- Email: [email protected]
Inorganic pyrophosphatase activity in human cell cultures with depressed alkaline phosphatase
Copyright Ail riahrs
0 1972 by Academic Press, Inc. of rrproduction in any form reseraed
Experimental
INORGANIC
Cell Research 74 (1972) 307-310
PYROPHOSPHATASE ACTIVITY WITH DEPRESSED ALKALINE
IN HUMAN CELL CULTURES PHOSPHATASE
F. HERZ and E. KAPLAN Departments of Pediatrics, Sinai Hospital, Baltimore, Md 21215, and The Johns Hopkins University School of Medicine, Baltimore, Md 21205, USA
SUMMARY A comparison is made of the effect of serum on inorganic pyrophosphatase and alkaline phosphatase in HeLa S3 and other human cell lines. When the cells are grown in human serum, alkaline phosphatase is maximally depressed to lesss than 5 36 of the activity of control cultures. By contrast, serum exerts only a slight effect on inorganic pyrophosphatase. The significance of these results is related to observations indicating that both activities are associated with a single protein.
The activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) in certain established cell lines of human origin is dependent on the concentration of human serum in the culture medium [l]. Enzyme activity is inversely proportional to the serum concentration and is lowest in cells grown in serum without the basal components of the culture medium. The term “depression” has been selected to describe the serum-dependent reduction in enzyme activity [2]. Several reports have indicated that preparations of alkaline phosphatase from various sources, including cultured mammalian cells, possess inorganic pyrophosphatase (pyrophosphate phosphohydrolase, E.C. 3.6.1.1) activity; thus, suggesting that both activities may be associated with a single enzyme [3-71. In this report it will be shown that in contrast to its striking effect on alkaline phosphatase, serum does not modulate the activity of inorganic pyrophosphatase in heteroploid cells to a significant extent.
MATERIALS
AND METHODS
The following serially propagated cell lines were employed: HeLa S3, Zimmer liver 4A and H.Ep Il. The alkaline phosphatase activity of the first two clonal strains is relatively low and that of H.Ep 11 cells is approx. 100 times higher [ 11.The activity of the three cell lines is depressed by serum. Puck medium N16 [S] with 10 % undialysed human serum was routinely used and cells grown under these conditions served as controls. The effect of serum was studied in cells grown in human serum without the basal components of Puck medium, except saline F [8]. The culture technique, harvesting and subsequent handling of the cells was carried out as previously described [ 1,9]. Alkaline phosphatase activity was measured by the hydrolysis of o-nitrophenvl ohosphate using~2-amind-2-m&hyl-l-~ropan&-H~I duffer at pH 10.6 and 38°C [91.Specific activitv was exaressed as -micromoles of p&dphenol liberated in 30 min at 38°C per mg of protein. Inorganic pyrophosphatase activity was assayed according to Cox et al. [4] and specific activity expressed as micromoles of pyrophosphate hydrolysed in 30 min at 37°C per mg of protein, the latter determined according to Lowry et al. [IO].
RESULTS Table 1 compares the specific activities of alkaline phosphatase and inorganic pyrophosphatase of three human cell lines at the end of one 7-day growth cycle in Puck medium Exptl Cell Res 74 (1972)
308
F. Herz & E. Kaplan
Table 1. Effect of serum on alkaline phosphatase and inorganic pyrophosphatase of various cell lines
Cell lines HeLa S3 Zimmer liver Ii!&
II
Alkaline phosphatase
Inorganic pyrophosphatase
Puck medium i-10% Human serum serum
Puck medium +lO% Human serum serum
0.8
0.04
0.4
0.2
85.0 1.6
25.0 0.03
0.3 0.8
0.4 0.2
Cells were grown as monolayers for 7 days at 37°C in an atmosphere of 5 % CO, in air. Following dispersion with 0.05 % trypsin and three washings with 0.15 M NaCl, cells were disrupted by ultrasonication [9]. Enzyme activity was measured in duplicate on replicate cultures. Specific activity expressed as indicated in text.
