DNA Damage Response Proteins and Oxygen Modulate Prostaglandin E2 Growth Factor Release in Response to Low and High LET Ionizing Radiation

Abstract

Common cancer therapies employ chemicals or radiation that damage DNA. Cancer and normal cells respond to DNA damage by activating complex networks of DNA damage sensor, signal transducer, and effector proteins that arrest cell cycle progression, and repair damaged DNA. If damage is severe enough, the DNA damage response (DDR) triggers programed cell death by apoptosis or other pathways. Caspase 3 is a protease that is activated upon damage and triggers apoptosis, and production of prostaglandin E2 (PGE2), a potent growth factor that can enhance growth of surviving cancer cells leading to accelerated tumor repopulation. Thus, dying tumor cells can promote growth of surviving tumor cells, a pathway aptly named Phoenix Rising. In the present study, we surveyed Phoenix Rising responses in a variety of normal and established cancer cell lines, and in cancer cell lines freshly derived from patients. We demonstrate that IR induces a Phoenix Rising response in many, but not all cell lines, and that PGE2 production generally correlates with enhanced growth of cells that survive irradiation, and of unirradiated cells co-cultured with irradiated cells. We show that PGE2 production is stimulated by low and high LET ionizing radiation, and can be enhanced or suppressed by inhibitors of key DDR proteins. PGE2 is produced downstream of caspase 3 and the cyclooxygenases COX1 and COX2, and we show that the pan COX1-2 inhibitor indomethacin blocks IR-induced PGE2 production in the presence or absence of DDR inhibitors. COX1-2 require oxygen for catalytic activity, and we further show that PGE2 production is markedly suppressed in cells cultured under low (1%) oxygen concentration. Thus, Phoenix Rising is most likely to cause repopulation of tumors with relatively high oxygen, but not in hypoxic tumors. This survey lays a foundation for future studies to further define tumor responses to radiation and inhibitors of the DDR and Phoenix Rising to enhance the efficacy of radiotherapy with the ultimate goal of precision medicine informed by deep understanding of specific tumor responses to radiation and adjunct chemotherapy targeting key factors in the DDR and Phoenix Rising pathways.

Keywords: DNA damage response; apoptosis; caspase; growth factor; radiotherapy.

Figure 1

(A) Phoenix Rising pathway of accelerated tumor repopulation. The cascade is initiated by cleavage and activation of caspase 3, which also promotes programed cell death by apoptosis. Activated caspase 3 cleaves and activates phospholipase 2 that hydrolyzes fatty acid phospholipid bonds, releasing arachidonic acid and lysophospholipids. Arachidonic acid is converted to PGH₂ by COX1 and COX2 peroxidases in the presence of oxygen; this step can be blocked by COX1–COX2 inhibitor indomethacin or hypoxia. Prostaglandin synthases generate the family of prostanoids, including PGD₂, PGE₂, and PGF₂. PGE₂ (and possibly other PGs) excreted from dying cells promote growth of surviving cells, accelerating tumor repopulation. (B) The DDR regulates cell fate after radiation damage. Proteins involved in DNA repair and damage checkpoint pathways crosstalk with programed cell death pathways to determine a variety of short- and long-term cell fates. Phoenix Rising and the DDR are linked through apoptosis and possibly other processes.

Figure 2

PGE₂ production and short-term cell viability/proliferation in response to γ-rays or X-rays in the presence or absence of DDR or COX inhibitors. PGE₂ and cell growth were measured by ELISA and SRB assay in response to (A) 10 Gy γ-rays or (B) 3 or 10 Gy X-rays in the presence or absence of indomethacin (Indo). Data are averages ±SD for two to four replicates per treatment group (A) or single determinations (B). In this and all subsequent figures, statistical significance was determined by t-tests, * indicates p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

PGE₂ production in response to high LET IR ± DDRi. LC-MS/MS was used to quantify PGE₂ levels in supernatants from HT1080 and BJ1hTERT cultures after irradiation with 3 Gy carbon ions (70 keV/μm). (A) Absolute PGE₂ levels. (B) PGE₂ levels were derived by subtracting the baseline values (no IR, no drug); n.d., not determined.

Figure 4

(A) Transwell multiple-endpoint assay system. Cells were seeded into the top (500–1,500 cells) and bottom (feeder, 100,000 cells) sections of the transwell system and incubated for 24 h. The top section was transferred to a separate dish while the bottom section was irradiated, and then replaced. PGE₂ was measured in the media 3 and 6 days after IR; growth of unirradiated cells was determined 7–9 days after IR. Apoptotic endpoints (caspase 3/7 activation, Annexin V, and membrane changes by propidium iodide staining) were measured at 3, 6, or 9 days post IR in irradiated feeder cells. When used, DDR or cyclooxygenase inhibitors were present during the entire experiment. (B) HeLa cells assayed for growth stimulation using the transwell assay system after irradiation with 10 Gy low LET γ-rays. Indomethacin (Indo) inhibits COX1–2 and suppresses PGE₂ production.

Figure 5

PGE₂ production and stimulated proliferation of unirradiated HeLa cells in response to high LET carbon ions. (A) ELISA measurements from media in transwells containing HeLa cells 6 days after 0 or 4 Gy carbon ion irradiation. (B) Relative growth of unirradiated cells in transwells with or without DDR inhibitors or indomethacin, 6 days after irradiation of feeder cells. (C) As in (B) but without feeder cells. Data are averages (±SEM) for two determinations per condition.

Figure 6

Flow cytometric analysis of apoptotic phenotypes of feeder cells (from Figure 5) irradiated with high LET carbon ion IR. Duplicate plates were irradiated in parallel for assays 3 days post IR. Feeder cells co-cultured with transwell inserts were assayed 6 days post IR. Each sample was split at the time of harvest for two assay endpoints. Caspase cleavage and activation was assayed in live cells (A) 3 or (D) 6 days post IR using a fluorescent caspase 3/7 peptide fragment. Annexin V/propidium iodide co-staining of fixed cells discriminates between early, mid, or late apoptotic stages and was assayed (B) 3 or (E) 6 days post IR. No IR controls for the annexin V/PI assay (C) 3 or (F) 6 days post IR; ~10,000 cells were interrogated per determination. Unstained cells served as negative controls.

Figure 7

Caspase activation and relative growth in two HNSCC cell lines after exposure to 4 Gy high LET IR. (A) Caspase cleavage and activation was assayed in live cells 48 h post IR. (B,D) Relative growth of primary tumor cells from transwells 6 days post IR with or (C,E) without irradiated feeder cells. Data represent the averages and SDs for three replicate measurements per determination.

Figure 8

IR-induced PGE2 production is suppressed by hypoxia. PGE₂ levels were determined by ELISA 3 days after X-ray, silicon ion, or carbon ion radiation at indicated LET and doses. Cells were incubated under normoxic, or hypoxic (1% O2) conditions. Data represent the averages and SDs for nine replicate measurements per determination.