Andrew Kolozsvary

Potentiation of radiation response in human carcinoma cells in vitro and murine fibrosarcoma in vivo by topotecan, an inhibitor of DNA topoisomerase I.

DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the radiation effects on human carcinoma cells in culture and murine fibrosarcoma in mice. Cell culture studies showed that a dose dependent reduction in cell survival was obtained with a 4 hr exposure of the drug following irradiation of cells. No enhancement of cell killing was seen when cells were treated with the drug before irradiation. Preliminary in vivo tumor studies showed a significant radiosensitizing effect of topotecan that was both drug dose (20 mg/kg) and time sequence (4 hr before irradiation) dependent. There was no enhanced skin reaction following the combined treatments.

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

PURPOSE To demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. METHODS AND MATERIALS Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses of irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. RESULTS The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 micrograms/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 +/- 0.1 and 1.3 +/- 0.1, respectively. Exposure of cells to 10 micrograms/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 +/- 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. CONCLUSION An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors.

Ramipril mitigates radiation-induced impairment of neurogenesis in the rat dentate gyrus

BackgroundSublethal doses of whole brain irradiation (WBI) are commonly administered therapeutically and frequently result in late delayed radiation injuries, manifesting as severe and irreversible cognitive impairment. Neural progenitors within the subgranular zone (SGZ) of the dentate gyrus are among the most radiosensitive cell types in the adult brain and are known to participate in hippocampal plasticity and normal cognitive function. These progenitors and the specialized SZG microenvironment required for neuronal differentiation are the source of neurogenic potential in the adult dentate gyrus, and provide a continuous supply of immature neurons which may then migrate into the adjacent granule cell layer to become mature granule cell neurons. The extreme radiosensitivity of these progenitors and the SGZ microenvironment suggests the hippocampus as a prime target for radiation-induced cognitive impairment. The brain renin-angiotensin system (RAS) has previously been implicated as a potent modulator of neurogenesis within the SGZ and selective RAS inhibitors have been implicated as mitigators of radiation brain injury. Here we investigate the angiotensin converting enzyme (ACE) inhibitor, ramipril, as a mitigator of radiation injury in this context.MethodsAdult male Fisher 344 rats received WBI at doses of 10 Gy and 15 Gy. Ramipril was administered beginning 24 hours post-WBI and maintained continuously for 12 weeks.ResultsRamipril produced small but significant reductions in the deleterious effects of radiation on progenitor proliferation and neuronal differentiation in the rat dentate gyrus following 10 Gy-WBI, but was not effective following 15 Gy-WBI. Ramipril also reduced the basal rate of neurogenesis within the SGZ in unirradiated control rats.ConclusionsOur results indicate that chronic ACE inhibition with ramipril, initiated 24 hours post-irradiation, may reduce apoptosis among SGZ progenitors and/or inflammatory disruption of neurogenic signaling within SGZ microenvironment, and suggest that angiotensin II may participate in maintaining the basal rate of granule cell neurogenesis.

Preferential radiosensitization of 9L glioma cells transduced with HSV-tk gene by acyclovir

The antiviral drug acyclovir, an analogue of purine,was found to selectively enhance the radiosensitivity ofrodent tumor cells which were transduced with theherpes simplex virus thymidine kinase gene (HSV-tk). 9Lrat glioma cells transduced with HSV-tk and treatedwith acyclovir (20 μg/ml) for 24 hr beforeor after irradiation were highly sensitive to radiation,as compared with non-transduced glioma cells. When 9Lcells transduced with HSV-tk gene were exposed toacyclovir and radiation, the sensitizer enhancement ratio (SER)was 1.6. In vivo, a significant increase inthe median survival time of rats with 9L-tktumors was observed when acyclovir was administered beforeand after single-dose irradiation, relative to the survivaltime of similar rats receiving radiation alone. Theresults show that an antiviral agent can selectivelyenhance cell killing by radiation in cells transducedwith the HSV-tk, and suggest that the additionof HSV-tk gene therapy to standard radiation therapywill improve the effectiveness of treatment for braintumors.

Fractionated radiosurgery for 9L gliosarcoma in the rat brain.

