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Keywords:

  • reconstituted nucleosome;
  • nucleosome cores;
  • nucleosome linker;
  • cisplatin analogues;
  • nitrogen mustard analogues;
  • 5S rRNA gene

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The interaction of anti-tumour drugs with reconstituted chromatin has been investigated using defined nucleosomal complexes. This allowed the effect of nucleosome cores on drug-induced DNA damage to be assessed for four nitrogen mustard analogues, dimethylsulphate and three cisplatin analogues. A defined nucleosomal complex was employed that contained two precisely positioned nucleosome cores. The construct was then subjected to drug treatment, and the resulting DNA damage was quantitatively analysed using a Taq DNA polymerase stop assay. At the sites of damage, densitometric comparisons between purified and reconstituted DNA were used to evaluate the influence of nucleosomal core proteins on specific drug–DNA interactions. Results were combined with previous data obtained for other DNA-damaging drugs investigated using the same nucleosomal construct. For most of the DNA-damaging agents studied, this method revealed protection at the positioned nucleosome cores and indicated that the preferred site of DNA binding for these compounds was in the linker region of the construct. Statistical analyses confirmed the significant level of damage protection conferred by the nucleosome cores and revealed differences between the examined compounds. Larger compounds generally displayed a greater tendency to target the linker region of the nucleosomal DNA and were impeded from damaging nucleosomal core DNA. In contrast, smaller molecules had greater access to nucleosomal core DNA.

Abbreviations:
2AcC3PtenCl2

dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-2-carboxamide)platinum(II)

4AcC3PtenCl2

dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II)

9-amino AcC3PtenCl2

dichloro(N-[3-[(2-aminoethyl)amino]propyl]-9-aminoacridine-4-carboxamide)platinum(II)

C3-AA

9-aminoacridine attached nitrogen mustard analogue (with C3 chain)

C20-AMSA

amsacrine attached nitrogen mustard analogue (with C2 chain)

C50-AMSA

amsacrine attached nitrogen mustard analogue (with C5 chain)

carboplatin

cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)

cis-[PtCl2(C6H11NH2)2]

cis-bis(cyclohexylamine)dichloroplatinum(II)

cisplatin

cis-diamminedichloroplatinum(II)

DMS

dimethyl sulphate

DNase I

deoxyribonuclease I

L/C Ratio

linker-core ratio

trans-diamminePtCl-phenazine-1-carboxamide

N-(2-aminohexyldiamminoplatinum(II))-phenazine-1-carboxamide

Covalent DNA-binding compounds constitute one of the largest classes of anti-cancer agents. Most DNA lesions induced by anti-tumour drugs have been investigated using purified DNA (1–3). However, such studies neglect the complex nature of the interaction that takes place between the drug and DNA within the cell (4). In a cellular environment, DNA associates with many proteins and is generally present in the form of chromatin. Therefore, to gain access to DNA inside cells, DNA-damaging anti-tumour drugs must surmount this protein barrier.

The fundamental repeating unit of chromatin is the nucleosome, which has been found to consist of two major structural domains: a core particle and linker DNA (5). A high-resolution X-ray crystal structure of the nucleosome core has been determined (6,7). In most eukaryotic organisms, the average core particle consists of 146 base pairs of DNA coiled approximately 1.75 times around a central protein histone octamer protein core (two each of H2A, H2B, H3 and H4) (5). Studies show that the strong histone–DNA associations in the nucleosome render the core DNA relatively resistant to digestion by many different nucleases. The remainder of the nucleosome unit is composed of linker DNA (average length 20–70 bp) which joins adjacent nucleosome cores (8).

In a previous study (9), we employed the well-established nucleosome footprinting agents deoxyribonuclease I (DNase I) and bleomycin to precisely map the position of two nucleosome cores on a 319-bp DNA sequence containing the somatic 5S rRNA gene of Xenopus borealis DNA sequence. Both agents produced two large protein footprints in the same regions of the construct. These protected regions ranged between 114 and 135 bp in size and were located, on average, between base pairs 1–132 and 198–319 of the construct. Having established the number and location of positioned nucleosomes in the construct, various anti-tumour compounds could then be assessed for their nucleosome footprinting capacity in vitro.

