CD44 Splice Research Articles

CD44-independent hepatocyte growth factor/c-Met autocrine loop promotes malignant peripheral nerve sheath tumor cell invasion in vitro.

Malignant peripheral nerve sheath tumors (MPNSTs) are invasive peripheral nerve neoplasms that express both the receptor tyrosine kinase c-Met and its ligand hepatocyte growth factor (HGF). The combined expression of these proteins has been implicated in tumor cell growth and metastasis. However, HGF/c-Met autocrine activity requires the presence of a serine protease, the HGF activator (HGFA), and, in some cells, the CD44 transmembrane glycoprotein. Here, we found that HGFA, HGF, c-Met, and CD44 are coexpressed in MPNSTs but their localization did not correlate with increased cell proliferation. The ST8814 MPNST cell line also expresses all of these proteins, can convert pro-HGF to active HGF, and exhibits constitutive c-Met phosphorylation. Blocking c-Met activity or expression inhibits the invasive behavior of these cells but not their proliferation. Interestingly, although a CD44 splice variant contributes to MPNST cell invasion and interacts with c-Met and HGF in ST8814 cells, it is not required for c-Met activation. These data indicate that an HGF/c-Met autocrine loop can promote MPNST invasion through a CD44-independent mechanism and suggest that c-Met, HGFA, and HGF are potential molecular targets to inhibit MPNST metastasis.

Coprocytobiology: on the nature of cellular elements from stools in the pathophysiology of colonic disease.

The gastrointestinal epithelium is known to undergo constant and rapid renewal resulting in millions of cells being shed into the fecal stream every day. The conventional wisdom was that these cells disintegrate upon exfoliation and will not survive the transit through the intestinal tract. In 1990, we (P.N.) made the discovery that a significant number of these cells remain intact and viable and that they can be isolated. The implications of this important discovery became apparent when we demonstrated that these cells are exclusively of colonic origin, are anatomically representative of the entire colon, and can be used for clinical investigations of disease processes. The term coprocytobiology (CCB) was coined to encompass the broad range of applications of this new technology. The somatic cell sampling and recovery (SCSR) process involves the isolation of exfoliated colonocytes from a small sample of stool ( approximately 1 g) collected and transported in a unique medium at ambient temperature, providing cells for the detection of a number of biomarkers of disease propensity. These exfoliated colonocytes express cytokeratins indicating epithelial lineage as well as colon-specific antigen. Over the years, the study of exfoliated colonocytes has provided striking new insights into the biology of colon cancer and inflammatory bowel disease, including detection of p53 gene mutations, reverse transcriptase polymerase chain reaction amplification, and identification of CD44 splice variants, neoplasia-associated specific binding of plant lectins, and expression of COX-2, the inducible form of cyclooxygenase. The functional diversity of cells isolated by SCSR is revealed by the demonstration of cell surface markers such as secretory component, IgA, and IgG on the one hand and the amplification and cloning of the human insulin receptor and the expression of the multidrug resistance gene mdr-1 on the other hand. This review portrays the immense potential of CCB as a powerful tool for investigating the pathophysiology of disease, identifying genetic variants in pharmacogenetics, assessment of mucosal immunity, and several other applications that use somatic cells.

Regulation of proximal tubular epithelial cell CD44-mediated binding and internalisation of hyaluronan

Background: Increased expression of the connective tissue polysaccharide hyaluronan (HA) in the renal corticointerstitium is associated with progressive renal fibrosis. Numerous studies have demonstrated involvement proximal tubular epithelial cells in the fibrotic process and in the current study we have characterised their expression of the HA receptor, CD44, and examined changes in CD44 expression and function in response to either IL-1β or glucose. Methods: Characterisation of CD44 splice variant expression was carried out in primary cultures of human proximal tubular cells (PTC) and HK2 cells. Binding and internalisation HA was examined by addition of exogenous of fluorescein-HA (fl-HA), and expression of CD44 examined by immunoblot analysis and flow cytometry. Alteration in “functional” CD44 was determined by immunoprecipitation of CD44 following stimulation in the presence of fl-HA. Results: PTC, both primary culture and the PTC cell line, HK2, express at least 5 CD44 splice variants, the expression of which are not altered by addition of either IL-1β or 25 mM d-glucose. Addition of either stimulus increased cell surface binding and internalisation of fl-HA and increased expression of functionally active CD44. Increased binding and internalisation of fl-HA, was blocked by anti-CD44 antibody, and by the inhibition of O-glycosylation. Conclusions: The data demonstrate that stimuli inducing PTC HA synthesis also regulate PTC–HA interactions. Furthermore increased HA binding and internalisation is the result of post-translational modification of CD44 by O-glycosylation, rather than by alteration in expression of CD44 at the cell surface, or by alternate use of CD44 splice variants.

