[Cancer Research 60, 2576-2578, May 15, 2000]
© 2000 American Association for Cancer Research
Effects of the Multidrug Transporter P-Glycoprotein on Cellular Responses to Ionizing Radiation1
Adam C. Ruth and
Igor B. Roninson2
Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607
 |
ABSTRACT
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Ionizing radiation induces apoptosis, mitotic catastrophe, and
senescence-like terminal proliferation arrest in tumor cells. We
investigated the effect of the MDR1
P-glycoprotein (Pgp), recently shown to inhibit caspase-mediated
apoptosis, on cellular responses to radiation. Pgp strongly inhibited
radiation-induced apoptosis in a HeLa-derived cell line with inducible
MDR1 expression and in NIH 3T3 cells transduced with a
MDR1-expressing retroviral vector. The inhibition of
apoptosis by Pgp was associated, however, with increases in
radiation-induced mitotic catastrophe and senescence and produced only
a marginal change in the survival of irradiated cells. Pgp had no
effect on radiation responses in apoptosis-resistant HT1080 cells.
These results indicate that Pgp inhibits radiation-induced apoptosis,
but this effect of Pgp provides no substantial increase in radiation
resistance of the tested cell lines because apoptosis-resistant cells
die from mitotic catastrophe or undergo senescence-like terminal
proliferation arrest.
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Introduction
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Ionizing radiation inhibits the growth of tumor cells
through both cytotoxic and cytostatic mechanisms. Radiation
cytotoxicity has been associated with two mechanistically and
morphologically distinct forms of cell death: programmed cell death
(apoptosis), characterized by nuclear condensation and DNA degradation;
and mitotic catastrophe, characterized by the formation of multiple
micronuclei (1)
. Ionizing radiation (as well as treatment
with cytotoxic drugs or differentiating agents) also induces permanent
cytostatic proliferation arrest, which is accompanied by phenotypic
markers of senescence, including enlarged and flattened morphology and
expression of
SA-ß-gal3 activity detectable at pH 6.0 (2)
. The relative
contributions of these responses to the overall growth-inhibitory
effects of radiation have not yet been characterized. In the present
study, we investigated the effect of Pgp, the product of the multidrug
resistance (MDR1) gene, on radiation responses in three cell
lines. Pgp acts as an efflux pump for various lipophilic compounds
(3)
. Multidrug-resistant cell lines that overexpress Pgp
were variably reported to show no change (4)
, a decrease
(5
, 6)
, or an increase (7
, 8)
in their
radiation resistance; the resistance to radiation in the latter cases
has been attributed to Pgp-unrelated events that had occurred in the
course of drug selection. Pgp recently was shown, however, to inhibit
caspase-mediated apo-ptosis (9
, 10)
. The mechanism of
this Pgp effect is as yet unknown; it was suggested to involve the
efflux of some unknown mediator of apoptosis by Pgp or putative effects
of Pgp on intracellular pH (9
, 10)
. Because ionizing
radiation is a known inducer of apoptosis, we tested whether Pgp
expression would affect cellular radiation response in three
fibroblastoid or epithelial cell lines. Our results indicate that Pgp
inhibits radiation-induced apoptosis but has no significant effect on
radiation resistance because the inhibition of apoptosis is associated
with a concurrent increase in mitotic catastrophe and senescence in
radiation-damaged cells.
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Materials and Methods
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Cell Lines.
NIH 3T3 cells were from ATCC. The derivations of HT1080 subline E14,
which expresses the murine ecotropic receptor (11)
, and
the HeLa subline HtTA, which expresses tetracycline-inhibited
transactivator (12)
, have been described previously.
Retroviral vector LMDR1, which expresses the human MDR1 cDNA
(13)
, was introduced into the NIH 3T3 and HT1080 cells by
ecotropic retrovirus transduction. Pure populations of LMDR1-transduced
cells were isolated by FACS after indirect immunofluorescence labeling
with a monoclonal antibody UIC2, specific for the MDR1 Pgp
(14)
, and designated NIH 3T3-MDR1 and HT1080-MDR1,
respectively. HtTA cells were transfected with plasmid pUHDMDR1, which
carries the full-length coding sequence of the human MDR1
gene in the tetracycline-regulated vector pUHD15-1 (12)
,
and a stable transfectant HtTA-MDR1 was isolated. MDR1
expression in HtTA-MDR1 cells was inhibited by 48-h incubation with 1
µg/ml tetracycline and induced by removing tetracycline for at least
48 h.
Irradiation, Drug Treatment, and Cellular Assays.
The J.L. Shepherd Model 143 irradiator was used for 
-irradiation.
Cells were plated at a density of 2 x 105 per 3.5-cm plate and irradiated 24 h
after plating. For colony assays, 250 cells were plated in a 3.5-cm
plate in triplicate. The plating efficiency for the HtTA, NIH 3T3, and
HT1080 cell lines were 69, 79, and 73%, respectively; these values
were unaffected by MDR1 expression. Twenty-four h later,
cells were irradiated or exposed continuously to vinblastine-containing
medium, and after 8 days, colonies were stained with crystal violet.
