Tumor induction by an Lck-MyrAkt transgene is
delayed by mechanisms controlling the size
of the thymus
Scott Malstrom*†‡, Esmerina Tili*‡, Dietmar Kappes§, Jeffrey D. Ceci¶, and Philip N. Tsichlis*?
*Kimmel Cancer Center, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA 19107;¶Department of Human
Biology and Genetics, Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, TX 77555; and§Fox Chase Cancer Center,
Philadelphia, PA 19111
Communicated by Hilary Koprowski, Thomas Jefferson University, Philadelphia, PA, September 4, 2001 (received for review March 23, 2001)
Transgenic mice expressing MyrAkt from a proximal Lck promoter
construct develop thymomas at an early age, whereas transgenic
mice expressing constitutively active Lck-AktE40K develop primar-
ily tumors of the peripheral lymphoid organs later in life. The
thymus of 6- to 8-week-old MyrAkt transgenic mice is normal in
size but contains fewer, larger cells than the thymus of nontrans-
genic control and AktE40K transgenic mice. Earlier studies had
shown that cell size and cell cycle are coordinately regulated. On
the basis of this finding, and our observations that the oncogenic
potential of Akt correlates with its effect on cell size, we hypoth-
esized that mechanisms aimed at maintaining the size of the
thymus dissociate cell size and cell cycle regulation by blocking
MyrAkt-promoted G1progression and that failure of these mech-
anisms may promote cell proliferation resulting in an enlarged
neoplastic thymus. To address this hypothesis, we examined the
cell cycle distribution of freshly isolated and cultured thymocytes
from transgenic and nontransgenic control mice. The results
showed that although neither transgene alters cell cycle distribu-
tion in situ, the MyrAkt transgene promotes G1 progression in
culture. Freshly isolated MyrAkt thymocytes express high levels of
cyclins D2 and E and cdk4 but lower than normal levels of cyclin D3
and cdk2. Cultured thymocytes from MyrAkt transgenic mice, on
hypothesized organ size control mechanisms may down-regulate
the expression of this molecule. Primary tumor cells, similar to
MyrAkt thymocytes in culture, express high levels of cyclin D3.
These findings support the hypothesis that tumor induction is
caused by the failure of organ size control mechanisms to down-
regulate cyclin D3 and to block MyrAkt-promoted G1progression.
tidylinositol-3-kinase-dependent mechanism (for review, see ref.
1 and references therein). Akt1 (or c-akt) is the cellular homolog
of the retrovirus-transduced oncogene v-akt. The virus carrying
(1) and causes T cell lymphomas when inoculated into newborn
AKR mice (1). Moreover, v-akt, but not c-akt, is highly onco-
genic when expressed in the nononcogenic rat T cell lymphoma
line 5675 (1). v-akt contains an amino-terminal myristoylation
signal and is constitutively active. Given that c-akt with a
Src-derived myristoylation signal (MyrAkt1, referred to as
MyrAkt hereafter) is also constitutively active (1), we asked
whether MyrAkt is also oncogenic. To address this question, we
constructed MyrAkt and c-akt transgenic mice expressing the
transgene in the thymus from a proximal Lck promoter con-
struct. A constitutively active Akt mutant (AktE40K) was used
as a control.
In this report, we show that MyrAkt induces thymic lympho-
mas with a short latency, whereas AktE40K induces lymphomas
that arise primarily in peripheral lymphoid organs later in life.
The fact that, despite the expression of constitutively active Akt,
lymphomas develop later in life suggested that oncogenesis by
he Akt protooncogene encodes a serine?threonine protein
kinase that is activated by a variety of signals by a phospha-
the constitutively active Akt transgenes is a multiple-step pro-
cess. The experiments presented here were designed to address
the nature of subsequent events in tumor induction, focusing
primarily on the MyrAkt transgenic mice. First we examined the
size and cellularity of the transgenic and normal control thymus
in young preleukemic mice. These results showed that thymo-
cytes expressing the MyrAkt transgene are larger than normal
control thymocytes or thymocytes expressing the AktE40K
transgene. Despite the increase in cell size induced by the
MyrAkt transgene, however, the size of the thymus of young
MyrAkt transgenic mice was the same as that of young normal
mice and AktE40K transgenic mice. This outcome is because the
MyrAkt transgenic thymus contains fewer, larger cells. This led
us to explore the correlation between cytomegaly and oncogen-
esis. The main questions we addressed were (i) what is the mech-
anism responsible for the low cellularity of the MyrAkt thymus,
and (ii) is tumor induction caused by failure of the regulatory
mechanisms aimed at preserving the size of the thymus?
