[CANCER RESEARCH 37, 670-677, March 1977]

Work done in the past has indicated that MTW9 tumorregression cannot be explained by either an alteration of themetabolic rate, a difference in the number of cells releasedfrom the tumor, or a homograft type of rejection. The general metabolic activity, as measured by blood flow andoxygen and glucose consumption per unit weight, wasfound to be equal or higher in regressing than in growingtumors (11). The number of neoplastic cells shed into thecirculation by either a growing or a regressing tumor isalmost the same and of the order of 3 to 4 x 106cells/day/gtissue (3). There was no extensive invasion of lymphomonocytic types of cells to suggest that MTW9 regression was animmunological type of tissue rejection (10).

Rather, tumor regression seems to be due to an intracellular digestion characterized morphologically by an intensive increase in the number of phagosomes and biochemically by an increase of lysosomal enzymes (12, 20).

If cellular digestion is a major event in tumor regression(16), clarification of the interdependence between hormoneremoval and increment of digestion is necessary. The hypothesis that hormonal deprivation has a direct stimulatoryaction on the lysosomes and that tissue destruction is theconsequence of their increased “aggressiveness―is contradicted by 3 observations. First, the increment of lysosomalenzyme activity in MTW9 was observed only on the 3rd dayafter hormone withdrawal when regression was well underway and a 50% size reduction had already been observed inmany specimens (20). Second, lysosomal enzymes werenever found in the interstitial fluid of MTW9, not even whena sharp increase of their activity occurred inside the cellsduring regression (12). Finally, the ratio of free:bound lysosomal enzyme activities (4, 20) did not change significantlyup to 72 hr after hormonal deprivation when the reductionin size was about 50%.

Since the increment in the number of lysosomes and inthe activity of lysosomal enzymes during MTW9 regressionseems to be the consequence and not the cause of regression and since lysosomes constitute the quality controlapparatus of the cells (6), we hypothesized that the largeincrease of lysosomal activity during regression might bedue to a larger work load caused by an increased availabilityof substrate, a consequence of hormone withdrawal.

Inthis paperwe reportthat, indeed, cytosol proteins froma regressing MTW9 tumor are more extensively digested byproteolytic enzymes than the same proteins from a growingtumor. Increased availability of substrate due to an increased lability of the proteins could be responsible for the

670 CANCERRESEARCHVOL. 37

Increased Susceptibility of Cytosol Proteins to ProteolyticDigestion during Regression of a Hormone-dependent MammaryTumor1

Maurice Rouleau2 and Pietro M. Gullino3

Laboratory of Pathophysiology, National Cancer Institute, NIH, Bethesda, Maryland 20014

SUMMARY

Regression of MTW9 mammary carcinoma, which consistently follows withdrawal of mammotropic hormones,was characterized by a rapid decrease of thymidine incorporation into DNA but only a slight reduction of uridineincorporation into RNA and amino acid incorporation intoproteins. Within 24 hr of hormone withdrawal, cytosol proteins of MTW9 became more easily degraded by trypsin, a-chymotrypsin, or subtilisin BPN'. Labilization of cytosolproteins occurred much earlier than any change in the levelof protein synthesis or lysosomal enzyme activity. The datashowing increased susceptibility to proteolysis could not beexplained either by the presence of endogenous proteases,by the destruction of the exogenous proteases used in theassay, or by the existence of protease inhibitors. Nor wereany differences detected either in the distribution of radioactive precursor among the cytosol proteins from growingor regressing tumors or in the electrophoretic pattern of thesame proteins. Preincubation of the cytosol proteins withdithiothreitol orwith prolactin, 17/3-estradiol, progesterone,and hydrocortisone did not modify the susceptibility to proteolysis. However, after heat denaturation, cytosol proteinsof regressing and growing tumors became equally susceptible to proteolysis. It is suggested that regression of MTW9mammary carcinoma occurs not only because cell reproduction is arrested, but also because susceptibility ofcytosol proteins to proteolysis is increased.

INTRODUCTION

About 30% of human mammary carcinomas regress aftercastration and/or hypophysectomy of the host (5). Hormonedependency of tumor growth can be studied in animalmodels which mimic reasonably well the human disease.MTW9, a Wistar/Furth mammary adenocarcinoma (17), represents such an experimental tumor, since it grows onlywhen stimulated by pituitary mammotropins and regressespromptly after the stimulus is removed (23).