and in human serum. As can be seen,alkaline phosphatase activity of HeLa S3 and Zimmer liver 4A grown in serum was characteristically reduced to less than 5 % of the activity of control cultures. By comparison, growth in serum had only a slight effect on inorganic pyrophosphatase activity. It should be noted that whereas the alkaline phosphatase of control H.Ep II cells was 100 times higher than that of HeLa S3, inorganic pyrophosphatase activity was only two times greater. Since the specific activity of alkaline phosphatase increases continuously during a growth cycle in regular medium [I l] and declines progressively in serum [l], a comparison of the influence of serum on the levels of alkaline phosphatase and inorganic pyrophosphatase activity was made with cells harvested at 24h intervals. As can be seen in table 2, in cells cultured in regular medium, alkaline phosphatase activity increased 12fold between 48 and 144 h after cell transfer, and decreased from 0.11 at 48 h to 0.02 at 168 h in cells grown in serum. Inorganic pyrophosphatase activity in cells growing in Exptl Cell Res 74 (1972)
Puck medium increased at a relatively slower rate from 0.1 at 48 h to 0.3 at 144 h. In cells cultured in serum the activity of this enzyme did not follow the pattern of alkaline phosphatase, but showed a slight increase towards the end of the growth cycle. No alterations in the specific activity of acid phosphatase and no reduction in total cell protein were noted in these experiments. Essentially similar results were obtained with HeLa S3 cells. When the nutrient environment was changed in the middle of the growth cycle from Puck medium to serum and vice versa, a dissimilar response in the activities of both enzymes was also discernible. Thus, whereas alkaline phosphatase activity diminished progressively upon switching from regular medium to serum, inorganic pyrophosphatase activity increased slightly, reaching the levels of cells continuously growing in Puck medium. While a similar pattern was observed with inorganic pyrophosphatase following the shift from serum to regular medium, the specific activity of alkaline phosphatase increased 20-fold within 72 h (table 3). Further evidence that both activities did Table 2. Effect of growth in Puck medium and in human serum on alkaline phosphatase and inorganic pyrophosphatase of Zimmer liver 4A cells Puck medium
Human serum
Alkaline Hours of phosphatase growth
Inorganic Alkaline pyrophos- phosphatase phatase
Inorganic pyrophosphatase
48 72 96 120 144 168
0.1 0.2 0.2 0.3 0.3 0.3
0.1 0.1 0.1
0.14 0.47 0.89 1.32 1.68 1.58
0.11 0.07 0.04 0.03 0.03 0.02
84 0:2
Cells suspended in saline F were inoculated into Puck medium and into human serum. Duplicate cultures were harvested at 24 h intervals. Enzyme activity was determined in triplicate and related to cell protein.
Pyrophosphatase in cell cultures Table 3. Effect of changing the nutrient environment on alkaline phosphatase and inorganic pyrophosphatase of Zimmer liver 4A cells From Puck medium From serum to to serum Puck medium Hours after medium change
Alkaline phosphatase
Inorganic Alkaline pyrophos- phosphatase phatase
Inorganic pyrophosphatase
24 48 12 96
0.35 0.24 0.11 0.11
0.2 0.3 0.3 0.3
0.2 0.3 0.3 0.3
0.05 0.54 0.88 1.07
Cells suspended in saline F were inoculated into Puck medium and into human serum. After 3 days, cells were rinsed 3 times with saline F; serum was added to cells growing in regular medium and the latter to cells growing in serum. Beginning 24 h later, duplicate cultures from both sets were harvested at 24 h intervals. Enzyme activity was determined in triplicate.
