1990, The British Journal of Radiology, 63, 585-588
Proceedings of the British Institute of Radiology Radiobiology proffered papers Abstracts of papers presented at a meeting of the BIR at 36 Portland Place, London W1 N 4AT, on Friday, 23 February 1990 Radiosensitivity of human tumour cell lines assessed in the adhesive tumour cell culture system, by C. S. Parkins and G. G. Steel The effect of LET on the radiation response of human tumour cell lines of differing radiosensitivity, by J. Peacock, C. S. Parkins, G. G. Steel, R. Britten and H. Warenius. Abstract not received The acute response of mouse kidney cells after fractionated irradiation assayed by colony formation in primary culture, by Y.-M. Jen and J. H. Hendry Pathogenesis of radiation nephropathy. morphological studies in the pig, by M. Wooldridge, M. E. C. Robbins, M. Rezvani, S. J. Golding and J. W. Hopewell The effect of myeloablation on the subsequent^engraftment and homing of haemopoietic precursor cells in long-term cultures, by J. G. Bierkens, J. H. Hendry and N. G. Testa Radiosensitization in vivo: oral (po) dosing with RSU 1069 or RB 6145 maintains potency but reduces toxicity compared with parenteral (ip) administration, by S. Cole, I. J. Stratford, E. Wright, J. Nolan and G. E. Adams Repair, redistribution and repopulation: the relative importance in determining the acute response of the skin to fractionated irradiation, by G. J. M. J. van den Aardweg and J. W. Hopewell Dose and field-size related changes in the thickness of the dermis after ^-irradiation in the pig, by V. K. Sieber, J. C. Heryet and J. W. Hopewell Proliferation in CaNT tumours during and after fractionated radiotherapy assessed by local control, clonogenic survival and cell kinetics, by J. J. Pavy, A. Rojas, R. J. Hodgkiss, G. D. Wilson, J. M. Collier, E. J. Kelleher, C. A. Whitsed and M. C. Joiner Radiation and drug sensitivity measured with the MTT assay, by J. A. Hanson, D. P. Bentley, A. E. Bean, J.-A. Garnet and J. L. Moore Cellular and molecular approaches to intrinsic radioresistance in human malignant glioma, by G. Ross, R. Brown, T. Wheldon, R. I. Freshney, R. Rampling and A. Barrett Radiosensitivity of human tumour cell lines assessed in the adhesive tumour cell culture system C. S. Parkins and G. G. Steel Institute of Cancer Research, Sutton, Surrey The adhesive tumour cell culture system (ATCCS) has been proposed as a predictive test for radiosensitivity because of its short culture period, selective growth of tumour cells and because it is thought to yield a result which reflects the clonogenic cell response. We have compared the radiosensitivity of a range of human tumour cell lines using the ATCCS and clonogenic survival. We found that the optical method of assessing cell number and radiosensitivity gave good agreement with clonogenic response at 2 Gy, although the ATCCS response showed unusually high radioresistance at high doses. The relationship between the cell number seeded and optical density was not always linear within individual assays and we prefer to use the term "slope ratio" for the derived radiation response, e.g. SR2 and not SF 2 . The assay could distinguish the radiosensitivity of cervix carcinoma cells (SF2 = 0.7; SR2 = 0.74, 0.80) from neuroblastoma cells (SF2 = 0.3; SR2 = 0.30, 0.41). The use of the cell-adhesive-matrix (CAM) coating on the supplied culture surface did not significantly improve colony-forming efficiency. Results are also presented for the growth and radiosensitivity of xenograft and biopsy tumour samples.
former and 2-5 Gy for the latter. The underlying cause for this difference could be the time available for cellular repair prior to the fixation of potentially lethal damage. Using primary culture and a clonogenic assay, mouse kidney cells after single dose, 2-, 8- and 16-fraction irradiations were assayed for their survival 12 h after single or fractionated irradiations. When the data sets were fitted separately to a linear-quadratic model the a component was constant for all regimens of irradiation. Using a direct analysis, the a//? ratio was 4.4+ 1.3 Gy for the three fractionated regimens plus the single-dose data set, and 2.8 +0.9 Gy for the three fractionated regimens only. The difference was significant (p < 0.0001) and the fitting excluding the single-dose data set was a better fit. The result indicates an cc/p value within the range of values reported using functional kidney assays, and also suggests that the time available for repair prior to assay does not account for the low a/j? ratio observed in mouse kidney.
