No abstract

A tumour-specific polypeptide designated U90 is one of a set of polypeptides which are encoded by the host cell and are specific for the transformed cell state, being immunoprecipitated by the sera of tumour-bearing animals. The interest in these tumour-specific polypeptides centres on the finding that they are also recognized by antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, implying some role for HSV-2 in tumorigenesis. The peptide map of HSV-2-induced U90 is indistinguishable from that of U90 present in uninfected tumour cells, including mouse cells transformed by human papillomavirus type 16. In tumour cells, U90 is located principally in the plasma membrane fraction and cannot be induced by heat shock, glucose starvation, or treatment with tunicamycin or calcium ionophore. U90 is not related to either the heat shock protein of Mr 90000 (HSP90) or the glucose-related polypeptide of Mr 94000 (GRP94) as determined by peptide mapping and the use of monospecific, monoclonal and antipeptide antibodies. This suggests that U90 is a novel transformationspecific protein which can be induced by infection with HSV-2.Macnab et al. (1985) have shown that a specific set of polypeptides of Mr 90000 (a doublet), 40000 and 32000 are immunoprecipitated from transformed cells by antisera (TBS) from tumour-bearing animals or sera raised against cells infected with herpes simplex virus type 2 (HSV-2) strain HG52, or by a mouse monoclonal antibody (MAb), TG7A. This polypeptide set could not be immunoprecipitated from control embryo cells.In this study we have characterized the 90000 Mr polypeptide doublet identified by immunoprecipitation, described its localization in the cell, and demonstrated that it is not induced by heat shock and does not possess any similarity to stress proteins such as heat shock protein, HSP90 (Mr 90000), or the glucose-regulated polypeptide, GRP94 (Mr 94000). In addition, it is shown that infection of transformed Bn5T cells with HSV-2 (HG52) increases the amount of the upper polypeptide band (U90) of the 90K doublet by 2.5-to fivefold, whereas the amount of the lower polypeptide band (L90) does not increase.For these experiments we employed rat embryo (RE) cells as control cells and Bn5T cells (a cell line derived from a tumour induced by transformation of RE cells with the BgllI n fragment of HSV-2) as transformed cells, and the assay used to derive the results was immunoprecipitation of L-[ 35S]methionine_labelled polypeptides (Macnab et al., 1985). The immunoprecipitates were trapped using Protein A-Sepharose (Sigma) or 0001-0270 © 1991 SGM Pansorbin (Calbiochem), and the radiolabelled proteins were visualized by autoradiography after separation by electrophoresis on 7 or 7.5% polyacrylamide gels. Polypeptide extractions were carried out in RIPA buffer (Macnab et al., 1985) containing three protease inhibitors, 0.034 mg/ml PMSF, 0-03 mg/ml benzamidine and 0.1 mg/ml phenanthroline.Polyclonal antisera were raised in mice against HSV-2 (HG52)-infected BHK C13 cells and in r...

Anti-sera raised against HSV-2-infected cells (WI) and the sera of animals bearing tumours (TBS) to HSV-2 transformed cells contain antibodies to a set of tumour-specific cell-coded polypeptides. The specificity of these polypeptides for tumour cells is monitored by the ability of [35S]-L-methionine labelled proteins to be immunoprecipitated by these anti-sera, in contrast to control cells from which the polypeptides are not precipitated. The polypeptides which share an epitope and are co-precipitated are of MWs 90,000 (a doublet), 40,000 and 32,000. The upper 90,000-MW polypeptide (U90) is induced by HSV-2 infection. This communication deals with the 40,000-MW polypeptide which was shown to be immunoprecipitated by TBS and a monoclonal antibody (MAb) raised to the DNA-binding proteins of HSV-2-infected cells. Immunological and biochemical studies reveal that the 40,000-MW protein which is immunoprecipitated comprises more than one polypeptide, and that the proteins may need to interact to produce the peptide pattern specific for the tumour form of the immunoprecipitated 40,000-MW protein. WI antisera and TBS both recognise antigens specific for tumour cells in sections of cervical-carcinoma tissue. Sera from patients with cancer of the cervix contain antibodies to a cell-coded polypeptide of MW 40,000, which by peptide analysis is indistinguishable from the 40,000-MW polypeptide induced by HSV-2 infection and immunoprecipitated by WI and TBS.