Influence of epitopes CD44v3 and CD44v6 in the invasive behavior of fibroblast-like synoviocytes derived from rheumatoid arthritic joints.

To investigate the functional implications of CD44 splice variant expression in fibroblast-like synoviocytes (FLS) obtained from patients with rheumatoid arthritis (RA). FLS were isolated from synovial tissue obtained from both diseased and nondiseased joints. The expression of splice variants containing exons v3 and v6 was analyzed using immunocytochemistry with exon-specific antibodies and reverse transcription-polymerase chain reaction followed by Southern blotting. The invasive capacity of the cells was studied in a transwell invasion assay. FLS obtained from RA joints expressed various CD44 splicing combinations containing the variant exons v3 and/or v6. These cells were highly invasive, whereas cells from normal tissues, which lacked expression of CD44 splice variants, were not. Variant exons CD44v3 and CD44v6 were instrumental in matrix invasion in vitro, with cells enriched for CD44v3 and v6 exhibiting greater invasion and antibodies that specifically recognize CD44v3 and v6 abrogating this capacity to invade. Invasive cells showed a reduced expression of CD44v7/8, and antibodies against this epitope had no significant effect on cellular infiltration of the matrix. The antibodies had no effect on cell migration into the porous section of the transwell. FLS obtained from patients with RA express CD44 splice variants and are highly invasive, whereas cells obtained from healthy tissue do not express these variants and are not invasive. Expression of the epitopes CD44v3 and CD44v6 is instrumental in the invasive capacity but not in cell migration. This finding highlights a functional implication for the expression of CD44 splice variants at the level of matrix degradation.

Clinicopathologic significance of expression of CD44s and CD44v6 isoforms in squamous cell carcinoma of the supraglottic larynx.

CD44 splice variants are assumed to have a critical role in the malignant progression of many human tumors. However, the clinical significance of CD44 expression is not yet understood. The aim of this study was to investigate the prognostic significance of expression of CD44s and CD44v6 isoforms in squamous cell carcinomas of the supraglottic larynx. CD44s and CD44v6 expression was determined by immunohistochemical analysis of paraffin-embedded tissue specimens from 101 patients. There was a significant correlation between decreased CD44s or CD44v6 expression and a poorer histologic differentiation. No relationship was observed with T stage or nodal metastasis. Decreased CD44s expression, but not CD44v6 expression, correlated with increased recurrence rates. There was no correlation between the decreased expression of any isoform tested and survival. These data confirm a reduction of CD44s and CD44v6 expression in poorly differentiated tumors. However, these changes do not offer a useful adjunct to current prognostic indicators.

Analysis of the Gene Expression Profile Activated by the CC Chemokine Ligand 5/RANTES and by Lipopolysaccharide in Human Monocytes

The gene expression profile induced by the CC chemokine ligand (CCL) 5/RANTES in human monocytes was examined using the oligonucleotide array technology. Of 5600 transcripts examined, 42 were consistently induced by CCL5, and none were suppressed. Chemokine-inducible transcripts could be clustered in functional groups, including selected cytokines and receptors (e.g., IL-1beta, CCL2/monocyte chemotactic protein-1, and the CCL5 receptor CCR1) and molecules involved in extracellular matrix recognition and digestion (e.g., CD44 splice transcripts, urokinase-type plasminogen activator receptor, matrix metalloprotease (MMP)-9, and MMP-19). Transcript expression, confirmed by quantitative real-time PCR analysis for selected genes, was associated with protein induction for some (e.g., CCL2), but not all (e.g., IL-1beta), transcripts examined. The chemokine-induced gene profile was distinct from that activated by LPS, a prototypic phagocyte activator. Although certain transcripts were stimulated by both agonists (e.g., IL-1beta and CCL2), others were induced only by either LPS (e.g., TNF-alpha and IL-6) or CCL5 (e.g., MMP-19) or were divergently regulated (e.g., CCR1). Thus, CCL5, a prototypic CC inflammatory chemokine, activates a restricted transcriptional program in monocytes distinct from that induced by the prototypic pathogen-derived proinflammatory stimulant LPS. Chemokine-induced chemokines production could represent a novel amplification loop of leukocyte recruitment, while a subset of chemokine-inducible transcripts could be involved in monocyte extravasation and tissue invasion.