PURPOSE Fractionated radiosurgery is being carried out in the clinic to improve the therapeutic ratio of single-dose radiosurgery using various fractionation schemes. Because there is a paucity of experimental radiobiological data in the literature on the tumor response and late-responding normal tissue of critical intracranial structures to radiosurgery, the present animal study was designed to compare the response following a single high dose of radiation with that obtained from calculated fractionated doses of radiosurgery. METHODS AND MATERIALS Male Fischer rats with 9L gliosarcoma growing in their brains were stereotactically irradiated and assayed for the tumor control rate and brain tissue damage. The radiation dose needed for 50% tumor control (TCD50) was used as the endpoint of the efficacy of radiosurgery. Normal brain damage was measured histologically following a period of time over 270 days. Histological evaluation included hematoxylin-eosin (H & E), Luxol fast blue and periodic acid Schiff (LFB/PAS) for the presence of myelin and glial fibrillary acidic protein (GFAP) for the assessment of astrocytic re-activity. The optical density of optic nerves and chiasms staining with LFB/PAS was quantitatively measured using a computer image analysis to assess the magnitude of demyelination. RESULTS Radiosurgery (RS) was found to be more effective in curing small tumors than large tumors. The dose required to control 50% of the tumored animals for 120 days was 24, 31, and 40 Gy for 2-, 6-, and 12-day-old tumors, respectively. Using 12-day-old brain tumors, two fractions of 23.5 Gy and three fractions of 18.5 Gy were found to be equivalent to the single dose of 35 Gy for tumor control. For normal brain damages, the visual pathways including optic nerves and chiasm were found to be highly radiosensitive structures. A single dose of 35 Gy produced 100% severe optic neuropathy. The fractionated RS regimens spared substantial optic nerve damage. CONCLUSION The present data provide a strong radiobiological rationale for the use of fractionated RS in the treatment of tumors located near critical normal structures, including visual pathways. The sparing effect of fractionated RS is greater for late-responding tissues, relative to the rapidly proliferating tumor tissues. This report also characterizes the dose/time tolerance relationship of optic neuropathy after single and fractionated RS.

Magnetic resonance imaging of perfusion in rat cerebral 9L tumor after nicotinamide administration

PURPOSE To investigate the effect of nicotinamide on normal brain and 9L tumor blood flow in the rat using magnetic resonance imaging (MRI) and arterial spin tagging. METHODS AND MATERIALS Using MRI at 7 Tesla, measurements of blood perfusion were determined from two-dimensional maps of intracerebral 9L rat tumors and normal Fischer rat brains. The spatial and temporal influence of nicotinamide, 500 mg/kg i.p., on cerebral blood flow (CBF) was studied in normal brain and tumors between 5 and 21 days after tumor implantation. The MRI CBF measurements employed a variable tip-angle-gradient-recalled echo (VTA-GRE-CBF) readout of the magnetization of the tissue slice. The VTA-GRE-CBF required 8 minutes for a blood flow image with inplane resolution of 250 microm x 500 microm x 2 mm. RESULTS Normal brain blood flow decreased following the administration of nicotinamide. In contrast, tumor blood flow remained unaffected in the time following nicotinamide administration. Consequently, the blood flowing in the tumor relative to that in normal brain demonstrated a significant and selective increase in response to nicotinamide administration. Relative tumor blood flow increased at 10 minutes after nicotinamide injection compared with predrug levels and remained elevated for at least 1 hour. CONCLUSION The results suggest that nicotinamide will not enhance radiosensitivity of brain tumors. The results support the use of nicotinamide to improve delivery of anticancer therapeutics through its ability to selectively increase tumor blood flow relative to that in normal brain.

SELECTIVE IN VIVO RADIOSENSITIZATION BY 5-FLUOROCYTOSINE OF HUMAN COLORECTAL CARCINOMA CELLS TRANSDUCED WITH THE E. COLI CYTOSINE DEAMINASE (CD) GENE

PURPOSE The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy. METHODS AND MATERIALS WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone. RESULTS Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization. CONCLUSION The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers.

