From the Department of Molecular Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The expression of P-glycoproteins encoded by the
mdr gene family is associated with the emergence of the
multidrug resistance phenotype in animal cells. mdr
expression can be induced by many extracellular stimulants including
cytotoxic drugs and chemical carcinogens. However, little is known
about the mechanisms involved. Here, we report that the expression of
the rat mdr1b can be induced by anticancer drug
daunorubicin. Further analysis identified a bona fide p53-binding site
spanning from base pairs 199 to
180 (5'-GAACATGTAGAGACATGTCT-3') in
the rat mdr1b promoter that is essential for basal and
daunorubicin-inducible promoter activities. In addition, our results
show that wild-type p53 can up-regulate not only the promoter function
but also endogenous expression of the rat mdr1b. To the
best of our knowledge, this is the first report showing that a specific
p53-binding site is involved in the transcriptional regulation of
mdr gene by wild-type p53. Since p53 is a sensor for a wide
variety of genotoxic stresses, our finding has broad implications for
understanding the mechanisms involved in the inducible expression of
mdr gene by anticancer drugs, chemical carcinogens, UV
light, and other DNA-damaging agents.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multidrug resistance (MDR),1 a major obstacle to the effective chemotherapy of many human malignancies, is characterized by the increased survival of cells in the presence of cytotoxic drugs with unrelated structures. A major mechanism for the development of MDR phenotype is overexpression of P-glycoproteins which are encoded by the MDR gene family (for reviews, see Refs. 1 and 2). The MDR gene family contains two members in humans and three in rodents. However, only one human (MDR1) and two rodent (mdr1a and mdr1b) mdr genes are functionally related to the MDR phenotype. High mdr mRNA levels are seen in certain tumor types before chemotherapy and, in some cases, are associated with relapse following chemotherapy (for reviews, see Refs. 1 and 3).
Increased mdr gene expression occurs in cultured cells selected by continuous exposure to both anticancer drugs and other cytotoxic agents, in which gene amplification is believed to be often associated with the overexpression of mdr genes (4, 5). However, increased mdr gene expression preceding gene amplification has been observed in early passages of drug-selected cells (6). Transient exposure of cells to different cytotoxic agents such as antitumor drugs (7-10), chemical carcinogens (11-19), and UV irradiation (20), etc. is also able to activate mdr expression, indicating that increased mdr expression is mediated by complex mechanisms.
The precise mechanisms of the induction of mdr gene expression by anticancer drugs, chemical carcinogens, UV, and other DNA-damaging agents remain unknown. It has been suggested that both post-transcriptional and transcriptional mechanisms are involved (7). A possible role for the cytoskeleton in post-transcriptional stabilization of mdr1 mRNA in rat hepatocytes treated with certain agents was suggested (21). On the other hand, in rat liver cells, it was found that doxorubicin-mediated mdr1 mRNA induction was fully inhibited by actinomycin D, suggesting that transcriptional regulation is involved (10). Nuclear run-off and transfection analyses showed that AAF-, methylcholanthrene-, aflatoxin B1-, methyl methanesulfonate-, or mitoxantrone-induced mdr1 expression is also associated with increased rates of transcription (9, 11, 15).
Here, we show that the expression of the rat mdr1b can be
induced by anticancer drug daunorubicin. Further analysis demonstrates that a bona fide p53-binding site (5'-GAACATGTAGAGACATGTCT-3') located
within bp 199 to
180 of rat mdr1b promoter is essential for not only basal but also daunorubicin-inducible promoter functions. We also provide evidence indicating that both the promoter activity and
endogenous expression of the rat mdr1b could be modulated by
wild-type p53. Although the modulation of mdr expression by either mutant or wild-type p53 has been noted, no p53-binding sites
have been identified previously (22-27). The present report represents
the first evidence that a specific p53-binding site is involved in the
transcriptional regulation of the mdr gene. Since p53 is
responsive to a variety of genotoxic stresses (for reviews, see Refs.
