Inhibitors of Topoisomerases As Anticancer Drugs: Problems and Prospects

Indian Journal of Experimental Biology Vol. 42, July 2004, pp. 649-659

Review Article

Inhibitors of topoisomerases as anticancer drugs: Problems and prospects

l l l 2 B S Dwarakanath *, Divya Khaitan & Rohit Mathur • 'Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Marg, Delhi 110054, India

2Dr B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India

DNA topoisomerases, which solve topological problems associated with various DNA transactions, are the targets of many therapeutic agents. Various topoisomerase inhibitors especially, topo-poisons, camptothecin (topo-I) and etoposide (topo-ll) are some of the drugs that are used in the current treatment protocols, particularly for the treatment of leukemia (AML, ALL etc). However, tumor resistance, normal and non-specific tissue cytotoxicity are the limitations for successful development of these drugs as one of the primary therapeutic agents for the treatment of tumors in vitro. This brief review presents the current understanding about cytotoxicity development and outlines various approaches to overcome the limita­ tions for enhancing the efficacy of topo-poison based anticancer drugs.

Keywords: Camptothecin, Cleavable complex, 2-Deoxy-D-glucose, Etoposide, Topoisomerases IPC Code: Int Cl 7 A61 K

5 6 Topoisomerases are ubiquitous and evolutionarily and no~al cells • . On the other hand, approaches conserved nuclear enzymes, which modulate the that selectively modify the drug accumulation and topological state of DNA by passing an intact DNA consequences of drug-induced primary lesions in tu­ helix through a transient single (topo-I) or double mor cells, while sparing cells from the normal tissue, (topo-II) strand break, facilitating vital nuclear trans­ could be equally effective. Pre-target events including actions such as transcription, replication, chromosome intracellular drug accumulation and distribution, drug­ l segregation, mitotic division etc . Since the level of target interactions influenced by enzyme levels, se­ topoisomerases is generally elevated in malignant quence specificity and activity, status of nuclear or­ (cancer) and actively growing cells, these enzymes ganization etc., and post-target events like macromo­ have been the specific targets of several anti-tumor lecular synthesis, DNA repair, cell cycle perturbations 2 drugs • Currently used topo~somerase inhibitors exert and apoptosis, collectively contribute to the drug­ their cytotoxic effects either by stabilizing the cova­ induced cytotoxicity 7.8. Therefore, an understanding lent complexes between the enzyme and DNA of the molecular mechanisms involved in the re­ (cleavable complex) or by inhibiting the catalytic ac­ pair/reversal of cleavable complexes as well as the tivitlA (Fig. I). Despite the development of specific interactions of topoisomerases with DNA repair ma­ and more effective inhibitors, with enhanced clinical chinery, besides the induction of cell cycle perturba­ activities, drug resistance by tumors as well as lack of tions and apoptosis will greatly facilitate the optimal specificity resulting in normal tissue toxicity has con­ use of topoisomerases as anticancer drugs. tinued to limit the success of cancer therapy using topoisomerase poisons. Therefore, there is a great Topoisomerases as molecular targets for antican­ need to develop newer approaches, which selectively cer therapy-The long stretch of DNA in eukaryotic enhance the drug-induced toxicity in cancer cells. cells is packed very efficiently to fit into nucleus as a · One of the approaches to achieve this objective result; the chromosomal DNA is twisted extensively. would be to synthesize novel, sequence specific DNA Therefore selected regions of DNA need to become ligands, with associated topoisomerase inhibitory aC­ sufficiently untangled and relaxed to allow vital nu­ tivity, that exhibit differential effects between tumor clear transactions viz., transcription, replication, chromosome strand separation, mitotic division, re­ 9 *Correspondent author combination' and chromatin remodeling • Topoi- Fax (+91) 11-2391-9509, [email protected] 650 INDIAN J EXP BIOL, JULY 2004

l5 somerases maintain the topology and regulate the dy­ similar throughout the cell cycle ,16 and distributed namics of DNA structure by not only relaxing, un­ homogenously throughout the nucleus 17. More recent knotting, interlinking of strands, decatenation of studies have mapped the enzyme linked cleavable gapped or nicked DNA circles but also by performing complexes on the DNA loops bound to the nuclear catenation and knotting to facilitate the condensation matrix at matrix attachment regions (MARs). Topo II and packing of DNA into higher order structure 10. In ~ and topo I remained completely extractable addition, these enzymes fine-tune the steady-state throughout the cell cycle, while scaffold associated level of DNA supercoiling both to facilitate protein · topo II a does not appear to be involved in catalytic interactions with DNA and to prevent excessive su­ DNA turnover, although it may playa role in replica­ percoiling that is deleterious I. Topoisomerases are tion of centrioles, where it accumulates during M l8 known that specifically relax only negative supercoils, phase . that relax supercoils of both signs or that introduce Levels of topoisomerases are generally elevated in either negative (bacterial DNA gyrase) or positive malignantly transformed (cancer) and actively grow­ 5 l9 supercoils into DNA (reverse gyrase) 1 I. These en­ ing cells ,6 as compared to the non-malignant tissue , zymes accomplish this feat by either passing one which is partly attributed to the higher rate of DNA strand (type-I subfamily) or by passing a region of replication, transcription and other cellular processes duplex from the same or a different molecule through in transformed cells for a higher rate of growth and a double strand gap generated in DNA (type-II sub­ proliferation. Increased demand for a rate of cellular family) followed by religation of the strand break. proliferation increases the topo levels manifolds (up Several studies have demonstrated that topo I activity to 200 folds), thereby allowing a differential sensitiv­ is sensitive to physiological, environmental and ity of tumors to drugs targeting topoisomerases as pharmacological DNA modifications and can act as a compared to normal cells. Higher levels of topo pool l2 specific mismatch and abasic site nicking enzyme . sizes has also been attributed to the over-expression Recently, increased in vitro cleavage by topo II has of topoisomerase gene as a result of mutation during also been reported following the introduction of aba­ the process of transformation, although in most tumor l3 sic sites into DNA substrates , analogous to PARP, types it appears to be as a result of higher requirement which binds to single strands breaks and initiate DNA in proliferating tumor cells. However, topo pool sizes l4 repair . vary among different types of tumors, albeit to a Topo II a level changes during the cell cycle lesser extent and this marked heterogeneity of enzyme o 22 whereas, topo II ~ and topo I levels remain essentially levels has been correlated with drug sensitivit/ - .

