Acid Hydrolase Activity During the Growth, Necrosis, and Regression of the Jensen Sarcoma

[CANCER RESEARCH 28, 1938-1943, October 1968]

Acid Hydrolase Activity during the Growth, Necrosis, and Regression of the Jensen Sarcoma

RalphF. Kampschmidtand Dan Wells Biomedical Division, The Samuel Roberts Noble Foundation, Inc., Ardmore, Oklahoma 73401

SUMMARY MATERIALS AND METHODS The subcutaneously transplanted Jensen sarcoma grew to a Tumor and Method of Transplantation. The Jensen sarcoma weight of 50-60 gm in 16â€”18days and then regressed. During was transplanted into female Holtzman rats weighing approxi the period of growth the tissue could be divided into a region mately 200 gm. Only the outer non-necrotic portions of the tu of necrotic dead cells and a region of live cells by the lissamine mor were used for transplantation. These portions of the tumor green exclusion test. The activity of 4 acid hydrolases (cathep were forced through 24-mesh stainless steel wire cloth placed sin, aryl sulfatase, $-galactosidase, and acid phosphatase) were at the base of a 10-nil syringe. Two volumes of these cells were determined in both tumor regions during growth and the be added to 3 volumes of a solution containing 50 mg each of peni ginning of regression. Measurements were made of the total cillin G and streptomycin sulfate in 100 ml of 0.9% saline. An enzymatic activity and the free activity or that not bound to injection of 0.25 ml was given either s.c. or in the rectus fe large cellular particles under the uniform set of conditions moris. The relative growth rate of the s.c. tumors was deter employed. mined by multiplying together the largest measurement of the A high percentage of the activity of the 4 enzymes was free length, breadth, and thickness of each tumor. in the inner necrotic region of the tumor. The total activity Sampling and Description of the Tumor Tissue. The vascu of cathepsin and acid phosphatase declined in the necrotic por lar zone of the Jensen tumor was determined by giving an intra tion with tumor age. There was a marked increase in both the venous injection of lissamine green and observing the access of free and total activities of cathepsin, aryl sulfatase, and fi the dye to various regions of the tumor (17) . After numerous galactosidase in the outer portion of the tumor during the early trials we were able to separate the active, growing, outer layer periods of regression. Total acid phosphatase activity declined from the inner necrotic layer without using the dye. As a check in the outer portion of the tumor during regression. The data on our separation, the lissamine green dye exclusion test for suggest that acid hydrolases are involved in the degradation of cell death as described by Goldacre and Whisson was used (18). tumor cells during both necrosis and regression of the tumor. On the basis of these criteria, the tissue used for analysis was classified as either the outer, actively dividing population or INTRODUCTION the inner, necrotic area of the tumor. The extremely necrotic During tumor necrosis or regression a large amount of ma or fluid portions found at the center of the tumors had very terial must be degraded and reutiized or excreted by the host. low enzymatic activity and were not routinely sampled. One possible step in this process could be the digestion of these The percentage of live tumor cells in each area of the tumor materials by the acid hydrolases of the lysosome. was estimated by gently teasing the cells apart and using the The lysosome was first recognized by de Duve et cii. (12) as dye exclusion test (18) . The distribution of tumor cells, leuko a sac-like structure containing a variety of acid hydrolases. cytes, and erythrocytes was determined after teasing apart re These particles have been shown to occur in a wide variety of peated samples of tissue and applying Wright's stain. animal cells and tissues (10) . The maintenance of the acid hy Fractionation into Free and Total Activities. The Jensen drolases in a state of latency inside a membrane has made it tumor cells were found to be more difficult to rupture than nor possible to show or suggest many diverse functions for the lyso mal liver cells. Considerable modification of the fractionation some in the economy of the cell (13) . Several different types of procedures used by de Duve et at. (12) to study intracellular tissue injury have been reported to decrease the stability of distribution of acid hydrolases in liver will, therefore, be neces the lysosomal membrane and release the acid hydrolases (28). sary for tumor tissue. For our present purpose we wished only The Jensen tumor was chosen for this study, since it would, to study the total amount of enzyme activity and to assess how upon subcutaneous transplantation of a small number of cells, readily these enzymes were released at various stages of tumor grow to a relatively large size and then regress. This provided growth. The tumor tissue samples were diluted 1:10 with ice an opportunity to study the free, or released, enzyme and the cold 025 M sucrose and mixed with 3 strokes of the Potter acid hydrolases bound to the lysosome during tumor growth Elvehjem tissue homogenizer. An aliquot for total enzymatic and regression. activity was given an additional 20 strokes after adding 0.2% Triton X-100. The total activity obtained was equal to that Received September 15, 1967; accepted May 18, 1968. found after either repeated freezing and thawing and then add

