1. No category

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1995 85: 2870-2876
Overexpression of cyclin D2 in chronic B-cell malignancies
A Delmer, F Ajchenbaum-Cymbalista, R Tang, S Ramond, AM Faussat, JP Marie and R Zittoun
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Overexpression of Cyclin D2 in Chronic B-Cell Malignancies
By Alain Delmer, Florence Ajchenbaum-Cymbalista, Ruoping Tang, Sylvie Ramond, Anne-Marie Faussat,
Jean-Pierre Marie, and Robert Zittoun
Tumor progression in B-cell chronic lymphocytic leukemia
(B-CLLI is thoughtt o result from thegradual accumulation
of small resting GOIGI phase lymphoid cells rather than the
proliferation of actively dividing cells. The recent identification ofG I cyclins that are likely t o control both the
progression through GO and G I phase and t h e G l l S transition
prompted ust o study themRNA expression of D-typecyclins
in the peripheral blood lymphocytes from 34 patients with
B-CLL, 7 patients with lymphoplasmacytic lymphoma (LPL),
and 2 patients with mantlecell lymphoma (MCL). Cyclin D2
mRNA was, on average, 5- t o IO-fold overexpressed in most
of the samples studied (B-CLL, 29/34; LPL, 717; MCL, 0121 as
compared with normalresting B lymphocytes, in which
cyclin D2 mRNA was barely detectable. In situ hybridization
with cyclin D2 digoxigenin-labeled mRNA probe showed
that all the cells from a given sample were stained with
approximately the same intensity. Cyclin D3 was never detected in any of the samples tested, whereas cyclin D1 was
expressed in only the 3 cases (1 LPL and 2 MCL) bearing a
t(11;14) translocation. A trisomy 12 was found in 4 of 19
(21%) B-CLLor LPL casesfor whichcytogenetic analysis was
available. Although the cyclin D2 gene has been mapped
t o chromosome 12~13,there was no apparent correlation
between trisomy 12 and the level of cyclin D2 expression.
Cell cycle analysis by flow cytometry
after staining with propidium iodide consistently showed that more than969’0 of
the cells were in GO/G1 phase, whatever the importance of
cyclin D2 overexpression was, and that cyclin D2 overexpression in B-CLL was not associated with any modifications
of the cell cycle repartition. No consistent overexpression of
cyclin D2 was found in acute myeloid leukemias. In conclusion, overexpression of cyclin D2 mRNA was found t o be an
almost constant feature in B-CLL and LPL. Therefore, it led
us t o hypothesize, with thesupport ofdata from some transfection experiments previously reported in murine hematopoietic cell lines, that cyclin D2 might play a role in B-CLL
pathogenesis, possibly by preventing cells from
programmed cell death.
0 1995 by The American Societyof Hematology.
M
GUS tran~ition.’”’~ The
cell cycle regulation has appeared
even more complex since
therecentidentification
of cdk
inhibitor^,'^.'^ one of which being deletedin numerous cancer
cell lines and tumor samples, including
The clinical behavior of patients withB-cell
chronic
lymphocytic leukemia (B-CLL)andsome rebated chronic
lymphoproliferative disordersis quite heterogeneous.” Most
patients experience a prolonged, indolent clinical course for
many monthsor years, whereas otherspresent with-or
rapidly develop-a more aggressivedisease. The prognostic
value of the lymphocyte doubling timehas been established
in B-CLL.” Nonetheless, the parameters that have
been used
so far to study the kinetics of B-CLL are crude and
related to
late phases of the cell cycle. For example, even in advanced
disease, cell cycle analysis by r3H]thymidine uptake or flow
cytometryandimmunocytochemistrywithKi67antibody
consistently showed that most malignant cells are in GO/GI
phase
and
very few none
or
in S
Therefore, the
tumor progression is likely to be more the consequence of
a progressive accumulation of small resting GO/Gl lymphoid
cells than the proliferation of actively dividing cells. This
abnormally prolonged survival of malignant cells in vivo is
a major biologic characteristic of B-CLL and is presumably
related to deregulated programmed cell death.” Neverthethegenes involved in the
less, no consistentanomalyof
control of apoptosis has been described so far in this disease.
