.JPG?ad=18503&adview=true)
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. P., Y. U., Y. P.]; Molecular Pharmacology Department, St. Jude Children’s Hospital, Memphis, Tennessee 38105 [J. L. W., J. L. N.]; and Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033 [N. A. L., A. E. P.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Recent studies have shown that a variety of DNA alterations originating from endogenous and exogenous sources (abasic sites, uracil misincorporations, nicks, oxidized bases, UV photolesions, and carcinogenic adducts) induce the formation of Top1 cleavage complexes (reviewed in Ref. 12 ). Moreover, we recently demonstrated that Top1 trapping by the nucleoside analogue 1-ß-D-arabinofuranosylcytosine might also contribute to the cytotoxicity of this widely used anticancer agent (13) .
DNA methylation represents a cytotoxic lesion that is produced by alkylating agents such as methyl sulfates (dimethyl sulfate), methyl sulfonates, methyl nitrosamines (dimethylnitrosamine), or methyl nitrosamides (methylnitrosourea and MNNG) that are present in the environment as byproducts of industrial processes (14 , 15) . DNA methylation is also generated endogenously by nonenzymatic mechanism involving S-adenosyl-L-methionine (16) . Methylating agents are electrophilic compounds that alkylate guanines at position N3 , N7, and O6 (15) . When O6MG is generated, it tends to base pair preferentially to thymine rather than cytosine (17 , 18) , which results in G:C to A:T transition. Such mutations are probably responsible for the development of various types of tumors (19 , 20) . O6MG is specifically removed by the AGT, which catalyzes the irreversible transfer of the alkyl group of the guanine to an internal cysteine of the enzyme (Cys-145 in humans; Refs. 21, 22, 23 ). Alkylators of the O6 position guanines are highly potent drugs used in chemotherapy. Dacarbazine and temozolomide specifically methylate the O6 position of guanines and are used in the treatment of advanced melanoma, brain tumors, and lymphomas (24) . Chloroethylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea represent standard treatments for malignant gliomas (25) and generate O6-chloroethyl adducts that are also repaired by the AGT enzyme (15) . However, the cytotoxicity of methylating agents does not seem to be attributable to their mutagenicity, and the mechanism by which alkylation of DNA leads to cell death remains unclear.
In this study, we show that the presence of O6MG in DNA can induce Top1 cleavage complexes in vitro and in MNNG-treated cells lacking the AGT enzyme. We also demonstrate that Top1 trapping by alkylated DNA may in part contribute to the cytotoxicity of MNNG.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
-32P]Cordycepin 5'-triphosphate was
purchased from DuPont NEN (Boston, MA), and terminal deoxynucleotidyl
transferase and T4 polynucleotide kinase were from Life Technologies,
Inc., (Grand Island, NY). Polyacrylamide was purchased from Bio-Rad,
Inc. (Richmond, CA). Human recombinant Top1 was purified from Sf9 cells
using a baculovirus construct for the
NH2-terminal truncated human Top1 cDNA as
described previously (26)
.
Cell Lines.
CHO cells expressing the human alkyl guanine transferase (CHO/pCMV-AGT)
or transfected with the empty pCMV-neo vector (CHO/pCMV; Ref.
27
) were cultured in RPMI 1640 (Life Technologies, Inc.,
Gaithersburg, MD) containing 10% heat-inactivated FCS, 2
mM glutamine, and 1 mg/ml geneticin in a 5%
CO2 incubator at 37°C.
Oligonucleotide Labeling and Annealing Procedures.
High-performance liquid chromatography-purified oligonucleotides were
purchased from The Midland Certified Reagent Company (Midland, TX).
