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[Cancer Research 60, 7114-7118, December 15, 2000]
© 2000 American Association for Cancer Research
Lower Cyclin H and Cyclin-dependent Kinase-activating Kinase Activity in Cell Cycle Arrest Induced by Lack of Adhesion to Substratum1
Leobaldo Solorzano2,
Mary Strasberg Rieber2 and
Manuel Rieber3
IVIC, Centre of Microbiology and Cell Biology, Caracas 1020 A, Venezuela
 |
ABSTRACT
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Knowledge
about adhesion checkpoints is important to counteract dissemination of
cells from solid tumors. Lack of anchorage in adherent cells is
associated with growth arrest and inhibition of cyclin-dependent
kinases (cdks) required to drive cell cycle progression. Because
cyclin-cdk complex activation requires CDK-activating kinase
comprising cdk7 and cyclin H, we now investigated their relationship to
decreased proliferation by lack of cell spreading. This report shows
that either UV irradiation on an adhesive substrate or culture on a
nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition
of proliferation by UV primarily induced the cdk inhibitor p21WAF1
without a significant effect on cyclin H and cdk7. In contrast, lack of
adhesion to substratum decreased cyclin H but not cdk7 with
accumulation of a slower migrating, presumably unphosphorylated cdk4
isoform. These results were paralleled by decreased cdk7-mediated
phosphorylation of GST-cdk2 and lower activation of a
baculovirus-derived cdc2-cyclin B kinase complex. This is the first
report showing that cyclin H-mediated down-regulation of cdk-activating
kinase activity is involved in growth arrest induced by lack of
anchorage.
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INTRODUCTION
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Although both primary and immortalized adherent cells undergo
growth arrest in suspension, the nature of this arrest is markedly
different. Primary cells exhibit growth inhibition involving rapid
cessation of DNA synthesis. In contrast, immortalized nontumorigenic
cells show growth arrest as measured by thymidine incorporation, but
cells that have entered S-phase continue into
G2-M and accumulate as a 4N population
(1)
. Many cell type-specific differences have been
disclosed between not only normal and transformed cells but also
between adherent cell lines, the proliferation of which is strictly
anchorage dependent. These apparent discrepancies are seen in
established cell lines, most probably because of adaptive events that
have occurred during in vitro culture (2)
.
Among the molecular mediators of growth arrest by lack of anchorage,
cyclin A, which associates with cdc2 and
cdk24
to drive cell
cycle progression into S-phase and mitosis, is not expressed in
suspended primary keratinocytes (2)
. A similar cyclin A
alteration is found in unanchored NIH3T3 cells, but overexpression of
cyclin D1 restores expression of cyclin A in these cells and rescues
them from cell cycle arrest (3)
. Also, the cyclin
E-CDK2 complex, which is required for the
G1-S transition of the cell cycle, is activated
in the late G1 phase in attached human
fibroblasts but not in fibroblasts maintained in suspension, except in
transformed fibroblasts in which the complex is active regardless of
attachment. The lack of cyclin E-CDK2 activity in suspended normal
cells was reported to result from increased expression of the p21Cip1
(p21) and p27Kip1 (p27) CDK2 inhibitors (4)
. Also,
cytoplasmic sequestration of these cdk inhibitors away from the nucleus
by cytoplasmic cyclin-cdk complexes is also seen in anchorage
independent cells (5)
. Others have also shown that
nonadherent cells fail to phosphorylate the retinoblastoma protein
(Rb), but enforced expression of cyclin D1 rescues Rb phosphorylation
and entry into S-phase when G1 cells are cultured
in the absence of substratum. Nonadherent cells also fail to activate
the cyclin E-associated kinase, and this effect can be linked to an
increased association of the cdk inhibitors, p21 and p27
(6)
.
