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Experimental Therapeutics |
Molecular Pharmacology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Contrary to current clinical practice, there is no reason to expect that the type of lesion creating genetic instability would necessarily correlate with the tissue of origin of a tumor (3) . Indeed, lesions creating a genetic instability (for example, mismatch repair defects) occur in a wide variety of tumors from different tissues of origin (1) . We propose, therefore, that identification of the particular mutations creating genetic instability in a tumor will be more predictive of the chemotherapeutic sensitivity of that tumor than will be its tissue of origin. In cases in which chemotherapy is effective, a conjunction of chemotherapeutic agent and tumor vulnerability may have been achieved fortuitously. Additionally, by using model organisms, defects that lead to genetic instability could be used to identify new selective agents.
For this approach to work, one would need to correlate vulnerability to chemotherapeutic agents with particular DNA damage response defects. Here, we used yeast cells with defined genetic defects in DNA damage response elements to see whether currently used chemotherapeutic agents have specificity for individual DNA damage response defects. The agents for which such a specificity exists are candidates for correlating effectiveness in human cancer with defects in DNA damage response elements in those same tumors.
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MATERIALS AND METHODS |
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), yMP10691
(rad14::HIS3), SP50248
(mlh1::TRP1), yMP11082
(pms1::LEU2), yMP10467
(mag1::URA3), yMP10590
(apn1::LEU2), yMP10612
(mgt1::LEU2), 9170-Tx1a
(rad6::LEU2), yMP10425
(rad18::LEU2), SP50280
(rev1::LEU2), yM10382
(rev3::LEU2), yMP11406
(rad50::hisG), yMP10383
(rad51::LEU2), yMP10630 (rad52-1),
yMP10535 (rad9::LEU2), yMP10485
(rad17::LEU2), yMP10913 (mec1-1),
yMP10605 (mec2-1), SP50262
(mad1::LEU2), yMP11372
(mad3::LEU2), and SP50265
(sgs1::LEU2). For treatment with pentostatin, all
strains were as above except for SP50646
(mgt1::kan). All strains are of the genotype
MATa ade2 ade3–130 leu2 trp1 ura3–52 cyh2
SCR::URA3, except for yMP10485
(rad17::LEU2) and yMP10636 (rad1),
which are CYH2+; yMP10630
(rad52-1) and yMP11406 (rad50::hisG),
which are MAT
; and SP50646
(mgt1::kan), which is MAT and lacks
SCR::URA3. SCR::URA3 is a cassette
consisting of two halves of ADE3 separated by URA3 for
measuring sister chromatid recombination (4)
. The alleles
rev1::LEU2, mad1::LEU2,
sgs1::LEU2, and mgt1::kan
were generated by PCR fragment-directed gene replacement
(5)
using plasmids pJJ252 or pJJ250 (6)
for
amplification of the LEU2 marker gene and pUCkanMX
(7)
for amplification of the kan gene, and they
generally lack greater than 90% of the open reading frame. Strains
harboring the PCR fragment-directed gene replacements
apn1::LEU2 and mad3::LEU2
were obtained from Amanda Paulovich, and mlh1::TRP1 was
obtained from Brian Thornton. Strains harboring the point mutations
rad52-1 (8)
, mec1-1 (9)
, and mec2-1
(9)
and the gene replacements
pms1::LEU2, rad18::LEU2,
mgt1::LEU2, mag1::URA3,
rev3::LEU2, rad50::hisG,
rad51::LEU2, rad14::HIS3,
rad6::LEU2, rad1,
rad9::LEU2, and rad17::LEU2 were
obtained from Amanda Paulovich.
Drugs.
Chemotherapeutic drugs were obtained from the Developmental
Therapeutics Program at the National Cancer Institute; cytarabine
monophosphate was also obtained from Sigma. Drugs were stored at
–80°C in aliquots and used only once. Camptothecin sodium salt and
idarubicin solutions were prepared for each experiment.
Toxicity Assays.
