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The significance of CD44 in human pancreatic cancer: I. High expression of CD44 in human pancreatic adenocarcinoma.

The CD44 cell surface glycoprotein, which is the adhesion molecule of lymphocytes, has been suggested to be an important factor for the metastatic potential and invasive ability of cancer. We demonstrated the expression of CD44 in human pancreatic adenocarcinoma cells and normal cells by using flow cytometry (FACS-can) and immunohistological staining. CD44 was highly expressed in human pancreatic adenocarcinoma cells, while it was little expressed in normal human pancreas cells. These results indicate that the quantitative analysis of CD44 expression may be a useful diagnostic tool for pancreatic cancer.

Adenocarcinoma↗

Transient absence of CD44 expression and delay in development by anti-CD44 treatment during ontogeny: a surrogate of an inducible knockout?

Because the lack of some adhesion molecules induced by site-directed mutagenesis has been described to be lethal, whereas the lack of others apparently has no effect, we were interested in seeing whether the developing organism might gradually adapt to the absence of adhesion molecules. Therefore, we chose a form of transient interference by i.v. injection of antibody into pregnant rats. As a model, we selected CD44, which has been reported to play a key role during embryogenesis. Rats received either an antibody recognizing an epitope on the CD44 standard isoform (CD44s) or on exon v6 (CD44v6). In the presence of anti-CD44s, delivery was frequently delayed, and intrauterine abortions were often observed. The fetuses were smaller, particularly the anlage of the hair follicle of the whisker, and the formation of alveoli in the lung, of the tubular system of the kidney, and of villi in the gut was delayed. The development of fetuses receiving anti-CD44v6 was hampered until days 16-18 of gestation. Immunodetection revealed a weaker expression of the target molecules at the implantation site and the complete absence of CD44s and CD44v6 expression in fetal tissue until day 12. During the late stages of gestation, the expression pattern of CD44 resembled that of 2-3-day-younger fetuses of untreated rats. Interestingly, degradation of hyaluronate was also delayed, particularly in the kidney. Thus, the diaplacental antibody passage was very efficient and should make it possible to obtain a clearly defined and differentiated concept of the requirements for the CD44 molecule during ontogeny and also for fail-safe mechanisms. Both experiences may be missed in the knockout proper.

Animals↗

Progression in gastric carcinoma relative to the ratio of CD44 epithelial variant transcript to CD44 hematopoietic variant transcript.

BACKGROUND: It has been suggested that CD44 splice variants play a role in the progression of certain epithelial cancers and non-Hodgkin's lymphoma. In this study, we investigated the epithelial variant/hematopoietic variant (E/H) ratio (the amount of the CD44 epithelial variant transcript relative to the CD44 hematopoietic variant transcript) in human gastric carcinoma compared with normal gastric mucosa. METHODS: The ratio was determined for tumors and adjacent noncancerous mucosa from 30 gastric carcinoma patients using reverse transcription-polymerase chain reaction and Southern blotting. We also determined the tumor (T)E/H--noncancerous mucosa (N)E/H (the difference between E/H ratios of tumor tissue and adjacent noncancerous mucosa) and examined these measurements for correlations with the pathologic features of gastric carcinoma, as well as for their usefulness as an indicator of tumor progression. RESULTS: The E/H ratio in tumor tissue was significantly higher than in adjacent noncancerous mucosa (P < 0.01). The TE/H--NE/H in patients with lymph node metastases was 0.16 +/- 0.11, compared with 0.07 +/- 0.08 in cases without lymph node metastases (P < 0.05). Significant correlations also were observed between the TE/H--NE/H and the depth of invasion, blood vessel invasion, and lymphatic vessel invasion (P < 0.03, P < 0.03, and P < 0.01, respectively). CONCLUSIONS: Our results suggest that increases in the E/H ratio may be a useful indicator of progression in gastric carcinoma.

Adult↗

Induction of CD44 expression in stab wounds of the brain: long term persistence of CD44 expression.

We studied the expression of the hyaluronan receptor protein CD44 in the mouse brain in response to stab injuries. CD44 expression was strongly activated in the area surrounding the injury within 2 days and then persisted for over 2 months. The expression extended in a direct line the depth of the actual wound inflicted. It appears that CD44 may be involved in the wound healing processes following injury to the brain.

