Radiosensitivity of new and established human melanoma cell lines: comparison of [3H]thymidine incorporation and soft agar clonogenic assays.
Seven new low-passage melanoma lines were developed in this laboratory from clinical melanoma specimens and characterised for chromosome complement, DNA ploidy and S-phase content. The radiosensitivity of these lines was compared with that of eight established melanoma cell lines, FME, MM-96, SK-MEL-5, SK-MEL-28, SK-MEL-2, MALME-3M, M19-MEL and LOX-IMVI, using a 96-well microculture assay technique. Dose-response curves were determined using a 5-day incubation period and 6-h terminal [3H]thymidine-labelling period. Radiation (60Co source) was carried out under a lead wedge to provide a radiation dose range of 0-10 Gy, or by irradiating part of the plate (radiation dose 0 or 2 Gy). Data for a range of cell densities in a single 96-well plate were combined into a single regression equation incorporating linear quadratic terms for radiation dose and cell density. SF2 values were defined as the amount of thymidine incorporated following a radiation dose of 2 Gy, expressed as a fraction of that of unirradiated cells, and varied from 0.36 to 0.93. The reproducibility in repeat assays, as defined by the standard error of determinations at different passage numbers, was +/- 0.04. The newly developed lines exhibited a similar range of radiosensitivity to that of the established lines, and melanin content did not correlate with resistance. For nine of the lines, radiation parameters were also determined using a modified Courtenay clonogenic soft agar assay technique, and the results compared with the thymidine incorporation results, and a significant linear correlation was found between SF2 and SF2' (r = 0.89). The linear (alpha) and quadratic (beta) terms of the best-fit linear quadratic dose-response curves, were significantly correlated between the two assays. It is concluded for this series of human melanoma lines that proliferation assays in 96-well plates provide radiosensitivity parameters comparable to those using clonogenic assays.
Marshall
E S
ES
Cancer Research Laboratory, University of Auckland School of Medicine, New Zealand.
Matthews
J H
JH
Shaw
J H
JH
Nixon
J
J
Tumewu
P
P
Finlay
G J
GJ
Holdaway
K M
KM
Baguley
B C
BC
D000328:Adult; D000368:Aged; D002470:Cell Survival / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005260:Female; D006801:Humans; D008297:Male; D008545:Melanoma / Q000378:metabolism / Q000532:radiotherapy*; D008875:Middle Aged; D011237:Predictive Value of Tests; D011836:Radiation Tolerance; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000528:radiation effects; D014410:Tumor Stem Cell Assay
D013936:Thymidine