[CANCER RESEARCH 46, 5649-5654, November 1986]

Influence of the Walker 256 Carcinosarcoma on Muscle, Tumor, and Whole-BodyProtein Synthesis and Growth Rate in the Cancer-bearing Rat1

John A. Tayek,2 Nawfal W. Istfan, Catherine T. Jones, Karim J. Hamawy, Bruce R. lustrina, ' and

George L. BlackburnLaboratory of Nutrition/Infection, Cancer Research Institute, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215

ABSTRACT

The in vivo rates of protein synthesis were assessed in tumor tissue,skeletal muscle, and whole body of rats bearing the Walker 256 carci-nosarcoma. Estimates of protein synthesis in the nontumorous tissueswere compared to tumor-free controls. Changes in size of the wholeanimal and tumor (i.e., growth) were measured, and fractional rates ofgrowth, synthesis, and breakdown were estimated. Muscle protein synthesis and whole-body growth were significantly reduced in rats bearinglarger tumors, and both were negatively correlated with tumor size (r =-0.723 and -0.825, respectively; /' < 0.01). Furthermore, whole-body

and muscle protein synthesis were positively correlated with body growth(r = 11.3811and 0.563, respectively; /' < 0.05). Tumor growth followed

first-order kinetics between days 7 and 13 following implantation, with amean rate constant of 34.3%/day for the larger tumors and 27.7%/dayfor the small tumors. The difference in tumor growth became statisticallysignificant over the final 3 days of tumor volume measurements. Fractional protein synthesis was significantly lower in the larger compared tothe smaller tumors (48.6 versus 84.8%/day; P < 0.05) as measured onday 14. This finding indicates a lower protein breakdown rate for thelarger tumors (14.3 versus 59.0%/day; P < 0.01) and suggests that theprocess of protein breakdown could play a significant role in determiningtumor size, leading support to the theory of tumors acting as nitrogentraps.

INTRODUCTION

The cachexia syndrome is associated with high rates of mortality in cancer patients. (1) Several mechanisms have beenoffered to explain the weight loss associated with the presenceof cancer. These include insufficient dietary intake and absorption, (2) enhanced tumor nutrient requirements, (3) alterationsin the host metabolism, (4) or a combination of these processes.(5)

Weight loss in cancer cachexia has been explained mechanistically by increases in total body energy expenditure and reductions in muscle-protein synthesis that lead to muscle wasting.(6) Studies by Lundholm and others have shown that skeletalmuscle protein synthesis is reduced with the onset of cancercachexia (7) and may be reduced further with increasing tumorsize. (8) Previous studies have utilized the technique of constantinfusion of labeled amino acids and its specific activity in theplasma amino acid pool to estimate plasma protein flux, oxidation, and whole-body protein synthesis. This method mayunderestimate intracellular amino acid oxidation and whole-body protein synthesis. In comparison, using the specific activity of the intracellular amino acid pool to determine whole-body protein flux and synthesis assumes the intracellular pool

Received 12/26/85; revised 5/14/86, 7/14/86; accepted 7/17/86.The costs of publication of this article were defrayed in pan by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported in pan by grants CA09459 and CA35352 awarded by the National

Cancer Institute, CM 24206 awarded by the National Institute of General MedicalSciences, and grants from Travenol Laboratories.

2Present address: Division of Endocrinology, Harbor-UCLA Medical Center,

1000 West Carson Street, Terranee, CA 90509.3To whom requests for reprints should be addressed, at Laboratory of Nutri

tion/Infection, New England Deaconess Hospital, 194 Pilgrim Road, Boston,MA 02215.