OVER EXPRESSION OF CD44V8-10 IN URINARY EXFOLIATED CELLS AS AN INDEPENDENT PROGNOSTIC PREDICTOR IN PATIENTS WITH UROTHELIAL CANCER

CD44 is a widely expressed cell surface adhesion molecule, of which various isoforms arise from alternative RNA splicing mechanisms. Over expression of specific CD44 splice variants, namely CD44v8-10, is evident in various malignant tumors and is considered to be associated with disease progression. In this study, we investigated whether the transcriptional level of CD44v8-10 relative to that of the standard CD44 isoform would predict the extent and prognosis of urothelial cancer. The CD44v8-10- to -standard CD44 ratio was measured in the tissue (40 urothelial cancer specimens and corresponding normal urinary tissue) and spontaneously voided urine samples of 150 patients with urothelial cancer and 50 with benign urological disease by reverse transcriptase-polymerase chain reaction using the set of primers capable of amplifying all CD44 splice variant isoforms. Initially any CD44 variant isoforms were barely detectable in normal urinary tissues, whereas CD44v8-10 was predominantly expressed in most urothelial cancer specimens. Furthermore, the CD44v8-10- to -standard CD44 ratio in urothelial cancer was closely associated with tumor progression. We then compared the ratio in urothelial cancer tissue and urinary exfoliated cells, and noted a linear and significant correlation of these 2 values in the same patients. Therefore, we investigated whether the CD44v8-10- to -standard CD44 ratio in urinary exfoliated cells would predict the prognosis and disease progression. The mean ratio in the urinary exfoliated cells of patients with invasive urothelial cancer was significantly higher than in those with superficial urothelial cancer. Of the patients with superficial bladder cancer disease-free survival rate of those with an elevated versus a normal ratio was significantly lower. Moreover, of the patients with advanced urothelial carcinoma who underwent complete resection disease-free survival of those with an elevated CD44v8-10- to -standard CD44 ratio was significantly lower than that of patients with a normal ratio. These results indicate that CD44v8-10 is strongly expressed in tumor tissue and evident at high levels in urinary exfoliated cells of patients with invasive versus superficial urothelial cancer. An elevated CD44v8-10- to -standard CD44 ratio in urinary exfoliated cells may serve as a novel prognostic predictor and indicator of disease extent in patients with urothelial cancer.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

Expression of CD44s and CD44 splice variants in human melanoma

The ability of tumor cells to adhere and detach from extracellular matrix and endothelial cells, is a crucial step in the metastatic process and may alter the clinical prognosis of some human tumors such as melanomas. CD44, the major cell surface receptor for hyaluronate, has been implicated in cell adhesion and in tumor progression. We studied the expression of standard CD44 molecule (CD44s) and its variants v3 and v6 in 57 human primary melanoma biopsies, without previous treatment. We analyzed the association between CD44 expression and the principal clinicopathological features, including survival. Fifty-six of 57 tumors expressed CD44s, associated to the cytoplasmic membrane. No expression of CD44v3 or CD44v6 was detected. No association between CD44s expression and prognostic factors such as tumor thickness, growth type, stage or anatomic site of the lesion was found. However, a positive correlation between CD44s expression and Clark level (Spearman, p<0.001) was found. While only 33.3% of melanomas Clark I + II showed high expression of CD44s (more than 50% of positive cells), 82.6% of melanomas Clark IV + V did so. Kaplan-Meier analysis revelead that patients whose melanomas had high expression of CD44s showed a reduced relapse free survival (RFS) rate, though without statistical significance. No difference between the level of CD44 expression and overall survival (OS) was found. We conclude that melanomas only expressed CD44s, and that its level was associated with Clark's stage. CD44s seems not to be useful as a tumor marker, because it does not predict either RFS or OS.

