Affiliation of authors: Department of Radiation Oncology, Experimental Division, University of California at Los Angeles School of Medicine.
Correspondence to: Frank Pajonk, M.D., Ph.D., Department of Radiation Oncology, Roy E. Coats Research Laboratories, University of California at Los Angeles School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095-1714 (e-mail: fpajonk{at}ucla.edu).
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ABSTRACT |
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INTRODUCTION |
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Activation of NF-B does not require protein synthesis. Homodimers and heterodimers
of its subunits p50, p52, p65/RelA, c-Rel, and Rel-B are located in the cytosol preformed and
bound to inhibitor molecules of the I
B family (I
B
, I
Bß,
I
B
, Bcl-3, p100, and p105). Activation of NF-
B requires that I
B is
phosphorylated at two serine residues (Ser-32 and Ser-36) by I
B kinases, polyubiquitinated,
and subsequently degraded by the 26S proteasome. This process frees NF-
B for
translocation to the nucleus and activation of its target genetic programs [reviewed in (5)].
Although it is widely accepted that ionizing irradiation can cause a typical inflammatory
response by activating NF-B, the role of this transcription factor as a survival factor for cells
after ionizing irradiation remains unclear. In general, activation of NF-
B has been reported
to protect cells from apoptosis (programmed cell death) (6-8). However,
this is not always the case; cells from patients with ataxia telangiectasia are one exception (9). Although radiation therapy-induced apoptosis has been reported to be
associated with radiotherapeutic cure of murine tumors (10,11), its
contribution in radiation therapy remains controversial. In most cases, cells in a tumor survive
initial damage caused by therapeutic doses of radiation therapy and traverse several cell cycles
before finally dying or producing clonogenic survivors (12) that cause
tumor recurrence. The success of cancer treatment depends mainly on eliminating these tumor
stem cells, by whatever pathway.
Activation of NF-B and consequent inhibition of apoptosis might be expected a
priori to increase cell survival after irradiation, but the possible relationships of these events
to the elimination of clonogenic stem cells after irradiation need further clarification. In this study,
we investigated the role of NF-
B in modulating the intrinsic radiosensitivity of two human
cancer cells lines, PC3 prostate cancer cells and HD-MyZ Hodgkin's lymphoma cells.
These cell lines were chosen because, for different reasons, they have high constitutive levels of
NF-
B that might confer relatively high resistance to radiation therapy.
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MATERIALS AND METHODS |
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Cultures of PC3 human prostate carcinoma (American Type Culture Collection, Manassas, VA) and of the Hodgkin's cell line HD-MyZ (DSMZ, Braunschweig, Germany) were grown in 75-cm2 flasks (Falcon Becton Dickinson and Co., Lincoln Park, NJ)) at 37 °C in a humidified atmosphere of 5% CO2-95% air. Dulbecco's modified Eagle medium (Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) was used supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (Life Technologies, Inc.).
Transduction Experiments
The recombinant replication-deficient adenoviruses Ad5-IB and Ad5-LacZ were
provided by R. Batra (University of California/West Los Angeles-Veterans Administration
Medical Center). The vectors had been generated and quality tested at the Vector Core at the
Gene Therapy Center of the University of North Carolina School of Medicine and are described
elsewhere (13). Ad5-I
B contains a gene for I
B
, an
NF-
B superrepressor, under control of a cytomegalovirus promoter/enhancer. The encoded
I
B
contains serine-to-alanine mutations at positions 32 and 36, preventing the
phosphorylation, ubiquitination, and subsequent degradation by the proteasome. Ad5-LacZ is a
control virus that contains the gene for ß-galactosidase instead of IkB
. Cells were
plated in 10-cm culture dishes (Falcon Becton Dickinson and Co.). After 24 hours, the medium
was changed and viral vectors containing the nonphosphorable I
B
or
ß-galactosidase gene were added at a multiplicity of infection (MOI) of 1000. After 2 hours
of incubation, the virus-containing medium was replaced by fresh medium, and cells were
incubated for an additional 48 hours to allow gene expression. Successful transduction was
confirmed by staining with 5-bromo-4-chloro-3-indolyl ß-D-galactoside.