28 and 29), which also induce mdr gene expression, our
finding has important implications for understanding mechanisms
involved in the inducible expression of drug-resistant genes by
DNA-damaging agents.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents--
Reagents were purchased from the following
companies: [-32P]dNTPs, [
-32P]UTP,
and [14C]chloramphenicol from ICN Biomedicals (Costa
Mesa, CA); poly(dI-dC)·poly(dI-dC) and acetyl-coenzyme A from
Pharmacia/LKB (Upsala, Sweden); oligonucleotides from Genosys Inc.
(Houston, TX); and rabbit polyclonal antibodies against c-Jun and p65
subunit of NF-
B from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)
and p53 antibody PAb421 from Calbiochem (Cambridge, MA). All other
reagents were purchased from Sigma.
Plasmids--
Wild-type (pCMVp53) and mutant
(pCMVp53248) p53 expression vectors were
generously provided by Dr. G. Lozano of M. D. Anderson Cancer
Center. Rat mdr1b-CAT reporter constructs (1288 RMICAT,
243 RMICAT,
163 RMICAT, and
243 RMICAT-
m) were constructed as
described previously (30, 31).
214 RMICAT,
214 RMICAT-m1, and
214
RMICAT-m2 were constructed by a PCR method using
1288 RMICAT as the
template and 5'-TCCATTTTAGCTTCCTTAG-3' as the 3' primer in combination
with each of the following 5' primers: 1) 5'-GGGGGTACCATATGGAGAGTTACCTGAAC; 2)
5'-GGGGTACCATATGGAGAGTTACCTGAATCGGTAGAGACATGTCTGT; 3)
5'-GGGGGTACCATATGGAGAGTTACCTGACATGTAGAGAACCGTCTGTGTTAATG.
All three 5' primers contained a KpnI site (underlined). The
PCR products were digested with KpnI and XbaI and
inserted into the KpnI/XbaI sites of a CAT vector
(18).
Cell Culture, DNA Transfection, and Chloramphenicol Acetyltransferase (CAT) Assay-- The rat hepatoma H-4-II-E cells were purchased from the American Type Culture Collection (ATCC 1548). Human osteosarcoma SAOS-2 cells, low-passage rat embryonic fibroblasts (REFs), A1-5 cells, and T101-4 cells were generously provided by Dr. G. Lozano. All the cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (Life Technologies, Inc.), 1 mM glutamine, and 50 µg of neomycin/ml in a humidified incubator containing 5% CO2. Prior to treatment, cells were grown in the medium to 70-80% confluence. Then cells were treated with daunorubicin (7 µg/ml) for various periods of time and harvested for the preparation of nuclear extracts and RNA.
The calcium phosphate precipitation method (33) was used to transfect cells with DNA. In brief, 2 h before transfection, cells in the exponential growth phase (approximately 70-80% confluence) were plated in Corning six-well plates. DNA-CaPO4 precipitate was added to the medium and incubated for 5-6 h. After cells were shocked with 15% glycerol for 30 s, washed with phosphate-buffered saline, cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Stable transfectant A1-5 and H-4-II-E cells were established by co-transfecting the cells with the reporter constructs and pcDNA3 (Introgen, Carlsbad, CA) at 5:1 ratio followed by selection with G418 (400 µg/ml, Life Technologies, Inc.). Pools of G418-resistant cells were collected and used for further analysis. In transient transfection assays, cells were directly treated with daunorubicin (7 µg/ml) 24 h after transfection. After 20-24 h of drug exposure, cells were harvested. CAT activities in the cell extracts were measured by a previously described method (34) using total protein extract (measured by the Bio-Rad protein assay kit) as a reference. Relative CAT activity levels were calculated by a PhosphorImager (model 400S, Molecular Dynamics) in terms of the conversion of [14C]chloramphenicol into acetylated chloramphenicol.Preparation of Nuclear Extracts and GMSA-- Nuclear extracts were prepared from H-4-II-E cells by the method of Digman et al. (35) with modifications as described previously (31). GMSAs were performed with approximately 5 µg of nuclear proteins in a total volume of 20 µl of binding mixture containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.5 mM dithiothreitol, 10% glycerol, 0.2% Nonidet P-40, 3 µg of poly(dI-dC)·poly(dI-dC), and radiolabeled DNA probe at room temperature for 20 min.
