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Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells
Open accessPublished version of a preprint
Abstract
The Mdm2 oncogene is a negative regulator of the p53 tumor suppressor and recently identified inhibitor of DNA break repair. Nutlin-3 is a small-molecule inhibitor of Mdm2–p53 interaction that can induce apoptosis in cancer cells through activation of p53. Although this is a promising therapy for those cancers with wild-type p53, half of all human cancers have inactivated p53. Here, we reveal that a previously unappreciated effect of Nutlin is inhibition of DNA break repair, stemming from its ability to increase Mdm2 protein levels. The Nutlin-induced increase in Mdm2 inhibited DNA double-strand break repair and prolonged DNA damage response signaling independent of p53. Mechanistically, this effect of Nutlin required Mdm2 and acted through Nbs1 of the Mre11–Rad50–Nbs1 DNA repair complex. In ovarian cancer cells, where >90% have inactivated p53, Nutlin combined with the genotoxic agents, cisplatin or etoposide, had a cooperative lethal effect resulting in increased DNA damage and apoptosis. Therefore, these data demonstrate an unexpected consequence of pharmacologically increasing Mdm2 levels that when used in combination with genotoxic agents induces synthetic lethality in ovarian cancer cells, and likely other malignant cell types, that have inactivated p53. Implications: Data reveal a therapeutically beneficial effect of pharmacologically increasing Mdm2 levels combined with chemotherapeutic agents for malignancies that have lost functional p53. Mol Cancer Res; 13(8); 1197–205. ©2015 AACR.
Overview
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Written from the paper’s full text.
TL;DR
Nutlin-3 increases Mdm2 protein stability independently of p53, and the resulting elevated Mdm2 inhibits DNA double-strand break repair and delays DNA damage signaling through Nbs1. In p53-inactivated ovarian cancer cells, combining Nutlin with cisplatin or etoposide cooperates to increase DNA damage and apoptosis.
Key findings
- Nutlin increased Mdm2 protein stability in p53−/− MEFs: after cycloheximide, high Mdm2 levels remained at 20 minutes in Nutlin-treated cells, whereas Mdm2 was barely detectable in vehicle controls.
- Nutlin inhibited DNA break repair in a Mdm2- and Nbs1-dependent, p53-independent manner: in 293T cells, 60.1% (±1.57%) of Nutlin-treated cells had DNA damage remaining 60 minutes post γ-radiation versus 29.7% (±2.35%) of DMSO-treated cells; in p53−/−Mdm2−/− MEFs, Nutlin no longer inhibited repair.
- Nutlin delayed DNA damage signaling: in p53−/− MEFs, γH2AX foci were 31% fewer at 10 minutes and significantly increased at 150 minutes post γ-radiation; pS/TQ foci were 28% fewer immediately and 62% more at 240 minutes.
- In ovarian cancer cells with inactivated p53, Nutlin plus genotoxic agents cooperated: SKOV-3 repair at 90 minutes was 57.6% with Nutlin versus 74.5% with vehicle; Nutlin plus 5 µM cisplatin inhibited growth by 51.5% (±0.49%) versus 35.0% (±1.08%) for cisplatin alone, and Nutlin plus 5 µM etoposide inhibited growth by 60.2–75.3% versus 49–53% for etoposide alone.
Methods
- Cell models: p53−/−, p53−/−Mdm2−/−, and Nbs1ΔB/ΔB murine embryonic fibroblasts; 293T cells; and ovarian cancer lines SKOV-3, OVCAR-5, and OVCAR-8.
- Treatments and assays: Nutlin (10 µM), cisplatin, etoposide, and γ-radiation; Western blotting with cycloheximide for Mdm2 half-life, neutral comet assay, immunofluorescence for γH2AX and pS/TQ foci, MTT proliferation/viability assays, and cleaved Caspase 3 Western blotting.
- Analysis: at least three independent experiments for most analyses, with student’s t-tests or 95% confidence intervals; γH2AX-positive assays after cisplatin or etoposide removal were performed two times.
