Arginase Activity in Solid Mouse Tumor Transplants and a Comparison with Ascites Tumors and Normal Tissues*

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The Histological Distribution of Arginase Activity in Solid Mouse Tumor Transplants and a Comparison with Ascites Tumors and Normal Tissues*

HARRY MALMOREN AND BENGT SYLVI~.N

(The Cancer l~search Division of Radiumheramet, Karolinska Instituter, Radiurahemmet, Stockholm 60, Sweden)

The first arginase assays on tumor tissues led tein synthesis has been discussed by Kochakian to the suggestion that such material in general et al. (10, 11), by Smith and Richterich (16) contains a greater arginase activity than do nor- and, with reference to the gluconeogenesis of the mal tissues (e.g., 8). The postulated increase in mammary gland, by Folley (4). arginine metabolism--together with the increased However, all arginase data on tumors so far glycolytic reaction--was even believed to repre- represent average figures on extracts from very sent basic characteristics of malignant growths large populations of tumor cells including cells in general (1~). These and other controversial with greatly different growth capacities and cyto- results (19) regarding the arginase activity of chemical characteristics. When it is considered tumors have been reviewed (17). By means of (el. ~) that solid tumors axe mainly built up of improved technics and comparable normal control a nongrowing cell type (B) and necrotic tissue, tissues, Greenstein, Jenrette, Mider, and White while the rapidly growing and multiplying typical (8, 9), in their macrochemical assays on liver and tumor cells (A type) represent only a small part manarnary tumors and also on lymphomas, found of the whole tumor mass, it will be understood that that the arginase levels in tumors were variable; more refined methods for the sampling and study in transplanted hepatomas as well as in embryonic of the particular cellular characteristics are needed. liver the values were markedly lower than in If the scale of sampling and assay were diminished normal adult livers, whereas the levels of spon- from a fresh weight of about 1,000 rag. (about taneous mammary tumors in mice were consider- gS0 mg. of protein content) to approximately ably higher and, in the case of lymphomas, slightly 0.01 rag. of total protein content per sample, higher than those in the comparable normal con- the enzymic data obtained could possibly be cor- trol tissue. Other tumors, e.g., a mouse melanoma, related with the cytological types and numbers a mouse Sarcoma 180, and the Jensen rat sarcoma of tumor cells per sample. This has recently been yielded high arginase levels similar to those of attempted in the study of the regional distribution the hepatomas and lymphomas. These results of the total dipeptidase and catheptic activity serve as a basis for the present concept of the in unicentric solid mouse tumor transplants (18). arginase activity of tumor tissue (6, 7). Subse- In this paper similar data will be reported on quently, few new results on the arginase activity the quantitative histological distribution of ar- of different tumor types have been reported. The ginase activity in a few representative tumor high activity levels in spontaneous C3H mouse types. Correlations with the regional cell types mammary adenocarcinomas and mammary glands and cell numbers per sample will be attempted. during lactation as compared with those in preg- nancy were recently studied on a fresh-weight A limited amount of data from some normal mouse basis (16). The arginase activity figures were cor- and rat tissues has been added for comparison. related with the tissue concentrations of RNA, The reason why an amidase like arginase was cho- DNA, and total nitrogen content. The possible sen for study was that this enzyme could be relationship between arginase activity and pro- assumed to play some essential role during cellular * This investigation was supported by grants from the growth and protein metabolism and, further, that Jubilee Fund of King Gustaf V, the Swedish Cancer Society, a suitable micromethod for assay was available. and the Jane Coffin Childs Memorial Fund for Medical Re- In addition, the previous conflicting findings and search, all of which is gratefully acknowledged. ideas as outlined above suggest that differences Received for publication December 28, 1958. in arginase activity occur between different tumor 5~5

