Carcinogenesis vol.17 no.l pp.
31-35, 1996
The anti-cancer drug camptothecin inhibits elongation but
stimulates initiation of RNA polymerase II transcription
Mats Ljungman and Philip C.Hanawalt1
Department of Radiation Oncology, Division of Cancer Biology, University
of Michigan Medical Center, 1331 East Ann Street, Ann Arbor,
MI 48109-0582 and 'Department of Biological Sciences,
Stanford University. Stanford, CA 94305-5020, USA
Camptothecin is a widely used anti-tumor drug that
specifically inhibits DNA topoisomerase I. It is believed
that topoisomerase I participates in the process of transcrip-
tion by relaxing torsional stress induced in the duplex DNA
by the elongating RNA polymerase. We have assessed the
effects of camptothecin on RNA polymerase II transcription
from the dihydrofolate reductase (DHFR) gene in Chinese
hamster ovary (CHO) cells. Using in vivo [3H]uridine pulse
labeling and in vitro nuclear run-on techniques to estimate
relative rates of transcription, it was found that campto-
thecin stimulated RNA synthesis from promoter-proximal
sequences of the DHFR gene, while transcription from
promoter-distal sequences was reduced. Furthermore,
camptothecin caused a significant accumulation of RNA
polymerases in the 5'-end of the DHFR gene. The effect of
camptothecin on transcription was reversible, resulting in
a wave of RNA synthesis recovery in a 5' to 3' direction
through the DHFR gene following a chase with campto-
thecin-free medium. We conclude that camptothecin stimu-
lates initiation but inhibits elongation of the RNA poly-
merase II transcribed DHFR gene.
Introduction
Camptothecin is a plant alkaloid possessing a broad spectrum
of anti-tumor activity (1). Treatment of cells with camptothecin
has been shown to result in the inhibition of the synthesis of
both DNA (2) and RNA (3-5) as well as the induction of the
tumor suppressor protein p53 (6) and the fragmentation of
chromosomal DNA (7). Camptothecin also selectively kills
cells in the S-phase (8) and arrests cells in the G2 phase of
the cell cycle (9). It is well established that the underlying
mechanism responsible for these cellular effects of campto-
thecin is the inhibition of DNA topoisomerase I (10,11).
The principal function of DNA topoisomerase I is to
relax DNA torsional stress induced by DNA replication fork
movement and by transcriptional elongation (for reviews see
1,12). The mechanism of relaxation has been shown to involve
the formation of a transient DNA single-strand break through
which the other DNA strand is passed, followed by the
rejoining of the transient break (12-14). Camptothecin spe-
cifically interferes with the rejoining step in the topoisomerase
reaction which results in the trapping of the topoisomerase I
enzyme in a reversible cleavable complex (10,11). It is thought
that this trapped topoisomerase complex is a barrier for
RNA polymerase elongation (15). It has been shown that the
Abbreviations: DHFR, dihydrofolate reductase; CHO, Chinese hamster
ovary cells.
Oxford University Press
elongation but not initiation of transcription of the ribosomal
genes by RNA polymerase I is inhibited by camptothecin
in vivo (16).
It has been shown that topoisomerase I is involved in
both the initiation (17,18) and elongation (19-21) of RNA
polymerase II transcribed genes. The aim of this study was to
assess the effect of camptothecin on transcription from the
polymerase II transcribed dihydrofolate reductase (DHFR*)
gene. Transcription from three different regions of the DHFR
gene in Chinese hamster ovary (CHO) B11 cells was assessed
both in vivo by [3H]uridine pulse labeling followed by DNA
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015
filter excess hybridization, and in vitro by the nuclear run-on
technique. Results show that in cells treated with camptothecin,
transcription was affected differentially throughout the DHFR
gene. While transcription from promoter-distal sequences of
the gene was reduced, transcription from the 5'-end of the
DHFR gene was stimulated in the presence of camptothecin.
