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Vol. 1, 631-636, June 1995 Clinical Cancer Research 631

Antitumor Activity of Antifolate Inhibitors of Thymidylate and

Purine Synthesis in Human Soft Tissue Sarcoma Cell Lines

with Intrinsic Resistance to Methotrexate’

Wei-Wei Li, William P. Tong,

and Joseph R. Bertino2

The Laboratory of Molecular Pharmacology, Memorial Sloan-

Kettering Cancer Center, New York, New York 10021

ABSTRACT

We examined the antitumor effects of two antifolate

inhibitors of thymidylate synthesis, N-(5-[N-(3,4-dihydro-

2-methyl-4-oxoquinazolin-6-yhnethyl)-N-methylamino]-2-

thenoyl-L-glutamic acid (D1694; Tomudex) and 1843U89 as

well as a folate-based inhibitor of purine synthesis, 5,10-

dideazatetrahydrofolic acid (DDATHF) on human soft tissue

sarcoma cell lines having intrinsic resistance to methotrexate

(MTX) due to impaired accumulation of polyglutamates of

MTX (HS-16 and HS-42 cells) and to increased levels of dihy-drofolate reductase and thymidylate synthase activity (HS-18

cells). Growth inhibition studies showed that ED� values for

D1694 and 1843U89 after a 24-h exposure were 11-19-fold and

22-222-fold lower, respectively, than those for MTX in HT-

1080, a MTX-sensitive cell line, and the three MTX-resistant

cell lines. In contrast, DDATHF was less cytotoxic than

MTX in both the MTX-sensitive and the three resistantsarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF

was 2.5-4.5-fold higher than MTX in these sarcoma cell

lines. However, D1694 and 1843U89, unlike MTX, accumu-

late in HS-16 and HS-42 cells as polyglutamate forms, reach-

ing 70% of the total intracellular drug level after 24 h.

DDATHF polyglutamates (9.4-24%) were less in the same

cell lines. Much lower Km values for D1694 and 1843U89 as

compared to MTX for folylpolyglutamate synthase were

measured in the sarcoma cell lines, with Vmax values equal to

or slightly higher than those obtained with MTX.

D1694 and 1843U89 are significantly more cytotoxicthan MTX in intrinsically MTh-resistant sarcoma cell linesas a result of extensive formation of polyglutamates. These

two thymidylate synthase inhibitors should be evaluated in

patients with soft tissue sarcomas.

INTRODUCTION

Soft tissue sarcomas remain one of the most difficult hu-

man cancers to treat by chemotherapy. For example, MTX3 has

been shown to have only marginal activity against this disease,

although this drug is effective in the treatment of other cancers

which include acute lymphocytic leukemia, choriocarcinoma,

head and neck cancer, and breast cancer (1, 2). Impaired MTh

transport, decreased accumulation of long chain MTX polyglu-

tamates, and increased DHFR levels, are causes of resistance to

MDC in soft tissue sarcomas (3-5). Among these mechanisms,

decreased accumulation of long chain polyglutamates of MTX

was found to be the major mechanism of intrinsic resistance.

New antifolates such as trimetrexate and lO-ethyl-lO-de-

azaaminopterin are in clinical trial, based on improved antitu-

mor properties compared to MTX in experimental tumors and

some human cancers. 10-Ethyl-10-deazaaminopterin is better

transported and polyglutamylated than MTX, and trimetrexate

does not require polyglutamylation for retention (6, 7). How-

ever, these drugs also act on DHFR, and their effects on soft

tissue sarcomas are limited by toxicity or may share similar

resistant mechanisms as MTX (5, 8).