Although rearrangements of bcl-2 gene havebeen only occasionallyreportedinB-CLL,”
the malignant cells usually
contain higher levels (up to 25-fold) of bcl-2 protein than
do normal lymphocytes.” However, the relevance of such a
finding to thepathogeny of the diseaseis still unclear.Therefore, studies investigating the role of genes that potentially
control G1 progression andthe Gl/S transition are warranted.
Some data suggest that the deregulation of GI cyclins
may be involved in oncogenesis. Cyclin D l was cloned (initially under the name of PRADl) from itsoverexpression in
AJOR INSIGHTS have been accomplished during the
last few years in the
understanding of cell cycleregulation in eukaryotic cells. Progression through the cell cycle
results from the sequentialand well-orderedactivation of
several components that play a key role in the GUS and the
G2/M transitions.’,’ Cyclins have appeared as major regulators in this network, because their association to the cyclindependent kinases (cdks) allows the subsequent activation
of the cyclidcdk complexes andtheir catalytic activity. Five
distinct classes of mammalian cyclins (A, B, C, D, and E)
have been identified. Cyclin A is produced in late GI and
is required for the onset of DNA synthesis, whereas B-type
cyclins control the passage through M phase. The regulation
is
of the GUS transition appears to be more complex and
not fully understood yet.
The putative role of GI cyclins,
E, in such issuehas
mainlyD-type
cyclins andcyclin
emergedfrom
transfection experiments in diverse cell
Cyclin E is expressed during late G1 and seems to
be required forentryinto S p h a ~ e . ~D-type
. ’ ~ cyclinsare
synthesized earlier in G1 phase and seem to be induced in
response to external agents that promote entry into the cell
cycle. D-type cyclins are also likely to be regulators of the
From the hbaratoire de Cinne‘tiqueet Culture Ceilulaires, Service
d’Hne‘matologie, and the Laboratoire de Cytogenne‘tique,H6tel-Dieu,
Paris, France.
Submitted June 6, 1994; accepted January 4, 1995.
Supported in part by grants from Association pour laRecherche
contre le Cancer(ARC),Villejug France, and Comitne‘ deParis,
Ligue Nationale Contre Le Cancer, France.
Address reprint requests to Alain Delmer, MD,Service d’Hne‘matologie, l place du Parvis Notre-Dame,75181 Paris Cedex 04, France.
The publication costs ofthis article were
defrayed in part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8510-0020$3.00/0
2870
Blood, Vol 85, No 10 (May 15), 1995:pp 2870-2876
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2871
OVEREXPRESSION OF CYCLIN D2 IN B-CLL
benign parathyroid tumors.29Its encoding gene (CCND1) is
mapped on chromosome llq13 in humans.' This region is
amplified in several solid turn or^^^.^' or rearranged in some
hematologic malignancies, such as mantle cell lymphoma
(MCL)32or splenic lymphoma with villous lymphocyte^.^^
The vin-Igene first identified as the common site of provirus
integration in retrovirus-induced rodent T-cell leukemias was
recently shown to be identical to the cyclin D2 gene.34The
possible role of vin-Ilcyclin D2 gene in oncogenesis is suggested by the overexpression that results from provirus integration.
The goal of the present study is to investigate the expression of D-type cyclins in chronic B-cell malignancies.
MATERIALS AND METHODS
Materials. Circulating blood lymphocytes from patients with
leukemic chronic B-cell malignancies and from healthy donors were
collected after informed consent was obtained. Mononuclear cells
were isolated by Ficoll Hypaque gradient density and were depleted
of monocytes by adherence in plastic flasks (IO6 celldml in RPMI
1640) for 1 hour at 37°C; samples of 10 to 20 X IO6 cells were
resuspended in GITC solution (4m o m guanidinium isothiocyanate,
0.5% sarcosyl, 25 mmol/L Na citrate) with 1% p-mercaptoethanol
and stored at -80°C until RNA extraction.
B lymphocytes from normal donors were separated from T lymphocytes by sheep erythrocyte r~setting'~
before storage under the
same conditions.
Cytospin slides of freshly isolated lymphoid cells were prepared
for insitu hybridization (ISH) using pretreated slides, as further
described.
Additional peripheral blood or bone marrow samples from patients
with acute leukemia were also analyzed.
