Beyond DNA Cross-Linking: History and Prospects of DNA-Targeted Cancer Treatmentâ€”Fifteenth Bruce F

Home , Nitrogen mustard, Topoisomerase inhibitor

[CANCERRESEARCH56, 5533-5546, December 15, 1996] Special Lecture

Beyond DNA Cross-Linking: History and Prospects of DNA-targeted Cancer Treatmentâ€”Fifteenth Bruce F. Cain Memorial Award Lecture'

Kurt W. Kohn2 Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892

Abstract tional nitrogen mustard tri(2-chloroethyl)amine revealed an unusually selective inhibition of reproduction in yeast (relative to a lesser The origin of cancer chemotherapy can be traced to the wartime potency of inhibition of respiration), and this selective inhibition of discovery ofthe lymphotoxic action ofnitrogen mustards These and other reproduction was lost upon hydrolysis of the mustard (10). The bifunctional agents were later found to produce various types of DNA cross-links, and some of these agents continue to be mainstays of current significance of this clue to an alkylation chemistry, however, was not therapy. The cellular pharmacologyofthese drugs wasStUdiedextensively yet appreciated. During this time period, mustard gas was applied during the @970sand 1980s by means of DNA filter elution methodology topically to tumors or injected intratumorally, mainly in animal ex In the course ofthese investigations, DNA topoisomerases were discovered periments, but a few clinical cases were included, and some local to be targets of anthracyclines and several other classes of anticancer tumors were eradicated (1 lâ€”l3).@ Intravenous mustard gas inhibited drugs. DNA cross-linkers and topolsomerase blockers have generally the growth, and even caused regression, of implanted tumors in rats similar cytotoxic mechanisms, which depend on DNA damage detection, (13). DNA repair, cell cycle arrest, and cell death by apoptosis. The molecular Clinical antitumor trials with nitrogen mustard, however, were control of these processes, involving oncogenes and tumor suppressor genes, is being revealed by current research. Cancer cells often have begun only after a later military disaster during World War II, when defects within these control systems, and these defects may confer selective evidence pointedto a selective lymphotoxic action by mustardgas in sensitivity to appropriately designed drug therapy. Panels of human tu humans (14). The wartime studies of mustard gas and nitrogen mus mor cell lines may serve to link the molecular defects with specific drug turd were shrouded in secrecy and were reported in summary after the sensitivities. Such correlations could guide the selection of drugs for war (15â€”21). Gilman and Philips, in their landmark paper in 1946 therapy based on molecular diagnosis of individual tumors. (15), surmised correctly the alkylation chemistry of the sulfur and nitrogenmustardsand summarizedthe biological findings in experi Introduction mental animals and humans. They cited the work of many researchers The era of cytotoxic anticancer treatment is now just 50 years old, in the United States and Great Britain whose wartime reports were having begun with the discovery of the antitumoraction of bifunc still classified and unavailable in the open literature. Evidence for tional alkylating agents, some of which remain to this day among the actions on chromosomes was reportedin 1943 in documents of the most useful anticancerdrugs. The key mechanistic feature of these Chemical Board,Ministryof Supply [London;cited by Elmoreet a!. and related drugs is their ability to cross-link DNA. This somewhat (20)].Studiesofthereactionmechanismsofnitrogenmustardwere personalreview will trace our understandingof DNA cross-linking reported in 1946 by Joseph S. Fruton, Max Bergmann, Calvin Golum agentsfromtheirdiscovery50 yearsago to theirrole in the currentera bic, and their coworkers in a series ofeight papers (J. Org. Chem., 11: of molecular biology and will consider also the cell biologically 518â€”591),andthe alkylation mechanismwas defined (22). related DNA topoisomerase blockers. Other historical details were The clue that led to the first extensive therapeutictrial against included in another recent review (1). lymphomas was the lymphotoxic effect noted in victims of the World War II tragedy at the Italian port of Ban. The events at Ban were kept History hushed up for many years but were eventually described in detail by Infield (14). The exposure to mustard gas on that occasion differed It is one of those paradoxes of our chaotic world that the origin of from the usual World War I experience in that many of the victims cancer chemotherapy traces back to the use of poison gas in war (Fig. received prolonged exposure over much of the body surface to low 1A). As early as 1917, mustard gas was noted to suppress the bone concentrations of the lipid-soluble alkylating agent dissolved in fuel marrow(2), although its cytotoxic action was attributedto the pro oil. It was the aftermath of a surprise German air attack that destroyed duction of hydrochloricacid in hypotheticalintracellularregionsthat a large number of ships in the crowded harbor, including among them were presumed to be hypersensitive to acidity (3â€”5).Thebone mar an American ship that was carrying mustard gas bombs. (The Allies row in human casualties showed suppression of granulocytic compo nents, involving not merely local necrosis, but inhibited regeneration evidently brought in poison gas on the possibility that the other side, (6, 7). Additionally,thrombocytopeniawas noted in these early stud in desperation,mightuse such weapons.) Severalfactorscombinedto ies. Intravenous administration of small doses of mustard gas in yield evidence of a specific lymphotoxic action of mustard gas that animals produced leukopenia and impaired antibody response, and was the basis for the clinical trials: the unusual nature of the exposure; these effects were thoughtto resemblethe effects of ionizing radiation the improved pathology techniques; and, very significantly, the in sight and devotion of the medical and research staff who investigated (8, 9). Between the world wars, studies of mustard gas and of the trifunc the event at the time (14). Nitrogen mustards were easier to handle than mustard gas because Received 9/16196;accepted 10/15/96. I Presented at the 87th Annual Meeting of the American Association for Cancer 3 Adair and Bagg (1 1) state that the antitumor testing of mustard gas was suggested to Research, Apnl 24, 1996, Washington, D.C. themin 1927 by Dr.JamesEwing. who hadbeen impressedby the peculiarand specific 2 To whom requests for reprints should be addressed. Phone: (301) 496-2769; Fax: nature of mustard gas burns while serving with the United States Army Medical Museum (301) 402-0752; E-mail: [email protected]. duringthe war. 5533