I Topo I I TopoD

3 Topo-I Topo-II "'110,,11"'" ,cA_I~'[".·?·.·.] ,...... ·41 • ...... ~~.~::.:~.~ .. ]~ ~ ------~~

Fig. I-Action of topoisomerase I and II and their inhibition DW ARAKANATH et al.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS 651

Higher levels of topoisomerase being a selective and city28.30. On the other hand, collision of topo poisons differential marker of transformed cells make these mediated stabilized cleavable complexes with tran­ enzymes an attractive molecular target for the devel­ scription bubble leads to the generation of gapped opment of antineoplastic chemotherapy. Currently DNA fragments and hence the formation of single 3 several anti-tumor drugs like etoposide, doxorubicin, strand breaks !, teniposide and camptothecin specifically target topoi­ Earlier studies have suggested the stabilization of somerase enzyme2. cleavable complexes as a prerequisite for cytotoxicity of topoisomerase poisons with a direct correlation between cleavable complexes and cytotoxicity, as Types of topoisomerase inhibitors and development well as indirect evidences relating the rate of repair of cytotoxicity--Topoisomerase inhibitors include and the formation of cleavable complexes in the dam­ catalytic inhibitors of the enzyme and topoisomerase age response32. Many studies have revealed that the poisons. Topoisomerase poisons exert their cytotoxic damage by topo poisons is processed similar to frank effects by stabilizing the covalent complexes between double strand breaks; but there are also evidences to enzyme and DNA (cleavable complex). These com­ believe that DNA-Erotein cross-links are probably pounds interfere in the religation step of the enzyme handled differently 3. An unequivocal damage re­ catalysis, thereby, leaving the DNA strand breaks un­ 4 sponse pathway to topo poison induced cleavable ligated ,lO. 23 . The protein-DNA strand breaks thus cre­ complexes has not emerged so far34, ated are not efficiently repaired and induce apopto­ DNA damage by UV irradiation and photoproducts sis24. On the other hand, the catalytic inhibitors inhibit formation has shown an increase in the levels of to po the enzyme catalysis activity by not allowing the en­ I-DNA complexes and p53 simultaneousll5. Al­ zyme to function itself and therefore do not allow to­ though, the role of p53 in response to DNA damage poisomerases to create strand break, Several com­ has been established, its interaction with endogenous pounds, membrone, suramin, bisdioxopiperazines topo I has also been implicated in repair of damaged (ICRF), are known catalytic inhibitors of topoisomer­ genome. Recent studies have shown that although ase, while epipodophyllotoxins like etoposide (topo-II MCF-7 cells with wild type p53 showed an elevation inhibitor) and camptothecin (topo-I inhibitor) are well in topo I-DNA complexes, p53 mutant SK-BR-3 cells known topoisomerase poisons 2. However, these com­ and null HL60 cells do not show such induction. A pounds differ in the mechanism of their action as well physical interaction between p53 and topo I has also as the cytotoxicity, for example, ICRF-193 does not been investigated and recruitment of topo I by p53 has have a significant cytotoxicity, while topoisomerase been proposed to cause an additional damage to ge­ poisons like etoposide and camptothecin (CPT) have nome, thereby committing cells to apoptosis36. considerable amount of cytotoxicity. Not only the concentrations of DNA-protein cross links, but their Cellular responses to topo poisons-There are life-time and stability also contribute to the cytotoxic­ enough evidences to believe that, while the stabilized ity, Hypersensitive response to CPT induced by cleavable complexes can be repaired to a certain ex­ Cockayne's syndrome has been correlated with the tent, moderate levels of the complex can mediate increased life-time of double strand breaks (DSBs) other cellular responses like cell cycle arrest, induc­ and cleavable complexes, predominantly at the site of tion of apoptosis, mitotic cell death and senescence replication in nascent DNA25 , Although, cleavable (Fig.2f' 8, 32. However, necrosis and non-specific cy­ complexes are formed in all phases of cell cycle, S­ totoxicity are observed at higher doses of topo poi­ phase cells are found to be more sensitive particularly sons37. Indirect evidences have shown that cleavable to topo poisons26. There is enough evidence that nas­ complexes can be repaired by non-homologous end cent DNA binds to the nuclear matrix is an important joining (NHEJ) repair pathway38, while the role of site of cleavable complex formation, and these com­ homologous recombination is equivocal. Cells defi­ plexes inhibit DNA synthesis by blocking the move­ cient in NHEJ repair pathway (Ku deficient) are more ment of nascent DNA away from replication sites on sensitive to topo poisons, which is correlated with the nuclear matrix27 . It is well established that topo higher amount of cleavable complexes. Cells deficient poisons mediated stabilized cleavable complexes col­ in double strand break (DSB) repair (ATM deficient) lide with replication fork leading to the formation of also sensitize them to topo poisons39 . Inhibition of lethal double strand breaks and hence causes cytotoxi- patch strand repair also increases the cytotoxicity of 652 INDIAN J EXP BIOL, JULY 2004

topo poisons. The induced expression of DNA dam­ to be deficient in DNA repair RAD6 pathway and also 44 age responsive genes DIN3 and RNR3 in TOP 1+ defective in its polymerization . 40 camptothecin treated yeast cells and deletion of Brief exposure to topo poisons at moderate con­ RAD52 gene, which is required for recombinational centrations mediate a higher mitotic cell death in syn­ repair of DSB, enhances cell sensitivity to camptothe­ chronized M phase cultures, leading to the formation cin. The protein linked DSB doubled when incubated of micronuclei, which correlate well with the amount 45 with 3-aminobenzamide (inhibitor of poly ADP ribose of cleavable complexes • In asynchronous cell cul­ 41 polymerase) in addition to camptothecin , shows that ture most of the topoisomerase inhibitors arrest the

