This study was undertaken to assess the influence of DNA supercoiling on transcription in vivo. Eukaryotic topoisomerases I and II remove both positive and negative supercoils, resulting in relaxed DNA (7
). When these enzymes are inhibited by drugs, the superhelical density of the affected DNA is altered. To examine the transcriptional response to topoisomerase inhibition, cells were incubated with camptothecin, a topoisomerase I inhibitor, or adriamycin (doxorubicin) to inhibit topoisomerase II. The transcriptional response of specific genes to drug treatment was monitored by RNase protection and by nuclear run-on, and conformational and/or topological changes were visualized by in vivo footprinting and Southern blotting.
Two cell lines were used for these experiments. Raji cells are a B-lymphocyte Burkitt lymphoma cell line in which one c-myc
gene is translocated near an immunoglobulin gene [chromosome, t(8, 14)] (20
). In these cells the translocated my
c allele is highly transcribed, while the wild-type copy is repressed (41
). The translocation breakpoint is at position −1398 relative to the c-myc
P2 promoter start site (9
). The translocated c-myc
allele also has scattered mutations within exon 1, intron 1, and exon 2 (53
The second cell line, Raji pMYC/CAT, was a Raji cell line stably transfected with the plasmid pREP9/GAL45
MYC/CAT (pMYC/CAT). In this EBV-based autonomously replicating, neomycin-selectable plasmid, 2.9 kb of c-myc
genomic DNA, starting 2.3 kb upstream of promoter P1, was fused with the CAT coding sequence (CAT
). Similar MYC EBV-based plasmids have previously been shown to bear nucleosomes positioned identically as seen for the endogenous c-myc
). Raji pMYC/CAT was used to follow myc
promoter function. Driven by neighboring c-myc
upstream and downstream sequences, and properly assembled into chromatin, the episomal c-myc
promoter was expected to recapitulate many features of c-myc
regulation; the high-copy-number plasmid was expected to yield an amplified signal.
Polymorphous response of RNA synthesis to inhibition of topoisomerases I and II.
To assess the effect of topoisomerase inhibitors on mRNA abundance, the levels of the transcripts of several genes were directly measured by using RNase protection (Fig. ). Cells were treated with camptothecin and adriamycin separately or together for 4 h. (Camptothecin inhibits topoisomerase I enzyme, and adriamycin inhibits topoisomerase II.) The influence of the histone deacetylase inhibitors butyrate and trichostatin A was also observed (35
). Typically, histone deacetylase inhibition leads to increased chromatin acetylation and conditions conducive for increased gene activity. Each of the mRNAs tested displayed a distinctive profile in response to topoisomerase inhibition:
FIG. 1 The steady-state level of each mRNA displays a distinctive profile in response to inhibition of topoisomerase I or II, by monitoring RNase protection. The c-myc exon 2 versus CAT probes used in this experiment distinguish between the c-myc RNAs encoded (more ...) hsp70
mRNA, which was high in all control samples (Fig. , lanes 7 to 9), was decreased by camptothecin (Fig. , lanes 10 to 12) but increased by adriamycin (Fig. , lanes 13 to 15), butyrate (Fig. , lane 19), and trichostatin A (Fig. , lane 20). The hsp70
mRNA half-life is 50 min (69
message, which was quite low in the control cells (Fig. , lanes 7 to 9), was strongly increased by 5 and 10 μM camptothecin (Fig. , lanes 11 and 12) and was raised by both butyrate (Fig. , lane 19) and trichostatin A (Fig. , lane 20). Induction of c-fos
transcription by these concentrations of camptothecin has been reported previously (66
). In contrast, adriamycin decreased c-fos
mRNA (Fig. , lanes 13 to 15).
