Biochimica et Biophysica Acta, 11)94 ( 1991)263-268
263
© 1991 ElsevierScience PublishersB.V. All rightsreserved0167-4889/91/$03.50 ADONIS 016748899100242K
BBAMCR 13015
Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells Shuji N a k a n o l, T a t s u h i k o K o g a J, l c h i r o l c h i n o s e ~, H i d e n o r i Y a m a d a 2 and Yoshiyuki Niho t First Department of Internal Medic;he. Faculty of Medicine. Kyushu Unit't'~vitv Ftlkuoka (Japan) and 2 Faculty of Pharmaceutical Science, Kyushu Unicersity. Fukuoka (Japan)
(l'.cceived 4 February 1991) (Revised r~lanuscriptreceived 13June 199I) Keywords: Thymidine:Density-inhibition:Nuclcotide: Degradation Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell lille. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient ( T K - ) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK - or TK-proficie:~, (TK +) cells was examined. As a result, the growth of T K - cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK + cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the superaatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in conflueni culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant ~anvth in the presence of certain growth inhibitors produced by density-inhibited V79 cells. Introduction
Proliferative potential of normal cells in vitro is stringently controlled by two major mechanisms, i.e., direct cell-to-cell interaction and soluble factors released by the cells [1-4]. In contrast, neoplastic cells escape from such growth regulations as a result of an aberrant growth control mechanism [5,6]. However, even neoplastic ceils, when saturated in cell density, show a depression of growth potential. Such densitydependent growth inhibition in tumor cells is not likely to be due to cell-to-cell interaction, but rather to the
Abbreviations:FCS,fetal calfserum;PBS,phosphate-bufferedsaline: TFA, trifluoroacetic acid; FPLC; fast protein liquidchromatography; HPLC. high-performanceliquid chromatography;dThd, thymidine; dTMP, thymidine monophosphate;dTDP. thymidine diphosphate; d'ITP, Ihymidinetriohosphate;cAMP, cyclic adenosine 3'.5'-monophosphate;TK. thymidinekinase. Correspondence:S. Nakano, First Departmentof Internal Medicine, Faculty of Medicine, Kyushu UniversiW,3-1-1 Maidashi. Fukuoka 812, Japan.
soluble inhibitor shed into the medium, since frequent medium replacement induces a substantial proliferation of tumor cells, resulting in a piling-up phenomenon of cellular growth. Therefore, the possibility remains that soluble inhibitory factors may be important in the density-dependent regulation of neoplastic cells. This is particularly important in considering the possibility of growth regulation of cancer cells by such inhibitory factors. Recently, we found that V79 Chinese hamster cells, which proliferate vigorously in a pile up fashion in vitro, come to a drastic arrest in growth if medium replacement is delayed over the critical period. We found in the supernatant a potent growth inhibitory activity which reversibly inhibits the cellular growth and DNA synthesis [7,8]. This inhibitory activity is apparently mediated by soluble inhibitory factors produced endogenously from density-inhibited V79 cells, and two molecular weight classes of growth inhibitory activities have been identified, with the major activity in low-molecular-weight fractions ranging 500-2000 [9]. This partially purified inhibitor was further separated into two distinct inhibitory activities. One of these was
264 already purified to apparent homogeneity and proved to be an oligopeptide inhibitor acting on a wide variety of normal and malignant cells [10]. We describe herein the purification of another low-molecular-weight growth inhibitor, which was finally demonstrated to be thymJdine (dThd). The biological study suggested that growth-inhibitory level of dThd would he accumulated in the confluent culture of V79 cells. The mechanism of dThd accumulation at density-inhibited state is discussed. Materials and Methods
Cell cultures and preparation of conditioned medium Cells were grown at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air in Dulbecco's modified Eagle's medium (Nissui Pharm., Tokyo) supplemented with 5% heat inactivated fetal calf serum (FCS) (Gibco Laboratories, Chagrin Falls, OH). V79 cells, established from Chinese hamster lung fibroblasts, were demonstrated to produce fibrosarcoma when injected into athymic nude mice at a dose of 1 • 107. Thymidine kinase (TK) deficient V79 mutant clones were obtained by mutagenizing V79 cells with 10 mM N-methyl-N'n~tro-N-nitrosoguanidine (Sigma, St. Louis, MO) and exposing them to 2 mM dThd after expression time for 3 days. Actively growing colonies were clonally isolated and propagated in medium containing 2 mM dThd. These TK-deficient mutant cells had lost TK activity, as evidenced by the lack of growth in HAT medium containing 1-10 -4 M hypoxanthine, 4 . 1 0 -7 M aminopterin, 1.6 • 10 -5 M dThd. The lack of TK activity was also demonstrated by an extremely low level of the incorporation of [3H]dThd into cellular DNA. A subclone of Balb/c 3T3 A31 cells [11], was used as the target cells for measurement of the inhibitory activity of the fractionated samples.
Preparation of supematants of density-inhibited V79 cells Since we found that the growth inhibitory activity of the supernatants was maximal at the early period of density-inhibited state [9], supernatants were prepared by exposing 10 ml of growth medium for 24 h to the cultures (100 ram, Falcon, Oxnard, CA) immediately after confluence as previously described [9,10].
Chemicals Thymine, thymidine, thymidine monophosphate (dTMP), thymidine triphosphate (dTTP) and cyclic AMP (cAMP) were purchased from Sigma, St. Louis, Mo.
Reverse-phase chromatography The low-molecular-weight growth inhibitory fractions ranging between 500-2000, obtained from Sephadex G-25 gel filtration chromatography of a 30 ml 20-fold concentrate of supernatants [9], were corn-
bined and lyophilized. The redissolved residue with one twentieth volume of distilled water was applied to a reverse-phase Lobar column chromatography (Lichroprep RP-18, Merck). Chromatography was carried cut with a Pharmacia fast protein liquid chromatography (FPLC) equipment, using distilled water containing 0.1% trifluoroaeetie acid (TFA). The column eluate was monitored with a fluorescent stream detector set at 240 nm with a flow rate of 1.0 ml/min. Approx. 70 fractions (10 ml) were collected by the fraction collector (FRAC-100, Pharmacia) and 1 ml of each fraction was lyophilized, resuspended in growth medium, and assayed for growth inhibitory activity.
