5 research outputs found
Dose fractionation effects in primary and metastatic human uveal melanoma cell lines
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
Changes in epidermal radiosensitivity with time associated with increased colony numbers
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
Cellular radiosensitivity of primary and metastatic human uveal melanoma cell lines
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)
Determination of colony numbers in pig epidermis as an estimate for radiosensitivity. A rapid assay based on in vitro BrdU-labelling
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
Single dose irradiation response of pig skin: a comparison of brachytherapy using a single, high dose rate iridium-192 stepping source with 200 kV X-rays
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