RNA Degradation by Bleomycin, a Naturally Occurring Bioconjugate

Mar 23, 1994 - zole moiety of bleomycin, indicating that BLM would bind to tRNA. At a high (0.3 mM) concentration of activated. Fe-BLM, limited degrad...
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Bioconjugate Chem. 1994, 5, 513-526

REVIEWS RNA Degradation by Bleomycin, a Naturally Occurring Bioconjugate Sidney M. Hecht Departments of Chemistry and Biology, University of Virginia, Charlottesville, Virginia 22901. March 23, 1994 The antitumor antibiotic bleomycin (BLM) has now been the focus of structural ( l ) synthetic , (2-7)) mechanistic (8-101, and therapeutic studies (11, 12) for 30 years. At a biochemical level, the most intensively studied property of bleomycin is its ability to mediate the oxidative destruction of DNA, a transformation that involves several steps including metal ion binding, dioxygen binding and activation, DNA binding, and the actual chemical transformation of DNA in a sequenceselective fashion (8-10). Detailed analysis of the structural elements in bleomycin responsible for the individual steps leading to DNA degradation have made it clear that BLM is a bioconjugate; as indicated in Figure 1, the N-terminus of this polypeptide-derived antibiotic is responsible for metal binding as well as oxygen binding and activation, while the C-terminus participates in DNA binding (8-10). The disaccharide moiety may also provide a metal ligand (10) and may possibly be involved additionally in cell surface recognition by bleomycin. At the level of noncovalent binding, BLM also participates in the formation of other conjugates, i.e., with metal ions such as Fe and Cu, with 0 2 , and ultimately with its polynucleotide substrates. While early mechanistic studies considered several potential therapeutic targets for bleomycin, including DNA and RNA polymerases, DNA ligase, and DNA and RNA nucleases (13-16)) the discovery that BLM could mediate DNA cleavage both in vitro (8-10) and in vivo (17-19) led to increasing efforts in the characterization of this facet of bleomycin action. The subsequent finding that DNA cleavage was sequence-selective, and could result in both single- and double-stranded breaks, prompted intensive efforts to understand the molecular basis for this sequence-selective cleavage. It has seemed reasonable to assume that the principles so derived could be used for the design of new structural classes of antitumor agents that employ the same biochemical strategy as bleomycin. An inevitable consequence of the focus on DNA as a therapeutic target for bleomycin is that less attention has been given to other biochemical and biological effects of the drug. For example, BLM mediates lipid peroxidation (20-23), a logical consequence of its characterized properties in small molecule oxidatiodoxygenation (24-26). It has also been shown that a bleomycin analog rendered dysfunctional for DNA cleavage by chemical modification was able to inhibit the growth of cultured mammalian cells in the presence of the local anesthetic dibucaine (27). Of special interest in this regard are studies of RNA degradation by bleomycin. Early studies failed to detect RNA cleavage by bleomycin (28-32). In retrospect, this is not entirely surprising as much of this work was done before it was appreciated that BLM-mediated polynucleotide degradation requires a metal ion cofactor and oxygen. Also noted was the inability of certain RNA's to 1043-1802/94/2905-0513$04.50/0

Metal Binding/ Oxygen Activation

Received

DNA Binding

I Ho

kor }

Disaccharide

Figure 1. S t r u c t u r e of bleomycin Az. T h e functional domains of t h e molecule are indicated.

inhibit BLM-mediated DNA degradation, suggesting that bleomycin did not bind to RNA (32). The first evidence in support of the ability of FeBLM to degrade RNA was reported by Magliozzo et al. (33). They demonstrated that titration of solutions containing 20 pM bleomycin with tRNA or DNA (0.2-0.6 mM) gave comparable quenching of the fluorescence of the bithiazole moiety of bleomycin, indicating that BLM would bind to tRNA. At a high (0.3 mM) concentration of activated FeBLM, limited degradation of yeast tRNApheand a few other tRNA isoacceptors was noted. A SURVEY OF RNA CLEAVAGE BY BLEOMYCIN

Transfer RNA's and tRNA Precursor Transcripts. The first systematic study of RNA cleavage by BLM was carried out by Carter et al. (34-361, using Fe(II).BLM A2 that was activated aerobically in the absence of any added reducing agent. A survey of several tRNA precursor transcripts and mature tRNA's indicated that most were not cleaved by activated FeBLM; however, a 118nucleotide Bacillus subtilis tRNAHisprecursor transcript was cleaved efficiently in the presence of 3 pM Fe(II).BLM Az, Le., under conditions comparable to those required to produce DNA damage. Relative to the cleavage of B-DNA by Febleomycin, which is selective for S-G-pyr3' sites, but typically results in the production of many DNA lesions with varying efficiencies even in duplexes of modest length, the cleavage of the tRNAHisprecursor was remarkable in two ways. First, the RNA was cleaved only a t a single site a t the lowest concentration of FeBLM employed; even a t higher concentrations of added drug, cleavage a t this site predominated. The other unusual feature associated with this substrate became apparent when the site of cleavage was identified by RNA sequence 0 1994 American Chemical Society

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Bioconjugate Chem., Vol. 5, No. 6, 1994

AAAUAAAAAUUGAAUU 3' C 5'GAAUACAAGCUUUAUCT\AUAUGEUUU6G- C C- G G- C G-U U-A

\

Hecht

C A C -G G3' C -G C U -G U A U-A C-G C 5 1G AAUACAAG -CAGGCCAG U A A AAGCAUUACCCG -C U-A G-C G-C U- A G-C G-C G- C G-CCUUCC

