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Division of Experimental Hematology, Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105
ABSTRACT
We examined the patterns of induction of apoptosis, Fas expression, and the influence of the status of the p53 tumor suppressor gene, in response to treatment of human colon carcinoma cell lines to 5-fluorouracil (FUra) combined with leucovorin (LV) under conditions of both DNA-directed (HT29, VRC5/c1, and RKO) and RNA-directed (HCT8 and HCT116) cytotoxicity. Acute apoptosis was induced in cell lines expressing wtp53 (RKO, HCT8, and HCT116), independent of the mechanism of FUra action. In HT29 cells that expressed mp53, apoptosis was a delayed event. Cell lines undergoing DNA-directed FUra cytotoxicity demonstrated marked accumulation of cells in S-phase (HT29 and RKO), whereas those lines undergoing RNA-directed cytotoxicity (HCT8 and HCT116) demonstrated marked cell cycle phase arrest in G2-M, both reversible by dThd. dThd partially protected HCT8 and HCT116 cells from FUra-LV-induced apoptosis but had no influence on FUra-LV-induced loss in clonogenic survival. In cells expressing wtp53, the Fas death receptor was induced in response to FUra-LV treatment. FUra-LV sensitized RKO cells to the anti-Fas monoclonal antibody CH-11 that was completely reversed by dThd, demonstrating the involvement of DNA damage in FUra-LV-induced, Fas-dependent sensitization to CH-11. In contrast, FUra-LV sensitized HCT116 cells to CH-11-induced apoptosis, which was not dThd reversible. Transduction of HT29 cells with Ad-wtp53 induced elevated Fas expression and sensitized the cells to FUra-LV-induced apoptosis. Data indicate that the presence of a wtp53 gene determines FUra-LV-induced Fas expression, the kinetics of FUra-LV-induced apoptosis and not the extent of apoptosis induced, both being independent of the mechanism of FUra action. Therefore, in colon carcinomas that express wtp53, the approach to sensitize tumors to Fas-mediated apoptosis may be further enhanced from the effect of FUra-LV in elevating Fas expression in a p53-dependent manner.
INTRODUCTION
FUra3 combined with LV, which targets FUra to the TS locus, is the most effective therapy for the treatment of colon carcinoma. It is well known that FUra induces cytotoxicity by two predominant mechanisms:
(a) FUra inhibits TS after extensive metabolism, where the
active metabolite, FdUMP, forms a covalent ternary complex with the
enzyme and the reduced folate cofactor, 5,10-methylenetetrahydrofolate,
used in the normal catalytic reaction. The induction of a thymineless
state and subsequent DNA damage follows, and the cells die by a process
of thymineless death (1, 2, 3)
. We demonstrated previously
that thymineless death is regulated via the Fas death receptor in colon
carcinoma cells selected for TS deficiency (TS-)
and is closely correlated with the induction of apoptosis
(4)
. Furthermore, induction of Fas expression by the
cytokine recombinant human IFN-
in human colon carcinoma cell lines
has sensitized these cells to Fas-mediated and FUra-LV-mediated
cytotoxicity and apoptosis, dependent upon FUra-LV-induced DNA damage
and independent of the status of the p53 tumor suppressor
gene (2)
.
(b) The second mechanism of induction of FUra cytotoxicity
is following metabolism of the drug to ribonucleotides with subsequent
incorporation of FUTP into RNA, resulting in aberrant processing of RNA
species. This has been associated with the toxicity of FUra to normal
gastrointestinal tissues in preclinical models (5
, 6)
.
There are human colon carcinoma cell lines that demonstrate a cytotoxic
response to FUra by this mechanism including HCT8 (7
, 8)
and HCT116 (9)
, supported by the finding of lack of dThd
reversibility, and the inability of IFN- to potentiate the cytotoxic
response of FUra-LV, as determined by clonogenic survival
(2)
. Classically, cells treated with FUra are considered
to undergo substantial arrest in S or at the G1-S
boundary, although this is not always the case (8)
. In
addition, the relationship between the mechanism of FUra action, cell
cycle arrest, influence of p53, and induction of apoptosis remains
poorly understood.
