Radiation-induced Neoplastic Transformation Research Articles

Genomic Loss and Epigenetic Silencing of the FOSL1 Tumor Suppressor Gene in Radiation-induced Neoplastic Transformation of Human CGL1 Cells Alters the Tumorigenic Phenotype In Vitro and In Vivo.

The CGL1 human hybrid cell system has been utilized for many decades as an excellent cellular tool for investigating neoplastic transformation. Substantial work has been done previously implicating genetic factors related to chromosome 11 to the alteration of tumorigenic phenotype in CGL1 cells. This includes candidate tumor suppressor gene FOSL1, a member of the AP-1 transcription factor complex which encodes for protein FRA1. Here we present novel evidence supporting the role of FOSL1 in the suppression of tumorigenicity in segregants of the CGL1 system. Gamma-induced mutant (GIM) and control (CON) cells were isolated from 7 Gy gamma-irradiated CGL1s. Western, Southern and Northern blot analysis were utilized to assess FOSL1/FRA1 expression as well as methylation studies. GIMs were transfected to re-express FRA1 and in vivo tumorigenicity studies were conducted. Global transcriptomic microarray and RT-qPCR analysis were used to further characterize these unique cell segregants. GIMs were found to be tumorigenic in vivo when injected into nude mice whereas CON cells were not. GIMs show loss of Fosl/FRA1 expression as confirmed by Western blot. Southern and Northern blot analysis further reveals that FRA1 reduction in tumorigenic CGL1 segregants is likely due to transcriptional suppression. Results suggest that radiation-induced neoplastic transformation of CGL1 is in part due to silencing of the FOSL1 tumor suppressor gene promoter by methylation. The radiation-induced tumorigenic GIMs transfected to re-express FRA1 resulted in suppression of subcutaneous tumor growth in nude mice in vivo. Global microarray analysis and RT-qPCR validation elucidated several hundred differentially expressed genes. Downstream analysis reveals a significant number of altered pathways and enriched Gene Ontology terms genes related to cellular adhesion, proliferation, and migration. Together these findings provide strong evidence that FRA1 is a tumor suppressor gene deleted and epigenetically silenced after ionizing radiation-induced neoplastic transformation in the CGL1 human hybrid cell system.

Modeling Radiation-Induced Neoplastic Cell Transformation In Vitro and Tumor Induction In Vivo with the Local Effect Model.

Ionizing radiation induces DNA damage to cycling cells which, if left unrepaired or misrepaired, can cause cell inactivation or heritable, viable mutations. The latter can lead to cell transformation, which is thought to be an initial step of cancer formation. Consequently, the study of radiation-induced cell transformation promises to offer insights into the general properties of radiation carcinogenesis. As for other end points, the effectiveness in inducing cell transformation is elevated for radiation qualities with high linear energy transfer (LET), and the same is true for cancer induction. In considering DNA damage as a common cause of both cell death and transformations, a worthwhile approach is to apply mathematical models for the relative biological effectiveness (RBE) of cell killing to also assess the carcinogenic potential of high-LET radiation. In this work we used an established RBE model for cell survival and clinical end points, the local effect model (LEM), to estimate the transformation probability and the carcinogenic potential of ion radiation. The provided method consists of accounting for the competing processes of cell inactivation and induction of transformations or carcinogenic events after radiation exposure by a dual use of the LEM. Correlations between both processes inferred by the number of particle impacts to individual cells were considered by summing over the distribution of hits that individual cells receive. RBE values for cell transformation in vitro were simulated for three independent data sets, which were also used to gauge the approach. The simulations reflect the general RBE systematics both in magnitude and in energy and LET dependence. To challenge the developed method, in vivo carcinogenesis was investigated using the same concepts, where the probability for cancer induction within an irradiated organ was derived from the probability of finding carcinogenic events in individual cells. The predictions were compared with experimental data of carcinogenesis in Harderian glands of mice. Again, the developed method shows the same characteristics as the experimental data. We conclude that the presented method is helpful to predictively assess RBE for both neoplastic cell transformation and tumor induction after ion exposure within a wide range of LET values. The theoretical concept requires a non-linear component in the photon dose response for carcinogenic end points as a precondition for the observed enhanced effects after ion exposure, thus contributing to a long debate in epidemiology. Future work will use the method for assessing cancer induction in radiation therapy and exposure scenarios frequently discussed in radiation protection.