Animals↗

CD44 and melanocytic tumors: a possible role for standard CD44 in the epidermotropic spread of melanoma.

CD44 is a polymorphic family of cell membrane glycoproteins that mediate cell-matrix and cell-cell interactions involved in the mechanisms of tumor invasion and metastasis, and are subject to differential regulation during normal and malignant cell growth. We have investigated immunohistochemically the expression of CD44S and the variant isoforms CD44v3 and CD44v6 in paraffin-embedded tissue from 5 Spitz nevi, 3 compound melanocytic nevi, 2 blue nevi, 6 primary melanomas, 15 cutaneous metastases (three epidermotropic, nine dermal and three ulcerated) and 10 lymph node metastases of melanoma. Melanocytes were extensively positive for CD44S in primary melanomas and benign melanocytic proliferations. Among 15 cases of cutaneous metastases of melanoma, the three epidermotropic metastases, as well as one of the three ulcerated ones were positive for CD44S. CD44S expression was diminished or totally absent in six of the nine dermal metastases, in two of the ulcerated metastases and in seven of the ten lymph node metastases. CD44v3 and CD44v6 melanocytic expression was absent in all the lesions studied. According to our results, selective retention of CD44S expression by melanocytes in epidermotropic metastases of melanoma seems to indicate that preservation of CD44S may contribute to the intraepidermal spread of melanoma.

Antibodies, Monoclonal↗

Hyaluronan increases RANKL expression in bone marrow stromal cells through CD44.

UNLABELLED: HA activates CD44 to stimulate RANKL expression in bone marrow stromal cells. HA stimulation of RANKL is blocked by anti-CD44 antibody and is absent in cells from CD44(-/-) mice. CD44(-/-) mice exhibit thicker cortical bone and a smaller medullary cavity, but indices of bone resorption are not affected. INTRODUCTION: Hyaluronan (HA), the major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, functions in part through its receptor, CD44, to stimulate a series of intracellular signaling events that lead to cell migration, adhesion, and activation. To determine whether HA activation of CD44 influences RANKL and osteoprotegerin (OPG) expression and whether CD44 is functionally important in bone metabolism, we studied whole bone and bone marrow stromal cells (BMSCs) from wildtype and CD44(-/-) mice. MATERIALS AND METHODS: BMSCs from wildtype and CD44(-/-) mice at 7 weeks of age were cultured and treated with either HA or anti-CD44 antibody. The levels of mRNA of RANKL, OPG, CD44, alkaline phosphatase (ALP), osteocalcin (OC), and alphaI collagen (COLL) were determined by quantitative real-time RT-PCR. Levels of RANKL and CD44 protein were measured by immunoblotting, and expression of CD44 in whole bone was determined by immunohistochemical staining. Double immunofluorescence staining and confocal microscopy were used to study colocalization of Cbfa1, CD44, and HA. Tibias were imaged using muCT, and cancellous and cortical parameters were measured. Osteoblast and osteoclast surface in the distal femoral metaphysis and osteoclast on the endocortical surface at the tibio-fibular junction were measured using quantitative histomorphometry. Differences were analyzed using ANOVA and the Newman-Keuls test. RESULTS: Addition of HA dose-dependently increased RANKL mRNA (3.6-fold) and protein (3-fold) levels in BMSCs. Stimulation of RANKL by HA could be blocked with anti-CD44 antibody. Treatment of cells with HA or anti-CD44 antibody had no significant effect on OPG mRNA levels. Both CD44 and HA localized on the plasma membrane in cells expressing Cbfa1. HA localization on the cell membrane disappeared when cells were preincubated with anti-CD44 antibody. Compared with control mice, cortical bone of CD44(-/-) was thicker, and medullary area was smaller at both 7 and 17 weeks, but at 7 weeks, indices of bone resorption were normal. At 17 weeks of age, tibial mass of CD44(-/-) mice was higher than control mice. CD44(-/-) animals expressed less RANKL in whole bone (-30%) and in BMSCs (-50%). Cells from CD44(-/-) animals failed to respond to either HA or CD44 antibody treatment. CONCLUSIONS: HA can increase RANKL expression in BMSCs through CD44.