Reverse-phase and gel filtration HPLC The more hydrophobic growth inhibitory fractions eluting at 8% acetonitrile concentration (pool B) on reverse-phase Lobar column chromatography (Fig. 1) were pooled, lyophilized, resuspended in 0.5 ml of distilled water and injected on a TSK gel ODS 120A reverse-phase column (Toyosoda, Tokyo), using the Hitachi HPLC system equipped with variable-wavelength detectors. The sample was chromatographed using a 2% isoeratie gradient in 0.1% TFA at a flow rate of 0.8 ml per min, and the effluent was monitored at 210 nm. Impurities included in a single fraction containing inhibitory activity from a reverse-phase HPLC were eliminated by repeating the same chromatography. The inhibitory peak was lyophilized, resuspended in 100 /.d of 0.1% TFA, applied to a gel filtration column (0.8 × 50 era) of Cellulofine GCL-25M (Seikagaku Kogyo, Tokyo) using a Hitachi HPLC system, equilibrated and eluted with 0.1% TFA at a flow rate of 0.6 ml per ml at room temperature. The column eluate was monitored with a UV detector set at 210 nm. Each peak was lyophilized, resuspended in growth medium and assayed for growth inhibitory activity.
Assay of growth inhibitory activity The growth inhibitory activities of fraetionated materials were determined by the measurement of cell growth and DNA synthesis in 3T3 cells, as described [9,10]. Briefy, 2.104 3T3 cells were inoculated into replicate plates (35 mm, Falcon) and incubated for 18 h for attachment. Medium was then replaced with growth medium containing each lyophilized and resuspended sample, and incubation was continued for an additional 2 days. Cell were trypsinized and counted on a Coulter Counter (ID).
Stability of the inhibitor Aliqunts from the inhibitor purified by gel filtration HPLC were tested for their sensitivity to trypsin and heat, as described [10]. Hydrolysis was performed by incubating an aliquot in 6 M HCI at 110°C for 120 rain.
Results
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Purification of the inhibitory acti~'ity T h e low-molecular-weight fractions obtained by gel filtration c h r o m a t o g r a p h y of a c o n c e n t r a t e d supern a t a n t were fractionated over a reverse-phase L o b a r column c h r o m a t o g r a p h y . As shown in Fig. 1, three distinct inhibitory p e a k s a p p e a r e d . Since the first peak eluting at a 0 % acetonitrile c o n c e n t r a t i o n contained e n o r m o u s a m o u n t s of salts, the second a n d the third inhibitory peaks were considered to be inhibitors, a n d designated as pool A a n d pool B, respectively. Pool A has b e e n subsequently purified to homogeneity a n d f o u n d to be a n oligopeptide inhibitor with its molecular weight 2000 [10]. T h e m o r e hydrophobic inhibitor (pool B) eluting at a 8 % acetonitrile (fraction 4 0 - 5 5 ) reversibly inhibited growth of the 3T3 cells (data not shown). T h e pool B was f u r t h e r purified in a process including reverse-phase H P L C a n d gel filtration H P L C . W h e n pool B was c h r o m a t o g r a p h e d using a 2% isocratic elution of acetonitrile, growth inhibitory activity was detected in a single p e a k (Fig. 2a), a n d this single p e a k was purified to homogeneity by gel filtration H P L C as a single s h a r p p e a k (Fig. 2b). W h e n unconditioned m e d i u m was similarly processed for a series of c h r o m a t o g r a p h y , n o significant p e a k s a p p e a r e d .
Physicochemical analysis of purified inhibitor T h e purified inhibitor was heat-stable a n d trypsinresistant but was inactivated by hydrolysis ( d a t a not shown). Both the native a n d hyctrolyzed purified inhibitors h a d a p e a k ultraviolet a b s o r b a n c e at 260 nm, suggesting a nucleic acid composition in the primary structure.
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~'ltJtion t i m e Fig. 2. (a) Reverse-phase HPLC of partially purified inhibitor (pool B). Pool B (Fig. 1), was lyophilized, resuspcnded in 0.5 ml of distilled water and subjected to reverse-phase HPLC. The sample was chromatographed as describ-d in Materials and Methods. Fraction aliquots were lyophilizcd, resuspended in growth medium and tested for inhibitory activity on cell growth in 3T3 cells. (b) Gel filtration HPLC of the inhibitory peak fraction from reverse-phase HPLC. Each peak was lyophilized, resuspended and tested for inhibitory activity on DNA synthesis.
Biological actit,ity of the purified inhibitor The growth inhibitory activity of the purified inhibitor was c o m p a r e d with the activity of synthetic inhibitory nucleoside analogues, including d T h d , d T M P a n d c A M P , since these c o m p o u n d s have an antiproliferative activity [12-15]. T h e purified inhibitor restricted growth activity to much the same extent as seen with d T h d a n d h a d a slightly higher activity t h a n d T M P or c A M P (Table I).
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Fig. 1. Reverse-phase Lobar column chromatography of concentrated low-molecular-weight growth inhibitory fractions obtained from Sephadex G-25 gel filtration chromatography of the supernatants. The ¢lution sequence of aeetonitrile was 0 to 15% stepwise gradient for 600 mL One fifth volume of each fraction was lyophilized, resuspended in growth medium and tested for growth inhibitory activity on sparse, proliferating culture of 3T3 cells, as described in Materials and Methods.
Comparison of tile growth ildlibitoo' acticity of tile purified inhibitor with activities of synthetic nucleosideanalogues Each substance was dissolved with PBS into the concentration showing approximately the same ultraviolet absorbance at 260 nm (A2sj) as that of the purified inhibitor, and 40 u.I of each solution was added into 2 ml of culture medium of the assay of growth inhibitory activity. Data are expressed as the percentage ±S.D. of triplicate plates. Treatment Control medium Purified inhibitor Thymidine dTMP cAMP
A zsl 1.46 1.35 1.54 1.46
Percent cell growth 100±5 84 + 2 83 ± 4 95 ± 9 91 ± I
266 Comparison by elution pattern In order to distinguish the inhibitor from other nucleic acid compounds, elution patterns on reversephase HPLC were compared. As shown in Fig. 3, the inhibitor differed from cAMP, dTMP and dTI'P, but the elution patterns of native and hydrolyzed inhibitors were identical and coinciden~-~;~'d~-the peaks of dThd and thymine, respectively. This coincidence was also noted when injecting the purified inhibitor and dThd together to form a large single peak, thus indicating that the purified inhibitor is dThd.
