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Histone phosphorylation and DNA synthesis are linked in synchronous cultures of HTC cells
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HISTONE PHOSPHORYLATION AND DNA SYNTHESIS ARE LINKED IN SYNCHRONOUS CULTURES OF HTC CELLS
Rod Balhorn +, John Bordwell*, Larry Sellers*, Daryl Granner* & Roger Chalkley + Departments of Biochemistry(+) and Internal Medicine (*) University of Iowa College of Medicine Iowa City, Iowa 52240
Received December 30, 1971 SUMMARY: We have examined histone phosphorylation in synchronized cultures of rapidly dividing HTC cells to determine which histone groups are phosphorylated and at what time in the cell cycle phosphorylation occurs relative to DNA synthesis. Whole histone was resolved into the five major groups by electrophoresis on polyacrylamide gels. Phosphorylation of both F 1 and F2a 2 (not F2b) occurred coincidentally with DNA synthesis, whereas F3, F2b and F2a I were not phosphorylated to any significant level at any time in the cell cycle. Phosphorylation of mammalian lysine-rich histones has been documented both in vivo and in vitro (1-9).
A substantial body of evidence can be mar-
shalled in favor of the idea that phosphorylation of lysine-rich histones is always observed in cells which are actively dividing (2,7,10-13).
Recently we
have reported that histone phosphorylation is dramatically increased during rapid growth in Ehrlich ascites tumor cells (5), during hepatic regeneration induced by partial hepatectomy (6), and further, that the high degree of lysine-rich histone phosphorylation observed in exponentially growing HTC cells is abolished when the cells move into stationary phase (14). Shepherd, Noland and Hardin (15) have previously argued that phosphorylation of lysine-rich histones continues steadily throughout the cell cycle in synchronized Chinese hamster ovary cells, and that histone F2b is phosphorylated in conjunction with DNA synthesis.
However, the method employed grossly
overestimates actual phosphorylation because of co-isolation of 32p-labeled contaminants
(6).
We have investigated this problem, employing a gel electropkoretic analy-
1326 Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sis of histone phosphorylation which avoids the problem of contamination while providing a sensitive and accurate separation of various phosphorylated histones (6).
In contrast to previous studies, we find that histone fractions F 1
and F2a 2 are phosphorylated in synchronous cultures of HTC cells, and that both are ph0sphorylated strictly coincidentally with DNA synthesis. MATERIALS AND METHODS Cell Culture:
HTC cells, derived from the ascites form of Morris hepa-
toma 7288C (16), grow in suspension culture with a doubling time of approximately 24 hours at 37°C and stay in exponential growth at cell densities between 2 x 105 and 8 x 105 cells/ml.
Up to 600 ml of cells were grown in 1
liter Erlenmeyer flasks in modified medium S-77 (17). Synchronization of HTC Cells:
Exponentially growing HTC cells were sus-
pended in fresh medium supplemented with 2.41 mM CaCI 2 and 107 cells were inoculated into Blake bottles.
36-42 hours later this medium was decanted and
replaced with similar medium containing 0.i ~M colcemid.
To collect mitotic
cells, the bottles were placed in an upright position 5-8 hours later and the medium was decanted off the top surface of the bottle.
The cells were main-
tained in mitosis for several hours in the presence of colcemid or were allowed to enter G 1 after their removal from colcemid medium by centrifugation. narily 1-1.5 x 106 mitotic cells/bottle were harvested.
Ordi-
The mitotic index was
determined by counting at least 200 cells which had been stained with 2% acetoorcein.
Experiments yielding less than 90% synchronized cells were discarded.
Incorporation of 32p into Histones and Fractionatipn by Gel Electrophoresis:
After pulse labeling the HTC cells for three hours by incubation with
0.5 mCi 32p-phosphoric acid (carrier free) per 105 cells, the nuclei were isolated and purified from 107 synchronized HTC cells using 1.0 gram of rat liver as a carrier.
