Hereditas 86: 103-106 (1977)
Lysosomal enzymes in rat sarcomas induced by 7,12-dimethylbenz(a)anthracene and Rous sarcoma virus BJoRN HULTBERG and FELIX MITELMAN Dqurtments of' Clinicul Chemistry und Clinicril Genetics, University Hospitul, Lund. Sweden
HULTBERG, B. and MITELMAN. F. 1977. Lysosomal enzymes in rat sarcomas induced by 7,12dimethylbenz(a)anthracene and Rous sarcoma virus. Herediras 86: 103-106. Lund, Sweden, ISSN 0018-0661. Received February 2 3 , 1977 -
The lysosomal apparatus in normal rat fibroblast cultures and in DMBA and RSV induced rat sarcoma cell cultures was compared. Both DMBA and RSV induced sarcoma cells showed relative increase of lysosomal enzyme activity outside the cells in the medium compared to the activity within the cells. An early equilibrium of enzyme activity level was reached in the medium from RSV sarcoma cells, whereas this was not seen in cultures of normal fibroblasts and DMBA sarcoma cells. There was a difference in the intracellular content of lysosomal enzymes between RSV and DM BA sarcoma cells. This was most clearly seen for N-acetyl-P-glucosaminidase. These findings and the possible role of lysosomal enzymes in malignant cells are discussed. Felix Mitelman, Department q / Clinical Genetics, University Hospiral, S-22185 Lund, Sweden
The invasion of normal tissue by malignant cells is usually characterized by the destruction of normal tissue components in the vicinity of the invading tumor cells. It is well established that lysosomal enzymes are important for the breakdown of protein and glycoconjugates and there is growing evidence that lysosomal enzymes may play a significant role in tumor invasion (POOLE1973).The specific activities of lysosomal enzymes are often significantly elevated in tumor cells as well as in extracellular environments, and correlations between enzyme level and invasive growth have been demonstrated (BOSMANN1972;POOLE1973). An increasing amount of data suggest a causal relationship between the chromosomal constitution and the malignant character of tumor cells (for ref. see MITELMAN and LEVAN1976). In experimental tumors it has been shown that histologically indistinguishable sarcomas induced by Rous sarcoma virus (RSV) and 7,12-dimethylbenz(a)anthracene (DMBA) are characterized by different nonrandom karyotypic changes (MITELMAN et al. 1972; LEVAN and MITELMAN 1976). On the other hand, isozyme
pattern studies (LEVANet al. 1973), ultrastructural analyses (LINDBERG and MITELMAN 1974) and biochemical studies of deoxyadenylate regions in DNA (PEROet al. 1975) revealed striking similarities between DMBA and RSV tumors. It was therefore of interest to compare also the lysosomal apparatus between normal fibroblasts and the two tumor types.
Material and methods Control fibroblast cultures were established from rat fetuses of an inbred Wistar/Furth strain. Primary tumors were induced in rats of the same inbred strain by subcutaneous injection of Rous sarcoma virus strain Schmidth-Ruppin and DMBA, respectively, as described previously (MITELMAN1971; MITELMAN and LEVAN1972). From these studies, one DMBA and two RSV sarcomas were selected and transplanted to rats of the same inbred strain by inoculating 0.2ml of a finely minced 1.5 suspension of the thawed tumor in Hanks' solution. After
