Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment

Up-regulation of CD44 variant isoforms has been linked to the progression of epithelial tumors and the metastatic phenotype. Here we report a functional role for CD44 variant isoforms in colorectal cancer metastasis. An antisense mRNA approach was used to down-regulate CD44 variant isoforms containing CD44 variant 6 (v6) in the metastatic colorectal tumor cell line HT29. Cell lines stably expressing antisense CD44 exon 10 (v6) showed reduced expression of alternatively spliced CD44 variant isoforms but no significant change in expression of CD44 core protein, as judged by immunohistochemical analysis using CD44 domain-specific monoclonal antibodies. Expression of antisense exon 10 (v6) had no effect on HT29 tumor cell proliferation in vitro or the ability of the cells to bind immobilized hyaluronan, but it resulted in a reduced capacity to form liver metastases in nude mice following intrasplenic injection. Metastases were not detected in nude mice inoculated with antisense CD44 exon 10 (v6)-expressing cell lines after 4 months, against a background of a 30% metastasis rate in the control HT29 parental and vector alone transfected lines. Furthermore, whereas 82% of mice intrasplenically injected with control HT29 parental and vector alone cell lines developed tumors in incisional wound sites, none of the mice injected with antisense exon 10 expressing HT29 cells developed similar tumors. This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecule generated through alternative mRNA splicing while allowing expression of core domains to remain unaffected. Furthermore, these results provide direct evidence for a functional role of CD44 variant isoforms in the metastasis of human colorectal tumor cells and may suggest a critical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.     

Preparing physicians for indicatory physical examination of the nervous system

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Tissue localization of bovine dander allergen Bos d 2

We examined serum levels of a CD44 splice variant that contained variant exons 8-10 (CD44v8-10) as a tumor marker in colorectal cancer patients. We performed enzyme-linked immunosorbent assays in 81 sera obtained from 71 colorectal cancer patients and 10 healthy controls. Serum CD44v8-10 levels were significantly higher in the colorectal cancer patients than in the healthy controls (0.209 +/- 0.098 versus 0.114 +/- 0.019 OD; P < 0.01). There was a close correlation between immunohistochemical expression and serum CD44v8-10 levels. Surgical resection of the tumors resulted in a reduction of serum CD44v8-10 levels. There was no significant correlation between serum CD44v8-10 level and serosal invasion or histologic type. However, a significant correlation was observed between serum CD44v8-10 level and lymphatic or venous invasion. In addition, serum CD44v8-10 levels were significantly higher in carcinomas associated with lymph node or liver metastasis than in those without metastasis. These findings suggest the usefulness of serum CD44v8-10 level in the prediction of colorectal cancer metastasis.     

Expression of CD44 variant isoforms in peripheral blood leukocytes in malignant lymphoma and leukemia: inverse correlation between expression and tumor progression

In a variety of human tumors, including high grade Non-Hodgkin's lymphoma (hgNHL), a linkage between expression of CD44 variant isoforms (CD44v) and tumor progression has been described. In search of an easily accessible diagnostic parameter, expression of CD44 standard (CD44s) and CD44 variant isoforms (exons v5, v6, v7 and v10) in peripheral blood lymphocytes (PBLs) of patients with hematological malignancies was evaluated by fluorescence activated cell scanning. The analysis of 30 blood samples of healthy donors and patients with non-malignant diseases and of 183 blood samples of patients with malignant hematological disorders revealed that only in patients with malignant disorders did a measurable proportion of PBLs express CD44 variant isoforms, mostly exons v5, v6, v7 and, less frequently, exon v10. Elevated levels of CD44v expression were noted in PBLs of patients with acute and chronic myeloid leukemia (AML: 16%, CML: 25%), Hodgkin's disease (HD: 17%), multiple myeloma (MM: 22%), polycythemia vera (PV: 33%), acute lymphoid leukemia (ALL: 23%) and, most frequently, in PBLs of patients with non-Hodgkin's lymphoma (NHL:54%). CD44v expression was not restricted to the malignant phenotype, but instead was also noted in T cells, B cells and monocytes, preferentially in a subpopulation of large cells. Furthermore, expression of CD44v in PBLs was not linked to the histological grading or clinical staging. There was, however, an inverse correlation with tumor progression, whereas response to therapy was frequently accompanied by upregulation of CD44v. Thus, expression of CD44v in the PBLs of patients with NHL mainly reflected immune responsiveness. Since NHL manifests itself primarily in lymphoid organs, its progression is difficult to follow. Monitoring of CD44v in PBLs could be used as an additional and convenient parameter for surveying the course of disease.     

