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

[CANCER RESEARCH56, 5533-5546, December 15, 1996]

Special Lecture

Beyond DNA Cross-Linking: History and Prospects of DNA-targeted CancerTreatmentâ€”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

The origin of cancer chemotherapy can be traced to the wartimediscovery ofthe lymphotoxic action ofnitrogen mustards These and otherbifunctional agents were later found to produce various types of DNAcross-links, and some of these agents continue to be mainstays of currenttherapy. The cellular pharmacologyofthese drugs wasStUdiedextensivelyduring the @970sand 1980s by means of DNA filter elution methodologyIn the course ofthese investigations, DNA topoisomerases were discoveredto be targets of anthracyclines and several other classes of anticancerdrugs. DNA cross-linkers and topolsomerase blockers have generallysimilar cytotoxic mechanisms, which depend on DNA damage detection,DNA repair, cell cycle arrest, and cell death by apoptosis. The molecularcontrol of these processes, involving oncogenes and tumor suppressorgenes, is being revealed by current research. Cancer cells often havedefects within these control systems, and these defects may confer selectivesensitivity to appropriately designed drug therapy. Panels of human tumor cell lines may serve to link the molecular defects with specific drugsensitivities. Such correlations could guide the selection of drugs fortherapy based on molecular diagnosis of individual tumors.

Introduction

The era of cytotoxic anticancer treatment is now just 50 years old,having begun with the discovery of the antitumoraction of bifunctional alkylating agents, some of which remain to this day among the

most useful anticancerdrugs. The key mechanistic feature of theseand related drugs is their ability to cross-link DNA. This somewhatpersonal review will trace our understandingof DNA cross-linkingagentsfromtheirdiscovery 50 yearsago to theirrole in the currenteraof molecular biology and will consider also the cell biologicallyrelated DNA topoisomerase blockers. Other historical details wereincluded in another recent review (1).

History

It is one of those paradoxes of our chaotic world that the origin ofcancer chemotherapy traces back to the use of poison gas in war (Fig.1A). As early as 1917, mustard gas was noted to suppress the bonemarrow(2), although its cytotoxic action was attributedto the production of hydrochloricacid in hypotheticalintracellularregions thatwere presumed to be hypersensitive to acidity (3â€”5).The bone marrow in human casualties showed suppression of granulocytic components, involving not merely local necrosis, but inhibited regeneration(6, 7). Additionally,thrombocytopeniawas noted in these early studies. Intravenous administration of small doses of mustard gas inanimals produced leukopenia and impaired antibody response, andthese effects were thoughtto resemblethe effects of ionizing radiation(8, 9).

Between the world wars, studies of mustard gas and of the trifunc

Received 9/16196;accepted 10/15/96.I Presented at the 87th Annual Meeting of the American Association for Cancer

Research, Apnl 24, 1996, Washington, D.C.2 To whom requests for reprints should be addressed. Phone: (301) 496-2769; Fax:

(301) 402-0752; E-mail: [email protected].

tional nitrogen mustard tri(2-chloroethyl)amine revealed an unusuallyselective inhibition of reproduction in yeast (relative to a lesserpotency of inhibition of respiration), and this selective inhibition ofreproduction was lost upon hydrolysis of the mustard (10). Thesignificance of this clue to an alkylation chemistry, however, was notyet appreciated. During this time period, mustard gas was appliedtopically to tumors or injected intratumorally, mainly in animal experiments, but a few clinical cases were included, and some localtumors were eradicated (1 lâ€”l3).@ Intravenous mustard gas inhibited

the growth, and even caused regression, of implanted tumors in rats(13).

Clinical antitumor trials with nitrogen mustard, however, werebegun only after a later military disaster during World War II, when

evidence pointedto a selective lymphotoxic action by mustardgas inhumans (14). The wartime studies of mustard gas and nitrogen musturd were shrouded in secrecy and were reported in summary after thewar (15â€”21). Gilman and Philips, in their landmark paper in 1946

(15), surmised correctly the alkylation chemistry of the sulfur andnitrogenmustardsand summarizedthe biological findings in experimental animals and humans. They cited the work of many researchers

in the United States and Great Britain whose wartime reports werestill classified and unavailable in the open literature. Evidence foractions on chromosomes was reportedin 1943 in documents of theChemical Board,Ministryof Supply [London;cited by Elmore et a!.(20)].Studiesof thereactionmechanismsof nitrogenmustardwerereported in 1946 by Joseph S. Fruton, Max Bergmann, Calvin Golumbic, and their coworkers in a series ofeight papers (J. Org. Chem., 11:518â€”591),and the alkylation mechanismwas defined (22).

The clue that led to the first extensive therapeutictrial againstlymphomas was the lymphotoxic effect noted in victims of the WorldWar II tragedy at the Italian port of Ban. The events at Ban were kepthushed up for many years but were eventually described in detail byInfield (14). The exposure to mustard gas on that occasion differedfrom the usual World War I experience in that many of the victimsreceived prolonged exposure over much of the body surface to lowconcentrations of the lipid-soluble alkylating agent dissolved in fueloil. It was the aftermath of a surprise German air attack that destroyeda large number of ships in the crowded harbor, including among theman American ship that was carrying mustard gas bombs. (The Alliesevidently brought in poison gas on the possibility that the other side,in desperation,mightuse such weapons.) Several factorscombinedtoyield evidence of a specific lymphotoxic action of mustard gas thatwas the basis for the clinical trials: the unusual nature of the exposure;

the improved pathology techniques; and, very significantly, the insight and devotion of the medical and research staff who investigatedthe event at the time (14).

Nitrogen mustards were easier to handle than mustard gas because

3 Adair and Bagg (1 1) state that the antitumor testing of mustard gas was suggested to

them in 1927 by Dr. JamesEwing. who hadbeen impressedby the peculiarand specificnature of mustard gas burns while serving with the United States Army Medical Museumduringthe war.

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HISTORYAND PROSPECTSOF DNA-TARGETEDCANCERTREATMENT

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they could be stored as stable crystalline hydrochiorides; upon neutralization, the nitrogen mustard free base had similar chemical andbiological properties to mustard gas (17, 24). Nitrogen mustards,therefore, were used in the first trials of systemic therapy. Immediately after the war, the first results with nitrogen mustards in thetreatment of neoplastic disease (chiefly lymphoma, leukemia, andallied conditions) were summarized in an Official Statement to themedical profession by Cornelius P. Rhoads as Chairman of a Committee of the National Research Council in New York (18). The fullpublished reports of the first treated patients soon followed (16, 17).Although tumors sometimes regressed (Fig. 1B), the regression wasonly temporary, and treatment was limited by hematological toxicity.Only hematological tumors responded. It was not clear whether nitrogen mustards were superior to radiation therapy, although radioresistant tumors sometimes responded to the new chemotherapy (17). It

is remarkable how many of the essential features of these drugs werediscerned correctly at this early date.

