6-Substituted purines: A novel class of inhibitors of endogenous protein degradation in isolated rat hepatocytes

ARCHIVES OF BIOCHEMISTRYAND BIOPHYSICS

Vol. 21’7,No. 1, August, pp. 282-294, 1982

6-Substituted

Purines: A Novel Class of Inhibitors of Endogenous Degradation in Isolated Rat Hepatocytes

Protein

PAUL B. GORDON AND PER 0. SEGLEN’ Norsk Hydra’s Institute

for Cancer Research, Department HospU,

of Tissue Culture, The Nonvegian Radium Mon&beUo, Oslo 3, Norway

Received December 11, 1981, and in revised form February 23, 1982

About 100 different purine derivatives and analogs were tested for their effect on protein synthesis and protein degradation in isolated rat hepatocytes. These included 6-aminopurines (adenine and adenosine analogs), 6-mercaptopurines, chloropurines, oxypurines, cytokinins, methylxanthines, methylindoles, benzimidazoles, and benzodiazepines. Most of the compounds were either inactive or inhibited protein synthesis as much as or more than they inhibited protein degradation. However, three methylated 6-aminopurines (3-methyladenine, 6-dimethylaminopurine riboside, and puromycin aminonucleoside) and four 6-mercaptopurines (6-methylmercaptopurine, 6-methylmercaptopurine riboside, 6-mercaptopurine riboside, and 2’,3’,5’-triacetyl-6-mercaptopurine riboside) had a markedly stronger effect on protein degradation than on synthesis, and might therefore be potentially useful as selective degradation inhibitors. None of the seven above-mentioned purines had any significant effect on the degradation of the exogenous protein, asialofetuin, and would therefore seem to selectively inhibit endogenous protein degradation. Since the degradation was not further affected by purines in the presence of amino acids or lysosomotropic amines, it is suggested that the purines exert their effect specifically upon the autophagic/lysosomal pathway. All the mercaptopurines significantly depressed cellular ATP levels, whereas the methylated aminopurines did not. For this reason, the latter are probably more useful as degradation inhibitors. 3-Methyladenine had no effect on protein synthesis at a concentration (5 mM) which inhibited protein degradation by more than 60%, and may therefore be regarded as a highly specific inhibitor of autophagy. Endogenous protein degradation is believed to take place both inside and outside lysosomes (l-3). The lysoscmutl pathway receives most of its material from autophagy, i.e., the process by which discrete regions of cytoplasm become sequestered as membrane-bounded corpuscles, autophagosomes, which eventually fuse with lysosomes (4-5). Lysosomes can also be fed by heterophagy, i.e., the uptake of exogenous material into heterophagosomes (endosomes, coated vesicles, receptosomes) which subsequently fuse with lysosomes (6, ‘0. 1Author to whom all correspondence should be addressed. 0003-9861/82/090282-13$02.00/O 282 Copyright All rights

Q 1982 by Academic Press, Inc. of reproduction in any form reserved.

The basic control of autophagy is exerted by amino acids, which appear to suppress the formation of autophagosomes (8-10). The pancreatic hormones, insulin and glucagon, have opposite effects on endogenous protein degradation in the liver (9, ll-17), insulin suppressing and glucagon stimulating the formation of autophagosomes (9, 18-20). The hormone effects are markedly dependent on the concentrations of amino acids, and may represent a modulation of the more basal amino acid control (9, 17). The nml~sosmml pathway, i.e., the fraction of endogenous protein degradation which is resistant to inhibitors of lysosomal function (l-3), may represent the

INHIBITION

OF PROTEIN

DEGRADATION

sum of the activities of several proteolytic systems. For example, the energy(oxygen)-dependent part of the nonlysosomal degradation in liver cells (3, 21) may to a certain extent reflect the activity of the hepatic ATP-dependent proteinase complex (22, 23), and enzymes such as the Ca’+-dependent proteinases (24) may contribute to the energy-resistant part. A provisional characterization of the pathways of endogenous protein degradation has been achieved through the use of various inhibitors effective on intact cells (l-3, 21). These include lysosomotropic amines (3, 25-27), microbial proteinase inhibitors (3, 21, 2%32), diazomethylketones (33), vanadate (34), cytoskeleton poisons (35, 36), and energy inhibitors (1,3). All of these agents inhibit the lysosomal pathway (only chymostatin and energy inhibitors inhibit nonIysosoma1 degradation too, cf. Ref. (21)), but they act upon late steps (lysosomal activity and/or fusion) which are common to autophagy and heterophagy. Rather than suppressing autophagy, most degradation inhibitors cause a paradoxical accumulation of autophagosomes, probably by preventing fusion of the latter with lysosomes (37). Apart from the natural regulators (amino acids and insulin) no inhibitors of autophagy have been available, an impediment which has greatly delayed the biochemical investigation of that process, In bacteria, amino acid starvation results in the accumulation of deacylated tRNA, which in turn stimulates the accumulation of a regulatory nucleotide, guanosine tetraphosphate (ppGpp),2 which both inhibits protein synthesis and stimulates protein degradation (38). Inhibitors of protein synthesis generally promote increased tRNA charging, reduced ppGpp levels, and an inhibition of bacterial protein degradation (44). Although eukaryotic cells do not contain ppGpp, protein synthesis inhibitors have been found to inhibit protein degradation in a variety of ’ Abbreviations used: DMR, 6-dimethylaminopurine riboside (N’,N”-dimethyladenosine); PAN, puromycin aminonucleoside; 3-MeAde, 3-methyladenine; ppGpp, guanosine tetraphosphate.

BY PURINE

DERIVATIVES

283

eukaryotic cell types (39-44), and there is some evidence suggesting that tRNA aminoacylation may regulate protein degradation in eukaryotes as well as in prokaryotes (45). However, evidence to the contrary has also been presented (39, 46-48). In a previous communication (49) we showed that the amino acid control of protein degradation in hepatocytes could take place independently of protein synthesis. The protein synthesis inhibitor, cycloheximide, affected protein degradation detectably only after a lag of 50-60 min, making it possible to distinguish between primary inhibition of protein degradation and effects secondary to inhibition of protein synthesis. On this basis we could demonstrate that certain purines closely related to puromycin (puromycin aminonucleoside and 6-dimethylaminopurine riboside), which affected protein synthesis moderately, were strong inhibitors of endogenous protein degradation. The lack of effect of these purines on the degradation of exogenous protein (heterophagy) indicated that they might be inhibitors of autophagy, an assumption supported by quantitative electron microscopy (50). In the present study, we have investigated a large number of purine derivatives with the purpose of finding maximally selective inhibitors of autophagy, i.e., compounds with little or no effect on protein synthesis or heterophagy. During the initial screening, the effect of the purines on protein synthesis and endogenous protein degradation was compared. Compounds with a markedly stronger effect on the latter process were selected for further study, thus eliminating nonspecific and nonselective inhibitors as well as drugs which inhibited degradation only as a secondary consequence of synthesis inhibition. MATERIALS

