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40 protocols using Olaparib

To characterize how the tumor cells grew in the absence of treatment and when exposed continuously to different drug concentrations, we seeded cells in a 48 well flat-bottom plate (Costar Corning) and left them to attach overnight in 200µL culture medium. Subsequently, we aspirated the medium and replaced it with treated growth medium, containing 0, 1, 10, 25, 50 or 100µM Olaparib (AstraZeneca), and monitored their growth for 9 days. We carried out two versions of this assay: i) a “low-density” version in which we seeded cells at 5,000 cells per well, and ii) a “high-density” version in which each well started with 60,000 cells. In each case, 3 replicates were performed for each experimental condition. During the experiment the medium was changed every 3 days. We also tested changing the medium daily but found that this did not significantly change the growth dynamics (not shown).
To prepare the treated medium, we first created a stock containing 100µM of drug by dissolving Olaparib (AstraZeneca) in 1mL Dimethyl sulfoxide (DMSO), filtering the solution using a 0.22nm syringe filter, and dissolving it in our regular culture medium. Next, we diluted this maximum tolerated dose (MTD) stock with normal culture medium to obtain batches with 1–50µM Olaparib. We verified that the DMSO did not adversely impact the cells’ growth dynamics (not shown).
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Cells were seeded in 6-well plates (n = 3) and treated the following day. Dual drug treatment of cisplatin and olaparib (AstraZeneca, UK) was administered by adding 10 μM cisplatin on day 1 for 1 h, followed by 1 μM olaparib treatment for 6 days with media change every other day.
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The human colorectal cancer HCT116 WT and p53−/− cells were cultured in McCoy’s5A (Modified) Medium. The human glioblastoma cell lines, SF763 and U87-MG were cultured in Dulbecco’s modified Eagle’s minimum medium with Glutamax (Thermo Fisher Scientific). Cells were grown as monolayers in their respective medium, supplemented with 10% fetal calf serum (PAA) and 1% penicillin and streptomycin (ThermoFisher Scientific) in plastic tissue culture disposable flasks (TPP) at 37 °C in a humidified atmosphere of 5% CO2 in air. For PARP1 inhibition, cells were incubated one hour in medium supplemented with Olaparib (AstraZeneca, 20 µM diluted in DMSO) to a final concentration of 200 nM before irradiation or oxidative treatment. Untreated cells were exposed to the same volume of DMSO. For hydrogen peroxide (H2O2) treatment, H2O2 (Sigma Aldrich 216763, 100 mM in water) was added to culture medium to a final concentration of 1 mM and cells were left exposed for a 10 min incubation time at room temperature.
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Olaparib and AZD1775 were provided by AstraZeneca (Wilmington, DE). The chemical structure of AZD1775 has previously been described (22 (link)). Antibodies against phosphorylated CDK1 (Y15), Total CDK1, γH2AX, tubulin, and caspase 3 were purchased from Cell Signaling Technology (Danvers, MA).
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Olaparib (AZD-2281; AstraZeneca, Cambridge, RU) was purchased from Tebu-bio (Ref 21,910–2154–25 mg, Le Perray en Yvelines, France) and used in vitro at 2 μM in the culture medium, according to a dose response experiment (Sup Fig. 1). Olaparib powder was first dissolved in DMSO at 10 mM, diluted to its final concentration with culture medium, and finally added to the cells 2 h before irradiation and left in the cell culture medium for 24 h. Negative control samples were treated with the DMSO concentration that was used for the test samples (0.02%).

Experimental strategy combining PARP inhibition and cell irradiation.

In the clonogenic assay (top part), cells at 80% confluence were incubated with PARPi 2 h before irradiation, and after 24 h, cells were seeded in 6-well plates. The plates were left at 37 °C for 8 days, and the clones were stained and counted. In the CellTrace analysis (bottom part), cells at 50% confluence were first stained with CellTrace reagent 24 h before being incubated with PARPi. Two hours after PARPi incubation, the cells were irradiated, and after 24 h, they were diluted to allow cell growth without contact inhibition. The flasks were left at 37 °C for 5 days, and the cells were analyzed by flow cytometry.

Fig 1
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6

Cisplatin and PARP1-inhibitor Combination for Cervical Cancer

Cisplatin was used as a chemotherapeutic agent, since this is often used in the clinic to treat cervical cancer. Cells were treated continuously with 0.3–0.5 µM cisplatin (cDDP, Platosin®, Pharmachemie B.V., Haarlem, The Netherlands) based on the measured IC50 depending on the cell line.
PARP1-inhibition (PARP1-i) was induced using 4–5 µM olaparib (Lynparza®, AstraZeneca, Cambridge, UK), dissolved in medium continuously.
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Olaparib, durvalumab and tremelimumab will be supplied by AstraZeneca.
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We used 16 endometrial cancer cell lines (Table 1). HHUA was purchased from RIKEN Cell Bank (Tsukuba, Japan). AN3CA, KLE, HEC-1B and RL95-2 were purchased from American Type Culture Collection (Manassas, VA). Ishikawa3-H-12 was a generous gift from Dr. Masato Nishida (National Hospital Organization Kasumigaura Medical Center, Japan). The other 10 cell lines were established by Hiroyuki Kuramoto [23 (link)].
Histologically, only the HEC-180 cell line was classified as a serous adenocarcinoma; the other cell lines were classified as endometrioid adenocarcinomas. The culture conditions of the 13 endometrial cancer cell lines were described previously [13 (link)]. HEC-180, HEC-251, and HEC-265 cells were maintained in Eagle’s MEM with 10% FBS. HEC-6 cells stably expressing wild-type PTEN were generated by a retroviral infection, as described previously [13 (link)]. Phoenix cells were transfected with retroviral vectors (pFB-neo) that contained tandem affinity purification (TAP)-tagged wild-type PTEN using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and the resulting supernatants were used to infect HEC-6 cells. Drug selection was used to purify cell populations after infections by neomycin (500 μg/mL, 7 days).
Olaparib (AZD2281/KU0059436) was provided by AstraZeneca (London, UK). Olaparib was solved in DMSO, and the concentration of DMSO in each assay was 0.1%.
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The following chemical reagents were used throughout the study: AZD2461 (kindly provided by AstraZeneca), olaparib (kindly provided by AstraZeneca and Syncom (Groningen, The Netherlands)), talazoparib (Selleckchem; #S7048), cisplatin (Teva; #7680479980428), hydroxyurea (Sigma; #H8627), AZD0156 (kindly provided by AstraZeneca), AZD6738 (Selleckchem; #S7693), AZD1390 (kindly provided by AstraZeneca), Mirin (Sigma; #M9948), camptothecin (Selleckchem; #S1288), 5-Iodo-2’-deoxyuridine (IdU) (Sigma; #I7125), 5-Chloro-2’-deoxyuridine (CldU) (Sigma; #C6891), Ethynyl-2’-deoxyuridine (EdU) (Sigma; #A10044). LP and SP (Bachem; #4143690 and #4111111, respectively).
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U2OS expressing GFP and GFP-CtIP variants were transfected with siCtIP, seeded in low confluency and treated with Olaparib (AstraZeneca) at the indicated doses or mock treated with DMSO for 1 h. After treatment, cells were washed two times with PBS and cultured under standard conditions to allow colony formation for the next 8–10 days. Colonies were then counted after staining with 0.5% crystal violet/20% ethanol. The results were normalized to DMSO treatment.
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