Several reports have demonstrated that chemotherapeutic agents including cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin induce Fas expression in a variety of different cell lines, dependent on the presence of a wtp53 gene (10) . This has further been substantiated in studies in which the wtp53 gene has been transfected, leading to the induction of Fas and enhancement of apoptosis (11, 12, 13, 14) . In the current study, we have demonstrated that FUra combined with LV also induces the up-regulated expression of Fas, dependent on the presence of a wtp53 gene. Furthermore, induction of Fas in the presence of wtp53 occurs in colon carcinoma cell lines that demonstrate both DNA- and RNA-directed FUra cytotoxicity. Cell lines (HT29 and RKO) in which FUra-LV cytotoxicity is DNA-directed demonstrate marked accumulation of cells in S-phase, whereas RNA-directed FUra-LV activity (HCT8 and HCT116) leads to accumulation of cells in G2-M, both reversible by dThd. After FUra-LV treatment, acute apoptosis is induced in the presence of wtp53 independent of the mechanism of FUra action. However, in the presence of a mp53 gene, cells undergo delayed apoptosis. These data demonstrate that the presence of a wtp53 gene alters the kinetics of cell killing associated with FUra but not the overall level of cytotoxicity and apoptosis induced.
MATERIALS AND METHODS
Cell Lines.
The HT29, HCT8, and HCT116 human colon carcinoma cell lines were
obtained from American Type Culture Collection.
VRC5/c1 was established as reported previously
(15)
, and RKO was obtained from Dr. Michael Kastan (St.
Jude Children’s Research Hospital, Memphis, TN). Cells were
maintained under dThd-free conditions in the presence of folate-free
RPMI 1640 containing 10% dFBS (dThd-free) and 80 nM
[6RS]5-methyltetrahydrofolate (2)
.
Clonogenic Assays.
Cell lines were plated at a density of 1500 (HT29 and HCT116), 2000
(HCT8), and 3000 (VRC5/c1) cells/well in six-well
plates. After overnight attachment, cells were treated, in triplicate,
with FUra (0.1–30 µM) in the presence of LV (1
µM), either in the absence or presence of dThd (20
µM) for 72 h. Clonogenic survival was determined at
5–7 days (the equivalent of seven doublings) after removal of drug, as
described previously (2)
. Because RKO cells lacked the
ability to clone, cells were plated at a density of 1 x
105/well 24 h prior to a 72 h exposure
to drugs and were subsequently allowed to regrow for a period of 4 days
prior to elucidation of the influence of drug treatment on cell
numbers, enumerated using a Coulter particle counter (2)
.
Apoptosis and Cell Cycle Analyses.
All cell lines were plated at a density of 100,000–200,000 cells/well
in six-well plates. After overnight attachment, cells were treated for
periods up to 8 days with FUra (0.1–30 µM) + LV (1
µM), either in the absence or presence of dThd (20
µM), caspase inhibitors (10 µM Z-IETD-FMK
and 100 µM Z-VAD-FMK; Enzyme Systems Products), or the
anti-Fas MoAb CH-11 (200 ng/ml; MBL International Corp.). Both the
floating cells and attached cells were pooled after trypsinization,
fixed in 70% ethanol, and stored at -20° prior to analysis.
Apoptotic cells were detected as a sub-G1
fraction after propidium iodide staining and analysis using a Becton
Dickinson FACScan (16
, 17) . For evaluation of the cell
cycle distribution, samples were collected as described and immediately
centrifuged and resuspended in 0.005% propidium iodide containing
0.1% sodium citrate and 0.1% Triton X-100, filtered, and analyzed by
FACScan.
p53 Sequencing.
All 11 exons of the p53 gene in the
VRC5/c1 cell line were amplified by PCR of
genomic DNA using specific primers as described previously
(18)
. Sequencing of both strands of each of the PCR
products was carried out using standard methodology (19)
.