Many of the investigators who were involved in the nitrogenmustard studies during and immediately following World War H wenton to become leaders in cancer chemotherapy, hematology, and pharmacology: Alfred Gilman, Frederick S. Philips, Louis S. Goodman,Maxwell M. Wintrobe, William Dameshek, Cornelius P. Rhoads,Joseph H. Burchenal, C. Chester Stock, and David A. Karnofsky.

Since nitrogen mustard is a simple chemical structure, easily amenable to variation, hundreds of derivatives were tested, but the hope ofcurative therapy was unrealized. Nevertheless, some pharmacologicalimprovements were obtained in the form of cyclophosphamide, melphalan, and chlorambucil, which remain prominent in modem cancerchemotherapy. Attempts to progress by way of understanding ofmechanisms were limited by lack of basic knowledge and lack ofpowerful methodology.

Cross-Links between cDNA Strands

In the early search for chemical mutagens, mustard gas was testedbecause of observations that pointed to its interference with mitotic

activity in injured or hormonally stimulated tissues. It thus became thefirst known chemical that, like ionizing radiation, produced mutations

and chromosome damage (19). Mustard gas was presumed to damagethe genetic material, although the chemical nature of the genes wasnot yet established. The mutagenic properties of mustard gas werediscovered in 1942 but were not made public until after the war (19).Auerbach et al. (19) in 1947 inferred that the mustards â€œmightcombine with the materials composing the gene.â€•

Some of the early work yielded evidence that remains interestingeven now in the modern light of DNA damage and repair mechanisms.Mustard gas, unlike ionizing radiation, produced delayed mutations,as evidenced by mutant Drosophi!a males that produced mosaicoffspring (19). The chromosome breaks produced by nitrogen mustardmay occur preferentially in regions of heterochromatin (25), suggesting a selectivity of action that might still be profitably pursued.

The concept of cross-link formation (although not specifically ofDNA) emerged from the observation that the characteristic biologicalactions of the mustards required the presence of at least two alkylatinggroups in the molecule (23, 25, 26). In 1949, Goldacre et aL (23)envisioned that â€œthetwo [haloalkyll groups are required to permit themolecule to react at two distinct points, lying either on a single surfaceor fibre or, more especially, on two contiguous fibres.â€•Many nitrogenmustard derivatives were tested for their ability to prolong the survivalof mice bearing transplanted leukemia, but only those that had at leasttwo alkylating groups were active (27). The double-stranded structureof DNA and its way of replicating, as described by Watson and Crick3 years later, made DNA a natural candidate for cross-linking target.

An experimental test of the notion of DNA interstrand cross-linkingwas made possible by the concept of DNA helix-coil transition,developed by Marmur and Doty (28) and Doty et a!. (29) in 1959. Thehelix-coil transition (also called â€œDNAmeltingâ€•)was found to berapidly reversible, but only up to the end of the transition, at whichpoint the complementary strands were presumed to have separatedcompletely and could no longer reassociate rapidly. Separated strandscould, however, reassociate slowly with the concentration dependence

5534

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CH3Fig. 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.

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HISTORY AND PROSPECTS OF DNA-TARGETED CANCER TREATMENT

kinetics as a function of time exhibited a lag, consistent with therequirement for a sequence of two reactions, as would be expected inthe formation of a cross-link. Similar methodology revealed DNAinterstrand cross-link formation and repair in bacterial cells (31).

The site on DNA that is most readily alkylated was determined byBrookes and Lawley to be the guanine-N7 position (32, 33). Theseinvestigators also detected nitrogen mustard-linked guanine dimersthat could have come from interstrand or intrastrand cross-links.Several other classes of anticancer drugs also proved capable of DNAcross-linking, including mitomycin (34), cisplatin (35), and chloroethylnitrosoureas (36).

DNA Damage in Mammalian Cells

The fact that the reaction of a single nitrogen mustard moleculecould prevent the denaturation of an entire DNA molecule suggesteda way to measure interstrand cross-links in biological experiments. Incells treated with pharmacologically relevant drug doses, however, theextent of interstrand cross-linking was only on the order of 1 permillion bp, and DNA of this size could not be isolated by the usualmethods. To carry out DNA cross-linking studies in cells and tissues,therefore, novel methodology was needed.

A solution to this problem came out of an unexpected observationin 1971 while we were studying newly replicated DNA that wethought might be associated with structural components of the nucleusthat could be separated by filtration (37). When nuclear lysates weresubjected to mild controlled shearing and then dripped through membrane filters, we found newly replicated DNA to be selectively remined. The ratio of newly replicated relative to bulk DNA wasconsistently elevated in the filter-retained fraction and gradually returned to equivalency during a chase period. Thus, newly replicatedDNA appeared to be part of a detergent-resistant complex that couldbe retained on filters. The big surprise came when we used NaOH asthe lysis solution. Contrary to what we had seen with various detergent recipes, newly replicated DNA was now enriched in the flowthrough fraction of the lysate. We surmised that short nascent DNAsingle strands, released by alkali, could pass through filters, whereasintact mature DNA was retained. Using X-rays to generate singlestrand breaks, we confirmed that shortened DNA single-strand segments passed through filters selectively in alkali. Using a peristaltic

20'30'40'50'60'

Fig. 2. Evidenceof DNA interstrandcross.linking.Equilibriumsedimentationin CsC1of bacterial DNA treated with various concentrations of nitrogen mustard (HN2) and thenbriefly exposed to 0.02 NNaOH followed by neutralization. Native (N) and denatured (D)DNAfonneddistinctbandsthatcouldbequantitated.HN2wasseento inhibitthestrandseparation of a dose-dependent fraction of DNA molecules.

of bimolecular kinetics. The anticipated effect of an interstrand crosslink was that the linked strands would reassociate rapidly with uni

molecular kinetics. In 1961, I had the opportunity to work in PaulDoty's laboratory and to test this idea using the cutting-edge technology of the day: the analytical ultracentrifuge with UV optics. Briefexposure to 0.02 N NaOH normally denaturedall of the DNA (i.e.,converted all of the DNA to single strands). However, if the DNA waspretreated with nitrogen mustard, a fraction of the DNA molecules didnot denature (Fig. 2). The conversion of denaturable to undenaturableDNA exhibited first-order kinetics with respect to the amount ofnitrogen mustard bound to DNA, indicating that the reaction of asingle molecule of nitrogen mustard could prevent the strand separation of an entire DNA molecule of up to 30 kbp (30). Moreover, the

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IFig. 3. Kinetics of DNA alkaline elution (48). A,

cells labeled with â€˜4C.thymi&newere exposed tovarious doses of X-ray at 0'C then lysed withdetergent on filters, and the DNA was eluted atpHl2.0 (open symbols) or pHl2.8 (filled symbols).B, dependence of apparent elution rate constant onX-ray dose. Different symbols in B refer to 2 [odependent sets of experiments.

