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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 by-stander-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 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.

Gap junctional intercellular communication (GJIC) has been implicated in homeostasis, development, differentiation, wound healing or regeneration and adaptive responses of differentiated cells. The dysfunction of homologous or heterologous GJIC has been associated with the tumorigenic phenotype. Restoration of growth control and the suppression of the tumorigenic phenotype have been previously associated with the up-regulation of GJIC by various anti-tumorigenic chemicals or transfection of connexin genes into tumor cells. To test the hypothesis that 'tumor suppressor' genes may be associated with the up-regulation of GJIC, we tested clones of tumorigenic HeLa, several non-tumorigenic HeLa-normal human fibroblast somatic cell hybrids and a tumorigenic segregant of one of the non-tumorigenic hybrids for GJIC. The parental HeLa cells (D98 AH.2) had no detectable GJIC but expressed detectable connexin 43 transcripts, while the non-tumorigenic HeLa-human fibroblast hybrids, which contained the chromosome 11 from the normal human fibroblast (CGL-1, CGL-2, ESH15 and EHS15c1), expressed ample connexin 43 transcripts and showed proficient GJIC. The tumorigenic segregant (CGL-3) from the non-tumorigenic HeLa-human fibroblast hybrid showed no GJIC or connexin 43. These results show that the presence of GJIC is closely linked to the suppression of the tumorigenic phenotype in the HeLa-human fibroblast hybrid and further suggest that GJIC may be associated with the mechanisms of tumor suppression. The mechanism by which the tumor suppressor gene(s) on the normal chromosome in the HeLa-human fibroblasts induces the up-regulation of connexin 43 is not yet explained.

Most mammalian cells exhibit transient delays in the G 1 and G 2 phases of the cell cycle after treatment with radiation or radiomimetic compounds. p53 is required for the arrest in G 1 , which provides time for DNA repair. Recently, a role of p53 in the G 2 /M transition has also been suggested. However, it has been reported that the presence of functional p53 does not always correlate with the induction of these checkpoints. To precisely assess the role of p53 in activating cell cycle checkpoints and in cell survival after radiation, we studied the response of two isogenic human fibrosarcoma cell lines differing in their p53 status (wild type or mutant). We found that when irradiated cells undergo a wild-type p53-dependent G 1 arrest, they do not subsequently arrest in G 2 . Moreover, wild-type p53 cells irradiated past the G 1 checkpoint arrest in G 2 but do not delay in the subsequent G 1 phase. Furthermore, in these cell lines, which do not undergo radiation-induced apoptosis, the wild-type p53 cell line exhibited a greater radioresistance in terms of clonogenic survival. These results suggest that the two checkpoints may be interrelated, perhaps through a control system that determines, depending on the extent of the damage, whether the cell needs to arrest cell cycle progression at the subsequent checkpoint for further repair. p53 could be a crucial component of this control system.

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.

Elmore, E., Lao, X-Y., Kapadia, R. and Redpath, J. L. Threshold-Type Dose Response for Induction of Neoplastic Transformation by 1 GeV/nucleon Iron Ions. Radiat. Res. 171, 764ndash;770 (2009). Neoplastic transformation of HeLa × 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

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.

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.

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.

(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.Two spectra were derived from a solar UV simulator by using two filters: the first transmitted radiation with lambda284 nm, the second with lambda293 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 lambda284 nm and 1582 J m(-2) with lambda293 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.At equilethal doses, the oncogenic transformation frequency in the directly irradiated cells was greater by a factor of at least 7 for lambda284 nm irradiation compared with lambda293 nm. The micronucleus induction frequency was also significantly higher with the lambda284 spectrum. Consistent with our previous findings, no delayed micronucleus formation was found in the progeny of cells exposed to lambda293 nm, while a threefold elevation above controls was seen in the progeny of cells exposed to lambda284 nm irradiation. This was also the case for formation of micronuclei with a centromere.It was found that: (1). for equilethal doses the lambda284 nm spectrum was more biologically effective than the lambda293 nm spectrum for induction of oncogenic transformation and micronucleus formation; and (2). the higher effectiveness of the lambda284 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.

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.

