Human breast carcinoma antigen is immunologically related to the polypeptide of the group-specific glycoprotein of mouse mammary tumor virus.

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The T47D human mammary adenocarcinoma vell line in vitro releases viral particles as well as soluble proteins. Both fractions were shown to contain antigens that immunologically crossreact with the major glycoprotein (gp52) of mouse mammary tumor virus. The crossreacting antigens are located on polypeptides with apparent molecular weights of about 68,000 and 60,000. The larger one is present in viral particles whereas both are found in the soluble fraction. Both proteins are glycosylated. The human tissue culture proteins differ from gp52 not only in molecular weight but also in charge heterogeneity and in polypeptide profiles obtained after partial proteolysis. The results suggest that there is a restricted similarity between MMTV gp52 and the immunologically related T47D proteins.Evidence for an association of viral proteins and viral nucleic acid sequences with human mammary carcinomas has been accumulated increasingly over the past few years. Key similarities between the human malignancy and mouse mammary tumors have focused attention on the possible involvement of mouse mammary tumor virus (MMTV)-like retroviruses in the human disease (1, 2). An antigen immunologically related to the protein moiety of gp52 (a 52,000 Mr glycoprotein that constitutes the major envelope protein of MMTV) was detected in sections of human breast carcinoma tissue and in particulate fractions of human milk (3-5). The antigen also has been detected in established cell lines derived from pleural effusions of patients with breast cancer (6, 7). In addition, these cell lines, MCF-7 and T47D, have been shown to possess DNA sequences with partial homology to the genome of MMTV (8).The T47D cell line, which was established and subcloned in our laboratory (9), releases retrovirus-like particles (10). Therefore, it was of interest to determine whether these particles manifested proteins antigenically related to MMTV gp52. Since MMTV gp52 is shed from tumor cells in vivo in the mouse and enters the circulation, the tissue culture system also might serve as a model in the search for crossreacting antigens in vitro. An MMTV gp52 crossreacting antigen was recently found in both retrovirus-like particles and soluble proteins released by the T47D cell line (10).The present study was designed to identify and characterize these gp52-related proteins and also to determine their degree of similarity to MMTV gp52.MATERIALS AND METHODS Cells and Viruses. The T47D cell line (sub-line Cl-11) was used in this study. The cells were grown in RPMI 1640 medium containing 10% fetal calf serum, insulin (0.2 IU/ml), 2 mM glutamine, and antibiotics (complete medium). When cells were grown in the presence of steroid hormones, serum was omitted. For virus collection, the cells were seeded in RPMI 1640 with 10% fetal calf serum and incubated hr; the medium then was replaced with RPMI 1640 with 5% dialyzed serum and 1 nM 17(-estradiol. After 24 hr, the medium was replaced with RPMI 1640 containing the same amount of 17f3estradiol without serum. The next day...

Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of a protein, released by the T47D cell line, immunologically related to the major envelope protein of mouse mammary tumor virus.

Segev

Hizi

Kirenberg

et al. 1985

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“…More recently, using immunoperoxidase staining, Mesa-Tejada et al (5) found that antigen reacting with antibody to the envelope glycoprotein of MuMTV (gp52) could be detected in breast cancer tissue. Ohno et al (6) have now demonstrated that this crossreactivity is directed to the protein moiety ofgp52 and not the carbohydrate.…”

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confidence: 99%

Antibodies reactive with murine mammary tumor virus in sera of patients with breast cancer: geographic and family studies.