protein concealed strand breaks can be lethal lesions cell at G2/M cell cycle checkpoint, possibly facilitat­ 46 and intracellular activity of topoisomerase I and II ing the repair of cleavable complexes • G2/M cell may be regulated coordinately through poly ADP ri­ cycle arrest primarily arises because of alteration in bosylation. tyrosine dephosphorylation of p34cdc2 and cyclin B I cdC2 47 Ubiquitin (El enzyme)/26S proteasome-dependent leading to inactivation of cyclin B/p34 complex . degradation of cleavable complexes has been Short exposure to topo poisons initiate ATM respon­ sive p53 dependent, p21 and GAAD-45 mediated cell suggested to be a unique repair process for the repair 48 of topo I mediated DNA damage leading to its down cycle arrest . However, DNA synthesis is required at 42 the time of topo inhibitors treatment, since these cell regulation . Yong Mao et al. have shown that treatment of mammalian or yeast cells expressing cycle dependent changes are not observed when incu­ human DNA topo I with camptothecin induces bated simultaneous with aphidicolin (DNA synthesis covalent modification of topo I by SUMO-lISmt3p, a inhibitor). Cleavable complex has also been found to ubiquitin like protein. Since, Ubc9 mutant yeast cells induce cellular proliferation through induction of ex­ cision repair enzymes or otherwise p53/p21 mediated expressing hu~an DNA topo I are hypersensitive to CPT, it is suggested that UBC9/SUMOl may be Bax synthesis and its translocation to the mitochon­ involved in the repair of topo I mediated DNA dria resulting in membrane permeability transition 35 leading to cytochrome c release culminating in apop­ damage . The trimeric topo I-CPT-DNA cleavable 48 complex seems to be the signal for SUMO I tosis . conjugation with topo I, which is supported by the Topo poisons can also mediate senescence, which observation that majority of SUMO-l conjugated topo correlates to a cytostatic but not quiescent cell popu­ 8 1 is covalently linked to DNA 42 and mutant cell lines lation resting in cell cycle blocks . Induction of p53 (CPT-K5 and U-937/CR) are defective in CPT dependent expression of p21, which appears to be induced SUMO-l conjugation to topo 143. Further, a necessary for cell cycle arrest, can also induce senes­ 8 ubiquitin mutant with substitution at Lys-63 appears cence • These studies further substantiated that the loss of clonogenicity of cells does not necessarily re­ Cleavable complex sult from loss of cell viability. DNA degradation during etoposide induced apop­ ~ ?? Ubiquitination SUMO tosis results in formation of high molecular weight ~----{ Frank DNA breaks DNA fragments, but not oligonucleosomal DNA 49 fragments , which is accompanied by down­ regulation of caspase-activated DNase (CAD). Fur­ ther it is not affected by z-VAD-fmk (caspase inhibi­ tor), suggesting that caspase is not involved in the excision of DNA loop domains. It appears that topo II is involved in caspase-independent excision of DNA loop domains during apoptosis, and represents an al­ ternative pathway of apoptotic DNA disintegration different from CAD driven caspase-dependent oligo­ nucleosomal DNA cleavage. Furthermore, there is Inhibition of apoptosis 50 evidence that several oncogenes including ras , myb51 and p5352 interact with sequences within the Fig. 2-:-Schematic diagram showing cellular responses to cleavable complexes stabilized by topo poisons topo II a promoter. DWARAKANATH et at. : TOPOISOMERASE POISONS AS ANTICANCER AGENTS 653

Pre-clinical studies-Pre-clinical studies in differ­ higher doses. Among several factors that contribute to ent solid tumors have established etoposide as a pri­ the resistance of human tumors is decrease in intra­ mary treatment modality in lung tumors and leukemia cellular drug availability due to multiple drug resis­ especially in Hodgkin's and non-Hodgkin's Lym­ tance (MDR) gene product, P-glycoprotein (Pgp) me­ phoma. Complete response in 12 % of small cell lung diated drug efflux process appears to be common to tumors, significant response in acute amyloid leuke­ many drugs, although camptothecin, a topo I inhibitor 65 mia and Hodgkins lymphoma has also been ob­ seems to be an exception . Besides drug efflux, al­ 53 served . A overall response rate (14%) has been ob­ terations or mutations in drug's cellular target or 66 served in combination with cisplatinum. Studies with binding sites , distribution of cleavage sites, increase human xenografts have shown an unprecedented effi­ in efficient repair of drug-induced damage (cleavable cacy for 9-aminocamptothecin with complete remis­ complexes) and cross resistance to other structurally 54 sions in colon , breast and non small cell lung carci­ and mechanistically related drugs also contribute to noma as well as in melanoma cell lines 55. the drug resistance. Lower levels of topoisomerases in Clinical studies-A clear association between topo slow growing tumors and reduction of its expression 67 II a and tumor cell proliferation has been established due to hypermethylation have been reported . 56 in many of the human tumors . Higher levels of ex­ Ubiquitin mediated repair and mutations in critical pression qf to po I has been observed in colon, ovary, proapoptotic and antiapoptotic regulatory proteins like prostate, lung and colorectal tumors as compared to p53 and Bch family or alterations in cell cycle ma­ 57 60 the levels in the corresponding normal tissues - . chinery, which activate checkpoints and prevent ini­ Clinical studies with topo II inhibitor, etoposide have tiation of apoptosis can also be the cause of resis­ 68 reported good responses primarily in patients with tance . Coordinately regulated detoxifying systems, Ewing's sarcoma, Hodgkin's disease, neuroblastoma, such as DNA repair, glutathione S-transferase and rhabdomyosarcoma, small cell lung cancer, testicular cytochrome P450 mixed-function oxidases can also 69 cancer, gestational choriocarcinoma, acute myeloge­ confer resistance to drugs . Resistance can also result nous leukemia and Kaposi's sarcoma, besides a spec­ from defective apoptotic pathways. A brief summary 61 trum of recurrent malignant solid tumors • A com­ of the factors that limit topo inhibitors as effective posite single agent response rate of greater than 20% anti-cancer drugs and suggested approaches to over­ 62 has been observed in some of these tumors . Topo come them is presented in Table 1. inhibitors have also been used as one of the primary Some of the topo II poisons induce acute and de­ drugs among multi-drug therapy in lung, testicular layed toxicities in normal tissue that include bone cancer and leukemias. Combination therapy with cy­ marrow suppression, mild nausea or vomiting and clophosphamide and vincristine in recurrent and re­ hair loss with myelo-suppression being the dose lim­ fractory pediatric tumors have shown 95% responses iting toxicity. Mucosities, liver toxicity, fever and 2 with 35% complete responses, 40% very good partial chills are also observed with high dose regimens . 63 responses and 20% partial responses . Similarly, Although, most of the side effects occur within 1-10 86-95% responses have been observed in lung cancer davs of drug administration, delayed induction of sec­ 64 in combination with cisplatin . ondary tumors mainly in the form of leukemias have 70 Approaches for overcoming limitations-Thera­ been observed • De novo translocations leading to peutic efficacy of topoisomerase inhibitors is limited fusion proteins involving topo I and MLL break point primarily by the resistance of tumors at lower doses cluster regions71 and transcriptional activation of short and toxicity to the normal tissues at moderate to interspersed elements (SINES, like Alu elements) by