AND METHODS

Chemicals. [‘%lValine (CFB. 75) was obtained from The Radiochemical Centre, Amersham, Bucks, United Kingdom. [‘251JAsialofetuin was a gift from H. Tolleshaug, Institute for Nutrition Research, Oslo, Norway. 3-Deazaadenosine was purchased from Southern Research Institute, Birmingham, Alabama; 5,6-

284

GORDON AND SEGLEN

dichloro-la-D-ribofuranosylbenzimidazole from Calbiochem A.G., Lucerne, Switzerland; 2-methylbenzimidazole and 3-methyladenine (6amino-3methylpurine) from Fluka AG, Buchs, Switzerland, 2’,3’-isopropylidene-6-chloropurine riboside from P-L Biochemicals Inc., Milwaukee, Wisconsin; and 2,3’,5’-triacetyl-6-mercaptopurine riboside from Serva Feinbiochemica GmbH, Heidelberg, Federal Republic of Germany. erythro-9-(2-Hydroxy-3-nonyl)adenine was a gift from Burroughs Wellcome Company, Research Triangle Park, North Carolina, and the benzodiazepines were donated by F. Hoffmann-La Roche, AG., Basel, Switzerland. N,N-Dimethyl-g-(tetrahydro-2H-pyran-2-yl)adenine (NSC 35859), trimethyl[9-(tetrahydropyran-2-yl)-9~-purin-6-yl] chloride (NSC 53346), 6-(1-aziridinyl-g-(tetrahydroW-pyran-2-yl)-SH-purine (NSC 34491), and parahydroxybenzylidene puromycin (3’-deoxy-3’-[a-[@hydroxybenzylidene)amino] - p - methoxyhydrocinnamamidol-N,iV-dimethyladenosine, NSC 44647) were gifts from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland. Other biochemicals were obtained from Sigma Chemical Company, St. Louis, Missouri. Cell preparation and incubation. Hepatocytes were prepared by collagenase perfusion (51) from the liver of 250- to 300-g male Wistar rats, maintained on a controlled feeding and illumination schedule, and starved for 18 h prior to cell isolation. The cells were incubated as suspensions (0.4 ml) in shaking centrifuge tubes at 37“C, at a concentration of 50-75 mg wet wt/ml in suspension buffer (51) fortified with pyruvate (20 m&Q, Mgz+ (2 mM), and gentamicin (10 Fg/pl). For measurements of protein synthesis, the incubation medium was supplemented with a complete mixture of amino acids (lo), and for measurements of protein degradation, with 15 mM valine. Incubations were terminated by the addition of 0.1 ml 10% (w/v) perchloric acid. Protein synthesis. Protein synthesis was measured as the incorporation of [‘“C]valine of constant specific radioactivity (5 mM and 0.25 &i/ml) during a 60-min incubation in amino acid-supplemented medium. The rate of synthesis was calculated as described previously (52), and expressed as percentage per hour. Endogewus protein clegradaticnz. Endogenous protein degradation was measured as the release of [i4Clvaline (from protein labeled in viva for 24 h (3)) between 30 and 90 min of incubation in medium (buffer) supplemented with 15 mM valine. The degradation rate was expressed as percentage per hour of the total protein radioactivity present at 30 min. Degradation of exogenous protein (asialofduin). Degradation of an exogenous protein, asialofetuin, was measured by the method of Tolleshaug et al. (54). After endocytosis of [‘“Ilasialofetuin for 15 min at 3’7°C the hepatocytes were washed (3X at O’C) and

reincubated for 60 min at 37°C. The release of acidsoluble ‘%I was expressed as a percentage of the initial protein radioactivity. CeUulur ATP content. ATP was measured in neutralized perchloric acid extracts by means of a bioluminescence monitor (Luminometer, LKB-Wallac), using the luciferin/luciferase assay kit from LKBWallac. ATP content was expressed as micromoles per gram cellular wet weight, using the wet weight of the cells before incubation as reference base. Evaluation of inhibitor effects. During the initial screening (Table I), test compounds were added to the cells at 0.1 and 1.0 mM, with three parallel cell samples at each concentration. Standard errors averaged 2% for protein synthesis and 4% for protein degradation; effects in excess of 10% could therefore be regarded as significant. As an index of the selectivity of the inhibitors, the ratio between the rates of protein synthesis and degradation, relative to the control, was computed. Inhibitors affecting protein synthesis and degradation equally would thereby get a ratio of 1.0. Compounds selectively inhibiting protein synthesis would get a ratio below 1.0, whereas compounds selectively inhibiting protein degradation would get a ratio higher than 1.0. Compounds producing a ratio of 1.3 or higher were considered to be of interest as potentially specific inhibitors of degradation and were subjected to further testing over a wider concentration range. The initial screening would eliminate nonspecific (general toxicity, interference with energy metabolism etc.) and other nonselective effects (synthesis inhibited as much as or more than degradation) as well as those cases in which inhibition of degradation was secondary to synthesis inhibition (49). RESULTS

&Dirnethylamin~rine R&side: A Selective Inhibitor of Autophagic/ Lgsosmal Protein Degradation 6-Dimethylaminopurine riboside (DMR) (N6,N6-dimethyladenosine) and its structurally closely related analogue puromytin aminonucleoside (PAN) (N6,N6-dimethyl-[3’-amino-3’-deoxyladenosine) were previously found to inhibit protein degradation in isolated rat hepatocytes (49). Figure 1 shows that DMR was maximally effective at 0.1-0.2 mM, inhibiting degradation almost to the same extent as did either propylamine, a lysosomotropic weak base (26), or amino acids in a complete mixture designed to suppress autophagy (10). Since the effect of DMR was not significantly additive to any of the latter

