IiRCHIVES OF BIOCHEMISTRY 9ND BIOPHYSICS 144, 715-722 (1971)

Labeling of Rat Liver Glucose-l-Phosphate, Glucose-&Phosphate,

Uridine Diphosphate Glucose, and Glycogen During

Glycogen Synthesis

IKDRAJIT DAS, HSIEN-GIEH SIE, 9ND WILLIAM H. FISHMAX

Department of Pathology (Oncology), Tujte University School of fifedicine, Boston, Massachusetfs, and the Cancer Research Department, New England Medical Center Hospitals, Boston, Massachusetts 02111

Received February 1, 1971; accepted March 1, 1971

“C-Glucose was injected intravenously into rats and the livers were rapidly re-

moved at intervals from 2 to 30 min and homogenized in perchloric acid. Fractionation was carried out on Dowex-l-formate resin and glucose, glucose-l-phosphate, glucose- G-phosphate, uridine diphosphate glucose, and glycogen were isolated by repeated

chromatography. Both a comparatively high specific radioactivity and total radioac- tivity of glucose-l-phosphate as compared to glucose-6-phosphate persisted from the

earliest 2-min point to 10 min of incorporation. This indicated that formation of glu- cose-l-phosphate may be an early product of glucose phosphorylation during glycogen

synthesis in vivo from exogenous glucose units. It may also be an early product during hydrocortisone-induced glycogenesis in animals receiving 14C02 on t.he basis of a sec- ond series of experiments. The observations are explained on the basis of two separate

pools of glucose-G-P in metabolism.

Since Leloir and Cardini (1) showed that glycogen was synthesized from UDP- glucose’ by liver extracts, it has become commonly accepted that the normal mecha- nism of glycogen synthesis in liver and muscle involves the sequence of reactions, glucose + glucose-6-phosphate + glucose- l-phosphate --) UDP-glucose to glycogen (2, 3).

Beloff -Chain, Catanzaro, Chain, Masi, Pocchiari, and Rossi (4) have suggested that the hexokinase reaction is not an obligatory step in the synthesis of glycogen from glucose, and found that in rat dia- phragm glucose-l-P is a better precursor of glycogen than either glucose or glucose- 6-P (4, 5). The question of the essentiality of glucose-6-P for glycogen synthesis has

arisen from the work of Petrova (6) (rat liver slices), Figueroa and Pfeiffer (7) (rat liver homogenates), Threlfall (8) (rat liver

1 Abbreviations: glucose-l-P, glucose-l-phos- phate; glucose-6-P, glucose-g-phosphate; UDP- glucose, uridine diphosphate glttcose.

in viva), and Nigam (9) (hepatoma ascit,es cells).

The present investigation was under- taken to evaluate the relative roles of glucose-6-P and glucose-l-P in t,he syn- thesis of glycogen by rat liver in vivo. The experimental evidence is based on 14C- glucose labeling of the six-carbon phosphate precursors of liver glycogen as a fur&on of time after intravenous injection of a tracer dose of 14C-glucose into starved anesthetized rats. In addition, 14C0, was used to label the same intermediates during the course of hydrocortisone-induced gluconeogenesis and glycogenesis.

The most interesting result observed in a11 the experiments was the higher specific radioactivity of glucose-l-P as compared to that found for glucose-6-P. The bearing of this finding on current concepts of glucose utilization in glycogen synthesis is discussed in relation to the hypothesis of two glucose- 6-P pools.

715

716 DAS, SIE, AND FISHMAN

EXPERIMENTAL

Materials. [U-‘%?I-glucose and sodium bicar-

bonate-14C were obtained from New England Nu- clear Corp. Phosphate esters and enzymes for the

analytical determinations were obtained from Cal- biochem., Sigma Chemical Co., and Boehringer

and Soehne (Germany). Glucostat special enzyme was purchased from Worthington Biochemical Co. and Dowex-l-resin, 200400 mesh, chloride form,

was obtained from Baker Chemical Co. Except otherwise stated, the paper used for chromatog-

raphy was Whatman No. 1; 2,5-diphenyl oxazole, 1,4-bis-[2-(5-phenyl (oxazolyl))]-benzene and

napht,halene were of scintillation grade and were obtained from Pilot Chemical, and Packard In- strument Co. Scintillation grade dioxane was ob-

tained from Fisher Scientific Co. All other chemi- cals were obt,ained from commercial sources. Male

Wist,ar rats (200-400 g.) were obtained from Gof- moor Farm, Mass.