A A G CGAAGG~ u UC UCAG c I l l u CGUC A I I I I I

G ~ G GCCC A uu G I I I C GGG C A A ~ A

G-C C-G A- U G- C

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u

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U A GUA

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Figure 2. Structures of Bacillus subtilis tRNAHisprecursor (left)and Escherichia coli tRNA% precursor (right).The major FeBLM cleavage site is denoted with an arrow, the minor sites with asterisks. analysis. As illustrated in Figure 2, the primary site of cleavage was a t uridiness. While this nucleotide was part of a 5'-GU-3' cleavage site, it was in a region of the molecule believed to be single-stranded, by analogy with the folding of other structurally characterized tRNA's. Further, the minor sites of cleavage in the tRNAHis precursor substrate exhibited no clear sequence selectivity. Treatment of tRNAfis precursor with BLM congeners that produce more DNA damage than BLM Az, such as BLM Ag, gave enhanced cleavage of tRNAHisprecursor and resulted in the appearance of a few additional minor sites. Otherwise, these species produced the same effects as BLM A2 ( C . E. Holmes and S. M. Hecht, unpublished data). In comparison with the tRNAHiSprecursor, an in vitro RNA transcript corresponding to Escherichia coli tRNA% precursor was refractory to cleavage by activated FeBLM (34). In spite of the ostensible structural similarities between these two tRNA precursor transcripts (Figure 2), only minimal damage was noted for the tRNA% precursor, even when the BLM:tRNA ratio was 5000-fold greater than that required to produce cleavage of tRNAHiS precursor (C. E. Holmes and S. M. Hecht, unpublished data). That this apparent difference in susceptibility to BLM was not a n experimental artifact was verified by repeating the cleavage experiment using a reaction mixture that contained both tRNA precursor transcripts; again, only tRNAHiSprecursor was cleaved. Numerous additional tRNA precursor transcripts and mature tRNA's have now been tested as substrates for cleavage by Fe*bleomycin. Those species that were cleaved included a Schizosaccharomyces pombe amber suppresser tRNAserconstruct and mature E. coli tRNAIHis (Figure 3) (37), as well as E. coli tRNASeCysprecursor construct (37)and a yeast cytoplasmic tRNAASpprecursor construct (C. E. Holmes and S. M. Hecht, unpublished data). Hiittenhofer et al. (38)have reported their findings using yeast tRNAPheas a substrate for Fe-BLM and have also reported that a n E . coli tRNA@ precursor transcript and E. coZiZLeU were cleaved, although the sites of cleavage for the last two RNAs were not given. A comparison of the sites cleaved in these substrates indicated that cleavage involved 5'-GN-3' sites, but that

3'

-E

j'

A-U

C-G A-U

C -G

CA - U A U A CUA

A '' C C 5 ' G- C G -C U-A G-C G-C C-G U-A A-U S4U UACCCUA; I I I I I gD

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c*

G ' ' I 1 U G , gAGAGCC m7G 1(/ CU -- AG-I *I U G- C 1G - C

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Figure 3. Structures of Schizosaccharomyces pombe amber suppressor tRNASerconstruct (top) and mature E. coli tRNAIHis (bottom). The major sites of FeBLM-induced cleavage are indicated by arrows, the minor sites by asterisks.

other sequences (notably 5'-UU-3' in E. coli tRNAIHiS(37) and 5'-UG-3' in yeast tRNAPhe( 3 8 ) )were also cleaved.

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1 2 3

A

I I I I I I I I I

CGGCGUUGG5’

I2345678910

Figure 4. Bleomycin-mediatedcleavage of an RNA transcript corresponding to the 5’-end of HIV-1 reverse transcriptase mRNA. The 5’-32Pend-labeled RNA was prepared by in vitro transcription of a Scu I-linearized DNA plasmid; lane 1, RNA alone;lane 2,100 pM Fe(1I)BLM Az;lane 3,500 pM Fe(1I)BLM A2.

As in the case of B. subtilis tRNAHisprecursor, a number of cleavage sites were located at positions believed to occur at a junction between single- and double-stranded regions of the molecule, but some cleavage sites (see, e.g., Figure 3) were in regions that are nominally singlestranded. Other RNAs. Although most of the work reported to date has involved transfer RNAs and tRNA precursor constructs prepared by in vitro transcription, other types of RNA substrates have also been studied. This included a substrate 347 nucleotides in length corresponding to the %-endof HIV-1 reverse transcriptase mRNA that was prepared by in vitro transcription from an expression plasmid following linearization of the plasmid with restriction endonuclease Sca I ( 3 4 ) . As shown in Figure 4, this RNA was cleaved a t least in four places by Fe(IIPBLM A2. Recently, Dix et al. (39)have utilized activated FeOBLM as a reagent for characterizing structural changes in the iron regulatory element (IRE) of ferritin mRNA from bullfrog. The wild-type RNA was cleaved a t a single site (Ul,) within the IRE, at a 5’-GU-3’ sequence believed to be a t the junction between a single- and double-stranded region in the stem-loop structure. Interestingly, a mutant substrate associated with decreased translational regulation by the endogenous regulatory protein, in which the flanking region contiguous with the IRE was altered by disrupting a phylogeneticallyconserved triplet set of base pairs, was cleaved at A10 and A11 by Fe-BLM, rather than U17. The latter two sites were the first two bases within the double-stranded region on the opposite

Figure 5. Fe(I1)bleomycin A2-mediated cleavage of yeast 5s ribosomal RNA. The secondary structure of the yeast 5s rRNA is shown at the top of the figure, with the three sites of cleavage shown by arrows. The polyacrylamide gel at the bottom illustrates the cleavage of the 5’-32Pend-labeled RNA lane l, rRNA alone (-1 pM final nucleotide concentration);lane 2,250 pM Fe(I1)BLM A2; lane 3,125 pM Fe(I1)BLM A2; lane 4, alkalitreated RNA, lane 5, G-lane, lane 6, A > G lane; lane 7, U A lane; lane 8, 250 pM Fe(II).BLM A2 100 mM NaC1; lane 9, 250 pM Fe(I1)BLM A2 5 mM Mg2+; lane 10, 250 pM Fe(IIpBLMA2 1 mM Mg2+.

+

+

+

+

side of the stem-loop structure from that cleaved in the wild-type IRE. The cleavage of yeast 5s ribosomal rRNA by Fe(IIPBLM A2 has also been studied. This substrate was of interest both because it represented a member of the third major class of RNA molecules and also because the structure has been conserved evolutionarily and characterized in detail using chemical and enzymatic probes. As shown in Figure 5, treatment of this rRNA with Fe(IIPBLM A2 afforded three cleavage bands. RNA sequence analysis indicated that all three sites of cleavage involved the uridine nucleotide in a 5’-GUA-3’ sequence; all of these sequences also have a one-base bulge one or two nucleotides to the 3’-side of the cleavage site. It is interesting that the sites in the 5s rRNA cleaved by Feobleomycin represent three of the four 5’-GUA-3’ sequences in the RNA, all three are believed to be present within helical regions of the RNA and to contribute to stabilization of RNA tertiary structure (37,40). One additional RNA substrate of special interest is an RNA-DNA heteroduplex, a species that is formed during both forward and reverse transcription. An RNA-DNA heteroduplex suitable for study was prepared by reverse transcription of E. coli 5s rRNA using a suitable DNA

