Effect of in vivo carcinogen exposure on colony formation and growth of hamster buccal pouch keratinocytes in culture.

The effect of in vivo carcinogen exposure on colony formation and growth of hamster buccal pouch keratinocytes in culture was evaluated at various times after exposing the mucosal surfaces of buccal pouches to initiating regimens of 7,12-dimethylbenz(a)anthracene (DMBA) or N-methyl-N-benzylnitrosamine (MBN). Keratinocytes were isolated by enzymatic dissociation of intact sheets of buccal pouch epithelium (HBPE) and separated into five fractions of varying buoyant density by isopycnic centrifugation through Percoll. The number of keratinocyte colonies which formed in cultures of unfractionated and fractionated HBPE was evaluated at 1, 2, 3, 4, and 8 weeks after treatment with DMBA and its control vehicle, paraffin oil, and at 6, 8, and 10 weeks after treatment with MBN and its control vehicle, propylene glycol. The rate of colony growth expressed as population doublings per day was determined at these same time periods in unfractionated cultures of carcinogen and control treated HBPE. Unfractionated control (paraffin oil and propylene glycol) cultures gave rise to a uniform number of keratinocyte colonies with a growth rate of approximately 1 population doubling per day at each time period examined. Control cultures of fractionated HBPE exhibited considerable variation in their capacity to form keratinocyte colonies. Fraction 5 cultures which were composed almost exclusively of basaloid cells failed to form colonies. Cultures of unfractionated and fractionated DMBA and MBN-treated HBPE exhibited a marked and persistent suppression in the number and growth of keratinocyte colonies. In addition we observed the development of a morphologically unique type of keratinocyte colony that first emerged in the high density, basaloid-rich fraction 5 cultures, and which subsequently developed in all fractionated and unfractionated HBPE cultures. These unique keratinocyte colonies were never observed in control cultures. These results demonstrate that in vivo exposure of HBPE to DMBA or MBN causes a marked and prolonged suppression in the number and growth of keratinocytes colonies in culture. Furthermore the treatment regimens appeared to induce or select for a population of keratinocytes which persisted in vivo and which exhibited a unique colony morphology when grown in surface culture.