Incorporation studies: (a) %-glucose. The dose

of 14C-glucose was diluted into 11.0 mg of glucose to a final volume of 0.25 ml and was injected (2 set) into the femoral vein of male Wistar rats, ether

anest,hetized after 24-hr starvation. The duration of the experiment in minutes and the radioactive

dose (in microcuries) administered for four groups of two rats each were respectively; Group 1: 2, 20;

Grollp 2: 5, 10; Group 3: Rat 1, 10, 10; Rat 2, 10,5; Group 4: 30, 5.

(6) W-bicarbonate (10). Male Wistar rats, after

24 hr of fasting, were injected with hydrocortisone (5 mg/animal in saline microcrystalline suspen- sion) intraperitoneally. After 3 hr, %-sodium

bicarbonate (aqueous solution) was injected into the femoral vein of the ether-anesthetized rats; 50

pCi (5-min exp.) and 25 pCi (lo-min exp.). After intervals of 5 and 10 min the animals were killed.

Preparation of liver extracts. The livers were rapidly removed and immediately frozen in Dry

Ice. [Total elapsed time from excision of liver to complete freezing was 20 sec.] Next they were weighed and homogenized in a Potter-Elvehjem

homogenizer with 3 vol of ice-cold 0.6 N HClOd acid, and the homogenate was centrifuged in a refrigerated centrifuge at 18,000 rpm.

The sedimented residue was resuspended in 2 vol of 0.2~ HClOd and centrifuged. This sediment was reserved for the isolation of glycogen. The combined sypernatant solutions were neutralized with ice-cold 5.0 N KOH to pH 7.0, and the precipi- tated KCIOn was removed by centrifugation in the cold. Three portions of this extract were used to

determine the glucose-l-P, glucose-6-P, and UDP- glucose enzymatically.

Enzymatic Determination of Glucose-&P, Glu- cose-l-P, and UDP-Glucose. These methods were

applied to the extracts and purified samples as indicated later. The reaction mixture contained

(final concentrations) : Tris HCl, pH 7.5, 100 pM; MgC12, 10 PM; NADP+ 5 PM, excess glucose-6-P

dehydrogenase (EC 1.1.1.49). The volume was ad- justed when necessary to provide a total vol. of 1.0

ml. Three mixtures were prepared in cuvettes. One had buffered PCA extract and no enzyme added; the second contained buffered PCA extract plus

glucose-6-P dehydrogenase, and the third had PCA extract plus glucose-6-P dehydrogenase plus

phosphoglucomutase. Appropriate enzyme blanks were also included. The increase in extinction

measured at 340 nm gave the amount of glucose- 6-P. The increase at 340 rnp due to the addition of

phosphoglucomutase (EC 2.7.5.1) yielded the amount of glucose-l-P in a companion digest of the

same composition as that for glucose-6-P deter- mination.

The reaction mixture (1.0 ml) for UDP-glucose

assay contained glycine buffer 375 mM, pH 8.7,1.25 mM, NAD+, and excess UDP-glucose dehydrogen-

ase (EC 1.1.1.22). The increase in optical density measured at 340 rn# after the addition of enzyme gave the amount of UDP-glucose present.

Isolation of perchloric acid-insoluble glycogen. The glycogen remaining in the centrifuged sedi- ment after extraction with perchloric acid is re-

ferred to as PCA-insoluble and that recovered from the supernatant, as PCA-soluble. The PCA-

insoluble residue was digested with 30% KOH at 100” for 2 hr and centrifuged. The supernatant fraction was collected and 2 vol of 95% ethanol

were added to precipitate the glycogen which, after standing at 4’ overnight, was collected by centrifugation. It was purified by reprecipitation

with 2 vol of cold 95% ethanol or by extraction with 10% trichloracetic acid and reprecipitation with 5

vol of cold 95% ethanol. Specific radioactivity was determined by sepa-

rately measuring glycogen concentration and

radioactivity. Glycogen values were obtained by the cysteine-

sulfuric acid method of Dische (11). To 1 ml of the solution was added 4.5 ml H&O* (HPSOI, sp gr 1.84: water, 6:l by volume) and the mixture cooled in

ice. Then the tube was heated in a boiling water bath for 3 min, cooled in ice, and 0.1 ml of 3% t-cysteine hydrochloride was added. After 5 min at room temperature, the extinction at 415 nm was

measured and the glucose concentrations deter-

mined with the aid of a standard curve.