Animals↗

CD44: structure, function, and association with the malignant process.

CD44 is a ubiquitous multistructural and multifunctional cells surface adhesion molecule involved in cell-cell and cell-matrix interactions. Twenty exons are involved in the genomic organization of this molecule. The first five and the last 5 exons are constant, whereas the 10 exons located between these regions are subjected to alternative splicing, resulting in the generation of a variable region. Differential utilization of the 10 variable region exons, as well as variations in N-glycosylation, O-glycosylation, and glycosaminoglycanation (by heparan sulfate or chondroitin sulfate), generate multiple isoforms (at least 20 are known) of different molecular sizes (85-230 kDa). The smallest CD44 molecule (85-95 kDa), which lacks the entire variable region, is standard CD44 (CD44s). As it is expressed mainly on cells of lymphohematopoietic origin, CD44s is also known as hematopoietic CD44 (CD44H). CD44s is a single-chain molecule composed of a distal extracellular domain (containing, the ligand-binding sites), a membrane-proximal region, a transmembrane-spanning domain, and a cytoplasmic tail. The molecular sequence (with the exception of the membrane-proximal region) displays high interspecies homology. After immunological activation, T lymphocytes and other leukocytes transiently upregulate CD44 isoforms expressing variant exons (designated CD44v). A CD44 isform containing the last 3 exon products of the variable region (CD44V8-10, also known as epithelial CD44 or CD44E), is preferentially expressed on epithelial cells. The longest CD44 isoform expressing in tandem eight exons of the variable region (CD44V3-10) was detected in keratinocytes. Hyaluronic acid (HA), an important component of the extracellular matrix (ECM), is the principal, but by no means the only, ligand of CD44. Other CD44 ligands include the ECM components collagen, fibronectin, laminin, and chondroitin sulfate. Mucosal addressin, serglycin, osteopontin, and the class II invariant chain (Ii) are additional, ECM-unrelated, ligands of the molecule. In many, but not in all cases, CD44 does not bind HA unless it is stimulated by phorbol esters, activated by agonistic anti-CD44 antibody, or deglycosylated (e.g., by tunicamycin). CD44 is a multifunctional receptor involved in cell-cell and cell-ECM interactions, cell traffic, lymph node homing, presentation of chemokines and growth factors to traveling cells, and transmission of growth signals. CD44 also participates in the uptake and intracellular degradation of HA, as well as in transmission of signals mediating hematopoiesis and apoptosis. Many cancer cell types as well as their metastases express high levels of CD44. Whereas some tumors, such as gliomas, exclusively express standard CD44, other neoplasms, including gastrointestinal cancer, bladder cancer, uterine cervical cancer, breast cancer and non-Hodgkin's lymphomas, also express CD44 variants. Hence CD44, particularly its variants, may be used as diagnostic or prognostic markers of at least some human malignant diseases. Furthermore, it has been shown in animal models that injection of reagents interfering with CD44-ligand interaction (e.g., CD44s- or CD44v-specific antibodies) inhibit local tumor growth and metastatic spread. These findings suggest that CD44 may confer a growth advantage on some neoplastic cells and, therefore, could be used as a target for cancer therapy. It is hoped that identification of CD44 variants expressed on cancer but not on normal cells will lead to the development of anti-CD44 reagents restricted to the neoplastic growth.

Alternative Splicing↗

A developmental pathway involving four phenotypically and functionally distinct subsets of CD3-CD4-CD8- triple-negative adult mouse thymocytes defined by CD44 and CD25 expression.