To determine the precise site of DNA damage caused by the DNA-damaging agents, a polymerase stop assay was utilized (10,11). This procedure uses Taq DNA polymerase and repetitive thermal cycling to amplify the DNA products of interest, followed by electrophoresis alongside dideoxy sequencing reactions that serve as size and position markers. Subsequent densitometric analysis permits the relative intensity of damage at each site to be determined in purified DNA compared with reconstituted nucleosomal DNA (9). In our previous study (9), it was found that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. It was also found that the cisplatin analogues, carboplatin, 4AcC3PtenCl2 and cis-[PtCl2(C6H11NH2)2], preferentially damaged in the linker region of the nucleosome (9).

In this article, this nucleosome analysis has been extended to include four nitrogen mustard analogues, dimethyl sulphate (DMS) and three additional cisplatin analogues (Figure 1). These analogues include a series of DNA-targeted drugs that consist of a nitrogen mustard or Pt attached to an acridine or amsacrine intercalating chromophore (12–17). A total of 14 DNA-damaging agents have now been evaluated in this nucleosome system (9) and are summarized in this article. These comparisons have enabled correlations between various aspects of drug–DNA interactions and effective anti-tumour activity to be revealed. For example, properties such as molecular weight, sequence specificity, mechanism and mode of action were considered with respect to their relative bearing on the ability of different compounds to damage nucleosomal DNA. Insight gained from these studies is hoped to improve our current understanding of the mechanism of anti-tumour drugs and ultimately benefit the development of more efficient chemotherapeutic agents with less-harmful side effects.

image

Figure 1.  (A) The chemical structures of cisplatin analogues used in this study. Carboplatin is the only clinically successful cisplatin analogue among those examined. Cis-[PtCl2(C6H11NH2)2] is another cisplatin derivative that incorporates two cyclohexylamine groups, while DMS is a methylating agent commonly used to footprint protein–DNA interactions in intact cells and was used as a control compound in this study. (B) The chemical structures of nitrogen mustard analogues used in this study. C3-AA, C50-AMSA and C20-AMSA are DNA-targeted analogues of the nitrogen mustard derivative, chlorambucil. The DNA-targeting element of C3-AA is an attached acridine moiety. Similarly, both C50-AMSA and C20-AMSA target DNA through an amsacrine moiety that is attached by a polymethylene linker chain of length n5 or 2, respectively. (C) The chemical structures of platinum analogues used in this study. The DNA-targeted cisplatin analogues 4AcC3PtenCl2, 2AcC3PtenCl2 and 9-aminoAcC3PtenCl2 contain acridine or 9-aminoacridine moieties attached by polymethylene linker chains of length n3. Trans-diamminePtCl phenazine-1-carboxamide is a DNA-targeted transplatin analogue that incorporates a phenazine moiety attached by a polymethylene linker chain of length n6.

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Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Compounds utilized in this study

Nitrogen mustard analogues were synthesized as described previously for C3-AA (16), C20-AMSA and C50-AMSA (13), and kindly donated by Prof W. A. Denny, University of Auckland, New Zealand. These were dissolved in ethanol to give 1 mm stock solutions and stored at −70 °C. Chlorambucil was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA).

The cisplatin analogues dichloro(N-[3-[(2-aminoethyl)-amino]propyl]acridine-2-carboxamide)platinum(II) (2AcC3PtenCl2), dichloro(N-[3-[(2-aminoethyl)amino]propyl]-9-aminoacridine-4-carboxamide)platinum(II)(9-amino AcC3PtenCl2), N-(2-aminohexyldiamminoplatinum(II))-phenazine-1-carboxamide (trans-diamminePtCl-phenazine-1-carboxamide) were prepared using general methods reported elsewhere (11,17,18) and were a kind gift of D. W. McFadyen, School of Chemistry, University of Melbourne, Parkville, Victoria. Cisplatin analogues were dissolved in dimethylformamide to give 1 or 5 mm stock solution concentrations and were stored at −70 °C.

Nucleosome preparation

The plasmid pXbsF201 (19) consists of a 240-bp BamHI-HindIII fragment from the X. borealis somatic 5S rRNA gene inserted into the plasmid vector pUC9. The PCR amplification of a 319-bp DNA sequence, containing the X. borealis somatic 5S rRNA gene, was performed using REV (5′-aacagctatgaccatg) and SEQ (5′-tcccagtcacgacgt) primer oligonucleotides and the plasmid pXbsF201 as template (9). The histone H1-stripped chicken nucleosome cores, prepared from chicken erythrocyte nuclei, were reconstituted on the 319-bp PCR product using the octamer transfer method (9,20).