104
B. HULTBERG AND F.
Hereditas 86 (1977)
MITELMAN
Table I. Activity of lysosomal enzymes (mean and range) in control and sarcoma cells (pmoles/min/g protein), medium and saline (pmoles/min/l)
Control intracellular enzyme activity Cultured fibroblasts Control extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline DMBA intracellular enzyme activity Sarcoma cells DMBA extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline RSV intracellular enzyme activity Sarcoma cells RSV extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline
P-galactosidase
P-glucosidase
5 3 (2.4-7
0.8 (0.5-1.3)
71 110
I72 90
8)
(64 88) (100 120) ( I30 -200) (60--120)
1 8 (I0-22) 48 70 105
75
(44-50) (68 75) (96 112) (68-90)
5.0 (3.2-8.0)
7.0 (6.0-8.0) 7.1 (4.4-8.0) 6. I (4.0---8.0) 0.2 (0.12-0.30) 4.5 5.0 4.8 4.8
(4.0- 5.6) (4.0--6.0) (4.0--5.6) (4.2-5.1)
6 2 ( 2 0- I 1 2 ) 0.7 (0.5-1.0) 180
250 302 102
(147 270) (212-308) (245-368) (80-120)
5. I 7.0 6.8 5.0
one transplant generation in vivo, cell cultures were established by seeding finely minced material in Falcon plastic bottles. Cell cultures were split twice a week. All cultures were grown in basal medium Eagle supplemented with L-glutamin-penicillin-streptomycin and 10% fetal calf serum. Media were heated at 70°C for 2 h to inactivate endogenous lysosomal enzyme activity. The treated medium (15 ml) was left in contact with the cells for 70 h. Samples were withdrawn at 20, 40 and 70 h. Cells were removed from the surface by a rubber policeman and washed in saline (10 ml) once. The lysosomal enzymes P-galactosidase, P-glucosidase, a-fucosidase and Nacetyl-P-glucosaminidase were determined as previously described (HULTBERG et al. 1973, 1975) in 10 control fibroblast cultures, 5 cultures from each of the 2 RSV-sarcomas, and in 6 cultures from the DMBA sarcoma. Chromosome analysis of the primary DMBA and RSV sarcomas in this study have been published previously (MITELMAN 1971; MITELMAN and LEVAN 1972).
Results To elucidate any contribution by the hydrolytic activity of newborn calf serum, conditions which would inactivate the lysosomal enzymes were sought.
(4.4 -6.4) (6.0-8.0) (4.8---7.2) (4.2--6.8)
a-fucosidase
5 0 (3 5-7 71 123 175 65
N-acetyl-P-glucosaminidase
2)
(60-90) (80 -160) ( 1 20-200) (50-100)
1 5 (1 0-20)
88 105 120 52
(84 90) (104-1 12) (96-110) (48-62)
4 4 ( I 5-79) I88 220 200 45
65 (33--105) 2,300 (2.100--2.700) 2.800 (2.400--3.000) 5,800 (4,200-7,200) 1,100 (900 1.500) 45 (30--60) 3,100 (3,000 3.300) 6,300 (6.000--6.600) 11,100 (10,800-11.400) 1,900 (1,800 -2.100) 300 ( I 32-440)
15.400 (5,100--22,500) 34,200 (30.000-37.500) (208-255) ( I72 220) 48,000 (47,500 49,000) 5,000 (4.500- 5,700) (28-76) (I36-220) -
-
It was found that all four enzymes being monitored were rendered completely inactive by heating medium at 70°C for 2 h. As can be seen from Table I , P-galactosidase, 0-glucosidase, and a-fucosidase exhibited low values in the DMBA cell line. This may explain the low activity level in medium from these cells of P-galactosidase and a-fucosidase. P-glucosidase, however, showed low value in medium both from control and tumor cells and did not show any increase with time. N-acetyl-P-glucosaminidase exhibited rather low value in the DMBA cell line but the level in medium was increased. In both RSV cell lines P-galactosidase, P-glucosidase, and a-fucosidase showed normal levels of activity, but there was a faster increase of P-galactosidase and a-fucosidase activity level in medium compared to the control fibroblast culture. P-glucosidase activity in medium was equal to that of the control culture. N-acetyl-P-glucosaminidase was increased both in tumor cells and medium. The ratio of the enzyme activity (P-galactosidase, a-fucosidase, and N-acetyl-0-glucosaminidase) in medium/cells at 20, 40 and 70 h was increased in both the DMBA and RSV sarcoma cell lines compared to the fibroblast culture. N-acetyl-P-glucosaminidase showed the most pronounced increase of this ratio.
