Printed in (;=eat Britain. © 1985 Pcrgan~on Pre~s l a d .
T H E M O L E C U L A R B A S I S F O R T H E D I F F E R E N T I A L S E N S I T I V I T Y O F B A N D T L Y M P H O C Y T E S T O
G R O W T H I N H I B I T I O N B Y T H Y M I D I N E A N D 5 - F L U O R O U R A C I L
SUFIAN M. EL-ASSOULI
Department of Experimental Therapeutics, Rosweli Park Memorial Institute, New York State Department of Health, 666 Elm Street, Buffalo, NY 14206, U.S.A.
(Received 3 February 1984. Revision accepted 14 August 19841
Abstract--Cultured leukemic lymphocytes originating from patients with T, B and non-T, non-B (null) leukemia were tested for their sensitivity to thymidine and 5-fluorouracil. T cells were found to be 5-7 fold more sensitive to thymidine growth inhibition than B-cells. At 10 -3 M concentration of thymidine, T cells showed a progressive (up to 75o70) decline in the populating trypan blue- excluding cells, after 72 h. At this concentration of thymidine B cells showed slight inhibition at 24 and 48 h, then at 72 h the surviving cell level returned almost to the level of unperturbed cells.
Thymidine at 10- 5 M concentration, caused 40o70 cell growth inhibition of T cells, however, at this concentration it had little or no effect on B cells. 5-fluorouracil effects on B and T lymphocytes are opposite to that of thymidine. B cells were on an average 5-7 times more sensitive to 5-FU than T cells. 5-FU at 10 -~ M caused up to 45°70 inhibition of B-cell growth but at this concentration it had no effect on the growth of T cells. B-, T- and null-lymphocytes sensitivity to thymidine and 5-FU was correlated with the level of the catabolic enzyme thymidine phosphorylase. B cells had, on average, 5-fold more thymidine phosphorylase than T or null cells. Furthermore, the enzyme from the B-cell line (HR1K) chromatographed differently on DEAE-Sephadex than the normal peripheral blood lymphocytes enzyme. The normal enzyme from peripheral blood lymphocytes when adsorbed to DEAE-Sephadex was eluted at a salt concentration of 0.3 M KC1. Enzyme ac- tivities of HRIK did not adsorb to the DEAE-Sephadex column but were adsorbed to a phosphocelluiose column. Enzyme from normal and leukemic lymphocytes showed similar molecular weights of 130,1100 dalton as determined by gel filtration.
Key words: Thymidine phosphorylase, leukemia lymphocytes, thymidine, 5-fluorouracil.
INTRODUCTION
THE ANTIMETABOLITES dThd and 5-FU are two of the most p r o m i n e n t clinical a n t i t u m o r agents. Their use alone or in combination with other agents has produced objective responses in cancer patients, particularly those with carcinoma of the stomach, colon pancreas, breast or ovary [5, 6, 18, 25]. However, this effect is reversible after short periods of exposure and remissions once obtained are generally of short duration. Several investigators have reported that dThd is more toxic to some type of malignant cells in culture than to normal cells from the same tissue [22]. Similarly 5-FU showed differential inhibitory activites on human T- and B-lymphocytes in culture [20]. The cytotoxicity o f dThd is presumably due to disturbance in D N A synthesis through feedback inhibition of ribonucleotide reductase due to increased intracellular concentration of thymidine triphosphate. Most important is the inhibition of CDP reductase which results in depriva- tion of dCTP. The major growth-inhibitory effect of 5-FU has been associated with the
Abbreviations: LS, lymphosarcoma; ALL, acute lymphoblastic leukemia; MM, multiple myeloma; CML-BC, chronic myelocytic leukemia in blast crisis; PBL, peripheral blood lymphocytes or mononuclear cells; PE, cells in pleural effusion; null, non-T, non-B; dThd, thymidine; 5-FU, 5-fiuorouracil; 5dUMP, 5-fluoro-2- deoxyuridine monophosphate; FUTP, fluorouridine 5-triphosphate; FdUrd, 5-fluoro-2-deoxyuridine; DTT, dithiothreitol; IaMSF, phenylmethyl sulfonylfloride; CDP, cytidine diphosphate; dCTP, deoxycytidine triphosphate.
Correspondence to: S. M. EI-Assouli, Cancer Research Unit, King Fahd Medical Research Center, School of Medicine and Allied Sciences, King Abdulaziz University, P.O. Box 12653, Jeddah 21483, Saudi Arabia.