â€P̃art of this work has been presented at the Annual Meeting of the

American Association for Cancer Research, May 6 to 8, 1976, Toronto,Ontario, Canada (28). This is Paper 8 in a series on mammary tumor regression from the Laboratory of Pathophysiology, National Cancer Institute, NIH,Bethesda, Md.

2 Recipient of a Fellowship from Le Conseil de Ia Recherche en Sante du

Québecin1974 and 1975.3 To whom requests for reprints should be addressed.

Received September 7, 1976; accepted December 2, 1976.

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Labi!ity of Proteins during Mammary Tumor Regression

increment of lysosomal activity and, therefore, forthe tissuedigestion observed in the MTW9 mammary tumor followinghormone withdrawal.

MATERIALS AND METHODS

Tumor Model and Treatment. MTW9 mammary carcinomas (17) were transplanted into the inguinal fat pads ofinbred Wistar/Furth female rats about 2 months old andweighing 150 to 180 g. The high level of mammotropins(serum prolactin, 200 to 1000 ng/ml) needed for tumorgrowth was provided by a pituitary tumor, MtTW1O (13),transplanted into the interscapular region of the MTW9-bearing animal. The tumor-bearing rats, randomized beforeuse, were kept under the same lighting conditions and fedPurina rat chow with water ad !ibitum.

MTW9 tumors of 2 to 4 g were used. Removal of MtTW1Oand castration (under ether anesthesia) consistently resuited in MTW9 regression, i.e., about 50% size reductionby 72 hr after hormone withdrawal. Regression was monitored daily by comparing the average of the shortest andlongest diameters of the tumor measured with a Verniercaliper.

Tumors were removed after decapitation of the rats andice cooled immediately. A series of 1- to 2-mm slices weremade through the entire tumor to check for necrotic areas.Usually, the entire tumors were used, since the amount ofnecrotic tissue was negligible. The tumors were then thoroughly minced with scissors and homogenized.

Incorporation of Radioactive Precursors. The radioactive precursors used were: L-[4,5-3H]leucine (6 Ci/mmole),L-[14C]leucine (309 mCi/mmole), [5-3H]uridine (26 Ci/mmole), [methy!-3H]thymidmne (65 Ci/mmole), D-[U-14C]-glucose (240 mCi/mmole), and [3H]RPH4 (algal profile, 4mCi/mi) (Schwarz/Mann, Orangeburg, N. Y.).

All rad ioactive precursors were injected i .p . at 9 :00 a.m.and, unless otherwise indicated, the animals were killed 1hr later and the tumors were prepared as described above.The minced tumors (ca. 2 g) were homogenized in 10 ml ofice-cold water with a SS-mi glass-to-glass homogenizer (Arthur Thomas, Inc. , Philadelphia, Pa.). Immediately thereafter, cold perchloric acid was added to a final concentrationof 6% and the tubes were kept in ice for 30 mm beforecentrifugation at 12,000 x g for 10 mm at 4°.

The supernatant thus obtained is referred to as the acidsoluble pool. DNA, RNA, and proteins in the pellet wereextracted according to the methods of Schmidt and Thannhauser (31), Munro and Fleck (26), and Schneider (32),respectively, and measured by the diphenylamine methodof Burton (2), the orcinol procedure (32), and the method ofLowry et a!. (22), respectively.

The radioactivity in the different fractions was determined in a Packard Tri-Carb Model 3380 liquid scintillationspectrometer; 10 ml of Insta-Gel (Packard) were used as thescintillation cocktail. Quenching was determined by theautomatic external standardization method. All measurements were corrected to 100% counting efficiency and were

4 The abbreviations used are: [3H]RPH, tritiated reconstituted protein hy

drolysate; SDS, sodium dodecyl sulfate.

recorded as dpm. The counting time chosen was sufficiently long to reduce the counting error to less than 1.5%.Since alterations in the rates of precursor incorporation arenot necessarily true indices of macromolecular synthesisbut might represent changes either in the rates of penetration or of activation of the precursors, we made sure theincorporation was proportional to the acid-soluble poolcontent of labeled precursor. All estimates of macromolecular synthesis were then corrected accordingly.