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primer. The RNA and DNA strands of the heteroduplex were uniquely end labeled in parallel experiments so the cleavage of each could be studied. Treatment with Fe(1I)BLM under aerobic conditions resulted in cleavage of both the RNA and DNA strands a t a limited number of sites and a t comparable concentrations. Further, the sites of cleavage of the RNA strand of the heteroduplex were different than those of the rRNA from which it was formed by reverse transcription, indicating that cleavage of the RNA strand involved recognition and cleavage of the heteroduplex per se (41). Characteristics of Bleomycin-Mediated RNA Cleavage. At a descriptive level, there are several facets of BLM-mediated RNA strand scission that are worth noting. In addition to the observation made initially, Le., that not every RNA studied has been a substrate for cleavage by bleomycin, none of the substrate RNA’s have been cleaved a t large numbers of sites. Further, unlike DNA (oligonucleotide) substrates for Febleomycin, to date no RNA substrate has undergone double-strand cleavage or cleavage toward the end of an RNA strand (vide infra). Although most of the survey work carried out in the Hecht laboratory has employed relatively high concentrations of bleomycin to facilitate the identification of authentic substrates, not all of the substrate RNA’s so identified exhibit comparable susceptibility to cleavage. Three RNA’s studied thus far, including B. subtilis tRNAHi8precursor, yeast 5s rRNA, and the RNA-DNA heteroduplex formed by reverse transcription of E. coli 5s rRNA, were found to undergo cleavage readily a t 1-3 pM FeBLM concentrations when the final RNA nucleotide concentrations were -1-5 pM. The sensitivity of these RNA’s to FeBLM was thus a t least several-fold greater than that of the other RNA substrates. The experiments carried out with RNA have employed conditions less complex than those which obtain in a cellular environment. In order to ensure that the observations made in these experiments could be obtained under physiological conditions, the effects of agents such as NaCl, spermidine, and MgCl2 on BLM-mediated RNA cleavage have been studied (37, 42). It was found that the cleavage of most RNA’s diminished sharply in the presence of cations such as spermidine and Mg2+;the structural basis for this is discussed below. However, the three RNA’s that had been found to act as efficient substrates for FeBLM (vide supra) were still cleaved efficiently even a t > 1mM Mg2+concentration. Remarkably, as demonstrated convincingly for some RNA substrates (see, e.g., Figure 5), the presence of salt and Mgz+ actually further increased the selectivity of RNA cleavage by bleomycin. While no study of the intracellular cleavage of RNA has been reported thus far, it seems likely that it may prove to be a highly selective process. THE CHEMISTRY OF RNA CLEAVAGE BY BLEOMYCIN

Several lines of evidence suggest that the mechanism of Fe(1I)BLM-mediated RNA strand scission, like that of DNA, involves oxidative transformation of the polynucleotide. These include the observations that RNA cleavage by bleomycin was supported by Fe(II), but not Fe(III), and that cleavage was potentiated by reducing agents such as ascorbate and dithiothreitol (37). In common with DNA cleavage, Fe(I1)BLM-mediated RNA strand scission also required 0 2 . It has been shown by several investigators that Fe(II1)bleomycin can be activated for DNA strand scission via the agency of HzOz in a process formally analogous to the peroxide shunt mechanism established for cytochrome P450 (8-10). RNA cleavage was also observed following admixture of Fe(1II)BLM and HzOz; the sequence selectivity was the

Hecht

same as that noted following aerobic activation (C. E. Holmes and S. M. Hecht, unpublished data). Product Analysis. The actual chemical products of bleomycin-mediated RNA degradation have been shown to include the nucleic acid bases adenine and uracil, as shown by TLC (33)and HPLC analyses (34). By the use of a tRNAHisprecursor transcribed in the presence of [3HlUTP and then 5’-32Pend labeled via the agency of [y-32PlATP polynucleotide kinase, it has been shown that strand breaks a t U35 (34-37) were roughly stoichiometric with the release of free [3H]uracil (R. J. Duff, C. E. Holmes, and S. M. Hecht, manuscript in preparation). During the studies of bleomycin-mediated DNA degradation, one particularly effective tool involved the use of oligonucleotide substrates that underwent oxidative transformation a t one site, or a small number of sites, thereby facilitating product analysis (8-10). This was especially true for substrates that underwent cleavage near the ends of the oligonucleotide strand, as low molecular weight products amenable to direct analysis were thereby obtained (43,441. For example, by the use of this strategy, the self-complementary octanucleotide 5’-CGCTAGCG-3’ was found to undergo FeBLM-mediated degradation almost exclusively a t deoxycytidines and deoxycytidine,; strand scission a t the former position afforded the dinucleotide CPGPCH~COOH, in which the glycolate moiety must have been derived from C-4‘ and (2-5’ of the sugar moiety of deoxycytidines (Scheme 1). As noted in Scheme 1, Febleomycin-mediated DNA degradation also results in the formation of base propenals, which in this case is believed to contain C-l’, C-2‘, and C-3‘ of the sugar moiety of deoxycytidines. As indicated, it is possible to rationalize the formation of these products as arising from initial abstraction of C-4‘ H of deoxyribose (8-10). Another set of products involves the formation of a (2-4‘ OH apurinic acid (the alkali labile lesion (45-47)) with concomitant release of free base (Scheme 2). It may be noted that the latter pathway does not lead directly to strand scission; an additional chemical treatment is required to obtain cleavage (8-10, 4547). Although the strategy outlined in Scheme 1has worked well for the analysis of BLM-mediated DNA cleavage products, to date no RNA substrate has been observed to undergo cleavage near the end of an oligonucleotide, thus precluding the use of this approach for the analysis of the chemistry of RNA cleavage. In order to determine whether a ribonucleoside could undergo FeBLM-mediated oxidative damage analogous to that observed for deoxyribonucleosides,the chimeric oligonucleotides shown in Figure 6 were employed a s substrates for Fe(1I)BLM Az. As illustrated in Scheme 3, both of these oligonucleotides afforded CPGPCH~COOH, suggesting strongly that both had undergone oxidative transformation initiated by abstraction of C-4‘ H from the sugar moiety of ribocytidines and ara-cytidines, respectively (34). It may be noted that no base propenal formation was detected following tRNA degradation (33, 34), as expected. The hydroxylated base propenal, whose formation might have been anticipated following the degradation of the chimeric oligonucleotides shown in Figure 6, has not been detected. This is not entirely unexpected, as the hydroxylated base propenal might be anticipated t o be unstable, undergoing facile hydrolysis to afford the free nucleic acid base. In fact, the appearance of free bases has been reported (33,34);as noted above for B. subtilis tRNAHisprecursor, the formation of uridine was roughly stoichiometric with tRNA cleavage a t uridines:, (C. E. Holmes, R. J. Duff, and S. M. Hecht, manuscript in preparation). In the case of the chimeric oligonucleotides,

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Scheme 1. Strand Scission Products Resulting from Treatment of CGCTAGCG with Fe-Bleomycin

i

5'

Criegee -type

CGCTAGCG GCGATCGC

+ -

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rearrangement

I

3'

pTAGCG

0

o=;-6

anti-

1

elimination

0

? .