In recent years, newer antifolates including D1694,

1843U89, and DDAThF have been synthesized as inhibitors of

folate-dependent enzymes other than DHFR. The quinazoline

antifolate D1694 and benzoquinazoline antifolate 1843U89 are

inhibitors of TS (9, 10), and DDATHF inhibits de novo purine

biosynthesis by acting on GARFT (1 1). These drugs therefore

may circumvent MTX resistance due to an increased or an

altered DHFR enzyme. Also, these drugs are retained in cells

due to polyglutamate formation, a property that may enhance

cytotoxicity especially after short-term exposure to drugs (12-

14). D1694 has be shown to have significant antitumor activity

against colon and breast cancer in Phase II studies (15).

1843U89, scheduled for Phase I studies, has potent antitumor

effects versus human tumor cell lines (10). DDATHF is also

highly active against experimental tumors in vitro and in vivo

and is currently undergoing clinical trials (16). However, little

information is available concerning the cytotoxicity and mech-

anisms of action of these drugs in soft tissue sarcomas, espe-

cially in tumors with intrinsic resistance to MTX. In this article,

we demonstrate that D1694 and 1843U89 are more potent, and

DDATFIF is less potent than MTh in sarcoma cell lines intrin-

sically resistant to MTh.

Received 12/14/94; accepted 1/31/95.1 Supported by Grant P01-CA-47179 from the USPHS.

2 American Cancer Society Professor of Medicine and Pharmacology.

To whom requests for reprints should be addressed, at Laboratory ofMolecular Pharmacology, Memorial Sloan-Kettering Cancer Center,1275 York Avenue, New York, NY 10021.

3 The abbreviations used are: MTX, methotrexate; DHFR, dihydrofolate

reductase; DDATHF, 5,10-dideazatetrahydrofolic acid; TS, thymidylate

synthase; GARVF, glycinamide ribonucleotide transformylase; FPGS,

folylpolyglutamate synthase; D1694, N-(5-[N-(3,4-dihydro-2-methyl-4-

oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl-L-glutamic acid.

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632 Antitumor Activity of Antifolate Inhibitors in Cell Lines

MATERIALS AND METHODS

Chemicals. MTX was obtained from Lederle Laborato-

ries. [3H]MTX (20 Ci/pmol) was purchased from Moravek

Biochemical (Brea, CA). D1694 and [5-3HJD1694 (19.1 Ci/

pmol) were supplied by Zeneca Pharmaceuticals. D1694, in the

free acid form as a pale yellow powder, was dissolved in 50 m�i

NaHCO3, and the pH was adjusted to 9-9.5 using NaOH.

1843U89 and [3H]1843U89 (31 Ci/pmol) were gifts from Dr. R.

Ferone of Burroughs Wellcome. 1843U89 was dissolved in 10

mM NaOH and the pH adjusted to 7.0 using HC1. (6R)-

DDATHF and [‘4CJDDATHF (7.5 mCi/pmol) were supplied by

Lilly Laboratories. DDATHF was dissolved in 0.1 N NaOH, and

the pH was adjusted to 7.0. Polyglutamate standards were gifts

from Dr. R. Ferone (1843U89 + G1u1) and Dr. A. Jackman

(D1694 + G1u2 and G1u3). MTX polyglutamates (MTX +

G1u2_5) were purchased from B. Schireke Laboratories (Jona,

Switzerland). L-[2,3-3H]GlUtamic acid (NET-395, 25 Ci/pmol)

was obtained from New England Nuclear (Boston, MA). All

other chemicals were reagent grade and obtained from standard

commercial sources.

Cell Lines. HT-1080, a human fibrosarcoma cell line,

was obtained from American Type Culture Collection. HS-16

(mesenchymal chondrosarcoma), HS-18 (liposarcoma), and

HS-42 (mixed mesodermal tumor ranging from liposarcoma to

rhabdomyosarcoma) were established from untreated patients

(5). All cell lines were grown as monolayer cultures at 37#{176}Cin

a 5% CO2 humidified atmosphere in RPMI 1640 media con-

taming 10% fetal bovine serum. Doubling times for the four cell

lines were 26 h (HT-1080), 36 h (HS-16), 38 h (HS-18), and

34 h (HS-42). HT-1080 cells are relatively sensitive to MTX,

while HS-16, HS-18, and HS-42 cell lines are 16-, 10-, and

46-fold more resistant to MTX after short-term exposure (24 h)

as compared to HT-1080 cells (5).