Before cell division, cells synthesize macromolecules that will
be equally distributed between the daughter cells. Accumulation
of these molecules in the parental cells is responsible for a
gradual increase in cell size. This increase is monitored by the
dividing cells, which progress from the G1phase to the S phase
of the cell cycle only after they pass a minimal size checkpoint.
Under normal conditions, cell growth and cell cycle progression
are coordinately regulated (2–4). However, early genetic studies
in the yeast Saccharomyces cerevisiae (5, 6) and more recent
genetic studies in mammals (7–9) have shown that the two can
be dissociated. The yeast studies were particularly informative in
that they identified two classes of mutations, one that blocked
cell cycle progression but allowed cell growth to continue, and
a second one that coordinately blocked both cell growth and cell
division. The first class of mutations affected cell cycle regula-
tors, whereas the second class affected various biosynthetic
cycle progression can be dissociated but also that inhibition of
cell growth exerts a dominant effect on the cell cycle (10).
The preceding description of the relationship between cell size
and cell cycle progression applies to single cells. Multicellular
organisms and their organs sense either the total cell mass (11,
12) or the total cell number (13, 14) to initiate homeostatic
signals that are superimposed on the intrinsic cellular signals
regulating cell cycle progression (15–17) or cell death (18–20).
The purpose of these signals is to couple the total cell number
Abbreviations: SP, single-positive; DP, double-positive; HA, hemagglutinin epitope.
†Present address: Xenogen Corporation, Alameda, CA 94501.
‡S.M. and E.T. contributed equally to this work.
?To whom reprint requests should be addressed at: Kimmel Cancer Center, BLSB Rm. 539,
233 South 10th Street, Philadelphia, PA 19107. E-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
December 18, 2001 ?
vol. 98 ?
no. 26 ?
or the total cell mass in a given organ or organism with the
pathways that regulate cellular proliferation and death. The final
outcome varies. Thus, tetraploid salamanders contain cells that
are approximately double the size of the cells of diploid
salamanders. These animals sense the larger size of the tet-
raploid cells and initiate signals aimed at maintaining the
constancy of the total cell mass. As a result, the body size of
tetraploid and diploid salamanders is the same despite the fact
that tetraploid salamanders have only half the number of cells
(21). On the other hand, changes in the size of the cells in the
Drosophila wing and eye imaginal discs, induced by mutations of
Dp70S6K(13), or Dakt (14), result in larger adult structures for
these compartments, suggesting that the aim of signals resulting
from sensing these changes is to maintain the total number of
The signals that regulate organ size are either systemic or
originate in the organ itself. This was demonstrated by thymus
and spleen transplantation experiments in mice. All multiple
fetal thymuses transplanted into developing mice grow to adult
size (22), suggesting that their growth depends on control
mechanisms intrinsic to the thymus. Multiple fetal spleens
transplanted in developing mice, on the other hand, grow until
their total mass attains the size of one adult spleen (23).
Therefore, spleen growth differs from thymus growth in that it
depends on systemic factors. These local and systemic mecha-
nisms regulate organ size by initiating signals that are superim-
posed on intrinsic cellular controls that promote or inhibit
cellular proliferation or apoptosis.
The cell cycle distribution of freshly isolated MyrAkt trans-
genic thymocytes was similar to that of normal control thymo-
cytes and AktE40K thymocytes, suggesting a dissociation be-
tween the regulation of cell size and the regulation of cell cycle
progression in the MyrAkt transgenic thymus. This dissociation
was not observed in cultures of CD8 single-positive (SP) trans-
genic thymocytes from MyrAkt transgenic mice, which were
shown to contain fewer cells in G1 and more cells in S by
comparison with cultures of normal control and AktE40K
thymocytes. The expression of cyclin D3 and cdk2 was lower in
freshly isolated MyrAkt thymocytes than in freshly isolated
normal control and AktE40K thymocytes. However, MyrAkt
transgenic thymocytes in culture expressed cyclin D3 at levels
comparable to or higher than those of cultured control and
AktE40k thymocytes. These data collectively suggest that local
mechanisms regulating the size of the thymus do so by down-
regulating the expression of cyclin D3 and, perhaps, other cell
cycle regulatory molecules such as cdk2. These mechanisms,
which are not operating on CD8 SP cells in culture, may prolong
the cell cycle by resetting the cell size checkpoint so that cells
progress through the cell cycle only after they attain a larger size.
