Analysis of the Effects of Daunorubicin and WP631 on Transcription
José Portugall*, Beatriz Martínl, Alejandro Vaquerol, Neus Ferrerl, Silvia Villamarínl and Waldemar Priebe2
lDepartamento de Biología Molecular y Celular. Instituto de Biología Molecular de Barcelona, CSIC. Jordi Girona, 18-26; 08034 Barcelona, Spain
2 The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
Abstract: The proficiency with which anthracyclines and other DNA-binding drugs target certain sequences in eukaryotic promoters offers a potential approach to interfere with the mechanisms that regulate gene expression in tumor cells. An in vitro transcription assay has been used to compare the ability of the bisintercalating anthracycline WP631 and the monointercalating anthracycline daunorubicin in terms of their ability to inhibit initiation of transcription of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal transcription by RNA polymerase II. However, WP631 is ~15 times more efficient at inhibiting transcription initiation from an adenovirus promoter containing an upstream Sp1-protein binding site. The differences in the ability of each drug to inhibit transcription initiation appear to be related to the competition between Sp1 and the anthracyclines for binding to the same site. To see whether WP631’s strong effect on transcription can also be observed in cells, we compared the effects of WP631 and other anthracyclines on the transcription of the c-myc gene, which promoter contains Sp1 binding sites. The resulting data suggest that WP631 might circumvent some kinds of tumor resistance at rather low drug concentrations, inhibit c-myc expression in some cell lines, and exert its antitumoral effect by inducing apoptosis.
1
INTRODUCTION
vivo. Consequently, there has been increasing interest in obtaining information about the effects of drugs on DNA-
Gene expression in eukaryotic cells requires a methodical control of RNA synthesis [1-3]. Formation of the transcription initiation complex includes the interaction of several protein factors that bind to specific sequences located at the promoter regions of the eukaryotic genes [1, 2]. While several protein factors are common to all promoters, the expression of some genes requires additional factors known as transactivators [4-7]. Sp1 is a zinc-finger transcription factor that activates transcription in many cellular promoters [5, 6, 8]. It recognizes the consensus sequence 5’- (G/T)GGGCGG(G/A)(G/A)(C/T)-3’ [4]. Transcriptional activation by the Sp1 protein involves composite effects including conformational changes in DNA structure, the
protein complexes under more physiological conditions (e.g., during active transcription by RNA polymerase II). In this context, results obtained using HeLa nuclear cell extracts and in vitro transcription assays showed the relation between the capacity of the anthracyclines to modify in vitro transcription [17, 18] and their different cytotoxic effects observed in MCF-7 [19] and Jurkat T cells (Villamarín, S., Ferrer, N., Priebe, W., Portugal, J., manuscript in preparation); in short, the results suggested that DNA- binding ligands might regulate gene expression by interfering in the binding of protein transcription factors to DNA.
interaction with other protein factors, and specific DNA- protein contacts [5, 9].
WP631 is a bisanthracycline (Fig. 1A) whose rational design was based on the proximity and orientation of two daunorubicin molecules when they bind to DNA in crystal
The selective inhibition of transcription by the specific targeting of protein-binding sites in DNA first became an active field of research when it was discovered that small molecules (including several anthracyclines), which can bind to specific DNA sequences, could control gene expression by interfering with DNA-protein and protein-protein interactions [10-17]. The idea was that the study of this selective inhibition might help to understand how transcription antagonists might regulate gene expression in
*Address correspondence to this author at the Departamento de Biología Molecular y Celular. Instituto de Biología Molecular de Barcelona, CSIC. Jordi Girona, 18-26; 08034 Barcelona, Spain; Tel: 34-93- 400 61 76; FAX: 34-93- 204 59 04; E-mail: [email protected]
structures [20]. The hypothesis underlying this design was that the bisintercalating anthracycline WP631 (Fig. 1) would exhibit a binding constant of the same order of magnitude as some protein factors [20, 21] and thus bind to DNA strongly and effectively enough to inhibit RNA synthesis both in vitro [17] and in cell culture (Villamarín, S., Ferrer, N., Priebe, W., Portugal, J., manuscript in preparation). In fact, since WP631 could inhibit the interaction between some nuclear protein factors and their binding sites with extraordinary efficiency, it was thought that WP631 might even regulate gene expression by altering the binding of regulatory proteins [17]. In light of these studies showing the outstanding ability of WP631 to inhibit in vitro transcription, we therefore decided to explore further the likely
0929-8673/01 $28.00+.00 © 2001 Bentham Science Publishers Ltd.
