Mol. Cells, Vol. I, pp. 183-1 86
Relationship between Inositol Phospholipid Metabolism and
Initiation of DNA Synthesis during Early S Phase of Cell Cycle
Myung Ae Lee, Cheol O. Joe l *, Jae Hoon Chung l and Ke Won Kang l
Department of Molecular Biology, Seoul National University, Seoul 151-742, Korea;
'Department of Biological Science and Engineering, Korea Advanced Institute of Science and
Technology, Tacdon 305-701. Korea
(Received on Apri l 6, 1991)
Intracellular inositol phospholipid hydrolysis involved in the initiation of DNA synthesis
was examined. NIH 3T3 cells metabolically labeled with [ 3H]inositol were growth-arrested
by serum starvation and 3 mM hydroxyurea treatment. and cells were allowed to initiate
DNA synthesis in the norn1al media. The increased breakdown of inositol phospholipid
into inositol phosphates and diacylglycerol was observed preceding to the maximal DNA
synthesis which was measured by the [ 3H ]thymidine incorporation into DNA of cells
during the progression of cell into S phase. Phosphoryl ation of phosphatidylinositol into
phosphatidylinositol phosphates subsequent to the hydrolysis into inositol phosphates began to increase about 2 h before the onset of maximal DNA synthesis. These results
suggest that the change of inositol phospholipid metabolism is closely associated with
the initiation of DNA synthesis.
Protein ki nase cascade has been implicated as the
major control mechanism of cell proliferation. Certain
growth factors, of which epidern1al growth factor is
a typical exa mple, stimulate the activities of tyrosine
kinase and seri ne/ threonine kinase, and trigger the
protein kinase cascade (Ahn and Kreb 1990; Kawahara el al., 1988). The cascade of protein kinase activities is thought to stimulate the initation of DNA syn thesis and mitotic division.
In recent years, the receptor mediated hydrolysis of
inositol phospholipid received a great deal of attention
as the cellul ar mechanism regulating cell division and
DNA sy nthesis (MacDonald et al., 1987; Majerus el
aI., 1986; Whitman el al., 1988), The response of inositol phospholipid to the extracellul ar signals was reported initially in 1953 (Hokin and H okin, 1953). It is
now evident that receptor mediated hydrolysis of inositol phospholipid is a common mechanism by which
various extracellular signals such as those from hormones, peptide growth factors, neurotransmitters and
other biologically active substances are transduced across the cell membrane (Nishizuka, 1986; Majersus
et al., 1990). Upon binding these agonistors to specific
receptors, cells initiate the production of second messenger molecules. Low molecular weight effector molecules that ca n covalently interact with the regulatory
component of an enzyme, typically protein kinase, to
increase its specific activity, are generally referred as
second messengers. At least two different second messenger molecules, 1,2-diacylglycerol and inositol 1,4,5triphosphate (Ip)), are known to be produced as the
* To whom correspondence should be addressed
result of inositol phospholipid metabolism. Evidence
has been obtained th at 1,2-diacylglycerol activates protein kinase C a nd that IP 3 stim ulates Ca H release from
the endoplasmic reticulum to activate Ca H /ca lmodulin dependent protein kinases. These two protein kinase systems phosphorylate many different proteins
required for the cell cycle transition in eukaryotic cells
(Berridge and Irvine, 1989; Majerus et al., 1990).
While the extremely complex processes involved in
stimulation of cell proliferation leadi ng to increased
turnover of inositol phospholipid during mitotic cell
division have been studied in detail, biochemical mechanisms controlling the initiation of DNA synth esis
remai ns unclear. In the present study, the authors present data from the quantitative analysis of inositol
phospholipid metabolites and their hydrolysis products during the initiation period of DNA synthesis in
mouse fibroblast cells.
Materials and Methods
Chemicals
[MethyPH]thymidine (70-80 Ci/mmol), [ methyl- '''C]
thymidine (56 mCi/mmol), myo-[2-3H ] inositol (20.8
Cilmmol) were from Amersham. Anion excha nge resin (AG I X 8) in the formate forn1 (200-400 mesh)
was obtained from Bio-Rad. All other reagents were
of ana lytical grade.
The abbreviations used are: [P I, inositol I-monophosphate;
IP 2, inositol 1,4-bisphosphate; IP 3, inositol IA,S-triphosphate;
PI, phosphotidylinositol; PIP, phosphatidylinositol 4-phosphate: P[P2, phosphatidylinositol 4.5-bisphosphate.
