Here we report the isolation of the recombinant cDNA clone from rat macrophages, Kupffer cells (KC) that encodes a protein interacting with carcinoembryonic antigen (CEA). To isolate and identify the CEA receptor gene we used two approaches: screening of a KC cDNA library with a specific antibody and the yeast two-hybrid system for protein interaction using as a bait the N-terminal part of the CEA encoding the binding site. The liver is a common site for metastasis from various forms of primary malignancies. Both experimental and clinical results reveal that the presence of carcinoembryonic antigen (CEA) 1 enhances liver metastasis from colorectal carcinoma cells (1, 2). CEA is a highly characterized, cell surface glycoprotein overexpressed by various tumor cells and provides a tool for tumor tissue-specific targeting. Increasing amounts of CEA in the serum correlates with the development of metastatic recurrence after surgical removal of the primary tumor (3). Earlier we have shown that CEA production of human colorectal cancer cell lines directly correlates with the metastatic potential (4). Poorly metastatic colon cancer cell lines become highly metastatic when transfected with the cDNA coding for CEA (5, 6). As a member of the immunoglobulin supergene family, CEA is involved in intercellular recognition and may facilitate attachment of colorectal carcinoma cells to sites of metastasis. In an experimental metastasis model of colorectal carcinoma in athymic nude mice, systemic injection of CEA enhanced experimental liver metastasis and implantation in liver by weakly metastatic tumor cells (7,8).To successfully treat cancers, it is necessary to prevent the development of metastasis after the treatment or removal of the primary malignancy. Therefore, it is important to elucidate the mechanism by which CEA enhances metastatic potential. The molecular basis by which CEA can influence metastasis is only partly understood. We have shown that CEA is rapidly cleared from the circulation of experimental animals, accumulates in the liver, and is endocytosed in vitro by Kupffer cells. This initiates a series of signaling events that leads to tyrosine phosphorylation on at least two intracellular proteins (9) and is followed by induction of IL-1␣, IL-6, IL-10, and tumor necrosis factor-␣ cytokines (10). CEA uptake by Kupffer cells is independent of its carbohydrate composition and is mediated by an 80-kDa binding protein (11). CEA is recognized by this binding protein through a 5-amino acid sequence, Pro-Glu-Leu-Pro-Lys (PELPK), located at the hinge region (amino acids 108 -112) between the N-terminal and the first immunoglobulin loop domain (12). Molecular modeling studies have suggested that this region is exposed on the surface of the molecule. 2 We have recently shown in a subset of colorectal cancer patients that mutations in the PELPK region of CEA results in the accumulation of large amounts of CEA in the serum (13). To further study the interaction between the peptide sequence, PELPK, and rat Kupffer cells, the...
A strategy for rapid generation of chicken sex chromosome-Z painting probes has been developed using microdissection. Whole chromosome painting probes (WCPs) were prepared from 10–15 copies of mitotic metaphase chicken Z chromosomes. The microisolated chromosomes were subjected to PEG/proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed shuttle PCR (DOP-Shuttle-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis and smears of DNA were revealed that ranged in size from 200–800 bp, without any evidence of preferential amplification. Both specificity and complexity of the probes have been analyzed by Southern blot and fluorescence in situ hybridization (FISH). Non-specific hybridization was efficiently blocked by using chicken competitor DNA. Analysis of the WCPs produced shows that collectively they provide uniform hybridization signals along the entire length of the chicken Z chromosome. To demonstrate one possible application of these complex probes, we screened a large-insert bacterial artificial chromosome (BAC) chicken genomic library to select Z chromosome-specific clones. To address specificity of the selected clones and to physically map them to the Z chromosome, FISH analysis was used. Of the 3 clones initially tested, one clone (C3) carrying a 250-kb insert mapped to the distal portion of the short arm of the chicken Z chromosome. Therefore, this technique has provided appropriate probes for screening large-insert genomic libraries. Further application of these probes includes the analysis of chromosome rearrangements, studies of cases of heteroploidy involving the Z chromosome, positional cloning of Z-linked genes and studies on mechanisms of sex-chromosome evolution in birds.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.