Apoptosis-Related Gene and Protein Expression in Human Lymphoma Xenografts (Raji) After Low Dose Rate Radiation Using 67Cu-2IT-BAT-Lym-1 Radioimmunotherapy

Abstract

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to ⁶⁷Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, ⁶⁷Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 μg Lym-1, or 335-500 μCi ⁶⁷Cu on less than 400 μg Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-β₁ and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. ⁶⁷Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after ⁶⁷Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with ⁶⁷Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.