ANTIMICROBIAL AGoNTS AND CHEMOTHERAPY, May 1979, p. 730-734
0066-4804/79/05-0730/05$02.00/0
Vol. 15, No. 5
Antifolate Activity of Isoaminopterin in HeLa Cells HORTENCIA ROSEMOND-HORNBEAK* AND M. G. NAIR Departments of Microbiology/Immunology and Biochemistry, University of South Alabama College of Medicine, Mobile, Alabama 36688
Received for publication 16 January 1979
The antifolate activity and the transport characteristics of isoaminopterin (IA) in HeLa cells were studied and compared with those of methotrexate (MTX). Both IA and MTX inhibited the incorporation of [2-'4C]deoxyuridine into HeLa cell DNA by 50% at a concentration of 0.09 LM by 8 h postaddition of the compounds. Unlike MTX, the inhibition of DNA synthesis by IA was time dependent and reached a maximum at 24 h. IA-induced inhibition was due to interference with folate metabolism, since it could be completely reversed with N5-formyl-tetrahydrofolate. Competitive transport experiments between IA and either radiolabeled MTX or radiolabeled folate showed that IA preferentially uses the reduced folate/MTX transport system. IA inhibited MTX uptake by 50% at a concentration of 6.8 ,uM but had a negligible effect on folate transport.
The use of aminopterin and methotrexate (MTX) in the treatment of neoplastic diseases is well known (1, 2, 4, 13, 14, 16). MTX is used either alone or in combination with other drugs for the treatment of leukemia (1, 14), Burkitt's lymphoma (2), choriocarcinoma (13), and psoriasis (16), as well as for the suppression of the immune response (4, 15). In spite of a documented high level of toxicity and the development of resistance (6), MTX continues to be one of the most widely used anticancer drugs. As part of a continuing program aimed at developing less toxic and more specific antifolate drugs for the treatment of different forms of cancer, we have synthesized a number of classical analogs of folic acid and aminopterin which are altered in the C9-N10 bridge region (7-11). One of these compounds, isoaminopterin (IA; N[p- { ((2,4-diamino-4-deoxy-6-pteridinyl)amino)
methyl} -benzoyl]-L-glutamic acid) (11), has been evaluated for anticancer activity in L1210 leukemic mice at The National Cancer Institute, National Institutes of Health, as described by Geran et al. (3). At the maximum dose tested (4.0 mg/kg), IA showed significant activity (% T/C 2125/s42) against L1210 leukemia in mice. In vitro studies with IA indicate that it is a very powerful antifolate. Nair et al. (11) have shown that it inhibits the growth of two folate-requiring organisms, Lactobacillus casei (ATCC 7469) and Streptococcus faecium (ATCC 8043), and is an effective inhibitor of dichloromethotrexateresistant L. casei dihydrofolate reductase. In this study the antifolate activity of IA is evaluated in a mammalian cell line in relation to (i) its ability to inhibit the incorporation of [2'4C]deoxyuridine into DNA, (ii) the effect of N5-
formyl-tetrahydrofolate (N5-formyl-THF) in reversing the induced DNA synthesis inhibition, and (iii) its transport characteristics. The antifolate activity and the transport characteristics of IA are compared to those of MTX. MATERIALS AND METHODS Materials. [:H]folic acid (47 Ci/mmol) and [:'H]MTX (250 mCi/mmol) were purchased from Amersham/Searle, and [2-'4C]deoxyuridine (58.1 mCi/ mmnol) came from New England Nuclear Corp. Folic acid and MTX were purchased from Sigma. Both folic acid and MTX were repurified by diethylaminoethylcellulose chromatography. N5-formyl-THF was purchased from Sigma Chemical Co. as the sodium salt. IA was synthesized in our laboratory as previously described (11). The chemical structures of these compounds are shown in Fig. 1. Cells. HeLa cells were vurchased from Flow Laboratories and maintained in monolayer cultures. The cells were grown in Eagle minimum essential medium supplemented with 10% newborn calf serum, 50 IU of penicillin per ml, and 50 ug of streptomycin per ml. The pH of the medium on the monolayers was maintained with a bicarbonate buffer in tightly closed flasks or Blake bottles. The cells were tested for mycoplasma contamination by Flow Laboratories. Monolayers of HeLa cells in 25- or 75-cm2 flasks used throughout these studies were prepared as follows. HeLa cell stock flasks were treated with 0.125% trypsin-ethylenediaminetetraacetic acid. The cells were pooled and suspended at a concentration of 10' cells per ml in medium containing 10% newborn calf serum. Samples of 10 and 25 ml of suspended cells were added to 25- and 75-cm2 flasks, respectively. The medium was replaced on the following day, and the cells were incubated for 48 h before use. All flasks used in an experiment were prepared simultaneously. Determination of HeLa cell DNA synthesis. Monolayers of HeLa cells were pulse-labeled with [2-
