Radiation Sensitivity of Primary Fibroblasts from Hereditary Retinoblastoma Family Members and Some Apparently Normal Controls: Colony Formation Ability during Continuous Low-Dose-Rate Gamma Irradiation

Article (PDF Available)inRadiation Research 169(5):483-94 · June 2008with16 Reads
DOI: 10.1667/RR1333.1 · Source: PubMed
Abstract
We previously described an enhanced sensitivity for cell killing and G(1)-phase cell cycle arrest after acute gamma irradiation in primary fibroblast strains derived from 14 hereditary-type retinoblastoma family members (both affected RB1(+/-) probands and unaffected RB1(+/+) parents) as well as distinctive gene expression profiles in unirradiated cultures by microarray analyses. In the present study, we measured the colony formation ability of these cells after exposure to continuous low-dose-rate (0.5-8.4 cGy/h) (137)Cs gamma radiation for a 2-week growth period. Fibroblasts from all RB family members (irrespective of RB1 genotype) and from 5 of 18 apparently normal Coriell cell bank controls were significantly more radiosensitive than the remaining apparently normal controls. The average dose rates required to reduce relative survival to 10% and 1% were approximately 3.1 and 4.7 cGy/h for the Coriell control strains with normal radiosensitivity and approximately 1.4 and 2.5 cGy/h for the radiosensitive RB family member and remaining apparently normal Coriell control strains. The finding that a significant proportion of fibroblast strains derived from apparently normal individuals are sensitive to chronic low-dose-rate irradiation indicates such individuals may harbor hypomorphic genetic variants in genomic maintenance and/or DNA repair genes that may likewise predispose them or their children to cancer.
483
RADIATION RESEARCH
169, 483–494 (2008)
0033-7587/08 $15.00
2008 by Radiation Research Society.
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Radiation Sensitivity of Primary Fibroblasts from Hereditary
Retinoblastoma Family Members and Some Apparently Normal
Controls: Colony Formation Ability during Continuous Low-Dose-Rate
Gamma Irradiation
Paul F. Wilson,
a,b,1
Hatsumi Nagasawa,
a
Christy L. Warner,
a
Markus M. Fitzek,
c
John B. Little
d
and Joel S. Bedford
a
a
Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523;
b
Biosciences and
Biotechnology Division, Lawrence Livermore National Laboratory, Livermore, California 94551;
c
Department of Radiation Oncology,
Indiana University School of Medicine, Indianapolis, Indiana 46202; and
d
Center for Radiation Sciences and Environmental Health,
Harvard School of Public Health, Boston, Massachusetts 02115
Wilson, P. F., Nagasawa, H., Warner, C. L., Fitzek, M. M.,
Little, J. B. and Bedford, J. S. Radiation Sensitivity of Pri-
mary Fibroblasts from Hereditary Retinoblastoma Family
Members and Some Apparently Normal Controls: Colony
Formation Ability during Continuous Low-Dose-Rate Gamma
Irradiation. Radiat. Res. 169, 483–494 (2008).
We previously described an enhanced sensitivity for cell
killing and G
1
-phase cell cycle arrest after acute irradiation
in primary fibroblast strains derived from 14 hereditary-type
retinoblastoma family members (both affected RB1
/
pro-
bands and unaffected RB1
/
parents) as well as distinctive
gene expression profiles in unirradiated cultures by microar-
ray analyses. In the present study, we measured the colony
formation ability of these cells after exposure to continuous
low-dose-rate (0.5–8.4 cGy/h)
137
Cs radiation for a 2-week
growth period. Fibroblasts from all RB family members (ir-
respective of RB1 genotype) and from 5 of 18 apparently nor-
mal Coriell cell bank controls were significantly more radio-
sensitive than the remaining apparently normal controls. The
average dose rates required to reduce relative survival to 10%
and 1% were 3.1 and 4.7 cGy/h for the Coriell control
strains with normal radiosensitivity and 1.4 and 2.5 cGy/h
for the radiosensitive RB family member and remaining ap-
parently normal Coriell control strains. The finding that a
significant proportion of fibroblast strains derived from ap-
parently normal individuals are sensitive to chronic low-dose-
rate irradiation indicates such individuals may harbor hypo-
morphic genetic variants in genomic maintenance and/or
DNA repair genes that may likewise predispose them or their
children to cancer.
2008 by Radiation Research Society
INTRODUCTION
A phenotype of in vitro cellular hypersensitivity to ion-
izing radiation has been documented in many cancer pre-
1
Address for correspondence: P.O. Box 808, Mail Code L-452,
Lawrence Livermore National Laboratory, Livermore, CA 94551-0808;
e-mail: wilson208@llnl.gov.
disposition and chromosomal instability syndromes, most
notably ataxia telangiectasia (AT), ataxia telangiectasia-like
disorder (ATLD) and Nijmegen breakage syndrome (NBS),
spurring interest in the use of radiobiological techniques to
identify additional genes involved in DNA repair and re-
lated cell cycle regulatory pathways and their contribution
to the tumorigenic process (3–5). Aside from cells derived
from these patients (with mutations in ATM, MRE11 and
NBS1, respectively) and similarly hyper-radiosensitive re-
sponses documented in non-homologous end-joining
(NHEJ)-deficient rodent cells (6–8), the degree of radiosen-
sitivity documented in other genetic conditions (e.g., breast
cancer, Fanconi anemia, and hereditary-type retinoblasto-
ma) is more modest when measured by traditional radio-
biological assays (1, 9–22). To confound matters further,
the classification of ‘radiosensitive’ in these assays is
somewhat arbitrary, since the radiation responses of pa-
tients’ cells overlap with the lower end of a broad range of
responses measured in cells derived from apparently nor-
mal, disease-free individuals (1, 9–26). Radiosensitivity
surveys using cell strains derived from apparently normal
individuals available at national cell banks have indicated
that a significant proportion (10–20%) are moderately ra-
diosensitive (23–27), a testament to the presence of hypo-
morphic genetic variants in the general population that may
adversely affect DNA damage surveillance and repair sys-
tems (28–31). The cumulative effect of reduced enzymatic
activities in such individuals may account for variations in
both in vitro cellular responses to various types of DNA
damage and in vivo responses of patients receiving radio-
therapy treatment (32–34).