The influence of hormones on CD44 expression in endometrial and breast carcinomas

The expression of distinct variant isoforms of the cell surface glycoprotein CD44 (CD44v) has been found to be associated with metastatic potential of rodent adenocarcinoma cells and with an altered prognosis in several types of human cancer. In hormone-dependent gynecological cancers, different CD44v expression patterns have been observed. The influence of ovarian steroid hormones and their antagonists on CD44v expression is still unclear, since there are only retrospective correlation studies so far. Therefore, we examined the CD44 mRNA expression in a standardized stimulation experiment in a number of breast and endometrial carcinoma cell lines varying in estrogen receptor (ER) status. Higher CD44 overall expression was observed in ER positive endometrial and breast carcinoma cell lines when compared to corresponding ER negative cell lines. The number and composition of alternatively spliced isoforms showed no clear correlation to the ER expression status. Three CD44v isoforms were detected in all cell lines expressing CD44v, two of which have not been reported previously in normal endometrial cells. These isoforms may have specific functions in this type of carcinoma. In the second part of the study, the influence of (anti-) hormones on CD44 expression in endometrial carcinoma cell lines was examined. CD44 overall expression showed an increase when the cells were grown in medium containing fetal calf serum (FCS) as compared to cells maintained in medium-free of FCS. CD44 expression was transiently increased by estradiol (1 h). The CD44 splice pattern of endometrial cancer cell lines RL95-2 and Hec-1-A, after treatment with (anti-) hormones showed constant and high expression rates for distinct CD44v-isoforms such as CD44E (CD44v8-v10). Only certain weakly expressed isoforms changed their expression level during the experimental period, but no direct correlation to hormone treatment was observed. In conclusion, estradiol or FCS increase CD44 overall expression, but there seems to be no direct influence of ovarian steroid hormones on the CD44v splice machinery in endometrial carcinoma cell lines.

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.

Expression of CD44 splice variants in spontaneous murine tumors.

CD44 is a widely distributed set of cell surface glycoproteins expressed in several types of cells and tissues, implicated in cell-cell and cell-substrate interactions. This molecule plays a major role in cell differentiation, development and activation and has also been described as a potential marker of malignancy and metastasis. In the present study we investigated by RT-PCR followed by exon specific amplification the expression of CD44 splice variants in four different murine tumors as well as in the invaded organs in order to correlate the expression of CD44 variants with potential tumor invasiveness and their implications for growth. Our data showed deregulation in the expression of CD44 isoforms but no discernible correlation in isoform expression pattern. However, in all tumors studied isoforms presented by the primary tumor were detected in the invaded organs before metastasis could be demonstrated by histopathological analysis.

The CD44v7/8 Epitope as a Target to Restrain Proliferation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

CD44 is a receptor for the glycosaminoglycan hyaluronan. It exists in a large range of isoforms because of variability in the pattern of glycosylation (both N- and O-linked) and of multiple splice variants. Human fibroblast-like synoviocytes derived from patients with rheumatoid arthritis express certain CD44 splice variants and we have investigated the functional implications of their expression. We found that the rate of proliferation of fibroblast-like synoviocytes expressing the CD44v7/8 epitope (average doubling time 55 hours) exceeds those obtained from the same synovial specimen but lacking this particular epitope (69 hours). Antibodies against CD44v7/8, but not against other exons, inhibit cell proliferation with concomitant induction of the cell cycle inhibitors GADD45, GADD153 and the cyclin-dependent protein-kinase inhibitors p21Waf/Cip. These data show that expression of CD44v7/8 contributes to the transformed phenotype of fibroblast-like synoviocytes. More importantly, they reveal the presence of a target that might be amenable to pharmacological intervention in the treatment of rheumatoid arthritis.

Soluble CD44 splice variants and pelvic lymph node metastasis in ovarian cancer patients.

Alternative splicing of CD44 and aberrant levels of soluble CD44 protein in the serum of cancer patients has been correlated to tumor progression and metastasis. To examine the clinical value of CD44 serum levels (sCD44) in ovarian cancer we determined concentrations of the soluble, variable isoforms sCD44std, sCD44v5 and sCD44v6 with a sensitive ELISA. Pre-operative serum samples from 66 patients with histologically diagnosed invasive disease as well as sera taken from 40 healthy blood donors were analyzed. In sera of ovarian cancer patients we detected elevated concentrations of overall CD44 serum levels represented by sCD44std (p=0.001), but decreased levels of the specific isoforms CD44v5 (p=0.0002) and v6 (p=0.0001). This is the first report demonstrating that ovarian cancer patients with pelvic lymph node metastasis at the time of diagnosis showed specifically elevated sCD44v6 (p=0.073) serum concentrations in comparison to patients without lymph node involvement, whereas overall sCD44 serum levels did not differ. Decreased serum levels of sCD44v5 were found in progesterone receptor-positive tumors (p=0. 059) and postmenopausal patients (p=0.032). Increased concentrations of sCD44v6 were detectable in estrogen receptor-positive tumors but not significantly (p=0.138). Serum CD44v5 levels were associated with shortened relapse-free survival time. No association was found between serum CD44 isoforms and the classical clinicopathological parameters stage and grading or overall survival. CD44 splice variants are possibly involved in a complex interaction with the hormonal environment during tumorigenesis and metastasis of ovarian cancer.

