The Presence of Parvalbumin in a
Nonmuscle Cell Line Attenuates
Progression through Mitosis
Colin D. Rasmussen and Anthony R. Means
Department of Cell Biology
Baylor College of Medicine
Houston, Texas 77030
based largely on the effects of introduction of Ca2+chelating agents, such as EGTA, into cells or the media
surrounding cells in culture. We have addressed this
question in a novel way by expressing the Ca2+-binding
protein parvalbumin in mouse C127 cells using a bovine
papilloma virus (BPV)-based expression vector containing a complete rat parvalbumin cDNA (6). The physiological role of parvalbumin is that of a high affinity Ca2+
buffer in fast twitch vertebrate muscle (7). The presence
of parvalbumin in a cell line that normally does not
contain this protein would be expected to perturb the
normal mechanism of Ca2+-dependent signalling involved in the regulation of cell cycle progression by
buffering transient increases in intracellular free Ca2+.
The results indicate that the presence of parvalbumin
in C127 cells slows progression through mitosis. This
effect occurs predominantly in prophase and, therefore,
is most likely different from that of calmodulin (CaM),
which is thought to be required for the movement of
chromosomes to the spindle poles during anaphase
(7a).
Based on studies that have examined the effect of
calcium chelators on cells, it has been proposed that
this cation plays a role in regulating cell proliferation.
In this study a novel approach was used to indirectly
examine the role of calcium in cell cycle progression.
A cDNA for the Ca2+-binding protein parvalbumin
has been expressed in mouse C127 cells, using a
bovine papilloma virus-based expression vector.
The normal role of parvalbumin is that of a calcium
buffer in vertebrate fast twitch muscle, and the C127
cells do not normally express this protein. The presence of parvalbumin had several effects on the
growth of C127 cells. The most striking phenotype
was an increase in cell cycle duration which analysis
showed was the result of an increase the length of
d and mitosis (predominantly at prophase). Since
changes in cell cycle duration typically occur as a
result of changes in G^ duration, the observed increase in the length of mitosis is most unusual. The
present results indicate that the previously observed
increase in the rate of cell proliferation in cells with
elevated calmodulin levels is not the result of a
general increase in the level of cytoplasmic calciumbinding protein, but is specific to calmodulin. In
addition, the results suggest that calcium regulates
progression through mitosis by both calmodulin-dependent (metaphase transition) and -independent
(prophase) mechanisms. (Molecular Endocrinology
3: 588-596, 1989)
RESULTS
Expression of a Rat Parvalbumin cDNA in C127
Cells
To analyze the effect of the presence of the Ca2+binding protein parvalbumin on cell proliferation, a BPVbased eukaryotic expression vector was constructed
and used to transform mouse C127 cells. The vector
BPV-CaMPV (Fig. 1) consists of a complete rat parvalbumin cDNA clone (6) placed 3' to the chicken CaM
promoter used previously to overexpress a chicken
CaM gene in C127 cells (8). BPV-CaMPV also contains
the 69% transforming region of BPV and the plasmid
pUC-18 for propagation in £ coli.
Mouse C127 cells were transfected with BPVCaMPV DNA, and transformed foci were selected after
14 days. Two clonal cell lines (PV-1 and PV-2) that
carried the BPV-CaMPV plasmid and expressed high
levels of PV mRNA were selected for further analysis.
Two previously characterized cell lines transformed by
BPV alone (BPV-1 and BPV-2) or one that overex-
INTRODUCTION
Calcium functions as an intracellular second messenger
and mediates a large number of cellular responses. The
ability of Ca2+ to relay information occurs by transient
increases in intracellular levels of this cation (1). One
proposed role for Ca2+ is in regulation of cell proliferation and cell cycle progression. Several studies have
suggested a Ca2+ requirement for progression through
Gi (2, 3) and mitosis (4, 5). These conclusions were
0888-8809/89/0588-0596S02.00/0
Molecular Endocrinology
Copyright © 1989 by The Endocrine Society
588
589
Parvalbumin in a Nonmuscle Cell Line
1kb
CaM-PV
E
BPV
E KE
pUC 18
69T
0.1kb
0.8 kb
Fig. 1. Construction of a BPV-Based Parvalbumin Expression Vector
The vector BPV-CaMPV was constructed as follows. A Hind\\\/Fsp\ fragment of chicken DNA containing the CaM gene promoter
used previously (8) was ligated into a Hin6\\\/Hinc\\ fragment of a pUC-18 based plasmid containing the complete parvalbumin
cDNA clone previously described (6). This plasmid was digested with H/ndlll, and a eamHI/H/ndlll blunt-ended fragment of the
BPV-1 genome (BPV- 69 T) was ligated to produce the vector BPV-CaMPV. • , BPV sequences; D, CaM gene promoter; M,
parvalbumin coding sequence; D, parvalbumin cDNA 5' and 3' untranslated regions. Thin line, pUC-18. Restriction sites: E, EcoRI,
K, Kpn\; P, Pst\ "T", mRNA transcription start site for the expected 0.8-kb transcript. The arrow marks the position of the
polyadenylation site in the parvalbumin cDNA.
presses a chicken CaM minigene and has elevated
intracellular CaM levels (CM-1) have also been used in
this study (8).