The childhood cancer retinoblastoma results from germ-
line or somatic mutation of the retinoblastoma susceptibility
gene RB1 located on chromosome 13q14.2 (35). Heredi-
tary-type retinoblastoma (resulting from germline RB1 mu-
tation) is associated with a high incidence of radiotherapy
and chemotherapy treatment-related secondary cancers
484 WILSON ET AL.
(36–38), a wide range of cellular radiosensitivity [reviewed
in ref. (26)], and a similarly wide range of chromosomal
instability observed in both somatic and tumor cells (of
both primary and secondary cancers) from RB patients (15,
16, 21, 22, 39–41). This indicates that mutations in other
genes (other than RB1) are likely to be involved in this
disease and in the variability of radiation responses seen in
RB patient cells. The range of instability phenotypes ob-
served in pRB-deficient cells is a reflection of an ever in-
creasing list of functions ascribed to the RB protein and its
protein partners in the maintenance of key cellular regula-
tory mechanisms, including increasing evidence of a direct
role in mitotic fidelity and DNA repair (42–46). As such,
radiobiological studies using somatic cells derived from un-
affected parents or siblings in RB families that are likewise
considered radiosensitive could yield valuable evidence
concerning the nature and effect of additional genetic mu-
tations or polymorphisms on this phenotype in a wild-type
RB1
/
background.
Moderate cellular radiosensitivity in primary fibroblast
strains derived from unaffected first-degree relatives of he-
reditary-type RB patients has been reported (17, 24), most
recently by Fitzek and coworkers (1). This latter report doc-
umented hypersensitivity for acute (high-dose-rate) radia-
tion-induced cell killing and G
1
-phase cell cycle delay in
primary fibroblast strains derived from 14 members of five
unrelated hereditary-type RB families, both unaffected
RB1
/
parents and affected RB1
/
children. The same 14
RB family member fibroblast strains were examined in this
low-dose-rate radiation study, along with 18 age- and sex-
matched apparently normal control fibroblast strains ob-
tained from the Coriell Cell Repositories. Fibroblasts de-
rived from these unaffected RB family parents were also
shown to differ significantly in their gene expression pro-
files in the absence of radiation exposure by cDNA mi-
croarray and quantitative RT-PCR measurements compared
to the majority of the same apparently normal Coriell fi-
broblast strains (2). Principal component analyses of the
microarray data showed a number of genes involved in can-
cer development to be significantly down-regulated in the
unaffected RB1
/
parent cells, including NM23A, MCM5,
HOXB2, HOXC10/D10, PLK and E2F1, which are under
further investigation.
The continuous low-dose-rate irradiation protocol used
in the present study is similar to the approach used to iso-
late the radiation-sensitive CHO mutant irs-20 and later
identify the genetic mutation responsible for this phenotype
(a hypomorphic mutation in Prkdc, the gene encoding the
catalytic subunit of the DNA-dependent protein kinase,
DNA-PK) (47–50). Low-dose-rate radiation hypersensitiv-
ity has been documented in a variety of other DNA repair-
deficient cells that likewise show reduced dose-rate effects
(lack of dose-rate sparing) for the induction of chromosome
aberrations and mutations and for cell survival (6, 10, 19,
20, 23, 34, 51–65). Several groups including our own have
reported better discrimination of relative individual radio-
sensitivity using low-dose-rate exposures of G
0
/G
1
-phase
density-inhibited fibroblasts and unstimulated T lympho-
cytes compared to high-dose-rate exposures (10, 23, 34,
64–67). To avoid the complication of differential cell cycle
phase radiosensitivities and cell proliferation during the
long exposure periods associated with low-dose-rate ex-
posures, irradiations are traditionally conducted with these
quiescent, non-cycling cultures of cells (6, 10, 19, 20, 23,
34, 54–58, 60–70). However, the current assay requires cell
division (and transit of the entire cell cycle) during contin-
uous low-dose-rate irradiation (rather than after a specified
dose delivered at low dose rate) for a cell to successfully
proliferate and form a colony. Thus this type of continuous
low-dose-rate exposure assay is particularly suited for de-
tecting deficiencies in both primary DNA double-strand
break (DSB) repair pathways, non-homologous end joining
(NHEJ) and homologous recombinational repair (HRR), as
well as defects in DNA damage-dependent cell cycle
checkpoints
2
(65, 71–73).
The purpose of the present study was to determine
whether exposure to continuous low-dose-rate
137
Cs ra-
diation at dose rates of 0.5–8.4 cGy/h for a 2-week colony
formation period could result in a greater degree of growth
inhibition of the fibroblast strains derived from RB family
members compared to apparently normal Coriell strains and
provide a more sensitive assay for detecting more moderate
changes in radiosensitivity compared to high-dose-rate ex-
posures (1). We demonstrate that primary fibroblasts de-
rived from both the unaffected RB1
/
parents and affected
RB1
/
probands of the five RB families are radiosensitive
compared to the majority of the apparently normal Coriell
control strains. This implies a deficiency in the DNA dam-
age response and/or DSB repair capacity in these individ-
uals, which does not correlate with their RB1 genotype. In
addition to the unaffected, apparently normal parents of the
RB probands, a proportion of the apparently normal Coriell
fibroblast strains examined in this study are similarly ra-
diosensitive in this range of low dose rates.
MATERIALS AND METHODS
Cell Culture and Irradiations
Control apparently normal human primary fibroblast cell strains were
obtained from the Coriell Cell Repositories at the Coriell Institute for
Medical Research (Camden, NJ; coded ‘AG’ or ‘GM’’). Derivation of
the primary fibroblast strains from affected probands and unaffected par-
ents of five hereditary-type retinoblastoma families (coded ‘MF’’) was
described previously (1). All fibroblast strains were passaged regularly
and maintained in -MEM (Gibco/Invitrogen) supplemented with 15%
FBS, 50 U/ml penicillin, and 50 g/ml streptomycin sulfate (Sigma) in
a37C humidified incubator supplied with 5% CO
2
prior to each exper-
iment. A single lot of serum was used for the entire set of experiments
described in this report to reduce interexperimental variability. Cultures
were detached with 0.25% trypsin (Gibco/Invitrogen)/1 mM EDTA in
Ca
2
/Mg
2
-free PBS, counted with a Coulter Z-1 particle data counter
2
P. F. Wilson, Genetic factors affecting radiation sensitivity and ge-
nomic stability. Ph.D. Dissertation, Colorado State University (2006).