Unexpectedly, endogenous c-myc
and the episomal pMYC/CAT respond differently to the drugs tested. Endogenous c-myc
levels were high in the controls (Fig. , lanes 7 to 9) but were lowered by all of the drug treatments (camptothecin [lanes 10 to 12], adriamycin [lanes 13 to 15], butyrate [lane 19], and trichostatin A [lane 20]). In contrast, the c-myc
promoter driven CAT
mRNA was dramatically increased by camptothecin (Fig. , lanes 10 to 12), butyrate (lane 19), and trichostatin A (lane 20) but decreased by adriamycin (lanes 13 to 15). CAT
mRNA levels generally paralleled c-fos
. It is important to note that, although c-fos
messages both have short half-lives (54
), these genes responded very differently to drug treatment. Moreover, the half-life of CAT
mRNA is also short (3
). Because some of these RNAs increased, while others decreased during the 4-h drug treatment (long relative to the half-lives of these molecules), differential kinetics of RNA degradation do not explain these results unless these topoisomerase inhibitors differentially modify mRNA half-life.
Of all of the mRNAs analyzed, gapdh
was least affected by the drug treatments. It is clear from the behavior of these genes that there is no stereotypical response of mRNA levels after topoisomerase inhibition. In contrast, butyrate and trichostatin A increased hsp70
, and MYC/CAT
, as expected for histone deacetylase inhibition; only endogenous c-myc
decreased after histone deacetylase inhibition, as reported previously by others (45
The steady-state mRNA levels assayed by RNase protection indicate the net effect on RNA synthesis and degradation. If, for example, a message is increased, this may be due to an increase in synthesis or a decrease in degradation. One way to discriminate between these possibilities is to measure RNA synthesis directly with nuclear run-on assays. These assays allow a limited extension of RNA polymerases transcriptionally engaged in vivo. A battery of genes transcribed by RNA polymerase I, II, or III was analyzed with nuclear run-on assays. To assess structural changes at promoters after topoisomerase inhibition, several genes were examined by in vivo footprinting with the conformation-sensitive DNA reagent potassium permanganate.
Context-dependent response of the c-myc promoter to topoisomerase inhibition.
expression is very context dependent. In Burkitt lymphoma cells such as Raji cells, the translocated c-myc
allele is deregulated, while the unrearranged allele is underexpressed. How immunoglobulin regulatory sequences project their influence over vast stretches of DNA (sometimes exceeding hundreds of kilobases) to activate the translocated allele is unknown. Disturbances of c-myc
expression are also associated with far-3′-genetic irregularities in cis
at the PVT locus in some tumors (62
). To explore directly c-myc
promoter activity in different chromosomal contexts, chromosomal or episomal transcription was compared between Raji cells and Raji pMYC/CAT by using nuclear run-on assays. After 4 h of drug treatment, nuclei were harvested, and RNA labeled in a brief run-on reaction was hybridized with a panel of oligonucleotides derived from the genes of interest. The run-on findings showed that all of the drug treatments repressed endogenous c-myc
in agreement with the RNase protection results.
Labeled nascent transcripts from untreated Raji cells and cells treated with the vehicle DMSO hybridized similarly with antisense myc
oligonucleotides extending from the P1 promoter through the P2 promoter and into exon 2 (Fig. A, lanes 1 and 2). Little evidence of holdback of RNA polymerase at the P2 promoter was noted. (Holdback would be indicated by strong hybridization with slot 5, with declining signals in slots 6 to 10.) The loss of RNA polymerase holdback in Burkitt lymphoma has been previously described (10
). Camptothecin, even at the lowest dose, caused dramatic holdback of the RNA polymerase at the endogenous c-myc
P2 promoter (Fig. A, lanes 3 to 5). The RNA labeled during the 10-min run-on reaction hybridized only with the first P2 sequence (slot 5), indicating that the RNA polymerase is loaded at the promoter but progresses less than 50 nucleotides. Hybridization with P1 oligonucleotides was lost (slot 4), suggesting that this promoter is vacant after camptothecin treatment. Similar results were observed with nuclei from camptothecin-treated BJAB cells (that possess only untranslocated c-myc
), showing that this drug-induced holdback requires no immunoglobulin sequences (data not shown). Adriamycin (Fig. A, lanes 6 to 8), butyrate (lane 12), and trichostatin A (lane 13) all yielded uniformly weaker myc
signals than the DMSO control. With 5 μM adriamycin, only weak holdback at P2 was noted (Fig. A, lane 7).