Table I-Factors limiting the efficacy of topoisomerase poisons and approaches to overcome them. Factors Limitations Approaches Drug retention and localization Pgp mediated drug efflux Enhance drug retention Drug target interaction Chromatin structure Induction of alteration in chromatin condensation Cleavable complex formation Inadequate enzyme levels Over- expression of topoisomerase genes Repair of cleavable complex Efficient repair systems Inhibition of repair Initiation of cellular response: Mutation in functional genes Modifiers of cellular responses: Cell cycle perturbations. Mitotic death. Cell cycle progression. Apoptosis Apoptosis 654 INDIAN J EXP BIOL, JULY 2004 topo II poisons have recently been implicated for in­ repair of cleavable complexes, manifestation of cyto­ duction of secondary malignancies72. genetic damage and up-regulation of damage depend­ There has been a constant effort for the develop­ ent apoptosis as wen as alterations in damage related ment of newer class of drugs with higher sequence cell cycle delays. 3 specificit/ . Many different analogues of camptothe­ Interestingly, when 2-DG has been added along cin CPT-II have been evaluated in pre-clinical stud­ with the topo inhibitors, a small but significant de­ ies and are under clinical trials 74. Strategies using crease in the cell death is noticed in case of etoposide, MDR inhibitors to enhance intracellular drug reten­ while no effect is observed with camptothecin (Fig.3). tion 75 and agents that leads to induction of topo II lev­ Since functioning of topo II is ATP dependent, these 76 els ego Hoechst-33342 etc are under investigations . observations suggest that 2-DG, induced fall in en­ Combinations of to po inhibitors with NF-kB inhibi­ ergy status (A TP), may reduce the formation of tors or GM-CSF to reduce myelo-suppression and cleavable complexes if the two are added simultane­ 77 related toxicities are currently under investigations . ously or 2-DG before etoposide. Reduced etoposide Combinations of cyclophosphamide with etoposide toxicity in glucose regulated protein (GRP) induced and vincristine for pediatric solid tumors have been in cells following long exposure to 2-DG (4-6 hr) has 78 83 phase IIII clinical trial • Use of topo poisons also po­ also been reported . Irrespective of the mechanisms 79 tentiates the radioimmunotherapy of tumors . Com­ involved in reduced toxicity of etoposide by 2-DG, bination of 'proteasome inhibitors PS-341 with topo our results as well as earlier findings suggest that the poisons has also enhanced the efficacy of these drugs efficacy of topo II inhibitors can be enhanced by 2- with little or no normal tissue toxicity in vitro are cur­ DG, only when 2-DG is added (administered) after 8o rently under clinical trials . Etoposide, ifosamide and the cells are treated with the inhibitor, i.e. when sig­ cisplatin combination therapy has been in clinical use nificant amounts of cleavable complexes have been 81 for refractory childhood solid tumors . formed. Analysis of the DNA damage using the nuclear Energy-linked modification of cellular responses to halo assal4 revealed that under these conditions the topo poisons using the glycolytic inhibitor 2-deoxy-D­ presence of 2-DG significantly enhanced the halo di­ glucose-The repair of cleavable complex, the pri­ ameters of BMG-l cells implying a higher level of mary lesions responsible for cytotoxicity as well as DNA strand breaks under these conditions (Fig.4). the drug retention linked to Pgp pump mediated efflux Although, the mechanisms involved in the repair of process are both known to be energy dependent proc­ cleavable complex are not completely understood, esses. Since cancer cells derive a major portion of the cells defective in double strand break repair are found energy (A TP) through glycolysis, it has been sug­ to be more sensitive to topo II inhibitors 85,80. gested that inhibitors of glycolysis, like 2-deoxy-D­ glucose (2-DG) may selectively enhance the cytotox­ icity of topo poison in cancer cells. Studies carried out in human glioma (BMG-l & U- 87) and squamous carcinoma (4197 & 4451) cell lines c:: 0.8 have shown that the cytotoxicity of etoposide, a topo o · II inhibitor, camptothecin, a topo I inhibitor and bis­ .~

More recently, ubiquitination mediated protein (to­ responsible for chemosensitization by 2-DG in these poisomerases) degradation resulting in the formation cells. Interestingly, it has also been found that 2-DG of frank DNA strand breaks, facilitating the comple­ enhances the topo inhibitor induced delayed apopto­ tion of repair by strand break repair pathways has sis, particularly in case of topo II inhibitor etoposide been suggested35. 43. 45. Therefore, it appears that 2-DG (Fig.S c,e). However, no significant induction of early does not interfere in the conversion of cleavable com­ apoptosis can be observed under these conditions, plex into frank strand breaks, while it clearly inhibits implying thereby that the enhanced cytotoxicity is the repair of strand breaks. Inhibition of DNA double primarily due to an increase in the mitotic death. strand break repair and excision repair following UV Further investigations have also shown that 2-DG as well as ionizing radiation by 2-DG has been re­ significantly enhanced the cell cycle delay induced by 8792 ported earlier - • all the three topo inhibitors. Since the extent of cell . Studies carried out to investigate the role of cyto­ cycle delay arising on account of the functioning genetic damage clearly show that, 2-DG enhances the check points47 is related to the level of DNA damage, topo inhibitor induced micronuclei formation by 40% the additional delay observed also indicates the pres­ and 60% respectively with etoposide and H-342, ence of a higher level of DNA damage in cells treated while a 20% increase has been observed with camp­ with 2-DG following exposure to topo inhibitors. tothecin (Fig.S b,d). These observations are similar to Taken together, the available evidences so far indi­ the earlier results on the enhancement of radiation­ cate that the presence of 2-DG for a few hours fol­ induced micronuclei formation in tumor cell lines as lowing exposure to topoisomerase inhibitors can sig­ well as short-term organ cultures of tumors88.89.92-95. nificantly enhance their cytotoxicity, by inhibiting the Modifications of the topo inhibitor induced cell death reversibility of cleavable complexes, the primary le­ and micronuclei formation by 2-DG observed here sions responsible for the cell death. A higher level of (Fig.Sd) imply that enhanced mitotic death is partly DNA strand breaks accumulated under these condi-

a]

~ Core diameter ().tm) l-----~ Halo diameter (11m)

; ~ ~-= ' c '~ ~t~~-I- --=-:::~";; ; E:t ~ E ~2 ~iJ I [b] 30 J . fJ'J I o 25 -' ~ ...c:: 20 4-< o 15 o Z 10 5 - , o -t--fi-r--(1<"...,o-,r-n-,--G-r-IIFT-...... ,.-III--r----T"I:l-r-o .. ! o 4 8 12 16 20 24 28 32 36 40 Halo diameter ().tM)