Developing tumors express high levels of cyclin D3 similar to
cultured thymocytes from MyrAkt-expressing mice. These data,
combined, support a model according to which genetic changes
promoting cell cycle progression are not sufficient for tumor
induction because they are neutralized by control mechanisms
that regulate organ size. The balance established between the
two opposing forces, however, is unstable. Tumor induction may
result from disruption of this balance because of failure of the
mechanisms that regulate organ size. The model presented here
suggests a protective mechanism that explains why genetic
changes that transform cells in culture may not be sufficient to
induce tumors in animals.
Materials and Methods
Mice. Hemagglutinin epitope (HA)-tagged wild-type Akt,
MyrAkt, and AktE40K were cloned into the pLck-GH vector
(24). The linearized constructs were microinjected into oocytes
from (C57BL?6 ? C3H?He)F2 mice (25). Nine c-akt, 18
AktE40K, and 21 MyrAkt transgene-positive founders were
identified. Transgene-positive founders were crossed to
C57BL?6 mice. Two of the AktE40K (E-3 and E-4) and three of
the MyrAkt (M-1, M-9, and M-24) mice expressing high or low
levels of the transgenes (Fig. 1A) were maintained as transgenic
lines. The MyrAkt transgenic line M-24, which expressed the
highest levels of the transgene, was lost because mice died of
thymic lymphomas at a very young age. The c-akt transgenic
mice were indistinguishable from the nontransgenic controls and
they will not be discussed further in this report. The mice used
generations. Other experiments were carried out with mice from
the 5th to the 7th backcross generations.
Single-Cell Suspension and Culture of Mouse Thymocytes. Single-cell
suspensions of thymocytes were washed twice in Dulbecco’s
minimal essential media (DMEM) (BRL?GIBCO), supple-
mented with nonessential amino acids (100 ?M each), 2-
mercaptoethanol (50 mM), penicillin (100 units?ml), strepto-
mycin (100 ?g?ml), and fetal bovine serum (FBS; 10%).
Subsequently, single-cell suspensions of thymocytes in the cell
wash media (0.5 ? 106cells per ml), were stimulated with
concanavalin A (Con A) (50 ?g?ml). Twenty-four hours later,
the cells were washed and resuspended in the same medium
supplemented with IL-2 (50 units?ml) and conditioned medium
harvested from rat splenocytes stimulated with Con A for 24 h
(Con A Sup) (10%).
Antibodies, Flow Cytometry, and Western Blotting. FITC-labeled
anti-mouse CD4 and Cy-chrome or phycoerythrin (PE)-labeled
anti-mouse CD8 antibodies were purchased from PharMingen.
With the exception of horseradish peroxidase-conjugated anti-
mouse and anti-rabbit IgG, which were purchased from Life
Sciences, the rest of the antibodies were purchased from Santa
and E-4) transgenic mouse lines. Ntg, nontransgenic control. (Right) Western
blot of cell lysates derived from sorted mouse thymocytes probed with the
anti-HA antibody shows that the transgene is expressed in CD4?, CD8?, and
CD4??CD8?double-positive (DP) but not CD4??CD8?double-negative (DN)
cells. (B) Mortality curves of transgenic mouse lines.
Biological consequences of constitutively active Akt transgenes in
www.pnas.org?cgi?doi?10.1073?pnas.231467698 Malstrom et al.
CD4 and?or CD8 staining was done following the protocols of
labeled antibody suppliers. Flow cytometry was carried out on a
Coulter EPICS XL-MCL flow cytometer. Data were analyzed by
using the software program WIN MDI 2.8. To determine the cell
cells were stained with ethidium bromide (EtdBr) in staining
buffer (3.4 mM sodium citrate, pH 7?10 mM NaCl?0.1% Non-
idet P-40?75 ?M EtdBr) (26). The DNA content of stained
nuclei was determined by flow cytometry.
Pellets of freshly isolated or cultured mouse thymocytes were
lysed in 200 ?l of Nonidet P-40 (20 mM Tris?HCl, pH 7.5?150
mM NaCl?1% Nonidet P-40?10% glycerol) or RIPA (50 mM
Tris?HCl, pH 7.5?150 mM NaCl?1% Triton X-100?0.1% SDS?2
mM EDTA?0.5% sodium deoxycholate) lysis buffer. Western
blotting was carried out by standard procedures.