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HOURSOFELUT1ONFig. 5. DNA single-strand breaks during nucleotide excision repair in Uv-irradiated

human fibroblasts, as revealed by DNA alkaline elution. Open symbols, internal controlsshowing the elution of DNA from cells irradiated with a standard dose of X-rays. Filledsymbols:A, normal human fibroblasts, untreated; B, normal human fibroblasts 4 mm afterUv irradiation;C,xerodermapigmentosumcells(complementationgroupA),untreated;D, xeroderma pigmentosum cells 4 mm after UV irradiation. The result showed thatnormal cells but not xeroderma pigmentosum cells produce transient DNA single-strandbreaksduringnucleotideexcisionrepair.Thisexperimentwascarriedout by AlbertJ.Furnace, Jr., in our laboratory in 1975 (49).

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pump to control the flow of alkaline solution through the filter, wefound that the system could be made surprisingly sensitive and robustwith respect to the size of the DNA single-strand segments.

It was possible to adapt the DNA filter elution technique to avariety of DNA lesions (38â€”42). Assays were developed for single

strand breaks, double-strand breaks (43), base-free sites (44), interstrand cross-links, DNA-protein cross-links (45), and protein-associated DNA strand breaks (46, 47).

Alkaline elution of DNA single strands exhibited a simple loglinear behavior with an apparent rate constant that was proportional tosingle-strand break frequency (Fig. 3; Ref. 48). Moreover, the elutionrate was independent of the number of cells loaded (up to the point ofclogging the filter) and nearly independent of flow rate. This wassurprising in view of the high and variable viscosity in the filter poresthrough which the DNA was passing. Presumably, there are mutuallyopposing factors that tend to cancel each other out and thus give theobserved simple and robust behavior. After optimizing the parametersof the system, we soon had in our hands a method that could quantitatebiologically relevant levels of DNA single-strand breaks in cells andtissues.

From the dependence of alkaline elution kinetics on time and X-raydose and the fact that X-rays produce strand breaks that are distributedrandomly, we deduced the behavior of a homogeneous size populationof DNA single strands (42). The result seemed at first surprising,because the theoretical elution rate for a population of equal-sizestrands was a step function: the probability of release from the filteris constant up to a critical time, when all the strands have beenreleased (Fig. 4). If the strands are shorter, it takes less time to reachthe total elution point. Physically it would be as if the strands aregliding at a constant rate over a fulcrum, and the time at which a givenstrand is released depends on how that strand happened to be placedrelative to a fulcrum when the strand was initially deposited on thefilter (Fig. 4, inset A). Actually the situation must be more complex,as suggested by Fig. 4, inset B.

One of our first applications of the alkaline elution method wasto nucleotide excision repair. Single-strand break formation was anexpected part of the repair process, but it had not been clearlydemonstrated in mammalian cells. The high sensitivity of thealkaline elution method allowed this type of observation to be

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DNA-Protein Cross-Links

The UV repair study led in an unexpected direction. When wechecked whether the sensitivity of our strand break assay might havechanged during the repair process, we were surprised to find that theincreased elution caused by a standard dose of X-ray was reducedduring the repair period (50). We suspected that this might be due toDNA-protein cross-links, because UV was known to produce suchcross-links, and cell proteins might adsorb to the filters and preventthe elution of protein-linked DNA strands. We found that this wasindeed what was happening and, furthermore, that xeroderma cellswere incapable of repairing the UV-induced DNA-protein cross-links(50). This indicated that nucleotide excision repair acted not only onbase damage but also on DNA-protein cross-links.

We were able then to devise a very sensitive andquantitativeassay5536

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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 elutionrate would be constant up to the time when all the DNA has eluted, at which point theelution rate would fall to zero (42). Inset A, simple model of a DNA strand gliding at aconstant rate over the boundary between adjacent filter pores; inset B, a more realisticmodel of a DNA strand gliding between several filter pores.




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increased DNA alkaline elution with an agent that had been found toproduce DNA strand breaks by other techniques.

We knew that DNA-protein cross-links could obscure the detectionof DNA strand breaks, but we had no reason to believe that doxorubicin would produce DNA-protein cross-links. By that time, however,we had worked out the conditions to remove the effects of DNAprotein cross-links in the assay. When we applied deproteinizingconditions to doxorubicin, we were astounded by the results (46, 47).We now saw a classic alkaline elution profile indicative of randomlydistributed stand breaks; the pattern looked as if the cells had beenX-rayed. The DNA elution was log-linear, and the apparent rateconstant of elution was proportional to drug concentration over apharmacologically relevant dose range (Fig. 7).

That meant that doxorubicin did produce DNA strand breaks, but italso produced DNA-protein cross-links, and the latter completelyobscured the former in the nondeproteinized assay. For randomlydistributed DNA-protein cross-links to completely obscure the DNAstrand break signal, the DNA-protein cross-links would have to bepresent in large excess. Because our assays were quantitative, we wereable to determine the relative frequencies of the two types of DNAlesion. We found that the frequencies of the single-strand breaks andDNA-protein cross-links were equal within experimental error. Weconcluded, therefore, that the locations of the two types of lesioncould not be random (Fig. 7, top left). Rather, there must be oneDNA-protein cross-link within each DNA single-strand segment.There was only one plausible way for this to occur, which was that theproteins were linked consistently either to the 3' or the 5' termini ofthe breaks (Fig. 7, top right). This suggested that the linked proteinmight be an enzyme that produced the break. There was at the timeprecedent for this type of mechanism in J. C. Wang's nicking-closingactivity ofE. coli w protein (71), Blair and Helinski's DNA relaxationenzyme associated with colicinogenic plasmids (72), and Champouxand Dulbecco's DNA untwisting enzyme of mammalian cells (73,74), enzymes that we now call DNA topoisomerases. Several types of

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AFig. 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 ofnitrogen 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/breakparameter = 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 toindependentsetsof experiments.

for DNA-protein cross-links (45), and this was to become important inan unexpected way.

DNA Interstrand Cross-Links in Mammalian Cells

The alkaline elution rate of DNA single-strand segments fromX-irradiated cells could be reduced by either DNA-protein cross-linksor DNA interstrand cross-links. Because bifunctional alkylatingagents generally produce both types of lesions, a specific assay forinterstrand cross-links required a way to eliminate the effects of theDNA-protein cross-links. We soon learned how to prevent proteinadsorptionto the filters, andthis allowed us to devise a specific assayfor interstrand cross-links suitable for pharmacological studies inmammalian cells (Fig. 6; Ref. 39).