730
VOL. 15, 1979
ANTIFOLATE ACTIVITY OF ISOAMINOPTERIN
NH2 N
N
0 ~~~COOH 2C-NH-C 2OH
NH-CH (1)
CH2-CH 2-COOH
H2N
RR (2);
H
(3); R=-CH3 0 Hr1
H2 N
a
>-CH2-
i2
H
N
CHO
H2NLN
3OO
CHO N
_-
C-NH-C-H 0
COOH CH2- CH2-COOH
N° N CH2-CCH 2-NCOOH
(5)
FIG. 1. Chemical structures. (1) IA; (2) aminopter2; (3) MTX; (4) N5-formyl-THF; (5) 1O-oxaaminop-
terin. '4C]deoxyuridine (0.025 ,uCi/ml) at 37°C for 2.5 h at specific time intervals after addition of IA or MTX. At the end of each respective incubation period, the cultures were washed with ice-cold phosphatebuffered NaCI, harvested, and pelleted. The cells were suspended in 2.5 ml of distilled water. The DNA was precipitated with 0.25 N perchloric acid and collected on 0.45-,um Millipore membrane filters. The acid-precipitable counts were determined by liquid scintillation spectrophotometry. As control, the incorporation of radiolabel in untreated cells was determined. Effect of iA on the transport of [3H]folate and [3HiMTX. Monolayers of the Heba cells were washed twice with warm minimum essential medium without serum to remove most of the serum associated with the cells. The growth medium was replaced with medium without serum. A constant amount of either [3H]-folate or [3H]MTX (0.8 ,uM) and varying concentrations of IA were simultaneously added to monolayers of HeLa cells (2.2 Ax0s cells), and the cultures were incubated for 10 min at 37°C. At the end of the incubation period, the cells were washed twice with ice-cold phosphate-buffered NaCl,harvested, and solubiized in Unisol tissue solubilizer. The radioactivity associated with the cells was determined by liquid scintillation spectrophotometry.
731
As shown in Fig. 2, radiolabeled deoxyuridine is linearly incorporated into HeLa DNA for about 3 h. Therefore, a 2.5-h pulse-label period with radiolabeled deoxyuridine was used throughout this study to measure de novo DNA synthesis. The antifolate activity of IA compared to MTX was evaluated by determining the effect of the compounds on the incorporation of [2-'4C]deoxyuridine into acid-precipitable DNA. Semiconfluent monolayers of HeLa cells were incubated at 370C for varying periods of time in the presence of several concentrations of IA or MTX. At 0, 1.5, 5.5, and 21.5 h postaddition of the compounds, the cultures were pulse-labeled with [2-'4C]deoxyuridine. At the end of the incubation period, the cells were harvested, and the acid-precipitable radioactivity incorporated into DNA was determined by liquid scintillation spectrophotometry. Within 2.5 h, MTX inhibited the incorporation of [2-'4C]deoxyuridine into acid-precipitable counts by 88% at a concentration of 5 ,uM (Fig. 3A), whereas IA only produced a 45% inhibition at this concentration (Fig. 3B). However, at 24 h, MTX and IA were comparably effective in inhibiting DNA synthesis. The 50% inhibitory dose values for MTX and IA were 0.08 ,uM and 0.05 ,tM, respectively (Fig. 3). From these results it is apparent that IA is as effective as MTX in inhibiting HeLa cell DNA synthesis at 24 h postaddition of the compounds, and that IA is a slower-acting com-
pound.
RESULTS
The effect of folate analogs on DNA synthesis was monitored by measuring the incorporation of radiolabeled deoxyuridine into acid-precipitable counts. The rate of incorporation of deoxyuridine into HeLa cell DNA as a function of time was determined by incubating semiconfluent monolayers of cells in the presence of [2-w4C]deoxyubrdine for varying periods of time. At the end of the incubation period, the cultures were harvested, and the acid-precipitable radioactivity incorporated into DNA was determined.