© 1991 The Korean Society of Molecul ar Biology
184
Inositol Phospholipid Hydrolysis in DNA Synthesis
Mol. Cells
Cell culture
Murine fib roblast NIH 3T3 cells were cultured in
Dulbecco's Modifi ed Eagle's Medium supplemented
with 10% heat inactivated feta l calf serum . Cells were
maintained in a humidified CO 2 incubato r at 37 °C.
DNA synthesis rale
Cells were treated with 0.01 flC i o f [ '4C Jthymidine
per 1111 fo r 3 days befo re experiments were perfo rmed.
C ultures were rinsed with PBS twice and growth was
a rrested by pl acing the semico nfluent cultures in the
medi a containing 0.02% serum a nd 3 mM hydroxyurea for 24 h. The [ lH J thymidine inco rpo ratio n into
DNA was determined by incubating cells in th e presence of [ lH Jthymidine for 30 min during the progression of th e cell cycle into S ph ase as previously described by No rman et al. (1 986).
Inositol phospholipid metabolism
To qua nti fy th e inositol phospholipid metabolites,
cells were metabolically labeled fo r 24 h befo re the
growth arrest in a medium containg 20 flCi/mi [ lH J
inositol. Cells were havested at the specified time period after release from the growth arrest. The medium
was removed, and 2 ml of ice cold 20% trichlo roacetic
acid was added in 100 mm dishes placed on ice. Cell s
were scraped off fro m the dishes using a rubber po licema n. Inos itol phospho lipids were extracted fro m the
acid precipi tated cell pellet by the sequential additio n
of 0.1 N HC l and CH lOH/C HCh (37: 19) solution.
Labeled phosphatidylinositol phosphates were separated and identified by thin-l ayer chromatography as
p reviously described by Whitma n et aI., (1985). The
aqueous ph ase was coll ected a nd inositol phosph ates
were sepa rated using D owex AG 1 X 8 formate column (Kikuchi et al., 1986), and qua ntitated by liquid
scintill atio n counting.
8
4
(l
Post incubation (h)
Figure 1. Time courses of [lHJthymidine incorporation rate
into NIH 3T3 cells. Cells were growth alTested by serum
starvation and 3 mM hydroxyurea for 24 h. Cells were allowed to grow in the regular media supplemented with 10%
fetal calf serum. [lHJThymidine (3 IlCi/ml) was added at
each incubation time period for 30 min, and DNA synthesis
rate was measured radiometrically.
.
•
•
•
~--l
PI
~--;
PIP
Results
The p rogression o f cell cycle o f NIH 3T3 fibrobl ast
was stopped by depletio n o f growth facto rs in th e medium a nd by a n inhibito r o f replicative DNA sy nthesis, hyd roxyu rea. Incuba tio n o f the growth-a rrested cell s in the fresh med ia containing 10% ca lf se rum ca used a ma rked increase in [ lH Jthymidine into DNA
of NIH 3T3 cells, in dicating th at cell s are in the tra nsition stage fro m th e G o to S ph ase. To assure [ lH J thymi dine inco rporatio n as a measure fo r the DNA synthesis in th e same number of cells, cellul ar DNA
of N IH 3T3 cells were grown in [ '4CJ thymidine before
the growth. a rrest a nd then lH and 14C radioactivity
profile was performed. lH :14C ratio th at represe nts
DNA synth esis rate per unit time indicates that maxi mal DNA synthesis occurs at 3 h after growth release.
Each value was the mean of triplicate determinatio n
fro m the rep resentative experiment (Fig. I). To ana lyze
inositol phospholipid metabolites, li pid sampl es extracted from cell s were dried under nitrogen fl ow a nd
ascending ch ro matography was ca rried out o n th e cellul ose TLC pl ates. Figure 2 presents th e typical perturbatio n o f inositol phosph olipid metabo lism du ri ng the
•
A
B
c
Figure 2. Autoradiogram of phosphatidylinositol metabolites
separated on TLC plate. The phosphatidylinositol metabolites in NIH 3T3 cell s at 0 h (A), 0.5 h (8), and I h (C)
after the growth release. Phosphatidylinositol metabolites
were standardized with respect to total labeled inositol containing phospholipid.
initiation process o f DNA synthesis. Remakable phosph o l),lation of ph osphatidylinositol (PI) a nd ph osphatidylinositol 4-phosph ate (PIP) into p hosph atidylinositol 4,5-bisph osph ate (PIP 2) was seen in I h a fter gro-
Vol. I (1991)
Myung Ae Lee et at.
185
1 0.00 0
3.000
8.0 00
Ea.
Ea.