03P-0

I

ocn,mn

CHO

Scheme 2. Alkali-Labile Lesion Resulting from Treatment of CGCTAGCG with FeBleomycin

1

0

0&-6 I

i

5'

*O

CGCTAGCG GCGATCGC

3'

0&6 O I

I

w

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L

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b

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0.7-6?

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pTAGCG

L,

+ n

the amount of free cytosine released was in excess of that anticipated from the characterized oligonucleotide cleav-

age mechanisms (8-10, 43-47)and was studied further (vide infra).

Hecht

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How

“w C

? o=P-6

ported to date, some useful data fully consistent with the chemistry outlined in Scheme 3 have been obtained using yeast 5 s ribosomal RNA. As shown in Figure 5, the cleavage of this rRNA by activated Febleomycin was carried out using both 5’- and 3’-end-labeled rRNA’s in parallel experiments. In addition to verifying the absence of double-strand cleavage of this substrate (371,the analysis indicated that each of the three cleavage sites was a primary site. This experiment also provided some information about the chemical nature of the cleavage products. Scheme 4 illustrates the nature of the products that would form as a result of cleavage of the 5 s rRNA a t uridines0 if the chemical mechanism of cleavage were analogous to that which is obtained for DNA (cf. Schemes 1 and 3). In particular, it would be anticipated that the 5’-end-labeled rRNA would afford a product having a phosphoroglycolate moiety a t the 3’-terminus (34). If actually formed as a reaction product, this species would migrate farther on a polyacrylamide gel than the corresponding band in the sequencing lane which has a 2’,3’cyclic phosphate a t its 3‘-terminus. As shown in Figure 5, this was actually observed. Likewise, cleavage of the 3’-32Pend-labeled rRNA by Fe-BLM gave bands that migrated slightly faster than the corresponding bands in the U-lane of the sequencing gel, the latter of which are known to have 5’-OH termini. As shown in Scheme 4, this is entirely consistent with cleavage of the RNA by the same oxidative mechanism as DNA, which is known to afford 5’-phosphate termini (8-1 0). Oxidation of RNA at C-1’ of Ribose. Prior to the definitive characterization of C-4‘ of deoxyribose as the site of oxidation of DNA by activated FeaBLM, it was suggested that oxidation of C-1’ was reasonable from a chemical perspective and could potentially explain some

C

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I

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qAGCG

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Cs rib0 CGCTAGCG

C

C,

- Bra CGCTAGCG

Figure 6. Self-complementary chimeric octanucleotides used as substrates for Fe(I1)bleomycin.

As noted above, FeBLM-mediated DNA strand scission produces alkali-labile lesions in addition to strand breaks (45-47); both sets of products are believed to derive from a common C-4‘ deoxyribose radical (8-10). Because the strand scission products formed from RNA appear to be analogous to those formed from DNA, it is logical to think that RNA degradation may also proceed via a C-4‘ ribose radical. The possible existence of a second set of RNA products, analogous to the alkali lesion in DNA, seems not unlikely, but has not yet been demonstrated experimentally due to the inherent lability of RNA to treatment with alkali. Cleavage of Double-Labeled Ribosomal RNA. Although no detailed structural analysis of the FeBLMmediated cleavage of any RNA molecule has been re-

Scheme 3. Oxidative Degradation of Chimeric Octanucleotides Initiated by Abstraction of C-4 H

I

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0zp-o n

G

Criegee -type b

rearrangement

+

-

6n o=P-6 I qAGCG

-

b

asp-0

OCH,COOH

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formed from that substrate upon treatment with bleomycin. Because the amount of free cytosine released upon treatment of C3-ara CGCTAGCG was greater than would have been anticipated from the degradation processes shown in Schemes 1 and 2, and because this oligonucleotide is known to have a conformation different from that of B-DNA (55),we explored the possibility that it might undergo oxidation at (3-1'. The strategy, outlined in Scheme 5, involved capture of putative intermediate i, which would form via Criegee-type rearrangement of a C-1' hydroperoxide intermediate, with diaminobenzene. In fact, the derived quinoxaline was shown to form, a s judged by HPLC comparison with a n authentic synthetic standard. Further verification of the structure of the bleomycin-induced product, as well as quantification of the amount of quinoxaline formed, was accomplished both by analysis of the UV spectrum of the product, which has a Am= a t 320 nm ( E 59001, and also by the use of El4C1l,2-diaminobenzene of known specific activity. It was found that the pathway outlined in Scheme 5 afforded 58%of the products derived from degradation of cytidines in Cj-ara CGCTAGCG and about 10% of those produced from C3-ribo CGCTAGCG (56). Not yet resolved experimentally is the issue of whether C-1' oxidation is also accompanied by formation of a C-1' hydroxide intermediate analogous to that depicted in Scheme 2, as originally suggested for oxidative damage to DNA (48).

Scheme 4. Putative Chemistry of 55 rRNA Cleavage by Fe(II).Bleomycin, Illustrated for the Lesion Produced at Uridines0 "GGUU

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+

t

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ribonuclease Phy M

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uracil

of the experimental observations that had been made (48). In fact, this site has subsequently been shown to be oxidized by other DNA damaging agents (49-51). On the basis of the earlier observation (52) that poly(dA>poly(rU)gave more free adenine production (relative to adenine propenal) than would have been expected from oxidation of C-4' H of deoxyribose with bleomycin, as might be anticipated from analogous oxidation processes operating a t C-1' (48) (cf. Schemes 1 and 2), Absalon et al. (53)studied the possible involvement of C-1' chemistry in DNA degradation. For the substrates studied, they excluded the involvement of C-1' chemistry convincingly. Recently, Long et al. (54)have shown that structural alteration of a DNA substrate, in a fashion that leads to altered conformation, can alter the ratio of products

POLYNUCLEOTIDE BINDING AND DEGRADATION

In spite of the fact that bleomycin-mediated polynucleotide degradation has been studied intensively for a number of years, surprisingly little is known about the

Scheme 5. Oxidative Degradation of Chimeric Octanucleotides Initiated by Abstraction of C-1' H

?