DNA damage reactions

DNA damage reactions were carried out using approximately 7 ng of reconstituted nucleosomal or purified free DNA. Final drug concentration ranges were as follows: 1 – 2 mm DMS; 0.05 – 2.0 μm 2AcC3PtenCl2; 4 – 500 nm 9-aminoAcC3PtenCl2; 45 – 500 nmtrans-diamminePtCl phenazine-1-carboxamide complex; 0.005 – 1.0 μm C3-AA; 0.075 – 50 μm C50-AMSA; 0.05 – 1.0 μm C20-AMSA and 400 – 600 μm chlorambucil. Each 40 μL reaction mixture also contained H buffer (2 mm 4-(2-hydroxyethyl)-1-piperazineethane sulphonic acid, 10 mm NaCl and 10 mm EDTA, pH 7.3). Reactions were incubated overnight in the dark at 37 °C for 18 h. After samples were subjected to phenol/chloroform extraction, ethanol precipitation and two ethanol washes, the DNA was dissolved in 4 μL of TE (10/0.1). Samples were then amplified using the linear amplification protocol and electrophoresed on a polyacrylamide sequencing gel.

Linear amplification

The 5′-terminal-labelling of oligonucleotide primers with 32P was carried out as previously described (21). Linear amplification reactions consisted of 16.6 mm (NH4)2SO4, 67 mm Tris–HCl (pH 8.8), 6.7 mm MgCl2, 0.3 mm dNTPs (i.e. 300 μm each of dATP, dGTP, dCTP and dTTP), 0.05 pmol 5′-[γ-32P]-labelled oligonucleotide primer, 0.44 U Ampli Taq DNA polymerase and approximately 7 ng of DNA from the appropriate damage reaction in a final volume of 5 μL.

Dideoxy double-stranded DNA sequencing of untreated DNA was carried out using the same primers to indicate the precise location of damage sites and allow the position of protein footprints to be determined with respect to the sequence. Sequencing reactions employed the pXbsF201 plasmid as the template DNA and were set up as described previously, except with 2.5 μm dNTPs and the addition of one of either 10 μm ddGTP, 100 μm ddATP, 200 μm ddTTP or 100 μm ddCTP, to terminate chain polymerization (22).

Linear amplification and dideoxy sequencing reactions were then subjected to 20 consecutive cycles of 95 °C for 30 seconds, 50 °C for 1 min and 72 °C for 1.5 min, using a Perkin Elmer Cetus Thermocycler 480 (Perkin Elmer, Waltham, MA, USA.). DNA fragments were then separated on a 6% (w/v) denaturing polyacrylamide gel. The gel was subsequently dried and exposed to a phosphor storage screen. Following sufficient exposure, the screen was scanned using a Molecular Dynamics PhosphorImager and the resulting gel image analysed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA).

Densitometry and statistical analysis

Densitometric analysis of footprinting gels was performed to ascertain the relative level of protection conferred to the DNA template by positioned nucleosome cores in the reconstituted chromatin construct. Using ImageQuant software, this procedure entailed a direct comparison between damage band intensities across adjacent lanes containing treated free and treated reconstituted DNA samples. After subtraction of local background values, damage band intensities calculated for each lane were imported into a Microsoft Excel spreadsheet and normalized to give a fractional value ≤1. Individual damage band intensity values were divided by the respective lane normalization factor to give relative intensity values. The relative intensity value of each damage site in the reconstituted DNA sample was then divided by the corresponding value in the free DNA sample to determine a relative damage intensity ratio. Log10 reconstituted/free damage intensity ratios were plotted against nucleotide band sequence positions. (These positions were determined relative to the dideoxy sequencing reaction lanes on the same gel). This allowed the damage intensity ratio at each damage site to be viewed with respect to its base pair sequence position on the template DNA. Furthermore, negative or positive graph values signified regions of relative DNA damage protection or enhancement, respectively.

To make direct comparisons between different DNA damage experiments, a linker-core ratio, or L/C ratio, was devised to describe the ratio between the relative levels of damage incurred in the linker and nucleosome core regions of the construct. Using densitometric data, L/C ratios were determined for each compound by calculating the average reconstituted/free DNA ratio in the linker region divided by the average reconstituted/free DNA ratio in the nucleosome core regions. By this definition, higher L/C ratios were therefore associated with more distinct or pronounced footprints. L/C ratios determined for all DNA-damaging agents studied are presented in Table 1. When comparing different agents, higher L/C ratios thus marked compounds with a reduced ability to damage nucleosomal DNA, or a greater preference for damaging the protein-free linker region.

