LKB1 deficiency in T cells promotes the development of gastrointestinal polyposis.

Germline mutations in STK11, which encodes the tumor suppressor liver kinase B1 (LKB1), promote Peutz-Jeghers syndrome (PJS), a cancer predisposition syndrome characterized by the development of gastrointestinal (GI) polyps. Here, we report that heterozygous deletion of Stk11 in T cells (LThet mice) is sufficient to promote GI polyposis. Polyps from LThet mice, Stk11+/- mice, and human PJS patients display hallmarks of chronic inflammation, marked by inflammatory immune-cell infiltration, signal transducer and activator of transcription 3 (STAT3) activation, and increased expression of inflammatory factors associated with cancer progression [interleukin 6 (IL-6), IL-11, and CXCL2]. Targeting either T cells, IL-6, or STAT3 signaling reduced polyp growth in Stk11+/- animals. Our results identify LKB1-mediated inflammation as a tissue-extrinsic regulator of intestinal polyposis in PJS, suggesting possible therapeutic approaches by targeting deregulated inflammation in this disease.

Host-related carcinoembryonic antigen cell adhesion molecule 1 promotes metastasis of colorectal cancer.

Liver metastasis is the predominant cause of colorectal cancer (CRC)-related mortality in developed countries. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell adhesion molecule with reduced expression in early phases of CRC development and thus functions as a tumor growth inhibitor. However, CEACAM1 is upregulated in metastatic colon cancer, suggesting a bimodal role in CRC progression. To investigate the role of this protein in the host metastatic environment, Ceacam1(-/-) mice were injected intrasplenically with metastatic MC38 mouse CRC cells. A significant reduction in metastatic burden was observed in Ceacam1(-/-) compared with wild-type (WT) livers. Intravital microscopy showed decreased early survival of MC38 cells in Ceacam1(-/-) endothelial environment. Metastatic cell proliferation within the Ceacam1(-/-) livers was also diminished. Bone marrow-derived cell recruitment, attenuation of immune infiltrates and diminished CCL2, CCL3 and CCL5 chemokine production participated in the reduced Ceacam1(-/-) metastatic phenotype. Transplantations of WT bone marrow (BM) into Ceacam1(-/-) mice fully rescued metastatic development, whereas Ceacam1(-/-) BM transfer into WT mice showed reduced metastatic burden. Chimeric immune cell profiling revealed diminished recruitment of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs) to Ceacam1(-/-) metastatic livers and adoptive transfer of MDSCs confirmed the involvement of these immune cells in reduction of liver metastasis. CEACAM1 may represent a novel metastatic CRC target for treatment.

CEACAM1 creates a pro-angiogenic tumor microenvironment that supports tumor vessel maturation.

We have studied the effects of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) on tumor angiogenesis in murine ductal mammary adenocarcinomas. We crossed transgenic mice with whey acidic protein promoter-driven large T-antigen expression (WAP-T mice) with oncogene-induced mammary carcinogenesis with CEACAM1null mice, and with Tie2-Ceacam1 transgenics, in which the Tie2 promoter drives endothelial overexpression of CEACAM1 (WAP-T × CEACAM1(endo+) mice), and analyzed tumor vascularization, angiogenesis and vessel maturation in these mice. Using flat-panel volume computed tomography (fpVCT) and histology, we found that WAP-T × CEACAM1(endo+) mice exhibited enhanced tumoral vascularization owing to CEACAM1(+) vessels in the tumor periphery, and increased intratumoral angiogenesis compared with controls. In contrast, vascularization of CEACAM1null/WAP-T-derived tumors was poor, and tumor vessels were dilated, leaky and showed poor pericyte coverage. Consequently, the tumoral vasculature could not be visualized in CEACAM1null/WAP-T mice by fpVCT, and we observed poor organization of the perivascular extracellular matrix (ECM), accompanied by the accumulation of collagen IV-degrading matrix metalloproteinase 9(+) (MMP9(+)) leukocytes and stromal cells. Vascular instability and alterations in ECM structure were accompanied by a significant increase in pulmonary metastases in CEACAM1null/WAP-T mice, whereas only occasional metastases were observed in CEACAM1(+) hosts. In CEACAM1(+) hosts, intratumoral vessels did not express CEACAM1, but they were intact, extensively covered with pericytes and framed by a well-organized perivascular ECM. MMP9(+) accessory cells were largely absent. Orthotopic transplantation of primary WAP-T- and CEACAM1null/WAP-T tumors into all three mouse lines confirmed that a CEACAM1(+) host environment is a prerequisite for productive angiogenic remodeling of the tumor microenvironment. Hence, CEACAM1 expression in the tumor periphery determines the vascular phenotype in a tumor, whereas systemic absence of CEACAM1 interferes with the formation of an organized tumor matrix and intratumoral vessel maturation.

