Abstract: BACKGROUND: Prostate cancer (PCa) and bone cell interactions are critical in the metastatic phase. Kallikrein 4 (KLK4/hK4) is expressed in both PCa and mineralized tissues. We determined if KLK4/hK4 expression was associated with, and influenced by, the bone environment of metastatic PCa. METHODS: Immunohistochemistry, in vitro co-culture, cell migration, and attachment assays. RESULTS: hK4 was localized to tumor cells and osteoblasts in bone metastases. KLK4/hK4 increased in LNCaP and PC3 cells co-cultured with SaOs2 cells; SaOs2 KLK4/hK4 was unchanged. Co-culture did not affect cell proliferation but altered alkaline phosphatase activity/mRNA levels in SaOs2 cells. KLK4-transfected PC3 cells had increased migration towards SaOs2 conditioned medium and greater attachment to the bone-matrix proteins, collagens I and IV. CONCLUSIONS: hK4 expression and interaction with both tumor cells and osteoblasts suggests a role for hK4 in PCa bone metastasis. Whether this observation is unique to bone metastasis or reflects a role for hK4 in PCa metastasis generally is yet to be established.
Abstract: Some chelators of the pyridoxal isonicotinoyl hydrazone class have antiproliferative activity that is far greater than desferrioxamine (DFO). In this study, DFO was compared with one of the most active chelators (311) on the expression of molecules that play key roles in cell-cycle control. This was vital for understanding the role of iron (Fe) in cell-cycle progression and for designing chelators to treat cancer. Incubating cells with DFO, and especially 311, resulted in a decrease in the hyperphosphorylated form of the retinoblastoma susceptibility gene product (pRb). Chelators also decreased cyclins D1, D2, and D3, which bind with cyclin-dependent kinase 4 (cdk4) to phosphorylate pRb. The levels of cdk2 also decreased after incubation with DFO, and especially 311, which may be important for explaining the decrease in hyperphosphorylated pRb. Cyclins A and B1 were also decreased after incubation with 311 and, to a lesser extent, DFO. In contrast, cyclin E levels increased. These effects were prevented by presaturating the chelators with Fe. In contrast to DFO and 311, the ribonucleotide reductase inhibitor hydroxyurea increased the expression of all cyclins. Hence, the effect of chelators on cyclin expression was not due to their ability to inhibit ribonucleotide reductase. Although chelators induced a marked increase in WAF1 and GADD45 mRNA transcripts, there was no appreciable increase in their protein levels. Failure to translate these cell-cycle inhibitors may contribute to dysregulation of the cell cycle after exposure to chelators. (Blood. 2001;98:842-850)
Abstract: This study reports the immunohistochemical localization of TGF-beta receptor type II (T beta R-II) and type III (T beta R-III) in cells of the forming periodontal ligament (PDL) in rat first molar roots. Mandibular periodontium was obtained from 3, 6 and 12-wk-old rats. This represented tissue from the initial, pre-mature and post-mature stages of root and periodontal development, respectively. Mandibular bone chips and molar roots were used to isolate osteoblasts, fibroblasts and cementoblasts. Cells were obtained using a 2-step trypsinization and explant technique, and cultured in Dulbecco's modification of Eagle's medium (DMEM) under routine cell culture conditions. Cells were cultured on coverslips for the purpose of detecting TGF-beta receptors, and compared with whole tissue sections using the same detection method. Cells which stained positively for T beta R-II and T beta R-III on both paraffin sections and cultured cell slides were counted. Both receptors were expressed in the various periodontal tissue compartments. PDL fibroblasts, cementoblasts and osteoblasts were stained positively for T beta R-II and T beta R-III. Endothelial cells were noted to be positive for T beta R-II only. T beta R-II was more widely distributed in cells than T beta R-III, but T beta R-III was extensively localized in the extracellular matrix. Both receptors were expressed on the cell membrane and also localized in the cytoplasm. The findings for paraffin sections were consistent with the immunohistochemical staining of cultured cells. The percentage of cells which stained positively for T beta R-II was greater (approximately 85%) than that for T beta R-III (approximately 60%) in all major types of the PDL cells on both paraffin sections and cultured cell slides. Extensive location of TGF-beta receptors in both cells and extracellular matrix suggests that several binding sites are available for TGF-beta s to interact with target cells during development and following maturation of the periodontium.