Radioactivity was measured (Mark I Liquid

Scintillation Counter, Nuclear Chicago) on a 0.5-

ml sample of the glycogen solution after it was

mixed with 0.5 ml of hyamine hydroxide and 9 ml

of scintillation liquid prepared by mixing 2,5-

717

diphenyloxazole, 1,4-bis[2-(5-phenyloxazolyl)-ll- benzene and naphthalene in dioxane (12). Counts

were corrected to 100% efficiency from the stand-

ard quenching curve by the channel ratio method. Pw-ijication and deterrrlinalion of specific adit-

dies oj PCA-soluble glycogen, gltccose, ghcose-1 -P, glucose-&P, and UDP-glucose. Dowex-1 formate was prepared from Dowex-l-chloride resin hy

treating it with 3 M sodium formate, filtering, and washing with water until the filtrate was free of

chloride. After repeating this several times, the resin was mixed with 50yo formic acid and finally

washed free of acid (13). The perchloric acid extract of liver (60-70 ml)

was passed slowly through a column (1 X 20 cm) of Dowex-1-formate, previously washed with water, which was then eluted with water till the

effluent was free of glucose as indicated by a negative cysteine-sulfuric acid test. The total

water eluate was evaporated t.o dryness, dissolved in a small quantity of water (water eluate) and

used in part for the determination of total glucose by the glucostat special reagent and also for the purification of glycogen and glucose.

Glucose determination. Glucostat andchromogen solutions were mixed in 2:l proportions before

using. At least one standard of glucose was run with each set of samples under test. To 1 ml of

water eluate 9 ml of the mixed reagent were added, and the mixt,ure was incubated at room tempera- ture. Exact,lv 10minlater the reaction was stopped

by the addition of 1 drop of 10 M HCl, and the extinction at 405 nm measured.

PCA-soluble glycogen. This was precipitated by

adding 2 vol of cold 95yo ethanol to 1 vol of water eluate ; purification and specific activity were

determined in the same way as in the case of PCA- insoluble glycogen. The siipernatant fraction contains glucose and oligosaccharides.

Glucose. The above ethanolic supernatant

fraction was evaporated to dryness, and the solid

was dissolved in 30 ml of water. Charcoal (8 g) was added. After standing 30 min at room tempera-

ture, the charcoal was filtered off under suction on Whatman No. 50 paper and washed with 57, ethanol.

Glucose was purified furt,her from the filtrate

by descending paper chromatography in two solvent systems. The first was n-butanol-acetic acid-water (52:13:35) and the second was the top layer after a mixture of ethyl acetate-pyridine-

water (2:1:2) was permitted to settle. After 24

hr in the first solvent and 8 hr in the second, t.he

papers were taken out. and the standard spot was

developed with the aniline hydrogen phthalate

reaction. All paper chromatograms were developed

at, room temperature 22-24’. Portions of the paper

corresponding in position to the st,andard spots

were extracted with water and the specific ac-

tivity was determined by measuring the concen- tration of glucose by the glucostat reagent.

Glucose-l-P, glucose-d-P, and UDP-glucose.

These were &ted from the Dowex column with 0.5 M ice-cold ammonium formate, the first 40 ml of the eluent (collected in ice) constit,uting t,he

glucose-l-P plus glucose-6-P fraction. The next 95.ml fraction was rich in UDP-glucose. The frac-

tions were concentrated and lyophilized for 7 hr, the last 4 hr of which required the lise of an in-

frared lamp to remove ammonium formate. Both fractions were dissolved in small amounts

of water and purified by repeated descending paper

chromatography. UDP-glucose from the UDP-glucose fraction

was developed for 16 hr in 95% ethanol-l M ammonium acet,ate, pH 7.5, (5: 2 by volume) (14). Areas corresponding to t.he standard spots, located

under an ultraviolet light, were extracted and

rechromatographed in the same solvent to enhance purity. Specific activity was determined by as- saying concentration with UDP-glucose dehv-

drogenase and by counting the same solut,ion in the scint,illation counter.