to be a precursor pool for overall protein synthesis. The use ofintracellular measurements makes possible comparison of tumor and tumor-free protein synthesis rates and also allowsquantification of tissue-specific amino acid concentrationswhich may well be altered by disease. Expecting a rapid tumorturnover, similar to that reported earlier by Kawamura et al.,(9) we selected the flooding dose technique which uses the freeintracellular amino acid concentration as the precursor pool toinvestigate whole-body and tissue protein synthesis parameters.This technique provides a large dose of amino acid so that thespecific radioactivity in all possible precursor pools is nearlyequal, therefore minimizing the error in the determination ofthe specific radioactivity of the free amino acid at the site ofprotein synthesis, both in the individual tissues and for thewhole body. (10)

The purpose of this study was to explore the relationshipbetween tumor protein synthesis and the rate of tumor growthand to evaluate the influence of the tumor on the whole-bodyand muscle protein synthesis. Fractional synthetic rates, A,.were studied in tumor, muscle, and the whole body of youngWalker 256 carcinosarcoma-bearing rats. The fractional growthand breakdown rates of the Walker 256 carcinosarcoma weredetermined, and the effects of tumor size on whole-body andmuscle protein synthesis were examined. Our hypothesis wasthat the metabolic influence of cancer on the host's protein

synthesis would be dependent upon the size of the tumor, andthat the rate of tumor growth would correlate with tumorprotein-synthetic rates.

MATERIALS AND METHODS

Rats and Experimental Design. Twenty-four male Sprague-Dawleyrates (130-150 g) were obtained from Taconic Farms (Germantown,NY). The rats were maintained on a 12-h light, 12-h dark photoperiodat 27"C. The animals were fed a complete reconstituted powdered diet

with 2% agar (AIN-76; Dyets, Inc., Bethlehem, PA) containing 20%(w/w) casein and given water ad libitum for the entire study period.(11) Eighteen of the rats were inoculated s.c. in the right flank with 0.1ml of a Walker 256 carcinosarcoma (Arthur D. Little, Inc., Cambridge,MA) cell suspension of approximately 1 x 10* cells. Six rats received

a saline injection and served as controls. Food intake and weights weremeasured daily. Each day the site of tumor inoculation was inspected,and the length, width, and depth of the tumor were measured withcalipers. Estimates of tumor volume were made with the formula for aprolate spheroid

y = L W •D •r/6

where / is the length, If is the width, and D is the depth of the solidtumor. The measured tumor volume was used to estimate tumor weightbased on regression analysis for tumor volume and weight at sacrifice.Tumor regression was defined as a loss of greater than 40% of themaximum tumor volume during the study period, and this was notedin 3 of the 18 rats. In this study, the 18 tumor-bearing rats were dividedinto 2 groups of equal number dependent upon final tumor size (i.e..,large and small).

After an overnight fast on the 14th day of tumor growth, the ratswere given injections of L-[l-14C]leucine (50 mCi/mmol; ICN Radi-

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W256 INFLUENCE ON PROTEIN METABOLISM AND GROWTH RATES IN RATS

ochemicals, Irvine, CA). The fasting state was used to ensure a comparable metabolic state among the groups without altering the kineticsof the tumor tissue, which is relatively insensitive to host starvation.(12) The volume injected via the lateral tail vein was approximately 1.5ml, which provided 30 /¿Ciof labeled and ISO //mol of unlabeled leucineper 100 g body weight. At 10 min postinjection, the animals weresacrificed by decapitation, and mixed arterial-venous blood was collected in chilled heparinized tubes. Immediately after blood collection,the body was quickly dissected. The tumor, liver, and portions of theabdominus reclus muscle were removed. The whole tumor was weighedand immediately frozen in liquid nitrogen to halt all metabolic processes. Previous inspection of large tumors has failed to demonstratecentral areas of necrosis which could reduce the protein-bound specificactivity and underestimate protein-synthetic rate. A 1-g piece of musclewas placed in 5 ml of 10% SSA4 and frozen in liquid nitrogen. A 1-2-

