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Suppression of oncogenic transformation by hypothemycin associated with accelerated cyclin D1 degradation through ubiquitin-proteasome pathway
Life Sciences, Vol. 65, No. 4, pp. 381394 1999 Copyight 0 1999 Ekevia Sciahx Inc. k’t’intedin the USA. All rightaroLlcrved 0024-3205/99/S-see fiwt matter pII
ELSEVIER
SUPPRESSION ASSOCIATED
SoO24-3205(99)00259-3
OF ONCOGENIC
TRANSFORMATION
WITH ACCELERATED
BY HYPOTHEMYCIN
CYCLIN Dl DEGRADATION
UBIQUITIN-PROTEASOME
THROUGH
PATHWAY
Hikaru Sonoda*, Kazuo Omi*, Kanji Hojo, Kazuyo Nishida, Satoshi bmurap Shionogi Research Laboratories,
and Kenji Sugita
Shionogi & Co., Ltd., Osaka 553-0002, Japan
§The Kitasato Institute, Tokyo 108-8641, Japan
(Received in final form March 3 1, 1999)
Summary Hypothemycin
was originally
trichothecoides.
isolated as an antifungal
Here we report that treatment
cells (DT cells) with hypotbemycin Dl
protein
with concomitant
was
carried
2 (ubc2)
are the targets
ubiquitin-conjugation specific
inhibitor
accumulation
prolongation
is indicated growth
of hypothemycin
process. for
phenotype,
and reverted
morphology
Furthermore,
of Gl
phase in their cell cycle. rhp6+
or its downstream in the presence greatly
(S.
(homologue
ubiquitin-conjugating
hypothemycin
proteasome,
that hypothemycin
NIH3T3
mutant of Schizosuccaromycespombe
RAD6) and mammalian
of multi-ubiquitinated
terms of malignant
on v-K-ras-transformed
out to show that S. pombe
Succuromyces cerevisiue
of Hvpomyces
caused drastic decrease in amount of cyclin
Analysis using hypothemycin-resistant pombe)
metabolite
of
enzyme
molecules
in
of lactacystin, enhanced
a the
form of cyclin Dl in DT cells. Therefore, it
facilitates ubiquitinating hypothemycin
the morphology
still remained transformed
process of cyclin Dl. In
inhibited
of DT cells. in the additional
anchorage-independent On the contrary,
their
presence of lactacystin.
Our results suggest that cyclin Dl is a key molecule working downstream in rassignaling
and that the transformation
can activate ubiquitin-proteasome
can be inhibited
pathway including
by the compound
which
degradation of cyclin Dl
Key Words hypothemycin, cyclin Dl, DT cells, ubiquitin-proteasomepathway, lactacystin
Correspondence
should be addressed to H. Sonoda, Shionogi Research Laboratories,
& Co., Ltd., Sagisu S-12-4, Fukushima-ku, lFax]81-6-6458-0987,
Shionogi
Osaka 553-0002, Japan, [Tel]81-6-6458-5861,
[Email][email protected]
*First and second authors equally contributed
to this work.
382
Hypothemycinand Cyclin D 1
Vol. 65, No. 4, 1999
Cyclin Dl plays a central role in Gl progression in mammalian cell cycle and its expression is stimulated by upstream mitogenic signals (1). Following these signals, newly expressed cyclin Dl in Gl phase assembles with CDK4 to form active kinase complex and the
amount of the complex is titrated by cyclin-dependent kinase inhibitors such as ~16~~, p21wafl and p27%P1 (2). The resulting redundant kinase complex can be rate-limiting for driving cells into S phase. Therefore, such tight control of the amount of cyclin Dl should be important in order to precisely regulate the progression of cell cycle and to prevent cells from tumorigenesis. In fact, the chromosomal rearrangement, translocation or amplification which cause deregulated expression of cyclin Dl are thought to promote tumorigenesis in various human cancers (3). Moreover, in murine tibroblasts transformed by oncogenes such as ras, enhancement of Gl progression was observed indicating that some of the transforming signaling from oncogenes affect cell cycle in Gl phase (4). The accelerated Gl-S transition can be considered as one of the malignant phenotypes in these cells which include morphological change and ability to grow anchorage-independently. Hypothemycin have been known as product of Hypomvces trichothecoides having antifungal activity (S), and also been briefly mentioned to have cytotoxic activity on some tumor cell lines (6). In this report, we describe mechanistic analysis on anti-transforming activity of hypothemycin, that is, prolonged Gl phase associated with cyclin Dl downregulation, identification of ubc2 cDNA as a multicopy suppressor for hypothemycin-resistant S. pombe mutant, and cancellation by proteasome inhibitor of hypothemycin induced cyclin Dl degradation and transformation.
Methods CeN culture: All cell lines used in this work were maintained in Dulbecco’s modified Eagle medium (DMEM) containing fetal calf serum (FCS) (10%). In the case of synchronization of cell cycle, cells were cultivated in DMEM without FCS in order to arrest them in Gl phase. Colony forming assay: Detail procedure for this assay have been previously described (7). Briefly, cells were pretreated with 0.02, 0.1, 0.5, 1 pg/ml hypoihemycin in DMEM containing FCS (10%) for 24 hours. Each culture were divided in two, one was to test for the colony formation in liquid medium, another was to test in soft agar medium containing additional agarose (0.4%) to liquid medium. Same concentrations of hypothemycin as the pretreatment was added in both culture conditions. After 8 days of culture, the number of colonies in liquid and soft agar medium were counted by crystal violet staining and microscopic examination, respectively. The efficiency of colony formation was calculated as proportion of the colony number appeared in the medium from which the drug had been withdrawn. Cell cycle analysis: Cells to be tested were harvested by trypsinization and collected. Dissolution of cytoplasm, stabilization of nuclei and staining of DNA was carried out using
Vol. 65, No. 4, 1999
CycleTEST
Hypothemycin and Cyclin Dl
kit (Becton Dickinson).
