Inhibitors of topoisomerase II: Structure-activity relationships and mechanism of action of podophyllin congeners

INHIBITORS OF TOPOISOMERASE II: STRUCTURE-ACTIVITY RELATIONSHIPS AND MECHANISM OF ACTION OF PODOPHYLLIN CONGENERS BYRON H. LONG and DALE A. STRINGFELLOW Cancer Research, PharmaceuticalResearch and DevelopmentDivision, Bristol-MyersCorporation, Wallingford, CT 06492

INTRODUCTION Until recently, classical anticancer drugs could be divided into six general classes with respect to mechanism of action: antimetabolite, antimicrotubule, DNA breaking, DNA alkylating, DNA cross-linking, and a group with unknown mechanisms of action. Perhaps one of the most significant discoveries of the present decade in the field of cancer chemotherapy has been the revelation that many of the agents grouped under the category of unknown mechanisms of action are now recognized as potent inhibitors of either type I or type II topoisomerases. The topoisomerase II (EC 5.99.1.3) inhibitors can be subcategorized as DNA intercalators, which include anthracyclines, anthracenediones, ellipticines, acridines, and actinomycin D, and as nonbinders to DNA, which include the demethylepipodophyllotoxins VP16 and VM26 and fostriecin (1-10). It is not surprising that topoisomerase II presents an ideal target for chemotherapeutic agents, as has been illustrated in recent reviews on the subject ( 11-13). This is not only apparent in the anticancer field but also in the antibacterial arena as well, where the quinolone antibiotics, which are potent inhibitors of gyrase, a prokaryote topoisomerase II, are highly successful in combating bacterial infection. Studies originally conducted with yeast containing temperature-sensitive topoisomerase II have shown that this enzyme is required for cell proliferation (14-16), possibly through a requirement for the unique DNA strand passing activity of topoisomerase II that allows all of the euchromatin present in the interphase nucleus to untangle and retract to form the fully condensed chromosomes during early prophase. In fact, Uemura and Yanagida (15) and Holm et al. (16) have clearly shown that yeast cells maintained at the restrictive temperature contain chromosomes that have failed to completely segregate even though the formation of a septum was not inhibited. Topoisomerase II has also been implicated in the functions of the nuclear matrix and chromosome scaffold, 223

224

B.H. LONG and D. A. STRINGFELLOW

possibly serving as anchor sites for DNA or as regulators or facilitators of DNA replication or RNA transcription (17-21). Using an antibody probe to measure enzyme levels in several transformed and developmentally regulated normal cell types, Heck and Earnshaw (22) have recently shown that transformed cells have approximately 1 x 166 copies, dividing erythroblasts have 8 X 1 0 4 copies, and erythroblasts that have ceased to proliferate have less than 300 copies of topoisomerase II per cell. Other laboratories have found similar results and extended their studies to include topoisomerase II inhibitors (23-25). A direct correlation was found between cell proliferation rates and the number of inhibitor-induced, proteinassociated DNA breaks, topoisomerase II activity, and cytotoxicity to the inhibitors in some but not all cell lines (23-25). Furthermore, the enzyme levels vary within the cell cycle, with the highest levels of enzyme activity occurring during DNA replication (24). Studies by Ackerman et aL (26) and Sahyoun et al. (27) demonstrate that topoisomerase II activity can be substantially increased by phosphorylation of a serine residue on the enzyme. It is, therefore, possible that the elevated enzyme activities observed in rapidly dividing, transformed cells may be due, in part, to phosphorylated, and hence, activated enzyme. Thus, it is quite conceivable that the selectivity displayed by topoisomerase II inhibitors for cancer cells over normal cells may be due to either different levels of enzyme or altered enzyme activity or both. Topoisomerase II inhibitors are effective anticancer drugs in the clinic and response rates are high in most cases, but they fall prey to the same failings that befall other types of anticancer drugs, mainly the appearance of resistance to the cytotoxic effects of the drug, which translates into patient relapse. Several mechanisms have been proposed to account for resistance to topoisomerase II inhibitors. These include decreased drug accumulation (2830), decreased enzyme levels or activities (23-25, 31-35), mutated enzymes (36, 37), metabolic inactivation of the drug (32), and altered distribution of the drug within cells (38). By recognizing these shortcomings, it is then possible to devise resistance model systems that could serve as screens to test the ability of new chemotypes or new analogs of proven topoisomerase II inhibitors to overcome various mechanisms of resistance that may develop within a tumor. The demethylepipodophyllotoxins VP16 (etoposide) (Vepeside®) and VM26 (teniposide) are of particular interest because they do not intercalate into DNA or appear to bind strongly to DNA (6, 9) and because the structure is amenable to some chemical alteration, thus allowing for structure-activity relationship studies. The approach taken by our laboratory has been to identify their mechanism of action, define structure-activity relationships, identify naturally resistant cell lines, select for acquired resistant cell lines, characterize the resistant cell lines for cross-resistance properties and mechanisms of drug resistance, and select model cell lines with different mechanisms of resistance that could be used in our analog screening and

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225

evaluation program. Analog studies provide the added benefit of being able to substantiate or refute alternative mechanisms of action for a given class of drug. This laboratory provided the first evidence that topoisomerase was the primary target of VP16 and VM26 (5-7); defined an initial structure-activity relationship for cytotoxicity and topoisomerase inhibition (5-7, 39); and identified and characterized one naively resistant cell line (31) and six acquired resistant cell lines (32) that could be utilized in analog evaluation.

MATERIALS AND METHODS Cell culture and radioactive labeling. Human lung adenocarcinoma cell line A549, human small cell lung carcinoma lines SW 1271 and SW900, and human colon carcinoma cell line HCT116 were grown and maintained in McCoy's 5A tissue culture medium containing 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin and supplemented with pyruvate, essential and nonessential amino acids, and vitamins. Drug resistant cell populations A549(VP)28, A549(VM)28, HCTlI6(VP)35, and HCTl16(VM)34 were derived from parental cell lines by 1 hr exposures to the respective drug once each week, followed by recovery in drug-free medium. Each cycle was repeated for 28, 34 or 35 weeks as previously described (40). Cells intended for alkaline elution experiments were labeled separately for 3 days with 0.01 t~Ci/ml [~4C]thymidine in 60 cm z plastic culture dishes to be used for experimental purposes or with 0.1 ~Ci/ml [3H]thymidine in 75 cm 2 plastic culture flasks to be used for reference purposes (41). Cytotoxicity assay. The cytotoxic effects of the different analogs were determined by assessing inhibition of cell proliferation in 25 cm 2 flasks 5 to 9 days following a 1-hr drug exposure, at which time flasks containing cells that had never been incubated with drug and served as controls were 50 to 80% confluent. Following the cell proliferation period, the surviving cells were released from flasks with trypsin-EDTA, fixed with formaldehyde, and counted using a Coulter counter. Average counts obtained from duplicate flasks of cells exposed to a given drug concentration were expressed as a percentage of the average number of untreated control cells from 4 flasks for each experiment. These percentages were plotted as probit values versus log of drug concentration to obtain the concentration of drug necessary to inhibit cell proliferation by 50% (IC50 concentration). Alkaline elution assays for single and double strand DNA breaks. Single and double strand DNA break production was quantified by standard alkaline elution techniques (42, 43). Logarithmically growing cells, containing ~4CDNA, were exposed to various concentrations of VP16, VM26, or analogs for 1 hr or for the length of time described in the Results. Cells were harvested and

226

B. H. LONGand D. A. STRINGFELLOW

layered onto polycarbonate filters with reference cells containing 3H-DNA and exposed to 300 rads of gamma-radiation as an internal elution standard. Cells were lysed with 2% SDS, 25 mM (pH 9.6) and the DNA remaining on the filters was slowly eluted by pumping at 40 ~l/min using a buffer composed of tetrapropylammonium hydroxide and EDTA, which was adjusted to pH 12.1 to reveal single strand DNA breaks or to pH 9.6 to reveal double strand DNA breaks. Quantification of single and double strand DNA breaks was achieved by relating slopes to DNA elution curves obtained following drug treatment to those obtained following exposure to different doses of gamma radiation and calculation of true double/single strand break ratio was achieved, as previously described (7, 31, 44). The true number of single strand DNA breaks per double strand DNA breaks was calculated from apparent ratios as previously described (45).