We applied this methodology to cell pharmacology studies of avariety of DNA cross-linkers, including nitrogen mustards (51â€”56),platinumcomplexes (53, 55â€”62),psoralen(63), andchloroethylmtrosoureas (51, 64). The latter compounds were particularly interestingbecause of their manner of producing cross-links slowly after alkylation at guathne-O@ positions and because of the inverse relationshipwe found between cross-linking and the activity of the guanine-06-alkyltransferase repair enzyme in different cell types (65). Drugsensitivity was dependent, in part, on the extent of cross-link formation (65â€”67).Further clinical development of new chloroethylatingagents was proposed (68â€”70).

Protein-associated DNA Strand Breaks: An Effect ofTopolsomerase-targeted Drugs

An unanticipated development occurred in 1978 in the course ofalkaline elution studies of doxorubicin. Because doxorubicin had beenreported to produce DNA strand breaks in alkaline sucrose gradientstudies, we anticipated no difficulty in demonstrating these breaks byalkaline elution. However, using treatments that should have producedDNA strandbreaks,we saw no increase in DNA elution whatsoever.This was puzzling because it was the first time we failed to find

5537



HISTORY AND PROSPECIS OF DNA-TARGETED CANCER TREATMENT

Fig. 7. Protein-associated DNA strand breaks produced in mouse leukemia cells by doxorubicin (@iconcentration as indicated; treatment time, I h). Deproteinizing alkaline elution conditions were used. Without deproteinization, no increase in elution occurred.The DNA of treated cells was prelabeled with â€˜4C-thymidine. An intemal standard of I3Hlthymidine-labeled cells irradiated with 300 rad was added, and thisserved as a normalized time scale on the horizontalaxis. This experiment was carried out by Warren E.Ross in our laboratory in 1978(47). Top left, randomlydistributed single-strand breaks and DNA-proteincross-links. Top right, proteins linked consistently toone terminus at each single-strand break.

DNA intercalating drugs were found to produce protein-associatedDNA strand breaks, as we then called these lesions, and were therefore hypothesized to be topoisomerase blockers (47, 75â€”78).

Support for the idea that protein-associated DNA strand breaks are

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Fig. 8. Kinetics of protein-associatedbreak formation and reversal in mammaliancells.ThisexperimentwascarriedoutbyLeonardA.Zwellinginourlaboratoryin 1981(75)andused 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 (202,203).]Cellswereexposedto theindicatedconcentrationsofdnigfor60 miii,at whichtimethe dnig was washed away and the cells were incubated further in the absence of dnig. Atvarious times, aliquots of cells were subjected to alkaline elution under deproteinizingconditionsto determinesingle-strandbreak frequencies.Under nondeproieinizingconditions,there was no increase in DNA alkaline elution (data not shown) because of the presence ofcovalent DNA-protein complexes. Assays for DNA-protein cross-links (data not shown)indicated a nearly 1:1 ratio of DNA-protein cross-links and single-strand breaks.

enzyme induced came from quantitative studies in cells. Severalobservations were consistent with an enzymatic origin (75): (a) afteraddition of drug, the lesion frequencies climbed to a steady-state levelthat was dependent on drug concentration (Fig. 8); (b) the level oflesions showed saturation kinetics with increasing drug concentration,indicating that there was a limiting factor governing the number oflesions that were formed; (c) the lesions reversed rapidly upon drugremoval; and (cOboth the formation and the reversal of lesions werestrongly temperature dependent. In all of these experiments, the strandbreaks and DNA-protein cross-links appeared and disappeared together, supporting the idea that they were different aspects of the samelesions. Even more telling, the formation and reversal of breaks wasdemonstrable in isolated cell nuclei without AlP, showing that thereversal was not a DNA repair process (79â€”82).

In attempting to identify the responsible enzyme, we found that theactivity could be extracted from isolated nuclei with 0.35 MNaC1 andthat the activity could be restored by adding the extract back to theisolated nuclei (83). Using this assay, the extract was fractionated toisolate the active component. Specific identification of the enzyme,however, had to await the characterization of topoisomerase II byLeroy Liu et a!. (84, 85), and it was in Liu's laboratory that drugeffects on the purified enzyme were first demonstrated (86â€”88).Weverified that the component responsible for the drug effects in thefilter assays was in fact topoisomeraseH (89).

In addition to DNA intercalators, the epipodophyllotoxin derivatives etopside (VP16) and teniposide (VM26), which are not strongDNA binders,were found to produceprotein-associatedDNA strandbreaks and were shown to be topoisomerase-targeted drugs (90â€”94).It is instructive that these chemical modifications of the naturalproduct, podophyllotoxin, retain the cytotoxic potency of the parentbut change the target from tubulin to topoisomerase H (95â€”97);naturesometimes exhibits coherence in unexpected ways.

Whereas topoisomerase H is inhibited by a wide variety of corn

TIME FOLLOWINGDRUG ADDITION (minutes)

5538

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Fig. 9. Stacking model of drug-stabilized topoisomeraseH-DNAcomplexes.Basesequencepreferencefor the locationof the siteson the DNAdiffersfordifferentdrugclasses.Fordoxorubicin and related anthracycines, the strongest base preference is for an A at the immediately upstream flank of theDNA strand break, as shown.Ellipticinesprefer a T, andetoposides prefer a C at this position. Amsacrine prefers an A attheimmediatelydownstreamflankofthebreak.TopoisomeraseH is a homodimer conferring diadic symmetry to the complexin which there can be a pair of single-strand breaks separated bya 5' overlap of 4 bp. To simplify the diagram, the two monomers of a topoisomerase homodimer are shown as separatecircles. Drug molecules are represented by filled rectangles.

by Yi Fan et a!. in our laboratory4 has indicated recently that astructure with camptothecin stacked against a base pair at a topoisomerase I cleavage site is in accord with a variety of experimentalevidence regarding structure-activity relationships (125), directinteraction between camptothecin derivatives and DNA purines,especially at the cleavage site (126, 127), and drug-resistant topoisomerase I mutations (1 12).

Toward Better Drugs through MOdified Mechanisms

After 50 years, some of the most useful anticancer drugs stillinclude DNA cross-linking agents, such as cyclophosphamide andcisplatin, and topoisomerase blockers, such as doxorubicin and camptothecin derivatives. One path toward improved drugs would bethrough chemical structure modification based on knowledge ofchemical mechanisms. It may be asked, for example, whether thedrugs react in a DNA sequence-dependent manner, whether drugs of

a given class differ in their preferred DNA reaction sites, and, if so,whether such differences have consequences relevant to therapy.When modified drugs show major alterations in mechanism in theabsence of clear information on the biological consequences of suchalterations, it may be justified to consider the modified drug as if itwere a new chemical structure and to move toward clinical trial of themodified drug in comparison with its parent.