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CH3 Fig. 1.A, gas shell explosion somewhere in France, 1918 (201). B, the first published radiograms of tumor regression following systemic chemotherapy: Hodgkin's disease treated with nitrogenmustard(17). Left,before treatment;right,9 weeks afterstartof treatment.Structuresof mustardgas (A) and nitrogenmustard(B) are also shown.

they could be stored as stable crystalline hydrochiorides; upon neu activity in injured or hormonally stimulated tissues. It thus became the tralization, the nitrogen mustard free base had similar chemical and first known chemical that, like ionizing radiation, produced mutations biological properties to mustard gas (17, 24). Nitrogen mustards, and chromosome damage (19). Mustard gas was presumed to damage therefore, were used in the first trials of systemic therapy. Immedi the genetic material, although the chemical nature of the genes was ately after the war, the first results with nitrogen mustards in the not yet established. The mutagenic properties of mustard gas were treatment of neoplastic disease (chiefly lymphoma, leukemia, and discovered in 1942 but were not made public until after the war (19). allied conditions) were summarized in an Official Statement to the Auerbach et al. (19) in 1947 inferred that the mustards â€œmight medical profession by Cornelius P. Rhoads as Chairman of a Com combine with the materials composing the gene.â€• mittee of the National Research Council in New York (18). The full Some of the early work yielded evidence that remains interesting published reports of the first treated patients soon followed (16, 17). even now in the modern light of DNA damage and repair mechanisms. Although tumors sometimes regressed (Fig. 1B), the regression was Mustard gas, unlike ionizing radiation, produced delayed mutations, only temporary, and treatment was limited by hematological toxicity. as evidenced by mutant Drosophi!a males that produced mosaic Only hematological tumors responded. It was not clear whether ni offspring (19). The chromosome breaks produced by nitrogen mustard trogen mustards were superior to radiation therapy, although radiore may occur preferentially in regions of heterochromatin (25), suggest sistant tumors sometimes responded to the new chemotherapy (17). It ing a selectivity of action that might still be profitably pursued. is remarkable how many of the essential features of these drugs were The concept of cross-link formation (although not specifically of discerned correctly at this early date. DNA) emerged from the observation that the characteristic biological Many of the investigators who were involved in the nitrogen actions of the mustards required the presence of at least two alkylating mustard studies during and immediately following World War H went groups in the molecule (23, 25, 26). In 1949, Goldacre et aL (23) on to become leaders in cancer chemotherapy, hematology, and phar envisioned that â€œthetwo[haloalkyll groups are required to permit the macology: Alfred Gilman, Frederick S. Philips, Louis S. Goodman, molecule to react at two distinct points, lying either on a single surface Maxwell M. Wintrobe, William Dameshek, Cornelius P. Rhoads, or fibre or, more especially, on two contiguous fibres.â€•Manynitrogen Joseph H. Burchenal, C. Chester Stock, and David A. Karnofsky. mustard derivatives were tested for their ability to prolong the survival Since nitrogen mustard is a simple chemical structure, easily ame of mice bearing transplanted leukemia, but only those that had at least nable to variation, hundreds of derivatives were tested, but the hope of two alkylating groups were active (27). The double-stranded structure curative therapy was unrealized. Nevertheless, some pharmacological of DNA and its way of replicating, as described by Watson and Crick improvements were obtained in the form of cyclophosphamide, mel 3 years later, made DNA a natural candidate for cross-linking target. phalan, and chlorambucil, which remain prominent in modem cancer An experimental test of the notion of DNA interstrand cross-linking chemotherapy. Attempts to progress by way of understanding of was made possible by the concept of DNA helix-coil transition, mechanisms were limited by lack of basic knowledge and lack of developed by Marmur and Doty (28) and Doty et a!. (29) in 1959. The powerful methodology. helix-coil transition (also called â€œDNAmeltingâ€•)was found to be rapidly reversible, but only up to the end of the transition, at which Cross-Links between cDNA Strands point the complementary strands were presumed to have separated In the early search for chemical mutagens, mustard gas was tested completely and could no longer reassociate rapidly. Separated strands because of observations that pointed to its interference with mitotic could, however, reassociate slowly with the concentration dependence 5534

kinetics as a function of time exhibited a lag, consistent with the 6@iMHN2 requirement for a sequence of two reactions, as would be expected in the formation of a cross-link. Similar methodology revealed DNA interstrand cross-link formation and repair in bacterial cells (31). The site on DNA that is most readily alkylated was determined by 20' Brookes and Lawley to be the guanine-N7 position (32, 33). These investigators also detected nitrogen mustard-linked guanine dimers that could have come from interstrand or intrastrand cross-links. 30' Several other classes of anticancer drugs also proved capable of DNA cross-linking, including mitomycin (34), cisplatin (35), and chloro 40' ethylnitrosoureas (36). 50' DNA Damage in Mammalian Cells The fact that the reaction of a single nitrogen mustard molecule could prevent the denaturation of an entire DNA molecule suggested 60' a way to measure interstrand cross-links in biological experiments. In cells treated with pharmacologically relevant drug doses, however, the 2K extent of interstrand cross-linking was only on the order of 1 per million bp, and DNA of this size could not be isolated by the usual D N methods. To carry out DNA cross-linking studies in cells and tissues, Fig. 2. Evidenceof DNA interstrandcross.linking.EquilibriumsedimentationinCsC1 therefore, novel methodology was needed. of bacterial DNA treated with various concentrations of nitrogen mustard (HN2) and then A solution to this problem came out of an unexpected observation briefly exposed to 0.02 NNaOH followed by neutralization. Native (N) and denatured (D) in 1971 while we were studying newly replicated DNA that we DNAfonneddistinctbandsthatcouldbequantitated.HN2wasseentoinhibitthestrand separation of a dose-dependent fraction of DNA molecules. thought might be associated with structural components of the nucleus that could be separated by filtration (37). When nuclear lysates were subjected to mild controlled shearing and then dripped through mem of bimolecular kinetics. The anticipated effect of an interstrand cross brane filters, we found newly replicated DNA to be selectively re link was that the linked strands would reassociate rapidly with uni mined. The ratio of newly replicated relative to bulk DNA was molecular kinetics. In 1961, I had the opportunity to work in Paul consistently elevated in the filter-retained fraction and gradually re Doty's laboratory and to test this idea using the cutting-edge technol turned to equivalency during a chase period. Thus, newly replicated ogy of the day: the analytical ultracentrifuge with UV optics. Brief DNA appeared to be part of a detergent-resistant complex that could exposure to 0.02 N NaOH normally denaturedall of the DNA (i.e., be retained on filters. The big surprise came when we used NaOH as converted all of the DNA to single strands). However, if the DNA was the lysis solution. Contrary to what we had seen with various deter pretreated with nitrogen mustard, a fraction of the DNA molecules did gent recipes, newly replicated DNA was now enriched in the flow not denature (Fig. 2). The conversion of denaturable to undenaturable through fraction of the lysate. We surmised that short nascent DNA DNA exhibited first-order kinetics with respect to the amount of single strands, released by alkali, could pass through filters, whereas nitrogen mustard bound to DNA, indicating that the reaction of a intact mature DNA was retained. Using X-rays to generate single single molecule of nitrogen mustard could prevent the strand separa strand breaks, we confirmed that shortened DNA single-strand seg tion of an entire DNA molecule of up to 30 kbp (30). Moreover, the ments passed through filters selectively in alkali. Using a peristaltic

