Overview of Radiosensitivity of Human Tumor Cells to Low-Dose-Rate Irradiation

For Peer Review O

nly

Genotype-dependent, Time-dependent Radiosensitivity of Human Tumor Cells to Low Dose-rate Irradiation: Implications for Clinical Application.

Journal: Acta Oncologica

Manuscript ID: draft

Manuscript Type: Original Manuscript

Date Submitted by the Author:

n/a

Complete List of Authors: Williams, Jerry; Loma Linda Medical Center, Radiation Medicine Zhang, Yonggang; Johns Hopkins Medical Institution, Oncology Zhou, Haoming; Johns Hopkins Medical Institution, Oncology CenterRussell, James; Memorial Sloan Kettering Cancer Center, Radiation Medicine Gridley, Daila; Loma Linda University and Medical Center, Radiation Medicine Koch, Cameron; University of Pennsylvania, Radiation Medicine Slater, James; Loma Linda Medical Center, Radiation Medicine Dillehay, Larry; Johns Hopkins Medical Institutes, Oncology Little, John; Harvard School of Public Health, John B. Little Center

Keywords: low dose-rate , radiosensitivity , TP53, cell-cycle redistribution

URL:http://mc.manuscriptcentral.com/SONC

Acta Oncologica

For Peer Review O

nly

1

Genotype-dependent, Time-dependent Radiosensitivity of Human Tumor Cells to Low Dose-rate Irradiation: Implications for Clinical Application.

Jerry R. Williams ScD1, 2, Yonggang Zhang MD2, Haoming Zhou2, , Daila S. Gridley, PhD1, James Russell Ph.D2,3, James M. Slater MD1, Cameron J. Koch PhD 4, John B. Little, MD 5

1. Radiobiology Program, Department of Radiation Medicine, Loma Linda University Medical Center Loma Linda CA

2. Laboratory of Radiobiology Johns Hopkins School of Medicine, Baltimore MD 3. Present address: Memorial Sloan Kettering Cancer Center, New York, NY4. Department of Radiation Oncology, University of Pennsylvania Philadelphia PA. 5. Center for Radiation Sciences and Environmental Health, Harvard School of Public Health, Boston MA.

Key words: Human tumor cells, radiosensitivity models, low dose-rate, TP53, CDKN1A,

14-3-3σ, inverse dose-rate effect.

Abbreviations: LDR, low dose-rate; HDR, high dose-rate.

This research was supported by NIH grant PO-CA79862 to JRW and by the Radiation Medicine

Department of the Loma Linda Medical Center.

Running title: Low dose-rate radiosensitivity

Address and email of corresponding author:

Dr. Jerry R. Williams, Department of Radiation Medicine, 11175 Campus Street, Chan Sun

Pavilion, Rm. A-1010, Loma Linda CA 92350-1700.

Email: [email protected]

Page 1 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

2

Abstract:

Purpose: We measure radiosensitivity to low-dose-rate (LDR) irradiation in 27 cell lines

that vary in genotype and in radiosensitivity to high-dose-rate (HDR) irradiation. We define

susceptibility to LDR-induced redistribution in the cell cycle in 15 of these cell lines.

Methods: We compare clonogenic survival after durations up to 96 hours of LDR (0.25

Gy/hr) irradiation and compare these to clonogenic survival after irradiation HDR. We measure

LDR-induced changes in cell-cycle redistribution using flow-cytometry. We extract coefficients

that describe radiosensitivity over different dose-segments for both dose-rates and seek

correlation between cell lines on the basis of genotype.

Results: Radiosensitivity to LDR and HDR irradiation fall into four common

radiosensitivity groups based on cell genotype. Radiosensitivity to LDR, HDR for all cell lines

correlates at lower doses (circa 2 Gy HDR, circa 6 Gy LDR), but not at higher doses (HDR > 4

Gy; LDR durations > 6 Gy). LDR radiosensitivity changes in a genotype-dependent manner at

circa 24 hours with sensitive cells becoming more resistant and resistant cells becoming more

sensitive. Redistribution induced by LDR irradiation shows distinct changes in kinetics at 24

hours.

Conclusions: Human tumor cells segregate into four major radiosensitivity groups for

both HDR and LDR irradiation that cluster with specific genotypes: mutATM; wtTP53;

mutTP53; and an unidentified gene in radioresistant glioma cells. LDR radiosensitivity and LDR

induced redistribution are time-dependent changing at circa 24 hours. We propose a genotype-

dependent, time-dependent model to explain the inverse-dose-rate effect and suggest possible

clinical application.

Page 2 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

3

Introduction:

Tumors are treated in the clinic with ionizing radiation delivered over a range of dose-

rates including low dose-rates (LDR) delivered by radioactive implants or radio-labeled

antibodies. We have reviewed the response of tumors and tumor cells to irradiation delivered at

different dose-rates, particularly as these data suggest possible clinical application, Gridley et al

[1]. The mechanisms by which LDR induces cell killing and redistribution in the cell cycle are

not well documented over multiple cell lines that vary in genotype and in sensitivity to high

dose-rate (HDR) irradiation and this is the primary goal of the studies presented here.

We have recently shown that radiosensitivity to high-dose-rate (HDR) irradiation in multiple

human tumor cells falls into four radiosensitivity groups, each group associating with a specific

genotype: mutATM; wtTP53; mutTP53; and an unidentified gene that associates with

radioresistant glioblastoma cells that we will refer to as “glio” [2, 3]. We now seek to

determine whether these cell lines segregate with the same genotypes when irradiated with LDR.

We have also demonstrated that redistribution in the cell cycle after a single acute dose of HDR

irradiation is genotype-dependent and time-dependent [4] and we now seek similar data for

redistribution during LDR irradiation.

Radiation-induced loss of clonogenic survival by LDR is complicated when durations of

exposures are long compared to times over which induction of gene expression and redistribution

in the cell cycle can occur. Such extended durations of exposure have been proposed to result in

differences in survival due to repair processes [5] and redistribution in the cell cycle [6, 7].

Studies using cultured cell systems have been used to demonstrate the sparing effect of LDR and

some of these studies suggest redistribution in the cell cycle is responsible for some changes in

radiosensitivity observed during protracted irradiation [8-16]. Mitchell et al. [12] first identified

more complex patterns for cell killing when cells were irradiated with a series of dose-rates circa

Page 3 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

4

0.37 Gy/hr to 1.54 Gy/hr. These data showed that for some cell lines and for some ranges of

dose and dose-rate, monotonic increases in dose-rate did not produce a monotonic increase in cell

killing. In specific cell lines, over specific dose-rate ranges, increasing dose-rate did not increase

but actually reduced cell killing. This was referred to as the “inverse dose-rate-effect”. We will

propose a mechanistic description of this effect.

A major variable in our studies is tumor cell genotype, focusing on TP53. The relationship

between TP53 and radiosensitivity to HDR irradiation has been reviewed [17-20]. While these

studies generally suggest that cells that express wtTP53 are more sensitive than cells that express

mutTP53, this finding is not universal and in some cases contradictory. Our recently published

studies [2, 3] better define the relationship between TP53 expression and HDR radiosensitivity

and we now extend these studies to LDR irradiation including cell lines that express four forms

of TP53: wildtype, mutant, null and abrogated. We also study genetically-manipulated cell lines

that vary in TP53, CDKN1A (p21), 14-3-3σ, K-ras, and DNA mismatch repair.

