J.L. Redpath

Low doses of diagnostic energy X-rays protect against neoplastic transformation in vitro.

Purpose : To investigate the effect of low doses of 60 kVp X-rays on in vitro transformation frequency. Materials and methods : HeLa ×skin fibroblast human hybrid cells were used to assay transformation from the non-tumorigenic to the tumorigenic phenotype. Subconfluent cultures of cells were exposed to a range of doses of 60 kVp X-rays and seeded for assay of transformation after 24 h post-irradiation holding. Experiments were repeated at least three times and the data pooled for analysis. Transformation frequencies were compared with those of sham-irradiated controls. Results : At doses < 1 cGy, the observed transformation frequencies were significantly less than those seen in unirradiated cells. Conclusion : Low doses (< 1 cGy) of 60 kVp X-rays protect HeLa ×skin fibroblast human hybrid cells against neoplastic transformation in vitro.

The effect of dose rate on radiation-induced neoplastic transformation in vitro by low doses of low-LET radiation.

Abstract Elmore, E., Lao, X-Y., Kapadia, R. and Redpath, J. L. The Effect of Dose Rate on Radiation-Induced Neoplastic Transformation In Vitro by Low Doses of Low-LET Radiation. Radiat. Res. 166, 832–838 (2006). The dependence of the incidence of radiation-induced cancer on the dose rate of the radiation exposure is a question of considerable importance to the estimation of risk of cancer induction by low-dose-rate radiation. Currently a dose and dose-rate effectiveness factor (DDREF) is used to convert high-dose-rate risk estimates to low dose rates. In this study, the end point of neoplastic transformation in vitro has been used to explore this question. It has been shown previously that for low doses of low-LET radiation delivered at high dose rates, there is a suppression of neoplastic transformation frequency at doses less than around 100 mGy. In the present study, dose–response curves up to a total dose of 1000 mGy have been generated for photons from 125I decay (approximately 30 keV) delivered at doses rates of 0.19, 0.47, 0.91 and 1.9 mGy/min. The results indicate that at dose rates of 1.9 and 0.91 mGy/min the slope of the induction curve is about 1.5 times less than that measured at high dose rate in previous studies with a similar quality of radiation (28 kVp mammographic energy X rays). In the dose region of 0 to 100 mGy, the data were equally well fitted by a threshold or linear no-threshold model. At dose rates of 0.19 and 0.47 mGy/min there was no induction of transformation even at doses up to 1000 mGy, and there was evidence for a possible suppressive effect. These results show that for this in vitro end point the DDREF is very dependent on dose rate and at very low doses and dose rates approaches infinity. The relative risks for the in vitro data compare well with those from epidemiological studies of breast cancer induction by low- and high-dose-rate radiation.

Low doses of very low-dose-rate low-LET radiation suppress radiation-induced neoplastic transformation in vitro and induce an adaptive response

Abstract Elmore, E., Lao, X-Y., Kapadia, R., Giedzinski, E., Limoli, C. and Redpath, J. L. Low Doses of Very Low-Dose-Rate Low-LET Radiation Suppress Radiation-Induced Neoplastic Transformation In Vitro and Induce an Adaptive Response. Radiat. Res. 169, 311–318 (2008). The purpose of this study was to determine whether adaptation against neoplastic transformation could be induced by exposure to very low-dose-rate low-LET radiation. HeLa × skin fibroblast human hybrid cells were irradiated with ∼30 kVp photons from an array of 125I seeds. The initial dose rate was 4 mGy/day. Cell samples were taken at four intervals at various times over a period of 88 days and assayed for neoplastic transformation and the presence of reactive oxygen species (ROS). The dose rate at the end of this treatment period was 1.4 mGy/day. Transformation frequencies and ROS levels were compared to those of parallel unirradiated controls. At the end of 3 months and an accumulated dose of 216 mGy, cells treated with very low-dose-rate radiation were exposed to a high-dose-rate 3-Gy challenge dose of 137Cs γ rays, and the effects compared with the effect of 3 Gy on a parallel culture of previously unirradiated cells. Cells exposed to very low-dose-rate radiation exhibited a trend toward a reduction in neoplastic transformation frequency compared to the unirradiated controls. This reduction seemed to diminish with time, indicating that the dose rate, rather than accumulated dose, may be the more important factor in eliciting an adaptive response. This pattern was in general paralleled by a reduction of ROS present in the irradiated cultures compared to controls. The very low-dose-rate-treated cells were less sensitive to the high challenge dose than unirradiated controls, suggesting the induction of an adaptive response. Since there was a suggestion of a dose-rate threshold for induction suppression, a second experiment was run with a fresh batch of cells at an initial dose rate of 1 mGy/day. These cells were allowed to accumulate 40 mGy over 46 days (average dose rate = 0.87 mGy/day), and there was no evidence for suppression of transformation frequency compared to parallel unirradiated controls. It is concluded that doses of less than 100 mGy delivered at very low dose rates in the range 1 to 4 mGy/day can induce an adaptive response against neoplastic transformation in vitro. When the dose rate drops below ∼1 mGy/day, this suppression is apparently lost, suggesting a possible dose-rate-dependent threshold for this process.