Radiation-Induced Malignant Transformation of Preneoplastic and Normal Breast Primary Epithelial Cells

Radiation is used in multiple procedures as a therapeutic and diagnostic tool. However, ionizing radiation can induce mutations in the DNA of irradiated cells, which can promote tumorigenesis. As malignant transformation is a process that takes many years, there are intermediate stages of cells that have initiated the process but have not yet evolved into cancer. The study here aimed to investigate the effect of ionizing radiation on normal and partially transformed human mammary epithelial cells. Breast primary epithelial cells were derived from normal breast tissue from two different donors and modified by transduction with the SV40 small and large T antigen and hTERT genes to obtain partially transformed cells and also with HRAS to completely and experimentally transform them. After exposure to different doses of ionizing radiation, oncogenic features were analyzed by means of an anchorage-independent growth assay and 3D cell culture. The addition of radiation exposure resulted in an increase in the number and size of colonies formed in each of the conditions analyzed and in the reduction of the capacity of partially transformed cells to form properly polarized 3D structures. Moreover, partially transformed cells require lower doses of radiation than healthy cells to enhance anchorage-independent growth capacity. Although cells from different donors have a different degree of sensitivity in the response to radiation, a higher sensitivity to the radiation-induced cell transformation process was observed in those cells that had already initiated the oncogenic process, which require higher doses of radiation to complete the transformation process. IMPLICATIONS: Individuals carrying accumulation of genetic alterations may have an increased susceptibility to radiation-induced neoplastic transformation.

Abstract 4368: Vitamin E and the green tea active agent EGCG suppress radiation-induced carcinogenesis by different molecular mechanisms

Abstract Treatment induced secondary malignancies after successful radiation and/or chemotherapy cure of primary tumors is a growing area of concern. The identification of chemopreventive compounds that reduce radiation-induced secondary malignancy but are both nontoxic and tolerated with long-term use is a critical need. We investigated the chemopreventive potential and mechanism of action of Vitamin E and EGCG (the active agent in green tea) with the human CGL1 radiation neoplastic transformation assay. The molecularly characterized CGL1 assay allows both alterations of the quantitative neoplastic transformation as well the potential underlying molecular processes of the chemoprevention to be determined. We found long-term treatment with 15 microM EGCG or 50 microM Vitamin E beginning 72 hours after 7 Gy irradiation, significantly reduce radiation-induced neoplastic transformation frequency by a factor of 2.3. We determined that Vit E suppressed radiation-induced carcinogenesis by the induction of a p53 and pro-apoptotic Bax dependent apoptosis in the progeny of the irradiated cells. In addition, we demonstrated that EGCG does not reduce radiation-induced carcinogenesis by increased apoptosis, but rather by the onset of p16 dependent senescence in the irradiated CGL1 progeny. Vit E and EGCG are excellent candidate nontoxic chemopreventive agents for radiation-induced carcinogenesis that work by two distinct molecular mechanisms. We propose that this information should also aid in the development of next generation chemopreventive compounds. These studies were supported by a grant from the DOD awarded to MSM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4368.

Abstract A36: Combinatorial attack on radiation-induced neoplastic transformation by phenylbutyrate: Involvement of Hsp90 molecular chaperone and histone deacetylase

Abstract A36 Phenyl fatty acids like phenylacetate (PA) and phenylbutyrate (PB) have shown anti-cancer efficacy in clinical trials and in both in vitro and in vivo models. The mechanism of action of these agents is not fully understood however. In vitro studies have demonstrated that PB treatment induces apoptosis and growth rate effects in a time- and dose- dependent manner in the p21ras-expressing hematopoietic cells, Ras-DC and the human chronic myeloid leukemia blast crisis cells, (CML-BC) K562 cells. Both leukemia cell types are characterized by deregulated activity of the oncoprotein tyrosine kinase known as Bcr-Abl frequently observed in radiation-associated leukemias. Molecular studies are underway to characterize the effect of PB on Bcr-Abl expression in these leukemia cells since Bcr-Abl is a well known client protein for Hsp90. Furthermore, gene expression profile analysis of PB treatment of leukemia cells and human osteoblast cells (HOS) demonstrated that PB down-regulated Hsp90 suggesting that PB may affect molecular chaperoning. The anti-leukemic activity of PB was investigated using the cobalt radiation-induced neoplastic transformation model, HOS cells. HOS cells were exposed to a total of 2 Gy (60Co, 50 cGy/exposure) and transformed foci were formed within 35 days. Cellular incubation with PB either pre- or post-radiation significantly suppressed the radiation-induced transformation. Molecular studies also showed that PB induced acetylation of histones H3 and H4 and apoptosis in this cell model. A radiation leukemia rodent model is currently being employed to evaluate PB’s anti-leukemia efficacy. Although preliminary, these data suggest that PB involvement in both Hsp90 molecular chaperoning and inhibition of histone deacetylase is mechanistically related to its anti-leukemia efficacy. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A36.