485LOW-DOSE-RATE SURVIVAL OF RETINOBLASTOMA CELLS
FIG. 1. Results of the continuous low-dose-rate irradiation colony formation assay for the 13 apparently normal Coriell fibroblast strains with normal
radiosensitivity (see Table 1). Data points represent means SEM for 2–12 independent experiments per fibroblast strain, and the fitted dashed lines
represent the upper and lower limits of survival responses for this group.
(Beckman Coulter, Inc.), and plated as single cell suspensions into rep-
licate T-75 flasks (Grenier Bio-One) with 25 ml of pH-equilibrated
-MEM containing 25 mMN-2-hydroxyethylpiperazine-N-2-ethanesul-
fonic acid (Hepes, Gibco/Invitrogen) to ensure pH equilibration at 7.2–
7.5 without supplied CO
2
. Seeded flasks were then capped tightly and
placed in a 37C warm room overnight to allow for cell attachment.
After overnight incubation in the 37C warm room, the flasks were
transferred to the Colorado State University MRB012 low-dose-rate ir-
radiation facility, which is maintained at 37C without CO
2
supply
2
(47).
Flasks were stacked into 34 columns with 35 flasks per column (for more
than 4 m
2
of culturable surface area) onto a large shelf located 56 cm
above a 79-cm-diameter source array of 12 2.2-mCi (81.4-MBq)
137
Cs
sources. Odd-numbered flasks (1–35) contained either 25 ml of culture
medium and cells or 25 ml of water to ensure consistent attenuation,
while even-numbered flasks (2–34) were empty. Once arranged, four
sources were exposed and the cells were irradiated continuously for 7–9
days with dose rates attenuating exponentially from 8.4 cGy/h at the
bottom of the columns to 0.5 cGy/h at the top of the columns.
2
The flasks
were then removed from the irradiation facility; the cell medium was
exchanged with fresh -MEM with 10% FBS, antibiotics, and 25 mM
Hepes buffer; the flasks were returned to the irradiation facility for an
additional 7–9 days, for a total exposure time of approximately 14–18
days. After irradiations were completed, the flasks were removed from
the irradiation facility and the culture medium was aspirated. The flasks
were rinsed twice with 0.9% w/v sodium chloride, fixed with 95% eth-
anol, and stained with a 0.1% w/v crystal violet/methanol solution. Col-
onies with 50 viable cells were scored as survivors.
Dosimetry was performed using a Radcal Corporation Model 2025 ion
chamber equipped with a 12-cm-diameter pancake probe, calibrated to
NIST-traceable standards, enclosed in a T-150 cell culture flask (Grenier
Bio-One) to ensure appropriate buildup at the cell-flask interface. The
dose rates delivered to cells within the culture flasks (in cGy/h) was
calculated by correcting the measured ion chamber exposure rates (in
R/h) for the temperature and pressure of the irradiation chambers at the
time of measurement and the mass absorption coefficients of muscle and
air for 662 keV -ray photons
2
(74).
Data and Statistical Analyses
Because of the number of cells plated per flask and the variable cloning
efficiencies of the various fibroblast strains examined, the detection limit
for survivors in this assay is approximately 1 in 1000 (10
3
). Estimates
for absolute survival were calculated as the average number of colonies
counted per number of cells plated per dose rate (75, 76). The logarithm
of these survival estimates were averaged for 2–12 independent experi-
ments per fibroblast strain and fitted with a linear (y ax c) least-
squares-weighted regression function for survival data measured for dose
rates of 3 cGy/h only, where y is the average absolute survival for a
dose rate x. If the calculated intercept (c) was 5% different from the
measured zero-dose-rate cloning efficiency (CE) for a particular strain,
the calculated intercept value c was used as the zero-dose cloning effi-
ciency to calculate the average relative surviving fraction (i.e., the ab-
solute surviving fractions corrected for the zero-dose cloning efficien-
cies); otherwise the average measured CE was used to normalize the
absolute survival data. Standard errors of the means (SEM) were calcu-
lated by combining the standard deviations of the absolute surviving frac-
tions by the square root of the sum-of-the-squares method and dividing
this value by the square root of the number of experiments (75, 76) and
constitute the error bars in Figs. 1–3 and Tables 1–3. The average survival
responses for the radiosensitivity categories in Table 4 are reported as
arithmetic means and standard deviations (SD) of relative colony for-
mation ability after continuous low-dose-rate irradiation for the fibroblast
strains in each category.
Statistical significance was tested by Student’s t tests for two indepen-
dent sample distributions (SigmaPlot). Correlations between different ra-
diosensitivity parameters were assessed using the Pearson product-mo-
ment correlation coefficient, fitted by the least-squares method to the re-
spective data sets being compared (SigmaPlot). Since no parameter-based
486 WILSON ET AL.
FIG. 2. Results of the continuous low-dose-rate irradiation colony formation assay for the five radiosensitive apparently normal Coriell fibroblast
strains (see Table 2). Data points represent means SEM for 2–5 independent experiments per fibroblast strain, and the fitted dashed lines represent
the upper and lower limits of survival responses for this group. The gray-hatched area indicates range of response of the 13 apparently normal Coriell
fibroblast strains with normal radiosensitivity from Fig. 1.
FIG. 3. Results of the continuous low-dose-rate irradiation colony formation assay for the 14 radiosensitive RB family member fibroblast strains
(see Table 3). Data points represent means SEM for 2–6 independent experiments per fibroblast strain, and the fitted dashed lines represent the
upper and lower limits of survival responses for this group. The gray-hatched area indicates range of response of the 13 apparently normal Coriell
fibroblast strains with normal radiosensitivity from Fig. 1. Solid symbols, proband children; open symbols, mothers; gray symbols, fathers.