Fig. (1). A: Chemical structure of bisanthracycline WP631. B: Scheme of the G-less template used in the analysis of the effects of daunorubicin and WP 631 in the Sp1-activated transcription experiments in vitro. C: Nucleotide sequence of the promoter and the G-less cassette displayed in panel B. The Sp1-binding site is shown underlined. The transcription initiation site is nucleotide +1.
relationship between this inhibitory ability and the efficacy of WP631 in cultured cells.
We have recently adapted an in vitro transcription assay [17] which uses both a strong well-defined promoter and a ~190 bp template that lacks cytidine residues on the transcribed strand (a G-less cassette) [29, 30]. In experiments
BISANTHRACYCLINE WP631 BISINTERCALATES INTO DNA
There is evidence that bisanthracycline WP631, which resulted from a new approach to the structure-based design of DNA binding agents, might be a promising antitumor drug [17, 20]. WP631 expands the inherent groove binding of daunorubicin, building a molecule with characteristic intercalating and groove-binding moieties [20]. Structural studies on the binding of WP631 to a short oligonucleotide have revealed that the two aglycones of WP631 bisintercalate between two CpG steps in a way reminiscent of the binding of two daunorubicin molecules [22, 23]. Since WP631, Fig. 1, has a binding constant similar to that of some protein factors [20, 21], it is assumed that it binds tightly to DNA and, thus inhibits RNA synthesis.
TRANSCRIPTION ASSAYS IN VITRO
Several laboratories have long been interested in targeting antitumor drugs toward DNA regulatory elements or DNA-protein complexes and in evaluating the effects of those drugs on transcription initiation or drug-induced termination [11, 12, 14-16, 24-27]. All of these groups consider that interfering in transcription offers a way to study drug cytotoxicity in vitro. The specific inhibition of transcription by RNA polymerase II would indicate whether the anthracyclines might regulate gene expression in eukaryotic cells [17, 26, 28].
in which the strong adenovirus major late (AdML) promoter and a G-less cassette were used to compare the effects of daunorubicin and WP631 on transcription by RNA polymerase II, this template permitted direct monitoring of specific RNA synthesis. Reactions were performed in the presence of RNAse T1 and the chain terminator 3′-0-methyl- GTP. In these conditions the ~190 bp-long RNAse T1- resistant transcript, resulting from the accurate initiation of transcription, is the unique RNA that accumulates. Using this approach we were able to readily measure basal transcription in the presence or the absence of a DNA- binding drug. Moreover, using a promoter that we constructed by inserting an Sp1 binding site close to the AdML promoter (AdSP01 promoter) (Fig. 1), we sought to determine the influence of WP631 versus daunorubicin (which has a lower DNA binding constant [31]) on an Sp1- transactivated promoter in vitro [17, 32] by comparing their effects on the basal promoter. Since the Sp1 site contains G/C rich tracts, including CpG steps [4], we expected both anthracyclines to bind to the same regions but with different binding affinities. From a practical point of view then, this means that we have been able to distinguish unambiguously between effects that are due to the decrease in transcription initiation and those due to elongation. Indeed, the use of DNA templates bearing a G-less cassette is becoming a very useful tool for analyzing drug-DNA interactions under conditions of active transcription by RNA polymerase II [16, 17, 33].
Effects of Daunorubicin and WP631 Current Medicinal ,Chemistry 2001, Vol. 8, No. 1 3
Table 1. Effects of Daunorubicin and Bisanthracycline WP631 on Basal and Sp1-Activated Transcription Initiation vitro
Promoter C50 [mM] (a) C100[mM ](b)
Daunorubicin WP631 r 50 (c) Daunorubicin WP631 r 100(c)
AdML 0.68 0.48 1.42 ~ 4.5 ~ 2.5 1.8
AdSP01 0.84 0.060 14.00 ~ 4.5 ~ 0.5 9.0
(a)values adapted, with permission, from ref. [17]
(b)C50 indicates the drug required to decrease the transcription initiation by 50 %. C100 is the drug concentration required to total ly inhibitit transcription initiation.
(c)r50 is the ratio of the drug concentrations required to inhibit the in vitro transcription from each promoter by 50%. r100 is the ratio of the concentrations required to totally inhibit transcription initiation from each promoter.
