The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex.

We have previously described the generation of a monoclonal antibody recognizing a novel cholangiocyte marker, designated BD.1, that is expressed by fetal and adult rat cholangiocytes but not hepatocytes or the hepatic progenitor cells known as oval cells. In the present report, we have undertaken a comprehensive examination of BD.1 expressed by long-term cultures of bile duct epithelial cells (BDEC) and prostate epithelial cells (PEC). We show that with continued passage, the levels of BD.1 expressed by BDEC and PEC drop significantly, a decrease that is temporally associated with transition from a diploid to an aneuploid karyotype. Cell cycle analysis revealed cell cycle dependent expression of BD.1 characterized by decreased BD.1 levels within the first 10 h after release from serum starvation followed by reacquisition as cells entered S phase. MAb BD.1 recognized a 170 kDa protein in Western blots and showed strong reactivity with a 170 kDa band in blots prepared from phosphoproteins isolated by metal affinity chromatography. Analysis by mass spectrometry of tryptic peptides generated from BD.1 purified by continuous elution electrophoresis identified the plus end microtubule-binding protein, CLIP170, in the fraction reactive with MAb BD.1. Double immunofluorescence with MAb BD.1 and a MAb specific for CLIP170 showed that both were reactive with intrahepatic bile ducts. However, overexpression or siRNA knockdown of CLIP170 in 293T cells did not significantly alter BD.1 levels, indicating that CLIP170 and BD.1 were distinct, co-migrating proteins. Immunoprecipitation analysis with MAb BD.1 and anti-CLIP170 antibodies showed that under microtubule depolymerizing conditions the two proteins could be co-precipitated with both antibodies, leading us to conclude they were capable of forming stable complexes. Two different protocols were devised to enrich for the CLIP170 binding protein recognized by MAb BD.1. Analysis of tryptic peptides by LC-ESI-MS/MS identified BD.1 as eIF3a, the largest subunit of the elongation initiation factor 3 (eIF3) complex. This identity was confirmed by the simultaneous knockdown of both BD.1 and eIF3a by eIF3a-specific siRNAs and by the strong reactivity of MAb BD.1 with the 170 kDa protein immunoprecipitated with the anti-eIF3a antibody, 5H10. Based on these findings, we concluded that the BD.1 antigen was identical to eIF3a, a multifunctional subunit of the eIf3 complex shown here to associate with microtubules through its interactions with CLIP170.

Carcinoembryonic antigen-related cell adhesion molecule 1a-4L suppression of rat hepatocellular carcinomas.

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CEACAM1) is a member of the CEA family of immunoglobulin-like adhesion molecules with two major splice variants, CEACAM1(a)-4L and CEACAM1(b)-4S, differing in the length of their COOH-terminal cytoplasmic tail. Both forms are down-regulated in prostate and liver carcinomas relative to normal tissues. We have previously shown in a nude mouse xenograft model that restoration of CEACAM1(a)-4L expression in human prostate carcinoma cells (PC-3) suppresses tumorigenicity, an effect observed with carcinomas from several other tissues but never established for hepatocellular carcinomas. In this report, we have examined the effect of CEACAM1(a)-4L on tumorigenicity of 1682A, a rat hepatocellular carcinoma that grows on the omentum when injected into the peritoneal cavity. Results show that restoration of CEACAM1(a)-4L expression at levels 13- and 0.45-fold compared with negative controls or normal hepatocytes, respectively, completely suppressed the formation of 1682A tumor nodules on the omentum at 3 weeks after injection. In contrast, 1682A cells infected with CEACAM1(b)-4S or an empty retroviral vector formed multiple clusters of tumor nodules. Although tumor nodules of 1682A cells positive and negative for CEACAM1(a)-4L did not display significant differences in histologic organization, aggregates formed in vitro by 1682A-L were smaller in size and displayed enlarged intercellular spaces relative to their 1682A-V counterparts. Restoration of CEACAM1(a)-4L expression did not elevate levels of apoptosis but seemed to cause an increase in the length of G1. This is the first demonstration of CEACAM1(a)-4L-induced tumor suppression in liver carcinomas using a quantifiable i.p. syngeneic transplantation model.