INHIBITION

OF PROTEIN DEGRADATION

285

BY PURINE DERIVATIVES

TABLE I EFFECT OF VARIOUS PURINES ON PROTEIN SYNTHESIS AND DEGRADATION IN ISOLATED RAT HEPATOCYTES

Compound Adenosine analogs Adenosine 1-Methyladenosine 3-Methyladenosine N’-Methyladenosine 6Dimethylaminopurine riboside Puromycin aminonucleoside Puromycin Parahydroxybenzylidene-puromycin N6-Hydroxyadenosine 1-N6-Ethenoadenosine 2’-0-Methyladenosine 3’-0-Methyladenosine 2’-Deoxyadenosine 3’-Deoxyadenosine (cordycepin) N-Benzoyl-2’-deoxyadenosine 2’3’-0-Isopropylideneadenosine Adenine arabinoside 3-Deazaadenosine 7-Deazaadenosine (tubericidin) Formycin B Kinetin riboside 6-(y,y-Dimethylallylamino)purine riboside 6-Benzylaminopurine riboside Zeatin riboside Adenine derivatives Adenine 1-Methyladenine 2-Methyladenine 3-Methyladenine N6-Methyladenine 6-Dimethylaminopurine 6-(@Hydroxyethylamino)purine 6-n-Hexylaminopurine 6-Histaminopurine 2,6-Diaminopurine 8Azaadenine erythro-9-(2-hydroxy-3-nonyl)adenine N,N-Dimethyl-9-(tetrahydro-Wpyran-2-yl)adenine (NSC 35859) Trimethyl[S-(tetrahydropyran-2-yl)SH-purin-6-yllammonium chloride (NSC 53346) 6-(1-Aziridinyl)-9-(tetrahydro-2Hpyran-2-yl)-9H-purine (NSC 34491) Kinetin 6-Benzylaminopurine

Percentage effect on protein synthesis

Percentage effect on protein degradation

Relative synthesis/ degradation ratio

0.1 mM

1 mM

0.1 mM

1 mM

0.1 mM

1 mM

-5 -14 0 -15 -27 -2 -92 -99 -6 -6 -3

-24

-6 +1 -6 -7 -48 -14 -24 -28 +11 -8 -3 -8 -2 -12 -12 -9 0

-31 -27

1.0 0.9 1.1 0.9 1.4 1.1 0.1 0.0 0.9 1.0 1.0 1.0 1.0

1.1 1.2 1.2

-11

-15 -5 -85 -91 -31 -100 -100

-46 -9 -14 -68 -62 -96 -30 -28 -58 -23 -97 -13

+2 -56 +5 -4 -32 -8 -21 -4 -38

-100

-28 +9 -38

-40 -32

-100 -97

-45 -22

-18

-23

-5 -8 +2 +5 -14 +7 -6 -13 -13 +6 -3 -6

-1 -12

0.5 1.2

-10

1.1

-34 -18 -76

0.7

0.4 0.3 1.9 0.0 0.0 0.8 1.1 1.0 1.0

0.7 0.2 0.8 0.8 0.6 0.9

-80

1.0 1.1 0.9 1.0 1.1 0.9 0.9

0.0 -

-10

-29 -8 -27 -7 -42 -41 -41 -32 -95 -51 +3 +4 -66

0.9 1.0 1.1

1.2 0.9 1.2 0.9 0.9 0.9

1.1 1.2 1.2 1.8 1.3 1.5 1.1 0.0

-100

f3 -12 -6 -13 -9 -12 +3 -6 -6 -5 +2 -4

1.1 1.0 1.0

1.0 0.9 0.0

-8

-49

-2

-41

0.9

0.9

-10

-41

0

-39

0.9

1.0

-7 -9 -17

-33 -77 -100

-6 -13 -6

-18 -70 -

1.0 1.0 0.9

0.8 0.8 0.0

+8 +6 -25 -12 -22 -100

-85 +7 -6

-10

-19

-65 -72 -62 -63 -74 -34 -18 -15 -69 -47 -75 -12

+19

0.1

0.7 0.0

1.1

0.3

286

GORDONANDSEGLEN TABLE I-Continued Percentage effect on protein synthesis

Percentage effect on protein degradation

Relative synthesis/ degradation ratio

0.1 mM

1 mM

0.1 mM

1 mM

0.1 mM

-12 -14 -9 -8 +3 0 -6 +2 -3 -3

-17 -30 -48 -92 -98 -85 -76 -13 -17 -8

-8 -6 +6 -6 -8 -12 -18 -6 -9 -6

-16 -50 -50 -68 -84 -84 -77 -24 -11 +1

1.0 1.4 1.0 0.3 0.1 0.9 1.0 1.1 0.9 0.9

-3 -2 -15 -31

-17 -27 -37 -71

-6 -4 -14 -49

-21 f12 -59 -82

1.1 0.7 1.5 1.6

-8

-25

-12

-47

+5 -12 -6

-22 -31 -17

-5 -11 -10

-47 -19 -19

-6 -3 -9 -18 -7 -3

-14 -70 -10 -82 -47 -50

-6 +1 -7 +2 0 -3

-22 -68 -4 -77 -43 -55

-3 ‘-8

-43 -49

-53 -38

6-Oxypurines 6-Methoxypurine 6-Ethoxypurine 6-n-Propoxypurine 6-Isopropoxypurine 6-Methoxypurine riboside

+3 +2 -15 -13 +5

-5 -18 -88 -93 -15

-24 -19 -50 -53 -18

Other purines Purine riboside Guanosine Hypoxanthine arabinoside Inosine 1-Methylinosine %Methylinosine 6-Methylpurine 1-Methylhypoxanthine

-8 -6 -6 +1 -3 0 -4 0

-97 -9 -15 +4 +5 -6 -8 -10

-79 -6 -14 +2 -11 -1 -7 -17

Compound 6-Mercaptopurines 6-Mercaptopurine 6-Methylmercaptopurine 6-Ethylmercaptopurine 6-n-Propylmercaptopurine 6-n-Butylmercaptopurine 6-Benzylmercaptopurine Azathioprine 6-(4-Carboxybutyl)mercaptopurine 6-Carbethoxymethylmercaptopurine 6-Carboxymethylmercaptopurine 6-Carboxymethylmercaptopurine hydrazide 2-Amino-6-methylmercaptopurine 6-Mercaptopurine riboside 6-Methylmercaptopurine riboside 2’,3’,5’-Triacetyl-6-mercaptopurine riboside 2,3’-Isopropylidene-6-mercaptopurine riboside 6-Mercaptoguanosine C-Mercaptopurine arabinoside 6-Chloropurines 6-Chloropurine 2,6-Dichloropurine 2,6-Dichloro-7-methylpurine 2,6,8-Trichloropurine 2,6,8-Trichloro-7-methylpurine 6-Chloropurine riboside 2’,3’-Isopropylidene-6-chloropurine riboside 6-Chloroguanosine