Becton Dickinson
383
Stained DNA was analyzed
FACscan according to the manufacture’s
by flow cytometry
Western blot analysis: Cells were harvested with cell scraper followed by washing phosphate-buffered
saline and collected, then Iysed with RIPA buffer consisting
HCl pH7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton pepstatin,
X-100 1 unit/ml
supplemented aprotinin
with
0.5 mM
phenylmethylsulfonyl
obtained
polyacrylamide
gel (14%), transferred onto nitrocellrose
antibody
Santa Cruz Biotechnology,
or anti-cyclin
immunoprecipitation
E polyclonal experiment,
the suspended
anti-cyclin
Dl monoclonal
at 4°C and centritiged
was washed four times with RIPA buffer and then suspended
sample buffer followed by boiling for 5 minutes. Polyclonal in Western ubiquitin
blotting (kind
were anti-cyclin
gift
from
monoclonal Inc.). For the antibody
Inc.) was added in 1 ml RIPA buffer containing
cell lysate. The mixture was gently rotated overnight precipitate
antibodies
Reacted
bands
were
(sc-
100 mg of
by 8000g. The
in 20 ml SDS-PAGE used for these samples
Dl (~~-753, Santa Cruz Biotechnology,
S. Ohmi).
40 pg/ml
and probed with anti-cyclin
D2 (Ab-1, Calbiochem)
(06-459, Upstate Biotechnology
agarose-conjugated
246AC, Santa Cruz Biotechnology,
fluoride,
cells by 8000g was loaded on SDS
membrane
Inc.), anti-cyclin antibody
of 10 mM Tris-
In each case, equal amount of protein in
the supematant D1 (~0246,
once with
0.1% sodium dodecyl sulfate (SDS) and
and 10 pg/ml leupeptin.
after centritiging
with a
procedure.
detected
Inc.) and anti-
using
ECL
system
(Amersham).
Cloning of multicopy suppressor cDNA for S. pombe Is mutant: The mutant ( h- rhp6d134K leul-32)
cells were transfected with a S.pombe cDNA library (8), and incubated on MMA plates
first at 26’C for 24 h and then at 36’C for 4-5 days. Colonies formed were isolated and then examined
by the
instability
revertants.
Complementing
test to distinguish
authentic
transformants
from
phenotypic
plasmids were recovered in Escherichia coli. The recovered cDNA
clones were tested for their activity by re-transfection
into the mutant and the sequences
were
determined.
Results In the course of our screening hypothemycin,
condition,
(7, 9, 10, 1 l), we found that
trichothecoi&
having antifungal
DT cells (12) in Gl phase in their cell cycle at the concentration
to the similar extent as in case of serum deprivation
little influence
(Fig.
1A). In the same
on the cell cycle of parental NIH3T3 cells was observed (Fig. 1B). In
order to know the mechanism expression
compound
which was known as product of Hrpomvces
activity, arrested asynchronous of 0.5 pg/ml
of anti-tumor
of this ras-specific
inhibition,
the effect of hypothemycin
of Gl cyclin in DT cells was examined. DT cells were synchronized
serum starvation for 24 hours, and then released from arrest by addition presence or absence of 0.5 pg/ml hypothemycin.
on the
in Gl phase by
of 10% serum in the
Western blot analysis using their extract and
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin D 1
384
A
LIT cells Serum Hypothemycin
Gl S G2lM
i
+
-I
_
71 .O% 28.1 0.9
44.9% 47.5 7.6
73.9% 21.4 4.7
B NIH3T3 cells _
Serum
i
+
Hypothemycin
Gl
S G2lM
+
47.7% 34.1 18.2
88.2% 5.4 6.4
47.9% 35.0 17.1
C No drug hours
0
1
2
4
+ Hypothemycin 8 24
0
1
2
4
8 24 +
cyclin Dl
+
cyclin E
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin Dl
D
No drug
385
+ Hypothemycin
60 v) ?j 40 8
i’ “O--O..
20
20
0
5
10
.....
(j
....
.............
‘0
0
10 hours
5
Fig. 1 Hypothemycin transformed
inhibits the cyclin Dl expression and blocks Gl-S progression cells. DT cells
(A) and NM3T3
Dulbecco’s modified Eagle medium containing asynchronous hypothemycin, cytometry.
manner
@) were
in
cultured
10% fetal calf serum (KS)
in
in an
for 24 hours with (right) or without (center) 0.5 t.&/ml
then harvested
and their DNA content
As a control of Gl-arrest,
hours is shown (left). Percentages graphs.
cells
(C) Gl-arrested
was analyzed
by flow
data from cells starved for serum for 24
of cells in each phase are shown under the
DT cells by serum deprivation
for 24 hours
released by addition of 10% FCS with or without hypothemycin,
were
then cultivated
for 1, 2,4 or 8 hours and whole protein was extracted. Western blots using these extracts and anti cyclin Dl antibody and cyclin E were carried out. (D) DNA content from the same time course experiments cytometry,
as C was determined
by flow
and percentages of cells in Gl (Cl) and S (0) phases are shown, Data
were from duplicate experiments.
anti-cyclin
Dl monoclonal
antibody showed that the amount of cyclin Dl protein was greatly
reduced four to eight hours after serum stimulation
in the presence of hypothemycin,
no such reduction was observed in the absence of hypothemycin
whereas
(Fig. 1C). Little influence
on
the level of cyclin E, another Gl cyclin, was detected (Fig. 1C). At that time, DT cells were still kept in Gl phase in the presence of hypothemycin absence of hypothemycin
level of cyclin Dl and the deficiency Thinking of the significance the prolonged cyclin D 1.
although they could enter S phase in the
(Fig. 1D). Thus, the correlation
was observed between
in entering into S phase in hypothemycin-treated
of the amount of cyclin Dl in Gl progression
Gl phase by the addition
of hypothemycin
the reduced DT cells.
as mentioned
above,
is caused by the reduced amount of
Vol. 65, No. 4, 1999
Hypomemycin and Cyclin Dl
386
In order to examine what mechanism used hypothemycin-resistant
mutant
mediated the biological effects of hypothemycin,
of S. pombe
which was obtained
presence of 100 pg/ml of the drug. Wild type of S. pombe 2A). The mutant also displayed
temperature-sensitive
diploid, the mutant was recessive analysis demonstrated
could not grow in that condition
for both the drug-resistant
and the ts phenotypes.
(Fig.
Genetic
that both these phenotypes were caused by single chromosomal
to be rhp6+ which was previously
in the
(ts) growth (Fig. 2A). In heterozygous
(data not shown). cDNA clones which could complement identified
spontaneously
we
the ts phenotype
characterized
mutation
of this mutant were
as a homologue
of S. cerevisiae
FUD6 (13), a member of ubc2 family (14). Direct sequencing
of PCR fragment of rhp6+ gene
obtained
continuous
from the
corresponding
mutant’s
genome
revealed
that three
nucleotides
to Lysin 134 residue were deleted (data not shown). Therefore, it was indicated
that this single amino acid deletion in the rhp6+ gene product was responsible resistance and the ts growth of the mutant. Furthermore, but also HHR6A
expression of not only wild type rhp6+
as well as growth at non-permissive
These data strongly suggest that rhp6 protein of S. pombe or its downstream Destruction (for review,
for the drug-
or HHR6B (both encode human ubc2) cDNA in the mutant
restored hypothemycin-sensitivity
mammalian
(AAG)
molecules to which hypothemycin
rhp6dl34K
temperature
and mammalian
(Fig. 2B).
ubc2 are the targets
is directed at least in S. pombe.