Whole histone was extracted as previously described (18), dis-
solved in 0.9 N acetic acid containing 15% sucrose, and was electrophoresed in 2.5 M urea gels for 3.5 hours at 130 V by the method of Panyim et al. (18). After fixing with Amido Schwarz (4 hours) and subsequent destaining, 2 mm gel
1327
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
slices were dried on planchets and counted in a Biospan Planchet Counter at 40% efficiency. RESULTS Lysine-rich histones isolated from rapidly dividing HTC cells show considerable electrophoretic heterogeneity as is shown in Figure i.
Cells grown
for three hours in the presence of 32p-sodium phosphate have discrete, labeled bands within the lysine-rich system as is also indicated in Figure i.
The
fastest moving lysine-rich histone shows essentially no 32p incorporation, whereas the two more slowly migrating bands are both labeled to approximately the same level.
Since the slowest band has about one-half the intensity of
the other 32p-labeled band, it is clear that the specific activities of the lysine-rich phosphohistones increase in the ratio 1:2 with an attendant decrease in rate of electrophoretic migration.
Previous studies (6,14) have
documented that A) the bands which contain 32p are phosphorylated histones;
0
u
ii,
,
,
i
+
LYSINE - RICH
Figure i. High resolution gel electrophoretic pattern and 32p-phosphate labeling of HTC lysine-rich his tone. Electrophoresis was performed at 2 mA/gel and 200 V for 65 hours at O°C.
1328
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B) these bands can be abolished by phosphatase treatment; C) bands of higher specific activity and slower mobility contain more than one phosphate group per F 1 molecule; and D) that they are absent in the stationary phase of cultures of these tumor cells (14).
2--
x
1
]•
U
[]
+
~b
0.5M UREA i
2L
o
x:
O
+ F2a2
6.0M UREA Figure 2. Short polyacrylamide gel electrophoretic patterns and 32p-phosphate labeling of whole histone electrophoresed in gels containing 0.5 M and 6.0 M urea. Electrophoresis was performed at 2 mA/gel and 130 V for 3.5 hours at 25°C.
1329
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Radioactive label is also found associated with histone fraction F2a 2. Since the presence of a single phosphate residue on this histone decreases its mobility by approximately 3%, we expected that phosphorylated F2a 2 would migrate coincidentally with F2b in our normal electrophoretic system.
To ensure
that the radioactivity is indeed associated with F2a 2, and not with F2b , we exploited an earlier observation which showed that the mobility of F2b can be modified considerably by changing the urea concentration within the gels (19). Figure 2 shows that F2b migrates with F 3 in 0.5 M urea, whereas in 6.0 M urea F2b moves more rapidly and co-migrates with F2a 2.
The radioactivity does not
follow the peregrinations of F2b but rather is invariably found at a position with approximately 3% slower mobility than F2a2, indicating that the labeled histone is phosphorylated F2a 2. Although our previous studies showed that histone phosphorylation was positively correlated with exponential growth in HTC cells (14), they gave no clues as to when in the cell cycle this event occurred.
Synchronized cultures
of HTC cells were employed to study the temporal relationship of DNA synthesis with histone phosphorylation.
The cells were pulsed with 32p-sodium phos-
phate and 3H-thymidine, in separate containers, at various times after the release of mitotic arrest. scribed above.
Histones and DNA were isolated by procedures de-
Biosynthesis of DNA (S phase) was measured by 3H-thymidine in-
corporation and histone phosphorylation by the radioactivity (32p) associated with specific histone fractions after electrophoresis.
The data of Figure 3
show that during both mitosis (M) and G 1 there is little incorporation of 32p into any histone fraction.
Phosphorylation of both histone F 1 and F2a 2 begins
to increase dramatically near the end of GI, peaks at the same time that DNA synthesis is at its maximum, and declines once more at the end of the DNA synthetic phase of the HTC cell cycle.
Histones F2b , F 3 and F2a I show very low
levels of 32p incorporation at all stages of the cell cycle. DISCUSSION Our observations on the temporal relationship of DNA synthesis and his-
1330
Vol. 46, No. 3, 1972 14-
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10-
-7
12
a
8-
-5 ×
I / i, I/ I/
Z
o
x -4
/"//
8
I; o NI
~
ij
i,/,,'.,
_ e/
• 2-
\
,'"'7 o
-2 ?
