Patterns of α- and β-catenin and E-cadherin expression in colorectal adenomas and carcinomas

Previous in vitro and in vivo model studies have shown that when E-cadherin expression in carcinoma cells is reduced, invasive behaviour ensues. The situation in human cancer in vivo, however, appears to be more complex, as immunohistochemically determined E-cadherin expression in various carcinomas, including colorectal cancer, does not always correlate with invasive growth. Loss of cell adhesion during invasion in spite of E-cadherin expression might be associated with a defective cadherin–catenin complex. The expression of α- and β-catenin in comparison with E-cadherin was therefore examined in colorectal adenomas and carcinomas and in lymph node and liver metastases. In normal colonic mucosa, α- and β-catenin immunoreactivity occurred along the lateral plasma membranes of the epithelial cells, in a pattern identical to E-cadherin staining. A similar pattern was found in colorectal adenomas and in most malignancies. In general, in neoplastic epithelia, the majority of the cancer cells displayed a normal (matching) pattern of E-cadherin and catenin expression. It is concluded that the patterns of expression of E-cadherin and α- and β-catenin are very similar in colorectal neoplasms. This observation indicates that invasion in colorectal cancer is not paralleled by consistent loss of expression of the components of the cadherin–catenin complex. © 1997 John Wiley & Sons, Ltd.     

E-cadherin and α-, β-, and γ-catenin protein expression in relation to metastasis in human breast carcinoma

In the metastatic process, various cell–cell adhesion molecules seem to play an important role. E-cadherin, a transmembrane protein with an extracellular and an intracellular domain, is one of the key players involved in cell–cell adhesion. The function of E-cadherin in preventing metastasis in tumour development is believed to be dependent on intracellular catenins. In a previous study, the expression of E-cadherin was examined in a series of human breast carcinomas. In that study, down-regulation of E-cadherin failed to correlate with lymph node and/or distant metastasis. In the present study, the expression of α-, β-, and γ-catenins has been examined in a subset of the same tumours in order to evaluate their possible role in breast cancer metastasis. Tumour tissues from 90 primary breast carcinomas were immunostained for α-, β-, and γ-catenins. Reduced or absent immunoreactivity in the tumour tissue was seen in 63 (70·0 per cent) for α-catenin, in 50 (55·6 per cent) for β-catenin, and in 50 (55·6 per cent) for γ-catenin. Reduced expression of each of the catenins alone failed to correlate to metastasis. However, when all of the four proteins (E-cadherin, α-catenin, β-catenin, and γ-catenin) were analysed as one group, a significant association was seen between reduction in immunoreactivity of at least one of these four proteins and the presence of metastases. These results indicate that if one of these proteins is down-regulated, the function of the others in suppressing metastasis is altered. A significant association was seen between lobular invasive tumours and β-catenin expression. © 1998 John Wiley & Sons, Ltd.     

Developmentally regulated expression of the neural cell adhesion molecule (NCAM) by mouse thymocytes

The expression of the neural cell adhesion molecule (NCAM) has been investigated during thymus ontogeny. NCAM mRNA was readily detectable at day 19 of gestation, the youngest age studied. Its level declined after birth to become undetectable at 3 weeks of age. Cell surface expression of NCAM protein was detected on 14% of day 15 fetal thymocytes and peaked during the perinatal period, when around 40% of the thymocytes expressed low to medium levels of NCAM. At postnatal day 2, the vast majority of the NCAM+ cells were also CD4+ and CD8+. At embryonic day 15, NCAM appeared also to be expressed by CD4- thymocytes since 14% of the cells were already NCAM+ whereas CD4 was virtually undetectable. In frozen section of the newborn thymus, surface staining for NCAM was present on a subpopulation of cells in the cortex, rare in the medulla and absent from the sub-capsular area. In conjunction with other cell adhesion molecules, NCAM could play a role in cell interactions during thymic development.     