We have subdivided mouse CD4-CD8-CD3- triple-negative (TN) thymocytes into four subsets based upon expression of CD44 and CD25, including CD44+CD25-, CD44+CD25+, CD44-CD25+ and CD44-CD25-. Characterization of these cells revealed several features distinct to each subset, in particular the expression of high levels of c-kit (the receptor for stem cell factor) by CD44+CD25-TN and CD44+CD25+TN but not by CD44-CD25+TN and CD44-CD25-TN. The CD44+CD25+TN subset also included the IL-7 and stem cell factor-responsive cells, whereas only minimal responsiveness was observed by the CD44- populations. These subsets also showed differential cytokine production potential (CD44+CD25- > CD44+CD25+ > CD44-CD25+ > CD44-CD25-) after stimulation with calcium ionophore, PMA and IL-1. The repopulation potential of these subsets in 2-deoxyguanosine-treated fetal thymic lobes supports the following maturation sequence: CD44+CD25- -->CD44+CD25+ -->CD44-CD25+ -->CD44-CD25-. Furthermore, the sequence of progression from CD44+CD25+ to CD44-CD25+ cells was confirmed by their TCR beta-chain gene configuration. The former population exhibits germ-line TCR beta-chain configuration, whereas the latter subset shows a rearranged pattern.

Animals↗

CD44 isoforms containing exons V6 and V7 are differentially expressed on mitogenically stimulated normal and Epstein-Barr virus-transformed human B cells.

CD44, a cell adhesion molecule, exists in multiple isoforms that are generated by RNA alternative splicing. CD44 isoforms containing exon V6 (CD44 V6) have been associated with tumorigenesis and metastasis. We investigated the association between human B-cell activation and CD44 V6 isoform expression by analysing its expression in resting and mitogenically stimulated B cells. Results showed that resting B cells expressed the CD44 H (no variable exon) isoform alone. Activation of B cells [phorbol myristate acetate (PMA), surface immunoglobulin cross-linking alone or in the presence of interleukin-2 (IL-2)] induced CD44E (variable exon V8-10), R2 (VIO) and CD44 isoforms containing exons V6 and/or V7 (CD44 V6/V7). Epstein-Barr virus (EBV) infection of B cells, an alternative method of B-cell activation, induced the expression of CD44 E and R2 but not CD44 V6/V7. These results indicate that CD44 V6/V7 expression depends on the mode of activation. CD44 isoform expression was also investigated in a panel of EBV-negative and EBV-positive Burkitt's lymphoma (BL) B-cell lines. EBV-negative BL cells did not express CD44. In contrast, EBV-positive BL cells expressed CD44 H, R2 and E but not CD44 V6/V7 isoforms, suggesting an association between EBV infection and CD44 isoform induction. To determine directly the role of EBV in CD44 isoform induction, an EBV-negative BL cell line, BL30 (negative for all isoforms of CD44), BL30 infected in vitro with the EBNA-2-defective P3HR1 (BL30/P3HR1), and the wild-type B95-8 strain of EBV (BL30/B95-8) were examined. The parental BL30 cells infected with the wild-type EBV strain, but not with the P3HR-1 strain, expressed CD44 H, R2 and E isoforms, as seen in EBV-immortalized B cells. These studies suggest that (1) alternative splicing of CD44 isoforms is differentially regulated depending on the mode and state of cell activation, and that (2) the CD44 V6/V7 isoforms may represent B-cell activation antigens that are induced by mitogenic stimulation but not following EBV infection.

B-Lymphocytes↗

CD44 and hyaluronan expression in the development of experimental crescentic glomerulonephritis.