Ionizing radiation induces perturbation in the progression of cells through the intermitotic

cycle. This perturbation is thought to involve the induction, suppression, or both, of gene

products that are needed for traversing certain critical “checkpoints” in the intermitotic cycle

[21]. The effects of ionizing radiation on cell progression have been reviewed [22-24]. We have

recently shown that redistribution in the cell cycle after HDR irradiation associates with specific

genes: wtTP53, mutTP53, mut ATM, abrogated CDKN1A and abrogated 14-3-3σ [4]. We now

study changes in redistribution during extended durations of LDR irradiation.

Our experimental strategy is to define a statistically-validated data base for clonogenic

survival in 12 cell lines selected on the basis of their HDR radiosensitivity and variation in

genotype and then determine whether an additional 15 cell lines, including genetically-

manipulated cell lines, correlate with these patterns of radiosensitivity. Similarly we define

Page 4 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

5

kinetics of redistribution in the cell cycle for nine selected cell lines and then compare these

results to an additional six lines, including the same genetically-manipulated lines used for

survival studies.

Material and methods:

Cell lines: The human glioblastoma cell lines U251 and U87 were obtained from the American

Type Culture Collection (Rockville MD). HCT116, 80S4, 379.2, 14-3-3 σ-/- , DLD1, 19S186

were obtained from Dr. Bert Vogelstein of Johns Hopkins. SW1222 was obtained from Dr.

James Russell at Memorial Sloan Kettering Cancer Center. T98G cells were from Dr. John B.

Little of Harvard School of Public Health. Most cell lines were maintained in McCoy's medium

(exceptions being SW1222 in RPMI 1640, U251 and U87 in F12/DMEM, T98G in MEM).

Medium was supplemented with 10% fetal bovine serum and 1% penicillin plus streptomycin

(Gibco Long Island NY, USA). 14-3-3 σ-/- cells were cultured in the presence of G418 (0.5

mg/ml) an aminoglycoside antibiotic. Stock cultures were grown in plastic flasks in a 37oC

incubator with a 5% CO2-95% air atmosphere and subcultured to maintain cells in exponential

growth.

Radiation and Clonogenic assay. For survival studies using HDR irradiation, cells were

exposed to high-dose-rate radiation in a Gammacell 40 (Nordion Ottawa ONT Canada) at

approximately 0.7 to 0.8 Gy/min. For these studies, single cells were plated 18 hours before

irradiation. After exposure plates were incubated for 8-14 days depending on specific growth

and colonies stained with crystal violet. Colonies with more than approximately 50 cells were

counted. For each cell line we performed controls to account for possible proliferation during

the period between plating and irradiation. This control consisted of plating 105 cells in separate

plates when replicates of cells were plated for colony formation. When irradiation was

performed on the plates for colony formation, the microcolony plates were stained and the

Page 5 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

6

number of cells per colony measured. The average number of cells per colony was below 1.20

cells per microcolony for all cell lines and did not vary significantly between cell lines.

Irradiation for LDR studies were carried out in a custom LDR irradiator that uses Cs-137 sources

attenuated to produce a dose-rate of 0.25 Gy/hr. Cells were plated at numbers that allow each

cell line to grow exponentially for 96 hours without reaching confluence. These exponential

cultures were irradiated and explanted, counted and plated for colony formation at appropriate

densities to produce between approximately 50 to 150 colonies per flask.

Cell-cycle Redistribution Assay: 2 x 105 cells were plated 18 hours before irradiation. Cells

were harvested at designated times by trypsinizing into single cell suspensions. Cells were then mixed

with 0.5ml Magic Solution (stock solution: 12 ml phosphate buffered saline, 20ml 0.1% NP-40/PBS,

3.6ml 37% Formaldehyde and 0.4ml 1mg/ml Hoechst 33258), vortexed and stored in refrigerator

(4oC) for no more than 4 days, before UV excitation analysis (FACSVantage SF, Becton Dickson).

Regression stripping was used to estimate the prevalence of cells that contain DNA content

corresponding to G1, S or G2.

Derivation of Coefficients that Describe Radiosensitivity: From each set of survival curves

we derive four coefficients, two from HDR data and two from LDR data. The coefficient of the

slope from 0 to 2.0 Gy HDR is defined as α(HDR) and that for the slope from 0 to 6.0 Gy LDR

is defined as α(LDR). The coefficient of the slope for three survival points: 0.0 Gy, 2.0 Gy HDR

and 6.0 Gy LDR is defined as α(LDR/HDR). The coefficient from the slope derived by best-fit

linear regression for HDR response between 5 and 10 Gy is defined as ω(HDR) and the

coefficient for best-fit linear regression of LDR data at 12 Gy and higher is defined as ω(LDR).

Analytical units: Data were tested for significance using Student’s t-test and the Mann-Whiney

test. A p value of < 0.05 was used to indicate statistical significance. All survival data is

Page 6 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

7

expressed as logarithms to the base 10 and radiosensitivity as logs10 cells killed per Gy. All error

bars represent the standard error of the mean unless otherwise specified.

Results:

We defined a basic data set of clonogenic responses to LDR in 12 unmanipulated cell lines that

vary in HDR radiosensitivity and genotype. These cell lines were selected from four radiosensitivity

groups based on clonogenic response to HDR irradiation [1, 2]. Clonogenic survival curves for these

cell lines after irradiation with either HDR or LDR are shown in figure 1.

Insert figure 1.

Data in figure 1 show that survival patterns after LDR fall into the same rank-order as do

the same four radiosensitivity groups treated with HDR irradiation. The mean value for survival

at each dose point, for each radiosensitivity group, and for both LDR and HDR radiation is

significantly different from the mean of cell groups above and below it (p< 0.05) [1, 2]. All

HDR responses are convex, with changes at higher doses (> 4 Gy) that indicate increased cell

killing e.g. ω(HDR) > α(HDR). For LDR survival curves, two radiosensitivity groups, R and

VR, are clearly convex also but two are concave, S and to a lesser extent VS, with increasing

radioresistance at later times. Thus for LDR irradiation, R and VR cells become more sensitive

and S and VS cells become more resistant. Interception of the two lines extrapolated from

α(LDR) and ω(HDR) occur at durations of exposure that would correspond to changes in

radiosensitivity occurring between 20 and 30 hours.

The similarities and differences between HDR and LDR radiosensitivity patterns for each

of the four radiosensitivity groups are more clearly seen when they are compared directly in the

same panel. In figure 2 we show a direct comparison of HDR, LDR responses for each

radiosensitivity group showing dotted and dashed lines representing α(HDR) and α(LDR).

Page 7 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

8

Insert figure 2.

When data are plotted as shown in figure 2, several important observations can be made.

