(Received for publication, September 26, 1995; and in revised form, December 12, 1995)
From the
Adriamycin is known to specifically induce DNA interstrand cross-links at 5`-GC sequences. Because 5`-GC sequences are a predominant feature of 5`-untranslated regions (transcription factor-binding sites, promoter, and enhancer regions), it is likely that adriamycin adducts at GC sites would affect the binding of DNA-interacting proteins. Two model systems were chosen for the analysis: the octamer-binding proteins Oct-1, N-Oct-3 and N-Oct-5, which bind to ATGCAAAT and TAATGARAT recognition sites, and Escherichia coli RNA polymerase binding to the lac UV5 promoter. Electrophoretic mobility shift studies showed that adriamycin adducts at GC sites inhibited the binding of octamer proteins to their consensus motifs at drug levels as low as 1 µM, but no effect was observed with a control sequence lacking a GC site. Adriamycin adducts at GC sites also inhibited the binding of RNA polymerase to the lac UV5 promoter. Adriamycin may therefore function by down-regulating the expression of specific genes by means of inactivation of short but critical motifs containing one or more GC sites.
The mode of action of the anticancer drug adriamycin has been examined extensively over the past 20 years. Many different studies have cited the interaction of the inherently reactive drug with cell membranes, DNA, proteins, metal ions, and molecular oxygen, leading to an apparently complex interplay of the mechanism of antitumor action, the major determinants of which may differ according to the properties of target cancer cells (Myers et al., 1988). Although the reactivities of adriamycin with a range of cellular constituents are well known, the specific cellular mechanisms involved and the ultimate cause of tumor cell death are still to be elucidated. One possible physiological action in tumor cells involves the altered regulations of DNA-binding proteins in actively transcribed DNA (i.e. the open DNA regions in nuclear matrix attachment sites (Ciejek et al., 1983)). Covalent attachment of the drug chromophore to specific DNA consensus sequences required for recognition by DNA-binding proteins may lead to altered levels and modes of binding by these proteins.
The majority of adriamycin administered to sensitive
tumor cells is known to rapidly localize in the nucleus (Gigli et
al., 1988). The drug has a high affinity for DNA, thus providing
the driving force for further nuclear uptake. It is well known that
intercalation is the immediate form of interaction, and there is an
extensive body of evidence to show that one of the first cellular
responses is the impairment of topoisomerase II activity (see for
example reviews by Liu (l989); Holm et al. (l99l); Capranico
and Zunino (l992); Sinha (l995)). However, upon reductive activation
the drug can also bind covalently to DNA (Moore, 1977; Sinha, 1980).
The sequence specificity of this interaction in vitro is
highly GC selective (Cullinane and Phillips, 1990), and it appears that
an interstrand cross-link occurs at this site (Cullinane et
al., 1994b; Cutts and Phillips, 1995). Adriamycin-induced
cross-links have also been reported in HeLa S cells
(Skladanowski and Konopa, 1994a), but it remains to be seen whether the
same in vitro sequence selectivity applies, although studies
with other sequence-specific damaging agents suggest that this is
likely (Murray and Martin, 1985; Hartley et al., 1992;
Cullinane and Phillips, 1994) .
Once a DNA-drug adduct is formed, it is widely accepted that the nature of the interaction impedes cellular functions that involve DNA (i.e. replication and transcription), particularly in the event of damage to both strands by the formation of an interstrand cross-link (Hopkins et al., 1991). It follows that DNA-binding proteins would be affected to varying degrees by their modified substrate. Although many intercalating drugs have been repeatedly shown to disrupt the actions of topoisomerase II, recently some covalently binding agents have also been shown to disrupt the binding of transcription factors to their specific consensus sequences (Broggini and D'Incalci, 1994; Welch et al., 1994; Sun and Hurley, 1994). If these adducts prevent binding of transcription factors to DNA in tumor cells, then the sequence selectivity of the particular drug will determine which transcription factors are affected, and hence which genes are inhibited. The net effect of this process is that gene-specific inhibition may occur, depending on the sequence specificity of the particular drug adducts.
The availability of in vitro electrophoretic mobility shift assay (EMSA) ()systems
for the octamer family of transcription factors provides a model system
for the study of adriamycin-induced inhibition of protein-DNA binding
and avoids the denaturation, heating, or alkaline treatments required
in other types of DNA damage detection, all of which contribute to
instability of adriamycin adducts (van Rosmalen et al., 1995).
