Characterization of mineral deposits formed in cultures of a hamster tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell line (CCP).
We have established tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) double-positive cell lines (CCP-2, CCP-7, CCP-8) from hamster bone marrow. Accumulation of mineral deposits was observed on the dishes when the clones were cultured in McCoy's 5A medium supplemented with 20% fetal calf serum. The materials were dissolved in 0.05 N HCl, and proteins found in the acid extracts were identified by N-terminal amino acid sequencing. The major components were bovine fetuin and prothrombin precursor. In addition, several cell-derived proteins, such as high mobility group 1 protein (HMG1), secretory leukocyte protease inhibitor (SLPI) and EPV20, a 2.0-kDa milk glycoprotein, were identified. HMG1 was detected, by immunostaining, on the cell surface of all the CCP clones. Metabolically labeled cellular sphingomyelin, sialyllactosylceramide, and proteoglycans were also found in the mineral deposits. Reverse transcription/polymerase chain reaction of CCP-2 mRNA revealed that the cells synthesized alkaline phosphatase, bone sialo protein, and osteonectin, but not matrix Gla protein, osteopontin, and type I collagen. CCP-2 cells formed tumors when injected subcutaneously into nude mice. In the tumor tissue, Alizarin-red-positive nodules surrounded by TRAP- and ALP-positive cells were observed, indicating CCP-2 cells can also induce calcification in vivo.
Sakiyama
Hisako
H
Division of Oncology and Biology, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku Chiba 263-8555, Japan. sakiyama@nirs.go.jp
Nonaka
Takashi
T
Masuda
Riako
R
Inoue
Naokazu
N
Kuboki
Yoshinori
Y
Iijima
Mayumi
M
Hirabayasi
Yoshio
Y
Takahagi
Masahiko
M
Yoshida
Kazuko
K
Kuriiwa
Kazuko
K
Yoshida
Michiteru
M
Imajoh-Ohmi
Shinobu
S
D016475:3T3 Cells; D000135:Acid Phosphatase / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D002113:Calcification, Physiologic; D002460:Cell Line; D006224:Cricetinae; D024243:HMGB1 Protein / Q000032:analysis; D007279:Injections, Subcutaneous; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D051379:Mice; D008819:Mice, Nude; D008903:Minerals / Q000737:chemistry / Q000378:metabolism*; D015676:Osteonectin / Q000096:biosynthesis; D011509:Proteoglycans / Q000032:analysis; D000071681:Tartrate-Resistant Acid Phosphatase
D015415:Biomarkers; D024243:HMGB1 Protein; D007527:Isoenzymes; D008055:Lipids; D008903:Minerals; D015676:Osteonectin; D011509:Proteoglycans; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase