Changes in tumor‐specific antigen expression during passage <i>in vitro</i> and <i>in vivo</i> of newly derived methylcholanthrene‐induced sarcomas of BALB/c mice

249

105

A tumor-specific transplantation antigen has been purified to homogeneity from the cytosol of a methylcho- We now report on the purification to homogeneity and the biochemical characterization ofa cytosolic TSTA, from Meth A, that consists of two polypeptide isoforms of similar size and charge. Sequence analysis of the two isoforms indicates that a large portion of the sequenced NH2-terminal region of both proteins, as well as a CNBr fragment, shares extensive sequence homology with the Drosophila melanogaster 83-kDa (7) and yeast 90-kDa heat shock protein (8).Upon stress, cells from bacteria to mammals decrease the synthesis of most proteins but transiently increase the synthesis of several heat shock, or stress, proteins (9, 10). One of the major heat shock proteins is an acidic cytosolic protein whose apparent molecular mass in the mouse is [85][86][87][88][89][90]12 MATERIALS AND METHODSPurification of TSTA. The Meth A TSTA was purified from 2.5 x 1010 Meth A ascites cells as described (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. 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 3121The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Section: -mentioning

confidence: 99%

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

Ullrich¹,

Robinson²,

Law³

et al. 1986

249

105

“…This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. (22), and mKSA, a simian virus 40-induced sarcoma of BALB/c (23). These three tumors were used in the present study as specificity controls.…”

mentioning

confidence: 99%

High-frequency cotransfer of the transformed phenotype and a tumor-specific transplantation antigen by DNA from the 3-methylcholanthrene-induced Meth A sarcoma of BALB/c mice.

Hopkins¹,

Besmer²,

DeLeo³

et al. 1981

We transformed BALB/3T3 mouse cells with cellular DNA extracted from the Meth A sarcoma, a 3-methylcholanthrene-induced tumor ofBALB/c mice, and asked whether foci arising in the transfection possess the previously defined Meth A tumor-specific transplantation antigen (TSTA). Five of eight foci selected from one experiment possessed Meth A TSTA. DNA extracted from one of the five TSTA-positive clones was used in secondary rounds oftransfection transformation. Four out of five foci tested from the secondary transfections possessed Meth A TSTA. These results suggest that in the Meth A sarcoma a transforming gene and a genetic determinant of TSTA are intimately related: they may be identical or very closely linked; alternatively, a particular transforming gene might induce the expression of a particular TSTA. Another possible explanation for these results is that the cotransfer of certain cellular genes by DNA transfection is considerably higher than predicted from the limited studies presently available.An intriguing class oftumor antigens is the tumor-specific transplantation antigens (TSTAs) of carcinogen-induced murine sarcomas. TSTAs are defined by the ability of tumor cells and tumor cell particulates or solubilized antigens to immunize a syngeneic host against subsequent challenge by live cells of the same tumor (1-6). Each chemically induced sarcoma appears to possess a unique TSTA even if the tumors were induced by the same agent in the same animal. The genetic basis ofTSTAs ofcarcinogen-induced tumors is unknown, as is the relationship of the antigens to the transforming events that induced the tumor.To investigate the relationship between the genetic determinants of TSTAs and the transformed phenotype, we transformed BALB/3T3 mouse cells with DNA from the 3-methylcholanthrene-induced Meth A sarcoma (5), using the calcium phosphate precipitation technique (7-10), and asked whether the transformants possess the previously defined Meth A TSTA (5,6,11,12). Because the stable expression of mammalian cell genes after DNA transfection is very inefficient, one would expect the frequency of cotransfer of the two phenotypes, transformation and TSTA, to be only 0. 01-0. 001 ifthey are specified by unlinked genes (13,14). However, if the two phenotypes have common or linked determinants, one would expect a high frequency of cotransfer.The Meth A sarcoma was selected for these experiments because its TSTA has been well characterized immunologically and biochemically and shown to be distinct from TSTAs of all other syngeneic neoplasms assayed to date (5,6,11,12 Recipient cells were a subclone of BALB/3T3 clone A31 (18) designated BALB/3T3 clone 7 and were grown in Dulbecco's modified Eagle's medium with 10% calf serum.DNA was precipitated with calcium phosphate essentially as described (7,20). Specifically, recipient cells were seeded at 1-1.5 X 106 per 10-cm dish for some experiments or 4 X 105 per 6-cm dish for others, and 40 Ag of high molecular weight DNA was sheared by two passages through a 20 gauge hyp...

“…Meth A and CI-4, two antigenically distinct MCinduced BALB/c sarcomas (3,13), were maintained in ascitic form by serial passage in female BALB/c mice. The ascitic form of both tumors was used exclusively for antigen preparation.…”

Section: Methodsmentioning

confidence: 99%

“…The ascitic form of both tumors was used exclusively for antigen preparation. The Meth A line passaged in vivo does not express murine leukemia virus or its antigens (13,14), in contrast to CI-4 which is a virus producer (13). The MC-induced BALB/c sarcomas CI-3, CII-5, and CII-10 and the simian virus 40-induced BALB/ c sarcoma mKSA were used as specificity controls.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Dubois¹,

Law²,

Appella³

1982

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.