The consensus recognition sequence for these proteins is an 8-bp
ATGCAAAT element and involves a single GC dinucleotide within the
octamer element that participates directly in binding of the Oct
proteins through the POU-specific region of the POU DNA-binding domain
(Klemm et al.(1994); reviewed in Herr and Cleary(1995)). An
alternative recognition sequence for some octamer-binding proteins is
the TAATGARAT motif present in herpes simplex virus immediate early
gene promoters (O'Hare and Goding, 1988; Preston et al.,
1988; Baumruker et al., 1988), but because this site does not
include a GC dinucleotide in the binding site, it serves as an
excellent control to assess the significance of adriamycin-induced
interstrand cross-links at GC sites.
DNA-dependent polymerases are also potential targets for inhibition when site-specific DNA adducts are formed in promoter regions. Simple gel retardation assays for the detection of binding of eukaryote RNA polymerases are not available. However, the interaction between Escherichia coli RNA polymerase and the lac UV5 promoter has been extensively studied, and a simple assay for this interaction is well documented (Straney and Crothers, 1985; Gray and Phillips, 1993). Although there is a high frequency of GC sites in this region, they do not appear to play a role in direct contact with the RNA polymerase (von Hippel et al., 1984; Brodolin et al., 1993). This system therefore provides an opportunity to study the indirect effect of adducts on protein binding within the promoter region.
In this study we show that the covalent binding of low concentrations of adriamycin to GC sequences interferes with the ability of octamer proteins to bind at these sites. The binding of prokaryote RNA polymerase was also inhibited in its binding to the lac UV5 promoter.
Fig. 1shows that there is an adriamycin concentration-dependent inhibition of octamer binding with the N-Oct-3, N-Oct-5, and Oct-1 proteins, with all following similar inhibition patterns. At 10 µM adriamycin, the binding of Oct-1, N-Oct-3, and N-Oct-5 is inhibited by approximately 25, 40, and 30% respectively (Fig. 1B). To confirm that this effect was due to covalent binding of the drug chromophore to the consensus sequence (or whether it was merely an effect of drug intercalation), 10 µM of adriamycin was reacted with the DNA probe for increasing times prior to exposure to the nuclear extract (Fig. 2, A and B). The yield of adriamycin-DNA adducts is known to be optimal after a reaction time of approximately 40 h with a half-life of formation of adducts being about 15 h (Cullinane et al., 1994b), and this effect is consistent with that shown in Fig. 2B where inhibition of protein binding is maximal after 60-80 h with a half-life of inhibition of all three octamer proteins being less than 20 h. Because the percentage inhibition of protein binding increased with drug-DNA reaction time, this confirms that the inhibition is due to specific adriamycin-induced adducts rather than other nonspecific effects such as intercalation. All three octamer-binding proteins were also inhibited (but to a lesser extent) by exposure of the DNA to 1 µM adriamycin for 40 h (Fig. 2, C and D.).
Figure 1:
Gel retardation assay of inhibition of
binding by octamer proteins with increasing adriamycin concentration. A, the H2B probe was exposed to 0.5-100 µM adriamycin in the presence of 20 µM FeCl for an incubation period of 24 h. Control samples were incubated
in the presence of 20 µM FeCl
(denoted Fe) or in the absence of both FeCl
and adriamycin
(denoted C). The reacted probe was exposed to nuclear extract
from A2058 cells. Electrophoretically retarded bands denote protein-DNA
complexes due to Oct-1, N-Oct-3, and N-Oct-5 proteins present in the
nuclear extract. B, PhosphorImager quantitation of the Oct-1
(
), N-Oct-3 (
), and N-Oct-5 (
) complexes show the
trends of protein binding with increasing adriamycin concentration.
Band intensities were calculated relative to the Oct-1 band in the 0.5
µM adriamycin lane.
Figure 2:
Inhibition of octamer protein binding as a
function of drug reaction time. The H2B probe was reacted for
0-96 h with 10 µM adriamycin and 20 µM FeCl (A), 1 µM adriamycin and 20
µM FeCl
(C), or 10 µM adriamycin in the absence of FeCl
before exposure to
the A2058 nuclear extract (E). Band intensities were
quantitated for A, C, and E as a percentage
of the total band intensity (i.e. total end-labeled probe) in
each lane, and the results are shown in B, D, and F, respectively. The time-dependent inhibition was fitted to a
single exponential decay.