Integrin α1β1 (VLA-1) mediates adhesion of activated intraepithelial lymphocytes to collagen

Intraepithelial lymphocytes (IELs) from human intestinal epithelium are memory CD8+ T cells that bind to epithelial cells through human mycosal lymphocyte (HML)-1 and to mesenchymal cells through very late activation antigen-4 (VLA-4). Their binding of extracellular matrix proteins and the mechanism involved were tested. Activated 51Cr-labelled lymphocytes were incubated in protein-coated microwells with various additives. After washing, the adherent cells were detected by radioactivity. The percentages of activated IELs that bound to collagen types I and IV were 20 and 31%, respectively; fewer bound to fibronectin or laminin. Compared to interleukin-2-activated peripheral blood CD8+ T lymphocytes, more IELs bound collagen IV and fewer bound fibronectin. IEL adhesion to collagen (but not fibronectin or laminin) was up-regulated by antibody ligation of CD2 or by protein kinase C stimulation by phorbol ester; staurosporine reduced binding, while herbimycin, phytohaemagglutinin and CD3 ligation had no effect. Antibody-blocking of integrin VLA-1 subunits alpha1 (CD49a) and beta1 (CD18) inhibited adhesion to collagen type I by 82+/-6% and to type IV by 94+/-1% (P<0.001), implicating VLA-1 as the main collagen receptor for IELs. Cell adhesion was dependent on extracellular divalent cations, a characteristic event of VLA-1 never before shown for IELs: manganese and magnesium ions supported binding in a dose-dependent manner; calcium ions inhibited their effectiveness. Therefore, IELs bind collagen through integrin alpha1beta1 after protein kinase C activation. Adhesion is modulated by divalent cations.     

Cross talk between αvβ3 and α4β1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand α4β1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between α4β1 and VCAM-1 in vivo. Here we show that the lymphocyte αβ3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte αvβ3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of α4β3 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This αvβ3-mediated enhanced migration of lymphocytes via α4β1 is likely to depend on the interaction of αvβ3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between αvβ3 integrin and α4β1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.     

Expression of the neural cell adhesion molecule (NCAM) during second- and third-molar development in the mouse

Distribution of the neural cell adhesion molecule (NCAM) during the development of the mandibular second- and third-molars of the mouse was studied by indirect immunofluorescence techniques. At the initial stage, NCAM was intensely expressed by the mesenchymal cells surrounding the dental lamina, and by the cap stage NCAM expression by the mesenchymal cells became restricted to the dental follicle. After that, in addition to the follicular mesenchyme, some cells in the basal part of the dental papilla showed NCAM-immunoreactivity for a while after the hard tissue formation had started. During root formation, the follicular cells lost NCAM first from the level of the cervical root and later from the coronal part, while an additional NCAM positive area appeared deep in the dental papilla. Even after the teeth had erupted, NCAM was expressed in the tissue surrounding the apical root and in the pulp core. During the initial and bud stages, the pattern of NCAM expression in the second and third molars was different from that in the first molar, where NCAM was found only after the late bud stage; while from the cap stage onward, it changed in the same sequence as in the first molar. The different pattern of NCAM expression implies that there is a difference in developmental events between the early stages of the first and the other two molars. On the other hand, the common sequence of NCAM expression in the tooth germs later than the cap stage suggests that NCAM plays an essential role in the formation of the basic structure of the teeth and periodontal tissues.     

Expression of neural cell adhesion molecule (NCAM) during the first molar development in the mouse

NCAM, the neural cell adhesion molecule, was immunolocalized in the mandibular first molar tooth germ of the mouse. NCAM was first detected in the tooth germ of the late bud stage, where only the cells in the outer part of the condensed mesenchyme (primitive dental follicle) exhibited faint immunoreactivity. The entire dental follicle was intensely immunostained for NCAM from cap stage to the stage when root formation started. During root formation, NCAM disappeared from the follicular tissue surrounding the cervical root as well as from the part covering the crown top. This loss of NCAM proceeded in the direction of the root apex, but even after the tooth had achieved functional occlusion, NCAM was still expressed by the mesenchymal cells adjacent to the root apex. On the other hand, NCAM was negative in the dental papilla until birth. After birth, NCAM-immunoreactivity appeared in the basal portion of the dental papilla, but this NCAM-positive area gradually diminished in width during the root elongation. Instead, another NCAM-positive zone appeared in the core of the pulp during root formation. Even in the tooth that had already erupted, the pulp core contained cells that were strongly positive for NCAM immunostaining. In addition to its expression in the above two mesenchymal cell lineages, NCAM was transiently expressed by epithelial components of the tooth germ, some of the cells of the dental lamina and the enamel organ. The results suggest that NCAM participates in several processes of tooth development.     