INHIBITION OF BASAL AND SP1-ACTIVATED TRANSCRIPTION BY DAUNORUBICIN AND WP631 IN VITRO
analyzed by high-voltage electrophoresis on denaturing polyacrylamide gels and autoradiography [17].
As the results suggest, the inhibition of transcription from
The effects of daunorubicin and WP631 on the transcription initiation of AdML and AdSP01 promoters have been measured using HeLa nuclear extracts and supercoiled plasmids (an additional advantage of using a G- less cassette as a template). Radiolabeled transcripts were
either promoter by increasing amounts of daunorubicin or WP631 is apparently due to the effect of each drug on transcription initiation by RNA polymerase II. About 4.5 mM daunorubicin fully inhibited transcription from the AdML promoter [17] while about 2.5 mM WP631 was required to
Fig. (2). Influence of daunorubicin and WP631 on Sp1-activated transcription from the AdSP01 promoter. The top panels show a representative transcription assay in vitro. In all cases, an unrelated RNA was added as a recovery and loading control (indicated by an asterisk). Lanes 1 and 8, transcripts obtained using a HeLa cell extract; lanes 2 and 9, Sp1-activated transcription obtained using whole HeLa extract plus added pure Sp1; lanes 3-7, effect of increasing amounts of daunorubicin on the Sp1-activated transcription (0.74, 1.48, 2.96, 3.70, and 4.40 mM, respectively); lanes 10-14, effect of increasing amounts of WP631 (0.022, 0.044, 0.22, 0.44, and 0.88 mM, respectively). The bottom panels present a quantitative analysis of the effects of daunorubicin and WP631 on Sp1-activated transcription of the AdSP01 promoter. The C50 values (i.e., the drug concentrations required to reduce transcription by 50%) were derived by fitting an exponential decay curve to plots of transcription (%) versus drug concentration. The data represent the mean of at least three independent experiments (mean value ± SD), (reproduced, with permission, from ref. [17]).
produce the same effect (Table 1). These results suggest that both drugs might block the formation of the transcription initiation complex. WP631 was more efficient at preventing transcription factors in the HeLa cell extract from binding to the promoter. At first glance, these results might be related to the tighter WP631 binding to DNA that is due, at least in part, to its bisintercalating nature [20-23]. Moreover, since there are no CpG or GpC steps in the G-less cassette neither drug had preferred intercalating sites in the transcribed region and thus they should have no effect during the elongation step. DNase I footprinting experiments have confirmed that there are no strong WP631 binding sites within the G-less cassette [17].
effect of an intercalating agent on transactivated initiation of transcription. In fact, despite the present lack of data on the effects of WP631 on living organisms, it is worth noting that the C50 of WP631 (60 nM) clearly falls within the range of concentrations associated with the clinical use of some anthracyclines [28].
DIRECT EVIDENCE THAT WP631 AND Sp1 MIGHT BIND TO THE SAME DNA SEQUENCE
More direct evidence about the high inhibitory effects of the two anthracyclines on Sp1 binding has been obtained by gel retardation (band-shift) and DNase I footprinting [17]. In
As Table 1 shows, daunorubicin and WP631 differentially influenced the initiation of AdML promoter
transcription in vitro. Concentrations of 0.68 mM daunorubicin and 0.48 mM WP631 decreased transcription by 50% (C50). Furthermore, the ratio of these inhibiting concentrations (around 1.5) seemed to be consistent with differences in the DNA binding of the monointercalator daunorubicin versus the bisintercalator WP631.
When analyzed using the AdSP01 promoter, the ability of WP631 to inhibit Sp1-activated transcription was outstanding in the low nanomolar range [17]. WP631 was also more efficient at inhibiting activated transcription than basal transcription. The C50 was only 0.060 mM for WP631 versus 0.84 mM for daunorubicin (see Fig. 2 and Table 1 for data from the quantitative analysis). Since both anthracyclines bind to CG-rich regions in DNA [21, 31], differences in the transcription of the Sp1-activated promoter were considered to be closely related to the different affinities of the two drugs for the putative DNA sequence. Furthermore, these results could be compared with the basal promoter data described above, thus providing a way to discriminate between the undefined effects of daunorubicin or WP631 on other protein-DNA complexes and their defined effects on Sp1-DNA complexes. A WP631 concentration as low as 0.5 mM totally inhibited Sp1- activated transcription.
