Impact of fractionated cisplatin and radiation treatment on cell growth and accumulation of DNA damage in two normal cell types differing in origin

Evidence on the impact of chemotherapy on radiotherapy-induced second malignant neoplasms is controversial. We estimated how cisplatin modulates the in vitro response of two normal cell types to fractionated radiation. AHH-1 lymphoblasts and VH10 fibroblasts were irradiated at 1 Gy/fraction 5 and 3 times per week during 12 and 19 days, respectively, and simultaneously treated with 0.1, 0.2, 0.4, 0.8, 1.7 and 3.3 µM of cisplatin twice a week. Cell growth during treatment was monitored. Cell growth/cell death and endpoints related to accumulation of DNA damage and, thus, carcinogenesis, were studied up to 21 days post treatment in cells exposed to radiation and the lowest cisplatin doses. Radiation alone significantly reduced cell growth. The impact of cisplatin alone below 3.3 µM was minimal. Except the lowest dose of cisplatin in VH10 cells, cisplatin reduced the inhibitory effect of radiation on cell growth. Delayed cell death was highest in the combination groups while the accumulation of DNA damage did not reveal a clear pattern. In conclusion, fractionated, concomitant exposure to radiation and cisplatin reduces the inhibitory effect of radiation on cell proliferation of normal cells and does not potentiate delayed effects resulting from accumulation of DNA damage.


Supplementary resultsdifferential sensitivity of AHH-1 and VH10 cells to cisplatin and ionizing radiation following single and fractionated exposure
In order to check if the sensitivity of AHH-1 and VH10 cells to cisplatin and ionizing radiation was different following single and repeated exposure, MTT assay for assessing cell metabolic activity was carried out after exposure to cisplatin and clonogenic cell survival was measured after exposure to ionizing radiation.The results were compared to cells growth measured during fractionated treatment with cisplatin and radiation.

Radiation sensitivity to single doses assessed by clonogenic survival assaymaterials and methods
The radiosensitivity of AHH-1 lymphoblasts was assessed using the soft agarose colonyforming assay 2 .Cells were seeded at cell densities ranging from 1000 -4000 cells in duplicates in six-well culture dishes, irradiated with single doses of 1, 2, 3, and 4 Gy (0.32 Gy/min, 137 Cs, Scanditronix, Uppsala Sweden) and incubated for seven days.The radiosensitivity of VH10 fibroblasts was assessed using the agarose overlay colony formation assay described above.
Cells were seeded at a cell density of 5000 cells in 100-mm diameter cell culture dishes, and 24 hours later, growth media was discarded and replaced with 3 ml of agarose mixture as described above, followed by pouring 7 ml of growth media over the agarose overlay.The cells were irradiated at 2, 4, 6, 8 Gy (0.767 Gy/min, 137 Cs, Gammacell 40, Theratronix, Canada) and incubated for twenty-one days.

Radiation sensitivity to fractionated doses assessed by clonogenic survival assaymaterials and methods
The sensitivity to fractionated doses of cisplatin and radiation was assessed by analysing population doublings.The methodology including the treatment doses is described in materials and methods of the article.Curves were fitted to linear functions as shown in Fig 2 of the article.In order to compare growth curve slopes of both cell types, the control slope of each cell type was set to 1 and slopes of treated cells were normalized to the control.

Results -single dose exposure
AHH-1 cells were more resistant to cisplatin than VH10 cells: the half maximal inhibitory concentration (IC50) of cisplatin for cell viability measured with the MTT test was

Results -fractionated exposure
The sensitivity of AHH-1 and VH10 to cisplatin did not differ: while the slopes of the growth curves decreased with cisplatin dose, no consistent difference in the pattern was seen between both cell types.The differential sensitivity to radiation was the same as for single dose exposure, with AHH-1 cells showing a higher sensitivity (lower growth rate) than VH10 cells.

Figure S1 .
Figure S1.Panel A: cell viability of AHH-1 and VH10 cells exposed to single doses of cisplatin.Panel B: clonogenic cell survival following single dose exposure to ionizing radiation.IC50: half maximal inhibitory concentration, LD50: lethal dose at 50%.Error bars represent standard deviations.

Figure S2 .
Figure S2.Relative population doublings representing growth curves of AHH-1 and VH10 cells exposed to fractionated doses of cisplatin (cDDP) and ionizing radiation (IR).Slopes of treated cells were normalised to the respective control that was set to 1. cDDP doses on the right margin illustrate the order of curves for both cell types.

table S2 .
P values and respective effect sizes (Cohen´s d values) for selected treatment pairs contained in figures 4-6.Included are treatment pairs relevent for analysing the interacion of IR and cDDP.One way ANOVA was carried out for data sets included in a figure and, if relevant, the day of observation, by all pairwise multiple comparison procedures with Holm-Sidak post-hoc test.Cohen´s effect size test was carried out for each pair of results (treatment 1 vs treatment 2).cDDP: cisplatin, IR: ionising radiation.cDDP doses are given in µM.Significant p values and large and very large effects are marked in colour according the key given in the right column on page 2 of the table.