Parvalbumin mRNA and Parvalbumin in BPVCaMPV-Transformed Cells
Parvalbumin mRNA expression was examined by
Northern blot hybridization. The parvalbumin cDNA
used in the BPV-CaMPV vector contains a polyadenylation signal within the 3' untranslated region. Correct
transcription and processing of the PV cDNA should
produce a single mRNA species of 0.8 kilobases (Kb).
Equal amounts of cytoplasmic RNA from BPV-1, PV-1,
and PV-2 cells were hybridized to an oligo-labeled
EcoR\(Pst\ fragment which comprises a portion of the
coding sequence of the rat parvalbumin cDNA clone
used to construct BPV-CaMPV. No hybridization to
BPV-1 mRNA was observed, indicating that the C127
cells do not express endogenous parvalbumin mRNA
(Fig. 2, lane BPV-1). However, in both PV-1 and PV-2
cells, a hybridization signal corresponding to a single
0.8-kb mRNA was observed (Fig. 2, lanes PV-1 and
PV-2). Therefore, transcription of the PV cDNA within
the BPV-CaMPV vector produced high levels of a correctly processed cytoplasmic parvalbumin mRNA.
Since significant levels of parvalbumin mRNA were
present in both BPV-CaMPV-transformed cell lines,
Western blot analysis was used to determine whether
this resulted in the synthesis of parvalbumin. Heatstable extracts from equal numbers of BPV-1, PV-1, or
PV-2 cells were resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose. Parvalbumin was detected
using an affinity-purified rabbit antirat parvalbumin anti-
sera (obtained from M. Berchtold). As a positive control,
HPLC-purified rat parvalbumin was included on the
same gel. The results show that a single protein species
of identical mol wt to HPLC-purified rat parvalbumin
standard was recognized in both PV-1 and PV-2 cells
(Fig. 3a). The antisera failed to detect parvalbumin in
BPV-1 cells, consistent with the absence of parvalbumin mRNA in this cell line observed by Northern blot
analysis.
Parvalbumin levels were quantitated immunologically
by dot-blot analysis. Dilution series of heat-stable extracts from BPV-1, PV-1, and PV-2 cells and known
amounts of the HPLC-purified parvalbumin standard
were applied to nitrocellulose using a dot-blot apparatus
(Schleicher and Schuell, Keene, NH). For the dilution
series of parvalbumin standards, heat-stable extract
from BPV-1 cells was added to the sample to maintain
an equal protein concentration compared to that in the
extracts from PV cells. Parvalbumin was detected by
the same procedures used for Western blot analysis
(Fig. 3b). Quantitation was performed by densitometric
scanning of the autoradiogram. The relative signals
obtained for the parvalbumin standards were used to
construct a standard curve to permit quantitation (correlation coefficient of standard curve was r = 0.996; df
= 3). Based on this standard curve we estimate that
PV-1 and PV-2 cells contain 960 and 480 ng parvalbumin/106 cells, respectively. The parvalbumin concentration in PV-1 cells represents a potential increase in
Ca2+-binding protein of twice the Ca2+-binding capacity
of endogenous CaM.
The cellular distribution of parvalbumin in PV-1 cells
was evaluated by indirect immunofluorescence microscopy. BPV-1 cells were examined to verify the specificity of the immunofluorescence signal. The results show
MOL ENDO-1989
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28S
18S
0.8kb
Fig. 2. Northern Analysis of Parvalbumin mRNA in BPV-Transformed Cell Lines
Equal amounts of cytoplasmic RNA from BPV-1, PV-1, and
PV-2 cells were analyzed by Northern blot hybridization for
the presence of parvalbumin mRNA as described in Materials
and Methods. The blot was hybridized to a radiolabeled EcoRI/
Pst\ fragment comprising most of the translated sequence of
the parvalbumin cDNA (see Fig. 1).
that no fluorescence was observed in BPV-1 cells,
confirming that parvalbumin is normally absent in C127
cells (Fig. 4A). However, a strong immunofluorescence
signal was observed in interphase PV-1 cells. Parvalbumin appeared to be distributed throughout the cytoplasm and showed no apparent localization to subcellular structures (Fig. 4B). There was also no localization
of parvalbumin to structures in mitotic cells. In fact, the
protein appeared to be excluded from the region occupied by chromosomes (shown by arrow in Fig. 4C).