487LOW-DOSE-RATE SURVIVAL OF RETINOBLASTOMA CELLS
TABLE 1
Results of the Continuous Low-Dose-Rate (0.5–8.4 cGy/h) Irradiation Colony Formation
Assay for the 13 Apparently Normal Coriell Fibroblast Strains with Normal
Radiosensitivity
a
Fibroblast
strain Sex Age
No. of
experiments
Cloning efficiency
SEM (range)
Dose rate (cGy/h) for
relative survival of:
0.10 (10%) 0.01 (1%)
AG01521 Male 3 days 4 0.35 0.15 (0.20–0.66) 2.3 4.6
AG01522 Male 3 days 4 0.30 0.06 (0.16–0.49) 3.6 4.9
GM01652 Female 11 years 4 0.03 0.01 (0.01–0.06) 2.8 5.1
GM02149 Female 54 years 4 0.04 0.01 (0.03–0.05) 3.4 5.2
GM02673 Male 33 years 3 0.16 0.05 (0.01–0.30) 2.8 4.2
GM03377 Male 19 years 2 0.10 3.2 4.9
GM03440C Male 20 years 3 0.18 0.04 (0.05–0.25) 2.3 3.9
GM05757 Male 7 years 1 0.32 2.5 4.1
GM08333B Male 5 months 3 0.29 0.03 (0.24–0.40) 3.0 4.3
GM08400 Female 37 years 4 0.06 0.01 (0.01–0.07) 3.2 5.0
GM08402 Male 32 years 3 0.03 0.01 (0.01–0.04) 4.4 5.6
GM08429 Male 1 day 12 0.28 0.07 (0.05–0.65) 3.1 4.6
GM08680 Male 5 months 3 0.23 0.11 (0.10–0.45) 4.0 5.2
a
Radiosensitivity classifications are based on arbitrary cutoff values for continuous-irradiation dose rates required
to reduce relative survival to 0.01 (1%): fibroblast strains requiring 3.5 cGy/h are classified as having normal
radiosensitivity and those requiring 3.5 cGy/h are classified as radiosensitive.
TABLE 2
Results of the Continuous Low-Dose-Rate (0.5–8.4 cGy/h) Irradiation Colony Formation
Assay for the Five Radiosensitive Apparently Normal Coriell Fibroblast Strains
a
Fibroblast
strain Sex Age
No. of
experiments
Cloning efficiency
SEM (range)
Dose rate (cGy/h) for
relative survival of:
0.10 (10%) 0.01 (1%)
GM00041C Female 3 months 2 0.08 0.01 (0.07–0.08) 1.0 2.0
GM00969D Female 2 years 3 0.06 0.01 (0.04–0.11) 1.9 2.9
GM04503B Female 31 years 2 0.09 0.02 (0.06–0.11) 1.2 2.3
GM04505A Female 20 years 2 0.08 0.03 (0.04–0.12) 1.1 2.1
GM05756A Male 2 months 5 0.11 0.05 (0.04–0.28) 2.3 3.3
a
Radiosensitivity classifications are based on arbitrary cutoff values for continuous-irradiation dose rates required
to reduce relative survival to 0.01 (1%): fibroblast strains requiring 3.5 cGy/h are classified as having normal
radiosensitivity and those requiring 3.5 cGy/h are classified as radiosensitive.
radiobiological model exists for this type of continuous low-dose-rate
exposure (coupled with variable cell growth and differential dose-rate-
dependent cell cycle phase sensitivities), best-fit lines for the data sets in
Figs. 1–3 have been drawn for illustrative purposes only. Best-fit lines
for survival data at dose rates 3 cGy/h were drawn using a least-squares
fit linear-exponential regression curve. Curves were then best fitted by
eye to the survival data for dose rates 3 cGy/h. The continuous radiation
dose rates required to reduce relative survival to 0.10 (10%) and 0.01
(1%) are the radiosensitivity parameters reported for individual fibroblast
strains and groups thereof in Tables 1–4. A fibroblast strain’s radiosen-
sitivity classification was based on an arbitrary cutoff value for the dose
rate required to reduce relative survival to 0.01 (1%); strains requiring
3.5 cGy/h to reduce relative survival to 1% were considered to have
normal radiosensitivity and strains requiring dose rates 3.5 cGy/h to
reduce relative survival to 1% were considered radiosensitive.