In preliminary experiments, appropriate concentration ranges were
determined for each drug and each strain. Exponentially growing yeast
cultures in complete minimal medium were diluted to 7.4 x 104 cells/ml, and 135 µl were dispensed
into each well of flat-bottomed 96-well plates. Drugs were dispensed in
seven 2-fold serial dilutions in 5% DMSO or 5% ethanol (thiotepa and
lomustine) or in H2O (cisplatin, mitomycin,
cytarabine, cytarabine phosphate, hydroxyurea, and fluorodeoxyuridine),
and 15-µl aliquots were added in triplicate to the yeast-containing
wells. Fifteen µl of the appropriate solvent were also added
in triplicate in control wells. Plates were incubated for 18 h at
30°C, and the A660 of the
cultures was read in a Bio-Tek Instruments EL340 microplate reader.
Slowly growing strains, especially rad6 and rad52
mutants, were incubated until the A660
of control wells read at least 0.4 but no more than 0.8. For three
agents, IC50 values were determined by measuring
colony-forming units in the case of lomustine and cytarabine
phosphate because this method resulted in greater sensitivity or in the
case of mitoxantrone because the color of the drug interfered with the
A660 reading. In this case, after
incubation, aliquots from each well were plated onto rich medium agar
plates. Sulfanilamide (0.1 mg/ml) was added to the growth medium for
methotrexate and trimetrexate assays. Each experiment was carried out
three times with yeast strains freshly streaked out from –80°C
stocks.4
X-irradiation.
Yeast were grown in liquid culture, cell aggregates were dispersed by
sonication, and serial dilutions of cells were plated onto YEPD
plates. The plates were irradiated for times ranging from 10 to 400 s
at a dose rate of 106 rads/s. from 10 to 400 s at 106
rads/s. After incubation at 30°C for 2 days, colony-forming unit were
determined, and the percentage of surviving cells was calculated
relative to unirradiated control plates.
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RESULTS |
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There are 76
FDA5
-approved anticancer agents, and of these, 44 are expected to be toxic
to yeast. In preliminary experiments, we determined that 31 of these
agents showed at least minimal toxicity in yeast. In two cases,
cytarabine (NSC-63878) and topotecan (NSC-609699)/irinotecan
(NSC-616348), we used analogues that were active yeast. The initial
step in the activation of cytarabine in human cells is its conversion
to cytarabine monophosphate. Because yeast lack the required
deoxycytidine kinase, we used cytarabine monophosphate (NSC-99445),
which is taken up efficiently and shows activity. Likewise, we used
camptothecin sodium salt (camptothecin sodium, NSC-100880) in
place of topotecan and irinotecan. Here we report the
IC50 data for these agents in the damage
response-deficient yeast strains. Fig. 1
shows the profiles of the alkylating and cross-linking agents. Fig. 2
shows the antimetabolites and nucleotide analogues. Finally, Fig. 3
shows topoisomerase poisons, X-rays, bleomycin, and actinomycin D. Most
of the DNA repair and DNA damage checkpoint pathways are represented by
more than one mutant in the strain panel. In a majority of the profiles
(e.g., cisplatin: Fig. 1a
), the various mutants
representing a particular pathway show a similar sensitivity. This
coherent response indicates that the observed sensitivity is a property
of a pathway defect rather than a property of the individual mutant
strain. Although many of the drugs have been studied previously in
various yeast mutants (14
, 15)
, this report is the first
comprehensive examination of the relative sensitivities of damage
response-defective yeast strains to anticancer agents.
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). Second, there are agents that show
toxicity in many pathway defects indicating the introduction of
multiple types of DNA damage (e.g., thiotepa, mitomycin C,
hydroxyurea, and X-rays: Figs. 1b and 1e
, 2c
and 3c
). Third, there are agents that lack
selective toxicity between the various mutant strains and wild type.
Such profiles indicate that although the agents are toxic, cell killing
occurs by a mechanism that is probably independent of DNA damage
(e.g., methotrexate: Fig. 2e
).
Selective Agents.
Five agents (cisplatin, cytarabine phosphate, camptothecin
sodium, mitoxantrone, and idarubicin) showed a high degree of
specificity.
Cisplatin (Fig. 1a
) is specifically toxic to strains
defective for the poorly understood Rad6/Rad18-controlled pathway of
damage tolerance during S-phase. It is known, however, that Rad6 and
Rad18 are required for the completion of S-phase when a damaged DNA
template is present (i.e., daughter-strand gap repair; Ref.