Materials and methods
Cell culture
The methotrexate-resistant Chinese hamster cell line, CHO Kl Bll [0.5],
containing an ~50-fold amplification of the DHFR gene (22), was plated at a
density of 1.5 X 106 cells per 60 cm2 culture dish in minimal essential
medium (MEM) supplemented with 10% dialyzed fetal bovine serum, 2 mM
glutamine, 1 X non-essential amino acids, 100 IU penicillin, 100 u.g/ml
streptomycin, 0.25 |ig/ml amphotencin and 5000 Bq/ml [methyl-3H]thymi-
dine (nuclear run-on experiments) or 150 Bq/ml [methyl-l4C]thymidine
(Amersham) ([3H]uridine-labeling experiments). Methotrexate (Calbiochem
Corp, La Jolla, CA) was included in the medium at a final concentration of
0.55 |iM to select for cells maintaining the amplified DHFR gene. The medium
was removed 24 h after seeding the cells and the cells were rinsed with PBS.
The cells were then supplied with a medium containing 0.1% fetal bovine
serum but lacking labeled nucleotides After 4 days of incubation in the low
serum medium (serum starvation), the cells were stimulated by exchanging the
medium with medium containing 15% fetal bovine serum. The camptothecin
experiments were performed 15 h after the serum addition to allow for
maximal transcription of the DHFR gene (23,24).
In vivo transcription
The measurement of in vivo synthesis of nascent RNA was performed as
previously described (24) except for the labeling period which was shortened
to 15 min. CHO Bll cells were either washed twice in PBS and supplied
with camptothecin-free media (chase experiments) or left in 0.8 ml of
medium containing 20 |iM camptothecin. RNA labeling took place in 1 ml
of labeling solution consisting of 800 uJ conditioned medium (with or
without camptothecin) and 200 |il of an aqueous solution containing 200 (iCi
(7.4 MBq) of [5-3H]uridine (Amersham). After the completion of RNA
labeling (15 min), the cells were put on ice and rinsed several times with ice-
cold PBS. The RNA was then partially hydrolyzed in cold alkali to produce
RNA fragments in the size range of 200 bases (25). These RNA fragments
were hybridized at 65C for 48 h to 24 ng of denatured and sonicated DNA
probes immobilized on Hybond N+ membranes (Amersham). Following a
stringency wash at 65C in O.lxSSPE and 0.1% SDS for 60 min, the
membranes were dried and 3H was determined by scintillation counting.
The values obtained were corrected for the number of cells present in each
sample which was estimated from the relative DNA content measured from
counting I4C following the DNase I digestion step in the RNA extraction
protocol (24).
In vitro nuclear run-on transcription
The nuclear run-on procedure was performed as previously described (24).
Nuclei were isolated from - I 0 7 cells and heterogeneous nuclear RNA was
labeled in vitro in the absence of camptothecin with 32P-labeled UTP for
31
M.Ljungman and P.C.Hanawalt
15 min at 30C. RNA was then isolated, partially hydrolyzed in cold alkali
(25) and hybridized at 65C for 48 h to 2-A |ig of DNA probes immobilized
on Hybond N+ membranes. Membranes were stringency washed at 65C in
0.1 X SSPE and 0.1% SDS for 60 min. Autoradiographs were scanned using
a scanning densitometer (Helena Laboratories), and peak values were corrected
for cell input by measuring the tritium count of isolated nuclei prior to the
run-on protocol (24).
Results
Camptothecin enhances transcription in the 5'-end of the
DHFR gene in vivo
First we estimated the relative rate of transcription in campto-
thecin-treated CHO Bll cells using an approach in which
nascent RNA was labeled in vivo with [5-H]uridine (Figure
1). Three DNA probes complementary to RNA synthesized
from the 5'-end, the middle portion or the 3'-end of the DHFR
gene were used to detect any differential effect on transcription
throughout the DHFR gene. In addition, a DNA probe comple-
mentary to the 3 '-half of the ribosomal RNA transcriptional unit
was used to assess the effect of camptothecin on polymerase I
transcription (Figure 2).
