Radiation-Induced Chromosomal Aberrations and Immunotherapy: Micronuclei, Cytosolic DNA, and Interferon-Production Pathway

Abstract

Radiation-induced chromosomal aberrations represent an early marker of late effects, including cell killing and transformation. The measurement of cytogenetic damage in tissues, generally in blood lymphocytes, from patients treated with radiotherapy has been studied for many years to predict individual sensitivity and late morbidity. Acentric fragments are lost during mitosis and create micronuclei (MN), which are well correlated to cell killing. Immunotherapy is rapidly becoming a most promising new strategy for metastatic tumors, and combination with radiotherapy is explored in several pre-clinical studies and clinical trials. Recent evidence has shown that the presence of cytosolic DNA activates immune response via the cyclic GMP-AMP synthase/stimulator of interferon genes pathway, which induces type I interferon transcription. Cytosolic DNA can be found after exposure to ionizing radiation either as MN or as small fragments leaking through nuclear envelope ruptures. The study of the dependence of cytosolic DNA and MN on dose and radiation quality can guide the optimal combination of radiotherapy and immunotherapy. The role of densely ionizing charged particles is under active investigation to define their impact on the activation of the interferon pathway.

Keywords: chromosome aberrations; cyclic GMP–AMP synthase; micronuclei; particle therapy; radioimmunotherapy; stimulator of interferon genes.

Figure 1

Micronuclei in the cytoplasm of a human umbilical vein endothelial cell exposed to X-rays. Two micronuclei are visible in this binucleated cell stained in DAPI. Photo courtesy of Dr. Alexander Helm.

Figure 2

Micronuclei originate from radiation-induced chromosome aberrations. Acentric fragments in irradiated cells are shown in this panel. (A) A dicentric chromosome and associated acentric fragment between chromosome 1 and 11 in a human peripheral blood lymphocytes exposed to heavy ions. Image visualized by mFISH. (B) Formation of a radiation-induced ring and its associated acentric fragment visualized by R × FISH (cross-species color banding). A normal chromosome 3 is shown in the middle, the centric ring on the right, and the acentric fragment resulting from the joining of the two residual fragments in the two different arms on the left. (C) A terminal deletion induced by heavy ions in human lymphocytes. The lack of telomere signal indicates that a fragment has been lost. (D) An interstitial deletion at the first mitosis following exposure of G0-phase mouse fibroblasts to X-rays. Interstitial deletions appear often as double minutes. In this photomicrograph of Giemsa-stained mitotic chromosomes, it is clear their nature of acentric rings resulting from the asymmetrical intra-arm intrachange.

Figure 3

Examples of dose-response curves for the induction of micronuclei (MN) per binucleated (BN) cell. The blue curve is the weighted mean of the calibration curves used in 10 different European laboratories for MN biodosimetry using human peripheral blood lymphocytes (PBL) within the RENEB project (78). The equation is Y = 0.016 + 0.0508·D + 0.0155·D², where Y is the frequency of MN per BN cell and D the ⁶⁰Co γ-ray dose in gray. The red curve refers to human neonatal dermal fibroblasts (HNDF) exposed to ⁶⁰Co γ-ray (59). The equation used by the authors to fit their data was Y = 0.033 + 0.1423·D + 0.0041·D². The green line refers to human PBL exposed to ²³⁹Pu α-particles (61). Data points (all at doses <1 Gy) were fitted by the function Y = 0.098 + 0.418·D.

Figure 4

Collection of measured relative biological effectiveness (RBE) data for the induction of MN per BN cell after exposure to protons, α-particles, or energetic heavy ions. RBE was calculated as the ratio of the initial slope of the dose-response curves for ions and photons as measured by the same authors. Different symbols refer to different cell types. HaCaT = spontaneously immortalized adult human keratinocytes (63); NHPK = normal human neonatal epidermal keratinocytes (63); HUVEC = human umbilical vein endothelial cells (67); HNDF = human neonatal dermal fibroblasts (59); FRTL-5 = Fischer rat thyroid cell line (60); PBL = human peripheral blood lymphocytes (61); SCCVIII = mouse squamous cell carcinoma (64); V79 =Chinese hamster fibroblasts (62); Cl-1 = Chinese hamster fibroblasts (46). The blue line is a guide for the eye.

Figure 5

Dose-response curves for the induction of MN in HUVEC cells exposed to X-rays or accelerated carbon ions at two different energies. A curvature at high doses can be observed. Figure reproduced with permission from Ref. (67).

Figure 6

Ionizing radiation can induce cytosolic DNA, thus triggering the cyclic GMP–AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, in two different ways. If cells progress to mitosis carrying chromosome aberrations, MN (from acentric fragments) can be produced at the first mitosis along with nucleoplasmic bridges (NPB, from dicentrics). These micronuclei can be incorporated in the cytoplasm of the daughter cells and, following the collapse of the nuclear envelope (NE), can be sensed by the cGAS. Alternatively, even if the cell is blocked or delayed in the cell-cycle, radiation-induced DNA fragments can leak through a damaged NE. This effect can be more likely for very small fragments induced by densely ionizing radiation, and NE rupture can be triggered by the enhanced mobility of the cells following radiation exposure.