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 5~6 Cancer Research Vol. 19, June, 1959 cell generations; this would explain the variations All transplants were actively growing without in the previous macroscale figures. histological or clinical signs of regression (cf.18). The present micromethod, as well as the com- For comparison, a series of ascites tumors has mon macrochemical procedures, provides chemical been studied, comprising the hyperdiploid Ehrlich- data referring to the in situ characteristics, viz., Landschtitz and tetraploid Ehrlich strains. no distinctions are made between contributions Normal control material.--H om o gen a tes of larg- derived from the tumor cells per se and the er samples and serial sections of punched-out inherent stromal and interstitial fluid compart- cylinders were studied from normal mouse muscle, ments. This point will be further discussed below. subcutaneous connective tissue, and liver of the Furthermore, the term arginase activity refers same strains. Data on whole blood, plasma, eryth- to the total activity obtained with the present rocytes, and leukocytes, separated according extraction method. The actual total amounts of to the technic of Bucldey et al. (1) were also added. enzyme cannot, as usual, be evaluated. These latter materials are of importance for the evaluation of the enzymic activities residing in MATERIALS AND METHODS the stromal compartments of tumors. The diffi- General procedure.--The main steps in the sam- culty of getting a pure fibroblast material has pling of fresh tumor material and normal mouse further prompted some assays on the Earle strain tissues intended for serial sectioning were as fol- L fibroblasts grown in in vitro suspensions. lows: Unicentric intramuscular tumor transplants Extraction method.--Serial sections intended for enzymic were used. From a block of fresh tumor tissue, assay were lysed and extracted for 1 hour at room temperature excised from the living animal under narcosis in 5 or 7 gl. of 0.05 per cent aqueous sodium deoxyeholate solution containing 25 per cent glycerol (18). The extent of tumor and rapidly frozen in isopentane chilled by liquid cell cytolysis was estimated microscopically at about 80 per air, small cylinders were punched out and then cent. All cell debris was left with the extracts in the small ex- stored in isopentane chilled by solid carbon dioxide. traction vessels during the subsequent incubation with the Precautions were taken so that the punched out substrate. Fresh normal tissues and isolated blood cells were tumor cylinder included a good deal of the sur- homogenized for about 20 minutes with continuous cooling in small motor-driven homogenizers in a similar solution of rounding normal connective tissue and muscle, sodium deoxycholate, and the homogenates were then ex- the peripheral growing tumor zone, and some tracted for another hour without further stirring. of the more or less necrotic tumor center. The The relative efficiency of various hypotonic extraction length of such punched-out cylinders was in gen- media was previously tested (18). With reference to arginase it was found that about 50 per cent more activity per total pro- eral about 6 mm., and the diameter of the punch tein content was extracted from serial sections of mouse liver was 8 mm. The cylinder was then frozen on to lysed in deoxycholate solution than was obtained following the the microtome head with a drop of water and usual homogenization of larger liver samples in deoxycholate subjected to serial sectioning in a cryostat at solution (Table 3). The use of a lytic agent in the extraction about - 15 ~ C. The serial sections were each about fluid seems of advantage only when mechanical homogenization of tissue sections cannot be accomplished. 10-1~/~ thick, 3 mm. in diameter, and contained Arginase assay.--The arginase assay, according to a micro- on an average a total amount of protein of about adaption of Greenberg's method (5), is based upon the de- 8-1~ gg. The average volume of these samples termination of urea liberated during the enzymic reaction was about 0.08 ~1. catalyzed by Mn ++ ions. The urea is precipitated with xanthydrol, and the dixanthyl urea (DXU) is determined colori- Successive serial sections were then used al- metrically at 430 m/~. ternately for histology and cell counting, deter- A volume of 5 gl. of the enzyme (tissue extract) was first mination of the amount of protein mass, and preineubated for about 3 hours at 40~ C. with 5 gl. of 0.05 M peptidase and arginase assays. Sections for his- MnSO, containing 0.05 Mmaleate at ptI 7. Then 5 gl. of 0.85 M tology were dried, while still frozen, on glass arginine solution at pH 9.5 was added. The final pit was 9.4. After 10 minutes' incubation at 40~ C. the reaction was stopped slides in air or in a desiccator. Sections used for by addition of 20 gl. of 87 per cent acetic acid. The urea was chemical analyses were transferred, while still fro- precipitated by addition of 3 gl. of a 5 per cent xanthydrol zen, to precooled small glass tubes and thawed solution, and the mixture was left in the refrigerator overnight. when the extraction medium was added. DXU was washed once with a saturated DXU solution in These procedures have recently been described methanol, and twice with a saturated DXU solution in methanol-water (3:1). After being dried in vacuo the DXU was in detail, and the technical errors have been dissolved in 200 gl. 50 per cent sulfuric acid. After 20-30 evaluated (18). minutes the color intensity was read at 430 mtt against 50 per Tumor strains.~Tumor transplants have been cent sulfuric acid in 10-ram. Beckman euvettes. investigated from Sarcoma 37 grown in common A control for the reagent blank was prepared by adding the acetic acid to the arginine-arginase mixture immediately after albino mice, from melanoma Harding-Passey in incubation instead of after 10 minutes. The blank value was inbred CBA and albino stock mice, and from small, amounting to an extinction of 0.02-0.04 per tissue mammary carcinomas in ABC and CBA strains. section.