Table 1.   Data summary for all DNA-damaging agents examined. From the top, DNA-damaging compounds are ranked in order of descending average linker-core ratios (L/C ratios). For all DNA-targeted compounds, the associated DNA-binding moieties are indicated, along with the number of residues in the linker molecule. For all compounds studied, average L/C ratios and Student’s t-test p-values are presented
DNA damaging compoundDescriptionMolecular weightDNA-targeting moietyTargeting moiety linker lengthL/C ratio Nucleosome 1L/C ratio Nucleosome 2Average L/C ratioAverage Student’s t-test p-value
  1. aCompound that forms covalent adducts with DNA.

  2. bCompound that causes strand breaks in DNA.

trans-DiamminePtCl phenazine-1-carboxamideDNA-targeted cisplatin analoguea686Phenazine carboxamide64.932.223.504.05 × 10−10
9-AminoAcC3PtenCl2DNA-targeted cisplatin analoguea6039-Amino acridine33.112.162.525.17 × 10−13
4AcC3PtenCl2DNA-targeted cisplatin analoguea588Acridine32.061.921.995.73 × 10−5
BleomycinGlycosidic antibioticb15001.71.821.761.97 × 10−7
DNase IEndonucleaseb310001.871.491.684.74 × 10−8
C50-AMSADNA-targeted nitrogen mustard analoguea719Amsacrine51.601.711.656.01 × 10−11
C3-AADNA-targeted nitrogen mustard analoguea525Acridine31.721.551.636.30 × 10−10
C20-AMSADNA-targeted nitrogen mustard analoguea662Amsacrine21.481.541.514.93 × 10−7
CarboplatinCisplatin analoguea3711.251.421.347.57 × 10−4
CisplatinAnti-tumour agenta3001.321.311.323.36 × 10−4
2AcC3PtenCl2DNA-targeted cisplatin analoguea606Acridine31.471.161.306.64 × 10−5
cis-[PtCl2(C6H11NH2)2]Cisplatin analoguea4641.121.271.201.74 × 10−3
DimethylsulfateAlkylatora1261.031.021.022.46 × 10−1
ChlorambucilNitrogen mustard analoguea2590.841.010.931.81 × 10−1

For all compounds studied, Student’s t-tests (two-sample, assuming unequal variances) were performed to determine whether the net difference in damage intensities between linker and nucleosomal regions was significant (p-value <0.001).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The nucleosome footprinting capacity of four nitrogen mustard analogues, DMS and three platinum complexes was investigated in this study. Products from linear amplification reactions with free and reconstituted nucleosomal DNA were electrophoresed on polyacrylamide sequencing gels alongside appropriate dideoxy sequencing lanes. For all analyses, damage levels in negative (no drug) control lanes were negligible compared to those observed in drug-treated samples.

Nitrogen mustard analogues

In Figure 2, the gel image shows damage patterns induced in free and reconstituted DNA by the following nitrogen mustard analogues: C3-AA (a 9-aminoacridine-tethered analogue), C20-AMSA (an amsacrine-tethered analogue with linker chain length n = 2), C50-AMSA (an amsacrine-tethered analogue with linker chain length n = 5) and chlorambucil (a non-DNA-targeted nitrogen mustard control). Note that C3-AA, C20-AMSA and C50-AMSA are derivatives of chlorambucil. All four nitrogen mustard analogues exhibited similar sequence specificities, and damage occurred mainly at runs of consecutive purine residues, as previously found (15,23–25).

image

Figure 2.  Damage induced by nitrogen mustard analogues in free and reconstituted DNA (SEQ strand) – PAGE analysis. Lanes labelled “FREE” contain free DNA, while lanes labelled ‘REC’ contain reconstituted DNA. Lanes 1–4 are free and reconstituted H2O blanks (no drug); lanes 5–8 contain DNA treated with 0.005, 0.0075, 0.5 and 1.0 μm C3-AA, respectively; lanes 9–12 contain DNA treated with 0.075, 0. 1, 1.0 and 50 μm C50-AMSA, respectively; lanes 13–16 contain DNA treated with 0.05, 0.1, 0.5 and 1.0 μm C20-AMSA, respectively; lanes 17–20 contain DNA treated with 400, 600, 400 and 600 μm chlorambucil, respectively; dideoxy sequencing lanes G, A, T and C indicate the sequence of the template strand (SEQ); sequencing blanks are marked B.