RNase Protection Assay--
A 162-nucleotide antisense RNA probe
(37 to +125) was synthesized using T7 RNA polymerase as described
previously (30). Of total RNA from cells, either 20 µg (for the
mdr1b probe) or 1 µg (for the 18 S rRNA probe) was
hybridized with 32P-labeled antisense RNA probes (2 × 105 cpm) and subjected to RNase protection assays as
described previously (17, 30). The protected RNA products were analyzed
on a 7% denaturing polyacrylamide gel and quantified using a Personal Densitometer SI (Molecular Dynamics).
Reverse Transcriptase-PCR Amplification and DNA Sequencing-- Two micrograms of total RNA isolated from H-4-II-E cells was used for reverse transcriptase reaction. On completion of the reverse transcriptase reaction, the enzyme was inactivated by heating to 94 °C for 56 min. Ten picomoles of each 5' primer (5'-CCTGAAGACTGGATAACTGTCATGGAGGAT) and 3' primer (5'-AGAGGGGGCCGAGTACTATCTACAAGGTAA) were used in a PCR to amplify rat p53 cDNA (30 cycles of 1 min at 94 °C, 2 min at 45 °C, and 3 min at 72 °C). PCR products were electrophoresed on an agarose gel, purified, and subjected to automated sequencing (ABI PRISM).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Daunorubicin Induces mdr1b Expression in Rat Hepatoma Cells-- To investigate whether the expression of the rat mdr1b gene expression is regulated by the anticancer drug daunorubicin, we treated rat hepatoma H-4-II-E cells with daunorubicin (7 µg/ml). At various time intervals, cells were harvested and mdr1b mRNA levels were measured by the RNase protection assay. As shown in Fig. 1, the steady-state mdr1b mRNA levels in these cells were elevated after exposure to daunorubicin for 12 and 24 h. Increases of about 3-5-fold were seen in three independent experiments. Similar results were obtained in cells treated with adriamycin and chemical carcinogen 2-AAAF (data not shown). These results demonstrated that rat mdr1b expression can be induced by these cytotoxic agents in rodent cells.
|
Rat mdr1b Promoter Responds to Daunorubicin Treatment--
To
investigate the possible involvement of transcriptional regulation in
the induction of the rat mdr1b gene expression by daunorubicin and, if so, to identify DNA sequences responsible for the
daunorubicin induction of mdr1b expression, we generated a
set of 5' deletion mutant CAT constructs and transfected them into
H-4-II-E cells following treatment with or without daunorubicin. When
1288 RMICAT,
243 RMICAT, and
214 RMICAT reporter constructs containing 1288, 243, and 214 bp of the rat mdr1b upstream
sequences, respectively, plus 125 bp downstream from the transcription
start site, were transiently transfected into H-4-II-E cells, CAT
activities increased an approximately 1.6-1.9-fold in daunorubicin
treated versus untreated cells (p < 0.05)
(Fig. 2). However, when
163 RMICAT,
which contains additional deletion to
163 bp was transfected, basal
transcriptional activities were reduced more than 80%. More importantly, the deletion also abolished daunorubicin inducibility (Fig. 2). Together, these results indicated that the rat
mdr1b promoter can respond to daunorubicin treatment and
that the sequence from bp
214 to
163 is essential for the
promoter's daunorubicin responsiveness.
|
214 to
177 bp Is Sufficient to Confer mdr1b Promoter
Inducibility by Daunorubicin--
To further substantiate the above
observations, we generated two additional constructs by inserting
sequences from bp
214 to
127 (containing NF-
B site) or
214 to
177 (containing no NF-
B site), respectively, into a
pBLCAT2 vector containing the tk basal promoter
and a CAT reporter gene. These constructs were then transiently
transfected into H-4-II-E cells, and CAT expression was measured. As
shown in Fig. 3, both constructs are
capable of responding to daunorubicin treatment, giving rise to
comparable levels of induction, whereas daunorubicin did not have
effects on the tk promoter. These results suggested that
NF-
B site is dispensable for the inducibility of mdr1b
promoter, and that the sequence from bp
214 to
177 may contain
important cis-acting elements responsible for the induction
of mdr1b promoter activity by daunorubicin.
|
Daunorubicin Induces Formation of a Specific Protein-DNA Complex
Within bp 201 to
177--
To test whether daunorubicin treatment
could induce protein DNA binding at sequences within bp
214 to
177,
we prepared nuclear extracts from H-4-II-E cells treated with or
without daunorubicin and performed GMSAs. As shown in Fig.