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Effects o f supernatant on T K - 1/79 mutant cells Next, we investigated whether dThd play a part in the growth inhibition of V79 cells at the density inhibited state. It was difficult to directly measure the dThd concentration in the supernatants, which probably include many nucleic acid analogues that interfere with dThd in the assay. Therefore, we examined whether
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Fig. 4. Growth inhibition of TK +- and TK--V79 ceils by conditioned medium from density-inhiblted cultures of V79 cells and the effect of exogenous thymidine on the growth of TK +- and TK--V79 cells (inset). Open circles, TK+-eells; dosed circles. TK--V79 cells. The data were expressed as a percentage of the number of cells incubated in each medium relative to that of cells incubated in fresh medium. Data are mean ± S.D. of duplicate determinations.
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Fig. 3. Comparison of elution patterns of the purified inhibitor B with various synthetic nucleic acid compounds including cAMP, thymldine, thymine, dTMP and dTTP. The chemicals were adjusted to approximately the same concentration of the purified inhibitor by absorbance at 260 nm and injected on a TSK gel ODS ]20A reverse-phase column with a UV detector set at 260 nm. The samples were chromatographed by the method described for Fig. 2a. Elulion patterns of the purified inhibitor (a), its hydmlysate (b), cAMP (c), dTTP (d), dTMP (e), thymidine (f) and thymine (g).
there was a difference in the growth inhibitory effect of the supernatants between TK + and T K - cells. It is expected that T K - cells are not inhibited by the excess of dThd because of the inability to convert dThd to dTI'P which exerts as an end-product feedback inhibitor of DNA synthesis [12-14]. As shown in Fig. 4, growth inhibition of the TK + ceils gradually became potent as the percentage of supernatants added to the growth medium increased from 30% and complete suppression was observed at 100% supernatants. In contrast, growth of the T K - V79 cells which were resistant to toxic level of dThd (Fig. 4, inset) was stimulated nearly 50% over the non-treated control, when the supernatants were added to the culture, up to 60% concentration. This enhancement of. growth does not seem to be due to the influence of growth factors produced by V79 cells, because similar growth enhancement was not seen in TK + cells. At the concentration of supernatants over 60%, the stimulated growth of TK- cells gradually declined and neither stimulation nor inhibition was observed at 85%. At this range, however, growth of TK + V79 cells was substan-
267 tially suppressed by 25-54%. Since the influence of other inhibitory factors are not considered bccausc growth inhibition of TK- cells is nil up to 60-85% concentration, it is likely that dThd is responsible for the growth inhibition of T K ' cells at this concentration. At concentrations over 85%, the growth of both TK + and TK- V79 cells was abruptly depressed in a dose-dependent manner, suggesting an extreme deftciency of nutrients or presence of inhibitory factors other than dThd. Evidence that growth inhibition by supernatants is always less in TK- cells also suggests that dThd appears to be contributory to the growth inhibition of TK ÷ V79 cells. By extrapolating the results from the growth inhibition curve obtained by known concentration of dThd (Fig. 4, inset), the coneentration of dThd in the supernatant is approx. 25 /~M, based on the assumption that there are no positive or negative effectors which interfere with dThd in the 50% concentration of supernatants.
Viability To test the possibility that dThd included in superratants might primarily derive from the degradation products of the denatured DNA produced by dead cells at the density-inhibited state, viability of the cells was examined at the time of harvest of the supernatants, using trypan blue dye exclusion. The cell viability at 24 h after confluence was 94%. When the medium was changed at this time and the the culture was incubated for an additional 24 h incubation, the viability was 88%. These data indicate that degradation products from the denatured DNA, as a source of dThd, appears unlikely. Discussion In our previous study, the supernatant of density-inhibited, tumorigenic V79 cells was shown to contain two distinct, low-molecular-weight growth inhibitory factors (peak A and B), one of which (peak A) was subsequently demonstrated to be an oligopeptide which reversibly inhibits the growth of a wide variety of normal and malignant cells [10]. In this report, we identified the another inhibitory factor (peak B) as dThd. Also, using TK- V79 cells, we have obtained evidence that V79 supernatants conta;,a dThd at such concentration that inhibits the actively growing V79 cells. It is well known that dThd is a nucleic acid precursor in the salvage pathway, dThd can be readily phosphorylated to dTMP by thymidine kinase, a salvage pathway enzyme, and dTMP then undergoes two additional phosphorylations to dTTP. In excess concentration of dThd, high level of dTTP ensues and exerts an allosterie inhibition of the de novo and salvage pathway enzymes, resulting in shut-off of the DNA synthesis [12-14]. Thus, an excess of exogenous
dThd is lethal to mammalian cells and has been used as a potential cancer chemotherapeutic agent [16,17]. The possibility of degradation product of DNA as a source of dThd is unlikely. Therefore, dThd appears to derive from pyrimidine nucleotides which are synthesized by de novo pathway, degraded and excreted into the medium, dThd has been demonstrated in the supernatant of cell culture or tissue homogenates. Various nucleic acid compounds, including dThd are present in small molecular weight fractions from tissues [18-22]. dThd is an inhibitor of [3H]dThd incorporation in the supernatants of macrophage and lymphocyte cultures but these supernatants had no effect on cell proliferation [ 18-21 ]. The suppression of [ 3H]dThd incorporation seen in these studies was ascribed to the dilution effect of cold dThd, a degradation product from the DNA of the dead cells [18-20]. Moreover, it has been reported that 10-20-times higher concentrations of dThd accumulate in the medium of TK- cells than TK ÷ cells [23]. However, to our knowledge, the accumulation of dThd in the medium up to the level that inhibits the growth of actively growing tumor cells as described in this report has not been reported. The mechanism by which dThd accumulates to the inhibitory level in density-inhibited V79 cell culture is not known. It is known that dThd secretion results from rapid turnover of d'Iq'P in cells [24]. Reichard and coworker [25] examined the fate of [5-3H]-cytidine incorporated into the cells in culture in the presence of an inhibitor of DNA synthesis, aphidicolin. They found that the synthesis of deoxyribonucleoside triphosphates during S phase was larger than their demand for DNA synthesis and that the cells degraded the surplus material to deoxyribonucleosides. When DNA replication was inhibited, deoxyribonucleotide synthesis continued and all materials was degraded and excreted into the medium as deoxyribonucleosides, leading to an accumulation of deoxyribonucleosides in the medium. Since certain inhibitors for DNA replication were demonstrated in the supernatants of V79 cells [9,10], a similar mechanism might occur in the density-inhibited culture of tilese cells. Therefore, an accumulation of dThd in the confluent culture medium is supposed to derive from the dThd triphosphate pool that is actively synthesized de novo, dephosphorylated and excreted into the medium rather than incorporated into DNA. This hypothesis is supported by the evidence that the growth of TK- cells is not inhibited but greatly enhanced by the supernatants despite their significant inhibitory activities on TK ÷ cells. The growth enhancement of TKcells by supernatants could be explained i f T K - V79 cells have an increased de novo DNA synthetic activity to compensate for the loss of dThd uptake through salvage pathway, since supernatants are considered to contain abundant substrates for de novo DNA synthesis.