We found that treatment with 20 (J.M camptothecin for
30 min at 37C reduced the rate of total RNA synthesis to
in vitro nuclear run-on in vitro nuclear run-on
control camp
in vivo [3H]uridine labeling
lptothecin (30 min)
chat* &-30 min
Fig. 1. Exerimental design. In vitro nuclear run-ons were performed for
15 min at 30C using nuclei isolated from either untreated cells (controls)
or cells treated with camptothecin. In vivo [ 3 H]undine labeling was
performed for 15 min at 37C using untreated cells (controls),
camptothecin-treated cells labeled in the presence of camptothecin (+camp),
camptothecin-treated cells labeled in the absence of camptothecin (chase 0-
15 min), and camptothecin-treated cells chased 15 min with drug-free media
then labeled in the absence of camptothecin (chase 15-30 min).
10 kb
DHFR
pZH4 pB13-7
S-and tf-end
Fig. 2. Gene maps showing the locations of the DNA probes used to
hybridize RNA synthesized from the DHFR gene and rRNA genes. The
probes are: pZH4, a 1.9 kb coRIflgfll fragment covering the second
intron and the third exon ( - 1 - 2 . 9 kb downstream of the DHFR initiation
codon) (39); pB6-14, a 5.3 kb BamHI fragment covering part of the third
intron and the fourth exon (41); pB13-7, a 2.1 kb BamHI fragment covering
the last, sixth exon (40); pZH8, a 4.8 kb Satl-EcoR\ fragment
complementary to most of the 28S rRNA gene (a gift from G.Spivak and
I.Mellon).
32
-30% of that in untreated control cells as measured by liquid
scintillation counting of a small aliquot of isolated total RNA
(Figure 3). This inhibitory effect on total RNA synthesis by
camptothecin is consistent with results obtained by others
(3,5). When studying the effect of camptothecin on the RNA
polymerase II transcribing DHFR gene, it was found that
transcription was differentially affected throughout the gene.
RNA synthesis was enhanced in the 5'-end of the DHFR gene
to -150% of that of untreated control cells, while RNA
synthesis was severely inhibited in the middle portion and the
3'-end to 20% and 10% of that of untreated controls, respect-
ively. RNA synthesis from the 3'-half of the rRNA transcrip-
tional unit was virtually abolished in the presence of
camptothecin, a result which supports earlier findings (16).
Nuclear run-on technique reveals a dramatic increase in
transcription signal from the 5'-end of the DHFR gene
The effect of camptothecin on DHFR transcription was also
evaluated using the nuclear run-on technique. In this technique,
the in vivo distribution of RNA polymerases on a particular
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015
gene is scored by allowing engaged RNA polymerases to
elongate in vitro in the presence of [32P]UTP (25,26). The
results obtained support the results from the uridine-labeling
experiments in that the DHFR RNA synthesis was enhanced
in the 5'-end (Figure 4A,B). However, the enhancement of the
in vitro nuclear run-on labeling was much greater. In fact, the
[32P]UTP-incorporation in the run-on transcripts from the 5'-
end was 10-15 times higher in nuclei from camptothecin-
treated cells than in untreated control cells. This is to be
compared to the 50% increase of transcription in the equivalent
experiment using the in vivo uridine-labeling technique (see
Figure 3). Another discrepancy between the results obtained
using the two different techniques was that the marked inhibi-
160-1
control, n = 3-5
+camp. n - 3-5
total RNA 5-end middle rRNA
Fig. 3. Levels of in vivo transcription from the DHFR and rRNA genes in
cells treated with 20 |iM camptothecin at 37C for 30 min. Nascent RNA
was labeled in vivo for 15 min in untreated cells in the absence of
camptothecin (control) or in camptothecin-treated cells in the presence
of 20 nM camptothecin (+camp). RNA was isolated and hybridized to
DNA probes described in Figure 2. The 3H counts were measured in
a scintillation counter and the c.p.m. values were in the range of
-50400 c.p.m. over background for the DHFR sequences and
-600-8000 c.p.m. for the rRNA sequence. The values are expressed relative
to untreated control cells (100%) and represent the mean of n independent
experiments with bars showing the sample standard deviation.