RNA extraction and Northern blotting. RNA was extracted by
the guanidinium thiocyanate-phenol-chloroformmethod as described
by Chomczynski and S a c ~ h iTen
. ~ ~micrograms of total RNA was
subjected to 1% MOPS/formaldehyde agarose gel electrophoresis
and blotted onto nitrocellulose membranes. The blots were hybridized overnight with "P-labeled cDNA probes. Filters were washed
twice in 2X SSC, 0.1% sodium dodecyl sulfate (SDS) at 50°C for
15 minutes and then twice in 0.2X SSC, 0.1% SDS at 50°C for 20
minutes. Autoradiography was performed for 3 to 8 days at -80°C
using intensifying screens.
Murine cyclins Dl, D2, and D34 and CDM" cDNAs were kind
gifts from Dr C.J. Sherr (St Jude Children's Research Hospital,
Memphis, TN). Inserts were labeled with (Y[~*P]~CTP
by the hexanucleotide primer technique. The oligonucleotide sequences of the
coding regions of human and murine cyclins Dl, D2, and D3 have
approximately 90% h~mology.~
Control of RNA loading was achieved by the subsequent rehybridization of membranes with a 26 mer oligonucleotide complementary
to 28s ribosomal RNA (rRNA) labeled with y[32P]ATP.37
The murine erythroleukemic cell line MEL, the lymphoid cell line
BaF3, and the myeloblastic cell line BTU3, which constitutively
express cyclins D2 and D3, served as positive controls. BTU3 cells
that derive from a myeloblastic tumor obtained after in vivo engraftment of a Friend murine leukemia virus (F-MuLV)-induced
myeloblastic cell line3*were also used as positive control for cyclin
Dl expression.
Cyclin D2 RNA slot-blot and computerized densitometric analysis.
One microgram and 5 pg of total RNA of each sample were heat
denatured and loaded onto nitrocellulose membrane using a slotblot apparatus (Hoefer Scientific Instruments, San Francisco, CA).
Membranes were hybridized with a 32P-labeledcyclin D2 probe and
subsequently with a 28s rRNA probe to normalize the amount of
RNA loaded. Autoradiographs were analyzed with a Stanvise software on a Compaq Deskpro 286 microcomputer (Imstar, Paris,
France). Each value was expressed as the ratio between the cyclin
D2 and the corresponding 28s rRNA signals. Samples from normal
B lymphocytes were run in parallel for each experiment.
In situ RNA hybridization. Slides for cytospin preparations were
pretreated with 3-triethoxysilyl-propylamineand acetone, post-fixed
in phosphate-buffered saline (PBS) with 1% glutaraldehyde for 1
minute, and then stored at +4"C in 70% ethanol.
Murine cyclin Dl, D2, and D3 cDNAs subcloned in Bluescript
plasmids were used to generate RNA probes. Sense and antisense
single-strand RNA probes were synthesized by in vitro transcription
using T3 and 'I7 RNA polymerases and digoxigenin-labeled UTP
(DIG RNA labeling kit; Boehringer Mannheim Biochemica, Mannheim, Germany).39For ISH purpose, RNA probes were fragmented
by partial alkaline hydrolysis. Before hybridization, slides were
treated with proteinase K for 15 minutes at 37"C, were then treated
with triethanolamine, and were thereafter dehydrated in graded ethanol. Hybridization (150 ng of RNA probe per slide) was performed
overnight at 42°C. After hybridization, the slides were washed twice
in 2 x SSC at 50°C for 5 minutes and then in 0 . 2 ~SSC for 15
minutes. Hybridized probes were detected by enzyme-linked immunoassay using anti-DIG-alkaline phosphatase conjugate antibody
and color reaction with 5-bromo-4-chloro-3-indolyl phosphate
(BCIP) and nitroblue tetrazolium salt (NBT) (DIG nucleic acid detection kit; Boehringer Mannheim Biochemica). Hybridization with
sense RNA probe was performed in each experiment and served as
negative control.