Hrreditas 86 (1977)
LYSOSOMAL ENZYMES IN RAT SARCOMAS
A
105
C 5
B
D
roa
30
A
3
4
5
6
7
3
4
5
6
7
PH
Fig. 1. pH-curves of P-galactosidase (A), P-glucosidase (B), a-fucosidase (C), and N-acetyl-P-glucosaminidase (D) in normal rat fibroblasts and medium. Activity expressed as pmoles/min/g protein in fibroblasts and pmoles/min/l in medium. Outer scale refers to fibroblasts ( 0 - 0 ) and the inner scale to medium (w-m).
pH-dependency curves from the normal rat fibroblasts and media taken after 70 h in contact with the cells are given in Fig. 1. The different acid hydrolases exhibited the same pH-dependency in fibroblast homogenate and medium. Both types of tumor cells and their media exhibited the same pH-dependency as shown in Fig. 1 . Also saline used to wash the harvested cells exhibited the same pH-profile as in Fig. 1.
Discussion The prospect that lysosomal enzymes released into the growth medium by malignant cells may alter the
architecture of the outer cell membrane and thus contribute to the uncontrolled growth characterizing these cells in vitro has recently been advocated (ELLIGSEN et al. 1975). There is also much evidence that lysosomal enzymes may play an important role for tumor invasion in vivo (POOLE1973). In the present experiment both DMBA and RSV induced sarcoma cells showed relative increase of enzyme activity outside the cell in the medium compared to the activity within the cell. In the medium from RSV sarcoma cells, an early equilibrium of enzyme activity level was reached for 0-galactosidase and a-fucosidase. The optimum level was almost reached after 40 h in contact with the cells. This early equilibrium was not seen in cultures of normal fibroblasts and DMBA sarcoma cells. N-acetyl-P-
106
B. HULTBERG AND F. MITELMAN
glucosaminidase activity showed a linear increase with time in all cultures, while P-glucosidase was not increased at all in any culture. The relevance of this extracellular pool of acid hydrolases has been discussed by DINGLEet al. (1969). They suggest that in living connective tissue the extracellular enzymic hydrolysis occurs initially in the immediate micro-environment of the cell and is followed by endocytosis of partially degraded material, the digestion of which is completed within digestive vacuoles; this has been termed the “two stage” digestion of extracellular macromolecules. Current evidence strongly suggests that the activity level of extracellular lysosomal enzymes is determined not only by release but also by enzyme degradation, enzyme denaturation and enzyme uptake. The hypothesis of NEUFELDand co-workers (1972, 1974) suggests that in certain cells (e.g. fibroblasts) lysosomal enzymes synthesised by the cell are first secreted extracellularly and afterwards engulfed by the cell to form secondary lysosomes. In view of these data “uptake” - not release would have to be considered to be the most important factor. A great amount of data relating to cytogenetic events associated with tumor induction by RSV and DMBA have been collected in various materials. In both rats and the Chinese hamster, tumor induction by the two oncogens result in distinctly different nonrandom karyotypic changes ( MITELMAN et al. 1972). With the aid of Giemsa banding technique the characteristic deviation in DMBA- and RSVinduced rat sarcomas have recently been identified as trisomy A2 (LEVANet al. 1974) and trisomy B7 (LEVANand MITELMAN 1976), respectively. In the present study there was a difference in the intracellular content of lysosomal enzymes between RSV and DMBA sarcoma cells. This was most clearly seen for N-acetyl-0-glucosaminidase. The main result - a relative increase of these enzymes outside the cells - was, however, similar in the two tumor types. Thus, in spite of the marked difference in chromosomal constitution between the virally and chemically induced tumors, no difference in the distribution of lysosomal enzyme activity could be demonstrated. As mentioned initially, analyses of other parameters - ultrastructure, isoenzymes, and deoxyadenylate regions in DNA - have also failed to demonstrate any clearcut difference between RSV- and DMBA-induced sarcomas.