TIME (hr.)
FIG. 2. Incorporation of [2- "C]deoxyuridine into acid-precipitable DNA as a function of time. A constant amount of [2-'4C]deoxyuridine (0.025 ,iCi/ml) was added to monolayers of HeLa cells (3 x Itt cells per flask), and the cultures were incubated at 37°C. At the indicated time intervals the cells were harvested, and the amount of acid-precipitable DNA in each flask was determined as described in the text. The results of a representative experiment are shown. Each point represents the average incorporation of duplicate cultures.
732
ROSEMOND-HORNBEAK AND NAIR
ANTIMICROB. AGENTS CHEMOTHER.
layers of HeLa cells were preincubated for 4 h at 37°C in medium containing 0.45 ,iM MTX or IA. Under these experimental conditions, DNA synthesis was inhibited by 75% and 60% in MTXand IA-treated cells, respectively. At the end of 60 the 4-h incubation period, varying concentrations of N5-formyl-THF were added to the cull tures. The cells were pulse-labeled with [2-14C]deoxyuridine at 33 h postaddition of the compounds. As controls, parallel cultures were incubated under the same experimental conditions 0: in the absence of N5-formyl-THF. As shown in V~~~~~~ Fig. 4, the inhibition of DNA synthesis induced by both IA and MTX was completely reversed by N5-formyl-THF at a concentration of 1.0 1iM. 80 These results indicate that IA-induced inhibition of HeLa cell DNA synthesis is due to interLn ference with folate metabolism. HeLa cells have different transport systems for MTX and folic acid (12). Competitive transport experiments between IA and either radio20labeled MTX or radiolabeled folate were performed to determine the transport pathway(s) 1-7 i0-8 10-6 used by IA to enter the cell. Parallel cultures of COMPOUND CONCENTRATION(M) HeLa cells were washed free of serum with miniFIG. 3. Effect of MTX and IA on HeLa cell DNA mum essential medium and incubated for 10 min synthesis. The indicated concentrations of either in the presence of varying concentrations of IA MTX or IA were added to semiconfluent monolayers and a constant amount (0.8 ,uM) of either radioof HeLa cells (3 x lit cells per flask). The cultures labeled MTX or radiolabeled folate. This conz
0
0066-4804/79/05-0730/05$02.00/0
Vol. 15, No. 5
Antifolate Activity of Isoaminopterin in HeLa Cells HORTENCIA ROSEMOND-HORNBEAK* AND M. G. NAIR Departments of Microbiology/Immunology and Biochemistry, University of South Alabama College of Medicine, Mobile, Alabama 36688
Received for publication 16 January 1979
The antifolate activity and the transport characteristics of isoaminopterin (IA) in HeLa cells were studied and compared with those of methotrexate (MTX). Both IA and MTX inhibited the incorporation of [2-'4C]deoxyuridine into HeLa cell DNA by 50% at a concentration of 0.09 LM by 8 h postaddition of the compounds. Unlike MTX, the inhibition of DNA synthesis by IA was time dependent and reached a maximum at 24 h. IA-induced inhibition was due to interference with folate metabolism, since it could be completely reversed with N5-formyl-tetrahydrofolate. Competitive transport experiments between IA and either radiolabeled MTX or radiolabeled folate showed that IA preferentially uses the reduced folate/MTX transport system. IA inhibited MTX uptake by 50% at a concentration of 6.8 ,uM but had a negligible effect on folate transport.
