Denaturation and Renaturation of a ß-l,6;l,3-Glucan, Lentinan, Associated withExpression of T-Cell-mediated Responses
Yukiko Y. Maeda, Sumiyo T. Watanabe, Chie Chibara, and Makoto RokutandaThe Tokyo Metropolitan Institute of Medical Science, 3-18, Honkomagome, Bunkyoku, Tokyo, 113 Japan
ABSTRACT
Correlation between the higher structure and biological functions of(entinan, a #-l,6;l,3-glucan capable of potentiating T- and non-T-cell-
resulted in the recovery of [a)D values. Measurements of optical rotatorydispersion in the spectral region between 600 and 200 nm showed thechange in the higher structure of lentinan more clearly. Denaturation andrenatural inn of lentinan using urea and dimethyl sulfoxide were associatedwith the decrease and the recovery of antitumor activity against P-815mastocytoma and vascular dilation and hemorrhage-inducing activity,found to be I -cell-mediated responses. Lentinan was also denatured by
NaOH and the transition of [a|D values and optical rotatory dispersioncurves were seen in the manner of two concentration-dependent phases.
Removal of NaOH led to the recovery of optical rotation of lentinan andits antitumor and vascular dilation and hemorrhage-inducing activity.
However, recovery of these bioactivities was more difficult in the case ofthe higher concentrations of NaOH above 2% than the lower ones.During the process of renaturation of lentinan, random aggregation maytake place. An increase of serum acute phase proteins, a non-1 -cell-
mediated response caused by lentinan, was not affected by the change ofthe higher structure of lentinan.
INTRODUCTION
Lentinan is a /3-1,3-glucan having two /3-1,6-glucopyranosidebranches for every five /3-1,3-glucopyranoside linear linkagesisolated from a Japanese edible mushroom, Lent iñusedades (1,2). Its average molecular weight is in the range of 300,000 to800,000 according to gel permeation chromatography and quas-ielastic light scattering measurement (3). Until now, manyinteresting biological activities of lentinan have been reportedsuch as ATA1 against murine solid type-tumors (4) and activa
tion of nonspecific inflammatory responses like APP increase(5) and VDH (6) in vivo as well as activation and generation ofhelper and cytotoxic T-cells (7-9) and augmentation of someimmune mediators like interleukins 1 and 3, colony stimulatingfactor(s), and the migration inhibitory factor (10-12). However,the reaction mechanisms governing the appearance of thesebiological activities of lentinan have as yet to be completelyexposed.
At the first stage of interaction between lentinan and thehost, cellular or humoral, the higher structure of the lentinan-molecule is thought to be very important for the followingexpression of its biological activities, such as for proteins. Ourgroup has proposed the importance of the higher structure ofpolysaccharide for the expression of ATA; that is, althoughpachyman obtained from Porta cocus Wolf has a small amountof /3-1,6-linked branches besides /3-l,3-!inkages in its main chainand is entirely devoid of ATA, a slight chemical modification
Received 4/9/87; revised 10/6/87; accepted 11/4/87.The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1The abbreviations used are: ATA, antitumor activity; APP(s), acute phase
of pachyman to a linear ß-l,3-glucan induced marked ATA(13). Pachyman treated with urea at 70°Calso could induce
ATA without any change of the primary structure (14). In ourprevious report, the addition of urea showed both a decrease inATA and a change of specific rotation at 589 nm, [a]D, oflentinan (15). Until now there have been very few reportsconcerning a possible correlation between the structure andactivity of neutral polysaccharides, probably because of the fewpolysaccharides that have been fully purified and well characterized.
MATERIALS AND METHODSMice. ICR and DBA/2 female mice were purchased from Charles
River Japan Breeding Laboratory (Atsugi, Japan) and used at 6 to 9weeks of age.
Tumor. P81S mastocytoma cells were obtained from AjinomotoCentral Laboratories (Kawasaki, Japan) and maintained in continuoussuspension cultures using RPMI 1640 medium supplemented 10% fetalbovine serum (GIBCO Laboratories, New York, NY). The cells werepassaged once in an ascites form in DBA/2 mice before use in theantitumor assay.
Reagents. Lentinan (lot No. 2H103AO) isolated from L. edades(Berk.) by the method described previously (1) was obtained by Ajinomoto Central Laboratories. Urea, DMSO, and NaOH were obtainedfrom Wako Pure Chemical Industries (Osaka, Japan).