268 Conversely, w h e n d e novo synthesis is inhibited by hydroxyurea, d e g r a d a t i o n o f d e o x y r i b o n u e l e o t i d e s d e c r e a s e d greatly, a n d d e o x y r i b o n u c l e o s i d e s a r e imp o r t e d into t h e cells f r o m t h e m e d i u m [26]. Such bala n c e b e t w e e n t h e r e a c t i o n s l e a d i n g to synthesis o r d e g r a d a t i o n m a y be a l t e r e d in V 7 9 cells as a n a b e r r a n t g r o w t h regulation, r e s u l t i n g in a h i g h level o f d T h d a c c u m u l a t i o n in t h e m e d i u m . C o n s e q u e n t l y , h i g h conc e n t r a t i o n s of d T h d will r e g u l a t e actively p r o l i f e r a t i n g V79 cells. T h e r e f o r e , d T h d is n o t r e g a r d e d as a n i n d u c e d r e g u l a t o r . R a t h e r , i n c r e a s e d levels o f d T h d in t h e m e d i u m m a y only b e a c o n s e q u e n c e o f t h e active g r o w t h potential ot~ n e o p l a s t i c cells w h i c h e s c a p e f r o m normal growth regulatory mechanisms.
Acknowledgments W e t h a n k M. O h a r a for comments on the m a n u s c r i p t . T h i s s t u d y w a s s u p p o r t e d by a G r a n t - i n - A i d for Scientific R e s e a r c h ( C ) (03670327) f r o m T h e M i n istry o f E d u c a t i o n , S c i e n c e a n d C u l t u r e , J a p a n a n d by a grant from the Fukuoka Foundation for Developm e n t o f Clinical M e d i c i n e .
References 1 Holley, R. W. (1980) J. Supramol. Struct. 13, 191-197. 2 Hsu. Y.. Barry, J. M. and Wang, J. L. (1986) Proc. Natl. Acad. Sci. LISA 81. 2107-2111. 3 Keski-Oja, J. and Moses, H.L (1987) Med. Biol. 65, 13-20. 4 Lieberman, M.A. and Olaser, L. (1981) J. Membr. Biol. 63, l - l l . 5 Pardee, A.B.. Dubrow, R., Hamlin, J.L. and Kletzien, R.F. (1978) Annu. Rev. Biochem. 47, 715-750.
6 Abercrombie. M. (1979) Nature (Lond.) 281,259-265. 7 Nakano, S.. Yamagami, H. and Takaki, R. (1979) Mut. Res. 62, 369-381. 8 Nakano. S.. Koga, T., Nagafuchi, S., Nakamura, M., Kounoue. E., Niho, Y. and Takaki, R. (1985) Mutation Res. 146, 271-276. 9 Koga, T., Nakano, S., Nakayama, M.. Kounoue, E.. Nagafuchi. S.. Niho, Y. and Yamada, H. (1986) Cancer Res. 46, 4431-4437. I0 Koga. "i'., Nakano, S., Ichinose. I., Yamada. H. and Niho, Y. (1988) Cancer Res. 29, 3737-3741. It Kakunaga, T. (1973) Int. J. Cancer 12, 463-473. 12 Moore, E.C. and Hurlbert, R.B. (1966) J. Biol. Chem. 241, 4802-4809. 13 Adams. R.L.P. (1969) Exp. Cell Res. 56. 55--58. 14 Bjursell. G. and Reichard. P. (1973) J. Biol. Chem. 24, 3904-3909. 15 Paslan, I.H., Johnson, G.S. and Anderson, W.B. (1975) Annu. Rev. Bioehem. 44, 491-522. 16 Blumenreich, M.S., i.Voodcock, T.M.. Andreeff, M.. Hiddenmann, W., Coo, T.C., Vare, K., O'Hehir, M., Clarkson, B.D. and Young, C.W. (1984) Cancer Res. 44, 2203-2207. 17 Lockshln, A., Mendoza, J.T., Giovanella, B.C. and Stehlln, J.S.. Jr. (1984) Cancer Res. 44, 2534-2539. 18 Opits, H.G.. Niethammer, D., Jackson, R.C., Lemke, H., Huget, R. and Flad, H.G. (1975) Cell. Immunol. 18, 70-75. 19 Evans, R. and Booth. C.G. (1976) Cell. Immunol. 26, 120-126. 20 Kasahara, T. and Shioiri-Nakano, K. (1976) J. lmmunol. 116, 1251-1256. 21 Lenfant, M.. Garcia-Giralt. E.. Thomas, M. and DiGuisto, L. (1978) Cell Tissue Kinet. 11,455-463. 22 Sekas, G. and Cook, R.T. (1976) Exp. Cell Res. 102, 422-425. 23 Chan, T.-S., Meuth, M. and Green, H. (1974) J. Cell. Physiol. 83, 263-266. 24 Plagemann, P.G.W., Richey, D.P., Zylka, J.M. and Erbe, J. (1974) Exp. Cell Res. 83, 303-310. 25 Nicander, B and Rcichard, P. (1985) J. Biol. Chem. 260, 92109222. 26 Bianchi, V., Pontis, E. and Rcichard, P. (1986) Proc. Natl. Acad. Sci. USA 83, 986-990.
