Novel mechanisms of resistance to inhibitors of DNA topoisomerases

Advan. Enzyme Rep/., Vol37, pp. 17-26,1997

Pergamon

CopyrightQ 1997ElsevierScienceLtd Printedin GreatBritain. All rightsreserved 0065-2571/97/$32.00

PII:SOO65-2571(96)00024-6

NOVEL MECHANISMS OF RESISTANCE TO INHIBITORS DNA TOPOISOMERASES

OF

WILLIAM T. BECK*?, TAYEB KHkLIFA*, HIROKI KUSUMOTOS, YIN-YUAN MO*, QUEEN RODGERS& JUDITH S. WOLVERTON§ and QINJIAN WANG* *Cancer Center, University of Illinois at Chicago, Chicago, Illinois, U.S.A. $Medical Institute of Bioregulation, Kyushu University, Beppu, Oita, Japan §St Jude Children’s ResearchHospital, Memphis, Tennessee,U.S.A. INTRODUCTION

DNA topoisomerases are enzymes that are essential for DNA synthesis and chromosome replication during cell division. Their breaking-rejoining function is unique and potentially lethal to the cell if the DNA breaks are not resealedproperly. Topoisomerasesrelieve torsional stresson DNA during the processesof transcription, replication and cell division, and possibly in DNA repair, by introducing transient single- or double-strand breaks in the DNA, thereby allowing another strand of DNA to passthrough the cleavedstrand(s) (reviewed in Refs (1) and (2)). Key steps are (i) the cleavage reaction, as the enzyme is covalently linked to both ends of broken DNA, making it a potentially lethal form; (ii) the rejoining reaction, which must occur to restore the integrity of the DNA; and (iii) the catalytic step in which ATP is consumed and the enzyme is released from the DNA (3,4, and referencestherein). In addition to (and because of) their important biological roles, the topoisomerasesare desirable targets for chemotherapy, as their inhibition can lead to cell death. All inhibitors of mammalian cell topoisomerases block the catalytic activity of the enzymes, but the mechanism(s) by which they do so are fundamentally different. As has been detailed, inhibitors of the type II enzymes, the drugs either stabilize cleaved DNA-protein complexes (5, 6), ultimately preventing enzyme action, or block the catalytic activity (7, 8), most likely by ‘locking’ the enzyme in a ‘closed clamp’ conformation around the DNA (9). Thus, the complex-stabilizing inhibitors (epipodophyllotoxins, anthracyclineq aminoacridines, etc.) appear to act by blocking the religation TAuthor for correspondence at: Cancer Center (m/c 569), College of Medicine, University of Illinois at Chicago, 9005, Ashland Avenue, Chicago, IL 60607-7173, U.S.A. 17

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step (5, 6) whereas agents such as merbarone, fostreicin, and the bis(dioxo)piperazines, ICRF-193 and ICRF- 187, all appear to block the catalytic activity of the enzyme (7-9). Further analysis reveals more fundamental differences: catalytic inhibitors such as the ICRF compounds can, apparently through their block of topo II, prevent chromosome segregation, leading to the appearance of long, unseparated chromosomes (10, 11). Despite these mechanistic differences, the effects of these agents on cell cycle progression appear to be similar: they all block cells in the G21M phase of the cell cycle (12). These data permit the suggestion that these drugs reveal the M-phase action of topo II, which has been shown to be essential in yeast (13). How any of these actions are translated into a cytotoxic event remains unclear. After treatment of cells with topoisomerase inhibitors, c-jun is activated (l&17), as is AP-1 binding activity (14-l 7), suggesting that activation of novel genesmay be involved in the cytotoxic response (18). Other factors involving cell cycle proteins and gene rearrangements may be involved (19-21), and may include a G2 checkpoint arrest (22). Further support for the involvement of novel genesin the cytotoxic response comes from the observations that their activation by drugs is attenuated in drug-resistant cells (17). Thus, in order to assessthe role(s) of these genes in the cytotoxicity of the topoisomerase inhibitors, we have selected cell lines for resistance to complex stabilizing inhibitors of topo II, as well as to catalytic inhibitors of topo II. Our results reveal some novel and unanticipated actions of these drugs that may offer insights into their cytotoxic mechanisms, and also into the bases by which tumor cells express resistance to these agents. MATERIALS