The procedures for measuring relative cell growth by methylene blue
staining, FACS analysis of cells with sub-G1 DNA
content, and morphological assays for SA-ß-gal expression and
micronuclei formation have been described previously (2)
.
Microscopic analysis of apoptosis was performed after
4',6-diamidino-2-phenylindole staining. The fractions of
apoptotic, micronucleated, or SA-ß-gal-positive cells were
determined by scoring 300 cells in each sample.
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Results and Discussion
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To study the effects of Pgp without potential artifacts associated
with drug selection of Pgp-expressing cells, we generated three
Pgp-expressing cell lines by retroviral transduction or by an inducible
expression vector. The first two lines were human HT1080 fibrosarcoma
and mouse NIH 3T3 fibroblasts that we transduced with a Pgp-expressing
retroviral vector; we isolated 100% Pgp-expressing cell populations by
flow sorting. For inducible MDR1 expression, the
HeLa-derived line HtTA (12)
was transfected with the human
MDR1 cDNA in a tetracycline-regulated vector, pUHD15-1. The
transfectant cell line HtTA-MDR1 does not express Pgp when grown in the
presence of tetracycline, but the removal of tetracycline induces Pgp
expression (Fig. 1A
) and the resulting resistance to a Pgp-transported drug,
vinblastine (Fig. 1B
).
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Fig. 1. Effects of tetracycline-regulated
MDR1 expression on radiation response in HtTA-MDR1 cell
line. A, FACS analysis of the reactivity of HtTA-MDR1
cells with the MDR1-specific antibody UIC2 and with
UPC10 isotype control in the presence (+Tet) and absence
(-Tet) of 1 µg/ml tetracycline. B,
clonogenic assays for vinblastine resistance of HtTA-MDR1 cells in the
presence (•) and absence ( ) of tetracycline; bars,
SD. C, effects of different doses of radiation on
the percentages of apoptotic, micronucleated, and SA-ß-gal+ HtTA-MDR1
cells arising 3 days after irradiation in the presence (•) or absence
() of tetracycline. The Poisson SD (bars) was
calculated as the square root of counted events and expressed as
percentage of abundance. D, time course of changes in
the percentages of apoptotic, micronucleated, and SA-ß-gal+ HtTA-MDR1
cells after exposure to 9 Gy of -irradiation in the presence
(•) or absence () of tetracycline; bars,
SD.
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To analyze the effects of MDR1 induction in HtTA-MDR1 cells
on radiation-induced apoptosis, HtTA-MDR1 and the control parental HtTA
cells were exposed to different doses of radiation in the presence or
absence of tetracycline, and cellular responses to radiation were
analyzed by morphological criteria (Fig. 2
). Radiation-induced apoptosis was strongly decreased in HtTA-MDR1 in
the absence of tetracycline (Fig. 1C
). The presence or
absence of tetracycline had no detectable effect on the percentage of
apoptotic cells induced by 9 Gy of radiation in untransfected HtTA
cells (data not shown). The removal of tetracycline also inhibited the
induction of apoptosis by Fas antibody or tumor necrosis factor
(data not shown), confirming the reported effects of Pgp on apoptosis
induced in other cell lines by the latter agents (9
, 10)
.
The inhibition of apoptosis by Pgp was also detected by a 2-fold
reduction in the percentage of cells that developed a decreased
(sub-G1) DNA content, as determined by FACS
analysis of cellular DNA content (data not shown). Whereas Pgp
expression inhibited apoptosis, it increased, at the same time, the
percentages of cells undergoing radiation-induced mitotic catastrophe
or senescence-like growth arrest (Fig. 1C
), as scored by
morphological and cytochemical criteria (see Fig. 2
). We also analyzed
the time course of the induction of apoptosis, mitotic catastrophe, and
senescence in HtTA-MDR1 cells exposed to 9 Gy in the presence or
absence of tetracycline (Fig. 1D
). Whereas the inhibition of
apoptosis by Pgp was already apparent 1 day after irradiation,
Pgp-dependent increases in mitotic catastrophe and senescence were
observed only 3 and 2 days after irradiation, respectively (Fig. 1D
), suggesting that the latter changes could be secondary
to the effect of Pgp on apoptosis. Similar results were obtained in the
analysis of the effects of MDR1 transduction on NIH 3T3
cells (Fig. 3, A and B
).
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Fig. 2. Morphological analysis of apoptosis, mitotic catastrophe,
and senescence. HtTA-MDR1 (A–C) and NIH 3T3-MDR1
(D–F) cell lines were exposed to 9 Gy of
-irradiation. The corresponding morphology for HT1080 cells has been
presented elsewhere (2). A and
D, apoptotic cells (a) were identified by
chromatin condensation and photographed at x400 magnification after
4',6-diamidino-2-phenylindole staining. B and
E, cells undergoing mitotic catastrophe were identified
by the appearance of micronuclei (mn) and photographed
at x400 magnification after H&E staining. C and
F, cells with the senescent phenotype (s)
were identified after staining for SA-ß-gal activity and photographed
at x400 magnification.