gel retardation experiments carried out with pure Sp1 protein and an oligonucleotide containing a putative Sp1 binding site, both drugs inhibited (to different extents) the interaction between Sp1 and the binding site (Fig. 3) [17, 32]. The differing susceptibility of the Sp1-DNA complex to daunorubicin or WP631 is consistent with the results observed in the in vitro transcription assays described above. WP631 appeared to work more efficiently as an inhibitor, although the concentrations required to inhibit transcription in vitro and to disrupt the Sp1-DNA complex were clearly different (60 nM in the first case versus 670 nM in the second [32]). Moreover, DNase I footprinting analysis of the binding of WP631 and Sp1 to the AdSP01 promoter has shown that pure Sp1 protein produces a prominent footprint located at the Sp1-binding site [17]. When Sp1 and WP631 were added into reaction together, the protein and the drug seemed to compete, to some extent, for binding to the Sp1 binding site; the evidence was a composite footprint in which the protein appeared to bind more tightly at the 5′ end
In terms of inhibitory activity, it appears that the newly designed bisanthracyclines are superior to monointercalating anthracyclines. The smaller C50 for WP631 (60 nM), as well as the ratio between the C50 of the two drugs (Table 1), is noteworthy since the C50 is in the nanomolar range and WP631 apparently competed efficiently with transcription factors. Clearly, WP631 inhibited the Sp1-activated transcription in vitro even more efficiently than it inhibited basal transcription. It is also worth noting that the strong effect of WP631 on AdSP01 is reasonably distinct, and consequently rather specific [17], whereas the daunorubicin concentrations that inhibit the transcription of either promoter are similar (Table 1).
Consequently, the amount of drug required to inhibit transcription initiation appears to depend on the competition between daunorubicin, or WP631, and protein factors for binding to CpG sites in the promoter. While previous reports have noted a direct effect of some CG-binding drugs on DNA-Sp1 interaction [12, 14, 15, 24], the present results are, to our knowledge, the first unambiguous example of a direct
Fig. (3). Gel retardation (band-shift) analysis of the effect of daunorubicin and WP631 on the binding of 10 ng of pure Sp1 protein to an end-labeled oligonucleotide containing its consensus binding sequence. The concentrations of daunorubicin or WP631 added to the binding reactions are indicated in the figure. Tracks labeled C and +Sp1 contained the oligonucleotide alone or in the presence of 10 ng pure Sp1, respectively.
Effects of Daunorubicin and WP631 Current Medicinal Chemistry, 2001, Vol. 8, No. 1 5
of the target sequence and WP631 appeared to bind to the other end of the putative Sp1 binding site [17].
CGCCCCGCCCCG-3′ sequence in AdSP01. It follows then that the binding of WP631 to the AdSP01 promoter is
The AdSP01 promoter contains a Sp1 binding site (Fig. 1C) that was designed for two purposes: to match the consensus Sp1 binding site [4, 34] and to resemble, as
facilitated by an alteration in DNA structure that occurs after protein binding and somewhat reduces the distance between the CpG steps.
closely as possible, the preferred WP631 binding site [21, 22]. There are many nucleotide variations among the in vivo Sp1-responsive elements and consequently a wide range of relative Sp1 binding affinities [4, 35]. These sites range from those with high affinity (e.g, those in the DHFR [dihydrofolate reductase] promoter or some SV40 promoters) to those with relatively lower affinity (e.g., the promoter for HSV.TK [herpes simplex virus thymidine kinase]) [4]. None of these Sp1 binding sequences, however, equates exactly with those in AdSP01. From a practical point of view, this means that WP631 might be able to bind to natural promoters with presumably lower, or perhaps somewhat higher, affinities than to AdSP01. Nevertheless, WP631 should be better than monoanthracyclines at interfering with any of those Sp1 binding sites, as it was in the case of the AdSP01 promoter. Consequently, new DNA- binding drugs could be designed with ultratight binding to the sequence CGGGGC, which is the most abundant among the tighter Sp1-binding sites [34]. Such drugs could be even more efficient than WP631 at inhibiting transcription since WP631 shows some preference for the CGATCG sequence [22, 23].
