HUMAN HT-29 COLON-CARCINOMA CELLS - MUCIN PRODUCTION AND TUMORIGENICITY IN RELATION TO GROWTH PHASES

The main aim of this study was to determine whether changes in mucin p roduction/secretion during the growth phases of a human adenocarcinoma cell line, HT-29, are associated with a different tumorigenic potenti al. HT-29 cells cultured in DMEM supplemented with 10% FCS were harves ted in the exponential and post-confluent phases of growth. Metabolic labeling of the cells was performed using [H-3]-glucosamine. Following a 24 hr-incubation period, radioactivity was measured in 1 ml-aliquot s of cell cytosol and culture medium. Concurrently mucin synthesis was assessed by size exclusion chromatography of [H-3]-glucosamine-labele d glycoprotein in a Sepharose CL-4B column. Clonigenic assay in soft a gar of pre- and post-confluent HT-29 cells was determined by scoring v iable colonies according to size and number using phase-contrast micro scopy. To assess in vivo tumorigenicity, pre- and post-confluent HT-29 cells (4 x 10(6)) in 0.2 ml DMEM were inoculated into nude mice. Tumo r size and volume were recorded for 31 days. H-29 cells grew as multil ayers of unpolarized, undifferentiated cells. Colony forming efficienc y was similar at all growth stages. A significant increase in mucin sy nthesis was noted in HT-29 cells harvested in the exponential phase of growth compared to the stationary phase (148.3+/-41.9 versus 49.1+/-5 .0, mean+/-SE, dpm/4 x 10(3) cells, p < 0.05). Mucin secretion was not significantly changed. Tumorigenicity in nude mice was consistently h igher when the cells were injected in the exponential phase of growth. On day 31 after cell inoculation the average tumor volume/site was 33 2.7 mm3 in mice injected with HT-29 cells in log phase compared to 142 .7 mm(3) (p<0.01) in animals which received post-confluent cells. Tumo r weights were 0.36 g and 0.22 g respectively (p<0.05). The present re sults indicate a definitive correlation between growth phases of HT-29 cells and mucin synthesis. mucin production was significantly higher in exponentially growing cells. The cloning efficiency in soft agar, a marker of in vitro tumorigenicity, was similar in HT-29 cells irrespe ctive of the growth stage. A main finding of the present study was the ina eased in vivo tumorigenicity of colon cancel cells inoculated int o athymic mice in the log phase of growth compared to cells harvested at post-confluence. These results are consistent with the view that mu cin plays an important contributory role in determining tumorigenicity .