1.0 1.0 1.0 0.8 0.9 1.0

1 mM

1.1 0.9 0.9 0.8 0.9 1.1 1.2 0.8

1.1 1.1 0.8 0.9 1.1

INHIBITION

OF PROTEIN DEGRADATION

287

BY PURINE DERIVATIVES

TABLE I-Continued

Compound Methylxanthines 3-Methylxanthine 1,‘7-Dimethylxanthine 1,3-Dimethylxanthine 3,7-Dimethylxanthine 7-Methylxanthosine Aminophylline

(theophylline) (theobromine)

Methylindoles 2-Methylindole 3-Methylindole 5-Methylindole ‘I-Methylindole Benzimidazoles Benzimidazole 2-Methylbenzimidazole 5,6-Dichloro-l-b-Dribofuranosylbenzimidazole Benzodiazepines Diazepam Bromazepam Chlordiazepoxide Clonazepam Flurazepam Medazepam Nitrazepam

Percentage effect on protein synthesis

Percentage effect on protein degradation

Relative synthesis/ degradation ratio

0.1 mM

1 mM

0.1 mM

1 mM

0.1 mM

1 mM

-14 -11 -11 -11 -11 -4

-5 -15 -8 -16 -6 -38

-10 -3 -10 -5 -10 -10

-10 -23 -17 -11 -11 -31

1.0 0.9 1.0 0.9 1.0 1.1

1.1 1.1 1.1 0.9 1.1 0.9

-9 -17 -11 -17

-96 -77 -89 -74

-12 -17 -11 -9

-81 -62 -64 -45

1.0 1.0 1.0 0.9

0.2 0.6 0.3 0.5

-4 -7

-22 -28

-3 -5

-19 -32

1.0 1.0

1.0 1.1

-8

-59

+4

-51

0.9

0.8

-21 -5 -10 -12 -7 -21 -16

-95 -26 -84 -75 -81 -96 -59

-6 -9 -10 -10 -5 -7 -3

-71 -26 -60 -62 -66 -74 -51

0.8 1.0 1.0 1.0 1.0 0.8 0.9

0.2 1.0 0.4 0.7 0.6 0.2 0.8

Note. Hepatocytes were incubated at 37°C with 0.1 or 1.0 mM of the test compound. Protein synthesis was measured as the incorporation of [14C]valine into protein during a 60-min incubation in the presence of an amino acid mixture (8), and protein degradation (in a separate experiment) as the release of [14C]valine (from in t&o-labeled protein) between 30 and 90 min of incubation in an amino acid-free medium. The effect of the test compounds is given as % inhibition (-) or stimulation (+) relative to inhibitor-free controls; the latter averaged 0.7%/h for the rate of protein synthesis (0.69 + 0.02; n = 18 Expts) and 4.3%/h for the rate of protein degradation (4.27 rf 0.14, n = 21 Expts). Each value is the mean of three to six cell samples from one or two experiments; SE in individual experiments averaged 2% for synthesis and 4% for degradation. Changes greater than 10% can therefore be regarded as significant in both cases. The specificity of inhibition is expressed by the synthesis/degradation ratio in the presence of the test compound relative to the control ratio (defined as 1.0).

treatments, it seems reasonable to assume that the purine inhibits the autophagic/ lysosomal pathway of degradation (3). In accordance with this assumption, DMR was found to have much less effect (26% inhibition at 0.13 InM, Ref. (53)) on the degradation of short-lived (l-h labeled) protein, which is assumed to be degraded

predominantly by nonlysosomal mechanisms (l-3). At 0.13 mM, DMR inhibited endogenous protein degradation by about 65% after a time lag of lo-15 min (Fig. 2A); yet it had no effect on the degradation of an exogenous protein, asialofetuin (Fig. 2B). Since asialofetuin is known to be degraded

288

GORDONANDSEGLEN

in lysosomes after heterophagic ingestion (adsorptive endocytosis) (54, 55), the lack of effect of DMR indicates that the purine acts upon a prelysosomal step unique to autophagy, as is the case with amino acids (10,16). Electron microscopic studies suggest that DMR inhibits the earliest morphologically recognizable step in the autophagic sequence, i.e., the formation of autophagosomes (50). Eflect of Llimeth$amkqnwine Riboside on Protein Synthesis and Cellular ATP 0.15 0.05 0.10 DIMETHYLAMINOPIJRINE

0.50

0.20 RIBOSIDE

lmt.4)

FIG. 1. Dose-response curve for inhibiton of endogenous protein degradation by 6-dimethylaminopurine riboside: effect of propylamine and amino acids. Hepatocytes labeled in wivo for 24 h were incubated at 3’7°C and the net release of [14C]valine from radioactive protein was measured between 30 and 90 min of incubation in the presence of various concentrations of 6-dimethylaminopurine riboside and other additions as indicated. 0, Control; 0, +l X amino acid mixture (8); A, + propylamine (10 mM). Each value is the mean + SE of six cell samples from two different experiments, normalized to the same (average) control value.

ENDOGENOUS

20

The effect of DMR on protein synthesis was moderate compared to its effect on degradation, i.e., only a 30-35s inhibition of synthesis at 0.13 mM (Fig. 3). Higher DMR concentrations increasingly inhibited protein synthesis (Fig. 4) while having little additional effect on degradation (Fig. 1); 0.13 mM was therefore chosen as the standard working concentration of this inhibitor. Figure 4 shows that DMR inhibited protein synthesis similarly in the absence and presence of added amino acids. The latter stimulate protein synthesis

EXOGENOUS PROTEIN IASIALOFETU~N)

LO

60

60 INCUBATION

20 TIME

LO

60

so

(min.1

FIG. 2. Effect of 6-dimethylaminopurine riboside on the degradation of endogenous and exogenous protein. Hepatocytes were incubated at 37°C in the presence (0) or absence (0) of 0.13 rnM 6dimethylaminopurine riboside. (A) Endogenous protein degradation was measured as the release of [“Cjvaline from protein labeled for 24 h in wivo. (B) The degradation of the exogenous protein, [‘2SI]asialofetuin, was measured as the release of acid-soluble i%I from protein ingested during a 15min preincubation of the cells. The amount of protein degraded is expressed as percentage of the total acid-insoluble radioactivity at the beginning of incubation. Each value is the mean rt SE of six samples from two different experiments. Some of the standard errors are too small to be seen.

INHIBITION

20

OF PROTEIN

I

I

I

LO

60

60

INCUBATION

TIME

fmin

DEGRADATION

100 )

FIG. 3. Effect of 6-dimethylaminopurine riboside on protein synthesis. Hepatocytes were incubated at 37°C in the presence (0) or absence (0) of 6-dimethylaminopurine riboside (0.13 mM), and protein synthesis was measured as the incorporation of [14Cjvaline (5 mM). Each value is the mean + SE of six cell samples from two different experiments.

