Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells.

A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

Rodent cell transformation and immediate early gene expression following 60-Hz magnetic field exposure.

Some epidemiological studies suggest that exposure to power frequency magnetic fields (MFs) may be associated with an elevated risk of human cancer, but the experimental database remains limited and controversial. We investigated the hypothesis that 60-Hz MF action at the cellular level produces changes in gene expression that can result in neoplastic transformation. Twenty-four hour 200 microT continuous MF exposure produced negative results in two standard transformation systems (Syrian hamster embryo cells and C3H/10T1/2 murine fibroblasts) with or without postexposure to a chemical promoter. This prompted a reexamination of previously reported MF-induced changes in gene expression in human HL60 cells. Extensive testing using both coded and uncoded analyses was negative for an MF effect. Using the same exposure conditions as in the transformation studies, no MF-induced changes in ornithine decarboxylase expression were observed in C3H/10T1/2 cells, casting doubt on a promotional role of MF for the tested cells and experimental conditions.

Induction of neoplastic transformation in C3H/10T1/2 cells by 2.45-GHz microwaves and phorbol ester.

C3H/10T1/2 cells were exposed to 2.45-GHz microwaves for 24 h and/or 1.5 Gy of 238-kVp X rays at 3.75 Gy/min. Transformation frequency and cell survival were measured with or without postirradiation addition of the tumor promoter tetradecanoyl-phorbol-13-acetate (TPA) at 0.1 microgram/ml. We previously reported (Carcinogenesis 6,859-864, 1985) an enhancement of transformation frequency when 10T1/2 cells exposed to a special sequence of microwaves and X rays were subsequently cultured in TPA. The same sequence of microwaves and X rays without promotion resulted in a transformation response similar to that induced by X rays alone. We now report statistically significant (at P greater than 0.999) enhancement of transformation response by TPA in cells exposed to 2.45-GHz microwaves (SAR = 4.4 W/kg). Microwaves alone had no effect on transformation. Plating efficiency and cell survival were not affected by TPA or microwave treatments.

Survival and oncogenic transformation of C3H/10T1/2 cells after extended X irradiation.

We performed two independent series of experiments aiming at establishing dose-response curves for lethality or oncogenic transformation in vitro following acute and protracted X-ray doses between 0.25 and 2 Gy. In the first series of experiments, we measured the survival of C3H/10T1/2 CL8 fibroblasts and their transformed counterparts (MCA TCL15) as a function of X-ray dose delivered at 0.49 Gy/min. In addition, 1- and 2-Gy doses were split into four fractions separated by 3-h intervals. The accuracy of survival fraction measurements was about 2%. We found that the dose-response curve at 0.49 Gy/min was a linear function of the dose for both cell lines with a negative slope of 0.171 +/- 0.007 Gy-1 or 0.160 +/- 0.003 Gy-1 for 10T1/2 or MCA cells, respectively, indicating a similar initial radiosensitivity of normal and transformed 10T1/2 cells. Dose fractionation resulted in a significant increase of the survival, relative to that measured when X-ray dose was delivered in a single fraction. The enhancement of survival was not, however, significantly different for 10T1/2 and MCA cells, indicating similar cellular repair abilities. In the second series of experiments, we measured oncogenic transformation in vitro (along with the survival) of C3H/10T1/2 cells, using a constant exposure time technique in which the dose rate was proportional to the total dose so that the repair time was equal at all dose levels. The dose-response curves for oncogenic transformation in the low-dose range between 0.25 and 2 Gy were consistent with a linear response with a positive slope 2.50 +/- 0.11 X 10(-4), 1.50 +/- 0.03 X 10(-4), or 0.87 +/- 0.05 X 10(-4) Gy-1, for acute, 1-h, or 3-h protracted exposures, respectively. Hence, relative to the acute irradiation, the 1- or 3-h protraction of the X-ray dose reduced the slopes by 0.60 +/- 0.03 or 0.35 +/- 0.03, respectively. These results indicate that in the dose range between 0.25 to 2 Gy, the dose-response curves for survival or oncogenic transformation were linear and can be modified by the temporal distribution of the X-ray dose.

Neoplastic transformation of C3H/10T1/2 cells following exposure to 120-Hz modulated 2.45-GHz microwaves and phorbol ester tumor promoter.