Abstract: BACKGROUND: A well-characterized cell culture model for cementoblasts is essential to understand the mechanisms of periodontal ligament (PDL) reattachment and regeneration. Whether cementoblasts express alkaline phosphatase (ALP) activity in vivo and in vitro remains to be determined. METHODS: Using a 2-step method of enzyme digestion/explant culture, osteoblasts, gingival/PDL fibroblasts, and cementoblasts were obtained from alveolar bone, gingiva, and the root surface of rat first molars and cultured. Initially, bone sialoprotein (BSP) was immunolocalized on tissue sections of periodontium and on cultured cells to distinguish mineral-forming cells from fibroblasts. Proteins were extracted from these cells to assess ALP activity by using an enzyme assay. RNA was extracted from the same cell source to detect ALP mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Cultured PDL/gingival fibroblasts were spindle shaped. Osteoblasts were irregularly shaped, and cell clusters/nodules were observed as they approached confluence. The cementoblasts manifested a polygonal shape and had two morphotypes: osteoblast-like and cuboidal or stratified. BSP was localized within the mineralized tissues and in osteoblasts and cementoblasts in culture and in tissue sections. The highest level of ALP activity was found in osteoblasts, a moderate level in PDL fibroblasts, and the lowest level in gingival fibroblasts. The cementoblasts lacked ALP activity, and this was reflected by a very weak signal (or no signal at all) for ALP mRNA in the cementoblasts. CONCLUSIONS: These studies indicate that cells consistent with a cementoblast-like phenotype may be successfully cultured, and that they lack ALP activity.
Abstract: Transforming growth factor-beta 1 (TGF-beta1) has been reported to be expressed within several tissue compartments of developing molar crowns and therefore is implicated in tooth development. Additionally, TGF-beta1 may also play a crucial role in tissue repair and regeneration. The aim of this study was to determine the distribution of TGF-beta1 in the developing periodontal attachment apparatus (cementum, periodontal ligament, and alveolar bone) in Lewis rats. Animals aged 3, 6, and 12 wks were killed, their mandibles removed, fixed, demineralized, and processed in paraffin. The localization of TGF-beta1 in tissues was detected by polyclonal goat antibodies against human TGF-beta1 by means of immunoperoxidase techniques. TGF-beta1 messenger RNA was detected by in situ hybridization with a cocktail oligonucleotide probe. Cell counts were determined for analysis of the percentage of cells stained positive for TGF-beta1. Results revealed that TGF-beta1 was expressed in the developing alveolar bone, periodontal ligament, and cementum at all stages of tissue development studied. Staining was stronger at sites of cementum and alveolar bone compared with the periodontal ligament. Intensity of the positive staining, based on 3 grades, indicated a similarity between the tissues obtained from different ages, but varied between several cell types. Cementoblasts and osteoblasts stained more strongly than fibroblasts. Large numbers (approximately 90%) of the osteocytes in developing bone expressed TGF-beta1; however, in mature bone, fewer osteocytes stained for TGF-beta1. The percentages of positively stained cementoblasts, osteoblasts, and fibroblasts in the periodontal space were greater at the apical portion than at the cervical portion of the root. TGF-beta1 mRNA was expressed in osteoblasts, some bone marrow cells, cementoblasts, and fibroblasts. This study indicates that TGF-beta1 may play an important role in the modulation of tissue formation and development of the periodontium.
Abstract: The distribution of basic fibroblast growth factor (bFGF) in periodontal ligament (PDL) tissue was investigated in samples which were obtained from freshly extracted human teeth. The PDL tissue was collected by scraping, and bFGF was identified and localized by immunohistochemistry. Fibroblasts, endothelial cells, some fibrocytes and extracellular matrix (ECM) stained positively for bFGF. It was observed that cells from healthy PDL stained more intensely than those from PDL of teeth associated with chronic periodontitis; histological cell counts revealed that the numbers of fibroblasts was greater (p < or = 0.0005) in healthy PDL than in diseased PDL tissue. The results of this study show that bFGF is produced primarily by PDL fibroblasts and endothelial cells in the PDL and that bFGF levels may be decreased in tissue associated with chronic periodontal lesions.