Clear Cell Carcinoma of the Ovary: Characterization of Its CD44 Isoform Repertoire

Objective. CD44 is a cell surface receptor implicated in tumor metastases. We have previously shown that there is a loss of CD44 splice control in clear cell carcinoma (CCCa) of the ovary. Our aim is to characterize the expression of CD44-3v, -5v, -7v, and -10v in clear cell ovarian tumors and to determine their prognostic value.Methods. Twenty-two cases of ovarian CCCa were studied for CD44-3v, -5v, -7v, and -10v expression by immunocytochemistry.Results. The primary tumors showed expression of CD44-3v, -5v, -7v, and -10v in 44, 55, 61, and 39% of the cases, respectively. We were able to compare the expression of CD44 in the primary tumor and metastatic sites from the same patient in 7 cases (metastatic sites n = 16). We observed decreased immunoreactivity of CD44-3v, -5v, -7v, and -10v in 67, 100, 93, and 92% of the sites, respectively. CD44-3v and -10v expression was absent in 100% of the nonaffected contralateral ovaries while -7v and -10v were expressed in 1/11 (9%) of them. When CD44-10v was not expressed in the primary tumor, only 18% of the women recurred or died of disease; in contrast, of the cases where it was present, 71% of the women recurred or died of disease (P = 0.049).Conclusions. There is aberrant alternative mRNA splicing in the development of CCCa of the ovary when compared to the contralateral nonaffected ovary. The expression of CD44-10v correlates with survival. Larger series are needed to further understand the role of CD44 isoforms in ovarian cancer metastases.

CD44 isoform expression in synovial sarcoma correlates with epitheliogenesis but not prognosis.

We immunohistochemically determined the expression of CD44 standard and splice variant isoforms in a series of synovial sarcomas and sought correlations with histological subtype and clinical parameters including outcome. From 39 patients, a total of 56 formalin-fixed and paraffin-embedded tumour samples (including initial, recurrent and metastatic tumours) were used to immunohistochemically evaluate the expression of epitopes encoded by CD44s and the CD44 splice variants CD44v3-v10. Significance of proposed prognostic indicators was evaluated in relation to the survival, time to local recurrence and time to metastases using log-rank analysis. Sixty-four percent of synovial sarcomas expressed CD44s and 46% expressed CD44v3-v10 (var). Synovial sarcomas with epithelial or epithelioid areas preferentially expressed CD44s in these areas when compared with the spindle cell element. There were no correlations with clinical parameters or outcome. The expression of CD44 was not found to correlate with survival, local recurrence or metastatic ability. In synovial sarcoma, CD44 and variant isoform expression appears to be associated with the degree of epithelial differentiation. CD44 expression in synovial sarcoma shows interesting similarities to CD44 expression in embryological epitheliogenesis.

Limitations of CD44v6 amplification for the detection of tumour cells in the blood of colorectal cancer patients.

Based on the important role of CD44 splice variants in colorectal cancer progression and metastasis, we evaluated the use of CD44v6 expression to detect and assess the metastatic potential of colorectal tumour cells circulating in peripheral blood. A nested amplification was designed that allowed to detect 10–100 colon cancer cells. This assay was applied to blood samples from healthy donors. Strong signals were detected in all cases, indicating that it cannot be used to detect colorectal carcinoma cells in whole blood. We then included an enrichment step based on the use of an anti-epithelial cells monoclonal antibody (BerEP4) coupled to magnetic beads. The CD44v6 reverse transcription polymerase chain reaction (RT PCR) assay was performed on cDNA synthesized from blood samples treated with these beads. We analysed 18 samples from 12 patients with a gastrointestinal disease, and 36 samples from ten patients with a colorectal cancer. None of the patients used as negative controls were found to contain epithelial cells in their blood as determined by cytokeratin 19 RT-PCR. By contrast, CD44 transcripts containing exon v6 were detected in nine out of the 18 samples tested (50%). For the colorectal cancer patients, six out of the seven samples (85.7%) that were cytokeratin 19-positive were CD44v6-negative, whereas ten samples out of the 29 not containing epithelial cells were CD44v6-positive (34.5%). This is probably due to the persistence of CD8+ leucocytes in the enriched preparations, as determined by PCR analysis of the CD8 α-chain. We conclude that detection of CD44v6 transcripts using a sensitive nested RT-PCR assay has no potential value to detect and characterize colorectal cancer micrometastases from blood, even following an initial enrichment step. © 2000 Cancer Research Campaign

The expression and clinical significance of CD44v in human gastric cancers.

