Neoplastic transformation in vitro by mixed beams of high-energy iron ions and protons.

The radiation environment in space is complex in terms of both the variety of charged particles and their dose rates. Simulation of such an environment for experimental studies is technically very difficult. However, with the variety of beams available at the National Space Research Laboratory (NSRL) at Brookhaven National Laboratory (BNL) it is possible to ask questions about potential interactions of these radiations. In this study, the end point examined was transformation in vitro from a preneoplastic to a neoplastic phenotype. The effects of 1 GeV/n iron ions and 1 GeV/n protons alone provided strong evidence for suppression of transformation at doses ≤5 cGy. These ions were also studied in combination in so-called mixed-beam experiments. The specific protocols were a low dose (10 cGy) of protons followed after either 5-15 min (immediate) or 16-24 h (delayed) by 1 Gy of iron ions and a low dose (10 cGy) of iron ions followed after either 5-15 min or 16-24 h by 1 Gy of protons. Within experimental error the results indicated an additive interaction under all conditions with no evidence of an adaptive response, with the one possible exception of 10 cGy iron ions followed immediately by 1 Gy protons. A similar challenge dose protocol was also used in single-beam studies to test for adaptive responses induced by 232 MeV/n protons and (137)Cs γ radiation and, contrary to expectations, none were observed. However, subsequent tests of 10 cGy of (137)Cs γ radiation followed after either 5-15 min or 8 h by 1 Gy of (137)Cs γ radiation did demonstrate an adaptive response at 8 h, pointing out the importance of the interval between adapting and challenge dose. Furthermore, the dose-response data for each ion alone indicate that the initial adapting dose of 10 cGy used in the mixed-beam setting may have been too high to see any potential adaptive response.

Threshold-type dose response for induction of neoplastic transformation by 1 GeV/nucleon iron ions.

Neoplastic transformation of HeLa x skin fibroblast human hybrid cells by doses of 1 GeV/nucleon iron ions in the range 1 cGy to 1 Gy to exposed cultures has been examined. The data indicate a threshold-type dose-response curve with no increase in transformation frequency until doses above 20 cGy. At doses <10 cGy, not all exposed cells receive a direct traversal of an iron-ion track core, but all exposed cells receive up to several mGy of low-LET radiation associated with the delta-ray penumbra. It is proposed that the threshold-type response seen is a consequence of an adaptive response associated with the delta-ray exposure. For comparison purposes, the dose response for (137)Cs gamma rays over the same dose range was examined using the same experimental procedure. As we have shown previously, the dose response for (137)Cs gamma radiation was J-shaped. The iron ions were 1.5 to 1.7 times more biologically effective than the gamma radiation over the dose range examined.

Low doses of very low-dose-rate low-LET radiation suppress radiation-induced neoplastic transformation in vitro and 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.

Detrimental and protective bystander effects: a model approach.

This work integrates two important cellular responses to low doses, detrimental bystander effects and apoptosis-mediated protective bystander effects, into a multistage model for chromosome aberrations and in vitro neoplastic transformation: the State-Vector Model. The new models were tested on representative data sets that show supralinear or U-shaped dose responses. The original model without the new low-dose features was also tested for consistency with LNT-shaped dose responses. Reductions of in vitro neoplastic transformation frequencies below the spontaneous level have been reported after exposure of cells to low doses of low-LET radiation. In the current study, this protective effect is explained with bystander-induced apoptosis. An important data set that shows a low-dose detrimental bystander effect for chromosome aberrations was successfully fitted by additional terms within the cell initiation stage. It was found that this approach is equivalent to bystander-induced clonal expansion of initiated cells. This study is an important step toward a comprehensive model that contains all essential biological mechanisms that can influence dose-response curves at low doses.

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

Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects.