OzP-6

AwGCriegee -type

0

rearrangement

O&d

I

-

I

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+ -

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-

I

O - C : D

520 Bioconpggate Chsm., Vol. 5, No. 6, 1994

strategy employed by BLM to select and destroy its polynucleotide substrates. Although the preferential cleavage of DNA, and to some extent RNA, a t certain 5’Gpyr-3’ sites has been well documented (8-10, 57-59), it is uncertain why those preferred sites are chosen. Further, while FeBLM-mediated damage to DNA occurs in the minor groove (8-10), it is still not certain whether the mode of association of RLM with DNA involves minor groove binding (60),intercalation (61-63), or both. Nonetheless, on the basis of the accumulated data, a number of inferences can be drawn about the way in vhich BLM selects its preferred binding sites and associates with them prior to their oxidative destruction. On the assumptioii that the recognition and binding of RNA by BLM is fundamentally analogous to that of DNA (vide infra),recent experiments with RNA allow further observations to be made. Characteristics of Polynucleotide Binding by Rleomycin. The equilibrium binding constant for the association of bleomycin and certain metallobleomycins *vith DNA is on ihe order of lo5 M-I, as demonstrated under a variety of experimental conditions (10). The lifetimes of the DNA complexes have been measured for Cu(II).BLM and Fe(III).BLM; they were 0.1 and 22 s, respectively (64). (Metal1o)bleomycinscan unwind DNA and cause helix elongation, although it is not certain whether this results from (partial) intercalation of the bithiazole moiety, ionic interactions between bleomycin and the phosphate ester backbone of DNA, or both (65). The preference of bleomycin for cleaving DNA a t 5’GC-3’ and 6’-GT-3’sites has been attributed to a specific hydrogen bonding interaction between the bithiazole moiety of BLM and the guanine nucleotide at the cleavage site (60). However, recent studies of the intrinsic preference 3f thc bithiazole moiety for specific sites on DNA suggest that it has little sequence specificity and that any preference that it may have is unlike that of bleomycin (66, 67). Further, a t least three lines of evidence suggest strongly that it is the metal-binding domain of BLM that is responsible for the sequence selectivity of DNA cleavage by bleomycin. These include (i) the observation that BLM congeners having the same C-terminus, but altered metal-binding domains, exhibited altered strand selectivity of DNA cleavage (441, (ii) the finding that a series of deglycobleomycinanalogs in which the metal-binding domain and bithiazole moiety was separated by semirigid spacers of increasing length all cleaved DNA substrates the same site, albeit with varying efficiencies (68), and (iii) the discovery that PMAH, a model for the metal-binding domain of BLM, exhibited similar sequence selectivity of DNA cleavage to that of RLM itself (69). It may be noted, however, that other analogs having metal-binding domains similar to that of BLM failed to produce sequence selective cleavage (7, 70). In the context of the foregoing observations, it may be pertinent to note that 5’-Gpyr-3‘sequences constitute the widest part of the minor groove of B-DNA (71),which is the preferred substrate for BLM (8-10, 72). Molecular modeling studies in the Hecht laboratory suggest that the chelated metal binding domain of bleomycin may be somewhat too large to fit within the (unperturbed) minor groove of B-form DNA; the somewhat greater width of the minor gToove a t GC as compared with AT sequences (-6 A vs 4 A, respectively) could well facilitate the DNA binding of the BLM metal binding domain a t 5’-GC-3’. Consistent with this suggestion were the observations that FeBLM cleaved DNA preferentially a t the sites of bulges (73) and (partially) perturbed duplexes (74) regardless of DNA sequence, as such sites would necessarily

Hecht

have somewhat wider minor grooves. As noted above, a number of the sites a t which activated FeBLM has been noted to cleave RNA involve one-nucleotide bulges (37) or the junction between single- and double-strand regions; the “minor groove” of RNA in such regions is also very likely to be somewhat wider than that of a normal A-form duplex. Factors That Limit the Efficiency of Cleavage of DNA and RNA by Bleomycin. Depending on the way that individual experiments are performed, any of several factors can limit the amount of polynucleotide damage mediated by bleomycin. These include the omission, or use of a suboptimal amount, of some cofactor required for cleavage such as metal ions, oxygen, or a reducing agent (8-10, 75). The facility of substrate degradation can also be influenced by factors such as pH (75, 76), temperature (35, 36, 761, and the timing and order of addition of reagents (77). Ironically, it has also been shown that large amounts of DNA substrate can inhibit cleavage by bleomycin (75, 78-82 1, apparently reflecting the fact that activation of Fe(II).BLM under aerobic conditions occurs more readily in solution before the activated species binds to DNA (81). Even under conditions optimal for substrate degradation, one or more steps must limit the efficiency of the overall process. A few lines of evidence suggest that one of the limiting steps involves the actual chemical transformations that lead to DNA and RNA degradation. These include the observations that (i) some polynucleotide substrates are cleaved with efficiencies much greater than others of ostensibly similar structure (34, 37,43,44), (ii) there is a primary isotope effect associated with the abstraction of C-4’ H from deoxyribose by FeBLM (82) which can vary from site to site on DNA (83),(iii) the associatioddissociation of BLM from DNA would seem to be fast relative to cleavage (641, and (iv) there appear to be BLM molecules bound to DNA a t sites that do not lead to (efficient) DNA degradation (84, 85). Recently, we carried out an experiment to define the reasons for the greater selectivity of RNA cleavage by BLM as compared with DNA cleavage; the results were surprising and provided important insights into the nature of BLM-polynucleotide interaction. Cleavage of a tRNA Precursor and Its Corresponding tDNk The initial observation that B. subtilis tRNAHisprecursor was cleaved a t a single major site by activated FeBLM, and that a number of other tRNA’s and tRNA precursor constructs were not cleaved a t all, suggested that bleomycin-mediated cleavage of RNA must differ fundamentally from that of DNA. The results of the survey of RNA cleavage summarized above have tended to lessen the distinction, as both DNA and RNA are cleaved primarily at 5’-Gpyr-3’sites, but the cleavage of RNA clearly does occur in a much more highly selective fashion than that of DNA. While it seemed a t first that the differences in bleomycin-mediated cleavage must be due to the fact that DNA and RNA are constituted from different types of mononucleotides, it was also recognized that DNA’s do not ordinarily assume secondary and tertiary structures analogous to those believed to obtain for the tRNA’s and tRNA precursors that had been studied most intensively as substrates for Fe-BLM. Accordingly, we prepared a DNA identical in primary sequence with B. subtilis tRNAHlsprecursor. Although there was no direct evidence for the secondary and tertiary structure of either of these species, it seemed highly likely that they would be quite similar based on reports of the behavior of pairs of tRNA’s and tDNA’s studied earlier. For example, E. coli tRNAfMet