Growth Characteristics of PV Cells
The growth characteristics of both PV-1 and PV-2 cells
were examined. Both cell lines attained a saturation
density at plateau phase that was significantly greater
than that of the control BPV-1 cells and nearly equal to
that observed for the CM-1 cell line that overproduces
CaM (Table 1). This suggests that plateau cell density
may be altered by the Ca2+-binding capacity of the cell
and cannot be specifically attributed to CaM. However,
unlike CM-1 cells both PV-1 and PV-2 cells had generation times longer than that of control BPV-1 cells (Table
1). These data suggest that the presence of a Ca2+buffering protein in C127 cells can slow the rate at
which cells progress through the cell cycle. These data
also show that the observation that elevated CaM levels
increase the rate of cell proliferation (8) is specifically
related to CaM activity and is not the result of a general
increase in cytoplasmic Ca2+-binding protein.
Previous studies have shown that Gi duration in BPVtransformed cells is strongly correlated with intracellular
CaM levels. Since PV-1 and BPV-1 cells contain equivalent intracellular CaM levels, we initially expected that
the lengths of G^ would be similar in the two cell lines.
However, since expression of parvalbumin was correlated with an increase in cell cycle duration, the length
of GT was measured to determine whether the difference in cell cycle length was due entirely to a difference
in the length of Gi. This cell cycle compartment is
generally accepted to be the most variable, and differences in cell cycle length are usually the result of
variability in the duration of Gi (9); overexpression of
CaM shortens this phase exclusively (8). To determine
d duration, synchronous populations of PV-1 cells
were obtained by the mitotic shake-off procedure (10)
and replated into culture medium containing [3H]thymidine. At various times after replating, cells were isolated, and the net incorporation of [3H]thymidine into
DNA was determined. The point at which significant
accumulation of label into DNA occurs marks the onset
of the S-phase, and the time from M to the onset of
DNA synthesis is a direct measure of the length of Gi.
The results obtained showed that Gi lengths in the
control BPV-1 and BPV-2 cell lines were 3.0 and 3.4 h,
respectively, compared with 3.8 h in PV-1 cells (Table
2). The observation that G^ in PV-1 cells was extended
by 0.8 h, relative to that in BPV-1 cells, suggests that
the presence of parvalbumin retards progression of
cells through Gi. However, while the difference in Gi
duration in the control cell lines can completely account
for the difference in generation times, the difference in
d duration between the PV-1 and the control cell lines
leaves 0.8 h unaccounted for (Table 2A), indicating that
some other cell cycle phase must be lengthened in PV1 cells.
Preliminary flow cytometric analysis indicated that
there was an increase in the G2/M population in exponentially growing PV-1 cells (data not shown). To determine whether this was a result of an increase in the
proportion of cells in mitosis, the mitotic index of PV-1,
BPV-1, and CM-1 cells was determined (Table 2B). The
data show that the mitotic index for PV-1 cells was
increased relative to that of the other cell lines, suggesting that the length of mitosis is increased in PV-1
cells. To determine if this accounted for the residual
0.8-h difference in cell cycle duration between BPV-1
and PV-1 cells, the length of mitosis was calculated
from the mitotic indices by the following equation: length
of M (h) = mitotic index x cell cycle length (h). The
results show that the length of mitosis was not significantly different in BPV-1 and CM-1 cells, but was 0.8 h
longer in PV-1 cells. Thus, the increased cell cycle
length in PV-1 cells is a result of an increase in both d
and mitosis.
It has been previously shown in Chinese hamster
ovary (CHO) cells that CaM levels double abruptly at
Parvalbumin in a Nonmuscle Cell Line
591
• „.
•»
»;
14kd
a.
Cell #(x10 5 )
ng PV Stds
10
2500
4
1000
1.6
400
0.6
160
0.3
64
0.1
25
\ '
\ '
\
/
\
Fig. 3. Expression of Parvalbumin in BPV-Transformed Cell Lines
a, The presence of parvalbumin in heat-stable extracts from equal numbers ( 2 x 1 0 6 ) of BPV-1, PV-1, and PV-2 cells was
analyzed by Western blot analysis as described in Materials and Methods. The marker (14kd) shows the observed mol wt of HPLCpurified rat parvalbumin. b, Parvalbumin levels were quantitated by dot-blot as described in the text and Materials and Methods.
