(CANCER RESEARCH 48. 5193-5202. September 15, 1988] 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 Patient1 Gary P. Frenette, Thomas E. Carey, James Varani, Donald R. Schwartz, Suzanne E. G. Fligiel, Raymond W. Ruddon, and Barry P. Peters2 Departments of Pharmacology [G. P. F., R. W. R., B. P. P.], Otolaiyngology [T. E. C., D. R. S.J, and Microbiology fJ. V., S. E. G. F.], The University of Michigan Medical School, Ann Arbor, Michigan 48109 ABSTRACT proteoglycan, and glycoprotein components (1-7). Laminin, a M, 950,000 glycoprotein constituent of the basal lamina, is Laminin biosynthesis was compared in four pairs of human squamous composed of three disulfide-linked subunits, A (M, 400,000- cell carcinoma cultures derived from primary and recurrent or metastatic 440,000), B, (Mr 205,000), and B2(Mr 200,000). Laminin binds tumors in four patients with cancer of the larynx and hypopharynx to to other constituents of the basal lamina, specifically type IV determine if changes in laminin production accompany tumor progression. collagen and heparan sulfate, via distinct binding domains (8- Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes. Pulse-chase bio- 11). The role of laminin in vivo is not completely understood, synthetic labeling of the cultures with [3SS|methionine established that but there is evidence to indicate that it promotes attachment of all of the squamous carcinoma cell lines synthesize immunoreactive A epithelial and endothelial cells to basal laminae (11-14), the (\l, 400,000), B, (M, 205,000), and ».(M, 200,000) laminin subunits; structure that separates normal epithelial cells from the con assemble them to form the intact laminin molecule (M, 950,000); and nective tissue stroma (15-17). The interaction between epithe secrete a portion of the laminin they produce into the culture media. lial cells and the basal lamina is thought to participate in the One aspect of laminin expression unique to keratinocytes, both malig nant and nonmalignant, was the occurrence of three additional glycopro- control of cell shape (18, 19), differentiation (20, 21), and cell tein forms (M, 195,000, 170,000, and 160,000) in the laminin immuno- anchorage (22). precipitates. In contrast to the laminin subunits, these glycoproteins were Malignant epithelial neoplasms are often associated with not immunoreactive with the anti-laminin antiserum on Western blots. anomalous basal lamina morphology, including gaps and punc Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electropho- tate distributions of matrix (23-27), suggesting aberrant basal resis without and with reduction of disulfide bonds revealed that the lamina deposition in human cancers. This loss of structural and laminin immunoprecipitates contained a family of oligomeric molecules. functional integrity of the basal lamina is a key event in the These ranged in apparent molecular weight from 370,000 to 950,000 and progression of epithelial cancers from carcinoma in situ to were composed of laminin subunits and the glycoprotein forms linked by invasive carcinoma, an early step in the metastatic cascade. interchain disulfide bonds. Two possible but not mutually exclusive mechanisms that could The malignant keratinocyte cell lines from different patients were account for basal lamina disruption in malignant tumors are distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (a) increased matrix degradation and (/>) alterations in the the rate of (3SSlmethionine incorporation into laminin during the pulse- synthesis and deposition of matrix components. Malignant labeling, the fraction of (39S]methionine-labeled laminin secreted into the neoplasms have been shown to produce higher levels of cell- medium during the chase incubation, and the absolute amount of laminin associated and secreted proteolytic enzymes capable of degrad secreted into the culture medium as determined by enzyme-linked im- ing normal basal laminae when compared to benign tumors or munosorbent assay. However, cell lines established from primary and adjacent uninvolved tissue (28-32). For example, the level of metastatic or recurrent cancer in the same patient were indistinguishable expression of type IV collagenase is related to invasive or in their profile of laminin biosynthesis and secretion. In comparison to metastatic potential in some tumor systems (29, 31, 33, 34). primary cultures of nonmalignant foreskin basal keratinocytes, the ma lignant cells secreted into the culture medium a larger fraction of the Alternatively, decreased synthesis or deposition of basal lamina laminin that they produce. This is an indication that the malignant components by adherent epithelial cells could compromise the keratinocytes in culture deposited a less stable basal lamina-like extra integrity of this extracellular structure. For example, trans cellular matrix than their malignant counterparts. The diminished integ formed