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The relative effect of nucleosome cores on drug–DNA interactions was evaluated by comparing the damage induced in free and reconstituted samples. In this analysis, the three DNA-targeted nitrogen mustard analogues, C3-AA, C20-AMSA and C50-AMSA, produced prominent protein footprints in the reconstituted samples. These protected regions corresponded with the established locations of positioned nucleosomes in the construct. In contrast, DNA damage patterns arising from the control nitrogen mustard, chlorambucil, did not contain such zones of consistent damage protection. Densitometric comparisons between free and reconstituted samples reflected these observations and are graphically depicted in Figure 3.

image

Figure 3.  REV and SEQ strand densitometry plots representing DNA damage induced by nitrogen mustard analogues. A comparison between free and reconstituted DNA treated with (A) C3-AA, (B) C50-AMSA, (C) C20-AMSA and (D) chlorambucil. Separate plots are shown for REV and SEQ strands. Negative values on the vertical axis represent regions of the DNA that are relatively protected from damage, while positive values indicate areas of relative damage enhancement. Ovals, N1 and N2 indicate approximate positions of the reconstituted nucleosome cores.

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Graphs derived from damage incurred by the three DNA-targeted analogues depict large protected regions in the vicinity of the two reconstituted nucleosome cores (Figure 3). Regions of relative damage enhancement were also pronounced and corresponded well with the established linker DNA location. In contrast, densitometry plots derived from the chlorambucil data did not indicate consistent regions of damage protection or enhancement.

Densitometric damage data from the REV strand of the construct were used to examine the effect of Nucleosome 1 (N1), while SEQ strand data were employed to investigate the Nucleosome 2 (N2) region. For each compound, reconstituted/free damage intensity ratios from five replicate damage experiments were averaged. These data were then partitioned into two groups: damage occurring within the linker region and damage occurring within the nucleosome core regions. Student’s t-tests comparing the linker and nucleosome data arrays were then performed. In Table 1, the resulting t-test p-values are shown separately for nucleosomes 1 and 2, and then averaged for each compound.

On average, higher L/C ratios (and lower t-test p-values) were produced by C50-AMSA and C3-AA, followed closely by C20-AMSA. More specifically, the compound with the longest linker chain, C50-AMSA, also produced the largest L/C ratio. Conversely, C20-AMSA had the shortest linker length and incurred the smallest L/C ratio. These results suggest that different intercalating moieties and linker lengths influence the damaging activity of DNA-targeted nitrogen mustard analogues to different extents. In previous studies, the influence of linker chain length on the specificity of DNA damage has been determined (26). Furthermore, chlorambucil, which does not possess a tethered DNA-targeting group, produced the lowest L/C ratio of all four nitrogen mustard analogues. The statistical insignificance of this L/C ratio was supported by the relatively high t-test p-value obtained for this agent (Table 1). Together, these results imply that histone–DNA interactions have a negligible effect on the level or pattern of damage incurred by chlorambucil. Other investigations into the effect of protein–DNA interactions on nitrogen mustard analogue damage also found that chlorambucil produced less-pronounced protein footprints compared to C3-AA, C20-AMSA and C50-AMSA (24). Overall, the comparative footprinting ‘efficiency’ of each nitrogen mustard analogue examined here correlates well with the outcomes of previous studies (24,25).

Cisplatin analogues

Three different DNA-targeted cisplatin analogues and a non-Pt reference compound, DMS, were also tested for their ability to footprint nucleosomes within the reconstituted construct. The three platinum compounds were 2AcC3PtenCl2, 9-aminoAcC3PtenCl2 and trans-diamminePtCl phenazine-1-carboxamide. Sequencing gel images showed that the sites of adduct formation were generally consistent with those described in previous investigations (11,17,18,27,28). For all three analogues examined, sites of relative damage protection in the regions of positioned nucleosome cores were revealed.

Densitometric data produced two distinct nucleosome footprints for all agents except the reference compound, DMS. Densitometry plots are shown in Figure 4 and L/C ratios in Table 1. A Student’s t-test was performed to assess the relative significance of observed nucleosome footprints and support the interpretation of L/C ratio values (Table 1).

image

Figure 4.  REV and SEQ strand densitometry plots representing DNA damage induced by DNA-targeted cisplatin analogues. A comparison between free and reconstituted DNA treated with (A) dimethyl sulphate, (B) 2AcC3PtenCl2, (C) 9-aminoAcC3PtenCl2 and (D) Trans-diamminePtCl phenazine-1-carboxamide complex. Separate plots are shown for REV and SEQ strands. Negative values on the vertical axis represent regions of the DNA that are relatively protected from damage, while positive values indicate areas of relative damage enhancement. Ovals, N1 and N2 indicate approximate positions of the reconstituted nucleosome cores.

