Abstract

   Hyperthermia has been used as an adjuvant treatment for radio- and
   chemotherapy for decades. In addition to its effects on perfusion and
   oxygenation of cancer tissues, hyperthermia can enhance the efficacy of
   DNA-damaging treatments such as radiotherapy and chemotherapy. Although
   it is believed that the adjuvant effects are based on
   hyperthermia-induced dysfunction of DNA repair systems, the mechanisms
   of these dysfunctions remain elusive. Here, we propose that elevated
   temperatures can induce chromatin trapping (c-trapping) of essential
   factors, particularly those involved in DNA repair, and thus enhance
   the sensitization of cancer cells to DNA-damaging therapeutics. Using
   mass spectrometry-based proteomics, we identified proteins that could
   potentially undergo c-trapping in response to hyperthermia. Functional
   analyses of several identified factors involved in DNA repair
   demonstrated that c-trapping could indeed be a mechanism of
   hyperthermia-induced transient deficiency of DNA repair systems. Based
   on our proteomics data, we showed for the first time that hyperthermia
   could inhibit maturation of Okazaki fragments and activate a
   corresponding poly(ADP-ribose) polymerase-dependent DNA damage
   response. Together, our data suggest that chromatin trapping of factors
   involved in DNA repair and replication contributes to heat-induced
   radio- and chemosensitization.

   Keywords: hyperthermia, DNA repair, DNA replication, chromatin, PARP

1. Introduction

   Hyperthermia is an anti-cancer treatment that involves tumor heating
   using an exogenous energy source. Technological advancement has widened
   the therapeutic window of hyperthermia to 40–45 °C [[44]1,[45]2].
   Hyperthermia combined with radio- or chemotherapy improves treatment
   outcomes which can be explained by multiple factors [[46]3,[47]4]. One
   mechanism of the sensitizing effect of elevated temperature is the
   induction of DNA repair dysfunction. Several in vitro, in vivo, and
   clinical studies have demonstrated that hyperthermia can enhance the
   beneficial effects of DNA-targeting therapeutic strategies by altering
   DNA damage response (DDR) pathways [[48]5,[49]6]. However, the
   molecular mechanisms of this DDR-inhibiting effect of hyperthermia and
   the complete list of hyperthermia-affected DDR factors remain unknown.

   It has recently been proposed that the anticancer cytotoxicity of
   DNA-binding small molecules can be attributed to their chromatin
   destabilization properties [[50]7,[51]8]. Curaxins, some
   anthracyclines, and anthraquinones alter chromatin structure by
   destabilizing nucleosomes and inducing histone eviction from chromatin.
   Such chromatin destabilization can promote abnormal trapping of
   proteins to chromatin (c-trapping) and thus lead to the exhaustion of
   the functional protein pool [[52]8]. This phenomenon was first studied
   on curaxin-induced c-trapping of the FAcilitates Chromatin
   Transcription (FACT) complex [[53]9,[54]10]. In this study, we
   questioned whether elevated temperatures could induce c-trapping of
   essential factors, particularly those involved in DNA repair, and thus
   stimulate the sensitization of cancer cells to DNA-damaging
   therapeutics.

   Using mass spectrometry (MS)-based proteomics, we identified an
   extensive list of proteins that could potentially undergo c-trapping in
   response to hyperthermia. Functional analyses of several identified
   factors involved in DNA repair demonstrated that c-trapping could
   indeed be the mechanism of hyperthermia-induced transient inactivation
   of DDR pathways. In addition, we found new evidence for the devastating
   effect of hyperthermia on DNA replication. Based on the proteomics data
   obtained, we hypothesized and verified that hyperthermia inhibits
   maturation of Okazaki fragments and provokes corresponding
   poly(ADP-ribose) polymerase (PARP)-dependent DDR. Together, our data
   suggest that chromatin trapping of factors involved in DNA repair and
   replication underlies heat-induced radio- and chemosensitization.

2. Materials and Methods

2.1. Antibodies

   The primary antibodies used for immunofluorescence or Western blot
   hybridization were SUPT16H (mouse, Abnova, Taipei, Taiwan), Ku80
   (mouse, Abcam, Cambridge, UK), MDC1 (mouse, Abcam), Mre11 (rabbit,
   Novus Biologicals, Centennial, CO, USA), 53BP1 (rabbit, Santa Cruz
   Biotechnology, Dallas, TX, USA), XRCC1 (rabbit, Abcam), TopBP1 (mouse,
   Santa Cruz Biotechnology), Lamin B1 (mouse, Abcam), histone H3 (rabbit,
   Abcam), PAR (rabbit, Trevigen, Gaithersburg, MD, USA). The secondary
   antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 594 were
   purchased from Invitrogen (Karlsbad, CA, USA); the horseradish
   peroxidase-conjugated anti-mouse and anti-rabbit IgG were purchased
   from GE Healthcare (Chicago, IL, USA).

