Doxorubicin and daunorubicin induce IFN by an ATM-dependent mechanism. Doxorubicin has pleiotropic effects on cells. Among its activities, it is a topoisomerase II poison that intercalates into DNA, resulting in double-strand DNA breaks (DSB) (24, 45). Ataxia-telangiectasia mutated (ATM), a member of the phosphoinositide 3-kinase- like family of serine/threonine protein kinases, is activated in response to DNA DSB (25, 46–48). ATM has also been linked to stimulation of innate immune signaling pathways (26, 49–52). This prompted us to examine the role of ATM in doxorubicin- and daunorubicin-mediated activation of the IFN- ␤ promoter. We treated control-FF or VP35–FF cells with an ATM kinase inhibitor (Ku55933) (53) or with mirin, an inhibitor of the Mre11-Rad50-Nbs1 (MRN)-ATM pathway, which is essential for sensing and signal- ing in response to double-strand DNA breaks. Mirin prevents MRN-dependent activa- tion of ATM without affecting ATM protein kinase activity and inhibits Mre11-associated exonuclease activity (54). Each inhibitor signiﬁcantly dampened, in the presence or absence of VP35, the IFN- ␤ promoter activity induced by doxorubicin or daunorubicin compared to DMSO treatment (Fig. 4A). To further implicate the ATM pathway in the response to doxorubicin, short hairpin RNA (shRNA) knockdown of ATM was performed. Relative to a scrambled shRNA, targeting ATM decreased the doxorubicin-mediated IFN induction in control-FF and VP35-FF cells relative to mock-treated controls (Fig. 4B). In contrast, neither pharmacological inhibition nor shRNA knockdown signiﬁcantly af-
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Whatever the real rate of unique positives seen during mammalian cell screening, there are some genotoxic positives that cannot be readily explained by their inherent DNA reactivity e.g. by any normal structural alert relationship for mutagenicity. These compounds are unlikely to directly bind to DNA, hence it is far more likely that any observed genotoxicity is induced by indirect DNA targets such as mitotic spindle (aneugencity), reactive oxygen species (ROS) generation or topoisomerase II inhibition. For pharmaceutical research, topoisomerase II inhibition is a particularly interesting genotoxic mechanism, as the enzyme is used as a target for both oncology and anti-infective drugs. Furthermore, some topoisomerase II poisons, be they either cancer drugs or antibiotics, are known to be very potent in vitro mammalian cell mutagens (Boos and Stopper, 2000; Smart, 2008a). Whilst there has been a great deal of research into topoisomerase II inhibition, it is surprising that the direct relationship between enzyme poisoning and genotoxicity has not been firmly established. For example, with anti-infectives, there is a known correlation between compounds that target bacterial gyrase and positive responses in mammalian cell genotoxicity assays, but attempts to quantify topoisomerase II inhibition and related genotoxicity have shown that the methods to measure topoisomerase II are either insensitive or (possibly) other genotoxic mechanisms are in play (Lynch et al., 2003). To help with the discovery of safe and efficacious new medicines to tackle the unmet need in such serious conditions as tuberculosis and hospital acquired infections it would clearly be useful to gain a better understanding of the relationship between off- target mammalian cell topoisomerase II inhibition and genotoxicity. Accordingly, a robust method for determining topoisomerase II inhibition in mammalian cells would be a valuable adjunct to available tools and may also help to elucidate the mechanism of genotoxicity for at present unexplainable positive agents.