263
© 1991 ElsevierScience PublishersB.V. All rightsreserved0167-4889/91/$03.50 ADONIS 016748899100242K
BBAMCR 13015
Evidence for the involvement of endogenous thymidine in the density-inhibition of tumorigenic Chinese hamster V79 cells Shuji N a k a n o l, T a t s u h i k o K o g a J, l c h i r o l c h i n o s e ~, H i d e n o r i Y a m a d a 2 and Yoshiyuki Niho t First Department of Internal Medic;he. Faculty of Medicine. Kyushu Unit't'~vitv Ftlkuoka (Japan) and 2 Faculty of Pharmaceutical Science, Kyushu Unicersity. Fukuoka (Japan)
(l'.cceived 4 February 1991) (Revised r~lanuscriptreceived 13June 199I) Keywords: Thymidine:Density-inhibition:Nuclcotide: Degradation Two distinct low-molecular-weight growth inhibitory activities were isolated from supernatants of a density-inhibited, tumorigenic V79 Chinese hamster cell lille. By chromatographic analyses, one of these was purified to homogeneity and eventually proved to be thymidine (dThd). In order to investigate the biological role of dThd in a density-inhibited culture of these cells, a dThd-kinase deficient ( T K - ) clone resistant to the excess of dThd was isolated from V79 cells and the effect of the supernatants on growth of these TK - or TK-proficie:~, (TK +) cells was examined. As a result, the growth of T K - cells was not inhibited but enhanced by the supernatant at the concentrations which significantly inhibited the growth of TK + cells. Such TK-dependent differential responses to supernatants suggest the presence of deoxyribonucleosides including a high level of dThd in the superaatants. Since it is unlikely that dThd might derive from denatured DNA of dead cells, an accumulation of endogenous dThd in conflueni culture appears to be responsible for dThd triphosphates which are synthesized de novo, degraded and excreted into the medium rather than incorporated into DNA as a consequence of aberrant ~anvth in the presence of certain growth inhibitors produced by density-inhibited V79 cells. Introduction
Proliferative potential of normal cells in vitro is stringently controlled by two major mechanisms, i.e., direct cell-to-cell interaction and soluble factors released by the cells [1-4]. In contrast, neoplastic cells escape from such growth regulations as a result of an aberrant growth control mechanism [5,6]. However, even neoplastic ceils, when saturated in cell density, show a depression of growth potential. Such densitydependent growth inhibition in tumor cells is not likely to be due to cell-to-cell interaction, but rather to the
Abbreviations:FCS,fetal calfserum;PBS,phosphate-bufferedsaline: TFA, trifluoroacetic acid; FPLC; fast protein liquidchromatography; HPLC. high-performanceliquid chromatography;dThd, thymidine; dTMP, thymidine monophosphate;dTDP. thymidine diphosphate; d'ITP, Ihymidinetriohosphate;cAMP, cyclic adenosine 3'.5'-monophosphate;TK. thymidinekinase. Correspondence:S. Nakano, First Departmentof Internal Medicine, Faculty of Medicine, Kyushu UniversiW,3-1-1 Maidashi. Fukuoka 812, Japan.
soluble inhibitor shed into the medium, since frequent medium replacement induces a substantial proliferation of tumor cells, resulting in a piling-up phenomenon of cellular growth. Therefore, the possibility remains that soluble inhibitory factors may be important in the density-dependent regulation of neoplastic cells. This is particularly important in considering the possibility of growth regulation of cancer cells by such inhibitory factors. Recently, we found that V79 Chinese hamster cells, which proliferate vigorously in a pile up fashion in vitro, come to a drastic arrest in growth if medium replacement is delayed over the critical period. We found in the supernatant a potent growth inhibitory activity which reversibly inhibits the cellular growth and DNA synthesis [7,8]. This inhibitory activity is apparently mediated by soluble inhibitory factors produced endogenously from density-inhibited V79 cells, and two molecular weight classes of growth inhibitory activities have been identified, with the major activity in low-molecular-weight fractions ranging 500-2000 [9]. This partially purified inhibitor was further separated into two distinct inhibitory activities. One of these was
264 already purified to apparent homogeneity and proved to be an oligopeptide inhibitor acting on a wide variety of normal and malignant cells [10]. We describe herein the purification of another low-molecular-weight growth inhibitor, which was finally demonstrated to be thymJdine (dThd). The biological study suggested that growth-inhibitory level of dThd would he accumulated in the confluent culture of V79 cells. The mechanism of dThd accumulation at density-inhibited state is discussed. Materials and Methods
Cell cultures and preparation of conditioned medium Cells were grown at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air in Dulbecco's modified Eagle's medium (Nissui Pharm., Tokyo) supplemented with 5% heat inactivated fetal calf serum (FCS) (Gibco Laboratories, Chagrin Falls, OH). V79 cells, established from Chinese hamster lung fibroblasts, were demonstrated to produce fibrosarcoma when injected into athymic nude mice at a dose of 1 • 107. Thymidine kinase (TK) deficient V79 mutant clones were obtained by mutagenizing V79 cells with 10 mM N-methyl-N'n~tro-N-nitrosoguanidine (Sigma, St. Louis, MO) and exposing them to 2 mM dThd after expression time for 3 days. Actively growing colonies were clonally isolated and propagated in medium containing 2 mM dThd. These TK-deficient mutant cells had lost TK activity, as evidenced by the lack of growth in HAT medium containing 1-10 -4 M hypoxanthine, 4 . 1 0 -7 M aminopterin, 1.6 • 10 -5 M dThd. The lack of TK activity was also demonstrated by an extremely low level of the incorporation of [3H]dThd into cellular DNA. A subclone of Balb/c 3T3 A31 cells [11], was used as the target cells for measurement of the inhibitory activity of the fractionated samples.
Preparation of supematants of density-inhibited V79 cells Since we found that the growth inhibitory activity of the supernatants was maximal at the early period of density-inhibited state [9], supernatants were prepared by exposing 10 ml of growth medium for 24 h to the cultures (100 ram, Falcon, Oxnard, CA) immediately after confluence as previously described [9,10].
Chemicals Thymine, thymidine, thymidine monophosphate (dTMP), thymidine triphosphate (dTTP) and cyclic AMP (cAMP) were purchased from Sigma, St. Louis, Mo.
Reverse-phase chromatography The low-molecular-weight growth inhibitory fractions ranging between 500-2000, obtained from Sephadex G-25 gel filtration chromatography of a 30 ml 20-fold concentrate of supernatants [9], were corn-
bined and lyophilized. The redissolved residue with one twentieth volume of distilled water was applied to a reverse-phase Lobar column chromatography (Lichroprep RP-18, Merck). Chromatography was carried cut with a Pharmacia fast protein liquid chromatography (FPLC) equipment, using distilled water containing 0.1% trifluoroaeetie acid (TFA). The column eluate was monitored with a fluorescent stream detector set at 240 nm with a flow rate of 1.0 ml/min. Approx. 70 fractions (10 ml) were collected by the fraction collector (FRAC-100, Pharmacia) and 1 ml of each fraction was lyophilized, resuspended in growth medium, and assayed for growth inhibitory activity.