Preparation of Urea, DMSO, and NaOH-treated Lentinan. Lentinansolution (2 mg/ml of distilled water) was prepared by autoclaving at121"(' for 20 min for solubili/ut ion and sterilization. Urea or DMSO
solution at several concentrations was mixed with lentinan solution ofthe same volume for 30 min and then a portion was dialyzed againstdistilled water at room temperature. For the preparation of lentinan in6 M urea or 100% DMSO, 25 mg of lentinan were dissolved directlywith 10 ml of urea or DMSO. For preparation of NaOH-treatedsamples, lentinan solution was mixed with NaOH solution at 0.4 to10% of the final concentration at 25°Cfor indicated periods from 10min to 48 h or at 0°Cfor 30 min, followed by neutralization withhydrochloric acid and dialysis against distilled water at 3*C. Sugar
contents of all the dialyzed samples were measured by the anthrone-sulfuric acid method.
Measurements of Optical Rotation. Measurements of [a]D and ORDwere performed on the 1)11' 140 polarimeter using a 10-cm cell and theJ-20C recording spectropolarimeter using a 1.0 cm cell (Japan Spectro-scopic Co., Tokyo, Japan), respectively. [a]D and ORD of lentinan in 6M urea and 100% DMSO and their dialyzed samples were measured ata concentration of about 0.25%, and measurements for other sampleswere made at the lentinan concentration of about 0.1 %. All the solutionswere maintained at 25°Cthroughout the experimental run.
Assay for VDH. As described previously, lentinan induces VDH invery localized areas such as ears, feet, and tails of normal ICR mice,and the response reaches maximum between days 3 and 5 postadministration (6). In order to test VDH-inducing activity of lentinan samples,the maximum responses of blood vessels at the ears (seven ICR mice/group) were observed for 7 days after an i.p. administration of thesamples. Fig. 1 shows the four grades of VDH.
Fig. 1. Grading of VDH induced by lentinan at ears of ICR mice. -, no induction; +, a few small spots of hemorrhage at the edge of the ears; ++, vascular dilationand intermediate degree of hemorrhage; +++, marked reddishness and swelling of the whole ears and extensive diffuse hemorrhage.
Antitumor Assay. P815 mastocytoma cells (2 x 106/rnouse) were
transplanted s.c. to DBA/2 mice and each test sample was injected i.p.on days 8, 10, 15, and 17 after the tumor transplantation. At 4 weeksposttransplantation, the tumors were extirpated and weighed, and thetumor growth inhibition ratio was calculated by the following formulaafter both the biggest and the smallest tumors were neglected
Concentration of Urea (M)0 05 1 3 6
Inhibition ratio (%) =A - B
x 700
where A and B showed average tumor weights of the control andexperimental groups, respectively.
Gradient Polyacrylamide Gel Electrophoresis. An aliquot of 9 n\ ofserum samples was applied to a 4 to 30% gradient polyacrylamide gel,PAA 4/30, in gel electrophoresis apparatus EL-4 (Pharmacia FineChemicals, Uppsala, Sweden) and electrophoresis was run at a constant125 V for 15 h at 3'C. The electrode buffer was 0.09 M Tris-borate
buffer (pH 8.35) containing 0.003 M disodium EDTA. The gel wasstained with 0.7% amido black 10B in 7% acetic acid and destainedwith 7% acetic acid.
RESULTS
Effect of Urea and DMSO on |a]D of Lentinan. Addition ofurea or DMSO decreased [a]D of lentinan in a concentration-dependent manner (Fig. 2). [a]D of lentinan showing a value of25°in water changed to 2°in 6 M urea and -16° in 100%
DMSO without any change of the primary structure of thelentinan molecule. Removal of urea and DMSO by dialysisagainst water resulted in the recovery of [a]D of lentinan, from2 to 25°and from -16 to 17°,respectively. Besides, both
Changes of [a]Dof Lentinan by Treatment with NaOH. Fig. 3shows the results of [a]D measurements as a function of NaOHconcentration for lentinan. Lentinan samples in NaOH solutions were prepared 10 min before the measurement at 25°C.