Additionally, up-regulation of p27 Kip1 has been reported to correlate
inversely with anchorage-independent growth of human cancer cell lines
(7)
. Another effect associated with anchorage-independent
inhibition of growth is a concomitant decrease in phosphorylation of
CDK2 on threonine 160 (4)
. Also, Evi-transformed
fibroblasts are anchorage independent, have an abbreviated
G1 phase of the cell cycle, and a reduced
requirement for serum mitogens for S-phase entry. Evi-mediated
transformation is paralleled by elevated cyclin A-cdk2 activity,
associated with a combination of increased accumulation and
stabilization of cyclin A bound to a faster-migrating species of cdk2
believed to be the active threonine 160-phosphorylated form
(7)
. This phosphorylation of cdk2 and cdk4 is dependent on
CAKs, the activity of which depends on the association of cdk7 and
cyclin H (8)
. In human diploid fibroblasts, phorbol esters
added within 5 h of serum stimulation inhibited
G1-S cell cycle progression and cyclin A-cdk2
activity indirectly by inhibiting message levels for both cdk7 and
cyclin H (8)
.
More recently, a TGF-ß1-mediated G1 arrest of
HepG2 cells was reported to involve down-regulation of a
Mr 45,000 CAK activity, known to
activate Cdk2 also via Thr-160 phosphorylation
(10)
. Therefore, we now investigated whether growth
inhibition by lack of anchorage in susceptible cells also interferes
with cdk activity by modulating CAK activation (9
, 10)
.
This was now investigated not only by measuring substrate-dependent
changes in CAK kinase activity (9
, 10)
but also by
determining differential protein expression in cyclin H or cdk7 induced
by lack of anchorage in K1735 melanoma cells.
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MATERIALS AND METHODS
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Cell Culture.
K1735 nonmetastatic melanoma cells were cultured in DMEM supplemented
with 10% serum (11)
. For studies involving growth arrest
by lack of anchorage, cells were seeded on polystyrene bacterial plates
(V W R # 25384) coated with 10 mg/ml BSA in PBS (pH 7.2) to
prevent cell attachment to substratum (12)
.
Total Cell Lysates.
Cells were collected by scraping with a rubber policeman after washing
in ice-cold PBS (pH 7.2) containing 0.1 mM sodium vanadate
and 1 mM phenylmethylsulfonyl fluoride. Cells were kept at
-70°C until lysis, which was carried out in 50 mM
Tris-HCl (pH 7.4), 120 mM NaCl, 2 mM EDTA, 50
mM NaF, 0.1 mM
Na3VO4, 0.5 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 5 µg/ml
leupeptin, 10 µg/ml soybean trypsin inhibitor, 1 µg/ml
N-tosyl-L-phenylalanine chloromethyl
ketone, and 0.4% NP40.
Immunoblotting.
Total cell lysates (100 µg) were run on 10–12% SDS-polyacrylamide
gels concurrently with prestained molecular weight markers (Amersham)
and were transferred passively to parallel replicate nitrocellulose
membranes (11)
. Blotted membranes were blocked with 5%
skimmed milk in Tris-buffered saline for 2 h at room temperature
and then incubated for 1 h in the same blocking solution
containing the specific antibodies. The antibodies used for specific
immune blotting of cyclin D3 (SC 182), p21WAF1 (SC-397), cyclin H (SC
609), and cdk7 (SC 529) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Immune detection of cdk2, cdk4, and cyclin D1 was
achieved with specific antibodies provided by Dr. M. Pagano (Mitotix,
Boston, MA and New York University, New York, NY). This was followed by
exhaustive washing in Tris-buffered saline containing 0.1% Tween 20,
subsequent incubation with protein A-peroxidase (Amersham) at 1:2000,
and development by ECL chemiluminescence (Amersham) and
autoradiography. Whenever indicated, the nitrocellulose membranes were
stripped at 70°C for 30 min in 62 mM Tris (pH 6.8), 2%
SDS, and 100 mM ß-mercaptoethanol, followed by extensive
washing with Tris-buffered saline plus 0.2% Tween 20. Stripped blots
were blocked again and reacted with new antibodies and further reaction
with peroxidase conjugates and detection by ECL (Amersham). Specific
molecular weights for immune blotted bands were established by
comparison with coelectrophoresed Rainbow colored markers (Amersham).
CAK Activities.
