(Received for publication, March 13, 1995; and in revised form, June 7, 1995)
From the
Interferon- (IFN
) potentiates the antitumor activity
of 5-fluorouracil (FUra) in colon cancer in vitro, in
vivo, and clinically. A likely mechanism for this action is the
induction by IFN
of thymidine phosphorylase (TP), the first enzyme
in one pathway for the metabolic activation of FUra to
fluorodeoxyribonucleotides. To test this hypothesis, an expression
vector containing the TP cDNA was transfected into HT-29 human colon
carcinoma cells. Five stable transfectants were selected and analyzed.
All showed increased sensitivity to FUra cytotoxicity, ranging from a
2-fold to a 19-fold decrease in the IC
for FUra, compared
to wild-type cells. Levels of TP mRNA, protein, and enzyme activity
were elevated in the transfectants, and there was a significant
correlation between the relative increase in sensitivity to FUra and
both the increase in both TP mRNA levels and TP activity. Transfected
cells exhibited increased formation of FdUMP, but not the
ribonucleotides FUDP and FUTP, from FUra when compared to wild-type
cells. The changes in TP activity, FdUMP formation, and FUra
sensitivity in the transfected cells were comparable with those seen
after treatment of wild-type cells with IFN
. These studies provide
direct evidence for the role of TP in mediating the sensitivity of
colon carcinoma cells to FUra, and further support the importance of
the induction of TP in the biomodulating action of IFN
on FUra
chemosensitivity.
Thymidine phosphorylase catalyzes the reversible synthesis of
thymidine and inorganic phosphate from thymine, using
deoxyribose-1-phosphate as
co-substrate(1, 2, 3) . The enzyme is widely
expressed in many human tissues, including leucocytes and platelets,
where it may have as its primary role the regulation of plasma
thymidine levels(4, 5, 6) . In addition to
its presumed role in thymidine metabolism and homeostasis, TP()also catalyzes the conversion of the pyrimidine
antimetabolite 5-fluorouracil (FUra) to 5-fluoro-2`-deoxyuridine, the
first step in one pathway for the metabolic activation of the cancer
chemotherapeutic agent to deoxyribonucleotides(7) . More
recently, interest in TP has focused on the intriguing observations
that TP expression is inducible by interferon and other cytokines (8, 9) and evidence that the enzyme also has
angiogenic and endothelial cell chemotactic activities (10, 11, 12, 13, 14, 15) .
The level of expression of TP varies up to 15-fold in different
human tissues (4, 16, 17) and between
different individuals. ()Furthermore, in nearly all biopsies
examined from carcinomas of the stomach, colon, and ovary, TP levels
were elevated up to 10-fold when compared to non-neoplastic regions of
these organs(4, 16, 17, 19) , and
higher levels of TP were also found in plasma of tumor-bearing animals
and from cancer patients (20, 21) . Treatment of human
colon carcinoma cells with IFN
caused a 5-10-fold increase
in the levels of TP mRNA and enzyme activity (8, 22, 23) , and large increases in
expression have also been found in patients treated with
IFN
.
These findings distinguish this enzyme from
uridine phosphorylase, which is expressed at comparable levels in human
gastric or colorectal tumors compared to non-malignant tissue, and
which is not induced by IFN
in human colon carcinoma cell
lines(8, 24) . The induction of TP expression by
interferon was accompanied by an increase in the cellular levels of the
active metabolite of FUra, FdUMP, presumably due to the sequential
actions of TP and thymidine kinase on FUra(8, 25) .
Human TP has been reported to be identical to the angiogenic factor platelet-derived endothelial cell growth factor (PD-ECGF)(11, 12, 13) , and studies indicate that the catalytic activity of the enzyme is indispensable for its angiogenic effects(14, 15, 26) . When transfected into either MCF7 breast carcinoma cells or ras-transformed NIH 3T3 cells, TP/PD-ECGF was found to increase the both the vascularization and the growth of the tumors in nude mice after subcutaneous inoculation of the cells(10, 26) . The regulation of TP/PD-ECGF by platelet agonists suggests that its expression may contribute to the response to injury to the endothelial lining of blood vessels(6) .
In clinical trials of patients with metastatic
colorectal carcinoma treated with FUra and IFN, response rates
were seen that were greater than historical controls treated with FUra
alone(27, 28, 29, 30) . In vitro studies have suggested that the induction of TP expression by
IFN
is a potential biochemical mechanism for the modulation of the
antitumor activity of FUra by IFN
. In this report, we demonstrate
that transfection of TP into human colon carcinoma cells greatly
increases the sensitivity of the cells to FUra, and that this action is
mediated by the enhancement of the metabolic activation of FUra by
increased TP expression.
The role of TP in mediating the sensitivity of cells to FUra
was investigated by transfecting an expression vector (pcDNAI-Neo)
containing the human TP cDNA (pPL5) into HT-29 human colon carcinoma
cells. The HT-29 cells were chosen as targets because IFN has been
shown previously to induce TP activity and increase sensitivity to FUra
in these cells(8, 22) , and because colon carcinomas
are most often treated with FUra. Several colonies of cells were
obtained upon selection of transfected HT-29 cells in G418, and five
independent clones (designated tp1-tp5) were chosen for further
analysis. The rate of cell growth of the transfected cells did not vary
more than 20% from the growth rate of the wild-type cells (data not
shown). The sensitivity of the cells to FUra growth inhibition was
evaluated by treating the cells with the fluoropyrimidine for 6 days.