Neoplastic Transformation In Vitro after Exposure to Low Doses of Mammographic-Energy X Rays: Quantitative and Mechanistic Aspects

Abstract Ko, S. J., Liao, X-Y., Molloi, S., Elmore, E. and Redpath, J. L. Neoplastic Transformation In Vitro after Exposure to Low Doses of Mammographic-Energy X Rays: Quantitative and Mechanistic Aspects. Radiat. Res. 162, 646–654 (2004). The induction of neoplastic transformation in vitro after exposure of HeLa × skin fibroblast hybrid cells to low doses of mammography-energy (28 kVp) X rays has been studied. The data indicate no evidence of an increase in transformation frequency over the range 0.05 to 22 cGy, and doses in the range 0.05 to 1.1 cGy may result in suppression of transformation frequencies to levels below that seen spontaneously. This finding is not consistent with a linear, no-threshold dose– response curve. The dose range at which possible suppression is evident includes doses typically experienced in mammographic examination of the human breast. Experiments are described that attempt to elucidate any possible role of bystander effects in modulating this low-dose radiation response. Not unexpectedly, inhibition of gap junction intercellular communication (GJIC) with the inhibitor lindane did not result in any significant alteration of transformation frequencies seen at doses of 0.27 or 5.4 cGy in these subconfluent cultures. Furthermore, no evidence of a bystander effect associated with factors secreted into the extracellular medium was seen in medium transfer experiments. Thus, in this system and under the experimental conditions used, bystander effects would not appear to be playing a major role in modulating the shape of the dose–response curve.

Further Studies on the Radiation-Induced Expression of a Tumor-Specific Antigen in Human Cell Hybrids

The neoplastic transformation of human cell hybrids (HeLa x skin fibroblasts) is accompanied by the expression of a cell surface protein for which monoclonal antibodies have been raised. The gamma-radiation-induced neoplastic transformation of these cells has been studied where the expression of this cell surface protein, as detected by immunoperoxidase staining, has been used as an end point. The yield of foci of positively staining cells has been shown to increase with increasing time postirradiation at which the assay is done and decrease with increasing density of viable cells plated postirradiation. The time of plating postirradiation is also an important parameter with transformation frequencies increasing over the first 6 h of postirradiation holding at confluence, followed by a gradual decrease.

Delayed heritable damage and epigenetics in radiation-induced neoplastic transformation of human hybrid cells.

The HeLa x skin fibroblast human hybrid cell system has proven to be an excellent model system for quantitative studies of radiation-induced neoplastic transformation in vitro. A unique aspect of this system is the reexpression of a cell surface protein p75/150 with tumorigenicity. The identification of p75/150 as intestinal alkaline phosphatase (IAP) allowed for the recent development of a more simplified, rapid, and sensitive screening method than the previous p75/150 antibody-based staining procedure. The new method directly detects neoplastically transformed, IAP-expressing cells by staining with the alkaline phosphatase chromogenic substrate, Western Blue (WB). Earlier studies with the antibody-based immunoperoxidase assay indicated that, while no foci with tumor-associated antigen (p75-positive) were evident 15 days after irradiation, the number of foci rose quickly and leveled off between Day 19 and Day 23. This late appearance of the IAP-positive foci suggested that the neoplastic transformation process was not an immediate consequence of radiation damage. The mechanism underlying this observation was unknown. The possibility existed that very small foci and/or foci expressing a low level of IAP were being missed at earlier expression times. The increased sensitivity of the WB staining technique has allowed for the reinvestigation of the kinetics of induction of radiation-induced foci in this system. Experiments were performed where parallel groups of transformation flasks were stained at Days 7, 9, 11, 13, 15, 17, 19, and 21 days after irradiation. The data clearly indicate that the radiation induction of IAP-positive foci is indeed delayed in this system with the vast majority of the foci beginning to appear after Day 9 after irradiation. The delay is not the result of a lack of ability to detect small IAP-positive foci since foci with as few as 15 IAP-positive cells were discernible. We have reported previously that under identical experimental conditions both the establishment of plateau phase and the onset of the expression of lethal mutations also occur after Day 9. We therefore propose that radiation-induced neoplastic transformation of HeLa x skin fibroblast hybrid cells is a consequence of the delayed expression of heritable damage under epigenetic control with a resultant loss of tumor-suppressor function.