First the slopes of the lines for α(HDR) and α(LDR) that have been extended linearly from

lower doses are essentially coincident in two radiosensitivity groups, R and S cells (panels B and

C). VR cells (panel A) show an increase in α(LDR) compared to α(HDR), suggesting increasing

radioresistance. In VS cells, survival patterns are more variable and LDR response at higher

doses falls into the same range of cell killing predicted by extrapolation of cell killing at lower

doses. However compared to HDR irradiation, VS cells show reduced toxicity with a suggestion

at doses above 12 Gy of increasing radiosensitivity. We will compare these coefficients for all 27

cell lines subsequently. The differences between the rates of cell killing at higher doses, ω(HDR)

and ω(LDR), are distinctly different for each radiosensitivity group. Importantly, these data show

that both VR and R cells become more sensitive and S cells, and to a lesser extent VS cells,

become more resistant at times circa 24 hours. While radiosensitivity for S and R cells is still

diverging at HDR doses of 10 Gy, for LDR irradiation radiosensitivity in absolute terms is

converging at 24 Gy LDR. Based on the data in figures 1 and 2, our earlier studies of the

kinetics of apoptosis and redistribution after HDR irradiation [4] and data presented subsequently

for redistribution during LDR irradiation, we will use the term “circa 24 hours” to denote the

common time for these several changes.

We extracted coefficients that describe cell killing at lower and higher doses as described

in the material section from both HDR and LDR survival curves for these 12 cell lines. We

performed similar experiments on an additional 15 cell lines that varied in histological type and

status of several genes: TP53, CDKN1A, 14-3-3σ, Ki-ras and genes involved in DNA mismatch

repair (data not shown). From these 54 survival curves, we calculate 108 coefficients that

describe the slope of cell killing at lower doses and higher doses for both LDR and HDR. These

Page 8 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

9

coefficients are: α(LDR), α(HDR), ω(LDR) and ω(HDR). These coefficients are listed in table 1

on the basis of histological type, radiosensitivity group, cell line, genetic characteristics and

TP53 status.

Insert Table 1.

The size of this data base validates the use of scatter diagrams to examine clustering and

correlation. Using this approach we are able to compare coefficients among the 27 cell lines and

identify those cell lines that share common values and any cell line that exhibits exceptional expression

of the four coefficients. In figure 3 we compare distribution of cell lines on the basis of TP53 status for

four pairs of coefficients that describe radiosensitivity at lower and higher doses for both HDR and

LDR.

Insert figure 3.

Data in all four panels show strong clustering for cells express wtTP53 distinct from cells that

express mutTP53. In table 2 below we analyze statistical variation for all four coefficients between

cells that express wtTP53 and mutTP53. In figure 3 we have identified several cell lines that lie

outside two standard deviations from other cells in their radiosensitivity group, further the cell line

SW1222 falls at the extreme diagonal for all four comparisons. We tested these several cell lines that

appear to be “outliers” using the t-test to determine whether they are significantly different from the

cell lines of the same genotype (wtTP53 or mutTP53) using comparisons of replicate survival data.

The VS line SW1222 is a true biological outlier as evidenced by its peripheral location in all four

panels and confirmed by comparison of replicate survival data. HCT116 is not a true outlier in that

it is not significantly different form other clones of the same parent line. For PPC-1 we conclude it

is an outlier based on its value for the parameter ω(LDR). 14-3-3σ-/- and LNCap are not true

outliers but appear so by random variation in the two parameters used to describe them.

Page 9 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

10

The data in panel A show strong correlation between α(HDR) and α(LDR) over all cell

lines. The data in panel B shows clustering between wtTP53 cells and mutTP53 cells based on

values for ω(HDR), but there is no correlation between ω(LDR) and ω(HDR). Similarly, the data in

panel C show clustering based on values of α(LDR) but there is no correlation with ω(LDR). The

data in panel D shows correlation between α(HDR) and ω(HDR and these data can be resolved

further into genotype-dependent radiosensitivity groups [1, 2]. We estimated the significance of

differences between means of wtTP53 and mutTP53 cells using Student’s t-test for each of the four

coefficients and these are shown in table 2.

Insert table 2.

Thus, three coefficients are significantly different between cells that express wtTP53 and

mutTP53 but not ω(LDR).

Although there was no correlation between the rate of LDR cell killing at lower doses

represented by α(LDR) at 6 Gy and ω(LDR), it was clear from figures 1 and 2 that there was a

tendency in S cells to become more resistant and R cells to become more sensitive. In figure 4

we show a global overview of changes in radiosensitivity presented as changes of cell killing at

24 hours compared to the rate of cell killing based on the slope of the line from 48 hours to 72

hours.

Insert figure 4. (Note old figure 4 is deleted).

These data show that protracted irradiation can produce a global shift in radiosensitivity

that is quite significant when compared to the rate of cell killing at 24 hours shown as dotted

lines that represent the mean of this coefficient for S cells (upper line) and R cells (lower line).

Some cell lines in figure 4 show little change over the duration from 24 and 72 hours and this

reflects a relative constant rate of radiosensitivity over this duration of exposure, not a deficit in

Page 10 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

11

variance of the measurements. We also tested whether the values of α(LDR) for each cell line

predicted the rate of change in radiosensitivity shown in figure 4 by testing correlation between

these two variables over all 27 cells lines.. There was only a modest correlation coefficient of

R2= 0.4623 but the mean rate of change between wtTP53 cells compared to the values for

mutTP53 cells was significant using the Whitney-Mann test, p< 0.05 with the former being

positive and the latter being negative.

We measured clonogenic survival and redistribution in the cell cycle as a function of time

of irradiation at 0.25 Gy/hr for 13 cell lines. In figure 5 we show relative patterns for cells that

represent three radiosensitivity groups: VS, S and R using similar cell lines for which we have

recently published kinetic measurements for redistribution a single dose of HDR irradiation [3].

SW1222 is an important positive control since it represents a unique radiosensitivity

characterized by dysfunctional redistribution in the cell cycle and hypersusceptibility to

apoptosis [4].

Insert figure 5. (Previously figure 6).

Page 11 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

12

Data in Figure 5 show that LDR irradiation induces changes in cell-cycle redistribution

with no significant differences between S and R cells for the first 24 to 30 hours but significant

differences after that time. At times greater than this time, radiosensitivity also changes in all

three cell groups with S cells becoming more resistant and R cells becoming more sensitive.

SW1222 shows earlier perturbation due to the absence of G1 arrest in these cells. S cells have an

elevated level of cells in the G1 phase compared to R cells. There is a general correlation

between increases or decreases in G1-phase cells and G2-phase cells that correlate generally with

changes in LDR radiosensitivity for the three groups of cells. When corrected for elevated G1

prevalence before irradiation in S cells, there are no significant differences in % of cells in each

of the phases of the cell cycle for R and S cells until after 24 hours. At the same time period,

radiosensitivity of both S and R cells change. These data are generally consistent with the

hypothesis that increases in G1-phase cells and decreases in G2-phase could render cells more

resistant and the opposite patterns of accumulation, increase in G2 and decrease in G1, could

render cells more sensitive. However the changes in the VS line compared to the S cell lines is

not consistent with this hypothesis, with the increases in G2 and the decreases in G1 not

consistent with quantitative changes in radiosensitivity shown in the upper panel. Figure 5 does

show the VS cell line is exceptional both in radiosensitivity and cell-cycle redistribution during

LDR irradiation.