The status of the p53 gene in all other cell lines has been
published (20, 21, 22, 23)
.
Expression of Fas.
Fas expression was determined during treatment of colon carcinoma cell
lines at IC50 levels of FUra (0.3–6
µM) + LV (1 µM), either in the absence or
in the presence of dThd (20 µM) for 48 h. Fas was
measured in cell extracts by a standard ELISA assay, as reported
previously (2)
, that correlated with the expression of Fas
mRNA as determined by reverse transcription. Levels of the protein were
linear in the range of 25–400 pg using purified Fas as a standard.
Cell surface-associated Fas was also determined by FACS analysis using
a phycoerythrin-conjugated DX2 anti-Fas MoAb (PharMingen), using
standard procedures.
Adenoviral Delivery of wtp53.
HT29 cells were plated as described and subsequently transduced with a
wtp53 adenovirus (Ad-wtp53; Genetic Therapy Inc.) at MOIs of 2, 5, 10,
50, and 100, either alone or in combination with FUra (10
µM) + LV (1 µM) for 72 h. Apoptosis
was determined by FACS analysis as described. Additional HT29 cells
were plated and transduced with a replication-incompetent adenoviral
vector control containing the basic vector backbone sequences (Ad-VC;
MOI, 100; Ref. 4
) or Ad-wtp53 (MOI, 100). Fas expression
was subsequently determined at 48 h by FACS analysis as described.
RESULTS
Sensitivity to FUra-LV.
The sensitivity of five human colon carcinoma cell lines to FUra
(0.1–30 µM) combined with LV (1 µM) is
shown in Fig. 1
, together with the
influence of coincubation with dThd (20 µM). As
demonstrated by clonogenic survival (HT29,
VRC5/c1, HCT8, and HCT116) or regrowth (RKO)
assays, HT29, VRC5/c1, and RKO were sensitive to
FUra at low drug concentrations, survival being reduced to <40% at
concentrations of 
3 µM. RKO was particularly sensitive
to FUra-LV, with <5% survival at a FUra concentration of 0.5
µM. At these low FUra concentrations, the cytotoxic
action of FUra was completely reversed in the presence of dThd in
VRC5/c1 and reversed to 80% of control in HT29
and RKO. The dependence of the cytotoxic action of FUra upon DNA damage
was evident in these three cell lines. At higher FUra concentrations
(10 µM), clonogenic survival was not reversible by dThd
in HT29, indicating a secondary mechanism of cytotoxicity under these
conditions. Similar results were obtained in
VRC5/c1 and RKO (data not shown). HCT8 and HCT116
were only sensitive to FUra-LV at higher FUra concentrations (
5
µM), where FUra cytotoxicity was not dThd reversible.
However, it is known that these cell lines demonstrate an RNA-mediated
mechanism of FUra cytotoxicity (7, 8, 9)
, and current data
are consistent with these observations.
|
). Although clonogenic survival was
reduced to <10% in HT29 and VRC5/c1 under these
conditions, apoptosis was increased only modestly (10–15%) at 72 h. In contrast, the RKO colon carcinoma cell line that also
demonstrated dThd-reversible FUra-LV-induced cytotoxicity exhibited
>50% apoptotic cells under these conditions. In the presence of 6
µM FUra + LV, 35–40% loss in clonogenic survival was
obtained in HCT8 and HCT116 at 72 h, and this resulted in a
substantial increase in the percentage of cells undergoing apoptosis,
in particular in HCT116 cells, in contrast to data derived in HT29 and
VRC5/c1. Thus, the percentage of cells undergoing
apoptosis increased from 4 to 26% in HCT8 and from 4 to 
70% in
HCT116 during 72 h of FUra-LV exposure. Of interest was that in
HT29, RKO, VRC5/c1, and HCT8 cell lines,
apoptosis induced by FUra-LV during 72 h of exposure could be
reversed by dThd, and in HCT116 cells, this was partially reversible.
|
.
Effect of FUra-LV on Fas Expression.