Limitations
- The paper does not state a limitations section.
- Evident from the text: the work is primarily in cultured MEFs and ovarian cancer cell lines, with no in vivo tumor or clinical data reported.
- Evident from the text: some assays had limited replication, including the γH2AX-positive assays after cisplatin or etoposide removal, which were performed two times; the Nutlin concentration refers to the entire mixture, with only half representing the active enantiomer A.
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Claims the paper makes
19 Own interpretation · 19 Own result · 12 Not checked yet
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our data established that in the absence of functional Nbs1, Nutlin was unable to inhibit double-strand DNA break repair.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
boosted- Asserted: passes — The authors put it forward as true.
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In the paper
“Similar results were obtained with the p53 mutant ovarian cancer cell lines, OVCAR-5 and OVCAR-8 (Figure 4B and 4C, respectively and Supplemental Figure S3B and S3C).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Delay of DNA damage signal in ovarian cancer cells caused by Nutlin
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“When p53−/−Mdm2−/− MEFs were used, Nutlin had neither an impact on the formation nor the resolution of the pS/TQ foci (Figure 2D and Supplemental Figure S2B).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Using this method, we observed an increased percentage of ovarian cancer cells that were γH2AX positive after incubation with Nutlin or cisplatin alone, but significantly more when cells were treated with both Nutlin and cisplatin (Figure 6E).”
- Also stated in: § Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents · plain
“Analogous results were obtained when etoposide was used (Figure 6F).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Also stated in: § Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“However, treatment with both 1 µM of cisplatin and 10 µM of Nutlin resulted in a 26.9% +/− 0.62% reduction in cell growth at 72 hours compared to vehicle treated cells (Supplemental Figure S4A).”
- Also stated in: § Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents · plain
“This cooperative effect was more apparent between Nutlin and 5 µM cisplatin, which combined inhibited growth by 51.5% +/− 0.49%, while 5 µM cisplatin alone only inhibited 35.0% +/− 1.08% (Figure 5A).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Also stated in: § Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“When Mdm2 was not present, Nutlin no longer inhibited the formation or the resolution of γH2AX foci compared to DMSO treated cells (Figure 2B and Supplemental Figure S1B).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
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A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“For all three cell lines, treatment with either Nutlin or 1 µM etoposide had a very slight effect on the expansion of the cells, but when combined, cell growth was significantly inhibited (35.9–56.2%) (Supplemental Figure S4B).”
- Also stated in: § Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents · plain
“Similarly, when treated with Nutlin and 5 µM etoposide, cells were inhibited by 60.2–75.3% compared to only 49–53% when 5 µM etoposide alone was used (Figure 5B).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents
plaindirectn = 3- Asserted: passes — The authors put it forward as true.
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- Also stated in: § Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Sixty minutes after γ-radiation, p53−/− MEFs with Nutlin had reduced repair of double-strand DNA breaks resulting in significantly more cells harboring damaged DNA compared to cells treated with vehicle control (Figure 1C).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“When Nutlin was combined with genotoxic agents in ovarian cancer cells, a significant increase in DNA damage and apoptosis independent of p53 was induced.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
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A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“These results indicate Nutlin-induced increase in Mdm2 results in a delay in double-strand DNA break repair in ovarian cancer cells that contain deleted or mutated p53.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair in ovarian cancer cells
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
- Rests on an assumption: Nutlin increases Mdm2 levels in ovarian cancer cells by stabilizing the protein, as it does in mouse embryonic fibroblasts.
- The quote only partly states this claim. 2
- It may state two separate claims as one. 24
- Probably not a substantial claim. 5
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Similar results were obtained with the OVCAR-5 and OVCAR-8 mutant p53 ovarian cancer cells (Figure 5A and Supplemental Figure S4A).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Inhibition of ovarian cancer cell expansion with combined treatment of Nutlin and genotoxic agents
plaincorrelational- Asserted: passes — The authors put it forward as true.