Under the experimental conditions described above, an ex- melanoma were serially sectioned along their longi- tinction of 0.100 corresponded to 0.13 per cent hydrolysis of the tudinal axis, and the l~-g-thick sections were substrate. A linear relationship between enzyme concentration and extinction (E) was found up to at least E = 1.5 under the subdivided by microdissection. These parts were experimental conditions mentioned above (cf. 5). then separately extracted in deoxycholate solution Peptidase assay.--The total, over-aU peptidase activity of and assayed for arginase activity. Cell counts alternate intermediate sections was determined according to were not made, but the total protein mass of the microtechnic developed by LinderstrSm-Lang and Holter each subdivision was determined on the adjacent (18) with alanylglycine used as a substrate (for details see Ref. 18). It was found that the dipeptidase activity of tumor tissue section dissected in the same way. No measurable was a valuable indicator of the regional vitality and distribu- arginase activity was found in the thin zones tion of tumor tissue, and, hence, these data are included for of changed connective tissue immediately outside comparison. the tumor borders. The activity in the external Mass determinations.---Since the sections were too sma]l for accurate routine weight determinations, the necessary mass muscle layer did not exceed the normal range. correlations were obtained on alternate intermediate sections Most of the observed arginase activity resided by using a chemical method for the determination of the total protein content per section, according to Nayyar and Glick TABLE 1 (14; cf. 18). tIistology of sections.AOne section out of each sequence of RELATIVE /~GINASE ACTIVITY OF A AND B CELLS six to eight serial sections was dried and stained for histology. IN THE INVESTIGATED SOLID MOUSE TUMORS The pertinent tumor cell types and numbers per section were Average activity figures obtained from eleven different recorded (of. 18), together with the results on the general cases of serially sectioned tumor cylinders (cf. Charts 1-3). morphology and vitality of the tumor tissue, the extension of Arginase activity refers to l0 minutes' incubation time; the stromal compartment, etc. To reach a graphic reconstruc- original figures per section have been recalculated per 10,000 tion of the tissue components contained in the sectioned tumor tumor cells. cylinders, the approximate areas in per cent of the total section A cells refer to the young, growing, and rapidly multiply- area covered by the main components, such as muscle (at the ing tumor cells at the extreme periphery of a unicentric trans- periphery), connective tissue, vital and necrotic tumor tissue, plant. B cells is a label common for the more central, larger, etc., were roughly estimated under the microscope. The sur- and no longer multiplying, but still vital, tumor cells. For face ratios of these components of each section were then again cytological characteristics cf. text and references Nos. 2 and 18. assembled in due order to form a so-called histogram as illustrated in Figures 1-3. The histograms do not represent pie- tures of a longitudinal section of the tumor cylinders. If, in- ARGINASE ACTIVITY PER stead, the histograms are read together with the other graphs 10,000 a'v~oR C~LLS in a vertical direction, the approximate surface ratios of the TUMOR MA TERIAL8 No. CASES component parts at each level along the cylinder can be A cell regions B cell regions ascertained. (E)* (E)* Owing to the regular texture and centrifugal growth pattern of these unicentric transplants, the youngest and most actively Sarcoma 37 1.0 0.?,8 growing and invading tumor generations were always found at Transplanted mam- the extreme periphery of the tumor nodules, while the older mary carcinoma 0.71 0.~.~ Melanoma Harding- cell generations made up the interior of the transplants. It can Passey 0.65 O. ~5 be estimated that the older cell generations constituted about 80-90 per cent of the whole mass of most tumor cylinders investigated. * E = 0.100 corresponds to 0.13 per cent hydrolysis. Cell counts were performed in each section intended for histology. The approximate numbers of tumor cell nuclei per in the peripheral tumor region, which contained section have been plotted in the graphs. The method for mak- ing the tentative correlations between the observed enzymic about 10 times more activity per gg. protein data per section and the particular microscopic characteristics content than the vital B cell zones. A relatively of adjacent sections used for histology in the various tumor reliable average figure was obtained from the regions have been described in detail (18). The deductions and B cell zones, namely, E = 0.8-0.5 per 10 #g. obvious corrections for contributions to the total enzymic tumor protein. These dissection attempts have activity per section derived from constituents other than tumor tissue will be illustrated below (cf. 18). been thus far successful only in the case of melanomas in which the pigmented tumor cells RESULTS could be clearly distinguished. It is provisionally The relative arginase activity figures of the assumed that the conditions are also the same in main tumor cell types are presented in Table tumor transplants of other types. 1. The original data from the serially sectioned Data available from the three types of solid tumor materials are further illustrated in detail tumor transplants thus far investigated suggest in Charts 1-3, which for comparison also contain that the peripheral tumor zones containing the data on the total dipeptidase activity per section. young and growing tumor cells of A type appear In order to analyze the main tissue compart- to carry at least 3-4 times more arginase activity ments separately, fresh punched-out cylinders of per cell than the more centrally located tumor

