Detection of differentially expressed genes in HeLa x fibroblast hybrids using subtractive suppression hybridization

To understand genetic differences and similarities between tumorigenic and nontumorigenic HeLa x fibroblast hybrid cells, subtractive suppression hybr idization (SSH), based on suppression PCR and a combination of normalizatio n and subtraction in a single procedure, was used. Using the nontumorigenic CGL1 and tumorigenic CGL3, forward (CGL1-CGL3) and reverse (CGL3-CGL1) sub tracted libraries mere constructed. Among 192 clones, seven were identified as differentially expressed genes specific for either CGL1 or CGL3. All se ven were not reported previously as differentially expressed genes in this hybrid system, In the forward subtraction, p16 was isolated, indicating the involvement of the loss of tumorigenic phenotype, Subsequent transfection of wild-type p16 to the tumorigenic CGL3 showed growth suppression in colon y formation assay; however, no tumor suppression was observed when the tran sfectant was inoculated into nude mice. These results indicate that: (a) SS H is a suitable method to identify differentially expressed genes in two ty pes of cells; and (b) although p16 plays some roles in growth suppression, the p16-transfected CGL3 is still capable to proliferate in vivo.