The extent of formation
of adriamycin-induced adducts with DNA is known to be catalyzed by the
presence of FeCl with approximately 5-fold more adducts
being formed when DNA is reacted with adriamycin for 50 h in the
presence of 10 µM FeCl
compared with the
absence of added FeCl
(Cullinane et al., 1994b).
This catalytic role is shown in Fig. 2E where Oct-1,
N-Oct-3, and N-Oct-5 proteins were scarcely affected when FeCl
was absent but resulted in extensive inhibition of binding
capacity of all three proteins when FeCl
was present during
the reaction of drug with DNA (Fig. 2, A and B). Overall, the inhibition of binding of octamer proteins
correlates well with the known characteristics of formation of
adriamycin-induced adducts. Although these trends are quite clear and
highly reproducible, there is some variation in the absolute level of
formation of octamer protein-DNA complexes (5-17% in Fig. 2, B, D, and F), and this is due
dominantly to the varying concentration of transcription factors
present in individual nuclear extracts (Thomson et al., l993).
Figure 3:
Octamer protein binding to the alternative
motif TAATGAATT. A, the OA25 probe was reacted with increasing
concentrations of adriamycin (0.5-100 µM) in the
presence of 20 µM FeCl prior to exposure to
the A2058 nuclear extract. B, the quantitation is
shown.
Figure 4:
Lambda exonuclease digestion of OA25 and
H2B probes following reaction with adriamycin. Exonuclease digestion
was performed after reaction with 1 µM adriamycin (lanes 5), 1 µM adriamycin and 20 µM FeCl (lane 6), 10 µM adriamycin (lane 7), 10 µM adriamycin and 20 µM FeCl
(lane 8), in the absence of adriamycin
and FeCl
(lane 3), and with only 20 µM FeCl
(lane 4). Lanes 1 and 2 denote samples reacted in the presence and the absence of
FeCl
, respectively, but not subjected to lambda exonuclease
digestion. Maxam-Gilbert G sequencing lanes G and octamer
binding sequences are shown for each probe.
Figure 5: Quantitative analysis of lambda exonuclease blockage sites in OA25 (A) and H2B (B) probes. The histograms show the results for lane 7 of Fig. 4(10 µM adriamycin). Only bands representing greater than 2% of the mole fraction of total radioactivity in the lane are shown. The boxed sequences represent the complementary strand to the consensus sequence of the alternative motif (A) and the sequence of the wild-type consensus motif (B). The numbering scheme employed is arbitrary.
The time dependence of formation of adriamycin adducts (prior to exposure to polymerase) under these conditions is shown in Fig. 6A. There is a decreasing amount of the DNA-RNA polymerase complex with increasing time of reaction of adriamycin with the DNA fragment (Fig. 6A) or with increasing adriamycin concentration (Fig. 7A), and the relative amount of DNA existing in the complexed form is shown in Fig. 6B and 7B, respectively. The extent of binding is reduced 5-fold with saturation at 10 µM drug concentration. To confirm that the promoter region was being alkylated, the DNA was probed by digestion with lambda exonuclease for DNA reacted for increasing times with 5 µM adriamycin. Sequence-specific blockages are evident in the image shown in Fig. 8, and the location of the blockages are shown in Fig. 9. Every potential adriamycin GC cross-linking site is alkylated, as indicated by inhibition of digestion several nucleotides 5` to each GC site. More importantly, adducts are evident at every GC site in the RNA polymerase-binding site.
Figure 6:
Gel retardation assay of the inhibition of
formation of the RNA polymerase-promoter complex by adriamycin. A, end-labeled 188-bp fragment containing the lac UV5
promoter was incubated with 5 µM adriamycin and 40
µM FeCl for 0-48 h (denoted by +Adr) before a phenol cleanup and exposure to RNA
polymerase. Samples reacted in the absence of adriamycin (C)
for 0 and 48 h are also shown. DNA probe not exposed to RNA polymerase
is indicated by P. B, the percentage of labeled probe
associated with the RNA polymerase-promoter complex with increasing
time of reaction with adriamycin is shown.
Figure 7:
Adriamycin concentration-dependent
inhibition of binding of RNA polymerase to the lac UV5
promoter. A, the 188-bp fragment was reacted with 0.5-50
µM adriamycin in the presence of 40 µM FeCl prior to the addition of E. coli RNA
polymerase. The DNA fragment was then subjected to electrophoresis, and
the retarded DNA-RNA polymerase band was detected by PhosphorImager
analysis. B, quantitation of the percentage RNA polymerase
bound is shown.
