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The β1Cintegrin is an alternatively spliced variant of the β1subunit that contains a unique 48-amino-acid sequence in its cytoplasmic domain. We have shown previously that β1Cis a potent inhibitor of cell proliferation and that in vivoits expression is downregulated in prostate and breast carcinoma. In this study, we describe a panel of specific monoclonal antibodies that react with the β1Ccytodomain. We show by immunoblot analysis that the newly generated monoclonal antibodies specifically recognize the β1Ccytodomain expressed as glutathione S-transferase fusion protein. The specificity of the antibodies to β1Cwas confirmed in competition studies by immunoblotting using β1C-specific synthetic peptides. These monoclonal antibodies reacted, in enzyme-linked immunosorbent assays, with the β1C785–808 peptide but failed to bind the β1C778–794, β1C805–825, or β1A765–798 peptides. Thus, the epitope recognized by the antibodies is located within the Q795-F804β1Ccytoplasmic sequence; this region overlaps the previously described Q795-Q802domain necessary for β1Cto inhibit cell proliferation. To our knowledge, these are the first monoclonal antibodies specific for a β1cytoplasmic isoform. The monoclonal antibodies described here will be useful tools for dissecting functional differences, among β1integrin variants, as well as for the study of the role of β1Cin prostate and breast epithelial cell proliferation.

β1Cintegrin is an unspliced form of the integrin β1subfamily, which has been shown to inhibit cell proliferation in vitro. Using an affinity-purified rabbit antibody, we have investigated 283 previously untreated breast carcinomas, with the aim of ascertaining the actual prevalence of β1Cexpression in these tumors and of defining its pathological correlates. Immunoblotting and reverse transcriptase-polymerase chain reaction experiments have also been performed in selected cases, to confirm the immunocytochemical findings. Overall, β1Cimmunoreactivity was down-regulated (ie, expressed in < 50% of the neoplastic cells) in 114 cases (40.3%). Down-regulation of β1Cexpression in breast carcinomas correlated significantly with the tumor grade, the proliferative fraction (as evaluated by Ki-67 immunostaining with the MIB-1 monoclonal antibody), the estrogen and progesterone receptor status, and the tumor size (pT classification) and marginally with the node status. In a multivariate analysis with all available measures fitted simultaneously, tumor grade (P= 0.004), Ki-67 immunolabeling (P= 0.01), and pT categories (P= 0.04) were significantly associated with β1Cimmunoreactivity. Although the short follow-up time (2–3 years) of the current series of patients does not allow the performance of survival analyses, the correlation of β1Cexpression with tumor size, grade, and proliferative fraction and its alleged role as an upstream regulator of p27kip1make this integrin variant a likely novel prognostic parameter for invasive carcinomas of the breast.

Alternative splicing of the integrin β1subunit mRNA generates a variant form, β1C, with a unique cytoplasmic domain that differs from β1for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1cells transiently transfected with cDNAs coding for human integrin β1Cor β1subunits or mutants containing truncated forms of the β1Ccytoplasmic domain. A differential effect of β1Cand β1on cell proliferation was observed. Expression of wild type β1was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of β1C. Cells expressing the β1Cmutant truncated at Leu794and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three β1Cdeletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of β1Cexpression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing β1C, full-length or deletion mutants, or β1equally adhered to a functionally inhibitory monoclonal antibody against human β1integrin. Expression of β1Cinversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of β1Cwas induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.

Trop-2 is a novel calcium signal transducer expressed at high levels by most human carcinomas. To develop an animal model to study the function of this molecule in vivo, we have cloned the murine Trop2 gene. Using human TROP2 primers, we amplified by PCR a segment of murine Trop2. This was used as a probe to clone a full-length gene by hybridization of a genomic library. The cloned murine Trop2 gene is functional, as indicated by sequencing and by expression after transfection. The murine Trop2 is 87.4% similar to its human homologue, with the highest conservation in the extracellular region between residues 86 and 157. Essentially all cysteines are conserved between the human and the murine genes, suggesting conservation of the Trop2 disulfide bridges and of its overall structure. Intriguingly, the cytoplasmic tail of Trop2 shows a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2)-binding sequence, which overlaps with a protein kinase C phosphorylation site. Thus, we speculate that PIP2 might regulate the phosphorylation state of Trop2 and play a role in its signal transduction. Murine Trop2 mRNA is detected in normal kidney, lung, ovary and testis, similarly to the human gene. Interestingly, the highest levels of expression are found in immortalized keratinocytes. Since Trop2 is undetectable in undifferentiated spindle cell carcinomas, this suggests a preferential expression at early stages of tumor progression. Int. J. Cancer 75:324–330, 1998. © 1998 Wiley-Liss, Inc.

The expression of the β1Cintegrin, an alternatively spliced variant of the β1subunit, was investigated in human adult and fetal tissues. In the adult, β1Cimmunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, β1Cwas undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed β1Cin vivoand in vitro, but no β1Cwas seen in basal cells, which are proliferating cells. Fetal prostate expressed β1Cin differentiated glands that had a defined lumen, but not in budding glands, indicating that β1Cis a marker of prostate epithelium differentiation. The β1Cand the common β1Avariants are differentially distributed: β1Awas found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, β1Cand β1Awere coexpressed in biliary epithelium, whereas vascular cells expressed only β1A. Because we found β1Cin nonproliferative and differentiated epithelium, we investigated whether β1Ccould have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a β1C, but not of a β1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitroresults, β1Cappeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of β1Cintegrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.

Signaling through Gαi2induces hypertrophy and can counterbalance pathways that promote muscle wasting.