(56) and would be expected to eliminate the need for amino acids derived from endogenous degradation (52); the persistence of the DMR inhibition therefore indicates that the effect of the purine on protein synthesis is independent of its effect on degradation. Table II shows that DMR did not interfere with hepatocytic ATP levels. It is therefore not likely that the effect of the inhibitor on protein synthesis or degradation is due to any nonspecific disturbance of cellular energy metabolism. In the absence of inhibitors, the hepatocytes maintained a constant ATP level throughout 90 min of incubation (2.8 + 0.2 pmol ATP/g wet wt; mean f SE of six experiments).

BY PURINE

289

DERIVATIVES

A number of other adenosine derivatives and analogs were tested at 0.1 and 1 InM (Table I). Except for some strong inhibitors of protein synthesis (puromytin, parahydroxybenzylidenepuromycin, 3’-deoxyadenosine (cordycepin), 7-deazaadenosine (tubericidin), kinetin riboside, 6 - [y,y - dimethylallylamino]purine riboside, 6-benzylaminopurine riboside, and adenine arabinoside), none of the compounds showed much activity at 0.1 mM. At 1 mM, adenosine itself inhibited both protein synthesis and degradation moderately (25-30s). Other compounds inhibited protein synthesis moderately, i.e., 30% or less (1-methyladenosine, 1-N6-ethenoadenosine, 2’-0-methyladenosine, N6benzoyl-2-deoxyadenosine, 2’,3’-O-isopropylideneadenosine, 3-deazaadenosine, zeatin riboside), or strongly, i.e., 45% or more (N’-methyladenosine, N6-hydroxyadenosine, 3’-0-methyladenosine, 2’-deoxyadenosine). As would be expected (49), the synthesis-inhibitory adenosine derivatives also had a pronounced inhibitory effect on protein degradation, although usually somewhat lower than on synthesis. The lack of a quantitative correlation was particularly evident with the most specific

Other Adenosine Analogs PAN differs from DMR only in having a hydroxyl group replaced by an amino group in the ribose moiety (49). PAN shared the specificity of DMR as an inhibitor of autophagic/lysosomal degradation, showing only a moderate effect on protein synthesis and no reduction in asialofetuin degradation or ATP levels (Table II); however, approximately IO times higher concentrations were required when PAN was used.

I

I 0.1

I 0.2

DIMETHYLAMINOPURINE

I 0.3

I 01 RIBOSIDE

I 0.5 ImM)

FIG. 4. Dose-response curve for inhibition of protein synthesis by 6-dimethylaminopurine riboside: effect of amino acids. Hepatocytes were incubated for 60 min at 3’7°C with various concentrations of 6-dimethylaminopurine riboside, and protein synthesis was measured as the net incorporation of [i4C]valine (5 mrd) during this period. 0, Control; 0, + amino acid mixture (8). Each value is the mean + SE of 12 cell samples from three different experiments, normalized to the same (average) control value.

GORDON

AND SEGLEN

TABLE

II

EFFECTSOF SOMEMETHYLATED 6-AMINOPURINESAND 6-MERCAPTOPURINES ONENDOGENOUSAND EXOGENOUSPROTEINDEGRADATION,PROTEINSYNTHESISANU CELLULARATP CONTENT Percentage effect on

Compound Methylated 6-aminopurines 6-Dimethylaminopurine riboside Puromycin aminonucleoside 3-Methyladenine

Endogenous protein degradation

Asialofetuin degradation

Protein synthesis

content

0.13 1.0

-67

-7

5.0

-61 -66

fl

-36 -26 +3 -8

+3 +12 +3 +6

-7

1.5

-50 -68 -55 -75

1.5

-59

2.0

-65 -54 -63

-22 -34 -29 -35 -27 -33 -27 -33

-25 -48 -16 -28 -24 -35 -23 -15

Concn (mM)

10.0 6-Mercaptopurines 6-Methylmercaptopurine

1.0 1.5

6-Mercaptopurine riboside 2’,3’,5’-Triacetyl-6-mercaptopurine riboside 6-Methylmercaptopurine riboside

1.0

0.13 0.20

-60

0

-8

+6 +8

ATP

Note. Hepatocytes were incubated at 3’7°C with inhibitors at the concentrations indicated, and endogenous protein degradation (30-90 min), asialofetuin degradation (O-60 min), protein synthesis (O-60 min), and cellular ATP content (at 60 min) were measured as described under Materials and Methods. The effect of each inhibitor is given as % inhibition (-) or stimulation (+) relative to the inhibitor-free control. Values are the means of one to nine experiments, each with three parallel cell samples. The average SE was below 3% for every parameter measured, i.e., changes of 10% or more can be considered significant.

protein synthesis inhibitors, such as puromycin which inhibited protein synthesis and degradation 92 and 24%) respectively, at 0.1 mM. In many other cases, the concomitant inhibition of protein synthesis and degradation might be a nonspecific effect, which would not be unexpected at a concentration as high as 1 mM. Adenine Derivatives Adenine itself had no effect at either 0.1 or 1 mM, and several derivatives (l-methyladenine, 2-methyladenine, 2,6-diaminopurine, 8-azaadenine) were likewise relatively inactive (Table I). No derivatives showed any activity at 0.1 mM, but several exhibited nonspecific inhibition (i.e., of both protein synthesis and degradation) at 1 mM (6j&hydroxyethylaminolpurine, 6 -n- hexylaminopurine, 6 - histaminopu rine, erythro-9-[2-hydroxy-3-nonylladenine, NSC 35859, NSC 53346, and NSC

34491). More interestingly, both N6-methyladenine and N6,N6-dimethyladenine (6dimethylaminopurine) inhibited protein degradation by about 40% at 1 mM, while protein synthesis was inhibited only by 25 and 12%) respectively. However, at higher concentrations, the inhibition of protein synthesis increased relatively more than the inhibition of protein degradation (61 and 66%) respectively, at 10 mM N6-methyladenine and 66 and 69%, respectively, at 5 mM N6,N6-dimethyladenine). Although neither of the compounds reduced asialofetuin degradation or ATP levels at these concentrations, they are obviously not sufficiently selective to be useful as degradation inhibitors. 3-Methyladenine (3-MeAde), on the other hand, showed a unique selectivity in being the only purine derivative (among about 100 tested) found to be capable of inhibiting protein degradation (42% inhibition at 1 mM) without inhibiting pro-