The acute response of mouse kidney cells after fractionated irradiation assayed by colony formation in primary culture Yee-Min Jen and Jolyan H. Hendry Department of Radiobiology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester
The pathophysiological mechanisms responsible for the development of radiation nephropathy are ill defined. Studies of changes in renal morphology should help ascertain the cell type(s) at risk and, in conjunction with functional data, provide a basis for the development of rational protocols for the treatment and/or prevention of radiation nephropathy. Both kidneys of 13 mature 10-month-old pigs were irradiated with a single dose of 9.8 Gy of ^Co y rays. The pigs were serially killed at periods up to 24 weeks after irradiation. Kidneys were perfusion-fixed, embedded in paraffin-wax and 5 /an thick sec-
It has been shown that the a//? ratios for early- and lateresponding tissues are different, being around 5-20 Gy for the
Vol. 63, No. 751
Pathogenesis of radiation nephropathy: morphological studies in the pig M. Wooldridge, M. E. C. Robbins, *M. Rezvani, fS. J. Golding and *J. W. Hopewell MRC Radiobiology Unit, Chilton, *CRC Normal Tissue Radiobiology Research Group, Research Institute, University of Oxford and ^Oxfordshire Regional CT Unit, Churchill Hospital, Oxford OX3 7LJ
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Proceedings of The British Institute of Radiology tions taken for evaluation. Changes were observed in nonglomerular and glomerular tissue within 2 weeks of irradiation. Non-glomerular lesions initially consisted of focal areas of interstitial fibrosis or tubular atrophy, predominantly in the corticomedullary region or along the path of medullary rays. Maximal damage, affecting over 50% of the cortex, was observed at 6 weeks after irradiation, and consisted of severe interstitial fibrosis, tubular atrophy and inflammatory cell infiltrate. Interstitial fibrosis involved a complete band of subcapsular tissue, with "fingers" of fibrotic tissue extending deep into the cortex. Similar changes were seen up to 24 weeks after irradiation. Glomerular changes consisted of progressive increases in PAS positive staining material in the mesangial matrix, glomerular hypercellularity with cell clumping, thickening of the capillary basement membrane and a decrease in capillary lumina. Between 12 and 24 weeks, damage increased in severity, leading to the development of completely sclerotic end-stage glomeruli which were predominantly subcapsular or juxtamedullary. There was no apparent change in the vasculature. These findings show that (a) there is no one target or dose-limiting cell and (b) the vasculature does not play a major role in the primary development of radiation nephropathy. The effect of myeloablation on the subsequent engraftment and homing of haemopoietic precursor cells in long-term cultures J. G. Bierkens, J. H. Hendry and *N. G. Testa Departments of Radiobiology and * Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital Withington, Manchester M20 9BX Long-term marrow cultures (LTBMCs) reconstitute in vitro the privileged microenvironment which is characteristic for the intact bone marrow and which is required for prolonged haemopoiesis. As such LTBMCs provide a model to study the various aspects of bone marrow transplantation. In the present experiments, the effect of 20 Gy y-irradiation as a modality of myeloablation on the subsequent engraftment of stromal layers in LTBMCs has been assessed and compared with the "homing" ability of haemopoietically inactive cultures. Irradiated stromal layers subsequently re-inoculated with bone marrow cells, displayed a lower supportive capacity for haemopoiesis as compared with corticosteroid deprived stromal layers. This was expressed both as reduced numbers of CFU-S and GM-CFC progenitors and a fall in non-adherent cell production. These observations may be related, at least in part, to a reduced cell homing of haemopoietic cells on the irradiated stroma. A significant decrease by 35% in the cell adhesion of factor dependent FDCP-Mix-A4 cells onto irradiated stromal layers was observed within a dose range of 10 to 20 Gy. Significantly higher doses were required to affect the sulphated GAG content in fully established stromal layers. It is therefore unlikely that the reduced capacity of irradiated stromal layers to home haemopoietic precursor cells is uniquely related to the alterations observed in the sulphated GAG content in these layers. Radiosensitization in vivo: oral (po) dosing with R S U 1069 or RB 6145 maintains potency but reduces toxicity compared with parenteral (ip) administration. S. Cole, I. J. Stratford, E. Wright, J. Nolan and G. E. Adams MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 0RD RSU 1069 is a nitroimidazole hypoxic cell radiosensitizer, which is also directly cytotoxic when reductively metabolized. RB 6145 is a prodrug of RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for
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C3H/He mice was 80mg/kg (0.38 mmol/kg) ip. This MTD increased four-fold to 320mg/kg (1.5 mmol/kg) with po administration. The MTDs of RB 6145 were 35Omg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 towards bone marrow stem cells was also reduced approximately four-fold after po administration; 20 mg/kg ip RSU 1069 and 80 mg/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Half of the mice given the highest po dose of 320 mg/kg of RSU 1069 developed cystic kidneys, with dilation, necrosis and fibrosis of the distal tubules but no nephrotoxicity resulted from the ip administration of 80 mg/kg of RSU 1069. Even relatively low doses of RSU 1069 reduced the number of spermatids visible in histological sections of testes 4 weeks later. There was some dose sparing (about two-fold) after po administration. For RSU 1069 and RB 6145, administered by either route, the maximum reduction in the survival of clonogenic cells from intradermal, murine KHT sarcomas occurred, when the drugs were given 45-60 min before 10 Gy of X rays. A range of doses of RSU 1069 or RB 6145 was administered po or ip 45 min before 10 Gy. The degree of radiosensitization by both compounds was largely independent of the route of administration. Preliminary studies with 3 H-RSU 1069 suggested that efficacy correlated with peak blood level of label and concentration in the tumour at the time of irradiation, which were reduced less than two-fold by po compared with ip administration. Toxicity tended to correlate with total exposure over time, which was reduced two to four-fold by po administration. Oral administration of RSU 1069 or RB 6145 may give therapeutic benefit, since doselimiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was retained. Repair, redistribution and repopulation: the relative importance in determining the acute response of the skin to fractionated irradiation G. J. M. J. van den Aardweg and J. W. Hopewell CRC Normal Tissue Radiobiology Research Group, Research Institute (University of Oxford), Churchill Hospital, Oxford OX3 7LJ The influence of the overall treatment time in fractionated irradiation was studied in the epidermis of the pig. The absence or presence of moist desquamation was assessed after /?