16
). Rad6- and Rad18-mediated survival does not require
Rad50, Rad51, Rad52, Rad1, Rad14, Rev1, or Rev3 (17)
. The
sensitivity of these strains reflects the additional role of nucleotide
excision repair (Rad1 and Rad14), recombinational repair (Rad50, Rad51,
and Rad52) and error-prone damage tolerance (Rev1, Rev3, and Rad6) in
the repair of inter- and intrastrand DNA cross-links as well as
mono-adducts formed by cisplatin. The importance of the Rad6 and Rad18
function is evidenced by the greater than 10-fold increase in
sensitivity in rad6 and rad18 strains relative to
the other sensitive strains (e.g., rad50). With
the exception of the magnitude of the rad6 and
rad18 sensitivity, the profiles for cisplatin and
mechlorethamine are highly similar, indicating a common response to
cross-links induced by platinum adduct formation and alkylative
cross-linking. Thiotepa, which is not generally thought to create DNA
cross-links, is also similar.
The nucleotide analogue cytarabine monophosphate (Fig. 2a
)
shows highly selective toxicity in the sgs1 mutant strain.
This profile does not resemble those of agents causing known types of
DNA damage. Cytarabine inhibits DNA synthesis, is incorporated into
DNA, and has also been reported to be recombinogenic in yeast
(18)
. The sgs1 mutant and the homologous
Bloom’s and Werner’s syndrome defects in human cells cause
hyperrecombination (19)
. It is therefore possible that the
observed selectivity of cytarabine phosphate is due to an effect on
recombination and that the Sgs1 protein is required for tolerance. We
found that a sgs1 rad52 double mutant is less sensitive to
cytarabine phosphate compared to the sgs1 single mutant,
corroborating a possible role for recombination in the mechanism of
toxicity (data not shown). We have observed differential growth
inhibition of the sgs1 mutant by adenine arabinoside
phosphate and fludarabine phosphate, indicating that the arabinose
moiety of these agents may be important for selectivity (data not
shown). The profile of fludarabine (Fig. 2b
) shows a greater
sensitivity of the rad50 and mec2 mutants than
that of cytarabine phosphate.
The selective category includes, in addition to cisplatin and
cytarabine monophosphate, three topoisomerase poisons. These agents
stabilize the covalent complex of topoisomerase and DNA ends during DNA
relaxation and lead to single-strand breaks and DSBs for topo I and
topo II poisons, respectively. The single-strand breaks caused by topo
I poisons are converted into DSBs during DNA replication (20
, 21)
. The camptothecin analogue, a topo I poison, kills DSB
repair mutants with high specificity. The DNA damage checkpoint mutants
also contribute to survival, albeit to a lesser degree (Fig. 3a
).
The topo II poisons mitoxantrone (Fig. 3b
) and idarubicin
(Fig. 3e
) also showed selectivity for DSB repair defects. It
is noteworthy that cells deficient in the DNA damage checkpoint are not
sensitized to these topo II poisons. This can be explained by the
observation that topo I poison-induced damage requires ongoing DNA
synthesis to create DSBs, thus largely limiting the damage to S-phase
when DNA damage-induced and DNA synthesis checkpoints can contribute to
cell survival. topo II poisons, in contrast, create DSBs throughout the
cell cycle, with the majority occurring in mitosis, when the DNA
damage-induced and synthesis checkpoints are no longer capable of
protecting cells. Interestingly, two other topoisomerase poisons,
daunorubicin and doxorubicin, show profiles that are nonselective,
suggesting the presence of non-topo II-mediated cellular damage (Fig. 3, f and g
).
Broadly Selective Agents.
Nine agents (mitomycin C, thiotepa, lomustine, carmustine,
streptozotocin, mechlorethamine, bleomycin, hydroxyurea, and X-rays)
show selective toxicity in a large number of DNA damage
response-defective strains. Most of these responses are consistent with
the known mechanisms of action of the agents. Mag1 (3-methyladenine
glycosylase) is involved in the removal of damage created by alkylation
of adenine (22)
, and as expected, the mag1
mutant is sensitive to thiotepa (Fig. 1b
). The
rad1 and rad14 strains are sensitive to agents
that introduce DNA cross-links (e.g., cisplatin and
mechlorethamine: Fig. 1, a and c
). Thiotepa,
however, is a non-cross-linking alkylating agent, and the sensitivity
of these strains was unexpected (23)
. Lomustine
(Fig. 1f
) and carmustine (Fig. 1g
) produce broad
profiles; although both are capable of forming DNA cross-links
(24)
, nucleotide excision repair-defective strains
(e.g., rad1 and rad14) were not
sensitive. These differences demonstrate a continuum of pathway
responses to cytotoxic drugs and underscore the importance of
identifying the critical toxic lesions for agents that are capable of
inducing different forms of DNA damage. Streptozocin (Fig. 1d
), a methylating agent, also yielded a broad profile,
although the mgt1 strain was particularly sensitive. This
was expected, because the Mgt1 protein, the yeast homologue of
human MGMT, is required for
O6-methylguanine reversal (25
, 26)
.