Plating efficiency as a function of time postirradiation: evidence for the delayed expression of lethal mutations.

The plating efficiency (PE) of a gamma-irradiated (7 Gy) human cell hybrid line (HeLa X skin fibroblast, designated as CGL1) has been measured as a function of time postirradiation and compared to that of unirradiated cells at similar cell densities and under the same growth conditions. The results indicate that following irradiation, the PE of the irradiated cells initially increases but never returns to that of unirradiated cells during the experimental period that we have examined. Furthermore, after a period of 9 to 10 days (equivalent to at least 10 cell doublings) postirradiation and plating, the PE of the irradiated cells begins to decrease and continues to do so over the next 5 days. A decrease does not occur in unirradiated cells until much later (i.e., Day 15) corresponding to at least 5 additional cell doublings. The data are discussed in terms of a delayed expression of lethal mutations. The possible impact of these observations on the estimation of radiation-induced transformation frequencies is also considered.

Neoplastic transformation of human hybrid cells by gamma radiation: a quantitative assay.

Human skin fibroblasts are extremely refractory to neoplastic transformation by ionizing radiation [C. Borek, Nature 283, 776-778 (1980); M. Namba, H. Nishitani, and T. Kimoto, J. Exp. Med. 48, 303-311 (1978)] and are therefore unsuitable for quantitative studies of dose-effect relationships. We show here that a nontumorigenic human hybrid cell line (HeLa X skin fibroblast) can be neoplastically transformed by treatment with gamma radiation. Furthermore, a dose-response relationship has been established. We propose that this human hybrid cell line may be a useful system for mechanistic studies of transformation from the preneoplastic to the neoplastic state by ionizing radiation and other agents.

Neoplastic transformation in vitro induced by low doses of 232 MeV protons

The aim was to define the dose – response curve for high-energy proton-induced neoplastic transformation in vitro. The HeLa × skin fibroblast human hybrid cell assay was used to determine the frequency of neoplastic transformation following doses of 232 MeV protons (mean linear energy transfer, LET = 0.44 keV μm – 1) in the range 5 – 600 mGy. Proton irradiations were carried out at the Loma Linda University Proton Treatment Facility, CA, USA. The data indicate no evidence for induction of transformation below a dose of 100 mGy. At doses of 5 and 50 mGy, there is evidence for a possible suppression of transformation frequencies below that for spontaneous transformation. The shape of the dose – response curve for high-energy proton-induced transformation of the human hybrid cell line CGL1 does not follow a linear no-threshold model and shows evidence for a threshold as well as for possible suppression of transformation at doses < 100 mGy, similar to that seen for other low-LET radiations.

High and low dose rate irradiation have opposing effects on cytokine gene expression in human glioblastoma cell lines

Effects of radiation on five cytokine expressing human glioblastoma cell lines were studied. In comparison to unirradiated controls, IL-1 beta and IL-6 mRNAs were generally reduced after low (LDR, 1.0 cGy/min) and very low (VLDR, 0.35 cGy/min) dose rate irradiation. In contrast, high (HDR, 200 cGy/min) and intermediate (IDR, 4.1 cGy/min) dose rates increased steady-state levels of IL-1 beta and IL-6 mRNAs. The surviving fraction was generally inversely proportional to the dose rate; however, these glioma cells were unusually susceptible to LDR. In the two cell lines tested, IDR was less cytotoxic than either HDR or LDR irradiation. Although cytokine gene expression had no clear effect on radiation survival in vitro, autologous cytokines could be important to radiation response in vivo by affecting immune response, tumour stroma, vasculature or surrounding tissues. Adjusting dose rates to account for inverse dose rate effects and altered gene expression may be a useful strategy in optimising radiation therapy of glioblastomas.