g piece of liver, and a second piece of muscle were frozen in 5 ml ofsaline for total nitrogen measurements. Finally, the entire carcass,including the unused liver portion, was frozen in liquid nitrogen andall of the tissues were stored at -25°C until the time of analysis. The

entire process required 3 min.Analytical Methods. The heparinized blood was centrifuged at 2500

rpm for 15 min, following which time the plasma was removed andstored at -25"C. The plasma was thawed at time of analysis, and 1 mlwas di-protei ni/ed with 0.2 ml of 30% SSA for measurement of plasma

leucine concentration and specific activity. The deproteinized serumwas vortexed, incubated at 25"C for 30 min, and centrifuged for 15 minat 4000 rpm. Fifty n\ were analyzed for total leucine content using o-phthalaldehyde with precolumn derivatization and HPLC (Waters Associates, Milford, MA). The supernatant (0.6 ml) was further treatedwith 0.2 ml of 30% H2O2 and incubated at 37°Cfor 60 min and thenat ?()'(' for 30 min, after which 250 /il were placed in a scintillation

vial with 5 ml of commercial scintillant (Instagel; United TechnologiesPackard, Downers Grove, IL) and analyzed for total I4C radioactivity

with a Beckman LS-8000 liquid scintillation spectrometer.The frozen carcass and tumor were brought down to -180°C with

liquid nitrogen and each was wrapped in cloth and crushed with amallet. The small pieces were then placed in a Waring blender withsolid ( '( ), to keep the tissue from thawing and were ground to a fine,

crystalline power. Portions of the tumor (1-2 g) were placed in 5 ml of10% SSA and 5 ml of saline. Portions (1-3 g) of the whole-body crystalswere placed in duplicate in 5 ml of 10% SSA, and a 1-3-g portion was

placed in 5 ml of saline.All tissue samples in 10% SSA (muscle, tumor, and whole body)

were homogenized (Polytron homogenizer; Brickmann Instruments,Westbury, NY) and centrifuged at 4000 rpm in order to separate theprotein (precipitate) and the free intracellular (acid-soluble) amino acidsfor determination of lem-ine specific activities in these two fractions.

The supernatant was further spun down at 15,000 rpm to remove anycontamination from the protein-bound fraction, and 50 /il were analyzed by HPLC for leucine concentration. An additional 1 ml wasfurther treated with 0.2 ml of 30% (v/v) H2O2 to eliminate radioactivityfound in a-ketoisocaproate (13) and incubated at 35°Cfor 60 min and70°Cfor 30 min. This was then centrifuged, and 0.5 ml of the super

natant was added to 10 ml of commercial scintillant (Instagel) andanalyzed for I4C radioactivity (Beckman LS-8000 liquid scintillationspectrometer). Intracellular-free, leucine-specific activity, Si, was thencalculated from the radioactivity counts and leucine concentration inthe SSA-soluble fraction.

The precipitate was washed three times with 2% SSA, and afterbeing dried in an 100'C oven overnight, 30-40-mg samples were

solubilized in 2-3 ml quaternary ammonium hydroxide (Soluene-350;Packard). Following a 48 li soluhi li/ai ion. 10 ml of commercial scintillant (Betafluor; National Diagnostics, Somerville, NJ) and 5 drops ofglacial acetic acid were added and the sample was analyzed for UCradioactivity with a Beckman LS-8000 liquid scintillation spectrometer.A second dried sample (30-50 mg) was analyzed for total nitrogen bymicro-Kjeldahl digestion, as previously described. (14) The protein-bound specific activity, Sb, of leucine was then calculated from the

radioactivity counts, the measured percentage of nitrogen in the precipitated fraction, and the average percentage of leucine content in ratproteins, as reported from our laboratory. (15)

The total protein content of each tissue was determined by thawingand homogenizing samples frozen in saline and spectrophotometricallyanalyzing for total nitrogen after a micro-Kjeldahl digestion.