AND

METHODS

Cell lines. We have utilized the human leukemic cell line, CCRF-CEM (CEM), and have selected drug-resistant variants from it. Discussed in this paper are the cell lines selectedfor resistance to the complex-stabilizing topo II inhibitor, teniposide (VM-26) (23 and referencestherein), and the catalytic inhibitor, merbarone (24). The selections involved growth in sublethal concentrations of the respective drug and subsequent cloning of the cells by limiting dilution. The details of the selection processeshave been published (23,24). Cytotoxicity and growth-inhibition assays. Growth-inhibition assays(both 4% and 72-hr drug exposure) were performed as described previously (24). We also showed that the growth-inhibition assayscorresponded well with clonogenie assays(24). Immunoblots. Immunoblotting for topoisomerases was done as described previously (23, 24). Antibodies against topo II a, l3 (24), and both a and p

19

RESISTANCE TO TOPOISOMERASE INHIBITORS

(23), as well astopo I (TI-1; 25) were developed and used as described.Antibodies against several proteins involved in signaling, especially jun kinase-1 (JNK-I), were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz. CA), and used according to manufacturer’s instructions. Northern blots. cDNA probes were used to assessthe expression of several genes, especially c-&n (17) and JNK-1 (26). Expression of specific mRNAs was assessedby Northern analysis, as detailed previously (17). Immunostaining. Immunocytochemical staining of cytospin preparations of CEM cells with TI-1 antibody was done as described (25) but using a peroxidase-tagged second antibody, rather than an FITC-tagged second antibody. RESULTS

AND

DISCUSSION

Resistance and Cross-resistance of Teniposide- and Merbarone-resistant Cells

CEM

The resistance and cross-resistance profiles of teniposide-resistant CEM/ VM- 1 cells and merbarone-resistant CEM/Bl cells are shown in Table 1. Both cell lines are resistant and cross-resistant to VM-26, VP- 16, doxorubicin, and amsacrine. Of interest, both the teniposide- and merbarone-resistant cell lines are coflaterally sensitive to the catalytic inhibitor of topo II, ICRF-187. This was initially a rather surprising finding for the merbarone-resistant cells, but easily interpretable in light of the topo II levels of the cells (see below). Both merbarone and ICRF-187 are catalytic inhibitors of topo II. That the merbarone-resistant cells are collaterally sensitive to ICRF-187 suggeststhat merbarone is not a pure catalytic inhibitor, but rather, has other actions. Moreover, the cross-resistanceof the merbarone-resistant cells to the inhibitor of topo I, SN-38, probably reflects the anti-top0 I actions of merbarone as described in very early reports of this agent (27). TABLE 1. RELATIVE CROSS-RESISTANCE OF TENIPOSIDE- AND MERBARONERESISTANT HUMAN LEUKEMIC CELL LINES Cell Line CEM CEMNM- 1 CEM/BI

Teniposide

Merbarone

Drug SN-38

1 47” 33d

1 2.9a 5.9d

1 nd. 7.9d

*Data from Ref. (11). bData from Ref. (41). ‘W. T. Beck and T. L. Cadre, unpublished data. dData from Ref. (24). n.d., not done.

mAMSA

ICRF-187

1 30h 7.9d

1 0.67’ O&P

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Topoisomerase II Activities and Expression in Drug-sensitive and -resistant Cell Lines

We have shown previously that, compared to the drug-sensitive cells, the activity of topo II is decreasedin both the CEM/VM-1 (23) and CEM/Bl (24) cell lines. The likely basis for this decreasein topo II activity in the CEM/VM-I cells is the presenceof two mutations in the topo IIa( gene (28) resulting in an altered enzyme, whereas the likely basis for the decreased activity in the CEM/Bl cells is the decreasedexpression of the enzyme (24). The decreased expression of topo II CLin the merbarone-resistant cell lines was an unexpected finding, and ongoing experiments are aimed at understanding the basis for this phenomenon (MO and Beck, manuscript in preparation). Cytotoxic Signaling by Topoisomerase Inhibitors

It has been shown by several laboratories that treatment of tumor cells with inhibitors of topoisomerases-those that stabilize DNA-topoisomerase complexes-initiates a pathway of programmed cell death (PCD) or apoptosis (17, 29-31). Recent work also suggeststhat treatment of cells with topoisomeraseinhibitors activates proteasesinvolved in PCD (32). Associated with this PCD is the increased expression of the protooncogene, c-jun (14-17), activation of AP-1 complexes (14-17), and activation of the signaling pathway involving GrbUSos (33). Whether there is a causal relationship among these events is not clear. Indeed, activation of c-jun and PCD may be unrelated events, and c-jun activation may be a consecmencerather than cause of PCD (34). Whether activation of these protooncogenes and PCD pathways is also seenwith the catalytic inhibitors of topo II is a subject of intense investigation in our laboratory and will be discussed briefly below in the context of drug resistance. Novel Mechanisms of Resistance to Inhibitors of Topoisomerase II: Attenuated Cytotoxic Signal Transduction in Drug-resistant Cells