1938 CANCER RESEARCH VOL. 28

Jensen Sarcoma Acid Hydrolase Activity

ing various detergents or after thoroughly blending the tissue in a cooled Waring blendor. Another aliquot was centrifuged at 25,000 x g for 30 mm, and the activity determined in the supernatant was called the free activity. The free activity, I00 therefore, contained not only the enzyme released from the lysosome particle but also the activity of the microsomes. The homogenizing technic which we routinely used ruptured 80â€”90% of the tumor cells. Increases in free activity may, therefore, 80 represent increased fragility of whole cells as well as the lyso somal particles. E Enzymatic Activity. Cathepsin was determined on a 4% w/v 4;@ 60 hemoglobin substrate in 02 M acetate buffer at pH 3.6. The E

reaction was stopped with 5% trichioroacetic acid. This is a 0 slight modification of the method of Anson (1) . Catheptic ac > @ tivity is expressed as @molesof tryrosine/gm tissue/mm. Aryl 40 E sulfatase activity was measured by the method of Roy (24), I- using nitrocatechol sulfate as the substrate in 0.5 M acetate buffer at pH 5.4. The results are expressed as @molesofnitro @>20 4 cateehol sulfate/gm tissue/mn. A slight modification of the z method of Sellinger et al. (25) was used for ,6-galactosidase. The substrate o-nitrophenyl galactopyranoside was buffered in 02 M acetate at pH 5.0, and the reaction was stopped by add 5 10 15 20 25 ing 0.4 M glycine adjusted to pH 10.8 with sodium hydroxide. TUMOR AGE, DAYS The method of Lowry et at. (19) was used to determine acid phosphatase. The substrate was p-nitrophenylphosphate and Chart 1. The relative rate of growth and regression of the the buffer 02 M acetate at pH 5.5. These results are expressed subcutaneous Jensen sarcoma. The points show the mean and the at ,@moles of p-nitrophenol/gm tissue/mm. bars the standard error for a group of 50 rats. The relative Macrophages and Polymorphonuclear Leukocytes. Alveolar volume was obtained by multiplying the highest measurement ob macrophages were obtained from rat lungs by adapting the tamed for the length, breadth, and thickness of the individual technic of Myrvik et at. to the rat (21) . Polymorphonuclear tumors. leukocytes were obtained from peritoneal exudates produced by the injection of 50 ml of a 0.9% saline solution containing 0.1% shellfish glycogen, 3000 units of penicillin G, and 80 mg 17th and 20th days after tumor injections. The same amount of streptomycin into 200-gm Holtzman rats. Approximately 16 of tumor injected into the rectus lemons grew progressively and hr later the animals received 25â€”SOml of saline containing 50 killed the host about 2 weeks after injection. units of heparin. This fluid was removed after 1 hr and the A description of the Jensen tumor at various stages of sub leukocytes counted, centrifuged, washed with 0.34 M sucrose, cutaneous growth is presented in Table 1. Even while the tumor and diluted with 0.34 M sucrose to a concentration of 5 X 10@ was small it had a liquid center. The fluid core increased as the cells/mi. The macrophages and polymorphonuclear leukocytes tumor grew and represented approximately 50% of the weight were treated in the following manner to assure maximum re when the tumor attained maximum size. The blood leukocytes lease of enzymatic activity. The sucrose solution was drawn into were increased 2 to 3-fold above normal, and the most noticeable and forcibly expelled from a pipet at a rapid rate for 90 sec. change in differential count was the increase in monocytes with It was then frozen in a dry ice-acetone mixture and thawed tumor age. @ at 37Â°C in a water bath. The freezing and thawing was re The total number of cells in the fluid portion of the tumor peated 6 times as rapidly as possible. This method of rupturing decreased steadily with tumor growth. This was primarily due the polymorphonuclear leukocytes and lysosome membranes is to a decrease in the number of erythrocytes. Although the per similar to the one described by Cohn and Hirsch (8) and was centage of leukocytes to total cells in the fluid was high in the found to give the highest and most consistent enzyme activity. large or regressing tumors, the actual number of leukocytes was never more than 18,000/cu mm. RESULTS The solid portion of the tumor was divided into the inner necrotic area and the outer proliferating area of the tumor. Description of the Growing and Regressing Tumor. The Most of the tumor cells from the inner portion of the tumor relative rate of growth and regression of the subcutaneous Jensen were stained by lissamine green. The tumor cells from the outer sarcoma is shown in Chart 1. There was considerable variation portion of the tumor did not stain, indicating that our selection in the ultimate size attained by individual tumors and among of necrotic and living tumor regions was fairly accurate. During groups of tumor-bearing animals, but the time when regression tumor regression the layer of live cells, those not staining with started remained fairly constant. The first indications of a de lissamine green, became thinner and could not be found beyond crease in tumor size uniformly occurred some time between the the 21st day after transplantation. There was a marked in