At first, with increasing concentrations of NaOH, [a]D of lentinan decreased markedly and showed the lowest value of -2°
at 1% NaOH. However, it then proceeded to rise at still higherconcentrations; that is, [a]D of lentinan at 2, 4, 8, and 10%NaOH was 11, 10, 14, and 13°,respectively. For renaturation
of lentinan, each mixture of lentinan and NaOH prepared at25°Cfor 10 min or at 0°Cfor 30 min was subjected to neutral
ization with hydrochloric acid followed by dialysis againstwater. [a]D values of all samples were restored in a rangebetween 17 and 20°.
Subsequently, to investigate whether the change of [a]D oflentinan in NaOH solution was due to alkaline degradationtaking place in a stepwise manner at the reducing end of themolecule through the production of metasaccharinates (18, 19),
-20125 25 50 100
Concentration of DMSO (%)
Fig. 2. Effect of urea and dimethyl sulfoxide on specific rotation at 589 nm(|n|n) of lentinan at 25"C. Equal volumes of lentinan solution (2 mg/ml of
distilled water) and urea (•)or dimethyl sulfoxide (O) at several final concentrations were mixed at room temperature for 30 min, and their [a]D was measuredat the lentinan concentration of 0.1% except for 6 M urea and 100% dimethylsulfoxide, with which lentinan was dissolved directly at 2.5 mg/ml. Renaturedlentinan samples were prepared by removal of 6 M urea (•)and 100% dimethylsulfoxide (LJ) by dialysis against distilled water. Values are means for tripledeterminations. Bars, SD.
10
»1.1 21 8 10
0.4 05 Concentration of NaOH (ÃŽ)
Fig. 3. Effect of sodium hydroxide on specific rotation at 589 nm (|a]D) oflentinan at 25'C. Lentinan solution (2 mg/ml of distilled water) and sodiumhydroxide at 0.4 to 10% of the final concentration were mixed at 25'C for 10min (•)and their [a]D was measured at the lentinan concentration of 0.1%.Renatured lentinan samples were prepared by neutralization with hydrochloricacid and dialysis against distilled water after mixing with sodium hydroxide atO'C for 30 min (O), or 25'C for 10 min (D). Values are means for triple
determinations. Bars, SD.
the changes of [a]D of lentinan at NaOH concentrations of 0.4,1, and 4% at 25°Cwere followed over a time course. As shown
in Fig. 4, no changes were seen for at least 6 h incubation, withthe decreases then detected 24 and 48 h later assumed to bedue to alkaline degradation. Thus, the remarkable changes of[a]o of lentinan treated with NaOH in a relatively short time
Fig. 4. Time course effect of sodium hydroxide on specific rotation at 589 nm([«]D)of lentinan. Equal volumes of lentinan solution and sodium hydroxide atfinal concentrations of 0.4% (•),1.0% (O), and 4% (D) were mixed at 25'C for
48 h and their [a)D lentinan concentration of 0.1 % was measured at the indicatedtime. Values are means for triple determinations. Bars, SD.
-500
200 eoo300 1(00 500
Wavelength (nm)Fig. 5. Optical rotatory dispersion curves of lentinan in (/) water, (2) 3 M
urea, (3) 6 M urea, and (4) 100% dimethyl sulfoxide, and after the removal of (5)6 M urea and (6) 100% dimethyl sulfoxide at 25'C.
are considered to be due to destruction of the higher structurebut not the primary structure.
min in order to prevent even the slightest chance of alkalinedegradation. Renatured lentinan after treatment with an NaOH
«a(fi
-100
-150
-50
200 300 aoo 500 eooWavelength (nm)
Fig. 6. Optical rotatory dispersion curves of lentinan in (/) water, and (2)0.5%, (3) 1%, (4) 2%, and (5) 8% sodium hydroxide, and after the removal of (6)0.5%, (7) 1%, («)2%, and (9) 8% sodium hydroxide at 25'C.
concentration of 1%, where marked changes were observed inspecific rotation and ORD curves of lentinan, expressed highVDH responses and ATA. However, lentinan samples treatedwith NaOH concentrations of 2 to 4% induced low titers ofVDH and ATA. Doses more than 4-fold of lentinan treatedwith 2% NaOH express almost the same grade of VDH asnontreated lentinan (Fig. 7). Random aggregation can bethought to take place during renaturation of the lentinan samples while still retaining a considerable number of active sitesprobably determined by the primary structure. Thus, it followsfrom these results that the higher structure of lentinan is relatedto the expression of its biological activities. In addition, a closecorrelation between the expressions of VDH and ATA was alsoobserved.
