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Mitochondrial enzyme activity in “senescent” and virus-transformed human fibroblasts
Q 1968
by Academic
Experimenlal
Press
Inc.
Cell Research
53, 135-138
133
(1968)
MITOCHONDRIAL ENZYME ACTIVITY IN “SENESCENT” AND VIRUS-TRANSFORMED HUMAN FIBROBLASTS’ NASROLLAH University
of Washington,
HAKAM12
and
School of Medicine, Seattle, Wash. 98105, Received
March
D. A. PIOUS3 Department USA
of Pediatrics,
13. 1968
T,IE limited in vitro lifespan of diploid cells in culture has been postulated to be caused by loss of a nonchromosomal self-replicating organelle, necessary for cell survival, whose rate of replication is slower than that of the nucleus [4, 5]. The only nonchromosomal organelle for which there is substantial evidence for self-replication at present is the mitochondrion [Cl]. Loss of mitochondria might reasonably be expected to lead to reproductive death of the cell because of the central role of mitochondria in energy production. It is therefore of interest that the specific activity of a mitochondrial enzyme, cytochrome oxidase, \vas found to be more than twice as high in the permanent cell line HeLa as in diploid human fibroblasts [Sj. This finding is compatible with the possibility that senescence in diploid cells occurs because of loss of mitochondria, and that cell line permanence is engendered by an elevated mitochondrial content, arising through mutation-selection or a similar mechanism. An alternative interpretation is that the difference in cgtochrome oxidase activities between HeLa and fibroblasts reflects histotypic differences related to the different tissues of origin of the two cultures. To investigate these possibilities, \ve followed the cytochrome oxidase specific activity of a fibroblast strain from shortly after its in vitro cultivation to the cessation of division (“Phase III” of Hayflick and Moorhead). In addition, \ve measured the cytochrome oxidase activity of a virus-transformed human fibroblast permanent cell line, and compared it to the activity of nontransformed fibroblast strains, in order to evaluate the possible role of elevated mitochondrial enzyme activity on the ability to divide indefinitely in culture. 1 Supported 2 Postdoctoral Public Health 3 Research Development,
in part by US Public Health Service Grant Number GB115583. trainee, National Institute of Child Health and Human Development, US Service. (Address reprint requests to Dr Nasrollah Hakami.) Career Development Awardee, National Institute of Child Health and Human US Public Health Service. Experimental
Cell Research
53
136
Nasrollah
MATERIALS
Hakami and D. A. Pious AND
METHODS
Cell cultures and methods.-Cell cultures were initiated and maintained by methods described previously [ 81. TC 12266, a fibroblast strain derived from newborn foreskin. TC S V SDC CZ,, an established cell line derived from an SV40-transformed human fibroblast strain [IO], kindly supplied by Doctor G. Todaro. This cell line (designated “Strain A” in reference [lo]), has undergone over 100 cell doublings following its transformation by virus at the 68th generation in vitro [3]. Enzyme assay.-Cytochrome oxidase activity was determined spectrophotometritally by the rate of disappearance of reduced cytochrome c following the addition of a lysate of whole cells prepared with deoxycholate [6]. Protein was determined by the method of Lowry et al. [7]. Enzyme specific activities are expressed as m,uuMoles of cytochrome c oxidized per mg of protein per min.
