ABSTRACT
Los factores celulares y moleculares que regulan los procesos de formación y mineralización del cemento son poco conocidos hasta la fecha. Principalmente esto se debe a que no existe un marcador biológico de este tejido. Nosotros recientemente hemos aislado, clonado y expresado una proteína derivada del cemento radicular humano llamada proteína del cemento. Esta proteína es expresada por cementoblastos y células progenitoras del periodonto. El objetivo de este trabajo fue el de expresar la proteína del cemento en células de Drosophila melanogaster para en un futuro, determinar sus modificaciones postraduccionales. Nuestros resultados muestran que hemos establecido una línea celular que expresa constitutivamente y de forma estable la proteína del cemento. Esto es de singular importancia, ya que en un futuro mediato, estas células serán el vehículo para la producción de la proteína del cemento en cantidades suficientes para determinar su papel durante el proceso de la formación del periodonto de novo en modelos animales y de un modo predecible.
To the present date, molecular and cellular factors which regulate cement formation and mineralization processes are not well known. This is mainly due to the lack of a biological marker for this type of tissue. We have recently isolated, cloned and expressed a protein derived from human radicular (root) cement, called cement protein. This protein is expressed by cementoblasts as well as periodontium progenitor cells. The aim of the present work was to express cement protein in Drosophila melanogaster cells, so as to determine, in the future, its post-translational modifications. Our results show we have established a cellular line which expresses protein cement in an essential and stable fashion. This fact is of unique importance, since in the mediate future these cells will become the vehicle for cement protein production in sufficient amounts to determine in a predictable manner, its role during the process of de novo periodontium formation process in animal models.
ABSTRACT
Introducción: Las proteínas CEMP1 y CAP presentes en los cementoblastos y sus progenitores contribuyen a los procesos de mineralización en tejidos del ligamento periodontal, incluyendo la migración y la proliferación de fibroblastos gingivales; sin embargo su papel y relación con procesos neoplásicos no se han estudiado a profundidad. Para lograr un mejor entendimiento de la posible contribución de estas proteínas en los procesos tumorales, particularmente en las metástasis óseas, se investigó su expresión y localización en tejidos y líneas celulares de cáncer humano. Materiales y métodos: Trece casos de cáncer de próstata y mama que desarrollaron enfermedad metastásica ósea fueron analizados por medio de inmunohistoquímica; mientras que la expresión de las proteínas en dos líneas celulares de carcinoma de próstata (PC-3) y mama (MCF-7) se estudió por medio de ensayos de Western Blot. Resultados: Los tejidos de cáncer revelaron expresión citoplasmática y ocasionalmente nuclear de CAP en células tumorales y estructuras glandulares pequeñas, así como en el citoplasma de los fibroblastos estromales adyacentes al frente de invasión tumoral. En lo correspondiente a CEMP1, su expresión se localizó en el citoplasma de las células tumorales de 5 casos, pero no en el estroma. Ensayos de Wester Blot mostraron expresión de CEMP1 en las células PC-3 y MCF-7; y de CAP en las MCF-7. Conclusiones: Los resultados muestran que las proteínas de cemento radicular CEMP1 y CAP se expresan en tejidos neoplásicos y células neoplásicas, y que posiblemente contribuyen en ciertas condiciones patológicas como el cáncer metastásico en humanos.
Introduction: CEMP1 and CAP are recognized as cementum proteins, they appear to be limited to cementoblasts and their progenitors, and participate in the mineralization process of periodontal ligament tissues, including the proliferation and migration of periodontal ligament fibroblasts. However, their contribution in neoplastic processes had not been explored. In the present study, we investigated their protein expression and localization in cancer tissues and cells. Materials and Methods: CEMP1 and CAP expressions were analyzed immunohistochemically in 13 cancer cases with bone metastasis. In addition, Wester Blot essays were use to detect expression of the proteins in the prostate (PC-3) and mama (MCF-7) cancer cell lines. Results: CAP expression was detected in all tissues examined. Strong cytoplasmatic and rarely nuclear staining was found in small tumor nests, glandular structures and, in the stromal fibroblasts at the immediate vicinity of the tumor nests. CEMP1 was found in the cytoplasm of tumor cells in 5 cases, but its expression was negative in the stromal tissues. Also, cancer lines PC-3 and MCF-7 showed CEMP1 expression; however, CAP expression was observed only in MCF-7 cells. Conclusions: The results suggest that CEMP1 and CAP are present in tissues other that cementum and possibly contribute to pathological conditions such as metastatic cancer.
Subject(s)
Humans , Male , Female , Middle Aged , Aged, 80 and over , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Proteins/metabolism , Blotting, Western , Dental Cementum/cytology , Immunohistochemistry , Biomarkers, Tumor , Cell Adhesion Molecules/metabolism , Bone Neoplasms/secondary , Breast Neoplasms/pathology , Prostatic Neoplasms/pathology , Core Binding Factor alpha Subunits/metabolismABSTRACT
This study evaluated the attachment, chemo-attractive, proliferative and mineralization inductive potential of a bovine cementum extract (CPE) on newborn murine dental follicle cells (MDFC) in vitro. Cementum extract was partially purified by DEAE-cellulose chromatografy. A band representing and Mr of 55,000 was excised form the fel and the protein (s) were electroeluted. Attachment assays revealed that CPE (1.0 µg/ml) promoted MDFC attachment by 96 percent in comparison with collagen type I (5 µg/ml), and was five-fold greater compared with serum-free media (SFM), (p<0.05). Between 1 and 5 days CPE at 1.0 µg/ml and collagen type I at 5 µg/ml sustained more than 75 percent attachement and spreading of MDFC when compared to SFM (P<0.05). Contrary to other reports, fibronectin (0.5 µg/ml) was more potent than CPE in promoting MDFC chemoattraction (P<0.05). MDFC proliferation was stimulated by CPE (0.125 µg/ml), but this response was elicited only when CPE was used together with 10 percent FBS (37.3 percent) or 0.2 percent FBS (76 percent) (p<0.05). Alkaline phosphatase expression by MDFC was increased by CPE (1.0 µg/ml), in comparison to the control. Calcium deposits were detected by von Kossa staining in 14-day MDFC cultures treated with CPE. Nodule formation and its mineralization in long-term MDFC cultures were induced by CPE (1.0 µg/ml). Molecules(s) contained in CPE appear to regulate various biological activities in MDFC, indicating that CPE could play a key role in selecting progenitor cells required for the process of cementogenesis during development
Subject(s)
Animals , Cell Adhesion Molecules , Chemotaxis/drug effects , Dental Cementum/chemistry , Dental Sac/cytology , Dental Sac/drug effects , Cell Division , In Vitro Techniques , Proteins/pharmacology , Tissue Extracts/pharmacologyABSTRACT
Hertwing's epithelial root sheath (HERS) cells were isolated and recombined with ectomesenchymal cells in vitro utilizing extracellular matrix components as substrate. After 14 days in culture, HERS cells were differnetiated and exhibited a stratified organization. These features resembled those observed in vivo as epithelial rests of Malassez. A mineralization process was also present in HERS cells, in which calcium salts were deposited. This mineralization was correlated with the strong immunoexpression of osteopontin by HERS. The results obtained add support to the possible role of HERS in the secretion of Hypocalcified material on the root during early cementogenesis