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Topoisomerase II (topo II) is essential for the survival of proliferating cells, and it is involved in recombination, chromosome condensation, and decatenation of sister chromatids before anaphase of mitosis . In cancer che- motherapy, topo II is a major target for various antican- cer drugs. These drugs can be divided into two types based on their mode of action. One type is the topo II poisons, so named because of their severe cytotoxicity resulting from their ability to stabilize enzyme-DNA co- valent complexes and activates DNA damage checkpoint [2,3]. Topo II catalytic inhibitors do not stabilize topo II-DNA covalent complexes. Instead, these compounds block the enzyme before DNA cleavage or at the last step of the catalytic cycle after religation [4,5]. An example of this latter type of inhibitors is ICRF-193 (bisdioxopi- perazine), which blocks the opening of an already closed clamp by inhibiting ATPase activity of the enzyme and preventing DNA decatenation of the replicated chromo- somes by it, thus arresting cell proliferation [6,7]. These catalytic inhibitors inhibit topo II before the transient
Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA cova- lent intermediate. This intermediate can be stabilized by a class of drugs termed Top2 poisons, resulting in massive DNA damage. Thus, Top2 activity is a double-edged sword that needs to be carefully con- trolled to maintain genome stability. We show that Uls1, an adenosine triphosphate (ATP)-dependent chromatin remodelling (Snf2) enzyme, can alter Top2 chromatin binding and prevent Top2 poisoning in yeast. Deletion mutants of ULS1 are hypersensitive to the Top2 poison acriflavine (ACF), activating the DNA damage checkpoint. We map Uls1 s Top2 interaction domain and show that this, together with its ATPase activity, is essential for Uls1 function. By performing ChIP-seq, we show that ACF leads to a general in- crease in Top2 binding across the genome. We map Uls1 binding sites and identify tRNA genes as key regions where Uls1 associates after ACF treatment. Importantly, the presence of Uls1 at these sites pre- vents ACF-dependent Top2 accumulation. Our data reveal the effect of Top2 poisons on the global Top2 binding landscape and highlights the role of Uls1 in antagonizing Top2 function. Remodelling Top2 bind- ing is thus an important new means by which Snf2 enzymes promote genome stability.
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and efficacy are still limited. There are few immunogenic cell death–inducing (ICD-inducing) drugs available that can kill cancer cells, enhance tumor immunogenicity, increase in vivo immune infiltration, and thereby boost a tumor response to immunotherapy. So far, the ICD markers have been identified as the few immunostimulating characteristics of dead cells, but whether the presence of such ICD markers on tumor cells translates into enhanced antitumor immunity in vivo is still being investigated. To identify anticancer drugs that could induce tumor cell death and boost T cell response, we performed drug screenings based on both an ICD reporter assay and a T cell activation assay. We showed that teniposide, a DNA topoisomerase II inhibitor, could induce high-mobility group box 1 (HMGB1) release and type I IFN signaling in tumor cells and that teniposide-treated tumor cells could activate antitumor T cell response both in vitro and in vivo. Mechanistically, teniposide induced tumor cell DNA damage and innate immune signaling, including NF-κB activation and stimulator of IFN genes–dependent (STING-dependent) type I IFN signaling, both of which contribute to the activation of dendritic cells and subsequent T cells. Furthermore, teniposide potentiated the antitumor efficacy of anti-PD1 in multiple types of mouse tumor models. Our findings showed that teniposide could trigger tumor immunogenicity and enabled a potential chemoimmunotherapeutic approach to potentiating the therapeutic efficacy of anti-PD1 immunotherapy.
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The results showing that the simultaneous loss of Siz1p and Siz2p activity is epistatic to the top2 SNM al- leles and yields an unmodified Top2p suggest that the role of SUMO E3 in chromosome segregation could be limited to the Smt3p modification of a specific Top2p subpopulation. As sumoylation of topoisomerase II in S. cerevisiae (data not shown) and vertebrates (by SUMO-2) (A zuma et al. 2003) peaks in mitosis, it is conceivable that the sumoylated pool of Top2p plays an important role in mitotic chromosome segregation. As the sumoy- lated pool of Top2p is very small (Figure 3C), it is likely that this subset of Top2p molecules participates in centromere–kinetochore dynamics. Indeed, utilizing a novel approach of modeling sumoylated proteins by direct fusion of targets to SUMO (constitutive SUMO modification), we were able to show that the modified pool of Top2p is enriched at the centromeres (Figure 5C). The exact function of this pool is still unknown, but our genetic data (Figure 4) and disruption of pericen- tromeric sister chromatid cohesion by hypersumoyla- tion of Top2p (B achant et al. 2002) suggest that Top2p, when sumoylated at the physiological level, is involved in establishing or maintaining the bipolar kinetochore orientation.