Assay for topoisomerase 11 inhibition. The in vitro catenation activity of topoisomerase II, partially purified from Novikoff hepatoma cells as previously described (6), was used to assess inhibition of the enzyme by the different podophyllin congeners. The assay was conducted in 20 #1 of assay buffer containing ATP, histone I, and supercoiled, covalently closed, circular PM2 DNA, which was used as the substrate, with or without drug. The assay mixture was incubated at 37°C, with the reaction beginning upon addition of enzyme and ending upon addition of stop buffer after 1 hr, and the total vol was subjected to electrophoresis in 1% agarose gels, as described in detail elsewhere (6). VP16 andporfiromycin influx and efflux. Cells plated at a density of 5 × 10 6 cells in each 75 cm z flask on the previous day were incubated at 37°C with 5/~M [3H]VPI6 or [3H]porfiromycin for 0 to 30 min. The cells were immediately rinsed 4 times with ice cold phosphate-buffered saline at the end of the incubation period, then lysed with 0.1 M NaOH. For efflux studies, cells were incubated with radiolabeled drug for 30 min, washed once with warm, drugfree medium, and incubated at 37°C for 0 to 30 min, then rinsed and processed as above. Intracellular contents of drug expressed in pmoles/106 cells were calculated from the specific radioactivities of each drug. Cell vol were determined by suspending 1 × 107 cells in warm medium containing 5 #Ci of 3H20 and incubating at 37°C for 30 min, after which 2 #Ci of [14C]mannitol was added, the cells were sedimented by centrifugation, and the pellets were dissolved in 0. I M NaOH. Radioactivities for 3H and 14C were determined by scintillation counting. Cell vol were calculated from the formula: cell vol/106 cells [(3H dpm in pellet/total 3H dpm/ml in suspension) - (~4C dpm in pellet/total 14C dpm/ml in suspension)] / (total cell number × 10-6). Intracellular drug concentrations were calculated by dividing the intracellular contents by the cell vol (46).

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RESULTS AND DISCUSSION Structure-activity relationship for cytotoxicity. Podophyllotoxin is a naturally occurring antibiotic that inhibits assembly of microtubules into the mitotic apparatus by binding to the tubulin dimer (47, 48). Its failure in the clinic as an anticancer agent due to toxic side effects (49) prompted chemists at Sandoz to chemically modify podophyllotoxin in attempts to obtain a more tolerated agent. This program resulted in the synthesis of well over 100 analogs, of which only VP16 and VM26 demonstrated significant activity in initial clinical trials (50, 51). We have studied over 20 structurally related podophyllotoxin analogs obtained from Sandoz (52), our own chemists at Bristol-Myers, the NCI (Dr. Suffness), and other sources for structureactivity relationship studies (7, 32, 39) (see Fig. 1 for structures). Although cytotoxic effects of 48 analogs on P-815 mouse mastocytoma ceils in vitro were reported by Keller-Jusl6n et al. (52), it was necessary to determine the cytotoxic effects of the analogs we were studying on the human lung adenocarcinoma cell line A549 under similar conditions. These results are presented in Table 1 along with literature values for antimicrotubule activity of many of the congeners. It is clear from Table 1 that glycosylation greatly diminished antimicrotubule activity, yet many of the glycosylated congeners were still cytotoxic. For example, glucosylation of podophyllotoxin resulted in an increase of the Ki for tubulin binding from 0.5 to 180 ~M (53, 54). Therefore, another mechanism must account for observed cytotoxicity of some of the glycosylated congeners.

Structure-activity relationship for single strand DNA breakage. It is now recognized that VP16, VM26, and DNA intercalating drugs inhibit topoisomerase II by stabilizing a covalent intermediate that normally forms between the enzyme and its DNA substrate, as part of its strand passing activity. In this situation, one or both subunits of the enzyme becoming covalently linked to the DNA through a phosphodiester bond formed between a tyrosine moiety in the active site of the enzyme and a 5' phosphate at a cleavage site introduced by the enzyme (57) (Fig. 2). This intermediate is unstable and readily reversible in the absence of drug (57), upon removal of drug from the ceils (7, 9, 32, 39, 44, 45, 57, 58), or by raising the salt concentration or diluting the reaction mixture when purified enzyme is incubated with DNA in the presence of inhibitor (4) (Fig. 2). However, the complex is stabilized by strong denaturing conditions such as highly basic pH or strong detergents like sodium dodecyl sulfate (1-4, 8, 9) (Fig. 2). Therefore, the DNA breaks associated with the enzyme-DNA complex stabilized by drug could be observed under proper conditions. The first study of the DNA breakage produced by podophyllin analogs was conducted by Loike and Horwitz (59). Although these studies provided an idea of what structural features were required for this class of drugs to produce

228

B. H. LONG and D. A. STRINGFELLOW

Podophyllotoxin Code

Rnaloss

Consenor

R1

Ra

R~

R4

~1~R3

O~ IB-PEL O-pe|tatin

OH

H

H

CHs CH$

OOC

deoxypodophyl |otoxin

H

H

H

P

podophyl lotoxin

H

H

OH

CHg

OlIP

4,-d*im*lthy |podophy| | otoxin

H

H

OH

H

rl~p8

4 '-delta thy lpodophy I ) o toxin H 4-~-O-g |U¢oside 4'-deltethylpodophyl;otoxin %6-0- H benzy i idi he_~_D_sluc osid e

H

6

H

H

88

4-epipodophy|lotoxi.

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H

DI1PBE

I~P

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H3C 0 ~

~r~'O CH3 OR4

H

Uhore|

/r~--O~

CH3

6

H

H

EP6

4-apipodophy| D-O-glucoside |otoxin

H

8

H

CHs

OMFP

4 '-delte thy I-4-epipodophy I | o toxin

H

OH

H

H

DIIEP8

4 °-delethy ]-4-epipodophy I | o toxin 9-O-s|ucoside

H

~

H

H

" / 0- ~ ' -~ 0 ., .

H

mt

'1~/~

88

Etoposide Code

/

Rnaloss

Con~enor

R;

R|

Rs

H

BeVP

4'-denthylb=nzyl-4-.pipoaophyli.-

HIC-CHr~CHg

IrPF8

tOXin 4,6-O-ethylidine 9|ucosiae 4-epipodophyllotoxin 4,6-0-

HIC-

CH3

CHl

~

CHt

CHs

[PT8

ethyl idine-~-O-sl u¢°side 4-epipodophyllotoxin 4.6-0-

VP16

4'-dole thy l-4-epipodophy I lo tox in

theny I idine-~-D-sl ucas ide

Rf-~ ~ f ~~ - -

H

0

benzy I idine-~-O-glucoside 4 ~6-0-e thy I idine-~)-D-sl uco$ide V11~6 4 '-delle thy l-4-epipodophy I 1otoxin 4,6-O-thenyl idine-~-O-91 ucoside DI1EPB8 4'-deaethyl-4-epipodophyllotoxin

4'&-O-benzYI idi ne-~-O-9] u¢°side d|OHVP 3'.4'-didel.ethyl-4-epipodophyllotoxin e thy | idine-~-O-9| u¢o$ ide d|OHVlt 3, ,4 ' - d i d a l l thy |-4-epipodophy I 1otoxin the~y I idine-~-D-9 lu¢ oside

HsC- H

CHI

~

H

CH3

H

CH~I

H

H

H

H

~

H3C~--

H3 C

/

Other

Etoposide

Code

ConBoner

R;

d|QVP

3',4'-diquinone 4na|o9 of diOHVP

HsC-

Other

Podophyl|otoxin

H

RI-~ ~Hu~v-~O0

Analogs

Rn&|o9

OR3 ORp

(

0

Code C o n B e n o r PP

p;cropodop~y I lotox;n

[I

~1

H3C ~

CH3 OCH3

FlG. I. Molecularstructures of VP16, VM26, podophyllotoxin, and congeners.

229

SAR OF TOPOISOMERASE II INHIBITION TABLE 1. CYTOTOXICITY OF A549 HUMAN LUNG ADENOCARC1NOMA CELLS PRODUCED BY PODOPHYLLIN CONGENERS

Congener P PP DOP a-PEL b-PEL EP EPG EPEG EPTG EPBG DMP DMPG DMPBG DMEP DMEPG DMEPEG (VP16) DMEPTG (VM26) DMEPBG BeVP diOHVP diQVP diOHVM diQVM

Inhibition of microtubule assembly (IDso)(#M)

Tubulin binding (Ki)(#M)

0.6 30 0.5 0.5 0.7 5

0.5 10 0.5

0.5

0.6

2 >>100 >>100

0.1 12

Cytotoxicity (ICso)(#M) 3 11 <1 0.1 <0.1 2 >>100 >>100 3 1.5 3 >>100 >~10 <1 250 8.4 0.5 0.4 160 36 72 14 17

Values for inhibition of microtubule assembly were from (55, 56), values for tubulin binding were from (53, 54), and cytotoxicity values resulting from a 1-hr exposure of A549 cells to drug were from (32, 39).

the observed DNA breakage, the alkaline sucrose sedimentation procedure they utilized to obtain their results was neither as quantitative nor as sensitive as afforded by alkaline elution techniques that were only being evolved about that time. Using these newer methods, we have been able to more accurately quantify the amount of DNA breakage produced in cells by VP 16, VM26, and their related analogs. As can be seen from Table 2, P, PP, DOP, ~- and B-PEL, EP and EPG were essentially inactive toward inducing DNA breakage at 100 #M. The presence of a free hydroxyl group at the 4' position of the pendent ring resulted in some DNA breakage (DMP, vs. P). Therefore, a free hydroxyl group at this position was not required for inducing DNA breakage, and hence, inhibiting topoisomerase II (BeVP, EPEG, EPTG, EPBG). This breakage activity was substantially more if the 4 position is epimerized (DMEP, DMEPG, DMEPBG vs. DMP, DMPG, DMPBG). Glycosylation of the 4 position reduced the effect of the drug (DMPG, DMEPG vs. DMP, DMEP), but DNA breakage activity was greatly enhanced if the glucose

230

a.H. LONG and D. A. STRINGFELLOW CO

I +SDS

÷ o

•

-p PK

>

I ~q~.