,L.. j
--,,, .,.-(,,o-"y ////
/ /"
/7-. ~. e/x
/ "'-a'
O-
I -5
I
i
i
l
I 0
h
1
l
l
I
~ I
5
i
i
J I0
i i
i i
[ 15
I
i
1
1
I
I
I
20
HOURS AFTER RELEASE OF COLCEMID BLOCK
Figure 3. Correlation of DNA synthesis and 32p-phosphate incorporation into histones of synchronized HTC cells. DNA synthesis was estimated by incubating 2-4 x 105 cells in 0.2 pCi 3H-thymidine (New England Nuclear) for 30 min. Methods of cell synchrony, 32p labeling and fractionation of histones are described in the text. tone phosphorylation disagree with the report by Shepherd et al. (15). These authors found that F 1 histones were phosphorylated continuously throughout the cell cycle, and that phosphorylation of histone F2b began just prior to DNA synthesis and declined with the latter.
In contrast, we have shown that F 1
and F2a 2 (not F2b) are both phosphorylated only when DNA is synthesized.
It
seems probable that the cause of this disagreement lies in the methods used to analyze for phosphorylation.
Not only did these authors use a continuous pulse
of radioactivity throughout the cell cycle, but direct counting of 32p-radioactivity associated with lysine-rich histones prepared by the method of Johns (20) is likely to be an unreliable estimate of protein phosphorylation since
1331
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
histone so isolated is grossly contaminated with 32p-containing material which is not covalently bound to histone (6).
In the electrophoretic system used in
this study, such 32p-labeled non-histone material migrates in the opposite direction to histone, and even if a part of this were covalently associated with histone it would so effectively change the histone mobility that it would migrate much more slowly than the band systems used in the analysis described in this paper. The level of F I phosphorylation in exponential phase H~C cells is such that about 70% of the parent F I histone is phosphorylated with either one or two phosphate groups (see Figure i).
However, F I isolated from stationary
phase cells shows essentially no phosphorylation (14) as also is the case for the adult rat liver (6). The actual level of F I phosphorylation in adult liver is exceedingly low when care is taken to avoid nucleic acid contamination (6) and probably reflects a small endogenous population of more rapidly dividing cells in the otherwise non-dividing tissue.
We feel that these observa-
tions pose very serious questions to those proponents of the theory which states that phosphorylation of histones is used as a device to control differential levels of gene activity.
Certainly the liver is an active organ which
is capable of considerable control in the response of its genome to a variety of hormonal and environmental stimuli and yet these are functions which it can apparently perform quite adequately without significant histone phosphorylation. Finally we note that the study of histone phosphorylation derived its initial impetus from observations of i n ~ i t r o enzymatic phosphorylatlon of histone (4,8).
The tissue most utilized for the isolation of "histonekinases" has
been the adult rat liver (8,9,21), a tissue which under normal physiological conditions does not phosphorylate hiatones (3,4,6).
This raises the qmestion
of whether the earlier work was conce~nedwith a true histone kinase, or perhaps with a more general protein kinase.
On the other hand, it may be that
the enzyme studied was indeed histone kinase but that in the cell it is either inactive or unable to interact with the chromosomal material.
1332
The observations
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
described in this paper suggest a means for resolving some of these difficulties.
A search should be undertaken for a protein kinase which is active dur-
ing exponential growth of Pfrc cells, is active only during S phase of the cell cycle, but is inactive during the stationary phase of growth.
If such an en-
zyme could he isolated, it would seem to be a likely candidate for a specific histone kinase.
