Abstract
Cells of the intervertebral disc exist in an unusual environment compared to those of other tissues. Within the disc there are low levels of nutrients available, low oxygen levels and it is an acidic environment due to high lactate levels. Apoptosis (programmed or controlled cell death) has been reported in intervertebral discs, as well as necrosis (uncontrolled cell death). This study has focused on examining the sensitivity of nucleus pulpo-sus (NP) cells to several stimuli, in comparison to two other cells types.
Ultra violet (UV) irradiation, serum starvation (with no foetal calf serum) and treatment with 2mM hydrogen peroxide were used to induce apoptosis in cultured bovine NP cells, HeLa (cancer cell line) and 293T cells (human embryo kidney derived) cells. Apoptosis was identified by nuclear morphology following staining with fluorescent Hoechst 33342 dye and propidium iodide; the incidence was measured at 24, 48 and 72 hours. Untreated controls were used for each treatment and at each time point.
The incidence of apoptosis increased with time for all treatments. After 72 hours, UV treatment produced the highest levels of apoptosis with levels of apoptosis occurring in the order of HeLa (94%) > NP cells (29%) > 293T cells (15%). Treatment with hydrogen peroxide and serum starvation induced apoptosis at lower levels in all three cell types (maximum of 30%). Serum starvation induced apoptosis in only 10% of NP cells at 72 hours, compared to 20% in HeLa cells. None of the controls contained apoptotic cells.
NP cells are stimulated to apoptose in response to UV irradiation, hydrogen peroxide and serum starvation. However, levels of apoptosis are much lower after UV treatment in comparison to HeLa cells (3 times lower), suggesting that they may have a protective mechanism to this apoptotic stimulus, compared to HeLa cells. The low levels of apoptosis observed in NP cells with serum starvation may be due to the low nutrient environment that they exist in normally.
Correspondence should be addressed to Mr Carlos Wigderowitz, Senior Lecturer, University Department of Orthopaedic and Trauma Surgery, Ninewells Hospital and Medical School, Dundee DD1 9SY.