The ribonucleotide reductase inhibitor hydroxyurea (Fig. 2c
)
blocks DNA replication by depleting the dNTP pool. This is consistent
with the observed sensitivity of the intra-S phase checkpoint-deficient
mec1 and mec2 strains (9
, 27)
.
Additional DNA damage in the form of DSBs is indicated by the
sensitivity of rad50, rad51, and rad52
strains. This result is consistent with the observation that stalled
replication forks result in DNA breaks (28)
. Since these
breaks occur exclusively during S phase, the Rad9 and Rad17 functions
may not be required, because the cell cycle would already be slowed
down in a MEC1-dependent fashion due to a decreased level of
dNTPs. X-ray treatment (Fig. 3c
) results in a profile
similar to the topo I poison camptothecin sodium (Fig. 3a
), except for the strong requirement of the Rad6 and Rad18
proteins for survival. X-rays are often referred to as a DSB-causing
agent, which is consistent with the strong sensitivity of the
recombinational repair mutants. The sensitivity of rad6 and
rad18 strains indicates that other forms of DNA damage are
also present in irradiated cells as expected. The profiles of
camptothecin sodium (Fig. 3a
) and mitoxantrone (Fig. 3b
) suggest that DSBs do not necessarily require Rad6 and
Rad18 functions for repair or tolerance.
Nonselective Agents.
Nine agents (methotrexate, trimetrexate, fluorouracil,
fluorodeoxyuridine, pentostatin, dacarbazine, actinomycin D,
daunorubicin, and doxorubicin), although toxic, showed only minor
differences in sensitivity among the various mutant strains and the
wild-type strain. These nonselective profiles, especially in the case
of agents that block key steps in DNA synthesis (i.e.,
methotrexate, trimetrexate, fluorouracil, and fluorodeoxyuridine), are
surprising because the induced damage must not require a DNA damage
response for survival. Methotrexate (Fig. 2e
) inhibits
dihydrofolate reductase, resulting in a block in the biosynthesis of
purines and thymidine. Our results suggest that the purine deficiency
might affect RNA synthesis more strongly than DNA replication.
Interestingly, the profile of the methotrexate analogue, trimetrexate
(Fig. 1f
) does show a moderate sensitivity of
mec1 and mec2 strains, as would be expected for a
block in DNA synthesis. Fluorouracil (Fig. 2g
) and
fluorodeoxyuridine (Fig. 2h
) inhibit thymidilate synthase,
resulting in a block in the synthesis of dTMP from dUMP. In this case,
only DNA synthesis would be affected, and it is surprising that
mec1 and mec2 strains are not more sensitive.
Pentostatin (Fig. 2d
) inhibits adenine deaminase, which is
involved in posttranscriptional modification of RNA and is not expected
to cause DNA damage (29)
. Dacarbazine, doxorubicin,
daunorubicin, and actinomycin D (Fig. 1 h
and Fig. 3
,
f–h) showed very similar, virtually nonselective profiles.