-irradiation and dose-effect curves were fitted to the quantal data using probit analysis. Experiments were compared on the basis of ED 50 values: the dose required to produce moist desquamation in 50% of the irradiated skin fields. With an overall treatment time of ^ 1 5 days the iso-effect doses (ED50) for moist desquamation were well correlated (r = 0.99) with the number of fractions (in the range of 1-48 F) when the data were plotted on a log-log scale and when full account was taken for any incomplete repair of sublethal damage. The slope of this Strandquist-plot was represented by a fraction-exponent (N) of 0.42. This is not significantly different from those obtained for so-called late-responding normal tissues. The results of fractionated irradiation given in 1-10 F were well fitted by a Fe-plot suggesting an a//? ratio of 8.7 + 0.5 Gy. However, the data for irradiation given in 6-48 F suggested a significantly lower a//J ratio of 0.85 + 0.3 Gy. Comparison of the ED 50 values for irradiation given as 2 F/14 days with 2 F/38 days indicated a positive time factor with an increase in the iso-effect dose of nearly 40%, owing to repopulation. In contrast, 28 F/38 days compared with 28 F/14 days indicated a negative time factor, i.e. a dose reduction of nearly 30%. This could be explained by the redistribution of the target cells
The British Journal of Radiology, July 1990
Proceedings of The British Institute of Radiology within the cell cycle. Fractionation studies on pig skin using X-irradiation also indicated that repopulation and redistribution are counteracting processes. The overall effectiveness of each varying with the overall treatment time. The presence of a significant degree of re-distribution of cells in the cell cycle conflicts with the basic concept of equal effects per equal dose fraction. Dose and field-size related changes in the thickness of the dermis after {{-irradiation in the pig V. K. Sieber, J. C. Heryet and J. W. Hopewell CRC Normal Tissue Radiobiology Research Group, University of Oxford, Churchill Hospital, Oxford 0X3 7LJ The late response of dermal tissue to jS-irradiation, from sources of 1-40 mm diameter, was assessed in pig skin 2 years after irradiation. Sources of 1 and 2 mm diameter were used to represent "hot particles" of the type encountered in accidental occupational exposures to radiation, whilst the larger sources (5-40 mm) represented the lower end of a range that might be used therapeutically. Single doses of /3-irradiation, in the range 13-230 Gy, were given from '"Sr/^Y plaques of diameter of 1.0, 2.0, 5.0, 11.0, 15.0, 22.5 and 40.0 mm. At 2 years after irradiation samples of skin were fixed, 5 /an histological sections were prepared and the thickness of the dermis in irradiated skin was measured from a projected image of these sections. Comparisons were made with adjacent unirradiated control sites. The thickness of an irradiated area was expressed as a percentage of adjacent dermal thickness. A dose-related reduction in dermal thickness was found, irrespective of the diameter of the source, with a maximum, 60% reduction in dermal thickness. The dose required to produce this maximum reduction in dermal thickness for the 40 mm diameter source was 23.4 Gy whilst for the 1 mm diameter source it was 1050 Gy. After even higher doses there was a less severe reduction in the extent of dermal thinning; this was attributed to the presence of invasive fibrosis. In contrast to the acute response where a field size effect was seen using sources of 5-22.5 mm diameter there was no field-size effect for sources of 5 mm. The mean ED 50 for ^ 30% reduction in dermal thickness following irradiation by sources 5 mm diameter was 18.00 + 0.65 Gy. For the "hot particles" ( l m m and 2 mm diameter) a field-size effect was demonstrated as the ED 50 for ^ 30% reduction in dermal thickness following irradiation with a l m m diameter source was 129.04+1.17 Gy and 41.62+ 1.17 for a 2 mm diameter source. The ED 50 for the early radiation response of the skin, moist desquamation, following irradiation with the 22.5 mm diameter source was 27.3 + 0.7 Gy whilst exposure with the small sources (1 mm and 2 mm diameter) produced an acute ulceration rather than desquamation with ED 50 values of 253.7 + 31.0 Gy and 119.3 + 9.0 Gy, respectively. The ED5Os for the later dermal response occurred after exposure to lower doses of radiation than did those for acute response. Therefore, it is the late dermis tissue response that determines an acceptable level of exposure of the skin to radiation. Proliferation in CaNT tumours during and after fractionated radiotherapy assessed by local control, clonogenic survival and cell kinetics J. J. Pavy, A. Rojas, R. J. Hodgkiss, G. D. Wilson, J. M. Collier, E. J. Kelleher, C. A. Whitsed and M. C. Joiner Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, P.O. Box 100, Northwood, Middlesex HA6 2JR The effect of overall time on tumour proliferation, during and after a course of fractionated X rays, was studied using three
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methods for assessing repopulation, in the mouse mammary carcinoma CaNT. Local control: 10x3.4Gy, calculated to produce half the damage required to control 50% of tumours, were given in air over 3V3, 5 or 10 days. This partial amount of damage was "topped-up" with neutrons 1 or 8 days after the end of each regime. Preliminary analyses of the data show no significant repopulation during treatment when the overall time was extended from 3 to 10 days. This is compatible with no appreciable repopulation occurring over a 10 day treatment period or that repopulation was effectively counterbalanced by reoxygenation. By contrast, if an interval of 8 days was left, more neutron dose was required to produce the same level of effect, especially for the 10 F/10 days. Clonogenic cell survival: an in vivo-in vitro excision assay was performed using 10 F/5 days. Contrary to what was seen in vivo, no significant increase in tumour clonogens was detected during the first week after treatment. The total dose of 34 Gy reduced the relative clonogens per tumour down to ~ 10" 3 , which was less than expected to cause half the full effect. Cell kinetics: using the same schedule as for the excision assay, the labelling index (LI) was determined using flow cytometric analysis of tumour cells pulse labelled with BUdR. An immediate reduction in LI during treatment was followed by a prompt but partial recovery. Control values were not achieved even by 20 days after end of treatment. An increase of G 2 cells was observed, persisting over the same period. The interpretation of the kinetic data is complicated because clonogenic cells cannot be distinguished from doomed cells.