Low Doses of Very Low-Dose-Rate Low-LET Radiation Suppress Radiation-Induced Neoplastic TransformationIn Vitroand Induce an Adaptive Response

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 x skin fibroblast human hybrid cells were irradiated with approximately 30 kVp photons from an array of (125)I 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 (137)Cs gamma 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 approximately 1 mGy/day, this suppression is apparently lost, suggesting a possible dose-rate-dependent threshold for this process.

Radiation-Induced Neoplastic Transformation and Cancer Risk Assessment at Low Doses of Low-LET Radiation

Epidemiologic studies of cancer incidence are insensitive at low doses (<10 cGy). For this reason it is necessary to turn to relevant experimental systems in order to shed light on what may be happening regarding cancer risk at these low doses. In terms of in vitro studies, a cancer-relevant endpoint is neoplastic transformation. Over the past 10 years we have investigated the effects of a range of doses for a variety of sources of low-LET radiation with the goal of determining the shape of the dose-response curve, and underlying biologic responses, at low doses. The first conclusion we have arrived at is the dose-response for high dose-rate radiation is not linear but J-shaped. The transformation frequency only begins to rise above background at doses above 10 to 20 cGy. Secondly, we have evidence that the low dose suppression is most likely a consequence of multiple mechanisms (upregulation of DNA repair and antioxidant status as well as hyper-radio-sensitivity of cells destined to become neoplastically transformed). Thirdly, we have evidence that this suppression of transformation also occurs for radiation delivered at low (mGy/min) to very low (mGy/day) dose-rates. In addition, we have shown that exposure to very low dose-rate radiation (mGy/day) over 2 to 3 months confers a resistance to neoplastic transformation by a subsequent challenge dose, i.e. an adaptive response as classically defined. It would therefore appear that low doses of low-LET radiation mount responses that protect cell populations against neoplastic transformation in vitro. Clearly, in vitro systems have limitations compared the in vivo situation, nonetheless, they have historically given comparable conclusions at high radiation doses to those seen in vivo in terms of the effect of dose-fractionation, dose-rate, LET and chemical modifiers. A priori, there is therefore no reason to doubt that the same will be true at low doses. Indeed, there are several in vivo studies that would support this contention. Additionally, the relative risk for neoplastic transformation determined from our studies agrees well with those for cancer incidence from radiation epidemiology in the case of breast cancer and leukemia. Based on the above, and other extensive evidence in the literature, it is unlikely that cancer risk from low-LET radiation has a linear-no-threshold dose-response and that threshold, or even hormetic, responses are more probable. Acknowledgement. Supported by the U.S. Department of Energy Low Dose Radiation Research Program.

Radiation-Induced Neoplastic Transformation in vitro, Hormesis and Risk Assessment

Dose-response curves for various low-LET radiation sources have consistently been demonstrated to be J-shaped for the cancer-relevant endpoint of neoplastic transformation in vitro. Most of these studies have been performed where the radiation has been delivered at intermediate to high dose-rates (30-3000 mGy/min), where the threshold dose for induction of neoplastic transformation is around 100-200 mGy. Below these doses, the transformation frequency is less than that seen spontaneously, indicative of a hormetic effect. More recently, data have been obtained for low dose rates (<0.5 mGy/min) of low-LET radiation, and again hormetic effects are apparent but with threshold doses now being >1000 mGy. Similar trends have been reported in animal experiments as well as in human epidemiologic studies. Indeed, the relative risks for induction of neoplastic transformation in vitro in the dose range 1 to 1000 mGy agree well with those for incidence of radiation-induced breast cancer and leukemia in humans. These findings support the notion that the endpoint of neoplastic transformation in vitro is a plausible endpoint to not only study mechanisms involved in response to low doses of radiation, but also to provide information of potential importance to risk assessment.