Hereditus 86 (1977)
Swedish Cancer Society, and the John and Augusta Persson Foundation for Medical Research. We are grateful to Mrs Munevera Mirazovic for technical assistance.
Literature cited BOSMANN, H. B. 1972.Elevated glycosidases and proteolytic enzymes in cells transformed by RNA tumour virus. Biochem. Biophys. ACIN 264: 339-343 DINGLE,J. T . 1969.The extracellular secretion of lysosomal enzymes. --.-In Lysosomcs in Biology untl Patho1og.y. Val. 2 ( E d . J. T. DINGLE and H. B. FELL), North-Hoi/und, Amsterdum, p. 421 --436 ELLIGSEN. J. D.. THOMPSON,J . E. and FREY,H. E. 1975. An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells. - ESP. Cell Res. Y2: 87-94 HICKMAN, S. and NEUFELD.E. F. 1972. A hypothesis for I-cell disease: Defective hydrolases that d o not enter lysosomes. Biochem. Biophys. Rer. Commun. 4Y: 992-999 HICKMAN.S., SHAPIKO,L. J. and NEUFELI),E. F. 1974. A recognition marker required for uptake of a lysosomal Biochem. Biophys. Res. enzyme by cultured fibroblasts. Commun. 57: 55-61 HULTBERG,B., S J ~ B L A DS., and OCKERMAN, P. A. 1973. Properties of five acid hydrolases in human skin fibroblast cultures. Possible use in the diagnosis of inborn lysosomal Act0 Paediut. Scand. 62: 474-480 diseases. HULTBEKG,B., SJBBLAD,S. and OCKERMAN, P. A. 1975. Glycosidases in human skin fibroblast cultures. a-fucosidase, a-galactosidase, ac-glucosidase. p-mannosidase, and N-acetyla-glucosaminidase, Acta Paediat. Scund. 64: I23-- I3 I LEVAN.G. and MITELMAN, F. 1976.G-banding in Rous rat sarcomas during serial transfer: Significant chromosome aberrations and incidence of stromal mitoses. ~-Heretiitus -
~~
-
-
84: 1--18
LEVAN,G.. MITELMAN, F. and MAKK,J. 1973. lsozymes of experimentally induced primary sarcomas in the rat. Hereditas 73: 318--321 LEVAN,G . , AHLSTROM,U. and MITELMAN, F. 1974. The specificity of chromosome A2 involvement in DMBAinduced rat sarcomas. - Hereditas 7 7 263--280 LINDBERG. L. G. and MITELMAN, F. 1974. Morphometric Luh. analysis of Sarcomas with different karyotypes. Invest. 31: 90-95 MITELMAN, F. 1971.The chromosomes of fifty primary Rous rat sarcomas, -- Hereditas 6Y: 155-186 MITELMAN, F. and LEVAN,G. 1972. The chromosomes of primary 7,12-dimethylbenz(a)anthracene-induced rat sarcomas. - Hereditas 71: 325-334 MITELMAN, F. and LEVAN,G. 1976.Clustering of aberrations t o specific chromosomes in human neoplasms. 11. A survey of 287 neoplasms. -- Hereditas 82: 167- 174 MITELMAN, F., MARK.J., LEVAN,G. and LEVAN,A. 1972. Tumor etiology and chromosome pattern. Science 176: 1340- 1341 PERO,R. W., BRYNGELSSON, T., MITELMAN. F. and LEVAN, G. 1975. Changes in the deoxyadenylate regions of rat DNA in sarcomas induced by 7,12-dirnethylbenz(u)anthracene and Rous sarcoma virus. Hereditus 80: 153- I55 POOLE,A. R. 1973.Tumour lysosomal enzymes and invasive growth. In Lymsomes in Biology and Pathology, Vol. 3 (Ed. I. T. DINGLE),Norrh-HoNand, Amsterdam, p. 303-337
-~
~
~
~
Acknowledgments. -- This work was financially supported by grants from the Swedish Medical Research Council, the