On the other hand, if an enzyme with these temporal properties
cannot be identified, one might suspect that in fact the enzyme is present at all stages of growth but that its activity is curtailed in some way at appropriate stages of the cell cycle and during stationary phase. ACKNOWLEDGMENTS This work was supported by USPHS Grant #CA-I0871 and #CA-12191 and by the American Cancer Society (Iowa Division) Grant #P-451. Roger Chalkley is a Research Career Development Awardee from the USPHS, Grant #GM-41460, and Daryl Granner is a Veterans Administration Clinical Investigator. REFERENCES i. Ingles, C.J. and Dixon, G.H., Proc. Nat. Acad. Sci. U.S., 58, i011 (1967). 2. Kleinsmith, L.J., Allfrey, V.G. and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 55, 1182 (1966). 3. Langan, T.A., Proc. Nat. Acad. Sci. U.S., 6_~4, 1276 (1969a). 4. Langan, T.A., J. Biol. Chem., 244, 5763 (1969b). 5. Sherod, D., Johnson, G. and Chalkley, R., Biochemistry, 9, 4611 (1970). 6. Balhorn, R., Rieke, W.O. and Chalkley, R., Biochemistry , i0, 3952 (1971). 7. Sung, M.T., Dixon, G.H. and Smithies, O., J. Biol. Chem., 246, 1358 (1971). 8. Langan, T.A., Science, 162, 579 (1968). 9. Langan, T.A., Rall, S.C. and Cole, R.D., J. Biol. Chem., 246, 1942 (1971). i0. Georgatsos, J.G., Rammos, G., .Palavradzi, D. and Symeonides, A., Europ. J. Cancer, 4, 313 (1968). Ii. Cross, M.E. and Ord, M.G., Biochem. J., 118, 191 (1970). 12. Gurley, L.R. and Walters, R.A., Biochemistry, IO, 1588 (1971). 13. Gutierrez-Cernosek, R.M. and Hnilica, L.S., Biochim. Biophys. Acta, 247, 348 (1971). 14. Balhorn, R., Cha!kley , R. and Granner, D., submitted to Biochemistry. 15. Shepherd, G.R., Noland, B.J. and Hardin, J.M., Biochim. Biophys. Acta, 228, 544 (1971). 16. Thompson, E.G., Tomkins, G.M. and Curran, J.F., Proc. Nat. Acad. Sci. U.S., 56, 296 (1966~. 17. Granner, D.K., Thompson, E.G. and Tomkins, G.M., J. Biol. Chem., 245, 1472
(1970). 18. Panyim, S., Bilek, D. and Chalkley, R., J. Biol. Chem., 246, 4206 (1971). 19. Panyim, S. and Chalkley, R., Biochem. Bioph~s. Res. Cotm~., 37, 1042 (1969). 20. Johns, E.W., Biochem. J., 92, 55 (1964). 21. Meisler, M.~I. and Langan, T.A., J. Biol. Chem., 244, 4961 (1969).
1333
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
HISTONE PHOSPHORYLATION AND DNA SYNTHESIS ARE LINKED IN SYNCHRONOUS CULTURES OF HTC CELLS
Rod Balhorn +, John Bordwell*, Larry Sellers*, Daryl Granner* & Roger Chalkley + Departments of Biochemistry(+) and Internal Medicine (*) University of Iowa College of Medicine Iowa City, Iowa 52240
Received December 30, 1971 SUMMARY: We have examined histone phosphorylation in synchronized cultures of rapidly dividing HTC cells to determine which histone groups are phosphorylated and at what time in the cell cycle phosphorylation occurs relative to DNA synthesis. Whole histone was resolved into the five major groups by electrophoresis on polyacrylamide gels. Phosphorylation of both F 1 and F2a 2 (not F2b) occurred coincidentally with DNA synthesis, whereas F3, F2b and F2a I were not phosphorylated to any significant level at any time in the cell cycle. Phosphorylation of mammalian lysine-rich histones has been documented both in vivo and in vitro (1-9).
A substantial body of evidence can be mar-
shalled in favor of the idea that phosphorylation of lysine-rich histones is always observed in cells which are actively dividing (2,7,10-13).
Recently we
have reported that histone phosphorylation is dramatically increased during rapid growth in Ehrlich ascites tumor cells (5), during hepatic regeneration induced by partial hepatectomy (6), and further, that the high degree of lysine-rich histone phosphorylation observed in exponentially growing HTC cells is abolished when the cells move into stationary phase (14). Shepherd, Noland and Hardin (15) have previously argued that phosphorylation of lysine-rich histones continues steadily throughout the cell cycle in synchronized Chinese hamster ovary cells, and that histone F2b is phosphorylated in conjunction with DNA synthesis.
However, the method employed grossly
overestimates actual phosphorylation because of co-isolation of 32p-labeled contaminants
(6).