Growth Inhibition Studies. Monolayer cells in 6-well

plates were exposed to various concentrations of antifolates. For

long-term exposure, drug exposure time was 5 days. For short-

term exposure, cells were incubated with the drug for 24 h, the

medium was aspirated, and the cells were washed twice with

cold PBS. Fresh drug-free medium was added and growth was

followed for an additional 96 h. Cells were counted using a

Model ZB Coulter Counter, and ED50 values were determined

from growth inhibition data (17).

Drug Uptake Studies. Exponentially growing cells were

harvested by centrifugation after trypsinization. Cells were sus-

pended in RPMI 1640 containing 10% fetal bovine serum at

density of i0� cells/ml and incubated at 37#{176}Cin the presence of

1 p.M [3H]D1694 (1020 cpm/pmol), [3H]1843U89 (1700 cpm/

pmol), or [‘4C]DDATHF (35 cpm/pmol), respectively. At spec-

ified times, 100-pA aliquots of cells were removed, added to 700

p.1 ice-cold 0.9% NaC1 solution and 200 p.1 9% sucrose in

microfuge vials, washed twice with cold 0.9% NaC1, boiled for

5 mm, and centrifuged for 2 mm at 15,000 X g. The radioac-

tivity in the supernatant fraction was then measured using a

liquid scintillation counter.

Analysis of Intracellular Levels of D1694, 1843U89, and

DDATHF Polyglutamates. Radiolabeled drugs (final spe-

cific activity, 680, 395, and 13.8 cpm/pmol for D1694,

1843U89, and DDATHF, respectively) were added to flasks of

exponentially growing cells to yield a 1.0 or 10 p.M concentra-

tion. At indicated times cells were harvested by scraping, cen-

trifuged (4#{176}C), and washed twice with ice-cold 0.9% NaC1

solution. The cell pellets were suspended in 500 p.1 of boiling 50

mM sodium phosphate (pH 5.5), boiled for 5 mm, and centri-

fuged at 20,000 X g for 10 mm at 4#{176}C.Supernatants were stored

at -20#{176}Cuntil analysis using HPLC. HPLC analysis of cell

extracts was performed using a modification of a previously

described method (12, 13, 18). Separations were done on an

absorbosphere C18 column (5-p.m particle size, 4.6 X 250 mm)

equipped with a 1-cm guard cartridge column of the same

material from Alltech Associates using a Spectra Physics 8800

HPLC gradient pump. A 100-pA sample was injected. For

D1694, the mobile phase consisted of 0.1 M sodium acetate

buffer (pH 5.5) and a linearly increasing gradient of acetonitrile

(4-14%) over 60 mm at a flow rate of 1 ml/min. For 1843U89,

the mobile phase consisted of 0.1 M sodium acetate buffer (pH

5.5) and a linearly increasing gradient of acetonitrile (3-30%)

over 50 mm at a flow rate of 1 ml/min. For DDATHF, the

mobile phase consisted of 0.1 M sodium acetate buffer (pH 5.5)

and a linearly increasing gradient of methanol/acetonitrile (10-

60%). Unlabeled standards were added to the samples to provide

internal controls. Radioactivity in each 1-ml fraction was mea-

sured by a Beckman LS-5801 liquid scintillation counter. The

effluent was monitored at 312, 265, and 260 nm for D1694,

1843U89, and DDATHF, respectively, by a Waters 99 photo-

diode array detector.

FPGS Activity Assay. FPGS activity in cell extracts was

determined as described by McGuire et al. (19). Each assay

contained, in a final volume of 250 p.1, 100 mr�i Tris-HC1 (pH

8.85), 10 mM AlT, 20 mM MgCl2, 20 m’vi KC1, 100 mM

�3-mercapto-ethanol, 4 mrvi [3H�glutamate (4.1 cpm/pmol), en-

zyme, and substrate (either D1694, 1843U89, or DDATHF).