+SDS >

I

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>

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FIG. 2. Model for DNA strand passing activityoftopoisomerase II. Topoisomerase II can exist in four different physical or chemical states regarding interaction with duplex DNA strands: not associated, physically associated, and chemically associated through either covalent bonding of one or two enzyme subunits to the 5'-phosphate of one or both DNA strands, respectively. DNA strand passage occurs when DNA strands are in close proximity, a condition that is artificially obtained in vitro by including either histone HI or spermidine to aggregate the DNA. Under alkaline elution conditions, the presence of sodium dodecylsulfate (SDS) denatures the enzyme and causes separate fragments only where both subunits of the enzyme are involved and proteinase K (PK) digests away the covalently linked enzyme to reveal frank breaks. Elution at pH 9.6 will reveal only double strand DNA breaks and at pH 12.1 will reveal both single and double strand DNA breaks. m o i e t y was m o d i f i e d by the f o r m a t i o n o f an O-(416) acetal t h r o u g h the r e a c t i o n with an a l d e h y d e ( D M P B G , D M E P E G , D M E P T G , D M E P B G vs. D M P G , D M E P G ) (7, 39). F i n a l l y , the s t r u c t u r a l g r o u p a s s o c i a t e d with the a l d e h y d e was very i m p o r t a n t , n o t only for D N A b r e a k a g e (Table 2), b u t also for a n t i t u m o r activity a n d c y t o t o x i c p o t e n c y , as s h o w n b y Keller-Jusl6n et aL (52) in their e x p e r i m e n t a l m o d e l systems. It s h o u l d be e m p h a s i z e d , h o w e v e r , that p o t e n c y d i d n o t go h a n d in h a n d with a n t i t u m o r activity, in t h a t the oc h l o r o b e n z y l i d i n e a n a l o g was one o f the m o s t p o t e n t congeners to be tested b u t h a d little a n t i t u m o r activity, whereas VP16, which was 10 fold less p o t e n t t h a n VM26, s h o w e d the best a n t i t u m o r activity (52).

Kinetics o f DNA break formation and disappearance. The i n d i c a t i o n that a n u c l e a r e n z y m e o r e n z y m e s m a y m e d i a t e the c y t o t o x i c effects o f V P I 6 a n d certain D N A i n t e r c a l a t i n g drugs was suggested b y the w o r k o f Zwelling et al. a n d W a z n i a k a n d Ross, w h o s h o w e d t h a t D N A b r e a k a g e c o u l d be p r o d u c e d in i s o l a t e d nuclei a n d t h a t this b r e a k a g e was i n h i b i t e d i n c u b a t i n g the nuclei

231

SAR OF TOPOISOMERASE II INHIBITION TABLE 2. SINGLE STRAND DNA BREAKS PRODUCED IN A549 HUMAN LUNG ADENOCARCINOMA CELLS BY PODOPHYLLIN CONGENERS Drug dose Congener

10-4M

10-SM

P PP DOP a-PEL b-PEL EP EPG EPEG EPTG EPBG DMP DMPG DMPBG DMEP DMEPG DMEPEG (VP16) DMEPTG (VM26) DMEPBG BeVP diOHVP diQVP diOHVM diQVM

<1 <1 1+ 1 1 +- 1 <1 <1 <1 18

<1

12+-2 3 +- 2 >30 >30 17 + 4

10 +- 1

2+- 1 <1 8 +- 3 12 +- 2 < <30

10-6M

10-7M

21+-6 44

2+-1 5

14-1 5+-2 12+_6 22+-6 <30

10-8M

<1 1+1 8+_3 16+-3

<1 1+-1

<1

20 7 28

17 8

Mean number of breaks per 108 nucleotides +_S.E. of 3 or more samples (7, 32, 39).

with d r u g at 4°C i n s t e a d o f 37°C (45, 58). F u r t h e r evidence s u p p o r t i n g the h y p o t h e s i s t h a t an enzyme m a y be the t a r g e t for VP16 a n d VM26 was the o b s e r v a t i o n that D N A b r e a k s r a p i d l y f o r m e d in cells u p o n a d d i t i o n o f d r u g until a d r u g c o n c e n t r a t i o n d e p e n d e n t p l a t e a u level was r e a c h e d t h a t was r e l a t i v e l y s t a b l e for m a n y h o u r s (7, 31, 39, 44, 45, 59) (Fig. 3). H o w e v e r , if the m e d i u m c o n t a i n i n g d r u g was e x c h a n g e d for fresh, drug-free m e d i u m , then the b r e a k s r a p i d l y d i s a p p e a r e d . S i m i l a r effects were o b s e r v e d when d o u b l e s t r a n d D N A b r e a k s were m o n i t o r e d (44) (Fig. 4). This d i s a p p e a r a n c e was n o t due to D N A r e p a i r , b e c a u s e classical D N A r e p a i r i n h i b i t o r s h a d no effect on the d i s a p p e a r a n c e o f the D N A b r e a k s (7). T h e s p e c u l a t i o n b y Ross et aL (60) t h a t the p r o t e i n a s s o c i a t e d b r e a k s p r o d u c e d b y a d r i a m y c i n m a y be the result o f a D N A m e t a b o l i z i n g e n z y m e such as a t o p o i s o m e r a s e , as well as the s u g g e s t i o n f r o m the kinetics d a t a for the r a p i d f o r m a t i o n o f an e q u i l i b r i u m state for D N A b r e a k a g e a n d their r a p i d reversal f o l l o w i n g d r u g r e m o v a l a n d the f o r m a t i o n o f d o u b l e s t r a n d b r e a k s led us to investigate the possibility t h a t t o p o i s o m e r a s e II was the target for these a n t i c a n c e r drugs.

232

13. H. LONG and D. A. STRINGFELLOW CO ILl a

r

25

B

0

I.IJ _.1 (.~

20

Z 0 ~, 69 ,< UJ rr 123 a Z

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15

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t

rr t,(,'9 UJ .J

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x J

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x

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0 2

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TIME, HRI FIG. 3. Formation and disappearance of single strand DNA breaks produced by VM26 (A) and VPl6 (B). A549 cells were incubated continuously in medium containing 0.1/~M VM26 or 1 /~M VP16 (X) or 0,5/~M VM26 or 4/~M VPI 6 (e) for various periods of time up to 4 hr or for 1 hr in medium containing 0.5 #M VM26 or 4 #M VPI 6, then incubation continued in drug-free medium for various periods of time up to 3 hr (o), after which DNA in the cells was subjected to alkaline elution at pH 12.1. Arrow marks removal of drug from the cells (44).

0

2

4

6

0

2

4

6

tume (HR) FIG. 4. Formation and disappearance of double strand DNA breaks produced by VM26 (A) and VP 16 (B). A549 cells were incubated continuously in medium containing 0.5/~M VM26 or 4/~M VP16 (o) or 1.0/~M VM26 or 10 #M VP16 (I) for various periods of time up to 6 hr or for 1 hr in medium containing 0.5 #M VM26 or 1/~M VP16 (o) or 1.0/~M VM26 or 10/~M VPI6 (D), then incubation (continued in drug-free medium for various periods of time up to 5 hr, after which DNA in the cells was subjected to alkaline elution at pH 12.1. Arrow marks removal of drug from the cells (44).

EP

FIG. 5. Inhibition of topoisomerase II catalyzed DNA catenation by congeners of podophyllotoxin. Supercoiled, covalently closed, circular PM2 DNA used as substrate was incubated with purified topoisomerase II, ATP, and histone H 1 in the absence or in the presence of different concentrations of podophyllotoxin congeners for I hr at 37”C, followed by electrophoresis in 1% agarose gel and visualization of DNA by UV light after staining with ethidium bromide (6).