HPLC-purified parvalbumin (Std.) was diluted in 2.5-fold increments, as were the heat-stable extracts from PV-1, PV-2, and BPV-1
cells.
the Gi/S boundary, and that this increase is correlated
with entry into the S phase (11). CaM levels were
measured during the cell cycle in mitotically synchronized BPV-1 and PV-1 cells to determine if CaM increases at the Gi/S boundary in BPV-transformed
C127 cells and whether the presence of parvalbumin
had an effect on cell cycle-dependent regulation of CaM
levels. The data show that CaM levels double as BPV1 cells progress through the cell cycle (Fig. 5). The
midpoint of the increase (2.8 h) occurred very near the
previously determined Gi/S boundary (3.0 h). This suggests that a doubling of CaM as cells traverse the Gi/
MOL ENDO-1989
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Table 1. Growth Characteristics of BPV-Transformed C127
Cells
Cell Line
CaM Levels
(ng/106 Cells)
BPV-1'
CM-1 a
PV-1
PV-2
410
1810
419
ND
Generation Time (h)
14.6
12.7
16.2
15.6
Plateau Density
(Cells/Dish)
2.29
4.21
4.10
4.10
x
x
x
x
106
106
106
106
The growth characteristics of BPV-transformed cell lines were
determined. Generation time was determined by regression
analysis of the exponential phase portion of growth curves.
Cell number determinations were made by direct counting of
appropriately diluted samples of isolated cells using a hemocytometer. Variability was typically less than 3% among parallel samples. The intracellular levels of CaM were determined
by RIA as previously described (23). The presence of parvalbumin had no significant effect on the intracellular CaM concentration. ND, Not determined.
a
BPV-1 and CM-1 cells have been previously characterized
(8). The data included here are for comparison to PV cells.
duration in PV-1 cells may be the result of buffering of
a Ca2+ signal before the Gi/S boundary.
In contrast to Gi, which can vary considerably (9),
the length of mitosis is usually constant. Thus, the
increase in the duration of mitosis in PV-1 cells is
unique. Because the length of M was increased in PV1 cells, the relative distribution of cells within M was
examined to determine whether this effect was due to
a general lenghthening of all parts of mitosis or an
increase in the duration of a particular compartment.
The frequency of cells within a particular portion of
mitosis is an indicator of the relative length of each
mitotic phase. Cells stained with aceto-orcein were
scored for the relative proportion of cells in each compartment of mitosis (Fig. 6). From the data in Fig. 6, the
length of each phase of mitosis was then calculated
indirectly as follows: length of phase = % of cells in
phase x length of mitosis (h). The results show that
while lengthening appears to occur in all phases of
mitosis, the most significant difference (0.5 h) occurs
as a result of an apparent increase in the length of
prophase (Table 3). Thus, the presence of parvalbumin
appears to retard the rate at which cells either progress
through or exit prophase.
Fig. 4. Immunofluorescence Microscopic Localization of Parvalbumin in BPV-Transformed Cell Lines
Parvalbumin localization was examined by indirect immunofluorescence microscopy as described in Materials and
Methods. A (interphase BPV-1 cells) and B (interphase PV-1
cells) were exposed for identical times in producing both the
negative and the final print. Similar fluorescence intensity was
observed for all cells in PV-1 cultures. C (also PV-1 cells) was
exposed for a shorter time to show the detail in a mitotic cell.
DISCUSSION
S boundary may be a general feature of proliferating
cells. In PV-1 cells, CaM levels also doubled (Fig. 5).
The midpoint of the increase occurred exactly at the
Gi/S boundary (3.8 h). Therefore, the normal temporal
relationship between an increase in CaM concentration
and entry into the S phase is not affected by parvalbumin. These results suggest that the difference in Gi
We have indirectly examined the role of cytosolic free
Ca2+ in the regulation of cell proliferation by using a
BPV-based expression vector to express a parvalbumin
cDNA in mouse C127 cells which normally do not
contain this Ca2+-binding protein. Parvalbumin was chosen because it is most abundant in vertebrate fast
twitch muscle, where it functions to buffer the transient
increase in intracellular Ca2+ levels that occurs during
muscle contraction (7). This protein binds Ca2+ with the
593
Parvalbumin in a Nonmuscle Cell Line
Table 2. Cell Cycle Parameters in BPV-Transformed Cells
A. Length of Gi and Generation Time
Cell Line
G, (h)
Difference (h)
Generation Time (h)
Difference (h)
Residual (h)
BPV-1
8PV-2
PV-1
3.0
3.4
3.8
0.4
0.8
14.6
15.0
16.2
0.4
1.6
0.8
B. Length of Mitosis
Cell Line
Ml (%)
Length of M (h)
Difference (h)
BPV-1
CM-1
PV-1
7.8
8.4
11.7
1.1
1.1
1.9
0.8
The length of Gi and generation times were determined as described in the text and Materials and Methods. The SES for generation
time and Gi determinations are less than ±0.2 h. The mitotic index was determined by microscopic examination of aceto-orceinstained cells. The length of mitosis was calculated from the mitotic index (Ml), as described in the text. The SE for this calculation
was less than ±0.1 h.