not respond alike to alterations of the culture conditions was provided by experiments in which H. Ep II cells were continuously grown in serum for several successive growth cycles. Under these conditions, alkaline phosphatase activity dropped to 2.5 “//o of control levels after 2 cycles and continued depressed for the remaining cycles in serum. Normal values were obtained following 2 growth cycles in Puck medium. On the other hand, inorganic pyrophosphatase was only affected to a limited extent. Following an initial reduction after one growth cycle in serum, the activity varied between 0.2 and 0.4 during the subsequent cycles. Upon transferring the cells to regular medium, pyrophosphatase activity returned to control levels after one week (table 4). The total protein content of cells cultured in serum was similar to that of cultures in Puck medium. DISCUSSION Inorganic pyrophosphate is formed as a byproduct in many important biosynthetic reac-
309
tions, including DNA and RNA polymerization, coenzyme synthesis and amino acid and fatty acid activation. It has been suggested that the hydrolysis of inorganic pyrophosphate by inorganic pyrophosphatase provides a mechanism whereby these biosynthetic reactions are made irreversible. Several reports have indicated that inorganic pyrophosphate may be a natural substrate for alkaline phosphatase and that alkaline phosphatase and inorganic pyrophosphatase may be associated with a single protein [3-71. Studies with kidney cells of rats kept on a phosphate-free diet have shown a parallel increase in the specific activity of both enzymes [5]. Cox et al. also found a nearly parallel rise in HeLa S3 and BSC-1 monkey kidney cells grown in the presence of agents which promote the induction of increased levels of alkaline phosphatase activity [4]. The results presented in this report do not rule out the possibility that alkaline phosphatase and inorganic pyrophosphatase activities are due to the same enzyme. However, they demonstrate that in heteroploid cells of human origin the specific activity of inorganic pyrophosphatase is not as stringently controlled by the concentration of serum in the culture medium as alkaline phosphatase activity. Although it has been established that the reduction in alkaline phosphatase activity is not due to extrinsic or intrinsic enzyme inhibitors [2] and that the factor(s) in serum responsible for enzyme depression appears to be thermo-stable and non-dialysable. its mode of action is not understood. Thus, the results do not distinguish a direct action on enzyme synthesis from an accelerated enzyme degradation or from conformational alterations of the protein. The latter possibility would be in accord with the concept of one enzyme possessing both activities and that serum or some of its components mediate modifications of the molecule to such an Exptl Cell Res 74 (I9721
3 10 F. Herz & E. Kaplan
Table 4. Effect of growth in serum on alkaline phosphatase and inorganic pyrophosphatase of H.Ep II cells Days following medium change
Cells grown in
Cell protein (mg/flask)
Alkaline phosphatase
Inorganic pyrophcsphatase
Initiala 7 14 21 28 35 42 49 56 63
Puck medium Human serum Human serum Human serum Human serum Human serum Human serum Human serum Puck medium Puck medium
1.03 1.72 1.27 0.92 1.19 1.11 1.34 0.92 0.90 1.13
83.4 26.4 2.1 3.1 1.0 2.5 4.4 2.8 39.6 80.0
0.8 0.4 0.2 0.2 0.2 0.2 0.4 0.3 0.8 0.8
H.Ep II cells originally grown in Puck medium were suspended in Saline F and inoculated into human serum. After 7 consecutive growth cycles in serum, cells were retransferred into regular medium. Cells were harvested weekly, washed thrice with 0.15 M NaCl and stored at - 20”. Enzyme activities were measured simultaneously at the end of the experiment. a Means of cell preparations from 8 consecutive 7-day growth cycles in Puck medium.
extent as to be converted into a less efficient alkaline phosphatase without significantly affecting its capacity to hydrolyze inorganic pyrophosphate. This interpretation is reminiscent of the enhanced catalytic efficiency of the hormonally-induced alkaline phosphatase in HeLa cells [12]. This work was supported by Research Grant HD 01461 from NIH. The technical assistance of Mr David A. Sevdalian is gratefully acknowledged.
REFERENCES
3. Cox, R P & Griffin, M J, Lancet 2 (1965) 1018. 4. Cox, R P, Gilbert, Jr, P & Griffin, M J, Biochem j 105 (1967) 155. 5. Melani, F & Farnararo, M, Biochim biophys acta 178 (1969) 93. 6. Griffin, M J, Arch biochem biophys 132 (1969) 299. 7. Miles, P L & Nayudu, P R V, Enzymologia 41 (1971) 27. 8. Puck, T T, Cieciura, S J & Robinson, A, Jexptl med 104 (1956) 615. 9. Herz, F & Nitowsky, H M, Arch biochem biophys 96 (1962) 506. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Nitowsky, H M & Herz, F, Nature 189 (1961) 756. 12. Cox, R T, Elson, N A, Tu, S-H & Griffin, M J, J mol biol 58 (1971) 197.
1. Herz, F & Sevdalian, D A, Arch biochem biophys
146 (1971) 1. 2. Herz, F, Kaplan, E & Sevdalian, D A, J cell physiol 74 (1969) 213.