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Fig. 3. Effects of MDR1 on radiation responses of
NIH 3T3 and HT1080 cell lines. A, effects of different
doses of radiation on the percentages of apoptotic, micronucleated, and
SA-ß-gal+ NIH 3T3 (•) and NIH 3T3-MDR1 () cells arising 3 days
after irradiation. B, time course of changes in the
percentages of apoptotic, micronucleated, and SA-ß-gal+ NIH 3T3 (•)
and NIH 3T3-MDR1 () cells after exposure to 9 Gy of
-irradiation. C, effects of different doses of
radiation on the percentages of apoptotic, micronucleated and
SA-ß-gal+ cells in HT1080 (•) and HT1080-MDR1 () populations,
measured 3 days after irradiation. Bars, SD.
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In contrast to HtTA and NIH 3T3, HT1080 cells, in agreement with
previously reported resistance of this cell line to apoptosis (2
, 15)
, showed only a weak apoptotic response to irradiation
(<10%) and strong induction of mitotic catastrophe and senescence.
Pgp had no detectable effect on the weak apoptotic response of HT1080
cells, and it did not affect their mitotic catastrophe or senescence
(Fig. 3C
). These results further confirm our interpretation
that the effects of Pgp on senescence and mitotic catastrophe are
secondary to its effect on apoptosis. Finally, we analyzed the effects
of Pgp on radiation survival in all three cell lines, using colony
formation (Fig. 4
) and cell growth inhibition assays (data not shown). Pgp expression
resulted in only a marginal increase in radiation survival of HtTA-MDR1
and NIH 3T3-MDR1 cells, detectable only at the highest doses (Fig. 4A
), and it had no effect in HT1080-MDR1 cells (Fig. 4, B and C
).
The results of the present study demonstrate that Pgp inhibits
radiation-induced apoptosis, in agreement with its reported ability to
inhibit caspase-mediated apoptosis induced by other agents (9
, 10)
. This effect of Pgp may potentially contribute to radiation
survival in those cell types where the ability to undergo apoptosis is
a determinant of radiation susceptibility, as may be the case in many
normal tissues (16)
or in hemopoietic malignancies
(17)
. Recently, Thevenod et al.
(18)
showed that stress-induced Pgp expression in kidney
proximal tubule cells protects them from cadmium-induced
apoptosis, a finding with potential implications for renal carcinomas
that commonly express Pgp. We found, however, that the inhibition of
radiation-induced apoptosis by Pgp has no significant effect on
radiation survival in the tested solid tumor-derived or immortalized
cell lines. This result parallels previous reports where overexpression
of the apoptosis suppressor BCL2 was used to inhibit
apoptosis induced by radiation (19)
, aphidicolin
(20)
, or etoposide (21)
in solid
tumor-derived cell lines. In these studies, BCL2 completely
or almost completely abolished the apoptotic response, but it had no
effect on cell survival. Importantly, inhibition of etoposide-induced
apoptosis was accompanied by an increase in the percentage of cells
undergoing mitotic catastrophe (21)
.
In the present work, we found that inhibition of apoptosis by Pgp
is associated with increases in the fractions of cells undergoing
mitotic catastrophe or senescence. Because the increase in the latter
responses appears to be secondary to the inhibition of apo-ptosis,
it seems most likely that the development of apoptosis masks the two
other drug effects in damaged cells. In other words, cellular damage by
drugs or radiation triggers mitotic catastrophe and senescence, as well
as apoptosis; if the latter process is inhibited, the damaged cells
still die from mitotic catastrophe or undergo senescence-like terminal
proliferation arrest. These findings suggest that strategies aimed at
augmenting mitotic catastrophe or senescence would be likely to improve
the efficacy of radiation therapy or chemotherapy in solid tumors.
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Acknowledgments
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We thank Dr. Roberta Franks and Kaihua Wang for constructing the
pUHDMDR1 vector and Drs. Eugenia Broude and Bey-Dih Chang for advice
and assistance with morphological assays.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants R37CA40333 and
R01CA62099 from the National Cancer Institute. 
2 To whom requests for reprints should be
addressed, at Department of Molecular Genetics (M/C 669), University of
Illinois at Chicago, 900 S. Ashland Avenue, Chicago, IL 60607-7170.
Phone: (312) 996-3486; Fax: (312) 413-8358; E-mail: roninson{at}uic.edu
3 The abbreviations used are: SA-ß-gal,
senescence-associated ß-galactosidase; Pgp, P-glycoprotein; FACS,
fluorescence-activated cell sorting.
Received 1/11/00.
Accepted 3/27/00.
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Top
ABSTRACT
Introduction