Amino Acid Analysis. The protein pellet obtained at theend of the Schneider procedure was dissolved in 0.05 NNaOH, dialyzed extensively against distilled water, and lyophilized. The lyophilized protein (Ca. 10 mg) was hydrolyzed under a vacuum of 100 j@min 2 ml of 6 N HCI (PierceChemicals, Rockford, Ill.) for 48 hr. After lyophilization, thedry residue was dissolved in 2 ml of sample buffer. Thecorresponding acid-soluble pool was lyophilized and theresidue was dissolved in sample buffer (1 ml for each 500mg of original tissue). Buffers used were those described bySpackman et a!. (35).

Aliquots of 0.8 ml were analyzed on a JEOL 6AH aminoacid analyzer equipped with a 50:50 split-stream divider.Alternate fractions of the elution buffers (0.84 mI/mm) werecollected (190 sec/fraction) and the radioactivity was determined in each fraction. The recovery of radioactivity wasalways greater than 93%. Radioactive peaks were identifiedby rerunning them on a Beckman 121-H amino acid analyzer.

Proteolysis Assay. Rats bearing MTW9 tumors weregiven i.p. injections of 0.5 mCi L-[4,5-3H]leucine. At 72, 96,and 120 hr after injection, 1 rat/time point was castrated toinduce regression. All rats were killed by decapitation onthe 6th day after injection, so that the tumors from castratedanimals had been deprived of hormones for 72, 48, and 24hr, respectively.

Tumors were homogenized in 0.05 M phosphate buffer,pH 7.6, containing 0.15 M NaCI (1:3, w/v) with a 30-miTeflon-to-glass homogenizer (A. Thomas, Inc.) and çentrifuged at 105,000 x g for 1 hr. Free labeled amino acid wasremoved from the supernatant fluids by passage through acolumn (1.9 x 45 cm) of Sephadex G-25 coarse (PharmaciaFine Chemicals, Inc., Piscataway, N. J.) equilibrated withthe same buffer. Protein fractions were pooled and, foreach proteolytic assay, these solutions (referred to as thecytosol protein solutions) were diluted with sufficient phosphate:NaCI buffer to contain the same amount of protein, asdetermined by their absorbance at 280 nm.

The proteases used for the assay were either trypsin (EC3.4.21.4) (Sigma type Ill), a-chymotrypsin (EC 3.4.21.1)(Sigma type II), or subtilisin BPN' (EC 3.4.21 .14) (Sigma typeVII); the E@I values used for their quantitation were 15.4(38), 20.5 (39), and 11.7 (25), respectively.

A 2-mi sample was incubated with an exogenous proteaseat 30°.At 5, 10, 20, 30, 40, and 50 mm, respectively, 200-idaliquots were removed and precipitated with 200 @Iof icecold 12% perchloric acid. The addition of 1 mg of bovineserum albumin as cold carrier was found to be unnecessary.From an identical sample without enzyme, three-200-jdaliquots were precipitated at zero time (average blank value)and at 50 mm (average autoproteolysis value). After 1 hr on

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M. Rou!eau and P. M. Gu!!ino

ice, precipitates were centrifuged for 5 mm (Beckman 152microfuge), and a 200-jtI aliquot of each supernatant wasused for scintillation counting. At the end of each assayperiod, four-200-pi aliquots were also removed from eachreaction mixture and counted directly. The average valueobtained was used to establish the number of dpm representing 100%.

The reproducibility of the test was checked and, for eachtime point, the standard deviation on the average percentage of hydrolysis was always less than 1.0.

Polyacrylamide Gel Electrophoresis. The cytosol proteinsolutions obtained after passage through Sephadex G-25were made 1% SDS, 1% glycerol, and 3% mercaptoethanol,then boiled for 3 mm and used for the electrophoresis.

The 10% separating and 3% stacking polyacrylamide slabgels were prepared in a Hoefer apparatus (Hoefer ScientificInstruments, San Francisco, Calif.) by the methbd described by Laemmli (19). The analytical (1.5 mm thick) andpreparative (3.0 mm thick) gels were run at 50 ma/gel for2.25 and 4 hr, respectively. Gels were stained overnight withCoomassie Brilliant Blue G-250 (Sigma Chemical Co. , St.Louis, Mo.), according to the method of Swank andMunkres (36), and destained electrophoretically in an E-Cdestainer (A. Thomas) at 2.5 amp/gel for 1 hr in 9% aceticacid. Gels were photographed with a Polaroid camera withthe use of an Eastman Kodak No. 25 gelatin red filter.