Transcription from the episome in Raji pMYC/CAT was also analyzed by using nuclear run-on (Fig. B). The intensity of the hybridization to sequences downstream of c-myc
P2 (slots 6 and 7) was increased in Raji pMYC/CAT compared with Raji cells (Fig. B, lanes 2 and 3 versus lane 1) as expected, due to the increased copy number of this template. Overexpression from the episome was confirmed by comparing the relative intensities of the hybridization with CAT
(slot 9) versus c-myc
exon 2 (slot 10); CAT
is specific for the episome whereas c-myc
exon 2 is specific for the endogenous gene. The relative transcription of sequences shortly downstream of P2 (Fig. B, lanes 1 to 3, slots 5 to 7) was increased in Raji pMYC/CAT compared with Raji cells, a finding indicative of greater utilization of P2 from the episome, whereas P1 usage was not increased (slot 4). The intensity of the c-myc
run-on signal from pMYC/CAT cells declined progressively further 3′ of the start site, suggesting that on the episome there is holdback of RNA polymerase near the c-myc
P2 promoter, and thus fewer polymerases are located distally. P2 utilization with promoter proximal holdback of episomal c-myc
has been noted previously (1
Although RNase protection of Raji pMYC/CAT cells showed simply that camptothecin induced CAT
, the run-on transcription from pMYC/CAT in the presence of increasing camptothecin gave a more complex pattern (Fig. B, lanes 4 to 6). The first P2 oligonucleotide (slot 5) hybridized more strongly than the 3′ sequences, indicating more RNA polymerase holdback, but the P2 distal sequences and CAT were also more intensely transcribed, suggesting that transcription penetrated further into the gene with less holdback. These two contradictory observations are reconciled by superimposition of the transcription profiles of the episomal and endogenous c-myc
promoters. As described above, camptothecin caused strong holdback of the endogenous gene at the first P2 oligonucleotide (Fig. A, lanes 3 to 5, slot 5). Therefore, increased holdback of the endogenous c-myc
gene overlying increased downstream transcription from the plasmid pMYC/CAT reconciles these results with the RNase protection data. Adriamycin strongly repressed the episomal c-myc
promoter just as it did the endogenous gene, abolishing hybridization with all probes (Fig. B, lanes 7 to 9). Interestingly, the run-ons obtained with both camptothecin and adriamycin most closely resembled those obtained with adriamycin alone (Fig. A, lanes 9 to 11, and B, lanes 10 to 12). Butyrate and trichostatin A caused upregulated transcription of all MYC/CAT sequences without altering their relative intensities (Fig. B, lanes 13 and 14). Adriamycin inhibition of transcription was not mitigated by butyrate (data not shown). If immobilized topoisomerase II recruited histone deacetylase to inhibit transcription (23
), then these drugs should have increased transcription in the presence of adriamycin; this did not occur.
Importantly, inclusion of adriamycin or camptothecin directly in the nuclear run-on reactions neither inhibited nor stimulated transcription of c-myc or other genes (data not shown); thus, the changes observed in these experiments reflect conditions set up in vivo. Run-on analysis of nuclei harvested during a time course of adriamycin drug treatment demonstrated full promoter responses in less than 2 h (Fig. B).
RNase protection and nuclear run-on experiments indicated that different functional states of the c-myc promoter, directly affecting transcriptional levels, exist in the chromosome versus the epsiome and in response to topoisomerase I versus topoisomerase II inhibition.
Context-dependent KMnO4 sensitivity of the c-myc promoter to topoisomerase inhibition.