391
392 SUFIAN M. EL-ASSOULI
anabolite, FdUMP, which in the presence of N' '°-methylenetetrahydropholate binds covalently to thymidylate synthetase and inactivates the enzyme, thereby blocks DNA synthesis [14]. 5-FU, after its conversion to FUTP is also incorporated into bacterial and mammalian mRNA, rRNA and tRNA and, thereby, produce a structurally or func- tionally deficient species of RNA [4, 231. Thus the conversion of 5-FU to nucleotide derivatives is a prerequisite for its antimetabolic and antineoplastic activity as in the case with dThd which has to be converted to dTTP. The failure to perform these conversions is thought to be the reason for the resistance to the drug. Investigators have observed that resistance to 5-FU in Ehrlich ascites cells and LI210 leukemia cell lines is due in part to a decrease in uridine kinase activity [1, 21]. Another mechanism of resistance for both dThd and 5-FU is the rapid cleavage of dThd and FdUrd by thymidine phosphorylase [8].
Thymidine phosphorylases are present in many normal and neoplastic cells [2, 13, 16, 24, 26]. There are two distinct pyrimidine nucleoside phosphorylases. First is thymidine phosphorylase (E.C. 2.4.2.4; thymidine: orthophosphate deoxyribosyltransferase), which catalyze the reversible reaction: Thymidine + Pi ~- Thymine + et-deoxyribofuranose-1- phosphate and is highly specific for deoxyribonucleosides in both eukaryotes and pro- karyotes; the second enzyme is uridine phosphorylase (E.C. 2.4.2.3; uridine: ortho - phosphate ribosyltransferase) which catalyzed the reversible reaction: uridine + Pi ~ uracil + ct-D-ribofuranose-l-phosphate. Thymidine phosphorylase acts primarily on thymidine, however, uridine phosphorylase may also cleave thymidine. The substrate specificity of uridine phosphorylase is uncertain. In these studie~ we investigated sensitivity of cell lines which have been originated from patients with B- , T- and null-leukemia- lymphoma to thymidine and 5-fluorouracil and characterized these cells for the level of the catabolic enzyme thymidine phosphorylase. We also described, in these studies, the partial biochemical characteristics of thymidine phosphorylase in lymphocytes from peripheral blood of normal donors and that of HR1K cells, a B cell isolated from a Burkitt's lymphoma.
MATERIALS AND METHODS
Cell line and peripheral blood lymphocytes
Seven cell lines with B-cell characteristics, 2 celt lines with T-cell characteristics and 3 cell lines with null-cell characteristics (Table 1) were used in these studies. All these cell lines were provided by Dr. J. Minowada (Roswell Park Memorial Institute, Buffalo, N.Y.) they have been described by Minowada et al. [17]. For cytotoxicity studies, all cell lines were maintained as suspension cultures. For thymidine phosphorylase partial purification and characterization, cells were grown in spinner cultures. All cells were grown in RPMI-1640
TABLE 1.
Cell line Diagnosis Source Cell line type
RPM1 1788 Normal P B L B
H R 1 K Burkitt's T u m o r B R A J I Burkitt's T u m o r B D A U D I Burkitt's T u m o r B
U-698-M LS T u m o r B
R P M I 8392 A L L P B L B
U-266 M M P B L B
R P M I 8402 A L L PBL T
M O L T 4 A L L P B L T
N A L M - I C M L - B C P B L Non-T, Non-B
K-562 C M L - B C PE Non-T, Non-B
KG-1 A L L P B L Non-T, Non-B
Sensitivity of B and T lymphocytes to dThd and 5-FU 393 medium (GIBCO, Grand Island, N.Y.) supplemented with 10070 heat inactivated fetal calf serum (GIBCO). The average generation time of cells under these conditions was 18 h. Peripheral blood lymphocytes were prepared with FicolI-Hypaque centrifugation (Pharmacia Fine Chemicals, N.J.) [3] from huffy-coat of normal human volunteers.
Thymidine and 5-fluorouracil cytoxicity
For these studies, cells in log phase growth in suspension cultures were diluted with fresh medium to 4 x 105 cell mi-' and were allowed to grow for 24 h, after that thymidine or 5-fluorouracil were added. All cultures were run in duplicate. A|iquots from each flask were removed on day 1, 2 and 3 for counting of total cells and viable cells (cell excluding 0.4% trypan blue dye). The drug concentrations that produced 50°70 inhibition of cell growth (ID50) were determined by plotting the number of viable cells on day 3 as a percent of control (cell which had not been treated with any drug), against drug concentrations.