DMEPBG

SAR OF TOPOISOMERASEII INHIBITION

233

Inhibition of topoisomerase H DNA strand passing activity. Topoisomerase II catalyzes an ATP-dependent catenation of covalently closed, circular DNA in vitro, resulting in a product that is easily separated from substrate by agarose gel electrophoresis (61, 62). Using partially purified topoisomerase II from Novikoff hepatoma cells we were able to provide the first demonstration of the inhibition of eukaryote topoisomerase II by VP16 and VM26 (5-7), When the reaction was conducted in the absence of drug, most of the supercoiled, covalently-closed, circular PM2 DNA (Form I) used as the substrate was converted into a catenated network of still covalently-closed circles of DNA that barely migrated into the gel (Fig. 5). When congeners of podophyUotoxin were added to the incubation mixture before the addition of enzyme, an inhibition of topoisomerase II activity was observed (Fig. 5) for only those congeners that were capable of inducing extensive DNA breakage in cells (Table 2). VM26, VP16, and D M E P B G produced an observable inhibition of form I DNA depletion at 1/.tM and almost complete inhibition of form I DNA depletion at 100/.tM whereas 100/.tM EP had no effect toward inhibiting this depletion and 10 to 30/.tM D M E P was required to produce the same effect as 1 #M VM26 (Fig. 5). The following comparison can be made from these and other unpublished results between cytotoxicity, DNA breakage activity, and inhibition of catenation produced by the different congeners of podophyllotoxin (Table 3). Of the different drugs, only those congeners having high antimicrotubule activity failed to display correspondence between cytotoxicity and DNA breakage activity. The close relationship between cytotoxicity, DNA breakage, and topoisomerase II inhibition provided strong evidence that topoisomerase II was the primary target by which VP16 and VM26 produced their cytotoxicity (5-7). However, the precise mechanism by which this inhibition results in cell death has yet to be determined. Unusual characteristics of the DNA breakage produced by the different congeners. Several interesting observations were made early during the DNA TABLE 3. COMPARISON OF CYTOTOXICITY,DNA BREAKAGE ACTIVITY,AND TOPOISOMERASEII INHIBITION BY THE DIFFERENT CONGENERS OF PODOPHYLLOTOXIN

Cytotoxicity DMEPBG, VM26, DMEP > EP > DMP >VPI6 >> DMEPG, DMPG DNA Breakage DMEPBG > VM26> VP16> DMEP>> DMEPG > DMP > DMPG > EP Inhibition of Catenation DMEPBG, VM26 > VP16 ~ DMEP > DMEPG > DMP, DMPG, EP Congeners with the greatest activities are on the left (7).

234

B. H. L O N G and D. A. S T R I N G F E L L O W

breakage studies of the podophyllin congeners. It was noticed that both VP 16 and VM26 produced obviously biphasic alkaline elution curves, even at moderate concentrations of drug (see Chart I of Ref. 44), whereas many of the analogs produced more linear alkaline elution curves (see Fig. 3 of Ref. 39). It was also observed that concentrations of VPI6 above 10/.tM and of VM26 above 2/.tM resulted in the release of large amounts of DNA in the cell lysis fractions (44), whereas this was not the case for many of the analogs. The 4'-methyl analogs provide a case in point. Figure 6 compares typical curves obtained by eluting DNA at pH 12.1 of cells incubated with different concentrations of either VM26 (Fig. 6A) or MeVM (EPTG) (Fig. 6B). The elution curve for concentrations of VM26 even as low as 0.2/.tM display a biphasic nature, yet the elution curve for 1/2M MeVM is linear and for 2 l,tM MeVM is only slightly biphasic, even though the slope is much greater than that observed for 0.2 ~M VM26. Differences are also seen when percentages of DNA occurring in the lysis fractions and the initial slopes and later slopes of alkaline elution curves obtained from cells incubated with different concentrations of VM26 and MeVM are compared (Table 4). Similar observations are made for VP16 and MeVP or DMEPBG and EPBG (unpublished results). Several features of Table 4 should be emphasized. First, only the higher concentrations of VM26,

100

7O

50

~.5

o

2o

u=

B

A I

100

5O

!

2O

10 100

50

2O

10

PERCENTOF TOTAL Lr31~-DNA REMAINING ON FILTER FIG. 6. Comparison o f p H 12.1 elution curves of D N A from cells incubated with different concentrations of VM26 (A) or MeVM (B). A549 cells were incubated with drug for 1 hr at 37°C then lysed on polycarbonate filters in the presence of proteinase K a n d the D N A was eluted at p H 12.1.

235

SAR OF TOPOISOMERASE II INHIBITION TABLE 4. COMPARISON OF ELUTION CURVES OF VM26, VPI6, AND 4'-METHYL ANALOGS

Concentration (#M)

Lysis fraction (% of total)

VM26

0.02 0.05 0.01 0.2 0.5 1.0 2.0 5.0 10 20 50 100

0.5 + 0.2 0.4 +_ 0.4 0.1 +_ 0.5 0.2 _+ 0.5 -0.1 + 1.0 1.0_+0,06 3.5 _+ 0.5 7.7 23 46 82 87

0.05 0.10 0.25 0.61 1.7 3.8 4.8 7.7 5.9 3.4

+ 0.04 + 0.08 +_ 0.14 + 0.21 +0.8 + 1.5 _+ 2.6

0.03 + 0,02 0.12 _+ 0.10 0.23 -+ 0.09 0.32 + 0.18 0.53_+0.20 0.51 +0.13 0.36 _+0.08

VP16

0.2 0.5 2.0 5.0 10 20 50 100

0.4 + 0.6 0.03 -+ 1.0 -0,3 + 0.9 -0.2 + 0.8 0,2+1.3 1,9 + 2.0 10,4 20 27

0.16 0.32 0.48 0.88 2.5 4.4 6.6 7.5 5.9

-+ 0.16 + 0.21 -+ 0.42 + 0.57 +-1.4 + 1.6

0.12+0.12 0.20+0.11 0.33 -+ 0.14 0.45 + 0.19 0.54 + 0.22 0.42 + 0.07

0.1 0.2 0.5 1.0 2.0 5.0 10

0.7 0.4 + 0.7 0.2 -+ 0.4 -0.1 + 0.1 -0.1+0.4 2.0 + 1.1 10.2

0.12 0.07 0.35 0.93 2.6 6.6 11.3

-+ 0.04 + 0.20 + 0.37 +1.7 -+ 2.5

0.06 0.12 0.36 0.51 0.71 0.56 0.23

+ 0.03 + 0.14 + 0.13 +0.21 + 0.09

0.27 0.37 0.37 0.92 2.2

+- 0.37 + 0.30 + 0.36 -+ 0.15 + 1.0

0.06 0.08 0.19 0.40 0.70

-+ 0.02 + 0.01 +- 0.04 + 0.01 +- 0.07

Congener

1.0

MeVM

MeVP

10 20 50 100 200

0.1 0.0 0.4 0.2 0.2

+ 0.6 -+ 0.8 + 0.8 -+ 0.6 + 0.6

Initial fraction

Fractions 5-9

(slope)

(slope)

Mean values with S.E. were obtained from 8 separate experiments for VM26 and VPI6 and from 3 separate experiments for MeVM and MeVP where each concentration was conducted in duplicate for each experiment.

VP16, and MeVM, which are highly cytotoxic congeners (Table 1), produce substantial release of DNA in the lysis fractions (Table 4). Second, the slopes of the late eluting fractions (5 to 9) reach a maximum, then decline. Third, the maximum slope values obtained by fractions 5-9 were 0.5 for VM26 and VP 16 but were 0.7 for MeVM and MeVP. Fourth, the slopes of the initial fractions also reached a maxima that occurred at concentrations of either VM26 or VP16 that were 10-fold higher than required for the maxima reached by fractions 5-9. Fifth, the maximum slope values obtained by the initial

236

B . H . LONG and D. A, STRINGFELLOW

fractions were less than 8 for VM26 and VP16 but exceeded 11 for MeVM and were unobtainable for MeVP. The appearance of DNA in the lysis fraction is an indication of double strand DNA breakage. Agents that produce exclusively single strand DNA breaks, such as camptothecin, a potent topoisomerase I inhibitor (63, 64), alkylating agents that induce excision repair, and ionizing radiation do not cause the release of DNA into the lysis fraction, even at excessively high concentrations, but cause very steep initial slopes (Long, manuscript in preparation). These results suggest that there is not only a difference in quantity but also in quality between the breaks produced by the demethylepipodophyllotoxin and the epipodophyllotoxin analogs.