We have investigated this problem, employing a gel electropkoretic analy-
1326 Copyright © 1972, by AcademicPress, Inc.
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
sis of histone phosphorylation which avoids the problem of contamination while providing a sensitive and accurate separation of various phosphorylated histones (6).
In contrast to previous studies, we find that histone fractions F 1
and F2a 2 are phosphorylated in synchronous cultures of HTC cells, and that both are ph0sphorylated strictly coincidentally with DNA synthesis. MATERIALS AND METHODS Cell Culture:
HTC cells, derived from the ascites form of Morris hepa-
toma 7288C (16), grow in suspension culture with a doubling time of approximately 24 hours at 37°C and stay in exponential growth at cell densities between 2 x 105 and 8 x 105 cells/ml.
Up to 600 ml of cells were grown in 1
liter Erlenmeyer flasks in modified medium S-77 (17). Synchronization of HTC Cells:
Exponentially growing HTC cells were sus-
pended in fresh medium supplemented with 2.41 mM CaCI 2 and 107 cells were inoculated into Blake bottles.
36-42 hours later this medium was decanted and
replaced with similar medium containing 0.i ~M colcemid.
To collect mitotic
cells, the bottles were placed in an upright position 5-8 hours later and the medium was decanted off the top surface of the bottle.
The cells were main-
tained in mitosis for several hours in the presence of colcemid or were allowed to enter G 1 after their removal from colcemid medium by centrifugation. narily 1-1.5 x 106 mitotic cells/bottle were harvested.
Ordi-
The mitotic index was
determined by counting at least 200 cells which had been stained with 2% acetoorcein.
Experiments yielding less than 90% synchronized cells were discarded.
Incorporation of 32p into Histones and Fractionatipn by Gel Electrophoresis:
After pulse labeling the HTC cells for three hours by incubation with
0.5 mCi 32p-phosphoric acid (carrier free) per 105 cells, the nuclei were isolated and purified from 107 synchronized HTC cells using 1.0 gram of rat liver as a carrier.
Whole histone was extracted as previously described (18), dis-
solved in 0.9 N acetic acid containing 15% sucrose, and was electrophoresed in 2.5 M urea gels for 3.5 hours at 130 V by the method of Panyim et al. (18). After fixing with Amido Schwarz (4 hours) and subsequent destaining, 2 mm gel
1327
Vol. 46, No. 3, 1 9 7 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
slices were dried on planchets and counted in a Biospan Planchet Counter at 40% efficiency. RESULTS Lysine-rich histones isolated from rapidly dividing HTC cells show considerable electrophoretic heterogeneity as is shown in Figure i.
Cells grown
for three hours in the presence of 32p-sodium phosphate have discrete, labeled bands within the lysine-rich system as is also indicated in Figure i.
The
fastest moving lysine-rich histone shows essentially no 32p incorporation, whereas the two more slowly migrating bands are both labeled to approximately the same level.
Since the slowest band has about one-half the intensity of
the other 32p-labeled band, it is clear that the specific activities of the lysine-rich phosphohistones increase in the ratio 1:2 with an attendant decrease in rate of electrophoretic migration.
Previous studies (6,14) have
documented that A) the bands which contain 32p are phosphorylated histones;
0
u
ii,
,
,
i
+
LYSINE - RICH
Figure i. High resolution gel electrophoretic pattern and 32p-phosphate labeling of HTC lysine-rich his tone. Electrophoresis was performed at 2 mA/gel and 200 V for 65 hours at O°C.
1328
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B) these bands can be abolished by phosphatase treatment; C) bands of higher specific activity and slower mobility contain more than one phosphate group per F 1 molecule; and D) that they are absent in the stationary phase of cultures of these tumor cells (14).
2--
x
1
]•
U
[]
+
~b
0.5M UREA i
2L
o
x:
O
+ F2a2
6.0M UREA Figure 2. Short polyacrylamide gel electrophoretic patterns and 32p-phosphate labeling of whole histone electrophoresed in gels containing 0.5 M and 6.0 M urea. Electrophoresis was performed at 2 mA/gel and 130 V for 3.5 hours at 25°C.