We examined LDR-induced redistribution in the cell cycle and LDR radiosensitivity for

six additional cell lines that have undergone specific manipulations of genotype. From these

comparisons (data not shown) we make two observations: 1) No genetic manipulations as listed

below altered either the rate of cell killing observed in the parent line for the first 24 hours or the

rate of redistribution in the cell cycle for the first 24 hours; and 2) subsequent to 24 hours there

are induced changes for some specifically manipulated genotypes for both radiosensitivity and in

Page 12 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

13

redistribution, however these changes do not show a general correlation. Cells abrogated in

TP53 subsequent to 24 hours show increased radioresistance compared to other S cells.

Redistribution in these cells shows decreased prevalence in G1-phase cells, increased prevalence

in S-phase cells and a small but significant increase inG2-phase cells. 2) Cells abrogated in 14-3-

3σ show slightly increased radiosensitivity after 24 hours and there are small modifications in

cell-cycle redistribution: G1-phase cells increase, S cells decrease and G2 cells decrease. 3).

Cells abrogated in CDKN1A in a wtTP53 background (80S4 cells) subsequent to 24 hours shows

no changes in radiosensitivity but do show increases prevalence of cells in S-phase, decreases

cells in G1-phase and results in a small decrease in G-2 phase cells. 4). Cells abrogated in

CDKN1A in a mutTP53 background (19S186), restitution of DNA mismatch repair by

chromosomal transfer (N6CH3), insertion of a K-ras vector (CaCo2-neoras) did not show

alterations either in radiosensitivity or redistribution in the cell cycle. All of these differences,

both positive and negative are statistically significant (p < 0.05) and we interpret these data to

suggest that in all these cell lines there is not a general correlation between radiosensitivity and

redistribution into sensitive or resistant phases of the cell cycle after 24 hours.

We directly tested the hypothesis proposed by several authors that there a significant

correlation between radiosensitivity of populations of cells and prevalence or cells in the cell

cycle. In figure 6, we compare the prevalence of cells in G1-phase and G2-phase of the cell

cycle after 48 hours of LDR irradiation with the rate of cell killing (logs10 SF/Gy) at the same

time for 15 cell lines.

Insert figure 6 (previously figure 7)

While there is clearly significant clustering between cells that express wtTP53 and

mutTP53, there is no significant correlation between prevalence of cells in G1-phase or G2-

phase of the cell cycle and radiosensitivity for the 15 cell lines.

Page 13 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

14

Discussion:

Our data provide a detailed description of LDR radiosensitivity and LDR-induced cell

cycle redistribution over dose, time, histological type and genotype for multiple human tumor

cells. The levels of survival that we observe in our several studies are similar to those published

by other authors [9-12, 16]. However we can make several novel conclusions:

1. Cellular radiosensitivity to LDR irradiation falls into genotype-specific

radiosensitivity groups. Data in figures 1 and 2 show that radiosensitivity to both HDR and

LDR irradiation segregates into four groups, survival levels in each group being statistically

different than those for other groups. The slopes of cell killing at 2 Gy HDR and 6 Gy LDR

correlate strongly and it is only at higher doses (> 4 Gy) that HDR response increases and only

after times subsequent to 24 hours does LDR radiosensitivity change in a genotype-dependent

manner (figures 1, 2 and 3). These studies show for the first time that radiosensitivity to LDR, as

we have previously shown for HDR segregate into four radiosensitivity groups that associate

each with one of four specific genotypes: mutant ATM, wildtype TP53, mutant TP53 and an

unidentified gene in some radioresistant glioblastoma cells, “glio”.

Our studies show that certain other genes do not modulate HDR or LDR radiosensitivity

before 24 hours: CDKN1A, 14-3-3σ, Ki-ras and several genes involved in DNA mismatch

repair.

2). There is a distinct change in LDR radiosensitivity and LDR-induced

redistribution at circa 24 hours. Our data demonstrate there are changes in LDR

radiosensitivity in all cells circa 20 to 30 hours and data in figure 6 also show there are also

changes in cell-cycle redistribution beginning at approximately the same times. We have

previously shown that there are distinct changes in cell cycle redistribution and changes in

expression of apoptosis at these same time frames [4]. While the extent of this change for longer

Page 14 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

15

durations is dependent on genotype, the time at which this transition occurs is independent of

genotype. The data in this paper reinforce our earlier hypothesis that cellular processing of

radiation damage can be considered to fall into two distinct time-dependent phases: an “acute

phase” (0 to ~24 hours) and a “post-acute response” (~ 24 to greater than 72 hours).

3) There is only partial correlation between changes in LDR radiosensitivity and

changes in LDR-induced redistribution in the cell cycle. Several authors have proposed that

changes in LDR radiosensitivity, at least at higher doses and longer exposure durations,

correlates with radiation-induced changes in the cell-cycle, particularly Knox et al. [16]. Our

data only partially supports this data and only for wtTP53 and mutTP53 cell lines. When other

cell lines that vary in genotype are considered (figure 6) there is no correlation between cell

killing circa 48 to 72 hours and prevalence of cells in G1 and G2 phases of the cell cycle at that

time (figure 6). During the fist 24 hours aspects of HDR radiosensitivity and LDR

radiosensitivity correlate strongly with the four genotypes specified above. After circa 24 hours,

there are distinct changes in LDR radiosensitivity and redistribution appear modulated by six

specific genotypes: mutATM, wtTP53, mutTP53, abrogated TP53, abrogated 14-3-3σ and

abrogated CDKN1A in a wildtype TP53 background. Changes in LDR radiosensitivity have not

been reported by others on the basis of cell genotype.

4). Relative radiosensitivity to both LDR irradiation and HDR irradiation suggest a

simple model of cellular radiosensitivity. Our data suggest a simple segmented response

model that describes LDR and HDR radiosensitivity and this is shown in figure 7.

Insert figure 7 (previously figure 8)

This model suggests a novel relationship between HDR and LDR radiosensitivity. This

model is based on our studies at a dose-rate of (0.25 Gy/hr) and we will describe the effect of

variation in dose-rate elsewhere. The model proposes response for 1-2 Gy HDR and 2-6 Gy

Page 15 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

16

LDR, expresses a common radiosensitivity, designated as α (HDR/LDR). This model proposes

this common radiosensitivity persists and determines survival until one of two events occur 1).

HDR irradiation induces a more sensitive component at dose between 2 to 4 Gy; or 2) at circa 24

hours of LDR irradiation there is a time-dependent change in radiosensitivity to ω(LDR). The

values of α(HDR/LDR), ω(HDR) and ω(LDR) are genotype-dependent.

While the model in figure 7 is highly conjectural, it is consistent with other data in the

literature. For the first time figure 7 outlines a method for quantitative prediction, using the

parameters in table 1, of total cell killing by LDR irradiation.

5. Possible explanation of the inverse dose-rate effect: We hypothesize that the inverse dose-

rate effect reflects genotype-dependent, time-dependent changes during LDR radiosensitivity.

