Cisplatin fastens chromatin irreversibly even at a high chloride concentration

Abstract

Cisplatin is one of the most potent anti-cancer drugs developed so far. Recent studies highlighted several intriguing roles of histones in cisplatin's anti-cancer effect. Thus, the effect of nucleosome formation should be considered to give a better account of the anti-cancer effect of cisplatin. Here we investigated this important issue via single-molecule measurements. Surprisingly, the reduced activity of cisplatin under [NaCl] = 180 mM, corresponding to the total concentration of cellular ionic species, is still sufficient to impair the integrity of a nucleosome by retaining its condensed structure firmly, even against severe mechanical and chemical disturbances. Our finding suggests that such cisplatin-induced fastening of chromatin can inhibit nucleosome remodelling required for normal biological functions. The in vitro chromatin transcription assay indeed revealed that the transcription activity was effectively suppressed in the presence of cisplatin. Our direct physical measurements on cisplatin-nucleosome adducts suggest that the formation of such adducts be the key to the anti-cancer effect by cisplatin.

Graphical Abstract

Cisplatin fastens chromatin irreversibly even at a high chloride concentration.

Figure 1.

Force-extension curves of DNA incubated with 3.3 mM cisplatin in buffers with different NaCl concentrations. The dotted lines are fits by the WLC model. For [NaCl] = 20 and 60 mM, the high- and low-tension regimes are fitted separately (pink and red for [NaCl] = 20 mM, and blue and cyan for [NaCl] = 60 mM with the first (second) of each pair covering the high- (low-) tension regime) as described by the bimodal modeling (25,26) while the entire range for [NaCl] = 90 mM can be nicely fitted with a single persistence length (green). For comparison, the force-extension curve of bare DNA (black circle) measured in [NaCl] = 180 mM was overlaid with a WLC model fit (black).

Figure 2.

Force-extension behavior of a N-DNA molecule under various ionic conditions. (A, E) Typical response (raw extension data, black; extension data averaged over 1 s, blue) of a bare DNA in PBS solution with respect to varying tension (red) (A) and the force-extension curves from the S–R cycle driven by increase and decrease of tension (marked by upward and downward arrows, respectively) (E). The OST in a bare DNA happens at F ∼ 65 pN (point ‘a’ in (A and E)). (B, F) Mechanical rupturing and relaxation of a N-DNA in PBS solution. Temporal extension of a N-DNA in a S–R cycle and the force-extension curves of the N-DNA are shown in (B and F), respectively. The curves clearly show the hysteresis. Here, the hysteresis appears in two different force regimes, high (60–75 pN) and intermediate (3–20 pN). (C, G) Salt-induced disassembly of a N-DNA via change of buffer to 3 M NaCl. Temporal extension (C) and the force–extension curves (G) of a N-DNA are acquired and displayed similarly to (B and F). The curves exhibit no hysteresis, similarly to the case of a bare DNA. This suggests that NCPs assembled onto DNA were fully disrupted by releasing histones into solution. Due to the high ionic strength, the onset force of OST was decreased to ∼60 pN (point ‘b’ in (C, G)). (D, H) Response of a DNA (or N-DNA) recovered from the salt challenge. The buffer was restored to PBS. Temporal extension (D) and the force-extension curves (H) of DNA are acquired and displayed similarly to the above-mentioned data. This result confirms that there are no more residual histones on the DNA strand and the DNA is just like bare DNA shown in (E).

Figure 3.

Physical change of single N-DNA molecules induced by cisplatin treatment. Here cisplatin was supplied at two different concentrations, (B, C) high (3.3 mM) and (E, F) low (0.1 mM), the latter being clinically relevant. (A) Temporal extension of a bare DNA (raw extension data, grey; extension data averaged over 0.5 s, black) with respect to varying tension (red). Because this DNA was nicked, its OST happened at F ∼ 65 pN. (B, C) Constant extension of a CBN-DNA regardless of applied tension indicates the structural fixation of the N-DNA incubated with 3.3 mM cisplatin in PBS solution (B) and even after changing the solution to 3 M NaCl (C). Here, the N-DNA molecule in (B) was formed by incubating NAP1-associated core histones to the same bare DNA molecule in (A). (D) (A)-like data for another bare DNA used in (E and F). (E, F) Response of a N-DNA incubated with 0.1 mM cisplatin in PBS solution (E) and after changing the solution to 3 M NaCl (F). Panels from (B) to (F) follows the same convention used in panel (A).

Figure 4.

S-R cycles of the CBN-DNA molecules shown in Figure 3. (A) The collapsed form of CBN-DNA after 3.3 mM cisplatin treatment exhibited neither the OST nor the hysteresis in a mild (red) and extremely high salt condition (blue). A typical S-R cycle for a bare DNA was shown in black for comparison. (B) A CBN-DNA prepared with 0.1 mM cisplatin was stretched by mechanical force. The hysteresis is prominent in the CBN-DNA molecule both in a mild (red) solution and under the extremely high salt condition (blue). The maximum extensions of the CBN-DNA achieved upon OST in both PBS (red) and 3 M NaCl (blue) solutions were shorter than that of its original bare DNA (black).

Figure 5.

In vitro transcription from cisplatin-treated DNA and chromatin templates. (A, D) Schematic representation of the in vitro transcription assay with cisplatin-treated (A) DNA and (D) chromatin. Bare DNA and reconstituted chromatin were treated sequentially with cisplatin, transcription factors (p53 for bare DNA; p53, p300 and AcCoA (acetyl coenzyme A) for chromatin), nuclear extracts derived from HeLa cells, and NTP in the indicated order and time. (B, E) Effect of cisplatin on in vitro transcription from bare DNA (B) and chromatin templates (E). Reaction conditions of each lane are indicated above the lane. (C, F) Relative transcription levels were quantitated by phosphorimager and normalized to that of p53-dependent transcription for bare DNA and p53- and p300-dependent transcription for chromatin (lane 2 in (C) and lane 4 in (F), respectively). The error bars in histograms represent the standard deviation of data in triplicates.

Figure 6.

Proposed mechanism of cisplatin's drug efficacy by chromatin fastening. (A) Cartoon of a N-DNA bearing several NCPs. (B) NCPs are mechanically disrupted by force, but lingering association of residual histones (yellow circle) is evident from the elevated level of OST onset force of ∼70 pN. Strong 3 M NaCl solution is capable of stripping N-DNA of core histones. (C) Structural fixation and modification of cisplatin-treated chromatin revealed by mechanical and ionic disturbances. Depending on the location of DNA nicks (red arrows), the OST may not be observed in our experimental setting (typically, maximum force applicable ∼100 pN) as shown in case (a) (case (a): DNA nicks hidden within cisplatin-fixed NCPs, and case (b): DNA nicks exposed in DNA linkers). The yellow, blue, and red circles indicate residual, released (cisplatin-free or loosely captured), and cisplatin-crosslinked histones, respectively.