Decreased connexin gene expression and loss of the capacity for either homologous or heterologous intercellular communication has been associated with neoplastic transformation. We tested the hypothesis that loss of gap junctional intercellular communication (GJIC) correlates with tumorigenic potential in the HeLa x skin fibroblast human hybrid cell system. Connexin gene expression, gap junction function and tumorigenicity were determined for the non-tumorigenic somatic hybrid cell line (CGL1) and a series of UVC-induced tumorigenic cell lines derived from CGL1. CGL1 and the parental skin fibroblasts express connexin43 (alpha1 gap junction gene) mRNA and protein, form gap junctional plaques and have functional gap junctions. UVC-irradiation of CGL1 cells produced a cell line (UV12) with an aggressive tumorigenic phenotype, which lost connexin43 expression as well as both homologous and heterologous GJIC and was in this respect similar to HeLa cells. However, the phenotype of UV12 cells exhibited some instability and revertants to a less aggressive tumorigenic phenotype were isolated. These cells expressed connexin43 mRNA and protein, and demonstrated homologous GJIC. Furthermore, cells reconstituted from a tumor derived from this revertant cell line retained significant connexin43 expression and homologous GJIC, although they exhibited an aggressive tumorigenic phenotype. Thus, functional homologous GJIC cannot be dissociated from tumorigenicity in this system. However, heterologous GJIC between these same UVC-induced tumorigenic cell lines and normal human skin fibroblasts was reduced, whereas the non-tumorigenic hybrid cells showed extensive heterologous GJIC. In summary, re-acquisition of connexin43 expression and homologous GJIC does not restore the non-tumorigenic phenotype in UVC-induced tumorigenic HeLa skin fibroblast human hybrid cells. However, reduction of heterologous GJIC does correlate with tumorigenicity in this cell system.

An adaptive response against spontaneous neoplastic transformation in vitro induced by low-dose gamma radiation has recently been reported using a clone of C3H 10T1/2 cells with a predisposition toward spontaneous transformation (Azzam et al., Radiat. Res. 146, 369-370, 1996). To test the generality of this observation, the HeLa x skin fibroblast human hybrid cell system was used to look for such an adaptive response using a similar experimental protocol. In the experimental protocol used, the frequency of neoplastic transformation of unirradiated cultures (Arm A) was compared to that of cultures which had been irradiated with 1 cGy of gamma radiation and either plated immediately (Arm B) or held for 24 h at 37 degrees C prior to plating (Arm C) for assay of neoplastic transformation. The pooled data from four separate experiments demonstrated a significantly reduced transformation frequency for Arm C compared to Arms A and B. This is indicative of an adaptive response in the case of postirradiation holding, in agreement with the earlier study using C3H 10T1/2 cells. However, with the exception of one experiment, the existence of the adaptive response was not clear on the basis of analysis of individual experiments. This points out the importance of sample size when looking at low-dose effects that are close in magnitude to the inherent variations in spontaneous transformation frequency.

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Previous studies have indicated that the nature of the interaction of radiation and Taxol may be dependent on the cell line, drug dose and treatment schedule. The present study represents a systematic attempt to examine the schedule dependence of the interaction of radiation and Taxol in HeLa cells. The protocol used was radiation treatment (7 Gy), followed by a variable interval (0-24 h), followed by exposure to Taxol (7.5 nM, 24 h), followed by plating for a colony-forming assay. Parallel samples were also taken for flow cytometric analysis of the distribution of cells in the phases of the cell cycle at the beginning and end of Taxol treatment. The results indicate that sub-additive, additive or supra-additive interaction can be seen depending on the interval between the radiation and Taxol treatments. Full radiation survival curves were determined for cells exposed to Taxol either immediately or 10 h after the completion of radiation treatment, corresponding to sub-additive and supra-additive interactions, respectively. An examination of the data revealed that maximum cell killing occurred when the percentage of cells in G1 phase was at a minimum at the time of addition of Taxol. Studies of Taxol-induced toxicity using cells synchronized in G1 phase with mimosine and then released and allowed to progress through the cell cycle confirmed this observation. The conclusion of this study is that prior radiation treatment can modify the effect of subsequent Taxol treatment through alterations in the distribution of cells in the phases of the cell cycle. This finding has important implications for the clinical scheduling of these two cancer treatment modalities.

