Response of Thyroid Follicular Cells to Gamma Irradiation Compared to Proton Irradiation: II. The Role of Connexin 32

Abstract

The objective of this study was to determine whether connexin 32-type gap junctions contribute to the "contact effect" in follicular thyrocytes and whether the response is influenced by radiation quality. Our previous studies demonstrated that early-passage follicular cultures of Fischer rat thyroid cells express functional connexin 32 gap junctions, with later-passage cultures expressing a truncated nonfunctional form of the protein. This model allowed us to assess the role of connexin 32 in radiation responsiveness without relying solely on chemical manipulation of gap junctions. The survival curves generated after gamma irradiation revealed that early-passage follicular cultures had significantly lower values of alpha (0.04 Gy(-1)) than later-passage cultures (0.11 Gy(-1)) (P < 0.0001, n = 12). As an additional way to determine whether connexin 32 was contributing to the difference in survival, cultures were treated with heptanol, resulting in higher alpha values, with early-passage cultures (0.10 Gy(-1)) nearly equivalent to untreated late-passage cultures (0.11 Gy(-1)) (P > 0.1, n = 9). This strongly suggests that the presence of functional connexin 32-type gap junctions was contributing to radiation resistance in gamma-irradiated thyroid follicles. Survival curves from proton-irradiated cultures had alpha values that were not significantly different whether cells expressed functional connexin 32 (0.10 Gy(-1)), did not express connexin 32 (0.09 Gy(-1)), or were down-regulated (early-passage plus heptanol, 0.09 Gy(-1); late-passage plus heptanol, 0.12 Gy(-1)) (P > 0.1, n = 19). Thus, for proton irradiation, the presence of connexin 32-type gap junctional channels did not influence their radiosensitivity. Collectively, the data support the following conclusions. (1) The lower alpha values from the gamma-ray survival curves of the early-passage cultures suggest greater repair efficiency and/or enhanced resistance to radiation-induced damage, coincident with the expression of connexin 32-type gap junctions. (2) The increased sensitivity of FRTL-5 cells to proton irradiation was independent of their ability to communicate through connexin 32 gap junctions. (3) The fact that the beta components of the survival curves from both gamma rays and proton beams were similar (average 0.022 +/- 0.008 Gy(-2), P > 0.1, n = 39) suggests that at higher doses the loss of viability occurs at a relatively constant rate and is independent of radiation quality and the presence of functional gap junctions.

Connexins: Key Mediators of Endocrine Function

“…Other experiments have revealed that early-passage cultures of rat thyroid cells, which express functional Cx32 channels, are more resistant to ␥ irradiation than later-passage cultures, which express a truncated, nonfunctional form of Cx32. Strikingly, this beneficial effect was not observed after proton irradiation (181). These observations may be related to those made on insulin-producing ␤-cells after exposure to drugs that experimentally reproduce the massive cell death observed in pancreatic islets at the onset of type I diabetes (5,265).…”

“…Cx32 and coupling are lost with passages in culture, together with the ability of the cells to form follicular structures (166,180,181,364,365). These alterations can be reverted by exposing cell monolayers to thyroid stimulating hormone (TSH) (365) or by transfection of communication-incompetent thyrocytes with Cx32, but not Cx43 (545).…”

Cellular and molecular effects of protons: Apoptosis induction and potential implications for cancer therapy

“…55 In another report on the effects of proton irradiation, activation of apoptosis in thyroid follicular cells was investigated by searching for different markers such as cytoplasmic DNA fragments and micronuclei. 56 These authors also reported a block of cell cycle at the G2 phase but didn't describe modifications of the mRNA phenotype nor the increase of ROS induced by proton irradiation. 56 It is interesting to note that also gamma irradiation of CCRF-CEM, a human lymphoblastic leukemia cell line, has been reported to induce arrest at the G2/M phase of the cycle.…”

Proton Irradiation Augments the Suppression of Tumor Progression Observed with Advanced Age

“…Preclinical in vivo studies on proton therapy are usually performed using young animals (2, 4). Since proton therapy is utilized for tumor treatment of children (due to the superior targeting advantages over gamma radiation) as well as middle-age to older patients (40, 41), it is important to investigate the efficacy of such therapy, along with the radiobiology of protons in general, as a function of host age, gender and genetics. We have demonstrated proton-induced tumor signaling to be modulated with age and found suppression of TGFβ to play a key role in inhibiting tumor growth in proton-irradiated old mice.…”