MyrAkt and AktE40K Transgenes Differ in Oncogenic Potential. Thy-
mocytes from AktE40K and MyrAkt transgenic mice were
stained with antibodies recognizing a series of developmentally
regulated membrane molecules. Flow-cytometric analysis of the
stained cells showed that the AktE40K and the MyrAkt trans-
genes induce similar shifts in thymocyte subpopulations. One of
these shifts involved the CD4 and CD8 SP cells, whose relative
numbers were increased by both transgenes (data not shown).
Despite the fact that the two transgenes exert similar effects on
thymopoiesis, MyrAkt mice die of lymphomas at a significantly
younger age than AktE40K mice (Fig. 1B). Lines expressing
higher levels of the transgene died at a younger age than lines
expressing lower levels of the transgene. Thus, M-1 mice died
earlier than M-9 and E-3 mice died earlier than E-4. Ninety-
seven percent of MyrAkt and 93% of AktE40K transgenic mice
were autopsied at the time of death. The results (Table 1)
revealed that, whereas MyrAkt transgenic mice die primarily of
thymic lymphomas, AktE40K transgenic mice die primarily of
lymphoid neoplasms arising in peripheral lymphoid organs. All
tumors carried clonal rearrangements in the T cell receptor
genes (data not shown). MyrAkt (M-9) transgenic mice, which
express lower levels of the transgene, occupy an intermediate
position between MyrAkt (M-1) and AktE40K transgenic mice
regarding the frequency of lymphomas arising in peripheral
lymphoid organs. This observation suggests that the phenotypic
differences between the tumors arising in MyrAkt and AktE40K
transgenic mice are likely to result from differences in specific
kinase activity between the products of the two transgenes (1)
rather than from qualitative differences between them. Histo-
logically, all tumors were lymphoblastic lymphomas. The tumor
surface phenotype was variable with some expressing a CD4 or
CD8 SP, and some expressing a CD4?CD8 DP phenotype.
Interestingly, an Lck-MyrAkt2 transgene is equally oncogenic
with the Lck-MyrAkt1 transgene described here (27). To deter-
mine why the MyrAkt and AktE40K transgenes differ in onco-
genic potential, we first examined whether they differ in their
ability to inhibit apoptosis induced by growth factor (IL-2 and
Con A Sup) withdrawal. The results showed that both transgenes
protect cells from apoptosis as expected (1). Earlier studies using
mice expressing v-akt transgene from a CD2 promoter construct
had shown that the transgene did not prevent antigen-induced
clonal deletion of thymocytes expressing P14, a class I MHC-
restricted T cell receptor transgene (28). This finding suggests
that the activated Akt transgenes are unlikely to promote tumor
induction by inhibiting negative selection of DP thymocytes.
The v-akt transgenic mice described in the preceding para-
graph exhibit T cell protection from Fas-induced apoptosis.
However, these mice do not develop tumors. Instead, when they
grow old, they develop an autoimmune syndrome that has been
attributed to enhanced T cell survival (29). The low oncogenic
potential of v-akt by comparison with MyrAkt in mice is remi-
niscent of the low oncogenic potential of v-akt in chickens (30).
Other factors that may contribute to the inability of the v-akt
transgene to induce tumors in mice may be the promoter used
and the level of expression of the transgene.
MyrAkt Transgenic Thymocytes Are Larger than AktE40K Transgenic
and Nontransgenic Control Thymocytes. To determine whether
expression of the MyrAkt or AktE40K transgenes affects the
cellularity of the thymus, thymuses of six MyrAkt (line M-1), six
AktE40K (line E-3), and six nontransgenic animals were
weighed and processed to generate single-cell suspensions. The
results showed that all thymuses were identical in size by weight
(Fig. 2A). However, the thymuses of MyrAkt transgenic mice
contained fewer cells (Fig. 2B). This result suggested that
thymocytes of MyrAkt transgenic mice are larger. This predic-
tion was confirmed by flow cytometry (Fig. 2C).
The size increase of the thymocytes derived from MyrAkt
transgenic mice could be the direct result of MyrAkt expression.