Fig. 4-Effect of 2-DG (SmM, 2hr) on etoposide induced DNA damage studied by nuclear halo assay in a human glioma cell line (BMG-l). [a] Photo-micrographs of nuclear halo [b] Frequency distribution of halo diameters following various treatments. 656 INDIAN J EXP BIOL, JULY 2004

60r------r======~ 25 a b ~ 50 (;' c 40 III :::J CT & 30 'Q) en U :::J .~ 20 C o 0. o o 10 ~ ~

Etoposide H-342 Camptothccin Etoposide H-342 Carnptothccin

Fig 5-Photomicrographs of DAPI stained BMG-l cells showing treatment-induced micronucleus (arrowhead) and apoptosis (arrow). [aj Control; [bj Etoposide (10 /-lM); [c] Etoposide (10 /-lM) 2-DG (5 mM) in a human glioma cell line (BMG-J). Enhancement of etoposide induced micronuclei formation [dj and apoptosis [e] by 2-DG observed at 48hr after treatment are also shown. tions and the significant increase in micronuclei for­ fiers that selectively enhance the sensitivity of neo­ mation lends support to this proposition. However, plastic cells. One of the challenges for the future is to maintenance of higher intracellular levels of topo in­ develop predictive assays and to individualize therapy hibitors, due to the inhibition of energy dependent, to benefit patients from the tumor specific therapeutic Pgp mediated process responsible for the drug efflux65 regimens. Further, development of appropriate in vi­ can also contribute to the enhanced cytotoxicity _ Pre­ tro and in vivo models to predict major toxicity is ex­ clinical studies in tumor bearing animals to investi­ pected to facilitate the development of agents and gate the effects of the combined treatment (topo in­ strategies for ameliorating treatment induced toxicity hibitors + 2-DG) on tumor control as well as systemic with topo poisons. toxicity responsible for the therapeutic efficacy are In conclusion, topoisomerase inhibitors form an warranted before contemplating clinical trials. Pre­ important class of anticancer drugs. They could be liminary studies in Ehrlich ascites tumor bearing mice expected to play a greater role in the future, when have indeed shown that a combination of etoposide limitations associated with their use are overcome by and 2-DG could be more effective than etoposide some of the approaches outlined here. 96 alone in achieving the tumor control , 97. Acknowledgement Future perspectives We are grateful to Gen T Ravindranath, Director Considerable progress has been made in the last INMAS for his keen interest and constant encourage­ decade towards the understanding of molecular and ment in this work. We thank Dr Seema Gupta and Dr cellular mechanisms underlying the cytotoxicity of Shailja Singh for helpful discussions during the topoisomerase inhibitors, as well as natural and ac­ prepration of this manuscript. These studies have been quired tumor cell resistance to these drugs. These ad­ supported by DRDO project (INM-280), Ministry of vancements should facilitate the design of new topo Defence, Govt. of India_ Mr. Rohit Mathur is thankful poisons as well as adjuvant biological response modi- to CSIR, New Delhi for awarding JRF. DW ARAKANATH et al.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS 657