Cell homogenate
All operations were carried out at 4°C. Cells were diluted in 6 vol of buffer A (20 mM KPO,; 1 mM DTT; 1 mM PMSF and 10% glycerol, pH 8.0) and homogenized in Dounce homogenizer with a tight fitting pestle. The homogenate was centrifuged at 39,100 × g for 1 h. The supernatant was dialysed overnight against 100 vol of buffer A to give fraction I.
Thymidine phosphorylase assay
Thymidine phosphorylase was routinely measured spectrophotometrically by a modification of Friedkin procedure [12l. The method is based on measuring the increase in absorbance at 300 nm resulting from the conversion of thymidine to thymine. The incubation medium contained l0 mM thymidine; 30 mM KPO,; pH 7;
0.3 mM DTT in a final volume of 0.3 ml and varying amounts of the enzyme preparation. The reactions were started by the addition of thymidine and tubes were incubated at 37°C for 30 rain, unless specified otherwise.
Reactions were stopped by incubating the tubes on ice and addition to each tube of 0.5 ml of ice cold 3070 perchloric acid. Tubes were spun down at 5000 rpm for 5 rain in JA-20 rotor and the supernatant was made alkaline by adding 2 ml of 0.3 N NaOH to each tube. Optical densities were measured at 300 nm against the control which was a reaction mixture stopped at 0 time without thymidine. All assays were done in duplicate.
One micromole of thymine formed from thymidine causes an increase of 3.6 in absorbance at 300 nm. The unit of enzyme was defined as the amount that catalyses the formation of one micromole of free thymine per hour at 37°C.
Ion exchange chromatography, hydroxylapatite and gel filtration
DEAE-Sephadex (G-25) was swollen in buffer B (10 mM KPO,; 1 mM EDTA; 1 mM DTT; 10% glycerol pH 8.0), packed in 5 × 21 cm column and equilibrated with the same buffer. Fraction 1 was loaded on the column. The column was washed with 2 bed vol of buffer B then eluted with 500 ml of 0-0.5 M KCI gradient in buffer B. Fractions which contained thymidine phosphorylase activity were pooled and dialysed against 100 vol of buffer B to give fraction II.
Fraction II was loaded on a phosphocellulose ( P l l ) column (5 x 21 cm) washed, eluted in the same way as DEAE-Sephadex column. Fractions from phosphocellulose which contained thyrnidine phosphorylase activity were dialysed against 100 vol of buffer C (20 mM KPO,; 1 mM DTT; 1007o glycerol pH 7.0) to give fraction IIl.
Fraction Ill was loaded on hydroxylapatite column (2.5 x 10 cm), which was equilibrated and washed with buffer C. Proteins were eluted from the column by 200 ml of 0-0.7 M KCI gradient in buffer C. Fractions containing thymidine phosphorylase activity were pooled, concentrated against 30% polyethylene glycol 20,000 in buffer D (20 mM KPO,; 1 mM DTT and 0.3 M KCI), Fraction of the concentrated enzyme was loaded on Sephadex G-200 column (2.5 × 93 cm) which was prepared and calibrated as described [10], column was eluted with buffer D.
R E S U L T S Thymidine sensitivity o f B, T and null lymphocytes
T w o B - c e l l l i n e s ( H R 1 K a n d R P M I 8 3 9 2 ) ; o n e T - c e l l l i n e ( R P M I 8 4 0 2 ) a n d t w o n u l l - c e l l l i n e s ( K G - I a n d K - 5 6 2 ) w e r e t e s t e d f o r t h e i r s e n s i t i v i t y t o t h y m i d i n e a t t h r e e d i f f e r e n t c o n - c e n t r a t i o n s l 0 -s, 10 -4 a n d 10 -3 M o v e r a p e r i o d o f 7 2 h . F i g u r e 1 s h o w s t h e f r a c t i o n a l s u r - v i v a l o f c e l l s a t 7 2 h a s a f u n c t i o n o f t h y m i d i n e c o n c e n t r a t i o n c o m p a r e d w i t h t h e m e a n s u r v i v a l o f c e l l s w i t h o u t t h y m i d i n e . B - c e l l l i n e s w e r e s i g n i f i c a n t l y less s e n s i t i v e t o l 0 -3 M t h y m i d i n e , m e a n w h i l e a t t h e s a m e c o n c e n t r a t i o n b o t h T - a n d n u l l - c e U l i n e s w e r e h i g h l y s e n s i t i v e . T h e a v e r a g e t h y m i d i n e I D 5 0 ' s f o r T a n d n u l l c e l l s w e r e 0 . 3 r a M . H R 1 K ( B - cell) s h o w e d l i t t l e o r n o s e n s i t i v i t y e v e n a t 1 m M t h y m i d i n e a n d R P M I 8 3 9 2 ( B - cell) s h o w e d o n l y 30~70 r e d u c t i o n i n cell v i a b i l i t y ( F i g . 1).
