Association of increased radiocurability of murine carcinomas with low constitutive expression of p21^WAF1/CIP1 protein☆

Abstract 

Purpose: The study investigated whether basal, constitutive levels of p21^WAF1/CIP1 protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21^WAF1/CIP1 protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line.

Methods and Materials: Protein levels of p21^WAF1/CIP1, p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis.

Results: Protein levels of these oncogenes varied among tumors, with p21^WAF1/CIP1 showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21^WAF1/CIP1expression: tumors with high levels of p21^WAF1/CIP1were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation (p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages of apoptosis were more curable by radiation. Despite a strong trend to correlation, (p = 0.15), p21^WAF1/CIP1 expression did not correlate significantly with radiation-induced apoptosis, which suggested that p21^WAF1/CIP1 influenced tumor radioresponse by mechanisms beyond that of apoptosis induction.

Conclusion: Our findings showed that murine tumors exhibit wide variation in constitutive levels of p21^WAF1/CIP1 which had a significant relationship with tumor radioresponse: tumors with high levels of p21^WAF1/CIP1 were less radiocurable than those with lower levels. These findings support the concept that p21^WAF1/CIP1 is a major determinant of tumor radioresponse in vivo, and may have important clinical implications. The pretreatment assessment of p21^WAF1/CIP1 protein could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality.

Keywords:  p21^WAF1/CIP1, Murine tumors, Radioresponse, Apoptosis