Nitrogen mustards have characteristic nucleotide sequence preferences for DNA alkylation (128â€”131),and topoisomerase blockers, asalready discussed, also vary in DNA site preferences. However, weknow very little about how such differences translate into effects oncells. Alkylating agents react with many biomolecules other thanDNA. Because DNA is the important target, side effects might bereduced by minimizing the non-DNA reactions. DNA targeting ofnitrogen mustards has been studied in compounds containing anacridine moiety capable of DNA intercalation (131, 132). Some ofthese compounds have unusual DNA sequence preferences, and in

4 Y. Fan, Y. Pommier, K. W. Kohn, and J. N. Weinstein. Molecular modeling studies

of the DNA-topoisomerase ternary complex with camptothecin, submitted for publication.

pounds, topoisomerase I thus far has a relatively narrow range ofspecific inhibitors (98, 99). Chief among these is camptothecin, a

fascinating natural product the discovery, chemical structure, andstructure-activity relationships of which were described by MonroeWall and Mansukh Wani in the Cain Memorial Award Lecture in1994 (100). The first observations on the effects of camptothecin onDNA were actuallymade by Susan Horwitz(101), who presentedtheCain Memorial Award Lecture in 1992. Camptothecin was shown toblock purified topoisomerase I specifically (102, 103) and to produceprotein-associated DNA strand breaks in cells (104). The role oftopoisomerases as drug targets in cells was confirmed by the isolationand characterization of drug-resistant mutant forms of topoisomeraseI or H from drug-resistantcells (105â€”112).

The different types of topoisomerase H blockers were found tostabilize DNA-cleavable complexes at different sites in DNA, dependent in parton DNA sequence (113, 114). The location of the blockedcomplexes on DNA in reconstituted chromatin depended in part onnucleosorne positioning (115). In cells, topoisomerase H DNA cleavage sites were found to occur frequently in regions of DNA that arenuclear matrix association regions (1 16) and in the c-myc gene whenit is actively transcribed (117, 118). Camptothecin-trapped topoisomerase I complexes in vivo tend to occur in transcriptionally activeregions (119) and in intemucleosomal linker regions (120).

Analysis of the nucleotide sequence contexts at sites of druginduced topoisomerase H cleavage complexes in purified DNA suggested a structural model for the complexes (121). The base pairs thatmost influenced site selection were those that were situated on theimmediate flank of a cleavage site. Depending on the drug, this basepair could be on the 5' or the 3' flank, and the base preference differed

for different drug classes (121 , 122). This suggested a model of thetrap@ complex in which the drug molecule stacks against a flankingbase pair at the cleavage site (Fig. 9; Ref. 121).

Similarly, we found for the interaction of camptothecin withtopoisomerase I that the strongest sequence preference was for thebases immediately flanking the cleavage site (123, 124). Thestacking model can therefore apply also to the camptothecintopoisomerase I trapped complexes. A molecular modeling study

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Ellipt icine VM26



9


some cases, the monofunctional alkylating derivatives are active,suggesting mechanistic differences worthy of clinical investigation.

The relationship between DNA site preferences and cytotoxicity fortopoisomerase-targeted drugs is also unknown. In some cell lines, thepotency of these drugs correlates with DNA and/or RNA syntheses(133â€”135),but it is not know how this relates to the genomic sites ofthe lesions.

Extensive structure-activity data on camptothecins have given acoherent picture that may help in the development of improvedversions of these drugs (125, 136, 137). Ability to block topoisomerase I was closely correlated with antitumor activity. The features ofthe molecule that are essential for activity include the a-hydroxylactone structure of ring E and the steric configuration of the asymmetric carbon on this ring (position 20; Fig. 10). Positions 9 and 10 onring A can be substituted without loss of activity, whereas substitutionof position 12 on the same ring destroys activity. This suggests thatthe region of the molecule near position 12 is juxtaposed against theDNA or topoisomerase in the complex, whereas the other side of themolecule, in the vicinity of positions 9 and 10 in ring A and position7 in ring B, is accessible to solvent. In searching for improvedcamptothecin derivatives, substitutions can therefore be made at positions 7, 9, 10, and sometimes I 1. Several such derivatives are inclinical use or under investigation (138).

What basis in mechanism might there be for improved activity ofsuch derivatives? One possibility emerged from the current partialunderstanding of how camptothecin produces potentially lethal lesions in cells. When camptothecin forms a complex with DNA andtopoisomerase I, one strand of the DNA helix is cleaved, and theenzyme is covalently bound via a tyrosine residue to a 3'-phosphateterminus. The drug may stabilize this cleaved complex, in part, byforming a labile covalent bond between an opened lactone ring (ringE) and a nucleophilic group on the enzyme (98, 139, 140). Covalencynotwithstanding, the complex can dissociate within minutes to re-formundamaged DNA. A potentially lethal lesion can result if a cleavedcomplex is encountered by a moving replication fork (141â€”143),which could have the consequence of producing a DNA double-strandbreak (Ref. 144; Fig. 11). Such lesions are considered to be onlypotentially lethal, because they could be subject to repair. How thenmight structure modification enhance the production of such lesions?A key parameter is the lifetime of the drug-DNA-topoisomerasecomplex: the longer its lifetime, the greater the probability of a fatalinteraction with a genomic event, such as a moving replication fork ortranscription process (135, 142, 145). In support of this picture,camptothecin derivatives were found to differ in the rate of reversibility of their complexes, and cytotoxic potency was found to increase with increasing lifetime of the complexes (146). With thisconceptual underpinning, camptothecin derivatives were preparedwith an alkylating substituent in the 7 position, which could then react

.5'

â€˜I

7

10

11

Et

HO

0Fig. 10. Structure of camptothecin. 5 M. Valenti, K. W. Kohn, and Y. Pommier, unpublished observations.

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camptothecin

@ ReplIcatIoncomplex

Fig.11.Howacollisionbetweena movingreplicationforkanda camptothecin-trappedtopoisomerase I complex may result in an irretrievable DNA lesion.

covalently with the enzyme or the DNA to make the complexes nearlyirreversible (127). These derivatives do form nearly irreversible cornplexes, and they have enhanced cytotoxic potency, which, unlike thatof other camptothecins, is not reversed by DNA synthesis inhibitors.5Clinical trials will be needed to determine whether enhanced cytotoxicpotency by this mechanism can translate into improved therapeuticresults.

Toward Better Therapy through Cell Biology

Although we know a great deal about the chemical lesions produced by anticancer drugs and about their repair, we still do not knowmuch about the origin of the antitumor activity: we do not know howcancer cells may be killed selectively or why the drugs work in somecases and not in others. The emerging detailed knowledge of how cellproliferation, differentiation, and apoptosis are regulated may opennew possibilities for therapy.