Cell killing by exposure to LDR radiosensitivity (0.25 Gy/hr and higher) for durations that

extend beyond 24 hours, can be resolved into two components of cell killing. Cell killing for the

first 24 hours is predicted as the product of the coefficient α(HDR/LDR) and dose. Additional

cell killing beyond 24 hours is predicted by the product of ω(LDR) and that increment of dose

delivered subsequent to 24 hours; this second component killing fewer or more cells per unit

dose than cell killing before transition depending on cell genotype. The inverse-dose-effect is

observed, according to our model, only in cells for which longer durations lead to increased cell

killing which would be R and VR cells. Our model would also predict this effect is observed

only over dose-rates that exceed approximately 0.25 Gy/hr and only for doses delivered for

durations significantly greater than 24 hours. Cell killing at these longer durations would reflect

increased cell killing in R and VR cells due to the increased killing per unit dose of the ω(LDR)

component. At higher dose-rates when total doses are delivered in less than 24 hours, the

additional cell killing per unit dose observed at longer durations would not contribute to overall

Page 16 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

17

survival. Therefore as dose-rates are increased survival levels would increase, the definition of

the inverse dose-rate effect.

6. Possible Clinical application of LDR: There is a large data base associating in vitro

radiosensitivity of tumor cells after HDR irradiation with radiocurability of tumors of the same

histological type [25, 26]. West et al [27] shows correlation between survival levels of explanted

tumor cells and clinical response of tumors from which cells have been explanted [27]. In vitro

radiosensitivity has also been used to predict tumor response of changes in clinical protocols

based on the concept of Biologically-effective-dose (BED) [28-30]. Thus, our studies on in vitro

radiosensitivity of tumor cells treated with LDR irradiation can be considered as a first

approximation of possible response of tumors treated with this type of irradiation. We have

described possible use of LDR in the clinic [1] and propose that there are distinct possible

applications of this radiotherapy modality either as direct cytotoxic therapy such as in

brachytherapy or as a tool to sensitize tumors to subsequent HDR fractions or hypofractions.

Possible clinical applications include:

Direct Cytotoxic Therapy Using LDR. Our data strongly suggest that tumors

comprised of cells that express genotypes that associate with R (mutTP53) and VR (glio)

radiosensitivity groups should become more sensitive to cell killing during protracted irradiation

while tumors comprised of cells that express wtTP53 would become more resistant. In tumors

such as prostate where there is an alternative between brachytherapy and external beam

fractionated therapy, our data suggest the status of TP53 expression might be considered in

making this choice. Tumors whose cells express wtTP53 may respond better to fractionated

external beam avoiding the increase in radioresistance seen at longer LDR irradiation, but tumors

whose cells express mutTP53 may respond better to brachytherapy since our data predict they

would become more sensitive under protracted irradiation. We will investigate these predictions

Page 17 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

18

elsewhere. This would only be valid if dose-rates were 0.25 Gy or higher and durations at this

dose-rate extends over durations greater than 24 to 48 hours. The extent of cell killing by such

therapies could be predicted from the model in figure 7. This prediction must be confirmed by

pre-clinical studies before considering clinical application.

Concomitant LDR Effects on Tumors and Normal Tissue: Another interesting concept

for possible therapeutic use of LDR should consider the possibility that longer durations of LDR

would sensitize tumors whose cells express mutTP53, but could simultaneously render normal

tissues more resistant if such tissues respond to extended LDR irradiation as would tumor cells

that express wtTP53. Thus LDR irradiation of tumors and the immediate surrounding normal

tissue might increase the therapeutic ratio. This implication can be tested in xenograft tumors

systems and LDR toxicity studies in appropriate animals.

LDR Sensitization of Tumors to HDR: Another strategy that we think should be

investigated further is the extent to which LDR irradiation can sensitize tumors to HDR fractions

or hypofractions. We have demonstrated that LDR at very low dose-rates (0.01 to 0.03 Gy/hr)

delivered over long durations (> 48 hours) can sensitize tumor cells preferentially to large

fractions of external beam [31, 32]. We have also shown that LDR in combination with HDR

and IUdR induces complete regression in xenograft tumors comprised of malignant glioma tumor

cells [33]. We will publish data elsewhere to show that radioresistant glioma cells may be

especially sensitive to LDR irradiation compared to other cell genotypes.

Predicting total cells killed by LDR irradiation. The analytical structure shown in

figure7, coupled with the quantitative structure of our data bases, shown in table 1 and references

1 and 2, can be used to make a quantitative prediction of relative differences in total cells killed

by conventionally-fractionated radiotherapy, hypofractionated radiotherapy or LDR radiotherapy.

Page 18 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

19

References:

1. Gridley DS, Williams JR, Slater J.M. 2005 Low-dose/low-dose-rate radiation: a feasible strategy

to improve cancer radiotherapy? Cancer Therapy 3:105-130, 2005.

2. Williams J.R., Zhang Y., Russell J., Koch C., Little J. 2007 Human Tumor Cells Segregate into

Radiosensitivity Groups that Associate with ATM and TP53 Status. Acta Oncologica; 46(5):628-

38.

3. Williams J, Zhang Y, Russell J, Gridley D, Koch C, Slater J, and Little J. Quantitative Overview of

Human Tumor Radiosensitivity Across Histological Type and TP53 status. International Journal

of Radiation Biology. In press.

4. Williams JR, Zhang Y, Zhou H, Russell J, Koch CJ. Little JB. 2007 Genotype-dependent

radiosensitivity: Clonogenic Survival, Apoptosis and Redistribution in the Cell Cycle.

International Journal of Radiobiology (in press).

5. Elkind MM, Sutton H. X-ray damage and recovery in mammalian cells in culture. 1959 Nature.

1959 Oct 24;184:1293-5.

6. Terasima T, Tolmach LJ. 1961. Changes in x-ray sensitivity of HeLa cells during the division cycle.

Nature. Jun 24;190:1210-11.

7. Lajtha LG, Oliver R. 1962. Cell population kinetics following different regimes of irradiation.

British Journal of Radiology. Feb 35:131-40.

8. Bedford JS, Hall EJ. 1963. Survival of HeLa cells cultured in vitro and exposed to protracted

gamma-irradiation. International Journal of Radiation Biology: Related Studies in Physics

Chemistry and Medicine 45, 377-383.

Page 19 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

20

9. Hall EJ, Bedford JS. 1964. Dose rate: its effect on the survival of HeLa cells irradiated with gamma

rays. Radiation Research 22, 305-315.

10. Bedford JS, Mitchell JB. 1973 Dose-rate effects in synchronous mammalian cells in culture.

Radiat Res. May; 54(2):316-27.

11. Hall EJ, Brenner, DJ. 1991 The dose-rate effect revisited: radiobiological considerations of

importance in radiotherapy. International Journal of Radiation Oncology Biology and Physics

21, 1403-1414. (1991).

12. Mitchell JB, Bedford JS. 1977. Dose-rate effects in synchronous mammalian cells in culture. II. A

comparison of the life cycle of HeLa cells during continuous irradiation or multiple-dose

fractionation. Radiation Research 71, 547-560.

13. Mitchell JB, Bedford JS, Bailey SM. 1979. Dose-rate effects on the cell cycle and survival of S3

HeLa and V79 cells. Radiation Research 79(3): 520-536.

14. Mitchell JB, Bedford JS, Bailey SM. 1979. Dose-rate effects in plateau-phase cultures of S3 HeLa

and V79 cells. Radiation Research 79(3):552-67.

