Constitutive expression of non-bone/liver/kidney alkaline phosphatase in human osteosarcoma cell lines

Abstract

Alkaline phosphatase (ALP) plays an important role in bone mineralization; high levels in differentiated osteoblasts allows their identification easily in vitro. It is generally assumed that the activity of ALP in osteosarcoma-derived cell lines commonly used in studies of bone cell biology is exclusively due to the bone/liver/kidney (BLK) isoenzyme. However, we noted that two human osteosarcoma cell lines, U-2 OS and U-393 OS, predominantly expressed a truncated 1.8 kb mRNA for BLK-ALP. This observation stimulated further investigation upon the ability of ALP to form functional protein. We found that, unlike the BLK-ALP of the Saos-2 osteosarcoma cell line, the activity of U-2 OS ALP was thermostable, unaffected by L-homoarginine and levamisole, but inhibited by L-phenylalanine; these properties are characteristic of the placental and/or placental-like (PL-/PL-like ALP) isoenzymes which are 98% homologous at the amino acid level. In the U-393 OS cell line, which expresses the normal-sized 2.5 kb mRNA in substantially higher levels than that produced by U-2 OS cells, the ALP activity had kinetic properties very similar to that produced by the Saos-2 line for all criteria tested. The HOS osteosarcoma cell line (also known as TE-85), which express the normal-sized 2.5 kb BLK-ALP mRNA only, exhibited ALP activity with kinetic properties of both the BLK and PL-/PL-like isoenzymes. The three test lines, U-2 OS, U-393 OS and HOS, produced PL-/PL-like ALP mRNA and protein constitutively, and levels of these increased in cells treated with 1 microM dexamethasone. However, dexamethasone treatment of cells did not alter the types of ALP isoenzyme expressed. Thus our results show that, like Saos-2 cells, U-393 OS cells produce active BLK-ALP exclusively, whereas U-2 OS cells produce PL-/PL-like ALP only, and the HOS cell line produces both. Our findings have important implications for phenotypic characterization of various human osteosarcoma cell lines, and suggest that the production of PL-/PL-like ALP may be a more common occurrence in osteosarcomas than was originally thought.

Possible prognostic role of IL-17R in osteosarcoma

“…However, we not exclude that the constitutively diVerent ability of the three cell lines to secrete VEGF might contribute to the degree of response to IL-17. U-2 OS presents the lowest diVerentiated osteoblast-like phenotype, followed by HOS, then MG63 (Scotlandi et al 1993;Clover and Gowen 1994;Ali et al 1996). Because malignancy and poor prognosis seem to depend on VEGF secretion (Kaya et al 2000;Oda et al 2006) and are associated with the degree of dediVerentiation of tumor cells (Axelson et al 2005;Staals et al 2006), we have compared the degree of diVerentiation in U-2 OS, HOS and MG63 by assaying intracellular Catepsin B and ALP enzymatic activities, which are accepted as opposite markers of diVerentiation.…”

Portland cement for bone tissue engineering: Effects of processing and metakaolin blends

“…21 ALP activity has been widely recognized as a marker for bone cell functionality. 27,34,35 Expression levels are usually elevated during dynamic extracellular matrix deposition. 36 ALP activity measurements did not reveal any potential influence of the MK content in the activity of this enzyme in the cell-culture medium, thus confirming that the introduction of MK primarily modulated cell compatibility but not cell synthetic activity.…”

“…ALP activity, an early marker of bone cell differentiation, 27 was evaluated in additional cell-culture experiments at days 2, 4, 6, and 8. Culture media was changed every 2 days.…”

Biochemical and proteomic investigation of bovine nasal secretion

“…While the liver has long believe to be the major site for tissue non-specific AP production, this enzyme has been considered to be present and expressed also in the mammalian kidney, bone and placenta (Goldstein et al, 1980;Hahnel et al, 1990;Ali et al, 1996;Prasad et al, 2005 andZimin et al, 2009). The tissue non-specific AP has also been reported to be present in brain tissue (Brun-Heath et al, 2011) and circulating B cells in human (Hossain and Jung, 2008).…”