alcian-blue and Chondrosarcoma

Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia. We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P. S., Fang, C. H., Leslie, M. P., and Di Cesare, P. E. (2000) J. Orthop. Res. 18, 345-350; Issack, P. S., Liu, C. J., Prazak, L., and Di Cesare, P. E. (2004) J. Orthop. Res. 22, 751-758). In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein. LRF bound directly to the NRE both in vitro and in living cells. Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF. LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells. In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining. LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity. LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation.

Topics: Alcian Blue; Amino Acid Motifs; Animals; Base Sequence; Cartilage; Cell Differentiation; Cell Line; Cell Line, Tumor; Cell Nucleus; Chondrosarcoma; Chromatin; Chromatin Immunoprecipitation; Cloning, Molecular; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression Regulation; Genes, Reporter; Glutathione Transferase; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoblotting; Immunoprecipitation; Magnetics; Mice; Mice, Inbred C3H; Molecular Sequence Data; Mutation; Plasmids; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Rats; Transcription Factors; Transcription, Genetic; Two-Hybrid System Techniques; Zinc Fingers

Chondrocytes from the Swarm chondrosarcoma, a transplantable rat tumor, have been difficult to maintain in tissue culture for extended periods due to a time-dependent alteration of the culture to a more fibroblastic phenotype. This feature precluded the use of these cultures to examine chronic conditions that may affect cell metabolism, and the homogeneity or heterogeneity of the tumor cells within the culture population could not be examined. Use of suspension culture in agarose stabilized the chondrocyte phenotype, permitting long-term culture. Clones of tumor chondrocytes were established in agarose and were examined over 2-3 weeks for evidence that the cells were accumulating a proteoglycan-rich extracellular matrix, as determined by positive staining by Alcian blue, and were undergoing cell division. Nearly 90% of the cloned cells exhibited a prominent extracellular matrix by day 7 of culture and greater than 99% did so by day 14. Cell division did not occur to any great extent until days 6-7 of culture. After this lag, the cells appeared to undergo logarithmic growth, with a cell generation time of about 12 days. By 20 days of culture, between 80 and 90% of the initial clones contained multiple cells, indicating that nearly all the cells were in, or had entered, the cell cycle. These results suggest that the chondrocytes from the rat chondrosarcoma form a homogeneous cell population with respect to their ability to synthesize an extra-cellular matrix and divide.

Topics: Alcian Blue; Animals; Cartilage; Cell Count; Cell Separation; Cells, Cultured; Chondrosarcoma; Clone Cells; Culture Media; Extracellular Matrix; Microscopy, Electron; Rats; Rats, Inbred Strains; Sepharose; Staining and Labeling

Five cases of undifferentiated soft tissue sarcomas have been studied with particular reference to the identification of their mucosubstances. The use of alcian blue in solutions of magnesium chloride is described. It is suggested that undifferentiated soft tissue chondrosarcomas may be missed owing to inadequate investigation.

Topics: Abdominal Neoplasms; Adolescent; Adult; Aged; Alcian Blue; Chondrosarcoma; Diagnosis, Differential; Extremities; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Soft Tissue Neoplasms; Staining and Labeling

The present investigation endeavors to characterize the mucosubstance content of 170 myxoid and chondromatous tumors and chordomas by histochemical methods. The results obtained using the critical electrolyte concentration (CEC) method as introduced by Scott and co-workers23,24 were compared with those obtained by staining with alcian blue and toluidine blue at different pH's with and without pretreatment with bovine testicular hyaluronidase. Tissues known biochemically to contain different heteroglycans were used as controls: synovial fluid and cock's comb (hyaluronic acid) stained with alcian blue up to a MgCl2 concentration of 0.1 M; fetal cartilage (chondroitin 4- and 6-sulphate) pulposus with notochordal remnants (keratan sulphate) up 10 1.0 M. The staining reaction of intramuscular myxoma and myxoid liposarcoma corresponded to that of synovial fluid and cock's comb (containing hyaluronic acid). Benign chondromatous tumors (osteochondroma, enchondroma, extraskeletal chondroma, chondromatosis in bursae, synovia, and tendon) as well as well-differentiated chondrosarcomas had a similar staining reaction to that of adult cartilage (containing keratan sulphate). However, the intensity of the reaction was lower in these tumors than in the adult cartilage, indicating that the keratan sulphate content of the tumors is lower. Most of the moderately well-differentiated chondrosarcomas, the poorly differentiated chondrosarcomas, and pulmonary metastases of chondrosarcoma, as well as mesenchymal chondrosarcoma and extra-skeletal chondrosarcoma possessed the same staining properties as fetal cartilage, known to contain chondroitin 4- and 6-sulphate but not keratan sulphate. A few of the moderately well-differentiated chondrosarcomas stained up to a MgCl2 concentration of 1.0 M. Three cases of poorly differentiated chondrosarcomas stained with alcian blue up to 0.35-0.45 M in the lowest differentiated areas, indicating the presence of sulphated heteroglycans, as chondroitin 4- and 6-sulphate. Most chordomas possessed the same staining properties as fetal cartilage; however, a few chordomas stained in the same way as notochordal remnants of nucleus pulposus (containing keratan sulphate), which are thought to be the origin of these tumors. The results of staining of the tumors in the present series with the Scott technique corresponds well with toluidine blue and alcian blue at different pH's with and without pretreatment of the sections with testicular hyaluronidas

Topics: Adult; Aged; Alcian Blue; Bone Neoplasms; Child; Chondroitin Sulfates; Chondroma; Chondrosarcoma; Chordoma; Glycosaminoglycans; Histocytochemistry; Humans; Hyaluronic Acid; Hyaluronoglucosaminidase; Hydrogen-Ion Concentration; Keratan Sulfate; Liposarcoma; Myxoma; Osmolar Concentration; Soft Tissue Neoplasms; Tolonium Chloride