Charles R. Geard

Taxol: A novel radiation sensitizer

The investigational antineoplastic agent, taxol, a natural product from the yew, Taxus sp. L., is currently being evaluated in a series of Phase II clinical trials. To date, the drug has shown activity against ovarian cancer, lung cancer, and melanoma. Taxol is a potent microtubule stabilizing agent that selectively blocks cells in the G2 and M phases of the cell cycle and is cytotoxic in a time-concentration dependent manner. It is well known from radiobiological principles that G2 and M are the most radiosensitive phases of the cell cycle. On the rationale that taxol could function as a cell-cycle selective radiosensitizer, we examined the consequences of combined drug-radiation exposures on the human grade 3 astrocytoma cell line, G18. Survival curve analysis shows a dramatic interaction between taxol and ionizing radiation with the degree of enhanced cell killing dependent on taxol concentration and on the fraction of cells in the G2 or M phases of the cell cycle. The sensitizer enhancement ratio (SER) for 10 nM taxol at 10% survival is approximately 1.8. These results obtained with cycling aerated radioresistant brain tumor cells indicate that significant advantage may derive from appropriate time-concentration dependent interactions in combined modality protocols.

The Bystander Effect in Radiation Oncogenesis: I. Transformation in C3H 10T½ Cells In Vitro can be Initiated in the Unirradiated Neighbors of Irradiated Cells

Abstract Sawant, S. G., Randers-Pehrson, G., Geard, C. R., Brenner, D. J. and Hall, E. J. The Bystander Effect in Radiation Oncogenesis: I. Transformation in C3H 10T½ Cells In Vitro can be Initiated in the Unirradiated Neighbors of Irradiated Cells. It has long been accepted that radiation-induced genetic effects require that DNA be hit and damaged directly by the radiation. Recently, evidence has accumulated that in cell populations exposed to low doses of α particles, biological effects occur in a larger proportion of cells than are estimated to have been traversed by α particles. The end points observed include chromosome aberrations, mutations and gene expression. The development of a fast single-cell microbeam now makes it possible to expose a precisely known proportion of cells in a population to exactly defined numbers of α particles, and to assay for oncogenic transformation. The single-cell microbeam delivered no, one, two, four or eight α particles through the nuclei of all or just 10% of C3H 10T½ cells. We show that (a) more cells can be inactivated than were actually traversed by α particles and (b) when 10% of the cells on a dish are exposed to α particles, the resulting frequency of induced transformation is not less than that observed when every cell on the dish is exposed to the same number of α particles. These observations constitute evidence suggesting a bystander effect, i.e., that unirradiated cells are responding to damage induced in irradiated cells. This bystander effect in a biological system of relevance to carcinogenesis could have significant implications for risk estimation for low-dose radiation.

MECHANISM OF RADIATION-INDUCED BYSTANDER EFFECTS: A UNIFYING MODEL

The radiation‐induced bystander effect represents a paradigm shift in our understanding of the radiobiological effects of ionizing radiation, in that extranuclear and extracellular events may also contribute to the final biological consequences of exposure to low doses of radiation. Although radiation‐induced bystander effects have been well documented in a variety of biological systems, the mechanism is not known. It is likely that multiple pathways are involved in the bystander phenomenon, and different cell types respond differently to bystander signalling. Using cDNA microarrays, a number of cellular signalling genes, including cyclooxygenase‐2 (COX‐2), have been shown to be causally linked to the bystander phenomenon. The observation that inhibition of the phosphorylation of extracellular signal‐related kinase (ERK) suppressed the bystander response further confirmed the important role of the mitogen‐activated protein kinase (MAPK) signalling cascade in the bystander process. Furthermore, cells deficient in mitochondrial DNA showed a significantly reduced response to bystander signalling, suggesting a functional role of mitochondria in the signalling process. Inhibitors of nitric oxide (NO) synthase (NOS) and mitochondrial calcium uptake provided evidence that NO and calcium signalling are part of the signalling cascade. The bystander observations imply that the relevant target for various radiobiological endpoints is larger than an individual cell. A better understanding of the cellular and molecular mechanisms of the bystander phenomenon, together with evidence of their occurrence in‐vivo, will allow us to formulate a more accurate model for assessing the health effects of low doses of ionizing radiation.

The Columbia University Single-Ion Microbeam

Abstract Randers-Pehrson, G., Geard, C. R., Johnson, G., Elliston, C. D. and Brenner, D. J. The Columbia University Single-Ion Microbeam. Radiat. Res. 156, 210–214 (2001). A single-ion microbeam facility has been constructed at the Columbia University Radiological Research Accelerator Facility. The system was designed to deliver defined numbers of helium or hydrogen ions produced by a van de Graaff accelerator, covering a range of LET from 30 to 220 keV/μm, into an area smaller than the nuclei of human cells growing in culture on thin plastic films. The beam is collimated by a pair of laser-drilled apertures that form the beam-line exit. An integrated computer control program locates the cells and positions them for irradiation. We present details of the microbeam facility including descriptions of the collimators, hardware, control program, and the various protocols available. Various contributions to targeting and positioning precision are discussed along with our plans for future developments. Beam time for outside users is often available (see www.raraf.org).

Past Exposure to Densely Ionizing Radiation Leaves a Unique Permanent Signature in the Genome

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.

Atm Inactivation Results in Aberrant Telomere Clustering during Meiotic Prophase

ABSTRACT A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm−/− mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly inAtm −/− mice. Numerous spermatocytes ofAtm −/− mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm −/− mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes ofAtm −/− and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.