CAK activities were assayed either by directly measuring the
phosphorylation of human GST-cdk2 (SC-4069; Santa Cruz Biotechnology)
by immune precipitates of cdk7 (SC 529; Santa Cruz Biotechnology) on
protein A-beads containing 50 mM Tris-HCl (pH 7.5), 10
mM MgCl2, 5 µCi of
[
-32P]ATP in 10 µM ATP, and 1
mM DTT or indirectly by measuring the ability of equal
protein concentration of cell extracts to activate human recombinant
cdc2 kinase (Promega V-2891) purified from insect cells infected with a
baculovirus vector carrying the genes for human p34cdc2 and cyclin B.
The latter assays measured the ability of cell extracts to activate the
phosphorylation of histone H1 (10 µg) by 5 units of the
baculovirus-derived cdc2-cyclin B complex in kinase buffer containing
50 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, 5 mM
MnCl2, 5 µCi
[-32P]ATP in 10 µM ATP, and 1
mM DTT.
Histone H1 and Rb Kinase Assays.
Histone H1 kinase assays after specific immune precipitation with
protein A/G agarose beads and either rabbit antibody to cyclin D3 (SC
182) or mouse antibody to cyclin D1 (SC-8396) were carried out as
described previously (13)
. For Rb kinase assays, histone
H1 was substituted by 1 µg of pRb (SC-4112; Santa Cruz
Biotechnology), and similar conditions were used for phosphorylation
(13)
.
Densitometric Analysis.
After chemiluminescence, data from Western blots were acquired with a
Hewlett-Packard Scan Jet IV scanner for quantitation of specific bands
with the Gel-Pro Analyzer software (Media Cybernetics, Silver Spring,
MD).
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RESULTS
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Unequal Regulation of p21WAF1 and D Cyclins in Growth Arrest
Induced by UV Radiation or Lack of Anchorage.
Preliminary experiments showed that culturing K1735 cells on BSA-coated
nonadhesive plates (12)
prevented cell spreading and
promoted cell aggregation, which mostly continued after 5 h of
transferring aggregated cells to an adhesive tissue culture plate.
Complete spreading occurred when these K1735 cells were directly
cultured on tissue culture plates, even after exposing cells to 10
J/m2
of UV radiation (Fig. 1
, upper panel). However,
UV-irradiated adherent cells and cell aggregates showed a similar
degree of growth arrest 2 days after each of these treatments (Fig. 1)
.
A comparison of these unequal growth arresting effects on expression of
the p21WAF1 cyclin-dependent kinase inhibitor and D cyclins revealed
that UV increased p21WAF1 levels by 3-fold, in agreement with results
showing that these cells harbor a wild-type p53 (11)
and
in agreement with the DNA-damaging effect of UV, which activates
p21WAF1 (14)
. No comparable induction occurred in cells
arrested by lack of anchorage, which revealed instead a preferential
increase in cyclin D3, which persisted even after a 5-h subculturing of
the cell aggregates on tissue culture plates. In contrast, no
comparable elevation was observed in cyclin D1, which decreased more
rapidly after transferring cell aggregates to tissue culture plates
(Fig. 2)
.
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Fig. 1. UV radiation or lack of anchorage inhibits K1735 cell
proliferation. Replicate cultures were seeded on tissue culture or
BSA-coated dishes for determination of relative cell numbers and
morphology after 1 or 2 days of culture. Left inset,
unanchored aggregates partly start reattaching within 5 h after
subculture on tissue culture plates.
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Fig. 2. Unequal regulation of p21Waf1 and D cyclins by UV
radiation or lack of anchorage inhibits K1735 cell proliferation.
Changes in D cyclins or p21WAF1 were analyzed by specific immune
blotting for digital imaging and quantitation. Note induction of
p21WAF1 by UV, a transient increase in cyclin D1 more reversible than
that of cyclin D3 after 5 h of readherence to tissue culture
plates.
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Decrease in the Fast-migrating Cdk4 Isoform in Growth Arrest
Induced by Lack of Anchorage.
In mammalian cells, two types of cdk2 isoforms may be detected, because
these enzymes occur as inactive unphosphorylated or as faster-migrating
forms when activated by CAK phosphorylation (9
, 10)
.