After the reaction was stopped at different time points with the

addition of 1 ml cold 5 mr��t glutamate (pH 7.5) containing 25 mrvi

2-mercoptoethanol, the unreacted [3H]glutamate was separated

from the polyglutamates by DE-52 minicolumn chromatogra-

phy. Enzyme activity is expressed as the incorporation of pmol

glutamate/h/mg at 37#{176}C.

RESULTS

Inhibition of Cell Growth by D1694, 1843U89, and

DDATHF. Mid-log sarcoma cells were exposed to antifolates

for 24 h or 5 days. ED50 values for D1694 and 1843U89 after

24-h exposure were 1 1-19-fold and 22-222-fold lower than that

for MTX, respectively, in HT-1080, a MTX-sensitive cell line,

and three MTX-resistant cell lines (Table 1). In contrast, higher

ED50 values for DDAThF were observed compared to MTX in

both the MTX-sensitive HT-1080 cell line and the three resistant

cell lines. Compared with 24-h exposure, 5-day exposure to the

antifolates resulted in more marked cell growth inhibition in all

four cell lines. ED50 values for D1694 and 1843U89 were lower

than that for MTX. However, ED50 values for DDATHF were

still higher than that for MTX in all cell lines.

Uptake of ICI-D1694, 1843U89, and DDATHF. Up-

take of radiolabeled D1694, 1843U89, and DDATHF in the four

cell lines at an extracellular concentration of 1 p.M is shown in

Fig 1. Uptake of the three drugs increased in a time-dependent



Fig. 1 Uptake of MTX, ICI-D1694, 1843U89,and DDATHF in soft tissue sarcoma cell lines.Cells were exposed to 1 p.M radiolabeled drugs.At the indicated times, the cells were assayed forradiolabeled activity as described in ‘ ‘Materials

and Methods.” 0, HT-1080 cells; #{149},HS-16cells; L�, HS-18 cells; A, HS-42 cells.

(�)

C.)N.0

0

E0�

0 10 � � � SO � 70

0 10 20 30 40 50 10 70 0 10 20 30 40 50 00 70

Clinical Cancer Research 633

Table 1 Inhibitory effects of MTX, D1694, 1843U89, and DDATHF on growth of sarcoma cell lines”

ED5() (nM)

24h Sday

D1694 1843U89 DDATHF MTX D1694 1843U89 DDATHFCell line MTX

HT-1080HS-16HS-18HS-42

a Mean of tw

170270016278000

0 to three d

9.0 15.7 3250 4.8 2.3 0.169.2 42.2 >100 p.M 24.0 8.0 0.4

65.4 74.1 9150 37.8 62.8 0.897.3 36.4 >100 p.M 7.1 6.5 0.6

ifferent experiments. SE is less than 20%. See ‘ ‘Materials and Methods’ ‘ for details.

8.446.3

53.713.5

time (mm)

manner in all four cell lines. The intracellular concentrations

achieved at 60 mm were 44.9, 37.5, and 28.1 pmol/107 cells for

D1694 and 27.0, 29.7, and 31.3 pmol/107 cells for 1843U89 in

HT-1080, HS-18, and HS-42 cells, respectively, which were

2.5-4.5-fold higher than that of MTX in the same cell lines.

Uptake of both D1694 and 1843U89 was lower in HS-16 cells

(12 and 12.3 pmol/107 cells, respectively). DDATHF uptake

was more rapid and reached levels of 54.4, 27.4, 56.2, and 54.7

pmol/107 cells in HT-1080, HS-16, HS-18, and HS-42 cell

lines.

