Role of apoptosis in low-dose hyper-radiosensitivity.

Little is known about the mode of cell killing associated with low-dose hyper-radiosensitivity, the radiation response that describes the enhanced sensitivity of cells to small doses of ionizing radiation. Using a technique that measures the activation of caspase 3, we have established a relationship between apoptosis detected 24 h after low-dose radiation exposure and low-dose hyper-radiosensitivity in four mammalian cell lines (T98G, U373, MR4 and 3.7 cells) and two normal human lymphoblastoid cell lines. The existence of low-dose hyper-radiosensitivity in clonogenic survival experiments was found to be associated with an elevated level of apoptosis after low-dose exposures, corroborating earlier observations (Enns et al., Mol. Cancer Res. 2, 557-566, 2004). We also show that enriching populations of MR4 and V79 cells with G(1)-phase cells, to minimize the numbers of G(2)-phase cells, abolished the enhanced low-dose apoptosis. These cell-cycle enrichment experiments strengthen the reported association between low-dose hyper-sensitivity and the radioresponse of G(2)-phase cells. These data are consistent with our current hypothesis to explain low-dose hyper-radiosensitivity, namely that the enhanced sensitivity of cells to low doses of ionizing radiation reflects the failure of ATM-dependent repair processes to fully arrest the progression of damaged G(2)-phase cells harboring unrepaired DNA breaks entering mitosis.

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks.

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.

Expression of proteins coincident with inducible radioprotection in human lung epithelial cells.

Human lung epithelial cells and many other cell lines are hypersensitive to low doses of ionizing radiation (<0.2 Gy). However, above a threshold dose of 0.4-0.6 Gy, an induced radioprotective response is triggered that protects cells at higher radiation doses. At 4 h, when maximal induced radioprotection is seen in these cells after low-dose priming, the two-dimensional gel protein expression pattern in 0.5-Gy-exposed cells is subtly altered, with seven proteins being 2- to 5-fold down-regulated and one being 2-fold up-regulated. They include: (a) the protein kinase C inhibitor 1, or histidine triad nucleotide-binding motif (HINT) protein; (b) substrates for protein kinase C activity including the chloride intracellular channel protein 1; and (c) a cytoskeletal protein degraded during apoptosis. In addition, a lung cancer-specific protein that binds to both telomeres and nascent mRNA molecules is down-regulated, as is interleukin 1alpha. Therefore, at least in human lung epithelial cells, radioprotection may be the result of signaling pathway switching, which results in the removal of damaged cells and the preparation for enhanced general transcription in surviving cells during a period in which cell proliferation is repressed. This combination of events may be cell-type-specific and may have implications for the protection of normal lung tissue during unavoidable radiation exposure such as in radiotherapy.

Radiogenetic therapy: strategies to overcome tumor resistance.

The aim of cancer gene therapy is to selectively kill malignant cells at the tumor site, by exploiting traits specific to cancer cells and/or solid tumors. Strategies that take advantage of biological features common to different tumor types are particularly promising, since they have wide clinical applicability. Much attention has focused on genetic methods that complement radiotherapy, the principal treatment modality, or that exploit hypoxia, the most ubiquitous characteristic of most solid cancers. The goal of this review is to highlight two promising gene therapy methods developed specifically to target the tumor volume that can be readily used in combination with radiotherapy. The first approach uses radiation-responsive gene promoters to control the selective expression of a suicide gene (e.g., herpes simplex virus thymidine kinase) to irradiated tissue only, leading to targeted cell killing in the presence of a prodrug (e.g., ganciclovir). The second method utilizes oxygen-dependent promoters to produce selective therapeutic gene expression and prodrug activation in hypoxic cells, which are refractive to conventional radiotherapy. Further refining of tumor targeting can be achieved by combining radiation and hypoxia responsive elements in chimeric promoters activated by either and dual stimuli. The in vitro and in vivo studies described in this review suggest that the combination of gene therapy and radiotherapy protocols has potential for use in cancer care, particularly in cases currently refractory to treatment as a result of inherent or hypoxia-mediated radioresistance.

Molecular approaches to chemo-radiotherapy.