RESULTS
The relative colony formation ability of the 32 apparently
normal and hereditary retinoblastoma primary fibroblast
strains examined in this study after 2 weeks of continuous
low-dose-rate
137
Cs irradiation is shown in Figs. 1–3 and
Tables 1–3. Figure 1 is a plot of the relative colony-forming
ability SEM for 13 Coriell fibroblast strains with normal
radiosensitivity (strains requiring 3.5 cGy/h to reduce rel-
ative survival to 1%). Figure 2 is a plot of the relative
colony-forming ability SEM for five radiosensitive Cor-
iell fibroblast strains (strains requiring 3.5 cGy/h to re-
duce relative survival to 1%). Figure 3 is a plot of the
relative colony-forming ability SEM for the 14 appar-
ently normal (RB1
/
) and affected (RB1
/
) RB family
member fibroblast strains. The legend symbols for Fig. 3
are coded identically: The affected proband children (either
sex) are solid symbols, mothers are open symbols, and fa-
thers are gray symbols, with a different symbol for each
family. Data for these groups are listed in Tables 1–3, re-
spectively, and include the sex, age, RB1 genotype (in the
case of the RB family members), number of experiments
conducted per strain, and the average dose rates required
488 WILSON ET AL.
TABLE 3
Results of the Continuous Low-Dose-Rate (0.5–8.4 cGy/h) Irradiation Colony Formation Assay for the 14
Radiosensitive RB1
/
and RB1
/
RB Family Member Fibroblast Strains
a
Fibroblast strain
(RB family) Sex Age RB1 genotype
No. of
experiments
Cloning efficiency SEM
(range)
Dose rate (cGy/h) for
relative survival of:
0.10 (10%) 0.01 (1%)
MF-2M (I) Female 33 years / 5 0.11 0.02 (0.01–0.20) 1.3 2.5
MF-3R (I) Female 14 months / 4 0.10 0.02 (0.03–0.20) 1.3 2.6
MF-4F (I) Male 33 years / 3 0.45 0.06 (0.27–0.63) 2.1 3.4
MF-6F (II) Male 24 years / 5 0.13 0.02 (0.02–0.30) 0.8 1.6
MF-7R (II) Male 14 months / 4 0.10 0.02 (0.03–0.23) 1.5 2.8
MF-10R (III) Male 4 months / 4 0.28 0.03 (0.11–0.42) 1.5 2.8
MF-11F (III) Male 41 years / 6 0.09 0.02 (0.01–0.23) 1.7 3.2
MF-12M (III) Female 36 years / 4 0.04 0.01 (0.01–0.11) 1.2 2.2
MF-13M (IV) Female 23 years / 5 0.13 0.03 (0.03–0.37) 1.0 2.0
MF-14R (IV) Female 4 months / 4 0.22 0.03 (0.05–0.38) 1.8 3.2
MF-15F (IV) Male 24 years / 5 0.16 0.02 (0.10–0.26) 1.2 2.4
MF-16M (V) Female 35 years / 3 0.15 0.06 (0.03–0.37) 1.4 2.2
MF-17R (V) Female 24 months / 2 0.02 0.01 (0.02–0.03) 1.1 1.9
MF-18F (V) Male 45 years / 5 0.19 0.06 (0.01–0.73) 1.0 1.7
a
Radiosensitivity classifications are based on arbitrary cutoff values for continuous-irradiation dose rates required to reduce relative survival to 0.01
(1%): fibroblast strains requiring 3.5 cGy/h are classified as having normal radiosensitivity and those requiring 3.5 cGy/h are classified as radio-
sensitive.
TABLE 4
Summary of the Continuous Low-Dose-Rate (0.5–8.4 cGy/h) irradiation Colony Formation Assay Results for
the 32 Coriell and RB Family Member Fibroblast Strains Examined in this Study, Grouped by
Radiosensitivity Category
a
Radiosensitivity group
a
No. of
fibroblast
strains
Range of
cloning
efficiencies
Dose rate (cGy/h; mean SD, range)
for relative survival of:
0.10 (10%) 0.01 (1%)
Significant difference
(P 0.05) from
normal controls
Apparently normal Coriell fibroblasts with normal
radiosensitivity 13 0.03–0.35 3.1 0.6 (2.3–4.4) 4.7 0.5 (3.9–5.6) Referent
Apparently normal Coriell fibroblasts with hyper-
radiosensitivity 5 0.06–0.11 1.5 0.6 (1.0–2.3) 2.5 0.6 (2.0–3.3) Yes
RB family member fibroblasts (RB1
/
and RB1
/
) 14 0.02–0.45 1.4 0.4 (0.8–2.1) 2.5 0.6 (1.6–3.4) Yes
RB1
/
RB family member fibroblasts (unaffected
parents) 8 0.04–0.45 1.3 0.4 (0.8–2.1) 2.3 0.6 (1.6–3.4) Yes
RB1
/
RB family member fibroblasts (affected parent
and probands) 6 0.02–0.28 1.5 0.3 (1.1–1.8) 2.8 0.5 (1.9–3.2) Yes
a
Radiosensitivity classifications are based on arbitrary cutoff values for continuous-irradiation dose rates required to reduce relative survival to 0.01
(1%): fibroblast strains requiring 3.5 cGy/h are classified as having normal radiosensitivity and those requiring 3.5 cGy/h are classified as radio-
sensitive.
to reduce relative survival to 0.10 (10%) and 0.01 (1%).
Each individual radiosensitive fibroblast strain (all RB fam-
ily members and the five radiosensitive Coriell controls),
and groups thereof, is significantly (P 0.05) more radio-
sensitive than the average response of the 13 Coriell fibro-
blast strains with normal radiosensitivity identified in Ta-
ble 1.
The effect of continuous low-dose-rate irradiation on the
retention of proliferative capacity of the 32 primary fibro-
blast strains examined in this study is summarized in Table
4 and Fig. 4. In Table 4, the Coriell and RB family member
fibroblast strains are grouped by radiosensitivity category
and RB1 genotype (for the RB family members), with the
number of fibroblast strains per group, their range of clon-
ing efficiencies (CE), and the mean SD and range of
dose rates required to reduce relative survival to 10% and
1%. A histogram of the continuous-irradiation dose rates
required to reduce relative survival to 1% for the 14 RB
family member and 18 Coriell control fibroblast strains is
shown in Fig. 4 (the MF, AG and GM prefixes have been
omitted for clarity). There is an obvious separation between
the 13 Coriell control strains having normal radiosensitivity
and the remaining radiosensitive Coriell and RB family
member strains, with the strains of normal radiosensitivity
requiring dose rates approximately twice those required for
the radiosensitive strains to achieve the same degree of
growth inhibition.
The continuous-irradiation dose rates required for 10%
489LOW-DOSE-RATE SURVIVAL OF RETINOBLASTOMA CELLS
FIG. 4. Histogram of dose rates (in cGy/h) required to reduce relative
colony formation ability to 0.01 (1%) in the continuous low-dose-rate
irradiation colony formation assay for the 18 apparently normal Coriell
fibroblast strains (open symbols) and 14 RB family member fibroblast
strains (RB1
/
parents, gray symbols; RB1
/
parent/probands, solid
symbols) examined in this study. The AG, GM and MF prefixes for each
strain identifier have been removed for clarity. Circles, females; squares;
males.
and 1% relative survival are significantly correlated (P
10
3
) with the unirradiated cellular cloning efficiency.
However, strains with nearly identical cloning efficiencies
(e.g., AG01521 and MF-4F, GM02673 and MF-15F) have
dramatically different responses in this assay, implying that
a cell’s intrinsic cloning efficiency is not itself predictive
of its response after irradiation (77). When the dose rates
required to reduce relative survival to 10% and 1% are
compared with the acute (high-dose-rate) irradiation D
0
sur-
vival values reported by Fitzek et al. (1) for the same fi-
broblast strains, only the continuous-irradiation dose rate
required to reduce relative survival to 1% is significantly
correlated with this acute-irradiation survival parameter (P
0.022). The general lack of correlation between the two
assay types is reflective of the significant differences be-
tween them. In an acute-irradiation survival assay, log-
phase or G
0
/G
1
-phase cells are irradiated with a single high-
dose-rate exposure (completed within minutes) and then
plated for colony formation without further radiation ex-
posure of cells in the growing colony. In the continuous
low-dose-rate irradiation survival assay, cells are irradiated
continuously at low dose rate as they attempt to proliferate,
with each successive round of daughter cells exposed to the
same genotoxic stress from the low-dose-rate irradiation as
the parental cells.