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Bioconjugafe Chen;., Vol. 5, No. 6,1994 521 3

5

7

9

1 1 1 3 1 5

Figure 7. Comparison of the cleavage of tRNAHisand tDNAHis precursor substrates by Fe(II).BLM. The reactions were run with the tRNA and tDNA substrates at 7-8 pM nucleotide concentrations; lane 1, tDNA only; lane 2,1.25 pM BLM A2; lanes 3-8, 0.25,0.5,1.25,2.5,25, and 250 pM Fe(II).BLM A2, respectively; lane 9, tRNA only; lane 10,1.25pM BLM Az; lanes 11-16,0.25, 0.5, 1.25, 2.5, 25, and 250 pM Fe(II).BLM A2, respectively.

and its correspondingtDNA, the latter of which contained a 3'-terminal riboadenosine, were both substrates for methionyl-tRNAsynthetase (86). It was also shown that tDNA analogs of E. coli tRNAPheand tRNALys were capable of inhibiting tRNA activation by the cognate aminoacyl-tRNA synthetases, and also acting as substrates for the same activating enzymes (87). Initially, B. subtilis tRNAHisprecursor and its correspondingtDNA were treated with Fe(II).BLM A2, under

aerobic conditions. Cleavage of the tDNA was detected readily following treatment with 500 nM Fe(II).BLMA2; tRNAHiscleavage was slightly less facile, being detectable at 1.25pM concentration and readily apparent a t 2.5 pM (Figure 7). Remarkably, at low concentrations of added Fe-BLM, cleavage of the tDNA occurred predominantly at a single site, identical with the primary site of cleavage of tRNAHis precursor (42)! This observation argues strongly that the recognition of DNA and RNA by activated Fe*BLMmust be fundamentally analogous and relies primarily on recognition of nucleic acid structure and conformation,rather than the nature of the constituent mononucleotides. Treatment of the tDNA with higher concentrations of Fe(II).BLM A2 resulted in increasing amounts of degradation and the appearance of additional cleavage bands. The additional sites of cleavage, summarized in Figure 8, reinforce the view that cleavage of the tDNA was analogous to that of tRNAHisprecursor, as several of the additional sites of cleavage of the tDNA were identical to the minor sites of cleavage of tRNAEisprecursor. Also similar was the inhibition of cleavage of the tDNA and tRNA substrates upon admixture of Mg2+,demonstrating that nucleic acid secondary and tertiary sti-uztme determine the susceptibility of a BLM substrate to inhibition by Mg2+(42). The cleavage of the tDNA and tRNA did differ in one important respect, however, i.e., in the results obtained when higher concentrations of Fe-BLM were employed. As shown in Figure 7, the tDNA (present at 7-8 pM nucleotide concentration) was consumed completely in the presence of 2.5 pM Fe(II).BLM A2; even more extensive degradation was apparent at 25 pM FeBLM. In comparison, the extent of tRNAqi3 cleavage obtained using 2.5 pM Fe(II).BLM did not change dramatically in the presence of greater concentrations of the drug, even when 250 pM Fe(II).BLM was employed (42). In the belief that this difference must reflect the greater affinity of Fe*BLMfor the tDNA, a competition experiment was carried out. In this experiment radiolabeled tDNA or tRNA substrates having the same specific activity were treated with Fe(II).BLM A2 in the presence of varying concentrations of anlabeled tRNAHis or tDNA. Unexpectedly, unlabeled tRNAHiswas much more effective than unlabeled tDNA in inhibiting the FeBLM-induced degradation of both radiolabeled sub-

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Figure 8. Structures of B. subtilis tRNAHisprecursor and a tDNA arbitrarily folded in the same secondary structure. In addition to the major sites of cleavage at U 3 5 (T35), denoted by large arrows, other significant sites of tDNA cleavage are indicated by small arrows. Minor sites of cleavage of both substrates are denoted by asterisks.

-

Hecht

Figure 9. Structures of 5’-CGCGAATTCGCG-3’, a B-DNA (88),and 5’-GGGGCCCC-3’, a n A-DNA (89).Both species are viewed along the minor groove at the site of cleavage actually established for the dodecanucleotide (left) in comparison with the corresponding GC sequence for the octanucleotide (right). The relevant C-1’ H’s are shown in red; the C-4‘ H’s are in blue.

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Figure 10. Structure of yeast cytoplasmic tRNAASp(90) with Febleomycin (92) bound in a n orientation consistent with the observed chemistry of C-4’ H abstraction.