Increasing concentrations of FUra caused progressively greater
cytotoxicity and growth inhibition of the cells; the concentration of
FUra producing a 50% reduction in the number of cells (IC
)
in the wild-type (wt) HT-29 cells was 2.5 µM ( Fig.1and Table 1). All five of the transfected cell lines
were more sensitive to the cytotoxic actions of FUra when compared to
HT-29/wt cells, with most of the cell lines having a 2-5-fold
decrease in the IC
, and one of the transfectants (tp3)
exhibiting a 19-fold increase in sensitivity to FUra with an IC
of 0.13 µM (Table1).
Figure 1:
Effect of TP
transfection on the cytotoxicity of FUra to HT-29 cells. Growth
inhibition was measured after treatment of cells with FUra for 7 days
in 96-well plates (4 10
HT-29 cells/well in RPMI
1640 medium with 10% dialyzed fetal bovine serum); increasing
concentrations of FUra were added after allowing for cell attachment
overnight. Cell numbers were quantitated by staining with
sulforhodamine B and are expressed relative to cells without FUra.
Experiments were done using wild-type HT-29 cells (
) and
TP-transfected HT-29 cell lines tp1 (
), tp2 (
), tp3
(
), tp4 (
), and tp5 (
). Data are means of three
experiments, each done in duplicate.
Expression of TP in
the transfected cells was then examined. A 1.8-kb mRNA for TP was
barely detectable in the HT-29/wt cells (Fig.2), similar to
previous studies of this cell line(23) . Somewhat higher levels
of mRNA expression were found in TP transfectants 1, 2, 4, and 5, and a
substantially higher abundance of TP mRNA was observed in the tp3
transfectant (Fig.2). The sizes of the predominant messages in
the wt and tp1 to tp4 cells were similar (1.7-1.8 kb), whereas
the mRNA in tp5 was smaller (1.3 kb). Two additional bands of 1.1 and
2.3 kb were also observed in the tp3 cells. TP mRNA levels were
quantitated by densitometric scanning and corrected for actin
expression (Table1). The relative increase in expression of the
1.3-1.8-kb mRNAs in the cell lines was found to be significantly
correlated (r = 0.98) with the relative decline in the
IC values for FUra.
Figure 2:
Effect of TP transfection on TP mRNA
expression in HT-29 colon carcinoma cells. Poly(A) RNA
was isolated from wild-type (wt) and TP-transfected (tp1-tp5) HT-29 colon carcinoma cells and 10 µg
fractionated on a formaldehyde-containing 1% agarose gel. RNA was
transferred to nitrocellulose and hybridized to a
P-labeled human TP cDNA probe (upperpanel). Each blot was then stripped and reprobed with
actin (lowerpanel).
Thymidine phosphorylase protein levels were determined on immunoblots after SDS-polyacrylamide gel electrophoresis of soluble cell extracts from wt and TP-transfected HT-29 cells (Fig.3). A band corresponding to the recombinant TP standard (lane7) was only detected in the tp3 cells, with an apparent size of approximately 50 kDa.
Figure 3: Effect of TP transfection on TP protein expression in HT-29 colon carcinoma cells. For analysis of TP protein levels, wild-type (lane1) and transfected HT-29 cells (lanes 2-6) were lysed, sonicated, centrifuged, and 50 µg of supernatant protein fractionated by SDS-polyacrylamide gel electrophoresis. The proteins were electroblotted onto membranes, and incubated with rabbit anti-human TP (1:2000) and a goat anti-rabbit IgG conjugated to alkaline phosphatase. Immunolabeled proteins were visualized using x-ray film after incubation with LumiPhos. Purified recombinant human TP (2.5 ng) was also analyzed (lane7).
TP enzyme activity
was measured in crude cell extracts using
[H]thymine as substrate and in the presence of
excess dR-1-P. Enzyme activity was higher in all the transfectants when
compared to the wild-type cells (Table1). The largest increase,
greater than 5-fold compared to HT-29/wt cells, was found in the tp3
cells, and the other transfected cell lines had increases of
approximately 2-fold. Comparison of the enzyme activity with
sensitivity to FUra cytotoxicity demonstrated that the cell line with
the highest enzyme active, tp3, had the greatest increase in
sensitivity to FUra. The other transfectants, which had intermediate
elevations in TP activity, had more modest increases in sensitivity to
FUra. There was a significant correlation (r = 0.94)
between the relative increase in TP activity and the relative decrease
in the IC
for FUra.
