Loss Of CD44 Research Articles

CD44 increases the efficiency of distant metastasis of breast cancer.

Metastasis is the predominant cause of death from cancer yet we have few biomarkers to predict patients at increased risk of metastasis and are unable to effectively treat disseminated disease. Analysis of 448 primary breast tumors determined that expression of the hylauronan receptor CD44 associated with high grade (p = 0.046), ER- (p = 0.001) and PR-negative tumors (p = 0.029), and correlated with increased distant recurrence and reduced disease-free survival in patients with lymph-node positive or large tumors. To determine its functional role in distant metastasis, CD44 was knocked-down in MDA-MB-231 cells using two independent shRNA sequences. Loss of CD44 attenuated tumor cell adhesion to endothelial cells and reduced cell invasion but did not affect proliferation in vitro. To verify the importance of CD44 to post-intravasation events, tumor formation was assessed by quantitative in vivo imaging and post-mortem tissue analysis following an intra-cardiac injection of transfected cells. CD44 knock-down increased survival and decreased overall tumor burden at multiple sites, including the skeleton in vivo. We conclude that elevated CD44 expression on tumour cells within the systemic circulation increases the efficiency of post-intravasation events and distant metastasis in vivo, consistent with its association with increased distant recurrence and reduced disease-free survival in patients.

Loss of CD44 and SOX2 expression is correlated with a poor prognosis in esophageal adenocarcinoma patients.

It has been suggested that markers associated with cancer stem cells (CSC) may play a role in esophageal cancer. Our aim was to investigate the expression pattern of proposed CSC markers ALDH1, Axin2, BMI1, CD44, and SOX2 in esophageal adenocarcinoma (EAC) and to relate their expression to survival. In this study we included 94 EAC patients and examined the expression of the above-mentioned markers by using immunohistochemistry on tissue microarrays. Expression was scored as positive or negative or categorized as low or high in terms of an immunoreactivity score (IRS). Expression rates were related to clinicopathologic characteristics and overall and disease-free survival (DFS). In a multivariate analysis, negative expression of CD44 and of SOX2 were both significant prognostic factors for DFS [hazard ratio (HR), 1.73; 95 % confidence interval (CI), 1.00-2.96; P = 0.046 and HR, 2.06; 95 % CI 1.14-3.70 P = 0.016). When CD44 and SOX2 expression were analyzed together, negative SOX2 expression was an independent prognostic factor for DFS (HR, 1.91; 95 % CI 1.05-3.46; P = 0.034). Low IRS scores for ALDH1 or Axin2 were associated with a reduced median survival (12.8 vs. 28.7 and 12.1 vs. 25.5 months, respectively). However, these markers and BMI1 were not prognostic factors for survival. Loss of CD44 expression and loss of SOX2 expression are prognostic factors of poor survival in EAC patients. This suggests a role of these proteins in EAC that requires further investigation.

Extranodal Nasal NK/T-Cell Lymphoma: A Rare Oral Presentation and FASN, CD44 and GLUT-1 Expression

Extranodal natural killer (NK)/T-cell lymphoma is an aggressive malignant tumor with distinctive clinicopathological features, characterized by vascular invasion and destruction, prominent necrosis, cytotoxic lymphocyte phenotype and a strong association with Epstein-Barr virus. Here is reported an extranodal nasal NK/T-cell lymphoma case, involving the maxillary sinus, floor of the orbit, and interestingly extending to the oral cavity through the alveolar bone and buccal mucosa, preserving the palate, leading to a primary misdiagnosis of aggressive periodontal disease. Moreover, this work investigated for the first time the immunohistochemical expression of fatty acid synthase (FASN) and glucose transporter 1 (GLUT-1) proteins in this neoplasia. FASN showed strong cytoplasmatic expression in the neoplastic cells, whereas GLUT-1 and CD44 were negative. These findings suggest that the expression of FASN and the loss of CD44 might be involved in the pathogenesis of the extranodal nasal NK/T-cell lymphoma, and that GLUT-1 may not participate in the survival adaptation of the tumor cells to the hypoxic environment. Further studies with larger series are required to confirm these initial results.