4A, a major DNA-protein complex was formed in the daunorubicin-untreated nuclear extracts when
a double-stranded oligonucleotide spanning bp
214 to
177 was used
as the probe (lane 1, C1). The binding activity of this complex remained largely unchanged after daunorubicin treatment (lanes 2-5 versus lane 1). However, a slow migrating
protein-DNA complex was induced 1 h after treatment (C2,
lane 2). The binding activity of this induced complex remained
elevated but gradually reduced throughout the 12-h induction period
(lanes 2-5).
|
Daunorubicin-induced DNA-binding Protein Is p53--
In examining
the DNA sequence of bp 201 to
177 (fragment B), we found within it
a sequence (bp
199 to
180) strikingly similar to the p53-binding
consensus sequence 5'-PuPuPuC(A/T)(A/T)GPyPyPy-3' (38), with only 2 base pair mismatches. A comparison of the putative mdr1b
p53-binding site with the p53 consensus sequence and the p53-binding
site from the gadd45 third intron (39) is shown in Fig.
5B. To determine whether the
sequence located between bp
199 and
180 was indeed a p53-binding
site, we carried out GMSAs using a double-stranded oligonucleotide
spanning bp
214 to
177 as the probe and nuclear extracts prepared
from daunorubicin-treated H-4-II-E cells in the presence of various
unlabeled oligonucleotides as competitors. As shown in Fig.
5A, daunorubicin-induced DNA-protein complex was efficiently
competed only by the gadd45 p53-binding sequences
(lane 2), but not by the mutated gadd45
p53-binding sequence (lane 3) and other unrelated sequences,
i.e. AP-1 (40) and NF-
B (41) (lanes 4 and
5). Instead, the protein binding activity was actually
enhanced by these unrelated oligonucleotides (compare lanes
3-5 to lane 1). The exact reasons for the enhanced binding activities are not clear at the present.
|
p53-binding Site Is Required for the Daunorubicin-inducible
promoter Activities--
To characterize the functional role of the
identified p53-binding site, the same mutations in Fig. 4C
were introduced within the context of the wild-type 214 RMICAT
construct, and resultant recombinants were designated
214 RMICAT-m1
and
214 RMICAT-m2. These mutant constructs were then transfected into
H-II-4-E cells following treatment with or without daunorubicin. As
shown in Fig. 6A, both
mutations abolished the daunorubicin responsiveness. Similar results
were obtained when the same mutations were introduced into heterologous
(tk) promoter constructs (Fig. 6B). These results suggested that the integrity of p53 binding is essential for the daunorubicin inducible-promoter function of the rat
mdr1b.
|
Wild-type but Not Mutant p53 Transactivates mdr1b Promoter
Activity--
To investigate whether p53 is able to regulate rat
mdr1b promoter function, A1-5 cells, which were derived
from primary REFs transformed by a p53 temperature-sensitive
mutant p53Val-135 (46), were stably transfected with
reporter constructs containing either a wild-type p53-binding site
(214 RMICAT) or a mutated p53 site (
214 RMICAT-m1). As expected,
when stably transfected A1-5 cells were shifted from the restrictive
(37 °C, cells contain mutant p53) to permissive (32.5 °C, cells
contain wild-type p53) temperature (46), CAT activity was clearly
induced in cells stably transfected with wild-type
214 RMCAT but not
those stably transfected with mutant
214 RMCAT-m1 (Fig.
7A).