strates. Unless the kinetics of binding are dramatically different for the two substrates, or there is some RNAspecific mechanism for BLM deactivation, the inescapable conclusion is that Fe*BLM binds more tightly to the R N A substrate than to its DNA counterpart. Why Is the Cleavage of RNA More Selective Than That of DNA? Given the likelihood that bleomycin actually binds more tightly t o RNA than t o DNA, the observation that RNA cleavage occurs less frequently is intriguing. There are two possible explanations for this phenomenon, either or both of which could account for the experimental observation. The first is that FeBLM produces RNA damage much of which does not lead to RNA strand scission. The second is that bleomycin binds t o RNA in an orientation not conducive to the production of strand breaks. Much of what is known about the actual chemistry of RNA cleavage at present relies on the experiments

described above involving the use of C3-riboCGCTAGCG as a substrate for Fe-BLM. Assuming that this is a valid model for RNA degradation, RNA strand scission results from initial abstraction of C-4’ H of ribose, i.e., the same mechanism employed for DNA strand scission. RNA may also be degraded by a process involving initial abstraction of C-1’ H of ribose, but this would probably not lead directly to strand scission (Scheme 5 ) . Both of the mechanisms for octanucleotide degradation that have been established experimentally involve hydroperoxy intermediates (cf. Schemes 1 and 5). For DNA, the initially formed C-4‘ radical resulting from H abstraction can also be converted to a C-4’ OH intermediate, which is believed to lead to the formation of the alkali labile lesion (Scheme 2) (8-10). In principle, C-4‘ and C-1’ ribose radicals in RNA could also afford the respective hydroxylated ribose intermediates. Collapse of such species would presumably occur without RNA strand scission (cf. Scheme 2 and ref 48);the unmasking of these possible lesions in BLM-treated RNA is complicated by the intrinsic lability of the RNA backbone to reagents such as alkali. At present, it is difficult to assess the extent to which the apparent selectivity of RNA cleavage by BLM may reflect the production of lesions that do not lead to strand scission. However, one may note that three of the foregoing four mechanisms for RNA degradation are predicted to afford free nucleic acid bases concomitant with production of the lesion, and the fourth (Scheme 3) would also do so by hydrolysis of a putative hydroxylated base propenal, which is anticipated to be unstable. Therefore, if the extent of strand breakage is compared with base release, the importance of lesions that do not lead to strand scission can be estimated. This has been done both for B. subtilis tRNAHisprecursor and for C3ribo CGCTAGCG. In both cases, the strand scission product would seem to be the major product (R. J. Duff, C. E. Holmes, and S. M. Hecht, manuscript in preparation) arguing that selectivity of RNA cleavage is not due primarily to the formation of lesions that do not lead to strand scission. On the other hand, given the conformational variability of RNA, it may be the case that some sites susceptible t o Fe-BLM-mediated oxidation do not produce strand breaks. The second possible reason for selectivity of RNA cleavage, i.e., the inability of BLM t o oxidize the RNA substrate from the orientation in which it is bound, is supported by a few experimental observations. These

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Bioconjugate Chem., Vol. 5, No. 6,1994 523

Figure 11. Model of the Zn(II).BLM &-d(CGCTAGCG)Z complex. BLM (light blue) is positioned in the minor groove of the B-form octamer (red). The six intermolecular NOES are shown as yellow lines; the apparent length differences are due to pergpective only. H bonding contacts are shown as yellow arrows pointing toward the hydrogen atoms. The metal ion (gold ball) is 3.3 A from the C7 H4‘ (red ball).

include the evidence that certain metallobleomycins bind to DNA at more sites than those at which they produce oxidative damage (84, 85). The finding of a primary isotope effect associated with the abstraction of C-4’ H from deoxyribose (82, 83) also argues that chemical transformation of the DNA substrate is rate-limiting. I t would not be surprising if RNA cleavage exhibited the same characteristics, limiting the amount of products that can be formed. In this context, it may be noted that RNA exists in an A-form conformation, which could be less conducive to oxidative transformation than B-DNA. This is illustrated in Figure 9, which shows the X-ray crystallographic structures determined for 5’-CGCGAATTCGCG-3’(88), a B-DNA structure, and 5’-GGGGCCCC-3’ (89), an ADNA structure. The structure of the dodecanucleotide, which is cleaved by FeBLM at cytidines and cytidine11 (M. Morgan, unpublished results), highlights the nature of the minor groove at the site actually cleaved by Fe-BLM; both the C-4’ H (blue) and C-1’ H (red) atoms are clearly accessible within the minor groove, although BLM apparently abstracts only C-4’ H (8-10, 53). In comparison, the minor groove of 5’-GGGGCCCC-3’at the analogous GC site is wider and shallower but has C-4’ H displaced out of the groove where it may be more difficult for BLM to abstract. Interestingly, C-1’ H is still prominent within the minor groove of this A-DNA and presumably also within A-RNA helices as well. Although the susceptibility of the C-1’ H to abstraction by BLM is clearly less than that of C-4’ H where both are available in the minor groove (8-10, 53), the lesser accessibility

of C-4’ H in the minor groove of A-DNA and RNA might permit the (occasional) abstraction of C-1’ H. This would be entirely consistent with the observation (vide supra) that Fe-BLM afforded a product from C3-ribo and C3-ara CGCTAGCG whose formation must involve abstraction of C-1’ H from the sugar moiety of cytidines (56). X-RAY CRYSTALLOGRAPHICALLY DEFINED RNA SUBSTRATES FOR BLEOMYCIN

An important goal in the study of bleomycin-mediated RNA degradation is better definition of the way in which the antitumor agent binds to RNA. In addition to more detailed structural analysis of known RNA substrates for bleomycin, it would also be helpful to determine whether Fe-BLM can mediate the cleavage of RNA’s whose structures are known at high resolution. In this regard, the recent report by Huttenhofer et al. (38)is of special interest, as it identifies two major sites of cleavage of yeast tRNAPheby Fe-BLM. In collaboration with the laboratory of Prof. Richard Giege, Universite Louis Pasteur, we have shown that yeast cytoplasmic tRNAASpis cleaved at a single major site, cytidine67,by FeOBLM. On the basis of this observation, as well as a consideration of the locations at which BLM cleaves a number of other tRNA and tRNA precursor substrates, we have constructed a model that provides a working hypothesis for the way in which FeBLM might bind to tRNAASp. As shown in Figure 10, this model employs the X-ray crystallographic coordinates for yeast tRNAAsp(90)and a structure for FeeBLM based on a set

524 Bioconjugafe Chem., Vol. 5, No. 6, 1994

of metal binding ligands described by Oppenheimer et al. (91). FeBLM was docked to the “minor groove” formed by the TVCG stem-loop structure, adjusting the interaction to permit potential hydrogen bonding interactions between the bithiazole N-atoms and N2 H of guanosine, as well as electrostatic interaction between the dimethylsulfonium moiety of the BLM A2 C-substituent and a phosphate oxygen anion. This orientation would permit the abstraction of (2-4‘ H from the ribose moiety of cytidines, in a spatially reasonable fashion (92). Although the structure proposed in Figure 10 is not supported a t present by direct physical measurement, the essential correctness of the mode of interaction is suggested by the recent report of the solution structure of Zn(I1)bleomycin AS bound to the DNA octanucleotide duplex 5‘-CGCTAGCG-3‘ (Figure 11) (93). The latter complex was characterized structurally using twodimensional NMR experiments and molecular dynamics calculations. RNA AS A THERAPEUTICALLY RELEVANT TARGET FOR BLEOMYCIN