Characterization of a major colon cancer susceptibility locus (Ccs3) on mouse chromosome 3.

Treatment of mice with the carcinogen azoxymethane (AOM) induces a number of lesions in the colon, including hyperplastic lesions, as well adenomas and carcinomas in situ. Inbred strains of mice show different responses to AOM-induced carcinogenesis. A/J mice are highly susceptible and develop a greater number of hyperplastic lesions and tumors (15-70 tumors per mouse) than resistant C57BL/6J mice (0-6 tumors per mouse). Susceptibility to AOM-induced tumors segregates as a co-dominant trait in (A x B6)F1 hybrids. Using a set of 23 AcB and BcA recombinant congenic mouse strains derived from A/J (susceptible) and B6 (resistant) parents, we observed that the number of hyperplastic lesions and tumors induced by AOM was under different genetic controls in AcB/BcA strains. The multiplicity of AOM-induced tumors is controlled by a major locus that we have mapped on the distal portion of chromosome 3, to which we have given the temporary designation colon cancer susceptibility locus 3 (Ccs3). B6 and A/J alleles at Ccs3 are associated with resistance and susceptibility, respectively. Haplotype analysis in key informative AcB/BcA strains restricts the size of the Ccs3 locus to a 14 Mb segment that contains 94 annotated genes. The expression level of all these genes in normal colon has been established by transcript profiling with microarrays, and has led to the identification of a subset of positional candidates that are expressed at high levels in this tissue. The 4q and 1p human chromosomal segments sharing syntenic homology with the mouse Ccs3 segment are known to be associated with inflammatory bowel diseases and colorectal tumors in humans, suggesting that the study of the mouse Ccs3 locus may help further the pathogenesis of these human conditions.

Intestinal tumor progression is promoted by decreased apoptosis and dysregulated Wnt signaling in Ceacam1-/- mice.

The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is downregulated in colonic and intestinal hyperplastic lesions as well as in other cancers, where it functions as a tumor suppressor. To investigate the functions of CEACAM1 in the normal intestine and in intestinal tumors, we generated a compound knockout mouse model and examined both Ceacam1(-/-) and Apc(1638N/+):Ceacam1(-/-) mice. Ceacam1(-/-) intestinal cells exhibited a significant decrease in apoptosis, with no change in proliferation or migration, however. Compound Apc(1638N/+):Ceacam1(-/-) mice demonstrated an increase in intestinal tumor multiplicity and tumor progression. Increases in intussusceptions and desmoid lesions were also observed. We have shown that CEACAM1-L associates with beta-catenin by co-immunoprecipitation and colocalization in CEACAM1-L-transfected CT26 and CT51 mouse colon carcinoma cells. Ceacam1(-/-) enterocytes displayed decreased glycogen synthase kinase 3-beta activity with corresponding nuclear localization of beta-catenin. Increased T-cell factor/Lef transcriptional activity was observed in CEACAM1-null CT51 colonic cells and in Caco2 colon cancer cells in which CEACAM1 was downregulated. A significant increased expression in c-Myc and cyclin D1 targets of the Wnt signaling pathway was also revealed in the Ceacam1(-/-) intestine. CEACAM1 therefore actively participates in Wnt signaling in intestinal cells and its downregulation in intestinal tissue contributes to malignancy by augmenting tumor multiplicity and progression.