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. Transpl.anted Mammary Carcinoma CBA Dipeptidase Activity A per section B per 10.000 tumor celts -r-8 o Zt~ 6 Q 20 ,-4 10

Arginase Activity C I per section D per 10.000 tumor celts

8 o.6t| ~ ,~} /~' 0.61 ~ 0./-, Oh "L 9 9 '"0.2 o21 9 e 9 , , 9 , ,

E Protein Content F C.t. Invasion MuscLe 1584 \ I L E ,_ 10 84 O ._(2 "~ ~1 Necrosis 201

160 200 (Series GM) Section No. A ceils 'B celts A cetis ' 1 :2 mm C~ART 1.--The graphs should be read in a vertical direction The following abbreviations have been used: in order to correlate all of the data for any given section of the AlaGly -- incubated with alanylglycine as a substrate. tumor. The histograms of the serially sectioned tumor cylinders C.t. = connective and adipose tissue. do not represent a longitudinal section of the cylinder. Instead, T.C.N./section = the approximate total tumor cell nuclear when the histogram is read vertically to the cylinder axis, the counts per section. linear extensions of the various tissue components are propor- A = A cells only. tional to the rough quantitative surface distribution of these B = B cells only. components in the corresponding sections. The approximate total tumor cell counts per section are plotted in a graph below Data from a l~-day-old unicentric transplant of a CBA the histogram. Below these plots, the sections which con- strain mammary carcinoma; the tumor diameters were about tained tumor tissue with only A or B cells, respectively, are ~0 X 15 X 15 ram. The first cylinder started with a wide zone indicated. The intermediate regions contained a mixed popula- containing dispersed tumor cells; the second part contained tion of A, intermediate A to B, and B cells. The typical grow- solid tumor tissue. The tumor periphery was cut obliquely. ing A cells are generally found only at the extreme periphery The second cylinder started far out in the surrounding muscle, of the transplant along its entire border to the connective which appeared normal, and ended at the tumor periphery tissue. In general, this region is represented diagrammatically which contained mainly tumor cells of the A type. as cross-hatched and labeled as the invasion zone. A fairly even In both cylinders the dipeptidase and arginase activity distribution of a small number of A cells is present along the curves were roughly parallel to each other. There was no direct invasion zone on the border to the surrounding host tissue, proportionality between activity values and tumor cell num- and consequently no maximum in the number of A cells can bers per section. The dipeptidase and arginase activities did not be said to exist. Correlations between enzymic data and tumor reach the same high levels as in the previous case. In the cell numbers are attempted only in sections containing more second sample the peptidase activity increased about 0.S mm. than 1,000 tumor ceils. For further explanations the readers outside the tumor border, while the arginase activity rose first are referred to our previous detailed presentation (18). at the tumor border. 528