Alternatively, it may represent the cellular response to cytokines
produced by the MyrAkt-expressing cells. To distinguish be-
tween these possibilities, we examined whether large size is a
thymocytes were stained with anti-CD4 and anti-CD8 antibod-
ies. Forward scatter analysis revealed that whereas the MyrAkt
DP and SP cell populations were enlarged, the double-negative
(DN) cells were not (Fig. 2D). Because the SP and the DP cells
also express the transgene (Fig. 1A), we conclude that the effect
of MyrAkt on the cell size is cell autonomous. Collectively, the
data in Fig. 2 suggest that the thymus senses the large size of the
cells expressing MyrAkt and initiates signals aimed at maintain-
ing the total thymic mass constant (22). As a result, the increase
in cell size is accommodated by a reciprocal decrease in cell
Table 1. Incidence, latency, and spectrum of lymphomas induced by the MyrAkt and AktE40K transgenes
mice Deaths Thymomas
Age of animals at
time of death, days
146.6 ? 131.0
127.8 ? 77.0
193.0 ? 97.8
66.5 ? 0.9
525.7 ? 165.3
264.0 ? 117.3
297.3 ? 119.5
499.5 ? 133.3
Mice were monitored for a total of 600 days. Ages are mean ? SD.
*This column includes mice that were discovered dead, and because of autolysis the cause of their death could not be determined objectively.
Malstrom et al. PNAS ?
December 18, 2001 ?
vol. 98 ?
no. 26 ?
The MyrAkt and AktE40K Transgenes Do Not Alter the Cell Cycle
Proteins in Freshly Isolated Thymocytes. Given that cell growth and
the cell cycle are normally coordinately regulated, we examined
whether the MyrAkt transgene stimulates cell cycle progression.
Single-cell suspensions of freshly isolated transgenic and non-
transgenic thymocytes were stained with ethidium bromide and
analyzed by flow cytometry. The results showed that the cell
cycle distribution of thymocytes was similar in all mice (Fig. 3
Left). We conclude that cell size and cell cycle regulation are
dissociated in the thymus of MyrAkt transgenic mice.
Previous studies had shown that constitutively active Akt
up-regulates the expression of G1cyclins by enhancing protein
translation (31) and stability (32). These studies were carried out
in cultured cells in which constitutively active MyrAkt promotes
cell cycle progression (31, 32). Because constitutively active
MyrAkt does not promote cell cycle progression in transgenic
thymocytes in situ (Fig. 3 Left), we proceeded to also examine the
expression of G1 cyclins in freshly isolated thymocytes. The
results showed that cyclins D2 and E were up-regulated, whereas
cyclin D3 and cdk2 were down-regulated, and that the down-
regulation of these molecules was more pronounced in the
MyrAkt transgenic line M-1, which expresses higher levels of the
transgene (Fig. 3 Right). cdk4 expression was also marginally
elevated in the M-1 MyrAkt transgenic mouse line. The unex-
pected down-regulation of cyclin D3 and cdk2 in the MyrAkt
transgenic thymus suggests that these molecules may be the
target of dominant inhibitory signals aimed at maintaining the
normal size of the thymus.
The MyrAkt and AktE40K transgenic mouse lines differed
with regard to the expression of G1cyclins, cdk2, and perhaps
cdk4. However, the expression of p27Kipdid not distinguish
between these lines in that it was down-regulated in all (Fig. 3
Right). Similarly, GSK3? phosphorylation and ?-catenin expres-
sion were up-regulated in all transgenic lines (data not shown).
The MyrAkt Transgene Promotes G1 Progression in Culture. To de-
termine whether the normal cell cycle distribution of MyrAkt
we examined the cell cycle distribution of transgenic and normal
when in culture, the thymocytes would not be subject to the
postulated inhibitory mechanisms. Thymocytes from MyrAkt and
AktE40K transgenic mice and nontransgenic controls (four mice
Sup. Five days later, growing CD8 SP thymocyte cultures had been
established (data not shown). Cells from these cultures were
subcultured in the same medium at 0.5 ? 106cells per ml.
Twenty-four hours later, 10% of the cells in each culture were
stained with ethidium bromide and analyzed by flow cytometry to
determine their cell cycle distribution. The results showed that
cultures of MyrAkt transgenic thymocytes consistently contained a
lower percentage of cells in G1and a higher percentage of cells in
S phase (Fig. 4A). Western blots of lysates from these cells revealed
that not only cyclin D2, but also cyclin D3, was up-regulated (Fig.