The Hyaluronic Acid Receptor CD44 Coordinates Normal and Metaplastic Gastric Epithelial Progenitor Cell Proliferation

The stem cell in the isthmus of gastric units continually replenishes the epithelium. Atrophy of acid-secreting parietal cells (PCs) frequently occurs during infection with Helicobacter pylori, predisposing patients to cancer. Atrophy causes increased proliferation of stem cells, yet little is known about how this process is regulated. Here we show that CD44 labels a population of small, undifferentiated cells in the gastric unit isthmus where stem cells are known to reside. Loss of CD44 in vivo results in decreased proliferation of the gastric epithelium. When we induce PC atrophy by Helicobacter infection or tamoxifen treatment, this CD44(+) population expands from the isthmus toward the base of the unit. CD44 blockade during PC atrophy abrogates the expansion. We find that CD44 binds STAT3, and inhibition of either CD44 or STAT3 signaling causes decreased proliferation. Atrophy-induced CD44 expansion depends on pERK, which labels isthmal cells in mice and humans. Our studies delineate an in vivo signaling pathway, ERK → CD44 → STAT3, that regulates normal and atrophy-induced gastric stem/progenitor-cell proliferation. We further show that we can intervene pharmacologically at each signaling step in vivo to modulate proliferation.

CD44 regulates vascular endothelial barrier integrity via a PECAM-1 dependent mechanism

Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan's Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.

Loss of CD44 attenuates aberrant GM-CSF signaling in Kras G12D hematopoietic progenitor/precursor cells and prolongs the survival of diseased animals

Loss of CD44 attenuates aberrant GM-CSF signaling in Kras G12D hematopoietic progenitor/precursor cells and prolongs the survival of diseased animals

Abstract 27: Chemotherapy-induced Akt survival signaling is regulated by CD44- Ezrin/Radaxin Moesin (ERM) scaffolding, dependent on EGFR activity

Abstract Akt survival signaling has emerged as a predictor of poor prognosis in tumors resistant to chemotherapy. Despite the firm correlation between enhanced Akt survival signaling in resistant tumor tissue, there is a poor understanding of the exact mechanisms which elicit this robust pathway activation. In the present study we sought to uncover the full pathway of Akt activation in response to chemotherapy in order to fully elucidate mechanisms and proteins involved in chemotherapy resistance. Using breast and ovarian cancer cells, immunoprecipitations revealed that the CD44 glycoprotein initiates a unique scaffolding effect with the Ezrin/Radixin/Moesin (ERM) proteins and Akt in response to the anti-tubulin chemotherapies, docetaxel and vincrisine. Knockdown of CD44 using siRNA revealed that the activation of Akt(ser473) and ERM(tyr353) by chemotherapy are abolished in a time and dose dependent manner. Flow cytometry, caspase activation and cell viability assays confirmed that loss of Akt signaling sensitizes cancer cells to chemotherapy-induced apoptosis and a similar sensitivity was observed in the absence of CD44 expression. Furthermore, immunoprecipitations revealed that inhibition of EGFR using the small molecule inhibitor, Erlotinib, prevented CD44/ERM/Akt scaffolding in response to chemotherapy. In summary, CD44 and ERM scaffolding is responsible for the activation of Akt survival signaling induced by chemotherapy, in-vitro. Loss of CD44 sensitizes cancer cells to apoptosis. This scaffolding effect is dependent on the activity of EGFR, suggesting that a unique interaction exists between this ErbB1 receptor tyrosine kinase and the CD44 glyco-protein. This is the first evidence for cell surface events which precipitate survival signaling in cancer cells as a consequence of chemotherapy. These findings elucidate novel biomarker interactions for translation in the clinical setting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 27. doi:1538-7445.AM2012-27