of some cyclins are known to be regulated by ubiquitin-proteasome
see ref. 15). Like other Gl
cyclins
pathway
such as cln2, cln3 in S. cerevisiae
cyclin E, cyclin Dl has been reported to be degraded through ubiquitination
To examine the relationship
between the hypothemycin-induced
level in DT cells and the proteasome hypothemycin mammalian
and lactacystin, proteasome
degradation
(16).
of cyclin Dl
pathway, DT cells were treated with both
which is known to bind covalently to and specifically
inhibit the
subunit X/MB1 (17). Western blot using their extract showed that, as
34 kD band of cyclin Dl grew fainter, the band corresponding form appeared, whereas no such accumulation were treated
rapid reduction
and
with lactacystin
to a higher molecular
weight
of the shifted band was observed when DT cells
alone (Fig. 3A). In order to know whether the shifted band
represented
ubiquitinated
monoclonal
antibody from DT cells were subjected to Western blotting using both anti-ubiquitin
and anti-cyclin
form
Dl polyclonal
is known to be necessary
ubiquitination
semi-solid phenotypes
the immunoprecipitate
with
anti-cyclin
of the protein by protcasome
of high molecular
and lactacystin
strongly
Dl
of a protein
in which the protein is
weight form of cyclin Dl in the presence
suggests that hypothemycin
of
could accelerate the
of cyclin Dl.
When NIH3T3 morphology
Dl,
antibody (Fig. 3B). Since the multiple ubiquitination
for recognition
degraded, the acummulation both hypothemycin
of cyclin
cells are transformed
by various
oncogenes,
they often change their
from flat to spindle one and obtain the ability to grow anchorage-independently media. Suppression
of their malignancy
(18). The anchorage-independent
can be estimated by the inhibition
in
of these
growth of DT cells in soft agar medium in the
Vol. 65,No.4,1999
Hypothemycin and Cyclin D 1
B Hm:rhp6-b134K
pcMVL-X pcMVL-rhp6 pcMVL-HHR6A pchlVL-HHRGB
Spot tests for hypothemycin
and temperature
sensitivity.
(A) Hypothemycin-
resistant and ts phenotypes of the ,I?.pombe strain rhp6d134. 32) and mutant (h- rhp6d134K
leul-32)
temperature)
phase in liquid minimum
to late exponential
supplemented (minimum
cells were grown at 26% (permissive-
with leucine. Cells were suspended
by stepwise
10 fold dilutions. medium
hypothemycin,
agar)
plates
medium
(MML) (8)
at 1 x lo7 cells/ml, followed
10 ml of aliquots
+leucine
Wild type (h- leul-
with
were spotted onto MMA or without
and incubated at 26’C or 36% (non-permissive
100 pg/ml temperature
of ) for
4-5 days. (Educts are seen as dots in the drug containing
plates.) (B) Suppression
of hypothemycin-resistance
of the rhp6d134K
by S. pombe transformed
rhp6+ and human by
cytomegalovirus
and temperature-sensitivity
indicated
ubc2 cDNAs.
plasmids.
The rhp6d134K
pchPJL-X
(containing
promoter and the LEUZ gene) is an expression
cells
cells were human
vector with no
insert and used as a control (33). S. pombe rhp6+ cDNA and its two distinct human homologue cDNAs, HHR6A and HHRBB, were inserted in the same expression vector as the control. Spot tests were performed as in (A) except that minimum
medium was not supplemented
with leucine.
388
Hypothemycin and Cyclin D 1
A
Hypothemycin + Lactacystin
Lactacystin
hours
0
1 2
4
Vol. 65, No. 4, 1999
8 24
0
1 2
4
8 24
t-shifted
+
B
Blot
Dl
cyclin Dl
cyclin Dl
Ub
Hypothemycin + Lactacystin
12
34 Fig. 3
Accumulation
of ubiquitinated
hypothemycin
and lactacystin.
cyclin
Dl
Gl-arrested
in DT cells cultivated
DT cells by serum deprivation
hours were released by addition of 10% FCS with 5 p&I lactacystin both 0.5 pg/ml hypothemycin
and 5 pM lactacystin.
Western blotting probed by anti-cyclin (B) DT cell lysate prepared without
(lane
monoclonal (lane
by
with (lane 2, 4) or with anti-cyclin
blotting using either anti-cyclin
(lane 3, 4) polyclonal
cyclin Dl, IgH; cross-reacted
alone or with
antibody was carried out.
after 8 hours serum induction
followed by Western
1, 2) or anti-ubiquitin
ubiquitinated
D 1 monoclonal
for 24
Time course analysis
1, 3) both drugs was immunoprecipitated
antibody
with both
antibody.
mouse immunoglobulin
Ub-cyclin
Dl Dl Dl;
heavy chain.
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin D 1
389
presence of hypothemycin
was examined (Fig. 4A). In a dose-dependent
gave rise to a significant
difference
in the efficiency
soft agar medium. At a concentration specifically
. Moreover,
inhibited
constitutively
of 0.5 pg/ml, the anchorage-independent hypothemycin
within 24 hours. It also flattened
growth was most
the morphology
of NIH3T3
in DT cells
cells transformed
by v-raf or
‘activated mutant of MEK 1 (19), both of which are activated forms of consecutive
of transformed approximately not shown).
phenotype
compete
the
reversion
reversion
to lactacystin
with hypothemycin
of their
induced
depended
morphology.
by hypothemycin
in response
malignant
phenotype
The
effect
was examined
blocked the morphological
to the transforming on the transformants
of proteasome on
the
(Fig. 4D). Although
of lactacystin
the
addition of lactacystin
did not induce any change in
it is suggested
signal
of ubiquitination
reversion induced by hypothemycin.
of NIH3T3 cells with lactacystin
(data not shown). Therefore,
expressed
on the activation
of cyclin Dl, the inhibition
was different among the cell lines, simultaneous
completely
On the other hand, treatment their morphology
reversion
as seen in the degradation
with the IC,, value of
of cyclin Dl expression as well (data
0.3 I_IMwhich corresponds in the inhibition If this morphological
morphological
of ras (20) (Fig. 4B, C). The suppression
in these cell lines showed dose-dependency
pathway by hypothemycin
sensitivity
between liquid and
induced the normal morphology
members in MAP kinase cascade working downstream
might
manner, hypothemycin
of colony formation
that the molecule(s)
from these oncogenes
might be destroyed
which
and conferred
the
in the ubiquitin-proteasome
pathway.
Discussion In cells once stimulated by growth factors to proliferate, the expression is constant through the cell cycle (21).