The shape of the dose-response curve for cancer induction by low doses of ionizing radiation is of critical importance to the assessment of cancer risk at such doses. Epidemiologic analyses are limited by sensitivity to doses typically greater than 50-100 mGy for low LET radiation. Laboratory studies allow for the examination of lower doses using cancer-relevant endpoints. One such endpoint is neoplastic transformation in vitro. It is known that this endpoint is responsive to both adaptive response and bystander effects. The relative balance of these processes is likely to play an important role in determining the shape of the dose-response curve at low doses. A factor that may influence this balance is cell density at time of irradiation. The findings reported in this paper indicate that the transformation suppressive effect of low doses previously seen following irradiation of sub-confluent cultures, and attributed to an adaptive response, is reduced for irradiated confluent cultures. However, even under these conditions designed to optimize the role of bystander effects the data do not fit a linear no-threshold model and are still consistent with the notion of a threshold dose for neoplastic transformation in vitro by low LET 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.

Neoplastic transformation in vitro after exposure to low doses of mammographic-energy X rays: quantitative and mechanistic aspects.

The induction of neoplastic transformation in vitro after exposure of HeLa x 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.

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.

Mechanisms of suppression of neoplastic transformation in vitro by low doses of low LET radiation.

Suppression of neoplastic transformation of HeLa x skin fibroblast human hybrid cells in vitro following low doses of low linear energy transfer radiation has been reported previously. The present study represents an exploration of two hypothesized mechanisms that may underlie this observed suppression. These are the up-regulation of reduced glutathione (GSH), a known antioxidant, and induction of DNA repair activity. The hybrid cells were found to have a high endogenous level of GSH and no induction following low doses of 60 kVp X-rays was observed. Buthionine sulfoximine (BSO), a GSH biosynthesis inhibitor, completely suppressed GSH levels in both unirradiated and irradiated cells. Furthermore, there was no significant impact of BSO-induced suppression of GSH on the neoplastic transformation frequency of either unirradiated or low dose irradiated cells indicating that glutathione levels play no role in the low dose suppression of transformation frequency. To assess the possible role of DNA repair in the low dose suppression of transformation the effect of 3-aminobenzamide (3-AB), a poly-ADP-ribose polymerase (PARP) inhibitor was examined. In these experiments, there was no significant effect of 3-AB on the transformation frequency at a dose of Cs-137 gamma rays of 0.5 cGy, however, at a dose of 5 cGy there was a significant increase (P < 0.05) in the transformation frequency in the presence of 3-AB. These findings suggest that the influence of DNA repair on the low dose suppression of transformation is significant at a dose of 5 cGy, but not at the lower dose of 0.5 cGy.

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 x 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 x skin fibroblast human hybrid cells against neoplastic transformation in vitro.

Differential effectiveness of solar UVB subcomponents in causing cell death, oncogenic transformation and micronucleus induction in human hybrid cells.

PURPOSE: (1). To determine the biological effectiveness of two solar ultraviolet (UVB) spectra with different lower wavelength thresholds for oncogenic transformation and micronucleus induction in CGL1 cells; (2). to investigate whether the action spectra for short- and long-term effects are similar; and (3). to investigate possible links between transformation and other delayed effects. MATERIALS AND METHODS: Two spectra were derived from a solar UV simulator by using two filters: the first transmitted radiation with lambda > 284 nm, the second with lambda > 293 nm. The resulting spectra have the same UVA, but different UVB components (lambda between 284 and 320 nm, 19 W m(-2), and lambda between 293 and 320 nm, 13 W m(-2)). CGL1 cells were irradiated with 466 J m(-2) with lambda > 284 nm and 1582 J m(-2) with lambda > 293 nm. These doses were approximately equilethal. The endpoints examined were oncogenic transformation, and centromere-positive and -negative micronucleus frequencies in the directly irradiated cells and in transtheir progeny. RESULTS: At equilethal doses, the oncogenic transformation frequency in the directly irradiated cells was greater by a factor of at least 7 for lambda > 284 nm irradiation compared with lambda > 293 nm. The micronucleus induction frequency was also significantly higher with the lambda > 284 spectrum. Consistent with our previous findings, no delayed micronucleus formation was found in the progeny of cells exposed to lambda > 293 nm, while a threefold elevation above controls was seen in the progeny of cells exposed to lambda > 284 nm irradiation. This was also the case for formation of micronuclei with a centromere. CONCLUSIONS: It was found that: (1). for equilethal doses the lambda > 284 nm spectrum was more biologically effective than the lambda > 293 nm spectrum for induction of oncogenic transformation and micronucleus formation; and (2). the higher effectiveness of the lambda > 284 nm spectrum found at equilethal doses for delayed effects in the progeny of irradiated cells resembles that found for transformation. The results suggest that the UVB action spectrum for cell killing is different from that of some delayed effects, and from that of transformation.