The preparative gels were frozen by contact with a blockof CO2 and sliced transversely into approximately 1-mmslices. Each slice was placed in a Packard Combusto-Cone,dried overnight at 55°,combusted in a Model 360 sampleoxidizer (Packard Instruments Co. , Downers Grove, Ill.).The radioactivity of each slice was counted for 10 mm.

RESULTS

Synthesis during Regression

The 1st group of experiments was aimed at defining thecapability of the MTW9 mammary tumor to synthesize nucleic acids and proteins after hormone withdrawal.

DNA Synthesis. Four of 14 animals used had a growingtumor, while the others had tumors deprived of hormonesfor 24, 48, or 72 hr. Each animal received 0.25 mCi of[3H]thymidmne i.p. 1 hr before sacrifice. Tumor DNA wasisolated and thymidine incorporation was measured.

Thymidine incorporation declined sharply as regressionprogressed (Chart 1). Tumors deprived of hormones for 72hr incorporated only 15% as many dpm into DNA as thegrowing tumors. This probably could reflect DNA synthesisin the cells of the stroma so that DNA synthesis in theepithelial cells of the regressing MTW9 practically ceasedby 72 hr.

RNA Synthesis. One hr before sacrifice, each tumorbearing rat received 0.5 mCi of [5-3H]uridine i.p. Total RNAwas extracted and the uridine incorporation was determined. In comparison with DNA, RNA labeling was reducedto a much lesser extent. At 72 hr after hormone withdrawal,the RNA synthesis as measured by uridine incorporationwas reduced by only 30%, although the reduction in tumorvolume was almost 50% (Chart 2).

0 24 48 72TIMEAFTERCASTRATION)hr)

(4)

!@°@

20

10@ \I)4)I

I I I

0 24 48 72TIME AFTER CASTRATION )hr)

Chart 1. Incorporation of [methyl-3H]thymidine into DNA during regression of MTW9 tumors. At the times indicated on the abscissa, each animalreceived 0.25 mCi of [3H]thymidine i.p. One hr later, rats were killed andtumors were analyzed. Results are expressed as dpm/mg DNA ±S.D. Numbars in brackets, numbers of tumors analyzed.

4za,E

a-a

Chart 2. Incorporation of [5-3H]uridine into RNA during regression ofMTW9 tumors. At the times indicated on the abscissa, each animal received0.5 mCi of [3H]uridine i.p. One hr later, rats were killed and tumors wereanalyzed. Results are expressed as dpm/mg RNA ±S.D. Numbers in brackets, numbers of tumors analyzed.

Protein Synthesis. The capability of the regressing tumorto synthesize proteins was ascertained by measuring theincorporation of [3H]RPH into total proteins during a 1-hrpulse. A mixture of 15 different 3H-labeled amino acids waschosentominimizeanyhormonallyinducedchange inpoolsize that might occur with any 1 amino acid, to increase theextent of overall protein labeling, and to eliminate the risk ofany 1 protein from not being labeled due to a lack of a singleamino acid. A 1-hr pulse was chosen because this shortperiod reflects largely the rate of protein synthesis, andinsufficient time will have elapsed for significant reutilization.

Up to 24 hr after hormone withdrawal, regressing MTW9tumors incorporated as much radioactivity into total pro

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Relative proportions of labeled amino acids found in the acidsoluble pool and in the protein hydrolysates of growingandregressing

MTW9 tumors, 1 hr after i.p. injection of (U-'4CJglucoseThe

distribution of radioactivity was determined on anaminoacidanalyzer as illustrated in Chart 4. Each horizontal line ofvaluesrefers

to 1 tumor.%

distributionAspartic

GlutamicSamplesUnknown acid Serine acidAlanineProteinsGrowing

13.5 14.6 3.1 28.440.412.614.3 4.0 23.145.910.614.0 3.5 29.142.8Reg-18H

13.3 15.0 2.6 26.842.39.516.6 2.1 26.845.010.715.9 2.9 27.443.2Reg-48H

11.6 13.9 4.3 24.645.610.214.8 3.7 25.445.9Acid-soluble

poolsGrowing66.8 6.0 0 15.911.3Reg-18H

64.8 5.5 0 16.313.463.25.2 0 15.815.8Reg-48H

62.8 5.8 0 14.5 14.5

60

@50

E40

230

20

10

Labi!ity of Proteins during Mammary Tumor Regression

teins as did growing tumors. The incorporation of labeledamino acids then decreased with time, but after 72 hr ofregression it was still 55% of the control value (Chart 3). Asfor RNA, protein synthesis proceeded at a high level despitehormone removal.