NCAM (neural cell adhesion molecules) expression in malignant mesotheliomas

Neural cell adhesion molecules (NCAM) are adhesion molecules expressed by neural and neuroendocrine tumors and a few biphasic tumors such as synovialosarcomas and breast phyllode tumors. To investigate NCAM expression in mesotheliomas, we studied 26 cases of epithelioid (n = 12), biphasic (n = 11), and sarcomatoid (n = 3) malignant mesotheliomas (MM), in comparison with normal mesothelium, and 50 primary non-small cell lung carcinomas (NSCLC) (25 adenocarcinomas [ADC] and 25 squamous cell carcinomas [SCC]), using electron microscopy as a gold standard for recognition of MM. NCAM reactivity using 123C3 antibody was compared with that of NE markers such as chromogranin A and synaptophysin. Although normal mesothelium remains negative, NCAM was expressed in 19 of 26 MM (73%) with a membranous staining on frozen or paraffin sections. In 6 of 12 epithelioid MM, the tumor cells expressed NCAM, whereas in 5 cases stromal fibroblasts showed a strong but focal staining. In 11 biphasic MM, 4 presented an NCAM reactivity of both epithelioid and spindle cell components, whereas in 7, only fusiform component was NCAM positive. Two of 3 sarcomatoid MM showed an NCAM expression. Chromogranin expression was never seen, whereas synaptophysin was noticed in 2 cases. No case of NSCLC showed membranous 123C3 staining, whereas 2 ADC weakly expressed synaptophysin. We conclude that NCAM expression in MM is reminiscent of its expression in mesoderm during fetal life and consistent with that reported in other biphasic tumors. These data show that NCAM expression occurs in 73% of MM, highly exceeding that observed in lung cancer.     

Gradient expression of neural cell adhesion molecule (NCAM) in the pontine migratory stream of fetal rats.

Immunohistochemical localization of a neural cell adhesion molecule (NCAM) was examined in the subpial pontine migratory stream of the fetal rat with a monoclonal antibody specific for the rat NCAM, MAb-AF11. Although the pontine cell strand showed weaker expression of NCAM than the parenchymal areas of the brainstem, within the cell strand, NCAM-immunoreactivity was somewhat greater in the anterior part of the stream near the neurons' destination than the caudal part. The weak NCAM gradient in the pontine migratory stream may contribute to formation of the pontine cell strand and the basal pontine gray.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

Integrins αvβ3 and α5β1 Mediate Attachment of Lyme Disease Spirochetes to Human Cells

Borrelia burgdorferi (sensu lato), the agent of Lyme disease, is able to cause chronic, multisystemic infections in human and animal hosts. Attachment of the spirochete to host cells is likely to be important for the colonization of diverse tissues. The platelet-specific integrin α_IIbβ₃ was previously identified as a receptor for all three species of Lyme disease spirochetes (B. burgdorferi sensu stricto, B. garinii, and B. afzelii). Here we show that B. burgdorferi also recognizes the widely expressed integrins α_vβ₃ and α₅β₁, known as the vitronectin and fibronectin receptors, respectively. Three representatives of each species of Lyme disease spirochete were tested for the ability to bind to purified α_vβ₃ and α₅β₁. All of the strains tested bound to at least one integrin. Binding to one integrin was not always predictive of binding to other integrins, and several different integrin preference profiles were identified. Attachment of the infectious B. burgdorferi strain N40 to purified α_vβ₃ and α₅β₁ was inhibited by RGD peptides and the appropriate receptor-specific antibodies. Binding to α_vβ₃ was also shown by using a transfected cell line that expresses this receptor but not α_IIbβ₃. Attachment of B. burgdorferi N40 to human erythroleukemia cells and to human saphenous vein endothelial cells was mediated by both α₅β₁ and α_vβ₃. Our results show that multiple integrins mediate attachment of Lyme disease spirochetes to host cells.     