Effect of Denatured and Renatured Lentinan in APP Production. ICR mice (three/sample) were given 5 mg/kg of non-treated, denatured, or renatured lentinan i.p. On 1, 4, 7, and 10days postadministration, the sera were collected from the miceand applied to gradient polyacrylamide gel electrophoresis.APP increase in each serum was measured by comparison of
Table 1 Antitumor and VDH-inducing activity of urea, dimethylsulfoxide, orsodium hydroxide-treated ¡entinan
Lentinan was mixed with urea, DMSO, or NaOH at several concentrationsand then a portion was subjected to dialysis or neutralization and dialysis asdescribed in "Materials and Methods."
* Maximum responses of VDH expressed at the ears (7 ICR mice/group) for
7 days after an i.p. injection of lentinan samples at a dose of 5 mg/kg.*P815 mastocytoma cells (2 x 10'/mouse) were transplanted s.c. to DBA/2
mice (7/group) and the samples were administered i.p. 8, 10, 15, and 17 daysafter the tumor transplantation at a dose of 5 mg/kg. Four weeks later, averagetumor weight and inhibition ratio of the tumor growth were calculated.'' LTN, lentinan; D, dialysis; ND, neutralization and dialysis; NDt, not deter
mined.
100
2 5 10 20LTN/NaOH2%(ND)
Dose (mg/kg)
Fig. 7. Dose-response effect of sodium hydroxide-treated lentinan on VDHinduction. ICR mice (7/group) were given injections i.p. of lentinan (LTN)/NaOH 2% neutralization and dialysis (ND) as shown in Table 1 and the maximumgrades of VDH were measured for 7 days postadministration. Grades of VDH:•,+++; &, ++; D, +; n, -.
bands of LA (haptoglobin-hemoglobin complex), LB (hemopexin), and LC (ceruloplasmin and haptoglobin) assigned previously (21). As shown in Fig. 8, all samples, including thenontreated, showed maximum APP increase in the 7-day sera;that is, APP increase, known as a non-T-cell-mediated responseseems not to need the specific higher structure of lentinan,unlike VDH and ATA.
Fig. 8. Increase of acute phase proteins by urea, dimethyl sulfoxide, or sodiumhydroxide-treated lentinan. An aliquot of 9 pi of each serum pooled from 3 ICRmice 7 days after an i.p. injection of 5 mg/kg of lentinan samples was applied on4 to 30% gradient polyacrylamide gel electrophoresis. The increase of serumacute phase proteins was detected by comparison of three bands, LA (haptoglob-lin-hemoglobin complex), LB (hemopexin), and LC (ceruloplasmin and haptoglo-bin). Lentinan samples: /, none; 2, lentinan; 3, lentinan/NaOH, 0.4% (neutralization and dialysis); 4, lentinan/NaOH, 1% (neutralization and dialysis); 5,lentinan/NaOH, 2% (neutralization and dialysis); ft, lentinan/NaOH 4% (neutralization and dialysis); 7, lentinan/urea, 6 M; 8, lentinan/urea, 6 M (dialysis); 9,urea; 10, lentinan/DMSO, 100%; //, lentinan/DMSO, 100% (dialysis); 12,DMSO as shown in Table 1.
Several neutral polysaccharides capable of potentiating immune responses including ATA have been found to possess thesingle or triple helical conformation from X-ray studies, sedimentation equilibrium, light scattering, and viscosity measurements, and a "C nuclear magnetic resonance study. Lentinanand curdlan, a linear /3-1,3-glucan isolated from the culture ofAlcaligenes faecalis var. myxogenes 10 C3K, are in the triplehelical structure (23, 24). Schizophyllan, a 0-1,3-glucan havingone /3-1,6-glucopyranoside branch for every three /3-1,3-gluco-pyranoside linear linkages isolated from Schizophyllum commune, also possesses the triple helical structure, which is notrecoverable once it is denatured in DMSO (25). Curdlan-typepolysaccharide 13140 from Alcaligenes faecalis var. myxogenesIFO 13140 (TAK) shows the single helical conformation in itsresilient gel or in 0.19 M NaOH solution with random coilconformation in 0.22 M NaOH solution (26). Whether thesingle or the triple helical conformation is necessary for theexpression of the bioactivities of neutral polysaccharides is aninteresting problem which awaits clarification.