Earlier work from this laboratory revealed that the activation of c-jun by a complex-stabilizing inhibitor of topo II, teniposide, was seento increase and then decreasein drug-sensitive cells (17); further, in drug-resistant cells, expression of c-jun was attenuated (17). Given that c-@n activity is regulated by phosphorylation through the actions of kinases (35-37), we asked if these enzymeswere altered following treatments with complex-stabilizing inhibitors of topo II. We therefore asked if a catalytic inhibitor of topo II, merbarone, had any effect on the activity of c-jun, and whether any such activity was altered in the merbarone-resistant cells. As seen in Figure 1, treatment of teniposide-resistant CEMNM-1 cells with VM-26 resulted in attenuated activation of c-jun, compared to CEM cells. Interestingly, we also found that treatment of CEM cells with merbarone also led to increased expression of c-&n transcription, but it peaked at a later

40 -

10 -

20 -

W-26

IO

15 25

30

0

Time after treatment (hr)

20

5

10

:5

Merbarone

20

25

FIG. 1, Effect of VM-26 or merbarone on expression of c-jun mRNA in CEM and CEMNM-I ceils. Cells were treated at time 0 with either 10 pi VM-26 or 200 pi merbarone and harvested at the times indicated. mRNA was prepared and Northern blots were probed for c-jun expression as detailed in Materials and Methods. Blots were exposed to X-ray film and the cjun messagewas quantitated using a Bio-Rad GS700 Densitometer and Molecular Analyst software. Shown is a representative gel.

d

830g

8

M

50 -

60-

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W. T. BECK et nl.

time than seen with VM-26. Moreover, this response was also attenuated in the CEM/VM- 1 cells, which are modestly cross-resistant to merbarone (Table 1). These data support the idea that c,jun activation may play a role in generating a cytotoxic responseby theseagents.Thesestudies will be detailed elsewhere (Khelifa et al., ms in preparation). Given that the activity of c-jun is regulated by phosphorylation, we examined the expression of jun kinase-1 (JNK-I) following treatment of GEM cells with VM-26. It can be seen in Figure 2 that this treatment caused increased expression of the JNK-1 protein, and this expression may also be due to increased phosphorylation, currently under investigation. Of interest, this increase in JNK-I protein was also attenuated in the drug-resistant cell lines (data not shown). Is a G2 Checkpoint Involved in the Cytotoxicity of Topo II Inhibitors? We suggestthat inhibitors of DNA topoisomerases,especiallytopo II, induce cytotoxic signaling pathways mediated by the inhibition of the enzyme (4, 37). The fact that both types of inhibitors+omplex-stabilizing as well as catalytic inhibitors-induce c-&n suggeststhat the inhibition of topo II initiates cytotoxic signaling. The time courses of c-jun expression induced by VM-26 and merbarone are consistent with their actions in causing G2/M block. Accordingly, we have proposed elsewherethat since both classesof topo II inhibitors block cells in G2/M (12), they induce a G2 checkpoint that is involved in the signaling process (37). Experiments in support of this notion will be detailed elsewhere(Wang et al., ms in preparation). Novel Mechanisms of Resistance to Inhibitors of DNA TopoisomeraseI The cross-resistance of merbarone-resistant cells to topo I inhibitors such as SN-38 (Table 1) was intriguing to us. It was shown in early studies that

JNK-1 t 1

2

3

6

Hours after VIM-26 FIG. 2. Effect of VM-26 on jun kinase- 1 protein expression in CEM cells. Cells were treated with 10 p VM-26, collected at the times indicated, and whole cell extracts were prepared and separated on loo/ polyacrylamide gels. Shown is a representative immunoblot using an antibody against JNK-I. See Materials and Methods for details.