INHIBITION

OF PROTEIN

DEGRADATION

tein synthesis at all (Table I). Even at higher concentrations (5-10 mM), which inhibited protein degradation by more than 60%, protein synthesis was essentially unaffected (Table II). 3-MeAde furthermore had no effect on either asialofetuin degradation or cellular ATP levels (Table II). Despite the relatively high concentration (5-10 mM) needed to produce a maximal inhibition of endogenous protein degradation, 3-MeAde ought to become very useful as a specific inhibitor of the autophagic/lysosomal pathway. Mercaptopurines 6-Mercaptopurine had very modest effects on protein synthesis and degradation even at 1 mM (approx 15% inhibition of both), but its methylated derivative 6methylmercaptopurine showed a strong and relatively selective inhibition of protein degradation (Table II). At 1.5 mM the effect of 6-methylmercaptopurine (68% inhibition of protein degradation; 34% inhibition of protein synthesis) was comparable to that of 0.13 mM DMR, as with the latter the selectivity was reduced at higher concentrations (results not shown). Other 6-substitutions produced compounds which were either relatively inactive (&carboxymethylmercaptopurine, 6-[ll-carboxybutyllmercaptopurine, 6-carbethoxymethylmercaptopurine) or which inhibited both protein synthesis and degradation strongly (6 - ethylmercaptopurine, 6-n propylmercaptopurine, B-n-butylmercaptopurine, 6-benzylmercaptopurine, azathioprine) at 1 mM (Table I). 6-Mercaptopurine riboside as well as its ribosyl-acetylated derivative 2’,3’,5’-triacetyl-6-mercaptopurine riboside inhibited protein degradation with a selectivity similar to that seen in the case of B-methylmercaptopurine, and in the same concentration range (optimal effects around 1.5 mM, cf. Table II). 6-Methylmercaptopurine riboside, on the other hand, was active in the same low-dose range as was DMR (63% inhibition of degradation and 33% inhibition of synthesis at 0.2 mM, cf. Table II). 2’,3’- Isopropylidene - 6 - mercaptopurine riboside was moderately selective at 1 mM

BY PURINE

DERIVATIVES

291

(Table I), but the selectivity disappeared at higher concentrations (results not shown). Some other mercaptopurines tested (2-amino-6-methylmercaptopurine, 6-mercaptoguanosine, 6-mercaptopurine arabinoside) showed only modest and nonselective effects (Table I). None of the four active mercaptopurines in Table II affected asialofetuin degradation appreciably, but they all caused significant depression of cellular ATP levels. For this reason, the mercaptopurines would seem to be less suitable than the aminopurines as selective inhibitors of endogenous protein degradation. Other Purine Derivatives and Analogs With the exception of 6-chloropurine and 2,6-dichloro-7-methylpurine, which were relatively inactive, all the tested chib ropurines (2,6-dichloropurine, 2,6,8-trichloropurine, 2,6,8-trichloro-7-methylpurine, 6-chloropurine riboside, 2’,3-isopropylidene-6-chloropurine riboside, and 6chloroguanosine) inhibited both protein synthesis and degradation relatively strongly at 1 mM (Table I). Among the oxypurines, only B-methoxypurine showed a moderate selectivity at 1 mM (Table I), which, however, disappeared at higher concentrations as the overall inhibition increased (results not shown). 6-n-Propoxy- and 6-isoproxypurine were strong inhibitors of protein synthesis at 1 mM, whereas 6-ethoxypurine and 6-methoxypurine riboside showed little activity. Several purines (guanosine, hypoxanthine arabinoside, 1-methylhypoxanthine, 6-methylpurine, and inosine and its methylated derivatives) were found to have little or no activity. Purine riboside, on the other hand, was a strong, nonselective inhibitor at 1 mM (Table I). With the exception of aminophylline (about 35% nonspecific inhibition at 1 mM), the methylxanthines tested (3-metnylxanthine, 1,7-dimethylxanthine, 2,3dimethylxanthine (theophylline), 3,7-dimethylxanthine (theobromine), and 7methylxanthosine) were all essentially inactive (Table I). Some methylindoles (2-, 3-, 5- and 7-

292

GORDONANDSEGLEN

methylindole), benzimidazoles (benzimidazole, 2-methylbenzimidazole, and 5,6dichloro-1-/Sribofuranosylbenzimidazole), and benzodiazepines (diazepam, bromazepam, chlordiazepoxide, clonazepam, flurazepam, medazepam, and nitrazepam) were tested because of the marked structural similarity between these groups of compounds and the purines. All of these substances were moderate (benzimidazole, 2-methylbenzimidazole, and bromazepam) or strong (the rest) inhibitors of both protein synthesis and degradation at 1 mM; no selective effects on degradation were seen (Table I). DISCUSSION

Considering the pivotal role of purines in energy exchange, nucleic acid metabolism, and numerous regulatory mechanisms, it is perhaps not surprising that many purine derivatives, at high concentrations, could be found to inhibit hepatocytic protein degradation. Most of these inhibited to an even greater extent protein synthesis, a process which is even more sensitive to energy deficiency and other general derangements than is protein degradation (57). Protein synthesis was therefore considered to be a relevant reference parameter for the evaluation of inhibitor selectivity. Unless the test compound showed a significantly greater inhibition of protein degradation than of synthesis, its effect was considered to be nonselective, although no attempt was made to elucidate the mechanism of general inhibitory effects during the coarse drug screening performed here. Among the compounds selected for more detailed investigation, seven were found to exhibit a reasonable selectivity for protein degradation vs synthesis. All of these would appear to act specifically upon autophagic degradation, as indicated by their lack of effect on asialofetuin degradation. This criterion has previously been found to hold true for the amino acids (10, 16), and preliminary electron microscopic studies indicate that the three inhibitory aminopurines (DMR, PAN, and 3-MeAde) all inhibit autophagosome formation to various degrees (Ref. (50) and unpublished experiments). The availability of reason-