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Two sections were obtained from each case, one for posi- tive test slide and the other for negative control (by omit- ting the primary antibody and non-immune serum is used instead of primary antibodies). Immunohistochemical staining was carried out using labeled streptavidin-biotin peroxidase complex technique Enzinger FM and Weiss SW 1988 (DAKO LSAB +Kit, HRP) DAKO-Dakopatts, Glostrup, Denmark(code no.k0679). After dewaxing and rehydration, sections were incubated for 5 minutes at 37°C in 0.05% protease type XXIV Sigma in phosphate buffered saline (PBS). This was followed by endogenous peroxidase block using 0.3% menthanolic H2O2. Sec- tions were then washed in PBS, and incubated for 5 min- utes in 1% Bovine Serum Albumin (BSA) to reduce non specific binding. The monoclonal antibodies for EBV CS1- 4 (Dakopatts, diluted at 1:50) that recognizes EBV- encoded LMP1 and mouse anti-human Topoisomerase II α protein (DAKO, clone: Ki-S1, isotype: IgG2a) were used. The bottle contains 1 ml of Topo II α antibody provided in liquid form as purified IgG diluted in 0.05 M Tris/HCL, 15 mM NaN, pH 7.2, 1% bovine serum albumin (BSA). (Bottle no. 2) was applied to 1:80 dilutions in 1% BSA in PBS. After overnight incubation at 4°C, sections were washed and treated with anti-rabbit, anti-mouse and anti- goat Ig in phosphate buffered saline (PBS) for 30 minutes at room temperature, then followed by streptavidin per- oxidase conjugate 1:300 at 37°c for 20 min. Sections were then washed in PBS, visualized with diamino-benzidene H 2 O 2 (DAB) and counterstained with Mayer's hematoxy- line. Positive and negative controls were included. For negative control slide, one vial (3 ml) of non-immune serum or immunoglobulins in PSA with 0.09% sodium azide was used.
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top2a mutants exhibit defects in cell cycle progression Topoisomerase II alpha genes are known to be critical for cell cycle progression. Therefore, we quantified, by flow cytometry, the distribution of blm cells within the major phases of the cell cycle. At 27 hpf, the proportion of dissociated blm cells in the G2/M phase (~31%) is approximately double that of wildtype siblings (~16%) consistent with defects in mitosis (Figure 3A). By immu- nohistochemistry, the total number of cells in the blm eye that express the G2/M marker phospho-histone H3 is lower than wildtype siblings (Figure 3B). However, consistent with the flow cytometry analyses (Figure 3A), the proportion of cells expressing the G2/M marker, normalized to the total number of nuclei, is significantly higher in blm mutants (8.7%) than in wildtype siblings (5.8%) (Figure 3C). At the transcript level, no significant difference is observed at 27 hpf for p21-like or ccnb1, G1 and G2 phase markers respectively, in blm mutants compared to siblings (Figure 3D). Equivalent levels of the post-mitotic retinal marker atoh7 were observed in blm and siblings indicating that retinogenesis had initiated in blm mutants (Figure 3D). With regards to cell death, a higher degree of apoptosis was observed in blm mutants at 24 hpf (Figure 3E). In summary, blm mutants exhibit an increased proportion of cells in G2/ M phase, consistent with delayed cell cycle progression.
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Molecular docking study was performed for comparing the mode of action of lapachol-TOP I and lapachol-TOP II using MOE 2010 software package. Crystal structures of TOP I-ligand complex (PDB entry: 1SC7, 3.0 Å) and TOP II-ligand complex (PDB entry: 1ZXM, 1.87 Å) were used. All water molecule ligands were removed and the docking active pockets were defined by the ligand mole- cules. The detail docking parameters of TOP I were as follows: placement method (Triangle Matcher), the first scoring function rescoring (Affinity dG), and the saved poses (100); the refinement (forcefield), the second re- finement scoring function rescoring (London dG) and the saved poses (30). The detail docking parameters of TOP II were as follows: placement method (Triangle Matcher), the first scoring function rescoring (London dG), and saved poses (30); the refinement (forcefield), the second refinement scoring function rescoring (none), the refinement saved poses (10). To verify whether MOE software was suitable for docking TOP I and TOP II, the ligand conformations of 1ZXM and 1SC7 were with- drawn and re-docked to the active pockets. The first 10 and 30 conformations of the docking scores were saved and the root-mean-square deviation (RMSD) values of docking conformation and initial conformation were cal- culated. Then, the ligand molecules in the crystal struc- tures were re-docked to the defined active pockets, and the scores of ligand conformation after docking and ori- ginal conformation in the crystal structures were calculated.