A per section B per 10,000 tumor celts

-r 8 O z 61 f ~ /r 20 10 ~,2 ~ AlaGly

Arginase Activity C per section D per 10.000 tumor cells

1.0

c 0.8 08 9 9 .9 L) "'"4 O~ .~- O. 0.6 9 9 9 " 9 x 0.4

0.2 0.2 , L . , i , i , , i

E Protein Content F Tumor 15- Y (/1 E c ~10- o o t_ o ul ~'5" Z (J _J i , i i 200i A cells B cells (Series GV) Section No. i i ' 1 '' 2 mm CHART 2.--See also general legend of Chart 1. One serially 1-17; cell numbers dotted) a few dispersed tumor cells oc- sectioned tumor cylinder from a 24-day-old intramuscular curred, roughly 100 cells per section, but isolated cells could not transplant of a CBA mammary carcinoma. The tumor size was be identified with certainty. about 40 X 30 X 25 ram. This tumor showed an extensive in- The data suggest that the regions containing only a limited vasion zone into the surrounding edematous connective tissue; number of growing cells of A type carry the highest per-cell the edema led to low protein figures per section in the first two enzymic activities. Both activities dropped to low magnitudes thirds of the sample. The external noninvaded muscle coat following the onset of necrosis. seemed normal. At the first part of this cylinder (sections No. 5~9

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. Melanoma Harding-Passey Dipeptidase Activity A per section B per 10.000 tumor celts 10 $ 81

Arginase Activity

i C J per section D per 10,000 tumor cells 1.0

c 0.8 .o 0.6 0.6- X 0./~ " 0.4"

0.2 0.2

9 II1 |

E Protein Content F MuscLe Tumor C.t. Tumor 15- ~n E ~.~ 10 c~ "C~.t. Invasion MuscLe ._~ Z5

~ 5"0 II]0' 150 B B cells (SeriesGB) Section No.

'= i )ram C~ART 8.--See also general legend of Chart 1. Two samples tumor cells of pure A and B type is indicated at the bottom of from a fairly rapidly growing 7-week-old I-Iarding-Passey Section F. In the first cylinder, the part of the tumor between melanoma in a CSI-I mouse. The tumor size was about 40 X the A and B cell regions was occupied by A and intermediate 80 X ~0 ram. The cylinders included the growing A cell zone A to B cells. The first part of the second cylinder, before the along the tumor periphery and the vital B cell zone in the in- B cells predominated, likewise contained A and intermediate terior of the tumor. The external connective tissue sheath was A to B cell types. edematous and partly invaded by bxmor cells. The high protein The regional distribution of arginase was markedly similar mass figures at the beginning of the first cylinder were due to to that of the dipeptidase activity per section. The correlated normal muscle. figures suggest that the growing A cells contain about ~-4 In the second cylinder the muscle layer was rather edema- times more arginase activity than the older B cells. tom but not yet invaded by tumor cells. The distribution of 530