CD44 is a widely expressed cell surface glycoprotein which is involved in both cell-matrix and cell-cell interactions which regulate a variety of processes, including leucocyte migration and activation. Therefore, we examined the expression of CD44, and its major ligand hyaluronan, during the induction and progression of experimental glomerulonephritis. Antibody staining of normal rat kidney showed constitutive CD44 expression by resident glomerular macrophages, parietal epithelial cells, medullary and occasional cortical tubules. There was a marked increase in CD44 expression over days 1, 7 and 21 of rat crescentic anti-glomerular basement membrane (GBM) glomerulonephritis. Infiltrating monocytes and lymphocytes were CD44+, with ultrastructural studies showing high levels of CD44 expressed on the surface of lymphocytes adherent to activated endothelium. Marked hyaluronan deposition was seen in areas of fibrosis on days 7 and 21, such as glomerular crescents and the periglomerular area. Hyaluronan deposition was accompanied by the presence of many CD44+ cells. Double immunohistochemistry showed that both CD44+ED1+ macrophages and CD44+ myofibroblasts (identified by expression of alpha-smooth muscle actin) were present in areas of fibrosis. There was also a dramatic increase in cortical tubular CD44 expression, which was most evident in areas of tubular damage. Although tubular epithelial cells expressed CD44 upon both the basolateral and luminal surface, CD44 expression was most prominent within tightjunctions, suggesting a role for CD44-CD44 interactions in cell-cell adhesion within the tubule. Analysis of CD44 isoforms by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that the standard form of CD44 predominated in both normal and diseased kidney. However, a series of alternatively spliced CD44 isoforms was also detected, whose expression was markedly increased during disease. At least seven isoforms containing the v6 domain were identified, with the smallest form representing activated T cells. In conclusion, CD44 is constitutively expressed in normal kidney and is dramatically up-regulated in rat anti-GBM disease, suggesting possible roles for the CD44-hyaluronan interaction in leucocyte recruitment, renal fibrosis and tubular cell-matrix and cell-cell interactions during the induction and progression of crescentic glomerulonephritis.

Animals↗

N-terminal and central regions of the human CD44 extracellular domain participate in cell surface hyaluronan binding.

CD44 molecules are cell surface receptors for hyaluronan (HA). To define regions of the extracellular domain of CD44 that are important for HA binding, we have studied the ability of HA-blocking CD44 mAbs to bind to CD44 from a variety of sources. Five CD44 mAbs (5F12, BRIC235, 3F12, BU-75, and HP2/9) of 21 studied were identified that at least partially blocked FITC-labeled HA (HA-FITC) binding to the standard form of CD44 (CD44S) in CD44-transfected Jurkat cells. Analysis of reactivity of HA-blocking CD44 mAbs defined three distinct epitopes. Lack of reactivity of mAb 5F12 with a CD44 fusion protein (CD44-Rg) containing an N-terminal truncation of 20 amino acids (aa), as well as reactivity of mAb 5F12 with an N-terminal CD44 synthetic peptide (CD44-9A), demonstrated that the N-terminal proximal region of CD44 (aa 1 to 20) was involved in mAb 5F12 binding. A mutant cell line, CEM-NKR, derived from the T-ALL cell line, CEM, did not bind mAb 5F12 nor bind HA, whereas wild-type CEM did bind mAb 5F12 and HA. Sequence analysis of wild-type CEM and CEM-NKR CD44 cDNA demonstrated a G to A point mutation at position 575 in the CD44 cDNA of CEM-NKR, resulting in an arginine to histidine mutation at aa position 154. Taken together, our studies demonstrated that there are three epitopes to which HA-blocking mAbs bind in the extracellular domain of CD44, and that the CD44 N-terminal proximal and central regions are two regions in the extracellular domain of CD44 that may interact and either mediate or regulate HA binding to cell surface CD44.

Amino Acid Sequence↗

Functional heterogeneity of CD44 molecules in ovarian cancer cell lines.

We have previously shown that CD44 partly mediates ovarian cancer cell attachment to peritoneal mesothelium through recognition of mesothelial-associated hyaluronate. CD44 is a major receptor for hyaluronate and exists as a standard 90-180-kDa form (CD44H), as well as several higher molecular mass variant forms produced by alternative splicing. To determine whether functional differences exist between CD44H and its variants we have investigated the relationship between CD44 isoform expression and mesothelial adhesion in 12 ovarian cancer cell lines. Eight lines were CD44 positive (range, 83-94%) and demonstrated significant binding to mesothelium and hyaluronate, whereas two lines showed reduced CD44 levels (3-13%) and demonstrated decreased binding. Interestingly, two other lines (OVC-3 and SW626) expressed CD44 in the majority of cells (>93%) and yet bound weakly to mesothelium. Mean linear fluorescence intensity of CD44 expressed by OVC-3 and SW626 cells was approximately one-half that of strongly binding cell lines, suggesting that the ability to adhere may be partly related to CD44 surface density. However, immunoprecipitation and immunoblot analyses revealed that standard CD44H represented only 23-31% of total CD44 in weakly binding cells, with the majority of species being comprised of CD44 variants. Indirect immunofluorescence of OVC-3 and SW626 cells confirmed the presence of CD44 variants containing exons v3, v6, and v9. In contrast, CD44H represented the majority (75-86%) of total CD44 expressed by strongly binding cell lines such as CAOV-3 and UPN36T. Transfection of CD44H cDNA into weakly binding OVC-3 cells restored significant mesothelial binding which was partly blocked by anti-CD44 antibody. These data suggest that the expression of CD44 is necessary but not sufficient for mediating attachment of ovarian cancer cells to mesothelium. Although CD44 variants may constitute the major CD44 species in certain ovarian cancer cell lines, it appears that these CD44 species are not always capable of mediating significant binding to mesothelium or hyaluronate. Rather, an adequate level of CD44H is the critical determinant of binding in this system. The role of CD44 variants in the process of ovarian cancer metastasis will require further investigation.