Cytogenetic analysis. Peripheral blood was incubated at 37°C
for 72 hours inthe presence of phorbol 12-myristate 13-acetate
(PMA) and lipopolysaccharide (LPS). Chromosome preparations
were made by standard techniques and were analyzed with R- or Gbanding. Karyotypes were described according to ISCN (1991).40
Cell cycle analysis b y j o w cytornetry. Lymphoid cells (1 X lo6)
were suspended in0.5 mL propidium iodide solution (50 pg/mL
propidium iodide, 0.1% sodium citrate, 0.1% Nonidet P40) and analyzed with a Facstar I cytometer (Becton Dickinson, Le Pont-deClaix, France). Cells were measured at a flow rate of 200 cellsls;
signals were recorded in list mode and analyzed with Consort 30
software (Hewlett Packard, Evry, France).
Culture of normal B cells. Freshly isolated monocyte- andTcell-depleted B cells from normal donors were cultured at a final
concentration of 2 X 106/mLin RPMI supplemented with 10%heatinactivated fetal calf serum (FCS), glutamine, and antibiotics in the
presence ofPMA (0.3 ng/mL) and ionomycin (0.12 mg/mL) and
incubated at 37°C with 5% COz for 24 hours. Cells were collected
at 6, 12, and 24 hours and stored in GITC solution at -80°C.
RESULTS
Patient population. Peripheral blood samples from 43
patients (pts) with chronic lymphoproliferative disorder (BCLL, 34 pts; lymphoplasmacytic lymphoma [LPL], 7 pts;
MCL, 2 pts) were studied, as well as B lymphocytes from
7 normal individuals. In each patient, the lymphocyte count
was more than 10 X 109/L at the time of collection. Apart
from appropriate clinical and cytologic features, the diagnosis of B-CLL relied on a characteristic phenotypic pattern
with CD5 positivity, weak expression of surface Igs, and,
for the most recent cases, CD23 positivity and lack of reactivity with FMC7 antibody, as shown byflow cytometry
analysis:' Half of the patients (B-CLL, 16 pts; LPL, 4 pts;
MCL, 2 pts) had previously required therapy and received
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DELMER ET AL
2872
$ $ $ $
c
7 kb
-
2 kb
-
a
m
c
n
n
A
B
C
D
E
F
G
H
cyclin D2
CDK4
28s
one or more regimens based on alkylating agents andor
anthracyclins.
The expression of cyclin D2 mRNA was also studied in
bone marrow or peripheral blood samples from 2 patients
with acute lymphoblastic leukemia (ALL) and 15 patients
with acute myeloid leukemia (AML) (AMLI. 2 pts; AML2,
6 pts; AMLA, 3 pts;andAMLS,
4 pts, according to the
French-American-British [FAB] classification). The circulating blast cell count was more than SO X 109/L in 1 I of 17
of these cases. Nine patients were studied at diagnosis and
8 at relapse.
Expression of cyclin D2 mRNA. By Northern blot, the
normal resting B lymphocytes from peripheral blood were
found to express a very low amount of cyclin D2 mRNA.
They were consistently negative by ISH, probably because
this method is slightly less sensitive. The exposure of normal
B lymphocytes to PMA and ionomycin resulted in a rapid
induction of cyclin D2 mRNA, reaching a plateau after 12
hours (Fig 1).
In chronic B-cell malignancies, an overexpression of
cyclin D2 mRNA was observed by Northern blot in all samples but 5 from B-CLL cases and the 2 from MCL patients,
in which a low level of cyclin D2 expression comparable to
that of normal resting B cells was found (Table 1). In each
case, there was only one 7-kb transcript (Figs 1 and 2) and
no aberrant mRNA signals were detected. By computerized
densitometry on slot blot autoradiographs and after norrnalization with 28s rRNA probe, the mean expression of cyclin
D2 in B-CLL and LPL circulating lymphocytes was 17-fold
(range, 1- to 93-fold) that of normal resting B lymphocytes;
in most samples the level of cyclin D2 overexpression ranged
from 5- to 10-fold (Fig 3).
ISH with cyclin D2 RNA probe was performed in 16
Fig 1. Northern blot analysis.
A filter containing total mRNA
from normal resting B lymphocytes (B-Ly), PMA- andionomycin-stimulated normal B lymphocytes after 6 (B-Ly 6h) and 12
hours (B-Ly 12hl of culture, and
peripheral blood lymphocytes
of
B-CLL patients (A through F) and
LPL (G and H) was cohybridized
with both cyclin D2 and CDK4
probes. RNA loading was controlled by subsequent hybridization with28s rRNA oligonucleotide probe. Cyclin D2mRNA 17
kb) was rapidly induced in normal B lymphocytes after exposure to PMA and ionomycin.