Although radiotherapy is used to treat many solid tumours, normal tissue tolerance and inherent tumour radioresistance can hinder successful outcome. Cancer gene therapy is one approach being developed to address this problem. However, the potential of many strategies are not realised owing to poor gene delivery and a lack of tumour specificity. The use of treatment-, condition- or tumour-specific promoters to control gene-directed enzyme prodrug therapy (GDEPT) is one such method for targeting gene expression to the tumour. Here, we describe two systems that make use of GDEPT, regulated by radiation or hypoxic-responsive promoters. To ensure that the radiation-responsive promoter is be activated by clinically relevant doses of radiation, we have designed synthetic promoters based on radiation responsive CArG elements derived from the Early Growth Response 1 (Egr1) gene. Use of these promoters in several tumour cell lines resulted in a 2-3-fold activation after a single dose of 3 Gy. Furthermore, use of these CArG promoters to control the expression of the herpes simplex virus (HSV) thymidine kinase (tk) gene in combination with the prodrug ganciclovir (GCV) resulted in substantially more cytotoxicity than seen with radiation or GCV treatment alone. Effectiveness was further improved by incorporating the GDEPT strategy into a novel molecular switch system using the Cre/loxP recombinase system of bacteriophage P1. The level of GDEPT bystander cell killing was notably increased by the use of a fusion protein of the HSVtk enzyme and the HSV intercellular transport protein vp22. Since hypoxia is also a common feature of many tumours, promoters containing hypoxic-responsive elements (HREs) for use with GDEPT are described. The development of such strategies that achieve tumour targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy.

The therapeutic advantage of combined X-rays and melphalan.

Early skin reactions on mouse feet and delay in growth of a mouse tumor (CA NT) were measured after combined treatments with X-rays and the cytotoxic drug Melphalan. The drug was given as a single dose (10 mg kg-1) with graded single doses of X-rays, either before or after irradiation with an interval of up to 4 days. In the mouse skin, addition of the drug increased the radiation response only slightly. The maximum Enhancement Ratio (ER) measured at a skin reaction of 1.5 (approximately 23 Gy) was 1.07 +/- 0.02 SEM for the schedule MEL 3 days before X-rays. ER's for all schedules tested were similar with a range of 1.00 to 1.07. For the tumor more enhancement was observed; the largest ER's were found when Melphalan was given before rather than after irradiation, with maximum ER's of 2.3 and 2.4 when the drug was given 3 days or 1 day before X-rays. This has been attributed to reoxygenation of hypoxic cells after drug treatment, rendering the tumor more radiosensitive. The range of ER over all schedules was 1.5-2.4. Since ER is greater for the tumor than skin for all schedules, a therapeutic advantage is indicated under these specific experimental conditions of single X-ray and drug doses.

Role of glutathione peroxidase in the radiation response of mouse kidney.

Glutathione peroxidase (GSH-Px) has been implicated in mediating the radioprotective effects of glutathione (GSH). This hypothesis was tested in vivo by determining the effect of GSH-Px depletion on the radiation response of murine kidneys. Renal GSH-Px levels were depleted to 17% of control values by feeding animals a selenium deficient diet for 6 weeks; this had no significant effect on renal levels of GSH or GSH-S-transferase (GST). However, we also tested the effect of direct depletion of GSH to 3-4% of control values, using a combination of DL-buthionine sulphoximine (BSO) and diethyl maleate (DEM). Kidneys with normal or depleted levels of GSH-Px and/or GSH were irradiated with 240kVp X rays (2 fractions, 7 days apart to minimize intestinal injury). Mice breathed 7% oxygen during irradiation. Renal damage was assessed at 20, 25, and 32 weeks after the first fraction of X rays, in terms of reduced hematocrit and renal clearance of 51Cr-EDTA. Depletion of GSH-Px levels to 17% of control did not alter renal radiosensitivity, but depletion of GSH to 3-4% of control values radiosensitized the kidney by a factor of 1.4. Depletion of both GSH and GSH-Px together did not radiosensitize the kidney any more than was achieved by GSH depletion alone.

Low-dose hypersensitivity: current status and possible mechanisms.

PURPOSE: To retain cell viability, mammalian cells can increase damage repair in response to excessive radiation-induced injury. The adaptive response to small radiation doses is an example of this induced resistance and has been studied for many years, particularly in human lymphocytes. This review focuses on another manifestation of actively increased resistance that is of potential interest for developing improved radiotherapy, specifically the phenomenon in which cells die from excessive sensitivity to small single doses of ionizing radiation but remain more resistant (per unit dose) to larger single doses. In this paper, we propose possible mechanisms to explain this phenomenon based on our data accumulated over the last decade and a review of the literature. CONCLUSION: Typically, most cell lines exhibit hyper-radiosensitivity (HRS) to very low radiation doses (<10 cGy) that is not predicted by back-extrapolating the cell survival response from higher doses. As the dose is increased above about 30 cGy, there is increased radioresistance (IRR) until at doses beyond about 1 Gy, radioresistance is maximal, and the cell survival follows the usual downward-bending curve with increasing dose. The precise operational and activational mechanism of the process is still unclear, but we propose two hypotheses. The greater amount of injury produced by larger doses either (1) is above a putative damage-sensing threshold for triggering faster or more efficient DNA repair or (2) causes changes in DNA structure or organization that facilitates constitutive repair. In both scenarios, this enhanced repair ability is decreased again on a similar time scale to the rate of removal of DNA damage.

