CRABP I expression and the mediation of the sensitivity of human tumour cells to retinoic acid and irradiation

Purpose: To examine the role cytoplasmic retinoic acid binding protein type 1 (CRABP I) and retinoic acid receptor beta2 (RAR-beta2) in mediating radiosensitization of human tumour cells in vitro by retinoic acid. Materials and methods: Human squamous cell carcinoma cell lines of different types were treated with retinoic acid followed by irradiation. Radiation response under drug treatment was detected by colony-formation assay. mRNA and protein expression levels of CRABP I, RAR-beta and cyclin D1 were investigated under different treatment conditions by room temperature polymerase chain reaction and Western blotting. The retinoic acid-sensitive cell line HTB35 was transfected for inducible CRABP I overexpression to test the role of this protein in modulating the sensitivity to retinoic acid and radiation as well as in regulating RAR-b2 and cyclin D1 expression. Results: The basal CRABP I level clearly correlated with the clonogenic survival of tumour cells and normal fibroblasts after treatment with retinoic acid and ionizing irradiation (IR). Cells expressing high basal CRABP I were more resistant to combined retinoic acid radiation treatment than cells with low basal expression. Overexpression of CRABP I in retinoic acid-sensitive HTB35 cells induced a retinoic acid-insensitive phenotype resistant to combined treatment with retinoic acid and radiation. This effect was independent of RAR-b2 expression. CRABP I overexpression resulted in stimulated cyclin D1 expression indicating the dependency of this cell cycle control protein on retinoic acid metabolism. Conclusion: CRABP I plays an important role not only in mediating the retinoid effects, but also in modulating the radiation sensitivity of tumour cells after combined retinoic acid radiation treatment.