Radiation and drug sensitivity measured with the M T T assay J. A. Hanson, D. P. Bentley, A. E. Bean, J.-A. Garnett and J. L. Moore Radiation Science Laboratory, Velindre Hospital, Cardiff CF4 7XL The MTT assay (Twentyman & Luscombe, 1987) has been used to determine cell response following in vitro exposure to radiation and drugs. Quantitatively, response measured in the MTT, colony forming and tritiated thymidine uptake assays varied, however qualitative ranking of cell lines was generally similar when assay time and conditions were optimized for each cell line. Radiation and adriamycin resulted in viable giant cells in some lines which may have been responsible for the observed plateau in response seen particularly with DAUDI cells. The MTT assay, along with a dye exclusion (D.Ex) assay, was further evaluated for the measurement of in vitro response of chronic lymphocytic leukaemia lymphocytes to chlorambucil. Response determined in the MTT assay on Day 6 and at a concentration of 1 ^g/ml (continuous exposure) correlated with that measured in the D.Ex assay in five pre-treatment patients, but not in four treatment patients. A correlation was further seen between the D.Ex response in four treatment patients and their WBC count, indicative of clinical response. Clinical response data for the pre-treatment patients are as yet not available; however, these initial results indicate that the MTT assay may be used in determining in vitro cell response in this leukaemia. Low control absorbance and cell loss during aspiration of MTT, with non-adherent cell lines, were limiting factors. Addition of sodium succinate at a final concentration of 20 mM was found to increase formazan production whilst maintaining linearity in both CHO and Molt4 cells with no change noted in dose-response. The use of SDS in place of DMSO was also studied and was found, in two non-adherent lines, to give similar dose-response curves to that obtained
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Proceedings of The British Institute of Radiology using DMSO; however, less response was found in two adherent lines. As no aspiration step is involved, SDS may be of use with non-adherent cell lines. Reference TWENTYMAN, P. R. & LUSCOMBE, M., 1987. British Journal of Cancer, 56,219.
Cellular and molecular approaches to intrinsic radioresistance in human malignant glioma ° Gillian Ross, R. Brown, T. Wheldon, R. I. Freshney, R. Ramphng and Ann Barrett Departments of Radiation and Medical Oncology, Glasgow University Progress in understanding mechanisms of cellular radioresistance is dependent on the identification of appropriate experimental models. Human malignant glioma lies at an extreme of clinical resistance, and it has been postulated that this may be attributable to "intrinsic" cellular radioresistance.
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We have assessed the acute radiosensitivity of six human glioma cell lines to megavoltage irradiation, using a monolayer colony forming assay. Data has been analysed using linearquadratic and multi-target models of radiation action. All lines generate survival curves with broad initial shoulder (mean, Dq, 2.83+0.42; n = 4.9+1.1), with values of alpha (mean, 0.1+0.02) and SF2 (mean, 0.7 + 0.05) suggestive of "intrinsic" cellular radioresistance, by comparison with such parameters obtained in other human tumour lines. There is evidence to implicate the rate and fidelity of rejoining of radiation-induced double strand breaks (DSB) as a determinant of radiosensitivity in mammalian cells. We are evaluati n g t w o experimental systems aiming to identify differences in D N A repair, in human tumour cells of varying cellular radiosensitivity. We are examining the ability of intact tumour cells o r t h e i r n u c i e a r extracts to rejoin plasmid DNA (plC20H; pTKHgH), after induction of DSB within a "reporter gene" by restriction endonuclease. Pilot studies have obtained successful "reporter gene" transfection into a panel of human tumour lines covering a significant range of cellular radiosensitivity (glioma; ovarian carcinoma; neuroblastoma).