Comparison of double strand DNA breaks produced by VM26 and Me VM. By conducting the alkaline elution at pH 9.6 instead of 12.1, it is possible to study double strand DNA breakage induced by the podophyllin congeners. Figure 7 shows typical pH 9.6 elution curves resulting from exposure of cells to VM26 or MeVM. For elution at pH 9.6, it was necessary to expose the reference cells to 4000 rads of gamma radiation in order to have appreciable elution of the 3H-DNA. It is evident from Figure 4 that 20- to 30-fold more MeVM is required to produce comparable double strand DNA breaks as induced by tr

O

(D 80

_z Z ~

70 i

:6o A ~7100 t-,, ~_~ 9o,

1.0

~

2.0

.~ 80

S

70 I

LL 0 I-I

u.I O

n,LU n

•

B lO0

! 90

80

PERC;ENT OF TOTAL REMAINING

70

60

r3H3 D N A

ON FILTER

F I G . 7. Comparison of pH 9.6 elution curves of D N A from cells incubated with different concentrations of VM26 (A) or MeVM (B). A549 cells were incubated with different concentrations of drug (~M) as described in Figure 6.

SAR OF TOPOISOMERASE II INHIBITION

237

VM26 whereas only 2- to 5-fold more was required to produce comparable single strand D N A breaks (Table 4), and there exists a 30-fold difference in potency toward cytotoxicity (Table 1). This qualitative difference becomes more obvious when the ratios of true single to double strand D N A break ratios are calculated (Table 5). It is necessary to calculate true single strand D N A breakage because alkaline elution at p H 12.1 actually measures both single and double strand D N A breaks. In this experiment, 0.1/,tM VM26 induced only about 3 true single strand breaks and MeVM induced almost 25 single strand breaks for each double strand D N A break. These results suggest that cytotoxicity and antitumor activity may be more closely related to the inhibition of both subunits rather than one subunit of topoisomerase II. Again, similar results were obtained when D M E P B G was compared with E P B G (results not shown). No double strand D N A breakage was induced by MeVP at the highest concentrations studied. TABLE 5. TRUE SINGLE TO DOUBLE STRAND DNA BREAK RATIOS INDUCED BY DIFFERENT CONCENTRATIONSOF VM26 AND MeVM

Drug VM26

MeVM

Concentration (#M)

Single strand DNA breaks (Breaks/108Nucleotides) (Rads)

Double strand DNA breaks (Rads)

True s/d

0.02 0.05 0.1 0.2 0.5 1.0

1.6 2.4 5.0 9.4 14.0 17.4

55 104 156 193

240 433 942 1535

2.6 2.8 1.3 0.6

0.2 0.5 1.0 2.0 5.0

4.0 7.9 16.7 22.3 25.4

185 248 282

139 416 2203

24.7 9.9 0.6

Values for s/d were calculated as described in Methods.

Correlation between cytotoxicity and single and double strand DNA break formation for different congeners. Pommier et al. (65) had demonstrated a correlation between double but not single strand D N A breaks and sister chromatid exchange numbers, mutations of the hypoxanthine:guanine phosphoribosyltransferase locus, and cytotoxicity produced by the topoisomerase II inhibitors 4'-(9-acridinylamino)methanesulfon-m-anisidide or 5iminodaunorubicin in Chinese hamster V79 cells. A similar correlation was found between cytotoxicity and double but not single strand D N A breaks produced in human lung adenocarcinoma A549 cells by the podophyllin analogs (32). These results are illustrated in Figure 8. Little correlation was

238

B. H. LONG and D. A. STRINGFELLOW

8°I

•

A

B

60OI

•

/;/' i40"

g g

•

..... i

O

20"

0,

i

"ib"

/

,

. '":

2b

•

/'o

/"

ab

"

50

SSB (Breaka/$Oe8 N u c l e o t l d e s )

,

Jj;'

..~.T

4b

/

500

•

/o

•

iobo 15bo 2obo 25bo aooo

DSB (Raa E Q u i v a l e n t s )

FIG. 8. Comparison of cytotoxicity with single and double strand DNA breakage in A549 cells produced by various concentrations of the different podophyllotoxin congeners VM26 (o), DMEPBG (m), VP16 (A), DMEPG (<)), MeVM (o), EPBG (o), BeVP (1), diOHVP 01% diQVP (~7), diOHVM (A), and diQVM (A) (32).

observed when cytotoxicity was plotted against single strand DNA breaks (Fig. 8A). In fact, some analogs were capable of inducing almost 20 single strand DNA breaks without producing appreciable toxicity. A much better correlation was observed between double strand DNA breaks and cytotoxicity (Fig., 8B). These results suggest that both subunits of topoisomerase II need to be inhibited for a cytotoxic event to occur (31, 32, 65). Cytotoxicity of VP16 and VM26 in resistant human carcinoma cell lines. Four different human lung carcinoma cell lines were characterized for sensitivity or resistance to VM26 and VPI6 and ICs0 values for cytotoxicity are presented in Table 6. This study revealed that SW1271 was the most sensitive line and SW900 was the most naively resistant of the four lines, being 4- to 6-fold more resistant than the SW1271 cells, when assessed following incubation of the cells with drug for 1 hr, as described in Materials and Methods. A549 and H 157 cells were intermediate in sensitivity, with H 157 appearing to be more resistant than A549 cells. Table 6 also demonstrates that VM26 was 5- to 10-fold more potent than VP16 in the 4 cell lines (31). DNA breakage by VP16 and VM26 in resistant human carcinoma cell lines. Typical p H 12.1 elution curves obtained after 1 hr incubation of SW1271, A549, H 157, and SW900 cells with different concentrations of VM26 or VP 16

239

SAR OF TOPOISOMERASE II INHIBITION TABLE 6. CELL PROLIFERATION RATES AND IC50 DRUG CONCENTRATIONS FOR THE INHIBITION OF CELL PROLIFERATION Cell line

Cell description

Doubling time (hr)

SW1271 A549 H157 SW900

small celllung carcinoma lung adenocarcinoma large cell lung carcinoma small cell lung carcinoma

34 28 50 52

Drug IC50 values VM26 VPt6 0.5 0.7 1.0 2.0

2.7 4.9 9.8 16

VP16/VM26 ratio 5.4 7.0 9.8 8.0

ICs0 values are median inhibitory drug concentrations expressed in ~M (31).

are presented in Figure 9. Relatively linear curves resulted from exposure of cells to low concentrations of either drug, but higher concentrations yielded biphasic elution profiles. SW1271 cells were clearly the most sensitive with respect to production of single strand DNA breaks in that detectable breaks were produced by either 10 nM VM26 or 100 nM VP16. On the other hand, SW900 cells required I 0-fold higher concentrations of either drug to produce detectable breaks (Fig. 9). No differences were observed between disappearance of single strand DNA breaks between the sensitive or the resistant cells upon substitution of drug-containing to drug-free medium, suggesting that neither repair of these inhibited topoisomerase complexes nor kinetics or the reversal of inhibition by drug were different in sensitive and resistant cells (31). IT 100 _J

~

_ ~

' OuM

'

7o

0.5 pMMPM

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0 pM

02pM

0.5 ~

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20 iJ~v Z <~ ~ 2a uA II

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M

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I

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7.J

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1.0 pM 2J0IJM 5.0 pM

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L

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5 m"~ io

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. . . . . .

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210

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TOTALEaH3DNA

50 REMAINING

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. . . .I .

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FIG. 9. Comparison of pH 12.1 elution curves of DNA from naturally sensitive and resistant cells incubated with different concentrations of VM26 (A-D) or VP16 (E-H) for 1 hr at 37°C. SW1271 (A,E), A549 (B,F), H157 (C,G) and SW900 (D,H) (31). AER--I

240

B. H. LONG and D. A. STRINGFELLOW

Double strand D N A breaks were detectable in all 4 cell lines that increased in a nearly linear relationship with drug dose, as previously described (31). A comparison of true single and double strand D N A breaks expressed in rad equivalents is presented in Table 7. This comparison reveals that exposure of SW1271 and A549 cells to low concentrations of either drug resulted in predominantly single strand D N A breaks, as evidenced by the high ratios of single to double strand D N A breaks. Increasing the concentrations to 0.2 to 0.5 #M for VM26 or 2 #M for VP16 resulted in high ratios of true single to double strand D N A breaks. However, H 157 and SW900 cells both contained TABLE 7. TRUE SINGLE TO DOUBLESTRANDDNA BREAK RATIOS INDUCED IN SENSITIVEAND RESISTANTCELL LINES BY DIFFERENT CONCENTRATIONSOF VM26 AND VPI6 Drug VM26

VPI6

Concentration Single/doublestrandDNAbreakratios (~M) SW1271 A459 H157 SW900 0.01 0.02 0.05 0.1 0.2 0.5

74.5 12.1 5.2 6.6 3.6

0.2 0.5 1.0 2.0

10.3 9.4 9.4 1.2

2.6 2.8 1.3

1.0 1.5 -0.5

-0.2 -0.4

7.7 3.1

0.3

0.5

True single to double strand DNA break ratios were calculated as described in Materials and Methods. Negativevaluesare due to the biphasicpH 12.1elution curvesyieldingunderestimations of the number of single strand DNA breaks (31). low ratios of true single to double strand D N A breaks, even at the lowest concentrations of drug required to measure single strand D N A breaks (31). Opposite results were observed by Goldenberg et al. for the effects of adriamycin on adriamycin-acquired resistant P388 murine leukemia cells (66). The significances of our observation and the different results obtained by the two laboratories are not yet understood but may be related to differences in resistance mechanisms that may exist between naive and acquired resistant cells a n d / o r levels of topoisomerase II within the nuclei (see below).