Some recent epidemiological studies have shown a positive association between cancer incidence and exposure to electromagnetic (EM) fields. Evidence from in vitro studies indicates that this effect could be due to synergistic interaction between EM fields and tumor promoters. However, no dose-response data related directly to carcinogenesis have been published. In this study, actively growing cultures of C3H/10T1/2 cells were exposed for 24 h to 2.45-GHz microwaves pulse-modulated at 120 Hz. Conditions of EM-field exposure were designed to simulate low-field exposures (specific absorption rate 0.1, 1, or 4.4 W/kg; the corresponding peak amplitudes were electric field 18, 56, or 120 V/m, magnetic field 0.09, 0.27, or 0.56 muT, respectively). In separate experiments, a 24-h EM-field exposure at 4.4 W/kg was preceded or followed by X irradiation at 0.5, 1, or 1.5 Gy. Cells were assayed for cell survival and neoplastic transformation with or without post-treatment administration of 0.1 micrograms/ml of 12-O-tetradecanoylphorbol-13-acetate (TPA) for the duration of the assay. The EM fields alone had no effect on cell survival or induction of neoplastic transformation. However, enhancement of transformation due to EM fields plus TPA was highly significant and ranged up to a level equivalent to that produced by 1.5 Gy of X rays. The frequency of neoplastic transformation was dependent on the level of EM exposure and was additive with doses of X rays given as a cocarcinogen.

Evaluation of nitrobenzimidazoles as hypoxic cell radiosensitizers.

Radiobiological and pharmacokinetic assays were performed to determine the potential of 2-nitrobenzimidazole (NBI) as a hypoxic cell radiosensitizing agent. As judged by comparing survival curve slopes of Serratia marcescens irradiated under aerated and hypoxic conditions, the NBI enhancement ratio (ER) at 2 mM concentration was 2.4 +/- 0.2, compared with an oxygen enhancement ratio of 3.3 +/- 0.3. 2,5-Dinitrobenzimidazole (DNBI) was investigated in vitro; its ER was 3.0 +/- 0.3 at 4 mM concentration. Very poor tissue penetration of DNBI precluded further testing in vivo. Acute toxic signs appeared in C3H/HeJ mice following ip injection of NBI at 100 mg/kg. These would be partly attributable to the stress caused by the high pH of the injection vehicle. The LD50 was estimated to be 125-150 mg/kg. Mammary adenocarcinoma tumors grown in the flanks of these mice exhibited maximum NBI levels at 5 min postinjection (ip). Peak tumor radiosensitization occurred in the interval between 5 and 10 min postinjection. The ER for tumor regrowth delay was 2.1 +/- 0.3 following 50 mg/kg injected into mice 5 min before irradiation. Functional evaluation up to 40 days after treatment revealed no evidence of neurological deficit.

In vitro action of continuous-wave ultrasound combined with adriamycin, X rays or hyperthermia.

We compared the ability of continuous-wave ultrasound to enhance cytotoxicity from X irradiation, hyperthermia or exposure to adriamycin. The survival of CHO cells exposed in culture medium to these agents was determined with and without continuous-wave ultrasound (1.62 or 1.765 MHz). In water-filled transmission exposure vessels with 2-cm-diameter Mylar end windows, 10-min insonation not producing cytotoxicity could produce .OH radicals (measured by electron paramagnetic resonance) even at 0.4 W/cm2. Ultrasound at intensities ranging between 1 and 2.5 W/cm2 increased the clonogenic cytotoxicity of adriamycin (P = 0.0023 by paired t test) but not of X rays (2-10 Gy) or hyperthermia (44 degrees C for 10-50 min). The only significant action of continuous-wave ultrasound under similar test conditions was the potentiation of adriamycin-induced clonogenic cytotoxicity, possibly mediated by cavitational activity.

Neutron dose-rate experiments at the AFRRI nuclear reactor. Armed Forces Radiobiology Research Institute.

Some in vitro and in vivo studies with neutrons have shown increased carcinogenic effectiveness following low-dose-rate or fractionated irradiation compared to acute exposure in the low-dose range. This would imply that the risk of cancer for persons exposed occupationally to low doses of neutrons is underestimated at present. The C3H 10T1/2 assay has played a major role in investigating neutron dose-rate effects. We describe three independent series of experiments addressing the question of the influence of dose rate using the AFRRI fission-neutron source with the C3H 10T1/2 cell transformation assay as well as with two mutation assays utilizing human-hamster hybrid AL cells. In the first two series, we focused on performing experiments with fission-neutron doses and dose rates similar to those for which enhancement of neoplastic transformation of C3H 10T1/2 cells was originally reported, and observed no discernible dose-rate effect. In the third series concurrent with the induction of neoplastic transformation in C3H 10T1/2 cells, we also measured mutagenesis at two loci in AL cells. Data for survival, neoplastic transformation, and mutation were obtained at two dose rates in the range of neutron doses 0.005 to 0.9 Gy. Dose-rate effectiveness factors expressed as ratios of the effect for low compared to high dose rate did not differ from one, indicating no influence of dose rate on these end points.