4B). These findings suggest that elimination of the intrathymic
inhibitory signals allows MyrAkt to up-regulate the expression of
cyclin D3. Alternatively, up-regulation of cyclin D3 occurs as the
cells mature to the SP stage, at which point they leave the thymus
to populate the peripheral lymphoid organs.
and nontransgenic control thymocytes. (A) The weight of the thymus of
age- and sex-matched mice from each group). (B) Thymuses of MyrAkt1
transgenic mice contain fewer cells than thymuses of nontransgenic or
AktE40K transgenic mice. (C) Cell size (forward scatter, FS) comparisons be-
tween nontransgenic (Non-Tg) control and AktE40K, nontransgenic control
and MyrAkt, and AktE40K and MyrAkt thymocytes. MyrAkt thymocytes are
larger. (D) The effect of MyrAkt on the cell size is cell autonomous. CD4 and
derived from nontransgenic control mice. DN, double-negative.
MyrAkt transgenic thymocytes are larger than AktE40K transgenic
isolated transgenic and nontransgenic control thymocytes. (Left) Cell cycle
distribution of freshly isolated thymocytes from nontransgenic, MyrAkt, and
AktE40K transgenic mice. There were four 6- to 8-week-old, age- and sex-
matched mice per group. (Right) Expression of cell cycle regulators in freshly
isolated thymocytes from nontransgenic control and transgenic mouse lines.
Lanes: 1, nontransgenic; 2, MyrAkt1 M-1; 3, MyrAkt1 M-9; 4, Akt1-E40K E-3;
and 5, Akt1-E40K E-4. For cyclin D3 and cyclin D2, the results of two indepen-
to cyclin D2 and cyclin D3. The intensity of individual bands was measured by
densitometry. Band intensity ratios were as follows: cyclin D3, nontransgenic
and 0.45; cyclin E, nontransgenic vs. MyrAkt M-1 transgenic thymocytes ?
0.25; cdk2, nontransgenic vs. MyrAkt M-1 transgenic thymocytes ? 2.89.
www.pnas.org?cgi?doi?10.1073?pnas.231467698Malstrom et al.
mice (six mice per group) growing in IL-2 and Con A Sup-
106cells per ml. Twenty-four hours later, the cells were starved of
IL-2 and Con A Sup for 10 h, at which point they were analyzed for
their cell cycle distribution. The remaining cells were restimulated
and they were harvested and analyzed for their cell cycle distribu-
tion at 2, 4, 6, and 12 h after restimulation. The results showed that
10 h after starvation, a high percentage of MyrAkt-expressing cells
continued to cycle. Restimulation promoted reentry of nontrans-
genic and AktE40K transgenic thymocytes into the cell cycle but
had a lesser effect on the cell cycle distribution of MyrAkt trans-
genic thymocytes (Fig. 4C). Lysates of cells harvested before
starvation and at 0, 2, 4, and 6 h after restimulation were analyzed
cyclins D2 and D3 were both expressed at higher levels in cultured
MyrAkt transgenic thymocytes and that they were only partially
down-regulated after starvation. We conclude that the MyrAkt
transgene enhances the expression of G1 cyclins and cell cycle
progression in culture.
Primary Thymomas from MyrAkt Transgenic Mice Express High Levels
of Cyclin D3. Despite the cell cycle stimulatory activity of the
MyrAkt transgene, which can be detected early in life, tumors
develop significantly later. On the basis of the preceding data, we
propose that the delayed induction of thymic lymphomas in the
transgenic mice may be caused by failure of cell cycle inhibitory
mechanisms aimed at maintaining the normal size of the thymus.
To test this hypothesis, we examined the expression of cyclin D3
in freshly isolated cells from six thymomas arising in MyrAkt
transgenic mice. The results (Fig. 5) showed that tumor cells are
similar to cultured thymocytes in that they express high levels of
cyclin D3. We conclude that the tumor cells did escape the
inhibitory signals down-regulating the expression of cyclin D3 in
thymocytes in situ.
Evidence presented in this report shows that two constitutively
active Akt transgenes, MyrAkt and AktE40K, expressed in the
thymus from a proximal Lck promoter construct, are oncogenic.