The equilibrium
level of cyclin Dl
of active synthesis and destruction
of
cyclin Dl protein may be kept stable because the half life of cyclin Dl is very short (22). It is already reported that such rapid turnover of cyclin Dl depends on phosphorylation residue by unknown
one possible mechanism
of hypothemycin
Dl might be the activation including
in enhancement
of the polyubiquitination
mutant
of S. pombe, hypothemycin
process of cyclin Dl directly, or alternatively
kinase
phosphorylates
machinery.
cyclin
Dl
One
hypothemycin
reason
we can
speculate
proteins are generally is that
activation
might made it hard to detect intermediately
resulting in preferential that their degradation
detection of highly ubiquitinated
in proteasome was inhibited.
may facilitate the
that the activity of the unknown
(16) may be controlled
However, we do not know the reason why the ubiquitinated
as a single band whereas the ubiquitinated bands.
can act on a ubiquitination
ubc2. Therefore, it can be speculated that hypothemycin
ubiquitination which
of cyclin
of such kinase. On the other hand, as seen in our genetic experiment
using hypothemycin-resistant machinery
on threonine
kinase other than CDK4 which is known to bind to cyclin Dl (16). Thus,
under
the ubiquitinating
cyclin Dl was detected
appeared as smear or ladder
of multi-ubiquitinating ubiquitinated
step by
form of cyclin Dl
form of the protein in the condition
390
Vol. 65, No. 4, 1999
Hypothemycinand Cyclin D 1
00
50
z
,o 8 7
0
0:02
0.1
0.5
1
pg/ml hypothemycin
B No drug DT
raf I NIH3T3
MEK / NIH3T3
&pothemvcin DT
raf / NIH3T3
MEK I NIH3T3
Vol. 65, No. 4, 1999
l@pohmycin and Cyclin D 1
391
D Hvpothemvcin
+ Lactacvstin
raf / NIH3T3
Fig. 4 Hypothemycin NIH3T3
inhibits malignant
phenotypes
cells. (A) Comparison
in ras-, raf- and MEK-transformed
of suppressive
activity
of hypothemycin
for
colony formation
of DT cells in liquid and soft agar media. The effkiency
colony formation
either in liquid (gray bar) or in soft agar medium (hatched bar)
is shown. Data were from triplicate activated mutant of MEK-transformed hypothemycin
Cell cycle progression
of DT, raf- and
and 5 PM lactacystin
(D) are
x25.
in some tumor cells has been known to be inhibited
GO/G1 and G2/M phases by lactacystin, be explained
Morphology
NIH3T3 cells with no drug (D), 0.5 pg/ml
(C) or both 0.5 l,tg/ml hypothemycin
shown. Original magnification
possibly
experiments.
of
a proteasome
by the accumulation
in both the
inhibitor (23, 24). This inhibition
of p21 waft (25) and ~27~n’
CDWcyclin
complexes, caused by the proteasome blockade. It might have discrepancy
proteasome
inhibitor
study inhibit
and ubiquitination
cell growth.
However,
activator such as hypothemycin as long as we have examined,
turnover by the treatment of hypothemycin the accumulation
for
that both
shown in the present the augmented
protein
was specific to cyclin Dl and we could not detect
any change in the level for the CDK inhibitors Dl inhibition,
could
(26), inhibitors
(data not shown). Therefore, in terms of cyclin
of the CDK inhibitors
caused by lactacystin
similar effect with the decrease in cyclin Dl level in response to hypothemycin. results have been reported for a immunosuppressant
rapamycin
might have the The consistent
(27) and for a all-trans
retinoic
acid (28). The phenotypic
normalization
of Gl phase, inhibition as its less influence the oncogenic
induced by hypothemycin
of anchorage-independent
in DT cells such as prolongation
growth and morphological
on parental NIH3T3 cells indicates that hypothemycin
signal transduction
which is necessary for ras-induced
reversion as well specifically
transformation
blocks
but not for
Hypohnycin and Cyclin D 1
392
normal growth. It is reasonable least in cell cycle regulating transformation NIH3T3
Vol. 65, No. 4, 1999
to think that cyclin Dl mediates such oncogenic machinery
caused an elevated level of cyclin Dl and acceleration
cells (4). Moreover,
transformation
of NIH3T3
the ectopic expression
cells (29). These findings
cyclin Dl is necessary for ras-transfoirnation.
of p21w*
of Gl progression
suppressed
indicate that up-regulated with hypothemycin
expression
of cell cycle, hypothemycin
signal transduction
of
(Fig. lA), whereas
cyclin Dl is not a sole target, but another target protein would be involved the oncogenic
in
ras-mediated
of cyclin Dl was restored at that time (Fig. 1C). So it can be speculated
Take together, from a viewpoint
at
that ras-
On the other hand, in our study, a number of cells
still remained in the Gl phase after 24 hours incubation the expression
signaling
in DT cells. In fact, it was demonstrated
in the Gl arrest.
inhibits transformation
from ras to Gl progression
that
by blocking
at cyclin Dl protein level at least
until 8 hours of the treatment. The oncogenic expression
activity
of cyclin Dl have been reported by showing
of cyclin Dl co-operated
Besides, in transgenic
with ras in transformation
models, cyclin Dl transgenic
crossed with ras transgenic
mice with incidence
transgene (31). Hence, the overexpression indicating
the existence
In case of NIH3T3
malignancy. necessarily
of another
result in malignant
phenotype
mice developed
(30).
tumors when they were a single
of cyclin Dl is not sufficient for full transformation, signaling
cells, introduction
transformation
pathway
necessary
of cyclin Dl expression
for higher
vector did not
(32). On the other hand, the morphology
still remained
of cyclin Dl was suppressed quite well.
of characteristic
of primary rat fibroblast
much higher than mice carrying
ras-mediated
arrested DT cells by serum depletion expression
in vitro that the
for higher malignancy,
refractive
(data not shown)
of Glalthough
Therefore, the refractive morphology, is responsible
for the molecule(s)
one
other than
cyclin Dl to which signaling may be branched between ras (possibly MEKI) and cyclin Dl, and the destruction observation
of the molecule(s) that
accelerated
molecule(s)
owing morphological
phenotypes
brought
chemotherapy
about
may be mediated through ubiquitin-proteasome
proteasomal by
degradation
of cyclin
change resulted in disappearance oncogenic
must exist in ubiquitin-proteasome
ras
suggests
that
Dl
and
pathway. Our putative
other
of many of the malignant
a novel
target
for
cancer
system.
Acknowledgments We thank Drs. M. Noda and K. Yanagihara for giving us DT cells and v-&-transformed NIH3T3 cells. We thank Drs. K.Okazaki and H.Okayama and cDNA library and for valuable discussions. Okazaki for providing us with anti-ubiquitin deeply appreciate Dr. Y. Terada for invaluable Nagata
for helpful
preparing manuscript.
discussions.
for providing us with S. pombe strains
We are deeply grateful to Drs. S. Ohmi and K. polyclonal
antibody and expression
suggestions.
plasmid. We
We thank Drs. T. Yoshioka and K.
We also thank Mr. T. Kawagoe
for excellent
assistance
in
Vol. 65, No. 4, 1999
Hypothemycin awl Cyclin D I
393
References 1.
