CD44 is a cell adhesion molecule with numerous isoforms created by mRNA alternative splicing. Expression of CD44 variants has been suggested to play a potential role in tumor progression and metastasis. We designed primers CD44V, CD44V6/7, CD44R1 and CD44V6-10 to analyze and compare the roles of each CD44 variants. Expressions of CD44 variants were investigated in normal colonic mucosa, the lymph nodes which was histopathologically free of cancer cell, and cancer tissues of 44 human colorectal cancer patients by RT-PCR method. The expression of CD44V was observed in 28 out of 39 (71.8%) tumors and 7 out of 11 (63.6%) N1 normal regional lymph nodes, and CD44V6/7 was observed in 28 out of 39 (71.8%) tumors and 9 out of 11 (81.8%) N1 normal regional lymph nodes. The expressions of CD44V and CD44V6/7 were most frequently observed compared with any other CD44 variants. In normal colonic mucosa, the expression of CD44 variants are low but in cancer tissue and its regional lymph node, the expression of CD44V and CD44V6/7 were significantly higher and more frequent than any other CD44 variants (p<0.05). These results suggest that CD44V and CD44V6/7 can be a molecular marker for colorectal cancer and its micrometastasis to the regional normal lymph node.
Alternative Splicing ; Antigens, CD44/genetics* ; Colorectal Neoplasms/pathology ; Colorectal Neoplasms/immunology* ; Gene Expression ; Human ; Lymph Nodes/pathology ; Lymph Nodes/immunology* ; Protein Isoforms/genetics

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.
Adult ; Agrin/genetics ; Antigens, CD44/genetics ; Base Sequence ; DNA Primers/genetics ; Gene Expression/radiation effects ; Glucuronidase/genetics ; Heparan Sulfate Proteoglycans/genetics/*metabolism ; Heparitin Sulfate/metabolism ; Humans ; Male ; Matrix Metalloproteinase 1/genetics ; N-Acetylglucosaminyltransferases/genetics ; RNA, Messenger/genetics/metabolism ; Skin/*metabolism/*radiation effects ; Skin Aging/genetics/physiology ; Syndecan-1/genetics ; Syndecan-4/genetics ; Ultraviolet Rays/*adverse effects ; Young Adult

Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.
Animals ; Antigens, CD44/genetics/metabolism ; Biomarkers, Tumor/genetics/metabolism ; Cell Transformation, Neoplastic/*genetics/metabolism ; Cyclooxygenase 2/*genetics/metabolism ; Dog Diseases/*genetics/metabolism ; Dogs ; Female ; Immunohistochemistry/veterinary ; Mammary Neoplasms, Animal/*genetics/metabolism ; Mammary Neoplasms, Experimental/*genetics/metabolism ; Neoplastic Stem Cells/*metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Retrospective Studies

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.
5'-Nucleotidase/metabolism ; Adipose Tissue/*cytology ; Adult ; Antigens, CD/metabolism ; Antigens, CD44/metabolism ; Antigens, Thy-1/metabolism ; Cell Differentiation/physiology ; Cells, Cultured ; Female ; Humans ; Immunoblotting ; Immunohistochemistry ; Immunophenotyping ; Mesenchymal Stem Cells/metabolism ; Muscle Development/genetics/physiology ; Osteogenesis/genetics/physiology ; Receptors, Cell Surface/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/metabolism ; Young Adult

CD44 is a glycoprotein expressed in a wide variety of cell types. Recently expression of some alternatively-spliced variants of CD44 transcripts (CD44v) has been suggested to play a potential role in tumor metastasis and the detection of CD44v containing exon 6 to 11 may be helpful for the diagnosis of cancers. Expressions of CD44v containing exon 6 to 11 were investigated in 20 human colorectal cancer samples, peripheral blood leukocytes isolated from colorectal cancer patients, and 4 colorectal cancer cell lines using reverse transcription-polymerase chain reaction and Southern blot analysis. The standard form of CD44 transcripts was expressed in all samples tested. CD44v containing exon 6 to 11 was expressed in 18 cases of colorectal cancers (sensitivity = 90%), 3 out of 4 cell lines, and one normal tissue (specificity = 95%). These results suggest that the expression of CD44v containing exon 6 to 11 can be regarded as tumor specific and that this marker may be helpful for the early diagnosis of colon cancers, if specimens from the early stage are available.
Adenocarcinoma/*genetics ; Adult ; Aged ; Antigens, CD44/*genetics ; Base Sequence ; Blotting, Southern ; Colorectal Neoplasms/diagnosis/*genetics ; DNA Primers ; Electrophoresis, Agar Gel ; Feces/chemistry/cytology ; Female ; Gene Expression Regulation, Neoplastic/genetics ; Human ; Male ; Middle Age ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA Splicing/*physiology ; RNA, Messenger/analysis ; Tumor Cells, Cultured/*physiology ; Tumor Markers, Biological