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Tumor Biology |
Department of Radiation Oncology, Stanford University Medical Center, Stanford, California 94305-5468 [H. L. M., A. J. G.], and Department of Radiation Oncology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157 [C. K.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fas, also know as CD95 or APO-1, is a cell surface protein belonging to the tumor necrosis factor receptor family. Binding of FasL3 to a cell expressing Fas receptor results in its death by apoptosis (7) . This interaction is thought to function in immune response termination, T cell activation-induced cell death, control of peripheral tolerance to self-antigens, and the maintenance of immune privilege in the eye and testes (8 , 9) . Whereas many tissues express the Fas receptor protein, FasL expression is limited to activated T lymphocytes, macrophages, and cells of the eye and testes (10 , 11) . Most recently, FasL has been reported to be expressed in human melanoma (12) , hepatocellular carcinoma (13) , and highly metastatic tumors of the colon (14) . Expression of FasL on tumor cells has been hypothesized to function in deletion of infiltrating immune cells and thus aid in tumor escape from immune surveillance (15) .
Several studies suggest that Fas transcriptional expression is p53 regulated (16, 17, 18) . A p53-responsive element was identified within the first intron of the Fas gene and three putative p53-responsive elements are located within the promoter (16) . In support of the direct transcriptional regulation of Fas by p53, K562 cells stably transfected with a plasmid containing a temperature-sensitive human p53 gene demonstrated a 4- to 6-fold up-regulation of cell surface Fas when p53 was in a wild-type conformation (19) .
The clinical importance of the cross-talk between the p53 and Fas-FasL pathways in modulating apoptosis has been suggested by several studies (20 , 21) . Although numerous genotoxic therapies used in cancer treatment activate both p53 and Fas signaling pathways, the contribution of each pathway to cell killing is not well defined. Clearly, treatment of leukemic (20) and hepatocellular carcinoma cell lines (21) with chemotherapeutic agents causes up-regulation of FasL. In addition, hepatoma cells have been shown to increase Fas surface expression in response to anticancer drugs (21) , and incubation of blocking F(ab')2 anti-Fas antibodies protects these cells from doxorubicin- and bleomycin-induced apoptosis. However, in many of the same tumors in which Fas and FasL are activated, p53 is also activated and can signal cell death or cell cycle arrest. Tumors with mutated or deleted p53 have been reported to be resistant to radiation- and chemotherapy-induced apoptosis (22) . It has been suggested that resistance to chemotherapy of p53 null tumors may in part be the result of their inability to elevate surface Fas in response to genotoxic stress (23) . For example, human hepatomas with wild-type p53 were found to up-regulate Fas and FasL in response to chemotherapeutic drugs, whereas hepatomas in which p53 was mutated or deleted were resistant to chemotherapy-induced apoptosis and failed to up-regulate Fas and FasL (21) .
Human cancers with a functional wild-type p53 genotype have also been shown to elevate their surface Fas expression in response to ionizing radiation (24) . In contrast, human cancer cell lines in which p53 is mutated or deleted fail to demonstrate this response (24 , 25) . Given the evidence for cross-talk between p53 and Fas-FasL and the clinical importance of these pathways in cancer treatment, we examined Fas inducibility by genotoxic and nongenotoxic stimuli known to induce p53, in cell lines genetically matched, differing only in their p53 status.
In a survey of cell lines, those cells with wild-type p53 had greater surface levels of Fas and FasL and were more sensitive to FasL-mediated killing. Furthermore, we found that oncogenically transformed cells with wild-type p53 transcriptionally repressed the expression of Fas and FasL over time in culture. Oncogenic transformation therefore may establish a cellular context that leads to either loss of p53 or Fas expression during cell proliferation, protecting against Fas-mediated apoptosis during tumor expansion.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Treatments.