Considering the necessity of a specific higher structure oflentinan for its biological activities, the possibility that lentinanbinds with cellular receptors or forms a complex with certainhumoral factors must be considered. Recently, Czop and Austen(27) have described that yeast glucan and zymosan particlespromote generation of leucotrienes by human monocytes uponstimulation of their /3-glucan receptor during phagocytosis.However, unlike zymosan, lentinan does not stimulate phagocytosis (16), although it activates the alternative complementpathway (28) and augments interleukin 1 from monocytes (10).In addition, zymosan and curdlan-type polysaccharide 13140act on polymorphonuclear leukocytes and induce tumorcidalactivity, functions not seen in lentinan and schizophyllan (29).It is as yet not clear whether different activities of severalpolysaccharides are mediated by the same /3-glucan receptorfound during phagocytosis. Further studies are required on thedetermination of the specific higher structure regulating T-cell-mediated responses as well as on the substances, cellular orhumoral, which bind selectively with lentinan and other neutralpolysaccharides, mediating these responses.
ACKNOWLEDGMENTS
We are most grateful to Profs. S. Natori of Meiji College of Pharmacy and K. Mori of Tokyo University for their help in the measurements of specific rotation and ORD.
REFERENCES
1. Chibara, G., Hamuro, J., Maeda, Y. Y., Arai, Y., and Fukuoka, F. Fraction-ation and purification of the polysaccharides with marked antitumor activity,especially lentinan, from I.entinas edades (Berk.) Sing, (an edible mushroom).Cancer Res., 30: 2776-2781, 1970.
2. Sasaki, T., and Takasuka, N. Further study of lentinan, an antitumor polysaccharide from Leminus edades. Carbohydr. Res., 47:99-104, 1976.
3. Suzuki, N., and Wada, A. Hydrodynamic behavior of lentinan molecules asstudied by quasielastic light scattering. Carbohydr. Res., 109:295-298,1982.
4. Chihara, G., Maeda, Y. Y., and Hamuro, J. Current status and perspectivesof immunomodulators of microbial origin. Int. J. Tissue React., 4:207-225,1982.
5. Maeda, Y. Y., Chihara, G., and Ishimura, K. Unique increase of serumproteins and action of antitumor polysaccharides. Nature (Lond.), 252: 250-252, 1974.
6. Maeda, Y. Y., Watanabe, S. T., Chihara, G., and Rokutanda, M. T<ellmediated vascular dilatation and hemorrhage induced by antitumour polysaccharides. Int. J. Immunopharmacol., 6:493-501, 1984.
7. Dennert, G., and Tucker, D. Antitumor polysaccharide lenlinan— a T-celladjuvant. J. Nati. Cancer Inst., SI: 1727-1729, 1973.
8. Dresser, D. W., and Phillips, J. M. The orientation of the adjuvant activitiesof Salmonella typhosa lipopolysaccharide and lentinan. Immunology, 27:895-902, 1974.
9. Hamuro. J., Röllinghoff,M., and Wagner, H. 0(1—»3)Glucan-mediatedaugmentation of alloreactive murine cytotoxic T-lymphocytes in vivo. CancerRes., 38: 3080-3085, 1978.
10. Fruehauf, J. P., Bonnard, G. D., and Herberman, R. B. The effect of lentinanon production of interleukin-1 by human monocytes. Immunopharmacology,5: 65-74, 1982.
11. I/a«a. M., Ohno, K., Akimura, K., and Hamuro, J. Lentinan augments theproduction of interleukin 3 and colony stimulating factor(s) by T-cells. In: T.Aoki, E. Tsubura, and I. Urushizaki (eds.), Manipulation of Host DefenceMechanisms, pp. 59-60. Amsterdam: Excerpta Medica, 1983.
12. Zákány,J., Chihara, G., and Fachet, J. Effect of lentinan on the productionof migration inhibitory factor induced by syngeneic tumor in mice. Int. J.Cancer, 26: 783-788, 1980.