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.

Kinetics of induction of reactive oxygen species during the post-irradiation expression of neoplastic transformation in vitro.

PURPOSE: To examine whether the delayed expression of cell death and neoplastic foci in irradiated HeLa x human skin fibroblast human hybrid cells correlates with the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells. MATERIAL AND METHODS: HeLa x human skin fibroblast human hybrid cells were irradiated and plated as for assay of neoplastic transformation. At regular time intervals during the post-irradiation expression period of 21 days, samples were harvested and assessed for the presence of oxyradical activity by measuring the oxidation of dichlorohydrofluorescein (DCFH) to dichlorofluorescein (DCF) using flow cytometry. This reagent principally detects hydrogen peroxide. The kinetics of production of ROS were compared with those previously measured using the identical experimental protocol for the expression of both cell death and neoplastic foci. RESULTS: The kinetics of production of ROS closely matched those for the onset of delayed cell death and delayed expression of neoplastic transformation. CONCLUSIONS: The induction of ROS is associated with delayed cell death and delayed neoplastic transformation in irradiated HeLa x human skin fibroblast human hybrid cells.

Solar UV radiation: differential effectiveness of UVB subcomponents in causing cell death, micronucleus induction and delayed expression of heritable damage in human hybrid cells.

PURPOSE: To determine the effectiveness of two UV spectra with different UVB components for cell kill and micronucleus induction in irradiated human HeLaxskin fibroblast (CGL1) hybrid cells and their progeny. To determine the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells at various post-irradiation times and their relationship with induced delayed biological effects. MATERIAL AND METHODS: A commercial solar ultraviolet simulator was used. Two different filters were employed: the first transmitted radiation with lambda>284nm and the second radiation with lambda>293nm. The resulting spectra have different UVB components (lambda between 284 and 320nm, 19 W/m(2), and between 293 and 320nm, 13 W/m(2)) and the same UVA component (lambda between 320 and 400nm, 135 W/m(2)). CGL1 cells were irradiated with various doses. Clonogenic survival and micronucleus formation were scored in the irradiated cells and their progeny. ROS were detected by incubation of cultures at various post-irradiation times with dichlorodihydrofluorescein diacetate followed by flow cytometric measurement of the final product, dichlorofluorescein. RESULTS: The biological effectiveness of the lambda>284nm spectrum was higher by a factor of 3 compared to the lambda>293nm spectrum for cell kill, and by a factor of 5 for micronucleus induction. No delayed cell death or micronucleus formation was found in the progeny of cells exposed to lambda>293nm, while a large and dose-dependent effect was found in the progeny of cells exposed to lambda>284nm for both of these endpoints. ROS levels above those in unirradiated controls were found only in the progeny of cells exposed to the lambda>284nm spectrum. CONCLUSIONS: The spectrum with lambda>284nm was more effective than that with lambda>293nm for induction of cell kill and micronucleus formation in the directly irradiated cells as well as induction of delayed effects in the progeny in the form of delayed reproductive death and micronucleus formation. The presence of ROS in the progeny of the irradiated cells may be the cause of the delayed effects.

