The P53-53BP1-Related Survival Of A549 And H1299 Human Lung ...

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Abstract

Radiation therapy is one of the main methods of treating patients with non-small cell lung cancer (NSCLC). However, the resistance of tumor cells to exposure remains the main factor that limits successful therapeutic outcome. To study the molecular/cellular mechanisms of increased resistance of NSCLC to ionizing radiation (IR) exposure, we compared A549 (p53 wild-type) and H1299 (p53-deficient) cells, the two NSCLC cell lines. Using fractionated X-ray irradiation of these cells at a total dose of 60 Gy, we obtained the survived populations and named them A549IR and H1299IR, respectively. Further characterization of these cells showed multiple alterations compared to parental NSCLC cells. The additional 2 Gy exposure led to significant changes in the kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci numbers in A549IR and H1299IR compared to parental NSCLC cells. Whereas A549, A549IR, and H1299 cells demonstrated clear two-component kinetics of DNA double-strand break (DSB) repair, H1299IR showed slower kinetics of γH2AX foci disappearance with the presence of around 50% of the foci 8 h post-IR. The character of H2AX phosphorylation in these cells was pATM-independent. A decrease of residual γH2AX/53BP1 foci number was observed in both A549IR and H1299IR compared to parental cells post-IR at extra doses of 2, 4, and 6 Gy. This process was accompanied with the changes in the proliferation, cell cycle, apoptosis, and the expression of ATP-binding cassette sub-family G member 2 (ABCG2, also designated as CDw338 and the breast cancer resistance protein (BCRP)) protein. Our study provides strong evidence that different DNA repair mechanisms are activated by multifraction radiotherapy (MFR), as well as single-dose IR, and that the enhanced cellular survival after MFR is reliant on both p53 and 53BP1 signaling along with non-homologous end-joining (NHEJ). Our results are of clinical significance as they can guide the choice of the most effective IR regimen by analyzing the expression status of the p53-53BP1 pathway in tumors and thereby maximize therapeutic benefits for the patients while minimizing collateral damage to normal tissue.

Keywords: DNA repair; ionizing radiation; non-small cell lung cancer; p53; radioresistance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Phase contrast microscopic images of…

Figure 1

Phase contrast microscopic images of parental and irradiation-surviving A549 ( a ) and…

Figure 1 Phase contrast microscopic images of parental and irradiation-surviving A549 (a) and H1299 (b) cells. Objective: 20x.
Figure 2

Figure 2

Plating efficiency of parental and…

Figure 2

Plating efficiency of parental and irradiation-surviving A549 ( a ) and H1299 ( …

Figure 2 Plating efficiency of parental and irradiation-surviving A549 (a) and H1299 (b) cells. Colonies of the small size were counted under light microscope. Colonies ≥ 50 cells were counted. Data are means ± SEM of more than three independent experiments.
Figure 3

Figure 3

Assessment of the proliferative activity…

Figure 3

Assessment of the proliferative activity in both parental (non-irradiated) and irradiation-surviving A549 and…

Figure 3 Assessment of the proliferative activity in both parental (non-irradiated) and irradiation-surviving A549 and H1299 cells using the Click-iTTM EdU test (cells were seeded in 96-well plates at concentrations of 1500 and 2000 cells/0.32 cm2, marked by black and grey columns, respectively). (a) Changes in the percentage of EdU-positive cells in A549 and A549IR cell populations. (b) Changes in the percentage of EdU-positive cells in H1299 and H1299IR cell populations. Cell counting was performed at objective 10×. Data are means ± SEM of more than three independent experiments.
Figure 4

Figure 4

Proliferation activity of parental and…

Figure 4

Proliferation activity of parental and irradiation-surviving non-small cell lung cancer (NSCLC) cells 24…

Figure 4 Proliferation activity of parental and irradiation-surviving non-small cell lung cancer (NSCLC) cells 24 h after exposure to different doses of X-rays. Changes in proliferation activity of A549 and A549IR cells (a) and H1299 and H1299IR cells (b) were analyzed 24 h after exposure to 2, 4, and 6 Gy of X-rays. * denotes significant differences between groups at p < 0.05. ** denotes significant differences between groups at p < 0.001. Data are means ± SEM of more than three independent experiments.
Figure 5

Figure 5

Cell cycle analysis of parental…

Figure 5

Cell cycle analysis of parental (A549 and H1299) and irradiation-surviving (A549IR and H1299IR)…

Figure 5 Cell cycle analysis of parental (A549 and H1299) and irradiation-surviving (A549IR and H1299IR) NSCLC cells 24 h after exposure to different single doses of X-rays. Proportion of A549 and A549IR cells in G0/G1 (a), S (b), and G2/M (c) cell cycle stages. Proportion of H1299 and H1299IR cells in G0/G1 (d), S (e), and G2/M (f) cell cycle stages. * denotes significant differences compared to unirradiated cells at p < 0.05. A total of 50,000 events were acquired for each sample. Data are means ± SEM of more than three independent experiments.
Figure 6

Figure 6

Assessment of apoptosis by YO-PRO-1…

Figure 6

Assessment of apoptosis by YO-PRO-1 in parental and irradiation-surviving NSCLC cell lines. ( …