MEF, RKO, and NCI-H1299 cells were incubated in a hypoxic anaerobic
chamber (Bactron Anaerobe Chamber, Cornelius, OR) at 0.01%
O2 for varying time periods to determine the
effect of hypoxia on Fas receptor induction and apoptosis. MEF, RKO,
and NCI-H1299 cells were irradiated with 200-1000 cGy at a dose rate of
281 cGy/min from a 137Cs source.
FACS Analysis.
To determine the surface levels of Fas and FasL on the MEFs,
1 x 106 cells were incubated on
ice for 30 min with 1 µg/ml biotinylated anti-Fas antibody, Jo2 (BD
PharMingen, San Diego, CA), anti-FasL antibody, Kay-10 (BD PharMingen),
or an irrelevant isotype control. After this incubation, the cells were
washed and incubated on ice for 30 min with
streptavidin-phycoerythrin (BD Immunocytometry Systems, San
Jose, CA). The cells were then washed and analyzed with a FACScalibur
flow cytometer (BD Immunocytometry Systems). This same protocol was
followed with the RKO and NCI-H1299 cells. The antihuman Fas antibody
was from BD Immunocytometry Systems and the antihuman FasL antibody was
NOK-1 (BD PharMingen). All experiments were conducted a minimum of
three times.
Apoptosis Assays.
Apoptosis was determined by staining cells with the APO-Direct Kit
(PharMingen) followed by analysis on a FACScalibur flow cytometer
(Becton Dickinson). The APO-DIRECT assay detects DNA strand breaks.
Apoptosis was then confirmed by staining a subpopulation of all treated
cells with Hoechst-propidium iodide followed by visualization on a
fluorescent microscope. The activating anti-Fas antibody used to induce
apoptosis in selected experiments was the antihuman clone DX2 (BD
PharMingen). All experiments were repeated three times.
51Cr Release Assays.
MEF or NCI-H1299 cells were plated at 10,000/per well of a 96-well
plate. Cells were loaded with 1 µCi/well with
51Cr and allowed to incubate at 37°C for 2 h. After this incubation, the cells were washed three times with DMEM
plus 10% FCS medium. After the last wash, 100 µl of fresh medium
were added to each well. Myoblasts from lpr mice expressing
FasL or control vector were added to cells at ratios of 20:1, 10:1, and
1:1 in a final volume of 100 µl of medium. Medium (100 µl) was
added to control wells measuring spontaneous release, and 100 µl of
0.1% Triton X-100 was added to wells measuring total lysis. Cells were
allowed to incubate for 12 h and then centrifuged for 5 min at
1200 rpm. Supernatant (50 µl) was harvested from each well and
counted on a ß-scintillation counter. All treatments were done in
triplicate, and experiments were repeated three times.
RNase Protection Assay.
RNA was extracted with TRIzol reagent (Life Technologies, Inc., Grand
Island, NY) according to the manufacturer’s direction. The extracted
RNA was then used in a BD PharMingen mAPO-3 kit, according to the
manufacturer’s instruction, to measure Fas mRNA levels. All RNase
protection assays were conducted three times to confirm results.
Selection Experiments.
The down-regulation of Fas inducibility as a function of cell
proliferation was tested by analyzing surface levels of both Fas and
FasL in p53 wild-type and null MEFs over time in culture. MEF cells at
passages 5, 10, 15, and 20 were tested both for their basal Fas and
FasL surface levels as well as their ability to up-regulate Fas and
FasL after 6 Gy ionizing radiation. Staining was repeated three
consecutive times to confirm results.
The human lung cancer cell line NCI-H1299 stably transfected with a tetracycline-inducible p53 construct was incubated in either the presence or the absence of doxycycline (200 ng/ml) to induce p53 expression. These cells were then treated for 12 h with the activating anti-Fas antibody, DX2, or FasL-expressing myoblasts to induce Fas-mediated apoptosis. These experiments were repeated three times to confirm results.
DNA Sequencing of p53.
