Figure 1.
Representative images of metaphase chromosomes in HLEC1 at CPD 14.5 (A) and WI-38 at CPD 44.7 (B).
Bars, 20 µm.
Figure 2.
Growth curve of HLEC1 (A) and WI-38 (B).
HLEC1 at CPD 5.0 and WI-38 at CPD 19.0 were plated and serially passaged with weekly replenishment. The population doubling times of HLEC1 and WI-38 were estimated to be 66.6 h at CPD 5.0–15.5 and 32.4 h at CPD 19.0–64.9, respectively. HLEC1 and WI-38 ceased to divide at CPD 17.1 and 69.1, respectively.
Table 1.
Karyotype.
Figure 3.
Clonogenic survival of HLEC1 (A) and WI-38 (B).
Within 1 h after exposure to indicated dose of X-rays at a dose rate of 0.43±0.01 Gy/min, cells were reinoculated for colony formation and incubated for 14 days at which time they were fixed and stained. At the time of reinoculation, CPD of HLEC1 and WI-38 was 11.0 and 32.0, respectively (n.b., CPD 11.0 was experimentally available youngest HLEC1 in our hand). The plating efficiency of HLEC1 and WI-38 was 1.6±0.6 and 19.5±3.6%, respectively. Curves were fitted to the exponential equation y = exp (−kx), and D0 and D10 were calculated as 1/k and [ln(1/0.1)]/k, respectively. R2, correlation coefficient square. The data are presented as means and SD of three independent experiments with quadruplicate measurements.
Figure 4.
Representative images of colonies arising from sham-irradiated HLEC1 (A), 6 Gy-irradiated HLEC1 (B), sham-irradiated WI-38 (C), and 6 Gy-irradiated WI-38 (D).
Shown are colonies that were formed during 14 days in 10-cm dishes and stained with crystal violet. Bar, 5 cm.
Figure 5.
The area of clonogenic colonies.
(A) The area distribution of clonogenic colonies arising from HLEC1 (left panel) and WI-38 (right panel) as a function of dose. The area of clonogenic colonies (45–68 colonies for HLEC1 and 54–93 colonies for WI-38 per dose point) was measured as described in Figure S1. Red bars indicate the means at each dose point. The horizontal lines indicate the mean+2SD area in sham-irradiated controls (41.7 mm2 for HLEC1 and 31.7 mm2 for WI-38). *0.01≤p<0.05 and **p<0.01 compared with sham-irradiated controls. See Figure S2 for the frequency distribution replotted as a function of the area. (B) The fraction of clonogenic colonies exceeding the mean+2SD area of sham-irradiated controls among all clonogenic colonies analyzed. (C) The fraction of clonogenic colonies exceeding the mean+2SD area of sham-irradiated controls among all plated cells. Curves were fitted to the exponential equation, though the data points for 1 Gy and 6 Gy for WI-38 were excluded because of zero values. R2, correlation coefficient square. When the intercept of the exponential curve for WI-38 was changed from 0.255 to 1, its slope changed from 1.032 to 0.716, and changed from 0.979 to 0.994. For Figure 5B and 5C, circles with solid lines and diamonds with dotted lines indicate the data for HLEC1 and WI-38, respectively.
Figure 6.
Changes in population doublings of HLEC1.
(A) Cell numbers in individual clonogenic colonies. Cell numbers in 218 countable clonogenic colonies (open circles) were directly counted under the stereomicroscope, and those in 99 uncountable colonies (blue-filled circles) were estimated as described in Figures S4, S5 and S6. Red bars indicate the means at each dose point. A horizontal line indicates the mean+2SD value (4,944 cells) in sham-irradiated controls. *0.01≤p<0.05 and **p<0.01 compared with sham-irradiated controls. (B) PDN of clonogenic cells experienced during colony formation. To calculate PDN, cell numbers in individual clonogenic colonies (shown in Figure 6A) were summed, divided by the number of clonogenic colonies (i.e., the number of clonogenic cells plated), and then transformed to the logarithm to base 2. (C) CPD of all cells in the whole population. For calculation of the data, cell numbers in clonogenic colonies (shown in Figure 6A) were first summed. Second, the number of all cells situated outside clonogenic colonies (i.e., cells in abortive colonies or other nonclonogenic cells) was directly counted under the stereomicroscope and summed. Third, the sum of these cell numbers was divided by the number of plated cells, and transformed to the base-2 logarithm. Finally, 11.0 (CPD when plated) was added to these numbers, and shown as open circles with a solid line. Gray-filled circles with a dotted line represent CPD of all cells calculated assuming that irradiation does not alter the proliferation of clonogenic cells (i.e., PDN of irradiated clonogenic cells is the same as PDN of sham-irradiated controls regardless of dose).
Figure 7.
The impact of cell numbers in clonogenic colonies on the surviving fraction in HLEC1 (left panel) and WI-38 (right panel).
The general “colony number-based” surviving fraction was calculated from the number of clonogenic colonies divided by the number of plated cells with correction for the plated efficiency. This was compared here with the “cell number-based” surviving fraction that was calculated as the sum of the integrated density of clonogenic colonies (i.e., cell numbers in clonogenic colonies given a linear relationship between the integrated density and cell numbers in each clonogenic colony) divided by the number of plated cells with correction for that at 0 Gy. Colony number-based survival curves (open symbols with solid lines) were taken from Figure 3, and the data of the integrated density of clonogenic colonies presented in Figure S5 were used to obtain cell number-based curves (closed symbols with dotted lines). The data represent means and SD of three independent experiments with quadruplicate measurements. *0.01≤p<0.05 compared between two types of survival curves at each dose. For WI-38, the difference between the colony number-based survival curve and the cell number-based survival curve was insignificant for all 5 dose points tested (p>0.10), indicating that the colony number-based survival fraction is akin to the cell number-based one. For HLEC1, the difference between two types of survival curves reached a statistical significance only at 4 Gy (p = 0.02), but there was clearly a tendency toward the increased surviving fraction at 2 Gy and 6 Gy.