Topoisomerase I and II inhibitors modify transcription of the endogenous and episomal c-myc
genes. What structural alterations of the template DNA occur concomitant with topoisomerase inhibition? In vivo footprinting was performed with KMnO4
to see whether the changes in transcription due to drug treatments are reflected in the conformational state of promoter DNA sequences. KMnO4
is most reactive with pyrimidines in single-stranded or otherwise conformationally distorted DNA (17
). Concomitant alterations or adjustments of protein contacts with melted or strained DNA would further modify the chemical reactivity of promoter and regulatory DNA. After treatment with permanganate, cellular DNA was extracted, and the phosphodiester backbone at the modified bases was cleaved with piperidine; ligation-mediated PCR was then performed with gene specific primers to amplify and display the sites of altered KMnO4
The endogenous c-myc promoter region demonstrated dramatic changes at the P2 promoter in response to topoisomerase II inhibition with adriamycin. Enhanced reactivity at multiple specific bases on both strands extended 87 bases, from −34 upstream of the P2 promoter transcription start site to +53 downstream (positions 2456 to 2542, with the mRNA start at position 2490 [accession number X00364]) (Fig. A and B, lanes 3). Sequences further 3′ displayed generalized hyporeactivity, presumably reflecting the depletion of elongation complexes and consequently fewer transcription bubbles to generate permanganate targets. Decreased sensitivity to permanganate was also seen at the c-myc P1 promoter start site in response to adriamycin. Similar adriamycin sensitivity was observed on both the endogenous and episomal c-myc sequences within the P2 promoter proximal region (Fig. A and B, lanes 3 and 10). These data indicate that adriamycin freezes a hyper-open complex at c-myc P2 transcription start site but depresses open DNA downstream. The promoter-frozen complexes are not activated during nuclear run-on experiments and so must either be inactivated or else have encountered an insurmountable barrier.
FIG. 4 Response of c-myc promoter structure to topoisomerase inhibition. In vivo potassium permanganate footprint. (A) Bottom DNA strand. (B) Top DNA strand. Cpt, camptothecin; Adr, adriamycin; TSA, trichostatin A. The cells were incubated with inhibitors for (more ...)
Camptothecin, in contrast to adriamycin, elicited only subtle changes in permanganate sensitivity from the chromosomal c-myc in Raji. Reactivity at several bases from positions 2450 to 2501 surrounding the P2 start site was slightly reduced after drug treatment (Fig. B, lane 2). Opposite to the endogenous gene, camptothecin enhanced the KMnO4 sensitivity of downstream-transcribed episomal template, indicating more elongating complexes (Fig. A, lane 9), a finding consistent with the increase of CAT mRNA (Fig. , lanes 10 to 12). Butyrate (Fig. B, lane 12) and trichostatin A (lane 13) augmented the permanganate reactivity of residue +58 relative to residue +53 on the nontemplate strand (positions 2547 and 2542, respectively) and increased the overall reactivity farther downstream on the template strand (Fig. A, lanes 12 and 13); this pattern often correlates with high-output states of c-myc. Thus, unlike the endogenous gene, histone deacetylase inhibition correlated with increased output and openness of episomal c-myc DNA. The footprints obtained upon combination of camptothecin and adriamycin most closely resembled those obtained with the latter alone (Fig. A and B, compare lanes 2, 3, and 4). Footprint changes after topoisomerase I or II inhibition were not simply a secondary result of transcription inhibition, since treatment with α-amanitin, which freezes transcription complexes in place, generated no specific response to KMnO4 at promoters (data not shown).
c-myc upstream CT element becomes conformationally stressed after topoisomerase II inhibition.
When stressed by torsional strain, particular DNA segments adopt altered DNA conformation or structure. FUSE and CT are two such elements upstream of active c-myc
genes that are peculiarly sensitive to KMnO4
). If topoisomerase inhibition perturbs the degree or distribution of torsional strain, then altered reactivity of FUSE and CT should follow. The permanganate footprint of the CT element, located 300 bp 5′ of the MYC P2 start site, was dramatically altered after adriamycin treatment. The endogenous c-myc
gene seen in the Raji footprint (Fig. A, lane 3) shows several dramatically darker DNA bands corresponding to the T nucleotides of the three CT repeats closest to the promoter; these same repeats are the preferred targets for hnRNP K binding in vitro. The two most upstream elements exhibited sharply reduced reactivity; these sites are the preferred binding sites for the transcription factor Sp1. Competition between hnRNP K and Sp1 for binding in this region has previously been reported (38
). Raji pMYC/CAT cells (Fig. A, lanes 7 to 9) showed only minor changes in this same region, due either to reduced reactivity in all plasmids or to heterogeneous CT reactivity between plasmids; the number of active episomes at any instant is unknown. (By comparing the chromosomal and episomal exposure times, it was estimated that these cells have at least 20 copies of the episome [Fig. ].) The increased reactivity of the CT element after adriamycin treatment might indicate a greater propensity toward melting, perhaps due to the failure of topoisomerase II to remove accumulated strain at the promoter and at the CT element 250 to 300 bp upstream of the promoter.