Total RNA was extracted from E1A/Ras-transformed MEF
p53+/+ passage 5 and 20 cells with TRIzol Reagent
(Life Technologies, Inc., Grand Island, NY), and reverse transcribed
with oligo(dT) as primer to produce cDNA. Full-length p53 was PCR
amplified from the above cDNA template with the following primers
(5'-GGTGTCACGCTTCTCCG; 3'-TCAGTCTGAGTCAGGCCC). The 1.2-kb p53 PCR
product was separated on a 1% agarose gel and sequenced on an ABI
PRISM 310 Genetic Analyzer (PE Applied Biosystems, Branchburg, NJ).
Cell Cycle Analysis.
p53 wild-type and null MEF cells, passage 5, transformed with E1A/Ras
as well as p53 wild-type MEF cells, passage 20, were exposed to 6 Gy
ionizing radiation and collected at 12 and 24 h after irradiation.
The cells were fixed in 70% ethanol at -20°C overnight. On the next
day, cells were washed and resuspended in 0.5 ml of PBS containing 50
µg/ml propidium iodide and 5.0 µg/ml RNase A. The cell suspension
was incubated at 37°C in the dark for 30 min and then analyzed on a
FACScalibur flow cytometer. These experiments were repeated twice more
to confirm results.
Western Blotting Analysis of p21.
Cell extracts were prepared from control and irradiated MEF cells at 3
and 6 h after 6 Gy ionizing radiation. Protein (50 µg) was
loaded onto each lane of a 12% Tris-glycine gel. After
electrophoresis, the proteins were transferred onto a polyvinylidine
difluoride membrane. The membrane was blocked in 5% BSA + 5% nonfat milk + 0.1% Tween 20 in Tris-buffered
saline and then incubated with antimouse p21 rabbit antiserum (Greg
Hannon, Cold Spring Harbor Laboratory) at a dilution of 1:2000 in
blocking buffer for 1 h. The membrane was washed and then
incubated with alkaline phosphatase-antirabbit IgG (Vector, Burlingame,
CA) at a dilution of 1:3000 in blocking buffer. The blot was washed
again and developed with enhanced chemofluorescence substrate (Amersham
Life Science, Little Chalfont, United Kingdom). The fluorescent product
was detected with a STORM Optical Scanner (Molecular Dynamics,
Sunnyvale, CA).
Analysis of Fas and FasL Expression in Late Passage Cells.
Genomic sequencing of Fas and FasL was conducted to determine whether
mutations occurred in these genes in the late passage wild-type MEFs.
Protein surface expression and mRNA levels of expression were analyzed
by FACS and RNase protection assay as described above. DNA acetylation
was analyzed by comparing mRNA levels of Fas and FasL in late passage
cells in the presence and the absence of the histone deacetylase
inhibitor, trichostatin A, administered at 25 ng/ml for 24 h.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Basal levels of Fas mRNA were 3-fold higher in wild-type p53 cells
and were also found to be induced 4-fold after 6 Gy of ionizing
radiation. Hypoxia, on the other hand, failed to up-regulate Fas in
either wild-type or null p53-containing cells.
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). Consistent with the Fas mRNA data, ionizing radiation induced Fas
levels in p53 wild-type cells while failing to induce surface Fas
elevation in cells lacking p53. In contrast to genotoxic insults,
hypoxia, a nongenotoxic stress known to induce p53, failed to induce
Fas in these cells regardless of their p53 status. This finding is
significant in light of the recent report of Fas being induced on
vascular smooth muscle cells transfected with a chimeric
p53–4-hydroxytamoxifen-sensitive estrogen receptor, following
4-hydroxytamoxifen exposure (17)
. Thus, whereas previous
studies suggest that Fas is induced by ectopic overexpression of
wild-type p53, we demonstrate that not all stresses known to induce p53
also induce Fas.
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). The basal surface levels of Fas were >7-fold greater in p53
wild-type cells and surface levels of Fas increased almost 4-fold over
basal levels following ionizing radiation. This induction of surface
Fas in p53 wild-type cells was also found to increase in response to
increasing radiation dose (Fig. 3
).
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).