2.2. Cell Culture, Drug Treatments, and Hyperthermia

   Human HeLa or HEK293 cells were obtained from ATCC. The cells were
   cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; PanEco, Moscow,
   Russia) supplemented with 10% fetal bovine serum (FBS; GE Healthcare)
   at 37 °C in a humidified CO2 incubator. For hyperthermia, cells were
   immersed in a precision-controlled water bath at 42–45 °C (±0.05 °C)
   for 30 min. To induce double-stranded DNA breaks (DSBs), cells were
   treated with 20 µg/mL etoposide (Sigma-Aldrich, St. Louis, MO, USA) for
   1 h; to induce single-stranded DNA breaks (SSBs), cells were treated
   with 200 µM peroxide hydrogen (Sigma-Aldrich) for 1 h; to induce
   replication stress, cells were treated with 10 mM hydroxyurea
   (Sigma-Aldrich) or with 10 µM aphidicolin (Sigma-Aldrich) for 1 h; for
   inhibition of lagging strand synthesis, cells were treated with 2 mM
   emetine (Sigma-Aldrich) for 1 h; for c-trapping induction, cells were
   treated with 10 µM curaxin CBL0137 (Selleckchem, Houston, TX, USA) for
   1 h. To label active replication sites, cells were incubated with 10 μM
   5-ethynyl-2’-deoxyuridine (EdU; Jena Biosciences, Jena, Germany) for 20
   min at 37 °C. Incorporated EdU were visualized using a Click−iT EdU
   Imaging Kit (Invitrogen) according to the manufacturer’s instructions.

2.3. Chromatin Enriching Salt Separation and Immunoblotting

   HeLa cells were incubated in a lysis buffer (LB; 10 mM Hepes-NaOH (pH
   7.5), 1.5 mM MgCl[2], 0.5 mM EDTA, 10 mM KCl, 0.5% NP40, phosphatase
   and protease inhibitors). Cells were incubated at 4 °C for 10 min and
   collected by centrifugation at 1000× g for 5 min. Cells were then
   incubated in an LB containing 100 mM NaCl. After incubation at 4 °C for
   10 min, the first soluble fraction (“0.1” fraction) was separated by
   centrifugation at 10,000× g for 10 min. Cells were then incubated in an
   LB containing 400 mM NaCl. After incubation at 4 °C for 1 h, the second
   soluble fraction (“0.4” fraction) was separated from the chromatin
   fraction by centrifugation at 8000× g for 10 min. The chromatin pellet
   (“insoluble” fraction) was then sonicated in an LB at 1/2 amplitude for
   30 s with a VirSonic 100 ultrasonic cell disrupter.

   Aliquots of each sample were separated by sodium
   dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and
   blotted onto polyvinylidenedifluoride (PVDF) membranes. The membranes
   were blocked for 1 h in 2% ECL Advance blocking reagent (GE Healthcare)
   or 2% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS containing 0.1%
   Tween 20 (PBS-T) followed by incubation overnight at 4 °C with a
   primary antibody diluted in PBS-T containing 2% blocking reagent or 2%
   BSA. After three washes with PBS-T, the membranes were incubated for 1
   h with the secondary antibodies (horseradish peroxidase-conjugated
   anti-rabbit or anti-mouse IgG) in PBS-T containing 2% blocking agent or
   2% BSA. The immunoblots were visualized using a Pierce ECL plus Western
   blotting substrate. Images of the full-length blots are presented in
   [55]Figures S1 and S2.