Role of CD44 in Endogenous Kras G12D-mediated Myeloproliferative Neoplasm

Abstract 1398Oncogenic KRAS mutations are identified in virtually all the cancer types, making KRAS one of the most frequently mutated genes in human cancers. Our lab established a mouse model of myeloproliferative neoplasm (MPN) initiated by oncogenic Kras mutation (Kras G12D) expressed from its endogenous locus. Because CD44, a cell adhesion molecule and a cell signaling regulator, was found to involve in regulating leukemic stem cells in chronic myeloid leukemia (Krause, Lazarides et al. 2006) and acute myeloid leukemia (Jin, Hope et al. 2006), we have generated compound mice expressing oncogenic Kras but deficient for CD44 (Kras G12D; CD44−/−) to examine the role of CD44 in endogenous oncogenic Kras- mediated MPN.Our preliminary data show that loss of CD44 prolonged survival of compound Kras G12D; CD44−/− mice and greatly downregulated GM-CSF signaling in hematopoietic stem/progenitor cells. However, CD44 deficiency did not significantly affect the development of MPN, suggesting that GM-CSF signaling is not essential for Kras G12D-initiated MPN and CD44 deficiency might not affect other cytokine signaling. We are currently investigating this issue.Because Kras G12D hematopoietic stem cells (HSCs) are required to initiate MPN phenotypes, we then examined whether loss of CD44 would affect the behavior of Kras G12D HSCs. Our preliminary results demonstrate that CD44 deficiency did not significantly alter HSC frequencies in bone marrow and spleen, nor their cell cycle profile and apoptosis status. When Kras G12D; CD44−/− bone marrow cells were transplanted into lethally irradiated mice by retro-orbital injection, ∼19% of recipient mice developed MPN, similarly to recipient mice transplanted with same number of Kras G12D cells (∼14%). These MPN mice simultaneously developed acute T-cell lymphoblastic leukemia/lymphoma. Our results suggest that CD44 is dispensable for the homing and engraftment of Kras G12D HSCs. We are currently investigating whether CD44 function is essential for the homing and engraftment of leukemic initiating cells of MPN. Disclosures:No relevant conflicts of interest to declare.

Extracellular ATP induces CD44 shedding from macrophage‐like P388D1 cells via the P2X7 receptor

The P2X7 receptor (P2X7R) is a nucleotide receptor expressed predominantly on hemopoietic, bone, and epithelial cells. The P2X7R can be activated by extracellular ATP and induces the influx of calcium, releases cytokines, and participates in cell proliferation and apoptosis. CD44 is an adhesion molecule. The effects of CD44 include cell-cell and cell-matrix adhesion interactions, lymphocyte activation, and cell migration. Many studies have shown that P2X7R and CD44 play important roles in hematological malignancies, but no study exists regarding the relationship between P2X7R and CD44. In the present study, we characterized P388D1 cells for the surface expression of CD44 and analyzed ATP-induced shedding. The data showed that P388D1 cells express CD44. Incubation of P388D1 cells with ATP induced a rapid loss of CD44 from the P388D1 cell surface. In addition, using a receptor inhibitor and P2X7R short hairpin RNA, we showed that the loss of CD44 is mediated via the P2X7R. Finally, we demonstrated that activation of P2X7R by ATP induces CD44 shedding.

CD44 Promotes Epithelial Mammary Gland Development and Exhibits Altered Localization during Cancer Progression

The basal cell layer has emerged as a critical player in cancer progression, and understanding the molecular contribution of specific cell types is important in treatment and prevention. The adhesion receptor CD44, which mediates epithelial-stromal and cell-cell interactions, has been shown to both promote and suppress tumor progression. To better understand the normal function of CD44, we have investigated its role in mouse mammary gland development and its expression in human breast and prostate cancer. We have found that CD44 is expressed in the myoepithelium of the developing mammary gland and modulates ductal development of FVB/N mice. The loss of CD44 results in defective luminal-myoepithelial cell-cell adhesion and promotes the mixing of luminal and myoepithelial layers, disrupting epithelial bilayer organization, and CD44-null mice experience delayed ductal outgrowth and impaired terminal end bud formation. The myoepithelial expression of CD44 is also relevant to its expression in cancer, as CD44 is expressed in the basal cells of early-stage breast and prostate cancer but exhibits altered localization with increasing tumorigenicity and is strongly expressed by tumor epithelium.

C-Met recruits ICAM-1 as a coreceptor to compensate for the loss of CD44 inCd44null mice

CD44 isoforms act as coreceptors for the receptor tyrosine kinases c-Met and VEGFR-2. However, Cd44 knockout mice do not show overt phenotypes, in contrast to Met and Vegfr-2 knockout mice. We hypothesized that CD44 is being compensated for by another factor in Cd44 null mice. Using RNAi technology and blocking experiments with antibodies, peptides, and purified ectodomains, as well as overexpression studies, we identified intercellular adhesion molecule-1 (ICAM-1) as a new coreceptor for c-Met in CD44-negative tumor cells and in primary hepatocytes obtained from Cd44 null mice. Most strikingly, after partial hepatectomy, CD44v6-specific antibodies inhibited liver cell proliferation and c-Met activation in wild-type mice, whereas ICAM-1-specific antibodies interfered with liver cell proliferation and c-Met activation in Cd44 knockout mice. These data show that ICAM-1 compensates for CD44v6 as a coreceptor for c-Met in Cd44 null mice. Compensation of proteins by members of the same family has been widely proposed to explain the lack of phenotype of several knockout mice. Our experiments demonstrate the functional substitution of a protein by a heterologous one in a knockout mouse.