FIG. 5 Topoisomerase II inhibition provokes conformational changes at the CT-element of the endogenus c-myc gene. (A) In vivo potassium permanganate footprint. Cpt, camptothecin; Adr, adriamycin. The cells were incubated with inhibitors for 4 h. (B) Nucleotides (more ...)
In addition the AT-rich FUSE element, found 1,700 bp 5′ of the MYC P2 start site, was examined in the Raji pMYC/CAT cell line (data not shown). Here adriamycin treatment showed subtle changes in the footprint. Several DNA bands with decreased sensitivity to permanganate were seen within the FUSE element, indicating a more closed, double-stranded character. The FUSE element in the parental Raji cell line was not examined because in the expressed c-myc
allele the FUSE element is translocated away from the myc
coding sequence. The decreased reactivity of the FUSE element after adriamycin treatment might be due to repression of the episomal MYC/CAT gene, since a closed FUSE sequence is associated with a repressed c-myc
). Camptothecin failed to modify the permanganate footprints of both the FUSE and the CT upstream elements, indicating that topoisomerase I was not active in these topological domains (Fig. A, lanes 2 and 6, and data not shown).
Adriamycin alters the structure of the c-fos and hsp70 promoters.
transcription was also examined by using nuclear run-on assays. Promoter proximal segments of c-fos
were undertranscribed relative to sequences that are more distal (Fig. A, lanes 1 and 2, and Fig. B, lanes 1 to 3, slots 12 to 14 versus slots 15 and 16), a finding consistent with the transcriptional pause reported in murine c-fos
intron 1 (44
). In fact, the signals arising from the 5′-most sequences of the transcript were difficult to discern above the background. Adriamycin induced a biphasic response of the c-fos
promoter. Within the first hour of treatment, transcription was increased, most noticeably at the 5′ end (Fig. B, lanes 2 and 3). Subsequently, transcription from the entire gene was shut down (Fig. B, lanes 4 to 6). Transient augmentation of c-fos
transcription by camptothecin was noted (Fig. A, lanes 2 to 6). Butyrate and trichostatin A slightly elevated transcription at the c-fos
start site (Fig. A, lane 12, and Fig. B, lanes 13 and 14).
The dramatic shutoff of c-fos due to adriamycin treatment was explored further by using KMnO4-LM-PCR in vivo footprinting. Dramatic alterations in the promoter conformation were caused by topoisomerase II inhibition. Increased reactivity within the immediate vicinity of the start site was prominent, while just 3′ of the start site on the bottom strand reactivity was diminished (Fig. A, lanes 4 and 9). (The DNA region shown in the c-fos footprint Fig. maps to slots 11 and 12 of the run-on transcription reactions shown in Fig. and .) Farther downstream, the same intron 1 segments holding paused RNA polymerases detected with nuclear run-on (Fig. , slots 15 and 16, and Fig. , slots 13 and 14), showed bases with altered reactivity (both increased and diminished), perhaps indicating that the drug treatment thwarted downstream transit of complexes (data not shown). Consistent with this interpretation, the DNA from adriamycin-treated cells became exclusively hyporeactive distal to the downstream pause region (data not shown). As with c-myc, topoisomerase II inhibition freezes complexes at the c-fos promoter and prevents RNA synthesis either by inactivating the transcription machinery or by imposing an insurmountable barrier such as accumulated torsional strain or drug-frozen topoisomerase II bound to the DNA template. It is also likely that unrelieved torsional strain perturbs the reactivity of downstream segments.