Lysosomal enzymes in rat sarcomas induced by 7,12-dimethylbenz(a)anthracene and Rous sarcoma virus BJoRN HULTBERG and FELIX MITELMAN Dqurtments of' Clinicul Chemistry und Clinicril Genetics, University Hospitul, Lund. Sweden
HULTBERG, B. and MITELMAN. F. 1977. Lysosomal enzymes in rat sarcomas induced by 7,12dimethylbenz(a)anthracene and Rous sarcoma virus. Herediras 86: 103-106. Lund, Sweden, ISSN 0018-0661. Received February 2 3 , 1977 -
The lysosomal apparatus in normal rat fibroblast cultures and in DMBA and RSV induced rat sarcoma cell cultures was compared. Both DMBA and RSV induced sarcoma cells showed relative increase of lysosomal enzyme activity outside the cells in the medium compared to the activity within the cells. An early equilibrium of enzyme activity level was reached in the medium from RSV sarcoma cells, whereas this was not seen in cultures of normal fibroblasts and DMBA sarcoma cells. There was a difference in the intracellular content of lysosomal enzymes between RSV and DM BA sarcoma cells. This was most clearly seen for N-acetyl-P-glucosaminidase. These findings and the possible role of lysosomal enzymes in malignant cells are discussed. Felix Mitelman, Department q / Clinical Genetics, University Hospiral, S-22185 Lund, Sweden
The invasion of normal tissue by malignant cells is usually characterized by the destruction of normal tissue components in the vicinity of the invading tumor cells. It is well established that lysosomal enzymes are important for the breakdown of protein and glycoconjugates and there is growing evidence that lysosomal enzymes may play a significant role in tumor invasion (POOLE1973).The specific activities of lysosomal enzymes are often significantly elevated in tumor cells as well as in extracellular environments, and correlations between enzyme level and invasive growth have been demonstrated (BOSMANN1972;POOLE1973). An increasing amount of data suggest a causal relationship between the chromosomal constitution and the malignant character of tumor cells (for ref. see MITELMAN and LEVAN1976). In experimental tumors it has been shown that histologically indistinguishable sarcomas induced by Rous sarcoma virus (RSV) and 7,12-dimethylbenz(a)anthracene (DMBA) are characterized by different nonrandom karyotypic changes (MITELMAN et al. 1972; LEVAN and MITELMAN 1976). On the other hand, isozyme
pattern studies (LEVANet al. 1973), ultrastructural analyses (LINDBERG and MITELMAN 1974) and biochemical studies of deoxyadenylate regions in DNA (PEROet al. 1975) revealed striking similarities between DMBA and RSV tumors. It was therefore of interest to compare also the lysosomal apparatus between normal fibroblasts and the two tumor types.
Material and methods Control fibroblast cultures were established from rat fetuses of an inbred Wistar/Furth strain. Primary tumors were induced in rats of the same inbred strain by subcutaneous injection of Rous sarcoma virus strain Schmidth-Ruppin and DMBA, respectively, as described previously (MITELMAN1971; MITELMAN and LEVAN1972). From these studies, one DMBA and two RSV sarcomas were selected and transplanted to rats of the same inbred strain by inoculating 0.2ml of a finely minced 1.5 suspension of the thawed tumor in Hanks' solution. After
