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 keratinocyte (HKc) growth and/or differentiation, we constructed a cDNA library by subtractive hybridization between primary HKc and FaDu head-and-neck squamous cell carcinoma cells. Among the first set of independent cDNAs that we have isolated, ten correspond to known genes, and two represent novel sequences. Nine of the ten known genes are expressed at significantly lower levels in the majority of the SqCC cell lines in comparison with primary HKc. These include cDNAs that encode keratins K5 and K14 which are cytoskeletal proteins normally expressed in lining epithelia, the 14-3-3 protein stratifin/HME-1, lipocortin-II and CaN19 which are calcium-binding proteins that may play a role in HKc differentiation by regulating protein kinase C, plasminogen-activator inhibitor-2 which is a serine-proteinase inhibitor, HBp17 which is a HKc-specific secreted inhibitor of fibroblast growth factors, integrin alpha 3 which plays a role in the anchoring of keratinocytes to basement membrane, and YL-8, a ras-like protein that probably mediates intracellular protein trafficking. In addition, we isolated two cDNAs, LIS-1 which encodes the 45-kDa intracellular subunit of the platelet-activating factor acetyl-hydrolase, and the unknown sequence HFBCB84 which showed reduced expression in only a small number of tumor lines as compared to HKc. Inactivation or loss of any of these proteins may confer a selective advantage onto squamous epithelial cells and contribute to their malignant transformation.