The percentage of leucine in tumor protein was determined byhydrolyzing two duplicate samples (200-300 mg) in 10 ml of 12 M-HC1 at 120°Cfor 3 h. The pH was adjusted to 2.0 by the addition of 7

ml of 2 M sodium hydroxide. Each sample was then diluted 1:10 witha 1.9% (w/v) lithium citrate buffer and leucine concentration wasanalyzed by HPLC. The leucine concentration for muscle and whole-body proteins of Sprague-Dawley rats of similar ages, has been determined previously. (15)

The injection of 150 //mol of leucine/100 g body weight was used toensure adequate distribution of leucine and specilli- radioactivity to theacid-soluble portion of tumor tissue, since the distribution of leucinethroughout the Walker 256 tumor tissue has not been previouslyreported. Measurement of [l4C]leucine incorporation into protein after

a large bolus dose has two theoretical advantages: (a) because of thereduction in the differences between the specific radioactivity of thefree amino acid in the plasma and tissue, the error from the inabilityto identify the specific radioactivity at the site of protein synthesis isminimized; (h) the isotopie steady state would be maintained for a shortperiod (10 min) of study so that label recycling, which underestimatesprotein-synthetic rate in rapidly turning over tissue, such as liver andtumor, would also be minimized. Recent evidence supports that recycling after prolonged infusions makes an important contribution toprotein flux and can lead to underestimates of protein flux of about25%. (16) Large doses of leucine over a short period of time have failedto stimulate nontumorous protein synthesis, (17) and it is assumed thattumor tissue would respond in a similar way.

Calculations. In order to estimate the rates of protein synthesis andbreakdown in the tumor, we used the model of Waterlow et al. (18)which accounts for changes in protein mass during non-steady states.In this model, synthesis, breakdown, and growth are represented byfractional constants and first order kinetics. Fig. 1 shows the growthcurve of the tumor, represented as mean values for the 15 tumor-bearing rats with nonregressing tumors. The weight of the tumor, as afunction of time, could be approximated by

= w¡e** (A)

In this equation, W, and II .•are the tumor weights at times I, and

W256 INFLUENCE ON PROTEIN METABOLISM AND GROWTH RATES IN RATS

respectively. Thus

Kg —K, —Kd (B)

Furthermore, total protein synthesis (Ts) between times t, and *2isexpressed by

f"Ts = J K, W(t) dt (Q

which can be solved by replacing W(t) and K, with their correspondingvalues in Equations A and B and by integration between times i, andt2.The resulting expression for total protein synthesis is

Ts (D)

where " is the protein in tumor tissue obtained from measurement oftumor weight and tissue analysis of nitrogen content. An estimate ofthe mean daily protein synthesis is then obtained by dividing A by thetime during which tumor or body growth took place. The fractionalsynthetic rate of tissues, in percentage per day (A,), is experimentallydetermined from the isotopie appearance of tracer leucine in the proteinbody fraction, as derived by the equation of McNurlan et al. (IO)

K,= SbSi T

100 (E)

where Sb is the specific radioactivity of leucine bound into protein aftertime T (days), Si is the acid-soluble, free leucine, specific radioactivitywhich has been shown previously (10) to fall slowly and linearly withtime. The fractional degradation rate (A,/) was determined from thedifference between K, and Kf.

Comparison between the three groups was done by one-wayanalysisof variance and significance defined by the Bonferroni test using acommonly available statistics package (BMDP-83, UCLA). Comparison within the tumor group was by a two-tailed Mesi where applicable.Linear and exponential regression analysis was performed by themethod of least squares. All data are represented as mean ±SEM.Significance was defined at P = 0.05.