Figure 8:
Lambda exonuclease digestion of adriamycin
reacted lac UV5 promoter region. The 188-bp fragment was
reacted with 5 µM adriamycin and 40 µM FeCl for 0-48 h, and the DNA was extracted twice
with phenol and once with chloroform prior to ethanol precipitation.
Samples were then digested with lambda exonuclease and characterized by
electrophoresis through an 8% denaturing polyacrylamide gel.
Maxam-Gilbert sequencing lanes are denoted as G.
Figure 9: Location of adducts in the lac UV5 promoter region. Only bands comprising greater than 1% of the mole fraction of lane 4 of Fig. 8are shown. The boxed sequence section represents the region covered by RNA polymerase when bound to the promoter in the open complex (Carpousis and Gralla, 1985).
In the TAATGARAT motif, chemical modification and mutation analyses suggest that the Oct-1 POU-specific domain contacts the 3`-GARAT sequence (R indicates purine), whereas the 5`-TAAT sequence is contacted by the Oct-1 POU homeodomain. As the exonuclease digestion pattern shows (Fig. 5A), there are no adducts in this region to cause direct disruption of binding. However, DEPC alkylation (which forms adducts with adenine and guanine bases) does result in disruption of the interaction between the Oct-1 protein and the TAATGARAT binding sequence (Cleary and Herr, 1995). These results collectively show that DEPC alkylation of the alternative motif probably interferes with octamer binding in a similar manner to adriamycin inhibition of octamer binding to ATGCAAAT. However, because the exonuclease digestion indicates that adriamycin has no capacity to form adducts at the TAATGAATT sequence, the adriamycin-induced inhibition must be directly due to adducts at the 5`-GC sequence. Previous studies of the characteristics of adriamycin-induced DNA adducts show that the nature of this sequence specificity to be due to an interstrand cross-link (Cutts and Phillips, 1995).
Overall, the results from this study indicate that the covalent adducts induced by adriamycin in DNA do have a potential inhibitory role in vivo with respect to protein binding to sequences containing 5`-GC sites. Because the octamer-binding proteins are able to recognize a variety of motifs with varying affinities of binding, it is likely that a selective drug-induced inhibition of some octamer proteins would also arise in vivo. It is questionable whether the extent of inhibition of octamer protein binding facilitated by adriamycin would be significant enough to lead to detectably altered regulation patterns of specific cellular proteins, but it is possible that the effects of adducts could be further magnified when the complex interactions of proteins required in transcription initiation are considered.
It is significant that many genes in the mammalian genome (and especially those genes such as protooncogenes associated with proliferation) have a high occurrence of GC runs in their 5` regions (Hartley et al., 1988). For example the c-Ha-ras oncogene contains seven GC runs in the 5`-flanking region, some of these runs being contained in Sp1 binding enhancer regions (Mattes et al., 1988). GC modifying drugs such as adriamycin are likely to favor these regions due to the sequence content and accessibility of these regions (the tight packaging of DNA in nucleosome structures appears to become relaxed and accessible when assembled on nuclear matix attachment regions for transcriptional processes to occur) (Workman and Buchman, 1993; Krajewska, 1992; Wolffe, 1992; Ciejek et al., 1983). There are a number of transcription factors that require a GC in their consensus DNA sequences, including CTF/NF1, AP2, SP1, and Oct-1 and Oct-2 proteins (Wingender, 1990; Williams and Tijian, 1991), and all of these sequences are therefore potential targets for adriamycin alkylation.
It is now important to establish if there is a correlation between adriamycin treatment and selective targeting of genes that are high in GC content. Although adriamycin has been a major inclusion in chemotherapy regimes for 20 years and appears to act as a topoisomerase II inhibitor, a major research effort is still required directed toward elucidating its basic modes of action in tumor tissue. It has recently been shown that the ability of a range of adriamycin derivatives to induce interstrand cross-links correlated well with their cytotoxicity (Skladanowski and Konopa, 1994b), and it has generally been assumed that this effect is related essentially to the impairment of the processive movement of DNA polymerase and RNA polymerase. The present results suggest that interstrand cross-links may well exert a separate earlier effect in inhibiting the binding of proteins such as transcription factors and RNA polymerase to the modified DNA and because of the short sequences (e.g. octamer) involved in promoter activity may confer gene specificity to adriamycin action.