tion of RNA synthesis from promoter-distal regions of the
DHFR gene observed with the in vivo [3H]uridine-labeling
approach was not that obvious when using the in vitro nuclear
run-on technique. However, synthesis of rRNA was severely
inhibited by camptothecin when measured with both transcrip-
tion assays.
Recovery of transcription bv the removal of camptothecin
In the next set of experiments, we wanted to address the
question of whether the effects of camptothecin on transcrip-
tion were reversible. Cells were treated with 20 yiM campto-
thecin for 30 min at 37C, and nascent RNA was [3H]uridine-
labeled in vivo for 15 min in camptothecin-free media (chase
0-15 min). In addition, some cells were allowed to incubate
in drug-free media for 15 min prior to the uridine-labeling of
the RNA in drug-free media (chase 15-30 min) (Figure 1).
The results show that a chase of camptothecin-treated cells
with drug-free media only partially restored synthesis of total
RNA from -30% to -50-60% of that synthesized in untreated
control cells (Figure 5). The same was true for rRNA synthesis
where in vivo [3H]uridine-labeling of nascent rRNA rebounded
from < 1 % to -50% of that of controls. The measurements of
1600-
B [J
g 1400-
anscr iption
1200-
j1
o
1000-
800-
1
1
> 600-
c H control, n = 3-5
1 400-
1 H camp, n = 3-5
relat
200-
n_ f=mA
total RNA 5-end middle 3-end rRNA
Fig. 4. Relative rate of in vitro nuclear run-on transcription. Nuclei were
prepared from cells treated with 20 JIM camptothecin at 37C for 30 min
and from untreated control cells, and nascent RNA was labeled with
[32P1UTP at 30C for 15 min. RNA was then isolated and hybridized to
DNA probes described in Figure 2. (A) Autoradiograph showing that
camptothecin enhanced the run-on signal in the 5'-region of the DHFR gene
and suppressed the signal in the 3'-half of the rRNA gene. (B) The results
from a number of experiments are summarized and the values are expressed
relative to untreated control cells and represent the mean of n independent
experiments with bars showing the sample standard deviation.
Effect of camptothecin on transcription
transcription from the 5'-end of the DHFR gene revealed that
transcription remained elevated at levels of -50% of that of
controls even after 15-30 min of incubation in drug-free
media. Transcription from the middle portion of the DHFR
gene rebounded from -20% to -60% during a 15 min chase
and to control levels during a 15-30 min chase. At the 3'-end
of the DHFR gene, the transcription showed no signs of
recovery during a 0-15 min chase but transcription was fully
recovered after a 15-30 min incubation in drug-free media.
Discussion
The effect of the anti-cancer drug camptothecin on RNA
polymerase II transcription of the DHFR gene was assessed
using both in vivo [3H]uridine-labeling and in vitro nuclear
run-ons to study nascent RNA synthesis. Although both assays
showed that RNA synthesis was enhanced by camptothecin in
promoter-proximal regions while reduced in promoter-distal
regions, the two transcription assays gave quantitatively differ-
ent results. While the in vivo pulse-labeling assay snowed a
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015
1.5-fold enhancement of promoter-proximal transcription by
camptothecin, the in vitro run-on assay revealed a 10- to 15-
fold increase in this region (compare Figures 3 and 4B).
Furthermore, transcription from promoter-distal regions
appeared more severely inhibited by camptothecin when using
the in vivo pulse-labeling technique in comparison to the
in vitro nuclear run-on technique (compare Figures 3 and 4B).