The British Journal of Radiology, July 1990
Proceedings of the British Institute of Radiology Radiobiology proffered papers Abstracts of papers presented at a meeting of the BIR at 36 Portland Place, London W1 N 4AT, on Friday, 23 February 1990 Radiosensitivity of human tumour cell lines assessed in the adhesive tumour cell culture system, by C. S. Parkins and G. G. Steel The effect of LET on the radiation response of human tumour cell lines of differing radiosensitivity, by J. Peacock, C. S. Parkins, G. G. Steel, R. Britten and H. Warenius. Abstract not received The acute response of mouse kidney cells after fractionated irradiation assayed by colony formation in primary culture, by Y.-M. Jen and J. H. Hendry Pathogenesis of radiation nephropathy. morphological studies in the pig, by M. Wooldridge, M. E. C. Robbins, M. Rezvani, S. J. Golding and J. W. Hopewell The effect of myeloablation on the subsequent^engraftment and homing of haemopoietic precursor cells in long-term cultures, by J. G. Bierkens, J. H. Hendry and N. G. Testa Radiosensitization in vivo: oral (po) dosing with RSU 1069 or RB 6145 maintains potency but reduces toxicity compared with parenteral (ip) administration, by S. Cole, I. J. Stratford, E. Wright, J. Nolan and G. E. Adams Repair, redistribution and repopulation: the relative importance in determining the acute response of the skin to fractionated irradiation, by G. J. M. J. van den Aardweg and J. W. Hopewell Dose and field-size related changes in the thickness of the dermis after ^-irradiation in the pig, by V. K. Sieber, J. C. Heryet and J. W. Hopewell Proliferation in CaNT tumours during and after fractionated radiotherapy assessed by local control, clonogenic survival and cell kinetics, by J. J. Pavy, A. Rojas, R. J. Hodgkiss, G. D. Wilson, J. M. Collier, E. J. Kelleher, C. A. Whitsed and M. C. Joiner Radiation and drug sensitivity measured with the MTT assay, by J. A. Hanson, D. P. Bentley, A. E. Bean, J.-A. Garnet and J. L. Moore Cellular and molecular approaches to intrinsic radioresistance in human malignant glioma, by G. Ross, R. Brown, T. Wheldon, R. I. Freshney, R. Rampling and A. Barrett Radiosensitivity of human tumour cell lines assessed in the adhesive tumour cell culture system C. S. Parkins and G. G. Steel Institute of Cancer Research, Sutton, Surrey The adhesive tumour cell culture system (ATCCS) has been proposed as a predictive test for radiosensitivity because of its short culture period, selective growth of tumour cells and because it is thought to yield a result which reflects the clonogenic cell response. We have compared the radiosensitivity of a range of human tumour cell lines using the ATCCS and clonogenic survival. We found that the optical method of assessing cell number and radiosensitivity gave good agreement with clonogenic response at 2 Gy, although the ATCCS response showed unusually high radioresistance at high doses. The relationship between the cell number seeded and optical density was not always linear within individual assays and we prefer to use the term "slope ratio" for the derived radiation response, e.g. SR2 and not SF 2 . The assay could distinguish the radiosensitivity of cervix carcinoma cells (SF2 = 0.7; SR2 = 0.74, 0.80) from neuroblastoma cells (SF2 = 0.3; SR2 = 0.30, 0.41). The use of the cell-adhesive-matrix (CAM) coating on the supplied culture surface did not significantly improve colony-forming efficiency. Results are also presented for the growth and radiosensitivity of xenograft and biopsy tumour samples.
former and 2-5 Gy for the latter. The underlying cause for this difference could be the time available for cellular repair prior to the fixation of potentially lethal damage. Using primary culture and a clonogenic assay, mouse kidney cells after single dose, 2-, 8- and 16-fraction irradiations were assayed for their survival 12 h after single or fractionated irradiations. When the data sets were fitted separately to a linear-quadratic model the a component was constant for all regimens of irradiation. Using a direct analysis, the a//? ratio was 4.4+ 1.3 Gy for the three fractionated regimens plus the single-dose data set, and 2.8 +0.9 Gy for the three fractionated regimens only. The difference was significant (p < 0.0001) and the fitting excluding the single-dose data set was a better fit. The result indicates an cc/p value within the range of values reported using functional kidney assays, and also suggests that the time available for repair prior to assay does not account for the low a/j? ratio observed in mouse kidney.