RESULTS
AND
DISCUSSION
For studies of mitochondrial enzyme function in senescent human tibroblasts, TC 12266 was maintained hv serial subcultivation at split ratios of l/S or l/l0 during the rapid growth period and at l/4 during the period of declining growth rate. A total of 25 passages (corresponding to an estimated 74 cell generations) was made, at which time growth ceased and further passages could not be made. Cvtochromc oxidase specific activity (activity per milligram cell protein) was assayed at intervals of two to three passages; representative results of these determinations are shown in Table 1. It can be seen from the data that the enzyme specific activities at various passages However, variation in activity is varied over a tvvo and a half fold range. clearly random with respect to the age of the culture. In particular, no evidence can be seen for loss of enzyme activity related to prolonged serial passage. The studies on the cell line derived by virus transformation of human tibroblasts are also shown in Table 1. The mean cytochrome oxidase specific activity for these cells, 6.9 & 1.7, is not significantly different from the mean of multiple determinations (5.6 + 0.9) obtained from a nontransformed tibroblast strain [6], nor from the sequential values for human hbroblasts reported here. However, it is significantly lower than the specific activity of the HeLa cell line [6]. Thus, no evidence was found in these studies of reduction in mitochondrial enzyme activity in senescent human tibroblasts. Although our data do not preclude the possibility of a reduction in the number of mitochondria in senescent cells, they do argue against a critical reduction in mitochondrial function. The findings by Cristofalo and Kritchevsky that respiration is unimExperimental
Cell Research
53
Mitochondrial
enzyme activity
in fibroblasts
paired in senescent human fibroblasts [l] are also in clusion. The data obtained from the SV,,-transformed alteration in mitochondrial enzyme function is likewise comitant of cell line permanence. Thus the difference activity between the HeLa line and human fibroblasts
accord with this concell line indicate that not a necessary conin cytochrome oxidase is more likely a reflec-
TABLE 1. The effect of serial passage and virus transformation cytochrome ozidase specific actiuity of human fibroblasts
Passage number
Cell strain TC TC TC TC TC TC
12266 1‘2266 12266 12266 12266 12266
TC SV SDC
3 5 11 16 21 2.5 Cl,
Generations in uifro (estimated)
135
on the
Cytochromc oxidase specific activity’
13 19 38 53 68 74
3.2 7.8 3.5 4.2 5.8 8.3
150
6.91-l.?
a m~.U of substrate oxidized mg protein-’ min-‘. * Standard error. TC 12266 is a nontransformed human fibroblast strain derived from human foreskin. Cytochrome oxidase specific activity was determined at intervals during the course of serial subcultivation of this strain. The last determination was made at the twenty-fifth passage, after which no further subcultivation could be made and no further growth was noted. TC SV SDC Cl, is a cell line derived by SV,, transformation of a human fibroblast strain. The cylochrome oxidase specific activity shown is the mean of three independent determinations.
tion of the difference in tissue of origin rather than a difference between cells with limited and unlimited capacity to grow in vitro. Whether selfreplicating nonchromosomal structures other than mitochondria, e.g. centrioles [‘L], are present and involved in senescence of human fibroblasts as postulated by Hayflick and hloorhead, or whether some other mechanism is operative, is at present unknown. SUMMARY
The possible role of changes in mitochondrial content in limiting the in uifro lifespan of human fibroblasts in culture and on the cell line permanence engendered by virus transformation was evaluated. The specific activity of a mitochondrial enzyme, cytochromc oxidase, was determined in a normal human fibroblast strain from shortly after its in vitro cultivation to the cessaExperimenfal
Cell Research
53
138
Nasrollah
Hakami
and D. A. Pious
tion of division. Enzyme specific activity was also determined in an S\‘40transformed permanent human fibroblast line. No age-dependent reduction in cytochrome oxidase specific activity was found in “senescent” human fibroblasts, nor was the specific activity of virus-transformed human fibroblasts significantly different than that of non-transformed cells. These data argue against changes in mitochondrial content as the basis for the limited in uitro lifespan of normal human fibroblasts, or for the cell line permanence of virus-transformed human fibroblasts.
REFERENCES V. S. and KRITCHEVSKY, D., J. Cell Physiol. 67, 125 (1966). S. and GIBORA, A., Prog. Nucl. Acid Res. Molec. Biof. 6, 143 (1967). H., Personal communication. HAYFLICK, L., Expfl Cell Res. 37, 614 (1965). HAYFLICK, L..and.iXoo~~~a~, P: S., kxpff bell Res. 25, 585 (1961). HAKAMI, N. and PIOUS, D. A., h’afure 216, 1087 (1967). LOWRY, 0. L., ROSEBROUGH, N. J., FARR, A. L. and RaivDALL, R. J., J. Biof. Chem. (1951). PIOUS, D. A., HAMBURGER, R. N. and MILLS, S., Expff Cell Res. 33, 495 (1964). REICH, E. and LUCK, D., Proc. Nafl Acad. Sci. 55, 1600 (1966). TODARO, G., WOLXIAN, S. and GREEN, H., J. Cell Comp. Phgsiof. 62, 257 (1963).
1. CRISTOFALO,
2. GRANICK, 3. GREEN, 4. 5. 6. 7. 8. 9. 10.
Experimenful
Cell
Rtsett. r I .iJ
193,265
by Academic
Experimenlal
Press
Inc.