Enhancement of tumor radiosensitivity and reduced hypoxia-dependent binding of a 2-nitroimidazole with normobaric oxygen and carbogen: a therapeutic comparison with skin and kidneys.

To evaluate the therapeutic potential of normobaric oxygen and carbogen as hypoxic-cell sensitizers, both radiosensitization in a mouse mammary carcinoma, mouse skin and kidneys, and the reduction in the proportion of hypoxic tumor cells were quantified in mice breathing air, oxygen, or carbogen. Local tumor control, acute skin reactions, reduced renal clearance, and hematocrit were used as assays. X rays as 10 fractions in 5 days were given to skin and tumors and 10F/12 days to kidneys. In the tumor study, the pre-irradiation breathing time was varied from 2 to 20 min. Hypoxic cells, before and during a 10F/5 day schedule, were quantified using a 2-nitroimidazole with a theophylline side chain. Bioreductively reduced metabolites of this probe were localized in hypoxic cells that were then stained using an immunofluorescent technique and analyzed by flow cytometry. The fraction of cells with high fluorescence intensity was 19% in air, 9% in oxygen, and 3% in carbogen-breathing mice. For all three gases, hypoxia-dependent binding was similar in non-irradiated tumors and those treated with four or nine fractions. Both gases significantly enhanced tumor radiosensitivity (ER = 1.3 to 1.6) and carbogen was slightly more effective than oxygen. With carbogen, maximum sensitization was observed with a 5 min pre-irradiation breathing interval. With oxygen, pre-irradiation breathing times of 2-20 min gave similar sensitization. In skin an enhancement ratio of 1.2 was observed, whereas enhancement ratios for both renal endpoints were significantly lower (1.0 to 1.07). Relative to both tissues, there was therefore a substantial therapeutic gain by irradiating CaNT tumors under both gases, especially with carbogen.

The translational research chain: is it delivering the goods?

PURPOSE: To address whether the translational research chain has influenced clinical practice in radiation oncology. METHODS AND MATERIALS: Merits and limitations of the various steps of the translational chain, i.e., in vitro studies, animal experiments, biomathematical modeling, Phase I and II trials, and randomized Phase III trials are briefly reviewed. The process and value of translational research in radiation oncology are addressed using dose fractionation and the time factor in tumors as examples. RESULTS: The examples show that translational research may indeed change clinical practice in radiation oncology. However, it takes several decades and considerable efforts to define and test new strategies. The "translational process" is by no means unidirectional but a continuing multiway dialog among basic scientists, applied scientists, clinical scientists, and clinical oncologists. CONCLUSION: Translational research works in radiation oncology, and it is difficult to conceive a better alternative for future improvement of therapy. The slow speed of the translational process indicates that there is a need for improving the various steps of the translational network and the interaction as a whole. Massive investments in one part of the network are likely to be at least partly wasted unless the other links are strengthened as well.

A comparison of the effects of p(62)-Be and d(16)-Be neutrons in the mouse kidney.