Most patients' samples overexpress cyclin D2
as
compared
with normal resting B lymphocytes after normalization t o 28s
probe. CDK4 signal (2 kb) has
roughlythe same intensity as
cyclin D2 signal in both stimulated normalB lymphocytes and
B-CLL samples.
cases. It showed that all cells from a given positive sample
were stained with approximately the same intensity (Fig 4).
This finding excluded the possibility that signals observed
in Northern blot or in slot blot might be related to monocytes
contamination or to overexpression in only a minority of
tumor cells. The intensity of staining observed for each sample in ISH experiments was in accordance with the level of
overexpression detected on autoradiographs.
In addition, cyclin D2 expression was also assessed in
several cases of acute leukemias at a time of highly proliferative disease. Only 7 of 1.5 AML and neither of the 2 ALL
samples had detectable expression of cyclin D2 by Northern
blot (data not shown).
Expression of other D-type cyclins and CDK4 mRNA.
All the samples from B-CLL or LPL cases (n = IS) and
normal resting B lymphocytes (n = 3) thathavebeen assessed for cyclin D3 mRNA expression by Northernblot
and ISH were negative (Fig 2).
Table 1. Expression of D-Type Cyclins in Chronic B-Cell
Malignancies and Acute Leukemias
Cyclin D2
B-CLL
LPL
MCL
ALL
AML
ND
29/34011
711
012
012
7115
Cyclin D l
Cyclin D3
0120
1/59
212'
ND
ND
0
0/5
ND
ND
In each column are denoted the number of samples in which an
overexpression of each cyclin D has been observed by Northern blot
(on left) and the number of samples tested (on right).
Abbreviation: ND, not done.
Karyotype included a t(11;14)(q13;q32) in the 3 cases in which
cyclin D l was expressed.
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2873
OVEREXPRESSION OF CVCLIN D2 IN B-CLL
E3
E
Fig 2. Northern blot analysis.
A filter containing total RNA extracts from B-CLL patients (l
through 7) and normal Blymphocytes (B-Ly) was cohybridized withboth
cyclin D2 and
cyclin D3 probes. Positive controls are MEL (on the left) and
BTU3 (on the right) murine cell
lines. No signal was detected
with cyclin D3 probe in normal B
lymphocytes or in B-CLLsamples. Positive control BTU3 cell
line shows a strong cyclin D3 s i g
nal sized2.2kb.
RNA loading
was controlled with 28s rRNA
oligonucleotide probe.
7 n7
r
m
1
2
3
4
5
6
7
3
&
7 kb
-
cyclin D2
2.2 kb
-
cyclin D3
28s
Cyclin D1 mRNA was not detected in patients withBCLL (n = 20) or in normal B cells. However, a high expression of cyclin D1 was observed in 1 of the 5 LPL samples
testedand in the 2 MCL samples. In these 3 cases, both
cyclin D1 transcripts were found by Northern blotat the
expected sizes of 4.5 and 1.8 kb.
Cohybridization of Northern blot filters with both cyclin
D2 and CDK4 probes showed that the intensity of CDK4
signals (1 single 2-kb transcript) roughly paralleled those of
cyclin D2 in normal resting and stimulated B lymphocytes
as in B-CLL samples (Fig l ) .
Correlation of cyclin D2 expression with clinical, cytogenetic, and kinetics findings. There was a trendtoward a
higher expression of cyclin D2 in lymphoid cells from patients with advanced B-CLL or LPL than in cells from newly
diagnosed andor untreated patients, but this was not statistically significant (Fig 3). Among the patients in whom a 20fold or greater overexpression of cyclin D2 was observed,
5 of 7 had a long-lasting, advanced, and resistant disease.
Nevertheless, in some instances, the tumor cells from patients with such clinical characteristics were found to express
a low level of cyclin D2 mRNA.
In AML, cyclin D2 expression did not appear to correlate
with either the white blood cell (WBC) count, the WBC
doubling time, or the status of the disease.