Irradiation
PC3 cells were trypsinized, counted, and diluted to a final concentration of 106 cells/mL. HD-MyZ cells were dislodged mechanically, counted, and diluted to a final concentration of 106 cells/mL. The cell suspensions were immediately irradiated at room temperature with a 137Cs laboratory irradiator (Mark I; J. L. Shepherd and Associates, San Fernando, CA) at a dose rate of 580 rads/minute. Corresponding control cells were sham irradiated.
Cell Extracts and Electrophoretic Mobility Shift Assays
For preparation of total cellular extracts, normal and treated cells were dislodged
mechanically, washed with ice-cold phosphate-buffered saline (PBS), and lysed in TOTEX buffer
(20 mM HEPES [pH 7.9], 0.35 mM NaCl, 20% glycerol,
1% Nonidet P-40 [NP40], 0.5 mM EDTA, 0.1 mM ethylene
glycol-bis(ß-aminoethylether)-N,N,N',N'-tetraacetic acid, 0.5 mM dithiothreitol [DTT], 50 µM phenylmethylsulfonyl fluoride
[PMSF], and aprotinin [90 trypsin inhibitor U/mL]) for 30 minutes on ice.
Lysate was centrifuged at 12 000g for 5 minutes at 4 °C. Protein
concentration was determined with the BCA protocol (Pierce Chemical Co., Rockford, IL).
Fifteen micrograms of protein from the resulting supernatant was incubated for 25 minutes at
room temperature with 2 µL of bovine serum albumin (10 µg/µL), 2 µL of
poly[d(I-C)] [poly-deoxyinosinic-deoxycytidylic acid] (1
µg/µL), 4 µL of Ficoll buffer (20% Ficoll 400, 100 mM HEPES,
300 mM KCl, 10 mM DTT, and 0.1 mM PMSF), 2 µL of buffer
D+ (20 mM HEPES, 20% glycerol, 100 mM KCl, 0.5 mM
EDTA, 0.25% NP40, 2 mM DTT, and 0.1 mM PMSF), and 1 µL
of the [-32P]adenosine triphosphate-labeled oligonucleotide
(Promega Corp., Madison, WI; NF-
B: AGTTGAGGGGACTTTCCCAGG). For a negative
control, unlabeled oligonucleotide was added to 50-fold excess. Gel analysis was carried out in
native 4% polyacrylamide/0.5x TBE (Tris-boric acid-EDTA) gels. Dried gels were
placed on a phosphor screen for 24 hours and analyzed on a phosphor imager (Storm 860;
Molecular Dynamics, Sunnyvale, CA).
Clonogenic Survival
Colony-forming assays were performed immediately after irradiation by plating an appropriate number of cells into culture dishes in triplicate. After 14 days, cells were fixed and stained with crystal violet, and the number of colonies containing more than 50 cells were counted. The surviving fraction was normalized to the surviving fraction of the corresponding control, and survival curves were fitted by use of a linear-quadratic model.
Determination of Apoptosis
Apoptotic cells were detected with an In Situ Cell Death Kit (Boeringer Mannheim GmbH, Mannheim, Germany). The manufacturer's protocol was followed with some minor modifications. Briefly, attached and detached cells were collected, centrifuged at 500g for 5 minutes at 4 °C, fixed in ice-cold 75% ethanol, washed with PBS, and pelleted by centrifugation at 500g for 5 minutes at 4 °C. Cells were permeabilized by resuspension in a solution of 0.1% Triton X-100 and 0.1% sodium citrate and incubated for 2 minutes on ice. Cells were washed twice in PBS, resuspended in TUNEL (terminal deoxynucleotidyltransferase-mediated-uridine triphosphate nick-end labeling) reaction mixture, and incubated for 60 minutes at 37 °C. After three washes with PBS, fluorescence was measured at 518 nm in a flow cytometer (FACScan System; Becton Dickinson Immunocytometry Systems, San Jose, CA) and analyzed with the CellQuest software (Becton Dickinson Immunocytometry Systems).
