https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(00)02034-1.pdf

Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial

Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.

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Targeting DNA topoisomerase II in cancer chemotherapy

The inhibitor of apoptosis (IAP) family of proteins are highly conserved through evolution. However, the mechanisms by which these proteins interfere with apoptotic cell death have been enigmatic. Recently, we showed that one of the human IAP family proteins, XIAP, can bind to and potently inhibit specific cell death proteases (caspases) that function in the distal portions of the proteolytic cascades involved in apoptosis. In this study, we investigated three of the other known members of the human IAP family, c-IAP-1, c-IAP-2 and NAIP. Similarly to XIAP, in vitro binding experiments indicated that c-IAP-1 and c-IAP-2 bound specifically to the terminal effector cell death proteases, caspases-3 and -7, but not to the proximal protease caspase-8, caspases-1 or -6. In contrast, NAIP failed to bind tightly to any of these proteases. Recombinant c-IAP-1 and c-IAP-2 also inhibited the activity of caspases-3 and -7 in vitro, with estimated Kis of <=0.1 microM, whereas NAIP did not. The BIR domain-containing region of c-IAP-1 and c-IAP-2 was sufficient for inhibition of these caspases, though proteins that retained the RING domain were somewhat more potent. Utilizing a cell-free system in which caspases were activated in cytosolic extracts by addition of cytochrome c, c-IAP-1 and c-IAP-2 inhibited both the generation of caspase activities and proteolytic processing of pro-caspase-3. Similar results were obtained in intact cells when c-IAP-1 and c-IAP-2 were overexpressed by gene transfection, and apoptosis was induced by the anticancer drug, etoposide. Cleavage of c-IAP-1 or c-IAP-2 was not observed when interacting with the caspases, implying a different mechanism from the baculovirus p35 protein, the broad spectrum suicide inactivator of caspases. Taken together, these findings suggest that c-IAP-1 and c-IAP-2 function similarly to XIAP by inhibiting the distal cell death proteases, caspases-3 and -7, whereas NAIP presumably inhibits apoptosis via other targets.

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The c‐IAP‐1 and c‐IAP‐2 proteins are direct inhibitors of specific caspases

Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.

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IAP-Family Protein Survivin Inhibits Caspase Activity and Apoptosis Induced by Fas (CD95), Bax, Caspases, and Anticancer Drugs

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay Camptothecin, teniposide, and 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle The method of detection of DNA strand breaks (3′-hydroxyl termini) individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker The protein-associated DNA breaks in the “cleavable” DNA-topoisoerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods

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Detection of DNA Strand Breaks in Individual Apoptotic Cells by the in Situ Terminal Deoxynucleotidyl Transferase and Nick Translation Assays

PERSPECTIVES AND SUMMARY .. . . ...... . ... 351 MAMMALIAN DNA TOPOISOMERASES . . . . . . . . . . . . ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 353 Mammalian DNA Topoisomerase I . . . . . .. 353 Mammalian DNA Topoisomerase II 356 MAMMALIAN DNA TOPOISOMERASE II POISONS . . . . . . ... . . . ..... . . . . . . . . . . . . . . . . . . . . . 358 Intercalative Antitumor Drugs . .. . . 358 Nortintercalative Antitumor Epipodophyliotoxirts .... 361 Altered Regulation of Topoisomerase II in Tumor Cells......... . . . . . ..... 363 MAMMALIAN DNA TOPOISOMERASE I POISONS-CAMPTOTHECINS . . . ....... 364 BACTERIAL DNA TOPOISOMERASE II POISONS-QUINOLONE ANTIBIOTICS . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 POSSIBLE MECHANISM(S) OF CELL KILLING BY MAMMALIAN TOPOISOMERASE POISONS . . . . . . . . . . . . . . . ....... . . . . . . . . . . . . . 368 Inhibition of DNA Synthesis . . 368 Inhibition of Cell Division .. . .. 369 DNA Degradation..... ... . . 370 OTHER CELLULAR RESPONSES AND DRUG RESISTANCE . . ...... 370

DNA topoisomerase poisons as antitumor drugs.

Topoisomerase-targeting antitumor drugs.

To investigate the role of topoisomerases in the production of sister chromatid exchanges, the effects of inhibitors of type I and II topoisomerases on baseline and mutagen-induced sister chromatid exchanges were compared. V79 cells were treated with VM-26 and m-AMSA, known inhibitors of type II topoisomerase, or with camptothecin, the only known inhibitor of type I topoisomerase. We observed that inhibitors of both type I and II topoisomerases induced high levels of sister chromatid exchanges at 10−6 M, and that the dose-response curves of these drugs were very similar. A clear heterogeneity in the distribution patterns of exchanges induced by inhibitors of topoisomerases was observed. We believe that this heterogeneity in response to these compounds is due to variation in sensitivity within the cell cycle. We also studied interactions of these agents with mitomycin-C and with PUVA (8-methoxypsoralen + UVA), both cross-linking agents and potent sister chromatid exchange inducers, and with x-rays, an agent that induces high levels of DNA strand breaks. No significant change in exchange levels was observed in interactions between topoisomerase inhibition and the levels induced by the agents studied. We conclude that double-strand break prevalence, known to be increased through inhibition of type II topoisomerase, is not the primary mechanism for induction of sister chromatid exchanges. We further conclude that acute inhibition of type I and type II topoisomerases does not influence substantially the induction of exchanges by other agents.

Induction of sister chromatid exchanges by inhibitors of topoisomerases.

A number of isoindolo[l ,2-b]quinazolines and some benzo[4,5]isoquinolino[l,2-b]quinazolines as structural modification analogues of the antitumor compound batracylin were synthesized and evaluated against HL-60 cell growth and in topoisomerase II-mediated DNA cleavage assays. Of the compounds studied, 10,12-dihydro-7,8-methy lenedioxyisoindolo[ 1,2-b] quinazolin-12(10H)-one (1d), 2-amino-10,12-dihydroisoindolo[l ,2-b]quinazolin- 12(10H)-one (1p), and 2-amino-7,8-methylenedioxy-10,12-dihydroisoindolo[l ,2-b]quinazolin-12(10H)-one (1ab) exhibited good inhibitory activities against HL-60 cell lines as well as induction of topo II-mediated DNA cleavage activities.

Leroy-Fong Liu

Papers

DNA topoisomerase poisons as antitumor drugs.

Topoisomerase-targeting antitumor drugs.

Induction of sister chromatid exchanges by inhibitors of topoisomerases.

A Structured–Activity Relationship Study of Batracylin Analogues