1, 25-Dihydroxyvitamin D₃, Recombinant Human Transforming Growth Factor-β₁, and Recombinant Human Bone Morphogenetic Protein-2 Induce In Vitro Differentiation of Canine Osteosarcoma Cells

Abstract

ABSTRACT. Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-β1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-β1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-β1. These agents are potential inducers of apoptosis.-KEY WORDS: BMP-2, differentiation, osteosarcoma, TGF-β1, vitamin D3.J. Vet. Med. Sci. 61(6): 649-656, 1999 1,25(OH)2D3 also enhanced antitumor chemotherapy both in vitro and in vivo [32]. TGF-β1, one of the most abundant growth regulatory factors in bone matrix, has complex effects. These include growth inhibition as well as a stimulation of both ALP activity and the production of bone matrix proteins such as collagen, osteonectin and fibronectin in osteoblasts [23]. BMPs (BMP-2 to BMP-15) belong to the TGF-β superfamily, and BMP-2, 3, 4 and 7 induce or enhance osteoblast differentiation [36]. Previously, four separate sublines of cells were established from the POS canine osteosarcoma cell line [10,24]. These sublines are immortal and vary from one another in morphology and ALP activity. The purpose of this study is to clarify the effects of 1,25(OH)2D3, TGF-β1 and BMP-2 on differentiation in these osteosarcoma cell lines. MATERIALS AND METHODS Cells:Four canine osteosarcoma cell sublines (POS53B, 53C, 53D, and 14A) were obtained from a POS parental cell line by limiting dilution as described previously [24]. POS53B forms chondroblastic-type tissue when transplanted into nude mice. Similarly, POS53C, 53D, and 14A form undifferentiated-, osteoblastic-, and fibroblastic-type tissues, respectively. Each cell line was maintained in RPMI 1640 medium (Nissui Seiyaku Co., Tokyo) supplemented with 10% fetal bovine serum (FBS) (Cell Culture Lab., Cleveland, OH), L-glutamine (Nissui) and antibiotics (50 mg/l gentamycin sulfate and 1.5 mg/l amphotericin B) at 37°C in a humidified atmosphere of 5% CO2.

Osteosarcoma Development and Stem Cell Differentiation

“…1B). This model is supported by the following facts: First, OS tumors exhibit the characteristics of undifferentiated osteoblasts [26,75,83,181,195,196,219,233,277]. Second, differentiation-promoting agents (eg, PPARc agonists and 9-cis-retinoic acid) induce osteoblast differentiation [75,76].…”

Osteogenic BMPs promote tumor growth of human osteosarcomas that harbor differentiation defects

“…32 Human OS tumors usually exhibit osteoblast-like features. 3,[33][34][35][36][37][38][39] We demonstrated that differentiation-promoting agents (eg, PPARg agonists and 9-cis retinoic acid) can induce osteoblast differentiation and inhibit OS cell proliferation. 3,40 Here, we investigate whether the disruption of osteoblast differentiation plays a role in OS development.…”

Hyperphosphatasemia and concurrent adrenal gland dysfunction in apparently healthy Scottish Terriers

“…3,4,6,13,14 Other causes of hyperphosphatasemia in adult dogs include increased osteoblastic activity associated with increased bone modeling (or remodeling) and lytic lesions of bone. 4,6,[14][15][16][17][18] Scottish Terriers reportedly have higher ALP activities than do other breeds. 19 It has been suggested that high ALP activity in otherwise healthy Scottish Terriers may possibly be attributable to excessive activity of the bone ALP isoform, which is the mechanism of benign familial hyperphosphatasemia, a rare inherited condition in Siberian Huskies.…”