Correlation between cytotoxicity and single and double strand DNA break formation for VP16 and VM26 in the different sensitive and resistant cells. When cytotoxicity produced by different concentrations of VP16 or VM26 was plotted against the resulting single or double strand D N A breakage, a much better correlation was seen using double strand D N A breaks (31) (Fig. 10). Similar results were obtained by Goldenberg et al. even though their resistant

I/A' ',,,,

241

SAR OF TOPOISOMERASE II INHIBITION

'

1'

,, ¢

i r,,, 0(~

/

ill 5

10

-r/or/ 15

2/

SINGLE STRAND DNA BREAKS ( BREAKS/10 8 NUCLEOTIDES}

21,50

1

1

~

/

DOUBLE STRAND DNA BREAKS {RAD EQUIVALENTS x 10"=)

FIG. 10. Comparison of cytotoxicity with single (A) and double (B) strand DNA breakage in naturally sensitive and resistant cells produced by different concentrations of VM26 (o, l , &, T) and VP16 ( o , n , A , V ) . SW1271 ( ), A549 ( ), H157 (. ), and

SW900 (

3 (31).

cells yielded the opposite results regarding true single to double strand DNA break ratios (66). These observations are similar to those made for DNA breakage and cytotoxicity produced by congeners of VP16 in A549 cells (Fig. 8) and the results of Pommier et al. comparing two different classes of topoisomerase inhibitors (65), and further emphasize the importance and significance of inhibiting both subunits in order to produce cytotoxicity and anti-tumor activity (see below). Localization of the resistance phenotype to nuclei of SW900 cells. The influences of the plasma membrane in governing intracellular drug concentrations, and hence, influencing topoisomerase inhibition can be avoided by conducting DNA cleavage studies using isolated nuclei. Studies with nuclei have the added advantage that DNA repair mechanisms and possible drug metabolism should not greatly interfere. Previous studies have shown that the DNA breakage induced in isolated nuclei by topoisomerase II inhibitors has the same requirement for magnesium ions and ATP as was previously recognized for topoisomerase II activity in vitro (67,68). Therefore, drug-induced breakage of DNA in isolated nuclei can be assessed by alkaline elution techniques and compared with that produced in intact cells to determine if the plasma membrane presents a significant barrier in SW900 cells to account for the observed resistance to VPI6 and VM26. Nuclei isolated

242

B.H. LONG and D. A. STRINGFELLOW

100 ~, so -"-30 ~'o

I~1lOOl

A

070

a . 2G

E

F

G

H

flue (HR.I FIG. I 1. Comparison of pH 12.1 elution curves of DNA from nuclei of SW1271 (A-D) and SW900 (E-H) cells incubated with different concentrations o f V P 16 in the presence or absence of ATP for 1 hr. Incubations were conducted (A and E) at 0°C with 100/~M ATP (o) or at 37°C with 0 (o), 1 (n), 10 (n), 100 (A), or 1000 (A) #M ATP; (B) at 0°C with no VP16 (o) or at 37°C with 0 (o), 0.3 (i), 1.0 (rn), 3 (A), or 10 (Z~) ~M VPI 6 without ATP; (C) same as but with 100 t~MATP; (D) with 100 I~M ATP without VP16 at 0°C (o) or at 37°C (o) or at 37°C with 1.0/~M VP 16 and ATP at 1 (n), 10 (n), 100 (A), or 1000 (A) ~M ATP; (F) at 0°C with no VPI6 (o) or at 37°C with 0 (o), 3 (a), 10 (D), 30 (A), or 100 (/',) #M VP16 without ATP; (G) same as F but with 100/~M ATP; and (H) same as D but with 30 instead of 1.0/~M VPI6 (31).

from SW 1271 and SW900 cells were incubated with different concentrations of VP16 in the absence (Fig. 11B and F) or in the presence (Fig. 11C and G) of 100 ~M ATP, and alkaline elution was conducted at pH 12.1. A comparison of these panels revealed that higher concentrations of VPI6 were required to produce DNA breakage in nuclei from SW900 cells comparable to that produced in SW1271 nuclei either in the absence or presence of ATP. In fact, the single strand DNA breakage produced by VP16 in isolated nuclei in the presence of ATP (Fig. 11C and G) was comparable to that produced in intact SW 1271 and SW900 cells (Fig. 6E and H). ATP alone up to 1 mM had no effect on DNA integrity (Fig. 11A and E), but the A T P concentration had more of an influence on the extent of DNA breakage in SW1271 nuclei than in SW900 nuclei (Fig. l i D and H). These results demonstrate that the natural drug resistance phenotype of SW900 cells is localized in their nuclei and suggest that this resistance is not due to differences in drug influx or efflux but, rather, to differences in topoisomerase II levels or activities (31).

243

SAR OF T O P O I S O M E R A S E II INHIBITION

Accumulation and efflux of VP16 in SW1271 and SIV900 cells. Influx of radiolabelled VP16 by SW1271 and SW900 cells was followed for 30 min after addition of 5/.tM VP16 to the cells (46). For efflux studies, cells were incubated in the presence of radiolabeled drug for 30 min then incubated in drug-free medium for various lengths of time. Figure 12 shows that SW900 cells accumulated VP 16 to a greater extent than did SW 1271 cells even though they were substantially more resistant to VP16. No differences in efflux rates were observed. Both the influx and efflux of drug were greatly diminished at 0°C (Fig. 12). Drug I~aoved Z 0 i..4 F< OC FZ W 0 Z 0 0

I

........................i~ilili~::::::::::$

t27t

3

OC

J _J W 0 OC FZ I-4

.............. : , : 0

iO

....... 20

30

-

.

40

50

-60

TIME (MIN) FIG. 12. Influx and efflux of VP 16 in SW 1271 (o) a n d SW900 (o) cells. Cells were incubated with 5/~M of radiolabeled VP 16 at 37°C ( ) or 0°C ( . . . . . . . . ) for various lengths of time up to 30 min or for 30 min then incubated in drug-free medium for at 37°C ( ) or 0°C ( . . . . . . . . ) for various lengths of time up to 30 min. lntracellular drug concentrations were calculated from accumulation expressed in ~tmoles and cell volume, as described in Materials and Methods (46).

Ability of podophyllotoxin analogs to overcome naive resistance. A comparison of the abilities of the different analogs to overcome naive resistance to VM26 and VP16 is presented in Table 8. None of the analogs showed any appreciable advantage over VP16 or VM26 toward overcoming the resistance displayed by SW900 cells towards this class of compounds. Interestingly, MeVM, EPBG, diOHVP, and diQVP were significantly more cytotoxic than VM26 and VP16 to A549 cells relative to SW1271 cells (32). Furthermore,just the opposite effect was observed for diOHVM and diQVM, which were much more cytotoxic to SW1271 cells than to A549 cells. The reversed hypersensitivity observed between these analogs occurring between A549 cells and SW1271 cells is difficult to explain at the present time.

B. H. LONG and D. A. STRINGFELLOW

244

TABLE 8. ABILITY OF ANALOGS TO OVERCOME NAIVE DRUG RESISTANCE Drug

Median cytotoxic concentrations (/~M) SW1271 A459 SW900

VP16 VM26 DMEPBG MeVM EPBG diOHVP diQVP diOHVM diQVM

7.2 0.5 0.6 8.4 5.8 200 270 1.7 8.2

8.4 0.5 0.4 3.0 1.5 36 72 14 17

50 2.2 2.6 32 24 >300 >300 54 80

A549/ SW 1271

SW900/ SW1271

1.2 1.0 0.7 0.4 0.3 0.2 0.3 8 2.1

6.9 4.4 4.3 3.8 4.1 --32 10

Cytotoxicity was assessed as inhibition of cell proliferation following a 1 hr exposure to drug, as described in Materials and Methods (32).