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Although DMS is commonly used to footprint protein–DNA interactions in cells (29,30), the alkylating agent did not appear to be inhibited by the positioned nucleosome cores, and distinct footprints were not observed (Figure 4). As a result, the average L/C ratio determined for DMS was almost 1. Student’s t-tests also indicated that there were no significant differences between average damage intensities in the linker and core regions of the construct (Table 1).

With regard to the DNA-targeted Pt complexes, both 2AcC3PtenCl2 and 9-aminoAcC3PtenCl2 were found to preferentially target runs of two or more G nucleotides and, occasionally, GA, AG and GC dinucleotides (17,31). However, trans-diamminePtCl phenazine-1-carboxamide displayed a more diverse sequence specificity, and the adduct sites were predominantly found at single guanine bases as expected for this mono-functional analogue (18).

The DNA-targeted Pt complexes proved to be highly effective nucleosome footprinting agents, mostly with high L/C ratio values and low p-values (Table 1). Densitometry plots of the damage incurred by these DNA-targeted cisplatin analogues are shown in Figure 4. Within these plots, clearly defined regions of DNA damage protection and enhancement correspond closely with the established nucleosome and linker positions, respectively. While all three analogues gave rise to distinct nucleosome footprints, the phenazine-tethered compound produced the highest L/C ratio, closely followed by the 9-aminoacridine-Pt complex and then 2AcC3PtenCl2 (Table 1). With average L/C ratios of 3.50 and 2.52, respectively, the phenazine-tethered compound and 9-aminoacridine-Pt complex both exhibited an exceptionally strong preference for targeting the linker region of the construct. At 1.30, however, the L/C ratio for 2AcC3PtenCl2 implied a footprinting capacity closer in magnitude to that of cisplatin and carboplatin (9).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

A defined chromatin construct was employed to investigate the effect of nucleosome cores on the DNA damage caused by nitrogen mustard and cisplatin analogues. DNA damage in reconstituted and non-reconstituted (free) DNA was detected using the linear amplification procedure and analysed on polyacrylamide DNA sequencing gels using densitometry. These methods allowed the precise sites of DNA damage to be determined and indicated the relative intensity of damage at each site. For each compound, comparisons between damage levels in the free and reconstituted DNA (L/C ratio) permitted the influence of chromatin structure on drug–DNA interactions to be assessed. Other workers have found a number of compounds that preferentially damage the linker DNA in reconstituted nucleosomes, and these include aflatoxin B1 (32), benzo[a]pyrene diol epoxide (33), bleomycin, neocarzinostatin and melphalan (34).

Effect of molecular weight on L/C ratios

Most of the 14 DNA-damaging agents examined within this project displayed a capacity for footprinting nucleosomes (Table 1). However, the relative degree of damage inhibition conferred by nucleosome cores (L/C ratios) varied between compounds. Student’s t-tests provided a statistical method for assessing the relative significance of each footprint, and generally reflected the trends already established by the L/C ratios (Table 1). Table 1 revealed several interesting relationships between the physical and biological properties of the agents and their ability to damage nucleosomal DNA (discussed in the following text). The implications of such findings may eventually influence the future design and development of improved chemotherapeutics.

One of the most prominent associations observed was the relationship between each compound’s molecular weight and nucleosome footprinting capacity (L/C ratio magnitude). Table 1 lists all 14 DNA-damaging agents in order of descending average L/C ratio, and includes information regarding the basic physical properties of each agent. In general, larger compounds were associated with higher L/C ratios, while smaller compounds exhibited lower values. Furthermore, the compounds could be roughly divided into two broad groups: those with lower L/C ratios (ranging from 0.9 to 1.4) and those with higher L/C ratios (ranging from 1.5 to 3.5). The average molecular weight of the six compounds in the lower L/C ratio category was approximately 354. In contrast, compounds with higher L/C ratios (excluding bleomycin and DNase I) had an average molecular weight of approximately 631 – that is, almost twice the average size of the lower L/C ratio compounds. This implies that histone steric hindrance is a major feature in determining whether a compound can access DNA in a nucleosome.