Mechanisms of radiation-induced skin injury and implications for future clinical trials

Abstract Purpose: To summarize current knowledge regarding mechanisms of radiation-induced skin injury and medical countermeasures available to reduce its severity. Advances in radiation delivery using megavoltage and intensity modulated radiation therapy have permitted delivery of higher doses of radiation to well-defined tumor target tissues. Although skin is not a radiation dose-limiting tissue, injury to skin poses substantial morbidity risks in the curative treatment of cancers, especially when radiation is administered in combination with chemotherapy. In the continuum of radiation-induced skin injury, late effects are most severe being characterized by sub-cutaneous fibrosis and morbidity. The principal pathogenesis is initiated by depletion of acutely responding epithelial tissues and damage to vascular endothelial microvessels. Emerging concepts of radiation- induced skin injury suggest that the recovery of stromal stem cells and tissue repair remain chronically impaired by long-lived free radicals, reactive oxygen species, and pro-inflammatory cytokines/chemokines resulting in progressive damage after radiation exposure. Conclusions: As pathways underlying the cellular and molecular mechanisms of radiation-induced skin injury are becoming better understood, novel approaches are being developed for mitigating or treating the associated pathogenesis.

Increased tumour response of a murine fibrosarcoma to low temperature hyperthermia and low dose rate brachytherapy

The present animal tumour study was carried out to determine the effectiveness of low temperature hyperthermia combined with low dose rate radiation based on the cell culture studies of our laboratory and others that demonstrated a significant radiosensitization obtained by low temperature hyperthermia and low dose rate radiation. Well-oxygenated murine fibrosarcoma Meth-A tumours growing in Balb/c mice were treated with heat (41 degrees C tumour temperature) by immersion of the tumour-bearing leg in a waterbath concurrently with low dose rate radiation. Radiation was delivered using 192Ir interstitial implantation at absolute dose rates of 0.416-0.542 Gy/h. The effect of heat alone on tumour growth and normal tissue was minimal. Tumour growth delay following 30 Gy radiation was 4.9 days. Significant delay in tumour growth was observed with the addition of low temperature hyperthermia delivered concurrently. Enhancement in radiation response was seen with increasing duration of heat treatment; tumour growth delays were 9.5 days following 4 h heat (41 degrees C) treatment and 16 days following 6 h treatment. Three sessions of fractionated hyperthermia 4 h/day during the course of low dose-rate radiation significantly delayed tumour growth to 18.6 days. The results indicate that fractionated heat treatment in conjunction with low dose rate radiation has potential for improving tumour response without adversely affecting normal tissue reaction. This in vivo study represents an extension of the cell culture data and provides further radiobiological basis for the combined use of low temperature hyperthermia and low dose rate radiation.

Time-Dependent Inhibition of Pan-Inflammatory Cytokines Mitigates Radiation-Induced Skin Injury in Mice

Radiation injury to skin poses substantial morbidity risks in the curative treatment of cancers and is also of concern in the context of radiological attack or nuclear accident scenarios. Late effects can be severe and are frequently characterized by subcutaneous fibrosis and morbidity. These experiments presented here assess the potential of MW01-2-151SRM (MW-151), a novel small-molecule inhibitor of microglial activation and associated proinflammatory cytokine/chemokine production, as a mitigator of radiation-induced skin injury. Groups of C57BL/6 mice received focal irradiation of the right hind leg at a dose of 30 Gy. Therapy was initiated either on day 3, day 7 or day 14 postirradiation and maintained subsequently for 21 days by intraperitoneal injections administered three times per week. The primary end point was skin injury, which was assessed three times a week for at least 60 days postirradiation and scored using a semi-quantitative scale. Secondary end points measured at selected times included histology (primarily H&E) and immunofluorescence labeling of various macrophage (F4-80) and inflammatory (TGF-β, TNF-α, MMP9) markers. Relative to untreated controls, mitigation of radiation-induced skin injury in mice receiving MW-151 was highly dependent on the timing of therapy initiation. Initiation on day 3 postirradiation had no discernable effect, whereas mitigating effects were maximal following initiation on day 7 and present to a lesser degree following initiation on day 14. The response to MW-151 therapy in individual animals was essentially all-or-none and the relative benefits associated with the timing of therapy initiation primarily reflected differences in the number of responders. These data support the hypothesis that proinflammatory cytokines/chemokines play complex roles in orchestrating the response to radiation-induced skin injury and suggest that there is a critical period during which they initiate the pathogenesis resulting in late effects.