Abstract
The human epithelial mucin which is the product of the MUC1 gene is expressed by many carcinomas, including those of breast, ovary, colon, and lung. The core protein is aberrantly glycosylated in the tumors resulting in the exposure or appearance of novel epitopes. To examine the possibility of using the MUC1 gene and its products in active immunization against breast and other carcinomas, we have developed a syngeneic mouse model, by transfecting the gene into the mouse mammary epithelial tumor cell 410.4. An 8.3-kilobase EcoRI fragment of the gene was transfected using the expression vector pEMSV scribe alpha 2. Transcripts of the correct size, initiating from the transcriptional start site seen in human cells, were observed in the transfectants. The mucin was expressed in the cytoplasm and in the membrane, and the glycosylation pattern appeared to be similar to that seen in human tumor cells, since the core protein epitopes recognized by antibodies HMFG-1, HMFG-2, and SM-3 were exposed. The 410.4 transfectants expressing the human mucin showed a reduction in tumor incidence at low inocula and a delay in tumor growth at higher inocula. Pretreatment with 10(4) transfected cells could inhibit the development of tumors from a subsequent inoculum of 10(6) transfectants, but had no effect on the tumor development of the untransfected 410.4 cells. Our results suggest that the human mucin expressed by the 410.4 cells may mobilize an immune response which inhibits tumor development. They also indicate that the mouse model will be useful for evaluation of efficacy of immunogens based on the MUC1 gene and its product.
Highlights
The human epithelial mucin whichis the productof the antibody SM-3 [6,7] has been mapped to a domain of the the MUCl gene is expressed by many carcinomas, in- core protein which consists of tandem repeats [8].Due to a cluding those of breast, ovary, colon, and lung
Sylation pattern appeared to be similar to that seen inThe core of the SM-3epitope in the tandem repeat domain human tumorcells, sincethe core protein epitopes rec-of the MUCl product is defined by the amino acid sequence ognized by antibodies HMFG-1, HMFG-2, and SM-3 Pro-Asp-Thr-Arg-Pro, and this sequence is located between were exposed
The gene coding for a mucin expressed by many human epithelial cells and designated MUCl has been successfully expressed in mouse cells
Summary
Isolation of Mouse Cell Lines, Expressing the Mucin MUCl-An 8.3-kb EcoRI fragment from a cosmid clone isolated from the pCos2EMBL libraryhas been shown to contain all of the coding sequences for PEM (except for t h e last 24 amino acids of the cytoplasmic tail), together with 3.1 kg of 5’ untranscribed sequence.’ This fragment was inserted into the pEMSVscribe a2 vector, which contains a polyadenylation signal as well as the Moloney sarcoma virus long terminarlepeat promoter [26], t o give the construct R1MUClpEMSVscribe. Isolation of Mouse Cell Lines, Expressing the Mucin MUCl-An 8.3-kb EcoRI fragment from a cosmid clone isolated from the pCos2EMBL libraryhas been shown to contain all of the coding sequences for PEM (except for t h e last 24 amino acids of the cytoplasmic tail), together with 3.1 kg of 5’ untranscribed sequence.’. Transcriptase and cold dNTPs toyield a doublet band which mapped 71 and 72 bp upstreamof the ATG (Fig. 3) This cap GCAT 1 2 3 site is identical to that obtained using RNAfrom BT20 cells (a human breast cancercell line) which naturally express the mucin [10].This result indicates that 3t.h1ekb of 5' sequence in the MUCglene fragment are apparently sufficient to direct 72 bp expression of the MUClgene from its normal start site in the. T o analyze the profile of epitopes expressed on thecell-associated mucin, each of the threeantibodies were used tostain fixed cultures of the ExpresMsiUoCn l in Mouse Cells
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