Maureen O. Weeks

Identification of a sarcoma virus-coded phosphoprotein in nonproducer cells transformed by Kirsten or Harvey murine sarcoma virus

A similar protein of 21,000 MW (p21) coded for by Harvey or Kirsten murine sarcoma virus has been identified in nonproducer cells transformed by these two viruses. Antisera prepared from rats bearing tumors induced by syngeneic transplantation of NRK cells transformed by Harvey murine sarcoma virus (Ha-MuSV) specifically precipitated the Ha-MuSV p21 from a nonproducer Balb/c mouse cell and a nonproducer dog cell transformed by Ha-MuSV. The same antisera also precipitated a similar protein, Ki-MuSV p21, from a nonproducer mink cell transformed by Kirsten murine sarcoma virus (Ki-MuSV). Both the p21 of Ha-MuSV and of Ki-MuSV are phosphoproteins. Previous studies have reported a virus-specific p21 polypeptide from translation of Ha-MuSV RNA in cell-free protein synthesis systems (W. P. Parks and E. M. Scolnick, 1977, J. Virol. 22, 711-719; T. Y. Shih, D. R. Williams, M. O. Weeks, J. M. Maryak, W. C. Vass, and E. M. Scolnick, 1978, J. Virol 27, 45-55). This p21 protein was specifically precipitated by the same anti-tumor sera. Similarly, a p21 polypeptide translated from Ki-MuSV RNA was also specifically precipitated by the antitumor sera. Therefore, it is concluded that the p21 of Ha-MuSV and Ki-MuSV are homologous proteins coded for bv homologous sequences found in the recombinant genomes of Ha-MuSV and Ki-MuSV.

Monoclonal Antibodies Reactive With Breast Tumor-Associated Antigens

Publisher Summary Monoclonal antibodies (MAbs) led to the identification of novel tumor-associated antigens (TAAs) from various human carcinomas, melanomas, leukemias, and lymphomas. There are approximately two dozen characterized monoclonals reactive with human breast tumors. Several of these are chosen to elaborate those phenomena that are most relevant for the use of MAbs in the management of human breast cancer as well as in the study of the biology of human mammary carcinoma cell populations. This chapter describes numerous MAbs that are reactive with human mammary carcinomas. They can be classified into four groups based on the immunogen used to generate the MAb. These include using breast tumor cell lines, milk fat globule membrane, and membrane-enriched extracts of breast carcinoma metastases as immunogen, and lymph nodes from mastectomy patients. Each of the MAbs, including those prepared by several different groups to milk fat globule membrane, appears to be unique with respect to percentage of reactive mammary tumors, percentage of reactive cells within tumors, location of reactive antigen within the tumor cell, or degree of reactivity with nonmammary tumors as well as normal tissues.

Markedly elevated levels of an endogenous sarc protein in a hemopoietic precursor cell line.

The src gene product of Harvey murine sarcoma virus is a 21,000-dalton guanine nucleotide-binding protein. We have recently shown that a wide variety of vertebrate cell strains and cell lines express much lower levels of an endogenous p21 immunologically related to the Harvey murine sarcoma virus-coded p21. In this report, we have examined the levels of endogenous p21 in a unique hemopoietic precursor cell line, 416B, which was originally described as a continuous cell line of a hemopoietic stem cell, CFU-S. The currently available 416B cells express markedly elevated levels of endogenous p21. The level of endogenous p21 in the 416B cells is 5- to 10-fold higher than the level of p21 in Harvey murine sarcoma virus-infected cells and more than 100 times higher than the level of endogenous p21 that we have observed in a variety of other fresh or cultured cells. The results indicate that marked regulation of the levels of an endogenous sarc gene product can occur, and speculation about a possible role for endogenous p21 in normal hemopoietic stem cells is discussed.

Human Breast Cancer Markers Defined by Monoclonal Antibodies

Numerous monoclonal antibodies (Mab) have been described that are reactive with human mammary carcinomas. In general, they can be classified into three groups based on the immunogen used to generate the Mab; these are Mab derived using (a) breast tumor cell lines, (b) milk fat globule membrane, or (c) membrane enriched extracts of breast carcinoma metastases as immunogen. Each of the Mab thus far described (Colcher et al., 1981a; Sloan and Omerod, 1981; Taylor-Papadimitriou et al., 1981; To et al., 1981; Arklie et al., 1982; Epenetos et al., 1982a,b; Foster et al., 1982; Gatter et al., 1982; Nuti et al., 1982; Rasmussen et al., 1982; Colcher et al., 1983a; Hand et al., 1983; Menard et al., 1983; Papsidero et al., 1983; Thompson et al., 1983) including those prepared by several different groups to milk fat globule membrane, appears to be unique with respect to either percent of reactive mammary tumors, percent of reactive cells within tumors, location of reactive antigen within the tumor cell, or degree of reactivity with nonmammary tumors as well as normal tissues.

