Cellular DNA was stained and flow cytometry was performed as described above

As opposed to these markers, appearance of the mitochondrial chaperone Hsp60 and t actin are unchanged throughout the differentiation process. Lastly, we also determined the relative quantity of cells in order Cyclopamine culture after six days of treatment using media containing FBS or RA to examine whether proliferative arrest was occurring during the differentiation process. Not surprisingly, serum withdraw and treat ment with RA reproducibly led to a,60% decrease in cell number, while combined treatment with RATPA made a 50% decrease in cell number for each neuroblastoma cell lines, Collectively, these data demonstrate that treatment of neuroblastoma cells with RA or RATPA produces most of the phenotypes consistent with neuronal differentiation. Differentiation Shifts Sensitivity of Neuroblastoma Cells to 6 OHDA in Cell Autonomous Vogue Differentiation of neuroblastoma cells toward a neuronal phenotype results in measurable improvements in susceptibility to oxidative Endosymbiotic theory stress, To demonstrate this change in oxidative stress resistance, we conducted dose-response survival assays on neuroblastoma cells using 6 OHDA. Undifferentiated SH SY5Y and SK N SH cells cultured in media containing FBS show an immediate decline in survival in reaction to escalating 6 OHDA concentration, using 50percent lethal dose toxicity values of 16. 562. 6 mM and 24. 262. 2 mM, respectively, Dif ferentiation over a six day timecourse with RA or RATPA, but, reproducibly encourages a shift in some OHDA resistance. In RA just ailments, SH SY5Y and SK N SH cells illustrate,LD50 values of 31. 462. 2 millimeter and 32. 862. 2 mM. The LD50 values order SL-01 are further increased by addition of TPA after three days to 43. 561. 9 mM and 44. 862. 9 millimeter, respectively. Importantly, these improvements in 6 OHDA sensitivity appear to result from a general resistance to oxidative stress as opposed to inhibition of mitochon drial function which in addition has been attributed to 6 OHDA, since differentiation of both cell lines had no influence on their sensitivity to some broad panel of mitochondrial electron transport chain inhibitors, Low cell independent factors secreted by differentiated cells, which may basically offer neuroprotective effects by stimu lating cell survival or scavengingdetoxifying oxidative species, wouldbe likely to guard undifferentiatedna ng neuroblasto ma cells from 6 OHDA toxicity.