Interaction between Radiation-Induced Adaptive Response and Bystander Mutagenesis in Mammalian Cells

Abstract Zhou, H., Randers-Pehrson, G., Geard, C. R., Brenner, D. J., Hall, E. J. and Hei, T. K. Interaction between Radiation-Induced Adaptive Response and Bystander Mutagenesis in Mammalian Cells. Radiat. Res. 160, 512–516 (2003). Two conflicting phenomena, the bystander effect and the adaptive response, are important in determining biological responses at low doses of radiation and have the potential to have an impact on the shape of the dose–response relationship. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, we reported previously that nonirradiated cells acquired mutagenesis through direct contact with cells whose nuclei had previously been traversed with either a single or 20 α particles each. Here we show that pretreatment of cells with a low dose of X rays 4 h before α-particle irradiation significantly decreased this bystander mutagenic response. Furthermore, bystander cells showed an increase in sensitivity after a subsequent challenging dose of X rays. Results from the present study address some of the pressing issues regarding both the actual target size and the radiation dose response and can improve on our current understanding of radiation risk assessment.

Effects of irradiated medium with or without cells on bystander cell responses

Recent studies have indicated that extranuclear or extracellular targets are important in mediating the bystander genotoxic effects of alpha-particles. In the present study, human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-mylar dishes 2-4 days before irradiation, depending on the density requirement of experiments. One side (with or without cells) was irradiated with alpha-particles (from 0.1 to 100 Gy) using the track segment mode of a 4 MeV Van de Graaff accelerator. After irradiation, cells were kept in the dishes for either 1 or 48 h. The non-irradiated cells were then collected and assayed for both survival and mutation. When one side with cells was irradiated by alpha-particles (1, 10 and 100 Gy), the surviving fraction among the non-irradiated cells was significantly lower than that of control after 48 h co-culture. However, such a change was not detected after 1h co-culture or when medium alone was irradiated. Furthermore, co-cultivation with irradiated cells had no significant effect on the spontaneous mutagenic yield of non-irradiated cells collected from the other half of the double-mylar dishes. These results suggested that irradiated cells released certain cytotoxic factor(s) into the culture medium that killed the non-irradiated cells. However, such factor(s) had little effect on mutation induction. Our results suggest that different bystander end points may involve different mechanisms with different cell types.

Radioresponse of human astrocytic tumors across grade as a function of acute and chronic irradiation

Astrocytomas make up the largest group of primary brain tumors of glial origin. Long term survival is rare with high grade tumors (grades 3 and 4), which recur despite subtotal resection, chemotherapy, and aggressive postoperative radiation therapy. In contrast, the 5-year survival for low grade astrocytomas (grades 1 and 2) following subtotal resection and postoperative radiotherapy approaches 50%. Variable sensitivity across grade may contribute to the difference in the behavior of these tumors. To investigate this possibility, the radioresponse of human glial tumors across grade as a function of the dose rate of irradiation was studied. Cell lines derived from a low grade astrocytoma (grade 1) and two high grade astrocytomas (grades 3 and 4) were established in culture. Clonal survival was determined following irradiation of the three cell lines with Cesium 137 gamma rays at high dose rate, 78 Gy/hr, and at low dose rate, range 14 cGy to 79 cGy/hr. The low grade astrocytoma was found to be more radiosensitive than either of the high grade tumors. The alpha/beta (Gy-1/Gy-2) values (linear quadratic model) were 0.35/0.082 for the grade 1 line and 0.20/0.036 and 0.30/0.045 for the grade 3 and 4, respectively. D0 (cGy) values (single-hit multi-target model) were 99, 144, and 117 for grades 1, 3, and 4, respectively. A dose rate effect was present for all three tumor lines irradiated from 14 cGy/hr to 78 Gy/hr. An inverse dose rate effect was also noted at 37 cGy/hr for each of the astrocytic lines. These findings may be useful in the development of strategies to treat astrocytic brain tumors which use high and/or low dose rate irradiation.

Radiation and taxol effects on synchronized human cervical carcinoma cells

PURPOSE To evaluate the effectiveness of the plant derived chemotherapeutic agent taxol alone and in combination with ionizing radiation on synchronous and asynchronous human cervical carcinoma cells and to define the mechanistic basis for this cytotoxic response. METHODS AND MATERIALS Asynchronous and synchronous cells (obtained by modified mitotic shake-off) derived from carcinomas of the human uterine cervix were treated with a range of concentrations of taxol (0, 1.0, 2.5, 5.0, 10.0 and 20.0 nM) for either 8, 24 or 48 h. Synchronized cell cycling was evaluated by counting mitotic indices and by uptake of bromodeoxyuridine (BrdUrd). Cells were irradiated (137Cs gamma rays at 1.12 Gy/min) alone and after taxol treatment and plating efficiencies and radiosensitivity determined. RESULTS Taxol treatment resulted in a dose time dependent loss of colony forming ability with 10 nM for 24 h producing about 10% cell survival. Irradiating taxol treated cells resulted in a strictly additive response in contrast to previous supra-additive results with astrocytoma and melanoma cells. Mitotically synchronized cells rapidly moved into G1 phase with a second mitotic peak at 28 h (total cycle time). Taxol treatment resulted in a continued accumulation of mitoses, and a failure and/or delay of entry of a fraction of cells into S phase after a G1 phase of at least 10 h. That is, taxol effects cell cycling at a stage other than G2/M. Irradiating (3 Gy) synchronized cells showed a 10-fold variation in sensitivity, with mitosis as the most sensitive phase with taxol alone resulting in some cytotoxicity and combined effects additive or less than additive. CONCLUSION Taxol effects these cervical carcinoma cells at other stages of the cell cycle than G2/M. This may explain the failure to obtain taxol radiosensitization with these cells and it may indicate that taxol has a multiplicity of actions with differences in effectiveness likely between cells of different origins.