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Regulation of lysosomal cysteine-proteinase activities in 3T3 and SV-3T3 cells
Cell Biology International Reports, VoL 10, No. 3, March 19864 REGULATION OF LYSOSOMAL CYSTEINE-PROTEINASE
209
ACTIVITIES IN 3T3 AND SV-3T3 CELLS.
Bonelli G., Tessitore L., Isidoro C., Musi M. and Baccino F.M. of General Pathology, corso Raffaello 30, 10125 Torino, Italy
Institute
Density-arrested 3T3 c e l l s are characterized by enhanced rates of protein degradation a n d higher cathepsin D activity when compared with exponentially growing c u l t u r e s ; by c o n t r a s t , in S V - 3 T 3 cells protein degradation rates and cathepsin D levels do not exhibit any modulation in relation to cell density (1,2). The growth-associated proteolytic regulation operating in non transformed 3T3 cells is due to activation of the lysosomal pathway of protein degradation (3). At least for some c e l l s or tissues, there are data indicating that, among lysosomal proteinases, the proteolytic potential of cysteine-proteinases largely exceeds that of cathepsin D (4). We thence measured the activity levels of cathepsin B, L, and H in 3T3 and SV-3T3 cells with respect to cell density. Cells were grown, by standard procedures, in D-MEM containing 10% heat-inactivated FCS. The cell nun~ber was determined w i t h a Coulter ZM cell counter. Cysteineproteinase activities were measured fluorometrically according to Barrett (5). To minimize the interference by cytosolie inhibitors and enzyme inactivation, cells were disrupted with 0.1% (w/v) Brij in the presence of i0 mM ditiothreitol, as enzyme activator,immediately followed by incubation with the appropriate substrate. In low density SV-3T3 cells the activity levels of the three enzymes were very similar to those measured in exponentially growing 3T3 cells at the beginning of the experimental period, and no change was detectable on attainment of high cell densities. By contrast, in 'normal' 3T3 cells the enzymatic activities progressively increased with cell density; the severalfold increase of cathepsin L is shown in the Figure. Therefore, the regulation of lysosomal proteinase activities in these cells parallels that of the lysosomalautophagic proteolytic mechanism. Changes in cell protein degradative rates are not necessarily associated with parallel changes in iysosomal proteinase activities. Yet, the elevation of the latter in 3T3 cells may be instrumental for Supported by CNR Special Project
conspicuous activation of the lysosomal pathway for protein degradation exhibited by these cells on growth arrest (I, the
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Figure: 3T3 (e) and SV-3T3 (y) cells grown for 7 days in D-MEM supplemented with 10% heat-inactivated fetal calf serum (plating density l0 s cells / 5 cm dish). Enzyme activity and cell number determined in parallel cultures every 2 days. Cathepsin L activity: arbitrary units per mg protein. Data are averages from duplicate determinations from three experiments (S.D./means < 10%). 'Oncology', MPI and AIRC.
References i. Lockwood T.D.,Minassian I.A. and Roux L. (1982) Biochem. J. 206, 239-249. 2. Lockwood T.D. and Minassian I.A. (1982) Biochem. J. 206, 251-258. 3. Baccino F.M., Tessitore L. and Bonelli G. (1984) Toxicol. Pathol. 12, 281-287. 4. Bohley P., Kirschke H., Langner J., Miehe M., Rieman S., Salama Z., Shon E., Wiederanders B. and Ansorge S. in: Biological Functions of Proteinases (Holzer H. & Tschesche H., eds.), pp. 17-34. Springer-Verlag, Berlin, 1979. 5. Barrett A.J. and Kirschke H. (1981) Methods in Enzymol. 80, 535-56
0309-1651/86/030209-01/$03.00/0
~ 1986 Academic Press Inc. (London) Ltd.
209
ACTIVITIES IN 3T3 AND SV-3T3 CELLS.
Bonelli G., Tessitore L., Isidoro C., Musi M. and Baccino F.M. of General Pathology, corso Raffaello 30, 10125 Torino, Italy
Institute
Density-arrested 3T3 c e l l s are characterized by enhanced rates of protein degradation a n d higher cathepsin D activity when compared with exponentially growing c u l t u r e s ; by c o n t r a s t , in S V - 3 T 3 cells protein degradation rates and cathepsin D levels do not exhibit any modulation in relation to cell density (1,2). The growth-associated proteolytic regulation operating in non transformed 3T3 cells is due to activation of the lysosomal pathway of protein degradation (3). At least for some c e l l s or tissues, there are data indicating that, among lysosomal proteinases, the proteolytic potential of cysteine-proteinases largely exceeds that of cathepsin D (4). We thence measured the activity levels of cathepsin B, L, and H in 3T3 and SV-3T3 cells with respect to cell density. Cells were grown, by standard procedures, in D-MEM containing 10% heat-inactivated FCS. The cell nun~ber was determined w i t h a Coulter ZM cell counter. Cysteineproteinase activities were measured fluorometrically according to Barrett (5). To minimize the interference by cytosolie inhibitors and enzyme inactivation, cells were disrupted with 0.1% (w/v) Brij in the presence of i0 mM ditiothreitol, as enzyme activator,immediately followed by incubation with the appropriate substrate. In low density SV-3T3 cells the activity levels of the three enzymes were very similar to those measured in exponentially growing 3T3 cells at the beginning of the experimental period, and no change was detectable on attainment of high cell densities. By contrast, in 'normal' 3T3 cells the enzymatic activities progressively increased with cell density; the severalfold increase of cathepsin L is shown in the Figure. Therefore, the regulation of lysosomal proteinase activities in these cells parallels that of the lysosomalautophagic proteolytic mechanism. Changes in cell protein degradative rates are not necessarily associated with parallel changes in iysosomal proteinase activities. Yet, the elevation of the latter in 3T3 cells may be instrumental for Supported by CNR Special Project
conspicuous activation of the lysosomal pathway for protein degradation exhibited by these cells on growth arrest (I, the
3). ~"
6
~
4
o
o
o 0 ,
,
J
i
f
J
J
,
,
J
,
,
L
,
4
~3
•,-I 2 oJ
5
a
0 2 4 time in cUlture
6 (days)
Figure: 3T3 (e) and SV-3T3 (y) cells grown for 7 days in D-MEM supplemented with 10% heat-inactivated fetal calf serum (plating density l0 s cells / 5 cm dish). Enzyme activity and cell number determined in parallel cultures every 2 days. Cathepsin L activity: arbitrary units per mg protein. Data are averages from duplicate determinations from three experiments (S.D./means < 10%). 'Oncology', MPI and AIRC.
References i. Lockwood T.D.,Minassian I.A. and Roux L. (1982) Biochem. J. 206, 239-249. 2. Lockwood T.D. and Minassian I.A. (1982) Biochem. J. 206, 251-258. 3. Baccino F.M., Tessitore L. and Bonelli G. (1984) Toxicol. Pathol. 12, 281-287. 4. Bohley P., Kirschke H., Langner J., Miehe M., Rieman S., Salama Z., Shon E., Wiederanders B. and Ansorge S. in: Biological Functions of Proteinases (Holzer H. & Tschesche H., eds.), pp. 17-34. Springer-Verlag, Berlin, 1979. 5. Barrett A.J. and Kirschke H. (1981) Methods in Enzymol. 80, 535-56
0309-1651/86/030209-01/$03.00/0
~ 1986 Academic Press Inc. (London) Ltd.