|
Endogenous mdr1b Expression Is Modulated by Wild-type p53-- To assess the regulation of endogenous mdr1b expression by p53, we examined mdr1b mRNA levels following temperature shift in A1-5 cells. We reasoned that if the mdr1b is a true p53 target gene, its expression should increase following wild-type p53 induction after temperature shift. As a control, we also measured mRNA levels in REFs and T101-4 cells following temperature shift. REFs has an endogenous wild-type p53, whereas T101-4 cells, like A1-5 cells, are derived from REFs but carry a non-temperature-sensitive p53 mutant (46). RNase protection assays revealed mdr1b mRNA levels increased a 3-6-fold in A1-5 cells after temperature shift from 37 °C (mutant p53) to 32.5 °C (wild-type p53) (Fig. 8, compare lanes 3 and 4 to 1 and 2). This induction was unlikely due to a nonspecific phenomenon by the temperature change, because no induction of mdr1b expression was observed in control REFs or T101-4 cells (compare lanes 7 and 8 to 5 and 6, and 11 and 12 to 9 and 10). These results indicated that the up-regulation of the mdr1b in A1-5 cells after temperature shift was induced by wild-type p53, and that p53 is indeed capable of modulating endogenous mdr1b gene expression.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we identified an authentic p53-binding site located
from bp 199 to
180 of the rat mdr1b that is important for both basal and daunorubicin-inducible promoter activities. We also
provided evidence showing that both the promoter function and
endogenous expression of the rat mdr1b can be modulated by wild-type p53. A bona fide p53 response gene should fit the following criteria (28): (i) the existence of p53-binding sites that can be
specifically recognized by p53; (ii) the ability of these sites to act
as a p53 response element, activating basal transcription in a
wild-type p53-dependent manner; (iii) the response of the element to p53 in the endogenous genomic promoter context; and (iv) the
induction of the target genes after cellular stress, such as DNA
damage, in cells containing wild-type but not mutant forms of p53. The
results presented in this study suggested that the rat mdr1b
meet all these criteria. Therefore, like p21/WAF1, mdm-2, GADD45, cyclin G,
bax, and IGF-BP3, etc., the rat mdr1b can be considered as a genuine p53 response gene.
Studies on the role of p53 in the regulation of the
mdr gene family have been quite controversial. Previous
studies have demonstrated that co-transfection with several mutant p53
expression vectors activated the human MDR1 and hamster
pgp-1 promoters, whereas co-transfection of a wild-type p53
expression vector had no effect or repressed the promoter activity
(22-26). Yet, no bona fide p53-binding sites were elucidated in these
studies. It has been shown that p53 can indeed repress activities of
many promoters without specific p53-binding sites (for review, see Ref.
28). The repression usually involves promoters containing the TATA box,
presumably through sequestering TATA-binding protein, transcription
activating factors, or interacting with other transcriptional
activators by p53. Paradoxically, the human MDR1 promoter is
a TATA-less promoter, therefore mechanisms involved in the repression
of the MDR1 promoter by wild-type p53 are unknown (26).
Similarly, it is also unclear how the p53 mutants gain the functions to
activate the human MDR1 promoter (24). In addition to
repressing it, wild-type p53 was also shown to stimulate the
MDR1 promoter in p53-null cell line in a transfection assay
(27). The reasons for the discrepancies among these studies are still
unknown but there are many plausible explanations: (i) p53 is a
multiple functional protein whose functions are regulated by a complex
network (48), its regulation of gene expression may differ not only
among cell types but also among physiological conditions under which
assays are performed; (ii) p53 can also bind transcriptional
coactivators such as CBP/p300 (49-51), which interacts with a battery
of other transcriptional regulators such as NF-B, Jun/Fos, nuclear
receptors, and their coactivators (for review, see Ref. 52). The
abundance of these transcriptional regulators may differ among
different cell settings and thereby influence the overall expression of transfected genes; (iii) different p53 expression vectors,
mdr reporter constructs, and time of analysis, may affect
the overall results. It should also be noted that even the transfection
procedures themselves may perturb endogenous p53 levels (53), affecting results of transient transfection assays. These considerations, taken
together, may explain the discrepancy of the transfection results
described above. In this regard, the identification of an authentic
p53-binding site in the mdr1b promoter region as described
herein is of particular importance, since it is the first time a
specific p53-binding site was elucidated to be implicated in the
transcriptional regulation of mdr gene expression.
