Differentiation of neuroblastoma cells promotes resistance to 6-OHDA toxicity.

<p><b>A</b>, SH-SY5Y cells were plated and allowed to adhere overnight, then differentiated with 10 µM retinoic acid only (six days) or with retinoic acid and 100 nM TPA (three days each). Cells were subsequently treated for 24 hours with different doses of 6-OHDA diluted in serum-free NBA/B27 media and analyzed for cellular viability. Relative cell number was normalized to untreated cells and plotted according to 6-OHDA concentration. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) comparing treated samples to untreated controls. <b>B</b>, SK-N-SH cells were treated and assayed as in A. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing treated samples to untreated controls. <b>C</b>, SH-SY5Y cells were plated and allowed to adhere overnight. They were subsequently treated with the indicated doses of 6-OHDA diluted either in fresh NBA media (alone or containing 10% FBS, 10 µM RA or 100 nM TPA), or in conditioned media harvested from SH-SY5Y cells that had been cultured in 10% FBS or differentiation media (RA only, TPA only or RA/TPA) for six days. After 24 hours the cells were analyzed for viability and relative cell number was determined relative to untreated controls. <b>D</b>, SK-N-SH cells were treated and assayed as in C. <b>E</b>, LD50 values ± SE for naïve SH-SY5Y and SK-N-SH cells cultured in fresh or conditioned media from each treatment paradigm. For all experiments, cells were treated and assayed as in C. Significance scores (<i>p</i>-value) are shown for comparisons between fresh and conditioned media (CM) for each condition. <b>F</b>, The ratio of LD50 values in conditioned versus fresh media for each media condition was calculated to determine whether media from conditioned cells contains factors that protect naïve/undifferentiated cells from 6-OHDA toxicity. Values >1 indicate a survival advantage in conditioned media, whereas values ≤1 indicate a lack of survival advantage or disadvantage in conditioned media.