CFTR is a negative regulator of γδ T cell IFN-γ production and antitumor immunity

CFTR, a chloride channel and ion channel regulator studied mostly in epithelial cells, has been reported to participate in immune regulation and likely affect the risk of cancer development. However, little is known about the effects of CFTR on the differentiation and function of γδ T cells. In this study, we observed that CFTR was functionally expressed on the cell surface of γδ T cells. Genetic deletion and pharmacological inhibition of CFTR both increased IFN-γ release by peripheral γδ T cells and potentiated the cytolytic activity of these cells against tumor cells both in vitro and in vivo. Interestingly, the molecular mechanisms underlying the regulation of γδ T cell IFN-γ production by CFTR were either TCR dependent or related to Ca²⁺ influx. CFTR was recruited to TCR immunological synapses and attenuated Lck-P38 MAPK-c-Jun signaling. In addition, CFTR was found to modulate TCR-induced Ca²⁺ influx and membrane potential (V_m)-induced Ca²⁺ influx and subsequently regulate the calcineurin-NFATc1 signaling pathway in γδ T cells. Thus, CFTR serves as a negative regulator of IFN-γ production in γδ T cells and the function of these cells in antitumor immunity. Our investigation suggests that modification of the CFTR activity of γδ T cells may be a potential immunotherapeutic strategy for cancer.     

Inverted Vδ1/Vδ2 ratio within the T cell receptor (TCR)-γδ T cell population in peripheral blood of heart transplant recipients

We investigated the levels of TCR-γδ T cells and their subpopulations Vδ1 and Vδ2 in the peripheral blood lymphocytes (PBL) of 28 heart transplant (HTx) patients. Patients (n = 10) receiving cyclosporin A (CsA) for treatment of a nephrotic syndrome (NS) and 10 healthy individuals served as controls. There was no difference in levels of TCR-γδ T cells between the different groups. However, an elevated proportion of Vδ1⁺γδ T cells was found in the PBL of HTx patients, especially when these cells were present in their graft-infiltrating lymphocyte (GIL) cultures. Vδ1⁺γδ T cells of HTx patients showed normal expression of CD45RO and lacked the activation markers CD25 and HLA-DR. After expanding in IL-2-containing medium, PBL cultures of HTx patients more often were dominated by Vδ1 cells than PBL cultures of controls, in which Vδ2 cells were predominantly grown. The aberrant composition of the TCR-γδ population in HTx patients was not a result of immunosuppressive medication, since the proportion Vδ1⁺γδ T cells was normal in the PBL of the NS patients receiving a similar dose of CsA. It is postulated that long-term antigenic stimulation by the graft, at low level, might be responsible for the altered composition of the γδ pool in the HTx patients. Since no donor HLA-specific γδ T cells have been detected, other ligands, such as heat shock proteins, may be involved.     

Localization of the L2 monoclonal antibody binding site on chicken neural cell adhesion molecule (NCAM) and evidence for its role in NCAM-mediated cell adhesion

Recent studies have described the localization of functional and structural domains on the neural cell adhesion molecule NCAM. In the present study we have extended these observations to examine the location of the carbohydrate epitope recognized by the L2 monoclonal antibody. This carbohydrate moiety is localized to the 65,000-dalton amino-terminal fragment of NCAM (previously designated Fr1), but is not present in the amino-terminal 25,000-dalton region of NCAM that contains the heparin-binding domain. We have also examined the role of this domain in NCAM-mediated cell adhesion, and have shown that incubation of an NCAM substratum with L2 monoclonal antibody inhibits cell attachment to this substratum. These data therefore suggest that the carbohydrate moiety recognized by L2 monoclonal antibody may be involved in the modulation of NCAM-mediated cell adhesion.