CD44 Regulates Tight-Junction Assembly and Barrier Function

Upon barrier disturbance, adult CD44 knockout (KO) mice show delayed recovery of epidermal barrier function. This correlates with the loss of apical polarization of lamellar body (LB) secretion. As tight junctions (TJs) are crucial for barrier function and regulate polarized targeting of vesicles, we hypothesized that CD44 regulates TJs and associated cell polarity complexes, which in turn contributes to altered skin barrier function in CD44 KO mice. We show a delay in embryonic barrier formation associated with a loss of apical LB localization in CD44 KO mice, which correlates with alterations in TJ proteins and Par3. Simultaneously, the activity of Rac1, a major regulator of TJ barrier function, was reduced. Importantly, normalization of barrier function at E18.5 coincided with the recovery of these proteins. Tape-stripping experiments revealed that the loss of CD44 also affected TJ proteins upon induced disturbance of the barrier in adult mice. In CD44 KO keratinocytes, cell polarization and TJ barrier function were impaired. An alteration of differentiation markers was also observed, but was less pronounced than alterations of TJ proteins. Taken together, the results reveal an important function for CD44 in the assembly and function of TJs, suggesting their involvement in the skin barrier phenotype of CD44 KO mice.

Induction of CD44 cleavage in articular chondrocytes

The hyaluronan receptor CD44 provides chondrocytes with a mechanism for sensing and responding to changes in the extracellular matrix. The purpose of this study was to document the fragmentation and loss of CD44 and to determine the likely mechanisms involved. A polyclonal anti-CD44 cytotail antibody was generated to detect CD44 fragmentation by Western blot analysis. Chondrocytes were isolated from human or bovine articular cartilage. Primary articular chondrocytes were treated with interleukin-1beta (IL-1beta), hyaluronan oligosaccharides, or phorbol myristate acetate or were passaged and subcultured in monolayer to induce dedifferentiation. Conditions that altered the capacity of CD44 to transit into lipid rafts, or pharmacologic inhibitors of metalloproteinase or gamma-secretase activity were used to define the mechanism of fragmentation of CD44. Chondrocytes from osteoarthritic cartilage exhibited CD44 fragmentation as low molecular mass bands, corresponding to the CD44-EXT and CD44-ICD bands. Following dedifferentiation of chondrocytes or treatment of primary chondrocytes with hyaluronan oligosaccharides, IL-1beta, or phorbol myristate acetate, CD44 fragmentation was enhanced. Subsequent culture of the dedifferentiated chondrocytes in 3-dimensional alginate beads rescued the chondrocyte phenotype and diminished the fragmentation of CD44. Fragmentation of CD44 in chondrocytes was blocked in the presence of the metalloproteinase inhibitor GM6001 and the gamma-secretase inhibitor DAPT. CD44 fragmentation, consistent with a signature pattern reported for sequential metalloproteinase/gamma-secretase cleavage of CD44, is a common metabolic feature of chondrocytes that have undergone dedifferentiation in vitro and osteoarthritic chondrocytes. Transit of CD44 into lipid rafts may be required for its fragmentation.

Abstract 1446: Effect of PF-03475952, a potent and neutralizing CD44 antibody, on cancer cell apoptosis in vitro and metastasis in two orthotopic models

