Purification and biochemical properties of tumor-associated transplantation antigens from methylcholanthrene-induced murine sarcomas.

Abstract

A tumor-associated transplantation antigen with an apparent molecular weight of 75,000 has been isolated from the cytosol of the BALB/c methylcholanthrene-induced sarcoma, Meth A. The antigen was purified either by preparative electrophoresis in the presence of NaDodSO4 or by immunoaffinity chromatography after hexylamine agarose chromatography, gel filtration, and hydroxylapatite chromatography. The 75-kilodalton (kDal) protein prepared by either of these methods effectively primed BALB/c mice to reject the Meth A tumor; such priming provided no protection against challenge by other independently derived sarcomas of BALB/c origin. A second protein, also 75 kDal, was isolated from the cytosol of the recently derived methylcholanthrene-induced sarcoma CI-4 by essentially the same chromatographic scheme. This protein also was immunogenic in the tumor rejection assay and provided protection only against CI-4 challenge. The antigens purified from the Meth A and CI-4 sarcomas appear to be closely related proteins. Both of them can be purified from the cytosol fraction and can be recognized by a rabbit antiserum prepared against the Meth A 75-kDal protein. The two proteins have approximately the same molecular weight, have similar but not identical amino acid compositions, and differ in their chromatographic behavior on hexylamine agarose and hydroxylapatite as well as in their isoelectric points. These results indicate that the individually specific transplantation antigens found in chemically induced sarcomas may be the products of a single multigene family or somatic derivatives of a single gene.

A mouse tumor-specific transplantation antigen is a heat shock-related protein.

“…The antigen was eluted with a 0.3-0.6 M gradient of NaCl in buffer A. Purity of TSTA was determined by two-dimensional PAGE, and TSTA activity was measured as described (3 …”

“…This protein will be referred to as the 85-kDa stress protein. Synthesis of this protein is decreased upon glucose deprivation (13) but is increased in virally infected cells (14, 15) and in early mouse embryos (16 (3,4) except for an additional step. The hydroxyapatite-purified antigen was equilibrated with 20 mM Tris Cl (pH 8.0) in 10% (wt/vol) glycerol (buffer A) and then injected into a Mono Q HR5/5 column (Pharmacia) equilibrated with buffer A.…”

“…In order to understand the molecular changes that render these antigens immunogenic and to elucidate their normal cellular function, Appella and coworkers have purified TSTAs from two methylcholanthrene-induced sarcomas, Meth A and CI-4 (3), as well as from a simian virus 40 (SV40)-induced sarcoma, mKSA (4). These antigens share several common properties: they are all acidic cytosolic proteins of 75-86 kDa; they are not glycoproteins (3), viral antigens (4,5), or alien histocompatibility antigens (6); and they are present at the cell surface (4). The normal cellular function of these antigens is not known.…”

“…The antigen was eluted with a 0.3-0.6 M gradient of NaCl in buffer A. Purity of TSTA was determined by two-dimensional PAGE, and TSTA activity was measured as described (3 Ci/mmol) in methionine-free Eagle's minimal essential medium with Earle's salts (Flow Laboratories), containing 5% dialyzed fetal bovine serum. Cell lysates and immunoprecipitates were prepared as described (23) and solubilized by the method of Garrels (24).…”

GRP94/gp96 Elicits ERK Activation in Murine Macrophages

“…A number of molecular chaperones, including GRP94/gp96, calreticulin (CRT), 1 and Hsp70, are capable of eliciting antitumor immune responses against their tumor of origin (1)(2)(3)(4). The efficacy of GRP94/gp96 in animal model studies of tumor rejection has spurred investigation into tumor-derived chaperones, principally GRP94/gp96, as immunotherapeutics for human cancers (5-7).…”