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The quote only partly states this claim.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Assessment of γH2AX foci formation showed that 10 minutes following γ-radiation, p53−/− MEFs in the presence of Nutlin formed 31% fewer foci than vehicle treated cells (Figure 2A and Supplemental Figure S1A).”
- Also stated in: § Nutlin inhibits the DNA damage signal through Mdm2 · hedged
“At 150 minutes post γ-radiation, there were significantly increased numbers of γH2AX foci in the Nutlin exposed MEFs compared to DMSO treated controls, indicating a delay in foci resolution.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Also stated in: § Nutlin inhibits the DNA damage signal through Mdm2 · hedged
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Ten minutes after γ-radiation, there were 29% fewer γH2AX foci in the Nutlin treated p53 deleted SKOV-3 cells compared to vehicle control (Figure 4A and Supplemental Figure S3A).”
- Also stated in: § Delay of DNA damage signal in ovarian cancer cells caused by Nutlin · plain
“At 240 minutes post γ-radiation, the Nutlin treated SKOV-3 cells had 46% more foci remaining compared to DMSO treated cells.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Delay of DNA damage signal in ovarian cancer cells caused by Nutlin
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
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It may state two separate claims as one.
- Also stated in: § Delay of DNA damage signal in ovarian cancer cells caused by Nutlin · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“SKOV-3 ovarian cancer cells have deleted p53 and showed increased levels of Mdm2 following exposure to Nutlin (Figure 3A).”
- Also stated in: § Nutlin inhibits DNA break repair in ovarian cancer cells · plain
“Only 57.6% of the cells treated with Nutlin were able to repair their DNA damage within 90 minutes, compared to 74.5% of cells exposed to vehicle control (Figure 3A).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair in ovarian cancer cells
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Rests on an assumption: The ovarian cancer cell lines used are representative models of high-grade serous ovarian cancer with inactivated p53.
- Also stated in: § Nutlin inhibits DNA break repair in ovarian cancer cells · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We observed at both 60 and 90 minutes following γ-radiation, Nutlin treated cells repaired their DNA to a similar extent as DMSO control (Figure 1D), whereas MEFs overexpressing Mdm2 had significantly reduced DNA repair.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Mdm2 protein remained present for longer in cells with Nutlin compared to vehicle control treated cells (Figure 1A).”
- Also stated in: § Nutlin increases Mdm2 protein stability independent of p53 · plain
“Specifically, there were high levels of Mdm2 protein remaining after 20 minutes with cycloheximide in the Nutlin treated cells, whereas in the vehicle control treated cells, Mdm2 was barely detectable.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin increases Mdm2 protein stability independent of p53
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
- Also stated in: § Nutlin increases Mdm2 protein stability independent of p53 · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We detected 60.1% (+/−1.57%) of the cells exposed to Nutlin had DNA damage remaining 60 minutes post γ-radiation, compared to only 29.7% (+/−2.35%) of DMSO treated cells (Figure 1B).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Nutlin treatment led to increased Mdm2 protein levels in both cell lines (Figure 3B and 3C).”
- Also stated in: § Nutlin inhibits DNA break repair in ovarian cancer cells · plain
“In addition, both lines exposed to Nutlin had significantly reduced DNA break repair at 60 and 90 minutes post γ-radiation (Figure 3B and 3C).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair in ovarian cancer cells
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Rests on an assumption: The ovarian cancer cell lines used are representative models of high-grade serous ovarian cancer with inactivated p53.
- Also stated in: § Nutlin inhibits DNA break repair in ovarian cancer cells · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“We observed fewer cells in addition to an increase in dead and dying cells in the cultures exposed to both Nutlin and cisplatin (Figure 6A and Supplemental Figure S5) as well as Nutlin and etoposide (Figure 6B and Supplemental Figure S5) in comparison to either drug alone.”