Development of cholinergic neurons in rat brain regions: dose-dependent effects of propylthiouracil-induced hypothyroidism

CD44 is an adhesion molecule involved in cell-to-cell and cell-to-matrix interactions. This transmembrane glycoprotein exists in either standard or variant forms, originated by alternative splicing. One of the isoforms (CD44V6) has been shown, in some systems, to modify the metastatic potential of tumor cells. To investigate the role of this biomarker as possible prognostic antigen in colorectal cancer, we immunohistochemically analyzed the distribution of CD44V6 expression on formalin-fixed, paraffin-embedded tissues from resected colorectal cancers of 34 patients. The monoclonal antibody VFF7 against the amino acid sequence encoded by exon CD44V6 was applied using the avidin-biotin-peroxidase method. For each resected specimen, normal (N), adenomatous (AD), and carcinomatous (CA) colonic mucosa were tested. In 68% of the resected cases, these areas were present in the same slide, and in 76% of cases, nodal or liver metastases (MT) were available for evaluation. Adenomatous polyp biopsy specimens of 10 carcinoma-free patients were also tested. In selected cases, CD44V6 expression was also determined using the Western blot immunoprecipitation technique. CD44V6 immunoreactivity was detected in 100% of the ADs, and in 91% of CAs, but was mostly weak in only 38% of MTs (n=26). In 49% (n=35) of ADs, 11% (n=34) of CAs, and 4% of MTs (n=26), the stain was moderate to strong. CD44V6 immunoreactivity was predominantly membranous in ADs and cytoplasmic in MTs. In the CAs, both staining patterns were noted. Interestingly, the normal mucosa had a weak subnuclear localization of the stain. In the cases evaluated by Western blotting immunoprecipitation analysis, the level of CD44V6 protein expression was similar to that obtained by immunohistochemistry. No correlation was found with tumor type, stage, or patient survival. The predominant CD44V6 expression in ADs and CAs, but not in MTs, suggests that, in many cases, the expression of this adhesion molecule may be lost during the acquisition of migratory function by the tumor cells.     

Expression of CD44 variants and its association with survival in pancreatic cancer

Since the CD44 variant 6(v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors, we examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 variants was evaluated immunohistochemically in paraffin-embedded pancreatic cancer tissues from 42 patients who were confirmed surgically and histologically to have received curative resection. An indirect immunoperoxidase method was used with monoclonal antibodies against epitopes of the standard (CD44s) portion, v6 and v2. Protein expression data were evaluated statistically for any correlations with the length of survival or with histological parameters. The expression of CD44v6 and v2 was observed only in tumor cells, if at all. On the other hand, expression of total CD44 (including CD44v, as well as CD44s) was observed in both tumors and adjacent normal sites. Tumor tissue from 21 (50%) and 16 (38%) patients showed positive immunoreactivity with mAb 2F10 (anti-CD44v6) and mAb M23.6.1 (anti-CD44v2), respectively. The expression of CD44v6 and v2 was correlated with decreased overall survival (P = 0.0160 and P = 0.0125, respectively). A significant correlation was obtained between CD44v2 peptide expression and vessel invasion (P = 0.026). These results suggest that CD44v2 and CD44v6 may be useful markers for poor prognosis in curatively resected primary pancreatic cancer.     

The splice-pattern of CD44 is altered in chronic pancreatitis exhibiting dysplastic changes

Recent studies have shown that several splice variants of CD44, might be involved in tumor progression. Since chronic pancreatitis is suggested to be a risk factor for pancreatic cancer we investigated the splice pattern of CD44 in chronic pancreatitis to elucidate the role of CD44 in pancreas tumorigenesis. The expression of CD44-isoforms was examined in 40 specimens of chronic pancreatitis and 12 specimens of normal pancreas by immunohistochemistry, Westernblotting and exon specific RT-PCR. Pancreatic cancer tissue from two patients who developed pancreatic cancer 2 and 3 years following surgery for chronic pancreatitis were analyzed. Strong expression of CD44s was found in all cells, whereas the expression of CD44v6 was restricted to ductal cells. Westernblotting revealed an overexpression of CD44v6 in chronic pancreatitis as compared to normal pancreas. Exon specific analysis revealed an altered splice pattern of CD44, similar to that in pancreatic cancer, in 12.5% of the chronic pancreatitis specimens. Both patients who developed pancreatic cancer after chronic pancreatitis exhibited this altered splice pattern in both, chronic pancreatitis and pancreatic cancer. These results suggest that variant forms of CD44-mRNA might be expressed in early dysplastic alterations in chronic pancreatitis.     

In vivo chiro-inositol metabolism in the rat: a defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro-[3H]inositol into phospholipid in the Goto-Kakizaki (G.K) rat

BACKGROUND: CD44 is a cell adhesion molecule often expressed in the form of various splice variants. The role of standard CD44 isoform (CD44s) and its variants in colorectal carcinogenesis is partly conflicting. Therefore, we compared the expression of CD44s (hermes-3) and its splice variants (v3 and v6) with traditional prognostic factors in 194 colorectal cancer patients treated at Kuopio University Hospital and followed up for a mean of 14 years. METHODS: Formalin-fixed, paraffin-embedded tissue sections from 194 patients with colorectal carcinoma were examined immunohistochemically to detect the expression of different forms of CD44. The hypothesis that CD44s, CD44v3, and CD44v6 expression intensities and distribution in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. RESULTS: In high-grade tumours CD44s and CD44v6 expression intensities and CD44s percentages were stronger than in low-grade tumours. CD44v6, CD44v3, and CD44s expression intensities in tumour epithelium were also stronger in Dukes C and D tumours than in A and B tumours. In the univariate survival analysis patients with strong CD44s, CD44v3, and CD44v6 expression intensities in tumour epithelium had lower cancer-related survival than the patients who had weak CD44s, CD44v3, and CD44v6 expression intensities. Recurrence-free survival was also shorter in patients with intense signals for CD44v3 and CD44v6 in tumour epithelium. In the multivariate analysis the CD44v6 expression intensity in tumour epithelium predicted independently both cancer-related and recurrence-free survival in T1-4N0-3M0 and T1-3N0M0 cases. In addition, the CD44v3 expression intensity in tumour epithelium was a significant predictor of RFS in T1-3N0M0 cases. CONCLUSIONS: These results strongly suggest that the CD44 splice variants v6 and v3 have prognostic significance in colorectal cancer.     