Of the 14 DNA-damaging agents, chlorambucil and dimethylsulphate were the only compounds that did not produce detectable nucleosome footprints. Consequently, the corresponding L/C ratios were very small (approximately 1.0). These findings thus implied that neither chlorambucil nor DMS preferentially target the linker or core regions of reconstituted chromatin. For both compounds, this effect has also been described in previous investigations (32,35). Furthermore, these two agents also had the lowest molecular weight (MW) values of all compounds examined. Although chlorambucil and cisplatin are similar in size, differences between their DNA-damaging mechanisms may account for the different nucleosome footprinting capacities observed here. DMS, on the other hand, has been successfully employed in footprinting analyses to study a range of protein–DNA interactions (30,36–38). However, several transcription factor binding studies have also reported DMS to be a less-effective footprinting agent than larger compounds, such as bleomycin and various nitrogen mustard analogues (24,25). Together, these observations suggest that DMS may not be an effective probe for examining all protein–DNA interactions.

Other drugs classified within the lower L/C ratio category included cisplatin, carboplatin, cis-[PtCl2(C6H11NH2)2] and 2AcC3PtenCl2. Unlike chlorambucil and DMS, however, these compounds produced distinct nucleosome footprints which signified their preference for binding to the linker region of the nucleosome. Interestingly, the clinically successful anti-tumour drugs, cisplatin and carboplatin, both had L/C ratios of 1.3. Hence, it may be advantageous for new anti-tumour drugs based on cisplatin to have a similar L/C ratio. On the other hand, the bis-cyclohexylamine platinum analogue (cis-[PtCl2(C6H11NH2)2]) produced a similar L/C ratio of 1.2 and yet has not been shown to exhibit clinically effective anti-tumour properties (39).

The acridine-tethered analogue, 2AcC3PtenCl2 (L/C ratio 1.3), was the only DNA-targeted compound (12) classified into the ‘low’ L/C ratio category. This compound is significantly larger than cis-[PtCl2(C6H11NH2)2] and at least twice the molecular weight of cisplatin. While 2AcC3PtenCl2 induces substantial damage levels in purified DNA, it has not exhibited significant anti-tumour activity in animal models (11,28,40). In comparison, other DNA-targeted analogues in this study produced higher L/C ratios (>1.5) and have previously demonstrated significant cytotoxic activity. These include 4AcC3PtenCl2, 9-aminoAcC3PtenCl2 (40), trans-diamminePtCl phenazine-1-carboxamide (41) and the nitrogen mustard analogues C3-AA (14,16), C20-AMSA and C50-AMSA (13). This observation suggests that the chemotherapeutic potential of compounds with DNA-targeting mechanisms may be enhanced when their L/C ratios are higher. Interestingly, while the L/C ratio for 4AcC3PtenCl2 was over 1.5 times the magnitude of the L/C ratio for 2AcC3PtenCl2, the only point of difference between these analogues is the position of the tethered acridine group. This infers that the site of attachment of an intercalating moiety may directly affect the L/C ratio and biological activity of a DNA-targeted drug.

Larger compounds that were classified into the higher L/C ratio category included bleomycin, DNase I, DNA-targeted cisplatin analogues (excluding 2AcC3PtenCl2) and all DNA-targeted nitrogen mustard analogues. Table 1 shows that the DNA-targeted cisplatin complexes (trans-diammine PtCl phenazine-1-carboxamide, 9-aminoAcC3PtenCl2 and 4AcC3PtenCl2) collectively produced the highest L/C ratios. These were closely followed in rank by bleomycin and DNase I (the established nucleosome footprinting agents), and then the DNA-targeted nitrogen mustard analogues (C50-AMSA, C3-AA and C20-AMSA). Within this group of eight compounds, a diverse range of molecular weights, chemical structures and mechanisms of action were represented. A common feature, however, was their strong preference for binding to inter-nucleosomal DNA. In the case of bleomycin and DNase I, their propensity for targeting linker DNA has already been documented comprehensively (5,34,42–44). In the context of the current study, the magnitude of their size warranted a pronounced distinction between their binding preferences and those of the remaining compounds, which were significantly smaller. However, the L/C ratios of bleomycin and DNase I were not the maximum values established in this study and instead were ranked directly between the ratios of DNA-targeted cisplatin and nitrogen mustard analogues (see Table 1). Thus, the extent to which the mode of action of these agents dictated their capacity for damaging nucleosomal core DNA was considered in further detail.