5 0 0 _ BPV-1
200,
3
6
9
12
Mrs. after Mitotic Selection
15
500
200,
3
6
9
12
15
Hrs. after Mitotic Selection
Fig. 5. CaM Levels during the Cell Cycle in BPV-1 and PV-1
cells
CaM levels were determined by RIA during a synchronous
cell cycle in mitotically selected cells. The results are expressed
as nanograms of CaM per 106 cells. The location of the Gi/S
boundary is marked. Top panel, BPV-1 cells; bottom panel,
PV-1 cells.
high affinity that is characteristic of members of the
superfamily of EF-hand Ca2+-binding proteins (12).
However, unlike other members of this group, such as
troponin-C and CaM, parvalbumin does not associate
with intracellular target proteins even in muscle. Therefore, the presence of parvalbumin should not have any
effect on cell proliferation other than those that occur
as a consequence of its Ca2+-buffering capacity.
In two independent cell lines that expressed high
levels of parvalbumin form the BPV-vector-containing
parvalbumin cDNA, the saturation density at the plateau
phase was increased relative to that in the control BPV1 cell line. This effect was also observed in cells with
elevated levels of CaM (8) (this study). Studies have
shown that an increase in extracellular Ca2+ levels
causes WI-38 fibroblasts to grow to a higher saturation
density (13). The observation that cells with elevated
CaM or parvalbumin reach a significantly higher plateau
density suggests that cessation of cell proliferation at
confluence may be affected by the ability of cells to
sequester Ca2+ by binding to high affinity Ca2+-binding
proteins.
BPV-CaMPV-transformed cells had an increased cell
cycle length relative to the control BPV-transformed
cells. Analysis shows that this is due to increases in the
length of both Gi and mitosis. Previous evidence has
suggested that differences in cell cycle duration are
usually the result of changes in the length of Gi (9).
Consequently, the observation that the length of mitosis
was increased in PV cells is novel.
Calcium has been proposed to regulate progression
at both the Gi/S boundary and during mitosis. This
cation is required for progression of Gi cells into the S
phase (2). WI-38 cells when deprived of extracellular
Ca2+ arrest at the Gi/S boundary (3). It has been
suggested that the role for Ca2+ in regulating Gi progression is mediated via CaM. Indeed, CaM levels
increase abruptly at the Gi/S boundary, and this increase is correlated with the onset of DNA synthesis
(11) (this study). Recent results have shown that a
constitutive increase in CaM levels shortens the length
of Gi (8), while a transient increase in CaM levels
accelerates cells past the Gi/S boundary (7a). In contrast, a reduction of CaM levels by expression of antisense RNA in C127 cells causes cells to arrest at the
MOL ENDO-1989
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40-
30-
CD
.2
20-
10-
Prophaae
Prometaphaaa
Mataphaa«
Anaphaaa
Talophaa*
Mitotic Phase
Fig. 6. Distribution of Cells in Mitosis in BPV-1 and PV-1 Cells
The distribution of cells within prophase, prometaphase, metaphase, anaphase, and telophase were determined by microscopic
examination of squashed cells stained with aceto-orcein to visualize chromatin morphology as previously described (11). The data
are shown in both bar graph and tabular form. D, BPV-1 cells; • , PV-1 cells.
Table 3. Length of Mitotic Phases in BPV-1 and PV-1 Cells
Mitotic Phase
BPV-1 (h)
PV-1 (h)
Difference (h)
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
0.26
0.28
0.36
0.10
0.10
0.76
0.33
0.48
0.18
0.18
0.50a
0.05"
0.12c
0.08"
0.086
The length of each phase of mitosis was calculated indirectly,
based on the data presented in Fig. 6. Calculations have a SE
of ±0.1 h.
a
P<0.05.
b
Not significantly different (P > 0.5).
c
0.5>P>0.2.
Gi/S boundary (7a), as does treatment of cells with
CaM antagonist drugs (11, 14, 15). Comparison of
these results with the present data leads us to speculate that the effect of parvalbumin on progression
through Gi is the result of buffering a Ca2+ signal acting
through CaM. Previous studies have shown that all of
the variation in the length of Gi resides in the part of
Gi that follows the serum restriction point (termed Gips)
(9). It is during Gi ps that Ca2+ is required for cells to
progress through the remainder of Gi and begin DNA
synthesis (2, 3).
In CHO cells and BPV-transformed C127 cells, intracellular CaM levels double at the Gi/S boundary (11)
(this study). In PV-1 cells, CaM levels increased normally, but the timings of both this increase and the d /
S boundary were delayed. Since the relationship between the increase in CaM levels and entry into the S
phase was maintained, we would suggest that the
increase in CaM that occurs coincident with the onset
of DNA synthesis might be triggered, either directly or
indirectly, by an increase in cellular free Ca2+ levels and
that the presence of parvalbumin buffers this signal.
Since at present no evidence for or against such a
mechanism exists, this proposal would be an interesting
topic for further study.
