CD99/MIC2 Constitutes a Differentiation Antigen of a Human Osteoblast Cell Line

Abstract

Background: The histological origin of the Ewing's family of tumors (EFT) is still not clear. Since these small cell bone tumors may originate from osteogenic stem cells, the presence of the CD99/MIC2 antigen, known to be overexpressed in EFT, was studied in a human osteoblast cell line in response to differentiation inducers.

Methods: The HBA-71 monoclonal antibody directed to the CD99/MIC2 antigen was used to stain a human osteoblast cell line as well as the two EFT cell lines KAL and EW-2 after pretreatment of the cells with the differentiation inducers calcitriol and the histone deacetylase (HDAC) inhibitors sodium butyrate (NaB), sodium phenylacetate (NaPA) as well as N, N'-hexamethylen-bis-acetamide (HMBA). Alkaline phosphatase (ALP) levels were determined as cellular differentiation marker.

Results: Significant expression of the CD99/MIC2 antigen, yielding a molecular weight of 32 kD in Western blotting, was found in the human osteoblast cell line. Pretreatment of the osteoblasts with calcitriol and HMBA increased ALP content and suppressed the CD99/MIC2 antigen. Calcitriol had no major effect on CD99/MIC2 expression of both EFT cell lines, but HMBA enhanced ALP activity in KAL cells and downregulated CD99/MIC2. EW-2 cells exhibited reduced levels of both CD99/MIC2 and ALP.

Conclusions: This study supports the role of CD99/MIC2 as differentiation antigen of osteoblasts and a Ewing's sarcoma cell line with neuroectodermal phenotype. Response to calcitriol is absent or low in the two EFT cell lines tested.

Keywords: CD99; Differentiation; Ewing’s sarcoma; HBA-71; Histone deacetylase inhibitor; MIC2; Osteoblast.

Figure 1

Flow cytometric analysis of CD99/MIC2 HBA-71 antigen expression of (A) KAL EFT cells and (B) AHTO-7 osteoblasts. Immunofluorescence of the cells stained with HBA-71 is shown in black, the NIC-1 isotype controls in grey (KAL) and white (AHTO-7), respectively.

Figure 2

CD99/MIC2 Western blot and effects of fixation, trypsin treatment and differentiation inducers on HBA-71 expression of AHTO-7 osteoblasts. The CD99/MIC2 antigen was blotted with the HBA-71 antibody using extracts of KAL and AHTO-7 cells, respectively. Protein amounts of β-actin are shown as controls. Relative fluorescence intensities are expressed as mean ± SD (n = 3). With the exception of NaB pretreatment, all differences to the control are statistically significant.

Figure 3

Effects of differentiation inducers on HBA-71 immunofluorescence of EW-2 and KAL EFT cells. Relative fluorescence intensities are expressed as mean ± SD (n = 3). All differences to the respective controls are statistically significant.

Figure 4

Changes in cellular ALP activity in response to differentiation inducers of AHTO-7, KAL and EW-2 cells. Activity of ALP is expressed as mean ± SD (n = 3). All differences to the control are statistically significant, except for 1, 25-vitD3 and NaB treatment of EW-2 cells.