Fig. 1A
shows the 36-bp duplex sequence used in standard
experiments. Suicide substrates shown in
Fig. 3A
were
generated by annealing a 3' end-labeled 21-mer upper strand
(5'-GATCTAAAAGACTTGGAAAA(A)-3', where (A)
corresponds to [-32P]-labeled cordycepin)
incorporated at the end of the oligonucleotide. 3'-Labeling and
5'-phosphorylation of single-stranded oligonucleotides and annealing
were performed as described previously (26)
.
|
|
|
50 fmol/reaction) were incubated with 5 ng of Top1
with or without CPT for the indicated times at 25°C in reaction
buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5
mM MgCl2, 0.1 mM EDTA,
and 15 µg/ml BSA]. Reactions were stopped by adding SDS (final
concentration, 0.5%). For the reversal experiments, the SDS stop was
preceded by the addition of 0.5 M NaCl. Kinetics of
cleavage and religation were performed as described previously
(13)
. 3.3 volumes of Maxam Gilbert loading buffer (80%
formamide, 0.01 M EDTA, 10 mM NaOH, 1 mg/ml
xylene cyanol, and 1 mg/ml bromphenol blue) were added to reaction
mixtures that were loaded on 16 or 20% denaturing polyacrylamide gels.
Quantitations were performed using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA).
Detection of Covalent Top1-DNA Complexes in Cells.
Top1-DNA adducts were isolated using the ICE Bioassay (13
, 28 , 29)
. Briefly, 106 treated or untreated
cells were pelleted and immediately lysed with 1 ml of 1% sarkosyl.
After homogenization with a Dounce, cell lysates were gently layered on
step gradients containing four different CsCl solutions (2 ml of each)
of the following densities: 1.82, 1.72, 1.50, and 1.45
(29)
. Tubes were centrifuged at 165,000 x g in a Beckman SW40 rotor for 24 h at 20°C. Half-ml
fractions were collected from the bottom of the tubes. Aliquots of each
fraction (100 µl) were diluted with an equal volume of 25
mM sodium phosphate buffer (pH 6.5) and applied
to Immobilon-P membranes (Millipore, Bedford, MA) using a slot-blot
vacuum manifold. Detection of Top1-DNA adducts was performed by Western
blotting using the Top1 monoclonal antibody C21 obtained from Dr.
Yung-Chi Cheng (Yale University, New Haven, CT) according to standard
procedures.
Yeast Strains and Drug Sensitivity Determination.
The yeast strains used in this study are derivatives of CH335
(30)
. CH335 was converted to leu2- by one-step
gene disruption. A Top1- derivative of CH335leu
was constructed by one-step gene disruption. The resulting strain is
termed CH335top1-. Strain CH335leu was
transfected with either yCP50 or pGALhTOP1. The strain carrying yCP50
served as a vector control, whereas pGALhTOP1 expresses human TOP1
under the control of the yeast GAL1 promoter (31)
. Drug
sensitivity determinations in yeast cells were performed as described
previously (32)
with the following modifications. Cells
were pregrown in synthetic complete medium without uracil, with
galactose as a carbon source (SC-ura/GAL). After overnight growth,
cells were diluted to 2 x 106
cells/ml in fresh SC-ura/GAL, and then appropriate concentrations MNNG
were added. Cells were incubated for 4 h at 30° with shaking;
then aliquots were removed, and diluted samples were plated to
synthetic complete agar lacking uracil, with glucose as carbon source.
Survival is expressed relative to the number of viable colonies at the
time of drug addition. For the comparison of the sensitivity of
top1- and
TOP1+ cells, the appropriate strains were
grown in yeast extract/peptone/dextrose/adenine medium as described
previously (32)
, exposed to MNNG for 2–4 h, and then
plated to yeast extract/peptone/dextrose/adenine agar to determine
surviving percentages.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
A,
arrowhead). Guanine modifications were introduced at the +1
position immediately downstream to the Top1 site (Fig. 1A)
.
