CLONING OF PUTATIVE GROWTH-REGULATORY GENES FROM PRIMARY HUMAN KERATINOCYTES BY SUBTRACTIVE HYBRIDIZATION

In order to isolate genes that might be involved in regulating human k eratinocyte (HKc) growth and/or differentiation, we constructed a cDNA library by subtractive hybridization between primary HKc and FaDu hea d-and-neck squamous cell carcinoma cells, Among the first set of indep endent cDNAs that we have isolated, ten correspond to known genes, and two represent novel sequences. Nine of the ten known genes are expres sed at significantly lower levels in the majority of the SqCC cell lin es in comparison with primary HKc. These include cDNAs that encode ker atins K5 and K14 which are cytoskeletal proteins normally expressed in lining epithelia, the 14-3-3 protein stratifin/HME-1, lipocortin-II a nd CaN19 which are calcium-binding proteins that may play a role in HK c differentiation by regulating protein kinase C, plasminogen-activato r inhibitor-2 which is a serine-proteinase inhibitor, HBp17 which is a HKc-specific secreted inhibitor of fibroblast growth factors, integri n alpha 3 which plays a role in the anchoring of keratinocytes to base ment membrane, and YL-8, a ras-like protein that probably mediates int racellular protein trafficking. In addition, we isolated two cDNAs, LI S-1 which encodes the 45-kDa intracellular subunit of the platelet-act ivating factor acetylhydrolase, and the unknown sequence HFBCB84 which showed reduced expression in only a small number of tumor lines as co mpared to HKc. Inactivation or loss of any of these proteins may confe r a selective advantage onto squamous epithelial cells and contribute to their malignant transformation.