A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas

Abstract

The keratin proteins of 9 cell lines cultured from human squamous cell carcinomas (SCC) of the epidermis and oral epithelium were examined. All of these lines contained the 4 major keratins (46 kd, 50 kd, 56 kd and 58 kd) found in normal keratinocytes cultured from these epithelia. A 40 kd protein was also present in the Triton-insoluble cytoskeletal fraction of 3 of the lines. The 40 kd protein had an isoelectric point of 5.3, near that of the 46 kd and 50 kd keratins. It was precipitated by an antiserum against total s. (stratum) corneum keratins, and also by an antiserum against purified 46 kd keratin. An antiserum raised against the purified 40 kd protein had a high titer against the 40 kd protein, detected by immunoprecipitation, and did not crossreact with the 46-58 kd keratins. The anti-40 kd serum stained, by indirect immunofluorescence, a cytoplasmic filament network in cells of the SCC lines that synthesize the 40 kd protein. These filaments were of a pattern indistinguishable from that stained by antiserum against total s. corneum keratins, and the pattern was not altered by pretreatment of the cells with colcemid. The anti-40 kd serum did not stain the SCC lines that do not synthesize the 40 kd protein; normal human newborn foreskin keratinocytes or fibroblasts; or mouse fibroblast 3T3, human cervical carcinoma HeLa or mouse teratoma-derived keratinocyte XB cells. Keratinocytes cultured from uninvolved epidermal or oral tissue of patients whose tumors had given rise to SCC lines that synthesize the 40 kd protein were also not stained by the anti-40 kd serum. Synthesis of the 40 kd protein was greatly reduced in tumors formed by the SCC lines that make this protein in culture. Partial proteolytic digestion maps of the 40 kd protein were unique, but very similar to those of the 46-58 kd keratins. These results constitute strong evidence that the 40 kd protein is a keratin, and is neither a breakdown product nor an incomplete translation product of a gene for one of the larger keratins. It is the smallest epidermal keratin yet to be described. The 40 kd keratin may be the product of a normally unexpressed gene or transcript that often becomes activated during the course of keratinocyte transformation. Like the larger keratins, the level of its expression is regulated and is different in cells growing in vivo and in culture.