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. MALMGREN AND SYLvf~mHistological Distribution of Arginase Activity 531 regions with vital cells of B type. The latter over the whole population was 0.00-0.04 per cent cell type was uniformly low in arginase activity hydrolysis per 10,000 cells per 10 minutes. This throughout all tumor types, irrespective of tumor range a~ees with that of Westfall et al. (20). age or size. The relative arginase activity runs In relative terms, the average activity of strain surprisingly parallel to that of the total dipep- L cells was about ~o of that of the liver cell argi- tidase activity per section (Charts 1-3). Necrotic nase level (Table 3). tumor zones have always shown little activity. Another interesting point is that the arginase DISCUSSION activity of the homogenized ascites tumor ceils As outlined above, the apparent differences (Table 2) was distinctly lower (one-third to one- in cytology (2) and growth activity between the fifth) than that of the B cell regions in solid peripheral A cells and the more central B cells tumors. This was also the case with the total imply that considerable biochemical and phys- catheptic activity, while the total dipeptidase ac- iological differences between these cell types exist tivity of ascites populations was previously found TABLE 3 to be as high as the B cell regions of solid tumors (15, 18). AVERAGE ARGINASE ACTIVITY OF SOME NORMAL The arginase activities of some normal mouse MOUSE TISSUES AND CELLS tissues, mouse blood, and blood cells are given Data on serial sections of fresh solid tissues and blood cell homogenates extracted in 0.05 per cent deoxycholate in Table 3. It can be seen that normal liver has solution. TABLE 2 ARGINASE ACTIVITY AVERAGE ARGINASE ACTIVITY OF ASCITES TUMOR CELLS Per cent hydrolysis per 10 minutes Samples of tumor cells, at various times after the inoculation, were homogenized in a 0.05 per cent deoxycholate solu- MA TERIAI~ Per 10 ~g, tion. Argilmse activity is expressed as the extinction per Per 10,000 protein Per /~l. I0,000 tumor cells per 10 minutes incubation time. All figures cells content are means of triplicate tests in samples of each three ascites tumor mice. The recorded ranges indicate the individual vari- Subcutaneous connective ations. tissue 0.3 Skeletal muscle 0.05 0.10 Liver 4.4" r Micrograms Mouse blood 0.25 Days afteri No. ceils >( of protein Arginase Mouse plasma 0.11 Tumor lines inocula- 10 TM per ml. per 10,000 activity Mouse erythrocytes 0.0002 tion (range) tumor cells (E)* Mouse white blood cells 0.04 (range)

~Iyperdiploid 200-300 1 .g-1.5 O. 04--0.06 * The corresponding average figure for homogemzed macro- Ehrlich- 200 2.2-3.4 0.04-0.1~ samples of liver was 3.0 per cent hydrolysis. Landschtitz 170--270 1.8--2.8 O. 05 ascites tumor in various respects (18). The topochemical data cells 12 200 2.2 O. 05 in Table 1 and Charts 1-3 indicate that the Fetraploid 0.02 arginase activity per serial section of the tumors Ehrlich 0.02 ascites is not proportional to the total numbers of tumor tumor cells cells contained in each section. The observations suggest that in the peripheral regions a larger * E--0.100 corresponds to 0.13 per cent hydrolysis. arginase activity is present either in the tumor cells per se or in other tissue components of this the highest activity. Growing solid tumor cells region. The serially sectioned cases and, in addi- reached approximately one-third to one-fourth tion, the dissection experiments reported above of the per-cell liver level. The B cells of solid tu- indicate that the adjacent and somewhat changed mors, and still more the ascites tumor cells, pre- stromal connective tissue just outside the tumor sented a much lower level of arginase activity contains insignificant arginase activity comparable (Tables 1 and 2). to that of other normal connective tissue. The red The fibroblast strain of Earle (NCTC clone blood cells present in the sections were not very 929 L), grown in in vitro suspensions with the numerous and carried little activity, as shown addition of 10 per cent blood serum, showed a by separate assays (Table 3). Another possibility still lower per-cell arginase activity. At the end is that additional enzymic activity could be de- of their growth period, when a cell titer of about rived from the interstitial fluid at the tumor 106 per cc. had been reached, the range of activity periphery. This has not yet been investigated~