The observation that the duration of mitosis was
increased in PV-1 cells suggests that Ca2+ buffering by
parvalbumin delays progression through mitosis. Several studies have indicated a central role for Ca2+ in the
control of mitosis. Transient increases in Ca2+ have
been associated with nuclear envelope breakdown and
chromatin condensation in sea urchin embryos (16) and
the onset of anaphase in cultured animal cells (17,18).
Progression through mitosis can be arrested before
nuclear envelope breakdown by microinjection of the
Ca2+-chelator EGTA (4, 5). Microinjection of inositol
trisphosphate, which causes release of Ca2+ from intracellular stores, induces premature nuclear envelope
breakdown and chromatin condensation (5). Analysis
of mitotic PV cells showed that an increase in the length
of prophase accounted for most of the increased duration of mitosis, suggesting the possibility that parvalbumin retards progression through mitosis by buffering
the transient increases in Ca2+ apparently required for
nuclear envelope breakdown and chromatin condensation. CaM is also required for progression through
mitosis. CaM antagonists block progression through
mitosis in both yeast (14) and CHO cells (15). Reduction
of CaM levels in C127 cells by expression of CaM
antisense RNA also arrests in mitosis (7a). However,
cells with reduced CaM levels arrest predominantly at
metaphase. This study suggests that Ca2+ may potentially regulate progression through mitosis by two distinct mechanisms, on CaM dependent (regulating the
onset of anaphase) and one CaM independent (regulating nuclear envelope breakdown and chromatin condensation).
Parvalbumin in a Nonmuscle Cell Line
The precise nature of the molecular mechanisms
underlying the effects of parvalbumin in proliferation of
C127 cells cannot be determined from the studies
presented here. Previous studies have shown that at
low free Ca2+ levels, such as those present in a resting
cell, the Ca2+-binding domains of parvalbumin are occupied by Mg 2+ (12). However, it is unlikely that the
effects of parvalbumin in C127 cells are a consequence
of binding Mg 2+ , since CaM also has two Ca2+-binding
domains that will bind Mg 2+ . Thus, CaM and parvalbumin would have similar, not different, effects on growth
if such an effect were the result of altered intracellular
Mg 2+ . While parvalbumin has not been shown to associate with intracellular proteins in fast twitch muscle
where it is the most abundant, the possibility exists that
the effects observed in C127 cells expressing parvalbumin may be the result of a fortuitous interaction with
a protein required for cell cycle progression. It will be
interesting to determine if parvalbumin binds specific
cellular proteins in C127 cells and if such an interaction
might be Ca2+-dependent.
The use of mammalian cells has provided insight into
roles for Ca2+ in cell proliferation. However, these cells
are of limited subsequent use because of the inability
to directly relate genotype to phenotype. Future experiments must focus on the future expression of parvalbumin in genetically tractable organisms, such as Schizosaccharomyces pombe or Aspergillus nidulans. The
isolation of mutations that suppress the Gi or mitotic
delay phenotypes should define gene products directly
involved in transduction of the Ca2+ signal that regulates
progression through the cell cycle.
595
0.5% Nonidet P-40, pH 8.3) and lysed for 10 min at 4 C with
periodic vortexing. The Nonidet P-40 lysate was centrifuged
for 2 min at 14,000 x g to pellet nuclei, and the supernate
was retained. The supernate was extracted with phenol-chloroform-iso-amyl alcohol, and the cytoplasmic RNA was precipitated as described previously (21). Cytoplasmic RNA was
analyzed by electrophoresis on formaldehyde-containing 1.2%
agarose gels, followed by capillary transfer to Biodyne A filters
(Pall) as described previously (20). After transfer filters were
air-dried, then baked for 3 h at 68 C.
Synthesis of Radiolabeled cDNA Probes
Radiolabeled cDNA probes were synthesized by the oligolabeling procedure exactly as described previously (22). The
specific activity of these probes was typically 1 - 2 x 109 cpm/
ng. Before hybridization the probe was denatured by heating
to 100 C for 7 min, followed by cooling on ice.
Hybridization of Radiolabeled Probes
Filters were prehybridized for 1-4 h at 42 C in 5 x SSC, 50%
formamide, 5 x Denhardt's, 50 HIM NaPO 4 ,1% SDS, and 200
/xg/ml yeast rRNA, pH 6.5 (21). Hybridization of labeled probes
was carried out for 18 h at the same temperature and in a
solution of the same composition as that used for prehybridization. Probe was added at a concentration of 5 x 106 cpm/
ml. After hybridization, filters were washed for 5 min at room
temperature through five washes of 1 x SSC-0.1% SDS, and
for 30 min at 55 C in 0.1 x SSC-0.1% SDS to remove
nonspecifically bound probe. Autoradiography was carried out
at - 7 0 C using Kodak XAR-5 film (Eastman Kodak, Rochester,
NY) with intensifying screens.
Quantitation of Intracellular CaM Levels
Determinations of CaM levels in cells were performed by RIA
as previously described (23).