- Also stated in: § Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents · plain
“However, cleaved Caspase 3 was readily apparent in ovarian cancer cells treated with the combination of Nutlin and 5 µM cisplatin, and was similar to the amount of cleaved Caspase 3 observed in cells treated with 10 µM cisplatin alone (Figure 6C).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents
plaindirectn = 3- Asserted: passes — The authors put it forward as true.
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- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one. The quote only partly states this claim.
- Also stated in: § Lethal combination for ovarian cancer cells of Nutlin and genotoxic agents · plain
A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Cells with Nutlin had 28% fewer foci immediately after γ-radiation, and 62% more foci at 240 minutes post γ-radiation compared to controls (Figure 2C and Supplemental Figure S2A).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
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It may state two separate claims as one.
In the paper
“DNA break repair is already delayed in the Nbs1ΔB/ΔB MEFs and addition of Nutlin did not further inhibit DNA repair (Figure 1E).”
- Also stated in: § Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53 · plain
“However, when a mutant of Nbs1 that cannot bind Mdm2 (mut 8; (11)) was expressed, Nutlin treatment did not result in a delay in DNA repair (Figure 1E).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53
What the sources say
- Not checked yetcites [11] Bouska, 2008, Mdm2 promotes genetic instability and transformation independent of p53, Mol Cell Biol source
plain- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
- Also stated in: § Nutlin inhibits DNA break repair through Mdm2 and Nbs1, independent of p53 · plain
In the paper
“Nutlin-3 is a small molecule inhibitor of Mdm2/p53 interaction that can induce apoptosis in cancer cells through activation of p53.”
- Also stated in: § Introduction · plain
“In cancer cells with wild-type p53, Nutlin can induce p53-mediated apoptosis (6).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Abstract
What the sources say
- Not checked yetcites [6] Vassilev, 2004, In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, Science source
plaindirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: fails — The paper’s conclusions may not depend on it.
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Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
- Rests on an assumption: Nutlin binds Mdm2 and blocks the Mdm2-p53 interaction.
- Also stated in: § Introduction · plain
None of the sources cited for this claim have been checked yet.
In the paper
“We have shown elevated Mdm2 levels act through Nbs1 of the Mre11/Rad50/Nbs1 (MRN) DNA repair complex to impair DNA damage response signaling, leading to inhibition of double-strand DNA break repair and increased genome instability independent of p53 (10,11).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Introduction
What the sources say
boosteddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results reveal Nutlin used in combination with cisplatin increases the efficacy of cisplatin, showing that in the presence of Nutlin, 2-fold lower concentrations of cisplatin are effective.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
In the paper
“Mdm2 is an E3 ubiquitin ligase that binds directly to p53, targeting it for proteosomal degradation (1).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Introduction
What the sources say
- Not checked yetcites [1] Eischen, 2014, The Mdm network and its regulation of p53 activities: a rheostat of cancer risk, Hum Mutat source
plaindirect- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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Probably not a substantial claim. It may state two separate claims as one.
In the paper
“Consequently, p53 is mutated or deleted in approximately half of all of human cancers, with some malignancies having higher p53 mutation rates (1).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Introduction
What the sources say
- Not checked yetcites [1] Eischen, 2014, The Mdm network and its regulation of p53 activities: a rheostat of cancer risk, Hum Mutat source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results indicate Nutlin delays the DNA damage signal leading to more DNA damage signaling at later times after the genotoxic drug.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Our results have significant therapeutic implications, as they reveal that pharmacologically modulating Mdm2 levels could be used to more effectively treat malignancies that have inactivated p53, as well as allow lower concentrations of toxic chemotherapeutic drugs to be used.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
hedgedconditional- Asserted: passes — The authors put it forward as true.
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- Load-bearing: passes — The paper’s conclusions depend on it.
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It may state two separate claims as one.
- Rests on an assumption: Findings in cell lines and mouse embryonic fibroblasts will translate to human tumors.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Therefore, Nutlin inhibits the initial DNA damage response signal leading to prolonged signaling, and this requires Mdm2 but is independent of p53.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
plaindirect- Asserted: passes — The authors put it forward as true.