The physiological responses of cells to a variety of DNA-damagingdrugs or topoisomerase blockers are remarkably similar in that treatedcells tend to arrest at certain points in the cell cycle and, in somecases, to undergo apoptotic death. The cell cycle arrest generally is nota manifestation of drug toxicity. Rather, it is a protective mechanismofcell cycle checkpoints in which the initiation of one cell cycle eventis inhibited while another is in progress (147â€”152).

In this context, a cell cycle checkpoint response to DNA damage isindicated when there is evidence that (a) treated cells tend to arrest orbecome delayed at a specific point in the cell cycle; (b) the arrest canbe prevented by adding an inhibitor; and (c) prevention of arrestincreases cytotoxicity.

Thus, a (12 checkpoint is indicated by the findings that (a) tumorcells that have sustained DNA damage often arrest in G2 (or very latein S); (b) this arrest can be prevented by caffeine, pentoxifylline orcertain protein kinase inhibitors which are not by themselves cytotoxic; 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 Mtransition in drug-treated or irradiated cells suggests a system ofmutually interacting kinases and phosphatases (Fig. 12; Refs. 150,152, 159â€”164).An interesting feature of this system is that it appearsto contain positive feedback loops, such that the components mayhave switch-like behavior and may not constitute a linear causal




These opposing actions of p53 can account for the conflictingresults in different cell systems that have been reported on the effectof p53 on drug sensitivities (157, 173â€”176).Cells that are inherentlyapoptosis competent would be expected to exhibit reduced sensitivityto DNA damage when p53 is inactivated by mutation. Lymphomacells, for example, generally undergo apoptosis very readily. Thus,most p53-mutant Burkitt's lymphoma cell lines were found to havereduced sensitivities to ionizing radiation, the DNA cross-linkingagents nitrogen mustard and cisplatin, and the topoisomerase inhibitoretoposide (179).

Some carcinoma cells, on the other hand, have weak or nonexistentcapabilities to undergo apoptosis, even when they have normal p53function. In such cases, cells can have increased sensitivity to DNAcross-linking agents when p53 function is disrupted (157). Withoutp53 function, treated cells then do not arrest in G1, but proceedthrough S and arrest in G2. Survival then becomes increasingly

dependent on an intact G2 checkpoint. When the G2 checkpoint isinhibited (e.g., by means of pentoxifylline or by means of a staurosporine derivative, UCN-Ol), survival is reduced further, and theselective toxicity against p53 mutant cells is increased (157, 158).

Another confounding factor in p53 disruption is chromosome instability, which arises in cells that cycle unchecked despite persistentDNA damage (183). Chromosome instability increases the heterogeneity of cell populations and favors the selection of rapidly growing ordrug-resistant malignant cells. Although p53 represents a well-definedmolecular and functional distinction between normal tissues and certamtumors,theutilizationofsuchdifferencestoselectivelyeradicatethese tumors by means of chemotherapy is a complex problem requiring a detailed understanding of integrated regulatory functions.

p53 affects the transcription of several genes that are believed tomediate its diverse cell biological effects (184, 185). Included amongthese are p2lwafl/cipl and gadd45, which inhibit the G1-S transitionand probably also S phase itself (186â€”188), and bax and bcl-2 thataffect the threshold for apoptosis ( 189â€”191). The logic of this systemmay be as follows. Increased levels of active p53 protein lower thethreshold for apoptosis in all cells that are inherently competent tocarry out the process. By increasing the transcription of p2lwafl/cipl,which blocks cyclin-dependent kinase cdk2, p53 at the same timecauses E2F activities to fall. Because E2F can cooperate with p53 toenhance apoptosis (192), the reduced E2F activity could prevent cellsfrom undergoing apoptosis despite the lowered threshold. Thus,Polyak et a!. (178) have recently shown that disruption of p2lwafl/cipl can convert the p53-induced response from@ arrest to apoptosis, indicating that p2lwafl/cipl can mediate cell cycle arrest andprotect against apoptosis. The functional value of this logic could bethat cells that inappropriately elevate E2F levels through expression ofoncogenes or viral genes would be eliminated. [As a countermeasure,some oncogenic viruses synthesize products that disrupt p53 function(193).]

Studies on a Panel of Human Tumor Cell Lines

Detailed knowledge at the level of molecular and cellular biologymay not immediately lend itself to clinical application. One formulation of the problem is that malignancy is driven by altered expressionof particular gene products and that such alterations could make cellsselectively sensitive to particular cytotoxic therapies. The objective isto develop cytotoxic therapies that would be tailored to tumors ofcertain types defined by their molecular characteristics. Some of theimportant molecular changes in common cancers may be preserved inappropriately chosen cell lines. The molecular alterations in humantumor cell lines could then serve to link preclinical and clinical

Mltoticevents

Fig. 12. Integrated switch model of the G2 to M phase transition (152). Mitotic eventsare stimulated by the hypophosphorylated cyclin B-cdc2 complex, which is the activeform of the kinase. During S or 02 phaseScells accumulate this kinase in an inactive formin which the cdc2 component is hyperphosphorylated. The conversion of inactive to activeform is accomplished by a phosphatase, cdc25C, the activity of which is enhanced byhyperphosphorylation, this phosphorylation being carried out by cydlin B-cdc2. Thus wehave a positive feedback loop that can have switch-like behavior. A small initial stimuluscan activate the switch. The switch is kept in check by ongoing DNA replication or repair,so that when these processes are completed, the switch is thrown. How the switch iscontrolled is, however, unknown. One controlling component is the weal kinase, whichtends to keep cyclin B-cdc2 hyperphosphorylated and inactive.

sequence of events (152). The inputs which control the switch and thecomponents that sense the presence of DNA damage however remainto be elucidated.

In early studies, arrest in G2 was the most prominent effect, becausethe tumor cells studied tended to be defective in tumor suppressor p53function. More recent studies in cells having intact p53 function showthat G@ arrest (arrest prior to the G1 to S transition) also is animportant feature of drug responses (165, 166).

Much of the complex regulatory network controlling the G1 to Stransition has now been revealed, including its component cyclins,protein kinases and phosphatases, kinase inhibitor proteins, transcription factors and their inhibitors, and promoter elements of genes forsome of these components (Fig. 13; Refs. 164, 167â€”169).It will bechallenging to integrate the vast amount of information accumulatingabout various parts of the network. Therapeutic implications currentlycenter on p53, which is defective in about one-half of human cancers(170, 171), and on the pathway involving cyclin D, tumor suppressorprotein p16, and pRb,6 one or another of which is altered in mostclinical tumors (Refs. 152, 157, 172â€”176;Fig. 13).

p53 and other regulatory components that control the G1 checkpointare also involved in the control of cell death by apoptosis (177, 178).

