Immortalized human bronchial epithelial cells (BEAS-2B) expressing the c-raf-1 and c-myc genes induced large cell carcinomas in athymic nude mice with neuroendocrine features. Both BEAS-2B cells transfected with either c-raf-1 (2B-raf) or c-myc (2B-myc) showed elevated MRNA levels of neuron-specific enolase (NSE) consistant with earlier findings of neuro-endocrine markers in nude mice tumors induced by c-raf-1 and c-myc. This effect was additive in BEAS-2B cells carrying c-raf-1 and c-myc (2B-raf/myc), and derived human tumor cell lines. Applying c-raf-1 or murine c-myc-specific antibodies the expression of proto-oncogenes was specifically demonstrated in immunoprecipitations of transfected BEAS-2B lines. Using a highly efficient retrovirus isolation/infection protocol, the reproducible induction of tumors with c-raf-1 in combination with C-MYC was confirmed by infecting 2B-raf or 2B-myc cells with amphotropic c-raf-1 or c-myc recombinant virus. Experiments analyzing the function of the c-raf-1 kinase in malignant transformation showed the highest activity in 2B-raf and 2B-raf/myc cells followed by 2B-myc, BEAS-2B and normal human bronchial epithelial (NHBE) cells. Tumorigenic BEAS-2B cells transfected with various mutant ras genes expressed elevated c-raf kinase in a p2l protein-dependent manner. The association of oncogenes, radiation resistance, and tumorigenicity was tested in the tumorigenic human laryngeal carcinoma, SQ-20B, by introduction of sense (raf) and anti-sense (far) human c-raf-I CDNA sequences. The malignant potential of SQ-20B and the radiation-resistant phenotype was significantly increased in the presence of raf and decreased in the presence of far sequences. Sl nuclease protection assays revealed a specific reduction of steady-state levels of the endogenous c-raf-I sense transcript in SQ-20B cells transfected with far DNA. In accordance, transfection of BEAS-2B cells with c-raf-I or v-Ha-ras but not c-myc induced the expression of a radiation-resistant phenotype.