Effect of an intercalator moiety on L/C ratios

Although a significant relationship between compound size and nucleosome footprinting capacity was observed in this study, L/C ratios did not increase proportionally with increasing molecular weights (see Table 1). However, a broader correlation between the compound classes, their DNA-damaging mechanisms and L/C ratios was distinct. For example, DNA-targeted compounds collectively gave rise to the highest L/C ratios and most prominent nucleosome footprints. For several of these agents, previous investigations have already demonstrated their ability to act as excellent probes of protein–DNA interactions in intact human cells (24,25,45–47). As these compounds intercalate with DNA via the tethered DNA-binding group, a large disruption of protein–DNA interactions would be required for binding to occur in the nucleosome core (48–50). Thus, DNA damage is expected to be significantly inhibited at the sites of positioned nucleosome cores compared to the inter-nucleosomal linker DNA (51,52).

Nitrogen mustard analogues were investigated in this to generate information about DNA-damaging agents with a different mechanism of action to that of the platinum-based compounds. Despite their structural differences, the three DNA-targeted nitrogen mustard analogues displayed very similar damage trends in the presence of positioned nucleosome cores. Table 1 shows the way in which the L/C ratios of these complexes are clustered together with respect to the ratios of other groups of compounds. This suggests that the DNA-damaging mechanism of action of these DNA-targeted nitrogen mustard analogues has a common effect on their interaction with reconstituted nucleosomal DNA, regardless of the type of DNA-intercalating moiety attached. In general, the damage data obtained for these agents also correlates well with observations made in previous investigations (24,25). Furthermore, in human cells, Temple and colleagues (25) found that C20-AMSA, C50-AMSA and C3-AA all produced more distinct protein footprints than either DMS or chlorambucil. This finding is consistent with qualitative and quantitative observations made in the current investigation (Table 1).

A prominent feature of the acridine and amsacrine-tethered nitrogen mustard analogues was that they gave rise to altered sequence specificities compared to the cisplatin analogues. Other DNA-damaging characteristics unique to each of the nitrogen mustard analogues were also observed. These differences were most readily apparent in the corresponding densitometry plots for each complex. In general, C3-AA produced ‘deeper’ and more even footprints than the amsacrine analogues, which gave rise to footprints containing greater fluctuations in the damage intensity ratios. Interestingly, this phenomenon was also described implicitly by Temple and colleagues (1997) who observed that C3-AA gave more even damage ratios and clearer footprints than C50-AMSA and other agents. In this study, however, C3-AA treatments also incurred greater ‘end effects’ in the resulting damage patterns, than either C20-AMSA or C50-AMSA. This was evidenced by distinct damage enhancement peaks within nucleosomal regions towards the ends of the construct, particularly in the SEQ strand (Figure 3). At these locations, the template DNA may not be as closely associated with nucleosomal proteins because of some degree of ‘unwinding’. The degree of constraint imposed on DNA at the periphery of the nucleosome structure is sometimes found to be sequence specific (34), and in this case, may facilitate the intercalation of acridine-rather than amsacrine-tethered complexes. Alternatively, binding of the acridine moiety may incur a higher level of nucleosome core dissociation and thus damage at these sites compared to amsacrine interactions. As L/C ratios alone give no indication of these damage trends, such effects should be considered when evaluating an agent’s capacity for damaging nucleosome core DNA.

Summary

In summary, these DNA modification studies have helped to construct a more detailed model for the mechanism of action of various DNA-damaging drugs in the presence of nucleosome structures. The findings reported here ultimately show that drugs with different chemical and chemotherapeutic properties can also display similar binding preferences in the presence of specific nucleosome cores. This information should be taken into account when designing novel anti-tumour drugs based on cisplatin or other DNA-damaging agents. Cisplatin (L/C ratio – 1.32), carboplatin (L/C ratio – 1.34) and bleomycin (L/C ratio – 1.76) were the only compounds examined in this project that are used clinically, but have very different L/C ratios. Hence, it could be argued that cisplatin analogues should have a low L/C ratio of about 1.3, while bleomycin analogues should have a higher L/C ratio of about 1.8. The development of chemotherapeutic agents with improved efficacy and reduced toxic side effects could benefit from such a rational approach.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported in part by grants from the NHMRC and the University of NSW. AMG was funded by an Australian Postgraduate Award.

References

  1. Top of page
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  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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