Discrepancies in the results obtained from in vivo [3H]uridine
pulse-labeling and in vitro nuclear run-on transcription have
been reported before (27). It is believed that the two assays
measure different aspects of transcription. While the in vivo
undine pulse-labeling technique is thought to reflect transcrip-
tion elongation in intact cells, the in vitro nuclear run-on
technique merely reflects the abundance of RNA polymerases
+camp, n = 3-5
chase 0-15 min, n= 2
chase 15-30 min. n = 2
total RNA 5-end middle 3'-end rRNA
Fig. 5. Reversal of the effect of camptothecin on RNA synthesis. Cells were
treated with 20 uM camptothecin for 30 min at 37C for 30 min followed
by the labeling of nascent RNA in vivo with [3H]uridine according to the
experimental design described in Figure 1. RNA was isolated and
hybridized to DNA probes described in Figure 2. The 3H counts were
measured in a scintillation counter and the c.p.m. values were in the range
of -50-400 c.p.m. above background for the DHFR sequences and
-600-8000 for the rRNA sequence. The values are expressed relative to
untreated control cells (100%) and represent the mean of n independent
experiments with bars showing the sample standard deviation.
33
M.Ljungman and P.C.Hanawalt
B
I C*mptoth0cln 30 mlnutmu
Chmmo O-15 mlnutms
Fig. 6. Cartoon illustrating the effect of camptothecin on the synthesis of
RNA from the DHFR gene. (A) Before the treatment with camptothecin,
RNA polymerases are spaced throughout the DHFR gene. (B) Following a
30 min treatment with camptothecin, the translocation of RNA polymerases
is blocked while free RNA polymerases are able to initiate transcription.
This leads to the accumulation of RNA polymerases at the 5'-region of the
gene. (C) When the effect of camptothecin is chased with a drug-free
medium, some of the RNA polymerases that accumulated during the drug-
treatment are permitted to elongate. (D) Following a longer chase, the
polymerases are once again spaced throughout the gene.
within a specific gene. Thus, the nuclear run-on assay is not
necessarily a good measure of rates of transcription in cells
(27). However, this technique is still informative on the
abundance and location of polymerases within a gene whether
they are elongating or not. The release of blocked polymerases
in the run-on assay may result from the preparation of nuclei
which introduces DNA nicks and modifies the interactions of
proteins with DNA.
Assuming that the in vitro run-on technique reflects the
abundance of RNA polymerases in a particular region and that
the in vivo pulse-labeling technique reflects the rate of RNA
synthesis in vivo (27), we conclude that a large number of RNA
polymerases accumulated in the promoter-proximal region of
the DHFR gene during the camptothecin treatment (Figure
6B). In support of this, we have recently obtained results using
a psoralen photocross-linking approach suggesting that the
accessibility of the DNA in the promoter region of the DHFR
gene is reduced following camptothecin treatment (unpublished
results). Furthermore, the majority of these polymerases were
not capable of elongating in vivo since the huge increase in
the in vitro run-on signal (10 to 15-fold) was not matched by
a comparable increase in the in vivo pulse-labeling (1.5-fold).
These results suggest that camptothecin enhances transcrip-
tional initiation but that the elongation of the polymerases is
blocked shortly after initiation, most likely by a topoisomerase
I complex trapped by camptothecin on the transcribed strand.
Following the removal of camptothecin we found that
34
synthesis of DHFR RNA recovered in a wave that progressed
in a 5' to 3' direction (Figure 6C). The rate of RNA synthesis
from the middle portion of the gene was approaching control
levels within the first 15 min of the chase, while little recovery
was detected in the 3'-end until after an additional 15 min of
chase (Figure 6D). Interestingly, the polymerases that had
accumulated at the 5'-end during drug treatment did not appear
to translocate down the gene all at once following drug
removal. Regarding the kinetics of RNA synthesis recovery
throughout the gene, it has been estimated that the rate of
elongation in vivo for mammalian RNA polymerases is -30
nucleotides per second (28). That means that it would take an
RNA polymerase -17 min to traverse the 30 kb long DHFR
gene from start to finish. We found in our study that the
synthesis of RNA from the 3'-end of the DHFR gene resumed
control levels following a 15-30 min chase in camptothecin-
free media. This result is in excellent accord with the estimated
elongation rate of the RNA polymerase (28) and suggests that
RNA synthesis following drug removal is either carried out
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015
by newly initiated polymerases or by polymerases that accumu-
lated at the 5'-end of the DHFR gene during the camptothecin
treatment.