FIG. 6 Topoisomerase II inhibition alters the structure of the c-fos promoter. (A) In vivo potassium permanganate footprint. Cpt, camptothecin; Adr, adriamycin. The cells were incubated with inhibitors for 4 h. (B) Nucleotides with altered intensities in the (more ...)
Also like c-myc
, camptothecin evoked no clear changes in the pattern of KMnO4
reactivity for c-fos
(Fig. A, lanes 3 and 8). Camptothecin perturbs c-fos
transcription by elevating basal expression (as detected with RNase protection [Fig. , lanes 10 to 12] and with run-on assays within 3 h of drug treatment [Fig. A, lanes 2 to 5]), while delaying and dampening induced expression (66
). These effects occur without disturbing promoter-DNA conformation. Therefore, it is likely that the primary effect of this drug is exerted at the level of elongation throughout the body of the gene.
Basal (non-heat shocked) hsp70 RNA levels were depressed by camptothecin (Fig. , lanes 10 to 12) but increased by adriamycin (Fig. , lanes 13 to 15) and HDAC inhibitors (Fig. , lanes 19 and 20). hsp70 nuclear run-on transcription increased transiently within the first hour of camptothecin or adriamycin treatment and then declined by 4 h (Fig. , lanes 2 to 6). Adriamycin developed an alternating hyphenated pattern of augmented and diminished permanganate reactivity throughout the region of the paused polymerase (Fig. A, lane 3). While on the opposite DNA strand, a single downstream residue (+9) (position 282, accession number M11717) became intensely reactive after adriamycin treatment (Fig. A, lane 10). Thus, as with c-myc and c-fos, adriamycin seemed to cause increased hsp70 promoter occupancy after a transient increase in promoter activity.
FIG. 7 Topoisomerase II inhibition alters the structure of the hsp70 promoter. (A) In vivo potassium permanganate footprint. An alternating pattern of increased and decreased intensity near the hsp70 transcription start site is seen. Cpt, camptothecin; Adr, (more ...)
gapdh is often employed as a normalization standard. Considering the relative stability of gapdh mRNA levels (Fig. ) and the modest response of gapdh nuclear run-on activity (Fig. and ) in the face of assorted pharmacological challenges, it may be likely that several molecular devices cooperate to enforce homeostasis on this gene.
Topoisomerase I inhibition forces downstream stalling in rRNA transcription units.
Humans have 200 to 300 copies of the 43-kb ribosomal DNA repeat unit, each transcribed from a single promoter. Each 13-kb primary transcript consists of a 3.6-kb 5′ leader sequence, followed by a 1.9-kb 18S
gene, a 1-kb spacer, a 0.15-kb 5.8S
gene, a 1.1-kb spacer, and a 5-kb 28S
gene; the transcript encoding segments are separated by a 30-kb spacer (accession number U13369
). The long half-life and large pool of ribosomes and rRNA precursors buffer rRNA levels from rapid fluctuation. Therefore, failure of camptothecin to depress rRNA levels as measured with RNase protection (by using a probe at the rRNA
transcription start) was not surprising (Fig. A, lanes 5 to 7), despite the well-established requirement of proper topoisomerase function for rRNA
). More perplexing was the dramatic increase of the rRNA
nuclear run-on signal seen in response to camptothecin (Fig. A, lanes 3 to 5, and Fig. B, lanes 4 to 6). Nascent rRNA synthesized by nuclear run-on was detected by hybridization with sequences at the proximal segment of the 18S rRNA
, ca. 4 kb downstream of the start site. The conformation of the rRNA
promoter before and after camptothecin treatment was assessed by using LM-PCR after permanganate oxidation of intact cells. Camptothecin induced no significant changes of the rRNA
promoter, suggesting that topoisomerase I inhibition provoked no alteration of transcription complexes at most of the rRNA
promoters (Fig. B, lanes 2 to 4). The most straightforward explanation of these data requires the stalling of elongation complexes within the proximal one-third of the rRNA
transcription unit after topoisomerase I inhibition. To test this possibility, nuclear run-on assays were performed by using nuclei from cells treated with camptothecin for various times; the nascent rRNA transcripts elongated in vitro were hybridized with a battery of oligonucleotide sequences derived from segments throughout the rRNA
transcription unit. Indeed, the predicted holdback was observed in the proximal one third of the transcribed region (Fig. A, lanes 2 to 6). Similar over-representation of proximal rRNA
gene sequences in nuclear run-on assays has been noted previously (75
). Initiated RNA polymerase I fails to penetrate very far into the body of the gene, perhaps due to accumulated torsional strain.