1329
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Radioactive label is also found associated with histone fraction F2a 2. Since the presence of a single phosphate residue on this histone decreases its mobility by approximately 3%, we expected that phosphorylated F2a 2 would migrate coincidentally with F2b in our normal electrophoretic system.
To ensure
that the radioactivity is indeed associated with F2a 2, and not with F2b , we exploited an earlier observation which showed that the mobility of F2b can be modified considerably by changing the urea concentration within the gels (19). Figure 2 shows that F2b migrates with F 3 in 0.5 M urea, whereas in 6.0 M urea F2b moves more rapidly and co-migrates with F2a 2.
The radioactivity does not
follow the peregrinations of F2b but rather is invariably found at a position with approximately 3% slower mobility than F2a2, indicating that the labeled histone is phosphorylated F2a 2. Although our previous studies showed that histone phosphorylation was positively correlated with exponential growth in HTC cells (14), they gave no clues as to when in the cell cycle this event occurred.
Synchronized cultures
of HTC cells were employed to study the temporal relationship of DNA synthesis with histone phosphorylation.
The cells were pulsed with 32p-sodium phos-
phate and 3H-thymidine, in separate containers, at various times after the release of mitotic arrest. scribed above.
Histones and DNA were isolated by procedures de-
Biosynthesis of DNA (S phase) was measured by 3H-thymidine in-
corporation and histone phosphorylation by the radioactivity (32p) associated with specific histone fractions after electrophoresis.
The data of Figure 3
show that during both mitosis (M) and G 1 there is little incorporation of 32p into any histone fraction.
Phosphorylation of both histone F 1 and F2a 2 begins
to increase dramatically near the end of GI, peaks at the same time that DNA synthesis is at its maximum, and declines once more at the end of the DNA synthetic phase of the HTC cell cycle.
Histones F2b , F 3 and F2a I show very low
levels of 32p incorporation at all stages of the cell cycle. DISCUSSION Our observations on the temporal relationship of DNA synthesis and his-
1330
Vol. 46, No. 3, 1972 14-
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
10-
-7
12
a
8-
-5 ×
I / i, I/ I/
Z
o
x -4
/"//
8
I; o NI
~
ij
i,/,,'.,
_ e/
• 2-
\
,'"'7 o
-2 ?
,L.. j
--,,, .,.-(,,o-"y ////
/ /"
/7-. ~. e/x
/ "'-a'
O-
I -5
I
i
i
l
I 0
h
1
l
l
I
~ I
5
i
i
J I0
i i
i i
[ 15
I
i
1
1
I
I
I
20
HOURS AFTER RELEASE OF COLCEMID BLOCK
Figure 3. Correlation of DNA synthesis and 32p-phosphate incorporation into histones of synchronized HTC cells. DNA synthesis was estimated by incubating 2-4 x 105 cells in 0.2 pCi 3H-thymidine (New England Nuclear) for 30 min. Methods of cell synchrony, 32p labeling and fractionation of histones are described in the text. tone phosphorylation disagree with the report by Shepherd et al. (15). These authors found that F 1 histones were phosphorylated continuously throughout the cell cycle, and that phosphorylation of histone F2b began just prior to DNA synthesis and declined with the latter.
In contrast, we have shown that F 1
and F2a 2 (not F2b) are both phosphorylated only when DNA is synthesized.
It
seems probable that the cause of this disagreement lies in the methods used to analyze for phosphorylation.
Not only did these authors use a continuous pulse
of radioactivity throughout the cell cycle, but direct counting of 32p-radioactivity associated with lysine-rich histones prepared by the method of Johns (20) is likely to be an unreliable estimate of protein phosphorylation since
1331
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
histone so isolated is grossly contaminated with 32p-containing material which is not covalently bound to histone (6).
In the electrophoretic system used in
this study, such 32p-labeled non-histone material migrates in the opposite direction to histone, and even if a part of this were covalently associated with histone it would so effectively change the histone mobility that it would migrate much more slowly than the band systems used in the analysis described in this paper. The level of F I phosphorylation in exponential phase H~C cells is such that about 70% of the parent F I histone is phosphorylated with either one or two phosphate groups (see Figure i).