Day¹,

Witkin²,

Sarkar³

et al. 1981

Sera of patients with breast cancer, of healthy women from the United States, East India, East Africa, and China, and of healthy women of American and Parsi families in which breast cancer occurred in several family members were assayed for levels of antibody reactive with the murine mammary tumor virus (MuMTV) by an enzyme-linked immunosorbent assay. Increased levels of antibody to MuMTV (absorbance -0.4) were found in sera of 18.6% ofAmerican patients with breast cancer and of 2.8% of healthy American women and in 38% of patients from India and 61.9% from East Africa (healthy, 26.9%). In contrast, antibody reactive with MuMTV was found in <5 of women with breast cancer from mainland China (healthy Chinese, 5.0%). Differences in serum MuMTV antibody levels between breast cancer patients in the four groups were found to be significant (P < 0.0001). Studies of two families from the United States and of one Parsi family from India with genetic propensity to breast cancer showed that high levels ofantibody to MuMTV were found in 33%, 71%, and 23% of healthy family members, respectively. The antibody to MuMTV was readily absorbed with purified MuMTV and gp52. In contrast, fetal calf serum, murine type C retroviruses, or erythrocytes from various species failed to absorb the antibody. A relationship between human breast cancer and murine mammary tumor virus (MuMTV) has been suggested from several observations. RNA partially homologous to the MuMTV genome was identified in human breast cancer tissue (1). Lymphocytes from individuals with breast cancer were shown to proliferate after stimulation by MuMTV (2, 3). Sera from breast cancer patients decreased the infectivitity of MuMTV in mice (4), suggesting the presence of antibodies against the virus. More recently, using immunoperoxidase staining, Mesa-Tejada et al. (5) found that antigen reacting with antibody to the envelope glycoprotein of MuMTV (gp52) could be detected in breast cancer tissue. Ohno et al. (6) have now demonstrated that this crossreactivity is directed to the protein moiety ofgp52 and not the carbohydrate.In prior studies using a virolytic assay with complement and antibody, we found that antibodies reactive with MuMTV are present in sera of approximately 20% of American women with breast cancer (7). Sera of age-matched healthy women and of patients with other cancers had such antibodies in lower frequency. We developed a method to detect these antibodies directly by using MuMTV and an enzyme-linked immunosorbent assay (ELISA) (8). With this method also we found that sera of approximately 25% of American women with breast cancer had antibodies to MuMTV. Approximately 10% ofwomen with benign cystic disease of the breast also had such antibodies in their sera (8). Reactivity was inhibited by the membrane proteins of MuMTV (gp52 and gp34) but not by the core antigen (p28) (9).We now report results on the specificity of the human antibody for MuMTV, the geographic distribution of the antibody in breast cancer patients, and analyses of families in...

“…Furthermore, an antigen immunologically related to the group-specific antigen gp52 (a 52,000-dalton envelope glycoprotein) of MMTV has been detected (8,9) in paraffin sections of human breast cancer by peroxidase immunocytochemistry. The fact that it is the polypeptide, rather than the polysaccharide, portion of gp52 that is responsible for the immunological reactivity with the human breast cancer antigen (10) adds additional significance to the biological similarities between the human and the murine mammary neoplasias.…”

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confidence: 99%

Properties of retrovirus-like particles produced by a human breast carcinoma cell line: immunological relationship with mouse mammary tumor virus proteins.

Keydar¹,

Ohno²,

Nayak³

et al. 1984

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Clonal derivatives 8 and 11 of the T47D human breast carcinoma cell line release particles that have the biochemical characteristics of a retrovirus. Particles recovered from cultures of [3H]uridine-labeled clone 11 had a density of 1.18 g/ml and contained 60-70S and 35S RNAs associated with reverse transcriptase activity. The production of these particles was steroid-dependent. Clone 8 particles had a higher density, 1.195 g/ml, and their production was independent of steroid hormone. By RIA, antigens crossreactive with the 52,000-dalton envelope glycoprotein gp52, the major external protein of mouse mammary tumor virus, were found associated with these particles and in the media. Most of the gp52-related antigen was in soluble form, but it was enriched in the particle preparation. A lesser amount of antigen was distributed within the cultured cells. Absorption of rabbit antibody to gp52 with clone 11 particle preparations eliminated the ability of this antibody to detect immunocytochemically a crossreactive antigen previously localized in tissue sections of human breast carcinoma. These results indicate that the particle isolates from T47D contain the same gp52-related antigen found in human breast carcinomas and constitute an excellent source for the purification and characterization of this antigen.Previous studies (1)(2)(3)(4) To this end, it was of interest to resolve certain issues regarding the nature of the crossreactivity observed between gp52 and the unique antigens found in human breast cancers. Our experience indicated that it was unlikely that the supply of surgically removed human tumors would provide sufficient antigen for the necessary biochemical studies. Therefore, we focused on the T47D cell line, which had been established from the pleural effusion of a patient with intraductal and invasive carcinoma of the breast (13).It is the purpose of the present paper to present data on the T47D cell line and its clonal derivatives that reproducibly secrete virus-like particles containing the gp52-related antigen. This situation solves the difficult logistic problem of making available a reliable source of material from which to purify the relevant breast cancer antigens with comparative ease. MATERIALS AND METHODSCells. The cells were grown in RPMI 1640 medium containing fetal calf serum (10%), insulin (0.2 unit/ml), glutamine (2 mM), streptomycin (100 pug/ml), penicillin (100 units/ ml), and mycostatin (25 ,g/ml). When the cells were grown in the presence of steroid hormones, the fetal calf serum was dialyzed.For virus collection, the cells were seeded in RPMI 1640 medium containing 10% fetal calf serum for 24 hr followed by medium containing 5% dialyzed fetal calf serum and 1 nM 17-estradiol. After one medium change, this medium was replaced by one containing 10 nM progesterone free of fetal calf serum (day 5 after passage). From day 6, medium was collected every day, and the cells were refed with progesterone-containing medium. The collected medium was clarified by centrifugation for 15 mi...