Fig. 3. Kinetics of DNA alkaline elution (48). A, cells labeled with â€˜4C.thymi&newereexposed to various doses of X-ray at 0'C then lysed with detergent on filters, and the DNA was eluted at pHl2.0 (open symbols) or pHl2.8 (filled symbols). I B, dependence of apparent elution rate constant on X-ray dose. Different symbols in B refer to 2 [ode pendent sets of experiments.

10 HOURS OF ELUTION X-ray Dose (R)

5535

pump to control the flow of alkaline solution through the filter, we 1.0 found that the system could be made surprisingly sensitive and robust t@ with respect to the size of the DNA single-strand segments. , â€˜Q

It was possible to adapt the DNA filter elution technique to a @â€˜ @ . variety of DNA lesions (38â€”42). Assays were developed for single â€˜t@ b@ strand breaks, double-strand breaks (43), base-free sites (44), inter . b @ strand cross-links, DNA-protein cross-links (45), and protein-associ Normal Normal @Jy ated DNA strand breaks (46, 47). 0.5 Alkaline elution of DNA single strands exhibited a simple log linear behavior with an apparent rate constant that was proportional to Lu

single-strand break frequency (Fig. 3; Ref. 48). Moreover, the elution U.. rate was independent of the number of cells loaded (up to the point of z 0 A C clogging the filter) and nearly independent of flow rate. This was 0.3 surprising in view of the high and variable viscosity in the filter pores Lu 1.0 through which the DNA was passing. Presumably, there are mutually @.1 opposing factors that tend to cancel each other out and thus give the z

observed simple and robust behavior. After optimizing the parameters U- 0 of the system, we soon had in our hands a method that could quantitate z biologically relevant levels of DNA single-strand breaks in cells and 0 I- tissues. C-) From the dependence of alkaline elution kinetics on time and X-ray dose and the fact that X-rays produce strand breaks that are distributed U- randomly, we deduced the behavior of a homogeneous size population of DNA single strands (42). The result seemed at first surprising, because the theoretical elution rate for a population of equal-size strands was a step function: the probability of release from the filter is constant up to a critical time, when all the strands have been released (Fig. 4). If the strands are shorter, it takes less time to reach the total elution point. Physically it would be as if the strands are gliding at a constant rate over a fulcrum, and the time at which a given strand is released depends on how that strand happened to be placed 5 10 15 5 10 15 relative to a fulcrum when the strand was initially deposited on the HOURSOFELUT1ON filter (Fig. 4, inset A). Actually the situation must be more complex, Fig. 5. DNA single-strand breaks during nucleotide excision repair in Uv-irradiated as suggested by Fig. 4, inset B. human fibroblasts, as revealed by DNA alkaline elution. Open symbols, internal controls One of our first applications of the alkaline elution method was showing the elution of DNA from cells irradiated with a standard dose of X-rays. Filled symbols:A, normal human fibroblasts, untreated; B, normal human fibroblasts 4 mm after to nucleotide excision repair. Single-strand break formation was an Uv irradiation;C,xerodermapigmentosumcells(complementationgroupA),untreated; expected part of the repair process, but it had not been clearly D, xeroderma pigmentosum cells 4 mm after UV irradiation. The result showed that demonstrated in mammalian cells. The high sensitivity of the normal cells but not xeroderma pigmentosum cells produce transient DNA single-strand breaksduringnucleotideexcisionrepair.Thisexperimentwascarriedout by AlbertJ. alkaline elution method allowed this type of observation to be Furnace, Jr., in our laboratory in 1975 (49).

A B made clearly (Fig. 5; Ref. 49). After irradiation with UV light, normal human fibroblasts exhibited a wave of single-strand breaks, n=1 k which peaked a few minutes following radiation and then slowly subsided. Xeroderma pigmentosum cells, which were thought in

C capable of nucleotide excision repair, exhibited no UV-induced w DNA strand-break signal. Lu @n=2 DNA-Protein Cross-Links z k/2 --- - 0 The UV repair study led in an unexpected direction. When we checked whether the sensitivity of our strand break assay might have -J I n4 Lu changed during the repair process, we were surprised to find that the k/8 --@-H----f---@---@increased elution caused by a standard dose of X-ray was reduced during the repair period (50). We suspected that this might be due to 1/k 2/k 4/k DNA-protein cross-links, because UV was known to produce such ELUTIONTIME(t) cross-links, and cell proteins might adsorb to the filters and prevent the elution of protein-linked DNA strands. We found that this was Fig. 4. Theoretical alkaline elution behavior of homogeneous populations of DNA indeed what was happening and, furthermore, that xeroderma cells strands of a given size. k is the elution rate of strands of unit length (n 1). Strands of twice that length would elute at one-half the rate k, and so forth. In each case, the elution were incapable of repairing the UV-induced DNA-protein cross-links rate would be constant up to the time when all the DNA has eluted, at which point the (50). This indicated that nucleotide excision repair acted not only on elution rate would fall to zero (42). Inset A, simple model of a DNA strand gliding at a constant rate over the boundary between adjacent filter pores; inset B, a more realistic base damage but also on DNA-protein cross-links. model of a DNA strand gliding between several filter pores. We were able then to devise a very sensitive andquantitativeassay 5536