The same pattern was obtained when L-[4,5-3H]leucine or[U-'4C]glucose, instead of [3H]RPH, was used to label thecellularproteins.

Utilization of Glucose

In a 2nd group of experiments, [U-14C]glucose was usedas a probe to test whether conversion of this metabolite intoamino acids followed by incorporation into proteins wasaltered during regression. Labeled glucose was selected,since MTW9 uses a relatively large amount of this sugar(10).

Each rat received 1 mCi of [U-'4C]glucose i.p. One hrlater, the rats were killed, the tumors were removed, and theproteins were isolated. The protein hydrolysates and thecorresponding acid-soluble pools were analyzed by chromatography to establish the relative proportions of theamino acids derived from labeled glucose. The percentageof distribution of these amino acids was found to be reasonably similar in 3 growing, compared with 5 regressing,tumors (Table 1). Serine, present in all protein hydrolysates,was never found in the acid-soluble pools of either growingor regressing tumors, and we did not investigate further thisparticular finding.

The large peak of radioactivity found in Fractions 10 to 15(Chart 4) could not be identified as it was nmnhydrin negative. Since this peak was also found in the protein hydrolysates prepared from extensively dialyzed protein solutions,it probably represented the carbohydrate residues of theglycoproteins.

The data of Table 1 indicate that conversion of glucoseinto amino acids and its incorporation into proteins as

Table 1

A70@

10 20 30 40

NUMBER OF FRACTIONS

50 10 20

NUMBER OF FRACTIONS

Chart 4. Chromatographic radioactivity profiles of the acid-soluble pooland of the protein hydrolysate of a MTW9 tumor after injection of labeledglucose. One hr before sacrifice, the animal received 1.0 mCi of [U-“C)glucosei.p. After lyophilization, the acid-soluble pool (A) and the proteinhydrolysate (B) were dissolved in sample buffer and analyzed on a JEOL 6AHamino acid analyzer. The eluant was collected (1.33 mI/fraction) andcounted. All the radioactivity was recovered in the 1st 45 fractions of theeluant from the column (Beckman AA15 resin; 40 x 0.9 cm) used to separatethe acidic and neutral amino acids. No labeled basic amino acids were found.Radioactive peaks were identified by a rerun on a Beckman 121-H amino acidanalyzer. ?, unknown;ASP, aspartic acid; GLU, glutamic acid;ALA, alanine;SEA, serine.

amino acids or sugar residues are not noticeably altered byhormone removal from MTW9 mammary tumors.

Proteolysis of Cytosol Proteins

The proteolysis assay was used to determine whether anincrease in protein lability occurred during MTW9 regression. Cytosol proteins were labeled and the assay was run

0 24 48 72

TIME AFTERCASTRATION)hr)

Chart 3. Incorporation of 3H-labeled amino acid into total proteins duringregression of MTW9 tumors. At the times indicated on the abscissa, eachanimal received 1 mCi of [3H]RPH i.p. One hr later, rats were killed andtumors were analyzed. Results are expressed as dpm/mg protein ±S.D.Numbers in brackets, numbers of tumors analyzed.

673MARCH 1977

(8) J)3)

@1@i(8)I@I

.@

5

@ 4wI-0

a.E

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M. Rouleau and P. M. Gu!lino

on the 105,000 x g supernatant fraction freed of labeledmetabolites (see “Materialsand Methods―).

Susceptibility to Trypsin. The susceptibility of cytosolproteins from growing and regressing tumors to proteolysisby trypsin is illustrated in Chart 5. The percentage of 3Hsolubilized by a fixed amount of trypsin was consistentlyhigher when cytosol proteins from regressing tumors wereused. The magnitude of this difference varied but was notdependent on the duration of regression. Using the cytosolproteins from the liver of the same animals (bearing either agrowing or regressing tumor), we have not been able todetect any difference in susceptibility to proteolytic attack(Chart 6). The extent of autoproteolysis, determined in theabsence of trypsin, was equal in all cytosol protein solutionstested and never exceeded 3% of the total number of dpm.