Isolation of Parvalbumin from Mouse C127 Cells
MATERIALS AND METHODS
Culturing Mouse C127 Cells
Mouse C127 cells are a nontransformed cell line, originally
derived from a mouse mammary tumor (19), and were obtained
from Dr. Dean Hamer of the NIH. C127 cells are cultured in
Dulbecco's Modified Eagle's Medium/high glucose (DMEM;
Gibco, Grand Island, NY) containing 10% fetal bovine serum
(FBS; Gibco) at 37 C in a humidified 95% air-5% CO2 atmosphere and passaged when 70-80% confluent. Cells were
maintained in T-flasks (75 cm2; Falcon, Oxnard, CA), while
cells for experiments were grown in plastic culture dishes
(either 60 or 100 mm; Falcon).
Transfection of C127 Cells and Selection of Transformed
Foci
Early passage C127 cells were transfected with BPV-CaMPV
DNA (circular form) by the calcium phosphate/DNA coprecipitate procedure as described previously (20). Transformed cells
were selected on the basis of focus formation 10 days after
transfection and clonal cell lines were established by the
limiting dilution method.
Isolation and Analysis of Cytoplasmic RNA
Cells were isolated by trypsinization and pelleted by centrifugation (500 x g; 3 min). The pellet was resuspended in lysis
buffer (10 mM Tris-HCI, 1.5 mM MgCI2, 140 mM NaCI, and
Cells were isolated by trypsinization and pelleted (500 x g; 3
min). The cell pellet was resuspended in homogenization buffer
(10 mM Tris-HCI, 1 mM dithithreitol, 1 mM EDTA, 0.2 mM
phenylmethylsulfonylfluoride, and 2 fig/m\ leupeptin, pH 7.5),
sonicated for 20 sec, heat treated at 80 C for 10 min, and
subsequently chilled for 30 min on ice. Denatured proteins
were pelleted by centrifugation (14,000 x g; 5 min), and the
heat-stable supernate, which included parvalbumin, was retained.
Western Blot Analysis
Proteins were resolved by SDS-PAGE, as described by Laemmli (24) and electrophretically transferred to nitrocellulose by
the method of Towbin ef al. (25). Nonspecific protein-binding
sites were blocked by incubation in blocking buffer [10 mM
Tris-HCI, 150 mM NaCI, and 5% nonfat dry milk (Carnation),
pH 7.3] for 1-2 h at room temperature with agitation. The filter
was incubated in blocking buffer containing 1 ngfm\ affinitypurified rabbit antirat parvalbumin for 2 h at room temperature.
Unbound antibody was removed by repeated washing in 10
mM Tris-HCI, 150 mM NaCI, and 0.05% Tween-20, pH 7.3.
The filter was then incubated in blocking buffer containing 1 x
106 cpm [125l]protein-A (ICN, Irvine, CA) for 1 h at room
temperature, and unbound radioactivity was subsequently removed by repeated washing as described above. The filter
was air dried and exposed to Kodak XAR-5 film overnight at
- 7 0 C.
For dot-blot quantitation of parvalbumin, heat-stable supernate from cells was applied to nitrocellulose using a dot-blot
MOL ENDO-1989
596
apparatus (Schleicher and Schuell, Keene, NH) according to
the manufacturer's instructions. Immunological detection of
parvalbumin was performed exactly as described for Western
transfers.
Parvalbumin Immunofluorescence
For indirect immunofluorescence localization of parvalbumin,
the procedure of Welsh et al. (26) was used with the following
modifications. Coverslips were incubated with primary antibody (1 M9/ml; affinity-purified rabbit antirat parvalbumin) for
60 min at 37 C. The secondary antibody was rhodamineconjugated goat antirabbit immunoglobulin G (Cappel, Cochranville, PA) at a dilution of 1:50. The coverslips were viewed
at x400 using appropriate filters in a Zeiss Axiophot microscope equipped for epifluorescence (Zeiss, New York, NY).
Cell Cycle Analysis
The lengths of mitosis, d , S, and G2 were determined as
described previously (8). Synchronous samples of mitotic cells
were obtained by mitotic shake (10). Mitotic cells were selectively shaken from a monolayer of exponentially growing cells
and retained in mitosis by placement in medium at 4 C. Cells
were released into the cell cycle by replating into 60-mm
culture dishes containing warm culture medium. Cell cycle
progression was monitored by incorporation of [3H]thymidine
(1.9 Ci/mmol; ICN) into trichloroacetic acid-precipitable DNA
as previously described (11). The length of G, was calculated
as described previously (8).
Acknowledgments
The authors wish to thank Elizabeth MacDougall and Charles
Mena for their excellent technical assistance, Dr. Paul Epstein
for providing a pUC-18 plasmid containing the parvalbumin
cDNA, Dr. Martin Berchtold (University of Zurich) for the antiparvalbumin antisera, Debbie Delmore and David Scarff for
the illustrations, and Lisa Gamble for preparing the manuscript.