2@ I I

I .@

A 0.5 HN2 Concentration 4@M)

Fig. 6. DNA interstrand cross-link assay. The alkaline elution assay was modified by using the less adsorptive polycarbonate filters, treating the cell lysate on the filter with proteinase K, and adding dodecyl sulfate to the eluting solution. A, increased elution rates at various doses of X-rays. B, reduced elution rates due to treatments with various doses of nitrogen mustard The cells were irradiated with 300 rad at OC before elution. C, linearization of the cross-linking effect with respect to drug concentration. Cross-link/break parameter = â€˜@/@lâ€”R0)/(l â€”R)l â€”I, where R,, and R are the fractions of DNA retained by control and drug-treated cells, respectively (39). Different symbols in C refer to independentsetsof experiments.

for DNA-protein cross-links (45), and this was to become important in increased DNA alkaline elution with an agent that had been found to an unexpected way. produce DNA strand breaks by other techniques. We knew that DNA-protein cross-links could obscure the detection DNA Interstrand Cross-Links in Mammalian Cells of DNA strand breaks, but we had no reason to believe that doxoru The alkaline elution rate of DNA single-strand segments from bicin would produce DNA-protein cross-links. By that time, however, X-irradiated cells could be reduced by either DNA-protein cross-links we had worked out the conditions to remove the effects of DNA or DNA interstrand cross-links. Because bifunctional alkylating protein cross-links in the assay. When we applied deproteinizing agents generally produce both types of lesions, a specific assay for conditions to doxorubicin, we were astounded by the results (46, 47). interstrand cross-links required a way to eliminate the effects of the We now saw a classic alkaline elution profile indicative of randomly DNA-protein cross-links. We soon learned how to prevent protein distributed stand breaks; the pattern looked as if the cells had been adsorptionto the filters, andthis allowed us to devise a specific assay X-rayed. The DNA elution was log-linear, and the apparent rate for interstrand cross-links suitable for pharmacological studies in constant of elution was proportional to drug concentration over a mammalian cells (Fig. 6; Ref. 39). pharmacologically relevant dose range (Fig. 7). We applied this methodology to cell pharmacology studies of a That meant that doxorubicin did produce DNA strand breaks, but it variety of DNA cross-linkers, including nitrogen mustards (51â€”56), also produced DNA-protein cross-links, and the latter completely platinumcomplexes (53, 55â€”62),psoralen(63), andchloroethylmtro obscured the former in the nondeproteinized assay. For randomly soureas (51, 64). The latter compounds were particularly interesting distributed DNA-protein cross-links to completely obscure the DNA because of their manner of producing cross-links slowly after alkyla strand break signal, the DNA-protein cross-links would have to be tion at guathne-O@ positions and because of the inverse relationship present in large excess. Because our assays were quantitative, we were we found between cross-linking and the activity of the guanine-06- able to determine the relative frequencies of the two types of DNA alkyltransferase repair enzyme in different cell types (65). Drug lesion. We found that the frequencies of the single-strand breaks and sensitivity was dependent, in part, on the extent of cross-link forma DNA-protein cross-links were equal within experimental error. We tion (65â€”67).Further clinical development of new chloroethylating concluded, therefore, that the locations of the two types of lesion agents was proposed (68â€”70). could not be random (Fig. 7, top left). Rather, there must be one DNA-protein cross-link within each DNA single-strand segment. Protein-associated DNA Strand Breaks: An Effect of There was only one plausible way for this to occur, which was that the Topolsomerase-targeted Drugs proteins were linked consistently either to the 3' or the 5' termini of An unanticipated development occurred in 1978 in the course of the breaks (Fig. 7, top right). This suggested that the linked protein alkaline elution studies of doxorubicin. Because doxorubicin had been might be an enzyme that produced the break. There was at the time reported to produce DNA strand breaks in alkaline sucrose gradient precedent for this type of mechanism in J. C. Wang's nicking-closing studies, we anticipated no difficulty in demonstrating these breaks by activity ofE. coli w protein (71), Blair and Helinski's DNA relaxation alkaline elution. However, using treatments that should have produced enzyme associated with colicinogenic plasmids (72), and Champoux DNA strandbreaks,we saw no increasein DNA elution whatsoever. and Dulbecco's DNA untwisting enzyme of mammalian cells (73, This was puzzling because it was the first time we failed to find 74), enzymes that we now call DNA topoisomerases. Several types of 5537

5,-.. __...3' 3,--- __..5' 3,..- _

Fbndom model Bound-to-one-terminus model

Fig. 7. Protein-associated DNA strand breaks pro duced in mouse leukemia cells by doxorubicin (@i concentration as indicated; treatment time, I h). Depro teinizing alkaline elution conditions were used. With out deproteinization, no increase in elution occurred. The DNA of treated cells was prelabeled with â€˜4C- thymidine. An intemal standard of I3Hlthymidine-la 0 beled cells irradiated with 300 rad was added, and this w served as a normalized time scale on the horizontal axis. This experiment was carried out by Warren E. Ross in our laboratory in 1978(47). Top left, randomly distributed single-strand breaks and DNA-protein cross-links. Top right, proteins linked consistently to one terminus at each single-strand break.