The glucose-1-P and glucose-6-P fractions were paper chromatographed using l-butanol-85y0 ammonia-water (60:30:10 by volume) as the solvent (15). Before use, the paper was washed

with 10 mM sodium borate solution and dried. After development for approximately 40 hr, the

solvent. heing allowed to overflow and drip from t,he serrated edge of the paper, glucose-l-P and

glucose-6-P were located by the phosphorus rea- gent of Hanes and Isherwood (16) as modified hy Bandurski and Axelrod (17). After extraction with

water glucose-l-P and glucose-6-P were separately rechromatographed in the same solvent but using

paper washed with 1 mM borate. After extraction from the paper the specific activity was deter- mined by enzymatic assay of the individual sugar

phosphates and by counting the same solution. The possibility t)hat, the radioactivity at the

position of glucose-l-P is due to a nonglucose radioactive contaminant was ruled alit as follows. First, the spot was eluted wit.h water, hydrolyzed with dilute acid, and then the mixture was chro-

matographed in aqueous phenol solvent (18) or the n-hutanol-acet,ic acid-water system men- tioned ahove. The area indicated by a glucose

standard was cut out, eluted, and the radioac-

tivity measrired. The specific radioactivity of

t.his gliicose was wit,hin experimental error of that

present in the original glucose-l-P.

Similar experiments were carried out for glir-

case-6-P after paper chromatography of purified

71s DAS, SIE, AND FISHMAN

labeled precursors. No evidence for a significant w&h glucose and metabolites isolated in the radioactive contaminant was found. early intervals.

RESULTS

Measurements made of the total amount of liver glucose, and its derivatives present under various experimental conditions (Table I) show in general that, with the exception of UDP-glucose, the amounts of each increases over the first 20 min after injection of 14C-glucose. During the 2-min interval the amounts of glucose-l-P and glucose-6-P were comparable but later the quantity of glucose-6-P became greater than that of glucose-l-P.

It was consistently found that the specific radioact,ivity of glucose-l-P was greater than Ohe corresponding value for glucose-6-P. Next, in most of the experiments, the specific radioactivity of UDP-glucose was less than that of glucose-6-P, except for the value at 10 min. Glycogen showed a very low specific radioactivity compared to glucose.

In order to isolate glucose and glycogen precursors with sufficient radioactivity to permit accurate counting, it was necessary to inject 14C-glucose of higher radioactivity in the shorter intervals of the experiment, 2 and 5 min. This st)ep explains the high radioact,ivity (Table II) which is associated

The ratios of the specific radioactivity of the sugar phosphates to glucose as a function of time after 14C-glucose injection are shown in Fig. 1. The curve of the specific activity ratio to glucose is a linear function of time in the case of glucose-6-P but in the case of glucose-l-P it rises steeply to a plateau. The UDP-glucose to glucose ratio starts with a slow rise then parallels the glucose- 1-P curve.

Anot’her view of the events taking place in the whole liver and its various pools of

TABLE I

TOTAL AMOUNTS OF GLUCOSE, GLUCOSE-~-P, GLUCOSE-B-P, UDP-GLUCOSE, AND GLYCOGEN PRESENT IN

MICROMOLES PER GRAM OF LIVER

Time (min)

0 2 5 10 30

Glucose 10.76 6.72 7.33 10.89 11.00 10.00 14.17 21.17 16.50 Glucose-l-P .068 0.055 0.056 0.106 0.100 0.128 0.100 0.036 0.045

Glucose-6-P .077 0.057 0.061 0.161 0.169 0.313 0.263 0.171 0.156 UDP-Glucose .310 0.36 0.38 0.38 0.35 0.35 0.39 0.37 0.347

Glycogen PCA-insoluble 1.98 .133 .145 7.77 6.44 1.50 1.64 PCA-soluble 0.46 0.061 0.055 1.17 1.22 8.50 0.57 1.88 1.17

TABLE II

SPECIFIC ACTIVITY OF GLUCOSE, GLUCOSE-~-P, GLUCOSE-~-P, UDP-GLUCOSE, AND GLYCOGEN IN COUNTS PER MINUTE PER MICROMOLE

Time (ruin)

2 Spec act of’injected glucose (cpm/~mo:Oe X 10s)

30

7.26 3.63 0.726 1.816 1.816

Glucose 65,126 70,073 6,475 10,327 740 2,225 1,579 2,055

Glucose-l-P 119,190 125,536 29,465 40,216 11,825 22,902 16,413 30,930 Glucose-6-P 23,130 21,841 4,825 12,722 1,679 3,211 8,374 13,723 UDP-Glucose 16,562 15,700 6,947 10,315 8,181 9,477 4,793 9,833

