Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer.

SummaryThe relationship between DNA double-strand break rejoining rates, inherent radiation sensitivity and tumour response to radiation therapy was determined for a group of 25 squamous cell carcinoma (SCC) and eight sarcoma (SAR) tumours. DNA double-strand break frequencies were measured by neutral filter elution in first passage following explant tumour samples after in vitro exposure to 100 Gy of 6OCo gammarays. There was no significant difference between SCC and SAR tumour cells in their sensitivity to break induction, but in a 1 h time period SAR tumour cells rejoined significantly fewer breaks than SCC tumour cells, consistent with the greater sensitivity of SAR and suggesting that differences in rates of break rejoining account for the different distributions of radiosensitivities seen when different tumour types are compared. The percentage of breaks rejoined in 1 h in these tumour samples correlated well with Do and with the ,B component of the survival curve, measured in vitro by clonogenic assay in tumour cell lines established from the tumour samples, but not with SF2 or the a component of the survival curve. The rates of DNA double-strand break rejoining therefore appear to influence the exponential portion of survival curves and probably the interactions between breaks. The percentage of breaks rejoined in 1 h was higher in SCC tumours that subsequently failed radiotherapy and, although the differences were not significant, they suggest that rates of break rejoining are an important component of tumour response to radiation therapy.Keywords: predictive assay; DNA double-strand break repair; squamous cell carcinoma; sarcomaThe inherent sensitivity of cells within a tumour is thought to be an important component of radiation response. Both Fertil and Malaise (1985) and Deacon et al. (1984) reported large variations in the initial portions of in vitro survival curves from tumour cells of various histological types that they suggested might be related to radiotherapy response in vivo. Tumour types that are more difficult to control by radiotherapy produced cell lines that were more resistant to radiation in vitro. Weichselbaum et al. (1988a) reported that head and neck squamous cell carcinoma (SCC) tumour cells from radiotherapy failures were more resistant to radiation, as measured by in vitro survival curve analysis, than tumour cells derived from SCC pretreatment samples. Several investigators have examined the predictive value of in vitro survival curve measurements, principally SF2, the survival level after a 2 Gy exposure, with varying success (Brock et al., 1990;West et al., 1992West and Hendry, 1993;Girinsky et al., 1992 were faster in the more radioresistant cell lines (Schwartz et al., 1988). We subsequently confirmed this observation measuring DNA double-strand break rejoining with pulsedfield gel electrophoresis (Giaccia et al., 1992) and showed that chromosome break rejoining kinetics were also faster in the more resistant cells (Schwartz and Vaughan, 1989). Our studies suggest that the...

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