Mechanistic modelling of genetic and epigenetic events in radiation carcinogenesis

Methodological problems arise on the way of radiation carcinogenesis modelling with the incorporation of radiobiological and cancer biology mechanistic data. The results of biophysical modelling of different endpoints [DNA DSB induction, repair, chromosome aberrations (CA) and cell proliferation] are presented and applied to the analysis of RBE-LET relationships for radiation-induced neoplastic transformation (RINT) of C3H/10T1/2 cells in culture. Predicted values for some endpoints correlate well with the data. It is concluded that slowly repaired DSB clusters, as well as some kind of CA, may be initiating events for RINT. As an alternative interpretation, it is possible that DNA damage can induce RINT indirectly via epigenetic process. A hypothetical epigenetic pathway for RINT is discussed.

The Effect of Dose Rate on Radiation-Induced Neoplastic TransformationIn Vitroby Low Doses of Low-LET Radiation

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 (125)I 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.

Health risks of low photon energy imaging

The major health risk associated with low photon energy imaging is thought to be the induction of cancer as a consequence of the radiation exposure and this is the focus of this paper. Low photon energy imaging typically involves exposure to a low dose (<50 mGy) of low linear energy transfer (LET) radiation delivered at high dose-rate. Since epidemiologic data cannot provide an accurate assessment of risk at the doses used in imaging, risk estimates are currently made by fitting a linear response to intermediate and high dose data for cancer induction in radiation-exposed human populations. This method assumes a linear no-threshold (LNT) response and implies that no dose of radiation is safe. This assumption is not borne out by many laboratory studies of cancer-related endpoints that would suggest that the risk at low doses is much less than would be estimated from linear extrapolation from intermediate to high doses. It is also well recognised that the dose-response from many epidemiologic studies could equally well be fit by threshold models. Through the study of radiation-induced neoplastic transformation in vitro J-shaped dose-response curves for a variety of low LET radiations, including those used in low photon energy imaging, have been demonstrated. The relative risks calculated from this data compare remarkably well with those for breast cancer and leukemia incidence in radiation-exposed populations. From this it is concluded that the LNT hypothesis is likely to overestimate the risk of cancer induction by low photon energy imaging, at least for certain tumors.

Suppression of Neoplastic Transformation in vitro by Low Doses of Low Let Radiation

A major concern of exposure to low doses of radiation is the risk of cancer induction. Epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <10 cGy. In order to gain insight into events at these low doses, laboratory-based studies of relevant endpoints are required. One such endpoint is radiation-induced neoplastic transformation in vitro. Such studies can provide quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. Data are presented that indicate that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below those seen spontaneously. Mechanisms involved include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contributions of these mechanisms is dose-dependent. The relevance of these observations to radiation risk estimation is discussed.

Nonlinear Response for Neoplastic Transformation following Low Doses of Low Let Radiation

There are now several independent studies that indicate that the dose-response for the endpoint of radiation-induced neoplastic transformation in vitro is non-linear for low linear energy transfer (LET) radiation. At low doses (<10 cGy) the transformation frequency drops below that seen spontaneously. Importantly, this observation has been made using fluoroscopic energy x-rays, a commonly used modality in diagnostic radiology, the practice of which is responsible for the majority of radiation exposure to the general public. Since the transformation frequency is reduced over a large dose range (0.1 to 10cGy) it is likely that multiple mechanisms are involved and that the relative contribution of these may vary with dose. These include the killing of a subpopulation of cells prone to spontaneous transformation at the lowest doses, and the induction of DNA repair at somewhat higher doses. Protective effects of low doses of low LET radiation on other cancer-relevant endpoints in vitro and in vivo have also been observed by several independent laboratories. These observations strongly suggest that the linear-nonthreshold dose-response model is unlikely to apply to the induction of cancer by low doses of low LET radiation in humans.

Radiation-induced neoplastic transformation in vitro: evidence for a protective effect at low doses of low LET radiation.