Fecal lactoferrin as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins

OBJECTIVES: 1) To investigate which neutrophil-derived proteins in feces most accurately reflect disease activity in inflammatory bowel disease. 2) To examine the extracellular release of these proteins by activated neutrophils and their stability in feces by in vitro study. METHODS: We studied 41 patients (91 samples) with ulcerative colitis (UC), 34 patients (105 samples) with Crohn's disease (CD), and 25 control subjects. Fecal levels of lactoferrin (Lf), polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase (MPO), and lysozyme (Lys) were measured by ELISA. We also measured fecal hemoglobin (Hb) and alpha 1-antitrypsin (alpha 1-AT), useful markers of disease activity in UC and CD, respectively. For the in vitro study, blood samples were stimulated with phorbol myristate acetate or latex beads. For the assessment of stability, homogenized stool samples were stored at 4 degrees C, 25 degrees C, and 37 degrees C for various periods. RESULTS: 1) Fecal Lf, PMN-E, MPO, and Lys concentrations were significantly increased in the active phase of the disease compared to the inactive phase in both UC and CD. 2) Fecal Lf, PMN-E, MPO, and Lys concentrations correlated significantly with fecal Hb concentration in UC, whereas fecal Lf, PMN-E, and MPO concentrations correlated significantly with alpha 1-AT concentration in CD. In UC, fecal Lf, PMN-E, MPO, and Lys concentrations were high in 15, 9, 14, and 14 samples, respectively, of 25 samples with normal Hb concentration. In CD, fecal Lf, PMN-E, and MPO concentrations were high in 19, 10, and 16 samples, respectively, of 30 samples with normal alpha 1-AT concentration. 3) The extracellular release of Lf was the most efficient and this molecule was the most stable in feces. CONCLUSIONS: Both our clinical and our in vitro studies suggested that Lf is the most suitable of these proteins to use as neutrophil-derived fecal marker of inflammation for clinical application.     

Complex CD44 splicing combinations in synovial fibroblasts from arthritic joints

CD44 is a broadly expressed cell surface glycoprotein which is the major cell surface receptor for the glycosaminoglycan, hyaluronan. In humans, alternative splicing of up to 9 variant exons (v2-v10) into CD44 mRNA, together with post-translational modification via glycosylation and chondroitin sulfate attachment has the potential of generating a large number of CD44 isoforms. Insertion of these various exons has the potential to change the functional capacities of the molecule and has implications in disease. We have analyzed CD44 splice variant expression in cultured VCAM-1-positive synovial fibroblasts isolated from patients with osteo- or rheumatoid arthritis and from normal synovium. Rheumatoid and osteoarthritic tissue express CD44 splice variants at the cell surface level. At the mRNA level exons v3, v6, v7, v8, v9 and v10 were detected in different splicing combinations. Rheumatoid tissue showed high expression, osteoarthritic tissues showed great variation. In contrast, non-inflamed tissue showed no splicing events. Our results indicate that the nature of CD44 splice variant expression may be linked to the inflammatory state of the synovial joint.     

Differential expression of CD44v6 in adenocarcinoma of the pancreas: an immunohistochemical study

Alternative splicing gives rise to numerous CD44 isoforms, some of which seem to have a role in tumour metastasis. Specifically, a variant form of CD44 with sequences encoded by exon v6 (CD44v6) confers metastatic potential when transfected into a nonmetastasizing cell line of rat pancreatic adenocarcinoma. This study has investigated standard CD44 (CD44s) and CD44v6 expression immunohistochemically in 6 samples of normal pancreatic tissue, 4 of tissue affected by chronic pancreatitis, and 24 of tissue from metastasizing and nonmetastasizing pancreatic adenocarcinomas. In addition, 18 samples from lymph node or visceral metastases were included in the study. CD44s was expressed in nonneoplastic tissue and in tissue from pancreatic adenocarcinomas. In contrast, CD44v6 was not detected in any of the normal tissue or chronic pancreatitis specimens, whereas 54% of pancreatic adenocarcinomas and 55% of metastases expressed this variant exon. Although it is not clear whether CD44 isoforms containing exon v6 play a part in malignant progression in the human exocrine pancreas, it seems plausible that the expression of multiple isoforms containing this and other variant exon confers a selective advantage on pancreatic adenocarcinoma.