Retinoic Acid Induces Apoptosis‐Associated Neural Differentiation of a Murine Teratocarcinoma Cell Line

: Incubation with all‐trans retinoic acid (RA) induces PCC7‐Mz1 embryonic carcinoma cells to cease proliferation and to develop into a tissue‐like pattern of neuronal, astroglial, and fibroblast‐like derivatives over a period of several days. Concomitant with the induction of differentiation by RA, a sizable fraction of the Mz1 stem cells detaches and dies, with the maximal level of cell death achieved after 10 h of RA treatment. This RA‐induced cell death fulfills all criteria of apoptosis, including nuclear condensation, intranucleosomal DNA degradation, expression of cysteine aspases (caspases), and the formation of apoptotic bodies. Apoptosis could be suppressed by the pan‐caspase inhibitor zVAD‐fmk (benzyloxycarbonyl‐valinyl‐alaninyl‐aspartyl fluoromethyl ketone). Induction of apoptosis required at least 2 h of incubation with RA and followed the same RA concentration (EC50 = 10−7M RA) and time dependence as the induction of differentiation as delineated by the expression of the neuron‐specific protein kinase C substrate GAP‐43. RA‐induced apoptosis increased with the plating density of PCC7‐Mz1 cells. This effect was not due to deprivation of an essential nutrient or factor from the medium because apoptosis was not significantly affected by an increase of the concentration of fetal calf serum. In addition to RA, apoptosis could be induced by DNA‐damaging treatment (UV light, cisplatin, methanesulfonic acid methyl ester) and cell cycle‐arresting agents (hydroxyurea) as well as by serum depletion. Because inhibition of transcription and translation caused cell death efficiently even in the presence of serum, the synthesis of apoptosis‐inhibiting factors by the cultured cells is indicated. Neither Apol/Fas antibody nor glutamate induced apoptosis. Mz1 cells that have entered a differentiation pathway in response to RA treatment become increasingly less sensitive to apoptosis. This may be due in part to the expression of the bcl‐2 proto‐oncogene, which was detectable on the mRNA and protein level beginning 4 days after the addition of RA. The intracellular signaling pathway leading to apoptosis does not involve conventional or novel members of the protein kinase C gene family. Neither activation of protein kinase C by phorbol esters (phorbol 12,13‐dibutyrate) nor inhibition by specific inhibitors (GF109203X, Gö 6976) and long‐term treatment with phorbol 12,13‐dibutyrate, in the presence or absence of RA, significantly influenced the amount or rate of apo