15. van Oostrum IEA, Erkens-Schulze S, Petterson M, Wils IS, Rutgers DH. 1990. The relationship

between radiosensitivity and cell kinetic effects after low- and high-dose-rate irradiation in five

human tumors in nude mice. Radiat Research 122, 252-261. (1990)

16. Knox, S. J., W. Sutherland, Goris ML. 1993. "Correlation of tumor sensitivity to low-dose-rate

irradiation with G2/M-phase block and other radiobiological parameters." Radiation Research

135(1): 24-31.

Page 20 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

21

17. Bristow RG Benchimol S and Hill RP 1996 The p53 gene as a modifier of intrinsic

radiosensitivity: implications for radiotherapy. Radiotherapeutic Oncology; 40:197-223.

18. Dahm-Daphi J p53: biology and role for cellular radiosensitivity. Strahlentherapie und Onkologie;

176:278-285.9 2000.

19. Matsui Y Tscuchida Y Keng PC 2001 Effects of p53 mutations on cellular sensitivity to ionizing

radiation. American Journal of Clinical Oncology. Oct;24(5):486-90.

20. Weber KJ, Wenz F. 2000 p53, Apoptosis and radiosensitivity –experimental and clinical data.

Onkologie; 25: 136-141.

21. Hartwell, LH. Wiener, TA 1989 “Checkpoints: Controls that ensure the order of cell cycle events”.

Science 246: 629.

22. Bernhard, E. J., A. Maity, Muschel RJ, McKenna WG. 1995. "Effects of ionizing radiation on cell

cycle progression. A review." Radiation and Environmental Biophysiscs 34(2): 79-83.

23. McIlwrath AJ, Vasey PA, Ross GM, Brown R. 1994 Cell cycle arrests and radiosensitivity of human

tumor cell lines: dependence on wild-type p53 for radiosensitivity. Cancer Research. Jul

15;54(14):3718-22.

24. Pellegata, NS, Antoniono RJ, Redpath JL, Stanbridge EJ. 1996. DNA damage and p53-mediated

cell cycle arrest: a reevaluation. Proceedings of the National Academy of Science U S A 93(26):

15209-14

Page 21 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

http://gateway.nlm.nih.gov/gw/Cmd?linkVars=SessionID%3D06111008432275600615360031%26BROWSER_STATE%3DGMResults%26ORBagentPort%3D14600%26GM2K_FORM%3DGMResults%26LAST_HIDDEN_TIMESTAMP%3D1163166296794%26UserSearchText%3DMcIlwrath%2Bp53%26sb_action%3DExpand%2BItem%2B%253A%2B4%26HIDDEN_TIMESTAMP%3D1163166301957

http://gateway.nlm.nih.gov/gw/Cmd?linkVars=SessionID%3D06111008432275600615360031%26BROWSER_STATE%3DGMResults%26ORBagentPort%3D14600%26GM2K_FORM%3DGMResults%26LAST_HIDDEN_TIMESTAMP%3D1163166296794%26UserSearchText%3DMcIlwrath%2Bp53%26sb_action%3DExpand%2BItem%2B%253A%2B4%26HIDDEN_TIMESTAMP%3D1163166301957

For Peer Review O

nly

22

25. Fertil B, Malaise EP. 1985 Intrinsic radiosensitivity of human cell lines is correlated with

radioresponsiveness of human tumors: analysis of 101 published survival curves. Int J Radiat

Oncol Biol Phys. Sep;11(9):1699-707.

26. Malaise EP, Fertil B, Chavaudra N. and Guichard M. 1986 Distribution of radiation sensitivities

for human tumor cells of specific histological types: comparison of in vitro to in vivo data.

International Journal of Radiation Oncology Biology and Physics. Apr;12(4):617-24.

27. West, C.M., Davidson, S.E., Roberts, S.A. & Hunter, R.D. 1997. The independence of intrinsic

radiosensitivity as a prognostic factor for patient response to radiotherapy of carcinoma of the

cervix. Br J Cancer 76, 1184-90

28. Jones B, Dale RG, Deehan C, Hopkins KI, Morgan DA. The role of biologically effective dose

(BED) in clinical oncology. Clin Oncol (R Coll Radiol). 2001;13(2):71-81.

29. Fowler JF. 2005 The radiobiology of prostate cancer including new aspects of fractionated

radiotherapy. Acta Oncol. 2005;44(3):265-76.

30. Haustermans K, Withers HR. The biological basis of fractionation. Rays. 2004 Jul-

Sep;29(3):231-6.

31. Williams JR, Zhang Y, Dillehay LE. 1992. Sensitization processes in Human Tumor Cells

During Protracted Irradiation: Possible Exploitation in the Clinic. International Journal of

Radiation Oncology Biology and Physics, 24(4), 699-704.

32. Williams JA, Williams JR, Xuan Y, Dillehay, L.E. 1998 Protracted exposure radiosensitization

of experimental human malignant glioma. Radiation Oncology Investigations., 6:255-263, 1998

Page 22 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

23

33. Yuan X, Dillehay LE, Williams JR, Williams JA. 2001 IUdR Polymers for Combined

Continuous Low-dose Rate Sensitization of Experimental Malignant Gliomas. International

Journal of Cancer (Radiotherapeutic Oncology Investigations) 96: 118-125.

Acknowledgements: We express our appreciation to Bert Vogelstein and Ken Kinzler for

discussions and providing colorectal tumor cells. We express our appreciation to Dr. Ted

DeWeese for providing data on prostate and bladder cell lines.

Figure and Table Legends:

Figure 1: LDR, HDR responses in four groups of human tumor cells that vary in genotype

and radiosensitivity to HDR irradiation as defined in Williams et al. [1, 2]. Panel A: HDR,

Panel B: LDR. In each panel data are shown for cells from four radiosensitivity groups

Page 23 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

24

and are designated as VR (very resistant) that includes three resistant glioblastoma cell line

(U251, T98, U87), R (resistant) that includes four cell lines that express mutant TP53

(DLD-1, WiDR, HT29, SW480), S (sensitive) that includes four cell lines that express

wildtype TP53 (HCT116, RKO, CBS, LS174T) or VS (very sensitive) that includes a single

cell line that is mutant in ATM. . Error bars represent the standard error of the mean

with n=9 for VR cells, n=12 for R cells, n=12 for S cells and n=3 for VS cells and are shown

where the error bars (SEM) are larger than the symbol.

Figure 2. LDR and HDR survival patterns for each of the four radiosensitivity groups:

Panel A: VS radiosensitivity group (n= 3). Panel B: S radiosensitivity group (n=12). Panel

C: R radiosensitivity group (n = 12). Panel D: VR radiosensitivity group (n =9). The slopes

for α(LDR) and α(HDR) are shown as dotted lines in each panel. Error bars represent

standard errors of the means.

Figure 3: Correlation patterns between four parameters that describe LDR and HDR

radiosensitivity for 27 cell lines. Panel A: α(LDR) versus α(HDR); Panel B: ω(LDR) versus

ω(HDR); Panel C: α(LDR) versus ω(LDR); Panel D: α(HDR) versus ω(HDR). Each point

is an individual measurement derived by linear regression of dose segments as described in

the text. Specific cell lines are identified for comparison between panels: U251 and U87, the

most resistant cell lines, SW1222 the most sensitive cell line and two manipulated lines,

379.2 is abrogated in TP53 and 14-3-3σ-/- is abrogated in that gene. Cell lines that lie

outside two standard deviations from other cells in their radiosensitivity groups are also

identified by name: Panel A, LNCap and 14-3-3σ -/-; Panel B, PPC-1 and 379.2; Panel C,

HCT-116 and Panel C, PPC-1.