The effect of reduced temperature (22 degrees C) or serum deprivation during low-dose-rate (0.66 cGy/min) gamma irradiation on cell killing and neoplastic transformation has been examined using the HeLa x skin fibroblast human hybrid cell system. The reduced temperature stops progression of these cells through the cell cycle while serum deprivation slows down cell turnover markedly. The data demonstrate an enhancement in both of the end points when cells are held at 22 degrees C compared to parallel experiments done at 37 degrees C. In operational terms, the decreased survival and increased neoplastic transformation are consistent with our earlier hypothesis of a higher probability of misrepair at reduced temperature (Redpath et al., Radiat. Res. 137, 323-329, 1994). The interpretation that this damage enhancement was associated with the reduced temperature, and not the fact that the cells were noncycling, was supported by the results of experiments performed with cells cultured at 37 degrees C in serum-free medium for 35 h prior to and then during the 12.24 h low-dose-rate radiation exposure. Under these conditions, cell cycle progression, as shown by reduction in growth rate and dual-parameter flow cytometric analysis, was considerably inhibited (cell cycle time increased from 20 h to 40 h), and there was no significant enhancement of cell killing or neoplastic transformation.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

A two- to threefold increase in the rate of neoplastic transformation in cells irradiated at a dose rate of 0.22 cGy/min with fission-spectrum neutrons compared to that at 10.7 cGy/min has been confirmed with the use of alkaline phosphatase chromogenic substrate Western Blue staining to detect foci of neoplastically transformed cells through their expression of a tumor-associated antigen, the end point of the HeLa x skin fibroblast human hybrid cell transformation assay. To investigate whether the inverse dose-rate effect is due to the existence of a period in the cell cycle in which cells are significantly more sensitive to neoplastic transformation than in the rest of the cell cycle, as has been postulated previously (Rossi and Kellerer, Int. J. Radiat. Biol. 50, 353-361, 1986; Brenner and Hall, Int. J. Radiat. Biol. 58, 745-758, 1990; Elkind, Int. J. Radiat. Biol. 59, 1467-1475, 1991), we compared the sensitivity of late mitotic/early G1-phase and mid-G1-phase cells with that of asynchronous cells. The rationale for examining these particular cell cycle phases was based on the fact that mitosis has been hypothesized to be a candidate for the extremely sensitive period, and on a preliminary report that mid-G1-phase C3H 10T1/2 cells may exhibit enhanced sensitivity for neutron-induced transformation. A nominal dose of 45 cGy of fission-spectrum neutrons was delivered at approximately 10 cGy/min. The data indicate that neither late mitotic/early G1-phase nor mid-G1-phase cells are significantly more sensitive than asynchronous cells. Further, the dependence on the phase of the cell cycle for neoplastic transformation of CGL1 cells induced by fission-spectrum neutrons is different from that previously demonstrated for gamma radiation, where late-mitotic cells were approximately five times more sensitive than mid-G1-phase and asynchronous cells (Redpath and Sun, Radiat. Res. 121, 206-211, 1990).

The effect of postirradiation holding at 22 degrees C on cell growth, progression of cells through the cell cycle, and the repair of sublethal, potentially lethal and potentially neoplastic transforming damage in gamma-irradiated HeLa x skin fibroblast human hybrid cells has been examined. Cell growth and cell cycle progression were essentially stopped at this reduced temperature. Cell survival was dramatically reduced by holding confluent cultures for 6 h at 22 degrees C, as opposed to 37 degrees C, after 7.5 Gy gamma radiation delivered at a rate of 2 Gy/min. Return of the cells to 37 degrees C for 6 h after holding at 22 degrees C did not result in increased survival. A similar effect was obtained when the cells were held at 22 degrees C between split-dose irradiation of log-phase cultures where no increase in survival was observed over a split-dose interval of 4 h. In this case a partial increase in survival was observed upon returning the cells to 37 degrees C for 3 h after holding at 22 degrees C for the first 3 h of the split-dose interval. Neoplastic transformation frequency was not enhanced by holding confluent cultures for 6 h at 22 degrees C after 7.5 Gy gamma radiation. This is consistent with previous observations that misrepair of potentially neoplastic transforming damage already occurs at 37 degrees C. The overall results are interpreted in terms of the reduced temperature favoring misrepair, rather than inhibition of repair, of sublethal, potentially lethal and potentially transforming radiation damage.

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.

