α2a-interferon-induced differentiation of human alveolar rhabdomyosarcoma cells: Correlation with down-regulation of the insulin-like growth factor type I receptor

Rhabdomyosarcoma, a tumor of skeletal muscle origin, appears developmentally arrested at an early stage in the myogenic differentiation pathway. The proliferation of an alveolar rhabdomyosarcoma cell line Rh30 is dependent on the insulin-like growth factor (IGF) II/IGF-I receptor (IGF-IR) signaling pathway and is highly sensitive to recombinant human IFN-α2a, which induces growth arrest and differentiation of these malignant myoblasts. IFN-α2a-induced growth arrest of Rh30 cells was observed within 48 h, and reduction in colony formation was obtained with an IC₅₀ of 0.81 IU/ml for 72 h exposure. Down-regulated expression of IGF-IR was apparent by 24 h after initiation of IFN-α2a treatment. Furthermore, an initial increase followed by reduced expression of MyoD, in concert with elevated expression of myogenin, increased frequency of skeletal muscle myosin-positive cells, and the formation of multinucleated cells, indicated an enhancement of differentiation of Rh30 cells in the presence of IFN-α2a. To probe the role of IGF-IR in the differentiation of Rh30 cells along the myogenic lineage, the effect of antisense RNA-mediated reduction of endogenous IGF-IR on growth and expression of muscle-specific proteins was determined. Rh30 cells transfected to stably express antisense IGF-IR (clone AS23) showed significant reduction in growth rate, decreased expression of IGF-IR protein, increased expression of MyoD, myosin heavy chain, and an increased number of multinucleated cells in comparison to the parental line. These data are consistent with overexpression of IGF-IR inhibiting differentiation. IFN- α2a treatment of AS23 cells further induced both MyoD end myogenin expression, thereby allowing cells to proceed further downstream of the differentiation pathway.