Human tumor cells synthesize and secrete alpha‐2‐macroglobulin in vitro

Abstract

In previous studies we showed that human sarcoma and melanoma cell lines synthesize and secrete into culture medium a glycoprotein, migrating in urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 140,000. It is not detected in cultures of the corresponding normal cells. Conditioned medium of the melanoma cell line HMB-2, producing among the cell lines tested the largest amounts of this glycoprotein, has now been used as a source for purification of the protein. NH2-terminal amino-acid sequence determination of the purified glycoprotein showed that it is identical to human alpha 2-macroglobulin (alpha 2M). Rabbit antibodies raised against the glycoprotein specifically reacted in immunoblotting and immunodiffusion tests with alpha 2M present in human plasma. Likewise, these antibodies immunoprecipitated from the conditioned media of 35S-methionine-labelled melanoma and osteosarcoma cell lines the protein which had a molecular weight corresponding to alpha 2M. alpha 2M was also synthesized and secreted by 2 strains of fetal lung fibroblasts but not by fetal skin fibroblasts or adult skin fibroblasts autologous to the osteosarcoma cell line.

Coexpression of tumor‐associated α2‐macroglobulin and growth factors in human melanoma cell lines

“…After separation, the proteins were transferred electrophoretically to nitrocellulose sheets (Schleicher-Schuell) according to established procedures [25]. For immunological staining of the transferred proteins, rabbit antiserum to TA-a,M [16] was used at a dilution of 1:1,000, and, after binding of peroxidaseconjugated swine anti-rabbit IgG (DAKO, Copenhagen, Denmark), the bands were visualized using 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as substrate.…”

“…We previously described detection and characterization of a,M synthesized and secreted by human tumor cells [15,16]. Our recent results showing the presence of tumor-associated a,M (TA-a,M) in human melanomas in vivo [ 171 prompted us to investigate the role of TA-a,M in carcinogenesis of human cells in more detail.…”

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

“…Expression of a1IM-specific mRNAs and production of mature protein in melanoma cell lines We have previously reported on a2M production by human melanoma cells (Bizik et al, 1986(Bizik et al, , 1989). Now we analyzed the a2M expression in more detail.…”

“…In a previous report, we described purification and characterization of tumor-associated a2M (TA-a2M) (Bizik et aL, 1986), secreted by cultured human melanoma and sarcoma cells. Rabbit antibodies prepared against this TA-a2M were used in the immunohistochemical study of tissue sections from human malignant melanomas .…”

Active transforming growth factor-β in human melanoma cell lines: No evidence for plasmin-related activation of latent TGF-β

“…Surprisingly, the HMB-2 cell line produced the highest amounts of active TGF-P. We have demonstrated previously [Bizik et al, 1986;Bizik et al, 19891 that this particular cell line secretes significant amount (300 ng/106 cells/ 24 h) of alpha-2-macroglobulin ( a Z M ) . There are numerous data on the involvement of azM in complexing of TGF-P and in this way modulating TGF-(3 activity [O'Connor-McCourt and Wakefield, 1987;Danielpour and Sporn, 19901.…”