Exptl Cell Res 74 (1972)
Received February 4, 1972
0 1972 by Academic Press, Inc. of rrproduction in any form reseraed
Experimental
INORGANIC
Cell Research 74 (1972) 307-310
PYROPHOSPHATASE ACTIVITY WITH DEPRESSED ALKALINE
IN HUMAN CELL CULTURES PHOSPHATASE
F. HERZ and E. KAPLAN Departments of Pediatrics, Sinai Hospital, Baltimore, Md 21215, and The Johns Hopkins University School of Medicine, Baltimore, Md 21205, USA
SUMMARY A comparison is made of the effect of serum on inorganic pyrophosphatase and alkaline phosphatase in HeLa S3 and other human cell lines. When the cells are grown in human serum, alkaline phosphatase is maximally depressed to lesss than 5 36 of the activity of control cultures. By contrast, serum exerts only a slight effect on inorganic pyrophosphatase. The significance of these results is related to observations indicating that both activities are associated with a single protein.
The activity of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1) in certain established cell lines of human origin is dependent on the concentration of human serum in the culture medium [l]. Enzyme activity is inversely proportional to the serum concentration and is lowest in cells grown in serum without the basal components of the culture medium. The term “depression” has been selected to describe the serum-dependent reduction in enzyme activity [2]. Several reports have indicated that preparations of alkaline phosphatase from various sources, including cultured mammalian cells, possess inorganic pyrophosphatase (pyrophosphate phosphohydrolase, E.C. 3.6.1.1) activity; thus, suggesting that both activities may be associated with a single enzyme [3-71. In this report it will be shown that in contrast to its striking effect on alkaline phosphatase, serum does not modulate the activity of inorganic pyrophosphatase in heteroploid cells to a significant extent.
MATERIALS
AND METHODS
The following serially propagated cell lines were employed: HeLa S3, Zimmer liver 4A and H.Ep Il. The alkaline phosphatase activity of the first two clonal strains is relatively low and that of H.Ep 11 cells is approx. 100 times higher [ 11.The activity of the three cell lines is depressed by serum. Puck medium N16 [S] with 10 % undialysed human serum was routinely used and cells grown under these conditions served as controls. The effect of serum was studied in cells grown in human serum without the basal components of Puck medium, except saline F [8]. The culture technique, harvesting and subsequent handling of the cells was carried out as previously described [ 1,9]. Alkaline phosphatase activity was measured by the hydrolysis of o-nitrophenvl ohosphate using~2-amind-2-m&hyl-l-~ropan&-H~I duffer at pH 10.6 and 38°C [91.Specific activitv was exaressed as -micromoles of p&dphenol liberated in 30 min at 38°C per mg of protein. Inorganic pyrophosphatase activity was assayed according to Cox et al. [4] and specific activity expressed as micromoles of pyrophosphate hydrolysed in 30 min at 37°C per mg of protein, the latter determined according to Lowry et al. [IO].
RESULTS Table 1 compares the specific activities of alkaline phosphatase and inorganic pyrophosphatase of three human cell lines at the end of one 7-day growth cycle in Puck medium Exptl Cell Res 74 (1972)
308
F. Herz & E. Kaplan
Table 1. Effect of serum on alkaline phosphatase and inorganic pyrophosphatase of various cell lines
Cell lines HeLa S3 Zimmer liver Ii!&
II
Alkaline phosphatase
Inorganic pyrophosphatase
Puck medium i-10% Human serum serum
Puck medium +lO% Human serum serum
0.8
0.04
0.4
0.2
85.0 1.6
25.0 0.03
0.3 0.8
0.4 0.2
Cells were grown as monolayers for 7 days at 37°C in an atmosphere of 5 % CO, in air. Following dispersion with 0.05 % trypsin and three washings with 0.15 M NaCl, cells were disrupted by ultrasonication [9]. Enzyme activity was measured in duplicate on replicate cultures. Specific activity expressed as indicated in text.
