SJG-136 is a pyrrolobenzodiazepine dimer and sequence-selective cross-linking agent that has shown activity in vitro in the NCI-60 panel as well as in vivo. The cytotoxicity of SJG-136 in a panel of 6 colon cell lines (HCT116, HT-29, SW620, HCT-8, HCT-15 and Colo205) has been shown to be time-dependent with the ratio of 1h/24h IC50s ranging from 39 to 105. The aims of this study were to evaluate the impact of SJG-136 on cell cycle distribution and to investigate the involvement of sensors of DNA damage such as ATM and ATR in addition to chk1 and chk2 (downstream kinases in DNA damage signalling) in relation to the duration of drug exposure. Cell lines were exposed to SJG-136 for 1h or 24h at their respective IC50s or to a fixed concentration of 1nM for 24h and were harvested at 6, 10, 24, 30, 48 and 72 hours after the beginning of drug exposure. When exposed to their respective IC50s, the most sensitive cell lines such as HCT116, HT-29, SW620 and Colo205 (IC50 = 21, 19, 13, 10 nM after a 1h exposure and 0.35, 0.3, 0.1 and 0.03 nM after a 24h-exposure, respectively) accumulated transiently in S phase then progressed to G2/M between 24 and 48h and to G0/G1 at 72h, while a sustained accumulation in S phase was observed in resistant cell lines. When the same cell lines were exposed to 1nM SJG-136 (IC70-IC90), the G2/M block was maintained up to 72h without any progression to G0/G1. Therefore cytotoxicity seems to be linked to a substantial and persistent block in G2/M. Western blotting analyses showed that ATR levels were high in both HCT-8 and HCT-15 but a decrease was observed in HCT-15 cells after exposure to SJG-136 for 24h at the IC50. In Colo205 and HT-29 cell lines the phosphorylation of chk2 was observed whatever the exposure but in HCT116 and SW620 only after 1nM SJG-136 for 24h. Chk2 protein could not be detected in HCT-8 and HCT-15 cells. The phosphorylation of chk2 could be linked to the G2/M block observed in the cell lines that were sensitive to SJG-136 and might be an important parameter to predict the extent of the cytotoxic effect. Chk1 phosphorylation was not seen in HT-29, HCT-8 or HCT-15 cells. In HCT116 no change in the level of phosphorylation was observed after exposure to SJG-136 while it decreased in SW620 cells. Chk1 phosphorylation does not seem to be directly linked to the cytotoxicity of SJG-136 but might be indirectly affected through a different pathway. Further studies are ongoing to address the role of chk2 phosphorylation in G2/M accumulation in these colon cell lines and to assess its predictive value for the cytotoxicity of SJG-136 and other cross-linking agents.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]