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DNA Topo II poisons induce DSB and filamin-A is required for efficient DSB repair through regulating HR and NHEJ. Thus, it was puzzling to us at the moment that filamin-A deficient cells have lesser cel- lular sensitivity to Topo II poisons than filamin-A proficient cells. To investigate the mechanism of this apparently conflicting phenomenon, we assessed the initial levels of DNA damage and the repair kinetics. Again, we used M2 cells that have spontaneously lost filamin-A expression and the A7 cells that have rein- stated filamin-A by stably expressing filamin-A in the M2 cells. After 2 hr exposure to 10 μM of etoposide or 0.25μg/ml of doxorubicin, the drug was washed out. At various time points as shown in Figure 2A, the numbers of DSB in these paired cells were assessed by measuring the number of γH2AX nuclear foci. Signif- icantly more γH2AX nuclear foci were induced in A7 cells than in M2 cells after 2 hours of etoposide incu- bation (50.7 and 33.3 foci/cell respectively, p<0.01), indicating that there is more initial DNA damage in
Although normal topo II action involves DSB induction, the DSBs are protein-associated and the DSB is usually religated soon after, so no overall damage will have occurred. One can assume that if something goes wrong during this process, a DSB might either remain unrepaired or topo II might mis-join the ends of the DSB. In S-phase, the replication fork can collide with the topo II cleavable complex, where the DSB is still bound to the dimer. This results in the enzyme being released from the DSB and this damage can cause sister chromatid exchanges (SCEs) and chromosome aberrations (CAs) 243 . Here, chromosome aberrations refer to breaks or gaps on both sister chromatids as well as dicentrics, rings and multi-centric chromosomes. Other groups have also shown that topo II poisons, such as amsacrine, etoposide, doxorubicin and ellipticine can produce CAs and SCEs 127,244 as the cleavable complex is often stabilised and DSB induction rather than religation is favoured. The SCE induction by these drugs is known to be formed through topo II activity because in drug-resistant cells, in which topo II activity and expression is reduced, CAs or SCEs were not created 127,244 .
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supercoiling domain data of Naughton et al. . Naughton et al. used biotinylated TMP (bTMP) incorp- oration into the DNA of human retinal pigment epithe- lial cells to show that chromosome-wide supercoiling, and more specifically supercoiling at TSS, requires the activity of RNA polymerase II as well as topoisomerase I and II proteins . After recapitulating these results at the TSS (Additional file 2: Figure S8), we asked whether supercoiling at CTCF sites also required RNA polymer- ase II and TOP2 proteins. Indeed, we found that DNA supercoiling at CTCF sites was: (1) lost after treatment with the RNA polymerase II inhibitor alpha-amanitin; (Fig. 7a); (2) is reduced in the presence of TOP2 (ICRF- 193) or TOP1 (campothecin) inhibitors (Fig. 7b); and (3) is not affected by topoisomerase inhibition when transcrip- tion is inhibited simultaneously (Fig. 7c). In contrast, no specific supercoiling pattern was observed at randomly se- lected genomic intervals (Fig. 7, dashed lines). Although TOP2 poisons affect both TOP2B and TOP2A, this ana- lysis together with the close association of TOP2B with CTCF raises the possibility that TOP2B can facilitate the remodeling of DNA supercoiling at CTCF sites.