MyrAkt induces primarily thymic tumors with a short latency,
whereas AktE40K exhibits long latency and induces primarily
lymphomas of the peripheral lymphoid organs. The delayed
appearance of MyrAkt and AktE40K-induced lymphomas sug-
gests that tumor induction by the constitutively active Akt
transgenes is a multiple-step process. The experiments presented
here were designed to address the potential mechanism by which
a second step may promote tumor induction in the MyrAkt
Earlier studies had shown that the size of the thymus in mice
is regulated by signals intrinsic to the thymus (22). In this report,
we presented evidence that these signals monitor the total
thymic cell mass. As a result, the increase in cell size induced by
a MyrAkt transgene expressed in the thymus is accompanied by
a decrease in the total cell number so that the total cell mass
remains constant. The mammalian thymus clearly differs from
the Drosophila wing and eye imaginal discs, where the aim of
genic mice. (A) Cell cycle distribution of cultured thymocytes from nontrans-
genic, MyrAkt, and AktE40K transgenic mice. Cells were grown in complete
(B) Expression of cyclin D3 and cyclin D2 in freshly isolated nontransgenic and
MyrAkt transgenic thymocytes, as well as in cultured MyrAkt and AktE40K
thymocytes. The same blot was probed with both antibodies. (C) Percent of
cells in S phase. Prestarved (ps) are cells growing exponentially in complete
medium. Data are combined from cultures of thymocytes derived from four
cycle regulators in cultured thymocytes from nontransgenic, MyrAkt, and
AktE40K transgenic mice.
Cell cycle distribution and expression of cell cycle regulators in
MyrAkt transgenic thymocytes, as well as from freshly isolated tumor cells
from six thymomas (three in each of two separate experiments) derived from
MyrAkt transgenic mice were probed with an antibody to cyclin D3. (B) Equal
loading was confirmed in the second experiment by reprobing with an anti
?-actin antibody (Sigma).
Primary thymomas from MyrAkt transgenic mice express high levels
Malstrom et al. PNAS ?
December 18, 2001 ?
vol. 98 ?
no. 26 ?
signals sensing changes in the cell size is to maintain the number
of cells constant. As a result, changes in cell size in these
compartments result in changes in organ size (13, 14). The
differences between the mammalian thymus and the Drosophila
wing and eye imaginal discs suggest that the regulation of organ
size differs between organs or organisms.
During cell cycle progression, cells increase in size as they
accumulate proteins and other macromolecules that are synthe-
the daughter cells will contain a full complement of macromol-
ecules, cells progress from G1to S only after they cross a minimal
size checkpoint (2–4). The coordinate regulation of the cell size
and the cell cycle predicts that a primary cell size-enhancing
signal induced by MyrAkt should speed up passage through the
minimal size checkpoint. This prediction had been confirmed in
MyrAkt-expressing cells in culture (33, 34). Stimulation of G1
progression in animal cells in situ if unchecked, however, would
increase the size of the organ containing these cells. Data
presented in this report suggest that this increase is prevented by
intrathymic signals, which are triggered by the increased size of
the cells and which elicit events aimed at maintaining the thymic
cell mass constant. These signals may limit the number of
thymocytes by any of three non-mutually exclusive mechanisms:
(i) they may increase apoptosis (18–20); (ii) they may reset the
minimal size checkpoint so that a larger cell size is required to
permit the transition from G1to S (7–11); or (iii) they may limit
the number of cell divisions T cell precursors may undergo
during maturation (35–38). MyrAkt exerts a strong anti-apopto-
tic effect (1) which makes the first possibility unlikely. On the
other hand, MyrAkt stimulates G1progression in primary cul-
tures of CD8 SP T cells derived from the thymus, but not in
thymocytes in situ, suggesting that the intrathymic signals lim-
iting the number of enlarged thymocytes operate by inhibiting
cell cycle progression.
Although we do not yet know the nature of the signals that
maintain the total mass of thymocytes constant, we addressed the
mechanism by which such signals inhibit cell cycle progression.
Monitoring the expression levels of several cell cycle regulators
revealed that cyclin D3 and cdk2 are down-regulated in MyrAkt
thymocytes in situ. However, cyclin D3 is up-regulated in MyrAkt
thymocytes in culture. This is specific for cyclin D3 and cdk2,
because cyclin D2 and cdk4 are up-regulated in MyrAkt transgenic
thymocytes in situ. The increase in cyclin D2 expression may result
from the direct stimulatory effect of constitutively active Akt on
protein translation or stability (31, 32).
On the basis of these findings, we hypothesized that the thymus
of MyrAkt transgenic mice is the battleground of two opposing
cell cycle progression and a second one that is triggered by the cell
size increase induced by the transgene and inhibits the cell cycle.
Failure of the cell cycle inhibitory signals will disrupt the unstable
balance between these forces and will allow an increase in thymic
cellularity and size. The correlation of cytomegaly with predispo-
sition to neoplastic transformation in MyrAkt transgenic mice
in tumor induction. This hypothesis is supported by data showing
cultured MyrAkt transgenic thymocytes in that they express high
levels of cyclin D3. It remains to be determined whether cyclin D3
(and other cell cycle regulators) synergize with the MyrAkt trans-
gene in oncogenesis.