Glycogen PCA-insoluble 71 198 5 17 125 101 PCA-soluble 909 885 10 13 1 1 8 8

‘4C-LABELING OF GLYCOGEN PRECURSORS 719

glucose and glycogen precursors is obtained that, incorporation into glucose-6-P was by examining the t,otal amount of incor- higher than glucose-l-P. On the other hand, poration of 14C-glucose into metabolites the radioact(ivity of UDP-glucose was higher per gram of liver (Table III). Here, the glu- than either glucose-6-P or glucose-l-P. case pool is most radioactive as expected. There was not much incorporation into Glucose-l-P radioactivity was much higher glycogen. than that of glucose-6-P and close to UDP- Table IV shows the level of liver glucose, glucose up to IO-min incorporation. After glucose-l-phosphate, glucose-6-phosphate,

UDP-glucose, and glycogen at 5 and i0 min after 14C sodium bicarbonate administration to hydrocortixone-treated rats.

I -

O 10 20

TIME IN MINUTES

The incorporations of isotopic 14C into glucose and its metabolites are presented in Table V. The specific radioactivity de- creases in the following order: G-l-P > UDPG > G-6-P > glucose > glycogen.

DISCUSSION

The present study attempts to evaluate glycogen synthesis in viva from glucose phosphates derived from exogenous glucose and from glycogen precursors which become 14-carbon-labeled from 14C-bicarbonate during hydrocortisone-induced gluconeo- genesis and glycogenesis.

14C-Glucose-labeling experiments. In de- veloping the experimental design, use was made of starved rats whose preexisting liver glycogen stores had become depleted, and observations were made at short in- tervals after inject’ing a tracer dose of 14C- glucose. The labeling pattern of liver glucose,

FIG. 1. Specific activity in counts per minute/ glucose-l-P, glucose-6-P, UDP-glucose, and

pmole/pcurie ratio 1 to glucose of the precursors glycogen were then examined as a function

after W-glucose administration. The values are of time. means of two determinations from Table III, glu- The results have been obtained by em- case-1-P; 0, glucosed-P, 0, UDP-glucose, 0. ploying the best, available methods for

TABLE III TOT~LAMOUNTOF~~C-GLUCOSE INCORPORATIONINCOUNTSPERMINUTEPER(;RAMOFLIVERINLIVER

GLUCOSE .QND METABOLITES CALCULATED FROM TABLES I AND II

2

20

Time (min)

5 10 10 30 Radioactivity (pCi)

10 2 3 5

Glucose 437,648 513,635 70,383 113,597 7,400 31,900 34,300 34,200 Glucose-l-P 6,555 7,030 3,123 4,022 1,514 2,290 591 1,392 Glucose-6-P 1,318 1,332 777 2,150 526 845 1,432 2,141 UDP-Glucose 5,962 5,966 2,639 3,610 2,860 3,700 1,770 3,440 Glycogen

PCA-insoluble 10 29 39 11 188 166 PCA-soluble 55 49 12 16 9 1 15 9

DAS, SIE, AND FISHMAN

TABLE IV

TOTAL AMOUNTS (MICROMOLES PER GRIM) OF GLUCOSE, GLUCOSE-~-P, GLUCOSE-~-P, UDP-GLUCOSE,

AND GLYCOGEN PRESENT IN LIVER

Time (min)

Glucose Glucose-l-P

Glucose-6-P UDP-Glucose

Glycogen PCA-insoluble

PCA-soluble

Anink no.

1 2 3 4 3

17.05 17.44 18.70 15.66 11.88 0.20 0.14 0.138 0.13 0.085

0.40 0.328 0.345 0.221 0.250 0.252 0.262 0.239 0.303 0.280

17.38 11.94

10 Animal no.

- 9.55 6.11 13.71 7.05 28.2

TABLE V

SPECIFIC ACTIVITY OF GLUCOSE, GLUCOSE-~-P, GLUCOSE-~-P, UDP-GLUCOSE, AND GLYCOGEN IN COUNTS

PER MINUTE PER MICROMOLE

Time (min)

Radioacti5vity (rCi) Radioact%y &Ci)

50 25 Animal No.