RESULTS

Dependent upon the final size of the tumor, two groups oftumor-bearing rats, in addition to the nontumorous controlgroup, were identified. In the rats bearing larger tumors, theaverage size of the tumor at sacrifice was 29.0 g (n = 9; SD 9.3;range, 20-50) while the smaller tumor group averaged 10.7 g(n = 9; SD 5.8; range, 5-19). Tumor weight represented 13 and5% of total body weight, respectively. The tumor volume atsacrifice correlated well with the weight of the tumor dissectedfree of the animal (r = 0.946, y = 0.737 + 1.078AO. In spite ofsimilar food intake in the 3 groups, there was a significantdecrease in the nontumorous weight gain in the rats bearinglarge tumors (P < 0.01) over the last 4 days in comparison tothe smaller tumor group and the controls. The average growthof the control rat was 7.7 g/day with a mean dietary intake of82 kcal/day (Table 1). Previous work with this tumor in ratshas shown that changes in dietary intake are not usually seenuntil the development of "progressive" tumor growth, which

occurs approximately 20 days after tumor implantation. (19)The fractional growth rate of the whole body was calculated

for the controls and tumor-bearing animals over the final 6-dayperiod. Plotting the nontumorous body weight of the animalsover this period showed that these rats were growing accordingto first-order kinetics (r2 = 0.981,0.976, and 0.892 for controlsand tumor-bearing rats with smaller and larger tumors, respectively). The control rats' growth rate (Kg) equaled 3.5%/day.

There was a significant reduction (P < 0.05) in the fractionalgrowth rate in rats bearing larger tumors to 1.8%/day. Total-body protein synthesis over the 6-day period of observation inthe rats bearing larger tumors [40.5 ±6.9 g] was reduced,compared to rats with smaller tumors and controls [56.2 ±6.9and 63.9 ±11.5 g (P = 0.14), respectively].

In addition to differences in tumor size and body weight,there were also differences in the weight of liver, which wassignificantly larger in the tumor-bearing rats, (P < 0.01) andthe size of the liver as percentage of nontumorous body weightcorrelated with tumor weight (r = 0.918; P < 0.001). Thepercentage of nitrogen composition of the liver was similar inboth control (3.04 ±0.22%) and tumor-bearing (3.15 ±0.15%)rats; consequently, total nitrogen content of the liver was significantly increased in the tumor-bearing rats.

Fractional synthetic rates for whole body and muscle arerepresented in Table 2. The whole-body protein-synthetic ratewas slightly but not significantly reduced in rats with largertumors from 34.7 to 21.7%/day (P < 0.07). This estimate ofwhole-body K, of 34.7%/day for the non-tumor-bearing 220-ganimals is similar to that measured by McNurlan and Garlick(20) using [14C]leucine by the flooding dose method. The ratefor whole-animal protein synthesis in g per day can be normalized to 5.0 g per 100 g nontumorous body weight/day, iscomparable with previous estimates using the methodology ofconstant infusion of labeled amino acid [4.7 g/day (21) 4.0 g/day (18)], and is slightly higher than the amount determinedusing a flooding dose in younger animals (3.1 g/day). (20) Themean daily muscle protein synthesized, assuming that skeletalmuscle represents 40% of nontumorous body weight, (18) wassignificantly reduced in the animals bearing larger tumors (Table 2).

In the tumor-bearing rats, whole-body protein synthesis andmuscle protein synthesis showed an inverse correlation with theweight of tumors at sacrifice (r = -0.523 and -0.723; P < 0.05and < 0.01, respectively). In addition, body growth (Ä"„)was

also negatively correlated with tumor weight (r = —0.848,P <0.01). In comparison, whole-body and muscle protein synthesisdirectly correlated with body growth: [(Kg); r = 0.380 and 0.563;P < 0.05 and < 0.01, respectively]. Our data suggest then thatthe Walker 256 carcinosarcoma influences whole-animal protein synthesis by reducing both skeletal muscle and whole-bodyprotein synthesis (Table 2). Tumor growth and protein kineticdata are listed in Table 3. Tumor Ks was significantly lower inthe large compared to the small nonregressing tumors (48.6%versus 84.7%/day; P < 0.05). The daily mean and final daytumor protein synthesis were similar in both tumor groups.Nitrogen content of the tumor averaged 2.05 ±0.11 % and didnot vary with tumor size. The tumor leucine concentration was7.66 ±0.50% of the total nitrogen content.