Cell Research
53, 135-138
133
(1968)
MITOCHONDRIAL ENZYME ACTIVITY IN “SENESCENT” AND VIRUS-TRANSFORMED HUMAN FIBROBLASTS’ NASROLLAH University
of Washington,
HAKAM12
and
School of Medicine, Seattle, Wash. 98105, Received
March
D. A. PIOUS3 Department USA
of Pediatrics,
13. 1968
T,IE limited in vitro lifespan of diploid cells in culture has been postulated to be caused by loss of a nonchromosomal self-replicating organelle, necessary for cell survival, whose rate of replication is slower than that of the nucleus [4, 5]. The only nonchromosomal organelle for which there is substantial evidence for self-replication at present is the mitochondrion [Cl]. Loss of mitochondria might reasonably be expected to lead to reproductive death of the cell because of the central role of mitochondria in energy production. It is therefore of interest that the specific activity of a mitochondrial enzyme, cytochrome oxidase, \vas found to be more than twice as high in the permanent cell line HeLa as in diploid human fibroblasts [Sj. This finding is compatible with the possibility that senescence in diploid cells occurs because of loss of mitochondria, and that cell line permanence is engendered by an elevated mitochondrial content, arising through mutation-selection or a similar mechanism. An alternative interpretation is that the difference in cgtochrome oxidase activities between HeLa and fibroblasts reflects histotypic differences related to the different tissues of origin of the two cultures. To investigate these possibilities, \ve followed the cytochrome oxidase specific activity of a fibroblast strain from shortly after its in vitro cultivation to the cessation of division (“Phase III” of Hayflick and Moorhead). In addition, \ve measured the cytochrome oxidase activity of a virus-transformed human fibroblast permanent cell line, and compared it to the activity of nontransformed fibroblast strains, in order to evaluate the possible role of elevated mitochondrial enzyme activity on the ability to divide indefinitely in culture. 1 Supported 2 Postdoctoral Public Health 3 Research Development,
in part by US Public Health Service Grant Number GB115583. trainee, National Institute of Child Health and Human Development, US Service. (Address reprint requests to Dr Nasrollah Hakami.) Career Development Awardee, National Institute of Child Health and Human US Public Health Service. Experimental
Cell Research
53
136
Nasrollah
MATERIALS
Hakami and D. A. Pious AND
METHODS
Cell cultures and methods.-Cell cultures were initiated and maintained by methods described previously [ 81. TC 12266, a fibroblast strain derived from newborn foreskin. TC S V SDC CZ,, an established cell line derived from an SV40-transformed human fibroblast strain [IO], kindly supplied by Doctor G. Todaro. This cell line (designated “Strain A” in reference [lo]), has undergone over 100 cell doublings following its transformation by virus at the 68th generation in vitro [3]. Enzyme assay.-Cytochrome oxidase activity was determined spectrophotometritally by the rate of disappearance of reduced cytochrome c following the addition of a lysate of whole cells prepared with deoxycholate [6]. Protein was determined by the method of Lowry et al. [7]. Enzyme specific activities are expressed as m,uuMoles of cytochrome c oxidized per mg of protein per min.
