Pierisin-1 is a potent apoptosis-inducing protein derived from the cabbage butterfly, Pieris rapae. It has been shown that pierisin-1 has an A⅐B structure-function organization like cholera or diphtheria toxin, where the ''A'' domain (N-terminal) exhibits ADPribosyltransferase activity. The present studies were designed to identify the target molecule for ADP-ribosylation by pierisin-1 in the presence of -[adenylate-32 P]NAD, and we found DNA as the acceptor, but not protein as is the case with other bacteria-derived ADP-ribosylating toxins. ADP-ribosylation of tRNAs from yeast was also catalyzed by pierisin-1, but the efficiency was around 1 ⁄10 of that for calf thymus DNA. Pierisin-1 efficiently catalyzed the ADPribosylation of double-stranded DNA containing dG⅐dC, but not dA⅐dT pairs. The ADP-ribose moiety of NAD was transferred to the amino group at N 2 of 2 -deoxyguanosine to yield N 2 -(␣-ADP-ribos-1-yl)-2 -deoxyguanosine and its  form, which were determined by several spectral analyses including 1 H-and 13 C-NMR and mass spectrometry. The chemical structures were also ascertained by the independent synthesis of N 2 -(D-ribos-1-yl)-2 -deoxyguanosine, which is the characteristic moiety of ADP-ribosylated dG. Using the 32 P-postlabeling method, ADP-ribosylated dG could be detected in DNA from pierisin-1-treated HeLa cells, in which apoptosis was easily induced. Thus, the targets for ADP-ribosylation by pierisin-1 were concluded to be 2 -deoxyguanosine residues in DNA. This finding may open a new field regarding the biological significance of ADP-ribosylation. P ierisin-1, a 98-kDa protein, is abundantly present in the fifth instar larvae and the early phase of pupae in the cabbage butterfly, Pieris rapae (1, 2). This protein has potent cytotoxic activity against various human cancer cell lines with a wide range of IC 50 values, inducing typical apoptotic cell death with characteristic morphological features, DNA fragmentation, and cleavage of poly(ADP-ribose) polymerase (2, 3). The N-terminal region of pierisin-1 has a partial regional sequence similarity with ADP-ribosylating toxins such as the A-subunit of cholera toxin, and disruption of this possible NAD-binding site by site-directed mutagenesis abolishes its apoptosis-inducing activity (4). Inhibitors of ADP-ribosyltransferase also significantly reduce its ability to induce apoptosis of cancer cells. Recently, we found that pierisin-1 is likely to have an A⅐B structure-function organization similar to that of cholera and diphtheria toxins, the ''A'' domain featuring the ADP-ribosyltransferase activity and the ''B'' domain binding to receptors in cell membranes, thereby incorporating the A domain into cells for ADP-ribosylation of intracellular target molecules. It is also suggested that the glycolipid in the membrane was a possible candidate for the receptor of pierisin-1, and the sensitivity of cancer cells to pierisin-1 depended on the presence of the receptor in cell membrane. Moreover, treatment using either the N-terminal polypeptide (amino acids...
We have previously reported that the cabbage butterf ly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines. In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished. PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081. An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS͞PAGE, the same as that of purified native pierisin. The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity. The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1. In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin. A glutamic acid residue at the putative NAD-binding site, conserved in all ADPribosylating toxins, was also found in pierisin. Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis. Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisininduced apoptosis. These results suggest that the apoptosisinducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells.
We report the long-term results of Tokyo Children's Cancer Study Group's studies L84-11, L89-12, L92-13, and L95-14 for 1846 children with acute lymphoblastic leukemia, which were conducted between 1984 and 1999. The value of event-free survival (EFS)±s.e. was 67.2±2.2% at 10 years in L84-11, which was not improved in the following two studies, and eventually improved to 75.0±1.8% at 10 years in L95-14 study. The lower EFS of the L89-12 reflected a high rate of induction failure because of infection and delayed remission in very high-risk patients. The L92-13 study was characterized by short maintenance therapy; it resulted in poor EFS, particularly in the standard-risk (SR) group and boys. Females did significantly better than males in EFS in the early three studies. The gender difference was not significant in overall survival, partly because 460% of the males survived after the testicular relapse. Randomized studies in the former three protocols revealed that intermediate-or high-dose methotrexate therapy significantly reduced the testicular relapse rate. In the L95-14 study, gender difference disappeared in EFS. Contrary to the results of larger-scale studies, the randomized control study in the L95-14 reconfirmed with updated data that dexamethasone 8 mg/m 2 had no advantage over prednisolone 60 mg/m 2 in the SR and intermediate-risk groups. Prophylactic cranial irradiation was assigned to 100, 80, 44, and 44% of the patients in the studies, respectively. Isolated central nervous system relapse rates decreased to o2% in the last two trials. Secondary brain tumors developed in 12 patients at 8-22 years after cranial irradiation. Improvement of the remission induction rates and the complete omission of irradiation are currently main objectives in our studies. Leukemia (2010) 24, 383-396; doi:10.1038/leu.2009 published online 24 December 2009 Keywords: acute lymphoblastic leukemia; children; long-term results; cranial irradiation; secondary malignancy IntroductionWe present here the long-term results of four studies for childhood acute lymphoblastic leukemia (ALL) of Tokyo Children's Cancer Study Group (TCCSG) conducted between 1984 and Treatment protocol for SR and IR of the L84-11 study 1,2 was based on the early St Jude's total therapy.3 ALL-BFM 81 4 protocol was modified and introduced to extremely high-risk group regimen for the first time. The protocols of the following three studies L89-12, 1,5 L92-13, 1,6 and L95-14, 7 were designed on the basis of the ALL-BFM framework. All the four protocols contained trials to reduce the number of patients who received irradiation, as had been reported in other studies. 8,9 The second point of analysis was on a gender difference [10][11][12] with respect to long-term event-free survival (EFS) and overall survival (OS). Randomized studies were mostly designed to test whether or not intermediate-dose methotrexate (ID-MTX) and high-dose methotrexate (HD-MTX) could replace the cranial irradiation. It is needed to describe the further long-term outcome of the patie...
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