Tumor Associated Antigens and Oncogene Products Defined by Monoclonal Antibodies

The advent of hybridoma/monoclonal antibody technology in 1975 (1) and gene, cloning procedures have drastically influenced both the understanding and practice of pathology. Using these methodologies and reagents, applications have been developed in the research laboratory, distributed to the clinical setting, and now often impact on the diagnosis and management of human disease. It is the authors’ intent to focus this chapter on some recently developed monoclonal antibody applications in human pathology using immunohistochemical techniques. The text will be roughly divided into three parts: background information and general principles, practical applications, and monoclonal antibodies as probes to investigate tumor biology and oncogene expression.

Monoclonal Antibodies in the Management of Breast Cancer

For many years antibodies have had a major role in the diagnosis, investigation and treatment of a wide variety of diseases. At the same time, immunologists have continued to develop methods of immunization that would produce large amounts of homogeneous antibodies (Krause, 1970; Haber, 1970). These approaches have not been generally applicable to the routine production of serologic reagents against many antigens. The identification of human tumor associated antigens (TAAs) and the development of specific immunologic reagents directed against these targets have long been sought (Pressmann and Korngold, 1953). In past decades, the classical method of generating antibodies to TAAs has been the hyperimmunization of an animal with an extract of a human tumor and the absorption of the resultant sera with a variety of normal tissues. For example, if an extract of human breast carcinoma was used as immunogen, then the serum has to be absorbed with extracts of normal breast, benign breast tumor, as well as extracts of normal human red blood cells, liver, etc. This absorbed hyperimmune serum has then to be tested for its ability to react with the immunogen (in this case breast carcinoma). All too often, only weak reactivity was observed and only enough sera were obtained to complete a finite number of tests; thus, the entire process has to begin over again. Furthermore, since hyperimmune sera are estimated to contain some tens of thousands of different types of immunoglobulins which differ in isotype, affinity and specifity, reproducibility of results from laboratory to laboratory was difficult if not impossible to achieve.

Monoclonal Antibodies Generated to a Synthetic Peptide Define Ras Gene Expression at the Single Cell Level in Human Colon and Mammary Carcinomas

Ras oncogenes were first recognized as the transforming genes of two rat-derived viruses, the Harvey (Ha) and Kirsten (Ki) strains of murine sarcoma virus (MuSV) (1). Molecular cloning studies have identified Ha-ras, Ki-ras, and subsequently, N-ras, as members of a gene family present in a wide range of species including humans (2). At least two mechanisms have been identified by which ras activation can mediate transformation of NIH 3T3 cells: (a) a point mutation in the ras gene resulting in an alteration of a single amino acid in the 21,000 dalton (p21) ras gene product (3), or (b) increased expression of the normal cellular p21 ras gene product (1,4,5).

Potential Clinical Application of a Monoclonal Antibody to a Tumor Associated Glycoprotein (TAG-72)

Monoclonal antibody (MAb) B72.3, generated using a membrane-enriched fraction of cells from a mammary carcinoma metastasis, recognizes a high molecular weight glycoprotein complex of approximately 220,000 to 400,000d, termed TAG (Tumor-Associated Glycoprotein) 72 (1,2). MAb B72.3 has a high degree of selective reactivity for human breast carcinomas versus normal adult tissues. For example, using the immunoperoxidase technique on formalin-fixed tissue sections and radioimmunoassays on cell extracts, MAb B72.3 reacts with approximately 40–46% of primary breast carcinomas and 62% of metastatic breast lesions while no reactivity was observed with a variety of normal human tissue. Ninety-four percent of effusion specimens from breast cancer patients contained tumor cells which express TAG-72, while no reactivity of MAb B72.3 was observed with mesothelial cells or macrophages in benign or malignant effusions. Analysis of the reactivity of MAb B72.3 to sections of formalin-fixed breast carcinomas and to the MCF-7 breast carcinoma cell surface demonstrated heterogeneity of expression of TAG-72. Treatment of MCF-7 cells with recombinant human leukocyte interferon (IFN-alpha A) enhanced the detection of TAG-72 in a dose-dependent manner, and reduced the amount of 125I-labeled MAb B72.3 required to bind 1000 cpm to MCF-7 cells by more than 20-fold. The results demonstrate a highly specific tumor antigen which is expressed selectively on human carcinoma cells versus adult normal tissue. Furthermore, these results indicate that (a) MAb B72.3 can be used to detect occult adenocarcinoma cells in human tissues and (b) biological response modifiers, such as IFN-alpha A may be used to alter the tumor cell surface antigenic phenotype and perhaps enhance the utility of MAb B72.3 in the immunodetection and therapy of breast cancer.