DNA intercalating drugs were found to produce protein-associated enzyme induced came from quantitative studies in cells. Several DNA strand breaks, as we then called these lesions, and were there observations were consistent with an enzymatic origin (75): (a) after fore hypothesized to be topoisomerase blockers (47, 75â€”78). addition of drug, the lesion frequencies climbed to a steady-state level Support for the idea that protein-associated DNA strand breaks are that was dependent on drug concentration (Fig. 8); (b) the level of lesions showed saturation kinetics with increasing drug concentration, indicating that there was a limiting factor governing the number of lesions that were formed; (c) the lesions reversed rapidly upon drug >- U removal; and (cOboth the formation and the reversal of lesions were z strongly temperature dependent. In all of these experiments, the strand 0 LU breaks and DNA-protein cross-links appeared and disappeared to U. gether, supporting the idea that they were different aspects of the same

LU lesions. Even more telling, the formation and reversal of breaks was demonstrable in isolated cell nuclei without AlP, showing that the 3 Z?r reversal was not a DNA repair process (79â€”82). 4 In attempting to identify the responsible enzyme, we found that the activity could be extracted from isolated nuclei with 0.35 MNaC1 and that the activity could be restored by adding the extract back to the isolated nuclei (83). Using this assay, the extract was fractionated to isolate the active component. Specific identification of the enzyme, however, had to await the characterization of topoisomerase II by Leroy Liu et a!. (84, 85), and it was in Liu's laboratory that drug effects on the purified enzyme were first demonstrated (86â€”88).We verified that the component responsible for the drug effects in the filter assays was in fact topoisomeraseH (89). TIME FOLLOWINGDRUG ADDITION (minutes) In addition to DNA intercalators, the epipodophyllotoxin deriva Fig. 8. Kinetics of protein-associatedbreak formation and reversal in mammaliancells. tives etopside (VP16) and teniposide (VM26), which are not strong ThisexperimentwascarriedoutbyLeonardA.Zwellinginourlaboratoryin1981(75)and DNA binders,were found to produceprotein-associatedDNA strand used the dnig m-AMSA. also known as amsacrine, which gave very clear and specific results. [m-AMSA was developed by Bnice F. Cain, who is commemoratedby these lectures(202, breaks and were shown to be topoisomerase-targeted drugs (90â€”94). 203).]Cellswereexposedtotheindicatedconcentrationsofdnigfor60miii,atwhichtime It is instructive that these chemical modifications of the natural the dnig was washed away and the cells were incubated further in the absence of dnig. At various times, aliquots of cells were subjected to alkaline elution under deproteinizing product, podophyllotoxin, retain the cytotoxic potency of the parent conditionsto determinesingle-strandbreak frequencies.Under nondeproieinizingconditions, but change the target from tubulin to topoisomerase H (95â€”97);nature there was no increase in DNA alkaline elution (data not shown) because of the presence of covalent DNA-protein complexes. Assays for DNA-protein cross-links (data not shown) sometimes exhibits coherence in unexpected ways. indicated a nearly 1:1 ratio of DNA-protein cross-links and single-strand breaks. Whereas topoisomerase H is inhibited by a wide variety of corn 5538

Fig. 9. Stacking model of drug-stabilized topoisomerase H-DNAcomplexes.Basesequencepreferenceforthe location of the siteson the DNAdiffersfordifferentdrugclasses.For doxorubicin and related anthracycines, the strongest base pref erence is for an A at the immediately upstream flank of the DNA strand break, as shown.Ellipticinesprefera T, and etoposides prefer a C at this position. Amsacrine prefers an A at theimmediatelydownstreamflankofthebreak.Topoisomerase H is a homodimer conferring diadic symmetry to the complex in which there can be a pair of single-strand breaks separated by a 5' overlap of 4 bp. To simplify the diagram, the two mono mers of a topoisomerase homodimer are shown as separate circles. Drug molecules are represented by filled rectangles.

Ellipt icine VM26

pounds, topoisomerase I thus far has a relatively narrow range of by Yi Fan et a!. in our laboratory4 has indicated recently that a specific inhibitors (98, 99). Chief among these is camptothecin, a structure with camptothecin stacked against a base pair at a topoi fascinating natural product the discovery, chemical structure, and somerase I cleavage site is in accord with a variety of experimental structure-activity relationships of which were described by Monroe evidence regarding structure-activity relationships (125), direct Wall and Mansukh Wani in the Cain Memorial Award Lecture in interaction between camptothecin derivatives and DNA purines, 1994 (100). The first observations on the effects of camptothecin on especially at the cleavage site (126, 127), and drug-resistant to DNA were actuallymade by SusanHorwitz(101), who presentedthe poisomerase I mutations (1 12). Cain Memorial Award Lecture in 1992. Camptothecin was shown to block purified topoisomerase I specifically (102, 103) and to produce Toward Better Drugs through MOdified Mechanisms protein-associated DNA strand breaks in cells (104). The role of topoisomerases as drug targets in cells was confirmed by the isolation After 50 years, some of the most useful anticancer drugs still and characterization of drug-resistant mutant forms of topoisomerase include DNA cross-linking agents, such as cyclophosphamide and I or H from drug-resistantcells (105â€”112). cisplatin, and topoisomerase blockers, such as doxorubicin and camp The different types of topoisomerase H blockers were found to tothecin derivatives. One path toward improved drugs would be stabilize DNA-cleavable complexes at different sites in DNA, depend through chemical structure modification based on knowledge of ent in parton DNA sequence (113, 114). The location of the blocked chemical mechanisms. It may be asked, for example, whether the complexes on DNA in reconstituted chromatin depended in part on drugs react in a DNA sequence-dependent manner, whether drugs of nucleosorne positioning (115). In cells, topoisomerase H DNA cleav a given class differ in their preferred DNA reaction sites, and, if so, age sites were found to occur frequently in regions of DNA that are whether such differences have consequences relevant to therapy. nuclear matrix association regions (1 16) and in the c-myc gene when When modified drugs show major alterations in mechanism in the it is actively transcribed (117, 118). Camptothecin-trapped topoi absence of clear information on the biological consequences of such somerase I complexes in vivo tend to occur in transcriptionally active alterations, it may be justified to consider the modified drug as if it regions (119) and in intemucleosomal linkerregions (120). were a new chemical structure and to move toward clinical trial of the Analysis of the nucleotide sequence contexts at sites of drug modified drug in comparison with its parent. induced topoisomerase H cleavage complexes in purified DNA sug Nitrogen mustards have characteristic nucleotide sequence prefer gested a structural model for the complexes (121). The base pairs that ences for DNA alkylation (128â€”131),and topoisomerase blockers, as most influenced site selection were those that were situated on the already discussed, also vary in DNA site preferences. However, we immediate flank of a cleavage site. Depending on the drug, this base know very little about how such differences translate into effects on pair could be on the 5' or the 3' flank, and the base preference differed cells. Alkylating agents react with many biomolecules other than for different drug classes (121 , 122). This suggested a model of the DNA. Because DNA is the important target, side effects might be trap@ complex in which the drug molecule stacks against a flanking reduced by minimizing the non-DNA reactions. DNA targeting of base pair at the cleavage site (Fig. 9; Ref. 121). nitrogen mustards has been studied in compounds containing an Similarly, we found for the interaction of camptothecin with acridine moiety capable of DNA intercalation (131, 132). Some of topoisomerase I that the strongest sequence preference was for the these compounds have unusual DNA sequence preferences, and in bases immediately flanking the cleavage site (123, 124). The stacking model can therefore apply also to the camptothecin 4 Y. Fan, Y. Pommier, K. W. Kohn, and J. N. Weinstein. Molecular modeling studies topoisomerase I trapped complexes. A molecular modeling study of the DNA-topoisomerase ternary complex with camptothecin, submitted for publication. 5539