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

The HeLa X skin fibroblast human cell hybrid system has been used to study the effect of fission-neutron dose rate on the induction of neoplastic transformation. Previously published data using neutrons from the JANUS reactor at Argonne National Laboratory are compared with some preliminary data obtained on the TRIGA reactor at the Armed Forces Radiobiology Research Institute. Comparable results were obtained on both reactors, both in terms of absolute values of transformation frequency and in terms of magnitude of the inverse dose-rate effect (a factor of 2.5 to 3).

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in genetically stable hybrids which are nontumorigenic and no longer express the HeLa tumor-associated antigen, intestinal alkaline phosphatase (IAP). Previous analysis of spontaneous segregants of the nontumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with reexpression of IAP and tumorigenicity. This observation suggests that a putative HeLa tumor suppressor gene(s) is located on chromosome 11 and that this gene may be a negative regulator of the IAP gene. We have isolated several gamma-ray-induced mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 that were specifically selected for reexpression of IAP to further investigate the potential linkage between IAP regulation and the putative tumor suppressor locus. The GIMs have a wide range of cell morphology and level of IAP expression (nearly a factor of 40). The tumorigenicity of the GIMs was examined by s.c. injection into nude mice and all were found to be tumorigenic. The tumor volume-doubling time is in the range of 4 to 8 days for all the cell lines; however, the lag time to reach 500 mm3 tumor volume was significantly longer when the GIM IAP activity was low (less than 20% relative activity), suggesting perhaps that there is a threshold level of IAP expression required for tumor formation and selection for high IAP expression in vivo. However, studies with tumor reconstitutes of the GIMs and transfection studies with an IAP complementary DNA expression vector indicate that high IAP expression alone is not sufficient to confer rapid tumor growth. Therefore, while the data lend strong support to the continued tight correlation between IAP reexpression and tumorigenicity and to our proposal that the tumor suppressor may negatively regulate the IAP gene, it suggests that selection for other gene activities may be responsible for aggressive tumor growth in this cell hybrid system.

We have previously reported that, when 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer was used in the growth medium to control pH fluctuations during the 21-day expression period of our human cell hybrid (HeLa x skin fibroblast) transformation assay, the yield of radiation-induced neoplastically transformed foci after 7 Gy of gamma-irradiation was suppressed. We now demonstrate that the observed suppression is not related to the presence of the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer per se but rather is a function of the growth medium pH. Detailed studies reveal that incubation of the irradiated cells during the entire 21-day expression period at pH 6.7-6.8 versus pH 7.0-7.2 significantly suppressed the transformation frequency after 7 Gy, from 4.4 x 10(-4) to 4.6 x 10(-5) (accumulated data). The endpoint fraction of flasks containing foci was also significantly reduced at the lower pH. Suppression was evident whether the growth medium pH was lowered from pH 7.0-7.2 to pH 6.7-6.8 by medium exchange on day 0, 1, or 9 or even up to 15 days post-irradiation. Growth curves revealed that the population doubling time of the cells is extended and the unirradiated and irradiated plating efficiencies are lowered by long term low pH exposure. We discuss possible mechanisms for the observed suppression, in terms of the influence of low extracellular pH on cell turnover, repair of radiation damage, cell toxicity, and activity of cellular proteases.

Previous studies have indicated that the nature of the interaction of radiation and Taxol may be dependent on the cell line, drug dose and treatment schedule. The present study represents a systematic attempt to examine the schedule dependence of the interaction of radiation and Taxol in HeLa cells. The protocol used was radiation treatment (7 Gy), followed by a variable interval (0-24 h), followed by exposure to Taxol (7.5 nM, 24 h), followed by plating for a colony-forming assay. Parallel samples were also taken for flow cytometric analysis of the distribution of cells in the phases of the cell cycle at the beginning and end of Taxol treatment. The results indicate that sub-additive, additive or supra-additive interaction can be seen depending on the interval between the radiation and Taxol treatments. Full radiation survival curves were determined for cells exposed to Taxol either immediately or 10 h after the completion of radiation treatment, corresponding to sub-additive and supra-additive interactions, respectively. An examination of the data revealed that maximum cell killing occurred when the percentage of cells in G1 phase was at a minimum at the time of addition of Taxol. Studies of Taxol-induced toxicity using cells synchronized in G1 phase with mimosine and then released and allowed to progress through the cell cycle confirmed this observation. The conclusion of this study is that prior radiation treatment can modify the effect of subsequent Taxol treatment through alterations in the distribution of cells in the phases of the cell cycle. This finding has important implications for the clinical scheduling of these two cancer treatment modalities.