What are the mechanisms for the enhanced initiation of the
DHFR gene by camptothecin? The role of DNA topoisomerase
I during the transcription process is to relax the torsional
strain induced by the elongating RNA polymerase (16,21,29).
Inhibition of topoisomerase I by camptothecin may result in
the escape of transcription-induced torsional tension. Although
transcriptional elongation is limited in the presence of campto-
thecin (15,16, this study), some transcription-induced super-
coiling may still be generated both in the wake of and ahead
of the polymerase (29). Since the DHFR promoter region is
located between two divergently transcribing genes (30), the
inhibition of topoisomerase I may lead to an accumulation of
transcription-generated negative torsional tension in this region.
Negative torsional tension has been found to enhance the early
stages of transcription both in vitro (31-33) and in vivo (34
37). We have previously found evidence for the presence of
negative torsional tension in the promoter region of the DHFR
gene in both human (38) and hamster cells (24).
In accordance with results from a previous study (16),
camptothecin was found to severely reduce the rate of RNA
synthesis from promoter-distal regions of the RNA polymerase
I transcribed ribosomal RNA genes (Figures 3 and 4). However,
the results obtained for the RNA polymerase II transcribed
DHFR gene following camptothecin treatment differ somewhat
from the results obtained on the transcription of the ribosomal
genes. Our nuclear run-on data from camptothecin-treated
cells suggest a much greater relative accumulation of RNA
polymerases at the 5'-end of the DHFR gene (10 to 15-fold)
than that reported for the 5'-end of the ribosomal genes (1.4-
fold) (16). Perhaps the high transcriptional activity of the
ribosomal genes in dividing cells does not allow for any
additional polymerases to accumulate onto the DNA template
even in the presence of camptothecin. In fact, a higher relative
accumulation of RNA polymerases at the 5'-end of the
ribosomal genes by camptothecin has been observed when
confluent cells were used (16).
In conclusion, camptothecin was found to enhance the
rate of transcriptional initiation while inhibiting the rate of
elongation by RNA polymerase II in the DHFR gene. This
resulted in the accumulation of RNA polymerases in the 5'-
region of the DHFR gene. Following drug removal, the RNA

synthesis returned in a 5' to 3' direction. A possible application
of a protocol of camptothecin treatment and reversal is that it
could be used in studies where a synchronized wave of
transcription from the 5'-end to the 3-end is desired. Of interest
would be to study the phenomena of transcription-coupled
DNA repair in different parts of a gene in which transcription
proceeds in such a synchronized manner. In addition, further
studies will determine how important the effects on RNA
synthesis are for the anti-tumor activity of camptothecin.
Acknowledgements
We would like to thank the members of our research group at Stanford
University for stimulating discussions and ideas that have been very valuable
for this study. We thank Joyce Hunt and Maria Furneri for technical assistance,
Robert Schimke for his generous gift of the CHO Bl 1 cell line and Graciela
Spivak. Isabel Mellon, Linus Ho and Lawrence Chasin for their generous
gifts of DNA probes We also thank Sara Ljungman and Al Rehemtulla for
critically reading this manuscript This work was supported by an outstanding
investigator award from the National Cancer Institute and a postdoctoral
fellowship from the National Institute of Health.
References
I.Chen,A.Y. and Liu.L.F. (1994) DNA topoisomerasesessential enzymes
and lethal targets. Annu. Rev. Pharmacol Toxicol., 34, 191-218.
2.Kessel,D., Bosmann.H. and Lohr.K. (1972) Camptothecin effects on DNA
synthesis in murine leukemia cells. Biochim. Biophys Acta, 269, 210-216.