Renal damage in the mouse was assessed after irradiation with p(62)-Be neutrons from the cyclotron at Clatterbridge, U.K., and compared with the renal response to d(16)-Be neutrons. One, 2, 4 and 8 fractions of radiation were given, or 8 low-dose fractions followed by a "top-up" dose of d(4)-Be neutrons at the Gray Laboratory. The use of both full course fractionation and the top-up technique in this study allowed ratios of isoeffective neutron dose at the two energies (NDR) to be measured over a wide range of neutron dose per fraction from 0.2 to 9.6 Gy. NDR (neutron dose ratio) is a direct way to specifying the relative RBE's of the two neutron beams. Radiation injury in the kidney was assayed with three methods: clearance of [51Cr]EDTA, reduction in haematocrit, and urine output. The dose-response curves obtained were resolved best at 31 weeks post-irradiation in these experiments. All three assays gave similar results. NDR was roughly constant at approximately 1.38 over the complete dose range. For equal effects in the kidney, the standard Hammersmith protocol for d(16)-Be neutrons of 17.6 Gy (N + gamma dose) given in 12 fractions would require 24.6 Gy in 12 fractions using the p(62)-Be beam at Clatterbridge. NDR for renal damage was greater than the NDR obtained previously for mouse skin (1.1-1.2) at all doses, indicating that kidneys would be spared in fractionated treatments with p(62)-Be neutrons compared with d(16)-Be neutrons if radiation doses were given to the same skin tolerance. Examination of the RBE of the two neutron beams relative to 240 kVp X-rays showed that this reflects a lower RBE compared with skin for p(62)-Be neutrons in the clinical dose-range, and a higher RBE compared with skin for d(16)-Be neutrons. These radiobiological data therefore favour the use of high energy neutron therapy if kidneys are included in the field, but this conclusion should not be extrapolated to other late responding normal tissues for which further experimental data should be accrued.

The influence of dose per fraction on repair kinetics.

The use of multiple fractions per day (MFD) in radiotherapy requires information about the rate of repair of radiation injury. It is important to know the minimum interval between fractions necessary for maximum sparing of normal tissue damage, whether rate of repair is dependent on the size of dose per fraction and if it is different in early and late responding tissues and in tumours. To address these questions, the rate of repair between radiation dose fractions was measured in mouse skin (acute damage), mouse kidney (late damage) and a mouse tumour (carcinoma NT). Skin and kidney measurements were made using multiple split doses of X-rays, followed by a neutron top-up. For skin, faster recovery was obtained with 4.4 Gy fractions (t1/2 = 1.29 +/- 0.35 h, 95% CL) than with 10.5 Gy fractions (t1/2 = 3.46 +/- 0.88 h). In contrast, kidney showed slower recovery at a low dose per fraction of 2 Gy (t1/2 = 1.69 +/- 0.39 h) than at a higher dose of 7 Gy per fraction (t1/2 = 0.92 +/- 0.1 h). These data show that repair rate is dependent on the size of dose per fraction, but not in a simple way. T1/2 values now available for many different tissues generally lie in the range of 1-2 h, and are not correlated with proliferation status or early versus late response to treatment. At the doses used currently in clinical MFD treatments, these data indicate that damage in almost all normal tissues would increase if interfraction intervals less than 6 h were used.(ABSTRACT TRUNCATED AT 250 WORDS)

The response of mouse skin to irradiation with neutrons from the 62 MV cyclotron at Clatterbridge, U.K.

Acute skin reactions on mouse feet were used to measure the effect of 62 MeV p-Be neutrons from the cyclotron at Clatterbridge, U.K. The results were compared with the response to 16 MeV d-Be neutrons from the cyclotron at Hammersmith, 4 MeV d-Be neutrons from the van de Graaff accelerator at the Gray Laboratory, and 250 kVp X-rays. Up to 16 equal radiation fractions were given alone, or 16 fractions followed by "top-up" doses of 4 MeV d-Be neutrons to study the effect of neutron doses less than 1 Gy per fraction. For equivalent skin reactions, 9-16% more dose (total neutron + gamma) was needed with p(62)-Be neutrons compared with d(16)-Be neutrons. This did not vary significantly between 1 and 16 fractions. The top-up studies indicated that this figure might rise to approximately 14-32% at very low doses of neutrons, the value depending on the method of analysis of the data. The data indicate that the "standard" clinical protocol of 1.47 Gy per fraction (N + gamma dose) in 12 fractions given at Hammersmith with d(16)-Be neutrons would correspond to a dose of 1.64 Gy per fraction (N + gamma) at Clatterbridge using a similar regime of p(62)-Be neutrons. d(4)-Be neutrons were more effective than d(16)-Be neutrons by a factor of 1.6 over the whole range of dose per fraction studied (0.05-14.5 Gy per fraction of d(4)-Be neutrons). Relative to X-rays, the RBE for p(62)-Be neutrons was 1.6 +/- 0.02 for a single X-ray dose of 30 Gy, rising to 2.9 +/- 0.04 for an X-ray dose per fraction of 4.6 Gy given 16 times. The full-course fractionation data and the top-up data together indicate an extrapolated limiting RBE at vanishingly small doses per fraction of 4.2-4.8 depending on the method of analysis. The X-ray data were well-fitted by a linear-quadratic (LQ) model of dose-fractionation, with alpha/beta = 8.6 +/- 1.5 Gy. The LQ model also provides a fairly good description of the neutron responses, alpha/beta being large (greater than 24) reflecting predominantly linear underlying dose-responses for all the neutron beams. This in turn reflects the small variation observed in the relative effectiveness between the 3 neutron beams with changes in dose per fraction.