The effect of reduced temperature (22 degrees C) or serum deprivation during low-dose-rate (0.66 cGy/min) gamma irradiation on cell killing and neoplastic transformation has been examined using the HeLa x skin fibroblast human hybrid cell system. The reduced temperature stops progression of these cells through the cell cycle while serum deprivation slows down cell turnover markedly. The data demonstrate an enhancement in both of the end points when cells are held at 22 degrees C compared to parallel experiments done at 37 degrees C. In operational terms, the decreased survival and increased neoplastic transformation are consistent with our earlier hypothesis of a higher probability of misrepair at reduced temperature (Redpath et al., Radiat. Res. 137, 323-329, 1994). The interpretation that this damage enhancement was associated with the reduced temperature, and not the fact that the cells were noncycling, was supported by the results of experiments performed with cells cultured at 37 degrees C in serum-free medium for 35 h prior to and then during the 12.24 h low-dose-rate radiation exposure. Under these conditions, cell cycle progression, as shown by reduction in growth rate and dual-parameter flow cytometric analysis, was considerably inhibited (cell cycle time increased from 20 h to 40 h), and there was no significant enhancement of cell killing or neoplastic transformation.

The nontumorigenic HeLa x skin fibroblast hybrid cell line, CGL1, can be induced to re-express HeLa tumor-associated cell surface antigen, p75-IAP (intestinal alkaline phosphatase), with resulting neoplastic transformation, by exposure to gamma radiation. This has allowed the human hybrid system to be developed into a quantitative in vitro model for radiation-induced neoplastic transformation of human cells. Recently, several gamma-ray-induced IAP-expressing mutants (GIMs) of the nontumorigenic HeLa x skin fibroblast hybrid CGL1 were isolated and all were tumorigenic when injected subcutaneously into nude mice (Mendonca et al., Cancer Res. 51, 4455-4462, 1991). Control cell lines which were negative for p75-IAP (CONs) were also isolated from irradiated populations, and none were found to be tumorigenic. We have now begun to investigate the molecular basis of radiation-induced neoplastic transformation in this system by studying the potential genetic linkage between p75/IAP expression, tumorigenicity and damage to a putative tumor suppressor locus on fibroblast chromosome 11. Previous analysis of rare spontaneous segregants has indicated that this locus is involved in the regulation of tumorigenicity and in the expression of the HeLa tumor-associated cell surface marker intestinal alkaline phosphatase (p75-IAP) in this system. Therefore, analysis by restriction fragment length polymorphism and chromosome painting have been performed for chromosome 11, and for chromosome 13 as a control, for the p75/IAP-positive GIM and p75/IAP-negative CON cell lines. We report that in five of eight of the GIMs large-scale damage to the fibroblast chromosome 11's is evident (four GIMs have lost one complete copy of a fibroblast chromosome 11 and one GIM has both copies of fibroblast chromosome 11 heavily damaged). None of the CONs, however (0/5), have lost a complete copy of either fibroblast chromosome 11. No large-scale damage to the control chromosome 13's was detected in the GIMs or CONs. The data further suggest that both copies of fibroblast chromosome 11 contain an active locus and that radiation-induced loss of either fibroblast chromosome 11 will result in neoplastic transformation in this system. We conclude that it is the loss of a putative tumor suppressor locus on fibroblast chromosome 11 which is responsible at least in part for radiation-induced neoplastic transformation of these human hybrid cells.

A two- to threefold increase in the rate of neoplastic transformation in cells irradiated at a dose rate of 0.22 cGy/min with fission-spectrum neutrons compared to that at 10.7 cGy/min has been confirmed with the use of alkaline phosphatase chromogenic substrate Western Blue staining to detect foci of neoplastically transformed cells through their expression of a tumor-associated antigen, the end point of the HeLa × skin fibroblast human hybrid cell transformation assay. To investigate whether the inverse dose-rate effect is due to the existence of a period in the cell cycle in which cells are significantly more sensitive to neoplastic transformation than in the rest of the cell cycle, as has been postulated previously (Rossi and Kellerer, Int. J. Radiat. Biol. 50, 353-361, 1986; Brenner and Hall, Int. J. Radiat. Biol. 58, 745-758, 1990; Elkind, Int. J. Radiat. Biol. 59, 1467-1475, 1991), we compared the sensitivity of late mitotic/early G1-phase and $\text{mid-}{\rm G}_{1}\text{-phase}$

A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use.