CD44 was originally implicated as a “homing” receptor directing the migration of recirculating lymphocytes[1]. CD44 expression has been confirmed not only in lymphocytes but also in a wide variety of epithelial tissues. It is considered as an important cell adhesion molecule for cell to cell interactions[2]. Molecular cloning and analysis of the genetic structure have revealed that the CD44 gene has at least 19 exons, of which 12 can be alternatively spliced to make up a wide variety of CD44 splice variants which have been found in various types of human malignancies and have been considered as a marker in tumor progression and metastasis[3]. We analysed the expression of aberrant CD44 transcripts with RT-PCR method in gastric carcinomas, and discussed the clinical significance of metastasis-related CD44v gene.

CD44 splice variant involvement in the chronic inflammatory disease of the spinal cord: HAM/TSP

Splice variants of CD44 molecule-harboring exon 10 (v6), often called v6 variants (v6v), are shown to confer tumor progressive, metastatic or invasive capacities. Furthermore, CD44 molecule on activated T-cells are shown to be required for infiltration of these cells into the inflammatory site and for accelerated immune response. Human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is caused by HTLV-I infection and characterized by spastic paraparesis and urinary disturbance with perivascular HTLV-I-infected and activated CD4+ T-cell infiltration. In order to explore the underlying mechanism causing the disease after HTLV-I infection, we analyzed CD44 variant expression on peripheral blood mononuclear cells (PBMC) and in the spinal cord specimens from patients with HAM/TSP, and compared them with those from other HTLV-I-infected individuals and controls. We found that v6v expression with special direct link of exons 10 (v6) and 14(v10) was highly expressed in PBMC from patients with HAM/TSP and that v6v and CD4 double positive T-cell infiltration into the spinal cord lesion of HAM/TSP. This combination of CD44 splice variant has not been previously reported in the study of chronic inflammatory disorders and may be a marker molecule for T-cells infiltrating into the central nervous system (CNS), especially the spinal cord.

Regulation of the cell adhesion molecule CD44 after nerve transection and direct trauma to the mouse brain

CD44 is a cell surface glycoprotein involved in cell adhesion during neurite outgrowth, leukocyte homing, and tumor metastasis. In the current study, we examined the regulation of this molecule 4 days after neural trauma in different forms of central and peripheral injury. Transection of the hypoglossal, vagus, or sciatic nerve led to the appearance of CD44-immunoreactivity (CD44-IR) on the surface of the affected motoneurons, their dendrites, and their axons. Fimbria fornix transection led to CD44-IR on a subpopulation of cholinergic neurons in the ipsi- and contralateral medial septum and diagonal band of Broca and colocalized with galanin-IR. Central projections of axotomized sensory neurons to the spinal cord (substantia gelatinosa, Clarke's column) also showed an increase in CD44-IR, which was abolished by spinal root transection. Nonneuronal CD44-IR was mainly restricted to sites of direct injury. In the crushed sciatic nerve, CD44-IR was found on the demyelinating Schwann cells and on infiltrating monocytes and granulocytes. Direct parasagittal transection of the cerebral cortex led to CD44-IR on resident astrocytes and on leukocytes entering the injured forebrain tissue. CD44-IR also increased on reactive retinal astrocytes and microglia after the optic nerve crush. Additional time points in the retina and hypoglossal nucleus (days 1, 2, and 14) and cerebral cortex (day 2) injury models also showed the same cell type pattern for the CD44-IR. Finally, polymerase chain reaction analysis confirmed the posttraumatic expression of CD44 mRNA and detected only the standard haematopoietic CD44 splice isoform both in direct and indirect brain injury models. Overall, the current study shows the widespread, graded appearance of CD44-IR on neurons and on nonneuronal cells, depending on the form of neural injury. Here, the ability of CD44 to bind to a variety of extracellular matrix and cell adhesion proteins and its common presence in different forms of brain pathology could suggest an important role for this cell surface glycoprotein in the neuronal, glial, and leukocyte response to trauma and in the repair of the damaged nervous system.