While the shape of the continuous low-dose-rate colony
formation assay dose-rate response appears to be similar to
an acute-irradiation colony formation assay dose response,
a number of important differences can be appreciated.
Overall population doubling times of these cell strains
ranged from 24 h to 100 h as the dose rate increased, and
differential dose-rate effects on cell cycle phase transitions
were observed using flow cytometry.
2
This makes the cal-
culation of the overall dose received by the original cell
generating the colony difficult (78). The total dose to the
flask can be calculated easily, but not the dose to the cells
in the flask generating colonies due to these dose-rate-spe-
cific and cell strain-specific growth characteristics. As such,
survival data are plotted on the ordinate in these series of
experiments as a function of dose rate, rather than dose, on
the abscissa, similar to other continuous low-dose-rate ex-
posure studies reported previously by this laboratory
2
(50).
Another important difference is the shape of the survival
curve for continuous low-dose-rate irradiation at dose rates
of 3 cGy/h or more. An asymptotic decrease in survival is
observed in these cells as dose rates approach the prolif-
eration-limiting low dose rate (the dose rate at which no
colonies form). This response is more typical of a chemical
dose–response curve rather than a radiation dose–response
curve fitted by an exponential or linear-quadratic function.
Cell survival on an exponential radiation survival curve is
assumed to decrease continuously as a function of dose,
implying that with enough cells plated (i.e. billions) one
could possibly recover a proliferating colony at high doses
based only on probability alone. Since the survival curve
for continuous low-dose-rate irradiation rapidly becomes
asymptotic as the dose rate approaches the proliferation-
limiting low dose rate, one would never expect to recover
a surviving colony at these dose rates, even with very large
numbers of cells plated. From Figs. 1–3, the proliferation-
limiting low dose rate for the Coriell strains with normal
radiosensitivity is approximately 5–6.5 cGy/h and ranges
from 2.5–4.5 cGy/h for the radiosensitive Coriell and RB
family member strains.
DISCUSSION
It is well accepted that individual genetic variation in
critical DNA damage response and repair pathways may
greatly influence DNA damage signaling threshold levels,
rates of repair, and in vitro cellular radiosensitivity. This
may be especially true for low-dose-rate exposures, where
such genetic variation could significantly alter the magni-
tude of dose-rate effects observed for different biological
end points. Exposure to low doses of radiation results in
levels of single-stranded DNA damage similar to those gen-
erated by cellular oxidative metabolism that can be easily
repaired by base and nucleotide excision repair pathways
(79–81). However, the frequency of complex, clustered
DNA lesions generated by radiation, including directly in-
duced DSBs and secondary DSBs generated by the pro-
cessing of closely localized single-stranded lesions (82, 83),
may be sufficient to significantly affect survival at low dose
rates in radiosensitive cells. Support for the biological ef-
fectiveness of low radiation doses delivered at low dose
rates comes from Loucas et al. (61), who demonstrated that
a 2-Gy dose of
137
Cs rays delivered at 4.8 cGy/h is suf-
490 WILSON ET AL.
ficient to generate complex chromosome exchanges (i.e.,
exchanges requiring three or more chromosome breaks in
two or more chromosome arms). This implies that nuclear
traversals by single electron tracks (separated in space and
time for low-dose-rate exposures) may occasionally be suf-
ficient to generate multiple DSBs and chromatin breaks
within multiple chromosome domains
2
(84).
The possibility of a low-dose, low-LET radiation thresh-
old for DNA DSB repair signaling has recently been re-
ported by Rothkamm and Lo¨brich (85). By examining
-H2AX focus kinetics in quiescent G
0
/G
1
-phase human fi-
broblasts (which were linearly correlated with the number
of DSBs measured by pulsed-field gel electrophoresis at
higher doses), the authors demonstrated that -H2AX foci
induced after exposure to 1 mGy of X radiation (approx-
imately 1 electron track per cell) may remain unrepaired
for several days. For higher doses, rapid repair and disap-
pearance of most foci to background levels was observed
by 24 h after irradiation. Other recent reports by Collis et
al. (86) and Ishizaki et al. (87) employing low-dose-rate
exposures observed significantly moderated activation of
the ATM protein (by phosphorylation of serine-1981) and
its downstream protein target Nbs1 (by phosphorylation of
serine-343) and almost no activation of Tp53 (by phos-
phorylation of serine-15) after continuous low-dose-rate ir-
radiation of 9.4 and 1.8 cGy/h, respectively, in normal, ma-
lignant and human TERT-immortalized human cells by
Western blot analysis. Cytologically, the formation of
-H2AX foci detected by immunofluorescence was like-
wise attenuated significantly in both studies. In another re-
port by Sugihara et al. (88), NIH 3T3 mouse cells (with
wild-type Tp53 status) transfected with a Tp53-promoter-
driven GFP-reporter plasmid demonstrated Tp53 activation
after 72 h of low-dose-rate exposure to radiation at 5
cGy/h but not at 1 cGy/h or 0.1 cGy/h.
Since nuclear -H2AX foci can provide a sensitive mea-
sure of DSBs at low radiation doses and dose rates, focus
levels were measured in density-inhibited G
0
/G
1
-phase cul-
tures of fibroblasts derived from the RB parents and ap-
parently normal Coriell controls irradiated at 10 cGy/h for
24h(65). This study again revealed significant differences
between the two groups, including a similar percentage of
Coriell control strains being moderately radiosensitive.
Measurements of chromosomal aberrations and -H2AX
focus levels were also made for these cells irradiated in the
G
2
phase of the cell cycle, where Rad51-mediated homol-
ogous recombinational repair contributes significantly to
the repair of radiation-induced DSBs along with NHEJ (71–
73). Fibroblast strains from the RB parents and some ap-
parently normal Coriell controls were shown to be radio-
sensitive using both the G
2
chromosomal radiosensitivity
assay
2
(65), in which chromatid-type aberrations are mea-
sured in mitotic cells irradiated 1.5 h previously in the G
2
phase, and a new extension of the G
2
assay in which
-H2AX foci are measured directly on mitotic chromo-
somes (65). Most importantly, the relative trends of radio-
sensitivity among these cell strains vary somewhat for cul-
tures irradiated in different phases of the cell cycle at high
or low dose rates. This implies that the responsible genetic
defect leading to these phenotypes may involve either of
the two major DNA DSB repair pathways (or both) and/or
the complex protein networks that relay DNA damage sig-
nals to them throughout the cell cycle and modulate DNA
damage processing.