Base pairing of O6MG with C results in the suppression of one hydrogen
bond, whereas 6TG can still form three hydrogen bonds as in normal G:C
base pairing (Fig. 1B
; Ref. 33
). As shown in
Fig. 2
A, incorporation of O6MG at the +1 position of the Top1
cleavage site resulted in an 8–10-fold increase of cleavage complexes
in the absence of camptothecin (Fig. 2
A, compare Lanes
2 and 7). A comparable increase was observed when O6MG
was base paired to T (data not shown). By contrast, incorporation of
6TG that retains all hydrogen bonds had no effect on Top1 activity
(Fig. 2B)
. We tested whether incorporation of O6MG at the +1
position could affect CPT activity. As shown in Fig. 2
A, the
presence of O6MG did not prevent further increase of Top1 cleavage
complexes by CPT (Fig. 2
A, compare Lanes 7 and
9).
Addition of salt to the Top1 reaction shifts the cleavage/religation
equilibrium toward religation, probably because it prevents binding of
the enzyme to the DNA once religation has taken place (3
, 34)
. Under such conditions, the effect of CPT is reversed (Fig. 2
A, Lanes 4 and 5). Top1 trapping by O6MG in the
absence of drug was also reversed in 0.5 M NaCl
(Fig. 2
A, compare Lanes 7 and 8).
Noticeably, reversal was only partial in the presence of CPT (Fig. 2
A, compare Lanes 9 and 10). Together,
these data demonstrate that presence of O6MG immediately downstream
from a Top1 cleavage site can enhance the formation of reversible Top1
cleavage complexes.
O6MG Incorporation Inhibits Religation of Top1 Cleavage Complexes
and Induces Top1-mediated Cleavage.
We then investigated the mechanism by which O6MG incorporation enhances
Top1 cleavage complexes. For this purpose, we used different
oligonucleotide substrates to analyze separately the effects of O6MG on
the induction and religation of Top1 cleavage complexes.
We first measured the effects of O6MG on the induction of the
Top1-mediated cleavage using a "suicide" substrate (Fig. 3A)
. This type of substrate is generally used (13
, 35, 36, 37)
to directly evaluate cleavage rates, assuming that the
3' end-labeled 7-mer product that is released cannot be religated
because of a complete loss of annealing after cleavage (Fig. 3
A, compare Lanes R and Top1 in the right
panel). Kinetics of cleavage revealed an 150-fold increase in
the rate of cleavage when O6MG was incorporated at the +1 position
relative to the Top1 site. This enhancement was not accompanied by an
increase of binding of the Top1 to DNA (data not shown).
We also investigated the effect of O6MG on the religation step of the
Top1 cleavage/religation reaction. Kinetics experiments were performed
using the donor-acceptor system as described in previous studies
(13
, 35
, 37)
. Briefly, acceptors are generated from
unlabeled suicide substrates (Fig. 3A)
and a 10-fold excess
of the 3' end-labeled complementary strand bearing either a G or an
O6MG at the 5'-end (called donor) is added to the mixture. The amount
of religated product (37-mer) is then measured as a function of time
(Fig. 3B)
. We estimated an 6-fold decrease in the initial
rate of Top1-mediated religation when O6MG was incorporated at the +1
position. Thus, O6MG at the +1 position inhibits the religation step of
the Top1 reaction. Together, these data show that Top1 trapping by O6MG
incorporation at the +1 position of a Top1 cleavage site is
attributable to a dual effect: induction of cleavage and an inhibition
of religation.
Induction of Top1 Cleavage Complexes in AGT-deficient and
AGT-transfected CHO Cells after MNNG Treatment.