Antigens, CD↗

CD44 isoform expression in primary and metastatic pancreatic adenocarcinoma.

CD44 is the transmembrane adhesion molecule which binds hyaluronate. The gene encoding CD44 is found on chromosome 11p and comprises 20 exons. Differential splicing of the 10 extracellular juxtamembranous exons (v1-10) generates the major isoforms of CD44. The major CD44 isoform found on hematopoetic cells (CD44s) contains none of the variably expressed exons, while the major isoform expressed on epithelial cells [CD44(v8-10)] contains exons v8-10. Metastasis-specific isoforms of CD44 were first documented in a model of rat pancreatic adenocarcinoma [CD44(v4-7), CD44(v6-7)] and subsequently in other cancers. This study is the first characterization of CD44 isoforms in primary and metastatic human pancreatic adenocarcinomas. CD44 isoforms were analyzed in specimens of 15 primary and 6 metastatic pancreatic adenocarcinomas as well as in 6 specimens of control pancreata by two different methods. Radiolabeled reverse transcriptase-PCR coupled with 8% PAGE allowed analysis of the major isoforms of CD44, while Southern blot hybridization with [alpha-32P]dCTP-labeled probes permitted analysis for metastasis-specific CD44 isoforms containing CD44(v6) or CD44(v8-10). No differences in the expression of CD44(v8-10) and CD44s were found among the primary and metastatic pancreatic adenocarcinomas, and control specimens of pancreata. However, a novel CD44(v6) isoform was found in metastatic lesions and may represent the human homologue of the rat pancreatic adenocarcinoma metastasis-associated CD44 isoform.

Adenocarcinoma↗

Hypermethylation-mediated regulation of CD44 gene expression in human neuroblastoma.

The CD44 adhesion receptor is silenced in highly malignant neuroblastomas (NBs) with MYCN amplification. Because its functional expression is associated with decreased tumorigenic properties, CD44 behaves as a tumor suppressor gene in NB and other cancers. Given that the precise mechanisms responsible for CD44 silencing are not elucidated, we investigated whether CD44 expression could be regulated by DNA hypermethylation. The methylation status of CD44 gene promoter and exon 1 regions was analyzed in 12 NB cell lines and 21 clinical samples after bisulfite genomic modification, followed by PCR and single-strand conformation polymorphism analysis and genomic sequencing. The results showed that almost all CD44-negative cell lines displayed hypermethylation in both regions, whereas all CD44-expressing cell lines were unmethylated. These observations correlated with the ability to restore CD44 mRNA and protein expression by treatment of CD44-negative cells with the 5-aza-2'-deoxycytidine demethylating agent. In contrast, no CD44 gene hypermethylation could be detected in 21 NB clinical samples of different stages, irrespective of CD44 expression. Although our results suggest that aberrant methylation of promoter and exon 1 regions is involved in CD44 silencing in NB cell lines, they also indicate that methylation of unidentified regulatory sequences or methylation-independent mechanisms also control the expression of CD44 in primary NB tumors and cell lines. We therefore conclude that CD44 silencing is controlled by complex and tumor cell-specific processes, including gene hypermethylation. Further investigation of other mechanisms and genes involved in CD44 regulation will be needed before demethylation-mediated reactivation of the CD44 gene can be considered as therapeutic strategy for neuroblastoma and perhaps other related cancers.