The increased susceptibility does not depend on the temperature at which the assay is run or on the amount ofenzyme used. The assay was conducted at 2 different temperatures (30°and 42°)and at 2 different concentrations oftrypsin (14 and 28 @g/ml).In both cases, the results wereessentially the same, the only difference being the percentage of protein hydrolyzed. The higher the temperature orthe larger the amount of enzyme used, the higher the percentage of hydrolysis. Results were not affected when thephosphate:NaCI buffer was supplemented with dithiothreitol and EDTA at a final concentration of 1 mM or substitutedby 50 mM Tris:1 mM CaCI2, pH 7.6. This seems to indicatethat the difference in susceptibility does not depend on adifference in disulfide bridges.

Susceptibility to Other Proteases. Subtilisin BPN' (9.2@g/ml)and a-chymotrypsin (11.4 @g/ml)were used as ex

ogenous proteases, and the results were essentially thesame as with trypsin. Not only was the increase of cytosolproteolysis observed in the regressing tumors, but the relative order of susceptibility to proteolysis remained the sameexcept that the percentage of hydrolysis varied due to the

100

80

10 20 30 40 50 10 20 30 40 50

TIME OF INCUBATION(mm) TIME OF INCUBATION (mInI

0

N

60D

0(I)I

2

0

0.

Chart 6. Susceptibility of 3H-Iabeled cytosol proteins of mammary tumorsand livers from the same rat to proteolysis by trypsin. Experimental detailsare described in the legend to Chart 5. The A280of each cytosol proteinsolution was adjusted to 1.9. Final concentration of trypsin was 13.6 @g/ml.The assay was performed at42°.0, animal bearing a 72-hr regressing tumor,., animalbearinga growing MTW9.

different specificities of the tested enzymes.Trypsin Activity and Stability during the Assay. To make

sure that the differential susceptibility to proteolysis was notdue to some factors affecting the exogenous enzyme usedfor assay, we studied the stability and activity of trypsinduring the test. At different times after the addition of trypsin to cytosol proteins prepared from growing tumors, auquots were withdrawn and assayed for their trypsin content.The esterolytic activity of trypsin was determined by monitoring the hydrolysis of a-N-benzoyl-L-arginine ethyl esterHCI at 253 nm in the manner described by Schwert andTakenaka (34). A decrease with time of this enzymatic activity would have revealed instability of the exogenous enzyme,butno change was observed.

To determine whether a trypsin inhibitor was present inthe cytosol of growing MTW9 (in which such an inhibitor isexpected to be maximal), we measured the residual trypsmnactivity after adding an increasing amount of cytosol to afixed amount of enzyme. The ratio of cytosol proteins totrypsmnvaried from 1 to 20, while the normal ratio used inthe proteolysis assay was about 10. The presence of aninhibitor would be expected to produce a decrease in freeenzyme activity, but none was observed. Furthermore,when trypsmnwas added to a mixture of cytosols from growing and regressing MTW9 (1:1), the proteolysis curve thusobtained was midway between the curves obtained for cytosols from growing and regressing tumors tested separately.

Should a trypsmninhibitor really exist, it must have a muchhigher K than the K5for a-N-benzoyl-L-arginine ethyl esterHCI (10@ M), since no displacement of the hypotheticalinhibitor-enzyme complex by the synthetic substrate wasseen at the beginning of the esterolytic assay. An inhibitorwith such a K, would be rather unusual, compared with allthe inhibitors known to date (37).

Comparison of Cytosol Proteins. In order to make surethat the preferential degradation of cytosol proteins fromthe regressing tumors was not due to some difference in theproteins used as substrates, 2 experiments were performed.First, the electrophoretic patterns of cytosol proteins werecompared with the use of a polyacrylamide gel containingSDS. No real difference could be detected between proteinsprepared from either growing tumors or from those de

15 30 45 60

TIME OF INCUBATION(mm)

0LuN-J

:3-J0U)

I

I-zLU(3

Lua.

Chart 5. Susceptibility of ‘H-labeledcytosol proteins of MTW9 mammarytumors to proteolysis by trypsin. Six days after i.p. injection of 0.5 mCi of[4,5-3H]leucine, tumors were homogenized (1:3, w/v) in phosphate bufferand centrifuged at 105,000 x g for 1 hr. Free labeled amino acid was removedfrom the supernatants by passage through a Sephadex G-25 column. The A,,@of each cytosol protein solution was adjusted to 1.8. At time zero, trypsin wasadded at a final concentration of 14.8 @g/ml.At the times indicated on theabscissa, an aliquot was precipitated with cold perchloric acid and thesoluble dpm were expressed as percentage of total dpm. The assay wasperformed at 30―and autoproteolysis (- - -) (digestion without exogenousprotease) values were obtained in the same way.