Statistics
All data are means ± 95% confidence intervals. A P value of <.05 from Student's t test was considered to be statistically significant. All statistical analyses were carried out with the JMP (version 3.2) software package from SAS (SAS Institute, Inc., Cary, NC) for Macintosh. All P values are two-sided.
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RESULTS AND DISCUSSION |
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As we have shown for HD-MyZ cells (Pajonk F, Pajonk K, McBride WH: unpublished
results), adenoviral vectors were highly efficient at inserting genes into PC3 prostate cancer cells.
Transduction rates in excess of 99%, confirmed by staining with
5-bromo-4-chloro-3-indolyl-ß-D-galactoside, were achieved at a MOI of 1000
(data not shown). This MOI caused no visible transduction-related toxicity after 48 hours, but
when PC3 cells transduced with the control vector were trypsinized and replated at that time,
their plating efficiency was slightly reduced to 29.5% ± 0.24% (78%
of the nontransduced control level [37.9% ± 5.63%]; P = .11; Student's t test). However, the clonogenicity of cells
transduced with the adenoviral vector containing the IB superrepressor gene was greatly
reduced to 7.4% ± 2.67% (19.6% of the nontransduced control level;
P = .010; Student's t test; Fig. 1,
A).
These findings are almost identical to our results for HD-MyZ cells (Pajonk F, Pajonk K, McBride
WH: unpublished results).
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DNA-binding activity of NF-B in HD-MyZ cells has been reported to be high in
comparison to other tumor cell lines (14) because of mutated I
B.
We had previously shown that NF-
B levels in HD-MyZ cells decreased after transduction
with the I
B super-repressor gene (Pajonk F, Pajonk K, McBride WH: unpublished results).
We examined whether PC3 cells responded in the same way. The same vector containing the gene
for ß-galactosidase was used as the control for changes in NF-
B activity caused by the
vector itself. DNA-binding activity of NF-
B was measured with a gel-shift assay. Radiation
therapy-induced (30 Gy, 3 hours after exposure; Fig. 2, A) and constitutive NF-
B activity
was dramatically decreased in PC3 cells transduced with the I
B super-repressor gene but
not in cells transduced with the ß-galactosidase gene 48 hours after transduction (Fig. 2,
B).
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So far, there has been only one report describing a possible relationship between NF-B
activity and the intrinsic radiosensitivity of human cancer cells (21). In
that study, the authors selected cell clones from p53-negative glioma cell lines with high-level
expression of wild-type I
B
messenger RNA after transfection with an expression
plasmid for this gene. Inconsistently, only two of the clones that were selected had both high
I
B
protein levels and increased sensitivity to radiation therapy. The possibility of
selection of radiosensitive clones rather than I
B-related radiosensitization cannot be
excluded as an explanation for these findings.
In this study, we used an adenoviral vector to insert a gene for the IB super-repressor
into PC3 prostate cancer cells and HD-MyZ Hodgkin's lymphoma cells; this I
B has
been shown to efficiently inhibit constitutive, radiation therapy-induced, and tumor necrosis
factor-
-induced activation of NF-
B (Pajonk F, Pajonk K, McBride WH: unpublished
data). Both cell lines most likely carry a mutated p53. Transduction rates of more than 99%
guaranteed inhibition of NF-
B in almost all cells. The data from this study show that
radiosensitivity of two human cancer cell lines with high levels of constitutively activated
NF-
B is not dependent on this pathway. Comparable results were recently reported for
NF-
B and cytotoxic drugs (22). However, inhibition of NF-
B
binding to DNA drastically decreased the clonogenicity in both cell lines, emphasizing the
importance of NF-
B activation for survival of these human cancer cells.
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Manuscript received May 9, 1999; revised September 7, 1999; accepted September 20, 1999.
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