It is likely that these agents kill yeast cells by a mechanism that
does not require a DNA damage response for survival. Daunorubicin,
doxorubicin, and actinomycin D, for example, are capable of generating
free radicals, which target membranes in addition to DNA
(30)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We chose the nonbiological, nonhormonal, FDA-approved agents to assess the relative sensitivity of the yeast mutants and were able to obtain IC50 values for 23 of the 44 agents tested. The profiles shown here emphasize the importance of damage response defects as the determinants of sensitivity to cytotoxic drugs and to X-rays. It is reasonable to expect, and our data support the idea, that molecular defects in the same pathway will have similar sensitivities (e.g., rad50, rad51, and rad52 treated with mitoxantrone). Three patterns of cellular sensitivity emerged from these profiles: agents that selectively target only one or few damage response defects (e.g., cisplatin, mitoxantrone, and cytarabine phosphate); those that show a broad spectrum of toxicities (e.g., thiotepa or streptozocin); and those that show little or no enhanced killing in DNA damage response-defective strains. Our results underscore the notion that there are two broad modes by which chemotherapeutic agents act. One class of agents has activity against a broad range of tumors or cell types because these agents lack specificity for any particular cellular context (e.g., X-rays). The current emphasis of clinical trials, to obtain as large a response as possible in patients who are grouped only by the organ site of their cancer, favors agents that act broadly. These agents however, precisely because they target a wide spectrum of defects, have the highest probability of affecting normal, noncancerous tissues. The second class, chemotherapeutic agents with greater specificity, offers the potential to improve the therapeutic response by targeting a narrowly defined cellular context (i.e., a specific pathway defect). Each cancer is likely to harbor genetic defects that were important for the etiology of that particular cancer, and these defects are probably not shared by all cancers from the same organ site. Agents of high specificity and therapeutic advantage (for a precisely defined class of tumors with specific genetic lesions) are almost certainly bypassed under current clinical trial procedures. The latter strategy, however, offers the greatest opportunity for selective targeting of cancer cells without killing normal cells.
The analysis of the FDA-approved cancer drugs using this isogenic panel of yeast addresses an important question in cancer therapy: do we need new drugs or better diagnostics? Agents, such as mitoxantrone, that selectively target a single pathway defect suggest that improved diagnosis may be efficacious. Cancers defective in DNA DSB repair may be hypersensitive to topo II poisons. The availability of better diagnostics that would allow the routine determination of pathway defects in individual tumors might greatly improve benefits from current chemotherapeutic agent. Conversely, the agents resulting in broad toxicities in multiple genetic backgrounds may lead to higher toxicity in noncancer cells. It is therefore important to identify agents with high specificity for unique genetic changes associated with cancers. Primary drug screens carried out in the context of a single genetic alteration may provide a valuable route to new selective drugs. Therapeutic agents could be screened for high toxicity in human cells carrying a particular damage response defect but low toxicity in isogenic wild-type control cells. This preliminary study has shown that many of the current cancer drugs (X-rays etc.) are relatively nonspecific and suggests that developing more specific therapeutics may be beneficial. At the same time some of the commonly used cancer drugs, such as cisplatin and the topoisomerase poisons, have significant specificity in their killing, and this provides strong evidence that new molecular diagnostics could improve their utility. The results described in this communication pertain to the activity of anticancer agents in yeast, and it should be noted that the biology of yeast and mammalian cells differs in several important aspects. Foremost is the absence of programmed cell death in yeast. Although programmed cell death or apoptosis may not alter the intrinsic sensitivity of cells harboring specific defects to particular agents, it may alter the response of the cell to damage (i.e., survival). In addition, the relative importance of alternative pathways for cellular response to cytotoxins can vary between the two systems. This is true for DNA DSB repair pathways, the major pathway of which in yeast is homologous recombination (represented by rad50, rad51, and rad52 in this study), whereas in mammalian cells, nonhomologous end joining is a more important repair pathway (31) . We expect, however, that sensitivity differences seen in yeast can, in many cases, be translated to mammalian cells with the same fundamental defect, such as a deficiency in DSB repair, regardless of the precise causative genetic defect. Finally, the examination of anticancer agents with non-DNA cellular targets in yeast can be limited by divergence of the target molecules in the two organisms. For example, the FDA-approved spindle poisons are not toxic to Saccharomyces cerevisiae, as these agents are not active against yeast tubulin; hence, it was not possible to include the spindle poisons in this study. Theoretically, the studies we performed in yeast could have been carried out using a human or rodent panel of matched pair cell lines. Although current limitations in generating a sizable panel (e.g., 21 deletions in diploid cells) of cell lines make this approach impractical, future studies in mammalian cells will focus on cellular sensitivity to drugs in the context of specific DNA damage response alterations. As it becomes more feasible to perform similar studies using larger isogenic panels of mammalian cells, the general utility of this approach should be even more potent in defining the particular patients who should best respond to various cancer treatments.
ACKNOWLEDGMENTS
We thank Andrew W. Murray, Graham C. Walker, Stephen J. Elledge,
Priscilla Cooper, Tom Petes, and Jan Hoeijmakers, who provided
invaluable advice; Amanda Paulovich and Brian Thornton for providing
yeast strains; John Lamb for comments on the manuscript; members of the
Byers lab at the Genetics Department of the University of Washington
for advice in X-irradiation; and Dr. Edward Sausville and members of
the Developmental Therapeutics Program National Cancer Institute for
helpful comments.
