Bone Abstracts

Ablation of osteosarcoma cells by sublethal hypothermia before radiation may increase sarcoma tissue sensitivity by inducing growth arrest. Normal cells that are not lethally damaged by hypothermia and radiation can undergo DNA repair thus promoting cell survival. Nevertheless, understanding of the response of normal bone forming osteoblast cells towards hypothermia is necessary before administering on osteosarcoma cells. In this study we evaluated the response of short-term moderate and severe hypothermia on Normal Human Osteoblast (NHOst) cell metabolism and growth markers. NHOst cells were exposed to moderate (35 °C) and severe (27 °C) hypothermia, and control at 37 °C for 12 h. NHOst cell metabolism was measured with MTS assay while rate of cell proliferation was calculated with Tryphan blue staining. Meanwhile changes in NHOst growth gene expression for Cdk1, Cdk2, Cdk4, and p21 (cell cycle progression), and Caspase 3, 8, 9, Bcl-2, and Bax (apoptosis) was quantitated using the RT² Profiler PCR Array. Flow cytometry further confirmed the rate of cell survival while phosphorylation of histone variant (H2AX Ser 139) as a marker for DNA damage was measured at 24 h. The mRNA fold change was statistically analyzed using Student’s t-test. NHOst cells remained metabolically active at 35 °C (103±0.32%) conversely at 27 °C (56.9±0.12%) cell metabolism was markedly inhibited (P<0.001). Results showed that NHOst proliferation was insignificantly reduced at 35 °C (0.76%) relative to control. Up-regulation of Cdk4 may suggest that hypothermia permits cell cycle re-entry (M/G1 phase). However, overexpression of p21, Cdk1, and Cdk2 mRNA tends to inhibit cyclin-dependent kinase activity signifying that cells are arrested at G1/S and S/G2 transitions. Both gene expression and flow cytometry results showed an increase in apoptosis at 27 °C. Although Caspase 3 was activated at 35 °C, the mRNA expression ratio between Bax and Bcl-2 was low (1.5:5.3). Flow cytometry data for 35 °C showed an increase of apoptosis by 3.54% relative to control. Interestingly, hypothermia did not induce DNA damage after 24 h. Our studies on moderate hypothermia (35 °C) at 12 h demonstrated a sublethal response towards normal bone cells by temporary arresting NHOst cells. Transient administration of moderate hypothermia on osteosarcoma cells before radiation may enhance radiosensitivity with minimal damage to normal cells.