3.Kessel,D. (1971) Effects of camptothecin on RNA synthesis in leukemia
LI210 cells. Biochim. Biophys. Acta, 246, 225-232
4.Wu,R , Kumar.A. and WarnerJ. (1971) Ribosome formation is blocked by
camptothecin, a reversible inhibitor of RNA synthesis. Proc. Nad Acad.
Set. USA, 68, 3009-3014.
5.Abelson,H. and Penman.S. (1972) Selective interruption of high molecular
weight RNA synthesis in HeLa cells by camptothecin. Nature New Biol.,
237, 144-146.
6.Nelson,W.G. and Kastan.M.B. (1994) DNA strand breaks: the DNA
template alterations that trigger p53-dependent DNA damage response
pathways. Moi. Cell. Biol., 14, 1815-1823.
7 Horowitz,M. and Horowitz,S. (1971) Intracellular degradation of HeLa
and adenovirus type 2 DNA induced by camptothecin. Biochem. Biophys
Res. Commun., 45, 723-727.
8. Hsiang,Y.-H , Lihou.M. and Liu.L. (1989) Arrest of replication forks by
drug-stabilized lopoisomerase I DNA cleavable complexes as a
mechanism of cell killing by camptothecin. Cancer Res , 49, 5077-5082.
9.Tsao,Y.P., Darpa,P. and Liu.L.F. (1992) The involvement of active DNA
synthesis in camptothecin-induced G2 arrestaltered regulation of p34cdc2
cyclin-B. Cancer Res., 52, 1823-1829.
10 Hsiang,Y.-H., Hertzberg.R., Hecht.S. and Liu.L. (1985) Camptothecin
induces protein-linked DNA strand breaks via mammalian topoisomerase
I. J. Biol. Chem., 260, 14873-14878.
I I.Hsiang,Y.-H. and Liu.L. (1988) Identification of mammalian topoisomerase
I as an intracellular target of the anticancer drug camptothecin. Cancer
Res., 48, 1722-1726.
12. WangJ.C. (1985) DNA topoisomerase. Annu. Rev. Biochem , 54, 665-697.
B.WangJ. (1971) Interaction between DNA and Escherichia coli protein
omega. J. Mol. Biol., 55, 523-533.
l4.ChampouxJ (1977) Strand breakage by the DNA untwisting enzyme
results in covalent attachment of the enzyme to DNA. Proc. Nail Acad.
Sci. USA, 74, 3800-3804.
15.Bendixen,C, Thomsen.B., AlsnerJ. and Westergaard.O. (1990)
Camptothecin-stabilized topoisomerase I-DNA adducts cause premature
termination of transcription. Biochemistry, 29, 5613-5619.
l6.Zhang,H., WangJ. and Liu.L. (1988) Involvement of DNA topoisomerase
I in transcription of human ribosomal RNA genes. Proc. Natl Acad. Sci.
USA, 85,1060-1064
l7.Merino,A., Madden,K., Lane.W., ChampouxJ. and Reinberg.D. (1993)
DNA topoisomerase I is involved in both repression and activation of
transcription. Nature, 365, 227-232.
18. Kretzscnmar.M., Meisterernst,M. and Roeder,R. (1993) Identification of
human DNA topoisomerase I as a cofactor for activator-dependent
transcription by RNA polymerase II. Proc. Natl Acad. Sci. USA, 90,
11508-11512.
l9.Gilmour,D. and Elgin.S. (1987) Localization of specific topoisomerase I
interaction within the transcribed region of active heat shock genes by
Effect of camptothecin on transcription
using the inhibitor camptothecin. Mol. Cell. Biol., 7, 141-148.
20. Stewart.A. and SchQtz,G. (1987) Camptothecin-induced in vivo topoiso-
merase I cleavages in the transcriptionally active tyrosine amino-
transferase gene. Cell, 50, 1109-1117.
21 Stewart.A.F., Herrera,R.E. and Nordheim.A. (1990) Rapid induction of c-
fos transcription reveals quantitative linkage of RNA polymerase II and
DNA topoisomerase I enzyme activity. Cell, 60, 141-149.
