http://repub.eur.nl/res/aut/1537/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/35976/ 2007-02-01T00:00:00Z Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csbm/mmice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csbm/mretina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csbm/mmice (as well as Csa-/-mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csbm/mretina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging. Copyright http://repub.eur.nl/res/pub/3205/ 2003-01-02T00:00:00Z Mutations in the CSB gene cause Cockayne syndrome (CS), a rare inherited disorder, characterized by UV-sensitivity, severe neurodevelopmental and progeroid symptoms. CSB functions in the transcription-coupled repair (TCR) sub-pathway of nucleotide excision repair (NER), responsible for the removal of UV-induced and other helix-distorting lesions from the transcribed strand of active genes. Several lines of evidence support the notion that the CSB TCR defect extends to other non-NER type transcription-blocking lesions, notably various kinds of oxidative damage, which may provide an explanation for part of the severe CS phenotype. We used genetically defined mouse models to examine the relationship between the CSB defect and sensitivity to oxidative damage in different cell types and at the level of the intact organism. The main conclusions are: (1) CSB(-/-) mouse embryo fibroblasts (MEFs) exhibit a clear hypersensitivity to ionizing radiation, extending the findings in genetically heterogeneous human CSB fibroblasts to another species. (2) CSB(-/-) MEFs are highly sensitive to paraquat, strongly indicating that the increased cytotoxicity is due to oxidative damage. (3) The hypersenstivity is independent of genetic background and directly related to the CSB defect and is not observed in totally NER-deficient XPA MEFs. (4) Wild type embryonic stem (ES) cells display an increased sensitivity to ionizing radiation compared to fibroblasts. Surprisingly, the CSB deficiency has only a very minor additional effect on ES cell sensitivity to oxidative damage and is comparable to that of an XPA defect, indicating cell type-specific differences in the contribution of TCR and NER to cellular survival. (5) Similar to ES cells, CSB and XPA mice both display a minor sensitivity to whole-body X-ray exposure. This suggests that the response of an intact organism to radiation is largely determined by the sensitivity of stem cells, rather than differentiated cells. These findings establish the role of transcription-coupled repair in resistance to oxidative damage and reveal a cell- and organ-specific impact of this repair pathway to the clinical phenotype of CS and XP. http://repub.eur.nl/res/pub/10236/ 2003-01-01T00:00:00Z PURPOSE: To investigate the effects of split-dose irradiation on primary and metastatic uveal melanoma cell lines, with a clonogenic survival assay. METHODS: Appropriate cell concentrations of four primary and four metastatic human uveal melanoma cell lines were cultured for irradiation with single doses and with two equal fractions separated by 5 hours. After irradiation, colony formation was allowed for 7 to 21 days. Two cutaneous melanomas were also tested for comparison. All survival curves were analyzed using the linear quadratic (LQ) model. Specific parameters for the intrinsic radiosensitivity (alpha-component, SF2), for the capacity of repair of DNA damage (beta-component), as well as the alpha/beta ratio were calculated. RESULTS: After single-dose irradiation a wide range in the values of the alpha- and beta-component was obtained for both primary and metastatic uveal melanomas, which resulted in a wide range of alpha/beta ratios. In contrast, calculations based on split-dose data, with which the beta-component could be estimated independent of the alpha-component, indicated that estimates for the capacity of sublethal DNA damage repair was very similar in all cell lines. This indicated that intrinsic factors dominated the radiosensitivity of these cell lines. Split-dose irradiation had little influence on the intrinsic radiosensitivity (alpha-component), but cell survival increased for all cell lines. For the two cutaneous melanomas comparable split-dose results were obtained. CONCLUSIONS: For both primary and metastatic uveal melanoma cell lines, data from single and fractionated doses indicate large variations in radiosensitivity, which are mainly dominated by the intrinsic radiosensitivities. Doses of approximately 8 Gy in five fractions would be sufficient to eradicate 10(9) cells (approximately 1 cm3) of the most radioresistant tumor cell lines, but this schedule is an overkill for the radiosensitive tumor cell lines. Based on specific morphologic and histologic tumor markers, more individualized dose fractionation schedules could improve the therapeutic ratio for uveal melanomas. http://repub.eur.nl/res/pub/9940/ 2002-01-01T00:00:00Z PURPOSE: To investigate the radiosensitivity of uveal melanoma cell lines by a clonogenic survival assay, to improve the efficiency of the radiation regimen. METHODS: Four primary and four metastatic human uveal melanoma cell lines were cultured in the presence of conditioned medium. After single-dose irradiation (0-12 Gy), colonies were allowed to form for 6 to 14 days. Two cutaneous melanomas cell lines were also tested for comparison. The survival curves were analyzed by the linear quadratic (LQ) model, and the surviving fraction at a dose of 2 Gy (SF(2)), the SF at 10 Gy (SF(10)), the ratio of initial irreparably damaged DNA (alpha-coefficient) to the capacity to repair sublethally damaged DNA (beta-coefficient), and the plating efficiency were calculated. RESULTS: The melanomas displayed a wide range of initial irreparable DNA damage (alpha-component), as well as a capacity for repair of sublethal DNA damage (beta-component), which ultimately resulted in a wide range of alpha/beta ratios. These findings were similar in both primary