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It may state two separate claims as one.
In the paper
“Primary chemotherapy for ovarian cancer involves the genotoxic platinum drugs, cisplatin or carboplatin (4).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Introduction
What the sources say
- Not checked yetcites [4] Kyrgiou, 2006, Survival benefits with diverse chemotherapy regimens for ovarian cancer: meta-analysis of multiple treatments, J Natl Cancer Inst source
plain- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
In the paper
“Although initial responses are good, platinum resistance occurs often, requiring the use of other genotoxic agents, such as etoposide (5).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Introduction
What the sources say
- Not checked yetcites [5] Rose, 1998, Prolonged oral etoposide as second-line therapy for platinum-resistant and platinum-sensitive ovarian carcinoma: a Gynecologic Oncology Group study, J Clin Oncol source
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: fails — The paper’s conclusions may not depend on it.
- Based: passes — The paper gives a reason why it holds.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
In the paper
“Moreover, since increasing Mdmx levels also leads to an inhibition of DNA break repair (14), compounds that stabilize Mdmx or both Mdm2 and Mdmx are also likely to have similar results.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
What the sources say
- Not checked yetcites [14] Carrillo, 2015, Mdmx promotes genomic instability independent of p53 and Mdm2, Oncogene source
hedgedconditional- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: unclear — It is unclear whether the paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“These results indicate Nutlin treatment has a comparable inhibitory effect on γH2AX foci formation and resolution as Mdm2 overexpression, and this effect is independent of p53, but requires Mdm2.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Nutlin inhibits the DNA damage signal through Mdm2
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
None of the sources cited for this claim have been checked yet.
In the paper
“Our data suggest that in addition to p53 activation, Nutlin is also stabilizing Mdm2 causing an increase in the levels of Mdm2, which dampens the p53 response to DNA damage, resulting in increased sensitivity to genotoxic drugs (22,23).”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
What the sources say
- Not checked yetcites [22] Li, 2014, Therapeutic efficacy of p53 restoration in Mdm2-overexpressing tumors, Mol Cancer Res source
- Not checked yetcites [23] Conradt, 2013, Mdm2 inhibitors synergize with topoisomerase II inhibitors to induce p53-independent pancreatic cancer cell death, Int J Cancer source
hedgeddirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Here our data reveal that stabilization of Mdm2 by Nutlin resulted in an inhibition in the DNA damage signal and delayed DNA break repair.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Discussion
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.
In the paper
“Therefore, Nutlin inhibits DNA repair and DNA damage response signals in ovarian cancer cells with inactivated p53, analogous to what was observed in MEFs.”
§ Pharmacologically increasing Mdm2 inhibits DNA repair and cooperates with genotoxic agents to kill p53 inactivated ovarian cancer cells > Results > Delay of DNA damage signal in ovarian cancer cells caused by Nutlin
plaindirect- Asserted: passes — The authors put it forward as true.
- Checkable: passes — It can be checked against evidence.
- Load-bearing: passes — The paper’s conclusions depend on it.
- Based: passes — The paper gives a reason why it holds.
It may state two separate claims as one.
The paper relies on this without support.
Nutlin binds Mdm2 and blocks the Mdm2-p53 interaction.
“Nutlin-3 is a small molecule inhibitor of Mdm2/p53 interaction that can induce apoptosis in cancer cells through activation of p53.”
“In recent years, the drug Nutlin-3 (Nutlin) was developed that binds in the p53-binding pocket of Mdm2, inhibiting the interaction between Mdm2 and p53.”
The paper relies on this without support.
The ovarian cancer cell lines used are representative models of high-grade serous ovarian cancer with inactivated p53.
“SKOV-3 ovarian cancer cells have deleted p53 and showed increased levels of Mdm2 following exposure to Nutlin (Figure 3A).”
“We also evaluated ovarian cancer cells that harbor mutant p53, OVCAR-5 (insertion at codon 224) and OVCAR-8 (deletion of codons 126–132) (19).”