However, F I isolated from stationary
phase cells shows essentially no phosphorylation (14) as also is the case for the adult rat liver (6). The actual level of F I phosphorylation in adult liver is exceedingly low when care is taken to avoid nucleic acid contamination (6) and probably reflects a small endogenous population of more rapidly dividing cells in the otherwise non-dividing tissue.
We feel that these observa-
tions pose very serious questions to those proponents of the theory which states that phosphorylation of histones is used as a device to control differential levels of gene activity.
Certainly the liver is an active organ which
is capable of considerable control in the response of its genome to a variety of hormonal and environmental stimuli and yet these are functions which it can apparently perform quite adequately without significant histone phosphorylation. Finally we note that the study of histone phosphorylation derived its initial impetus from observations of i n ~ i t r o enzymatic phosphorylatlon of histone (4,8).
The tissue most utilized for the isolation of "histonekinases" has
been the adult rat liver (8,9,21), a tissue which under normal physiological conditions does not phosphorylate hiatones (3,4,6).
This raises the qmestion
of whether the earlier work was conce~nedwith a true histone kinase, or perhaps with a more general protein kinase.
On the other hand, it may be that
the enzyme studied was indeed histone kinase but that in the cell it is either inactive or unable to interact with the chromosomal material.
1332
The observations
Vol. 46, No. 3, 1972
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
described in this paper suggest a means for resolving some of these difficulties.
A search should be undertaken for a protein kinase which is active dur-
ing exponential growth of Pfrc cells, is active only during S phase of the cell cycle, but is inactive during the stationary phase of growth.
If such an en-
zyme could he isolated, it would seem to be a likely candidate for a specific histone kinase.
On the other hand, if an enzyme with these temporal properties
cannot be identified, one might suspect that in fact the enzyme is present at all stages of growth but that its activity is curtailed in some way at appropriate stages of the cell cycle and during stationary phase. ACKNOWLEDGMENTS This work was supported by USPHS Grant #CA-I0871 and #CA-12191 and by the American Cancer Society (Iowa Division) Grant #P-451. Roger Chalkley is a Research Career Development Awardee from the USPHS, Grant #GM-41460, and Daryl Granner is a Veterans Administration Clinical Investigator. REFERENCES i. Ingles, C.J. and Dixon, G.H., Proc. Nat. Acad. Sci. U.S., 58, i011 (1967). 2. Kleinsmith, L.J., Allfrey, V.G. and Mirsky, A.E., Proc. Nat. Acad. Sci. U.S., 55, 1182 (1966). 3. Langan, T.A., Proc. Nat. Acad. Sci. U.S., 6_~4, 1276 (1969a). 4. Langan, T.A., J. Biol. Chem., 244, 5763 (1969b). 5. Sherod, D., Johnson, G. and Chalkley, R., Biochemistry, 9, 4611 (1970). 6. Balhorn, R., Rieke, W.O. and Chalkley, R., Biochemistry , i0, 3952 (1971). 7. Sung, M.T., Dixon, G.H. and Smithies, O., J. Biol. Chem., 246, 1358 (1971). 8. Langan, T.A., Science, 162, 579 (1968). 9. Langan, T.A., Rall, S.C. and Cole, R.D., J. Biol. Chem., 246, 1942 (1971). i0. Georgatsos, J.G., Rammos, G., .Palavradzi, D. and Symeonides, A., Europ. J. Cancer, 4, 313 (1968). Ii. Cross, M.E. and Ord, M.G., Biochem. J., 118, 191 (1970). 12. Gurley, L.R. and Walters, R.A., Biochemistry, IO, 1588 (1971). 13. Gutierrez-Cernosek, R.M. and Hnilica, L.S., Biochim. Biophys. Acta, 247, 348 (1971). 14. Balhorn, R., Cha!kley , R. and Granner, D., submitted to Biochemistry. 15. Shepherd, G.R., Noland, B.J. and Hardin, J.M., Biochim. Biophys. Acta, 228, 544 (1971). 16. Thompson, E.G., Tomkins, G.M. and Curran, J.F., Proc. Nat. Acad. Sci. U.S., 56, 296 (1966~. 17. Granner, D.K., Thompson, E.G. and Tomkins, G.M., J. Biol. Chem., 245, 1472
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