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FOOTNOTES |
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1 Supported by the Developmental Therapeutics
Program, National Cancer Institute. 
2 The first two authors contributed equally to
this work.
3 To whom requests for reprints should be
addressed, at Molecular Pharmacology Program, Fred Hutchinson Cancer
Research Center, 1100 Fairview Avenue North, Seattle, WA 98109.
E-mail: sfriend{at}fhcrc.org
4 Bar graphs for all 23 agents and X-rays can be
obtained from our Web site:
http://www.fhcrc.org/science/labs/seattle_project.
5 The abbreviations used are: FDA, Food and Drug
Administration; topo I, topoisomerase I; topo II, topoisomerase II;
DSB, double-strand break.
Received 7/30/99. Accepted 11/15/99.
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P. Fiorani, R. J. D. Reid, A. Schepis, H. R. Jacquiau, H. Guo, P. Thimmaiah, P. Benedetti, and M.-A. Bjornsti The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons J. Biol. Chem., May 14, 2004; 279(20): 21271 - 21281. [Abstract] [Full Text] [PDF]
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R. C.A.M. van Waardenburg, L. A. de Jong, F. van Delft, M. A.J. van Eijndhoven, M. Bohlander, M.-A. Bjornsti, J. Brouwer, and J. H.M. Schellens Homologous recombination is a highly conserved determinant of the synergistic cytotoxicity between cisplatin and DNA topoisomerase I poisons Mol. Cancer Ther., April 1, 2004; 3(4): 393 - 402. [Abstract] [Full Text] [PDF]
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M. R. Kelley, Y. W. Kow, and D. M. Wilson III Disparity between DNA Base Excision Repair in Yeast and Mammals: Translational Implications Cancer Res., February 1, 2003; 63(3): 549 - 554. [Abstract] [Full Text] [PDF]
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H. Zheng, X. Wang, A. J. Warren, R. J. Legerski, R. S. Nairn, J. W. Hamilton, and L. Li Nucleotide Excision Repair- and Polymerase {eta}-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links Mol. Cell. Biol., January 15, 2003; 23(2): 754 - 761. [Abstract] [Full Text] [PDF]
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C. Liu, J. J. Pouliot, and H. A. Nash Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1 PNAS, November 12, 2002; 99(23): 14970 - 14975. [Abstract] [Full Text] [PDF]
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 A Brieger, J Trojan, J Raedle, G Plotz, and S Zeuzem Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants Gut, November 1, 2002; 51(5): 677 - 684. [Abstract] [Full Text] [PDF]
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P. W. Schenk, A. W. M. Boersma, M. Brok, H. Burger, G. Stoter, and K. Nooter Inactivation of the Saccharomyces cerevisiae SKY1 Gene Induces a Specific Modification of the Yeast Anticancer Drug Sensitivity Profile Accompanied by a Mutator Phenotype Mol. Pharmacol., March 1, 2002; 61(3): 659 - 666. [Abstract] [Full Text] [PDF]
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 H. M. Dunstan, C. Ludlow, S. Goehle, M. Cronk, P. Szankasi, D. R. H. Evans, J. A. Simon, and J. R. Lamb Cell-Based Assays for Identification of Novel Double-Strand Break-Inducing Agents J Natl Cancer Inst, January 16, 2002; 94(2): 88 - 94. [Abstract] [Full Text] [PDF]
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 A. P. Gasch, M. Huang, S. Metzner, D. Botstein, S. J. Elledge, and P. O. Brown Genomic Expression Responses to DNA-damaging Agents and the Regulatory Role of the Yeast ATR Homolog Mec1p Mol. Biol. Cell, October 1, 2001; 12(10): 2987 - 3003. [Abstract] [Full Text] [PDF]
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S. Jacob, M. Aguado, D. Fallik, and F. Praz The Role of the DNA Mismatch Repair System in the Cytotoxicity of the Topoisomerase Inhibitors Camptothecin and Etoposide to Human Colorectal Cancer Cells Cancer Res., September 1, 2001; 61(17): 6555 - 6562. [Abstract] [Full Text] [PDF]
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