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Labi!ity of Proteins during Mammary Tumor Regression

prived of hormones even for as long as 72 hr (Fig. 1).Second, radioactivity profiles of the cytosol proteins separated by electrophoresis on polyacrylamide gel in the presence of SDS were compared. The proteins from growingand regressing tumors should be identically labeled if thepercentage of counts found in the acid-soluble fractionsafter trypsin digestion is to be used as the index of preferential degradation. Indeed, no difference in the radioactivity

*@p@-,

A B C DFig. 1. SDS gel electrophoresis (Laemmli's system) of cytosol proteins

from growing tumors (A) and tumors deprived of hormones for 24 (B), 48 (C),and 72 hr (D), respectively. Cytosol solutions were prepared as described inthe legend to Chart 5; they were made 1% SDS, 1% glycerol, and 3%mercaptoethanol and boiled for 3 mm. Twenty @.olof each solution (A280=2.20) were loaded onto a 1.5-mm-thick slab gel and subjected to electrophoresis for 2.25 hr at 50 ma/gel. The gel was stained with Coomassie brilliant G250 and destained electrophoretically.

0

(I,

a-0

0

Cl)

a-0..,@*-@@ —I

10 20 30 40 50 60 70 80 90

SLICE NUMBER

Chart 7. Electrophoretic radioactivity profiles of cytosol proteins of growing tumors and tumors deprived of hormones for 24, 48, and 72 hr. respectively. The cytosol solutions were prepared as described in the legend toChart 6: Preparative electrophoresis of 250 A1of each solution (A2,, = 1.92) inthe presence of SOS was run at 50 ma/gel for 4 to 4.5 hr. Gels were cut intoabout 1-mm slices, each slice was oxidized in a Model 360 sample oxidizer,and the cpm were plotted.

profiles was observed (Chart 7).The similarities of both the gel patterns (Fig. 1) and the

radioactivity profiles (Chart 7) indicate that the preferentialdegradation of cytosol proteins from regressing tumorscannot be explained by a difference in either the nature orlabeling pattern of the substrate.

Effect of Denaturation. If the difference in susceptibilityto proteolysis between growing and regressing cytosol proteins reflects an inherent property of the proteins, it shouldbe possible to eliminate this difference by denaturing theproteins. Cytosols of growing and regressing (72 hr) MTW9were compared before and after heating for 5 mm at 100°(no precipitate visible), followed by rapid cooling to prevent

675MARCH 1977

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M. Rou!eau and P. M. Gu!!ino

3) were similar as regression progressed, but the decreasewith time was much less pronounced than for DNA. It is notlikely that an arrest of protein and/or RNA synthesis couldbe the determining cause of regression since no significantchange in protein synthesis can be detected until 48 hr afterhormone withdrawal, and, at this time, regression is alreadyappreciable in many tumors. Similar results have also beenreported for the GR mouse model (33).

In many tissues undergoing involution under hormonalcontrol, an evolving lysosomal system has been found, andlysosomes were implicated in tissue degradation (21). Anincrease in lysosomal enzyme activity has been reported inMTW9, but the results do not provide evidence that MTW9regression is a consequence of lysosomal attack (4, 10, 20).

The major observation reported in this paper is an increased susceptibility of cytosol proteins to proteolyticdigestion after hormone withdrawal (Chart 5). This effect ispeculiar to the tissue requiring hormones for growth (Chart6) and was observed within 24 hr after hormonal deprivation, much earlier than any increment of lysosomal enzymeactivity or any change in protein synthesis (Chart 3) couldbe detected.

Several possibilities which could explain the increasedlab ility of cytosol protei ns fro m reg ressing tu mors havebeen ruled out, i.e. , presence of endogenous proteases(autoproteolysis values on Chart 5) or protease inhibitors;degradation of the exogenous proteases during the test;differences in the protein makeup (Fig. 1); alterations in thedisulfide bridges; changes in the distribution of radioactivity among proteins of the regressing tumors (Chart 7); ailosteric protection afforded by hormones; and metabolic alterations as tested by incorporation of [14C]glucose intoproteins either as amino acids or as carbohydrate residues(Table 1).