FIG. 8 Adriamycin, but not camptothecin, depresses rRNA promoter activity and alters promoter structure. (A) RNase Protection with a probe for the rRNA transcription start site. A 13-h Kodak XAR film exposure and a 2-h exposure (inset) are shown. (B) In vivo (more ...)
Adriamycin depressed rRNA levels in RNase protection studies (Fig. A, lane 8) and diminished rRNA synthesis as detected by nuclear run-on (Fig. A, lanes 6 to 8, Fig. B, lanes 7 to 9, and Fig. B, lanes 2 to 6). In contrast to topoisomerase I inhibition, this topoisomerase II inhibitor augmented KMnO4 reactivity at some nucleotides at the promoter while depressing oxidation at others, implying considerable reorganization of the template at the transcription start site (Fig. B, lane 5).
Transcription by RNA polymerase III of 7SK RNA in Raji cells was fully resistant to camptothecin and partly resistant to adriamycin as measured by nuclear run-on (Fig. A, lanes 3 to 8, and Fig. B, lanes 4 to 9).
Effect of topoisomerase I and II inhibition on the state of c-myc sequences in vivo.
To assess more directly the influence of adriamycin and camptothecin on DNA topology in vivo, the episome from drug-treated Raji pMYC/CAT was recovered and analyzed for linear, relaxed and/or nicked (relaxed/nicked), and supercoiled forms by Southern blotting. Plasmid pMYC/CAT extracted from bacteria provided the reference for this analysis. As expected, the plasmid linearized with Not
I migrated as a single band in the middle of the gel (Fig. B, lane 12). The untreated plasmid revealed three bands: the fastest-migrating band represented supercoiled plasmid; the band of intermediate mobility was the linearized plasmid, while the slowest species was the relaxed/nicked plasmid (Fig. B, lane 13). Figure C shows the DNA recovered from the Raji pMYC/CAT cell line electrophoretically separated, blotted to a membrane and probed with the plasmid-specific CAT sequence. DNA from untreated or DMSO-only treated cells displayed a mixture of supercoiled and relaxed/nicked episomal DNA with minimal linearization (Fig. C, lanes 15 and 16). Four hours of camptothecin treatment yielded the expected conversion from supercoiled to nicked plasmid (Fig. C, lane 17). Linear forms accrued only slowly because of the low probability that two closely spaced single-stranded nicks occurred on opposite DNA strands. In contrast to camptothecin, adriamycin had the opposite effect. Adriamycin treatment yielded more supercoiled and less relaxed episomal DNA (Fig. C, lane 18). These data suggest that under the conditions used in this study, adriamycin inhibited topoisomerase II prior to formation of the protein-DNA adduct cleavable complexes. (DNA fragmentation passes through a maximum and then declines as the concentration of adriamycin is increased [15
].) Such inhibition would result in hypersupercoiled episomal DNA in contrast to the linearized DNA expected from poisoned topoisomerase II-DNA complexes. The minimal linearization promoted by adriamycin on the endogenous c-myc
in Raji (Fig. A, lane 4) or on the episome recovered from Raji pMYC/CAT (Fig. C, lane 18) indicated that the transcriptional holdback and promoter remodeling occurring in response to this drug did not result from local DNA damage or repair complexes. Moreover, the rapidity of these changes might suggest direct involvement of topoisomerase II in maintaining a transcriptionally conducive chromatin environment.