Deletion of the carcinoembryonic antigen-related cell adhesion molecule 1 (Ceacam1) gene contributes to colon tumor progression in a murine model of carcinogenesis.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a glycoprotein that is part of the carcinoembryonic antigen and the immunoglobulin superfamilies. We have shown that it functions as a tumor suppressor and that this function depends upon the presence of the longer CEACAM1 cytoplasmic domain. In this report, we describe the generation of a Ceacam1-/- mouse. The Ceacam1-/- colon exhibits increased in vivo proliferation relative to the wild-type counterpart with a corresponding decreased expression of the p21(Cip1) and p27(Kip1) Cyclin D kinase inhibitors. The colonic villi undergo decreased apoptosis. Out of 35 litters of mice, no spontaneous tumors in any tissues normally expressing CEACAM1 were found over the lifespan of the animals, suggesting that CEACAM1 may not be involved in initiation of tumor development. However, when mice are treated with azoxymethane to induce colonic tumors, we find that Ceacam1-/- mice developed a significantly greater number of tumors than their littermate controls. Moreover, the tumor size was greater in the knockout mice relative to that in the wild-type mice. These results indicate that deletion of CEACAM1 favors progression of colon tumorigenesis.

Targeted disruption of the Ceacam1 (MHVR) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection.

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) and Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis in humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouse Ceacam1 gene in vivo. Manipulation of the Ceacam1 gene in mouse embryonic stem cells that contained the Ceacam1a allele yielded a partial knockout. We obtained one line of mice in which the insert in the Ceacam1a gene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p) Ceacam1a-targeted mice (Ceacam1aDelta4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV. Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.

The CEACAM1-L Ser503 residue is crucial for inhibition of colon cancer cell tumorigenicity.

CEACAM1 (also known as biliary glycoprotein, C-CAM or CD66a) is a cell adhesion molecule of the immunoglobulin family behaving as a tumor inhibitory protein in colon, prostate, liver, endometrial and breast cancers. Inhibition of tumor development is dependent upon the presence of the long 71-73 amino acid cytoplasmic domain of the CEACAM1 protein (CEACAM1-L). We have recently defined a number of cis-acting motifs within the long cytoplasmic domain participating in tumor cell growth inhibition. These are Tyr488, corresponding to an Immunoreceptor Tyrosine-based Inhibition Motif, as well as the three terminal lysine residues of the protein. In this study, we provide evidence that treatment with phorbol esters leads to increased phosphorylation of in vivo (32)P-labeled CEACAM1-L in mouse CT51 carcinoma cells, in the mouse 1MEA 7R.1 liver carcinoma cells and in 293 human embryonic kidney cells transfected with the Ceacam1-L cDNA. Basal level Ser phosphorylation was abrogated by treatment with the staurosporine inhibitor, but not by the protein kinase C-specific inhibitor calphostin C or other inhibitors such as H7 or sphingosine. Specific inhibitors of protein kinase A or calmodulin kinase had only minimal effects on the levels of basal or PMA-induced Ser phosphorylation. Furthermore, PMA treatment of the CT51 cells induced cell spreading and cellular relocalization of the CEACAM1-L protein. Since Ser503 has been described as a PMA-induced phosphorylation site in other cell systems, we investigated whether Ser503 was involved in these responses in mouse intestinal cells. No differences were noticed in the basal or the PMA-induced phosphorylation levels, kinase inhibitor sensitivity or the PMA-induced relocalization of the protein between the wild-type and the Ser503Ala mutant CEACAM1-L. However, we provide evidence that Ser503 participates in CEACAM1-L-mediated tumor inhibition as its mutation to an Ala led to in vivo tumor development, contrary to the tumor inhibitory phenotype observed with the wild-type CEACAM1-L protein.

The CEACAM1-L glycoprotein associates with the actin cytoskeleton and localizes to cell-cell contact through activation of Rho-like GTPases.