2.4. Preparative SDS-PAGE and In-Gel Trypsin Digestion

   Protein concentrations were determined using BCA Protein Assay Kit
   (Thermo Fisher Scientific, Waltham, MA, USA) according to the
   manufacturer’s standard protocol (bovine serum albumin was used as the
   standard). Equal amounts of biological samples (250 μg each) were
   separated via 9% (w/v) SDS-PAGE (20 × 20 cm) to prefractionate the
   proteins. After electrophoresis, the gel was divided into three
   fractions, each fraction was subjected to in-gel protein digestion, and
   extracted peptide samples were analyzed via LC-MS/MS for protein
   identification. Gel fractions were cut into small (1 × 1 mm) pieces and
   transferred into sample tubes. Protein disulfide bonds were reduced
   with 10 mM DTT (in 100 mM ammonium bicarbonate buffer) at 50 °C for 30
   min and afterward alkylated with 55 mM iodoacetamide (in 100 mM
   ammonium bicarbonate buffer) at room temperature for 20 min in the
   dark. After alkylation, the gel samples were destained with 50%
   acetonitrile (CAN) (in 50 mM ammonium bicarbonate buffer) and
   dehydrated by the addition of 100% ACN. After removal of the 100% ACN,
   the samples were subjected to in-gel trypsin digestion. The digestion
   buffer contained 13 ng/µL trypsin (in 50 mM ammonium bicarbonate
   buffer). The trypsin digestion proceeded overnight at 37 °C. The
   resulting tryptic peptides were extracted from the gel via the addition
   of two volumes of 0.5% trifluoroacetic acid (TFA) to the samples
   (incubation for 1 h) and then two volumes of 50% ACN (incubation for 1
   h). Finally, the extracted peptides were dried in vacuum and
   redissolved in 3% ACN with 0.1% TFA solution prior to LC-MS/MS
   analysis.

2.5. LC-MS/MS Analysis

   LC-MS/MS analysis was performed using the Q Exactive HF benchtop
   Orbitrap mass spectrometer (Thermo Fisher Scientific) which was coupled
   to the Ultimate 3000 Nano LC System (Thermo Fisher Scientific) via a
   nanoelectrospray source (Thermo Fisher Scientific). The HPLC system was
   configured in a trap-elute mode. Approximately 1 µg of tryptic peptides
   were loaded on an Acclaim PepMap 100 (100 µm × 2 cm) trap column and
   separated on an Acclaim PepMap 100 (75 µm × 50 cm) column (both from
   Thermo Fisher Scientific). Peptides were loaded in solvent A (0.2%
   (v/v) formic acid) and eluted at a flow rate of 350 nL/min with a
   following multistep linear gradient of solvent B (0.1% (v/v) formic
   acid, 80% (v/v) acetonitrile): 4–6% B for 5 min; 6–28% B for 91 min;
   28–45% B for 20 min; 45–99% B for 4 min; 99% B for 7 min; 99–4% B for 1
   min. After each gradient, the column was washed with 96% buffer B for 9
   min. Column temperature was kept at 40 °C. Peptides were analyzed on a
   mass-spectrometer, with one full scan (350–1400 m/z, R = 60,000 at 200
   m/z) at a target of 3 × 10^6 ions and max ion fill time 30 ms, followed
   by up to 15 data-dependent MS/MS scans with higher-energy collisional
   dissociation (HCD) (target 1 × 10^5 ions, max ion fill time 50 ms,
   isolation window 1.2 m/z, normalized collision energy (NCE) 28%,
   underfill ratio 2%), detected in the Orbitrap (R = 15,000 at fixed
   first mass 100 m/z). Other settings: charge exclusion—unassigned, 1,
   >6; peptide match—preferred; exclude isotopes—on; dynamic exclusion 30
   s was enabled.

2.6. LC-MS/MS Data Analysis

   Raw LC-MS/MS data from Q Exactive HF mass-spectrometer were converted
   to .mgf peaklists with MSConvert (ProteoWizard Software Foundation).
   For this procedure, we used the following parameters: “--mgf-filter
   peakPicking true”. For thorough protein identification, the generated
   peak lists were searched with MASCOT (version 2.5.1) and X! Tandem
   (ALANINE, 2017.02.01) search engines against UniProt Human protein
   knowledgebase with the concatenated reverse decoy dataset. The
   precursor and fragment mass tolerance were set at 20 ppm and 0.04 Da,
   respectively. Database-searching parameters included the following:
   tryptic digestion with one possible missed cleavage, static
   modification for carbamidomethyl (C), and dynamic/flexible
   modifications for oxidation (M). For X! Tandem we also selected
   parameters that allowed a quick check for protein N-terminal residue
   acetylation, peptide N-terminal glutamine ammonia loss, or peptide
   N-terminal glutamic acid water loss. Result files were submitted to
   Scaffold 4 software (version 4.0.7) for validation and meta-analysis.
   We used the local false discovery rate-scoring algorithm with standard
   experiment-wide protein grouping. For the evaluation of peptide and
   protein hits, a false discovery rate of 5% was selected for both. False
   positive identifications were based on reverse database analysis. We
   also set protein annotation preferences in Scaffold to highlight