The study of radiation-induced transformation in vitro has long been an experimental approach to examine mechanisms underlying radiation carcinogenesis. Even though the major concern of exposure to radiation is the risk of cancer induction at low radiation doses, most laboratory mechanistic studies have focused on high dose effects. This, coupled with the fact that epidemiologic data are rarely powerful enough to accurately discriminate this risk at doses <5 cGy, has led in recent years to an increased effort to study low dose effects using the endpoint of neoplastic transformation in vitro. Since transformation frequencies at low doses are typically low (< 10(-4)), such studies are, by necessity, large and labor intensive. However, they have yielded quantitative dose-response data, as well as insights into underlying cellular and molecular mechanisms. An interesting, and potentially important, finding is that low doses of low LET radiation can suppress neoplastic transformation in vitro to levels below that seen spontaneously. Mechanistic studies have revealed that multiple mechanisms are likely to be involved, and these include both the death of a subpopulation of cells prone to spontaneous neoplastic transformation and the induction of DNA repair. The relative contribution of these mechanisms appears to be dose-dependent. The relevance of in vitro studies to carcinogenesis in vivo is discussed.

Role of Vitamin D receptor gene in radiation-induced neoplastic transformation of human breast epithelial cell

1α,25-(OH) 2-Vitamin D 3, the physiologically active metabolite of Vitamin D is known for its pro-differentiating and antiproliferative activity on various cancer cell lines. It exerts its growth-regulatory effects through binding to the Vitamin D recepter (VDR), a member of the steroid/thyroid/retinoic acid receptor family, which functions as a ligand-dependent transcription factor. There is accumulating evidence that Vitamin D may be an important determinant of both the occurrence and progression of breast cancer. Since radiation is an important etiological factor for breast cancer progression, it is important to study the role of VDR gene in radiation-induced breast carcinogenesis. This study is focused on a human breast tumor model developed by irradiating the spontaneously immortalized MCF-10F cell line with graded doses of high-linear energy transfer (LET) radiation followed by treatment with estrogen. Study of VDR gene by restriction digestion with ApaI, BsmI and TaqI detected no polymorphism but direct sequencing analyses identified few single-base mutations within intron 8 and exon 9 of the gene. Over-expression of the VDR gene was noticed in irradiated and tumorigenic cell lines compared with control. Likewise, immunohistochemical data indicated a significant increase in VDR intensity in irradiated and tumorigenic cell lines. Considering all these evidence, it is likely that VDR can be used as a prognostic marker of tumor progression in radiation- and estrogen-induced breast carcinogenesis.

The shape of the dose-response curve for radiation-induced neoplastic transformation in vitro: evidence for an adaptive response against neoplastic transformation at low doses of low-LET radiation.

A dose-response curve for gamma-radiation-induced neoplastic transformation of HeLa x skin fibroblast human hybrid cells over the dose range 0.1 cGy to 1 Gy is presented. In the experimental protocol used, the spontaneous (background) frequency of neoplastic transformation of sham-irradiated cultures was compared to that of cultures which had been irradiated with (137)Cs gamma radiation and either plated immediately or held for 24 h at 37 degrees C prior to plating, for assay for neoplastic transformation. The pooled data from a minimum of three repeat large-scale experiments at each dose demonstrated a reduced transformation frequency for the irradiated compared to the sham-irradiated cells for doses of 0.1, 0.5, 1, 5 and 10 cGy for the delayed-plating arm. The probability of this happening by chance is given by 1/2(n), where n is the number of observations (5); i.e., 1/32 congruent with 0.031. This is indicative of an adaptive response against spontaneous neoplastic transformation at least up to a dose of 10 cGy of gamma radiation. The high-dose data obtained at 30 and 50 cGy and 1 Gy showed a good fit to a linear extrapolation through the sham-irradiated, zero-dose control. The delayed-plating data at 10 cGy and below showed a statistically significant divergence from this linear extrapolation.

Frequent allelic imbalance on chromosome 6 and 17 correlate with radiation-induced neoplastic transformation of human breast epithelial cells.