Our finding appears to be related to the conformationalstate of the proteins, since their preferential degradation isno longer observed (Chart 8) when they are first unfolded byheat denaturation.

Dice et a!. (7) have shown that a good correlation existsbetween the rate of protein turnover in vivo and their susceptibility to proteolysis by exogenous proteases in vitro. Itis also known that the rates at which specific proteins aredegraded vary with the physiological state; e.g. , the administration of tryptophan stops the degradation of tryptophanpyrrolase (30), starvation decreases the degradation of arginase (29) or increases the degradation of acetyl-CoA carboxylase (24), and a vitamin B6-deficient diet acceleratesthe degradation of pyridoxal enzymes (15). Thus the increased susceptibility of cytosol proteins to proteolysis thatwe report in this paper could reflect a change in the turnover rate of those proteins due to the new physiologicalstate created by the absence of hormones; it could alsoexplain the well-known increment of lysosomal activity inregressing tumors as a consequence of substrate availability.

Although steroids can bind to proteins (8) and inducestructural changes (18, 40), the addition of hormones to thecytosol proteins did not stabilize the latter against proteolytic attack. If the increased susceptibility to digestion is notdue to a lack of allosteric protection, the absence of hor

500

N

60:@

0U)

I

20

0.

10 20 30 40 50 10 20 30 40 50

TIME OF INCUBATION (minI TIME OF INCUBATION (minI

Chart 8. Susceptibility of 3H-labeled cytosol proteins of MTW9 mammarytumors to proteolysis by trypsin after heat denaturation. Experimental detailsare described in the legend to Chart 5. The A,50of each cytosol proteinsolution was adjusted to 1.7. Final concentration of trypsmnwas 13.9 pg/mI.The assay was performed at 30°.Left, before heat denaturation; right, after Smm in boiling water.

renaturation. Heat denaturation not only eliminated the difference in lability but also rendered the proteins more susceptible to the attack by trypsin (Chart 8), reaching 60%hydrolysis after 50 mm as compared to 39 and 48% beforeheating.

Effect of Hormones on the Stability of Cytosol Proteins.The susceptibility of cytosol proteins to trypsin digestionwas tested in the presence of a mixture of hormones.5 Ovineprolactin (0.25 @.tg/ml,final concentration), 17/3-estradiol,progesterone, and hydrocortisone (0.5 jig/mI, final concentration for each) were added to cytosols of growing and 72-hr regressing tumors. The cytosols were then incubated at 4Cfor 20 hr before being assayed. In either the presence orabsence of hormones, cytosol proteins from regressing tumors were more susceptible to trypsin digestion than thosefrom growing tumors; a 10-fold increase of hormone concentration did not alter the results.

DISCUSSION

Following hormone withdrawal, the incorporation of Iabeled thymidine into DNA of MTW9 mammary carcinomadecreased sharply as regression progressed (Chart 1). Asimilar result has been reported for a hormone-dependentmammary carcinoma of the GR mouse by Schüleinet a!.(33). They interpreted the decrease of DNA labeling aftercessation of hormone treatment as being due to a decreasein the cell production rate rather than to an increase in thecell loss rate (14). The same explanation may hold forMTW9, since no significant increment of cell loss via efferent blood was observed after hormone withdrawal (3), andwe now report a sharp decrease of label incorporation intoDNA.

The patterns of RNA and protein synthesis (Charts 2 and

5 Progesterone, 17 /3-estradiol (Calbiochem, La Jolla, Calif.), and hydro

cortisone (Sigma Chemical Co.) were solubilized in absolute ethanol at aconcentration of 0.25 mg/mI and added as such to cytosol protein solutionswhen indicated. Equivalent amounts of absolute alcohol were also added tothe controls. Ovine prolactin (a gift from the National Pituitary Agency, NIH)waspreparedin25mMTris-HCI,pH7.6,ataconcentrationof0.1mg/mI.

676 CANCERRESEARCHVOL. 37

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Lability of Proteins during Mammary Tumor Regression

677

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MARCH 1977

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1977;37:670-677. Cancer Res   Maurice Rouleau and Pietro M. Gullino  Mammary TumorDigestion during Regression of a Hormone-dependent Increased Susceptibility of Cytosol Proteins to Proteolytic

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