Accumulation of Intracellular Polyglutamates of

D1694, 1843U89, and DDATHF. Intracellular levels of

D1694, 1843U89, and DDATHF polyglutamates in the four cell

lines exposed to 1 or 10 p.M radiolabeled drugs for 24 h are

shown in Table 2. Intracellular D1694 and its polyglutamates

were 71.1 pmol/107 cells in HT-1080 cells and 32.8, 88.0, and

39.0 pmol/107 cells in HS-16, HS-18, and HS-42 cell lines,

respectively, after a 24-h exposure to the drug (1 p.M). Long

chain polyglutamates (Glu�6) formed were about 70-80% of

the total amount in all four cell lines, similar to the amount of




Table 2 Accumulation o f D1694, DDA THF, and 1843U89 and their polyglutamates i n four soft tissue sarcoma ell lines”

Cell line

Glutamate (pmol!107 cells)

N = 1 2 3 4 5 6 Total 3-6 (%)

ICI-D1694

HT-l080 12.1 7.1 16.5 33.1 2.3 1.1 71.1 53.0 (74.5)

HS-16 8.0 0.5 11.4 1 1.8 1.1 32.8 24.3 (74.0)

HS-18 9.3 3.7 16.3 54.8 2.8 1.1 88.0 75.0 (85.2)

HS-42 8.2 2.9 6.7 18.7 2.0 0.5 39.0 27.9 (71.5)

DDATHFHT-1080 160.6 5.9 16.6 22.2 13.4 25.3 227.4 77.5 (34.0)HS-l6 166.6 2.8 11.4 6.2 187.0 17.6 (9.4)HS-l8 117.6 8.0 17.3 12.7 7.3 3.7 166.6 41.0 (24.6)

HS-42 184.3 1 1.3 11.3 6.6 8.1 20.1 246.2 46.2 (18.7)

1843U89

HT-1080 7.8 149.2 6.1 6.7 170.7 12.8 (7.5)HS-16 12.2 31.3 43.5 0 (0)HS-18 5.8 71.9 5.9 5.7 89.3 11.6 (13.0)

HS-42 5.9 55.5 2.7 3.6 67.7 6.3 (9.4)1843U89

(4-h efflux)

HT-1080 1.4 89.0 9.0 0.5 101.6 9.5 (9.3)

HS-16 0.7 18.5 1.2 20.6 1.2 (5.9)HS-l8 1.3 26.2 8.0 5.3 40.0 13.3 (33.3)HS-42 1.2 11.6 3.2 6.1 22.1 9.3 (42.1)

MTX (10 p.M)

HT-1080 97.8 5.3 36.2 17.4 9.0 175.7 72.6 (41.1)HS-16 89.0 7.8 7.2 4.5 2.0 1 10.5 13.7 (11.9)HS-18 53.2 11.4 18.4 14.4 10.7 108.1 43.5 (40.0)HS-42 82.1 2.0 3.7 2.5 2.5 92.8 8.7 (9.4)MTX (1 p.M)”

HT-1080 23.2 9.0 11.7 8.8 3.2 59.9 27.7 (46.2)

HS-16 15.4 2.0 2.9 1.3 0.2 21.8 4.4 (20.0)HS-42 23.5 1.2 1.6 0.4 0.3 27.0 2.3 (8.5)

(‘ Logarithmically growing cells were exposed to [3H]D1694 (1.0 p.M), [3H]1843U89 (1.0 p.M), [‘4C]DDATHF (10 p.M), or [3H]MTX (10 JiM),

respectively, for 24 h and intracellular polyglutamate forms were analyzed using HPLC.

I) From Ref. 5.

Table 3 FPGS activity w ith MTX, D1694, 1843U89, and DDATHF as sub strates in abst racts for hu man soft tissue sarco ma cell lines

Activity (pmol!mg/h) Km’� (p.M)

Cell line MTX D1694 1843U89 DDATHF MTX D1694 1843U89 DDAThF

HT-l080HS-l6HS-l8

HS-42

490295415

650

387 439397 377688 370

482 421

308569315

257

18.531.910.6

51.6

0.20.20.7

0.4

0.30.30.1

0.4

2.31.41.5

1.7

“ Km values were calculated using Chou and Chou’s enzyme kinetic analysis (17). Values are means of two to three different experiments.