rat kidney cells exhibit a diminished capacity to deposit rity of the basal lamina matrix may be an important factor in the invasive in culture the laminin-containing extracellular matrix charac growth of human epithelial cancer. teristic of nonmalignant rat kidney cells (35). It may therefore be expected that more highly invasive cells may be impaired in their ability to produce laminin or deposit laminin in a cell- INTRODUCTION associated matrix. The basal lamina underlying normal epithelia is a continuous, Laminin may influence the malignant potential of tumor cells condensed macromolecular complex containing collagenous, in another way. Tumor cell attachment to the vascular endo- thelium, a key event in hematogenous metastasis, may be me Received 2/4/88: revised 6/20/88; accepted 6/21/88. The costs of publication of this article were defrayed in part by the payment diated by cell surface laminin. For example, pretreatment of of page charges. This article must therefore be hereby marked advertisement in B16-BL6 murine melanoma cells with intact laminin increased accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by USPHS Grants CA-41359, CA-35929, and the incidence of pulmonary métastasesfollowing tail vein in CA-28564 awarded by the National Cancer Institute, Department of Health and oculation in mice (36). In addition, metastatic potential has Human Services; by Grant PDT-324 awarded by the American Cancer Society; been directly correlated with cell-associated laminin in a murine by a grant from the Veterans Administration; and by a Predoctoral Fellowship (G. P. F.) from the Nancy Newton Loeb fund awarded by the University of fibrosarcoma cell line (37). Therefore, the ability of cells to fill Michigan Cancer Research Institute. surface binding sites (possibly the laminin receptors) (38-42) 2To whom requests for reprints should be addressed, at the University of Michigan Medical School, M6322 Medical Science I, Ann Arbor, MI 48109- with laminin they produce may influence their metastatic po 0626. tential. If so, it might be expected that more highly metastatic 5193 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1988 American Association for Cancer Research. LAMININ BIOSYNTHESIS IN CULTURED HUMAN KERATINOCYTES cells synthesize and retain more laminin on their surface than Ronnett et al. (48). The collected 4-h chase medium was adjusted to their less metastatic counterparts. the same concentration of PBS-Triton X-100-sodium deoxycholate- The parallel establishment of human squamous carcinoma SDS as the cell lysates by adding a 5-fold concentrated solution. The lysates were clarified by centrifugation for 60 min at 100,000 x cell lines from primary or metastatic sites from each of four g and were then immunoprecipitated overnight at 4"C using nonim patients allows for the first time a direct comparison of laminin mune serum or rabbit antiserum against mouse EHS tumor laminin [5 biosynthesis and deposition in these two types of lesions. In Ml/5 ml of lysate; antibody kindly provided by Dr. R. N. Knibbs and I. addition, squamous carcinoma cells are compared to primary J. Goldstein, Department of Biological Chemistry, The University of cultures of nonmalignant human foreskin basal keratinocytes Michigan (49)]. We have shown previously that this antiserum cross- and to JAR, a cell line derived from human gestational chorio- reacts with human laminin (43). Up to 80% of immunoreactive laminin carcinoma which we have previously characterized for laminin in JAR choriocarcinoma lysates was recovered in one round of immu biosynthesis (43). noprecipitation with this reagent (43). Immune complexes were adsorbed to protein A-Sepharose (15 /¿Iof packed beads/Ml antiserum), and the immunoprecipitated laminin forms MATERIALS AND METHODS were eluted from the washed protein A-Sepharose pellets by boiling for 3 min with an equal volume of 2-fold concentrated Laemmli SDS- Cell Lines. The human laryngeal and hypopharyngeal squamous PAGE sample buffer (50) with or without 2% 2-mercaptoethanol. In carcinoma cell lines were established from four patients (UM-10, UM- some cases, the washed protein A-Sepharose immunoprecipitates were 11, UM-17, and UM-22) as described previously (44) and cultured in split into two equal portions and incubated with or without 10 milIinnit.s MEM3 containing 1% nonessential amino acids and 15% fetal calf ofendo H (Miles) for 16 h at 37°Cin 0.5 ml of 0.1 M sodium citrate- serum. The cell lines were derived from either primary tumors (UM- 0.04% sodium azide, pH 5.3. The endo H had been pretreated with 2 SCC-10A, UM-SCC-11A, UM-SCC-17A, UM-SCC-22A), metastatic mM phenylmethylsulfonyl fluoride in the same buffer for 30 min at 4°C tumor sites (UM-SCC-11B, UM-SCC-17B, UM-SCC-22B), or a neo- to inactive residual protease activity. The endo H-treated immunopre plastic recurrence at the site of the primary tumor (UM-SCC-10B).