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Nasopharyngeal carcinoma (NPC) is closely associated with latent Epstein-Barr virus (EBV) infection. Although EBV infection of preneoplastic epithelial cells is not immortalizing, EBV can modulate oncogenic and cell death mechanisms. The viral latent membrane proteins 1 (LMP1) and LMP2A are consistently expressed in NPC and can cooperate in bitransgenic mice expressed from the keratin-14 promoter to enhance carcinoma development in an epithelial chemical carcinogenesis model. In this study, LMP1 and LMP2A were coexpressed in the EBV-negative NPC cell line HK1 and examined for combined effects in response to genotoxic treatments. In response to DNA damage activation, LMP1 and LMP2A coexpression reduced ␥ H2AX (S139) phos- phorylation and caspase cleavage induced by a lower dose (5 M) of the topoisomerase II inhibitor etoposide. Regulation of ␥H2AX occurred before the onset of caspase activation without modulation of other DNA damage signaling mediators, includ- ing ATM, Chk1, or Chk2, and additionally was suppressed by inducers of DNA single-strand breaks (SSBs) and replication stress. Despite reduced DNA damage repair signaling, LMP1-2A coexpressing cells recovered from cytotoxic doses of etoposide; however, LMP1 expression was sufficient for this effect. LMP1 and LMP2A coexpression did not enhance cell growth, with a moderate increase of cell motility to fibronectin. This study supports that LMP1 and LMP2A jointly regulate DNA repair signal- ing and cell death activation with no further enhancement in the growth properties of neoplastic cells.
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We firstly treated five cancer cell lines (LoVo, SW480, SW620, AGS, H446) with topoisomerase II inhibitors VP-16, ADM and topoisomerase I inhibitor CPT-11. Consistent with the anti-proliferative capacity [27–29], all these agents inhibited cell growth (Additional file 1: Figure S1). Subsequently, we performed migration and invasion assays. Although three TI inhibited the motility of AGS cells (Additional file 1: Figure S2A), the motility of SW620 cells was not affected (Additional file 1: Figure S2B). Interestingly, these TI promoted migration and in- vasion of LoVo, SW480, and H446 cells in a dose- dependent manner (Fig. 1a, b). To evaluate the influence of TI on cancer cell motility in vivo, we injected VP-16 or CPT-11-treated LoVo cells into caudal vein of NOD/ SCID mice. We noticed that TI-treated LoVo cells had stronger ability to form metastatic nodules on the sur- face of lungs (Fig. 1c). The Hematoxylin-eosin staining of sectioned lung tissues also showed that TI-treated groups had more metastases (Fig. 1d). These results sug- gest that VP-16, ADM and CPT-11 promote motility of a subset of cancer cells in vitro and in vivo.
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doublestranded breaks by stabilising a complex formed between topoisomerase II and DNA that is referred to as the cleavable complex. Ross et al. demonstrated this by showing that topoisomerase II was the most likely cellular target in the double stranded breaking activity of epipodophyllotoxin like Etoposide and Teniposide. 2 In parallel, Long and coworkers studied a range of related derivatives to establish a correlation between DNA cleaving or cytostatic activities of various molecules and the extent to which they inhibited topoisomerase II activity. In this way, he demonstrated that the cytotoxicity of analogues of Etoposide was associated with inhibiting DNA topoisomerase II by stabilising the covalent topo II- DNA cleavable complex . 3 Although Etoposide is active in the treatment of many cancers and is widely used in the therapy, it presents several limitations, such as moderate potency, poor water solubility, development of drug resistance, metabolic inactivation, and toxic effects . 4 Therefore in order to obtain better therapeutic agents and extensive synthetic efforts have been devoted to overcome these problems cited above. As highlights, a water-soluble phosphate ester prodrug of Etoposide, Etopophos , was launched in 1996 by Bristol-Myers Squib. This prodrug was readily converted in vivo by endogenous phosphatise to the active drug and exhibited similar pharmacological and pharmacokinetic properties as Etoposide.The in vivo bioavailability was increased from 0.04% to over 50% through this prodrug approach and thus constituted an improved formulation of Etoposide. 5 With increasing the information about its structure- activity relationships wide
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Immunohistochemistry, using monoclonal antibodies on formalin-fixed paraffin-embedded archival material has been widely used. Ki-67 and topoisomerase II α are most frequently evaluated immunohistochemically detectable proliferation antigens. Ki-67 is a nuclear non-histone pro- tein expressed in cells in G1, S, G2, and M cell cycle pha- ses, but absent from quiescent cells in G0. Type II DNA topoisomerases are nuclear enzymes that play a crucial role in DNA replication. They catalyze the relaxation of super- coiled DNA and separate intertwined DNA duplexes in an ATP-dependent process, through the generation of a double- stranded nick on the DNA during trascription. Topoisome- rase II α is expressed during the G1, S, G2, and M phases cell cycle (13).