Monoclonal antibody to novel cell surface epitope on Hsc70 promotes morphogenesis of bile ducts in newborn rat liver.

We previously described a cell surface reactive monoclonal antibody, MAb OC.10, which recognizes an epitope shared by rat fetal liver ductal cells, hepatic progenitor cells, mature cholangiocytes, and hepatocellular carcinomas (HCC). Here, intrasplenic injection of MAb OC.10 into newborn rats was shown by immunofluorescence microscopy to strongly label intrahepatic bile ducts. Furthermore, the in situ labeling of intrahepatic cholangiocytes by injecting MAb OC.10 increased the number of intraportal and intralobular bile ducts with well-defined lumens when compared to IgM-injected control animals. The antigen for MAb OC.10 was identified by mass spectrometry as Hsc70, a constitutively expressed heat shock protein belonging to the HSP70 family. Immunoblot analysis demonstrated that MAb OC.10 reacted with recombinant bovine Hsc70 protein, with protein immunoprecipitated from rat bile duct epithelial (BDE) cell lysates with monoclonal anti-Hsc70 antibody, and with Hsc70-FLAG protein over-expressed in human 293T cells. In addition, Hsc70-specific small interfering RNA reduced the amount of OC.10 antigen expressed in nucleofected BDE cells. Consistent with the specificity of MAb OC.10 for Hsc70, heat shock did not induce OC.10 expression in BDE cells, a characteristic of Hsp70. Immunofluorescence with BDE cells further suggested that MAb OC.10 binds a novel cell surface epitope of Hsc70. This was in contrast to a commercially available monoclonal anti-Hsc70 antibody that showed strong cytosolic reactivity. These findings demonstrate that presentation of the OC.10 epitope differs between cytosolic and surface forms of Hsc70 and may suggest distinct differences in protein conformation or epitope availability determined in part by protein-protein or protein-lipid interactions. Phage display and pepscan analysis mapped the epitope for MAb OC.10 to the N-terminal 340-384 amino acids of the ATPase domain of rat Hsc70. These findings suggest that MAb OC.10 recognizes an epitope on rat Hsc70 when presented on the cell surface that promotes morphogenic maturation of bile ducts in newborn rat liver. Furthermore, since we have shown previously that the OC.10 antigen is expressed on HCC subpopulations with oval cell characteristics, our current results indicate that Hsc70 has the potential to be expressed on the surface of certain tumor cells.

Cholangiocyte marker-positive and -negative fetal liver cells differ significantly in their ability to regenerate the livers of adult rats exposed to retrorsine.

We have used monoclonal antibodies against cell-surface developmental epitopes in combination with micromagnetic beads to isolate phenotypically defined subpopulations of cholangiocyte marker-positive fetal liver epithelial cells (CMP-FLEC). Differentiation potential was evaluated by injecting cell isolates from dipeptidyl peptidase IV (DPPIV) positive (DPPIV+) Fischer donor rats into the spleen of partially hepatectomized, DPPIV negative (DPPIV-) Fischer host rats exposed to retrorsine. At various time points, liver tissue was harvested and cells in DPPIV+ colonies were phenotyped by immunofluorescence and histochemical protocols. Functional differentiation and liver replacement were determined by comparing donor and host hepatocyte protein expression patterns and DPPIV enzyme activity in extracts from livers of host rats receiving CMP-FLEC. Our results showed that bipotentiality was retained during differentiation and maturation of CMP-FLEC, indicating that the acquisition of ductal morphology and phenotype were not indicative of lineage commitment. CMP-FLEC transplanted into the adult rat liver lost ductal and gained hepatocyte markers, and acquired protein expression patterns in 2D gels with a close similarity (>75% spot match) to host hepatocytes but differing significantly from the transplanted CMP-FLEC cell isolate (<25% spot match). The average size of donor hepatocyte colonies increased with time so that by 1 year, up to 70% of the host rat liver was replaced by CMP-FLEC derived DPPIV+ hepatocytes. Depletion of CMP-FLEC from fetal liver isolates resulted in a marked decrease in adult liver colonization, suggesting that a high percentage of the hepatocyte colonies in animals receiving total fetal liver isolates are derived from CMP-FLEC.

