The Eschenchia coli cytoplasmic membrane protein, p12, stimulates the protein translocation activity reconstituted with SecY, SecE and SecA. The gene encoding p12, which is located at 69 min on the E.coli chromosome, was deleted to examine the role of p12 in protein translocation in vivo. The deletion strain exhibited coldsensitive growth. Pulse-chase experiments revealed that precursors of outer membrane protein A, maltose binding protein and 3-lactamase accumulated at 20°C but not at 37°C. The deletion strain harboring a plasmid which carries the gene encoding p12 under the control of the araBAD promoter was able to grow in the cold when p12 was expressed with the addition of arabinose. Furthermore, the accumulated precursors were rapidly processed to the mature forms upon the expression of p12. Immunoblot analysis revealed the steady-state accumulation of precursor proteins at 20°C, whereas the accumulation was only marginal at 37°C, indicating that the function of p12 is more critical at 20°C than at 37°C. Finally, proteoliposomes were reconstituted with or without p12 to demonstrate that the stimulation of the activity by p12 increases with a decrease in temperature. From these results, we concluded that p12 is directly involved in protein translocation in E.coli and plays a critical role in the cold. We propose the more systematic name, SecG, for p12.

Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative. Amrubicin and its C-13 alcohol metabolite, amrubicinol, inhibited purified human DNA topoisomerase II (topo II). Compared with doxorubicin (DXR), amrubicin and amrubicinol induced extensive DNA-protein complex formation and double-strand DNA breaks in CCRF-CEM cells and KU-2 cells. In this study, we found that ICRF-193, a topo II catalytic inhibitor, antagonized both DNA-protein complex formation and double-strand DNA breaks induced by amrubicin and amrubicinol. Coordinately, cell growth inhibition induced by amrubicin and amrubicinol, but not that induced by DXR, was antagonized by ICRF-193. Taken together, these findings indicate that the cell growthinhibitory effects of amrubicin and amrubicinol are due to DNA-protein complex formation followed by double-strand DNA breaks, which are mediated by topo II.Key words: Amrubicin -Anthracycline -DNA-protein complex -Double-strand DNA break -DNA topoisomerase II DNA topoisomerase II (topo II) is a nuclear enzyme that regulates DNA topology through strand breakage, strand passage and religation. Thus, topo II is extensively involved in DNA metabolism, including replication, transcription, recombination and sister chromatid segregation.1) Mammalian topo II is the primary cellular target of a number of potent antitumor agents such as doxorubicin (DXR), daunorubicin (DNR), etoposide and amsacrine (m-AMSA).2) These agents interfere with the breakagereunion reaction of topo II by trapping a covalent enzyme-DNA complex, termed "cleavable complex," in which DNA strands are broken and their 5′ termini are covalently linked to the protein. In general, the cytotoxicity of these topo II poisons is dependent on stabilization of the cleavable complex followed by topo II-mediated DNA damage, rather than inactivation of topo II cellular functions. 3)Amrubicin is a novel, completely synthetic 9-aminoanthracycline derivative, and has antitumor activities in murine experimental tumor systems and human tumornude mouse systems. 4,5) Like other anthracycline derivatives, such as DXR and DNR, amrubicin is converted to its C-13 alcohol metabolite, amrubicinol.6) In contrast to doxorubicinol and daunorubicinol, amrubicinol has much higher antitumor activity than the parent drug in vitro. 6, 7)In addition, amrubicin showed much weaker cardiotoxicity than DXR in a rabbit chronic experimental model. 8)Phase II clinical trials of amrubicin for the treatment of malignant lymphoma, superficial bladder, small cell lung carcinoma, and non-small cell lung carcinoma are in progress. 9,10) Amrubicin showed substantial activity (response rate of 25%) against non-small cell lung cancer, and a response rate of 78.8% was obtained against small cell lung cancer.The mechanisms underlying the antitumor activities of amrubicin and amrubicinol are not well understood. Our present study has shown that amrubicin and amrubicinol target human topo II by stabilizing the cleavable complex. Furthermore, amrubicin and amrubicinol induced DNApro...