Because p53 is so often defective in clinical cancer (171), the effectsof p53 on drug sensitivities have been of intense recent interest. Thefunctions of p53, however, are now seen to be multiple and complex(177), and some of its different actions affect drug sensitivity inopposing directions (Refs. 157 and 179; Table 1). When p53 functionis disrupted, DNA damage does not cause cells to arrest in G1 (180),and DNA repair capability is reduced (157, 181). The enhancement ofDNA repair by p53 may be mediated in part through p53-inducedtranscription of GADD45 (182). The effects of p53 disruption indisturbing cell cycle arrest and DNA repair tend to reduce cellsurvival. On the other hand, the loss of the role of p53 in apoptosis

would tend to increase cell survival.

6The abbreviations used are: pRb, retinoblastoma protein; NCI, National CancerInstitute.

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 thisregulatory 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, dictatingeither 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 theprotein. 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. Thecyclin-cdk complexes hyperphosphorylate, and thereby block, the function of pRb. pRb, when not hyperphosphorylated, binds to and blocks E2F transcription factors. E2F stimulatesmany 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-Bcl2couple (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 essentiallyof (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 infuture 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

Impaired cell cycle checkpoint functionsImpaired DNA repairChromosome instability leading to lethal genotypes

Increased cell survival

Reduced apoptosisChromosome instability leading to selection of malignant

or drug-resistant cell populations

development of therapies for specific tumors based on moleculardiagnosis.

The NC! cell screen has generated a large amount of data on cellgrowth inhibition by many thousands of compounds tested against apanel of 60 human tumor cell lines representing a variety of tissues oforigin ( 194). Individual cell lines differ greatly in their drug responses. However, the pattern of responses of the set of 60 cell linesis strongly correlated with drug action mechanisms (195â€”197).Theresponse patterns, considered as 60-dimensional vectors, have beenanalyzed by means of a variety of statistical tools, including clusteranalysis, neural networks, and principle component regression. Theseanalyses have been done for large data sets of >40,000 compoundsand for a subset of compounds for which probable mechanisms ofaction could be inferred from experimental evidence or chemicalstructure. Cluster analysis over large sets of tested compounds (198,199) corroborates the observation that compounds related by structure

or by action mechanism usually group together. The coherence ofthese groupings is remarkable in view of the huge size of the data setand demonstrates the informational power inherent in the behaviorpatterns of a large panel of cell lines.

To test the ability of the cell screen data to predict mechanism ofaction, a set of 123 standard drugs were assigned probable mechanisms (196). The assignments were made without prior knowledge ofthe cell screen results. A neural network, trained on a subset of drugs,was then able to correctly assign the remaining drugs in nearly allcases. The strong relationship between mechanism of action and cellscreen response pattern was verified by other statistical methods(198â€”200).

The informational power of patterns encompassing sets of cell linesis now being extended to include molecular and cellular biologycharacteristics. The 60 cell lines of the NCI screen have been or arebeing characterized with respect to a variety of oncogenes, tumorsuppressor genes, and cell cycle responses. A variety of correlationsare being examined to determine how individual cell lines are relatedto each other and how molecular characteristics relate to drug responses. Assuming that the cell lines retain some of the molecularcharacteristics that are common in clinical tumors, these characteris

tics could guide clinical trials based on molecular diagnosis of thetumors. Thus drugs that act selectively against cell lines having certainmolecular characteristics could be tested in patients stratified withrespect to a molecular diagnosis of their tumors.

One phase of the cell line characterization studies has focused on

p53.7 Of 58 cell lines analyzed, 39 were found to be p53 mutant.Most, but not all, of the mutant lines showed elevated basal levels ofp53, and their responses to ionizing radiation were deficient withrespect to G@arrest and with respect to inducibility of p2lwafl/cipl,gadd45, and mdm2 transcription. Strong G1 arrest in response toionizing radiation occurred only in cell lines that exhibited stronginduction of p2lwafl/cipl mRNA. The p53 mutant lines exhibitedstatistically less growth inhibition than p53-normal lines to DNAcross-linking agents, topoisomerase inhibitors, and antimetabolites(l99).@ Apoptosis may therefore be an important factor in drug responses in the NC! screening assay. Because there was considerableoverlap in the sensitivity distributions, factors in addition to p53contribute to the growth inhibition, some of which may be identifiedas this work progresses. Antimitotic agents, such as taxol and vincristine, on the other hand, did not show any such dependence on p53,suggesting that these agents may have an advantage over other standard drugs in the treatment of p53-mutant tumors (l99).@

Postscript

The painting by Edouard Manet featured on the cover of this issueof Cancer Research (and Fig. 14) could be viewed in relation to thehalf-century quest for anticancer drugs. The painting, â€œGareSt.Lazare,â€•was brought to my attention in a recent Sunday afternoonlecture at the National Gallery of Art presented by the art historianJuliet Wilson-Bareau, to whom I am also indebted for an illuminatingresponse to my proposed use of the picture. Her lecture focused onâ€œthemystery behind the steamâ€•in the painting. The steam is billowing

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.Characterization of the p53 tumor suppressor pathway in cell lines of the NC! anticancerdrugscreenandrelationshipswithchemosensitivity,manuscriptin preparation.

5542




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

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6. Krumbhaar, E. B. Role of the blood and the hone marrow in certain forms of gaspoisoning. J. Am. Med. Assoc., 72: 39â€”41, 1919.

7. Knimbhaar, E. B., and Krumbhaar, H. D. The blood and bone marrow in YellowCross gas (mustard gas) poisoning. J. Med. Res., 40: 497â€”506,1919.

8. Pappenheimer, A. M., and Vance, M. The effects of intravenous injections ofdichioroethylsulfide in rabbits, with special reference to its leukotoxic action. J. Exp.Med., 31: 71â€”94,1920.

9. Hektoen, L., and Corper, H. J. The effects of mustard gas (dichlorethyl suiphid) onantibody formation. J. Infect. Dis., 28: 279â€”285, 1921.

10. Magne, H., and Remy, P. Les actions toxiques elementaires de quelques corpsgenerateurs d'acide chlorhydrique par hydrolyse. Bull. Soc. Chim. Biol., 19: 1092â€”1104, 1937.

I 1. Adair, F. E., and Bagg, H. J. Experimental and clinical studies on the treatment ofcancer by dichlorethylsuiphide (mustard gas). Ann. Surg., 93: 190â€”199,1931.

12. Kabelik, J. Experiences chimiotherapiques sur Ic carcinome d'Ehrlich de Ia souris.Compt. Rend. Soc. Biol., 110: 394â€”397,1932.

