Elizabeth K. Balcer-Kubiczek

Neoplastic Transformation of C3H/10T 1/2 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.

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.

Expression Analysis of Human HL60 Cells Exposed to 60 Hz Square- or Sine-Wave Magnetic Fields

Abstract Balcer-Kubiczek, E. K., Harrison, G. H., Davis, C. C., Haas, M. L. and Koffman, B. H. 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.

Repair Time for Oncogenic Transformation in C3H/10T1/2 Cells Subjected to Protracted X-irradiation

With exponential cultures of C3H/10T1/2 cells, we have investigated the effect of X-ray dose protraction on oncogenic cell transformation in the dose range 0.25-2 Gy. Within a particular experiment a constant exposure time was used. In different experiments exposure time varied between 1 and 5h. Cell transformation was analysed using the linear-quadratic relation, gamma (D) = alpha 1D + alpha 2D2, between transformation frequency per surviving cell and X-ray dose. Based on values of the linear coefficients, we developed an empirical formula for relating slopes of dose induction curves obtained at high or reduced dose rate condition. Our estimate of repair half-time for cell transformation with 95 per cent confidence limits is 2.4 (1.8, 3.0) h.

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.

Delayed expression of hpS2 and prolonged expression of CIP1/WAF1/SDI1 in human tumour cells irradiated with X-rays, fission neutrons or 1 GeV/nucleon Fe ions

PURPOSE Differences in gene expression underlie the phenotypic differences between irradiated and unirradiated cells. The goal was to identify late-transcribed genes following irradiations differing in quality, and to determine the RBE of 1 GeV/n Fe ions. MATERIALS AND METHODS Clonogenic assay was used to determine the RBE of Fe ions. Differential hybridization to cDNA target clones was used to detect differences in expression of corresponding genes in mRNA samples isolated from MCF7 cells irradiated with iso-survival doses of Fe ions (0 or 2.5 Gy) or fission neutrons (0 or 1.2 Gy) 7 days earlier. Northern analysis was used to confirm differential expression of cDNA-specific mRNA and to examine expression kinetics up to 2 weeks after irradiation. RESULTS Fe ion RBE values were between 2.2 and 2.6 in the lines examined. Two of 17 differentially expressed cDNA clones were characterized. hpS2 mRNA was elevated from 1 to 14 days after irradiation, whereas CIP1/WAF1/SDI1 remained elevated from 3 h to 14 days after irradiation. Induction of hpS2 mRNA by irradiation was independent of p53, whereas induction of CIP1/WAF1/SDI1 was observed only in wild-type p53 lines. CONCLUSIONS A set of coordinately regulated genes, some of which are independent of p53, is associated with change in gene expression during the first 2 weeks post-irradiation.

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.

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).

Single-Transducer Electrode Design for Beam Shaping in Biomedical Ultrasound

Abstruct-Single-electrode single-frequency continuous-wave transducers (radius 1.9 cm, frequency 1.7 or 1.75 MHz) were developed for producing desired circularly symmetric lateral intensity profiles at specified axial distances in water. The methods can be used to produce a variety of intensity profiles. The circ function was chosen as a prime objective since it has applications in therapeutic and diagnostic ultrasound as well as in ultrasonic bioeffects experimentation. Electrode shading parameterized by the Fermi-Dirac distribution was calculated to produce circ-function-like profiles at certain axial distances. Experimentally, circularly symmetric shading functions were approximated by an asteroidal design so that within each sector and at a given radius r, the proportion of electroded arc length was given by the shaded velocity potential V(r). Such electrodes were fabricated on PZT-4 (lead zirconium titanate) and PMN (lead metaniobate) transducers. Agreement between theory and experiment was obtained for PMN but not PZT-4, indicating that if low lateral crosstalk transducers are used, the asteroidal approximation to V(r) is valid. Inverse source calcula

Oncogenic transformation of C3H/10T1/2 cells by X-rays, fast-fission neutrons, and cyclotron-produced neutrons

Lethality and oncogenic transformation were measured in C3H/10T1/2 murine fibroblasts exposed to neutrons and X-rays at doses between 0.5 and 11 Gy. Transformation results with X-rays and low-energy, reactor-produced neutrons were used as a baseline to compare and evaluate the results obtained with high-energy, cyclotron-produced neutrons. The radiations were 100-kVp X-rays at 0.49 Gy min-1, reactor fission neutrons at 0.10 to 0.31 Gy min-1 with an 8 to 20 per cent gamma dose component, and cyclotron-produced neutrons at 0.51 Gy min-1 with mean energy 38 MeV and an 8 per cent gamma dose component. The radiobiological effectiveness (r.b.e.) for cell lethality was 2.4 +/- 0.2 for fission neutrons and 1.7 +/- 0.1 for high-energy neutrons. The maximum proportions of transformants per thousand surviving cells were, respectively, 3.7 +/- 0.8, 6.5 +/- 0.7, and 2.3 +/- 0.6 for X-rays, fission neutrons, and cyclotron-produced neutrons. The maximum observed r.b.e. for transformation induction was 3.8 for fission neutrons and 1.2 for cyclotron neutrons. Thus, high-energy neutrons exhibit a higher r.b.e. for cell killing capacity than for oncogenic transformation in C3H/10T1/2 cells.