V. BALDIN, J. LUKAS, M. J. MARCOTE, 7,812-821
M. PAGAN0
and G. DRAETTA,
Genes Dev.
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ELSEVIER
SUPPRESSION ASSOCIATED
SoO24-3205(99)00259-3
OF ONCOGENIC
TRANSFORMATION
WITH ACCELERATED
BY HYPOTHEMYCIN
CYCLIN Dl DEGRADATION
UBIQUITIN-PROTEASOME
THROUGH
PATHWAY
Hikaru Sonoda*, Kazuo Omi*, Kanji Hojo, Kazuyo Nishida, Satoshi bmurap Shionogi Research Laboratories,
and Kenji Sugita
Shionogi & Co., Ltd., Osaka 553-0002, Japan
§The Kitasato Institute, Tokyo 108-8641, Japan
(Received in final form March 3 1, 1999)
Summary Hypothemycin
was originally
trichothecoides.
isolated as an antifungal
Here we report that treatment
cells (DT cells) with hypotbemycin Dl
protein
with concomitant
was
carried
2 (ubc2)
are the targets
ubiquitin-conjugation specific
inhibitor
accumulation
prolongation
is indicated growth
of hypothemycin
process. for
phenotype,
and reverted
morphology
Furthermore,
of Gl
phase in their cell cycle. rhp6+
or its downstream in the presence greatly
(S.
(homologue
ubiquitin-conjugating
hypothemycin
proteasome,
that hypothemycin
NIH3T3
mutant of Schizosuccaromycespombe
RAD6) and mammalian
of multi-ubiquitinated
terms of malignant
on v-K-ras-transformed
out to show that S. pombe
Succuromyces cerevisiue
of Hvpomyces
caused drastic decrease in amount of cyclin
Analysis using hypothemycin-resistant pombe)
metabolite
of
enzyme
molecules
in
of lactacystin, enhanced
a the
form of cyclin Dl in DT cells. Therefore, it
facilitates ubiquitinating hypothemycin
the morphology
still remained transformed
process of cyclin Dl. In
inhibited
of DT cells. in the additional
anchorage-independent On the contrary,
their
presence of lactacystin.
Our results suggest that cyclin Dl is a key molecule working downstream in rassignaling
and that the transformation
can activate ubiquitin-proteasome
can be inhibited
pathway including
by the compound
which
degradation of cyclin Dl
Key Words hypothemycin, cyclin Dl, DT cells, ubiquitin-proteasomepathway, lactacystin
Correspondence
should be addressed to H. Sonoda, Shionogi Research Laboratories,
& Co., Ltd., Sagisu S-12-4, Fukushima-ku, lFax]81-6-6458-0987,
Shionogi
Osaka 553-0002, Japan, [Tel]81-6-6458-5861,
[Email][email protected]
*First and second authors equally contributed
to this work.
382
Hypothemycinand Cyclin D 1
Vol. 65, No. 4, 1999
Cyclin Dl plays a central role in Gl progression in mammalian cell cycle and its expression is stimulated by upstream mitogenic signals (1). Following these signals, newly expressed cyclin Dl in Gl phase assembles with CDK4 to form active kinase complex and the
amount of the complex is titrated by cyclin-dependent kinase inhibitors such as ~16~~, p21wafl and p27%P1 (2). The resulting redundant kinase complex can be rate-limiting for driving cells into S phase. Therefore, such tight control of the amount of cyclin Dl should be important in order to precisely regulate the progression of cell cycle and to prevent cells from tumorigenesis. In fact, the chromosomal rearrangement, translocation or amplification which cause deregulated expression of cyclin Dl are thought to promote tumorigenesis in various human cancers (3). Moreover, in murine tibroblasts transformed by oncogenes such as ras, enhancement of Gl progression was observed indicating that some of the transforming signaling from oncogenes affect cell cycle in Gl phase (4). The accelerated Gl-S transition can be considered as one of the malignant phenotypes in these cells which include morphological change and ability to grow anchorage-independently. Hypothemycin have been known as product of Hypomvces trichothecoides having antifungal activity (S), and also been briefly mentioned to have cytotoxic activity on some tumor cell lines (6). In this report, we describe mechanistic analysis on anti-transforming activity of hypothemycin, that is, prolonged Gl phase associated with cyclin Dl downregulation, identification of ubc2 cDNA as a multicopy suppressor for hypothemycin-resistant S. pombe mutant, and cancellation by proteasome inhibitor of hypothemycin induced cyclin Dl degradation and transformation.
Methods CeN culture: All cell lines used in this work were maintained in Dulbecco’s modified Eagle medium (DMEM) containing fetal calf serum (FCS) (10%). In the case of synchronization of cell cycle, cells were cultivated in DMEM without FCS in order to arrest them in Gl phase. Colony forming assay: Detail procedure for this assay have been previously described (7). Briefly, cells were pretreated with 0.02, 0.1, 0.5, 1 pg/ml hypoihemycin in DMEM containing FCS (10%) for 24 hours. Each culture were divided in two, one was to test for the colony formation in liquid medium, another was to test in soft agar medium containing additional agarose (0.4%) to liquid medium. Same concentrations of hypothemycin as the pretreatment was added in both culture conditions. After 8 days of culture, the number of colonies in liquid and soft agar medium were counted by crystal violet staining and microscopic examination, respectively. The efficiency of colony formation was calculated as proportion of the colony number appeared in the medium from which the drug had been withdrawn. Cell cycle analysis: Cells to be tested were harvested by trypsinization and collected. Dissolution of cytoplasm, stabilization of nuclei and staining of DNA was carried out using
Vol. 65, No. 4, 1999
CycleTEST
Hypothemycin and Cyclin Dl
kit (Becton Dickinson).
Becton Dickinson
383
Stained DNA was analyzed
FACscan according to the manufacture’s
by flow cytometry
Western blot analysis: Cells were harvested with cell scraper followed by washing phosphate-buffered
saline and collected, then Iysed with RIPA buffer consisting
HCl pH7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton pepstatin,
X-100 1 unit/ml
supplemented aprotinin
with
0.5 mM
phenylmethylsulfonyl
obtained
polyacrylamide
gel (14%), transferred onto nitrocellrose
antibody
Santa Cruz Biotechnology,
or anti-cyclin
immunoprecipitation
E polyclonal experiment,
the suspended
anti-cyclin
Dl monoclonal
at 4°C and centritiged
was washed four times with RIPA buffer and then suspended
sample buffer followed by boiling for 5 minutes. Polyclonal in Western ubiquitin
blotting (kind
were anti-cyclin
gift
from
monoclonal Inc.). For the antibody
Inc.) was added in 1 ml RIPA buffer containing
cell lysate. The mixture was gently rotated overnight precipitate
antibodies
Reacted
bands
were
(sc-
100 mg of
by 8000g. The
in 20 ml SDS-PAGE used for these samples
Dl (~~-753, Santa Cruz Biotechnology,
S. Ohmi).
40 pg/ml
and probed with anti-cyclin
D2 (Ab-1, Calbiochem)
(06-459, Upstate Biotechnology
agarose-conjugated
246AC, Santa Cruz Biotechnology,
fluoride,
cells by 8000g was loaded on SDS
membrane
Inc.), anti-cyclin antibody
of 10 mM Tris-
In each case, equal amount of protein in
the supematant D1 (~0246,
once with
0.1% sodium dodecyl sulfate (SDS) and
and 10 pg/ml leupeptin.
after centritiging
with a
procedure.
detected
Inc.) and anti-
using
ECL
system
(Amersham).