The HeLa × skin fibroblast human cell hybrid system has been used to study the effect of fission-neutron dose rate on the induction of neoplastic transformation. Previously published data using neu...

The dependence of γ-radiation-induced neoplastic transformation frequency on position in the cell cycle was measured for a human hybrid cell line (HeLa × skin fibroblast). The end point used was the induction of a tumor-associated antigen which in these cells correlates with tumorigenicity. Induction was measured in cells at G2, M, and mid-G1 phases and compared with the frequency induced in asynchronous cells. For studies of cells in G2 phase, the cells of an asynchronous population were collected for 3 h post-irradiation using the mitotic shake-off technique. For studies of cells in M and mid-G1 phases, cells were collected by mitotic harvest and then treated at the appropriate time. The data show that cells in G2 and M phase are very radiosensitive in terms of both cell killing and induction of neoplastic transformation compared to cells in mid-G1 or asynchronous populations. At a dose of 1 Gy, the transformation frequency was 10- to 20-fold higher for cells in M and G2 phase than for cells in mid-G1 o...

Les auteurs rapportent deux techniques d'isolement de cellules hybrides humaines (cellules HeLa croisees avec des fibroblastes du derme) associees a des tumeurs radio-induites basees sur le fait que les cellules tumorales expriment selectivement une proteine de surface qui peut etre reconnue par un anticorps monoclonal. De telles techniques permettent un enrichisement rapide du milieu de culture

Radiolysis of N2O-saturated solutions of transfer RNA (tRNA) and the arylacethydroxamic acids, N-hydroxy-N-2-acetylaminofluorene and N-hydroxy-N-4-acetylaminobiphenyl; their corresponding acetamides, 2-acetylaminofluorene and 4-acetylaminofluorene; or the O-glucuronide of N-hydroxy-N-2-acetylaminofluorene resulted in adduct formation of the nucleic acid with these carcinogenic arylamine derivatives. The yield of adducts on irradiation of the arylacethdroxamic acids with tRNA was greater than that for their corresponding acetamides or the O-glucuronide. The fluorenylacethydroxamic acid and acetamide were also more reactive than the biphenyl analogs. Adduct formation resulting from radiolysis of tRNA and the arylacethydroxamic acids or the O-glucuronide proceeded with retention of both the aromatic nucleus and the N-acetyl group. The yields of adducts were much greater for irradiated mixtures than for irradiation of either component alone followed by mixing. Evaluation of the data shows that initial modification of the tRNA or the carcinogen can lead to adduct formation. In the case of primary radical attack of the nucleic acid, it has been shown that short-lived reactive RNA intermediates are responsible for a major fraction of the observed yield of adducts in the irradiated mixtures. Comparative studies showed that irradiation under conditions that favor reaction of oxidizing radicals enhanced formation of the adducts. Oxygen was shown to protect RNA from irradiation-induced binding of the arylacethydroxamine acids due to competition of O2 with the carcinogen for the reactive RNA intermediates.

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.

Evidence is presented for the mutation of the tryptophan-requiring bacterial strain Escherichia coli WP2 uvrA from auxotrophy to prototrophy, and from streptomycin sensitivity to resistance, by Cerenkov emission associated with 137Cs gamma irradiation. Furthermore, the data strongly suggest a more than additive interaction between the gamma-induced damage and that induced by Cerenkov emission for both mutations scored. An additional observation is that mutant yields (expressed as mutants/10(7) survivors) show a dependence on the number of viable cells plated for both uv (254 nm) and Cerenkov-induced mutations, but not for those induced by gamma irradiation. This demonstrates another similarity between uv- and Cerenkov-induced damage.

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.

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.

8-Methoxypsoralen has been shown to act as a radiosensitizer of hypoxic bacteriophage and bacteria. Radiosensitization of bacteriophage requires irradiation in the presence of excess scavenger. Bacterial radiosensitization requires deficiencies in uvr and rec genes. For the drug to be effective it must be present during irradiation. Pulse radiolysis studies have shown that, like electron-affinic radiosensitizers, 8MOP can efficiently oxidize free radicals. Unlike oxygen and most electron-affinic radiosensitizers 8MOP does not act in a purely dose-modifying fashion, and can radiosensitize beyond the oxygen effect.