There have been several reports demonstrating that certain
chemotherapeutic agents may induce the expression of the cell surface
receptor Fas when wtp53 is expressed in cells (10
, 24
, 25)
. Therefore, we examined the expression of Fas in five human
colon carcinoma cell lines demonstrating either DNA- or RNA-directed
mechanisms of FUra cytotoxicity and expressing either wtp53
or mp53 genes (Fig. 3)
. Fas
expression was determined after 48 h of treatment with FUra-LV at
IC50s for each cell line. Under these conditions,
FUra-LV failed to induce Fas expression in HT29 or
VRC5/c1 (Fig. 3)
. In contrast, FUra-LV treatment
up-regulated Fas expression by 4–8-fold in HCT8, HCT116, and RKO.
FUra-LV-induced up-regulated expression of Fas correlated with the
presence of a wtp53 gene in these three cell lines, and the
inability of FUra-LV to induce Fas correlated with the expression of
mp53 genes in HT29 and VRC5/c1.
|
). At 72 h, <20% apoptosis was
induced in HT29, increasing to 34% at 5 days, and apoptosis was also
reversed by dThd. In contrast, acute apoptosis was induced in HCT116
cells. The percentage of cells undergoing acute apoptosis at 72 h
in response to 6 µM FUra was 48% and increased
to 76% at 5 days.
|
. In HT29, >80% of the cells were
accumulated in S-phase at 72 h and demonstrated <20% of cells in
the sub-G1 (apoptotic) compartment, indicating
substantial S-phase arrest in the absence of acute apoptosis. However,
60% of HCT116 cells were arrested in G2-M at
this time, whereas 48% were undergoing apoptosis. Data derived in RKO
were similar to those obtained in HT29 demonstrating substantial
accumulation of cells in S-phase (>80%), whereas HCT8 demonstrated
significant accumulation of cells (75%) in G2-M
after FUra-LV treatment (data not shown). S-phase accumulation of cells
after FUra-LV treatment was dThd reversible in HT29 (Fig. 5)
and RKO
(data not shown), and G2-M-phase accumulation was
also dThd reversible in HCT116 (Fig. 5)
and HCT8 (data not shown).
Hence, the DNA-directed mechanism of FUra action (HT29 and RKO) was
associated with substantial accumulation of cells in S-phase, whereas
the RNA-directed mechanism of FUra action (HCT8 and HCT116) was
associated with accumulation of cells in G2-M.
|
.
However, apoptosis increased significantly, with further incubation in
drug-free medium. The percentage of cells undergoing apoptosis
increased from 14% at the time of drug removal to 32% at 6 days
and to 46% at 8 days, indicating the irreversible commitment to
apoptosis during S-phase arrest. In HCT116 cells, 70% of the cells
were undergoing acute apoptosis at 72 h in response to 6
µM FUra + LV. When FUra-LV was removed at 72 h, the
apoptotic cells were also removed. Of the cells remaining,
approximately one-third underwent apoptosis 1 day after drug removal,
and this level of apoptosis remained constant for the remainder of the
incubation period.
|
. Ad-wtp53 did not induce apoptosis,
even at an MOI of 100. However, treatment of cells with FUra-LV alone
induced 20% apoptosis, which significantly increased to 35% as the
MOI of Ad-wtp53 was increased to 100.
|
Effect of Caspase Inhibitors on FUra-LV-induced Acute Apoptosis.
The mechanism of FUra-LV-induced cytotoxicity was DNA-mediated in RKO
cells and RNA-mediated in HCT116. However, the induction of acute
apoptosis was determined by the status of the p53 gene
(wtp53), independent of the mechanism of FUra action. The
effect of the caspase inhibitors Z-IETD-FMK and Z-VAD-FMK on acute
apoptosis induced by FUra-LV in these two cell lines with differing
mechanisms of FUra action was determined (Fig. 8)
. In both cell lines, apoptosis
increased as the concentration of FUra was increased during 72 h
exposure.
|
.