13. Chihara, G., Hamuro, J., Maeda, Y. Y., Arai, Y., and Fukuoka, F. Antitumour polysaccharide derived chemically from natural glucan (pachyman).Nature (Lond.), 225: 943-944, 1970.
14. Maeda, Y. Y., Ishimura, K., Takasuka, N., Sasaki, T., and Chihara, G.Antitumour polysaccharides and host defence against cancer. In: D. Mizuno,G. Chihara, F. Fukuoka, T. Vaniamolo, and Y. Yamamura (eds.). HostDefence against Cancer and Its Potentiation, pp. 181-197. Tokyo: Universityof Tokyo Press, 1975.
15. Hamuro, J., Maeda, Y. Y., Fukuoka, F., and Chihara, G. The significance ofhigher structure of polysaccharide lentinan and pachymaran with regard totheir antitumour activity. Chem.-Biol. Interact., 3:69-71, 1971.
16. Maeda, Y. Y., and Chihara, G. The effects of neonatal thymectomy on theantitumour activity of lentinan, carboxymethylpachymaran, and zymosan,and their effects on various immune responses. Int. J. Cancer, //: 153-161,1973.
17. Suga, T., Maeda, Y. Y., Uchida, H., Rokutanda, M., and Chihara, G.Macrophage-mediated acute-phase transport protein production induced bylentinan. Int. J. Immunopharmacol., X:691-699, 1986.
18. Corbet!, W. M., and Kenner, J. The degradation of carbohydrates by alkali.J. Chem. Soc., 1431-I435, 1955.
19. Whistler, R. L., and BeMiller, J. N. Alkaline degradation of polysaccharides.In: M. L. Wolfrom and R. S. Tipson (eds.). Advances in CarbohydrateChemistry. Vol. 13, pp. 289-329. New York: Academic Press, Inc., 1958.
20. Listowsky, I., Avigad, G., and Englard, S. Optical rotatory dispersion ofsugars. I. Relationship to configuration and conformation aldopyranoses. J.Am. Chem. Soc., 87: 1765-1771, 1965.
21. Manabe, T., Takahashi, Y., Okuyama. T., Maeda, Y. Y., and Chihara, G.Identification of mouse serum proteins increased by the administration ofantitumor polysaccharide lentinan, by micro two-dimensional electropho-resis. Electrophoresis, 4: 242-246, 1983.
22. Sasaki, T., Takasuka, N., Chihara, G., and Maeda, Y. Y. Antitumor activityof degraded products of lentinan: its correlation with molecular weight.Gann, 67: 191-195, 1976.
23. Bluhm, T. L., and Sar ko, A. The triple helical structure of lentinan. a linear/S-(l-»3)-D-glucan.Can. J. Chem., 55: 293-299, 1977.
24. Marchessault, R. H., Deslandes, Y., Ogawa, K.. and Sundararajan, P. R. X-ray diffraction data for 0-(l-»3)-D-glucan. Can. J. Chem., 55:300-303, 1977.
25. Norisuye, T., Yanaki, T., and Fujita, H. Triple helix of a Shizophyllumcommune polysaccharides in aqueous solution. J. Polymer. Sci., 18: 547-558, 1980.
26. Saito, H., Ohki, T., and Sasaki, T. A "< nuclear magnetic resonance studyof gel-forming (1—»3)-0-o-glucans.Evidence of the presence of single-helicalconformation in a resilient gel of a curdlan-type polysaccharide 13140 fromAlcaligenes faecalis var. myxogenes IFO 13140. Biochemistry, 16: 908-914,1977.
27. Czop, J. K., and Austen, K. F. Genaration of leukotriens by human monocytesupon stimulation of their ß-glucanreceptor during phagocytosis. Proc. Nati.Acad. Sci. USA, 82: 2751-2755, 1985.
28. Okuda, T., Yoshioka, Y., Ikekawa, T., Chihara. G.. and Nishioka, K. Anti-complementary activity of antitumour polysaccharides. Nature New Biol.,238: 59-60, 1972.
29. Morikawa, K., Takeda, R., Yamazaki, M., and Mizuno, D. Induction oftumoricidal activity of polymorphonuclear leukocytes by a linear ,¡1.1 nglucan and other immunomodulators in murine cells. Cancer Res., 45:1496-1501, 1985.
