Bunsyo Shiotani

Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest evoked by pierisin-1.

Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N2 of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G2-M-phase delay. In the colony survival assays, Rad17−/− DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM−/− DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling. (Mol Cancer Res 2006;4(2):125–33)

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces apoptosis in rat splenocytes and thymocytes by different mechanisms

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a potent carcinogen present in cooked meat. Although the target of this carcinogen is mainly in the liver, Trp-P-1 is distributed in the thymus and spleen as well as in the liver after administration. However, the cytotoxic effect of Trp-P-1 on lymphocytes has not been examined in detail. In the present study, we investigated the cytotoxicity of Trp-P-1 against rat splenocytes and thymocytes. Trp-P-1 reduced viability of both types of cells in the same manner, the LD(50) at 6h in culture was 15 microM, and the time for the 50% decrease in cell viability (t(1/2)) at 20 microM was 3h. In both types of cells, Trp-P-1 caused the activation of caspase-3-like proteases and the cleavage of poly(ADP-ribose) polymerase, both of which are biochemical markers of apoptosis. On the other hand, DNA fragmentation occurred in splenocytes, but not in thymocytes although Trp-P-1 activated 32-34kDa nucleases that may not be able to degrade DNA into nucleosomal units. These results indicated that Trp-P-1 induces apoptosis in both splenocytes and thymocytes by different mechanisms in which distinct apoptotic pathways may exist downstream of the caspase cascade.

Evoking Cytochrome P450 1A Activity Interferes with Apoptosis Induced by 3-Amino-1,4-dimethyl-5H-pyrido [4,3-b]indole (Trp-P-1) in Rat Hepatocytes under the Ex Vivo System

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is known as a dietary carcinogen and it requires metabolic activation by cytochrome P450 (CYP) 1A subfamily to have carcinogenicity. On the other hand, our previous report demonstrated that Trp-P-1 induces apoptosis in primary cultured rat hepatocytes, but the metabolically activated Trp-P-1 added extracelluarly to hepatocytes did not induce apoptosis. In this study, we focused on the intracellular status of CYPs and investigated apoptotic events induced by Trp-P-1 using hepatocytes isolated from rats treated with three chemical inducers for CYPs. In cultured hepatocytes from rats treated with 3-methylchoranthrene, which mainly induces CYP 1A, Trp-P-1-induced apoptosis was suppressed. In the same cultures, intact Trp-P-1 was decreased and its metabolites were increased. Phenobarbital and pyridine did not affect Trp-P-1-induced apoptosis. These results suggested that evoking CYP 1A activity might interfere with apoptosis induced by Trp-P-1 in rat hepatocytes under the ex vivo system.