~
~
.2=
2.000
.2=
o
2
4
5
Time (h)
Figure 3. Metabolism of inositol phospholipid during the
initiation period of DNA synthesis. PI (6 ), PIP (e), and
PIP 2 (0 ) from [lHJrnyo-inositol (4 IlCi/ml) prelabeled cells
were extracted and separated on TLC plate as described
in Materials and Methods.
wth release. The cell cyle dependent changes of inositol phospholipids are presented Figure 3. Cellular levels of PIP and PIP 2 increased rapidly, peak within
I h after growth commencement, and then gradually
fall down . In contrast to PIP and PIP 2 metabolites,
the level of PI was redltlced by 76% at 1 h and stabilized thereafter.
We also measured the breakdown of inositol phospholipid into inositol phosphates in the fibroblast cells
transiting from G to S phase. The changes of intracellular levels of inositol phosphates differ among three
spec ices of inositol phosphates. During the 5 h incubation time after growth release, intracellular level of
inositol l-monophosphate (IP 1) was gradually elevated,
so that the cellul ar content of IP 1 at 5 h was more
than 3 times higher than that of the cells under the
growth arrest. The content of inositol 1,4-bisphospate
(IP 2) was promtly increased in I h after the growth
release and then reached to new steady state level
at 2 h whereas the production of IP 3 rose more slowly
(Fig. 4). The data suggest that cell cycle progression
into S phase yields rapid hydrolysis of inositol phospholipid.
Discussion
Proliferation and division of cells follow orderly sequence of events which culminate in DNA synthesis.
A major regulatory control is exercised prior to the
entry of cells into S phase. The commitment of DNA
synthesis in eukaryotic cells is known to be completed
2 h before the onset of S phase (Pardee, 1987). After
this restriction point of commitment, serum or growth
factors are not required to complete the cell cycle.
It has been well described that inositol phospholipid
o
2
3
4
5
Time (h)
Figure 4. Time courses of lH incorporation into IP, IP 2, and
IP l from [lHJrnyo-inositol in NIH 3T3 cells during the initiation period of DNA synthesis. IP (e), IP 2 (6 ), and IP)
(0 ) were resolved by batch elution on Dowex (AG I X
8) formate (2 ml) columns.
turnover is closely linked with the cell proliferation
in receptor stimulated cells by extracellular signals
(Blackshear et al., 1985), or in transformed cells by
viral oncogenes (Kaplan et al., 1986; Kato et al., 1987).
However, cellular mechanisms for stimulating DNA
synthesis and the requirements for the initiation of
DNA synthesis as an early cellular phenomenon of
cell cycle progression remain obscure. Our results in
Figure 3 implicate that production of phosphorylated
phosphatidylinositols is the intracellular mechanism
preceeding the initiation of DNA synthesis. The inositol phospholipid is relatively minor component of cell
membrane comprising less than 10% of total phospholipids in eukaryotic cell membrane (Nishizuka et
al., 1984). Phosphorylation at inositol 4 hydroxyl group
takes place by phosphatidylinositol kinase and to a
lesser degree, by cyclcic AMP-dependent kinase (Farkars et al., 1984). The PIP is then subsequently phosphorylated at 5 hydroxyl position of inositol ring
moiety to form PIP 2 by PIP kinase (Nishizuka et al.,
1988, Pike and Eakes, 1987).
Apparently majority of enzyme activities is required
for the initiation of DNA synthesis. Pardee et al. (1986)
proposed that DNA replicase complex requires activation of one or more key existing enzymes in order
to initiate DNA synthesis. However, the involvement
of inositol phospholipid as a direct reactant in the
activation of the enzymes is not established. We observed the change of intracellular contents of inositol
phosph ates in the cells entering S phase (Fig. 4). The
results indicate that hydrolysis of phosphorylated inositol phospholipid takes place immediatly after phos-
186
Inositol Phospholipid Hydrolysis in DNA Synthesis
phOly lation of inositol phospholipid in the cells engaged in the initiation o f DNA synthesis. Since the meta bolic products of phospho rylated inositol ph osph olipid are known to serve as second messenger molecules
in a va riety o f receptor stimulated cell s, the cha nge
o f inosito l phospholipid meta bolism before th e o nset
o f DNA synth esis might provide effecto r molecules
necessa ry fo r the activatio n o f enzy mes respo nsible
fo r the initi atio n of DNA synth esis.
Our futu re study will esta blish cellula r role of these
seco nd messenger molecules o n the initi ation o f DNA
synthesis before th e o nset of S ph ase in eukaryotic
cells.
Acknowledgment
Thi s work was supported by gra nts fro m Ko rea
Science a nd Enginee ring Foundation, a nd fro m Science Resea rch Cente r fo r Cell Diffenti a tio n a t Seoul
N a tio nal University.
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