ably specific inhibitors of autophagy may represent a significant advance compared to the lysosome inhibitors now in use (1, 3, 21, 25-36). The mercaptopurines are antimetabolites which inhibit nucleic acid synthesis and antibody production, and which have therefore found some use as antineoplastic and immunosuppressive drugs (58). They may also chelate Cu2+ (59), interfere with uridine transport (60), and induce differentiation in erythroleukemia cells (61). These drugs are regarded as toxic at much lower concentrations than those used in the present study, and their interference with ATP levels as well as with protein synthesis should perhaps caution against their indiscriminate use as degradation inhibitors. However, for well-defined shortterm studies they may provide a useful alternative to the aminopurines. The structural similarity between the active mercaptopurines and two of the active aminopurines, DMR and PAN, is obvious. Apparently two factors can contribute to degradation-inhibitory specificity: methylation of the 6-substituent, and/or the presence of a ribose moiety. The nonribosylated, nonmethylated parent compounds-6-aminopurine (adenine) and 6mercaptopurine, respectively-are both relatively inactive. Methylation of the 6substituent confers both activity and good degradation specificity upon the mercaptopurine molecule (6-methylmercaptopurine); the specificity is somewhat less pronounced in the case of the 6-methylated aminopurines (N’-methyladenine and N6,N6-dimethyladenine). 9-Ribosylation alone similarly turns the mercaptopurine into a selective degradation inhibitor (&mercaptopurine riboside), whereas the corresponding aminopurine (adenosine) in this case is weakly active and nonselective. The combination of 6-substituent methylation and 9-ribosylation produces a potent and specific inhibitory mercaptopurine (6methylmercaptopurine riboside) and an equally potent and specific aminopurine (DMR). The monomethylated aminopurine riboside (N’-methyladenosine), however, inhibits protein synthesis too strongly to be selective. The dimethylamino or (methyl)mercapto

INHIBITION

OF PROTEIN

DEGRADATION

groups in the 6-position apparently cannot be replaced by other substituents such as methoxy or chlorine. Modifications of the 6-substituent other than methylation are likewise ineffective. The ribose moiety, on the other hand, can be modified to some extent; both multiple acetylations (2’,3’,5’triacetyl-6-mercaptopurine riboside) and replacement of one hydroxyl group with an amino group (PAN) are compatible with degradation specificity. Whether the presence of two important specificity-conferring molecular domains reflects the existence of a dual recognition mechanism for degradation control, or whether it is related to the effect on protein synthesis, or to transport and/or metabolism of the molecule, remains to be shown. DMR binds strongly to intracellular macromolecules (results not shown) but no attempt has yet been made to characterize the binding activity. The specificity of DMR, PAN, and the mercaptopurines is probably not sufficient to make elucidation of their mechanism of action facile. PAN is known to inhibit RNA synthesis and growth in some types of cultured cells (62, 63) and to promote differentiation in others (64). While DMR itself was not studied, both 6-dimethylaminopurine (65) and 6-methylaminopurine riboside (66) have been found to affect carbohydrate metabolism in rodent tissues. The stucture of the most specific of the degradation-inhibitory purines, 3-MeAde, is quite different from the 6-mercaptopurines and the h@-methylated aminopurines, and the possibility must be considered that it acts by a different mechanism. It may perhaps be of interest that l-methyladenine (which showed little or no selectivity in the present study), a maturation hormone for starfish oocytes (6’7), exerts its effect upon the plasma membrane rather than intracellularly (68). To the authors’ knowledge, no biological effects of 3-MeAde have been described; however, it can be formed intracellularly during the excision repair which follows damage to DNA by methylating agents (69). The high specificity of 3-MeAde makes it the most promising among the new inhibitors of autophagic/lysosomal protein

BY PURINE

DERIVATIVES

293

degradation discovered in the present study. The value of a specific autophagy inhibitor for experimental purposes is obvious; its potential as a therapeutic agent should also be considered. Although almost nothing is known about the role of autophagy in either health or disease, the process might play a role under conditions where excessive tissue wasting occurs (during cancer chemotherapy or glucocorticoid treatment, in starvation or cachexia, in muscular dystrophies and demyelinating diseases, lysosomal storage diseases, etc.). As yet 3-MeAde has only been tested on isolated rat liver cells. Studies on its tissue and species specificity will be of major importance for evaluating the possible therapeutic applicability of 3MeAde. ACKNOWLEDGMENT This work was generously supported from The Norwegian Cancer Society.

by a grant

REFERENCES 1. KNOWLES, S. E., AND BALLARD F. J. (1976) Bi@ them. J. 156,609-617. 2. A~~IIENTA,J. S., SARGUS, M. J., VENKATESAN, S., AND SHINOZUKA, H. (1978). J. Cell. Physiol. 94, ‘77-86. 3. SEGLEN, P. O., GRINDE, B., AND SOLHEIM, A. E. (1979) Eur. J. Biochem. 95, 215-225. 4. DEAN, R. T., AND BARRETT, A. J. (1976) Essays B&hem. 12,1-40. 5. PFEIFER, U. (1976) Verh. Dtsch. Ges. Path&. 60, 28-64. 6. GOLDSTEIN, J. L., ANDERSON, R. G. W., AND BROWN, M. S. (1979) Nature (London) 279,679685. 7. PASTAN, I. H., AND WILLINGHAM, M. C. (1981) Science 214, 504-509. 8. MITCHENER, J. S., SHELBURNE, J. D., BRADFORD, W. D., AND HAWKINS, H. K. (1976) Amer. J. P&ml 83,485-492. 9. SCHWORER, C. M., AND MORTIMORE, G. E. (1979) Proc. Nat Acad Sci USA 76,3169-3173. lo. Kov&x, A. L., GRINDE, B., AND SEGLEN, P. 0. (1981) Exp. Cell Res. 133, 431-436. 11. SEGLEN, P. 0. (1968) Hoppe-&&r’s Z. Physiol. Chewt. 349, 1229-1230. 12. MORTIMORE, G. E., AND MONDON, C. E. (1970) J. Biol. Chem 245,2375-2383. 13. WOODSIDE, K. H., WARD, W. F., AND MORTIMORE, G. E. (1974) J. Biol Chem. 249, 5458-5463. 14. HOPGOOD, M. F., CLARK, M. G., AND BALLARD, F. J. (1980) B&hem J. 186, 71-79.

294

GORDONANDSEGLEN

15. GRAMMELTVEDT, R., AND BERG, T. (1976) Hoppe-

+%&r’s 2. Physid

Chem 357,977-981.