Hence, we investigated whether growth arrest by either lack of
anchorage or UV irradiation (Fig. 1)
induced changes in cdk2 or cdk4
isoforms. Bidirectional immune blot (11)
revealed an
altered electrophoretic migration in cdk4 isoforms only with lack of
anchorage. Cell aggregates showed most of its cdk4 as a
slower-migrating band, in contrast to a faster-migrating cdk4 detected
in attached proliferating or UV-irradiated cells. On the other hand,
comparable levels of each cdk4 isoform were detectable within 5 h
of readherence of aggregates to tissue culture plates, implying a
correlation of lower mobility cdk4 and adhesion. However, no comparable
isoform changes were evident in cdk2, which rather showed a
quantitative increase with lack of adhesion (Fig. 3)
.
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Fig. 3. Reversible decrease in fast-migrating cdk4 with lack of
anchorage and readherence. Replicate blots obtained from bidirectional
transfer were assayed for changes in cdk2 and cdk4. Note only lower
cdk4-P in adherent cells, only upper cdk4 in unanchored cells, and
comparable levels of both upper and lower cdk4 isoforms by 5 h of
reattachment of aggregates to tissue culture plates.
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Cyclin H Is Preferentially Decreased in Unanchored Cell
Aggregates.
Because the cdk4 results suggested that lack of anchorage interfered
with cdk4 phosphorylation, which involves the cdk7-cyclin H complex
(10)
, we investigated whether this effect was associated
with alterations in any of these components. Whereas cdk7 levels were
essentially similar, cyclin H was unaffected by UV irradiation but
decreased preferentially in unanchored cell aggregates and partly
recovered within 5 h of reseeding cell aggregates on tissue
culture plates (Fig. 4)
.
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Fig. 4. Lack of anchorage is associated with a decrease in cyclin
H. Replicate blots obtained from bidirectional transfer were assayed
for changes in cdk7 and cyclin H. Note decline in cyclin H without a
comparable change in cdk7, with lack of adherence and its partial
increase by 5 h of reattachment of aggregates to tissue culture
plates. Results were normalized for protein loadings in each lane.
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Unequal CAK and Rb Kinase Activities in Adherent and Unanchored
Cells.
Because growth arrest by lack of anchorage (Fig. 1)
preferentially
correlated with a decline in cyclin H (Fig. 4)
and cyclin H is known to
activate the CAK activity in cdk7 (9
, 10)
, we investigated
whether Rb kinase associated with cyclin D1 or cyclin D3 and CAK
activity differed in adherent and nonadherent cells. For Rb kinase
studies, we measured Rb phosphorylation catalyzed by specific immune
precipitation of cyclin D1 or cyclin D3, followed by autoradiography
(13)
. This revealed a much lower cyclin D1-dependent Rb
phosphorylation in nonadherent cells but a comparable cyclin
D3-dependent Rb kinase in both adherent and nonadherent cells (Fig. 5
, left). A clearer
correlation of the cyclin H decline with growth arrest induced by lack
of anchorage was observed in CAK activity assays, which showed a
decreased cdk7-dependent phosphorylation of GST-cdk2 in nonadherent
cells, which also showed a decreased ability to activate a baculovirus
derived cdc2-cyclin B complex.
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Fig. 5. Lack of anchorage is associated with a decrease in CAK
activity and a decline in cyclin D1-dependent Rb kinase.
Left, the Rb kinase assay with replicate protein
concentrations (125 µg) from adherent and nonadherent cells were
either immune-precipitated with protein A/G agarose beads coupled to
either antibody to cyclin D1 or antibody to cyclin D3 to assay Rb
kinase. Note the decline in cyclin D1-dependent without a comparable
change in cyclin D3-dependent Rb kinase with lack of adherence.
Upper right, direct phosphorylation of GST-cdk2 by cdk7
immune precipitates from 200 µg of protein concentrations from
adherent and nonadherent cells. Lower right, indirect
CAK activation of baculovirus-derived cdc2-cyclin B complex using 125
µg of total protein from adherent and nonadherent cells to
phosphorylate histone H1. Note the decline in histone H1
phosphorylation with lack of adherence at equal levels of
baculovirus-derived cdc2-cyclin B complex.