T h e e x t e n t o f s e n s i t i v i t y o f t h e T - a n d n u l l - c e l l l i n e s w e r e d i r e c t l y p r o p o r t i o n a l t o t h e t h y m i d i n e c o n c e n t r a t i o n a f t e r l , 2 o r 3 d a y s o f a d d i n g t h e d r u g ( F i g s 1 a n d 2), h o w e v e r , t h i s w a s n o t t h e c a s e w i t h B - c e l l l i n e s . W h e n t h y m i d i n e s e n s i t i v i t y i n t h e B-, T - a n d n u l l -
394 SUFIAN M. EL-ASSOULI
~oo _"
" [ ~ , ~ K\
~_~ 6 o ~
I
, , ~
e.6 I \";,PM,8,o2
2o I %K- 62
nl
1 ["ixt6 ~
| x fO T M ! x16 3 IThymidine (M)
FIG. 1. Growth inhibition of B cells (HR1K and RPMI 8392), T cell (RPMI 8402) and null cells (KG-I and K-562) by varying amounts of thymidine.
cell lines were followed over a 3-day period (Fig. 2). On the first day, thymidine had little effect on the B-cell line ( R P M I 8392), reduction in cell viability was only 10070. However, thymidine cytotoxicity on the B-cell line ( H R I K ) , T-cell line (RPMI 8402) and the null-cell lines (K-562) were more dramatic in the first day and the reduction in cell viability was 30-50%. On the third day o f thymidine treatment the B-cell lines had returned almost to normal and showed 100% viability. T- and null-cell line viability on the third day was significantly reduced to 30% in RPMI 8402 and 10% in K-562. The molecular understan- ding o f the B-cell line adjustment to the effect o f thymidine is not clear at this point.
However, it is worth noticing that this adjustment is taking place only in the B-cell lines and not the T- or null-cell lines.
HRt K
cJ~ 80 I
N ,8 6o
0'~ 40
RPMI 8402
Z 20
K-562
0 I I I
! 2 5
Doy$ ( T h y m i d i n e |xt(~3M)
FIG. 2. Thymidine (10-3M) cytotoxicity as a function o f time to B ( H R ] K , R P M I 8392), T ( R P M I 8402) and null (K-562) ceils.
Thymidine phosphorylase level
Fraction I o f four B-cell lines (RPMI 1788, Raji, Daudi and U-698-M), one T-cell line
(MOLT-4), one null cell line ( N A L M - I ) and the peripheral blood lymphocyte were
assayed for its content o f the catabolic enzyme thymidine phosphorylase. Figure 3 shows
that the mean activity of thymidine phosphorylase was on the average 4-5 fold greater in
the B-cell lines which is less sensitive to thymidine than in the T, null or PBL.
Sensitivity of B and T lyrnphocytes to dThd and 5-FU
395
"• 2.2-
2.0
= q s
i 1 . 6 - t.44.2
,.o
l l
~ 0.8
!
0.4
".~- 0.2 . I =
~ o ~ ~, ~
o
m o -J-3 ? :e ,~
a. ~ z
c~
FIG. 3. The specific activities (units/mg protein) of thymidine phosphorylase in B (RPMI 1788, Raji, U-698-M); T (MOLT 4); PBL and null (NALM-1) cells.