The paper relies on this without support.
Findings in cell lines and mouse embryonic fibroblasts will translate to human tumors.
“Importantly, our data show the therapeutic benefit of pharmacologically increasing Mdm2 in conjunction with genotoxic agents as a means of inducing synthetic lethality in cancers with inactivated p53, such as ovarian cancer.”
“Our results have significant therapeutic implications, as they reveal that pharmacologically modulating Mdm2 levels could be used to more effectively treat malignancies that have inactivated p53, as well as allow lower concentrations of toxic chemotherapeutic drugs to be used.”
The paper relies on this without support.
Nutlin increases Mdm2 levels in ovarian cancer cells by stabilizing the protein, as it does in mouse embryonic fibroblasts.
“Nutlin treatment led to increased Mdm2 protein levels in both cell lines (Figure 3B and 3C).”
“Here our data reveal that stabilization of Mdm2 by Nutlin resulted in an inhibition in the DNA damage signal and delayed DNA break repair.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the citing paper
“They demonstrated that Nutlin-3a–induced increased levels of Mdm2 directly inhibit DNA damage response signaling and delayed DNA break repair in p53-null MEFs and in ovarian cancer cell lines that lacked p53 or that contained mutant p53 (48).”
From Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model (2015) · cites this paper as [48]
In the citing paper
“While our studies do not demonstrate a direct interaction of chromatin-associated Mdm2 with the MRN complex, increases in Mdm2 associated chromatin following Nutlin-3a or combination Nutlin-3a/carboplatin are consistent with studies of Eischen and colleagues.”
From Potentiation of carboplatin-mediated DNA damage by the Mdm2 modulator Nutlin-3a in a humanized orthotopic breast-to-lung metastatic model (2015) · cites this paper as [48]
Citations
Cites
Siegel, R., Ma, J., Zou, Z., & Jemal, A. (2014) · CA: A Cancer Journal for Clinicians · doi:10.3322/caac.21208 · Cited by 11,499
Integrated genomic analyses of ovarian carcinoma
Perou, C. M. (2019) · Carolina Digital Repository (University of North Carolina at Chapel Hill) · doi:10.17615/hvp3-wg08 · Cited by 7,927
Signet-Ring Cell Carcinoma of the Colon: A Case Report and Review of the Literature
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In Vivo Activation of the p53 Pathway by Small-Molecule Antagonists of MDM2
Vassilev, L. T., Vu, B. T., Graves, B., Carvajal, D., Podlaski, F., Filipovic, Z., Kong, N., Kammlott, U., Lukacs, C., Klein, C., et al. (2004) · Science · doi:10.1126/science.1092472 · Cited by 4,661
ATM and ATR Substrate Analysis Reveals Extensive Protein Networks Responsive to DNA Damage
Matsuoka, S., Ballif, B. A., Smogorzewska, A., McDonald, E. R., Hurov, K. E., Luo, J., Bakalarski, C. E., Zhao, Z., Solimini, N., Lerenthal, Y., et al. (2007) · Science · doi:10.1126/science.1140321 · Cited by 3,075
Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization
Zindy, F., Eischen, C. M., Randle, D. H., Kamijo, T., Cleveland, J. L., Sherr, C. J., & Roussel, M. F. (1998) · Genes & Development · doi:10.1101/gad.12.15.2424 · Cited by 1,245
Self-citationO'Connor, P. M., Jackman, J., Bae, I., Myers, T. G., Fan, S., Mutoh, M., Scudiero, D. A., Monks, A., Sausville, E. A., Weinstein, J. N., et al. (1997) · PubMed · Cited by 867
