Kenneth A Iczkowski1, Shan Bai, Cooley G Pantazis. 1. Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Pathology and Laboratory Medicine Service, Veterans Administration Medical Center, Gainesville, FL, USA. iczkoka@pathology.ufl.edu
Abstract
BACKGROUND: In prostate cancer, prior data show down-regulated immunohistochemical expression of cell adhesion protein CD44 standard (CD44s) and most variants (CD44v). MATERIALS AND METHODS: Expression of CD44 mRNA was studied by RT-PCR and cDNA sequencing in 19 prostate cancers and 10 benign controls. Immunohistochemical staining was performed with anti-CD44v7/8 in 80 prostatectomy specimens, and 12 were used for in situ hybridization for CD44v7 (exon 12). Western blotting with monoclonal antibody to CD44 standard, v6, v7/8, or v9 was performed using cancerous and benign prostate. RESULTS: Sequencing of RT-PCR products showed that benign tissue and cancer express CD44 standard and v10 mRNA at 482 base pairs (bp). In contrast, cancer tissues also overexpressed 800-1000 bp bands corresponding to v7-9 isoforms. In situ hybridization revealed increased CD44v7 signal in cancer compared to benign acini. Immunostaining for CD44v7/8 was increased, proportional to Gleason grade. By Western blot, cancer and benign tissue disclosed major bands of reactivity at 75-100 kD consistent with intact standard and variant isoforms. Tumors, however, had 6-45 kD bands for CD44 standard, v7/8 and v9, consistent with cleavage products. CONCLUSION: CD44 v7-9 isoform messenger RNA is increased in prostate cancer, and translation yields low molecular weight polypeptides of probable cleavage origin.
BACKGROUND: In prostate cancer, prior data show down-regulated immunohistochemical expression of cell adhesion protein CD44 standard (CD44s) and most variants (CD44v). MATERIALS AND METHODS: Expression of CD44 mRNA was studied by RT-PCR and cDNA sequencing in 19 prostate cancers and 10 benign controls. Immunohistochemical staining was performed with anti-CD44v7/8 in 80 prostatectomy specimens, and 12 were used for in situ hybridization for CD44v7 (exon 12). Western blotting with monoclonal antibody to CD44 standard, v6, v7/8, or v9 was performed using cancerous and benign prostate. RESULTS: Sequencing of RT-PCR products showed that benign tissue and cancer express CD44 standard and v10 mRNA at 482 base pairs (bp). In contrast, cancer tissues also overexpressed 800-1000 bp bands corresponding to v7-9 isoforms. In situ hybridization revealed increased CD44v7 signal in cancer compared to benign acini. Immunostaining for CD44v7/8 was increased, proportional to Gleason grade. By Western blot, cancer and benign tissue disclosed major bands of reactivity at 75-100 kD consistent with intact standard and variant isoforms. Tumors, however, had 6-45 kD bands for CD44 standard, v7/8 and v9, consistent with cleavage products. CONCLUSION:CD44 v7-9 isoform messenger RNA is increased in prostate cancer, and translation yields low molecular weight polypeptides of probable cleavage origin.
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