some cases, the monofunctional alkylating derivatives are active, suggesting mechanistic differences worthy of clinical investigation. The relationship between DNA site preferences and cytotoxicity for topoisomerase-targeted drugs is also unknown. In some cell lines, the potency of these drugs correlates with DNA and/or RNA syntheses (133â€”135),butit is not know how this relates to the genomic sites of the lesions. .5' Extensive structure-activity data on camptothecins have given a camptothecin coherent picture that may help in the development of improved versions of these drugs (125, 136, 137). Ability to block topoisomer @ â€˜I ReplIcatIon ase I was closely correlated with antitumor activity. The features of complex the molecule that are essential for activity include the a-hydroxy lactone structure of ring E and the steric configuration of the asym metric carbon on this ring (position 20; Fig. 10). Positions 9 and 10 on ring A can be substituted without loss of activity, whereas substitution of position 12 on the same ring destroys activity. This suggests that the region of the molecule near position 12 is juxtaposed against the DNA or topoisomerase in the complex, whereas the other side of the Fig.11.Howacollisionbetweenamovingreplicationforkanda camptothecin-trapped molecule, in the vicinity of positions 9 and 10 in ring A and position topoisomerase I complex may result in an irretrievable DNA lesion. 7 in ring B, is accessible to solvent. In searching for improved camptothecin derivatives, substitutions can therefore be made at po covalently with the enzyme or the DNA to make the complexes nearly sitions 7, 9, 10, and sometimes I 1. Several such derivatives are in irreversible (127). These derivatives do form nearly irreversible corn clinical use or under investigation (138). plexes, and they have enhanced cytotoxic potency, which, unlike that What basis in mechanism might there be for improved activity of of other camptothecins, is not reversed by DNA synthesis inhibitors.5 such derivatives? One possibility emerged from the current partial Clinical trials will be needed to determine whether enhanced cytotoxic understanding of how camptothecin produces potentially lethal le potency by this mechanism can translate into improved therapeutic sions in cells. When camptothecin forms a complex with DNA and results. topoisomerase I, one strand of the DNA helix is cleaved, and the enzyme is covalently bound via a tyrosine residue to a 3'-phosphate Toward Better Therapy through Cell Biology terminus. The drug may stabilize this cleaved complex, in part, by forming a labile covalent bond between an opened lactone ring (ring Although we know a great deal about the chemical lesions pro E) and a nucleophilic group on the enzyme (98, 139, 140). Covalency duced by anticancer drugs and about their repair, we still do not know notwithstanding, the complex can dissociate within minutes to re-form much about the origin of the antitumor activity: we do not know how undamaged DNA. A potentially lethal lesion can result if a cleaved cancer cells may be killed selectively or why the drugs work in some complex is encountered by a moving replication fork (141â€”143), cases and not in others. The emerging detailed knowledge of how cell which could have the consequence of producing a DNA double-strand proliferation, differentiation, and apoptosis are regulated may open break (Ref. 144; Fig. 11). Such lesions are considered to be only new possibilities for therapy. potentially lethal, because they could be subject to repair. How then The physiological responses of cells to a variety of DNA-damaging might structure modification enhance the production of such lesions? drugs or topoisomerase blockers are remarkably similar in that treated A key parameter is the lifetime of the drug-DNA-topoisomerase cells tend to arrest at certain points in the cell cycle and, in some complex: the longer its lifetime, the greater the probability of a fatal cases, to undergo apoptotic death. The cell cycle arrest generally is not interaction with a genomic event, such as a moving replication fork or a manifestation of drug toxicity. Rather, it is a protective mechanism transcription process (135, 142, 145). In support of this picture, ofcell cycle checkpoints in which the initiation of one cell cycle event camptothecin derivatives were found to differ in the rate of revers is inhibited while another is in progress (147â€”152). ibility of their complexes, and cytotoxic potency was found to in In this context, a cell cycle checkpoint response to DNA damage is crease with increasing lifetime of the complexes (146). With this indicated when there is evidence that (a) treated cells tend to arrest or conceptual underpinning, camptothecin derivatives were prepared become delayed at a specific point in the cell cycle; (b) the arrest can with an alkylating substituent in the 7 position, which could then react be prevented by adding an inhibitor; and (c) prevention of arrest increases cytotoxicity. Thus, a (12 checkpoint is indicated by the findings that (a) tumor 9 7 cells that have sustained DNA damage often arrest in G2 (or very late 10 in S); (b) this arrest can be prevented by caffeine, pentoxifylline or certain protein kinase inhibitors which are not by themselves cyto 11 toxic; and (c) when the G2 arrest is prevented, cell survival is reduced (152â€”158). A current view of the G2 checkpoint controlling the G2 to M transition in drug-treated or irradiated cells suggests a system of mutually interacting kinases and phosphatases (Fig. 12; Refs. 150, Et 152, 159â€”164).An interesting feature of this system is that it appears to contain positive feedback loops, such that the components may HO have switch-like behavior and may not constitute a linear causal 0

