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Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma.

Beumer JH, Fu KY, Anyang BN, Siegfried JM, Bakkenist CJ - BMC Cancer (2015)

Bottom Line: ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed.While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA. beumerjh@upmc.edu.

ABSTRACT

Background: ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.

Methods: We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.

Results: No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.

Conclusions: Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.

No MeSH data available.


Related in: MedlinePlus

No synergy was seen between gemcitabine and ATM kinase inhibitor. Exponentially dividing lung cancer cell lines were treated with increasing doses of gemcitabine and a fixed concentration of ATM kinase inhibitor KU55933 for 48 h and MTT reagent was then added. Representative examples from at least 3 replicate experiments are shown (mean, SD of 4 replicates)
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Fig4: No synergy was seen between gemcitabine and ATM kinase inhibitor. Exponentially dividing lung cancer cell lines were treated with increasing doses of gemcitabine and a fixed concentration of ATM kinase inhibitor KU55933 for 48 h and MTT reagent was then added. Representative examples from at least 3 replicate experiments are shown (mean, SD of 4 replicates)

Mentions: We were also interested in how cell killing by gemcitabine would be affected by ATM kinase inhibition. We reasoned that gemcitabine might induce DSBs in lung cancer cell lines due to acquired mutations that disrupt mechanisms that protect stalled replication forks. If this were the case then increased cell killing might be seen with gemcitabine and ATM kinase inhibitor but not gemcitabine and ATR kinase inhibitor. However, gemcitabine was not potentiated by ATM kinase inhibition (Fig. 4, Additional file 2: Table S2). Thus, the lesions induced by gemcitabine and ATM kinase inhibition do not interact in the lung cancer cell lines examined.Fig. 4


Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma.

Beumer JH, Fu KY, Anyang BN, Siegfried JM, Bakkenist CJ - BMC Cancer (2015)

No synergy was seen between gemcitabine and ATM kinase inhibitor. Exponentially dividing lung cancer cell lines were treated with increasing doses of gemcitabine and a fixed concentration of ATM kinase inhibitor KU55933 for 48 h and MTT reagent was then added. Representative examples from at least 3 replicate experiments are shown (mean, SD of 4 replicates)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4595318&req=5

Fig4: No synergy was seen between gemcitabine and ATM kinase inhibitor. Exponentially dividing lung cancer cell lines were treated with increasing doses of gemcitabine and a fixed concentration of ATM kinase inhibitor KU55933 for 48 h and MTT reagent was then added. Representative examples from at least 3 replicate experiments are shown (mean, SD of 4 replicates)
Mentions: We were also interested in how cell killing by gemcitabine would be affected by ATM kinase inhibition. We reasoned that gemcitabine might induce DSBs in lung cancer cell lines due to acquired mutations that disrupt mechanisms that protect stalled replication forks. If this were the case then increased cell killing might be seen with gemcitabine and ATM kinase inhibitor but not gemcitabine and ATR kinase inhibitor. However, gemcitabine was not potentiated by ATM kinase inhibition (Fig. 4, Additional file 2: Table S2). Thus, the lesions induced by gemcitabine and ATM kinase inhibition do not interact in the lung cancer cell lines examined.Fig. 4

Bottom Line: ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed.While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA. beumerjh@upmc.edu.

ABSTRACT

Background: ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.

Methods: We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.

Results: No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.

Conclusions: Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.

No MeSH data available.


Related in: MedlinePlus