Cloning of multicopy suppressor cDNA for S. pombe Is mutant: The mutant ( h- rhp6d134K leul-32)
cells were transfected with a S.pombe cDNA library (8), and incubated on MMA plates
first at 26’C for 24 h and then at 36’C for 4-5 days. Colonies formed were isolated and then examined
by the
instability
revertants.
Complementing
test to distinguish
authentic
transformants
from
phenotypic
plasmids were recovered in Escherichia coli. The recovered cDNA
clones were tested for their activity by re-transfection
into the mutant and the sequences
were
determined.
Results In the course of our screening hypothemycin,
condition,
(7, 9, 10, 1 l), we found that
trichothecoi&
having antifungal
DT cells (12) in Gl phase in their cell cycle at the concentration
to the similar extent as in case of serum deprivation
little influence
(Fig.
1A). In the same
on the cell cycle of parental NIH3T3 cells was observed (Fig. 1B). In
order to know the mechanism expression
compound
which was known as product of Hrpomvces
activity, arrested asynchronous of 0.5 pg/ml
of anti-tumor
of this ras-specific
inhibition,
the effect of hypothemycin
of Gl cyclin in DT cells was examined. DT cells were synchronized
serum starvation for 24 hours, and then released from arrest by addition presence or absence of 0.5 pg/ml hypothemycin.
on the
in Gl phase by
of 10% serum in the
Western blot analysis using their extract and
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin D 1
384
A
LIT cells Serum Hypothemycin
Gl S G2lM
i
+
-I
_
71 .O% 28.1 0.9
44.9% 47.5 7.6
73.9% 21.4 4.7
B NIH3T3 cells _
Serum
i
+
Hypothemycin
Gl
S G2lM
+
47.7% 34.1 18.2
88.2% 5.4 6.4
47.9% 35.0 17.1
C No drug hours
0
1
2
4
+ Hypothemycin 8 24
0
1
2
4
8 24 +
cyclin Dl
+
cyclin E
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin Dl
D
No drug
385
+ Hypothemycin
60 v) ?j 40 8
i’ “O--O..
20
20
0
5
10
.....
(j
....
.............
‘0
0
10 hours
5
Fig. 1 Hypothemycin transformed
inhibits the cyclin Dl expression and blocks Gl-S progression cells. DT cells
(A) and NM3T3
Dulbecco’s modified Eagle medium containing asynchronous hypothemycin, cytometry.
manner
@) were
in
cultured
10% fetal calf serum (KS)
in
in an
for 24 hours with (right) or without (center) 0.5 t.&/ml
then harvested
and their DNA content
As a control of Gl-arrest,
hours is shown (left). Percentages graphs.
cells
(C) Gl-arrested
was analyzed
by flow
data from cells starved for serum for 24
of cells in each phase are shown under the
DT cells by serum deprivation
for 24 hours
released by addition of 10% FCS with or without hypothemycin,
were
then cultivated
for 1, 2,4 or 8 hours and whole protein was extracted. Western blots using these extracts and anti cyclin Dl antibody and cyclin E were carried out. (D) DNA content from the same time course experiments cytometry,
as C was determined
by flow
and percentages of cells in Gl (Cl) and S (0) phases are shown, Data
were from duplicate experiments.
anti-cyclin
Dl monoclonal
antibody showed that the amount of cyclin Dl protein was greatly
reduced four to eight hours after serum stimulation
in the presence of hypothemycin,
no such reduction was observed in the absence of hypothemycin
whereas
(Fig. 1C). Little influence
on
the level of cyclin E, another Gl cyclin, was detected (Fig. 1C). At that time, DT cells were still kept in Gl phase in the presence of hypothemycin absence of hypothemycin
level of cyclin Dl and the deficiency Thinking of the significance the prolonged cyclin D 1.
although they could enter S phase in the
(Fig. 1D). Thus, the correlation
was observed between
in entering into S phase in hypothemycin-treated
of the amount of cyclin Dl in Gl progression
Gl phase by the addition
of hypothemycin
the reduced DT cells.
as mentioned
above,
is caused by the reduced amount of
Vol. 65, No. 4, 1999
Hypomemycin and Cyclin Dl
386
In order to examine what mechanism used hypothemycin-resistant
mutant
mediated the biological effects of hypothemycin,
of S. pombe
which was obtained
presence of 100 pg/ml of the drug. Wild type of S. pombe 2A). The mutant also displayed
temperature-sensitive
diploid, the mutant was recessive analysis demonstrated
could not grow in that condition
for both the drug-resistant
and the ts phenotypes.
(Fig.
Genetic
that both these phenotypes were caused by single chromosomal
to be rhp6+ which was previously
in the
(ts) growth (Fig. 2A). In heterozygous
(data not shown). cDNA clones which could complement identified
spontaneously
we
the ts phenotype
characterized
mutation
of this mutant were
as a homologue
of S. cerevisiae
FUD6 (13), a member of ubc2 family (14). Direct sequencing
of PCR fragment of rhp6+ gene
obtained
continuous
from the
corresponding
mutant’s
genome
revealed
that three
nucleotides
to Lysin 134 residue were deleted (data not shown). Therefore, it was indicated
that this single amino acid deletion in the rhp6+ gene product was responsible resistance and the ts growth of the mutant. Furthermore, but also HHR6A
expression of not only wild type rhp6+
as well as growth at non-permissive
These data strongly suggest that rhp6 protein of S. pombe or its downstream Destruction (for review,
for the drug-
or HHR6B (both encode human ubc2) cDNA in the mutant
restored hypothemycin-sensitivity
mammalian
(AAG)
molecules to which hypothemycin
rhp6dl34K
temperature
and mammalian
(Fig. 2B).
ubc2 are the targets
is directed at least in S. pombe.
of some cyclins are known to be regulated by ubiquitin-proteasome
see ref. 15). Like other Gl
cyclins
pathway
such as cln2, cln3 in S. cerevisiae
cyclin E, cyclin Dl has been reported to be degraded through ubiquitination
To examine the relationship
between the hypothemycin-induced
level in DT cells and the proteasome hypothemycin mammalian
and lactacystin, proteasome
degradation
(16).