Figure 4: Changes in the rate of cell killing in 27 human tumor cells during LDR

irradiation between 24 and 72 hours. Cell lines are identified individually. Solid symbols

Page 24 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

25

indicate values for α(LDR) for each cell line and open symbols indicate terminal rate of cell

killing at 72 hours. Arrows indicate the direction of change.

Figure 5: Comparison of changes in surviving fraction (top panel) and radiation-induced

changes in prevalence of cells in different phases of the cell cycle (lower three panels) as a

function of duration of irradiation at 0.25 Gy/hr. Redistribution is expressed as % of total

cells in G1, S and G2-phases of the intermitotic cycle. The line representing S cells is the

mean of four unmanipulated human colorectal tumor cells that express wtTP53 and the

line representing R cells is the mean of four unmanipulated human colorectal tumor cell

lines that express mutTP53. Redistribution data for S and R cells are the means of four cell

lines each (n=4), data for SW1222 cells are single measurements.

Figure 6: Prevalence of cells in the G1 phase of the cell cycle (upper panel) and in the G2

phase of the cell cycle (lower panel) at 48 hours of LDR irradiation compared to

radiosensitivity of each cell line at that time point. Regression lines are shown for all data.

Data points are individual measurements.

Figure 7: Cartoon of a simple multi-segment (MS) model for radiosensitivity to HDR and

LDR irradiation. The coefficient αS is the slope of the low-dose hyper-radiosensitive

response that is overcome at higher doses; α (HDR/LDR) is the common slope of the

response induced by HDR and LDR; ω(HDR) is the slope of an increased radiosensitivity

induced in all cells between 2-4 Gy HDR; and ω(LDR) is the LDR time-dependent

induction of increased resistance (S cells) or increased sensitivity (R cells). This model is

applicable for low dose-rates in the range of approximately 0.25 Gy/hr.

Table 1: Genetic and radiobiological characteristics of 27 cell lines. Each cell line is

described by histological type, radiosensitivity group, molecular characteristics, TP53

Page 25 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

26

expression status and four coefficients that describe HDR and LDR radiosensitivity.

Radiosensitivity groups are defined on the basis of values of α(HDR) as described in

Williams et al [1, 2].

Table 2: Statistical analysis of four coefficients that describe LDR and HDR

radiosensitivity at lower and higher doses. For wtTP53 cells n = 11; for mutTP53 cells, n =

12.

Page 26 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

27

Page 27 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Figure 1: LDR, HDR responses in four groups of human tumor cells that vary in

genotype and radiosensitivity to HDR irradiation as defined in Williams et al. [1, 2].

Panel A: HDR, Panel B: LDR. In each panel data are shown for cells from four

radiosensitivity groups and are designated as VR (very resistant) that includes three

resistant glioblastoma cell line (U251, T98, U87), R (resistant) that includes four cell

lines that express mutant TP53 (DLD-1, WiDR, HT29, SW480), S (sensitive) that

includes four cell lines that express wildtype TP53 (HCT116, RKO, CBS, LS174T)

or VS (very sensitive) that includes a single cell line that is mutant in ATM. . Error

bars represent the standard error of the mean with n=9 for VR cells, n=12 for R

cells, n=12 for S cells and n=3 for VS cells and are shown where the error bars

(SEM) are larger than the symbol.

HDR

-6

-5

-4

-3

-2

-1

0

0 2 4 6 8 10 (Gy)

Lo

g S

F

VR

R

S

VS

A LDR

-6

-5

-4

-3

-2

-1

0

0 6 12 18 24 (Gy)

Lo

g S

F

VR

RS

VS

B

Page 28 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Figure 2. LDR and HDR survival patterns for each of the four radiosensitivity

groups: Panel A: VS radiosensitivity group (n= 3). Panel B: S radiosensitivity

group (n=12). Panel C: R radiosensitivity group (n = 12). Panel D: VR

radiosensitivity group (n =9). The slopes for α(LDR) and α(HDR) are shown as

dotted lines in each panel. Error bars represent standard errors of the means.

C S

-8

-6

-4

-2

0

0 5 10 15 20 25 (Gy)

Lo

g S

F

α(LDR)α(HDR)

B R

-4

-3

-2

-1

0

0 5 10 15 20 25 (Gy)

Lo

g S

F

α(LDR)α(HDR)

A VR

-4

-3

-2

-1

0

0 5 10 15 20 25 (Gy)

Lo

g S

F

α(LDR)

α(HDR)

D VS

-8

-6

-4

-2

0

0 5 10 15 20 25 (Gy)

Lo

g S

F

α(LDR)

α(HDR)

HDR LDR

Page 29 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Page 30 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Correlation Between Coefficients that Describe LDR, HDR Radiosensitivity

Figure 3: Correlation patterns between four parameters that describe LDR and HDR radiosensitivity for 27 cell lines. Panel A: α(LDR) versus α(HDR); Panel B: ω(LDR) versus ω(HDR); Panel C: α(LDR) versus ω(LDR); Panel D: α(HDR) versus ω(HDR). Each point is an individual measurement derived by linear regression of dose segments as described in the text. Specific cell lines are identified for comparison between panels: U251 and U87, the most resistant cell lines, SW1222 the most sensitive cell line and two manipulated lines, 379.2 is abrogated in TP53 and 14-3-3σ-/- is abrogated in that gene. Cell lines that lie outside two standard deviations from other cells in their radiosensitivity groups are also identified by name: Panel A, LNCap and 14-3-3σ -/-; Panel B, PPC-1 and 379.2; Panel C, HCT-116 and Panel C, PPC-1.

α (LDR) and ω (LDR)

R2 = 0.0187

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.1 0.2 0.3

ω LDR

αLD

R

wt TP53 S

14-3-3σ -/-

SW1222

U251

379.2

HCT116

U87

Cα (HDR) and ω (HDR)

R2 = 0.4969

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.1 0.2 0.3 0.4 0.5 0.6

ω HDR

αH

DR

mt TP53 R null TP53

14-3-3σ -/-SW1222

U251

379.2

U87

PPC-1

D

α (LDR) and α (HDR)

R2 = 0.8764

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 0.1 0.2 0.3 0.4

α HDR

αLD

R

14-3-3σ -/-

SW1222

U251

379.2

LNCapU87

Aω (LDR) and ω (HDR)

R2 = 0.0543

0

0.05

0.1

0.15

0.2

0.25

0.3

0 0.1 0.2 0.3 0.4 0.5 0.6

ω HDR

ωLD

R

14-3-3σ -/-SW1222

U251

379.2

PPC-1

U87

B

Page 31 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

LS17

4T

HC

T116

40-1

6

RK

O

LoV

o

N6C

HR

3

80S

4

DLD

-1

SW

480

WiD

R

HT2

9

19S

186

SW

1116

SW

1222

379.