The acute response of mouse kidney cells after fractionated irradiation assayed by colony formation in primary culture Yee-Min Jen and Jolyan H. Hendry Department of Radiobiology, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester
The pathophysiological mechanisms responsible for the development of radiation nephropathy are ill defined. Studies of changes in renal morphology should help ascertain the cell type(s) at risk and, in conjunction with functional data, provide a basis for the development of rational protocols for the treatment and/or prevention of radiation nephropathy. Both kidneys of 13 mature 10-month-old pigs were irradiated with a single dose of 9.8 Gy of ^Co y rays. The pigs were serially killed at periods up to 24 weeks after irradiation. Kidneys were perfusion-fixed, embedded in paraffin-wax and 5 /an thick sec-
It has been shown that the a//? ratios for early- and lateresponding tissues are different, being around 5-20 Gy for the
Vol. 63, No. 751
Pathogenesis of radiation nephropathy: morphological studies in the pig M. Wooldridge, M. E. C. Robbins, *M. Rezvani, fS. J. Golding and *J. W. Hopewell MRC Radiobiology Unit, Chilton, *CRC Normal Tissue Radiobiology Research Group, Research Institute, University of Oxford and ^Oxfordshire Regional CT Unit, Churchill Hospital, Oxford OX3 7LJ
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Proceedings of The British Institute of Radiology tions taken for evaluation. Changes were observed in nonglomerular and glomerular tissue within 2 weeks of irradiation. Non-glomerular lesions initially consisted of focal areas of interstitial fibrosis or tubular atrophy, predominantly in the corticomedullary region or along the path of medullary rays. Maximal damage, affecting over 50% of the cortex, was observed at 6 weeks after irradiation, and consisted of severe interstitial fibrosis, tubular atrophy and inflammatory cell infiltrate. Interstitial fibrosis involved a complete band of subcapsular tissue, with "fingers" of fibrotic tissue extending deep into the cortex. Similar changes were seen up to 24 weeks after irradiation. Glomerular changes consisted of progressive increases in PAS positive staining material in the mesangial matrix, glomerular hypercellularity with cell clumping, thickening of the capillary basement membrane and a decrease in capillary lumina. Between 12 and 24 weeks, damage increased in severity, leading to the development of completely sclerotic end-stage glomeruli which were predominantly subcapsular or juxtamedullary. There was no apparent change in the vasculature. These findings show that (a) there is no one target or dose-limiting cell and (b) the vasculature does not play a major role in the primary development of radiation nephropathy. The effect of myeloablation on the subsequent engraftment and homing of haemopoietic precursor cells in long-term cultures J. G. Bierkens, J. H. Hendry and *N. G. Testa Departments of Radiobiology and * Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital Withington, Manchester M20 9BX Long-term marrow cultures (LTBMCs) reconstitute in vitro the privileged microenvironment which is characteristic for the intact bone marrow and which is required for prolonged haemopoiesis. As such LTBMCs provide a model to study the various aspects of bone marrow transplantation. In the present experiments, the effect of 20 Gy y-irradiation as a modality of myeloablation on the subsequent engraftment of stromal layers in LTBMCs has been assessed and compared with the "homing" ability of haemopoietically inactive cultures. Irradiated stromal layers subsequently re-inoculated with bone marrow cells, displayed a lower supportive capacity for haemopoiesis as compared with corticosteroid deprived stromal layers. This was expressed both as reduced numbers of CFU-S and GM-CFC progenitors and a fall in non-adherent cell production. These observations may be related, at least in part, to a reduced cell homing of haemopoietic cells on the irradiated stroma. A significant decrease by 35% in the cell adhesion of factor dependent FDCP-Mix-A4 cells onto irradiated stromal layers was observed within a dose range of 10 to 20 Gy. Significantly higher doses were required to affect the sulphated GAG content in fully established stromal layers. It is therefore unlikely that the reduced capacity of irradiated stromal layers to home haemopoietic precursor cells is uniquely related to the alterations observed in the sulphated GAG content in these layers. Radiosensitization in vivo: oral (po) dosing with R S U 1069 or RB 6145 maintains potency but reduces toxicity compared with parenteral (ip) administration. S. Cole, I. J. Stratford, E. Wright, J. Nolan and G. E. Adams MRC Radiobiology Unit, Chilton, Didcot, Oxon OX11 0RD RSU 1069 is a nitroimidazole hypoxic cell radiosensitizer, which is also directly cytotoxic when reductively metabolized. RB 6145 is a prodrug of RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for
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C3H/He mice was 80mg/kg (0.38 mmol/kg) ip. This MTD increased four-fold to 320mg/kg (1.5 mmol/kg) with po administration. The MTDs of RB 6145 were 35Omg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 towards bone marrow stem cells was also reduced approximately four-fold after po administration; 20 mg/kg ip RSU 1069 and 80 mg/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Half of the mice given the highest po dose of 320 mg/kg of RSU 1069 developed cystic kidneys, with dilation, necrosis and fibrosis of the distal tubules but no nephrotoxicity resulted from the ip administration of 80 mg/kg of RSU 1069. Even relatively low doses of RSU 1069 reduced the number of spermatids visible in histological sections of testes 4 weeks later. There was some dose sparing (about two-fold) after po administration. For RSU 1069 and RB 6145, administered by either route, the maximum reduction in the survival of clonogenic cells from intradermal, murine KHT sarcomas occurred, when the drugs were given 45-60 min before 10 Gy of X rays. A range of doses of RSU 1069 or RB 6145 was administered po or ip 45 min before 10 Gy. The degree of radiosensitization by both compounds was largely independent of the route of administration. Preliminary studies with 3 H-RSU 1069 suggested that efficacy correlated with peak blood level of label and concentration in the tumour at the time of irradiation, which were reduced less than two-fold by po compared with ip administration. Toxicity tended to correlate with total exposure over time, which was reduced two to four-fold by po administration. Oral administration of RSU 1069 or RB 6145 may give therapeutic benefit, since doselimiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was retained. Repair, redistribution and repopulation: the relative importance in determining the acute response of the skin to fractionated irradiation G. J. M. J. van den Aardweg and J. W. Hopewell CRC Normal Tissue Radiobiology Research Group, Research Institute (University of Oxford), Churchill Hospital, Oxford OX3 7LJ The influence of the overall treatment time in fractionated irradiation was studied in the epidermis of the pig. The absence or presence of moist desquamation was assessed after /?