Abstract Effect of PF-03475952, a potent and neutralizing CD44 antibody, on cancer cell apoptosis in vitro and metastasis in two orthotopic models Yuli Wang, Jean wang, Gary Li, Amy Jackson-Fisher, Brett Simmons, Eugenia Kraynov, Christy Ji, Baiyong Li, and David Pocalyko Pfizer Global Research & Development, San Diego, CA 92121 CD44 is a cell surface glycoprotein that has multiple functions. It can act as a receptor for ligands such as hyaluronic acid and osteopontin. Additionally, it can function as co-receptor to modulate the activation of growth factor receptors such as c-met & ERBB. CD44 can also influence cell signaling through interaction with components of the actin cytoskeleton such as the ERM protein. CD44 expression is increased in many types of human cancer. It is also a cell surface marker for cancer stem cells in breast cancer and HCC. In HCC, CD44 overexpression is a frequent event (∼30% based on several reports) and CD44 up regulation is associated with metastasis and poor patient survival. Down regulation of CD44 with RNAi knockdown or neutralizing antibody has been reported to reduce tumor metastasis, recurrence, and growth in different models. PF-03475952 is a fully human IgG2 anti-CD44 monoclonal antibody that was initially developed as a potential therapeutic agent for rheumatoid arthritis. It was shown previously that the PF-03475952 can disrupt the interaction of CD44 with its ligand, hyaluronic acid. The antibody binding induces loss of CD44 from the cell surface, and inhibits LPS-induced cytokine release from various leukocytes. In this report, we evaluated the potential of PF-03475952 as a cancer adjuvant therapy for HCC. HCC is the third most deadly forms of cancer world-wide with frequent early recurrence due to inter and extra hepatic metastasis. The effect of PF-03475952 on cancer metastasis was first demonstrated in an M24met melanoma model. The expression of CD44 and the effects of PF-03475952 on HCC cell lines were then characterized. In vitro, PF-03475952 was able to reduce cell adhesion and promote apoptosis in a CD44-dependent manner. More importantly, PF-03475952 can reduce lung metastasis in a CD44-positive HCC orthotopic model. The study suggested that the CD44 neutralizing antibody, PF-03475952, could be an efficacious agent as adjuvant therapy to prevent cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1446.

Y-Box Binding Protein-1 Induces the Expression of CD44 and CD49f Leading to Enhanced Self-Renewal, Mammosphere Growth, and Drug Resistance

Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

PF-03475952: a potent and neutralizing fully human anti-CD44 antibody for therapeutic applications in inflammatory diseases

CD44 is a cell adhesion molecule believed to play a critical role in T cell and monocyte infiltration in the inflammatory process. The reduction of CD44 expression or its ability to properly interact with its key ligand, hyaluronic acid (HA), inhibits migration and subsequent activation of cells within sites of inflammation. CD44-deficient mice exhibit decreased disease in a mouse arthritis model. Accordingly, we developed PF-03475952, a fully human IgG2 anti-CD44 monoclonal antibody (mAb). Binding of PF-03475952 to CD44 inhibits binding of HA and induces loss of CD44 from the cell surface. PF-03475952 also passed a series of safety pharmacology assays designed to assess the risk of the mAb to bind Fc gamma receptors, stimulate cytokine release from human whole blood, and stimulate cytokine release from peripheral blood mononuclear cells (PBMC) using plate-bound antibodies. The latter assay was designed specifically to evaluate the risk of cytokine storm that had been observed with TGN1412 (immunostimulatory CD28 superagonist mAb). PF-003475952 exhibits high-affinity binding to both human and cynomolgus monkey CD44, but does not cross-react with rodent CD44. Thus, a rat anti-mouse CD44 mAb was used to demonstrate a dose-dependent decrease of disease in mouse collagen-induced arthritis. Importantly, efficacy was correlated with >50% loss of cell surface CD44 on circulating cells. Loss of CD44 expression on CD3+ lymphocytes was monitored following a single dose of PF-03475952 in cynomolgus monkeys as a pharmacodynamic marker. The recovery of CD44 expression was found to be dose-dependent. PF-03475952 doses of 1, 10, and 100 mg/kg reduced CD44 expression below 50% for 218, 373, and >504 hours, respectively. Targeting of CD44 is a unique mechanism of action in the treatment of inflammatory diseases and is expected to reduce joint damage induced by inflammatory mediators, resulting in disease modification in inflammatory diseases such as rheumatoid arthritis.

