Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells

Abstract

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (−/−) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (−/−) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10–20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Tumor Cell Recovery from Senescence Induced by Radiation with PARP Inhibition

“…Our laboratories have confirmed that PARP inhibitors do not increase radiation-induced apoptosis in HCT-116 colon carcinoma cells, but instead markedly enhance growth arrest and senescence (18). In this context, a tumoristatic effect could be considered a less desirable outcome than a tumoricidal effect since we have further determined that the growth arrest induced by radiation with PARP inhibition is transient (as is also the case with that induced by radiation alone) (18) in at least a fraction of the cells.…”

“…In this context, a tumoristatic effect could be considered a less desirable outcome than a tumoricidal effect since we have further determined that the growth arrest induced by radiation with PARP inhibition is transient (as is also the case with that induced by radiation alone) (18) in at least a fraction of the cells. Although recovery of the culture as a whole could reflect outgrowth of a few undamaged or lightly damaged cells rather than proliferation of transiently arrested, senescent cells, we have succeeded in separating from the treated cultures β-galactoside-expressing cells that appear morphologically senescent, yet resume proliferation after a short delay (18). …”

“…These DSBs are largely replication-dependent, as reflected in the reported finding that blocking replication largely eliminates PARP inhibitor-mediated radiosensitization of most cells (17, 18). The downstream consequences, including modes of cell death or growth arrest, could thus differ substantially from those of DSBs induced directly by radiation alone.…”

“…While most published studies have not attempted to actually distinguish between the induction of DNA SSBs and DSBs, the detection of γ-H2AX foci that are not resolved is generally considered to be a reliable indicator of unrepaired DSBs (24, 25). Moreover, neutral comet assays, which provide a more direct and specific measure of DSBs than γ-H2AX foci, also indicate that PARP inhibition increases radiation-induced damage (18). Nevertheless, it might be relevant to monitor the DNA damage induced by radiation with PARP inhibition for extended periods of time to reliably establish that the breaks are prolonged and essentially irreparable.…”

“…Nevertheless, it might be relevant to monitor the DNA damage induced by radiation with PARP inhibition for extended periods of time to reliably establish that the breaks are prolonged and essentially irreparable. This suggestion relates to the fact that, as indicated below, the incorporation of PARP inhibition with radiation has generally not demonstrated cell killing that might be expected with unrepaired DNA DSBs; instead, studies have shown that this experimental strategy tends to lead to inhibition of tumor cell proliferation and, in a number of studies, to the induction of a state of senescence (18, 26–28). …”