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. 53~ Cancer Research Vol. 19, June, 1959 owing to the difficulties in sampling such fluid; ascites tumors were serially sectioned and assayed. moreover, it seems improbable that its enzymic The usual arginase activity levels (Table 1) of activity would attain figures high enough to ac- the A and B cell zones were then again demon- count for the observed increases in activity per strated. A similar peculiar shift in enzymic activity these microsamples (the total volume of each between ascites and solid forms of the same tumor serial section is only about 0.08 gl.). The most strain was also observed previously with reference probable explanation seems to be that the growing to the total per-cell catheptic activity (18). Fur- tumor cells in the peripheral regions carry more thermore, tissue fibroblasts, which in vivo contain arginase activity than do the older, nongrowing a measurable arginase activity (Table 3), seem B cell generations. This result parallels previous to lose most of this activity after prolonged in topochemical data on the distribution of dipep- vitro culturing. The recent data by Westfall et a/. tidases and cathepsins (18). (20) have been confirmed in the case of the strain Comparison between the tumor arginase figures L fibroblasts first isolated about 17 years ago and those of selected normal tissues may be made by Earle. (Table 3). Unfortunately, comparison is not pos- Thus, the results seem to imply, as expected sible between our tumor transplants and their from other lines of study, that we are concerned normal homologs. We can only state that the with a variety of tumor cell types depending cells in the actively growing tumor regions of upon environmental and other conditions. The the autonomous tumors investigated have high average enzymic characteristics of one cell type arginase activity, amounting to about one-third in solid tumors may not be the same as those to one-fourth of the activity observed in nor- of a similar cell type in the ascites form or in tumor mal mouse liver cells, tIomogenates from primary cultures. Such disparities between different tumor and transplanted hepatomas and from spontane- cell materials call for great caution against far- ous mammary adenocarcinomas have shown even fetched generalizations on the basis of the usual higher relative levels on a weight basis (cf. 8, macroscale assays generally involving heterogene- 9, 16). Therefore, we expect that growing ceils ous cell materials. from different tumor types may show a considerable range of variation in their relative arginase SUMMARY activity. The topographical distribution of arginase activ- If the observed arginase values have some sig- ity has been investigated at the histological level nificance in vivo, it seems possible that in the rate in solid unicentric transplants of three different of urea production and regeneration of ornithine types of autonomous mouse tumors. Assays have there are marked differences in the growing tumor been performed on freshly frozen serial microtome cells of A type as compared with those in the sections with an average protein content of about B cell generations. This would seem valid in most 10 gg. This technic permitted sufficient separation solid tumors. The reasons why the average ar- in space for a separate sampling of the peripheral ginase activity values of ascites tumor cell popula- tumor regions containing the young, growing ceils tions are surprisingly low are not clear. The ascites and the more central regions of older cell genera- tumor cells may represent specially adapted forms tions. in which the urea cycle is of less significance In continuous series of tumor sections there than in the solid tumors. The observed average was no proportionality between the observed ar- activity data do not mean that each ascites cell ginase activity and the number of tumor cells has the same quantitative enzymic pattern. A of a given sample. The young and rapidly growing small number of tumor cells may have a higher tumor cells had at least 3-4 times as much arginase arginase level (if they could be analyzed separate- activity per cell as the still vital, nongrowing, ly). This might provide an explanation for the central tumor cell types. Necrotic tumor regions variations in arginase activity with time after were almost completely lacking in arginase ac- inoculation; our data in Table ~ suggest that the tivity. per-cell arginase activity is higher during the early The histological distribution of arginase paral- growth phase of the ascites tumor than later on. leled closely the dipeptidase activity of the same It is possible that the ratio of growing cells is tumors. greater during this first period. The arginase activity of solid tumors was com- It is natural to think that nutritional conditions pared with that of ascites tumor cells and of some are primarily responsible for the low arginase normal mouse tissues. The activity of the investi- activity of the ascites population. This view is gated growing tumor cells was about one-third supported by other experiments in which solid to one-fourth that of normal mouse liver. The aver-