OCTOBER 1968 1939

Table 1

sizeBloodieukocytesotal/cu age Total (%)Solid GroupTumorMI7â€”8 (days) (gin)Tumor (gm)Fluid (ml) TL mmDifferential NE 12II10.-il 65119,00043 43 2 15III12â€”13 149526,00052 30 3 17IV16-18 27161138,00051 30 2 18V19-21 51262524,00049 31 2 20GroupPercent 2214822,00046 32 2 fluid tumor tumorP@@nt live (%)TCell distribution (%)Percent live Cell distribution (%)Outer live distribution RW@I574tumor cellsbTumor RWT tumor cellsbInner RWT tumor cellsbCell 33468II533 9511863 3010m431 95.2864 3606560 3510IV271 8613149 4926555 2416V4456075546 5445060 3826560 24 31 Adescription of the Jensen sarcoma at various stages of tumor growth. L, lymphocytes; N, neutrophils; E, esinophils; M, mono cytes; T, tumor cells; R, erythrocytes; W, leukocytes. CEach group contained 3-7 animals with tumors in the age range indicated. b Percent not staining with lissamine green by method of Goldacre and Whisson (18). crease in the number of leukocytes in the outer tumor area at in the outer portion of the tumor. The free activity in the the start of the regression period. inner necrotic part of the tumor was higher throughout tumor Activity of Acid Hydrolases in Jensen Tumor. The free and growth than that found in the outer portion. total activity of cathepsin during growth and regression of the The $-galactosidase activity (Table 4) followed a pattern Jensen sarcoma is shown in Table 2. Results with the i.m. similar to the other 2 enzymes. There was a higher percentage tumor are presented at only 10 days, since no major change of the free activity in the inner portion of the tumor compared in enzymatic activity was observed during tumor growth. The to the outer portion. The total activity was significantly in total catheptic activity was lower in the inner portion of both creased during tumor regression in the outer portion of the the i.m. and s.c. tumor. The total activity remained fairly tumor. constant in the outer portion until the tumor started to regress. The activities of free and total acid phosphatase during Only one value is shown because 3-4 days after the start of tumor growth and regression are shown in Table 5. Acid phos regression, the outer layer of live cells disappeared and the phatase activity responded differently from the other 3 en total enzyme activity of the entire tumor was similar to that zymes to tumor growth and regression. There was a steady shown for the inner portion. The proportion of the activity decrease in total enzymatic activity in the inner portion of the found free was always higher in the inner layer than in the tumor during growth and regression. The outer portion con outer portion of the tumor. taming the live cells showed only minor changes during tumor The activity of aryl sulfatase is shown in Table 3. The total growth. When the Jensen tumor regressed, the total acid phos activity remained fairly constant in the inner and outer por phatase activity declined. tion of the tumor until the tumor started to regress. The re Acid Hydrolase Activity in Leukocytes. The increased total greasing tumor had a significantly higher free and total activity enzymatic activity of cathepsin, aryl sulfatase, and fl-galac