Expression of dose in neutron therapy.

A pragmatic approach in neutron dosimetry is to consider the energy spectrum to consist of a neutron and a gamma component. The relationship between the two components of dose in neutron radiotherapy has been investigated for energies currently in clinical use. Changes in the neutron component itself are not dealt with. Because the two components are given simultaneously there is some interaction between them so that the gamma fraction is more effective than if the neutron and gamma doses were separated in time. This interaction has been accounted for using a well-proven extension of the linear-quadratic (LQ) equation. Using values of the LQ parameters alpha and beta measured recently in vivo, we have modelled the total effect from the neutron and gamma dose-contributions in terms of an equivalent neutron dose. This allows a comparison of different methods of expressing the measured physical dose with the biologically effective dose. The current practice in neutron therapy is to give the total (neutron plus gamma) dose, quoting also the gamma contamination. In all cases within the range of energies used for therapy, the total dose will give an overestimate of the biologically effective dose by approximately 4% for each 5% of gamma contamination. Expression of the neutron dose only (ignoring the gamma component) will give an underestimate of the biologically effective dose by approximately 1.5% for each 5% of gamma contamination, i.e. the error is approximately three times less for neutron dose than for total dose.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of very low radiation doses on the human bladder carcinoma cell line RT112.

Human RT112 cells were exposed to single doses of X-rays (0.05-5 Gy), and cell survival was measured using a Dynamic Microscopic Imaging Processing Scanner (DMIPS) with which individual cells can be located in tissue-culture flasks, their positions recorded, and after an appropriate incubation time the recorded positions revisited to allow the accurate scoring of survivors. The response over the X-ray dose range 1-5 Gy showed a good fit to a Linear-Quadratic (LQ) model. For X-ray doses < 1 Gy, an increased effect of X-rays was observed with cell survival below the prediction from the LQ model extrapolated from higher doses. Several arguments suggest that this phenomenon could reflect an induced radioresistance so that in this cell line, low single doses of X-rays are more effective per Gray than higher doses in reducing cell survival because only at higher doses, above a threshold, is there sufficient damage to trigger radioprotective mechanisms.

Recovery kinetics in mouse skin and CaNT tumours.

Recovery kinetics and recovery capacity were studied in a fast proliferating normal tissue, skin, and in an anaplastic mouse mammary carcinoma, CaNT. Three fractions per day of X-rays, repeated over 5 days, were given at varying interfraction intervals from 0 to 8 h. The rate of recovery in tumours (t1/2 = 0.31 +/- 0.15 h for local control) was significantly faster than in skin (t1/2 = 0.96 +/- 0.10 h). By contrast, the fractionation sensitivity of CaNT tumours was less than that of skin (alpha/beta = 43.3 +/- 8.5 Gy vs. alpha/beta = 7.9 +/- 0.2 Gy). Tissues with recovery half-times similar to or longer than that determined for skin would be at risk if interfraction intervals less than 6 h are used in regimes which involve the use of two or more fractions per day. This would be especially true for tissues that show a greater sensitivity to dose fractionation, and hence more sparing of radiation damage with hyperfractionation.

Radiation response of murine eccrine sweat glands.

Following irradiation of the left-hind feet of mice, we measured the ability of the eccrine glands to secrete sweat following stimulation by pilocarpine. Silicone elastomer impression moulds of the foot pads gave repeatable, detailed localization of sweat ducts by retaining the impression of each emerging sweat droplet. Loss of gland function occurred rapidly following irradiation (within 2 weeks) and the rate of loss was dose-dependent, being over three times greater following a dose of 13.0 Gy than after 6.8 Gy. There was a dose-dependent nadir of function at around 8 weeks, followed by a gradual recovery that was complete by about 30 weeks after irradiation, leaving a dose-dependent residual functional deficit. Eccrine sweat glands are very radiosensitive organs compared with the epidermis. A single dose of 13 Gy resulted in complete loss of eccrine gland function at 8 weeks whilst about 23 Gy would be required to elicit transient moist desquamation, in oxygen-breathing mice. Substantial sparing was seen when two doses were split by intervals of up to 24 h.

The Klaas Breur Lecture. Radiation, hypoxia and genetic stimulation: implications for future therapies.