Fig. 10. Structure of camptothecin. 5 M. Valenti, K. W. Kohn, and Y. Pommier, unpublished observations. 5540

These opposing actions of p53 can account for the conflicting results in different cell systems that have been reported on the effect of p53 on drug sensitivities (157, 173â€”176).Cells that are inherently apoptosis competent would be expected to exhibit reduced sensitivity to DNA damage when p53 is inactivated by mutation. Lymphoma cells, for example, generally undergo apoptosis very readily. Thus, most p53-mutant Burkitt's lymphoma cell lines were found to have reduced sensitivities to ionizing radiation, the DNA cross-linking agents nitrogen mustard and cisplatin, and the topoisomerase inhibitor etoposide (179). Some carcinoma cells, on the other hand, have weak or nonexistent capabilities to undergo apoptosis, even when they have normal p53 function. In such cases, cells can have increased sensitivity to DNA cross-linking agents when p53 function is disrupted (157). Without p53 function, treated cells then do not arrest in G1, but proceed Mltotic through S and arrest in G2. Survival then becomes increasingly events dependent on an intact G2 checkpoint. When the G2 checkpoint is

Fig. 12. Integrated switch model of the G2 to M phase transition (152). Mitotic events inhibited (e.g., by means of pentoxifylline or by means of a stauro are stimulated by the hypophosphorylated cyclin B-cdc2 complex, which is the active sporine derivative, UCN-Ol), survival is reduced further, and the form of the kinase. During S or 02 phaseScells accumulate this kinase in an inactive form selective toxicity against p53 mutant cells is increased (157, 158). in which the cdc2 component is hyperphosphorylated. The conversion of inactive to active form is accomplished by a phosphatase, cdc25C, the activity of which is enhanced by Another confounding factor in p53 disruption is chromosome in hyperphosphorylation, this phosphorylation being carried out by cydlin B-cdc2. Thus we stability, which arises in cells that cycle unchecked despite persistent have a positive feedback loop that can have switch-like behavior. A small initial stimulus can activate the switch. The switch is kept in check by ongoing DNA replication or repair, DNA damage (183). Chromosome instability increases the heteroge so that when these processes are completed, the switch is thrown. How the switch is neity of cell populations and favors the selection of rapidly growing or controlled is, however, unknown. One controlling component is the weal kinase, which drug-resistant malignant cells. Although p53 represents a well-defined tends to keep cyclin B-cdc2 hyperphosphorylated and inactive. molecular and functional distinction between normal tissues and cer tamtumors,theutilizationofsuchdifferencestoselectivelyeradicate sequence of events (152). The inputs which control the switch and the these tumors by means of chemotherapy is a complex problem re components that sense the presence of DNA damage however remain quiring a detailed understanding of integrated regulatory functions. to be elucidated. p53 affects the transcription of several genes that are believed to In early studies, arrest in G2 was the most prominent effect, because mediate its diverse cell biological effects (184, 185). Included among the tumor cells studied tended to be defective in tumor suppressor p53 these are p2lwafl/cipl and gadd45, which inhibit the G1-S transition function. More recent studies in cells having intact p53 function show and probably also S phase itself (186â€”188), and bax and bcl-2 that that G@ arrest (arrest prior to the G1 to S transition) also is an affect the threshold for apoptosis ( 189â€”191).The logic of this system important feature of drug responses (165, 166). may be as follows. Increased levels of active p53 protein lower the Much of the complex regulatory network controlling the G1 to S threshold for apoptosis in all cells that are inherently competent to transition has now been revealed, including its component cyclins, protein kinases and phosphatases, kinase inhibitor proteins, transcrip carry out the process. By increasing the transcription of p2lwafl/cipl, tion factors and their inhibitors, and promoter elements of genes for which blocks cyclin-dependent kinase cdk2, p53 at the same time some of these components (Fig. 13; Refs. 164, 167â€”169).Itwill be causes E2F activities to fall. Because E2F can cooperate with p53 to challenging to integrate the vast amount of information accumulating enhance apoptosis (192), the reduced E2F activity could prevent cells about various parts of the network. Therapeutic implications currently from undergoing apoptosis despite the lowered threshold. Thus, center on p53, which is defective in about one-half of human cancers Polyak et a!. (178) have recently shown that disruption of p2lwafl/ @ (170, 171), and on the pathway involving cyclin D, tumor suppressor cipl can convert the p53-induced response from arrest to apopto protein p16, and pRb,6 one or another of which is altered in most sis, indicating that p2lwafl/cipl can mediate cell cycle arrest and clinical tumors (Refs. 152, 157, 172â€”176;Fig. 13). protect against apoptosis. The functional value of this logic could be p53 and other regulatory components that control the G1 checkpoint that cells that inappropriately elevate E2F levels through expression of are also involved in the control of cell death by apoptosis (177, 178). oncogenes or viral genes would be eliminated. [As a countermeasure, Because p53 is so often defective in clinical cancer (171), the effects some oncogenic viruses synthesize products that disrupt p53 function of p53 on drug sensitivities have been of intense recent interest. The (193).] functions of p53, however, are now seen to be multiple and complex (177), and some of its different actions affect drug sensitivity in Studies on a Panel of Human Tumor Cell Lines opposing directions (Refs. 157 and 179; Table 1). When p53 function is disrupted, DNA damage does not cause cells to arrest in G1 (180), Detailed knowledge at the level of molecular and cellular biology and DNA repair capability is reduced (157, 181). The enhancement of may not immediately lend itself to clinical application. One formula DNA repair by p53 may be mediated in part through p53-induced tion of the problem is that malignancy is driven by altered expression transcription of GADD45 (182). The effects of p53 disruption in of particular gene products and that such alterations could make cells disturbing cell cycle arrest and DNA repair tend to reduce cell selectively sensitive to particular cytotoxic therapies. The objective is survival. On the other hand, the loss of the role of p53 in apoptosis to develop cytotoxic therapies that would be tailored to tumors of would tend to increase cell survival. certain types defined by their molecular characteristics. Some of the important molecular changes in common cancers may be preserved in