Reverse-phase and gel filtration HPLC The more hydrophobic growth inhibitory fractions eluting at 8% acetonitrile concentration (pool B) on reverse-phase Lobar column chromatography (Fig. 1) were pooled, lyophilized, resuspended in 0.5 ml of distilled water and injected on a TSK gel ODS 120A reverse-phase column (Toyosoda, Tokyo), using the Hitachi HPLC system equipped with variable-wavelength detectors. The sample was chromatographed using a 2% isoeratie gradient in 0.1% TFA at a flow rate of 0.8 ml per min, and the effluent was monitored at 210 nm. Impurities included in a single fraction containing inhibitory activity from a reverse-phase HPLC were eliminated by repeating the same chromatography. The inhibitory peak was lyophilized, resuspended in 100 /.d of 0.1% TFA, applied to a gel filtration column (0.8 × 50 era) of Cellulofine GCL-25M (Seikagaku Kogyo, Tokyo) using a Hitachi HPLC system, equilibrated and eluted with 0.1% TFA at a flow rate of 0.6 ml per ml at room temperature. The column eluate was monitored with a UV detector set at 210 nm. Each peak was lyophilized, resuspended in growth medium and assayed for growth inhibitory activity.
Assay of growth inhibitory activity The growth inhibitory activities of fraetionated materials were determined by the measurement of cell growth and DNA synthesis in 3T3 cells, as described [9,10]. Briefy, 2.104 3T3 cells were inoculated into replicate plates (35 mm, Falcon) and incubated for 18 h for attachment. Medium was then replaced with growth medium containing each lyophilized and resuspended sample, and incubation was continued for an additional 2 days. Cell were trypsinized and counted on a Coulter Counter (ID).
Stability of the inhibitor Aliqunts from the inhibitor purified by gel filtration HPLC were tested for their sensitivity to trypsin and heat, as described [10]. Hydrolysis was performed by incubating an aliquot in 6 M HCI at 110°C for 120 rain.
Results
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Purification of the inhibitory acti~'ity T h e low-molecular-weight fractions obtained by gel filtration c h r o m a t o g r a p h y of a c o n c e n t r a t e d supern a t a n t were fractionated over a reverse-phase L o b a r column c h r o m a t o g r a p h y . As shown in Fig. 1, three distinct inhibitory p e a k s a p p e a r e d . Since the first peak eluting at a 0 % acetonitrile c o n c e n t r a t i o n contained e n o r m o u s a m o u n t s of salts, the second a n d the third inhibitory peaks were considered to be inhibitors, a n d designated as pool A a n d pool B, respectively. Pool A has b e e n subsequently purified to homogeneity a n d f o u n d to be a n oligopeptide inhibitor with its molecular weight 2000 [10]. T h e m o r e hydrophobic inhibitor (pool B) eluting at a 8 % acetonitrile (fraction 4 0 - 5 5 ) reversibly inhibited growth of the 3T3 cells (data not shown). T h e pool B was f u r t h e r purified in a process including reverse-phase H P L C a n d gel filtration H P L C . W h e n pool B was c h r o m a t o g r a p h e d using a 2% isocratic elution of acetonitrile, growth inhibitory activity was detected in a single p e a k (Fig. 2a), a n d this single p e a k was purified to homogeneity by gel filtration H P L C as a single s h a r p p e a k (Fig. 2b). W h e n unconditioned m e d i u m was similarly processed for a series of c h r o m a t o g r a p h y , n o significant p e a k s a p p e a r e d .
Physicochemical analysis of purified inhibitor T h e purified inhibitor was heat-stable a n d trypsinresistant but was inactivated by hydrolysis ( d a t a not shown). Both the native a n d hyctrolyzed purified inhibitors h a d a p e a k ultraviolet a b s o r b a n c e at 260 nm, suggesting a nucleic acid composition in the primary structure.
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~'ltJtion t i m e Fig. 2. (a) Reverse-phase HPLC of partially purified inhibitor (pool B). Pool B (Fig. 1), was lyophilized, resuspcnded in 0.5 ml of distilled water and subjected to reverse-phase HPLC. The sample was chromatographed as describ-d in Materials and Methods. Fraction aliquots were lyophilizcd, resuspended in growth medium and tested for inhibitory activity on cell growth in 3T3 cells. (b) Gel filtration HPLC of the inhibitory peak fraction from reverse-phase HPLC. Each peak was lyophilized, resuspended and tested for inhibitory activity on DNA synthesis.
Biological actit,ity of the purified inhibitor The growth inhibitory activity of the purified inhibitor was c o m p a r e d with the activity of synthetic inhibitory nucleoside analogues, including d T h d , d T M P a n d c A M P , since these c o m p o u n d s have an antiproliferative activity [12-15]. T h e purified inhibitor restricted growth activity to much the same extent as seen with d T h d a n d h a d a slightly higher activity t h a n d T M P or c A M P (Table I).
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Fig. 1. Reverse-phase Lobar column chromatography of concentrated low-molecular-weight growth inhibitory fractions obtained from Sephadex G-25 gel filtration chromatography of the supernatants. The ¢lution sequence of aeetonitrile was 0 to 15% stepwise gradient for 600 mL One fifth volume of each fraction was lyophilized, resuspended in growth medium and tested for growth inhibitory activity on sparse, proliferating culture of 3T3 cells, as described in Materials and Methods.
Comparison of tile growth ildlibitoo' acticity of tile purified inhibitor with activities of synthetic nucleosideanalogues Each substance was dissolved with PBS into the concentration showing approximately the same ultraviolet absorbance at 260 nm (A2sj) as that of the purified inhibitor, and 40 u.I of each solution was added into 2 ml of culture medium of the assay of growth inhibitory activity. Data are expressed as the percentage ±S.D. of triplicate plates. Treatment Control medium Purified inhibitor Thymidine dTMP cAMP
A zsl 1.46 1.35 1.54 1.46
Percent cell growth 100±5 84 + 2 83 ± 4 95 ± 9 91 ± I
266 Comparison by elution pattern In order to distinguish the inhibitor from other nucleic acid compounds, elution patterns on reversephase HPLC were compared. As shown in Fig. 3, the inhibitor differed from cAMP, dTMP and dTI'P, but the elution patterns of native and hydrolyzed inhibitors were identical and coinciden~-~;~'d~-the peaks of dThd and thymine, respectively. This coincidence was also noted when injecting the purified inhibitor and dThd together to form a large single peak, thus indicating that the purified inhibitor is dThd.