Heat-sensitive state of mouse mammary carcinoma cells in tumors.

The heat response of the SCK mammary carcinoma of A/J mice was studied in vivo and in vitro. Solid tumors or tumor cells in culture were heated in a water bath and cell survival was determined by clonogenicity in vitro. Cells in tumors were much more sensitive to heat than cells in culture. To eliminate vascular effects, tumors were dissociated into small fragments and the tumor fragments were heated in vitro, so that cells were heated while in contact with neighbors and in a complete medium. Cells in tumor fragments were as sensitive to heat as cells in tumors, even though vascular effects during heat exposure were excluded. The heat-sensitive tumor fragments gradually became heat resistant during 3 h of incubation in a complete medium at physiological temperature. The transition from a heat-sensitive to a heat-resistant state was not correlated with the development of thermotolerance or stress-related proteins. The transition was inhibited when the extracellular environment was made acidic or hypoxic but not when it was glucose and serum deprived. These results suggest that SCK tumor cells in vivo are sensitive to heat, and the heat-sensitive state appears to be established under the influence of the intratumor environment.

Expression analysis of human HL60 cells exposed to 60 Hz square- or sine-wave magnetic fields.

A total of 960 complementary DNA (cDNA) clones from an HL60 cell cDNA library were screened to discover genes that were differentially expressed in HL60 cells exposed to 60 Hz square-wave magnetic fields (MFs) compared to sham-exposed cells. Square-wave fields are rich in odd harmonic frequency content. We used a two-gel cDNA library screening method (BIGEL) to identify treatment-induced alterations in gene expression. Four cDNA clones were tentatively identified as differentially expressed after exposure to square-wave MFs at 2 mT for 24 h. BIGEL-identified genes (GenBank accession number) corresponding to these clones were: TI227H (D50525), EST Homo sapiens partial cDNA (Z17814), human ribosomal protein S13 (L01124), and AICAR transformylase mRNAs (D82348). The differences in mRNA levels were not confirmed in test compared to experimental cells by Northern analysis. In other experiments, we used concurrent exposure to 60 Hz sine- or square-wave MFs (0 or 2 mT, duration of 3 or 24 h, no postexposure delay). In addition to the four BIGEL genes, we also investigated MYC, HSP70, RAN and SOD1. In the case of MYC and HSP70, square-wave MFs appeared to exhibit more marked alterations when compared to sinusoidal waveforms, but the overall results indicated no effect of possible differential magnetic-field-induced expression of all eight genes. In contrast, alterations of mRNA levels were observed for seven genes after exposure to X irradiation, hyperthermia and TPA. These results are contrary to previously proposed similarities between the action of these agents and MF effects on gene transcription.

p53 mutational status and survival of human breast cancer MCF-7 cell variants after exposure to X rays or fission neutrons.

We assessed cytotoxicity of X rays or fission neutrons and the status of the p53 tumor suppressor gene in irradiated and unirradiated actively growing cultures of human breast cancer MCF-7 cells. One parental or wild-type (WT) and the other resistant to adriamycin (ADRR) were studied within the same experiment. We found that, relative to MCF-7 WT cells, MCF-7 ADRR cells exhibited a small but significant resistance to X rays, but not to fission neutrons. Single-strand conformation polymorphism analysis followed by DNA sequencing and immunohistochemical staining with a p53 protein-specific antibody performed on pooled polyclonal or monoclonal populations of MCF-7 WT or ADRR cells confirmed that wild-type cells have two normal copies of the p53 gene. We discovered p53 loss of heterozygosity and a point mutation in the remaining allele of the p53 gene in adriamycin-resistant cells. This mutation is a splice acceptor site change on the upstream border of exon 5 and results in p53 protein overexpression. No new p53 mutations were observed in MCF-7 WT or ADRR cells surviving either X or fission-neutron irradiations. Our results suggest that the mutant p53 allele affects cytotoxic outcomes of DNA damage from X rays but not from neutrons.

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields.

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

Gamma dose measurement in a d(80) + Be neutron beam.