Associations between plasma membrane-linked proteins and the actin cytoskeleton play a crucial role in defining cell shape and determination, ensuring cell motility and facilitating cell-cell or cell-substratum adhesion. Here, we present evidence that CEACAM1-L, a cell adhesion molecule of the carcinoembryonic antigen family, is associated with the actin cytoskeleton. We have delineated the regions involved in actin cytoskeleton association to the distal end of the CEACAM1-L long cytoplasmic domain. We have demonstrated that CEACAM1-S, an isoform of CEACAM1 with a truncated cytoplasmic domain, does not interact with the actin cytoskeleton. In addition, a major difference in subcellular localization of the two CEACAM1 isoforms was observed. Furthermore, we have established that the localization of CEACAM1-L at cell-cell boundaries is regulated by the Rho family of GTPases. The retention of the protein at the sites of intercellular contacts critically depends on homophilic CEACAM1-CEACAM1 interactions and association with the actin cytoskeleton. Our results provide new evidence on how the Rho family of GTPases can control cell adhesion: by directing an adhesion molecule to its proper cellular destination. In addition, these results provide an insight into the mechanisms of why CEACAM1-L, but not CEACAM1-S, functions as a tumor cell growth inhibitor.

cis-Determinants in the cytoplasmic domain of CEACAM1 responsible for its tumor inhibitory function.

CEACAM1, also known as C-CAM, BGP and CD66a, is a member of the carcinoembryonic antigen (CEA) family which is itself part of the immunoglobulin supergene family. CEACAM1 is involved in intercellular adhesion, signal transduction and tumor cell growth regulation. CEACAM1 is down-regulated in colon and prostate carcinomas, as well as in endometrial, bladder and hepatic tumors, and 30% of breast cancers. We have shown in a mouse colon tumor model that CEACAM1 with a long cytoplasmic domain inhibited the development of tumors whereas a splice variant lacking the cytoplasmic domain did not. In this study, we define the subregions of the long cytoplasmic domain participating in the tumor inhibition phenotype of CEACAM1. We show that a single point mutation of Tyr488, conforming to an Immunoreceptor Tyrosine Inhibition Motif (ITIM), was sufficient to reverse the in vivo tumor cell growth inhibition. Substitution or deletion of residues in the C-terminal region of the CEACAM1 cytoplasmic domain also led to reversal of tumor cell growth inhibition. This result is in agreement with our previous studies demonstrating the C-terminal region of the cytoplasmic domain influences the levels of CEACAM1 Tyr phosphorylation and its association with the protein Tyr phosphatases SHP-1 and SHP-2. Furthermore, removal of the N-terminal domain of CEACAM1, essential for intercellular adhesion, did not impair the tumor inhibitory effect. These results suggest that Tyr phosphorylation or dephosphorylation of the CEACAM1 cytoplasmic domain represents a crucial step in the control of epithelial cell proliferation.

Comparison of expression patterns and cell adhesion properties of the mouse biliary glycoproteins Bbgp1 and Bbgp2.

Biliary glycoproteins are members of the carcinoembryonic antigen (CEA) family and behave as cell adhesion molecules. The mouse genome contains two very similar Bgp genes, Bgp1 and Bgp2, whereas the human and rat genomes contain only one BGP gene. A Bgp2 isoform was previously identified as an alternative receptor for the mouse coronavirus mouse hepatitis virus. This isoform consists of two extracellular immunoglobulin domains, a transmembrane domain and a cytoplasmic tail of five amino acids. In this report, we have examined whether the Bgp2 gene can express other isoforms in different mouse tissues. We found only one other isoform, which has a long cytoplasmic tail of 73 amino acids. The long cytodomain of the Bgp2 protein is highly similar to that of the Bgp1/4L isoform. The Bgp2 protein is expressed in low amounts in kidney and in a rectal carcinoma cell line. Antibodies specific to Bgp2 detected a 42-kDa protein, which is expressed at the cell surface of these samples. Bgp2 was found by immunocytochemistry in smooth muscle layers of the kidney, the uterus, in gut mononuclear cells and in the crypt epithelia of intestinal tissues. Transfection studies showed that, in contrast with Bgp1, the Bgp2 glycoprotein was not directly involved in intercellular adhesion. However, this protein is found in the proliferative compartment of the intestinal crypts and in cells involved in immune recognition. This suggests that the Bgp2 protein represents a distinctive member of the CEA family; its unusual expression patterns in mouse tissues and the unique functions it may be fulfilling may provide novel clues about the multiple functions mediated by a common BGP protein in humans and rats.