The use of aminopterin and methotrexate (MTX) in the treatment of neoplastic diseases is well known (1, 2, 4, 13, 14, 16). MTX is used either alone or in combination with other drugs for the treatment of leukemia (1, 14), Burkitt's lymphoma (2), choriocarcinoma (13), and psoriasis (16), as well as for the suppression of the immune response (4, 15). In spite of a documented high level of toxicity and the development of resistance (6), MTX continues to be one of the most widely used anticancer drugs. As part of a continuing program aimed at developing less toxic and more specific antifolate drugs for the treatment of different forms of cancer, we have synthesized a number of classical analogs of folic acid and aminopterin which are altered in the C9-N10 bridge region (7-11). One of these compounds, isoaminopterin (IA; N[p- { ((2,4-diamino-4-deoxy-6-pteridinyl)amino)
methyl} -benzoyl]-L-glutamic acid) (11), has been evaluated for anticancer activity in L1210 leukemic mice at The National Cancer Institute, National Institutes of Health, as described by Geran et al. (3). At the maximum dose tested (4.0 mg/kg), IA showed significant activity (% T/C 2125/s42) against L1210 leukemia in mice. In vitro studies with IA indicate that it is a very powerful antifolate. Nair et al. (11) have shown that it inhibits the growth of two folate-requiring organisms, Lactobacillus casei (ATCC 7469) and Streptococcus faecium (ATCC 8043), and is an effective inhibitor of dichloromethotrexateresistant L. casei dihydrofolate reductase. In this study the antifolate activity of IA is evaluated in a mammalian cell line in relation to (i) its ability to inhibit the incorporation of [2'4C]deoxyuridine into DNA, (ii) the effect of N5-
formyl-tetrahydrofolate (N5-formyl-THF) in reversing the induced DNA synthesis inhibition, and (iii) its transport characteristics. The antifolate activity and the transport characteristics of IA are compared to those of MTX. MATERIALS AND METHODS Materials. [:H]folic acid (47 Ci/mmol) and [:'H]MTX (250 mCi/mmol) were purchased from Amersham/Searle, and [2-'4C]deoxyuridine (58.1 mCi/ mmnol) came from New England Nuclear Corp. Folic acid and MTX were purchased from Sigma. Both folic acid and MTX were repurified by diethylaminoethylcellulose chromatography. N5-formyl-THF was purchased from Sigma Chemical Co. as the sodium salt. IA was synthesized in our laboratory as previously described (11). The chemical structures of these compounds are shown in Fig. 1. Cells. HeLa cells were vurchased from Flow Laboratories and maintained in monolayer cultures. The cells were grown in Eagle minimum essential medium supplemented with 10% newborn calf serum, 50 IU of penicillin per ml, and 50 ug of streptomycin per ml. The pH of the medium on the monolayers was maintained with a bicarbonate buffer in tightly closed flasks or Blake bottles. The cells were tested for mycoplasma contamination by Flow Laboratories. Monolayers of HeLa cells in 25- or 75-cm2 flasks used throughout these studies were prepared as follows. HeLa cell stock flasks were treated with 0.125% trypsin-ethylenediaminetetraacetic acid. The cells were pooled and suspended at a concentration of 10' cells per ml in medium containing 10% newborn calf serum. Samples of 10 and 25 ml of suspended cells were added to 25- and 75-cm2 flasks, respectively. The medium was replaced on the following day, and the cells were incubated for 48 h before use. All flasks used in an experiment were prepared simultaneously. Determination of HeLa cell DNA synthesis. Monolayers of HeLa cells were pulse-labeled with [2-
730
VOL. 15, 1979
ANTIFOLATE ACTIVITY OF ISOAMINOPTERIN
NH2 N
N
0 ~~~COOH 2C-NH-C 2OH
NH-CH (1)
CH2-CH 2-COOH
H2N
RR (2);
H
(3); R=-CH3 0 Hr1
H2 N
a
>-CH2-
i2
H
N
CHO
H2NLN
3OO
CHO N
_-
C-NH-C-H 0
COOH CH2- CH2-COOH
N° N CH2-CCH 2-NCOOH
(5)
FIG. 1. Chemical structures. (1) IA; (2) aminopter2; (3) MTX; (4) N5-formyl-THF; (5) 1O-oxaaminop-
terin. '4C]deoxyuridine (0.025 ,uCi/ml) at 37°C for 2.5 h at specific time intervals after addition of IA or MTX. At the end of each respective incubation period, the cultures were washed with ice-cold phosphatebuffered NaCI, harvested, and pelleted. The cells were suspended in 2.5 ml of distilled water. The DNA was precipitated with 0.25 N perchloric acid and collected on 0.45-,um Millipore membrane filters. The acid-precipitable counts were determined by liquid scintillation spectrophotometry. As control, the incorporation of radiolabel in untreated cells was determined. Effect of iA on the transport of [3H]folate and [3HiMTX. Monolayers of the Heba cells were washed twice with warm minimum essential medium without serum to remove most of the serum associated with the cells. The growth medium was replaced with medium without serum. A constant amount of either [3H]-folate or [3H]MTX (0.8 ,uM) and varying concentrations of IA were simultaneously added to monolayers of HeLa cells (2.2 Ax0s cells), and the cultures were incubated for 10 min at 37°C. At the end of the incubation period, the cells were washed twice with ice-cold phosphate-buffered NaCl,harvested, and solubiized in Unisol tissue solubilizer. The radioactivity associated with the cells was determined by liquid scintillation spectrophotometry.