One tentative model of the possible binding mode for the bisintercalation of WP631 into the Sp1-binding DNA sequence considers that, after binding, Sp1 bends its putative binding site, thereby allowing the bisintercalation of WP631 because of a change in the geometry of DNA that facilitates the bisintercalation covering 7 bp, instead of 6 bp [17]. It is known that Sp1-binding can bend the DNA molecule [36]
and act synergistically with other protein factors [5, 6, 9]. Hence, it is conceivable DNA bending by Sp1 would promote the binding of WP631 to DNA. Such Sp1-induced binding of WP631 would explain the ability of WP631 to inhibit activated transcription better than basal. It might also explain why the concentration of WP631 (versus daunorubicin) required to inhibit transcription from the AdML promoter was only about 1.5 times lower during basal transcription but about 15 times lower in Sp1-activated transcription assays [17, 32]. The detection of a Sp1-DNA- WP631 ternary complex in DNase I footprinting experiments [17] further strengthens this model.
An additional explanation for the enhanced effect of WP631, and one still compatible with the idea of an Sp1- induced change in DNA conformation, is that the observed inhibition of transcription from the Sp1-activated AdSP01
A MODEL FOR WP631’S STRONG INHIBITION OF SP1-ACTIVATED TRANSCRIPTION IN VITRO
As already mentioned above, WP631 is a bisintercalating ligand that occupies 6 bp of DNA, while daunorubicin is a monointercalating ligand that occupies only 3 bp [21]. Its larger binding site should therefore make WP631 more selective than daunorubicin since there are fewer potential drug-binding sites on target DNA. A striking feature of the binding of WP631 to the AdSP01 promoter is that the C50 of WP631 (Table 1) is considerably lower than that required to inhibit the basal in vitro initiation of AdML promoter transcription [17, 32]. Although a WP631 binding site should ideally contain two CpG steps separated by about another 2 bp, the consensus Sp1-binding site is slightly longer. Hence, WP631 could apparently bind to the 5′-
promoter may be due to the longer residence time of WP631 versus daunorubicin in this particular GC-rich DNA sequence. This is supported by studies showing that drug dissociation kinetics from DNA is an important determinant of antitumor activity [37, 38]. Anthracyclines like WP631, which bind strongly but reversibly to DNA [21], may be as efficient as covalently bound drugs at inhibiting the progress of transcription along a DNA template [26, 33]. In our studies with the AdSP01 promoter, three CpG steps were available for intercalation (Fig. 1), though the occupancy of two CpG steps by one molecule would have excluded a second molecule from the binding region. It is worth noting that WP631 binds with high affinity (Kb > 2.7 x 10 11 M-1) to herring sperm DNA [20, 21] and that this binding constant is close to that calculated for Sp1 binding to a consensus recognition site [39].
Table 2. Comparative In vitro Cytotoxicity of WP631, Daunorubicin and Doxorubicin
Cell line IC50 (µM)(a)
Doxorubicin Daunorubicin WP631
MCF-7 0.9 ± 0.5(b) n.d. 4.8 ± 2.5(b)
MCF7/VP16 14.2 ± 0.8(b) n.d. 2.5 ± 3.4(b)
Jurkat T 0.13 ± 0.07(c) 0.083 ± 0.009(c) 0.018 ± 0.006(c)
(a)Drug concentrations required to inhibit cell growth by 50 % following 72 h exposure (values represent arithmetic means ± S.E., determined from dose-response curves).
(b)Adapted, with permission, from [20]
(c)(Villamarín,S., Ferrer,N., Priebe,W., Portugal,J. unpublished observations) n.d. non-determined
The results obtained with the bisanthracycline WP631 agree with a more general model in which the binding of a small molecule can regulate gene expression by modifying the binding of protein factors to their natural target sequences [10, 11, 14-17]. A ligand that is able to recognize a 7-bp sequence, as WP631 appears to do in the Sp1 binding site, may suffice for gene-specific regulation in vivo [16].
expression of the c-myc oncogene, whose transcription is activated by Sp1 (see above), might be differentially affected by these drugs. Interestingly, at least one study has corroborated the results displayed in Fig. 4, by showing that a drug-induced reduction of c-myc expression could arrest a majority of cells in the G2 /M phase [46].