Progress in the treatment of hepatocellular carcinoma (HCC), a common tumor worldwide, has been disappoint- ing. Inhibitors of topoisomerases are being widely studied as potential inducers of tumor cell apoptosis. Our aims were to determine whether topoisomerase-directed drugs would in- duce apoptosis in a human HCC cell line (Hep 3B) and, if so, to investigate the mechanism. The topoisomerase I poi- son camptothecin (CPT) induced apoptosis of Hep 3B cells in a time- and concentration-dependent manner. In con- trast, the topoisomerase II poison etoposide failed to induce apoptosis despite the apparent stabilization of topoiso- merase II–DNA complexes. Unexpectedly, CPT-induced apoptosis in this cell type occurred without any detectable cleavage of poly(ADP-ribose) polymerase or lamin B, poly- peptides that are commonly cleaved in other cell types un- dergoing apoptosis. Likewise, Hep 3B cell apoptosis occurred without a detectable increase in interleukin-1 b –converting enzyme (ICE)-like or cysteine protease P32 (CPP32)-like protease activity. In contrast, trypsin-like protease activity (cleavage of Boc-Val-Leu-Lys-chloromethylaminocoumarin in situ) increased threefold in cells treated with CPT but not etoposide. Tosyl-lysyl chloromethyl ketone inhibited the trypsin-like protease activity and diminished CPT-induced apoptosis. These data demonstrate that ( a) apoptosis is in- duced in Hep 3B cells after stabilization of topoisomerase I-DNA complexes but not after stabilization of topoisome- rase II-DNA complexes as measured by alkaline filter elution; (b) Hep 3B cell apoptosis occurs without activation of ICE- like and CPP32-like protease activity; and (c) a trypsin-like protease activity appears to contribute to apoptosis in this cell type. (J. Clin. Invest. 1996. 98:2588–2596.) Key words: camptothecin • CPP32 • etoposide • interleukin-1 b –convert-
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Multiple Topo I inhibitors block Zta transcription activa- tion and DNA replication function. To further validate the role of Topo I in mediating Zta function during lytic reactivation and DNA replication, we compared the abilities of two other Topo II inhibitors, irinotecan and topotecan, to function sim- ilarly to camptothecin (Fig. 5). While irinotecan and topotecan are derivatives of camptothecin, they are thought to have al- tered target specificities and may interfere with different steps of the topoisomerase cleavage-ligation reaction (20). We FIG. 2. Camptothecin inhibits Zta-dependent transcription activation. (A) ZKO-293 cells were transfected with Zta and then treated with 1 M camptothecin or mock treated. Zta binding to the viral promoters Hp, Rp, and Mp was assayed by ChIP with antibody to Zta ( ␣ Zta) or with control IgG. ChIP DNA was quantified by real-time PCR and presented as change over IgG. (B) Same as in A except that ChIP assays were performed with antibody to Topo I. (C) 293 cells were transfected with Hp-Luc with either Zta expression vector ( ⫹ ) or control vector ( ⫺ ). Cells were then treated with 0, 0.25, or 1.0 M camptothecin (CTN), as indicated. Luciferase assays were performed at 48 h posttransfection. (D) Same as in C except for the Mp-Luc reporter. (E and F) Western blot control for Zta expression levels for luciferase assays shown in C and D, respectively.
on two phase II clinical trials with a 30% response rate in patients with CTCL. Although response rates were similar to previously used therapies, vorinostat showed relatively higher relief from pruritus in comparison to other agents used in the advanced form of the disease. Vorinostat was generally well tolerated, with adverse side effects including diarrhea, fatigue, and nausea. Some patients experienced pulmonary embolism and thrombocytopenia, and there is evidence of long-term safety. 71–73 Similar response
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