Changes in tumor volume were used to calculate tumorgrowth which averaged 31.7%/day for the 15 nonregressingtumors. The regressing tumors (n = 3) followed complex growthcurves and were excluded from this calculation. A curve showing tumor growth, derived from the means of 15 data points, ispresented in Fig. 1 to illustrate the first-order kinetics patternfollowed by these tumors in the period between 7 and 13 daysfollowing implantation. Growth rate constants were obtainedfor individual tumors and showed a faster growth rate (34.3versus 27.7%/day) for the larger tumors as indicated in Table3.

Tumor Kd was determined by subtracting Kg from tumor Ks(Table 3). This estimate is determined from K, measurementsin the fasting state since it has been shown that tumor tissue is

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W256 INFLUENCE ON PROTEIN METABOLISM AND GROWTH RATES IN RATS

Table I Clinicalcharacteristicsof Walker256 carcinosarcoma-bearingand non-tumor-bearingratsFood intake and nontumorous weight gain are averaged over the last 4 days of the study period (days 9-13 post-tumor inoculation). Despite similar dietary intakes,

significant (/' < 0.05) reduction in nontumorous weight gain was observed in the larger tumor-bearing group. Liver weight and its percentage of nontumorous bodyweight were significantly increased in the rats bearing smaller tumors (/' < 0.01) and further increased in the larger tumor group (/' < 0.001). Comparison betweenthe 3 groups was by one-way analysis of variance and significance was defined by the Bonferroni test; P < 0.05.

Healthy control (6)Small tumor-bearing (9)Large tumor-bearing (9)Initial

body wt(g)143

±2"

148 ±3150 ±3Final

nontumorousbody wt(g)223

±6224 ±5202 ±10Tumor

wt(g)10.7

±1.729.0 ±3.2*Food

intake(g/day)22.4

±0.821.8 ±0.919.3 ±1.7Nontumorous

wt gain(g/day)7.4

±0.57.4 ±0.52.3 ±1.8eLiver

wt(g)6.9

±0.48.5 ±0.3*9.6 ±O.idLiver

wt(% of nontumorous

bodywt)3.1

±0.13.8 ±0.1*4.8 ±0.2J"

Mean ±SE."P

W256 INFLUENCE ON PROTEIN METABOLISM AND GROWTH RATES IN RATS

12«t

10Q\ö

8luK1

2Ol¡Ä

Tumor~~Q

WholeBody--_26%IM

MuscleHo27%I'2%>23%g

Non Tumor Small LargeControls Tumors Tumors

Fig. 2. Estimate of protein synthesis. Mean whole-body protein synthesis inhealthy rats was 10.7 g/day and was reduced to 6.7 g/day in rats with largetumors. Muscle protein synthesis was reduced significantly (p < 0.05) from 2.8to 1.7 g protein per day in the same group. Muscle protein synthesis in healthycontrol animals represented 26% of whole-body protein synthesis, and this wasreduced to 23% of whole animals (whole body plus tumor) protein synthesis foranimals with larger tumors. Tumor protein synthesis represented 8 and 10% oftotal protein synthesis, respectively, of the animals with nonregressing small andlarge tumors.

appears that the reduction in whole-body protein synthesis isnot completely explained by changes in muscle and tumorcompartments. Other sources of active protein synthesis, suchas the gastrointestinal tract, must also contribute to the overallreduction in whole-body protein synthesis. Evidence to supportthis comes from Radcliffe and Morrison, (26) who documentedover a 30% reduction in intestinal weight and presumablyprotein mass in rats of similar age and type after 20 days ofWalker 256 carcinosarcoma growth.

