KERATIN GENE-EXPRESSION IN NONEPITHELIAL TISSUES - DETECTION WITH POLYMERASE CHAIN-REACTION

Keratin filament are characteristically present in epithelial cells an d tumors, but have also been detected in many normal and neoplastic no n-epithelial cell types using immunohistochemical techniques. To inves tigate the validity of this seemingly aberrant protein expression, we applied the highly sensitive polymerase chain reaction (PCR) technique to study keratin gene expression in a variety of non-epithelial tissu es. Total RNA was extracted from nine samples of leiomyosarcoma, four non-Hodgkin's lymph seven normal bone marrows, normal lymph node, norm al peripheral blood cells, freshly isolated and cultured endothelial c ells, cultured skin fibroblasts, and the myeloid leukemia cell line HL -60. Amplification printers and probes for the three most primitive ke ratin types (8, 18, and 19) were synthesized using published gene sequ ences. RNA from the breast carcinoma cell line MCF-7, known to be rich in all three keratins, was used as positive control. Concurrently run actin primers were used to confirm RNA integrity. After an initial cy cle with reverse transcriptase, PCR amplification was performed for 30 cycles. Southern blots of the PCR products showed variably intense ba nds corresponding to keratin 8 and 18 gene products in all samples, of fering conclusive evidence of keratin gene expression in cells of both stromal and hematopoietic derivation. However, keratin 19 gene transc ription was not nearly so ubiquitous, being detected in normal fibrobl asts and endothelial cells, two of four non-Hodgkin's lymphoma and fou r of nine leiomyosarcoma, but not in normal lymph node, peripheral blo od cells, HL-60 cells, or any of the seven normal bone marrows examine d. Dilutional experiments showed PCR to be highly sensitive in the det ection of keratin 19 gene expression, capable of registering one MCF-7 cell in 10(6) HL-60 cells. These studies show that variable levels of keratin 8 and 18 gene expression may be detected by PCR in a wide var iety of non-epithelial tissues, supporting previous immunohistochemica l and phylogenetic studies. However, keratin 19 gene expression appear s to be more restricted and was not evident in any hematopoietic cells devoid of contaminating stromal elements. These findings suggest a ro le for PCR in the detection of epithelial micrometastasis in certain s ites, articularly bone marrow.