The development of human breast cancer is a complex multi-step process that depends on various exogenous and endogenous factors that modulate the transformation of normal human breast epithelial cells into neoplastic ones. Using a spontaneously-immortalized human breast epithelial (MCF-10F) cell line, we have shown previously that radiation, in combination with estrogen, induces a stepwise neoplastic transformation of this cell line. In the present study, we investigate the incidence of microsatellite instability and loss of heterozygosity using a battery of markers on chromosomes 6 and 17, we correlate the genetic alteration with the malignant transformation of the MCF-10F cell line ranging from altered morphology to increase in proliferative rate, anchorage independent growth and tumorigenicity in nude mice. Microsatellite markers were selected from the hot spot regions (6q21-q27, 17p12-p13.3 and 17q12-q21) of both chromosomes. We found that the frequency of allelic imbalance occurs at the different stages of tumor progression with a range of 21 to 50% depending on the marker studied. The relatively high rate of allele imbalance at all these loci suggests the presence and inactivation of one or more tumor suppressor genes in these regions. Thus, the present data will be useful for systematic studies to identify the cellular and molecular changes associated with radiation-induced breast carcinogenesis.

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects.

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence alpha particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 +/- 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 x 10(-6) in unirradiated controls to 2.3 x 10(-5) (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.

Clonal chromosomal aberrations in simian virus 40‐transfected human thyroid cells and in derived tumors developed after in vitro irradiation

In vitro model cell systems are important tools for studying mechanisms of radiation-induced neoplastic transformation of human epithelial cells. In our study, the human thyroid epithelial cell line HTori-3 was analyzed cytogenetically following exposure to different doses of α- and γ-irradiation and subsequent tumor formation in athymic nude mice. Combining results from G-banding, comparative genomic hybridization, and spectral karyotyping, chromosome abnormalities could be depicted in the parental line HTori-3 and in nine different HTori lines established from the developed tumors. A number of chromosomal aberrations were found to be characteristic for simian virus 40 immortalization and/or radiation-induced transformation of human thyroid epithelial cells. Common chromosomal changes in cell lines originating from different irradiation experiments were loss of 8q23 and 13cen-q21 as well as gain of 1q32-qter and 2q11.2-q14.1. By comparison of chromosomal aberrations in cell lines exhibiting a different tumorigenic behavior, cytogenetic markers important for the tumorigenic process were studied. It appeared that deletions on chromosomes 9q32-q34 and 7q21-q31 as well as an increased copy number of chromosome 20 were important for the tumorigenic phenotype. A comparative breakpoint analysis of the marker chromosomes found and those observed in radiation-induced childhood thyroid tumors from Belarus revealed a coincidence for a number of chromosome bands. Thus, the data support the usefulness of the established cell system as an in vitro model to study important steps during radiation-induced malignant transformation in human thyroid cells. © 2001 Wiley-Liss, Inc.

Clonal chromosomal aberrations in simian virus 40-transfected human thyroid cells and in derived tumors developed after in vitro irradiation.

In vitro model cell systems are important tools for studying mechanisms of radiation-induced neoplastic transformation of human epithelial cells. In our study, the human thyroid epithelial cell line HTori-3 was analyzed cytogenetically following exposure to different doses of alpha- and gamma-irradiation and subsequent tumor formation in athymic nude mice. Combining results from G-banding, comparative genomic hybridization, and spectral karyotyping, chromosome abnormalities could be depicted in the parental line HTori-3 and in nine different HTori lines established from the developed tumors. A number of chromosomal aberrations were found to be characteristic for simian virus 40 immortalization and/or radiation-induced transformation of human thyroid epithelial cells. Common chromosomal changes in cell lines originating from different irradiation experiments were loss of 8q23 and 13cen-q21 as well as gain of 1q32-qter and 2q11.2-q14.1. By comparison of chromosomal aberrations in cell lines exhibiting a different tumorigenic behavior, cytogenetic markers important for the tumorigenic process were studied. It appeared that deletions on chromosomes 9q32-q34 and 7q21-q31 as well as an increased copy number of chromosome 20 were important for the tumorigenic phenotype. A comparative breakpoint analysis of the marker chromosomes found and those observed in radiation-induced childhood thyroid tumors from Belarus revealed a coincidence for a number of chromosome bands. Thus, the data support the usefulness of the established cell system as an in vitro model to study important steps during radiation-induced malignant transformation in human thyroid cells.