The cellular stress response, whereby very low doses of cytotoxic agents induce resistance to much higher doses, is an evolutionary defence mechanism and is stimulated following challenges by numerous chemical, biological and physical agents including particularly radiation, drugs, heat and hypoxia. There is much homology in the effects of these agents which are manifest through the up-regulation of various genetic pathways. Low-dose radiation stress influences processes involved in cell-cycle control, signal transduction pathways, radiation sensitivity, changes in cell adhesion and cell growth. There is also homology between radiation and other cellular stress agents, particularly hypoxia. Whereas traditionally, hypoxia was regarded mainly as an agent conferring resistance to radiation, there is now much evidence illustrating the cytokine-like properties of hypoxia as well as radiation. Stress phenomena are likely to be important in risks arising from low doses of radiation. Conversely, exploitation of the stress response in settings appropriate to therapy can be particularly beneficial not only in regard to radiation alone but in combinations of radiation and drugs. Similarly, tissue hypoxia can be exploited in novel ways of enhancing therapeutic efficacy. Bioreductive drugs, which are cytotoxically activated in hypoxic regions of tissue, can be rendered even more effective by hypoxia-induced increased expression of enzyme reductases. Nitric oxide pathways are influenced by hypoxia thereby offering possibilities for novel vascular based therapies. Other approaches are discussed.

Growth of fibroblasts as a potential confounding factor in soft agar clonogenic assays for tumour cell radiosensitivity.

Soft agar clonogenic assays are considered to be a standard method for measuring tumour cell radiosensitivity and it has been widely reported that fibroblast contamination does not occur. We report here that human fibroblasts can proliferate to form colonies in a modified form of the Courtenay-Mills soft agar clonogenic assay. It was observed that early passage skin fibroblasts could form colonies in soft agar, although the plating efficiencies were reduced compared with growth on plastic. It was demonstrated that normal lung could proliferate in agar with similar plating efficiencies to fresh tumours and that fibroblastic cells were present in these cultures. Characterisation of primary lung tumour cultures also showed that fibroblastic cells were present in these cultures. Characterisation of primary lung tumour cultures also showed that fibroblastic cells were present which lacked epithelial features and which resembled closely the cells found in cultures of normal lung. This is an important finding for workers using soft agar assays to culture human tumour cells and is of interest in understanding the processes of normal growth control of human fibroblasts.

A therapeutic benefit from combining normobaric carbogen or oxygen with nicotinamide in fractionated X-ray treatments.

The ability of normobaric oxygen and carbogen (95% O2 + 5% CO2) combined with nicotinamide to enhance the radiosensitivity of two rodent adenocarcinomas and of mouse skin and kidneys, using a 10 fraction radiation schedule, was compared with the effect of radiation in air with and without the drug. Tumour response was assayed using local control and regrowth delay, and compared with acute skin reactions, decreased renal 51Cr-EDTA clearance and reduction in haematocrit. Nicotinamide increased the radiation sensitivity of CaNT tumours under all three different oxygen concentrations tested (21, 95 and 100% oxygen). The effect was statistically significant for oxygen and carbogen but not for air; the combination of nicotinamide with carbogen gave the greatest increase in tumour radiosensitivity. Relative to treatments in air without the drug, the enhancement ratios (ER) at the TCD50 level were 1.17, 1.65 and 1.83 for CaNT tumours irradiated in air, oxygen or carbogen and injected with nicotinamide 1 h before each fraction. The ER in CaRH tumours irradiated in carbogen plus the drug was 1.83, which was greater, but statistically not significantly different, to that seen with carbogen alone (ER = 1.68). In skin, relative to air without the drug, the increase in radiosensitivity by nicotinamide was greater in oxygen and carbogen than in air (1.29, 1.36 and 1.08, respectively). The ERs for both assays of renal damage were similar and lower than those in skin: less than or equal to 1.07, less than or equal to 1.13 and less than or equal to 1.16 for irradiations done in air, oxygen and carbogen plus nicotinamide, relative to air alone. A comparison of these results in the tumours and normal tissues showed that a significant therapeutic benefit was obtained with normobaric oxygen and carbogen combined with nicotinamide. This benefit is greater than observed with other radiosensitizers tested so far. Toxic side effects of the treatment are unlikely in a clinical situation, since prolonged administration of nicotinamide is well tolerated in man. The combination of normobaric carbogen with nicotinamide could be an effective method of enhancing tumour radiosensitivity in clinical radiotherapy where hypoxia limits the outcome of treatment.