731
As shown in Fig. 2, radiolabeled deoxyuridine is linearly incorporated into HeLa DNA for about 3 h. Therefore, a 2.5-h pulse-label period with radiolabeled deoxyuridine was used throughout this study to measure de novo DNA synthesis. The antifolate activity of IA compared to MTX was evaluated by determining the effect of the compounds on the incorporation of [2-'4C]deoxyuridine into acid-precipitable DNA. Semiconfluent monolayers of HeLa cells were incubated at 370C for varying periods of time in the presence of several concentrations of IA or MTX. At 0, 1.5, 5.5, and 21.5 h postaddition of the compounds, the cultures were pulse-labeled with [2-'4C]deoxyuridine. At the end of the incubation period, the cells were harvested, and the acid-precipitable radioactivity incorporated into DNA was determined by liquid scintillation spectrophotometry. Within 2.5 h, MTX inhibited the incorporation of [2-'4C]deoxyuridine into acid-precipitable counts by 88% at a concentration of 5 ,uM (Fig. 3A), whereas IA only produced a 45% inhibition at this concentration (Fig. 3B). However, at 24 h, MTX and IA were comparably effective in inhibiting DNA synthesis. The 50% inhibitory dose values for MTX and IA were 0.08 ,uM and 0.05 ,tM, respectively (Fig. 3). From these results it is apparent that IA is as effective as MTX in inhibiting HeLa cell DNA synthesis at 24 h postaddition of the compounds, and that IA is a slower-acting com-
pound.
RESULTS
The effect of folate analogs on DNA synthesis was monitored by measuring the incorporation of radiolabeled deoxyuridine into acid-precipitable counts. The rate of incorporation of deoxyuridine into HeLa cell DNA as a function of time was determined by incubating semiconfluent monolayers of cells in the presence of [2-w4C]deoxyubrdine for varying periods of time. At the end of the incubation period, the cultures were harvested, and the acid-precipitable radioactivity incorporated into DNA was determined.
TIME (hr.)
FIG. 2. Incorporation of [2- "C]deoxyuridine into acid-precipitable DNA as a function of time. A constant amount of [2-'4C]deoxyuridine (0.025 ,iCi/ml) was added to monolayers of HeLa cells (3 x Itt cells per flask), and the cultures were incubated at 37°C. At the indicated time intervals the cells were harvested, and the amount of acid-precipitable DNA in each flask was determined as described in the text. The results of a representative experiment are shown. Each point represents the average incorporation of duplicate cultures.
732
ROSEMOND-HORNBEAK AND NAIR
ANTIMICROB. AGENTS CHEMOTHER.
layers of HeLa cells were preincubated for 4 h at 37°C in medium containing 0.45 ,iM MTX or IA. Under these experimental conditions, DNA synthesis was inhibited by 75% and 60% in MTXand IA-treated cells, respectively. At the end of 60 the 4-h incubation period, varying concentrations of N5-formyl-THF were added to the cull tures. The cells were pulse-labeled with [2-14C]deoxyuridine at 33 h postaddition of the compounds. As controls, parallel cultures were incubated under the same experimental conditions 0: in the absence of N5-formyl-THF. As shown in V~~~~~~ Fig. 4, the inhibition of DNA synthesis induced by both IA and MTX was completely reversed by N5-formyl-THF at a concentration of 1.0 1iM. 80 These results indicate that IA-induced inhibition of HeLa cell DNA synthesis is due to interLn ference with folate metabolism. HeLa cells have different transport systems for MTX and folic acid (12). Competitive transport experiments between IA and either radio20labeled MTX or radiolabeled folate were performed to determine the transport pathway(s) 1-7 i0-8 10-6 used by IA to enter the cell. Parallel cultures of COMPOUND CONCENTRATION(M) HeLa cells were washed free of serum with miniFIG. 3. Effect of MTX and IA on HeLa cell DNA mum essential medium and incubated for 10 min synthesis. The indicated concentrations of either in the presence of varying concentrations of IA MTX or IA were added to semiconfluent monolayers and a constant amount (0.8 ,uM) of either radioof HeLa cells (3 x lit cells per flask). The cultures labeled MTX or radiolabeled folate. This conz
0
