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 hybridization (SSH), based on suppression PCR and a combination of normalization and subtraction in a single procedure, was used. Using the nontumorigenic CGL1 and tumorigenic CGL3, forward (CGL1-CGL3) and reverse (CGL3-CGL1) subtracted libraries were constructed. Among 192 clones, seven were identified as differentially expressed genes specific for either CGL1 or CGL3. All seven 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 colony formation assay; however, no tumor suppression was observed when the transfectant was inoculated into nude mice. These results indicate that: (a) SSH is a suitable method to identify differentially expressed genes in two types of cells; and (b) although p16 plays some roles in growth suppression, the p16-transfected CGL3 is still capable to proliferate in vivo.
Nishizuka
S
S
Department of Microbiology and Molecular Genetics, College of Medicine, University of California-Irvine, Irvine, CA 92697-4025, USA. satoshi@discover.nci.nih.gov
Tsujimoto
H
H
Stanbridge
E J
EJ
D000818:Animals; D005260:Female; D005347:Fibroblasts / Q000473:pathology / Q000502:physiology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D019942:Genes, p16 / Q000502:physiology*; D006367:HeLa Cells / Q000473:pathology / Q000502:physiology*; D006801:Humans; D006822:Hybrid Cells / Q000473:pathology / Q000502:physiology; D051379:Mice; D008819:Mice, Nude; D009693:Nucleic Acid Hybridization / Q000379:methods; D016133:Polymerase Chain Reaction; D014162:Transfection