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Effects o f supernatant on T K - 1/79 mutant cells Next, we investigated whether dThd play a part in the growth inhibition of V79 cells at the density inhibited state. It was difficult to directly measure the dThd concentration in the supernatants, which probably include many nucleic acid analogues that interfere with dThd in the assay. Therefore, we examined whether
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Fig. 4. Growth inhibition of TK +- and TK--V79 ceils by conditioned medium from density-inhiblted cultures of V79 cells and the effect of exogenous thymidine on the growth of TK +- and TK--V79 cells (inset). Open circles, TK+-eells; dosed circles. TK--V79 cells. The data were expressed as a percentage of the number of cells incubated in each medium relative to that of cells incubated in fresh medium. Data are mean ± S.D. of duplicate determinations.
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Fig. 3. Comparison of elution patterns of the purified inhibitor B with various synthetic nucleic acid compounds including cAMP, thymldine, thymine, dTMP and dTTP. The chemicals were adjusted to approximately the same concentration of the purified inhibitor by absorbance at 260 nm and injected on a TSK gel ODS ]20A reverse-phase column with a UV detector set at 260 nm. The samples were chromatographed by the method described for Fig. 2a. Elulion patterns of the purified inhibitor (a), its hydmlysate (b), cAMP (c), dTTP (d), dTMP (e), thymidine (f) and thymine (g).
there was a difference in the growth inhibitory effect of the supernatants between TK + and T K - cells. It is expected that T K - cells are not inhibited by the excess of dThd because of the inability to convert dThd to dTI'P which exerts as an end-product feedback inhibitor of DNA synthesis [12-14]. As shown in Fig. 4, growth inhibition of the TK + ceils gradually became potent as the percentage of supernatants added to the growth medium increased from 30% and complete suppression was observed at 100% supernatants. In contrast, growth of the T K - V79 cells which were resistant to toxic level of dThd (Fig. 4, inset) was stimulated nearly 50% over the non-treated control, when the supernatants were added to the culture, up to 60% concentration. This enhancement of. growth does not seem to be due to the influence of growth factors produced by V79 cells, because similar growth enhancement was not seen in TK + cells. At the concentration of supernatants over 60%, the stimulated growth of TK- cells gradually declined and neither stimulation nor inhibition was observed at 85%. At this range, however, growth of TK + V79 cells was substan-
267 tially suppressed by 25-54%. Since the influence of other inhibitory factors are not considered bccausc growth inhibition of TK- cells is nil up to 60-85% concentration, it is likely that dThd is responsible for the growth inhibition of T K ' cells at this concentration. At concentrations over 85%, the growth of both TK + and TK- V79 cells was abruptly depressed in a dose-dependent manner, suggesting an extreme deftciency of nutrients or presence of inhibitory factors other than dThd. Evidence that growth inhibition by supernatants is always less in TK- cells also suggests that dThd appears to be contributory to the growth inhibition of TK ÷ V79 cells. By extrapolating the results from the growth inhibition curve obtained by known concentration of dThd (Fig. 4, inset), the coneentration of dThd in the supernatant is approx. 25 /~M, based on the assumption that there are no positive or negative effectors which interfere with dThd in the 50% concentration of supernatants.
Viability To test the possibility that dThd included in superratants might primarily derive from the degradation products of the denatured DNA produced by dead cells at the density-inhibited state, viability of the cells was examined at the time of harvest of the supernatants, using trypan blue dye exclusion. The cell viability at 24 h after confluence was 94%. When the medium was changed at this time and the the culture was incubated for an additional 24 h incubation, the viability was 88%. These data indicate that degradation products from the denatured DNA, as a source of dThd, appears unlikely. Discussion In our previous study, the supernatant of density-inhibited, tumorigenic V79 cells was shown to contain two distinct, low-molecular-weight growth inhibitory factors (peak A and B), one of which (peak A) was subsequently demonstrated to be an oligopeptide which reversibly inhibits the growth of a wide variety of normal and malignant cells [10]. In this report, we identified the another inhibitory factor (peak B) as dThd. Also, using TK- V79 cells, we have obtained evidence that V79 supernatants conta;,a dThd at such concentration that inhibits the actively growing V79 cells. It is well known that dThd is a nucleic acid precursor in the salvage pathway, dThd can be readily phosphorylated to dTMP by thymidine kinase, a salvage pathway enzyme, and dTMP then undergoes two additional phosphorylations to dTTP. In excess concentration of dThd, high level of dTTP ensues and exerts an allosterie inhibition of the de novo and salvage pathway enzymes, resulting in shut-off of the DNA synthesis [12-14]. Thus, an excess of exogenous
dThd is lethal to mammalian cells and has been used as a potential cancer chemotherapeutic agent [16,17]. The possibility of degradation product of DNA as a source of dThd is unlikely. Therefore, dThd appears to derive from pyrimidine nucleotides which are synthesized by de novo pathway, degraded and excreted into the medium, dThd has been demonstrated in the supernatant of cell culture or tissue homogenates. Various nucleic acid compounds, including dThd are present in small molecular weight fractions from tissues [18-22]. dThd is an inhibitor of [3H]dThd incorporation in the supernatants of macrophage and lymphocyte cultures but these supernatants had no effect on cell proliferation [ 18-21 ]. The suppression of [ 3H]dThd incorporation seen in these studies was ascribed to the dilution effect of cold dThd, a degradation product from the DNA of the dead cells [18-20]. Moreover, it has been reported that 10-20-times higher concentrations of dThd accumulate in the medium of TK- cells than TK ÷ cells [23]. However, to our knowledge, the accumulation of dThd in the medium up to the level that inhibits the growth of actively growing tumor cells as described in this report has not been reported. The mechanism by which dThd accumulates to the inhibitory level in density-inhibited V79 cell culture is not known. It is known that dThd secretion results from rapid turnover of d'Iq'P in cells [24]. Reichard and coworker [25] examined the fate of [5-3H]-cytidine incorporated into the cells in culture in the presence of an inhibitor of DNA synthesis, aphidicolin. They found that the synthesis of deoxyribonucleoside triphosphates during S phase was larger than their demand for DNA synthesis and that the cells degraded the surplus material to deoxyribonucleosides. When DNA replication was inhibited, deoxyribonucleotide synthesis continued and all materials was degraded and excreted into the medium as deoxyribonucleosides, leading to an accumulation of deoxyribonucleosides in the medium. Since certain inhibitors for DNA replication were demonstrated in the supernatants of V79 cells [9,10], a similar mechanism might occur in the density-inhibited culture of tilese cells. Therefore, an accumulation of dThd in the confluent culture medium is supposed to derive from the dThd triphosphate pool that is actively synthesized de novo, dephosphorylated and excreted into the medium rather than incorporated into DNA. This hypothesis is supported by the evidence that the growth of TK- cells is not inhibited but greatly enhanced by the supernatants despite their significant inhibitory activities on TK ÷ cells. The growth enhancement of TKcells by supernatants could be explained i f T K - V79 cells have an increased de novo DNA synthetic activity to compensate for the loss of dThd uptake through salvage pathway, since supernatants are considered to contain abundant substrates for de novo DNA synthesis.