1 2 3 4 5

Glucose Glucose-l-P

Glucose-6-P UDP-Glucose

Glycogen PCA-insoluble PCA-soluble

832 743 779 582 811 9,840 8,859 5,399 38,061 45,338

3,315 2,526 954 7,812 10,493 6,145 4,006 2,200 21,108 22,921

- - - 6 8 2 1 1 3 7

separating and purifying mixtures of glucose, glucose-l-P, glucose-6-P, UDP-glucose, and glycogen. The effectiveness of these tech- niques was established to our satisfaction by repeated testing of recovery of mixtures of known composition. Moreover, the radio- active purity of the important key glucose- I-P- and glucose-6-P-labeled products were found to be adequate. Accordingly, we have followed Threlfall’s general approach (18) with the difference that measurements of glucose-l-P have now been added.

That glucose-l-P was labeled to an extent, greater than glucose-6-P was the consistent finding in all the experiments completed for this report.

Compared with that of glucose, the specific radioactivity of glucose-6-P shows a steady linear increase which is preceded by a more rapid rise in bhe relative specific radio-

activity of glucose-l-P. The latter occur before the elevation takes place in the value for UDP-glucose; this probably reflects the conversion of glucose-l-P to UDP- glucose in a simple precursor-to-product relationship.

It may be unprofitable to attempt to interpret in too detailed a fashion the fluctua- tions in UDP-glucose specific radioactivity in relation to glucose-6-P and glucose. For, as pointed out by a referee of this paper, at the 2-min interval the specific radio- activity of glucose is higher than its phos- phorylated products whereas at later inter- vals it is lower due to a declining blood 14C- glucose content. Interpretation could be more readily made in similar experiments in which a stable 14C-glucose radioactivity would have been maintained in the blood by continuous perfusion.

‘%-LABELING OF GLYCOGEN PRECURSORS 721

The results may be explained by postu- lating two pools for glucose-6-P (19) to explain the high glucose-l-P specific ac- t,ivities in relation to glucose and glucose-6-P. This, in turn, brings up two questions: (1) of the cellular inhomogeneity of liver which is made up of hepatic cells, Kupffer cells, endothelial cells, blood cells, and connective tissue, and (2) of these cell groups carrying on metabolic functions in more than one direction. Those cells carrying on glu- coneogenesis and glycolysis would contain a glucose-6-P pool relatively low in 1% where- as those concerned with glycogenesis should exhibit a more radioactive glucose-6-P pool. If the liver, as in the present experi- ments, is depleted of glycogen and is syn- thesizing very little new glycogen, the mixing of these two cell populations by homogenization could be expected to yield a mean 14C-glucose-6-P less than 14C-glucose- 1-P.

A steady state of precursors and products lends itself best to interpretation of metabolic pat,hways. This was not achieved nor sought, especially in the present experiments, as our interest, was directed to the precursors which were being labeled first.

The data do not, of course, exclude a route of glucose to glucose-l-P but neither do they offer unambiguous support.

The possibility t,hat radioactive glucose- 1-P might have originated from glyco- genolysis of radioactive glycogen seems remot,e, since the glycogen was essentially unlabeled by the administered 14C-glucose up to 10 min. Moreover, every effort was made to keep the time to a minimum re- quired t,o kill the animal, remove the liver, and freeze it in Dry Ice. This interval was never more than 20 sec.

W02-Labeling experiments. Despite con- siderable variation from one animal to another a consistent pattern emerges; the specific radioactivity of glucose-l-phosphate being higher than glucose-6-phosphate. This would appear to be incompatible with the known pathway for COZ fixation;

which requires COZ to be incorporated in G-6-P before G-l-P.

However, as in the case of the 14C-glucose- incorporation study, the similar results obtained in the 14C02 experiment are not inconsistent with the view that the glucose- 6-P pool in liver could be compartmentalized in such a way that the glucose-6-P par- ticipating in gluconeogenesis and glyco- genesis is separated from t,hat involved in glycolysis (lS21). Thus, the same explana- tion is offered for both set,s of experiments.

ACKNOWLEl)(;MENTS

Snpported in part by U.S. Public HealthService Research Grants AM-06073 and CA-07538 and Research Career Award K6-CA-K453 (to W.H.F.)

from the Nat,ional Cancer Inst,itltt*e, Nat,ional

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722 DAS, SIE, AND FISHMAN

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