Development of acquired resistance in human carcinoma cell lines. A c q u i r e d resistant cell p o p u l a t i o n s were d e v e l o p e d by p r e s s u r i n g t w o different p a r e n t a l cell lines with a l - h r e x p o s u r e to IC70 c o n c e n t r a t i o n s o f either V M 2 6 or VP16 o n a weekly basis f o r p e r i o d s o f time e x t e n d i n g f r o m 28 to 35 weeks. Th e p a r e n t a l cell lines used in these studies were H C T 1 1 6 h u m a n c o l o n c a r c i n o m a cells a n d A549 lung a d e n o c a r c i n o m a cells. T h e resulting resistant cell p o p u l a t i o n s possessed resistance not o n l y to the d r u g used to pressure the cells but also to o t h e r t o p o i s o m e r a s e II inhibitors, as well as an e x p a n d e d m u l t i d r u g resistance profile f o r at least one line, H C T 1 1 6 ( V M ) 3 5 . T h e crossresistance p r o p e r t i e s o f these lines a n d the SW1271 a n d SW900 lines, i n c l u d e d for c o m p a r i s o n p u r p o s e s , are p r e s e n t e d in T a b l e 9. M u l t i d r u g resistance type o f m e c h a n i s m was p r e d i c t e d f o r H C T 1 1 6 ( V M ) 3 5 , n o t o n l y because o f the TABLE 9. COMPARISON OF MEDIAN CYTOTOXIC CONCENTRATIONS OF COMMON ANTICANCER DRUGS IN NAIVE AND ACQUIRED RESISTANT CELL LINES VPI6

VM26

(/.tM)

(/./,M)

ADM (/~M)

HCT116 HCT116(VP)35 HCT116(VM)34

10 86 53

0.8 5.6 5.3

0.4 2.5 8.6

0.03 0.01 0.55

0.2 0.3 1.3

1.3 0.9 1.2

10 6 6

A549 A549(VP)28 A549(VM)28

8 65 67

0.5 4.1 3.4

0.7 2.3 2.3

0.06 0.09 0.03

0.3 0.4 0.3

1.3 0.2 2.6

26 2 9

SW1271 SW900

7 50

0.5 2.2

0.5 2.1

0.02 0.03

0.3 0.6

10 0.4

24 2

Cell line

ActD Col MMC (t~g/ml) (/~g/ml) (/aM)

CsPt (#g/ml)

Cytotoxicity was assessed as inhibition of cell proliferation following a 1hr exposure to drug, as described in Materials and Methods (32).

SAR OF TOPOISOMERASEII INHIBITION

245

observation that this cell population was resistant to all of the drugs shown in Table 9, except for cis-dichloro-diamino Platinum II (csPt), but also because it displayed a 20-fold increase in resistance to adriamycin, while displaying an 8-fold increase to VM26, the pressuring agent, both c o m m o n features of the multidrug phenotype. One interesting observation regarding this profile was the increase in sensitivity observed for cis-dichloro-diamino Platinum II (csPT) that accompanied resistance to VPI6 and VM26. This feature is especially pronounced when SW 1271 and SW900 cell profiles were compared. Evidence is presented below suggesting that this phenomenon is due, in part, to altered repair of the monadducts occurring before DNA cross-links were formed (46).

Accumulation and efflux of VP16 and porfiromycin in acquired resistant cells. Influx and effiux of radioactive VP16 and porfiromycin in the acquired resistant cells were studied as described above. Porfiromycin was used in place of mitomycin C because of the unavailability of radioactive mitomycin C and because of the similarities in structure (Fig. 13) and clinical properties of the two drugs (69). It can be seen from these studies that both cell lines had decreased accumulation of VPI6, which was especially true of HCTl16(VM)34 cells (Fig. 14). However, only the H C T l I 6 ( V M ) 3 4 line displayed o H2N~ , , , , ~ II II I~..

o •.. CH20ICINH2 I ~l~OCH3

o o H2N ~ . II , . II CH2OCNH2 ~ I~_. ~OCH 3

H3O" O~ "lil" ~I~.N.H

H3C" "11~ "N" ~1,I/"N'CH3 I

MITOMYCIN C (MMC)

PORFIROMYCIN (PFM)

o

o

H3CO~____I... CH20~NH2 I ~ ~'OCH3 H3C" y ",N" ~,~. 8

I

I- "~N-H

MITOMYCIN A (MMA)

o

H3CO~ L % ~ . ~ O ." ~ " ~ ~ H3C

CH2OCNH2 OH

RR-2

FIG. 13. Molecularstructures of mitomycinsC and A, porfiromycin,and RR2.

246

B.H. LONG and D. A. STRINGFELLOW

A I

...... . ............

, ......

3"

Z

2"

rr

t"

0 I-I I-I-Z

u

B

4-

Z 0

". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

t0

20

30

40

50

O

60

TIME (MIN)

FIG. 14. Influx and effiux of VP16 and porfiromycin in HCT116 (e), HCT116-(VP)35 (=), and HCTlI6(VM)26 (o) cells. Cells were incubated with 5/~M of radiolabeled VP16 (A) or porfiromycin (B), as described in Figure 12.

lower accumulation of porfiromycin, a finding consistent with the multidrug resistance properties of this line and the lack of multidrug resistance properties for the HCT116(VP)35 cell line (32). Role of monoadduct repair in conferring sensitivity to VP16-resistant cells. It is well recognized that VP16 and cis-dichloro-diammino Platinum II display a therapeutically synergistic effect on experimental murine tumors in vivo (70, 71), and these results appear to carry over to the clinic with encouraging results (72-74). The novel observation that cells resistant to VP16 show collateral sensitivity to cis Platinum (Table 9) may provide an explanation for the clinical observations. A similar observation of collateral sensitivity to mitomycin can be made for H C T I 16(VP-)35 and A549(VP)28, and especially SW900 cells. Furthermore, Tan et al. have reported that Burkitt's lymphoma cells selected for resistance to nitrogen mustard were 10-fold more resistant to that agent than the parental line and possessed 2- to 6-fold higher levels of

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topoisomerase II and were more sensitive to topoisomerase II inhibitors, including VP16 and VM26 (75). Influx and efflux studies comparing SW 1271 with SW900 (46) (Fig. 12) and HCTl16(VM)34 and HCT116(VP)35 with HCT-116 (Fig. 14) using radioactive porfiromycin showed that increased accumulation did not account for this sensitivity, but that decreased accumulation of porfiromycin in HCT 116(VM)34 cells possibly accounted for why this cell line did not show collateral sensitivity to mitomycin C, as it did for cis Platinum. A comparison of mitomycin C activation capabilities in SW900 and SW1271 revealed no differences, nor were there differences in glutathione S-transferase activity that could account for mitomycin C sensitivity (46). The role of monoadduct repair in conferring resistance to mitomycin C was investigated through the use of RR2, a mitomycin A analog lacking the carbamoyl group that is present in mitomycin A (Fig. 13). This analog is capable of alkylating DNA through reaction with DNA at the aziridine ring site, thus forming monoadducts, but cannot form interstrand DNA crosslinks. By using alkaline elution, we have been able to follow the formation and repair of DNA breaks produced as part of the DNA repair process that removes the monoadducts of RR2. These studies have revealed that monoadduct repair rates were much slower in SW900 ceils (46). Figure 15 shows that the DNA breaks rapidly disappeared in SW1271 cells, but

'" 2C z

nd z rr

m 0

1 2 3 REPAIR TIME (HR.)

4

FIG. 15. Formation and disappearance of single strand D N A breaks in cells exposed to RR2, a mitomycin C analog that is incapable of forming interstrand D N A cross-links. Cells were incubated for 1 hr at 37°C with concentrations of RR2 capable of producing comparable D N A breaks, then the medium was changed to drug-free medium and the incubation continued for various lengths of time in the absence of drug in order to assess rates of repair of RR2 monoadducts. SW1271 (o), SW900 (o), and HCT116 (×) (46). AER--I*

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continued to accumulate before decreasing in SW900 cells. HCT 116 cells were intermediate in repairing this lesion. These observations not only suggest that monoadduct repair capability was deficient in SW900 cells and elevated in SW1271 cells but also raised the possibility that topoisomerase II may be involved in the DNA repair process. It is quite conceivable that SW1271 cells, which have high levels of topoisomerase II and are sensitive to VPI6 because of the high level of target enzyme, are also able to repair monoadduct alkylation products, and thus are resistant to mitomycin C and cis Platinum, whereas SW900 cells, which have low levels of topoisomerase II that confer resistance to VP16, are unable to rapidly repair the monoadducts, and thus are hypersensitive to alkylating agents such as mitomycin C and cis Platinum.