-irradiation and dose-effect curves were fitted to the quantal data using probit analysis. Experiments were compared on the basis of ED 50 values: the dose required to produce moist desquamation in 50% of the irradiated skin fields. With an overall treatment time of ^ 1 5 days the iso-effect doses (ED50) for moist desquamation were well correlated (r = 0.99) with the number of fractions (in the range of 1-48 F) when the data were plotted on a log-log scale and when full account was taken for any incomplete repair of sublethal damage. The slope of this Strandquist-plot was represented by a fraction-exponent (N) of 0.42. This is not significantly different from those obtained for so-called late-responding normal tissues. The results of fractionated irradiation given in 1-10 F were well fitted by a Fe-plot suggesting an a//? ratio of 8.7 + 0.5 Gy. However, the data for irradiation given in 6-48 F suggested a significantly lower a//J ratio of 0.85 + 0.3 Gy. Comparison of the ED 50 values for irradiation given as 2 F/14 days with 2 F/38 days indicated a positive time factor with an increase in the iso-effect dose of nearly 40%, owing to repopulation. In contrast, 28 F/38 days compared with 28 F/14 days indicated a negative time factor, i.e. a dose reduction of nearly 30%. This could be explained by the redistribution of the target cells
The British Journal of Radiology, July 1990
Proceedings of The British Institute of Radiology within the cell cycle. Fractionation studies on pig skin using X-irradiation also indicated that repopulation and redistribution are counteracting processes. The overall effectiveness of each varying with the overall treatment time. The presence of a significant degree of re-distribution of cells in the cell cycle conflicts with the basic concept of equal effects per equal dose fraction. Dose and field-size related changes in the thickness of the dermis after {{-irradiation in the pig V. K. Sieber, J. C. Heryet and J. W. Hopewell CRC Normal Tissue Radiobiology Research Group, University of Oxford, Churchill Hospital, Oxford 0X3 7LJ The late response of dermal tissue to jS-irradiation, from sources of 1-40 mm diameter, was assessed in pig skin 2 years after irradiation. Sources of 1 and 2 mm diameter were used to represent "hot particles" of the type encountered in accidental occupational exposures to radiation, whilst the larger sources (5-40 mm) represented the lower end of a range that might be used therapeutically. Single doses of /3-irradiation, in the range 13-230 Gy, were given from '"Sr/^Y plaques of diameter of 1.0, 2.0, 5.0, 11.0, 15.0, 22.5 and 40.0 mm. At 2 years after irradiation samples of skin were fixed, 5 /an histological sections were prepared and the thickness of the dermis in irradiated skin was measured from a projected image of these sections. Comparisons were made with adjacent unirradiated control sites. The thickness of an irradiated area was expressed as a percentage of adjacent dermal thickness. A dose-related reduction in dermal thickness was found, irrespective of the diameter of the source, with a maximum, 60% reduction in dermal thickness. The dose required to produce this maximum reduction in dermal thickness for the 40 mm diameter source was 23.4 Gy whilst for the 1 mm diameter source it was 1050 Gy. After even higher doses there was a less severe reduction in the extent of dermal thinning; this was attributed to the presence of invasive fibrosis. In contrast to the acute response where a field size effect was seen using sources of 5-22.5 mm diameter there was no field-size effect for sources of 5 mm. The mean ED 50 for ^ 30% reduction in dermal thickness following irradiation by sources 5 mm diameter was 18.00 + 0.65 Gy. For the "hot particles" ( l m m and 2 mm diameter) a field-size effect was demonstrated as the ED 50 for ^ 30% reduction in dermal thickness following irradiation with a l m m diameter source was 129.04+1.17 Gy and 41.62+ 1.17 for a 2 mm diameter source. The ED 50 for the early radiation response of the skin, moist desquamation, following irradiation with the 22.5 mm diameter source was 27.3 + 0.7 Gy whilst exposure with the small sources (1 mm and 2 mm diameter) produced an acute ulceration rather than desquamation with ED 50 values of 253.7 + 31.0 Gy and 119.3 + 9.0 Gy, respectively. The ED5Os for the later dermal response occurred after exposure to lower doses of radiation than did those for acute response. Therefore, it is the late dermis tissue response that determines an acceptable level of exposure of the skin to radiation. Proliferation in CaNT tumours during and after fractionated radiotherapy assessed by local control, clonogenic survival and cell kinetics J. J. Pavy, A. Rojas, R. J. Hodgkiss, G. D. Wilson, J. M. Collier, E. J. Kelleher, C. A. Whitsed and M. C. Joiner Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, P.O. Box 100, Northwood, Middlesex HA6 2JR The effect of overall time on tumour proliferation, during and after a course of fractionated X rays, was studied using three
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methods for assessing repopulation, in the mouse mammary carcinoma CaNT. Local control: 10x3.4Gy, calculated to produce half the damage required to control 50% of tumours, were given in air over 3V3, 5 or 10 days. This partial amount of damage was "topped-up" with neutrons 1 or 8 days after the end of each regime. Preliminary analyses of the data show no significant repopulation during treatment when the overall time was extended from 3 to 10 days. This is compatible with no appreciable repopulation occurring over a 10 day treatment period or that repopulation was effectively counterbalanced by reoxygenation. By contrast, if an interval of 8 days was left, more neutron dose was required to produce the same level of effect, especially for the 10 F/10 days. Clonogenic cell survival: an in vivo-in vitro excision assay was performed using 10 F/5 days. Contrary to what was seen in vivo, no significant increase in tumour clonogens was detected during the first week after treatment. The total dose of 34 Gy reduced the relative clonogens per tumour down to ~ 10" 3 , which was less than expected to cause half the full effect. Cell kinetics: using the same schedule as for the excision assay, the labelling index (LI) was determined using flow cytometric analysis of tumour cells pulse labelled with BUdR. An immediate reduction in LI during treatment was followed by a prompt but partial recovery. Control values were not achieved even by 20 days after end of treatment. An increase of G 2 cells was observed, persisting over the same period. The interpretation of the kinetic data is complicated because clonogenic cells cannot be distinguished from doomed cells.