Table 2

TumortumorFreeTotalFreeTotali.m.1110219Â±4b29Â±40.18Â±0.01036Â±0.070.17Â±0.021.17Â±0.06s.c.88229Â±210Â±2022Â±0.030.72Â±0.14020Â±0.961.17Â±0.17s.c.1110240Â±516Â±30.17Â±0.020.55Â±0.080.22Â±0.04128Â±0.12s.c.138250Â±531Â±50.12Â±0.02Â°028Â±0.09â€•022Â±0.021.00Â±0.10s.c.169279Â±558Â±60.10Â±0.03c026Â±0.08c024Â±0.00Â°1.19Â±020s.c.'@19â€”2116252age of weight weight portion of tumorOuter portion of siteTumor (days)No. ratsBody (gm)Tumor (gm)Cathepsinactivity@Inner

Â±629 Â±5020 Â±0.030.62 Â±0.080.44 Â±0.05Â°2.03 Â±0.15c Changes in free and total catheptic activity during growth and regression of the Jensen tumor. a @smoles of tyrosine/gm tumor tissue/mm. b Mean Â± standard error. CSignificantly different from the mean of the i.m. tumor at a P value of 0.05 or less. d Regressing tumors.

1940 CANCER RESEARCH VOL. 28

Table 3

Aryl sulfatase activitya Tumor No. Body Tumor Tumor age of weight weight Inner portion of tumor Outer portion of tumor siteTotali.m. (days) rats (gm) (gm) Free Total Free 0.57Â±0.03s.c.11 16 223Â±4b 32Â±3 025Â±0.02 0.53Â±0.03 0.07Â±0.01 0.64Â±0.08s.c. 8 15 230Â±2 8Â±2 0.19Â±0.03 039Â±0.04 0.10Â±0.01 0.62Â±0.04s.c.11 19 242Â±3 15Â±3 025Â±0.01 0.57Â±0.03 0.09Â±0.01 0.64Â±0.04S.C.13 22 250Â±2 29Â±3 026Â±0.02 0.54Â±0.03 0.13Â±0.01Â° Â±0.06s.c.4 16 14 269Â±4 52Â±4 023Â±0.02 0.54Â±0.03 0.13Â±0.01c 0.69 0.08cChanges 19-21 21 254 Â±4 27 Â±5 031 Â±0.03 0.63 Â±0.06 027 Â±0.02c 0.99 Â± tumor.a in free and total aryl sulfatase activity during growth and regression of the Jensen tumor/min.bzmoles of nitrocatechol/gm wet weight of error.0Mean Â± standard less.dSignificantly different from the mean of the i.m. tumor at a P value of 0.05 or Regressing tumor.Table

4$-galactosidase

activityâ€¢ Tumor No. Body Tumor . @ Tumor age of weight weight lOller portiOn of tumor Outer portion of tumor Totali.m.site (days) rats (gm) (gm) Free Total Free 0.01S.C. 11 13 239 Â±2b 31JÂ±3 0.05 Â±0.01 0.15 Â±0.01 0.03 Â±0.01 0.18 Â± 020Â±0.01S.C. 8 10 229Â±5 5Â±1 0.06Â±0.01 0.17Â±0.01 0.05Â±0.01 0.01cs.c. 11 20 248 Â±3 15 Â±2 0.08 Â±0.01c 0.16 Â±0.01 0.05 Â±0.01C 024 Â± 0.01cs.c. 13 12 260 Â±3 26 Â±2 0.08 Â±0.01c 0.16 Â±0.01 0.05 Â±0.01 026 Â± 0.02Cs.c.': 16 10 282 Â±6 47 Â±4 0.08 Â±0.01c 0.15 Â±0.01 0.06 Â±0.01Â° 027 Â± 0.01â€•Changes19â€”21 25 268 Â±6 30 Â±4 0.08 Â±0.01C 0.15 Â±0.01 0.10 Â±0.01C 0.35 Â± tumor.a in free and total p-galactosidase activity during growth and regression of the Jensen tumor/min.bi@mo1es of o-nitrophenyl/gm wet weight of error.CMean Â± standard less.dSignificantly different from the mean of the i.m. tumor at a P value of 0.05 or Regressing tumors.Table