Model For Topoisomerase H Inhibition From this work and from the studies conducted in other laboratories, it is possible to understand some of the processes that may occur as a result of the exposure of a eukaryote cell to a topoisomerase II inhibitor, even though the mechanisms by which the DNA intercalating drugs or VPI6 and VM26 actually inhibit topoisomerase II and produce cytotoxicitystill remain elusive. Furthermore, the DNA cleavage patterns resulting from this inhibition appear to be unique for a given chemical class of drug but appear to be similar when different analogs within a given chemical class are compared (2, 4, 9). One possible mechanism by which VP16 and VM26 inhibit the enzyme may be through hydrogen bonding with the exposed nucleic acid bases that may become exposed during the strand passing procedure (Fig. 16). A similar mechanism has been proposed for the gyrase inhibitory activity of the quinolone class of bacterial antibiotics (76). Presence of the drugs in such a position would prevent re-ligation of the DNA and freeze the enzyme in a covalently inserted mode in the DNA, resulting in the DNA breakage observed through the alkaline elution methods. It should be noted, however, that our results indicate that both the interaction of the drug with its specific target and the enzyme inhibition are not static events but only represent equilibrium situations. This interpretation is made from kinetic studies where DNA breaks appear rapidly upon exposure of cells to drug until a plateau is reached (Fig. 3). These breaks disappear in an equally rapid manner upon removal of the drug from cells. Different concentrations of drug only affect the level of the plateau and not the rate the plateau is obtained (44, 45). Both the DNA intercalating agents and VM26 and VPI6 appear to require the inhibition of both topoisomerase II subunits to produce cytotoxicity, as evidenced by the direct correspondence between double strand DNA breaks and cytotoxicity (Figs. 8 and 10) (31, 32, 65, 66). The implications of this observation are presented in a proposed model (Fig. 17), where two

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3'

~OMERASE II

FIG• 16. Model depicting the possible interaction of VP16 with single strand DNA regions at the cleavage site of topoisomerase II facilitated through hydrogen bonding with the nucleotide bases. The actual number of drug molecules that may interact at the cleavage site is unknown (32).

GENOMIC ONA

FIG. 17. Heterologous subunit exchange model proposed to describe the actual cytotoxic event hypothesized to occur only when all four subunits of two proximally located topoisomerase II molecules are inhibited by VP16 (32). proximally localized topoisomerase II molecules u n d e r g o a p r o p o s e d heterologous subunit exchange, resulting in the excision o f the loop o f D N A (32, 65). This minicircle can then recombine at a n o t h e r site where a n o t h e r molecule o f enzyme is located. This type o f illegitimate recombination has been d e m o n s t r a t e d by Ikeda to occur b o t h in vivo and in vitro by inhibiting D N A gyrase with oxolinic acid (77-80)• Such a mechanism would have deleterious effects on the g e n o m e o f a cell, which would certainly be lethal for that cell. This model is supported by the observations o f Singh and G u p t a that both V P I 6 and VM26 are mutagenic to the H G P R T , adenosine kinase, and other loci of Chinese hamster ovary cells (81, 82). DeMarini et al. have recently reported that VM26 is strongly mutagenic and clastogenic to the

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thymidine kinase locus in L5178Y mouse lymphoma cells as well (83). Furthermore, topoisomerase inhibitors are effective inducers of sister chromatin exchanges (65, 82, 84). Finally, such a model is not only compatible with but is also supported by the information we now know about the role topoisomerase II activity levels play in conferring resistance to the cytotoxic effects of a topoisomerase II inhibitor. In most cases, drug resistance is expressed by the over production of the target enzyme, thus providing a surplus of enzyme above the level normally present, which can continue to function even though most of the activity is inhibited. Fostriecin, a topoisomerase II inhibitor tha t appears to prevent the association between the enzyme and DNA (10) and thus shows no DNA breakage or mutagenic activities (85) should manifest resistance in this more classical manner. The opposite is true in the case of topoisomerase II related drug resistance, as it pertains to those inhibitors that stabilize the covalent intermediate formed between the enzyme and DNA, in that low rather than high enzyme levels confer the resistance phenotype (23, 25, 31). The only mechanism that could account for such a result would be if some deleterious event occurs that is related to the level of inhibited enzyme rather than the lack of active enzyme. It follows that if enzyme levels are low within a cell then there would be less formation of inhibited enzyme produced by a given concentration of drug than would be formed if enzyme levels were higher, keeping in mind that the inhibition represents an equilibrium situation. With respect to the model presented in Figure 17, there would be a lower frequency of recombination events occurring in a cell that had lower enzyme levels. This model would also predict that these recombination events would continue to accumulate with time because of the dynamic aspect of the model, which is precisely what occurs in cells, even though the DNA cleavage plateau level remains unchanged for periods of continuous drug exposure up to and possibly beyond 6 hr (7, 31, 32, 44, 45). Also, one would expect that levels of drug below that level required to cause an effective saturation of the enzyme in a sensitive cell containing high levels of enzyme would yield a large number of single strand DNA breaks, even though the number of double strand breaks produced, reflecting enzymes with both subunits inhibited, would be sufficient to kill the cell, whereas this same level of drug could exceed the saturation level in a resistant cell with low enzyme levels, resulting in very low single to double strand DNA break levels, yet still not result in the number of recombination events necessary to kill the cell. We see such a scenario when the SW1271 and SW900 cells are compared (Table 7). It should be noted that for this discussion, we are not addressing the separate situation where a target enzyme has become altered through mutation to render it relatively insensitive to the drug. At least one example of this mechanism of resistance has been identified for topoisomerase II (36, 37). It is conceivable that topoisomerase II may be responsible for the genetic

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instability that is an inherent property of a transformed cell through a mechanism as outlined above. The presence of high levels of enzyme found in transformed cells, which is not the case for normal cells (21), could result in the spontaneous generation of recombination events that would occur at much lower levels in normal tissues. Also, the gene amplification generally associated with cancer drug resistance (86-90) may occur through a process similar to the mechanism outlined above, by randomly excising a replicating fork, which would continue to replicate in circles until it became reinserted somewhere into chromosomal DNA. Only those cells containing the required amplified genes or losing those genes coding for a drug target (topoisomerase II) would be favored during the next drug challenge.

SUMMARY The specific inhibition of eukaryote DNA topoisomerase II by the anticancer drugs VP16, VM26, and 21 other congeners of podophyllotoxin has been extensively studied in this laboratory through the use of alkaline elution and other techniques. A structure-activity relationship has been established for cytotoxicity, single and double strand DNA breakage, and inhibition of the DNA strand passing activity of topoisomerase II. Furthermore, topoisomerase inhibition was measured in four naturally sensitive and resistant human lung carcinoma cells by quantifying the amount of single and double strand DNA breakage produced by VP16 and VM26 in cells and isolated nuclei. A direct correlation between double but not single strand DNA breaks and cytotoxicity was observed for the analogs in A549 human lung adenocarcinoma cells. In fact, some analogs were capable of producing substantial single strand DNA breakage without producing cytotoxicity. A similar correspondence was observed between double strand DNA breaks and cytotoxicity produced by VP16 and VM26 in the naturally sensitive and resistant cell lines. Evidence is also presented suggesting that the association of the drug with enzyme-DNA intermediate complex and the formation of the enzyme-DNA complex alone both reflected equilibrium governed conditions that were readily reversible. These studies support a model based on the proposal that the actual cytotoxic events are genetic alterations caused by possible heterologous subunit exchanges occurring between adjacent enzyme molecules, which result from the stabilization of the intermediate complex, rather than the actual loss of topoisomerase II activity caused by the inhibition. The resistance of normal cells and cells with acquired resistance to the possible clastogenic effects of topoisomerase inhibition may be, in part, related to the low topoisomerase II levels found in such cells. Topoisomerase II may also play a role in gene amplification and tumor cell heterogeneity by serving as a vehicle through which genetic recombination events may occur.

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P o r t i o n s o f t h e r e s e a r c h p r e s e n t e d in this p u b l i c a t i o n w e r e s u p p o r t e d b y a g r a n t f r o m t h e U n i t e d States P u b l i c H e a l t h S e r v i c e , N a t i o n a l C a n c e r I n s t i t u t e , g r a n t no. C A - 4 0 4 4 9 . T h e e x c e l l e n t t e c h n i c a l a s s i s t a n c e o f S t e v e T. M u s i a l a n d L a u r a A. N e w h o u s e is g r a t e f u l l y a c k n o w l e d g e d .

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