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. M:ALMGREN AND SYLvaN--Histological Distribution of Arginase Activity 533 age arginase activity of two ascites tumor cell of Certain Tumors and Normal Control Tissues. J. Nat. populations as well as that of cultured strain Cancer Inst., 1: 687-706, 1940-41. 9. . The Relative Arginase Activity of Certain Tumors L fibroblasts was found to be very low. and Normal Control Tissues. J. Biol. Chem., 137:795-96, The biochemical differences between the two 1941. main classes of cells in solid tumor transplants 10. KOCHAKIAN,C. D.; KEUTMANN, E. H.; and GARBER, E. E. were emphasized. It is not yet known why the Studies on Blood Arginase. In: Conference on Metabolic older tumor cell generations in several respects Aspects of Convalescence, 17:187-203, 1948. 11. KOCHAKIAN, C. D., and ROBERTSON, E. Corticoids and present very low enzymic activities. Body and Organ Weights, Nitrogen Balance and Enzymes. J. Biol. Chem., 190:481-94, 1951. ACKNOWLEDGMENTS 12. K6HLER, K. Enzymologie der Tumorzelle. Ergebn. d. The authors wish to thank Professor G. Klein and Dr. L. Enzymforsch., 6-7:157-88, 1987. R~v~sz of Karolinska Institutet for the generous supply of 13. LINDERSTRSM-LANG, K., and HOLTER, H. Beitriige zur ascites tumor-bearing mice used in this study. Our thanks are enzymatischen Histochemie, II. Ueber die Peptidasever- also due to Associate Professor T. Gustafsson and Miss Elin teilung in Wurzel und Blattkeim des Malzkornes. Ztschr. Klein of the Wenner-Gren Institute for the cultivated strain L physiol. Chem., 204:15, 1932. fibroblasts. 14. NAYYAR,S. N., and GLICK, D. Studies in Histochemistry. XXXI. A Method for the Determination of Protein in REFERENCES Millimicrogram Quantities. J. Histochem., 2: 282, 1954. 1. Bvc~Y, E. S., JR.; POWELL, M. J.; and GmSON, J. G. 15. MAL~OREN, H.; SYLVAN, B.; and R~v~sz, L. Catheptic The Use of Fraction I for Separation of Erythrocytes and and Dipeptidase Activities of Ascites Tumour Cells. Brit. Leucocytes from Whole Blood. In: The Preservation of the J. Cancer, 9: 473-79, 1955. Formed Elements and of the Proteins of the Blood, p. 185. 16. SMITH, T. C., and RICHTERICH, B. Arginase Activity and Boston: Harvard Medical School, 1949. Nucleic Acid Content in Mammary Adenocarcinoma and ~. CASPERSSON, T., and SANTESSON, L. Studies on Protein Normal Homologous Tissues of C3H Mice. Cancer Re- Metabolism in the Cells of Epithelial Tumours. Acta search, 17:1006-9, 1957. Radiologica., Suppl., XLVI. Stockholm, 1943. 17. STERN, K., and WILLHEIM, R. The Biochemistry of 3. EDLBACHER, S., and MERZ, K. W. ~ber den Stoffwechsel Malignant Tumors. Brooklyn, N.Y.: Reference Press, der Tumoren. I. Mitteil. Ztschr. physiol. Chem., 171: 252- 1943. 63, 1927. 18. SYLVAN,B., and MALMOREN, H. The Histological Distribu- 4. FOLLEY, S. J. Biochemical Aspects of Mammary Gland tion of Proteinase and Peptidase Activity in Solid Tumor Function. Biol. Rev., 24:316-54, 1949. Transplants. A Biochemical Study on the Enzymic Char- 5. GREENBERG, D. M. Arginase. In: S. P. COLOW~CK and acteristics of the Different Tumor Cell Types. Acta N. 0. KAPLAN (eds.), Methods in Enzymology, 2:368-74. Radiologica, Suppl. 154. Stockholm, 1957. New York: Academic Press, Inc., 1955. 19. WALDSCHMIDT-LEITZ,E. ; McDoNALD, E. ; and co-workers. 6. GREENSTEIN, J. P. Enzymes in Normal and Neoplastic l~ber Enzyme in Tumoren. I. Ztschr. physiol. Chem., Animal Tissues. In: A.A.A.S. Research Conference on Can- 219:115-27, 1933. cer, pp. 192-222, 1945. 20. WESTFALL,B. B.; PEPPERS, E. V.; EvANs, V. J.; SANFORD, 7. --. Biochemistry of Cancer, 2d ed. New York: Aca- K. K.; HAWKINS, N. M.; FIORAMONTI, M. C.; KERR, H. demic Press, Inc., 1954. A.; HOBBS, G. L.; and EARLE, W. R. The Arginase and 8. GREENSTEIN, J. P.; JENRETTE, W. V.; MIDER, G. B.; and Rhodanese Activities of Certain Cell Strains after Long WHrrE, J. Chemical Studies on the Components of Normal Cultivation in Vitro. J. Biophys. & Biochem. Cytol., 4: and Neoplastic Tissues. V. The Relative Arginase Activity 567-70, 1958.

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1959 American Association for Cancer Research. The Histological Distribution of Arginase Activity in Solid Mouse Tumor Transplants and a Comparison with Ascites Tumors and Normal Tissues

Harry Malmgren and Bengt Sylvén

Cancer Res 1959;19:525-533.

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