Radiation and drug sensitivity measured with the M T T assay J. A. Hanson, D. P. Bentley, A. E. Bean, J.-A. Garnett and J. L. Moore Radiation Science Laboratory, Velindre Hospital, Cardiff CF4 7XL The MTT assay (Twentyman & Luscombe, 1987) has been used to determine cell response following in vitro exposure to radiation and drugs. Quantitatively, response measured in the MTT, colony forming and tritiated thymidine uptake assays varied, however qualitative ranking of cell lines was generally similar when assay time and conditions were optimized for each cell line. Radiation and adriamycin resulted in viable giant cells in some lines which may have been responsible for the observed plateau in response seen particularly with DAUDI cells. The MTT assay, along with a dye exclusion (D.Ex) assay, was further evaluated for the measurement of in vitro response of chronic lymphocytic leukaemia lymphocytes to chlorambucil. Response determined in the MTT assay on Day 6 and at a concentration of 1 ^g/ml (continuous exposure) correlated with that measured in the D.Ex assay in five pre-treatment patients, but not in four treatment patients. A correlation was further seen between the D.Ex response in four treatment patients and their WBC count, indicative of clinical response. Clinical response data for the pre-treatment patients are as yet not available; however, these initial results indicate that the MTT assay may be used in determining in vitro cell response in this leukaemia. Low control absorbance and cell loss during aspiration of MTT, with non-adherent cell lines, were limiting factors. Addition of sodium succinate at a final concentration of 20 mM was found to increase formazan production whilst maintaining linearity in both CHO and Molt4 cells with no change noted in dose-response. The use of SDS in place of DMSO was also studied and was found, in two non-adherent lines, to give similar dose-response curves to that obtained
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Proceedings of The British Institute of Radiology using DMSO; however, less response was found in two adherent lines. As no aspiration step is involved, SDS may be of use with non-adherent cell lines. Reference TWENTYMAN, P. R. & LUSCOMBE, M., 1987. British Journal of Cancer, 56,219.
Cellular and molecular approaches to intrinsic radioresistance in human malignant glioma ° Gillian Ross, R. Brown, T. Wheldon, R. I. Freshney, R. Ramphng and Ann Barrett Departments of Radiation and Medical Oncology, Glasgow University Progress in understanding mechanisms of cellular radioresistance is dependent on the identification of appropriate experimental models. Human malignant glioma lies at an extreme of clinical resistance, and it has been postulated that this may be attributable to "intrinsic" cellular radioresistance.
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We have assessed the acute radiosensitivity of six human glioma cell lines to megavoltage irradiation, using a monolayer colony forming assay. Data has been analysed using linearquadratic and multi-target models of radiation action. All lines generate survival curves with broad initial shoulder (mean, Dq, 2.83+0.42; n = 4.9+1.1), with values of alpha (mean, 0.1+0.02) and SF2 (mean, 0.7 + 0.05) suggestive of "intrinsic" cellular radioresistance, by comparison with such parameters obtained in other human tumour lines. There is evidence to implicate the rate and fidelity of rejoining of radiation-induced double strand breaks (DSB) as a determinant of radiosensitivity in mammalian cells. We are evaluati n g t w o experimental systems aiming to identify differences in D N A repair, in human tumour cells of varying cellular radiosensitivity. We are examining the ability of intact tumour cells o r t h e i r n u c i e a r extracts to rejoin plasmid DNA (plC20H; pTKHgH), after induction of DSB within a "reporter gene" by restriction endonuclease. Pilot studies have obtained successful "reporter gene" transfection into a panel of human tumour lines covering a significant range of cellular radiosensitivity (glioma; ovarian carcinoma; neuroblastoma).
The British Journal of Radiology, July 1990