RESULTS
AND
DISCUSSION
For studies of mitochondrial enzyme function in senescent human tibroblasts, TC 12266 was maintained hv serial subcultivation at split ratios of l/S or l/l0 during the rapid growth period and at l/4 during the period of declining growth rate. A total of 25 passages (corresponding to an estimated 74 cell generations) was made, at which time growth ceased and further passages could not be made. Cvtochromc oxidase specific activity (activity per milligram cell protein) was assayed at intervals of two to three passages; representative results of these determinations are shown in Table 1. It can be seen from the data that the enzyme specific activities at various passages However, variation in activity is varied over a tvvo and a half fold range. clearly random with respect to the age of the culture. In particular, no evidence can be seen for loss of enzyme activity related to prolonged serial passage. The studies on the cell line derived by virus transformation of human tibroblasts are also shown in Table 1. The mean cytochrome oxidase specific activity for these cells, 6.9 & 1.7, is not significantly different from the mean of multiple determinations (5.6 + 0.9) obtained from a nontransformed tibroblast strain [6], nor from the sequential values for human hbroblasts reported here. However, it is significantly lower than the specific activity of the HeLa cell line [6]. Thus, no evidence was found in these studies of reduction in mitochondrial enzyme activity in senescent human tibroblasts. Although our data do not preclude the possibility of a reduction in the number of mitochondria in senescent cells, they do argue against a critical reduction in mitochondrial function. The findings by Cristofalo and Kritchevsky that respiration is unimExperimental
Cell Research
53
Mitochondrial
enzyme activity
in fibroblasts
paired in senescent human fibroblasts [l] are also in clusion. The data obtained from the SV,,-transformed alteration in mitochondrial enzyme function is likewise comitant of cell line permanence. Thus the difference activity between the HeLa line and human fibroblasts
accord with this concell line indicate that not a necessary conin cytochrome oxidase is more likely a reflec-
TABLE 1. The effect of serial passage and virus transformation cytochrome ozidase specific actiuity of human fibroblasts
Passage number
Cell strain TC TC TC TC TC TC
12266 1‘2266 12266 12266 12266 12266
TC SV SDC
3 5 11 16 21 2.5 Cl,
Generations in uifro (estimated)
135
on the
Cytochromc oxidase specific activity’
13 19 38 53 68 74
3.2 7.8 3.5 4.2 5.8 8.3
150
6.91-l.?
a m~.U of substrate oxidized mg protein-’ min-‘. * Standard error. TC 12266 is a nontransformed human fibroblast strain derived from human foreskin. Cytochrome oxidase specific activity was determined at intervals during the course of serial subcultivation of this strain. The last determination was made at the twenty-fifth passage, after which no further subcultivation could be made and no further growth was noted. TC SV SDC Cl, is a cell line derived by SV,, transformation of a human fibroblast strain. The cylochrome oxidase specific activity shown is the mean of three independent determinations.
tion of the difference in tissue of origin rather than a difference between cells with limited and unlimited capacity to grow in vitro. Whether selfreplicating nonchromosomal structures other than mitochondria, e.g. centrioles [‘L], are present and involved in senescence of human fibroblasts as postulated by Hayflick and hloorhead, or whether some other mechanism is operative, is at present unknown. SUMMARY
The possible role of changes in mitochondrial content in limiting the in uifro lifespan of human fibroblasts in culture and on the cell line permanence engendered by virus transformation was evaluated. The specific activity of a mitochondrial enzyme, cytochromc oxidase, was determined in a normal human fibroblast strain from shortly after its in vitro cultivation to the cessaExperimenfal
Cell Research
53
138
Nasrollah
Hakami
and D. A. Pious
tion of division. Enzyme specific activity was also determined in an S\‘40transformed permanent human fibroblast line. No age-dependent reduction in cytochrome oxidase specific activity was found in “senescent” human fibroblasts, nor was the specific activity of virus-transformed human fibroblasts significantly different than that of non-transformed cells. These data argue against changes in mitochondrial content as the basis for the limited in uitro lifespan of normal human fibroblasts, or for the cell line permanence of virus-transformed human fibroblasts.
REFERENCES V. S. and KRITCHEVSKY, D., J. Cell Physiol. 67, 125 (1966). S. and GIBORA, A., Prog. Nucl. Acid Res. Molec. Biof. 6, 143 (1967). H., Personal communication. HAYFLICK, L., Expfl Cell Res. 37, 614 (1965). HAYFLICK, L..and.iXoo~~~a~, P: S., kxpff bell Res. 25, 585 (1961). HAKAMI, N. and PIOUS, D. A., h’afure 216, 1087 (1967). LOWRY, 0. L., ROSEBROUGH, N. J., FARR, A. L. and RaivDALL, R. J., J. Biof. Chem. (1951). PIOUS, D. A., HAMBURGER, R. N. and MILLS, S., Expff Cell Res. 33, 495 (1964). REICH, E. and LUCK, D., Proc. Nafl Acad. Sci. 55, 1600 (1966). TODARO, G., WOLXIAN, S. and GREEN, H., J. Cell Comp. Phgsiof. 62, 257 (1963).
1. CRISTOFALO,
2. GRANICK, 3. GREEN, 4. 5. 6. 7. 8. 9. 10.
Experimenful
Cell
Rtsett. r I .iJ
193,265