Survival of colorectal cancer cell lines treated with paclitaxel, radiation, and 5-FU: effect of TP53 or hMLH1 deficiency.

Clonogenic survival and early cell death during treatment of human colon carcinoma cells were investigated following X-irradiation (IR) alone, IR followed by 5-FU for 24 h, and Taxol administered 24 h before IR and 5F-U. The investigated cell lines were: HCT116, 40-16 clonally derived from HCT116, and two HCT116 variants: N6CHR3 expressing hMLH1, and TP53 null cells denoted HCT116 p53-/-. The objective was to determine efficacy of the combined treatment and to correlate response with constitutive levels of TP53, WAF1, and hMLH1 proteins, as well as with mRNA levels of the apoptosis-related genes survivin, BNIP3, and MYC. At the end of treatment with 5-FU, the proportion of viable cells was between 0.65 and 0.70 for all cell lines. Additional cell loss occurred in 40-16 and HCT116 p53-/- cells following administration of Taxol before IR and 5-FU. Radiation sensitivity was unaffected by combined treatments, except for Taxol, irradiation, and 5-FU sequence in the HCT116 p53-/- and 40-16 cell lines, where radiation sensitivity determined by clonogenic survival curve slopes was doubled or quadrupled, respectively. Under our present experimental conditions, treatment response did not correlate with TP53 or hMLH1 status, but was associated with apoptosis-related genes, most notably BNIP3. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 175-185 (2000).

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.

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.

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.

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.

In vitro mechanisms of chemopotentiation by tone-burst ultrasound.

We investigated in vitro enhancement of cytotoxicity of chemotherapeutic agents by tone-burst ultrasound. Survival of CHO cell exposed to chemotherapeutic agents in culture medium was determined with and without insonation (1.62 and 0.29 MHz, 10% duty cycle). Insonations up to 0.4 MPa peak pressure (5 kW/m2 spatial and temporal average) occurred in the middle of 1 h drug exposures. Cytotoxicity in ultrasound control groups was never observed. Ultrasound increased the clonogenic cytotoxicity of adriamycin (p = 0.00027 by paired t test) and diaziquone but not of cisplatin or mitomycin C. Potentiation of adriamycin depended on exposure time and tone-burst frequency. .OH production in water occurred at intensities as low as 0.4 kW/m2, but did not increase with added adriamycin. Ultrasound did not affect membrane fluidity, but moderately increased cellular adriamycin accumulation, possibly explaining the observed drug potentiation.

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.

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.

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.

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.

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.

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.

Uniform pulsed fields for ultrasonic bioeffect experimentation.

The rationale and methods are presented for producing high amplitudes of pulsed ultrasound distributed uniformly over several square centimetres. Pulsed fields (p = 1 MPa, 1-9 pulses per burst, up to 10(4) bursts per second, pulse-average intensity up to 39 W cm-2, centre frequencies 1, 2.5 and 3.5 MHz) were produced using high power pulsers and minimally damped 3.8 cm diameter transducers. Wave-forms and negative pressure distributions in water are displayed for these transducers. The lateral distributions of pressure amplitudes were uniform within +/- 15% over areas up to 4 cm2. These fields are suitable for use in bioeffect experiments to investigate possible health hazards of diagnostic pulsed ultrasound, as well as for possible therapeutic applications.

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.

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.

Absence of mutagenic effects of continuous and pulsed ultrasound in cultured (AL) human-hamster hybrid cells.

Mutagenic effects of continuous and pulsed ultrasound were looked for using an in vitro assay system, the AL hybrid, that is up to 100 times more sensitive for mutagens such as x-rays and neutrons than the assays used previously to evaluate ultrasound. Cells in suspension in rotated plastic test tubes were insonated with continuous wave ultrasound at 1 MHz, ISPTP = 0.62-40 W/cm2 for 0-40 min. Cells attached in the central region of culture flasks received pulsed exposures at fc = 2.5 MHz, PRF = 1 kHz, 2 and 8 cycles per pulse, with p- = 1.2 MPa (ISPTA = 31-180 mW/cm2) for 0-30 min. Although these exposures were cytotoxic (the plating efficiency was decreased to approximately 65% by the highest doses), induction of mutation, if any occurred, was less than would be expected in this test system from 10-30 cGy of x-ray.

Pulsed ultrasound and neoplastic transformation in vitro.

C3H/10T1/2 mouse embryo fibroblast cells grown as 1.2 cm diameter monolayers were assayed for cytotoxicity and neoplastic transformation following 30 min insonations with three types of pulsed ultrasound (US). Two of the US beams, with a repetition frequency of 1 kHz, center frequencies of 2.5 and 3.5 MHz and time-averaged intensities (ITA) of 31 and 6.7 mW/cm2, respectively, simulated biomedical pulse-echo imaging. The third US beam, with a repetition frequency of 5 kHz, center frequency of 2.5 MHz and ITA of 885 mW/cm2, simulated pulsed biomedical Doppler insonation. All three US beams exhibited negative pressure amplitudes in the range of 1 MPa over the cellular preparation. In order to provide uniform insonation for all exposed cells, all beams were unfocused; pressure amplitude variations did not exceed +/- 15% within a beam diameter of at least 1.5 cm, within which test samples of attached cells were confined. Ultrasound was delivered alone or in combination with x-rays (2 Gy, 240 kVp at 3.5 Gy/min given within 5 min before US) as a costressor. Under all treatment conditions, there was no significant affect of US on clonogenic survival or neoplastic transformation (cumulative probabilities ranging from 0.13 to 0.92).