References 19 Ohneseit P A, Wildemann A, Herskind C, Bambocg M & Rodemann H P, Differential response of tumor cells and Champoux J J, DNA topoisomerases: Structure, function and normal fibroblast to fractionated combined treatment with mechanism, Ann Rev Biochem, (2000) 369. topotecan & ionizing radiation, Int J Radiat Bioi, 78 (2002) 2 Hande K R, Clinical applications of anticancer drugs targeted 125. to topoisomerase II, Biochim Biophys Acta, 1400 (1998) 173. 3 Osheroff N, Effect of antineoplastic agents on the DNA 20 Turley H, Comley M, Houlbrook S, Nozaki N, Kikuchi A, cleavage/religation reaction of eukaryotic topoisomerase II: Hickson I D, Gatter K & Harris A L, The distribution and expression of two isoforms of DNA topoisomerase II in Inhibition of DNA religation by etoposide, Biochemistry, 28 (1980) 6157. normal and neoplastic human tissues, Br J Cancer, 75 ( 1997) 4 Ross W, Rowe T, Glisson B, Yalowich J & Liu L, Role of 1340. topoisomerase II in mediating epipodophyllotoxin induced 21 Dwarkanath B S & Jain V K, Effects of gamma rays and DNA cleavage, Cancer Res, 44 (1984) 5857. glucose analogs on the energy metabolism of a cell line de­ 5 Davies S M, Robson C N, Davies S L & Hickson I D, Nu- rived from human cerebral glioms, Indian J Biochem Bio­ clear topoisomerase II levels correlate with the sensitivity of phys, 28 (1991) 203. mammalian cells to intercalating agents and epipodophyllo- 22 Hussain I, Mohler J L, Seigler H F, & Besterman J M, Ele­ toxins, J Bioi Chem, 263 (1988) 17724. vation of topoisomerase I and mRNA, protein, and catalytic 6 Fry A M, Chresta C M & Davies S M, Relationship between activity in human tumors: Demonstration of tumor-type topoisomerase II level and chemosensitivity in human tumor specificity and implications for cancer chemotherapy, Can­ cell lines, Cancer Res, 51 (1991) 6592. cer Res, 54 (1994) 539. 7 Sinha B K. Topoisomerase inhibitors-A review of their 23 Andoh T & Ishida R, Catalytic inhibitors of DNA topoi­ therapeutic potential in cancer, Drugs, 49 (1995) II. somerase II, Biochim Biophys Acta, 1400 (1998) 155. 8 Han Z, , Dunawy S W, Darnowski J W, Calabresi p, Sedivy 24 Hsiang Y H, Hertzberg R, Hecht S & Liu L F, Camptothecin J, Hendrickson E A, Balan K V, Pantazis P & Wyche J H, induces protein linked DNA breaks via mammalian DNA to­ Role of p21 in apoptosis and senescence of human colon poisomerase I, J Biol Chem, 260 (1985) 14873. cancer cells treated with camptothecin, J Bioi Chem, 277 25 Squires S, Ryan A J, Stutt H L & Johnson R T, Hypersensi­ (2002) 17154. tivity of cockayne's syndrome cells to camptothecin is asso­ 9 Verga-Weisz P D, Wilm M, Bonte E, Dumas K, Mann M & ciated with the generation of abrlOrmally high levels of dou­ Becker P B, Chromatin remodelling factor CHRAC contains ble strand breaks in nascent DNA, Cancer Res, 53 (1993) the ATPases ISWIand topoisomerase II, Nature, 388 (1997) 2012. 593. 26 Horwitz S B & Horwitz M S, Effects of camptothecin on the 10 Chen A Y, Liu L F, DNA topoisomerases: Essential enzymes breakage and repair of DNA during the cell cycle, Cancer and lethal targets, Ann Rev Pharmacol Toxicol, 34 (1994) Res, 33 (1973) 2834. 191. 27 Lambet J M & Fernandes Double J, Topoisomerase II 11 Wang J C, DNA topoisomerases, Ann Rev Biochem, 65 cleavable complex formation within DNA loop domains, (1996) 635. Biochem Phamlacol, 60 (2000) 10 1. 12 Pourquier P, Uneg L M, Kohlhagen G, Mazumder A, Gupta 28 Hsiang Y H, Lihou M G & Liu L F, Arrest of replication M, Kohn K W & Pommier Y, Effects of uracil incorporation, forks in drugs stabilized topoisomerase I DNA cleavable DNA mismatches and abasic sites on cleavage and religation complexes as a mechanism of cell killing by camptothecin, activities of mammalian topoisomerase I, J Biol Chem, 272 Cancer Res, 49 (1989) 5077. (1997) 7792. 13 Kingma P S, Corbett A H, Burcham P C, Manett L J & 29 Lynn R M, Bjornsti A M, Caron P R & Wang J C, Peptide Osheroff N, Abasic sites stimulate double stranded DNA sequencing and site directed mutagenesis identify tyrosine- cleavage mediated by topoisomerase II. DNA lesions as en- 727 as the active site tyrosine of saccharomyces cerevisiae dogenous topoisomerase II poisons, J Bioi Chem, 270 (1995) DNA topoisomerase I, Proc Natl Acad Sci USA , 86 (1989) 21441. 3559. 14 Satoh M S & Lindahl T, Role of Poly (ADP-ribose) forma- 30 Holm C, Covey J M, Kerrigan D & Pommier Y, Differential tion in DNA repair, Nature, 356 (1992) 356. requirement of DNA replication for the cytotoxicity of DNA 15 Heck M M S & Earnshaw W C, Topoisomerase II: A specific topoisomerase I and II inhibitors in chinese hamster DC3F marker for cell proliferation, J Cell Bioi, 103 (1986) 2569. cells, Cancer Res, 49 (1989) 6365. 16 Isaacs R J, Davies S L, Redwood MIS C, Wells N J & 31 Muscarella D E, Rachlinski M K, Sotiriadis J & Bloom S E, Hickson I D, Physiological regulation of topoisomerases II, Contribution of gene-specific lesions, DNA-replication asso­ Biochim Biophys Acta, 1400 (1998) 121. ciated damage, and subsequent transcriptional inhibition in 17 Zini N, Santi S, Ognibene A, Bavelloni A, Neri L M, Val- topoisomerase inhibitor mediated apoptosis in lymphoma mori A, Mariani E, Negri C, Astaldi-Ricotti G C & Maraldi cells, Exp Cell Res, 238 (1998)155. N M, Discrete localization of different DNA topoisomerases 32 Subramanian D, Rosenstein B S & Muller M T, Ultraviolet in HeLa and K562 cell nuclei and subnuclear fractions, Exp induced DNA damage stimulates topoisomerase I-DNA Cell Res, 210 (1995) 336. complex formation in vivo: Possible relationship with DNA 18 Meyer K N, Kjeldsen E, Straub T, Knudsen B R, Hickson I repair, Cancer Res, 58 (1998) 976. D, Kikuchi A, Kreipe H & Boege F, Cell cycle-coupled relo- 33 Radford I R, Initiation of ionizing radiation-induced apopto­ cation of types I and II topoisomerases and modulation of sis: DNA damage mediated or does ceramide have a role? Int catalytic enzyme activities, J Cell Bioi, 136 (1997) 775. J Radial Biol, 75 (1999) 521. 658 INDIAN J EXP BJOL, JULY 2004