104
B. HULTBERG AND F.
Hereditas 86 (1977)
MITELMAN
Table I. Activity of lysosomal enzymes (mean and range) in control and sarcoma cells (pmoles/min/g protein), medium and saline (pmoles/min/l)
Control intracellular enzyme activity Cultured fibroblasts Control extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline DMBA intracellular enzyme activity Sarcoma cells DMBA extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline RSV intracellular enzyme activity Sarcoma cells RSV extracellular enzyme activity Medium at 20 h Medium at 40 h Medium at 70 h Saline
P-galactosidase
P-glucosidase
5 3 (2.4-7
0.8 (0.5-1.3)
71 110
I72 90
8)
(64 88) (100 120) ( I30 -200) (60--120)
1 8 (I0-22) 48 70 105
75
(44-50) (68 75) (96 112) (68-90)
5.0 (3.2-8.0)
7.0 (6.0-8.0) 7.1 (4.4-8.0) 6. I (4.0---8.0) 0.2 (0.12-0.30) 4.5 5.0 4.8 4.8
(4.0- 5.6) (4.0--6.0) (4.0--5.6) (4.2-5.1)
6 2 ( 2 0- I 1 2 ) 0.7 (0.5-1.0) 180
250 302 102
(147 270) (212-308) (245-368) (80-120)
5. I 7.0 6.8 5.0
one transplant generation in vivo, cell cultures were established by seeding finely minced material in Falcon plastic bottles. Cell cultures were split twice a week. All cultures were grown in basal medium Eagle supplemented with L-glutamin-penicillin-streptomycin and 10% fetal calf serum. Media were heated at 70°C for 2 h to inactivate endogenous lysosomal enzyme activity. The treated medium (15 ml) was left in contact with the cells for 70 h. Samples were withdrawn at 20, 40 and 70 h. Cells were removed from the surface by a rubber policeman and washed in saline (10 ml) once. The lysosomal enzymes P-galactosidase, P-glucosidase, a-fucosidase and Nacetyl-P-glucosaminidase were determined as previously described (HULTBERG et al. 1973, 1975) in 10 control fibroblast cultures, 5 cultures from each of the 2 RSV-sarcomas, and in 6 cultures from the DMBA sarcoma. Chromosome analysis of the primary DMBA and RSV sarcomas in this study have been published previously (MITELMAN 1971; MITELMAN and LEVAN 1972).
Results To elucidate any contribution by the hydrolytic activity of newborn calf serum, conditions which would inactivate the lysosomal enzymes were sought.
(4.4 -6.4) (6.0-8.0) (4.8---7.2) (4.2--6.8)
a-fucosidase
5 0 (3 5-7 71 123 175 65
N-acetyl-P-glucosaminidase
2)
(60-90) (80 -160) ( 1 20-200) (50-100)
1 5 (1 0-20)
88 105 120 52
(84 90) (104-1 12) (96-110) (48-62)
4 4 ( I 5-79) I88 220 200 45
65 (33--105) 2,300 (2.100--2.700) 2.800 (2.400--3.000) 5,800 (4,200-7,200) 1,100 (900 1.500) 45 (30--60) 3,100 (3,000 3.300) 6,300 (6.000--6.600) 11,100 (10,800-11.400) 1,900 (1,800 -2.100) 300 ( I 32-440)
15.400 (5,100--22,500) 34,200 (30.000-37.500) (208-255) ( I72 220) 48,000 (47,500 49,000) 5,000 (4.500- 5,700) (28-76) (I36-220) -
-
It was found that all four enzymes being monitored were rendered completely inactive by heating medium at 70°C for 2 h. As can be seen from Table I , P-galactosidase, 0-glucosidase, and a-fucosidase exhibited low values in the DMBA cell line. This may explain the low activity level in medium from these cells of P-galactosidase and a-fucosidase. P-glucosidase, however, showed low value in medium both from control and tumor cells and did not show any increase with time. N-acetyl-P-glucosaminidase exhibited rather low value in the DMBA cell line but the level in medium was increased. In both RSV cell lines P-galactosidase, P-glucosidase, and a-fucosidase showed normal levels of activity, but there was a faster increase of P-galactosidase and a-fucosidase activity level in medium compared to the control fibroblast culture. P-glucosidase activity in medium was equal to that of the control culture. N-acetyl-P-glucosaminidase was increased both in tumor cells and medium. The ratio of the enzyme activity (P-galactosidase, a-fucosidase, and N-acetyl-0-glucosaminidase) in medium/cells at 20, 40 and 70 h was increased in both the DMBA and RSV sarcoma cell lines compared to the fibroblast culture. N-acetyl-P-glucosaminidase showed the most pronounced increase of this ratio.