268 Conversely, w h e n d e novo synthesis is inhibited by hydroxyurea, d e g r a d a t i o n o f d e o x y r i b o n u e l e o t i d e s d e c r e a s e d greatly, a n d d e o x y r i b o n u c l e o s i d e s a r e imp o r t e d into t h e cells f r o m t h e m e d i u m [26]. Such bala n c e b e t w e e n t h e r e a c t i o n s l e a d i n g to synthesis o r d e g r a d a t i o n m a y be a l t e r e d in V 7 9 cells as a n a b e r r a n t g r o w t h regulation, r e s u l t i n g in a h i g h level o f d T h d a c c u m u l a t i o n in t h e m e d i u m . C o n s e q u e n t l y , h i g h conc e n t r a t i o n s of d T h d will r e g u l a t e actively p r o l i f e r a t i n g V79 cells. T h e r e f o r e , d T h d is n o t r e g a r d e d as a n i n d u c e d r e g u l a t o r . R a t h e r , i n c r e a s e d levels o f d T h d in t h e m e d i u m m a y only b e a c o n s e q u e n c e o f t h e active g r o w t h potential ot~ n e o p l a s t i c cells w h i c h e s c a p e f r o m normal growth regulatory mechanisms.
Acknowledgments W e t h a n k M. O h a r a for comments on the m a n u s c r i p t . T h i s s t u d y w a s s u p p o r t e d by a G r a n t - i n - A i d for Scientific R e s e a r c h ( C ) (03670327) f r o m T h e M i n istry o f E d u c a t i o n , S c i e n c e a n d C u l t u r e , J a p a n a n d by a grant from the Fukuoka Foundation for Developm e n t o f Clinical M e d i c i n e .
References 1 Holley, R. W. (1980) J. Supramol. Struct. 13, 191-197. 2 Hsu. Y.. Barry, J. M. and Wang, J. L. (1986) Proc. Natl. Acad. Sci. LISA 81. 2107-2111. 3 Keski-Oja, J. and Moses, H.L (1987) Med. Biol. 65, 13-20. 4 Lieberman, M.A. and Olaser, L. (1981) J. Membr. Biol. 63, l - l l . 5 Pardee, A.B.. Dubrow, R., Hamlin, J.L. and Kletzien, R.F. (1978) Annu. Rev. Biochem. 47, 715-750.
6 Abercrombie. M. (1979) Nature (Lond.) 281,259-265. 7 Nakano, S.. Yamagami, H. and Takaki, R. (1979) Mut. Res. 62, 369-381. 8 Nakano. S.. Koga, T., Nagafuchi, S., Nakamura, M., Kounoue. E., Niho, Y. and Takaki, R. (1985) Mutation Res. 146, 271-276. 9 Koga, T., Nakano, S., Nakayama, M.. Kounoue, E.. Nagafuchi. S.. Niho, Y. and Yamada, H. (1986) Cancer Res. 46, 4431-4437. I0 Koga. "i'., Nakano, S., Ichinose. I., Yamada. H. and Niho, Y. (1988) Cancer Res. 29, 3737-3741. It Kakunaga, T. (1973) Int. J. Cancer 12, 463-473. 12 Moore, E.C. and Hurlbert, R.B. (1966) J. Biol. Chem. 241, 4802-4809. 13 Adams. R.L.P. (1969) Exp. Cell Res. 56. 55--58. 14 Bjursell. G. and Reichard. P. (1973) J. Biol. Chem. 24, 3904-3909. 15 Paslan, I.H., Johnson, G.S. and Anderson, W.B. (1975) Annu. Rev. Bioehem. 44, 491-522. 16 Blumenreich, M.S., i.Voodcock, T.M.. Andreeff, M.. Hiddenmann, W., Coo, T.C., Vare, K., O'Hehir, M., Clarkson, B.D. and Young, C.W. (1984) Cancer Res. 44, 2203-2207. 17 Lockshln, A., Mendoza, J.T., Giovanella, B.C. and Stehlln, J.S.. Jr. (1984) Cancer Res. 44, 2534-2539. 18 Opits, H.G.. Niethammer, D., Jackson, R.C., Lemke, H., Huget, R. and Flad, H.G. (1975) Cell. Immunol. 18, 70-75. 19 Evans, R. and Booth. C.G. (1976) Cell. Immunol. 26, 120-126. 20 Kasahara, T. and Shioiri-Nakano, K. (1976) J. lmmunol. 116, 1251-1256. 21 Lenfant, M.. Garcia-Giralt. E.. Thomas, M. and DiGuisto, L. (1978) Cell Tissue Kinet. 11,455-463. 22 Sekas, G. and Cook, R.T. (1976) Exp. Cell Res. 102, 422-425. 23 Chan, T.-S., Meuth, M. and Green, H. (1974) J. Cell. Physiol. 83, 263-266. 24 Plagemann, P.G.W., Richey, D.P., Zylka, J.M. and Erbe, J. (1974) Exp. Cell Res. 83, 303-310. 25 Nicander, B and Rcichard, P. (1985) J. Biol. Chem. 260, 92109222. 26 Bianchi, V., Pontis, E. and Rcichard, P. (1986) Proc. Natl. Acad. Sci. USA 83, 986-990.