MTX polyglutamates formed in HT-1080 cells but 5.5- and

12-fold higher than the amount of MTX in HS-16 and HS-42

cell lines, respectively. Total amounts of DDATHF and its

polyglutamates accumulated were comparable to that of MDC

obtained in the MTX-resistant cell lines, but long chain poly-

glutamates were only 9.4-24.6% of the total amount of intra-

cellular DDATHF, even in cells exposed to 10 p.M DDATHF for

24 h. 1843U89 and its polyglutamates accumulated were 170.7,

43.5, 89.3, and 67.7 pmol/107 cells in HS-1080, HS-16, HS-18,

and HS-42 cell lines. However, the major form of 1843U89

polyglutamates was the diglutamate (more than 70% of the total

amount) in all cell lines, although some tn- and tetraglutamates

of 1843U89 were observed in HS-1080, HS-18, and HS-42

cells. In order to determine whether the diglutamate of 1843U89

was retained intracellularly, cells were incubated in drug-free

medium for an additional 4 h after exposure to 1 p.M 1843U89

for 24 h. Approximately 60%, 60%, 36%, and 21% of the

diglutamate of 1843U89 persisted in this form in HT-1080,

HS-16, HS-18, and HS-42 cells, respectively (Table 2).

FPGS Activity. FPGS activities were measured using

D1694, 1843U89, and DDATHF as substrates. As shown in

Table 3, FPGS activities using D1694, 1843U89, and DDATHF

as substrates were equal to or greater than with MTX as the

substrate. In contrast, Km values for D1694 were 0.2, 0.2, 0.7,

and 0.4 p.M for HT-1080, HS-16, HS-18, and HS-42 cell lines,

respectively; 15-185-fold lower than that for MTX. Similar Km



Clinical Cancer Research 635

values for 1843U89 comparable to those for D1694 were ob-

tamed (0.1-0.4 JiM). Km values for FPGS for DDAThF were

also lower than MTX Km values in HT-1080 and HS-16, HS-18

and HS-42 cells (8-, 23-, 6-, and 30-fold lower, respectively).

DISCUSSION

Our previous studies indicated that HS-16 and HS-42 cells

were intrinsically resistant to short-term (24-h) exposure to

MTX due to the lack of retention of this drug as a consequence

of decreased accumulation of long chain MTX polyglutamates

(5). Further studies in HS-16 cells showed that this difference

was likely due to an increased breakdown of MTX polygluta-

mates because of increased -y-glutamyl hydrolase activity (20).

The reason for low level accumulation of MTX polyglutamates

in HS-42 cells is still unclear as FPGS activity and hydrolase

activity are similar to the levels in HT-1080 cells (Table 3 and

Ref. 20). MTX resistance in HS-18 cells is associated with an

increase in DHFR enzyme activity and an increase in DHFR

mRNA levels; MTX polyglutamylation is equivalent to 1080

cells (21). The present study shows that D1694 and 1843U89

were 25-83-fold and 22-222-fold more potent inhibitors of

growth in these cell lines than MTX after a 24-h exposure to the

drugs (Table 1). To explain the more potent effects of D1694

and 1843U89, uptake and polyglutamate accumulation of these

drugs were determined. Uptake of D1694 and 1843U89 was

3-fold higher than MDC in HS-18 and HS-42 cells. Although the

uptake of D1694 and 1843U89 was similar to that of MDC in

HS-16 cells, a 6-fold higher level of long chain polyglutamates

of D1694 than that of MDC was found in this cell line, similar

to the intracellular D1694 polyglutamate levels measured in two

other MTX-resistant cell lines. Thus, higher levels of D1694,

compared to MTX, retained intracellularly as polyglutamate

forms, likely explain the increased growth inhibition noted with

this antifolate, since polyglutamates of this compound are re-

tamed longer and bind to TS enzyme more tightly than the

monoglutamate form (22). In contrast to D1694, both 1843U89

and its diglutamate form are tight binding inhibitors of TS (15).