We next investigated whether Top1 could be trapped by O6MG in cellular
DNA. For this purpose, we measured Top1 cleavage complexes in CHO cells
after MNNG treatment using the ICE assay (13
, 28) . CHO
cells have very little or no AGT activity (27)
. In the
CHO/pCMV control cells, treatment with 1 µg/ml MNNG induced the
formation of Top1 cleavage complexes as seen in the DNA-containing
fractions 7 to 10 (Fig. 4
, left panels). Top1 trapping reached an optimum after 30 min
of treatment and was reduced after 3 h. Conversely, there was no
increase in Top1 complexes in MNNG-treated CHO/pCMV-AGT cells in which
AGT activity was restored by transfection of the human repair enzyme
(Fig. 4
, right panel). These results demonstrate that Top1
trapping by MNNG treatment is linked to the formation of O6MG, because
the removal of the alkyl group by the AGT enzyme abrogated the
formation of the Top1-DNA complexes.
|
. Viability of cells was determined after plating MNNG-treated or
untreated cells onto agar plates. Survival is expressed relative to the
number of viable colonies at the time of drug addition. As shown in
Fig. 5
, cells that overexpressed the human Top1 were significantly more
sensitive to MNNG for concentrations <3 µg/ml (with a
P = 0.0251 when 1.67 µg/ml of MNNG was
used). Conversely, Top1 knockout yeasts showed the same sensitivity to
MNNG as the wild-type strains. Altogether, these results suggest that
Top1 poisoning is a cytotoxic mechanism for O6MG incorporation in the
DNA of cells treated with monofunctional alkylating agents such as
MNNG.
|
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
and data not shown). Conversely, 6TG:C-containing
duplex that retains normal base pairing did not affect Top1 cleavage
complex formation. Together, these data confirm the importance of
hydrogen bonding and subsequent alignment of the DNA 5'-hydroxyl with
the tyrosyl phosphodiester bond for optimum religation and catalytic
activity of Top1.
MNNG is relatively efficient at methylating the
O6 position of guanine
(47)
. Using the ICE assay, we were able to detect the
formation of Top1-DNA cleavage complexes in CHO cells treated with
MNNG. CHO cells are constitutively lacking the AGT repair enzyme that
specifically removes O6MG adducts. In CHO cells transfected with the
human AGT, however, there was no induction of Top1 cleavage complexes,
suggesting that Top1 trapping is prevented by the removal of the methyl
group by AGT as O6MG adducts are formed. The absence of cleavage
complexes in CHO cells expressing AGT may indicate that other DNA
adducts, such as 7-methylguanine and 3-methyladenine that are formed in
larger amounts than O6MG, may not function as Top1 poisons. It should
be noted that we did not measure the persistence of these adducts, and
that these adducts are subject to DNA repair pathways, which, unlike
AGT, are not deficient in the CHO cells used. Although alkylation at
the O6 position is known to play a
major role in the mutagenicity and the carcinogenicity of
methylating agents, little is known concerning the mechanism by which
presence of O6MG in DNA leads to cell death. It has been suggested
previously that cell killing by alkylating agents could be attributable
to futile attempts of removing mismatched bases incorporated in the
newly synthesized DNA by the mismatch repair system during replication
and/or to the recognition of the damaged bases by the mismatch repair
proteins directly signaling cell cycle arrest and apoptosis
(48)
. On the basis of our data, we propose that increased
Top1 cleavage at sites of O6MG adducts could also play a role in the
cytotoxicity of methylating agents. We show that yeast overexpressing
human Top1 is significantly more sensitive to MNNG than the
corresponding wild-type cells (see Fig. 5
). Similar results were also
obtained when the yeast cells overexpressed yeast Top1 (data not
shown). This observation is consistent with the biochemical data
indicating that Top1 can be trapped by O6MG in vitro.
However, yeast cells completely lacking Top1 had the same sensitivity
to MNNG as cells expressing wild-type levels of the enzyme. Certainly,
MNNG is capable of killing cells by pathways that do not depend on
Top1, which explains the absence of differential cytotoxicity at
high-dose MNNG for the normal versus the human
Top1-overexpressing yeast (Fig. 5)
. However, we suggest that eukaryotic
cells likely have pathways that can prevent Top1-mediated cleavage at
sites of DNA damage. One such pathway could be targeted degradation of
Top1 or a reversible modification, such as phosphorylation. Although
cells can deal with normal levels of Top1, overexpression of the enzyme
saturates the cells ability to prevent enzyme cleavage at sites of DNA
damage, ultimately increasing cellular sensitivity to alkylated bases.