16. SEGLEN, P. O., GORDON, P. B., AND POLI, A. (1986)

Biochim Biqvhys. Ada 630,103-118. 17. POLI, A., GORDON, P. B., SCHWARZE, P. E., GRINDE, B., AND SEGLEN, P. 0. (1981) J. CeU Sci 48, l18. 18. ASHFORD, T. P., AND PORTER, K. R. (1962) J. CeU Bid 12, 198-202. 19. DETER, R. L. (1971) J. CeU Bid 48,473-489. 20. PFEIFER, U. (1978) J. CeU Biol 78,152-167. 21. GRINDE, B., AND SEGLEN, P. 0. (1980) B&him

43. AMENTA, J. S., SARGUS, M. J., AND BACCINO, F. M. (1977) Biochem. J. 168,223-227. 44. WOODSIDE, K. H. (1976) Biochim Biuphgs Actu 421,70-79. 45. SCORNIK, 0. A., LEDBE~ER, M. L. S., AND MALTER, J. S. (1989) J. Biol Chem 255,6322-6329. 46. STANNERS, C. P., WIGHTMAN, T. M., AND HARKIN& J. L. (1978) J. Cell. Ph@ol 95.125-137. 47. GUNN, J. M. (1978) Exp CeU Res. 117,448-451. 48. CLARK, J. L., RABE, J., AND ARFIN, S. M. (1979)

J. CeU.Physd 49. Kovics,

Biophys. Acta 632,73-86. 22. ETLINGER, J. D., AND GOLDBERG, A. L. (1977)

Proc. Nat. Ad

Sci USA 74.54-58.

23. HERSHKO, A., CIECHANOVER, A., HELLER, H., HAAS, A. L., AND ROSE, I. A. (1989) Proc Nat

Acad Sci USA 77,1783-1786. 24. NELSON, W. J., AND TRAUB, P. (1981) Eur. .I Biothem 116,51-57. 25. DE DWE, C., DE BARSY, T., POOLE, B., TROUET, A., T~LKENS, P., AND VAN HOOF, F. (1974)

Biochem Pha rmacol 23,2495-2531. 26. SEGLEN, P. O., AND GORDON, P. B. (1980) Mel Pharmacd 18,468-475. 27. OHKUMA, S., AND POOLE, B. (1978) Proc Nat.

Acad Sk USA 75,3327-3331. 28. DEAN, R. T. (1975) Nature (London) 257,414-416. 29. HOPGOOD, M. F., CLARK, M. G., AND BALLARD, F. J. (1977) Biochem. J 164,399-407. 30. NEFF, N. T., DE MARTINO, G. N., AND GOLDBERG, A. L. (1979) J. Cell Physiol 101,439-458. 31. LIBBY, P., AND GOLDBERG, A. L. (1989) Biochew. .I 188,213-220. 32. TANAKA, K., IKEGAKI, N., AND ICHIHARA, A. (1981) Arch. Biochem Biophys. 208,296-304. 33. SHAW, E., AND DEAN, R. T. (1980) Biochem J. 186, 385-390. 34. SEGLEN, P. O., AND GORDON, P. B. (1981) J. Bid Chem 256, 7699-7701. 35. AMENTA, J. S., SARGUS, M. J., AND BACCINO, F. M. (1977) Biochem J. 168.223-227. 36. GRINDE, B., AND SEGLEN, P. 0. (1981) Hoppe-Sey-

k-r’s 2. Physid

Chem 362,549-556.

37. Kovjlcs, A. L., REITH, A., AND SEGLEN, P. 0. (1982) Exp. CeU Res. 137.191-201. 38. ST. JOHN, A. C., CONKLIN, K., ROSENTHAL, E., AND GOLDBERG, A. L. (1978) J. Bid Chem 253, 3945-3951. 39. EPSTEIN, D., ELIAS-BISHKO, S., AND HERSHKO, A. (1975) Biochemistry 14,5199-5204. 40. GUNN, J. M., BALLARD, F. J., AND HANSON, R. W. (1976) J. Biol Chem 251,3586-3593. 41. MCILHINNEY, A., AND HOGAN, B. L. M. (1974)

Biochim. Biophys. A&

372,35%+X5.

42. NEELY, A. N., NELSON, P. B., AND MORTIMORE, G. E. (1974) Biochim Biophys. Acta 388, 45%

472.

98,237~240.

A. L., AND SEGLEN, P. 0. (1981) Biochim

Biophys Acta 676,213-229. 59. Kovlics, A. L., MOLN~R, K., AND SEGLEN, P. 0. (1982) FEBS Lett. 134,194-196. 51. SEGLEN, P. 0. (1976) Methods Cell Bid 13.29-83. 52. SEGLEN, P. 0. (1978) Biochem J. 174,469-474. 53. SEGLEN, P. O., GORDON, P. B., GRINDE, B., SOLHEIM, A., Kovics, A., AND POLI, A. (1982) Acta

Biol Med Germ 40,1587-1598. 54. TOLLESHAUG, H., BERG, T., NILSSON, M., AND NORUM, K. R. (1977) B&him. Biophya Acta

499,73-84. 55. VAN ZILE, J., HENDERSON, L. A., BAYNES, W. J., AND THORPE, S. R. (1979) J. Bid Chem 254, 3547-3553. 56. SEGLEN, P. O., SOLHEIM, A. E., GRINDE, B., GOR-

57.

58.

59. 66. 61. 62.

DON, P. B., SCHWARZE, P. E., GJESSING, R., AND POLI, A. (1980) Ann N. Y. Acad Sci. 349,1-17. SEGLEN, P. 0. (1978) in Protein Turnover and Lysosome Function (Segal, H. L., and Doyle, D. J., eds.), pp. 431-453, Academic Press, New York. CALABRESI, P., AND PARKS R. E. (1975) in The Pharmacological Basis of Therapeutics (Goodman, L. S., and Gilman, A., eds.), 5th ed., pp. 1254-1397, Macmillan, New York. KELA, U., AND VIJAWARGIYA, R. (1981) Biochem J. 193,799-803. SHOHAMI, E., KANNER, N., AND KOREN, R. (1980) B&him Biophys Acta 601,206-219. GUSELLA, J. F., AND HOUSMAN, D. (1976) Cell 8, 263-269. STUDZINSKI, G. P., AND ELLEM, K. A. 0. (1968)

Cancer Res. 28,1773-1782. 63. CHOLON, J. J., KNOPF, R. G., AND PINE, R. M. (1979) In Vitro 15, 736-742. 64. LACOUR, F., HAREL, L., FRIEND, C., HUYNH, T., AND HOUAND, J. G. (1980) Proc. Nat. Ad

Sci USA 77,2740-2742. 65. HOFERT, J. F., AND BOUTWELL, R. K. (1963) Arch, Biochem Biophys. 103.338-344. 66. SOLJNESS,J. E., AND CHAGOYA DE SANCHEZ, V. (1981) FEBS L&t. 125,249-252. 67. KANATANI, H., AND HIRAMOTO, Y. (1970) E~J. CeU Res. 61,280-284. 68. DOR~E, M. (1981) Exp. Cell Res. 131,115-126. 69. CATHCART, R., AND GOLDTHWAIT, D. A. (1981)

Biochemistry 20.273-280.