The gamma-ray dose component of a d(80) + Be neutron beam was measured with a 6Li-shielded Geiger-Mueller counter in a tissue-equivalent liquid phantom. The proportion of gamma-ray dose to total dose increased from 0.07 near the surface to 0.09 at 25 cm depth. Due to thermal neutron sensitivity, the G-M count rate increased by 25% in the absence of 6Li shielding.

Dosimetric properties of p(90)+(Be + Ta) and p(101)+(Be + Al) neutrons.

Dosimetric data were obtained for p(90) + (Be + Ta) and p(101) + (Be + A1) radiation with field size 9 x 11 cm at a 125-cm SSD impinging on a tissue-equivalent liquid phantom with density 1.10. The radiation was unfiltered or filtered by 10 cm of polyethylene. Dose rates measured with a 0.5-cm3 ion chamber at a depth of 5 cm ranged from 0.046 Gy min-1 microA-1 for filtered p(90) + (Be + Ta) to 0.11 Gy min-1 microA-1 for unfiltered p(101) + (Be + A1). Neutron time of flight energy spectra in air averaged above 10 MeV indicate mean neutron energies ranging from 42.0-51.4 MeV for the four beams under study. The central axis depths at which 50% dose attentuation occurred ranged from 15 to 20 cm for these beams. At 3 cm depth the gamma dose component of the total p(101) + (Be + A1) dose was 14.5%, or 17% with filtration.

Lack of dose rate modification (0.0049 vs. 0.12 Gy/min) of fission-neutron-induced neoplastic transformation in C3H/10T1/2 cells.

Clonogenic survival and neoplastic transformation of asynchronous cultures of C3H/10T1/2 cells were used to assay the effect of dose protraction of reactor-produced fission neutrons. Cells were exposed to eight neutron doses ranging from 0.05 to 0.9 Gy delivered at 11.7 or at 0.49 cGy/min. For each dose level, high and low dose rate irradiations were performed on the same day. At each dose a similar effectiveness of fission neutron irradiation at high or low dose rates was measured for both cell survival and transformation. The combined high and low dose-rate data were analysed by two- or three-parameter models. Depending on the model used, values of the effectiveness per unit dose derived as parameters of linear terms of the respective dose-response curves were 0.9-1.2 Gy-1 for clonogenic survival and 5-8 x 10(-4) Gy-1 for neoplastic transformation. It is concluded that the modification of fission neutron dose-response curves by dose rate is negligible or absent in the range of doses and dose rates examined, in contrast to results with other sources of fission or fast neutrons.

Effect of X-ray dose protraction and a tumor promoter on transformation induction in vitro.

We have investigated the effects of X-rays given in a brief exposure (1 min or less) or protracted over 5 h, on cell survival and the induction of neoplastic transformation in C3H/10T1/2 cells with an emphasis on latent transformation damage remaining after protracted irradiation. This latent damage and its expression were investigated at accumulated doses of 0.25 to 4 Gy by chronic treatment with TPA (12-O-tetradecanoyl-phorbol-13-acetate or phorbol myristate acetate) at 0.1 microgram/ml beginning after irradiation. Transformation incidence from protracted as well as brief X-irradiations was linearly related to X-ray dose in the presence of 0.1 microgram/ml TPA/ml. In the absence of TPA, the best fits were obtained with cubic rather than quadratic functions. The effect-modifying factors due to dose protraction were similar with or without TPA and averaged 4.6 at low doses (up to 2 Gy). Also within this dose range average transformation enhancement due to TPA was approximately 4. Our results indicate that dose protraction does not change the shape the dose-response curve for transformation, and that the shape change induced by TPA is also independent of dose protraction.

Potentiation of chemotherapy by low-level ultrasound.

We have performed in vitro and in vivo tests to determine whether ultrasound (US) at levels lower than previously investigated by others could still potentiate chemotherapeutic cell killing. Positive results were obtained with adriamycin and diaziquone. Two types of low-level US were effective: tone-burst US (10% duty cycle, 1.765 MHz, ISATA = 0.25 W/cm2), and pulsed US (2.5 MHz centre frequency, 1 kHz repetition frequency, MPa-level pressure amplitudes), distributed uniformly over the biological target. These US beams were non-cytotoxic and produced negligible temperature elevation. Statistically significant US-induced increases in drug cytotoxicity were observed in CHO and MCF-7 WT but not V79 cells for 1-h drug exposures at several drug concentrations. The effects of combined drug and US treatments in vivo were studied by measuring post-treatment volume changes in uterine cervical squamous cell carcinoma implanted in the cheek pouch of the Syrian hamster. A statistically significant US-drug synergy in tumour volume reduction was observed with adriamycin and diaziquone.

