©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thymidine Phosphorylase Mediates the Sensitivity of Human Colon Carcinoma Cells to 5-Fluorouracil (*)

(Received for publication, March 13, 1995; and in revised form, June 7, 1995)

Edward L. Schwartz (§) Nicole Baptiste Scott Wadler Della Makower

From theDepartment of Oncology, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York 10467

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Interferon-alpha (IFNalpha) 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 IFNalpha 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 IFNalpha. 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 IFNalpha on FUra chemosensitivity.


INTRODUCTION

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(^1)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. (^2)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 IFNalpha 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 IFNalpha.^2 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 IFNalpha 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 IFNalpha, 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 IFNalpha is a potential biochemical mechanism for the modulation of the antitumor activity of FUra by IFNalpha. 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.


EXPERIMENTAL PROCEDURES

Materials

Human colon carcinoma HT-29 cells were maintained in RPMI 1640 with 10% heat-inactivated fetal bovine serum and gentamicin, in a humidified CO(2) incubator at 37 °C. Recombinant human thymidine phosphorylase (also known as platelet-derived endothelial cell growth factor) was obtained from R& Systems (Minneapolis, MN). It was greater than 97% pure as judged by SDS-polyacrylamide gel electrophoresis with silver staining. Human recombinant interferon alpha-2a (Roferon) was provided by Hoffmann-La Roche (Nutley, NJ).

Cell Transfections

HT-29 cells were stably transfected with a construct containing the human TP cDNA under the control of a cytomegalovirus promoter (pCMV-TPneo; kindly provided by Dr. Roy Bicknell, Oxford University)(26) . Cells (2.5 10^5/well in 12-well plates) were incubated with plasmid DNA (0.5 µg/well) and Lipofectamine (8 µl/well; Life Technologies, Inc.), using conditions recommended by the manufacturer. After 72 h, the cells were trypsinized and suspended in fresh medium containing G418 (Geneticin, Life Technologies, Inc.; 0.5 mg/ml). After an additional 3 weeks, wells with individual large colonies growing were transferred to flasks and were maintained in the presence of 0.2 mg/ml G418.

TP Enzyme Assay

TP activity was measured in extracts from wild-type and transfected HT-29 cells, and from wild-type cells treated with IFNalpha (500 units/ml). Cells were washed with phosphate-buffered saline, and resuspended in 50 mM Tris-HCl (pH 7.5) and 1 mM EDTA. The cells were sonicated on ice and centrifuged, and the supernatant was stored at -70 °C until assayed. Assays contained 30 mM Tris-HCl, pH 7.4, 1 mM EDTA, 5 mM MgCl(2), 100 µg of cell extract, and 0.25 mM (20 µCi/ml) [methyl-^3H]thymine (DuPont NEN). Reactions were stopped at 40 min by boiling, tubes were briefly centrifuged, and supernatants were spotted on silica gel thin layer chromatography plates. Separation of [^3H]thymine from [^3H]thymidine was done using chloroform:methanol:acetic acid (17:3:1), and activity was calculated based on the percent conversion of base to nucleoside. TP activity was proportional to both the amount of protein assayed and time.

Western (Protein) Immunoblots

Wild-type and transfected HT-29 cells were washed twice with phosphate-buffered saline and suspended in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA. The cells were sonicated on ice and centrifuged at 12,000 rpm for 15 min. 50 µg of supernatant protein was fractionated by SDS-polyacrylamide gel electrophoresis. The proteins were electroblotted onto nylon membranes, and incubated with rabbit anti-human TP (1:2000; kindly provided by Dr. A. Yoshimura, Kagoshima University) and a goat anti-rabbit IgG conjugated to alkaline phosphatase (Schleicher & Schuell), according to directions supplied by the manufacturer. Immunolabeled proteins were visualized using x-ray film after incubation with LumiPhos (Schleicher & Schuell).