Hrreditas 86 (1977)
LYSOSOMAL ENZYMES IN RAT SARCOMAS
A
105
C 5
B
D
roa
30
A
3
4
5
6
7
3
4
5
6
7
PH
Fig. 1. pH-curves of P-galactosidase (A), P-glucosidase (B), a-fucosidase (C), and N-acetyl-P-glucosaminidase (D) in normal rat fibroblasts and medium. Activity expressed as pmoles/min/g protein in fibroblasts and pmoles/min/l in medium. Outer scale refers to fibroblasts ( 0 - 0 ) and the inner scale to medium (w-m).
pH-dependency curves from the normal rat fibroblasts and media taken after 70 h in contact with the cells are given in Fig. 1. The different acid hydrolases exhibited the same pH-dependency in fibroblast homogenate and medium. Both types of tumor cells and their media exhibited the same pH-dependency as shown in Fig. 1 . Also saline used to wash the harvested cells exhibited the same pH-profile as in Fig. 1.
Discussion The prospect that lysosomal enzymes released into the growth medium by malignant cells may alter the
architecture of the outer cell membrane and thus contribute to the uncontrolled growth characterizing these cells in vitro has recently been advocated (ELLIGSEN et al. 1975). There is also much evidence that lysosomal enzymes may play an important role for tumor invasion in vivo (POOLE1973). In the present experiment both DMBA and RSV induced sarcoma cells showed relative increase of enzyme activity outside the cell in the medium compared to the activity within the cell. In the medium from RSV sarcoma cells, an early equilibrium of enzyme activity level was reached for 0-galactosidase and a-fucosidase. The optimum level was almost reached after 40 h in contact with the cells. This early equilibrium was not seen in cultures of normal fibroblasts and DMBA sarcoma cells. N-acetyl-P-
106
B. HULTBERG AND F. MITELMAN
glucosaminidase activity showed a linear increase with time in all cultures, while P-glucosidase was not increased at all in any culture. The relevance of this extracellular pool of acid hydrolases has been discussed by DINGLEet al. (1969). They suggest that in living connective tissue the extracellular enzymic hydrolysis occurs initially in the immediate micro-environment of the cell and is followed by endocytosis of partially degraded material, the digestion of which is completed within digestive vacuoles; this has been termed the “two stage” digestion of extracellular macromolecules. Current evidence strongly suggests that the activity level of extracellular lysosomal enzymes is determined not only by release but also by enzyme degradation, enzyme denaturation and enzyme uptake. The hypothesis of NEUFELDand co-workers (1972, 1974) suggests that in certain cells (e.g. fibroblasts) lysosomal enzymes synthesised by the cell are first secreted extracellularly and afterwards engulfed by the cell to form secondary lysosomes. In view of these data “uptake” - not release would have to be considered to be the most important factor. A great amount of data relating to cytogenetic events associated with tumor induction by RSV and DMBA have been collected in various materials. In both rats and the Chinese hamster, tumor induction by the two oncogens result in distinctly different nonrandom karyotypic changes ( MITELMAN et al. 1972). With the aid of Giemsa banding technique the characteristic deviation in DMBA- and RSVinduced rat sarcomas have recently been identified as trisomy A2 (LEVANet al. 1974) and trisomy B7 (LEVANand MITELMAN 1976), respectively. In the present study there was a difference in the intracellular content of lysosomal enzymes between RSV and DMBA sarcoma cells. This was most clearly seen for N-acetyl-0-glucosaminidase. The main result - a relative increase of these enzymes outside the cells - was, however, similar in the two tumor types. Thus, in spite of the marked difference in chromosomal constitution between the virally and chemically induced tumors, no difference in the distribution of lysosomal enzyme activity could be demonstrated. As mentioned initially, analyses of other parameters - ultrastructure, isoenzymes, and deoxyadenylate regions in DNA - have also failed to demonstrate any clearcut difference between RSV- and DMBA-induced sarcomas.