The carboxyl-terminal region of biliary glycoprotein controls its tyrosine phosphorylation and association with protein-tyrosine phosphatases SHP-1 and SHP-2 in epithelial cells.

Biliary glycoprotein (Bgp, C-CAM, or CD66a) is an immunoglobulin-like cell adhesion molecule and functions as a tumor suppressor protein. We have previously shown that the Bgp1 isoform responsible for inhibition of colonic, liver, prostate, and breast tumor cell growth contains within its cytoplasmic domain two tyrosine residues positioned in immunoreceptor tyrosine-based inhibition motif (ITIM) consensus sequences. Moreover, we determined that these residues, upon phosphorylation, associate with the protein-tyrosine phosphatase SHP-1. In this report, we have further evaluated the structural bases of the association of Bgp1 with Tyr phosphatases. First, we demonstrate that Bgp1 also associates with the SHP-2 Tyr phosphatase, but not with an unrelated Tyr phosphatase, PTP-PEST. Association of Bgp1 and SHP-2 involves the Tyr residues within the Bgp1 ITIM sequences, Val at position +3 relative to the second Tyr (Tyr-515), and the SHP-2 N-terminal SH2 domain. In addition, our results indicate that residues +4, +5, and +6 relative to Tyr-515 in the Bgp1 cytoplasmic domain play a significant role in these interactions, as their deletion reduced Bgp1 Tyr phosphorylation and association with SHP-1 and SHP-2 by as much as 80%. Together, these results indicate that both SHP-1 and SHP-2 interact with the Bgp1 cytoplasmic domain via ITIM-like sequences. Furthermore, they reveal that the C-terminal amino acids of Bgp1 are critical for these interactions.

Virus-receptor interactions and interspecies transfer of a mouse hepatitis virus.

Molecular mechanisms regulating virus xenotropism and cross-species transmission are poorly understood. Host range mutants (MHV-H2) of mouse hepatitis virus (MHV) strains were isolated from mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine astrocytoma (DBT) cells. MHV-H2 was polytrophic, replicating efficiently in normally nonpermissive BHK cells, Syrian and Chinese hamster (DDT-1 and CHO) cells, human adenocarcinoma (HRT), primate kidney (VERO) and in murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK), and porcine testicular (ST) cell lines. To study the effects of xenotrophic spread on virus receptor-interactions in the original host, murine DBT cells were pretreated with a monoclonal antibody (MAb) CC1, directed against the MHV receptor, MHVR, a biliary glycoprotein (Bgp1a). Under treatment conditions that completely ablated the replication of the parental MHV strains, CC1 antireceptor antibodies did not block MHV-H2 replication. Following expression of MHVR in normally nonpermissive ST and CRFK cells, infection with the parental MHV strains, but not MHV-H2 was observed. To characterize the molecular basis preventing the interaction between MHV-H2 and MHVR, revertants of MHV-H2 (MHV-H2R6, MHV-H2R11) were isolated following a persistent MHV-H2 infection in DBT cells. These revertant viruses efficiently recognized MHVR, however infection of murine cells was resistant to MAb CC1 blockade. In addition, MHV-H2 and the revertant viruses efficiently recognized other Bgp receptors for docking and entry. These data suggest that interspecies transfer may remodel normal virus-receptor interactions that may result in altered virulence, tropism or pathogenesis in the original host.

Regulation of mouse PECAM-1 tyrosine phosphorylation by the Src and Csk families of protein-tyrosine kinases.

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. Recently, it was observed that PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 was shown to associate with SHP-2, a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, we wished to understand better the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, we determined that mouse PECAM-1 could be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs, but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, we found that these PTKs were efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylated mouse PECAM-1 at one or more yet to be identified sites. In other studies, we demonstrated that phosphorylation of PECAM-1 by Src or Csk family kinases was sufficient to trigger its association with SHP-2. Moreover, it was able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicated that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances.