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DISCUSSION
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Prior studies have demonstrated down-regulation of G1 cyclins such
as cyclin D1 (6)
, cyclin E (4, 5, 6)
, and
cyclin A (1, 2, 3)
in growth arrest induced by lack of
anchorage in some cell types. The mechanism for this adhesion-dependent
regulation seems to differ in normal and immortalized cells
(1)
and may involve the cytoplasmic displacement of p21
and p27 cdk inhibitors in anchorage-independent cells (6)
,
which emphasizes the role of cdk inhibitors and G1 cyclins in mediating
anchorage-regulated growth arrest (5
, 7)
. To define novel
changes in cell cycle-controlling proteins specifically associated with
lack of adhesion, we searched for differential alterations induced by
either UV-mediated growth arrest in adherent cells or those occurring
without UV in unanchored cell aggregates cultured on a nonadhesive
substrate (12)
. UV-mediated growth arrest clearly
correlated with induction of the cyclin-dependent inhibitor p21WAF1
(11
, 14)
in K1735 cells, which harbor a wild-type p53
(11)
, with no such effect apparent with inhibition by lack
of anchorage.
Because cell cycle progression not only requires induction of G1
cyclins (1, 2, 3, 4, 5, 6)
but also requires CAK mediated by cdk7 and
cyclin H (9
, 10)
, we investigated whether any one of these
components was involved in adhesion-dependent growth arrest.
Previously, others demonstrated that inhibition of cell cycle
progression with phorbol esters (9)
or TGF-ß1
(10)
decreases CAK activity, but these reports did
not implicate preferential changes in cyclin H. Moreover, G1-arrested,
TGF-ß1-treated HepG2 carcinoma cells showing lower CAK activity
retained continued cyclin D:cdk4/6 activity but failed to increase
p21WAF1 (10)
. We now found that the cdk7 activator, cyclin
H, is preferentially decreased when growth arrest is induced in K1735
by lack of anchorage but not by UV radiation, demonstrating partial
recovery in cyclin H within 5 h of reseeding unanchored cell
aggregates on tissue culture plates. Cyclin H decline in nonadherent
cells correlated with decreased CAK activity measured by decreased
histone H1 phosphorylation of a baculovirus-derived cdc2-cyclin B
complex and lower GST-cdk2 phosphorylation, data compatible with
induction of a slow-migrating cdk4, believed to be the unphosphorylated
cdk isoform (8
, 10)
by lack of anchorage. Growth-arrested,
unanchored aggregates also showed a preferential decrease in cyclin
D1-dependent but not in cyclin D3-dependent Rb kinase activity and
higher cyclin D3 levels. This may be attributable to a possible role
for cyclin D3 in growth arrest induced by lack of anchorage, because a
cyclin D3 kinase but not a cyclin D1-associated kinase remains active
in nonadherent keratinocytes (15)
. Also, cyclin D3 was
implicated recently as a critical contributor to the irreversible exit
of differentiating myoblasts from the cell cycle (16)
.
To our knowledge, this is the first report describing cyclin
H-associated decrease in CAK activity with growth arrest induced by
lack of anchorage. Recently, others reported that IFN-
-induced
growth arrest involves higher levels of the p21WAF1 cdk inhibitor and
lower CAK activity, but lower cyclin H is not involved because CAK is
restored after immune depletion of the cdk inhibitor (17)
.
These results showing this novel involvement of cyclin H in
adhesion-dependent checkpoints should be valuable in developing
antisense strategies against cyclin H in cells from solid tumors, which
can become anchorage independent in secondary growth and metastasis.
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FOOTNOTES
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Research supported by Conicit Grant G-97000613.
L. S. is a trainee graduate student at the Centre of Advanced Studies,
IVIC. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at IVIC, Centre of Microbiology and Cell Biology,
Apartado 21827, Caracas 1020 A, Venezuela. Fax: 582-504-1382; E-mail: mrieber{at}pasteur.ivic.ve
4 The abbreviations used are: cdk,
cyclin-dependent kinase; cdc, p34 cell cycle-dependent kinase;
CAK, cdk-activating kinase; TGF, transforming growth factor; Rb,
retinoblastoma.
Received 2/18/00.
Accepted 10/13/00.
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ABSTRACT
INTRODUCTION
