TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of pr otein tyrosine phosphatases, on primary cultures of colonocytes and st romal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37 degrees C to prepare viable gland fragments and isolated cells. Cell p reparations were inoculated onto plastic substratum or cytodex-3 micro carriers in a defined maintenance medium or in 1% fetal calf serum med ia. Incorporation of sodium orthovanadate (greater than or equal to 50 mu M) in these media constantly enhanced the survival (cell enumerati on and trypan blue exclusion P<0.05) and the adhesion (up to four-fold by crystal violet staining, P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-alpha or alkaline phosphata se expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mM n-butyric acid did not promote cell adhesion and survival except fo r distal cells exposed to 2 mM sodium orthovanadate. Besides studies i n the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells. (C) 1997 Academic Press Limited.