Cytogenetic analysis from peripheral blood was performed in 21 patients from this series. No metaphases were
obtained in 3 cases and karyotype was normal in 3 cases. A
clonal change involving chromosome 12 was found in 5
cases. Among these, trisomy 12 was the sole abnormality in
4 cases (B-CLL, 3 pts; LPL. 1 pt) and in 1 LPL case, karyotype was complex with t(5;12)(p15;q13) and t(11;14)
(q13;q32) translocations, and malignant cells were found
to overexpress both cyclins D1 and D2. Other cytogenetic
abnormalities included del( 13)(q13;q21) in 2 cases,
del(1 l)(q22) in 2 cases, and miscellaneous changes in 4
cases. The 2 MCL cases had a complex karyotype including
a t(l I ; 14)(q13;q32). Although the number of cases bearing
a chromosome 12 abnormality was limited, thefinding of
trisomy 12 was apparently not correlated with a particular
pattern of cyclin D2 expression.
Analysis of the cell cycle repartition of peripheral blood
lymphocytes was performed by Row cytometry after staining
with propidium iodide in 18 B-CLL cases. It consistently
showed that 96% cells or more were in GO/GI phase, whatever the level of cyclin D2 overexpression was. These results
did not differ from what weobserved using the same method
in normal resting B lymphocytes.
DISCUSSION
In the present study, we investigated the mRNA expression of D-type cyclins in peripheral blood lymphocytes from
patients with chronic B-cell malignancies. Cyclin D2 mRNA
was found to be overexpressed in 29 of 34 B-CLL cases and
in all cases of LPL (n = 7). The level of cyclin D2 expression
in these disorders was, on average, 5- to IO-fold higher than
in normal resting B lymphocytes. Cyclin D3 was notdetected
in any sample from B-CLL or LPL patients, whereas cyclin
D1 was expressed only in the 3 cases (LPL, 1; MCL, 2)
associated with a t(l1; 14) translocation!'
We failed to demonstrate the expression of cyclin D2 protein in these samples
using in vivo ' 5 S methionine incorporation and immunoprecipitation. Such a failure was also reported by others in
various experiments onB-CLL cells, most likely because
they are resting cells and do not incorporate "S methionine
in short culture without mitogens. However, in stimulated
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DELMER ET AL
2874
1
uu
e
0
U
m
10
73
U
U
0
U
1
untreated
patients
(n = 14)
previously treated
patients
(n = 16)
Fig 3. Overexpression of cyclin D2 mRNA in lymphoid cells from
patients with B-CLL (0)
and LPL (0)
as compared with normal lymphocytes by computerized densitometry and normalization to 28s
rRNA probe (logarithmic Y axis). The amount ofcyclin D2 mRNA
tended to be higher in cells from previously treated patients (right
column) than in cells from untreated patients (left column), but this
was not statistically significant (Mann-Whitney test).
human normal T lymphocytes, the level of cyclin D2 protein
has been shown to be closely correlated with RNA levels.I6
There was a trend toward a higher cyclin D2 mRNA amount
in patients with advanced and resistant disease, but the number of patients tested was not sufficient to reach statistical
significance. In other hematologic malignancies, such as
AML or ALL, cyclin D2 mRNA was inconstantly detected
by Northern blot, and the level of cyclin D2 expression in
the positive cases was moderate, even though the diseases
were highly proliferative ones.
Because the human cyclin D2 gene (CCND2) has been
mapped to chromosome 1 2 ~ 1 and
3 ~ trisomy 12 is the commonest chromosomal change in B-CLL and immunocytowe looked for a possible relationship between the
presence of a chromosome 12 abnormality and the level of
cyclin D2 overexpression. A trisomy 12 was found in 4 of
19 (21%) of B-CLL and LPL cases for which cytogenetic
analysis was available, and a translocation involving the long
arm of chromosome 12 was observed in an additional LPL
case, but these samples did not exhibit a particular pattern
of cyclin D2 expression. In B-CLL, the trisomy 12 is usually
detected early in the course of the disease and does not
appear to be a secondary chromosomal change.44Among the
structural abnormalities of chromosome 12 also described in
B-CLL, few of them specifically involve the 1 2 ~ 1 regi0n.4~
3
Despite the frequency of chromosome 12 abnormalities in
B-CLL, no critical gene located on this chromosome has
been identified so far that could contribute to the pathogenesis of the disease. Cyclin D2 gene might be a potential candidate for such a role and deserves further studies.