DNA DSBs are considered the critical genetic lesion un-
derlying long-term cellular survival after radiation exposure
(32, 89, 90), especially given the possibility that a DSB
‘misrepair’ event (including a lack of repair) could result
in a chromosomal aberration with tumorigenic potential
(91, 92). Vilenchik and Knudson estimated that the level
of endogenous DSBs generated in cycling human cells
(generated primarily during DNA replication and the de-
catenation of sister chromatids in G
2
prior to mitosis) is
equivalent to levels induced at a continuous low dose rate
of 30 cGy/h (82). Interestingly, 30 cGy/h was reported as
the lower limit for maximal dose-rate sparing for the sur-
vival of G
0
/G
1
-phase AG01522 fibroblasts and HTB-35 cer-
vical carcinoma cells as well as mouse C3H 10T½ cells
(56, 69). Further decreases in dose rate below the ‘limit-
ing’ low dose rate of 30 cGy/h did not yield an increase
in survival for a given dose. Cytogenetic data for low-dose-
rate exposures of G
0
/G
1
-phase lymphocytes and fibroblasts
has also suggested that a limiting low dose rate for induc-
tion of chromosome aberration occurs at 10 cGy/h (58,
60, 61), where the majority of chromosome break pairs re-
quired to produce an interchange result from the traversal
of single electron tracks (so-called intratrack effects).
Similar to the calculations made by Vilenchik and Knud-
son, it is interesting to relate the number of DNA DSBs
generated per hour to overall cell survival in the range of
dose rates used in the current assay. With 1 Gy of
137
Cs
rays producing approximately 25 DSBs (and -H2AX foci)
in these fibroblast strains (65), a dose rate of 1 cGy/h would
generate 0.25 DSB/h. With population doubling times rang-
ing from 50–200 h for dose rates 3 cGy/h,
2
cells may
accrue 35–150 radiation-induced DSBs before attempting
to undergo DNA replication or mitosis if the rate of DSB
formation per hour is sufficiently below the putative acti-
vation threshold for DSB surveillance and repair suggested
by Rothkamm and Lo¨brich (85) and others. The fact that
the proliferation-limiting low dose rate for the apparently
normal Coriell strains with normal radiosensitivity is ap-
proximately 4 to 6 cGy/h implies that approximately one
radiation-induced DSB per hour is sufficient to completely
inhibit colony formation. The proliferation-limiting low
dose rate for the radiosensitive RB family member and re-
maining Coriell fibroblast strains ranges from 2.5–4.5
cGy/h, implying that the radiosensitive cell strains can han-
dle only approximately half of this additional DSB yield
before colony formation is completely inhibited during the
exposure period.
Vilenchik and Knudson also reviewed data for dose-rate
491LOW-DOSE-RATE SURVIVAL OF RETINOBLASTOMA CELLS
effects for mutation induction in proliferating somatic and
germline cells for dose rates encompassing those used in
this current study (93, 94). Their evaluation of the literature
on the induction of HPRT mutants by low-dose-rate radi-
ation in somatic cells and specific-locus mutations in male
mouse germline cells indicated that dose-rate effects for
mutagenesis in a variety of mammalian cell types reaches
an inflection point in the range of 1–10 cGy/h, with dose
rates below this resulting in greater mutagenicity per unit
dose (i.e., a shift from a direct to an inverse dose-rate ef-
fect). They suggested the induction of radiation-induced
DSBs at a dose rate of 1 cGy/h (well below the rate of
endogenous DSB production equivalent to 30 cGy/h) might
fail to signal the appropriate DNA repair response pathways
and potentially result in a mutation if the cell replicates and
permanently ‘‘fixes’’ the damage lesion. Increasing the dose
rate by an order of magnitude to 10 cGy/h results in a level
of DNA damage induction that is discernible from the en-
dogenous ‘noise’ and registering a robust induction of re-
pair and reduced mutagenicity (81, 93, 94). Since inverse
dose-rate effects are associated with low-dose-rate expo-
sures of cycling cell populations, it is especially noteworthy
that the inflection point for low-dose-rate mutagenesis in a
variety of human and rodent cells occurs at the same dose
rates that result in significant killing of radiosensitive hu-
man cells in this assay.
Interestingly, the response of a Coriell hereditary reti-
noblastoma strain, GM06419, in this assay was similar to
that of the 13 apparently normal Coriell controls with nor-
mal radiosensitivity identified in Table 1.
2
Despite being
classified as radiosensitive by Li et al. (95) for measure-
ments of acute-radiation cell survival (D
0
85 cGy), this
strain was reported to have normal radiosensitivity using
the G
2
chromosomal radiosensitivity assay
2
(21, 22). This
discrepancy among the radiation responses of GM06419
cells in these different assays, conducted with cultures in
different stages of the cell cycle, reinforces the fact the
cellular radiosensitivity is not correlated with RB1 genotype
status and that other genetic factors must be involved in
the variable responses of RB cells to radiation. Likewise,
the radiosensitive responses observed in fibroblasts derived
from the RB family members in the present study, both the
affected RB1
/
children and the unaffected RB1
/
parents,
casts doubt on any direct role that RB1 haploinsufficiency
may play in influencing radiation sensitivity. The average
dose rates required to reduce relative survival to 10% and
1% are 1.5 and 2.8 cGy/h for the affected RB1
/
family
members and 1.3 and 2.3 cGy/h for the unaffected RB1
/
family members, respectively (see Table 4; differences are
not statistically significant). The unaffected parents of the
affected proband children in the RB families are disease-
free, apparently normal individuals with no known genetic
abnormalities in their family histories. The moderate cel-
lular radiosensitivity of fibroblasts derived from these par-
ents suggests the presence of hypomorphic variants in ra-
diation-responsive pathways.