of cyclin Dl
pathway, DT cells were treated with both
which is known to bind covalently to and specifically
inhibit the
subunit X/MB1 (17). Western blot using their extract showed that, as
34 kD band of cyclin Dl grew fainter, the band corresponding form appeared, whereas no such accumulation were treated
rapid reduction
and
with lactacystin
to a higher molecular
weight
of the shifted band was observed when DT cells
alone (Fig. 3A). In order to know whether the shifted band
represented
ubiquitinated
monoclonal
antibody from DT cells were subjected to Western blotting using both anti-ubiquitin
and anti-cyclin
form
Dl polyclonal
is known to be necessary
ubiquitination
semi-solid phenotypes
the immunoprecipitate
with
anti-cyclin
of the protein by protcasome
of high molecular
and lactacystin
strongly
Dl
of a protein
in which the protein is
weight form of cyclin Dl in the presence
suggests that hypothemycin
of
could accelerate the
of cyclin Dl.
When NIH3T3 morphology
Dl,
antibody (Fig. 3B). Since the multiple ubiquitination
for recognition
degraded, the acummulation both hypothemycin
of cyclin
cells are transformed
by various
oncogenes,
they often change their
from flat to spindle one and obtain the ability to grow anchorage-independently media. Suppression
of their malignancy
(18). The anchorage-independent
can be estimated by the inhibition
in
of these
growth of DT cells in soft agar medium in the
Vol. 65,No.4,1999
Hypothemycin and Cyclin D 1
B Hm:rhp6-b134K
pcMVL-X pcMVL-rhp6 pcMVL-HHR6A pchlVL-HHRGB
Spot tests for hypothemycin
and temperature
sensitivity.
(A) Hypothemycin-
resistant and ts phenotypes of the ,I?.pombe strain rhp6d134. 32) and mutant (h- rhp6d134K
leul-32)
temperature)
phase in liquid minimum
to late exponential
supplemented (minimum
cells were grown at 26% (permissive-
with leucine. Cells were suspended
by stepwise
10 fold dilutions. medium
hypothemycin,
agar)
plates
medium
(MML) (8)
at 1 x lo7 cells/ml, followed
10 ml of aliquots
+leucine
Wild type (h- leul-
with
were spotted onto MMA or without
and incubated at 26’C or 36% (non-permissive
100 pg/ml temperature
of ) for
4-5 days. (Educts are seen as dots in the drug containing
plates.) (B) Suppression
of hypothemycin-resistance
of the rhp6d134K
by S. pombe transformed
rhp6+ and human by
cytomegalovirus
and temperature-sensitivity
indicated
ubc2 cDNAs.
plasmids.
The rhp6d134K
pchPJL-X
(containing
promoter and the LEUZ gene) is an expression
cells
cells were human
vector with no
insert and used as a control (33). S. pombe rhp6+ cDNA and its two distinct human homologue cDNAs, HHR6A and HHRBB, were inserted in the same expression vector as the control. Spot tests were performed as in (A) except that minimum
medium was not supplemented
with leucine.
388
Hypothemycin and Cyclin D 1
A
Hypothemycin + Lactacystin
Lactacystin
hours
0
1 2
4
Vol. 65, No. 4, 1999
8 24
0
1 2
4
8 24
t-shifted
+
B
Blot
Dl
cyclin Dl
cyclin Dl
Ub
Hypothemycin + Lactacystin
12
34 Fig. 3
Accumulation
of ubiquitinated
hypothemycin
and lactacystin.
cyclin
Dl
Gl-arrested
in DT cells cultivated
DT cells by serum deprivation
hours were released by addition of 10% FCS with 5 p&I lactacystin both 0.5 pg/ml hypothemycin
and 5 pM lactacystin.
Western blotting probed by anti-cyclin (B) DT cell lysate prepared without
(lane
monoclonal (lane
by
with (lane 2, 4) or with anti-cyclin
blotting using either anti-cyclin
(lane 3, 4) polyclonal
cyclin Dl, IgH; cross-reacted
alone or with
antibody was carried out.
after 8 hours serum induction
followed by Western
1, 2) or anti-ubiquitin
ubiquitinated
D 1 monoclonal
for 24
Time course analysis
1, 3) both drugs was immunoprecipitated
antibody
with both
antibody.
mouse immunoglobulin
Ub-cyclin
Dl Dl Dl;
heavy chain.
Vol. 65, No. 4, 1999
Hypothemycin and Cyclin D 1
389
presence of hypothemycin
was examined (Fig. 4A). In a dose-dependent
gave rise to a significant
difference
in the efficiency
soft agar medium. At a concentration specifically
. Moreover,
inhibited
constitutively
of 0.5 pg/ml, the anchorage-independent hypothemycin
within 24 hours. It also flattened
growth was most
the morphology
of NIH3T3
in DT cells
cells transformed
by v-raf or
‘activated mutant of MEK 1 (19), both of which are activated forms of consecutive
of transformed approximately not shown).
phenotype
compete
the
reversion
reversion
to lactacystin
with hypothemycin
of their
induced
depended
morphology.
by hypothemycin
in response
malignant
phenotype
The
effect
was examined
blocked the morphological
to the transforming on the transformants
of proteasome on
the
(Fig. 4D). Although
of lactacystin
the
addition of lactacystin
did not induce any change in
it is suggested
signal
of ubiquitination
reversion induced by hypothemycin.
of NIH3T3 cells with lactacystin
(data not shown). Therefore,
expressed
on the activation
of cyclin Dl, the inhibition
was different among the cell lines, simultaneous
completely
On the other hand, treatment their morphology
reversion
as seen in the degradation
with the IC,, value of
of cyclin Dl expression as well (data
0.3 I_IMwhich corresponds in the inhibition If this morphological
morphological
of ras (20) (Fig. 4B, C). The suppression
in these cell lines showed dose-dependency
pathway by hypothemycin
sensitivity
between liquid and
induced the normal morphology
members in MAP kinase cascade working downstream
might
manner, hypothemycin
of colony formation
that the molecule(s)
from these oncogenes
might be destroyed
which
and conferred
the
in the ubiquitin-proteasome
pathway.
Discussion In cells once stimulated by growth factors to proliferate, the expression is constant through the cell cycle (21).
