colonic carcinogenesis in epidemiological studies and animal and human cancer models. Our aim was to determine the effect of folate supplementation on patients with recurrent adenomatous polyps using rectal mucosal cell proliferation as a biomarker. PATIENTS AND METHODS: Eleven patients with recurrent adenomatous polyps of the colon were randomised into a treatment group (n=6) receiving a dietary supplement of 2 mg folic acid per day for three months and a control group (n=5) receiving a placebo. Rectal biopsies where taken at 10 cm from the anal verge prior to supplementation and repeated at four, 12, and 18 weeks from the start of the supplementation. Each biopsy was immediately incubated in culture medium enriched with bromodeoxyuridine (BrdU). The S phase cells which incorporated BrdU into their DNA were identified following immunohistochemical staining. Twenty five orientated crypts were identified for each time point and the number and position of BrdU positive and BrdU negative cells were counted. BrdU labelling indices (LIs) were calculated for the entire crypt and for each of five equal compartments running consequently from the base to the luminal surface. RESULTS: The LI of the treatment group (9.1 (6.7, 12.3)) and the control group (9.3 (7.8, 10.3)) were comparable at the start. Over the duration of the supplementation period, LI in the control group did not alter significantly (9.3 (7.8, 10.3) v 9.6 (8.9, 10.4)). However, LI of the folate treated group was lowered after 12 weeks of supplementation (9.1 (6.7, 12.3) v 7.4 (5.3, 9.6)). Analysis of the LI for compartments within the crypt showed that the most significant drop in number of proliferating cells was in the upper most regions of the crypt. CONCLUSION: These data indicate that (a) folate supplementation decreases colonic mucosal cell proliferation in a high risk group for colon cancer and (b) the most significant reduction takes place at the luminal aspect of the crypt.

Induction of apoptosis by mitomycin-C in an ex vivo model of bladder cancer.

OBJECTIVE: To examine mitomycin-C (MMC)-induced apoptosis in an ex vivo model of superficial TCC, and relate it to the in vivo response to chemotherapy. Materials and methods Dose- and time-response curves were constructed to determine the optimal conditions for the induction of apoptosis by MMC in an ex vivo model of superficial bladder cancer. Subsequently, 41 individual tumours were exposed to MMC in the model and the effects assessed by measuring of apoptosis before and after chemotherapy. The relationships between tumour grade and stage and the intrinsic and induced apoptotic counts were determined. In tandem, in a clinical study, the relationship between in vivo response of a marker tumour to MMC and the ex vivo induction of apoptosis was determined. RESULTS: In the ex vivo model, apoptosis was induced at a MMC concentration of 0.5 mg/mL after an incubation time of 8 h. In 41 tumours the intrinsic apoptotic index (AI) was higher with increased grade and stage of tumour (P = 0.048). There was no correlation between the intrinsic AI and the AI after treatment with MMC (induced AI). In 21 tumours (51%) the induced AI did not increase above a predetermined response threshold and these tumours were considered resistant to MMC. Resistance to MMC was related to tumour grade (P = 0.037) with a trend for G3 pT1 tumours to be resistant to the therapy. There was a significant association between ex vivo sensitivity and in vivo marker tumour response (P = 0.02). CONCLUSIONS: Apoptosis is differentially induced in an ex vivo incubation model of superficial TCC by MMC and evidence suggests that this response matches that seen in vivo. The measurement of apoptosis before therapy does not predict the apoptotic response of a tumour to chemotherapy. The ability to undergo apoptosis correlates with clinical outcome.

Validation of a rapid method to quantify apoptosis in superficial bladder cancer.

OBJECTIVES: To derive and validate a rapid method for calculating apoptotic indices in superficial transitional cell carcinoma (TCC) as a measure of chemosensitivity to mitomycin. MATERIALS AND METHODS: Apoptotic cells, identified by light microscopy in 20 superficial TCC specimens, were expressed as an index of the total tumour cell population within defined fields. For a given field, the total cell population was estimated by: (i) an exhaustive count of the total number of cells in the field and (ii) an abbreviated method in which the number of cells in a subfield was multiplied to provide an estimate of the total field number. Field and specimen estimates were compared using agreement statistics and the intra- and inter-observer reproducibility of apoptotic indices calculated. RESULTS: Cellularity and apoptotic indices obtained using method (ii) were correlated significantly with the true cell counts (P < 0.001). Agreement statistics showed that only 9.4% of counts fell outside two standard deviations (SD) from the mean in field analysis, and only 10% of counts fell outside 2 SD from the mean in specimen analysis. There was a fivefold variation in tumour cell counts among individual fields. CONCLUSIONS: The reported variation in cellularity among fields shows that the calculation of apoptosis must use the total cell population as the reference. The limits of agreement for the estimated and true cell counts are small enough to be confident that the shorter method to estimate cellularity can be used in place of counting all cells.