The results of the continuous low-dose-rate irradiation
colony formation assay presented in this report clearly il-
lustrate a greater sensitivity of this exposure scheme for
accentuating moderate differences in cellular radiosensitiv-
ity throughout the cell cycle compared to more traditional
acute or low-dose-rate exposures of quiescent G
0
/G
1
-phase
cells. The dramatic changes in survival and proliferative
capacity at dose rates of 0.5 cGy/h to 10 cGy/h in the
primary fibroblast strains examined in this report provide
additional support for a critical range of cellular radiation
responses in the mGy/h to cGy/h dose-rate region. To the
extent that the moderate radiosensitivity detected in this
assay relates to effects of concern for human risk assess-
ment after low-dose and low-dose-rate radiation exposures,
these results suggest that the high proportion of more ra-
diosensitive individuals may be an issue of concern for ra-
diation protection. More generally, if such mild genomic
maintenance defects affect germline mutation rates, such
genetic polymorphisms could be important underlying fac-
tors in determining the frequency of conditions predispos-
ing to cancer, such as heterozygosity for RB1 or other tumor
suppressor genes.
ACKNOWLEDGMENTS
The authors would like to acknowledge Don Young and Zane Story
for valuable technical assistance and Dr. James Durham and Chuck Sam-
pier for assistance with dosimetry of the Colorado State University
MRB012 low-dose-rate facility. This work was supported in part by grant
T32-CA09236 (JSB) from the National Cancer Institute, National Insti-
tutes of Health, U.S. Department of Health and Human Services and
grants DE-FG03-01ER63235 (JSB) and DE-FG02-05ER64089 (JBL)
from the U.S. Department of Energy Low Dose Radiation Research Pro-
gram. A portion of this work was performed under the auspices of the
U.S. Department of Energy by Lawrence Livermore National Laboratory
under Contract DE-AC52-07NA27344.
Received: January 5, 2008; accepted: February 1, 2008
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    • "Several studies have shown that cells typically release the G2 checkpoint with ~5–10 DSBs still present, meaning this unresolved/unrepaired damage continues into the next cell cycle (Lobrich and Jeggo 2007). While several radiobiological assays, such as the sister chromatid exchange (SCE) and G2 chromosomal radiosensitivity assays, are capable of partially discriminating differences in G2-phase radiosensitivity (Wilson et al. 2010a), this is best accomplished using a continuous low dose-rate colony formation assay in which cells are challenged to proliferate during chronic irradiation (typically ≤15 cGy h −1 ) (Wilson et al. 2008). Unlike the G0/G1‐phase foci assay, this highly discriminatory survival assay also tests the contribution of an individual's DDR capacities in the S and G2 phases to recognize and resolve IR-induced DNA damage, including homologous recombinational repair (HRR), the other primary DSB repair pathway in mammalian cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Biological responses of human cells and tissues to ionizing radiation (IR) are strongly influenced by dose and dose-rate. Unlike the robust activation of cellular DNA damage responses (DDR) seen after high IR doses, the efficiency of activation of DNA damage repair and signaling pathways after much lower doses and dose-rates varies appreciably among different individuals. Genomic and functional assays measuring low dose and dose-rate IR responses repeatedly show increased inter-individual variability when cells and tissues experience DNA damage levels comparable to those experienced endogenously (due to aerobic metabolism, diet, lifestyle, etc). Complicating matters for risk assessment are recent observations of dose-response non-linearity (hyper-linearity) in the low dose range. With both physical and biological factors strongly influencing individual responses to IR at low doses and dose-rates, further radiobiological research is required to assist regulatory agencies in determining appropriate radiological protection standards for such exposures.
    Full-text · Article · Mar 2016
    • "The majority of these in vitro studies employed relatively high doses of IR (≥50 cGy) because of limited experimental sensitivity at low doses. However, there is accumulating evidence of inter-individual variation for induced chromosomal aberrations after low IR doses [8][9][10] and for cell survival and - H2AX foci induction during continuous low dose-rate exposure [7,11,12]. Epidemiological studies indicate that cancer risk increases with IR exposure even at low doses [13][14][15][16]. Reviews of individual and pooled studies of populations that received accidental, medical, or occupational IR exposures support a linear increase in cancer risk with increasing dose, with large uncertainties of risk at low dose. "
    [Show abstract] [Hide abstract] ABSTRACT: DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of (137)Cs gamma-rays. Quiescent G(0)/G(1)-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV approximately 0.2) than the level of spontaneous foci, which ranged from 0.2-2.6 foci/cell (CV approximately 0.6; mean+/-SD of 1.00+/-0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.
    Full-text · Article · Nov 2009
  • [Show abstract] [Hide abstract] ABSTRACT: Objective: VEGF is a promoting angiogenesis factor in retinoblastoma. It is associated with the differentiation and extension of tumor. HXO-RB44 supernatant fluid is a RB cell supernatant fluid. Present study was to explore the effect of RB cell supernatant fluid on expression of VEGF in ARPE-19 cells, a type of retinal pigment epithelial cell, and its related mechanism. Methods: HXO-RB44 cells were cultured in RPMI-1640 medium containing 10% bovine serum, and ARPE-19 cells were cultured in MEM medium containing 10% bovine serum. 100 μL of HXO-RB44 supernatant fluid was added in ARPE-19 cells medium. The expression of VEGF mRNA in ARPE-19 cells was monitored by RT-PCR, and the expression of VEGF protein was detected using Western-blot analysis in 0, 4, 8, 12, 24 hours after effect of HXO-RB44 supernatant fluid. Immunocytochemistry was used to evaluate the expression of VEGF in ARPE-19 cells based on the fluorescence intensity. Results: The expression of VEGF mRNA was significantly different among various time points after addition of HXO-RB44 supernatant fluid(F = 195. 072 , P = 0.000). With the prolong of action time of HXO-RB44 supernatant fluid, the expression of mRNA in ARPE-19 cells was obviously increased(P <0.01). Expression of VEGF protein was significantly increased in ARPE-19 cells incubated with supernatant fluid of HXO-RB44 cells after 4, 8, 12, 24 hours, showing a considerable increase among different groups (F = 160.687, P = 0.000) and followed the same pattern to VEGF mRNA in the dynamical change to HXO-RB44 supernatant fluid (P < 0.05). The green fluorescence intensity was gradually higher with the time prolong of HXO-RB 44 supernatant fluid action. Conclusion: Supernatant fluid of HXO-RB44 cells can enhance the expression of VEGF in ARPE-19 cells.
    Article · Oct 2009
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