RESISTANCE TO TOPOISOMERASE INHIBITORS

23

merbarone, in addition to inhibiting topo II, also inhibited topo I (27), thereby providing a mechanistic connection for the cross-resistancedata. However, we found no differences in topo I levels in 12 independently-selected merbaroneresistant cell lines (24). We had previously confirmed that topo I is localized in the nucleolus (25). Additionally, recent studies suggested that cytotoxic drug treatment caused the loss of topo I from the nucleolus (38, 39). We also found this to be the case for the topo I inhibitors. SN-38 and topotecan, and that this delocalization was a function of drug concentration (40). Of much interest, we found that SN-38 causedthe delocalization of topo I in CEM cells (Fig. 3, right side), and found that this response was attenuated in the merbarone-resistant cells (data not shown) (40) suggestingthat this was associated with the drug resistant phenotype. These and other studies will be be reported in detail elsewhere(Wolverton, Kusumoto and Beck. ms in preparation). SUMMARY

AND

FUTURE

CONSIDERATIONS

The topoisomerases are critical enzymes necessaryfor transcription, DNA replication, and chromosome segregation; in addition, they are the targets for critically important and clinically useful anticancer agents.These drugs block the catalytic functions of the enzyme(s) and in so doing initiate a cytotoxic signaling pathway that leads to programmed cell death. That these agents all cause cell cycle arrest at G2/M suggeststhat a checkpoint may be involved in

Topo I in CEM cells

Control

SN-38

FIG. 3. Effects of SN-38 on the subnuclear distribution of DNA topoisomerase I in CEM cells. Immunostaining of cytospins was done with TI-I antibody, as described previously. SeeMaterials and Methods for references.The dark spots in the nuclei represent topoisomerase I in the nucleoli (25).

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this cytotoxic signaling pathway. Tumor cells expressresistance to the topoisomerase inhibitors through decreased activity that is a consequence of decreasedexpression or mutation of the gene.We suggestthat the consequence of decreased topoisomerase activity is attenuation of a cytotoxic signaling mechanism. Deciphering these signals may provide insights into the molecular basis by which these drugs kill tumor cells. ACKNOWLEDGEMENTS

This work began when the authors were at St Jude Children’s Research Hospital. It continues to be supported in part by: grants from the National Cancer Institute (research grants CA40570 and 30103 (WTB). It is also supported in part by the Association pour la Recherche sur le Cancer (TK) and in part by the Cancer Center of the University of Illinois at Chicago. Other support was from program grant CA23099; cancer center support (CORE) grant CA21 765); in part by American Lebanese Syrian Associated Charities, all at St Jude. We thank our former colleagues, especially R. Kim and M. Danks, for helpful discussion and input into earlier work in our laboratory. We are grateful to Tanya Ignatova and Christine Dingledine for their editorial comments and to Tina Cadre and Wu Hua Sun for excellent technical support. REFERENCES 1. J. WANG, DNA topoisomerases, why so many?, J Biol. Chem. X6,6659-6662 (1991). 2. J. WANG, P. CARON and R. KIM, The role of DNA topoismomerases in recombination and genome stability: a double-edged sword?, Cell 62,403-406 (1990). 3. N. OSHEROFF, A. CORBETT and M. ROBINSON, Mechanism of action of topoisomerase II targeted anti-neoplastic drugs, Adv. Pharmacol. 29B, 105-126(1994). 4. J. NITISS and W. BECK, Antitopoisomerase drug action and resistance, Eur. J: Cancer 32A, 958-966 (1996). 5. G. CHEN, L. YANG, T. ROWE, B. HALLIGAN, K. TEWEY and L. LIU, Noninteracalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II, 1 Biof. Chem. 259,13560-l 3566 (1984). 6. M. ROBINSON and N. OSHEROFF, Effects of antineoplastic drugs on the post-strandpassageDNA cleavage/religation equilibrium of topoisomerase II, Biochemistry 30, 18071813(1991). I. E DRAKE, G. HOFMANN, S. MONG, J. BARTUS, R. HERTZBERG, R. JOHNSON, M. MATTERN and C. MIRABELLI, In vitro and intracellular inhibition of topoisomerase II by the antitumor agent merbarone, Cancer Res.49,2578-2583 (1989). 8. R. ISHIDA, T. MIKI, T. NARITA, R. YUI, M. SATO, K. UTSUMI, K. TANABE and T. ANDGH, Inhibition of intracellular topoisomerase II by antitumor bis (2$-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors, Cancer Rex 51,4909-4916 (1991). 9. J. ROCA, R. ISHIDA, J. BERGER, T. ANDOH and J. WANG, Antiminor bisdioxopiperazincs inhibit yeast topoisomerase II by trapping the enzyme in the form of a closed protein clamp, Proc. Nat1 Acad. Sci. USA 91, 1781-1785(1994). 10. R. ISHIDA, M. SATO, T. NARITA, K. UTSUMI, T. NISHIMOTO, T. MORITA, H. NAGATA and T. ANDOH, Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidixation by uncoupling chromosome dynamics from other cell cycle events, 1 Cell Biol. 126,1341-1351 (1994).

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