RNA Extraction and Northern (RNA) Analysis

RNA was extracted from control and IFNalpha-treated cells with guanidinium thiocyanate, purified by centrifugation through CsCl, and poly(A) RNA isolated on oligo(dT)-cellulose using standard procedures. The RNA (10 µg) was fractionated on formaldehyde-containing agarose gels, transferred to nitrocellulose, probed with P-labeled human TP cDNA (pPL8, kindly provided by Dr. Carl-Henrik Heldin), washed, and exposed to Kodak X-Omat film at -85 °C. Blots were stripped and reprobed with a P-labeled human actin cDNA. Autoradiograms were scanned with densitometer to quantify band densities.

FUra Cytotoxicity Assays

Growth inhibition was measured after treatment of cells with FUra for 7 days in 96-well plates (4 10^3 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, as has been described previously (31) .

FUra Metabolism

Wild-type and transfected HT-29 cells were incubated with 1 µM [6-^3H]FUra (3 Ci/mmol; Moravek Biochemicals) with or without 500 units/ml IFNalpha. After 24 h, cells were washed once with cold phosphate-buffered saline and extracted with 0.5 N perchloric acid. After a brief centrifugation, the supernatants were removed and neutralized with 1.5 volumes of alamine/freon, lyophilized, and reconstituted in HPLC mobile phase(8) . An aliquot was analyzed by HPLC; fractions were collected and radioactivity determined by liquid scintillation counting. Separation of fluoropyrimidine nucleotides was done using an Adsorbosphere C18 column (Alltech) as described previously(8) . Recovery of radioactive material from the HPLC was greater than 95%. Identity of radioactive peaks was determined using authentic standards.

Incorporation of [6-^3H]FUra into RNA and DNA

Wild-type and transfected HT-29 cells were incubated with 1 µM [6-^3H]FUra for 24 h, as described above. Cells were lysed and RNA and DNA isolated using phenol/guanidine isothiocyanate (Trizol, Life Technologies, Inc.). Radioactivity in the RNA and DNA fractions were determined by liquid scintillation counting. The DNA fraction was exhaustively digested with RNase and ethanol-precipitated prior to measurement of radioactivity.

Thymidylate Synthase (TS) Catalytic Assay

TS activity was measured based on ^3H(2)O release from [5-^3H]dUMP in the presence of 5,10-methylenetetrahydrofolate(32) . The assay contained, in 150 µl, 50 mM Tris-HCl, pH 7.4, 10 µM [5-^3H]dUMP (0.33 Ci/mmol), and 250 µM 5,10-methylenetetrahydrofolate. Reactions were started by the addition of cell extract and were incubated for 10 min at 37 °C. Reactions were stopped by the addition of 0.8 ml of ice-cold 3% acid charcoal; after 10 min on ice, the samples were centrifuged (10 min at 10,000 rpm), and a 0.5-ml aliquot of the supernatant assayed for radioactivity in a liquid scintillation counter. Reactions were linear with respect to time and protein concentration and were dependent on reduced folate for activity.


RESULTS

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 IFNalpha 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 10^3 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 (bullet) 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 [^3H]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 [^3H]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 IFNalpha-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 IFNalpha-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 IFNalpha (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.




DISCUSSION

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 IFNalpha 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) .^2 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.^2 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 IFNalpha on FUra, the induction of TP expression likely provides a biochemical basis for other physiologic and therapeutic actions of IFN and other cytokines.


FOOTNOTES

*
This work was supported by Grant CA54422 and Cancer Center Support Grant CA13330 from the National Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Dept. of Oncology, Albert Einstein Cancer Center, 111 E. 210th St., Bronx, NY 10467.

^1
The abbreviations used are: TP, thymidine phosphorylase; FUra, 5-fluorouracil; IFNalpha, interferon-alpha; PD-ECGF, platelet-derived endothelial cell growth factor; HPLC, high performance liquid chromatography; wt, wild type; kb, kilobase(s); TS, thymidylate synthase.

^2
D. Makower, S. Wadler, H. Haynes, and E. L. Schwartz, submitted for publication.


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