34 Kauffmann S H, Cell death induced by topoisomerase­ 51 Brandt T L, Fraser D L, Leal S, Halandras P M, Kroll A R, & targeted drugs: More questions than answers, Biochirn Bio­ Kroll D 1, c-Myc trans-activates the human DNA topoi­ phys Acta, 1400 (1998) 195. somerase II alpha gene promoter, J Biol Chem, 272 (1997) 35 Mao Y, Sun M, Desai S D & Liu L F, SUMO-l conjugation 6278. to topoisomerase I: A possible repair response to topoi­ 52 Sandri M I, Isaacs R J, Ongkeko W M, Harris A L, Hickson I somerase mediated DNA damage, Proc Natl Acad Sci USA, D, Broggini M & Vikhanskaya F, p53 regulates the minimal 97 (2000) 4046. promoter of the human topoisomerase n alpha gene, Nucleic 36 Lekin N D & Jackson S, Regulation of p53 in response to Acid Res, 24 (1996) 4464. DNA damage, Oncogene, 18 (1999) 7644. 53 Smit E F, Carney D N, Harford P, Sleijfer D T, & Postmus P 37 Walker P, Smith C, Youdale T, Leblanc J, Whitfield J & E, A phase II study of oral etoposide in elderly patients with Sikorska M, Topoisomerase II reactive chemotherapeutic small cell lung cancer. Thorax, 44 (1989) 631. drugs induce apoptosis in thymocytes Cancer Res, 51 (1991) 54 Giovanella D C, Stehilin J S & Wall J S, DNA topoisomer­ 1078. ases I targeted chemotherapy of human colon cancer xeno­ 38 Karpinich N 0, Tafani M, Rothman R J, Russo M A & Far­ grafts, Science, 246 (1989) 1046. ber J L, The course of etoposide induced apoptosis from 55 Giovanella B C, Hinz H R, Koziiski A J, Stelin J SIr, Silber damage to DNA and p53 activation to mitochondrial release R, & Potmesil M , Complete growth inhibition of human of cytochrome c, J Bioi Chem, 277 (2002) 16547. cancer xenograft in nude mice treatment with 20-(S)­ 39 Gillano N, Ikhtiar A M & Malinovski ° V, The effects of camptothecin, Cancer Res, 51 (1991) 3352. inhibitors of DNA topoisomerase II on the radiosensitivity, 56 Lynch B I , Guinec D G & Holden J A, DNA topoisomerase repair and conformational changes in the chromatin in J[ a: A new marker of cell proliferation in invasive breast mammalian cells, Radiobiologia, 33 (1993) 189. cancer, Human Pathol, 28 (1997) 1180. 40 Nittis & Wang 1 C, DNA topoisomerase-targeting antitumor 57 Ohashi N, Fujiwara Y, Yamaoka N, Katoh 0, Satow Y & drugs can be studied in yeast, Proc Natl Acad Sci USA, 85 Yamakido M, No alterations in DNA topoisomerase I gene (1988) 7501. related to CPT-Il resistance in human lung cancer, Jpn J 41 Reid RID, Kauh E A & Bjornst M A, Camptothecin sensi­ Cancer Res, 87 (1996) 1280. tivity is mediated by the pleiotropic drug resistance network 58 Vander Zee A G J, Hollema H, Dejong S, Boonstra H, Gouw in yeast, J Bioi Chern, 272 (1997) 12091. A, Willemse P H B, Zijlstra J G & Vries E G E, P­ 42 Desai S D, Liu L F, Vazquez-Abad D & Arpa P D, Ubiquitin glycoprotein expression and DNA topoisomerase I and II ac­ dependent destruction of topoisomerase I is stimulated by the tivity in benign tumors of the ovary and in malignant tumors antitumor drug camptothecin, J Bioi Chem, 272, (1997) of the ovary, before and after platinum/cyclophosphamide 24159. chemotherapy, Cancer Res, 51 (1991) 5915. 43 Andoh T, Ishii K, Suzuki Y, Ikegami Y, Kusumoki Y & 59 Madden K R & Champoux 1 J, Overexpression of human Kokada T T, Characterization of a mammalian mutant with a topoisomerase I in baby hamster kidney cells: Hypersensi­ camptothecin resistant DNA topoisomerase I, Proc NaIL tivity to clonal isolates to camptothecin, Cancer Res, 52 Acad Sci USA, 84 (1987) 5565. (1992) 525. 44 Spence J, Saids S, Hass A L & Finley D, A ubiquitin mutant 60 Giaccone G, Ark-Otte J V, Scagliotti G, Capranico G, Valk P with specific defects in DNA repair and multiubiquitination, V, Rubio P V, Dalesio 0, Lopez 0 , Zunino F, Walboomers J, Mol Cell Bioi, (1995) 1265. & Pinedo H M, Differential experience of DNA topoisomer­ 45 Dwarakanath B S & Mathew T L, Approaches for improving ase in non-small cell lung cancer and normal lung, Biochim cancer chemotherapy using topoisomerase inhibitors, XXII Biophys Acta, 12 (1995) 337. Annual Conference of Indian Association of Biomedical Sci­ 61 Kung F H, Desai S J, Dickerman D J, Goorin A M, Harris M entisls held at IABMS Chennai (2001). B, Inoue S, Krischer J P, Murphy S B, Pratt C B & Toledano

46 Rao A P & Rao P N, The cause of G2 arrest in CHO cells S, Ifosamide/carboplatin/etoposide (ICE) for recurrent ma­ treated with anticancer drugs, J Natl Cancer Insl, 57 (1976) lignant solid tumors of childhood, a pediatric oncology group 1139. phase IIII study, J Pediat Hemalol Oncol, 17 (1995) 265. 47 Tsao Y P, Arpa P D & Liu L F, The involvement of active 62 Issell B F, Rudolph A, & Louie A C, A preview in etoposide DNA synthesis in camptothecin induced G2 arrest: Altered (VP-J6): Current status and new developments,. edited. by B cdc2 regulation of p34 /Cyclin B I. Cancer Res, 52 (1992) 1823. F Issell, (Academic Press, London) 1984. 48 Guillouf C, Grana X, Selvakumaran M, Hoffman B, Gior­ 63 Vinolas N, Graus F, Mellado B, Caralt L & Estape 1, Phase II dano A & Liebermann D A, Dissection of the genetic pro­ trial of cisplatin and etoposide in brain metastases of solid

grams of p53 G 1 growth arrest and apoptosis: Blocking p53 tumors, Bull J Neurooncol, 35 (1997) 145. induced apoptosis unmasks G 1 arrest, Blood, 85 (1995) 2691. 64 Hoff 1 V, Grier H E, Douglass E C & Green D M, Etoposide, 49 Solovyan V T, Bezvenyuk Z A, Salminen A, Austin C A & ifosfamide and cisplatin therapy for refractory childhood Courtney M 1, The role of topoisomerase II in the excision of solid tumors, Response and toxicity, Bull Cancer, 75 (1995) DNA loop domains during apoptosis, J Bioi Chern, 277 2966. (2002) 21458. 65 Gottesman M M, Fojo T & Bates S E, Multidrug resistance 50 Woessner R D, Chung T D Y, Hofmann G A, Mattern M R, in cancer: Role of A TP dependent transporters, Nature, 2 Mirabelli M R, Drake F H & 10hnson R K, Differences be­ (2002) 48. tween normal and ras-transformed NIH-3T3 cells in expres­ 66 Megonigal M D, Fertala J & Bjornsti M A, Alterations in the sion of the 170 KD and 180 KD forms of topoisomerase II, catalytic activity of yeast DNA topoisomerase I result in cell Cancer Res, 50 (1990) 2901. cycle and cell death, J Biol Chern, 272 (1997) 12801 . DW ARAKANATH et af.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS 659