Bleomycin is commonly thought to exert its therapeutic effects a t the level of DNA degradation. In fact, treatment of cultured mammalian cells with bleomycin causes extensive DNA damage, growth inhibition, and diminished clonigenic potential; these effects all increase with increasing amounts of BLM employed, or as the time of treatment is lengthened (17-19, 94). On the basis of numerous studies, it seems clear that DNA constitutes a critical locus for the therapeutic action of bleomycin. However, there are a number of observations that seem difficult to reconcile with the action of bleomycin a t this single locus. These include the relatively poor correlation between the extent of BLM-induced DNA damage and growth inhibition of KB cells for a series of BLM congeners (94) and the remarkably facile cellular repair of bleomycin-mediated damage to chromatin (94). The possible effects of the cell membrane on the expression of cytotoxicity by bleomycin are also of interest. Poddevin et al. (95)demonstrated that introduction of bleomycin into cultured Chinese hamster fibroblast cells by electropermeabilization greatly enhanced the cytotoxic potential of bleomycin. In addition to the implication that the nuclear membrane could also constitute a barrier to bleomycin, suggesting the involvement of some cytoplasmic target, the finding that bleomycin crosses the cell membrane inefficiently raises the possibility that the drug may actually react with the membrane. In fact, bleomycin has been shown to mediate lipid peroxidation, an effect that could contribute to cytotoxicity (20-23). It has also been shown that the local anesthetic dibucaine, which increases membrane fluidity, rendered cultured KB cells susceptible to inhibition by a bleomycin congener dysfunctional in DNA degradation (27). In this regard, RNA represents a potentially attractive therapeutic target for bleomycin for several reasons. RNAs are present in the cytoplasm of eukaryotic cells, where they would be more accessible to exogenous agents; representative examples of all three major classes of RNA’s, as well as an RNA-DNA heteroduplex, have been shown to act as substrates for cleavage by activated FeSBLM. RNA cleavage by bleomycin is more highly selective than that of DNA and is anticipated to be even more so under physiological conditions due to the effects of Mg2+ and polyamines noted above. In addition, the apparent paucity of mechanisms for RNA repair suggests that damage to one or more RNAs essential for cell

Hecht

function could create an insufficiency of an RNA, or some derived protein, required for cell viability. At present, it is not known whether the cytotoxic effects of bleomycin are mediated (in part) via oxidative damage to RNA. While the studies carried out to date in cell free systems have been helpful in defining the parameters conducive to RNA degradation, it is essential that the effects of BLM on RNA in cultured mammalian cells be determined. Likewise, since mRNA can undergo cleavage by bleomycin, it is also possible that the cellular level of proteins whose turnover is fast relative to mRNA production could also be reduced. This possibility should also be addressed experimentally. ACKNOWLEDGMENT

I acknowledge with gratitude the contributions of my co-workers to these studies. Their names appear in the references; special thanks are due to Dr. Barbara Carter, Dr. Robert Duff, Dr. Chris Holmes, Mr. Michael Morgan, and Dr. Richard Manderville. I also thank my collaborators, including Drs. Jacques van Boom and Gijs van der Mare1 (University of Leiden), Dr. Christine Debouck (SmithKline Beecham Pharmaceuticals), and Drs. Richard Giege and Catherine Florentz (Universite Louis Pasteur). This work was supported at the University of Virginia by Research Grants CA27603, CA38544, and CA53913, awarded by the National Cancer Institute, DHHS, and by NATO Travel Grant CRG900204. LITERATURE CITED (1) Takita, T. (1979) Review of the structural studies on bleomycin. Bleomycin: Chemical, Biochemical and Biological Aspects (S.M. Hecht, Ed.) pp 37-47, Springer-Verlag, New York. (2) Takita, T., Umezawa, Y., Saito, S., Morishima, H., Naganawa, H., Umezawa, H., Tsuchiya, T., Miyake, T., Kageyama, S., Umezawa, S., Muraoka, Y., Suzuki, M., Otsuka, M., Narita, M., Kobayashi, S., and Ohno, M. (1982) Total synthesis of bleomycin Az. Tetrahedron Lett. 23,521-524. (3) Aoyagi, Y., Katano, K., Suguna, H., Primeau, J., Chang, L.H., and Hecht, S. M. (1982) Total synthesis of bleomycin. J . Am. Chem. SOC. 104, 5537-5538. (4) Otsuka, M., Masuda, T., Haupt, A,, Ohno, M., Shiraki, T., Sugiura, Y., and Maeda, K. (1990) Man-designed bleomycin with altered sequence specificity in DNA cleavage. J. Am. Chem. SOC.112, 838-845. (5) Boger, D. L., Menezes, R. F., and Dang, Q. (1992) Synthesis of desacetamidopyrimidoblamic acid and deglyco desacetamidobleomycin Az. J . Org. Chem. 57,4333-4336. (6) Boger, D. L., and Honda, T. (1994) Total synthesis of bleomycin A2 and related agents. Synthesis of the disaccharide subunit: 2-0-(3-O-carbamoyl-a-D-mannopyranosyl)-~gulopyranose and completion of the total synthesis of bleomycin A2. J . Am. Chem. SOC.116, 5647-5656. (7) Hamamichi, N., Natrajan, A,, and Hecht, S. M. (1992) On the role of individual bleomycin thiazoles in oxygen activation and DNA cleavage. J . Am. Chem. SOC. 114, 6278-6291. (8) Hecht, S. M. (1986) The chemistry of activated bleomycin. Acc. Chem. Res. 19, 383-391. (9) Stubbe, J., and Kozarich, J. W. (1987) Mechanisms of bleomycin-induced DNA degradation. Chem. Rev. 87, 11071136. (10) Natrajan, A.,and Hecht, S. M. (1993) Bleomycin: mechanism of polynucleotide recognition and oxidative degradation. Molecular Aspects of Anticancer Drug-DNA Interactions (S. Neidle, and M. Waring, Eds.) pp 197-242, MacMillan, London. (11) Carter, S. K.,Crooke, S. T., and Umezawa, H., Eds. (1978) Bleomycin: Current Status and New Developments, Academic Press, New York.

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