Background Miriplatin (formerly SM-11355), a novel lipophilic platinum complex developed to treat hepatocellular carcinoma, is administered into the hepatic artery using an oily lymphographic agent (Lipiodol Ultra-Fluide ® ) as a carrier. We clariWed the usefulness of miriplatin as an agent for transarterial chemoembolization. Methods Platinum compounds released from miriplatin into serum, medium and Earle's balanced salt solution were examined. Then, miriplatin and cisplatin were administered to rats bearing hepatoma AH109A tumors in livers. Platinum concentrations in tissues and DNA were assessed. Results Miriplatin showed a more sustained release than cisplatin. Dichloro[(1R, 2R)-1, 2-cyclohexane diamine-N, NЈ]platinum, the most abundant platinum compound released from miriplatin, was as eVective as cisplatin in inhibiting the growth of cells. Miriplatin was selectively disposed of in tumors, maintained in tumors longer than cisplatin and caused apparent tumor regression inducing platinum-DNA adducts to form and massive apoptosis. Conclusion Miriplatin appears to be a suitable chemotherapeutic agent for transarterial chemoembolization.

SecG is an integral membrane component of E. coli protein translocase. However, a discrepancy exists as to the importance of SecG for protein translocation at 37°C between cells and reconstituted proteoliposomes; protein translocation in AsecG cells is defective at 20°C but normal at 37°C, indicating that SecG is dispensable at 37°C, whereas SecG remarkably stimulates protein translocation into reconstituted proteoliposomes at 37°C. In this study, protein translocation into membrane vesicles containing or not containing SecG was examined in the presence and absence of the proton motive force at 37°C and 20°C. We found that the absence of the proton motive force renders protein translocation strongly dependent on SecG even at 37°C. Protein translocation into proteoliposomes in the absence of the proton motive force thus required SecG whereas that in cells, which always generate the proton motive force, did not.

Miriplatin is a lipophilic platinum complex which contains myristates as leaving groups and diaminocyclohexane as a carrier ligand. In order to examine in vivo the antitumor activities of miriplatin suspended in an oily lymphographic agent (Lipiodol Ultra-Fluide ® , LPD) against human hepatocellular carcinoma (HCC) after the intra-hepatic arterial administration, we have developed a novel orthotopic model of HCC in which the human hepatoma cell line Li-7 was successively implanted and maintained in the liver of nude rats. Li-7 tumors established in nude rat livers displayed a trabecular structure similar to their original morphology, and were exclusively supplied by the hepatic artery, suggesting that they exhibited in part the conditions of human HCC. Miriplatin suspended in LPD (miriplatin/LPD) administered into the hepatic artery of this model dose-dependently inhibited the growth of Li-7 tumors without markedly enhancing body weight loss and caused a significant reduction in the growth rate at a dose of 400 mg/head compared to LPD alone. In addition, at the therapeutic dose, miriplatin/LPD as well as cisplatin suspended in LPD (400 mg/head) was shown to be more active than zinostatin stimalamer suspended in LPD (20 mg/head) against Li-7 tumors after a single intra-hepatic arterial administration. These results suggest miriplatin to be a suitable candidate for use in transarterial chemoembolization (Cancer Sci 2009; 100: 189-194) H epatocellular carcinoma (HCC) is one of the most common cancers, and its incidence is increasing worldwide due to the dissemination of hepatitis B and C viral infections.(1-3) In addition to surveillance, advances in imaging and serum tumor markers have made early diagnosis possible. However, the prognosis remains poor because curative treatments such as surgical resection, liver transplantation, and percutaneous ablation are applicable to only a small proportion of patients. Most patients with HCC are diagnosed at the intermediate to advanced stage with hepatic impairment caused by virus-associated cirrhosis or chronic hepatitis. For these patients, transarterial chemoembolization (TACE) is a treatment option with survival benefit. Recent randomized controlled trials indicated that TACE induced marked objective responses and improved the survival of selected patients with HCC. (4,5) The liver receives a dual blood supply from the hepatic artery and the portal vein, whereas HCC is fed by only the hepatic artery.(6-8) On the basis of this peculiar feature of HCC, the efficacy and selectivity of antitumor agents can be increased by injecting them in combination with embolic material or the oily lymphographic agent (Lipiodol Ultra-Fluide ® , LPD) through the hepatic artery. LPD is known to be selectively distributed and retained for a long period in hepatic tumor tissues after intrahepatic arterial injection.(9-12) However, few antitumor agents currently used in TACE utilize LPD as a solvent and carrier because of their water-solubility. Ideal antitumor agents for intraarterial inject...