In HCT116 cells, the profile was different. In contrast to RKO,
apoptosis in HCT116 with increasing FUra concentrations was partially
reversed by dThd, as indicated above, and reversible by both Z-IETD-FMK
and Z-VAD-FMK. Furthermore, the increased apoptosis obtained when CH-11
was combined with FUra-LV was not dThd reversible in contrast to the
effect observed in RKO cells (Fig. 8)
. Of interest was that
FUra-LV-induced apoptosis was partially but not completely reversed by
dThd, in contrast to FUra-LV-induced loss in clonogenic survival, which
was not reversible by dThd at any FUra concentration.
DISCUSSION
FUra is an antimetabolite used predominantly in the treatment of epithelial cancers that has been known for many years to possess a dual mechanism of action, dependent upon the concentration of the drug used and the cellular characteristics of target cells. The panel of human colon carcinoma cell lines used in this study demonstrate predominant characteristics of DNA-directed damage attributable to the inhibition of TS and the induction of thymineless death, which are reversible by dThd at low FUra concentrations (<3 µM) in HT29, VRC5/c1, and RKO cell lines. This mechanism of FUra action is independent of the status of the p53 tumor suppressor gene, because RKO expresses wtp53, whereas HT29 and VRC5/c1 are mutant for p53. However, RKO is the most sensitive cell line of the panel to FUra, where clonogenic survival is eliminated at concentrations of FUra of <1 µM when combined with LV. Other cell lines (HCT8 and HCT116) demonstrate predominant characteristics of RNA-mediated FUra cytotoxicity even in the presence of LV, which occurs only at higher FUra concentrations (>10 µM) in contrast to DNA-mediated FUra cytotoxicity, and is not reversible by dThd as determined by clonogenic assay. In the presence of a wtp53 gene, both FUra-LV-induced DNA damage (RKO) and RNA-mediated damage (HCT8 and HCT116) induce an acute apoptotic response. However, in the presence of a mp53 gene (HT29), DNA damage results in prolonged arrest of cells in S-phase, and apoptosis occurs as a late event after irreversible commitment during cell cycle phase arrest. Cells undergoing FUra-LV-induced DNA damage (HT29 and RKO) demonstrate substantial accumulation of cells in S-phase, whereas in cells demonstrating the RNA-mediated mechanism of FUra action (HCT8 and HCT116), accumulation occurred in G2-M; both are reversible by dThd. To date, we have not identified a human colon carcinoma cell line that undergoes RNA-mediated FUra cytotoxicity in the presence of a mp53 gene. However, it would be anticipated that such cells would not undergo acute apoptosis but would undergo delayed apoptosis, similar to HT29 in response to FUra-LV. Consistent with this hypothesis are the data reported by Bunz et al. (26) , who disrupted the p53 gene through homologous recombination in a human colon carcinoma cell line. The requirement for nonpharmacological concentrations of FUra (375 µM) in the presence of dThd-replete conditions (10% FCS) rendered FUra-directed DNA cytotoxicity impossible to evaluate. However, FUra-induced RNA-directed cytotoxicity in p53 -/- and p53 +/+ cells yielded results similar to those presented in the current study; lower accumulation of cells at the G1-S boundary and a higher level of apoptosis occurred in p53 +/+ cells, analogous to events occurring in HCT8 and HCT116. In contrast, p53 -/- cells demonstrated greater G1-S-phase arrest and did not undergo apoptosis at 72 or 96 h. Whether apoptosis was delayed was not addressed. However, the substantial G1-S-phase arrest and lack of acute apoptosis in p53 -/- cells undergoing RNA-mediated FUra cytotoxicity is similar to data derived in HT29 cells undergoing FUra-LV-induced DNA-directed cytotoxicity in the presence of mp53. Furthermore, intestinal cells in p53 +/+ mice underwent acute apoptosis in response to FUra treatment (27) , where FUra induced RNA-mediated toxicity (5 , 6) , in contrast to p53 -/- mice, where intestinal cells demonstrated reduced FUra-induced apoptosis.