5-Fluorouracil sensitivity
In contrast to thymidine, 5-FU showed to be more cytotoxic to B-cell line than T-ceU line. The median ID 50's for B-cells (U-266 and RPMI 8392) were 2.5 pM and for null cells (NALM-1 and K-562) were 4.25 pM, however, for T-cell lines (MOLT-4 and R P M I
8402) it was 13 pM (Fig. 4). 5-FU was effective at a 2--4 pM concentrations and the variable sensitivities of B-, T- and null-cell lines are not detected at a high concentration of 5-FU. At 2 x 10-' M, 5-FU seem to have similar cytotoxicity on B-, T- and null-cell lines.
2018 t B-Cells ,U. t 4 - t6
¢')
t 2 ~- M~i,=,~ t O F - zs~'a
6 4
' l l
0
el IZ
T-Cells
o. oc
Non-T, Non-B
Mldio~
4.~FM
1
Z
FIG. 4. 5-fluorouracil as a growth inhibitor of B (U-266, RPMI 8392), T (MOLT 4, RPMI 8402) and null (NALM-i, K-562) cells.
Physical properties of PBL thymidine phosphorylase
Fraction I prepared from P B L o f healthy volunteers was chromatographed on
D E A E - S e p h a d e x (G-25). Proteins were eluted from the first D E A E - S e p h a d e x and Frac-
tions which contained thymidine phosphorylase activities were pooled, dialysed and loaded
on a second D E A E - S e p h a d e x column. From the chromatogram o f the first and the second
column (Fig. 5a) there was only one thymidine phosphorylase peak which was eluted at
396 SUFIAN M. EL-ASSOULI
0.3 M KCI. Fractions which contained thymidine phosphorylase activities of the second DEAE-Sephadex column were concentrated by dialysis against PEG 20,000 and chromatographed on Sephadex G-200. The calibrated molecular weight for protein in the fractions contained the highest activity o f thymidine phosphorylase was found to be 125,000-130,000
Physical properties of ttR1K thymidine phosphorylase
Fraction I prepared from HRIK cells was chromatographed on DEAE-Sephadex column.
All thymidine phosphorylased activities were found in the flow-through fraction. When the column was eluted with 0-0.5 M KCI gradient, no further thymidine phosphorylase activity was released from the column. The flow through thymidine phosphorylase activity was loaded on phosphocellulose column ( P l l ) . Thymidine phosphorylase activity was bound and eluted from the column at 0.28 M KCI (Fig. 5b). Thymidine phosphorylase ac- tivity eluted from phosphocellulose was dialysed, chromatographed on hydroxylapetite column as described in Materials and Methods. One peak o f thymidine phosphorylase ac- tivity was eluted from the column at 0.3 M KCl. This activity when subjected to sephadex G-200 gel filtration was found to have an estimated mol. wt of approx. 120-130,000.
DEAE-~ PBL
3 -
.-- ~ 0 . 5
2 - 0.4
,'~o
D
14 . . . . b
12
~ o .
1 I
,.-~l°s
8 i L.-- -io.4
4 / t / 0.2
0 - ~ T - ' - - - ~ ' : ' J - " . . . " * " ' J I , " ~ = i 4 8 t2 46 20 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2
Fraction Number
Eic. 5. (a) Second DEAE-Sephadex chromatogram of the peripheral blood lymphocyte fraction I.
(b) Phosphocellulose (P! 1) chromatogram of HR1K thymidine phosphorylase. Activities from DEAE-Sephadex flow through, were pooled and subjected to phosphocellulose (P1])
chromatography.
DISCUSSION
In these studies we reported the effect of two chemotherapeutic agents, thymidine and
5-fluorouracil on the survival and growth of the human leukemia-lymphoma cell lines
listed in Table 1. These two chemotherapeutic agents exert differential inhibitory activities
against the human T and B lymphocytes in cultures. All T cells tested were more sensitive
to thymidine than B cells and all B cells tested were more sensitive to 5-fluorouracil than T
cells. Null cells showed uniform sensitivity to both agents.
Sensitivity of B and T lymphocytes to dThd and 5-FU 397 The basis for this differential sensitivity of T and B cells to thymidine and 5-fluoro- uracil is unknown. In our studies we tried to correlate the sensitivity to thymidine and 5-fluoro- uracil with the level of the catabolic enzyme thymidine phosphorylase in T and B cells.
The level of this enzyme was found to be higher in the more resistant B ceils than in the more sensitive T cells. This finding is in agreement with other studies by Fox et al. [11].