13. Visser, J., and de Vos, J. J. Over den invloed van mosterdgas op kwaadaardigegezwellen. Geneesk. Tijdschr. Nederlandsch-Indie., 75: 1363â€”1380,1935.

14. Infield, G. B. Disaster at Bad. New York: Macmillan, 1971.15. Gilman, A., and Philips, F. S. The biological actions and therapeutic applications of

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16. Goodman, L. S., Wintrobe, M. M., Dameshek, W., Goodman, M. J., and Oilman, A.Nitrogen mustard therapy. Use of methyl-bis(beta-chloroethylamine hydrochlorideand tris(beta-chlomethyl)amine hydrochloride for Hodgkin's disease, lymphosarcoma, leukemia and certain allied and miscellaneous disorders. J. Am. Med. Assoc.,132:126â€”132,1946.

17. Wilkinson, J. F., and Fletcher, F. Effects of j3-chlorethylamine hydrochlorides inleukemia, Hodgkin's disease, and polycythemia vera. Lancet, 1946: 540â€”545,1947.

18. Rhodes, C. P. Nitrogen mustards in the treatment of neoplastic disease. J. Am. Med.Assoc., 131: 656â€”658, 1946.

19. Auerbach, C., Robson, J. M., and Carr, J. G. The chemical production of mutations.Science (Washington DC), 105: 243â€”247,1947.

20. Elmore, D. T., Gulland, J. M., Jordan, D. 0., and Taylor, H. F. W. The reaction ofnucleic acids with mustard gas. Biochem. J., 42: 308â€”315, 1948.

21. Beanie, J. W., and Howell, L. H. Biological actions and therapeutic applications ofnitrogen mustard. Q. J. Med., New Series., 23: 231â€”254,1954.

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27. Burchenal, J. H., and Riley, J. B. Nitrogen mustards: the relationship betweenchemical structure and chemotherapeutic activity. Cancer Res., 9: 553â€”554,1949.

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Iâ€¢i@;14. 1tiniiri@ h\ II@U@it4i \1@nct. (â€˜i @!.Ii:', 1 ( I, @i@i1 Ii I pcl@@@)kt11pt@@rtl@@i1@\@lLI\l@L'l@i1@@ftl1L@@@ )L' L\L@!l@l.@ \\Itll

@ @i!,i;-I@:@/ . h@. I@J@uctJ \1iiict ( @ti t@ r@i@c 11c@crnc\ I 11 F1ICfli@\ t

n@tlicr. 1t!Nilic \\ l1t\c11lc\cL@ l)U Rtid 1 @IrtI\@c@.\ttiritI ( IIICF\ I \it.\\ t@hiiictn. D@( . H@ @. ii n @lH\t

up from locomotives that are passing below into and out of the Parisrailway station, which is just to the right beyond the bridge. In theupper left-hand corner, one can make out Manet's studio window. Thewoman seated on the ledge, with the studio across the tracks of thebroad railway cutting just behind her, served as model in many ofManet's paintings. The child, in a white dress with her back toward us,is looking through the steam in the direction of the studio, thus unitingthe two figures whose relationship to each other so puzzled the criticsof the time. The billows of steam can be likened to smoke and dust inother Manet paintings, most notably the smoke from the guns in hispainting, â€œTheExecution of the Emperor Maximilian.â€•The child,with her left arm extended, can be referenced to a symbol of hope inother art works, and thus could be looking through the steam in thehope of seeing a better world beyond the smokes of war. â€œGareSt.

Lazareâ€•was painted in 1873, shortly after the Franco-Prussian War,during which Paris was invaded. The era of cancer chemotherapybegan out of the gases of war; now, as the mists of ignorance clear atlast, we may look for better therapy through basic science.

Acknowledgments

It is a greatpleasureto acknowledgemy colleagues who hadmajorpartsinthe studies from my laboratory. Regina A. Ewig worked with me during thediscovery and development of the DNA alkaline elution technique. Majorcontributions to the variations and applications of the technique were made byLeonard C. Erickson, Albert J. Fomace, Jr., Matthews 0. Bradley, Warren E.Ross, andLeonardA. Zwelling.Manyof the earlytopoisomerasestudieswereconducted by Warren Ross, Leonard Zwelling, Donna Kerrigan, Jan Filipski,and John K. Minford and by Yves Pommier, who is leading the current studiesrelated to topoisomerase inhibitors in our laboratory. Most of the work onchloroethylnitrosoureas was done by Leonard Erickson, Neil Gibson, and JohnA. Hartley.The studies of DNA sequence-dependentalkylationincludedtheefforts of William B. Mattes, John Hartley, and Ann Orr. The cell cycle workwas and is continumg to be spearheaded by Patrick M. O'Connor. Theinformation-intensive approach to human tumor cell line panels and theirmolecular pharmacology is being developed under the leadership of John N.Weinstein.

The book by G. B. Infield about the Disasterat Bail was broughtto myattentionby Dr. ShaiIzraeli,who hadreviewedthebook fortheIsraelMedicalCorps Journal (22: 17â€”19,1987).

I thankGailFeigenbaum,Curatorof AcademicPrograms,NationalGallery5543




36. Kohn, K. W. Interstrand cross-linking of DNA by l,3-bis(2-chloroethyl)-l-nitrosourea and other l-(2-haloethyl)-l-nitrosoureas. Cancer Res., 37: 1450â€”1454,1977.

37. Kohn, K. W., and Ewig, R. A. Alkaline elution analysis, a new approach to the studyof DNA single-strand interruptions in cells. Cancer Res., 33: 1849â€”1853,1973.

38. Kohn, K. W. DNA as a target in cancer chemotherapy: measurement of macromolecular DNA damage produced in mammalian cells by anticancer agents andcarcinogens. Methods Cancer Res., 16: 291â€”345,1979.

39. Kohn, K. W., Ewig, R. A., Erickson, L. C., and Zwelling, L. A. Measurement ofstrand breaks and crosslinks in DNA by alkaline elution. In: E. C. Friedberg and P.C.Hanawalt(eds.),DNARepair:ALaboratoryManualofResearchTechniques,pp.379â€”401. New York: Marcel Dekker, 1981.

40. Kuhn, K. W. The significance of DNA damage assays in toxicity and carcinogenicity assessment. Ann. NY Acad. Sci., 407: 106â€”118,1983.

41. Kohn, K. W. DNA filter elution methods in anticancer drug development. CancerTreat Res., 36: 3â€”38,1987.

42. Kohn, K. W. Principles and practice of DNA filter elution. Pharmacol. Ther., 49:55â€”77,1991.

43. Bradley, M. 0., and Kohn, K. â€˜A'.X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. NucleicAcids Res., 7: 793â€”804,1979.

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