5Acid

phosphatase activity' Tumor No. Body Tumor . . Tumor age of weight weight Inner portion of tumor Outer portion of tumor siteTotaljill. (days) rats (gm) (gm) Free Total Free 4.70Â±029s.c. 11 24 235Â±2b 28Â±3 1.05Â±0.00 325Â±034 1.18Â±0.05 0.34Â°S.C. 8 16 232 Â±4 6 Â±1 1.41 Â±0.07c 4.49 Â±026â€• 138 Â±0.11â€• 5.52 Â± 4.47Â±021s.c. 11 18 248Â±4 15Â±2 0.88Â±0.06c 228Â±024c 131Â±0.07 4.65Â±031s.c. 13 8 ?59Â±5 27Â±3 0.58Â±0.05Â° 1.91Â±021â€• 1.35Â±0.08 036s.c.â€• 16 10 284 Â±6 48 Â±4 0.36 Â±0.04c 1.06 Â±0.09c 1.44 Â±0.07â€• 4.60 Â± 19â€”21 18 273 Â±5 35 Â±4 025 Â±0.03C 0.88 Â±0.07C 1.03 Â±0.09 423 Â±025Â° Changes in free and total acid phosphatase activity during growth and regression of the Jensen tumor. a @smoles of p-nitrophenol/gm wet weight of tumor/mm. b Mean Â± standard error. CSignificantly different from the mean of the i.m. tumor at a P value of 0.05 or less. 4 Regressing tumors.

tosidase during tumor regression might be the result of infiltra Table 6. The activity of all 4 enzymes was much higher in the tion of the tumor tissue with leukocytes. There was a marked macrophages than in the polymorphonuclear leukocytes. Un increase in the number of leukocytes in the outer region of the less the macrophages of the tumor tissue had still higher ac tumor during regression (Table 1) . The activity of the 4 acid tivities, it appears unlikely that sufficient leukocytes were hydrolases was, therefore, determined in polymorphonuclear present to account for the increased activity found in regress leukocytes and alveolar macrophages. A comparison of the en ing tumors. It would also be difficult to explain both the in zymatic activity in these leukocytes with the change in total creased cathepsin and the decreased acid phosphatase activity activity from growing to regressing s.c. tumors is presented in in the regressing tumors by an infiltration of leukocytes.

OCTOBER 1968 1941

Table 6

Activity in ,@niolea of substrate/unit tissue/mirt -DifferencebetweenEnzymePolymorphonuclear

and regreming leukocytes macrophagee tumor' tumor)Cathepsin0.061 (108 cells)Alveolar (108 cells)growing (gin 0.86Aryl Â±0.004â€•1.01 Â±027b+ 0.35p-Galactosidase0.011sullatase0.094 Â±0.002029 Â±0.12+ 0.15Acid Â±0.001021 Â±0.05+ phosphatase0.400 + 0.0201.30 Â±0.19â€” 129 Comparison of the enzymatic activities of polymorphonuclear leukocytes or alveolar macro phages with the changed activity observed in growing and regressing Jensen tumors. a The activity observed in the outer portion of the growing tumor at 8 days was subtracted from the activity in the outer portion of the regressing 19-day tumor. These values were taken from Tables 2.-S. b Mean Â± standard error.