Azacitidine↗

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation.

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.

Animals↗

Prognostic value of CD44 expression in invasive squamous cell carcinoma of the vulva.

OBJECTIVE: CD44 is a cell adhesion molecule that binds extracellular matrix. CD44 isoforms arising from alternative mRNA splicing are implicated in tumor metastases. The aim of our study is to investigate the expression of CD44 splice variants and its correlation to lymph node metastases and disease-free survival in squamous cell carcinoma (SCC) of the vulva. METHODS: Thirty-five cases of SCC of the vulva were evaluated for CD44 splice variants -3v, -4v, -5v, and -7v expression by immunocytochemistry. When available one nonmetastatic lymph node (LN) was also studied. In cases with LN metastases, the metastatic LN as well as a nonmetastatic LN from the same patient were evaluated. RESULTS: All CD44 variants studied were expressed in all epithelium: normal, dysplastic, and SCC. CD44 variants showed decreased immunostaining in the tumor cells when compared to normal epithelium. Furthermore, intensity of expression of the CD44 isoforms changed within the tissue containing invasive cancer. Interestingly, CD44-4v expression was downregulated in the most differentiated cells within the carcinoma, mainly in patients who had disease recurrence or died of disease (P = 0.004). Confirming prior publications, CD44-5v and -7v expression did not correlate with survival. One hundred percent of metastatic tumors to LNs were immunoreactive with CD44-3v and only 1/30 normal LN had CD44-3v expression. Eighty percent of metastatic tumors to LNs were immunoreactive for CD44-4v. However, 3 LNs without tumor were also immunoreactive with CD44-4v. CONCLUSION: CD44-4v is a potential molecular marker of disease recurrence in vulvar carcinoma. A larger multiinstitutional study is needed to evaluate the specificity of CD44-3v expression in LN metastasis. If a larger scale study confirms our findings, a CD44-3v antibody could be used for radioimmunoimaging of occult lymph node metastases in patients with vulvar cancer.

Adult↗

CD44 expression by leucocytes in rheumatoid arthritis and modulation by specific antibody: implications for lymphocyte adhesion to endothelial cells and synoviocytes in vitro.

Anti-CD44 MoAb IM7 induced the loss of CD44 from mouse leucocytes thereby inhibiting leucocyte migration and joint inflammation in murine arthritis. Thus, targeting CD44 with MoAb may have potential for the treatment of patients with inflammatory joint diseases. Expression of CD44 by peripheral blood (PB) and synovial fluid (SF) leucocytes from rheumatoid arthritis (RA) patients was compared and the ability of IM7 to modulate this expression determined. RASF lymphocytes showed increased CD44 expression compared with those in PB indicative of an activated phenotype. As inflammatory SF did not up-regulate CD44 expression on PB lymphocytes, the increased CD44 expression by SF lymphocytes was a result of the selective homing of CD44(high) cells to the synovium rather than an effect of the synovial environment. RASF granulocytes showed reduced CD44 expression compared with those in PB, again indicative of an activated phenotype. However, this reduction could be induced on PB granulocytes following culture with inflammatory SF and was inhibited by anti-TNF-alpha MoAb, implying that soluble factors in inflammatory SF such as TNF-alpha induced granulocyte activation and CD44 loss. IM7 induced the loss of CD44 from lymphocytes (both from PB and SF) and granulocytes in vitro, but was subsequently re-expressed after 24 h culture in the absence of the MoAb. This loss of CD44 was blocked by serine- and metalloprotease inhibitors implying that IM7 induced the proteolytic cleavage of CD44 by a mechanism similar to that reported for the loss of CD44 from PMA-activated granulocytes. Furthermore, IM7-treated CD44(low) lymphocytes showed reduced adherence to both an endothelial cell line and RA synovial fibroblasts in vitro. The unique ability of IM7 to reduce CD44 expression by lymphocytes suggests that it could prevent lymphocyte extravasation and synovial infiltration in RA as previously reported in murine arthritis.

Antibodies, Monoclonal↗