This difference in thymidine phosphorylase level may explain in part the resistance of B cells to thymidine, since the high level of thymidine phosphorylase will rapidly degrade thymidine to thymine which has no cytotoxic effect.
Thymidine phosphorylase also modulates the sensitivities of T and B cells to 5-fluoro- uracil making B cells more sensitive than T cells to 5-fluorouracil. B cells will generate more deoxyribose 1-phosphate than the T cells, consequently the intercoversion of 5-fluro- uracil to 5-fluorodeoxyuridine (FdUrd) which is the actual cytotoxic metabolite is more rapid in B cells.
The resistance of B cells to thymidine was developed upon exposure of these cells to 10 -3 M thymidine. B cells (HRIK and RPMI 8392) were sensitive the first and second day of exposure to thymidine, however, on the third day of exposure both cell lines HRIK and RPMI 8392 became very resistant and had viability similar to the untreated cells. Under the same conditions T (RPMI 8402) and null (K-562) cells showed increased sensitivities with time, up to the third day of exposure. The resistance to thymidine, as is clear from these studies, was developed through some adjustment mechanism which seems characteristic of B cells, but not of T or null cells. These adjustments could occur through induction of thymidine phosphorylase or through the depression of thymidine kinase, both mechanisms will result in a reduction of dTTP level which is the actual inhibitor of ribonucleotide reductase. We have not so far tested, for the level of thymidine phosphory- lase or thymidine kinase at different times during exposure to thymidine. Since, not all B cells tested had Epstein-Barr virus, it is unlikely that the virus is involved in the mechanism of resistance. It seems more likely that the observed difference in inhibitory effects of thymidine and 5-fluorouracil are biochemical differences inherent in the B and T cells.
The superior inhibitory effects of thymidine on T-cells culture are consistent with the therapeutic effects of this agent on acute lymphocytic leukemia, which is comprised largely of T- and null-cells phenotypes. Also the better activity of 5-fluorouracil on B-cell lines is consistent wiht its effectiveness of lymphoma patients [7, 9, 15, 19].
Thymidine phosphorylase from the thymidine resistant B-cell line (HRIK) chromato- graphed differently from thymidine phosphorylase of normal peripheral blood lymphocytes.
The peripheral blood lymphocytes enzyme of normal donors was adsorbed to DEAE- Sephadex column and was eluted from the column at 0.28 M KCI. However, the HR1K thymidine phosphorylase enzyme when loaded on similar DEAE-Sephadex column was not adsorbed and was found in the flow through fraction.
The enzyme activity unbound to DEAE-Sephadex was adsorbed to phosphocellulose (PI 1) column and was eluted from the column at 0.3 M KC1. Although T lymphocytes comprise 80°7o of the PBL lymphocytes, it is not clear at this point whether the PBL en- zyme activities was contributed by T or B lymphocytes. At any case, it is of interest to find that thymidine phosphorylase of HRIK and PBL are widely different in their ionic charge. This difference may play a role in the regulation of the enzyme activities. One possibility of this regulation could have been acquired through phosphorylation and dephosphorylation of the enzyme; this will enable the enzyme to serve a regulatory func- tion.
Better understanding of pyrimidine deoxyribonucleoside phosphorylases is essential for developing a class of pyrimidine deoxyribonucleoside analogs that are not substrates for thymidine phosphorylase and could be of potential therapeutic values.
Acknowledgements--The
author wishes to thank B. Sahai, G. Anderson and R. Hajella for their critical
review of this manuscript.
398 SUFIAN M. EL-ASsoULI
REFERENCES
I. ARDALAN B., MACDONALD J. S., COONEY D. &: SCHEIN P. (1979) Studies on mechanism of 5-fluorouracil resistance in murine and human tumors. Bull. Cancer, Paris 66, 55.
2. BLANK J. G. & HOFFEE P. A. (1975) Purification and properties of thymidine phosphorylase from Salmonella typhimurium. Archs Biochem. Biophys. 165, 259.
3. BOYUM A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. din. Lab. Invest. Supl.
97, 1.
4. CARRICO C. K. & GLAZER R. I. (1979) Augmentation by thymidine of the incorporation and distribution of 5-fluorouracil in ribosomal RNA. Biochem. biophys. Res. Commun. 87, 664.
5. CARTER S. K. (1972) Single and combination nonhormonal chemotherapy in breast cancer. Cancer 30, 1543.
6. CARTER S. K. (1976) Cancer treatment today and its impact on drug development, with special emphasis on phase. II. Clinical trial. J. hath. Cancer Inst. 57, 235.