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). Both p53 wild-type and null cells were found to express FasL. In
addition, the levels of FasL were substantially elevated on the surface
of cells with a wild-type p53 genotype in response to increasing
radiation dose. This induction of FasL in response to radiation coupled
with the elevation of surface Fas expression may provide an additional
explanation as to why p53 wild-type cells and tumors are more sensitive
to radiation-induced apoptosis. Thus, these results further support
the concept that the p53 genotype of a tumor cell could make it more
susceptible both to self-elimination through the Fas pathway and to
increased sensitivity to Fas-mediated killing by the immune system.
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). FasL-expressing myoblasts isolated from lpr mice (Fas
receptor null) were used as death effector cells, and the MEFs were
used as the target cells. Fas-expressing p53 wild-type MEFs were
sensitive to Fas-mediated killing, and this killing was found to
increase after exposure to ionizing radiation. MEF cells lacking p53
were protected from Fas-mediated killing both in their resting state
and in response to ionizing radiation.
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). The increased apoptosis observed in these cells in the presence of
doxycycline and activating Fas antibody was greater than what could be
explained by activation of p53 alone.
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) and
wondered whether this loss in sensitivity could be attributed to
changes in surface levels Fas and FasL. The down-regulation of Fas and
FasL inducibility as a function of cell proliferation (27)
was tested by analyzing surface levels of Fas and FasL over time in
culture (Fig. 8
). Indeed, surface levels of Fas and FasL decreased with increasing
passage. In addition, the surface induction of Fas and FasL after
radiation diminished in MEFs with increasing passage. This
down-regulation of Fas and FasL was most pronounced in the p53
wild-type cells, and the null cells were more stable in their
maintenance of radiation-induced elevation of FasL.
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). In contrast to the loss of Fas and
FasL surface expression as well as apoptotic sensitivity seen in the
late passage wild-type p53 cells, other p53 downstream targets and
functions remained intact (Fig. 9
). Late passage cells were shown to
possess an intact G2 cell cycle arrest that
prevented increases in cell ploidy after ionizing radiation (Fig. 9B
) as well as maintaining their ability to induce the
cyclin-dependent kinase inhibitor p21 (Fig. 9C
). Because the
cells were transformed with the adenovirus E1A gene that
inhibits Rb, the p53-modulated G1 arrest is
abrogated (28)
. Thus, the loss of Fas and FasL surface
expression is not due to loss of p53 functionality, but in Fas and FasL
expression and processing pathways.
Although Fas was found to be wild-type, we hypothesized that loss of
its expression may be due to transcriptional repression. To test this
hypothesis, we incubated late passage MEFs in the presence of the
histone deacetylase inhibitor, trichostatin A (25 ng/ml for 24 h),
which relieves transcriptional repression caused by histone
acetylation. Late passage p53 wild-type MEFs were stained for their
surface expression of Fas and FasL after ionizing radiation in the
presence and absence of trichostatin A (Fig. 10A
). Trichostatin A restored basal surface levels of Fas and
FasL (Fig. 10A
) as well as their ability to be elevated
after ionizing radiation.
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). Late passage
wild-type MEFs were indeed found to have greatly reduced levels of Fas
and FasL message which failed to be induced in response to ionizing
radiation. In contrast, late passage wild-type MEFs treated with
trichostatin A had elevated basal levels of Fas and FasL message and
induced these genes in response to ionizing radiation. This effect was
found to be specific to Fas and FasL in that message levels of FADD
remained unchanged after trichostatin A treatment. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Of interest was our finding that radiation, but not hypoxia, was capable of inducing an increase in Fas surface expression in cells with wild-type p53. Although both stresses are known to induce p53, only radiation resulted in an elevation of Fas surface expression. This result suggests that Fas is not a major contributor to p53-dependent apoptosis induced by hypoxia in the tumor microenvironment.
