Selective assembly of laminin variants by human carcinoma cells.
BACKGROUND
The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble basement membranes, the pattern of production of various laminin subunits remains to be explored.


METHODS
The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies. For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains.


RESULTS
All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was demonstrated by immunofluorescence staining. The cells contained both B1 and S chain laminin in the cell layer, but preferentially secreted the B1 chain into the culture supernatant as shown by Western blotting. The 300 to 400 kDa M chain immunoreactive band was found in laminin secreted into the culture medium of HU-1 cells. Immunoprecipitation of biosynthetically labeled proteins showed that the M chain was synthesized as a complex with B chains. Little or no A chain laminin was detected in the culture medium supernatant. HU-1 cells also synthesized the newly described laminin variant, epiligrin which was secreted into the medium. Thus, the HU-1 cells secreted two laminin variants: M-B1-B12 laminin and epiligrin into the culture medium. Immunostaining of HU-1 nude mice tumors showed that tumor basement membranes contained M, B1, and B2 laminin and epiligrin immunoreactivity but apparently no S chain.


CONCLUSIONS
All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion and deposition of laminin variants into tumor basement membranes. These data indicate that the tumor cells vary among themselves with regards to laminin production and that some of them, like HU-1 may produce essentially all laminin chains simultaneously.
Laboratory of Molecular Pathology, University Institute of Pathological Anatomy, Copenhagen, Denmark.
U M
UM
Wewer
E A
EA
Wayner
B G
BG
Hoffstrom
F
F
Lan
B
B
Meyer-Nielsen
E
E
Engvall
R
R
Albrechtsen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002277:Carcinoma / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D003001:Cloning, Molecular; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D015603:Keratinocytes / Q000378:metabolism; D007797:Laminin / Q000378:metabolism*; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009368:Neoplasm Transplantation; D010455:Peptides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D015342:DNA Probes; D018076:DNA, Complementary; D007797:Laminin; D010455:Peptides; D012333:RNA, Messenger; D000097794:Kalinin