Application of the constant exposure time technique to transformation experiments with fission neutrons: failure to demonstrate dose-rate dependence.

A direct comparison of the effectiveness of fission neutrons at high (11.0-31.3 cGy/min) or several low dose-rates (0.14-3.2 cGy/min) was carried out under identical conditions. Monolayers of exponentially growing C3H/10T1/2 cells were exposed at 37 degrees C to reactor-produced neutrons (fluence-mean energy En = 0.68 MeV, < or = 5% gamma component, frequency mean linear energy yF = 21 keV/micron, dose mean lineal energy yD = 42 keV/micron in an 8-micron spherical cavity). Survival or transformation induction were studied at five doses from 10.5 to 94 cGy. In low dose-rate irradiations, these doses were protracted over 0.5, 1, 3 or 4.5 h, resulting in 17 different dose-rates. Up to six experiments were performed at each of five exposure times. Concurrently with transformation we studied cell proliferation in control versus cells irradiated at 40 cGy (acute and a 4.5-h protraction) and found no evidence of a shift in the cell cycle distribution among these cells. At a given dose and dose-rate, the effect of dose protraction on survival or transformation was assessed by the dose-rate modifying factor (DRMF), defined as the low:high dose-rate effect ratio at the same dose. Survival or transformation induction curves were nearly linear with initial slopes, respectively, of about 6.5 x 10(-3) or 6.2 x 10(-6) cGy-1. Consistent with dose-response curves, DRMFs were independent of the dose and dose-rate. The mean values of the DRMF with their uncertainties and 99% confidence intervals, based on measurements in individual doses and dose-rates for survival or transformation were, respectively: 1.01 +/- 0.03 (0.92, 1.09) or 0.98 +/- 0.04 (0.83, 1.08) indicating a similar precision in determining DRMF for survival or transformation, and no dose or dose-rate influence on these end points.

Lack of inverse dose-rate effect on fission neutron induced transformation of C3H/10T1/2 cells.

Exponential and density-inhibited cultures of C3H/10T1/2 cells were exposed to a single dose of 0.3 Gy of fission neutrons delivered at rates ranging from 0.005 to 0.1 Gy/min. No discernible effect upon cell survival or transformation was observed by a lowering of the fission neutron dose rate in either exponential or plateau cultures. At the level of 2.3 x 10(-4) transformants per surviving cell, the RBE for neoplastic transformation was three at acute dose rates and ten at the lowest dose rate studied (0.005 Gy/min for neutrons and 0.01 Gy/min for X-rays).

Effects of WR-1065 and WR-151326 on survival and neoplastic transformation in C3H/10T1/2 cells exposed to TRIGA or JANUS fission neutrons.

We demonstrated the ability of aminothiols WR-1065 and WR-151326, each at concentration 1 mM, to protect C3H/10T1/2 cells against the transforming effects of fission neutrons under two distinct sets of experimental conditions. Experiments with WR-1065 were performed with stationary cultures of C3H/10T1/2 cells, and a TRIGA reactor-generated fission neutron field at the Armed Forces Radiobiology Research Institute (USA). Experiments with WR-151326 were performed with proliferating cultures of C3H/10T1/2 cells and a JANUS reactor-generated fission neutron field at the Argonne National Laboratory (USA). Radioprotectors were present before, during, and after irradiation for total-periods of 35 min (WR-151326; 10 min pre-incubation) or 1 h (WR-1065; 30 min pre-incubation). Bioavailability of WR-1065 and WR-151326 in extracellular medium under experimental conditions simulating those of the transformation experiments was studied by measuring oxidation rates in the presence of attached C3H/10T1/2 cells in plateau and exponential phase of growth for periods of up to 5 h. Estimated half-lives for autoxidation of WR-1065 or WR-151326 were approximately 8 min or 1 h regardless of the proliferative status of cells. In the absence of WR-compounds, dose-response data for transformation induction by neutrons from TRIGA and JANUS reactors were fitted to a common curve with a linear coefficient of about 7 x 10(-4)/Gy. WR-151326 and WR-1065 were found to provide significant radioprotection by factors of 1.79 +/- 0.08 and 3.23 +/- 0.19, respectively, against fission neutron-induced neoplastic transformation. No significant protection against neutron-induced cell lethality was observed.