Drugging the p53 pathway: understanding the route to clinical efficacy
Khoo, K. H., Verma, C. S., & Lane, D. P. (2014) · Nature Reviews Drug Discovery · doi:10.1038/nrd4236 · Cited by 742
Rose, P. G., Blessing, J. A., Mayer, A. R., & Homesley, H. D. (1998) · Journal of Clinical Oncology · doi:10.1200/jco.1998.16.2.405 · Cited by 386
Assembly and function of DNA double-strand break repair foci in mammalian cells
Bekker-Jensen, S., & Mailand, N. (2010) · DNA Repair · doi:10.1016/j.dnarep.2010.09.010 · Cited by 338
Cited by
Sorted by citations
Clinical Overview of MDM2/X-Targeted Therapies
Burgess, A., Chia, K. M., Haupt, S., Thomas, D., Haupt, Y., & Lim, E. (2016) · Frontiers in Oncology · doi:10.3389/fonc.2016.00007 · Cited by 315
Eischen, C. M. (2016) · Cold Spring Harbor Perspectives in Medicine · doi:10.1101/cshperspect.a026096 · Cited by 161
Self-citationKocik, J., Machula, M., Wisniewska, A., Surmiak, E., Holak, T. A., & Skalniak, L. (2019) · Cancers · doi:10.3390/cancers11071014 · Cited by 46
Tonsing-Carter, E., Bailey, B. J., Saadatzadeh, M. R., Ding, J., Wang, H., Sinn, A. L., Peterman, K. M., Spragins, T. K., Silver, J. M., Sprouse, A. A., et al. (2015) · Molecular Cancer Therapeutics · doi:10.1158/1535-7163.mct-15-0237 · Cited by 42
Self-citation
Metadata
- Authors
- Carrillo, A. M., Hicks, M., Khabele, D., & Eischen, C. M.
- Affiliations
- Vanderbilt University Medical Center (US)
- Corresponding author
- Eischen, Christine M.
- Venue
- Molecular Cancer Research
- Published
- 2015-08-16
- Volume
- 13
- Issue
- 8
- Pages
- 1197-1205
- ISSN
- 1541-7786 (ISSN-L)
- 1557-3125
- PMC
- PMC4543442
- OpenAlex
- W2162795905
- Semantic Scholar
- d974b803bd1b571fb7d52709897c1048b7268428
- Cited by
- 31
- References
- 30
- Citations by year
- 2026: 1 · 2024: 1 · 2023: 4 · 2022: 1 · 2021: 2 · 2020: 1 · 2019: 4 · 2018: 3 · 2017: 3 · 2016: 7 · 2015: 3
- FWCI
- 2.33
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Molecular Cancer Research (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Cancer Institute (T32CA093240, K08CA148887, R01CA181204, F31CA150546, T32CA009582)
- Topics
- Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
- Keywords
- MDM2, Nutlin-3, p53 inactivation, DNA double-strand break repair, DNA damage response, Mre11-Rad50-Nbs1 complex, NBS1, synthetic lethality, ovarian cancer, cisplatin, etoposide, DNA-damaging agents, apoptosis, p53-MDM2 interaction
- MeSH terms
- Animals, Antineoplastic Combined Chemotherapy Protocols, Cisplatin, DNA Damage, DNA Repair, Etoposide, Female, Fibroblasts, Humans, Imidazoles, Mice, Transgenic, Mutagens, Ovarian Neoplasms, Piperazines, Tumor Suppressor Protein p53, Apoptosis, Comet Assay, Cell Line, Tumor, Cell Proliferation, Mice, Proto-Oncogene Proteins c-mdm2, DNA Breaks, Double-Stranded, HEK293 Cells
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Cite CoChat for the checks on this page, and the paper for its own findings.
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- CoChat. (2026). Citation check: Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells. https://app.cochat.ai/papers/10.1158/1541-7786.mcr-15-0089
- The paper
- Carrillo, A. M., Hicks, M., Khabele, D., & Eischen, C. M. (2015). Pharmacologically Increasing Mdm2 Inhibits DNA Repair and Cooperates with Genotoxic Agents to Kill p53-Inactivated Ovarian Cancer Cells. Molecular Cancer Research. https://doi.org/10.1158/1541-7786.mcr-15-0089