6The abbreviations used are: pRb, retinoblastoma protein; NCI, National Cancer appropriately chosen cell lines. The molecular alterations in human Institute. tumor cell lines could then serve to link preclinical and clinical 5541

Fig. 13. General logic of the control of S phase and apoptosis in cells treated with DNA-damaging drugs. Tumor cells often have defects in one or another component of this regulatory system, and future therapies may be designed to take advantage of these defects (157, 173-175). This regulatory system makes a life-or-death decision for the cell, dictating either cell cycle arrest or apoptosis (178, 188, 204, 205). DNA damage or other forms of genotoxic stress cause p53 to accumulate, mainly as a result of decreased degradation of the protein. p53 causes p2lwafl/cipl to increase due to transcriptional activation. p21 inhibits the function of 01-cyclin-dependent kinases by binding to the cycin-cdk complexes. The cyclin-cdk complexes hyperphosphorylate, and thereby block, the function of pRb. pRb, when not hyperphosphorylated, binds to and blocks E2F transcription factors. E2F stimulates many genes that are required for and that may initiate S phase. p53 also cooperates with E2F to stimulate apoptosis (192), in part through its transcriptional control of the Bax-Bcl2 couple (189â€”191).Quotation marks indicate that related protein species may function similarly in the same or in parallel pathways. The best defined parallel pathways consist essentially of (a) cyclin D-cdk4, pRb, and E2F1, and (b) cyclin E-cdk2, p107, and E2F4. Tumor suppressor protein p16 also is frequently defective in tumor cells and may play a target role in future therapies (172); it competes with cyclin D for binding to cdk4 and thereby specifically blocks this pathway.

Table 1 Factors affecting cell survival when p53 function is disrupted Decreased cell survival Increased cell survival Impaired cell cycle checkpoint functions Reduced apoptosis Impaired DNA repair Chromosome instability leading to selection of malignant Chromosome instability leading to lethal genotypes or drug-resistant cell populations development of therapies for specific tumors based on molecular tics could guide clinical trials based on molecular diagnosis of the diagnosis. tumors. Thus drugs that act selectively against cell lines having certain The NC! cell screen has generated a large amount of data on cell molecular characteristics could be tested in patients stratified with growth inhibition by many thousands of compounds tested against a respect to a molecular diagnosis of their tumors. panel of 60 human tumor cell lines representing a variety of tissues of One phase of the cell line characterization studies has focused on origin ( 194). Individual cell lines differ greatly in their drug re p53.7 Of 58 cell lines analyzed, 39 were found to be p53 mutant. sponses. However, the pattern of responses of the set of 60 cell lines Most, but not all, of the mutant lines showed elevated basal levels of is strongly correlated with drug action mechanisms (195â€”197).The p53, and their responses to ionizing radiation were deficient with response patterns, considered as 60-dimensional vectors, have been respect to G@arrest and with respect to inducibility of p2lwafl/cipl, analyzed by means of a variety of statistical tools, including cluster gadd45, and mdm2 transcription. Strong G1 arrest in response to analysis, neural networks, and principle component regression. These ionizing radiation occurred only in cell lines that exhibited strong analyses have been done for large data sets of >40,000 compounds induction of p2lwafl/cipl mRNA. The p53 mutant lines exhibited and for a subset of compounds for which probable mechanisms of statistically less growth inhibition than p53-normal lines to DNA action could be inferred from experimental evidence or chemical cross-linking agents, topoisomerase inhibitors, and antimetabolites structure. Cluster analysis over large sets of tested compounds (198, (l99).@ Apoptosis may therefore be an important factor in drug re 199) corroborates the observation that compounds related by structure sponses in the NC! screening assay. Because there was considerable or by action mechanism usually group together. The coherence of overlap in the sensitivity distributions, factors in addition to p53 these groupings is remarkable in view of the huge size of the data set contribute to the growth inhibition, some of which may be identified and demonstrates the informational power inherent in the behavior as this work progresses. Antimitotic agents, such as taxol and vincris patterns of a large panel of cell lines. tine, on the other hand, did not show any such dependence on p53, To test the ability of the cell screen data to predict mechanism of suggesting that these agents may have an advantage over other stand action, a set of 123 standard drugs were assigned probable mecha ard drugs in the treatment of p53-mutant tumors (l99).@ nisms (196). The assignments were made without prior knowledge of the cell screen results. A neural network, trained on a subset of drugs, Postscript was then able to correctly assign the remaining drugs in nearly all cases. The strong relationship between mechanism of action and cell The painting by Edouard Manet featured on the cover of this issue screen response pattern was verified by other statistical methods of Cancer Research (and Fig. 14) could be viewed in relation to the (198â€”200). half-century quest for anticancer drugs. The painting, â€œGareSt. The informational power of patterns encompassing sets of cell lines Lazare,â€•was brought to my attention in a recent Sunday afternoon is now being extended to include molecular and cellular biology lecture at the National Gallery of Art presented by the art historian characteristics. The 60 cell lines of the NCI screen have been or are Juliet Wilson-Bareau, to whom I am also indebted for an illuminating being characterized with respect to a variety of oncogenes, tumor response to my proposed use of the picture. Her lecture focused on suppressor genes, and cell cycle responses. A variety of correlations â€œthemysterybehind the steamâ€•inthe painting. The steam is billowing are being examined to determine how individual cell lines are related to each other and how molecular characteristics relate to drug re 7 P. M. O'Connor, J. Jackman, I. Bae, T. 0. Myers, S. Fan, D. A. Scudiero, A. Monks, E. A. Sausville, J. N. Weinstein, S. Friend, A. J. Fornace, Jr., and K. W. Kohn. sponses. Assuming that the cell lines retain some of the molecular Characterization of the p53 tumor suppressor pathway in cell lines of the NC! anticancer characteristics that are common in clinical tumors, these characteris drugscreenandrelationshipswithchemosensitivity,manuscriptinpreparation. 5542

of Art, for her help in obtaining slides and permission to reproduce the Manet parnting that appears on the cover of this issue.

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Kurt W. Kohn

Cancer Res 1996;56:5533-5546.

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