and in human serum. As can be seen,alkaline phosphatase activity of HeLa S3 and Zimmer liver 4A grown in serum was characteristically reduced to less than 5 % of the activity of control cultures. By comparison, growth in serum had only a slight effect on inorganic pyrophosphatase activity. It should be noted that whereas the alkaline phosphatase of control H.Ep II cells was 100 times higher than that of HeLa S3, inorganic pyrophosphatase activity was only two times greater. Since the specific activity of alkaline phosphatase increases continuously during a growth cycle in regular medium [I l] and declines progressively in serum [l], a comparison of the influence of serum on the levels of alkaline phosphatase and inorganic pyrophosphatase activity was made with cells harvested at 24h intervals. As can be seen in table 2, in cells cultured in regular medium, alkaline phosphatase activity increased 12fold between 48 and 144 h after cell transfer, and decreased from 0.11 at 48 h to 0.02 at 168 h in cells grown in serum. Inorganic pyrophosphatase activity in cells growing in Exptl Cell Res 74 (1972)
Puck medium increased at a relatively slower rate from 0.1 at 48 h to 0.3 at 144 h. In cells cultured in serum the activity of this enzyme did not follow the pattern of alkaline phosphatase, but showed a slight increase towards the end of the growth cycle. No alterations in the specific activity of acid phosphatase and no reduction in total cell protein were noted in these experiments. Essentially similar results were obtained with HeLa S3 cells. When the nutrient environment was changed in the middle of the growth cycle from Puck medium to serum and vice versa, a dissimilar response in the activities of both enzymes was also discernible. Thus, whereas alkaline phosphatase activity diminished progressively upon switching from regular medium to serum, inorganic pyrophosphatase activity increased slightly, reaching the levels of cells continuously growing in Puck medium. While a similar pattern was observed with inorganic pyrophosphatase following the shift from serum to regular medium, the specific activity of alkaline phosphatase increased 20-fold within 72 h (table 3). Further evidence that both activities did Table 2. Effect of growth in Puck medium and in human serum on alkaline phosphatase and inorganic pyrophosphatase of Zimmer liver 4A cells Puck medium
Human serum
Alkaline Hours of phosphatase growth
Inorganic Alkaline pyrophos- phosphatase phatase
Inorganic pyrophosphatase
48 72 96 120 144 168
0.1 0.2 0.2 0.3 0.3 0.3
0.1 0.1 0.1
0.14 0.47 0.89 1.32 1.68 1.58
0.11 0.07 0.04 0.03 0.03 0.02
84 0:2
Cells suspended in saline F were inoculated into Puck medium and into human serum. Duplicate cultures were harvested at 24 h intervals. Enzyme activity was determined in triplicate and related to cell protein.
Pyrophosphatase in cell cultures Table 3. Effect of changing the nutrient environment on alkaline phosphatase and inorganic pyrophosphatase of Zimmer liver 4A cells From Puck medium From serum to to serum Puck medium Hours after medium change
Alkaline phosphatase
Inorganic Alkaline pyrophos- phosphatase phatase
Inorganic pyrophosphatase
24 48 12 96
0.35 0.24 0.11 0.11
0.2 0.3 0.3 0.3
0.2 0.3 0.3 0.3
0.05 0.54 0.88 1.07
Cells suspended in saline F were inoculated into Puck medium and into human serum. After 3 days, cells were rinsed 3 times with saline F; serum was added to cells growing in regular medium and the latter to cells growing in serum. Beginning 24 h later, duplicate cultures from both sets were harvested at 24 h intervals. Enzyme activity was determined in triplicate.