22 Kaufman.R. and Schimke.R. (1981) Amplification and loss of dihydrofolate
reductase genes in a Chinese hamster ovary cell line. Mol. Cell. Biol., 1,
1069-1076.
23.FederJ. (1990) Ph.D. thesis, Stanford University.
24.Ljungman,M. and Hanawalt,P.C. (1995) Presence of negative torsions]
tension in the promoter region of the transcriptionally poised dihydrofolate
reductase gene in vivo. Nucleic Acids Res., 23, 1782-1789.
25.Chen-Kiang,S. and Lavery.D. (1989) Pulse labeling of heterogeneous
nuclear RNA in isolated nuclei. Methods Enzymol., 180, 82-96.
26. Ausubel.F., Brent,R., Kingston.R., Moore.D., SeidmanJ., SmithJ. and
Struhl.K. (1993) Current Protocols in Molecular Biology. Wiley, New York.
27.Schilling,L. and Famham.P. (1994) Inappropriate transcription from the 5'
end of the murine dihydrofolate reductase gene masks transcriptional
regulation. Nucleic Acids Res., 22, 3061-3068.
28.Alberts3., Bray.D., LewisJ., Raff.M., Roberts.K. and WatsonJ. (1994)
Molecular Biology of the Cell. Garland, New York.
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015
29.Liu,L.F. and WangJ.C. (1987) Supercoiling of the DNA template during
transcription. Proc. Natl Acad. Sci. USA, 84, 7024-7027.
3O.Mitchell,P, Carothers.A., HanJ., HardingJ., Kas,E., Vernolia,L. and
Chasin.L. (1986) Multiple transcription start sites, DNase I-hypersensitive
sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.
Mol. Cell. Biol, 6, 425-440.
31.GoodrichJ.A. and Tjian.R. (1994) Transcription factors HE and IIH and
ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell,
77, 145-156.
32 ParvinJ.D. and Sharp.P.A. (1993) DNA topology and a minimal set of
basal factors for transcription by RNA polymerase-II. Cell, 73, 533540.
33 Mizutani.M., Ohta.T., Watanabe.H., Handa.H. and Hirose,S. (1991)
Negative supercoiling of DNA facilitates an interaction between
transcription factor-IId and the fibroin gene promoter. Proc. Natl Acad.
Sci. USA, 88, 718-722.
34 Harland,R.M., Weintraub,H. and McKnight,S.L. (1983) Transcription of
DNA injected into Xenopus oocytes is influenced by template topology.
Nature, 302, 38-43.
35.Schultz,M.C, Brill.SJ., Ju.Q.D., Sternglanz.R. and Reeder.R.H. (1992)
Topoisomerases and yeast rRNA transcriptionnegative supercoiling
stimulates initiation and topoisomerase activity is required for elongation.
Genes Dev., 6, 1332-1341.
36. Weintraub.H., Cheng.P.F. and Conrad.K. (1986) Expression of transfected
DNA depends on DNA topology. Cell, 46, 115-122.
37. Dunaway.M. and Ostrander.E.A. (1993) Local domains of supercoiling
activate a eukaryotic promoter in vivo. Nature, 361, 746-748.
38.Ljungman,M. and Hanawalt,P.C. (1992) Localized torsional tension in the
DNA of human cells. Proc. Natl Acad. Sci. USA, 89, 6055-6059.
39.Mellon,I., Spivak.G. and Hanawalt,P.C. (1987) Selective removal of
transcription-blocking DNA damage from the transcribed strand of the
mammalian DHFR gene. Cell, 51, 241-249.
40. Carothers.A., Urlaub,G., Ellis.N. and Chasin.L. (1983) Structure of the
dihydrofolate reductase gene in Chinese hamster ovary cells. Nucleic Acids
Res., 11, 1997-2012.
Received on July 21, 1995; revised on 13 September, 1995; accepted on
September 21, 1995
35
Downloaded from http://carcin.oxfordjournals.org/ at University of Georgia on June 20, 2015