How Uganda reversed its HIV epidemic.

Uganda is one of only two countries in the world that has successfully reversed the course of its HIV epidemic. There remains much controversy about how Uganda's HIV prevalence declined in the 1990s. This article describes the prevention programs and activities that were implemented in Uganda during critical years in its HIV epidemic, 1987 to 1994. Multiple resources were aggregated to fuel HV prevention campaigns at multiple levels to a far greater degree than in neighboring countries. We conclude that the reversed direction of the HIV epidemic in Uganda was the direct result of these interventions and that other countries in the developing world could similarly prevent or reverse the escalation of HIV epidemics with greater availability of HIV prevention resources, and well designed programs that take efforts to a critical breadth and depth of effort.

Identification of a novel protein, LYRIC, localized to tight junctions of polarized epithelial cells.

Tight junctions (TJ) are multiprotein complexes that function to regulate paracellular transport of molecules through epithelial and endothelial cell layers. Many new tight junction-associated proteins have been identified in the past few years, and their functional roles and interactions have just begun to be elucidated. In this paper, we describe a novel protein LYsine-RIch CEACAM1 co-isolated (LYRIC) that is widely expressed and highly conserved between species. LYRIC has no conserved domains that would indicate function and does not appear to be a member of a larger protein family. Data from analysis of rat and human tissue sections and cell lines show that LYRIC colocalizes with tight junction proteins ZO-1 and occludin in polarized epithelial cells, suggesting that LYRIC is part of the tight junction complex. LYRIC dissociates from ZO-1 when junctional complexes are disrupted, and as tight junctions reform, ZO-1 relocalizes before LYRIC. These results suggest that LYRIC is most likely not a structural component required for TJ formation, but rather is recruited during the maturation of the tight junction complex.

Two variable regions in carcinoembryonic antigen-related cell adhesion molecule1 N-terminal domains located in or next to monoclonal antibody and adhesion epitopes show evidence of recombination in rat but not in human.

In this paper, we have characterized the structure, evolutionary origin, and function of rat and human carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) multifunctional Ig-like cell adhesion proteins that are expressed by many epithelial tissues. Restriction enzyme digestion reverse transcriptase-PCR analysis identified three cDNAs encoding novel CEACAM1 N-domains. Comparative sequence analysis showed that human and rat CEACAM1 N-domains segregated into two groups differing in similarity to rat CEACAM1(a)-4L and human CEACAM1. Sequence variability analysis indicated that both human and rat N-domains possessed two variable regions, and one contained a major adhesive epitope. Recombination analysis showed that the group of rat but not human N-domains with high sequence similarity was derived at least in part by recombination. Binding assays revealed that three monoclonal antibodies with strong reactivity for the CEACAM1(a)-4L N-domain showed no reactivity with CEACAM1(b)-4S, an allele with a different N-domain sequence. CEACAM1(b)-4S displayed adhesive activity efficiently blocked by a synthetic peptide corresponding to the adhesive epitope in CEACAM1(a)-4L. Blocking analysis also showed that the adhesive epitope for rat CEACAM1 was located downstream from the equivalent human and mouse epitopes. Glycosylation analysis demonstrated O-linked sugars on rat CEACAM1(b)-4S from COS-1 cells. However, this was not the alteration responsible for the lack of monoclonal antibody reactivity. When considered together with previous studies, our findings suggest an inverse relationship between functionality and amino acid sequence similarity to CEACAM1. Like IgG, the N-domain of CEACAM1 appears to tolerate 10-15% sequence diversification without loss of function but begins to show either altered specificity or diminished functionality at higher levels.