Chemoprevention by difluoromethylornithine: correlation of an in vitro human cell assay with human clinical data for biomarker modulation.

The efficacy of difluoromethylornithine (DFMO) as a chemopreventive agent has been tested in vitro using a human epidermal cell (HEC) assay with growth inhibition and involucrin induction as endpoints. Suppression of polyamine content is currently being utilized as a biomarker in clinical trials for the chemopreventive efficacy of DFMO against colon cancer formation. We have now examined the effects of DFMO on suppression of polyamine content in the HEC assay. The findings indicate 1) the % change in spermidine to spermine ratio and the depletion of putrescine show excellent correlation with chemopreventive efficacy in vitro; 2) the effective concentrations in vitro overlap the plasma concentrations in the clinical trial. These observations serve as further validation of the usefulness of the HEC assay as a screen for chemopreventive efficacy.

Comparative tissue-specific toxicities of 20 cancer preventive agents using cultured cells from 8 different normal human epithelia.

Comparative toxicity was determined for twenty potential chemopreventive agents in the Human Epithelial Cell Cytotoxicity (HECC) Assay using epithelial cell cultures from eight different tissues including: skin, kidney, breast, bronchus, cervix, prostate, oral cavity, and liver. The endpoints assessed were inhibition of: growth at 3 and 5 days; mitochondrial function; and proliferating cell nuclear antigen or albumin expression. Difluoromethylornithine (DFMO), s-allylcysteine, dehydroepiandrosterone (DHEA) analogue 8543, l-selenomethionine, and vitamin E acetate were not toxic or only produced mild toxicity with all endpoints in all eight cell types. N-acetyl-l-cysteine, calcium chloride, DHEA, genistein, ibuprofen, indole-3-carbinol, 4-hydroxyphenylretinamide (4-HPR), oltipraz, piroxicam, phenylethyl isothiocyanate, 9-cis-retinoic acid, and p-xylylselenocyanate each showed at least a 10-fold decrease in their TC(50) (toxic concentration that inhibited growth by 50%) for at least one endpoint with one or more cell types. For some agents such as DHEA and piroxicam, the TC(50)s for growth inhibition were 10-fold lower after 5 days compared with 3 days. Unique tissue-specific toxicity was observed for each toxic agent suggesting that tissue-specific effects are the rule rather than the exception. The HECC Assay is effective in identifying tissue-specific toxicity for chemopreventive agents and may help to identify potential toxicity problems in phase I human clinical trials.

Interstitial deletion of 11q13 sequences in HeLa cells.

Previous cytogenetic and molecular genetic studies have shown that the HeLa (cervical carcinoma) cell line D98/AH-2 contains two apparently normal copies of chromosome 11 and additional 11q13-25 material translocated onto a chromosome 3 marker. To determine the 11q13 breakpoint, we performed fluorescence in situ hybridization (FISH) using 18 different 11q13 specific BAC (bacterial artificial chromosome) and cosmid probes spanning a 5.6 Mb interval. Markers localized to the multiple endocrine neoplasia type 1 (MEN1) gene (menin) were also included in the analysis. The FISH study identified an interstitial deletion between markers D11S449 and GSTP1, an interval of 2.3 Mb, in the marker chromosome. This deletion did not include the MEN1 gene. Because point mutations and methylations can inactivate the MEN1 gene, single stranded conformational polymorphism (SSCP) and Northern and Western blot analyses were performed with MEN1 specific probes and antibody. SSCP did not reveal mutations of the MEN1 gene in HeLa or in seven other cervical cancer cell lines. Northern and Western blot studies revealed normal levels of expression of this gene in the cervical cancer cell lines as well as in HeLa cell derived tumorigenic hybrids. Because deletions of tumor suppressor genes often occur in cancer progression, we hypothesize that the inactivation of a tumor suppressor gene other than MEN1, localized to the 2.3 Mb interval on 11q13, might play a role in the abnormal growth behavior of HeLa cells in vitro or in vivo.