Loss of Fas expression in tumors has been shown to occur during malignant progression (32) . We likewise found that p53 wild-type MEFs lost Fas as well as FasL surface expression and inducibility during proliferation in culture by transcriptionally repressing these genes. In expressing elevated levels of both Fas and FasL, early passage cells, by their very proximity, are under a selective pressure to lose either p53 or their ability to induce Fas as a means of escaping "self-induced" apoptosis and in the process become more apoptotically resistant. These results suggest that even under situations in which p53 is capable of transactivating gene expression, Fas desensitization may occur by transcriptional silencing. In support of this hypothesis, late passage p53 wild-type MEFs maintained other p53-dependent functions such as cell cycle arrest and p21 induction while demonstrating a loss of Fas inducibility and apoptotic sensitivity in response to ionizing radiation.
This selective desensitization of the Fas pathway during cell proliferation appears to be a specific p53 adaptation. Late passage MEFs, although exhibiting greatly reduced surface levels of Fas and FasL, as well as inducibility, still possess an intact p53-mediated G2 cell cycle arrest and are capable of inducing p21 following ionizing radiation. Of note, this loss of Fas induction with increasing passage failed to occur in the advanced passage p53 wild-type cancer cell line RKO (data not shown). In that RKO cells do not express surface FasL (data not shown), they are not under selective pressure in culture as are the MEFs, which need to inactivate either p53 or Fas to escape self-apoptotic elimination. Our results demonstrate that oncogenically transformed cells, with wild-type p53, are under a selective pressure during proliferation to down-regulate their sensitivity to Fas-mediated apoptosis (which might have already occurred in the advanced passage cell line RKO with the loss of surface FasL). These data suggest that in the process of tumor progression, a selective pressure may exist on the cell to either lose or mutate p53 or down-regulate Fas as a means of promoting survival and evading immune surveillance.
Complimentary to our results, a recent report described the pivotal role of cross-talk between Fas/FasL and p53 in the etiology and metastasis of UV radiation-induced skin tumors (18) . Keratinocytes from FasL-deficient mice exposed to UV radiation exhibited significantly less apoptosis coupled with a greatly enhanced frequency of p53 mutations than keratinocytes from wild-type mice. It was hypothesized that the survival of the p53-mutated skin cells exposed to UV radiation was due to their inability to be removed by the Fas pathway. On the basis of these results, it could be hypothesized that loss of Fas with the retention of FasL in p53 null tumors would result in enhanced survival both due to reduced self-apoptotic elimination as well as diminished immune-mediated killing.
In terms of cancer treatment, these results demonstrate that not only are p53 null cells more resistant to radiation-induced apoptosis but they also up-regulate FasL in response to genotoxic stress without increasing Fas expression. A potential outcome of such treatment could be the elimination of immune surveillance cells that possess Fas receptors with the retention of, and thus enhanced survival of, the tumor. In terms of experimental investigation of the relationship between p53 and the Fas pathway, our data stress the importance of using early passage cells in the exploration of the p53 and Fas relationship and caution against the use of late passage oncogenically transformed cells in studying the intersection of these two signaling pathways.
The applicability of these results to the tumor microenvironment still must be tested in vivo. However, our results provide yet another possible mechanism of how loss of p53 aids in tumor survival; i.e., by promoting the loss of surface Fas and Fas inducibility, elimination of p53 could enhance tumor survival through evasion of immune surveillance.
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FOOTNOTES |
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1 This work was supported by NIH Grants RO1
CA64489 and PO1 CA67166. 
2 To whom requests for reprints should be
addressed, at Stanford University School of Medicine, Department of
Radiation Oncology, CBRL GK220A, Stanford, CA 94305-5468. Phone:
650-723-7366; E-mail: giaccia{at}leland.stanford.edu (A. J. G.); or
3 The abbreviations used are: FasL, Fas ligand;
MEF, mouse embryonic fibroblast(s).
Received 11/ 3/99. Accepted 6/19/00.
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, p53 wild-type cells; 
,
p53 null cells. Wild-type cells have greater basal levels of Fas than
the nulls, and Fas is induced in the wild-type cells after ionizing
radiation, but not hypoxia. These experiments were repeated three times
to confirm results. PE, phycoerythrin.