Altered expression of CD44 and DKK1 in the progression of Barrett’s esophagus to esophageal adenocarcinoma

Barrett's esophagus (BE) is an acquired condition in which the normal lining of the esophagus is replaced by intestinal metaplastic epithelium. BE can evolve to esophageal adenocarcinoma (EAC) through low-grade dysplasia (LGD) and high-grade dysplasia (HGD). The only generally accepted marker for increased risk of EAC is the presence of HGD, diagnosed on endoscopic biopsies. More specific markers for the prediction of EAC risk are needed. A tissue microarray was constructed comprising tissue samples from BE, LGD, HGD, and EAC. Marker expression was studied by immunohistochemistry using antibodies against CD44, DKK1, CDX2, COX2, SOX9, OCT1, E-cadherin, and beta-catenin. Immunostaining was evaluated semi-quantitatively. CD44 expression decreased in HGD and EAC relative to BE and LGD. DKK1 expression increased in HGD and EAC relative to BE and LDG. CDX2 expression increased in HGD but decreased in EAC. COX2 expression decreased in EAC, and SOX9 expression increased only in the upper crypt epithelial cells in HGD. E-cadherin expression decreased in EAC. Nuclear beta-catenin was not significantly different between BE, LGD, and HGD. Loss of CD44 and gain of DKK1 expression characterizes progression from BE and LGD to HGD and EAC, and their altered expression might indicate an increased risk for developing an EAC. This observation warrants inclusion of these immunohistochemically detectable markers in a study with a long patient follow-up.

Peripheral T lymphocyte depletion by apoptosis after CD4 ligation in vivo: selective loss of CD44- and 'activating' memory T cells.

We have demonstrated that a single intravenous bolus of rat anti-CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive images analysis system and shown that the increase in apoptosis was sufficient to account for the observed depletion of the peripheral CD4+ T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or necrotic response in the tissues, indicating that this was unlikely to be Fc or complement-mediated antibody killing. The anti-CD4-induced depletion selectively removed CD44- T cells. Using mice previously immunized with yeast-derived HIV-1 p24 recombinant protein there was sparing of memory T cell function after in vivo anti-CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti-CD4 antibody resulted in the depletion of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4+ T cell depletion may be explained by loss of memory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HIV-associated disease which is associated with ligation of CD4 molecules in vivo.

Progression of intracranial glioma disrupts thymic homeostasis and induces T-cell apoptosis in vivo.

The thymus is the site where all T-cell precursors develop, mature, and subsequently leave as mature T-cells. Since the mechanisms that mediate and regulate thymic apoptosis are not fully understood, we utilized a syngenic GL261 murine glioma model to further elucidate the fate of T-cells in tumor bearing C57BL/6 mice. First, we found a dramatic reduction in the size of the thymus accompanied by a decrease in thymic cellularity in response to glioma growth in the brains of affected mice. There was a marked reduction of double positive subset and an increase in the frequency of CD4(+) and CD8(+) single positive T-cell subsets. Analysis of double negative thymocytes showed an increase in the accumulation of CD44(+) cells. In contrast, there was a marked loss of CD44 and CD122 expression in CD4(+) and CD8(+) subsets. The growth of intracranial tumors was also associated with decreased levels of HO-1, a mediator of anti-apoptotic function, and increased levels of Notch-1 and its ligand, Jagged-1. To determine whether thymic atrophy could be due to the effect of Notch and its ligand expression by glioma in vivo, we performed a bone marrow transplant experiment. Our results suggest that Notch-1 and its ligand Jagged-1 can induce apoptosis of thymocytes, thereby influencing thymic development, immune system homeostasis, and function of the immune cells in a model of experimental glioma.

A multivariate analysis of adhesion molecules expression in assessment of colorectal cancer

Adhesion molecules are implicated in the progression of colorectal cancer (CRC). Despite the evidence of association between their expression and patients' prognosis, the data have not been examined simultaneously in a same study; thus, the relative clinical value remained largely unknown. The aim of this study was to identify the adhesion factors that display the most significant prognostic value for CRC patients to guide clinical decision-making regarding appropriate treatment. We examined by immunohistochemistry, the expression of E-cadherin and its associated catenins, alpha(alpha)-catenin and beta(beta)-catenin, DCC, and CD44 and its partner, MT1-MMP in a series of 140 CRC tissues at intermediate Stage II and Stage III to determine their prognostic significance. Clinicopathological survey indicated an inverse relationship between E-cadherin expression and tumor differentiation, and an association between CD44 expression and venous invasion. Univariate and multivariate analyses showed that loss of expression of E-cadherin and CD44 significantly correlated to poor survival, especially in Stage II. Combination studies indicated that loss of E-cadherin and loss of CD44 had the worst impact on patient prognosis, particularly in colon cancer. Immunohistochemical staining of E-cadherin and CD44 may help to identify a subgroup of high-risk patients with Stage II CRC, especially in colon cancer, who may need intensive follow-up and appropriate therapeutic strategy.