The pattern of rescue from acute apoptosis induced by DNA-directed (RKO) or RNA-directed (HCT116) FUra-LV-induced cytotoxicity differed in the presence of Z-IETD-FMK, Z-VAD-FMK, or CH-11 + dThd. Z-IETD-FMK is considered to have higher specificity for the inhibition of caspase-8 activation, whereas Z-VAD-FMK is a general caspase inhibitor. In DNA-directed damage, Z-IETD-FMK was not inhibitory to FUra-LV-induced apoptosis, whereas Z-VAD-FMK was completely inhibitory in RKO cells. In contrast, both caspase inhibitors prevented FUra-LV-induced RNA-mediated toxicity in HCT116 cells and may reflect the different pathways by which apoptosis is induced by FUra-LV under these conditions. Furthermore, FUra-LV treatment enhanced the cytotoxic action of CH-11 in both RKO and HCT116 cells, which was reversible by dThd in RKO but not in HCT116. These data suggest that Fas is an integral component of the mechanism by which FUra-LV induces apoptosis after DNA damage in RKO cells. However, in HCT116, up-regulated expression of Fas after treatment with FUra-LV in the presence of wtp53 sensitized cells to CH-11, but this was independent of the mechanism of FUra action in RNA-directed cytotoxicity.
The relationship between apoptosis, p53, and the sensitivity of cancer cells to chemotherapeutic agents has received much attention in the recent literature (28) . The relationship between wtp53 and induction of apoptosis after DNA damage has been well established, particularly in oncogenically transformed normal cells, which appear to have a lower threshold for apoptosis induction after drug treatment, as well as in tissues of lymphoid origin (reviewed in Refs. 28 and 29 ). The presence of wtp53 can enhance sensitivity to DNA-damaging agents, and in general restoration of wtp53 function enhances drug responses (30) . However, it is evident in HCT8 and HCT116 cells that FUra-LV-induced RNA-mediated cytotoxicity also induced acute apoptosis in the presence of wtp53. Data derived in these two cell lines demonstrated that acute apoptosis could be partially reversed by dThd, indicating a partial effect of FUra-LV in inducing DNA damage in these cell lines. The accumulation of cells in G2-M was also reversible by dThd, which may therefore contribute to this effect. However, DNA damage was clearly not the predominant mechanism of FUra-induced cytotoxicity, because the overall survival of the population as determined by clonogenic assay was not influenced by dThd, and hence any DNA-directed damage was not of permanent significance to the overall survival of the population. Furthermore, FUra-LV-induced sensitization to CH-11 in HCT116 was not dThd reversible, in contrast to the dThd reversibility obtained in RKO.
There is increasing evidence that mp53 can affect the rate
of onset but not the overall extent of apoptosis induced (28
, 31) . Brown and Wouters (28)
demonstrated that p53
status may determine the threshold and kinetics of drug-induced
apoptosis but not overall survival in a treated cell population. Thus,
mouse embryo fibroblasts from p53 +/+ or p53 -/- mice that were
transformed with E1A and Ras, treated with VP-16 for 1 h, and
assayed by
2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
inner salt assay at 1 day or by clonogenic survival after 8 days
demonstrated enhanced survival in p53 -/- cells at 1 day but not at 8
days. Data derived in HT29 and VRC5/c1 human
colon carcinoma cell lines expressing mp53 alleles and
treated with FUra-LV are consistent with this model, in which apoptosis
occurs as a late event after irreversible commitment to apoptosis
during S-phase arrest. However, RKO, which also undergoes DNA damage in
response to FUra-LV, undergoes acute apoptosis at a lower threshold of
FUra in the presence of a wtp53 gene. Hence, all three cell
lines undergo apoptosis induced by thymineless stress, although the
kinetics are more rapid in RKO, accelerated by the presence of a
wtp53 gene. (A schematic representation of FUra-LV-induced
apoptotic responses in human colon carcinoma cell lines is shown
Fig. 9
.)