Received November 7, 1988. Revision received December
7,1988. Accepted December 7,1988.
Address requests for reprints to: Dr. Colin D. Rasmussen,
Department of Cell Biology, Baylor College of Medicine, 1
Baylor Plaza, Houston, Texas 77030.
This work was supported by Grant BC-326 from the American Cancer Society.
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REFERENCES
23.
1. Carafoli E 1987 Intracellular homeostasis. Annu Rev
Biochem 56:395-433
2. Hazelton B, Mitchell B, Tupper J 1979 Calcium, magnesium and growth control in the.WI-38 fibroblast cell. J Cell
Biol 83:487-498
3. Tupper JT, Kaufman J, Bodine PV 1980 Related effects
of calcium and serum on the Gi phase of the human Wl38 fibroblast. J Cell Physiol 104:97-103
4. Steinhardt RA, Alderton J 1988 Intracellular free calcium
rise triggers nuclear envelope breakdown in sea urchin
embryo. Nature (Lond) 332:364-366
5. Twigg J, Rajnikant P, Whitaker M 1988 Translational
control of InsPs-induced chromatin condensation during
24.
25.
26.
the early cell cycles sea urchin embryos. Nature (Lond)
332:366-369
Epstein P, Means AR, Berchtold MW 1986 Isolation of a
rat parvalbumin gene and full length cDNA. J Biol Chem
261:5886-5891
Heizmann CW 1984 Parvalbumin, an intracellular calciumbinding protein: distribution, properties and possible roles
in mammalian cells. Experientia (Basel) 40:910-919
Rasmussen CD, Means AR Calmodulin is required or
progression through Gi and mitosis. EMBO J, in press
Rasmussen CD, Means AR 1987 Calmodulin is involved
in regulation of cell proliferation. EMBO J 6:3961-3968
Zetterberg A, Larsson O 1985 Kinetic analysis of regulatory events in Gi leading to proliferation or quiescence of
Swiss 3T3 cells. Proc Natl Acad Sci USA 82:5365-5369
Terasima T, Tolmach LT1961 Changes in x-ray sensitivity
of HeLa cells during the division cycle. Nature (Lond)
190:1210
Chafouleas JG, Bolton WE, Hidaka H, Boyd III AE, Means
AR 1982 Calmodulin and the cell cycle: involvement in
•regulation of cell-cycle progression. Cell 28:41-50
Kretsinger RH 1980 Structure and evolution of calciummediated proteins. CRC Crit Rev Biochem 8:119-174
Praeger FC, Cristofalo VJ 1986 Modulation of WI-38 cell
proliferation by elevated levels of CaCI2. J Cell Physiol
129:27-35
Eilam Y, Chernichovsky D 1988 Low concentrations of
trifluoperazine arrest the cell division cycle of Saccharomyces cerevisiae at two specific stages. J Gen Microbiol
134:1063-1069
Sasaki Y, Hidaka H 1982 Calmodulin and cell proliferation.
Biochem Biophys Res Commun 104:451 -456
Poenie.M, Aldertson J, Tsien RY, Steinhardt RA 1985
Changes in free calcium levels with stages of the cell
division cycle. Nature (Lond) 315:147-149
Keith CH, Ratan R, Maxfield FR, Bajer A, Shelanski ML
1985 Local cytoplasmic calcium gradients in living mitotic
cells. Nature (Lond) 316:848-850
Ratan RR, Shelanski ML, Maxfield FR 1986 Transition
from metaphase to anaphase is accompanied by local
changes in cytoplasmic free calcium in PtK2 kidney epithelial cells. Proc Natl Acad Sci USA 83:5136-5140
Lowy DR, Rand E, Scolnik EM 1978 Helper independent
transformation by unintegrated Harvey sarcoma virus
DNA J Virol 26:291-298
Rasmussen CD, Simmen RCM, MacDougall EA, Means
AR 1987 Methods for analyzing bovine papilloma virusbased calmodulin expression vectors. Methods Enzymol
139:642-654
Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning—A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Feinberg AP, Vogelstein B 1983 High specific activity
labelling of DNA restriction endonuclease fragments. Anal
Biochem 137:266-267
Chafouleas JG, Dedman JR, Munjaal RP, Means AR 1979
Calmodulin: development and application of a sensitive
radioimmunoassay. J Biol Chem 254:10262-10267
Laemmli UK 1970 Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature
(Lond) 227:680-688
Towbin H, Staehelin T, Gordon J 1979 Electrophoretic
transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl
Acad Sci USA 76:4350-4354
Welsh MJ, Dedman JR, Brinkley BR, Means AR 1978
Tubulin and calmodulin: effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus.
J Cell Biol 81:624-634