Nevertheless, changes in whole-body and muscle-syntheticrates appear to be the major factors responsible for alterationsin growth rates in the Walker 256 cancer-bearing rats. Thisobservation is consistent with the earlier work of Millward etal., (27) which demonstrated that muscle mass is regulatedprimarily through alterations in protein synthesis. Whole-bodygrowth, with or without tumor, had a parallel change withreductions in the synthetic rates, which suggests that alterationsin growth may be dependent upon these changes.

The finding of reduced A',,in the larger tumors is of interest

since it suggests that a possible factor affecting tumor growthis regulated by the process of protein degradation. As indicatedin Table 3, the average growth rate of the group of large tumorswas faster than that of the small tumors. The fact that thisdifference did not achieve statistical significance is probablyrelated to the variability inherent in tumor volume measurements early in tumor growth. Indeed, using the last 3 days oftumor measurements, the growth rates are significantly different for the two groups of tumors (data not shown). It should benoted, however, that a conclusion about reduction of Kd inlarger tumors is possible only if assumptions of first-orderkinetics for both protein synthesis and breakdown hold throughout the period of observation. These assumptions have beendiscussed by Waterlow (18) with regard to the use of isotopetracer methods for measurement of protein synthesis. Thegrowth curve in Fig. 1 suggests that tumor growth of the Walker256 carcinosarcoma could be approximated reasonably wellwith First-order kinetics between days 7 and 13, thus makingestimation of breakdown rates feasible as described (Kd = K, -

It is also understood that for the calculated rates of proteinbreakdown to apply, the growth characteristics of the tumors

at the time synthesis was measured should be similar to thoserepresented by the growth curve from day 7 to 13. Theoretically,sudden and severe reductions in growth rates could have occurred on day 14 in a manner which would account in part forthe reduced synthesis rates in the animals with larger tumors.However, such changes were not obvious in our study andwould need to be further addressed by a different experimentaldesign. In fact, using the last 3 days of tumor growth forregression analysis (data not shown) results in A',,/A\ ratios of

0.46 and 0.85 (P < 0.01) in the large and small tumors,respectively. The reduction in protein synthesis in the largertumors is not inconsistent with previous literature, (21, 28) andprotein synthesis rates in tumor tissue have been negativelycorrelated with tumor volume. (28) Furthermore, low rates ofprotein breakdown have been reported in transformed cell lines(BEN bronchial carcinoma, MCF7 and T47D human breasttumors, and SV40-transformed 3T3 fibroblasts), which contribute to the rapid growth rate of cancer cells. (22) We conclude,therefore, that while this study is not conclusive, it does supportthe possibility that protein breakdown regulates tumor growthin the Walker 256 carcinosarcoma in rats.

Finally, these findings do not supersede the host of imniu-nological, hormonal, and metabolic factors that play vital rolesin the mathematical relationships defined in this study thatdetermined the onset and rapidity of tumor growth. Rather, thecombination of biochemical measurement of tumor A\ andanatomical measurements of tumor growth (AK) as done in thisstudy provides a method of determining the theoretical proteinbreakdown and its relationship to tumor growth. The reducedprotein breakdown observed in this study of cancerous tissuelends support to the theory of tumors acting as nitrogen traps(19) as well as providing insight into potential novel approachesto alter tumor growth. In particular, directing chemotherapeuticagents towards increasing tumor protein breakdown in solidtumors, in addition to the conventional one of altering proteinsynthesis by antimetabolites, may offer a new avenue of therapeutic approach.

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W256 INFLUENCE ON PROTEIN METABOLISM AND GROWTh RATES IN RATS

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1986;46:5649-5654. Cancer Res John A. Tayek, Nawfal W. Istfan, Catherine T. Jones, et al. Cancer-bearing Ratand Whole-Body Protein Synthesis and Growth Rate in the Influence of the Walker 256 Carcinosarcoma on Muscle, Tumor,

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