Suppression of anchorage-independent growth after gene transfection

Abstract

A novel procedure for isolating anchorage-dependent cells has been developed. It involves negative selection of cells growing in suspension followed by clonal replica screening for anchorage-dependent growth. Cells which have regained anchorage-dependent growth have been isolated from a library of the Chinese hamster ovary cell line, CHO-K1, transfected with pSV2neo and human genomic DNA. One anchorage-dependent clone, 1042AC, has been studied in detail. Anchorage-dependent growth of 1042AC is stable when cultured as adherent monolayers, but revertants appear rapidly when cultured in suspension. Suppression is unlikely to be due to loss or mutation of hamster genes conferring anchorage-independent growth as hybrids between 1042AC and CHO-K1 have the suppressed phenotype of 1042AC. Furthermore, a population of cells obtained from the hybrid by selecting for revertants to anchorage-independent growth showed selective loss of the transgenome derived from 1042AC. The growth suppression was not due to transfection of the human Krev-1 gene, which has previously been shown to restore anchorage-dependent growth, nor was there any evidence of alteration in the endogenous hamster Krev-1 gene. However, evidence for a human gene being responsible for the suppressed phenotype has not been obtained yet.