Hereditus 86 (1977)
Swedish Cancer Society, and the John and Augusta Persson Foundation for Medical Research. We are grateful to Mrs Munevera Mirazovic for technical assistance.
Literature cited BOSMANN, H. B. 1972.Elevated glycosidases and proteolytic enzymes in cells transformed by RNA tumour virus. Biochem. Biophys. ACIN 264: 339-343 DINGLE,J. T . 1969.The extracellular secretion of lysosomal enzymes. --.-In Lysosomcs in Biology untl Patho1og.y. Val. 2 ( E d . J. T. DINGLE and H. B. FELL), North-Hoi/und, Amsterdum, p. 421 --436 ELLIGSEN. J. D.. THOMPSON,J . E. and FREY,H. E. 1975. An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells. - ESP. Cell Res. Y2: 87-94 HICKMAN, S. and NEUFELD.E. F. 1972. A hypothesis for I-cell disease: Defective hydrolases that d o not enter lysosomes. Biochem. Biophys. Rer. Commun. 4Y: 992-999 HICKMAN.S., SHAPIKO,L. J. and NEUFELI),E. F. 1974. A recognition marker required for uptake of a lysosomal Biochem. Biophys. Res. enzyme by cultured fibroblasts. Commun. 57: 55-61 HULTBERG,B., S J ~ B L A DS., and OCKERMAN, P. A. 1973. Properties of five acid hydrolases in human skin fibroblast cultures. Possible use in the diagnosis of inborn lysosomal Act0 Paediut. Scand. 62: 474-480 diseases. HULTBEKG,B., SJBBLAD,S. and OCKERMAN, P. A. 1975. Glycosidases in human skin fibroblast cultures. a-fucosidase, a-galactosidase, ac-glucosidase. p-mannosidase, and N-acetyla-glucosaminidase, Acta Paediat. Scund. 64: I23-- I3 I LEVAN.G. and MITELMAN, F. 1976.G-banding in Rous rat sarcomas during serial transfer: Significant chromosome aberrations and incidence of stromal mitoses. ~-Heretiitus -
~~
-
-
84: 1--18
LEVAN,G.. MITELMAN, F. and MAKK,J. 1973. lsozymes of experimentally induced primary sarcomas in the rat. Hereditas 73: 318--321 LEVAN,G . , AHLSTROM,U. and MITELMAN, F. 1974. The specificity of chromosome A2 involvement in DMBAinduced rat sarcomas. - Hereditas 7 7 263--280 LINDBERG. L. G. and MITELMAN, F. 1974. Morphometric Luh. analysis of Sarcomas with different karyotypes. Invest. 31: 90-95 MITELMAN, F. 1971.The chromosomes of fifty primary Rous rat sarcomas, -- Hereditas 6Y: 155-186 MITELMAN, F. and LEVAN,G. 1972. The chromosomes of primary 7,12-dimethylbenz(a)anthracene-induced rat sarcomas. - Hereditas 71: 325-334 MITELMAN, F. and LEVAN,G. 1976.Clustering of aberrations t o specific chromosomes in human neoplasms. 11. A survey of 287 neoplasms. -- Hereditas 82: 167- 174 MITELMAN, F., MARK.J., LEVAN,G. and LEVAN,A. 1972. Tumor etiology and chromosome pattern. Science 176: 1340- 1341 PERO,R. W., BRYNGELSSON, T., MITELMAN. F. and LEVAN, G. 1975. Changes in the deoxyadenylate regions of rat DNA in sarcomas induced by 7,12-dirnethylbenz(u)anthracene and Rous sarcoma virus. Hereditus 80: 153- I55 POOLE,A. R. 1973.Tumour lysosomal enzymes and invasive growth. In Lymsomes in Biology and Pathology, Vol. 3 (Ed. I. T. DINGLE),Norrh-HoNand, Amsterdam, p. 303-337
-~
~
~
~
Acknowledgments. -- This work was financially supported by grants from the Swedish Medical Research Council, the