2

CaC

o-2

CaC

o2-n

eo-r

as

U87

U25

1

T98G

LNC

ap

PP

C-1

PC

-3

DU

145

TSU

Cell Line

Ch

ang

e in

Rad

iose

nsi

tivi

ty b

twee

n 24

and

72

hour

swt TP53 mt TP53 null TP53

wt TP53 mt TP53 null TP53

Colorectal ProstateGlio

72 hours

α (LDR)

average α of S Cells

average α of R Cells

Bladder

14-3

-3 σ

+/+

14-3

-3 σ

-/-

Figure 4: Changes in the rate of cell killing in 27 human tumor cells during LDR irradiation between 24 and 72 hours. Cell lines are identified individually. Solid symbols indicate values for α(LDR) for each cell line and open symbols indicate rate of cell killing at 72 hours. Arrows indicate the direction of change.

Page 32 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nlyLDR Radiosensitivity and Radiation-induced Cell-Cycle

Changes in Three Radiosensitivity Groups.

0102030405060708090

0 8 16 24 32 40 48 56 64 72 80 88 96 (hrs)

G1

(%)

010

203040

506070

8090

0 8 16 24 32 40 48 56 64 72 80 88 96 (hrs)

S (%

)

0

1020

3040

5060

7080

90

0 12 24 36 48 60 72 84 96 (hrs)

G2

(%)

S cells R cells SW1222

-6

-5

-4

-3

-2

-1

0

0 6 12 18 24(Gy)

Log S

F

Page 33 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nlyFigure 5: Comparison of changes in surviving fraction (top panel) and radiation-

induced changes in prevalence of cells in different phases of the cell cycle (lower

three panels) as a function of duration of irradiation at 0.25 Gy/hr. Redistribution

is expressed as % of total cells in G1, S and G2-phases of the intermitotic cycle. The

line representing S cells is the mean of four unmanipulated human colorectal tumor

cells that express wtTP53 and the line representing R cells is the mean of four

unmanipulated human colorectal tumor cell lines that express mutTP53.

Redistribution data for S and R cells are the means of four cell lines each (n=4),

data for SW1222 cells are single measurements.

Page 34 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nlyRadiosensitivity and Cell Cycle Prevalence

Radiosensitivity versus G1 Prevalence

R2 = 0.0046

0102030405060708090

0 0.05 0.1 0.15 0.2 0.25

%C

ells

inG

1(4

8hrs

)LD

R

379.2SW1222

Radiosensitivity versus G2 Prevalence

R2 = 0.1057

0102030405060708090

0 0.05 0.1 0.15 0.2 0.25

Radiosensitivity (LogSF/Gy) at 48 hours

%C

ells

inG

2(4

8hrs

)LD

R

wt p53 mt p53 null p53

SW1222

379.2

Figure 6: Prevalence of cells in the G1 phase of the cell cycle (upper panel) and in

the G2 phase of the cell cycle (lower panel) at 48 hours of LDR irradiation

compared to radiosensitivity of each cell line at that time point. Regression lines

are shown for all data. Data points are individual measurements.

Page 35 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Page 36 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly0 2 4 6 8 10 12 14 16 18 Dose

Log

Surv

ivin

gFr

actio

n

Dose-depenent change in HDR radiosensitivity

circa 2-4Gy

αs

Time-depenent change in LDR radiosensitivity

at 24 hrs

ω (HDR)ω (LDR)R cells

α (HDR, LDR)Genotype

dependent

ω (LDR)S cells

Figure 7: Cartoon of a simple multi-segment (MS) model for radiosensitivity to

HDR and LDR irradiation. The coefficient αS is the slope of the low-dose hyper-

radiosensitive response that is overcome at higher doses; α (HDR/LDR) is the

common slope of the response induced by HDR and LDR; ω(HDR) is the slope of an

increased radiosensitivity induced in all cells between 2-4 Gy HDR; and ω(LDR) is

the LDR time-dependent induction of increased resistance (S cells) or increased

sensitivity (R cells). This model is applicable for low dose-rates in the range of

approximately 0.25 Gy/hr.

Page 37 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nly

Molecular and Radiobiological Characteristics of Human Tumor Cell Lines

HistologicalType

RadGroup

CellLine Comments

TP53status

α(LDR)

α(HDR)

ω(LDR)

ω(HDR)

VS SW1222 Mutant in ATM null 0.30 0.38 0.23 0.56LS174T wt 0.26 0.23 0.14 0.42HCT116 wt 0.36 0.32 0.092 0.4240-16 Clone of HCT116 wt 0.21 0.21 0.15 0.41379.2 TP53 double knockout from 40-16 null 0.23 0.19 0.037 0.3614-3-3σ+/+ Clone of HCT116 wt 0.26 0.14 0.083 0.3714-3-3 σ-/- Double knockout from 14-3-3σ+/+ wt 0.24 0.32 0.19 0.37

N6CHR3HMLH1 on chromosome 3 added to HCT116

wt 0.28 0.24 0.078 0.34

80S4CDKN1A double knockout from HCT116 wt 0.28 0.24 0.070 0.40

RKO wt 0.21 0.15 0.13 0.36

S

LOVO wt 0.28 0.21 0.083 0.36DLD-1 p53: 241ser - phe mt 0.088 0.052 0.083 0.30

19S186CDKN1A double knockout from DLD-1

mt 0.064 0.078 0.099 0.32

SW480 p53:248 arg - his mt 0.14 0.10 0.18 0.27WiDR p53:273 arg - his mt 0.077 0.061 0.15 0.24HT29 p53:273 arg - his mt 0.11 0.096 0.21 0.22SW1116 mt 0.14 0.16 0.13 0.27CaCO-2 null 0.074 0.090 0.070 0.28

Colorectal

R

CaCO-2-neo-ras

k-ras-/- - k-ras+/+ null 0.10 0.070 0.075 0.29

U251 P53:273arg - his mt 0.066 0.031 0.13 0.098U87 wt 0.055 0.017 0.17 0.31Glioblastoma VRT98G mt 0.095 0.058 0.070 0.13LNCap wt 0.16 0.29 0.12 0.41PPC-1 mt 0.15 0.10 0.24 0.51SPC-3 mt 0.12 0.065 0.15 0.29Prostate

R DU145p53:274 pro - leu223 val - phe

mt 0.079 0.10 0.17 0.24

Bladder R TSU mt 0.094 0.11 0.19 0.30

Table 1: Genetic and radiobiological characteristics of 27 cell lines. Each cell line is described by histological type, radiosensitivity group, molecular characteristics, TP53 expression status and four coefficients that describe HDR and LDR radiosensitivity. Radiosensitivity groups are defined on the basis of values of α(HDR) as described in Williams et al [1, 2].

Page 38 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review O

nlywt TP53 versus mut TP53

Coefficient t values p values

α(LDR) 5.49 <0.001

α(HDR) 4.79 <0.001

ω(LDR) -1.45 0.163

ω(HDR) 3.47 0.002

Table 2: Statistical analysis of four coefficients that describe LDR and HDR radiosensitivity at lower and higher doses. For wtTP53 cells n = 11; for mutTP53 cells, n = 12.

Page 39 of 39


Acta Oncologica

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Overview of Radiosensitivity of Human Tumor Cells to Low-Dose-Rate Irradiation

Documents

Transcript of Overview of Radiosensitivity of Human Tumor Cells to Low-Dose-Rate Irradiation