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.

Ceramide accumulation is independent of camptothecin-induced apoptosis in prostate cancer LNCaP cells.

We have investigated to determine the source of ceramide produced during the genotoxic apoptosis induced by the anti-cancer drug, camptothecin (CPT), in human prostate cancer LNCaP cells by measuring the activities of acid and neutral sphingomyelinases (SMase) and by using fumonisinB(1) (FB(1)), the inhibitor of ceramide synthase involving de novo synthesis of ceramide. In contrast to time-dependent elevation of intracellular ceramide level after CPT-treatment, the activities of both SMases were not increased but rather decreased. Instead, pretreatment for 3 h with FB(1) (100 microM), an inhibitor of ceramide synthase, almost completely abrogated ceramide accumulation observed in cells exposed to CPT for 18 h. These results indicate that ceramide is produced via de novo pathway but not via sphingomyelin hydrolysis pathway. Furthermore, it is to be noted that the pretreatment with FB(1) did not affect the CPT-induced apoptosis as assessed by DNA ladder formation, Hoechst 33342 staining, flow cytometry, and mitochondrial potential thereby leading us to propose that ceramide accumulation is independent of apoptosis in this system.