HPLC analysis showed that the MTX-resistant sarcoma cell

lines polyglutamated 1843U89 to the diglutamate (70-80% of

total amount) or above after a 24-h incubation with this drug

(1 p,M). Substantial amounts of the diglutamate were retained in

the cells after a 4-h efflux, levels that were 180-290-fold higher

than that of the MDC diglutamate retained in the cells under the

same conditions (data not shown). Thus, unlike MDC digluta-

mate that effluxes rapidly from cells, the 1843U89 diglutamate

is slowly effluxed. DDATHF, a folate analogue GARFT inhib-

itor, is more potent than MTX against some tumor cells resistant

to MDC due to decreased transport (23). However, MTX-resis-

tant tumor cells with low FPGS activity were also resistant to

DDATHF (14, 24). In soft tissue sarcoma cell lines, higher

concentrations of DDATHF, compared with that of MTX, were

required for equivalent growth inhibition after short-term expo-

sure to this drug (24 h), especially HS-16 and HS-42. This result

was not due to a decreased uptake of DDATHF. However,

accumulation of long chain polyglutamates of DDATHF was

low (9-24% of total DDATHF). DDATHF polyglutamates are

100-fold more potent inhibitors of GARFT than DDAThF (25).

Synthesis of intracellular polyglutamates is a function of

FPGS activity (Vmax) and substrate affinity (Km) � this en-

zyme. Enzyme activity with all four drugs as substrates was

equivalent; however, Km values for D1694 and 1843U89 were

much lower compared to the Km values for MTX. Thus, exten-

sive and rapid accumulation of polyglutamates of D1694 and

1843U89 is likely due to the excellent substrate properties (Kcat)

of these drugs for FPGS. Km values for DDATHF were higher

than those for two TS inhibitors although lower than that for

MTX, explaining in part the lack of accumulation of DDATHF

polyglutamates in these cell lines.

Compared to the HT-1080 cell line, these MTX-resistant

cell lines are still relatively resistant to D1694, 1843U89, and

DDATHF (24-h exposure). Increased breakdown of antifolate

polyglutamates and efflux in HS-16 cells may explain the rel-

ative resistance to these drugs in HS-16 cells, as this cell line

contains high glutamyl hydrolase activity (20). Resistance to

D1694 and 1843U89 in HS-18 cells may be related to the

increased TS activity present in these cells, while decreased

uptake of DDATHF may result in resistance in this cell line.

Relative resistance to these drugs in the HS-42 cell line may

result from relatively low levels of polyglutamate formation.

Growth inhibition by MDC, D1694, 1843U89, and

DDATHF in all four cell lines, as expected, was time dependent.

As these cell lines have doubling times ranging from 26 to 38 h,

a longer exposure ensures that these cell cycle agents are present

during S-phase. 1843U89 was more potent than D1694 after a

5-day exposure, probably due to more potent binding of

1843U89 to TS (26). The marked increase in cytotoxicity of

DDATHF after a 5-day exposure may be mainly due to a

progressive depletion of ribonucleoside triphosphate pools by

prolonged inhibition of purine synthesis (27).

D1694 and 1843U89 are significantly more cytotoxic than

MDC in intrinsically MTX-resistant sarcoma cell lines as a

result of extensive formation of polyglutamates. These two TS

inhibitors should be evaluated in Phase II trials in soft tissue

sarcomas.

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1995;1:631-636. Clin Cancer Res W W Li, W P Tong and J R Bertino intrinsic resistance to methotrexate.purine synthesis in human soft tissue sarcoma cell lines with Antitumor activity of antifolate inhibitors of thymidylate and

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