not respond alike to alterations of the culture conditions was provided by experiments in which H. Ep II cells were continuously grown in serum for several successive growth cycles. Under these conditions, alkaline phosphatase activity dropped to 2.5 “//o of control levels after 2 cycles and continued depressed for the remaining cycles in serum. Normal values were obtained following 2 growth cycles in Puck medium. On the other hand, inorganic pyrophosphatase was only affected to a limited extent. Following an initial reduction after one growth cycle in serum, the activity varied between 0.2 and 0.4 during the subsequent cycles. Upon transferring the cells to regular medium, pyrophosphatase activity returned to control levels after one week (table 4). The total protein content of cells cultured in serum was similar to that of cultures in Puck medium. DISCUSSION Inorganic pyrophosphate is formed as a byproduct in many important biosynthetic reac-
309
tions, including DNA and RNA polymerization, coenzyme synthesis and amino acid and fatty acid activation. It has been suggested that the hydrolysis of inorganic pyrophosphate by inorganic pyrophosphatase provides a mechanism whereby these biosynthetic reactions are made irreversible. Several reports have indicated that inorganic pyrophosphate may be a natural substrate for alkaline phosphatase and that alkaline phosphatase and inorganic pyrophosphatase may be associated with a single protein [3-71. Studies with kidney cells of rats kept on a phosphate-free diet have shown a parallel increase in the specific activity of both enzymes [5]. Cox et al. also found a nearly parallel rise in HeLa S3 and BSC-1 monkey kidney cells grown in the presence of agents which promote the induction of increased levels of alkaline phosphatase activity [4]. The results presented in this report do not rule out the possibility that alkaline phosphatase and inorganic pyrophosphatase activities are due to the same enzyme. However, they demonstrate that in heteroploid cells of human origin the specific activity of inorganic pyrophosphatase is not as stringently controlled by the concentration of serum in the culture medium as alkaline phosphatase activity. Although it has been established that the reduction in alkaline phosphatase activity is not due to extrinsic or intrinsic enzyme inhibitors [2] and that the factor(s) in serum responsible for enzyme depression appears to be thermo-stable and non-dialysable. its mode of action is not understood. Thus, the results do not distinguish a direct action on enzyme synthesis from an accelerated enzyme degradation or from conformational alterations of the protein. The latter possibility would be in accord with the concept of one enzyme possessing both activities and that serum or some of its components mediate modifications of the molecule to such an Exptl Cell Res 74 (I9721
3 10 F. Herz & E. Kaplan
Table 4. Effect of growth in serum on alkaline phosphatase and inorganic pyrophosphatase of H.Ep II cells Days following medium change
Cells grown in
Cell protein (mg/flask)
Alkaline phosphatase
Inorganic pyrophcsphatase
Initiala 7 14 21 28 35 42 49 56 63
Puck medium Human serum Human serum Human serum Human serum Human serum Human serum Human serum Puck medium Puck medium
1.03 1.72 1.27 0.92 1.19 1.11 1.34 0.92 0.90 1.13
83.4 26.4 2.1 3.1 1.0 2.5 4.4 2.8 39.6 80.0
0.8 0.4 0.2 0.2 0.2 0.2 0.4 0.3 0.8 0.8
H.Ep II cells originally grown in Puck medium were suspended in Saline F and inoculated into human serum. After 7 consecutive growth cycles in serum, cells were retransferred into regular medium. Cells were harvested weekly, washed thrice with 0.15 M NaCl and stored at - 20”. Enzyme activities were measured simultaneously at the end of the experiment. a Means of cell preparations from 8 consecutive 7-day growth cycles in Puck medium.
extent as to be converted into a less efficient alkaline phosphatase without significantly affecting its capacity to hydrolyze inorganic pyrophosphate. This interpretation is reminiscent of the enhanced catalytic efficiency of the hormonally-induced alkaline phosphatase in HeLa cells [12]. This work was supported by Research Grant HD 01461 from NIH. The technical assistance of Mr David A. Sevdalian is gratefully acknowledged.
REFERENCES
3. Cox, R P & Griffin, M J, Lancet 2 (1965) 1018. 4. Cox, R P, Gilbert, Jr, P & Griffin, M J, Biochem j 105 (1967) 155. 5. Melani, F & Farnararo, M, Biochim biophys acta 178 (1969) 93. 6. Griffin, M J, Arch biochem biophys 132 (1969) 299. 7. Miles, P L & Nayudu, P R V, Enzymologia 41 (1971) 27. 8. Puck, T T, Cieciura, S J & Robinson, A, Jexptl med 104 (1956) 615. 9. Herz, F & Nitowsky, H M, Arch biochem biophys 96 (1962) 506. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Nitowsky, H M & Herz, F, Nature 189 (1961) 756. 12. Cox, R T, Elson, N A, Tu, S-H & Griffin, M J, J mol biol 58 (1971) 197.
1. Herz, F & Sevdalian, D A, Arch biochem biophys
146 (1971) 1. 2. Herz, F, Kaplan, E & Sevdalian, D A, J cell physiol 74 (1969) 213.
Exptl Cell Res 74 (1972)
Received February 4, 1972