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Abstract

In STU mice bearing metastasizing SV40-transformed 51A-232B-M tumors, an immune response against a cellular 60kDa protein (p60) developed in about 50% of the tumor-bearing animals, in addition to the response against SV40 large T-antigen and cellular protein p53. The anti-p60 auto-immune response could be observed as early as 11 days after tumor challenge and was strictly linked with metastatic spread but was not a prerequisite for metastasis. Anti-p60 antibodies could not be detected in sera of animals bearing metastasizing Rous-sarcoma virus-transformed or methylcholanthrene-induced tumors, or in sera from human cancer patients with clinically confirmed metastatic spread. The anti-p60 auto-antibodies showed a broad cross-reactivity against components of similar size in a great number of cell lines of various species and in normal mouse tissue. The p60 auto-antigen is a cytoplasmic protein which is neither phosphorylated nor glycosylated in vivo. Immunoblotting performed with fresh cell lysates under non-reducing conditions using tumor-bearer sera revealed a diffuse p60 double band, but under reducing conditions only one sharp p60 band was observed. The reaction of p60 with anti-p60 auto-antibodies could be completely blocked by pre-treatment of fresh cell lysates with N-ethylmaleimide or p-chloromercuriphenyl sulfonate, or by oxidation in air prior to immunoblotting, indicating that the anti-p60 autoimmune response was directed against an epitope sensitive to SH-group-blocking reagents. Immunofluorescence studies with tumor-bearer sera showed only a very weak cytoplasmic fluorescence, possibly due to the nature of the p60 SH-groups in situ being masked. Immunoprecipitates with monoclonal antibodies against SV40 large T-antigen and p53 obtained from fresh cell lysates of SV40-transformed tumor cells contained no associated p60 auto-antigen. The p60 auto-antigen was purified from tumor cell homogenates with an enrichment factor of about 2,000; its iso-electric point is at pH 6.8. Determination of the biological half-life of p60 yielded a value of about 28 hr. The p60 auto-antibodies in pools of tumor-bearer sera taken at day 40 after tumor challenge all belonged to the IgG1 subclass.