Figure 6 Assessment of apoptosis by YO-PRO-1 in parental and irradiation-surviving NSCLC cell lines. (a) A549IR cells demonstrated a higher rate of apoptotic (YO-PRO-1-positive/propidium iodide (PI)-negative) cells 24 h after exposure to IR compared to parental A549 cells; (b) H1299IR cells showed lower percentage of apoptotic (YO-PRO-1 positive/PI negative) cells after IR exposure compared to parental H1299 cells. A total of 50,000 events were acquired for each sample. Data are means ± SEM of more than three independent experiments.
Figure 7

Figure 7

Kinetics of γH2AX and phosphorylated…

Figure 7

Kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci changes in A549…

Figure 7 Kinetics of γH2AX and phosphorylated ataxia telangiectasia mutated (pATM) foci changes in A549 and A549IR cells and H1299 and H1299IR cells after exposure to an extra single dose of 2 Gy of X-rays. Comparative analysis of changes in the number of γH2AX foci in A549 and A549IR (a) and H1299 and H1299IR cells (c) after 2 Gy X-ray exposure; changes in the number of рАТМ foci in A549 and A549IR cells (b) and H1299 and H1299IR cells (d) after 2 Gy X-ray exposure. ↕ denotes significant differences between groups at p < 0.05. Data are means ± SEM of more than three independent experiments.
Figure 8

Figure 8

Changes in the number of…

Figure 8

Changes in the number of residual γH2AX, 53BP1 foci, and their co-localization was…

Figure 8 Changes in the number of residual γH2AX, 53BP1 foci, and their co-localization was analyzed in A549 and A549IR cells and H1299 and H1299IR cells 24 h after exposure to different single doses of X-rays. The number of residual γH2AX foci increased post-IR at extra single doses in A549 and A549IR cells (a) and H1299 and H1299IR cells (b). Changes in the number of residual 53BP1 in A549 and A549IR cells (c) and H1299 and H1299IR cells (d). Changes in the number of co-localized γH2AX/53BP1 in A549 and A549IR cells (e) and H1299 and H1299IR cells (f). Data are means ± SEM of more than three independent experiments.
Figure 8

Figure 8

Changes in the number of…

Figure 8

Changes in the number of residual γH2AX, 53BP1 foci, and their co-localization was…

Figure 8 Changes in the number of residual γH2AX, 53BP1 foci, and their co-localization was analyzed in A549 and A549IR cells and H1299 and H1299IR cells 24 h after exposure to different single doses of X-rays. The number of residual γH2AX foci increased post-IR at extra single doses in A549 and A549IR cells (a) and H1299 and H1299IR cells (b). Changes in the number of residual 53BP1 in A549 and A549IR cells (c) and H1299 and H1299IR cells (d). Changes in the number of co-localized γH2AX/53BP1 in A549 and A549IR cells (e) and H1299 and H1299IR cells (f). Data are means ± SEM of more than three independent experiments.
Figure 9

Figure 9

ABCG2 expression profile in parental…

Figure 9

ABCG2 expression profile in parental and irradiation-surviving NSCLC cell lines. ( a )…

Figure 9 ABCG2 expression profile in parental and irradiation-surviving NSCLC cell lines. (a) A549IR cells did not show any difference in ABCG2 expression 24 h after exposure to IR compared to parental A549 cells; (b) H1299IR cells showed statistically significantly higher level of ABCG2 expression after IR exposure compared to parental H1299 cells. ABCG2 signal normalized to the number of cells. * denotes significant differences between correspondent control groups at p < 0.05. Data are means ± SEM of more than three independent experiments.
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References

    1. Moore S., Leung B., Wu J., Ho C. Population-based analysis of curative therapies in stage II non-small cell lung cancer: The role of radiotherapy in medically inoperable patients. Radiat. Oncol. 2020;15 doi: 10.1186/s13014-020-1466-y. - DOI - PMC - PubMed
    1. Moeller B., Balagamwala E.H., Chen A., Creach K.M., Giaccone G., Koshy M., Zaky S., Rodrigues G. Palliative thoracic radiation therapy for non-small cell lung cancer: 2018 Update of an American Society for Radiation Oncology (ASTRO) Evidence-Based Guideline. Pract. Radiat. Oncol. 2018;8:245–250. doi: 10.1016/j.prro.2018.02.009. - DOI - PubMed
    1. Baker S., Dahele M., Lagerwaard F.J., Senan S. A critical review of recent developments in radiotherapy for non-small cell lung cancer. Radiat. Oncol. 2016;11 doi: 10.1186/s13014-016-0693-8. - DOI - PMC - PubMed
    1. Andersen B.L., Goyal N.G., Weiss D.M., Westbrook T.D., Maddocks K.J., Byrd J.C., Johnson A.J. Cells, cytokines, chemokines, and cancer stress: A biobehavioral study of patients with chronic lymphocytic leukemia. Cancer. 2018;124:3240–3248. doi: 10.1002/cncr.31538. - DOI - PMC - PubMed
    1. Chang L., Graham P., Hao J., Ni J., Deng J., Bucci J., Malouf D., Gillatt D., Li Y. Cancer stem cells and signaling pathways in radioresistance. Oncotarget. 2015;7 doi: 10.18632/oncotarget.6760. - DOI - PMC - PubMed
Show all 60 references

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