Our observation of the involvement of wild-type p53 but not mutant p53 in the regulation of the rat mdr1b expression may be relevant to the increased expression of the mdr1b during hepatocarcinogenesis. Although the expression of mdr1 is highly activated, mutation of p53 does not always occur during hepatocarcinogenesis, at least in its early stage of liver tumor development (54). In addition, it has been known that the mdr1b expression in rat liver can be rapidly activated by chemical carcinogens such as 2-AAF and aflatoxin B1 (12, 13), however, in rat hepatocellular carcinomas induced by these carcinogens, p53 mutations do not always occur (55, 56). More importantly, van Gijssel et al. (57) recently reported that p53 activity can be also induced by 2-AAF and aflatoxin B1 in rat liver. When rat hepatoma H-4-II-E cells (contain wild-type p53) were treated with 2-AAAF, p53 activity was also been induced.2 These results, taken together, suggested that the activation of the rat mdr1b during chemical hepatocarcinogenesis may be due to the elevated wild-type p53 activities.
In broader prospects, it has been known that p53 is a universal sensor
of genotoxic stress (58), and can be induced by a wide variety of
DNA-damaging agents such as UV, -irradiation, carcinogens, and
cytotoxic drugs (for reviews, see Refs. 28 and 29). Strikingly, many of
these agents are also known inducers of mdr gene expression,
suggesting that p53 may contribute to the induction.
The p53-binding site identified in this study lies in the previously
identified murine mdr1b enhancer region (59). It overlaps a
palindromic sequence recognized by two peptides (41 and 49 kDa) (30),
and adjoins a downstream NF-B-binding site which is also important
for the promoter function (31). It is believed that most inducible
cis-acting elements contain multiple, distinct transcription
factor-binding sites that are part of a combinatorial mechanism that
relies on cooperative binding, interaction of transcriptional activator
proteins, and transcriptional synergy (60). Our study of site-directed
mutations demonstrated that the full promoter activity of the rat
mdr1b requires the integrity of both the p53-binding site
(bp
199 to
180) and NF-
B-binding site (bp
167 to
158) (Fig.
2) (31), suggesting that cooperative mechanisms between these two
cis-acting elements are implicated in the regulation of the
rat mdr1b expression. More recently, coactivator CBP/p300 was shown to interact with both p53 (49-51) and NF-
B (61, 62), and
enhance p53- and NF-
B-dependent transactivation,
respectively. The activity of the rat mdr1b promoter was
also found to be enhanced by CBP/p300.2 Taken together,
these may suggest that the binding of p53 and NF-
B to the
mdr1b promoter may recruit CBP/p300 and basal
transcriptional machinery to form a higher order transcription enhancer
complex, similar to that proposed in interferon-
and E-selectin
promoters (61, 63), which modulates inducible expression of the rat mdr1b. However, since our knowledge is rather limited at
this moment, the validity of this model still needs to be further
tested.
Finally, we would like to stress that, although our present results clearly demonstrated the direct involvement of p53 in the rat mdr1b gene regulation, the roles of p53 in the evolution of drug resistance in cancers remain to be critically evaluated. In clinical setting, the loss of functional p53 has been reported to be well correlated with de novo resistance to radiation and anticancer drugs, and some tumors with wild-type p53 respond well to chemotherapeutic drugs (Refs. 64-66, for review, see Ref. 29). However, it is unknown whether the correlation of drug resistance and p53 mutations is directly due to the activation of mdr by mutant p53, or other mechanisms such as alterations in drug targets, transporters, metabolisms, or the expression of genes regulating cell death and/or survival. Further studies are required to elucidate the molecular insights into how p53 regulates clinical drug sensitivity in cancer chemotherapy.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Guillermina Lozano for providing cell lines, plasmid constructs, and helpful discussions. We appreciate the technical assistance provided by Xinhui Zhou. We also thank members of Dr. Kuo's laboratory for critical review of the manuscript, and Jude Richard for editorial help.
![]() |
FOOTNOTES |
---|
* This work was supported in part by NCI, National Institutes of Health Grants CA56846, CA72404, and CA16672 (institutional core).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Pathology, Box 89, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-3256. Fax: 713-794-4672; E-mail: t_kuo{at}path.mdacc.tmc.edu.
1 The abbreviations used are: MDR, multidrug resistance; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; GMSA, gel mobility shift assay; AAF, acetylaminofluorene; 2-AAAF, N-acetoxy-2-acetylamino-fluorene; bp, base pair(s); tk, thymidine kinase; REF, rat embryonic fibroblasts.
2 G. Zhou and M. T. Kuo, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|