The equilibrium
level of cyclin Dl
of active synthesis and destruction
of
cyclin Dl protein may be kept stable because the half life of cyclin Dl is very short (22). It is already reported that such rapid turnover of cyclin Dl depends on phosphorylation residue by unknown
one possible mechanism
of hypothemycin
Dl might be the activation including
in enhancement
of the polyubiquitination
mutant
of S. pombe, hypothemycin
process of cyclin Dl directly, or alternatively
kinase
phosphorylates
machinery.
cyclin
Dl
One
hypothemycin
reason
we can
speculate
proteins are generally is that
activation
might made it hard to detect intermediately
resulting in preferential that their degradation
detection of highly ubiquitinated
in proteasome was inhibited.
may facilitate the
that the activity of the unknown
(16) may be controlled
However, we do not know the reason why the ubiquitinated
as a single band whereas the ubiquitinated bands.
can act on a ubiquitination
ubc2. Therefore, it can be speculated that hypothemycin
ubiquitination which
of cyclin
of such kinase. On the other hand, as seen in our genetic experiment
using hypothemycin-resistant machinery
on threonine
kinase other than CDK4 which is known to bind to cyclin Dl (16). Thus,
under
the ubiquitinating
cyclin Dl was detected
appeared as smear or ladder
of multi-ubiquitinating ubiquitinated
step by
form of cyclin Dl
form of the protein in the condition
390
Vol. 65, No. 4, 1999
Hypothemycinand Cyclin D 1
00
50
z
,o 8 7
0
0:02
0.1
0.5
1
pg/ml hypothemycin
B No drug DT
raf I NIH3T3
MEK / NIH3T3
&pothemvcin DT
raf / NIH3T3
MEK I NIH3T3
Vol. 65, No. 4, 1999
l@pohmycin and Cyclin D 1
391
D Hvpothemvcin
+ Lactacvstin
raf / NIH3T3
Fig. 4 Hypothemycin NIH3T3
inhibits malignant
phenotypes
cells. (A) Comparison
in ras-, raf- and MEK-transformed
of suppressive
activity
of hypothemycin
for
colony formation
of DT cells in liquid and soft agar media. The effkiency
colony formation
either in liquid (gray bar) or in soft agar medium (hatched bar)
is shown. Data were from triplicate activated mutant of MEK-transformed hypothemycin
Cell cycle progression
of DT, raf- and
and 5 PM lactacystin
(D) are
x25.
in some tumor cells has been known to be inhibited
GO/G1 and G2/M phases by lactacystin, be explained
Morphology
NIH3T3 cells with no drug (D), 0.5 pg/ml
(C) or both 0.5 l,tg/ml hypothemycin
shown. Original magnification
possibly
experiments.
of
a proteasome
by the accumulation
in both the
inhibitor (23, 24). This inhibition
of p21 waft (25) and ~27~n’
CDWcyclin
complexes, caused by the proteasome blockade. It might have discrepancy
proteasome
inhibitor
study inhibit
and ubiquitination
cell growth.
However,
activator such as hypothemycin as long as we have examined,
turnover by the treatment of hypothemycin the accumulation
for
that both
shown in the present the augmented
protein
was specific to cyclin Dl and we could not detect
any change in the level for the CDK inhibitors Dl inhibition,
could
(26), inhibitors
(data not shown). Therefore, in terms of cyclin
of the CDK inhibitors
caused by lactacystin
similar effect with the decrease in cyclin Dl level in response to hypothemycin. results have been reported for a immunosuppressant
rapamycin
might have the The consistent
(27) and for a all-trans
retinoic
acid (28). The phenotypic
normalization
of Gl phase, inhibition as its less influence the oncogenic
induced by hypothemycin
of anchorage-independent
in DT cells such as prolongation
growth and morphological
on parental NIH3T3 cells indicates that hypothemycin
signal transduction
which is necessary for ras-induced
reversion as well specifically
transformation
blocks
but not for
Hypohnycin and Cyclin D 1
392
normal growth. It is reasonable least in cell cycle regulating transformation NIH3T3
Vol. 65, No. 4, 1999
to think that cyclin Dl mediates such oncogenic machinery
caused an elevated level of cyclin Dl and acceleration
cells (4). Moreover,
transformation
of NIH3T3
the ectopic expression
cells (29). These findings
cyclin Dl is necessary for ras-transfoirnation.
of p21w*
of Gl progression
suppressed
indicate that up-regulated with hypothemycin
expression
of cell cycle, hypothemycin
signal transduction
of
(Fig. lA), whereas
cyclin Dl is not a sole target, but another target protein would be involved the oncogenic
in
ras-mediated
of cyclin Dl was restored at that time (Fig. 1C). So it can be speculated
Take together, from a viewpoint
at
that ras-
On the other hand, in our study, a number of cells
still remained in the Gl phase after 24 hours incubation the expression
signaling
in DT cells. In fact, it was demonstrated
in the Gl arrest.
inhibits transformation
from ras to Gl progression
that
by blocking
at cyclin Dl protein level at least
until 8 hours of the treatment. The oncogenic expression
activity
of cyclin Dl have been reported by showing
of cyclin Dl co-operated
Besides, in transgenic
with ras in transformation
models, cyclin Dl transgenic
crossed with ras transgenic
mice with incidence
transgene (31). Hence, the overexpression indicating
the existence
In case of NIH3T3
malignancy. necessarily
of another
result in malignant
phenotype
mice developed
(30).
tumors when they were a single
of cyclin Dl is not sufficient for full transformation, signaling
cells, introduction
transformation
pathway
necessary
of cyclin Dl expression
for higher
vector did not
(32). On the other hand, the morphology
still remained
of cyclin Dl was suppressed quite well.
of characteristic
of primary rat fibroblast
much higher than mice carrying
ras-mediated
arrested DT cells by serum depletion expression
in vitro that the
for higher malignancy,
refractive
(data not shown)
of Glalthough
Therefore, the refractive morphology, is responsible
for the molecule(s)
one
other than
cyclin Dl to which signaling may be branched between ras (possibly MEKI) and cyclin Dl, and the destruction observation
of the molecule(s) that
accelerated
molecule(s)
owing morphological
phenotypes
brought
chemotherapy
about
may be mediated through ubiquitin-proteasome
proteasomal by
degradation
of cyclin
change resulted in disappearance oncogenic
must exist in ubiquitin-proteasome
ras
suggests
that
Dl
and
pathway. Our putative
other
of many of the malignant
a novel
target
for
cancer
system.
Acknowledgments We thank Drs. M. Noda and K. Yanagihara for giving us DT cells and v-&-transformed NIH3T3 cells. We thank Drs. K.Okazaki and H.Okayama and cDNA library and for valuable discussions. Okazaki for providing us with anti-ubiquitin deeply appreciate Dr. Y. Terada for invaluable Nagata
for helpful
preparing manuscript.
discussions.
for providing us with S. pombe strains
We are deeply grateful to Drs. S. Ohmi and K. polyclonal
antibody and expression
suggestions.
plasmid. We
We thank Drs. T. Yoshioka and K.
We also thank Mr. T. Kawagoe
for excellent
assistance
in
Vol. 65, No. 4, 1999
Hypothemycin awl Cyclin D I
393
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