DISCUSSION cubated for acid phosphatase activity showed that macrophages at sites of tumor rejection have more numerous, larger, and more The inner portion of the Jensen sarcoma indicates a continu intensely reacting lysosomes than macrophages seen in grafts ing process of cell death and necrosis (Table 1) . The percentage which are accepted. It is possible, therefore, that the macro of the total acid hydrolase activity which was free was gen phages which appeared in the tissue at the start of regression erally twice as high in the inner necrotic portions compared to in our studies had much higher acid hydrolase activity than the outer portions of the tumor. The high percentage of free the alveolar macrophages that we measured. Our results are in activity and the decrease in total activity in the necrotic por agreement with Carson and Donnenberg (7) , who found that tion of the tumor resembles similar findings in liver injury or pulmonary alveolar macrophages had higher hydrolytic en cirrhosis (22, 27) . A rapid release of acid hydrolases from the zyme activities than glycogen-induced peritoneal polymorpho lysosomes of the liver has been shown after ligation of the nuclear cells. blood vessels (1 1), starvation (2), or carbon tetrachloride Studies of tile cytologic changes during tumor regression by poisoning (2, 20) . An increase in free activity of the enzymes Brandes et al. (3â€”5) show an increase in the concentration determined in the present study during tumor necrosis was, and size of lysosomes during tumor regression. Histochemical therefore, not unexpected since lysosomal rupture seems to be tests with acid phosphatase indicated a release of the enzyme a rather general feature of autolysing tissue (9). from the enlarged lysosomes into the cytoplasm and inter Very few macrophages were observed in the necrotic area of cellular space during tumor regression. These changes in the the Jensen tumor. The contribution of leukocytes to the acid tumor cells occurred prior to the onset of other easily detect hydrolase activities observed in the necrotic area would be able cytoplasmic changes. very small. Gershon et al. (16) found that dead or dying tumor In addition to the changes which occur during tumor re cells from obviously necrotic regions showed no increase in gression, other investigations have shown alterations in acid stainable acid phosphatase. This finding suggested to them that hydrolase activity during carcinogenesis. Dzialoszynski et al. death of tumor cells was not due to autophagia. Our results (14)foundanincreaseinarylsulfataseinskin,stomach,colon, on the activity of acid hydrolases in the inner region of the or breast when they became cancerous. Shomberger and Ru tumor would also suggest that the acid hydrolase activity was dolph (26) observed increases in cathepsin, fl-glucuronidase, probably confined to the lysis of the dead tumor cells. and acid phosphatase in skin cancer compared to normal mouse The events which occurred during tumor regression were skin. An increase in lysosomes during tumor growth and rup different from those usually found during necrosis or autolysis ture during the terminal phase was found by Parry and of tissue. The increased total enzyme activity in the outer por Ghadially (23). tion of the tumor during regression might be from leukocytes or the tumor tissue itself. Studies by Fodor et al. (15) with REFERENCES the regressing Flexner-Jobling carcinoma showed a sharp in crease in catheptic activity during the course of regression. Acid 1. Anson, M. L. The Estimation of Pepsin, Trypsin, Papain and phosphatase showed only a slight increase during regression. Cathepsin with Hemoglobin. J. Gen. Physiol. 52: 79â€”89,1938. Thus, in both our studies and those of Fodor et cii. (15) acid 2. Beaufay, H., van Campenhout, E., and de Duve, C. Tissue Fractionation Studies. II. Influence of Various Hepatoxic phosphatase shows less change during tumor regression than Treatments on the State of Some Bound Enzymes in Rat some of the other acid hydrolases. This is rather unfortunate, Liver. Biochem. J., 73: 617-623, 1959. since acid phosphatase is the acid hydrolase used most fre 3. Brandes, D., and Anton, E. The Role of Lysosomes in Cellular quently in histochemical tests. Carr (6) showed that macro Lytic Processes. III. Electron Histochemical Changes in Mam phages increase in stainable acid phosphatase after stimulation. mary Tumors after Treatment with Cytoxan and Vitamin A. Studies by Gershon et at. (16) in which the tissues were in Lab. Invest., 15: 987â€”1006,1966.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1968 American Association for Cancer Research. Acid Hydrolase Activity during the Growth, Necrosis, and Regression of the Jensen Sarcoma

Ralph F. Kampschmidt and Dan Wells

Cancer Res 1968;28:1938-1943.

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