CHANGES IN STEM-CELL POPULATIONS OF RAT TRACHEAL EPITHELIAL-CELL CULTURES AT AN EARLY STAGE IN NEOPLASTIC PROGRESSION

  • 1 January 1985
    • journal article
    • research article
    • Vol. 45  (7) , 3322-3331
Abstract
The development of transformed colonies and concomitant changes in proliferative and nonproliferative cell compartments were studied in rat tracheal epithelial (RTE) cell cultures following exposure to N-methyl-N''-nitro-N-nitrosoguanidine (MNNG). Primary RTE cells were plated onto 3T3 feeder layers and treated with MNNG (0.25 .mu.g/ml) or solvent. Seven days later, the feeder cells were removed to select for enhanced growth variants, which are the transformants of the RTE cell system, usually scored 5 wk after carcinogen exposure. Most of the RTE cell colonies, which originally formed during the first 7 days of culture, diasppeared within 2 wk after feeder cell removal in control and MNNG-treated cultures. In control cultures, about 3% of the original colonies persisted, while in MNNG-treated cultures, a larger percentage (.apprx. 9%) of the colonies persisted. These percentages remained constant from 3-7 wk. Based on colony size, cell density and cell morphology, the persistent colonies were classified into transformed colonies (large colony size, high cell density, high nuclear: cytoplasmic ratio) and untransformed colonies (small size, low cell density, low nuclear: cytoplasmic ratio). In the MNNG-treated cultures, about 50% of all persistent colonies showed transformed morphology. Their frequency remained unchanged between 3 and 7 wk of culture. In contrast, only 10 to 15% of the persistent colonies in control cultures showed transformed morphology at 3 wk, but that proportion increased steadily between 3 and 7. Apparently, in control cultures, transformed colonies developed spontaneously as a function of time within untransformed colonies. Autoradiographic studies with [3H]thymidine showed that labeling indices in the early normal RTE cell colonies between Days 4 and 7 of culture were very high, ranging between 75 and 90%. In contrast, the labeling indices of persistent colonies, both those without and those with transformed morphology, were low, i.e., between 19 and 25%, indicating that a major proportion of cells was either noncycling or cycling very slowly. The relative compartment sizes of cells with stem cell characteristics and of cells with characteristics of transformed stem cells were estimated before and after transformed colonies appeared. Transformed colonies were composed of nonstem cells, nontransformed stem cells, and transformed stem cells; the proportion of stem cells with transformed growth characteristics increased steadily between 2 and 10 wk, as EG-variant colonies developed; and transformed stem cells with a capacity to attach and grow on plastic appeared at 5 wk but with a frequency of only .apprx. 1% of the total cell population in transformed cultures. Cells with this phenotype comprised 15% or more of the total cell population of transformed RTE cell cultures at passage 6. The heterogeneity of the cell populations comprising transformed cultures and the continuing selection occurring in the transformed cell population were demonstrated.