and metastatic melanomas and were comparable with data obtained from two cutaneous melanomas. CONCLUSIONS: Cell lines obtained from primary and metastatic human uveal melanomas displayed a wide range of radiosensitivity, similar to that published for cutaneous melanomas. Translating these data to the clinical setting indicates that a fractionated dose of 8 to 10 Gy administered in three to four fractions, as currently delivered in many centers, should be sufficient to eradicate tumors of approximately 1 cm(3). http://repub.eur.nl/res/pub/9646/ 2001-01-01T00:00:00Z Epidermal clonogenic cell survival and colony formation following irradiation were investigated and related to radiosensitivity. A rapid in vivo/in vitro assay was developed for the quantification of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. Bromodeoxyuridine (BrdU)-labelled cells in full thickness epidermal sheets were visualized using standard immunohistochemistry. In unirradiated skin, approximately 900 BrdU-positive cells mm(-2) were counted. In a time sequence experiment, BrdU-positive cell numbers increased from an average of 900 cells mm(-2) to approximately 1400 cells mm(-2) after BrdU-labelling for 2-24 h. In irradiated skin, colonies containing >/=16 BrdU-positive cells were seen for the first time at days 14/15 after irradiation. The number of these colonies per cm(2) as a function of skin surface dose yielded a cell survival curve with a D(0)-value (+/-SE) of 3.9+/-0.6 Gy. This relatively high D(0)-value is possibly due to a rapid fall off in depth dose distribution for the iridium-192 source and consequently a substantial contribution of hair follicular epithelium to colony formation. At 14/15 days after irradiation, the ED(50) level of 33.6 Gy for the in vivo response of moist desquamation corresponded with 2.7 colonies cm(-2). Surprisingly, the number of colonies increased with time after irradiation with an estimated doubling time of approximately 4 days, while the D(0)-value remained virtually unchanged. This increase in colony numbers could be due to migration of clonogenic cells, to the recruitment of dormant clonogenic cell survivors by elevated levels of cytokines, or to both. Although frequent biopsying caused increased cytokine levels, which had a systemic effect on unirradiated skin, it had no influence on colony formation in irradiated skin. Smaller colonies, containing 4-8 cells or 9-15 cells, were abundant, particularly after higher doses, which resulted in higher D(0)-values. The majority of these small colonies were abortive and did not progress to larger colonies. There was no statistical evidence for significant variations in the interanimal responses. http://repub.eur.nl/res/pub/9541/ 2000-01-01T00:00:00Z An experimental brachytherapy model has been developed to study acute and late normal tissue reactions as a tool to examine the effects of clinically relevant multifractionation schedules. Pig skin was used as a model since its morphology, structure, cell kinetics and radiation-induced responses are similar to human skin. Brachytherapy was performed using a microSelectron high dose rate (HDR) afterloading machine with a single stepping source and a custom-made template. In this study the acute epidermal reactions of erythema and moist desquamation and the late dermal reactions of dusky mauve erythema and necrosis were evaluated after single doses of irradiation over a follow-up period of 16 weeks. The major aims of this work were: (a) to compare the effects of iridium-192 (192Ir) irradiation with effects after X-irradiation; (b) to compare the skin reactions in Yorkshire and Large White pigs; and (c) to standardize the methodology. For 192Ir irradiation with 100% isodose at the skin surface, the 95% isodose was estimated at the basal membrane, while the 80% isodose covered the dermal fat layers. After HDR 192Ir irradiation of Yorkshire pig skin the ED50 values (95% isodose) for moderate/severe erythema and moist desquamation were 24.8 Gy and 31.9 Gy, respectively. The associated mean latent period (+/- SD) was 39 +/- 7 days for both skin reactions. Late skin responses of dusky mauve erythema and dermal necrosis were characterized by ED50 values (80% isodose) of 16.3 Gy and 19.5 Gy, with latent periods of 58 +/- 7 days and 76 +/- 12 days, respectively. After X-irradiation, the incidence of the various skin reactions and their latent periods were similar. Acute and late reactions were well separated in time. The occurrence of skin reactions and the incidence of effects were comparable in Yorkshire and Large White pigs for both X-irradiation and HDR 192Ir brachytherapy. This pig skin model is feasible for future studies on clinically relevant multifractionation schedules in a brachytherapy setting. http://repub.eur.nl/res/pub/9283/ 1999-01-01T00:00:00Z A rapid assay has been developed for the quantitation of colonies arising from surviving clonogenic cells in pig epidermis after irradiation. The number of surviving clonogenic cells per unit area was related to the epidermal in vivo response of moist desquamation. After irradiation with single doses, ranging from 20 to 36 Gy, skin biopsies were taken and incubated in dispase for enzymatic separation of the epidermis and dermis. Full thickness epidermal sheets were labelled with bromodeoxyuridine (BrdU) in vitro. Proliferating cells were visualized using standard immunohistochemical procedures. Cell groups containing > or = 16 cells were counted as colonies. These colonies were first seen on day 14/15 after irradiation. The number of colonies per cm2, as a function of skin surface dose, yielded a cell survival curve with a D0 (+/- SE) of 3.87 +/- 0.57 Gy. The ED50 for the epidermal in vivo reaction of moist desquamation corresponded with a colony density of 2.7 colonies per cm2. After higher doses, abundant smaller colonies of 4-8 BrdU-positive cells were seen and these were more radioresistant, as represented by higher D0 values.