Although there was a strong
correlation between TP activity and sensitivity to FUra among the cell
lines examined, other factors could conceivably contribute to the
changes in the IC values observed. The level of TS
expression has been shown previously to correlate with responsive to
FUra in vitro and in vivo, with high TS levels
conferring resistance to FUra by circumventing the inhibition of TS
that results from the binding of the FUra metabolite, FdUMP, to the
enzyme(33, 34) . To eliminate differences in TS as a
potential explanation for the increase in sensitivity of the HT-29/tp3
cells, TS levels were measured in these and the wild-type cells using a
catalytic assay. Rather than exhibiting a decrease in TS activity, the
HT-29/tp3 cells had an increase in activity, 0.312 ± 0.065
pmol/min/mg, compared to the value obtained in the wild-type cells,
0.226 ± 0.026 pmol/min/mg.
It was anticipated that the
increased expression of TP in the HT-29/tp3 cells would lead to greater
metabolic activation of FUra to deoxyribonucleotides. The intracellular
disposition of [H]FUra in the HT-29/tp3 cells was
therefore compared to that in HT-29/wt cells. The incorporation of FUra
into DNA and RNA was 10-20% lower in the HT-29/tp3 cells, and the
levels of total cellular fluoropyrimidines and fluoropyrimidine
ribonucleotides FUDP and FUTP were similarly unchanged in the HT-29/tp3
cells compared to the HT-29/wt cells (Table2). In contrast,
cellular FdUMP levels were found to be 2.2-fold higher in the
transfected cells.
The effect of the elevated TP expression in the
transfected cells on FdUMP levels and sensitivity to FUra cytotoxicity
was compared to these parameters in IFN-treated cells, which also
express elevated TP activity. The increase in TP activity was directly
correlated with FdUMP levels, and indirectly correlated with the
IC
for FUra (Fig.4). In all instances, the
greatest change was observed in the HT-29/tp3 cells compared to the
HT-29/wt cells, with IFN
-treated HT-29/wt cells having
intermediate degrees of change.
Figure 4:
Comparative effects of
interferon-treatment and TP transfection on HT-29 cells. TP activity,
formation of FdUMP, and the sensitivity to FUra were determined in
HT-29/wt cells (openbars), HT-29/wt cells after
treatment with IFN (500 units/ml for 24 h) (hatchedbars), and HT-29/tp3 cells (solidbars). TP activity and FUra cytotoxicity were determined
as described in Table1, and FdUMP levels as described in Table 2.
The changes observed in the TP-transfected cells provide
direct evidence that an increase in TP activity can increase the
sensitivity of cells to FUra via an increased formation of FdUMP, an
active metabolite of FUra. The fact that the relationship between TP
activity, FdUMP levels, and FUra cytotoxicity in interferon-treated
cells are quantitatively consistent with those seen in the transfected
cells strongly supports a role for the induction of the enzyme in the
mechanism of action of IFN in these cells. The data obtained in
the present study are also consistent with previously published
reports, which indicated that resistance to FUra can occur as a
consequence of the loss of TP activity. For example, mutants in
thymidine phosphorylase that are resistant to FUra can be isolated by
selection of microorganisms with FUra(35) . A human ovarian
cancer cell line derived from a biopsy of a FUra-resistant tumor was
found to have a 70% reduction in TP activity and FdUMP levels compared
to a cell line derived from the tumor in the same patient prior to the
development of FUra resistance(36) .
TP activity has been
found to vary considerably among different tissues and among different
individuals(4, 16) . This suggests that in
many instances, the level of TP expression in an individual may be an
important determinant of the degree of responsiveness of a particular
tumor to FUra, and a preliminary study of mononuclear cell TP in
patients undergoing FUra-based chemotherapy supports this
hypothesis.
Furthermore, since TP levels have been found to
be elevated in most colon carcinomas examined compared to non-malignant
tissues(4, 16, 17, 19) , its
expression may provide some degree of selectivity to the cytotoxic
effects of FUra and therefore contribute to the clinical efficacy of
the fluoropyrimidine in colon cancer. Other biochemical determinants of
response to FUra, most notably the cellular levels of thymidylate
synthase, have also been shown to be of value in predicting clinical
response to FUra(34, 37) , with tumors expressing high
levels of TS being more refractory to therapy.
TP has recently been
shown to be identical to the angiogenic factor PD-ECGF, and a study of
ovarian cancer biopsies demonstrated that TP/PD-ECGF was the only one
of four angiogenic factors examined whose mRNA level was elevated in
malignant tumors when compared to benign tumors, and expression levels
were found to be highest in regions of high blood flow within
individual tumors(38) . A recent study of 91 primary colorectal
carcinomas (who were not treated with chemotherapy) found that the
expression of TP in tumor specimens was statistically correlated with
depth of tumor invasion, frequency of metastasis, and a shorter
survival time, when compared to tumors that were found not to be
expressing TP(39) . Hence, the data suggest that elevated
TP/PD-ECGF expression predisposes to both aggressive disease and
improved response to fluoropyrimidine-based chemotherapy. Since
angiogenesis is also involved in embryogenesis, wound healing,
rheumatoid arthritis, and certain retinopathies, TP/PD-ECGF likely
exerts a range of biochemical actions that mediate both normal and
pathologic cellular functions (18) . While the present study
helps to define the role of TP in the modulating actions of IFN on
FUra, the induction of TP expression likely provides a biochemical
basis for other physiologic and therapeutic actions of IFN and other
cytokines.