CYTOTOXICITY OF WP631
Above, we have compared the capacities of WP631 and daunorubicin to inhibit Sp1-activated transcription in vitro. However, it remains to be established whether these effects can be reproduced in vivo. In general, for most anthracycline derivatives, there appears to be a correlation between DNA- binding affinity and antitumor activity [40]. Our own preliminary studies have shown that WP631 is apparently more active than monoanthracyclines against several breast carcinoma and leukemia cell lines including MCF-7, MCF- 7/VP-16 [20], and Jurkat T cells (Villamarín, S., Ferrer, N., Priebe, W., Portugal, J., unpublished observations) and has promising antitumor activity. WP631 apparently overcomes a specific form of multidrug resistance in MCF-7 breast cells [20] and, at nanomolar concentrations, has a strong effect on leukemic Jurkat T cells. In fact, WP631 was 5-8 times stronger than daunorubicin or doxorubicin in inhibiting the growth of the Jurkat cells (Table 2). Together, these results reveal the potential therapeutic advantages of using rationally designed bisanthracyclines like WP631 in vivo.
Fig. (4). Cell cycle distribution of Jurkat T cells after continuous treatment with either 182 nM daunorubicin or 60 nM WP631 over 36 hours. The distribution of the cells in the different phases of the cell cycle was revealed by flow cytometry analysis of cells stained with propidium iodide. The concentrations of daunorubicin and
INDUCTION OF APOPTOTIC CELL DEATH AND ALTERED GENE EXPRESSION BY WP631
WP631 used in this experiment corresponded to their IC (i.e.,
75
drug concentrations required to inhibit cell growth by 75%, as determined from dose-response curves).
Some currently used antitumor agents, including several anthracyclines, might exert their cytotoxic effects by inducing apoptosis [28, 41-44]. Therefore, using flow cytometry, we sought to determine whether WP631 is as efficient at provoking apoptosis in cultured cells as it is at inhibiting transcription in vitro. We found that WP631, at nanomolar concentrations, could induce cell arrest and apoptosis in Jurkat T cells (Fig. 4). The presence of apoptotic cells was confirmed by DNA fragmentation analysis according to the method of Skladanowski and Konopa [45]
and morphological examination of cultured cells (Villamarín, S., Ferrer, N., Priebe, W., Portugal, J., manuscript in preparation). It is noteworthy that, under the experimental conditions described in the legend to Fig. 4, daunorubicin induced a high level of apoptosis, while WP631 induced only a marginal amount of apoptotic cell death but a high level of G2 /M arrest. Moreover, in preliminary experiments performed with MCF-7 breast cancer cells, WP631 produced none of the hallmarks of apoptotic cell death (Villamarín, S., Ferrer, N., Priebe, W., Portugal, J., unpublished observations). It may be that WP631 failed to induce apoptosis in MCF-7 cells because those cells are intrinsically refractory to apoptotic cell death [19] or relatively insensitive to the bisanthracycline (Table 2). At present, we are analyzing the molecular origins of the differences in the behavior of daunorubicin and WP631. We are particularly interested in whether alterations in the
We are also interested in whether some genes that are known to be activated by Sp1 in vivo may be especially affected by WP631. One of these genes is the c-myc oncogene, whose expression plays an important role in the control of the cell cycle and whose overexpression results in unrestricted cell growth [47, 48]. For the sake of comparison, it is worth noting that c-myc is overexpressed in MCF-7 cells and that some monointercalating anthracyclines partially suppress its expression [19]. Because WP631 strongly inhibits Sp1-activated transcription in vitro (Fig. 2), we hypothesized that WP631 should also reduce Sp1 binding in tumor cell lines expressing c-myc. Moreover, since c-myc appears to be a component of the apoptotic cell death pathway [49] and Sp1 activates c-myc transcription [50], we have examined whether inhibition of an Sp1 binding site in the c-myc promoter might delineate a prime target for WP631 in vivo. Previous results obtained using monointercalators and minor-groove-binding drugs have shown that targeting Sp1-DNA or other protein-DNA complexes might affect in vivo transcription [13, 24, 27, 51- 53], though, in most cases, it remains unknown whether this is a direct consequence of drug binding or mainly due to an indirect (pleiotropic) effect. As shown by Northern blot analysis (Fig. 5), treatment of MCF-7 cells with WP631 resulted in a dose-dependent reduction in the amount of c- myc transcripts that corresponded closely with growth inhibition. These findings would suggest that inhibition of
Effects of Daunorubicin and WP631 Current Medicinal Chemistry, 2001, Vol. 8, No. 1 7
the c-myc expression may be connected to growth arrest in MCF-7 cells.
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