Biosynthesis and secretion of laminin and laminin-associated glycoproteins by nonmalignant and malignant human keratinocytes: comparison of cell lines from primary and secondary tumors in the same patient.
Laminin biosynthesis was compared in four pairs of human squamous cell carcinoma cultures derived from primary and recurrent or metastatic tumors in four patients with cancer of the larynx and hypopharynx to determine if changes in laminin production accompany tumor progression. Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes. Pulse-chase biosynthetic labeling of the cultures with [35S]methionine established that all of the squamous carcinoma cell lines synthesize immunoreactive A (Mr 400,000), B1 (Mr 205,000), and B2 (Mr 200,000) laminin subunits; assemble them to form the intact laminin molecule (Mr 950,000); and secrete a portion of the laminin they produce into the culture media. One aspect of laminin expression unique to keratinocytes, both malignant and nonmalignant, was the occurrence of three additional glycoprotein forms (Mr 195,000, 170,000, and 160,000) in the laminin immunoprecipitates. In contrast to the laminin subunits, these glycoproteins were not immunoreactive with the anti-laminin antiserum on Western blots. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis without and with reduction of disulfide bonds revealed that the laminin immunoprecipitates contained a family of oligomeric molecules. These ranged in apparent molecular weight from 370,000 to 950,000 and were composed of laminin subunits and the glycoprotein forms linked by interchain disulfide bonds. The malignant keratinocyte cell lines from different patients were distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the rate of [35S]methionine incorporation into laminin during the pulse-labeling, the fraction of [35S]methionine-labeled laminin secreted into the medium during the chase incubation, and the absolute amount of laminin secreted into the culture medium as determined by enzyme-linked immunosorbent assay. However, cell lines established from primary and metastatic or recurrent cancer in the same patient were indistinguishable in their profile of laminin biosynthesis and secretion. In comparison to primary cultures of nonmalignant foreskin basal keratinocytes, the malignant cells secreted into the culture medium a larger fraction of the laminin that they produce. This is an indication that the malignant keratinocytes in culture deposited a less stable basal lamina-like extracellular matrix than their malignant counterparts. The diminished integrity of the basal lamina matrix may be an important factor in the invasive growth of human epithelial cancer.
Frenette
G P
GP
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
Carey
T E
TE
Varani
J
J
Schwartz
D R
DR
Fligiel
S E
SE
Ruddon
R W
RW
Peters
B P
BP
D002294:Carcinoma, Squamous Cell / Q000378:metabolism*; D002460:Cell Line; D002822:Choriocarcinoma / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism*; D006801:Humans; D007012:Hypopharyngeal Neoplasms / Q000378:metabolism; D007633:Keratins; D007797:Laminin / Q000096:biosynthesis*; D007822:Laryngeal Neoplasms / Q000378:metabolism; D008970:Molecular Weight
D007797:Laminin; D007633:Keratins