The overexpression of any G1 cyclin by transfection into
fibrobla~ts’~.’’,~~
or myeloid cells15 results in a shortening of
the G1 phase, but the total length of cell cycle remains
unchanged because of a compensatory prolongation of S
phase. However, this effect is more obvious for the cyclins
D l , D3, and E than for cyclin D2. Cyclin D2 overexpression
Fig 4. ISH with cyclin D2 RNA
probe (original magnification
x 100). Lymphoid cells from a patient with advancedB-CLL are
strongly positive with the antisense probe (on the left),
whereas control with sense
probe is negative (on the right).
From www.bloodjournal.org by guest on November 10, 2014. For personal use only.
2875
OVEREXPRESSION OF CYCLIN D2 IN B-CLL
in B-CLL was not associated with such alterations in cell
cycle repartition. Our cell cycle analysis data confirmed the
results of previous kinetic studies in B-CLL by showing that
only a very low subset of B-CLL cells were in S phase,
whatever the level of cyclin D2 expression was.
Cyclin D2 is likely to exert a distinctive function among
G1 cyclins. We have previously shown that normal human
resting T lymphocytes contained very low levels of cyclin
D2 mRNA.I6 Both cyclin D2 mRNA and protein were upregulated very quickly in GI, as soon as 2 hours after mitogenic
stimulation, suggesting that cyclin D2 might play a role in
GO/Gl transition. Other G1 cyclins were not expressed in
GO lymphocytes (except cyclin C) and appeared much later
in G1. Similarly, in normal human B lymphocytes (our data)
and in mouse B lymphocytes (C. Sautts, personal communication, May 1994), cyclin D2 mRNA was rapidly induced
after stimulation by PMA plus ionomycin and anti-IgM or
LPS, respectively. Therefore, in B-CLL, the overexpression
of cyclin D2 might reflect the progression of lymphocytes
in an early G1 phase, without implicating further passage
into S phase. One can hypothesize that being in early G1
instead of in a GO state could allow these cells to be responsive to some external signals such as cytokines or to activate
a set of genes that play a role in survival.
In human normal lymphocytes or cancer lymphoid cell
lines, cyclin D l and cyclin D2 were found to bedifferentially
expressed, whereas cyclin D3, cyclin E, and all other cell
cycle regulatory genes were almost ubiquitously det e ~ t e d . ’Cyclin
~ . ~ ~ Dl was silent in cell lines that do not bear
1lq13 chromosomal rearrangements, whereas cyclin D2 was
expressed in various cell lines without any association with
peculiar chromosomal changes, the highest levels being observed in cell lines immortalized by Epstein-Barr virus?6
Both cyclins were growth factor dependent in different cell
models and could be considered good candidates for potential oncogenes.
Some transfection experiments have suggested that cyclin
D2 could act more as a survival factor than a proliferative
one. In the interleukin-3 (IL-3)-dependent 32D myeloid cell
line, cyclin D2 was quickly downregulated when the cells
were deprived in growth factor, and cells died through
apopto~is.’~
Constitutive overexpression of cyclin D2 or D3
by stable transfection into this cell line appeared to be able
to differ cell death after IL-3 deprivation. In the murine
erythroleukemic cell line MEL, exposure to HMBA induced
cell differentiation and was associated with loss of proliferation and ultimately cell death. In MEL cells stably
transfected with cyclin D2, HMBA still induced differentiation butcell viability was sustained for weeks:’ These experiments have provided evidence that overexpression of cyclin
D2 may alter the cell death program by preventing its normal
activation after growth factor deprivation or induction of
differentiation.
We observed that overexpression of cyclin D2 mRNA
was an almost constant feature in B-CLL and the closely
related lymphoproliferative disorder LPL, whereas its detection was far less consistent in other hematologic malignancies Characterized by a more proliferative activity such as
AML. As shown by ISH experiments, this overexpression
was uniformly detected in all B-CLL cells and not only a
subset of them. Whether it may play a prominent role in
pathogenesis and/or progression of the disease or represent
only a secondary change remains to be elucidated. Further
studies should allow us to know if cyclin D2 prevents BCLL cells from undergoing programmed cell death and allow
us to investigate its relationship with other partners involved
in the control of apoptosis.
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