|
In summary, we have demonstrated that the target of FUra-LV-induced
cytotoxicity, whether DNA- or RNA-directed, induces acute apoptosis in
human colon carcinoma cell lines in the presence of a wtp53
gene. When toxicity is DNA directed, acute apoptosis occurs at very low
concentrations of FUra (<1 µM; RKO), cells are
arrested in S-phase; however, in the presence of mp53, cells
undergo prolonged S-phase arrest followed by delayed apoptosis (HT29
and VRC5/c1). When toxicity is RNA directed,
cells accumulate in G2-M and undergo acute
apoptosis in the presence of wtp53, both of which are dThd
reversible. However, loss in clonogenic survival induced by FUra-LV is
not dependent on rapid apoptosis or cell cycle block and was not
reversible by dThd. Furthermore, FUra-LV has induced wtp53-dependent
Fas expression, which has sensitized colon carcinoma cell lines to
anti-Fas treatment. We have demonstrated previously that IFN-
induces the expression of Fas in human colon carcinoma cell lines,
dependent upon FUra-LV-induced DNA damage, independent of the status of
the p53 tumor suppressor gene, and sensitizes these cells to
Fas-mediated and FUra-LV-induced/DNA damage-mediated cytotoxicity
(2)
. Because p53 is mutated in >75% of colon carcinomas
(34)
, which die by the process of thymineless death,
IFN- has the potential to yield a selective therapeutic approach in
combination with FUra-LV for the treatment of colon carcinomas. In
those colon carcinomas that express wtp53, the approach to
sensitize tumors to Fas-mediated apoptosis by up-regulating the
expression of Fas may be further enhanced from the effect of FUra-LV in
elevating Fas expression in a p53-dependent manner.
FOOTNOTES
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 research was supported by NIH Awards RO1 CA
32613, the Cancer Center Support (CORE) Grant CA 21765, and by the
American Lebanese Syrian Associated Charities. I. P. was supported in
part by a scholarship from the Fulbright Program and by the First
Institute of Pathology and Experimental Cancer Research, Semmelweiss
University of Medicine, Budapest, Hungary. 
2 To whom requests for reprints should be
addressed, at Division of Experimental Hematology, Department of
Hematology/Oncology, St. Jude Children’s Research Hospital, 332 North
Lauderdale, Memphis, TN 38105. Phone: (901) 495-3456; Fax:
(901) 523-2622; E-mail: janet.houghton{at}stjude.org
3 The abbreviations used are: FUra,
5-fluorouracil; LV, leucovorin; TS, thymidylate synthase; dThd,
thymidine; MoAb, monoclonal antibody; FACs, fluorescence-activated cell
sorter; MOI, multiplicity of infection; wt, wild type.
Received 4/14/00; revised 8/15/00; accepted 8/15/00.
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 M. T. Tomicic, C. Friedrichs, M. Christmann, P. Wutzler, R. Thust, and B. Kaina Apoptosis Induced by (E)-5-(2-Bromovinyl)-2'-deoxyuridine in Varicella Zoster Virus Thymidine Kinase-Expressing Cells Is Driven by Activation of c-Jun/Activator Protein-1 and Fas Ligand/Caspase-8 Mol. Pharmacol., February 1, 2003; 63(2): 439 - 449. [Abstract] [Full Text] [PDF]
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L. S. Schwartzberg, I. Petak, C. Stewart, P. K. Turner, J. Ashley, D. M. Tillman, L. Douglas, M. Tan, C. Billups, R. Mihalik, et al. Modulation of the Fas Signaling Pathway by IFN-{gamma} in Therapy of Colon Cancer: Phase I Trial and Correlative Studies of IFN-{gamma}, 5-Fluorouracil, and Leucovorin Clin. Cancer Res., August 1, 2002; 8(8): 2488 - 2498. [Abstract] [Full Text] [PDF]
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 M. Embree-Ku, D. Venturini, and K. Boekelheide Fas Is Involved in the p53-Dependent Apoptotic Response to Ionizing Radiation in Mouse Testis Biol Reprod, May 1, 2002; 66(5): 1456 - 1461. [Abstract] [Full Text]
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