Together, our results suggest that in the context of high level of Top1
expression, such as in the case of various tumor types
(49)
, cells possess an efficient mechanism for
MNNG-induced killing involving Top1 trapping by alkylated bases.
Interestingly, we also found that the presence of O6MG further
stabilizes CPT-induced Top1 cleavage complexes (Fig. 2A)
.
CPT binding might further destabilize the alignment of the 5'-hydroxyl
of the cleaved strand downstream from the cleavage site and render
religation even more difficult. This effect is similar to what we
observed when nicks were present downstream from the Top1 cleavage site
(positions +4 to +6; Ref. 26
). Top1 trapping by nicks in
the DNA has been proposed as a potential explanation for the synergism
between ionizing radiation and CPT (28
, 50)
. Such a
synergism was also reported at high levels of cytotoxicity between
topotecan and various alkylating agents (51)
. Recently,
treatment with the AGT inhibitor
O6-benzylguanine has been shown to
sensitize cells to various Top1 inhibitors (52)
. Such a
potentiation could be explained by the increase of O6MG adducts,
leading to enhanced Top1 trapping and further CPT-induced irreversible
DNA breaks. The presence of Top1 cleavage complexes originating from
O6MG after exposure to alkylating agents may also contribute to the
increase in sister chromatin exchanges and other chromatin aberrations
that occurs in cells exposed to alkylating agents.
Altogether, these results suggest a role for Top I poisoning by alkylated bases in the antiproliferative activity of alkylating agents as well as in the DNA lesions resulting from endogenous and carcinogenic DNA modifications.
|
FOOTNOTES |
|---|
1 Supported by Grants CA52814 and CA21765 (to
J. L. W. and J. L. N.) from the National Cancer Institute and by
funds from the American Lebanese Syrian Associated Charities. 
2 To whom requests for reprints should be
addressed, at Laboratory of Molecular Pharmacology, National Cancer
Institute, NIH, Building 37, Room 4E28, NIH, Bethesda, MD 20892-4255.
Phone: (301) 496-5944; Fax: (301) 402-0752; E-mail: pommier{at}nih.gov
3 The abbreviations used are: Top1, DNA
topoisomerase I; O6MG, O6-methylguanine;
AGT, O6-alkylguanine-DNA alkyltransferase;
CPT, camptothecin; CHO, Chinese hamster ovary; ICE, in
vivo complex of enzyme; 6TG, 6-thioguanine; MNNG,
1-methyl-3-nitro-1-nitrosoguanidine.
Received 6/30/00. Accepted 11/13/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. S. Hackbarth, M. Galvez-Peralta, N. T. Dai, D. A. Loegering, K. L. Peterson, X. W. Meng, L. M. Karnitz, and S. H. Kaufmann Mitotic Phosphorylation Stimulates DNA Relaxation Activity of Human Topoisomerase I J. Biol. Chem., June 13, 2008; 283(24): 16711 - 16722. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mora, O. Cruz, S. Gala, and R. Navarro Successful treatment of childhood intramedullary spinal cord astrocytomas with irinotecan and cisplatin Neuro-oncol, January 1, 2007; 9(1): 39 - 46. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. H. Kushner, K. Kramer, S. Modak, and N.-K. V. Cheung Irinotecan Plus Temozolomide for Relapsed or Refractory Neuroblastoma J. Clin. Oncol., November 20, 2006; 24(33): 5271 - 5276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Tentori, C. Leonetti, M. Scarsella, A. Muzi, E. Mazzon, M. Vergati, O. Forini, R. Lapidus, W. Xu, A. S. Dorio, et al. Inhibition of poly(ADP-ribose) polymerase prevents irinotecan-induced intestinal damage and enhances irinotecan/temozolomide efficacy against colon carcinoma FASEB J, August 1, 2006; 20(10): 1709 - 1711. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z.-H. Miao, V. A. Rao, K. Agama, S. Antony, K. W. Kohn, and Y. Pommier 4-Nitroquinoline-1-Oxide Induces the Formation of Cellular Topoisomerase I-DNA Cleavage Complexes. Cancer Res., July 1, 2006; 66(13): 6540 - 6545. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. El-Khamisy and K. W. Caldecott TDP1-dependent DNA single-strand break repair and neurodegeneration Mutagenesis, July 1, 2006; 21(4): 219 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. A. M. van Waardenburg, L. A. de Jong, M. A. J. van Eijndhoven, C. Verseyden, D. Pluim, L. E. T. Jansen, M.-A. Bjornsti, and J. H. M. Schellens Platinated DNA Adducts Enhance Poisoning of DNA Topoisomerase I by Camptothecin J. Biol. Chem., December 24, 2004; 279(52): 54502 - 54509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Reardon, J. A. Quinn, J. N. Rich, S. Gururangan, J. Vredenburgh, J. H. Sampson, J. M. Provenzale, A. Walker, M. Badruddoja, S. Tourt-Uhlig, et al. Phase 2 trial of BCNU plus irinotecan in adults with malignant glioma Neuro-oncol, April 1, 2004; 6(2): 134 - 144. [Abstract] [PDF]
|
|
|
|
 |
|
 L. M. Wagner, K. R. Crews, L. C. Iacono, P. J. Houghton, C. E. Fuller, M. B. McCarville, R. E. Goldsby, K. Albritton, C. F. Stewart, and V. M. Santana Phase I Trial of Temozolomide and Protracted Irinotecan in Pediatric Patients with Refractory Solid Tumors Clin. Cancer Res., February 1, 2004; 10(3): 840 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. T. Wong and A. Berkenblit The Role of Topotecan in the Treatment of Brain Metastases Oncologist, February 1, 2004; 9(1): 68 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Soe and F. Grosse p53 stimulates human topoisomerase I activity by modulating its DNA binding Nucleic Acids Res., November 15, 2003; 31(22): 6585 - 6592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Furuta, H. Takemura, Z.-Y. Liao, G. J. Aune, C. Redon, O. A. Sedelnikova, D. R. Pilch, E. P. Rogakou, A. Celeste, H. T. Chen, et al. Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes J. Biol. Chem., May 23, 2003; 278(22): 20303 - 20312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Stephan, F. Grosse, and K. Soe Human topoisomerase I cleavage complexes are repaired by a p53-stimulated recombination-like reaction in vitro Nucleic Acids Res., December 1, 2002; 30(23): 5087 - 5093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. S. Friedman, S. Keir, A. E. Pegg, P. J. Houghton, O. M. Colvin, R. C. Moschel, D. D. Bigner, and M. E. Dolan O6-Benzylguanine-mediated Enhancement of Chemotherapy Mol. Cancer Ther., September 1, 2002; 1(11): 943 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-Y. Chang, J.-F. Liu, S.-H. Juang, T.-W. Liu, and L.-T. Chen Novel Mutation of Topoisomerase I in Rendering Cells Resistant to Camptothecin Cancer Res., July 1, 2002; 62(13): 3716 - 3721. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Turner, S. Gururangan, J. Eastwood, K. Bottom, M. Watral, R. Beason, R. E. McLendon, A. H. Friedman, S. Tourt-Uhlig, L. L. Miller, et al. Phase II study of irinotecan (CPT-11) in children with high-risk malignant brain tumors: The Duke experience Neuro-oncol, April 1, 2002; 4(2): 102 - 108. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP |
) or
yeast overexpressing Gal1-human Top1 (
) were treated with increasing
concentrations of MNNG for 4 h at 30°C. Survival is expressed
relative to the number of viable colonies at the time of drug addition.
Values are the means of three independent experiments;
bars, SD.