The cell adhesion molecule C-CAM is a substrate for tissue transglutaminase.

C-CAM, a ubiquitously expressed cell adhesion molecule belonging to the carcinoembryonic antigen family, appears as two co-expressed isoforms, C-CAM-L and C-CAM-S, with different cytoplasmic domains, that can form homodimers in epithelial cells. In addition, C-CAM-L has been found in large molecular weight forms suggesting posttranslational, covalent modification. Here we have investigated the possibility that the cytoplasmic domain of C-CAM-L can act as a transglutaminase substrate. Glutathione S-transferase fusion proteins of the cytoplasmic domains of rat and mouse C-CAM-L as well as free cytoplasmic domains, released by thrombin cleavage from the fusion proteins, were converted into covalent dimers by tissue transglutaminase. These results demonstrate that the cytoplasmic domains of rat and mouse C-CAM-L are substrates for tissue transglutaminase, and lend support to the notion that higher molecular weight forms of C-CAM-L are formed by transglutaminase modification.

Mutational analysis of the virus and monoclonal antibody binding sites in MHVR, the cellular receptor of the murine coronavirus mouse hepatitis virus strain A59.

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.

Optimal ratios of biliary glycoprotein isoforms required for inhibition of colonic tumor cell growth.

Rodent biliary glycoprotein (Bgp), also known as C-CAM, has recently been shown to function as a tumor suppressor in colon, prostate, and bladder cancers. This glycoprotein is a member of the carcinoembryonic antigen family and is one of the only proteins in this family to encode either a long (71-73 amino acids) or short (10 amino acids) cytoplasmic domain. We and others have shown that the growth-inhibitory properties of Bgp depend upon the expression of its long cytoplasmic domain. However, the two Bgp isoforms normally coexist in most cell types surveyed; the longer variant is usually present in lower amounts than the shorter one. In this study, we have examined the in vitro and in vivo growth properties of both mouse Bgp variants separately and in combination. To determine the physiologically relevant expression levels and ratios of the two Bgp variants, we have quantified the amount of the longer variant in normal colonic epithelial cells and showed that it constitutes 15-20% of total Bgp expressed in this tissue. To mimic the in vivo situation, we have generated double transfectant cell lines expressing the longer and shorter Bgp isoforms coordinately in tumorigenic CT51 mouse colonic carcinoma cells and demonstrated that the longer Bgp isoform exhibits a dominant tumor growth inhibition phenotype over that of the shorter variant within physiological levels of expression of Bgp. Unexpectedly, significant overexpression of the longer Bgp isoform alone led to reversal of the tumor inhibition phenotype. These results, therefore, suggest that there may be a limiting threshold of Bgp expression or Bgp-associating proteins mediating the tumor inhibition phenotype.

Use of a rat cDNA probe specific for the Y chromosome to detect male-derived cells.

A cDNA probe that exhibits specificity for the rat Y chromosome was generated by using a set of primers specific to the murine Sry gene, the sex-determining region of the Y chromosome. A 459-base pair (bp) DNA fragment was obtained by polymerase chain reaction (PCR) amplification from male, but not female, rat genomic DNA (EMBL Nucleotide Sequence Database accession number X89730). This DNA fragment was purified, cloned using a vector, and digested with EcoR1 to yield a 270-bp DNA fragment. This 270-bp cDNA fragment, when used as a probe in Southern blot analysis of rat DNA, was observed to bind to three separate bands of approximately 2.3, 5.0, and 7.0 kb in size. The binding was demonstrated with male, but not female, genomic DNA. Another set of primers was generated to sequences within the 270-bp fragment that produced a PCR product of 104 bp. This DNA fragment, when used as a probe in Southern blot analysis, enabled PCR detection of at least 0.1% male cells in a mixed population of female cells. These cDNA probes should prove useful in studies designed to track cell populations (e.g., tumor metastasis and hemopoietic cells after bone marrow transplantation) in syngeneic male/female pairs. In addition, a cDNA probe that is specific for the rat Sry gene might be valuable in studies of fetal male sexual development or the study of spermiogenesis.