The mechanism of inhibition of eukaryotic by itself has no effect on the binding of various forms of DNA to yeast DNA topoisomerase II. When both and ATP are present, however, the enzyme is converted to the closed form, as evidenced by the SV8 endoproteinase cleavage pattern as well as the characteristics of the DNAenzyme complexes. These results suggest that bis(2,6-dioxopiperazines) act by stabilizing eukaryotic DNA topoisomerase II in the closed-clamp form and preventing it from opening again. MATERIALS AND METHODS 1781The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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IDC-P in RP specimens was an independent risk factor for PFS and CSS and could predict clinical outcomes.

Abstract. , a novel noncleavable, complexstabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909--4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by . Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of ode2 kinase, spindle apparatus reorganization and disassembly and re, assembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes. topoisomerases (topo) t have been implicated in e maintenance of genetic processes such as repliation, transcription, and recombination by controlling the higher order chromosomal structure through the cell cycle (for review, see Wang, , 1987Cozzarelli and Address all correspondence to Ryoji Ishida,

Intraductal carcinoma of the prostate is an adverse prognostic factor in localized prostate cancer patients. However, whether it influences outcome of those patients with distant metastases discovered at initial diagnosis is unclear. Here, we evaluated whether the presence of intraductal carcinoma of the prostate in prostate needle biopsies is an adverse prognostic factor for cancer-specific survival and overall survival in such prostate cancer patients. We retrospectively enrolled 150 eligible patients. All patients received androgen-deprivation therapy and/or chemotherapy. Their age, performance status, pain, metastatic sites, clinical T stage, serum prostatespecific antigen, alkaline phosphatase, hemoglobin, Gleason score, and the presence of Gleason pattern 5 were analyzed. Primary end point was cancer-specific survival; secondary end points included prostate-specific antigen progression-free survival and overall survival. Fine and Gray's model and the Cox proportional hazards model were used as statistical tests. Intraductal carcinoma of the prostate was detected in 100 (67%) patients. At a median follow-up of 38 months, 79 patients (53%) had died of the disease and nine (6%) had died of other causes. The average time interval to cancer-related death was 28 months. On multivariate analysis, only intraductal carcinoma of the prostate was significantly associated with cancer-specific survival (P = 0.018) and overall survival (P = 0.001), and only the presence of Gleason pattern 5 was significantly associated with prostate-specific antigen progression-free survival (P = 0.026). The presence of intraductal carcinoma of the prostate was the only significant prognostic parameter for cancer-specific survival and overall survival in prostate cancer patients with distant metastasis at presentation. These results may prove useful in planning future treatments.

A temperature sensitive mutant of BHK21, tsBN2, showed a premature chromosome condensation (PCC) upon the temperature shift of 40.5 degrees, even in the absence of DNA replication. The induction of PCC requires new protein synthesis, but not necessarily new RNA synthesis. Our data suggested that the messenger RNA for chromosome condensation starts to be transcribed at the beginning of S phase. At the permissive temperature (33.5 degrees), the messenger RNA for chromosome condensation translated with a very slow rate during S phase and rapidly in G2-M phase. At the nonpermissive temperature (40.5 degrees), however, those messenger RNAs were translated anytime, so that various figures of PCC appeared depending on the cell cycle. On the way of PCC induction, ribosomal RNA synthesis was inhibited at first, as expected from mitosis. Our data suggested that the synthesis of protein(s) for chromosome condensation was regulated by the post-transcriptional mechanism, in which tsBN2 might be defective, especially at the translational level.

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To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase II␣ containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase II␣ immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase II␣ mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase II␣ through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase II␣ and DNA and analyzed by flow cytometry. Cells in the G 2 -M phase contained about double the PT1341-reacted topoisomerase II␣ than did cells in G 1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase II␣ antibody. Thus, threonine 1342 in topoisomerase II␣ is phosphorylated throughout the cell cycle.DNA topoisomerases are enzymes that play an important role in DNA replication and transcription by relieving torsional or interlocking constraints of DNA accumulating during processes of macromolecular syntheses (1-3). Topoisomerases I and II relax supercoiled DNA through transient single-and double-strand breaks, respectively, and topoisomerase II unknots or decatenates the knotted or catenated DNA. The latter activity of topoisomerase II is presumably related to chromosome dynamics in mitosis, where topoisomerase II is absolutely required (4 -7). In addition to catalytic activity, topoisomerase II may function to anchor chromosomal DNA loops to the nuclear scaffold (8, 9); topoisomerase II is a major non-histone protein present in nuclear scaffold fractions (10, 11), and DNA sequences that bind preferentially to the nuclear matrix/chromosomal scaffold (MAR/SAR) contain topoisomerase II cleavage consensus (12). In contrast, the role of topoisomerase II in the maintenance of chromosomes was not supported in studies done using Xenopus egg extracts or Xenopus embryos (13, 14). Topoisomerase II has been considered as one of the targets for anticancer drugs (15). Topoisomerase II inhibitors, such as etoposide or 4Ј-(9-acridinylamino) methane sulfon-m-anisidide, stabilize a catalytic reaction intermediate termed " cleavable complex" in which the enzyme binds covalently to 5Ј-phosphoryl termini of broken DNA (15). There are two known isoforms of topoisomerase II in mammalian cells, topoisomerase II␣ and II␤. They have molecular masses of 170 kDa and 180 kDa, respectively. These isoforms are mapped to different genes on chromosomes 17 and 3 (16), respectively,...