67 Fujimori A, Hoki Y, Popescu N C & Pommier Y, Silencing granulocyte colony-stimulating factor after ifosamide, carbo­ and selective methylation of the normal topoisomerase I gene platin and etoposide in children with recurrent or refractory in camptothecin resistance CEM/C2 human leukemia cells, solid tumors: A children cancer group report, J Pediat Oncol Res, 8 (1996) 295. Hematol, 23 (2001) 30. 68 Gudkov A V, Zelnick C R, & Zakarov A R, Isolation of ge­ 82 Dwarakanath B S, Adhikari J S, Divya Khaitan, Mathew T L netic suppressor elements, inducing resistance to topoi­ & Jain V K. Modification of cellular response to topoisomer­ somerase II-interactive cytotoxic drugs, from human topoi­ ase inhibitor for improving cancer therapy. XV Asia Pacific somerase II c-DNA, Proc Natl Acad Sci USA, 90 (1993) Cancer Conference held at Chennai, 12-15'h December, 3231. 1999. 69 Schuetz E G, Beck W T & Schuetz J D, Modulators and 83 Tomida A, Yun J & Tsuruo T, Glucose regulated stresses substrates of P-glycoprotein and cytochrome P-450 3A coor­ induce resistance to camptothecin in human cancer cell s, Int dinately upregulate these proteins in human colon carcinoma J Cancer, 68 (1996) 391. cells, Mol Pharmacal, 49 (1990) 311. 84 Roti Roti J L & Wright W D, Visualization of DNA loops in 70 Karp A J E & Smith M A, The molecular pathogenesis of nucleoids from HeLa cells: Assays for DNA damage and re­ treatment induced (secondary) leukemia: Foundations for pair, Cytometry, 8 (1987) 461. treatment and prevention, Semin On coL, 24(1997) 103. 85 Caldecott K, Banks G R & Jeggo P A, DNA double strand 71 Broeker P L S, Super H G, Thirman M J, Pomykala H, Yo­ break repair pathways and cellular tolerance to inhibitors of nebayashi Y, Tanabe S, Zeleznik-Le N, 7 Rowley J D, Dis­ topoisomerase II, Cancer Res, 50 (1990) 5778. tribution of IIq23 breakpoints within the MLL breakpoint 86 Jeggo P A, Caldecott K W, Pidsley S & Banks G R, Sensi­ cluster region in acute leukemia and in treatment related tivity of Chinese hamster ovary mutants defective in DNA acute myeloid de novo leukemia: Correlation with scaffold double strand break repair to topoisomerase II inhibitors, attachment regions and topoisomerase II consensus binding Callcer Res, 49 (1989) 7057. sites, Blood, 87 (1996) 1912. 87 Verma A, Shrama R & Jain V K, Energetics of DNA repair 72 Rudin C M & Thompson C B, Transcriptional activation of in UV irradiated peripheral blood leukocytes from chronic short interspersed elements by DNA damaging agents, Gelles myeloid leukamia patients. Plwtochem Photobiol 36 (1982) Chromosomes Callcer, 30 (200 I) 64. 1513. 73 Capranico G, Binashi M, Borgnetto M E, Zunino F, & 88 Jain V K, Kalia V K, Sharma R, Maharajan V & Menon M, Palumbo N, A protein mediated mechanism for DNA se­ Effects of 2-deoxy-D-glucose on glycolysis, proliferation ki­ quence specific action of isomerase topo II poisons, TIPS, 18 netics and radiation response of human cancer cells, lilt J (1997) 323. Radiat Oil coL Biol Phys, II (1985) 943. 74 Carbonero R G & Supko J G, Current prospectives of clinical 89 Dwarakanath B S & Jain V K, Energy linked modifications experience, pharmacology and continued development of the of the radiation response in a human cerebral glioma cell camptothecins, Clill Cancer Res, 8 (2002) 641. line, Int J Radiat Oncol Biol Phys, 17 (1989) 1033. 75 Dantzing A H, Reversal of P-glycoprotein mediated multi­ 90 Frankeberg S M, Harbich R & Frankenberg D, 2-deoxy-D­ drug resistance by a potent cyclopropyldibenzosoberane glucose inhibits rejoining of radiation induced DNA double modulator L Y339979, Cancer Res, 56 (1998) 4171. strand breaks in yeast, Int J Radiat Bioi, 61 (1992) 185. 76 Tolner B, Hartley J A & Hochhauser, Transcriptional regula­ 91 Jha B, & Pohlit W, Effects of 2-deoxy-D-glucose on DNA tion of topoisomerasc Il a. expression at confluence and double strand break repair, cell survival and energy metaboli sm pharmacological modulation of expression by bis­ in euoxic Ehrlich ascites tumor cells, Int J Radiat Bioi, 62 benzimidazole Drugs, Mol Pharmacal, 59 (2001) 699. (1992) 409. 77 Olver I N, Hercus T, Lopez A, Vadas M, Somogyi A A, 92 Dwarakanath B S, Zolzer F S, Chandna S, Bauch T, Adhikari Doyle I, Foster D J R, Keeefe D, Taylor A, Brown M, Bik J S, Muller W U, Streffer C & Jain V, Heterogeneity in 2- Tol, Cole 1, Rawling T, Cambareri B, Meyers M, Oleszewski deoxy-D-glucose induced modifications in energetic and ra­ N, Bastiras S, Senn C, Hey A, Verma M & Wigley P, A diation responses of human tumor cell lines. lilt J Radiat On­ phase I study of the GM-CSF antagonist E21R, Cancer col Bioi Phys, 51 (2001) 1151. Chemother PhannacoL, 50 (2002) 171. 93 Dwarakanath B S & Jain V K, Enhancement of radiation 78 Mantadakis E, Herrera L, Leavey P J, Bash R 0 , Winick N J damage by 2-deoxy-D-glucose in organ cultures of brain tu­ & Kamen B A, Fractionated cyclophosphamide and etop,' mors, Ind J Med Res, 82 (1985) 266. side with advanced or refractory solid tumors: Phase II win­ 94 Dwarakanath B S & Jain V K, Modification of the radiation dow study, J Clin Oncol, 18 (2000) 2576. induced damage by 2-deoxy-D-glucose in organ cultures of 79 Roffler S R, Chan 1 & Yeh MY, Potentiation of radioimmu­ human cerebral gliomas, lilt J Radiat Oncol Biol Phys, 13 no therapy by inhibitor of topoisomerase I, Cancer Res, 54 (1987) 741. (1994) 1276. 95 Dwarakanath, B S & Jain V K, In vitrf) radiation response of 80 Cusack Jr C, Chan J, Liu R, Houston M, Abendroth K & human intracranial meningiomas and its modification by 2- Elliot P J, Adams J, & Baldwin A S, Enhanced chemosensi­ deoxy-D-glucose, Ind J Med Res, 92 (1990) 183. tivity to CPT-II with Proteasome inhibitor PS-341 : Implica­ 96 Gupta S, Mathur R & Dwarakanath B S, Modification of tions for systemic nuclear factor KB inhibition, Cancer Res, etoposide induced toxicity by 2-deoxy-D-glucose in murine 61 (2001) 3535. tumors, Biomedicine, 2003 (Unpublished). 81 Cairo M S, Shen V, Krailo M D, Bauer M, Miser J S, Sato J 97 Gupta S, Mathur R & Dwarakanath B S, Modification of K, Blatt J, Blazer B R, Frierdich S, Mares W L, & Reaman G etoposide induced toxicity by 2-DG in murine tumors, Euro J H, Prospective randomized trial between two doses of Oncol (Unpublished).