7. CENTRY G. A., MORSE P. A. JR. & POTTER V. R. (1965) Pyrimidine metabolism in tissue culture cell derived from rat hepatoma. Cancer Res. 25, 517.
8. CHAUDHURI N. K., MUKHERJI~E K. L. & HEIDELaERGER C. (1958) Studies on fluorinated pyrimidines. VII.
The degredature pathway. Biochem. Pharmac. 1,328.
9. CLEAVER J. E. (1967) ThymidineMetabolism and CellKinetics, p. 43. John Wiley, New York.
10. ELASSOULI S. M. & MISHRA N. C. (1980) Neurospora crassa DNA polymerase: biochemical characterization, molecular heterogeneity and their biological roles. FEMS Microbiology 13, 181.
11. Fox R. M., PIDDINOTON S. K. &PtPER A. A. (1979) Thymidine phosphorylase deficiency in cultured human T-leukemic lymphocytes. Proc. Am. Ass. Cancer Res. 20, 262.
12. FRIEDKIN M. & DEWAYNE R. (1954) The enzymatic synthesis of nucleosides. 1. Thymidine phosphorylase in mammalian tissue. J. biol. Chem. 207, 245.
13. FRIEDKIN M. & KALCKAR H. (1961) Nucleoside phosphorylases. In The Enzymes, (BoYER P. E., LARDY H. &
MYRBACK D., Eds.), 2nd Edn., Vol. 5, p. 237. Academic Press, New York.
14. KLUBES P., CONNELLY K., CERNA 1. & MANDEL H. G" (1978) Effect of 5-fluorouracil on 5-fluorodeoxyuridine-5-monophosphate and 2-deoxyurdine 5-monophosphate pools and DNA synthesis in solid mouse LI210 and rat Walker 256 tumor. Cancer Res. 38, 3225.
15. LEE S. S., GIOVANELLA B. C. & STEHLIN J. S. (1977) Selective lethal effect of thymidine on human and mouse tumor cell. J. Cell Physiol. 92, 401.
16. MARSH J. C. & PERRY S. (1964) Thymidine catabolism by normal and leukemic human leukocytes. J. clin.
Invest. 43,267.
17. MINOWADA J., JANOSSY G., GREAVES M., TSUBOTA T., SRIVASTAVA B. I., MORIKAWA S. & TATSUMI E.
(1978) Expression of an antigen associated with acute lymphoblastic leukemia in human leukemia-lymphoma cell lines. J. natn. Cancer Inst. 60, 1269.
18. Moertel C. G. 0975) Clinical management of advanced gastrointestinal cancer. Cancer 36, 657.
19. MORSE P. A. JR. & POTTER V. R. 0956) Pyrimidine metabolism in tissue culture cells derived from rat hepatoma. Cancer Res. 25, 499.
20. OHNUMA T., ARKIN H., MINOWADA J. & HOLLAND J. (1978) Differential chemotherapeutic susceptibility of human T-lymphocytes and B-iymphocytes in culture. J. natn. Cancer Inst. 60, 749.
21. REICHARD P., SKLOD O. & KLEIN G. (1959) Possible enzymatic mechanism for the development of resistance against fluorouracil in ascites tumors. Nature, Lond. 183, 939.
22. Tsunol K. K. & K. WONG L. K. (1978) Antiproliferative agents and differential survival between normal and cancer cells. Cancer Res. 38, 3745.
23. WILKINSON D. S., TESTY T. D. & HAMES R. J. (1975) The inhibition of ribosomal RNA synthesis and maturation in Novikoff hepatoma cells by 5-fluorouridine. Cancer Res. 35, 3014.
24. WOODMAN P. W. (1979) Thymidine phosphorylase activity in plasma: a cancer marker or an artifact of ultrafiltration. Proc. Soc. exp. BioL Med. 162, 175.
25. YOUNG R. C., HUBBARD S. P. & DEVITA V. T. (1974) The chemotherapy of ovarian carcinoma. Cancer Treat. Rev. 1, 99.
26. ZIMMERMAN M. & SEIDENBERG J. (1964) Deoxyribosyl transfer. 1. Thymidine phosphorylase and nucleoside deoxyribosyl transferase in normal and malignant tissues. J. biol. Chem. 239, 2618.