In summary, the data presented here, support a hypothesis
transgene is delayed by conservative signals intrinsic to the
targeted organ and aimed at maintaining the normal size of the
organ. According to this hypothesis, tumor induction results
from failure of the unstable balance between the transgene and
the intrinsic inhibitory signals. Exploring the nature of these
inhibitory mechanisms may provide new insights into the pre-
vention and treatment of cancer.
Note Added in Proof. The larger size of mammalian cells constitutively
expressing Akt1 was also reported in a paper by Tuttle et al. (39) that was
published while the present paper was in press.
We thank Fuming Pan and Jugin Wang for technical assistance with the
generation of the transgenic mice. This work was supported by National
Institutes of Health Grant R01 CA57436. S.M. was supported by
National Institutes of Health Grant 5-T32-CA09678.
1. Chan, T. O., Rittenhouse, S. E. & Tsichlis, P. N. (1999) Annu. Rev. Biochem.
2. Stocker, H. & Hafen, E. (2000) Curr. Opin. Genet. Dev. 10, 529–535.
3. Su, T. T. & O’Farrell, P. H. (1998) Curr. Biol. 8, 687–689.
4. Mitchison, J. M., Novak, B. & Sveiczer, A. (1997) Cell Biol. Int. 21, 461–463.
5. Hartwell, L. H. (1971) J. Mol. Biol. 59, 183–194.
6. Nurse, P., Thuriaux, P. & Nasmyth, K. (1976) Mol. Gen. Genet. 146, 167–178.
7. Franch, H. A., Shay, J. W., Alpern, R. J. & Preisig, P. A. (1995) J. Cell. Biol.
8. Hemerly, A., Engler, J. d. A., Bergounioux, C., Van Montagu, M., Engler, G.,
Inze, D. & Ferreira, P. (1995) EMBO J. 14, 3925–3936.
9. Sheikh, M. S., Rochefort, H. & Garcia, M. (1995) Oncogene 11, 1899–1905.
10. Johnston, G. C., Pringle, J. R. & Hartwell, L. H. (1977) Exp. Cell. Res. 105, 79–98.
11. Weigmann, K., Cohen, S. M. & Lehner, C. F. (1997) Development (Cambridge,
U.K.) 124, 3555–3563.
12. Neufeld, T. P., de la Cruz, A. F., Johnston, L. A. & Edgar, B. A. (1998) Cell
13. Volarevic, S., Stewart, M. J., Ledermann, B., Zilberman, F., Terracciano, L.,
Montini, E., Grompe, M., Kozma, S. C. & Thomas, G. (2000) Science 288,
14. Verdu, J., Buratovich, M. A., Wilder, E. L. & Birnbaum, M. J. (1999) Nat. Cell
Biol. 1, 500–506.
15. de Nooij, J. C., Letendre, M. A. & Hariharan, I. K. (1996) Cell 87, 1237–1247.
16. Gao, F. B., Durand, B. & Raff, M. (1997) Curr. Biol. 7, 152–155.
17. Henrique, D., Hirsinger, E., Adam, J., Le Roux, I., Pourquie, O., Ish-Horowicz,
D. & Lewis, J. (1997) Curr. Biol. 7, 661–670.
18. Raff, M. C. (1992) Nature (London) 356, 397–400.
19. Calver, A. R., Hall, A. C., Yu, W. P., Walsh, F. S., Heath, J. K., Betsholtz, C.
& Richardson, W. D. (1998) Neuron 20, 869–882.
20. Bangs, P. & White, K. (2000) Dev. Dyn. 218, 68–79.
21. Fankhauser, G. (1952) Int. Rev. Cytol. 1, 165–193.
22. Metcalf, D. (1963) Aust. J. Exp. Med. Sci. 41, 437–448.
23. Metcalf, D. (1964) Transplantation 2, 387–392.
24. Perez, P., Lira, S. A. & Bravo, R. (1995) Mol. Cell. Biol. 15, 3523–3530.
25. Ceci, J. D., Kovatch, R. M., Swing, D. A., Jones, J. M., Snow, C. M., Rosenberg,
M. P., Jenkins, N. A., Copeland, N. G. & Meisler, M. H. (1991) Oncogene 6,
26. Grimes, H. L., Gilks, C. B., Chan, T. O., Porter, S. & Tsichlis, P. N. (1996) Proc.
Natl. Acad. Sci. USA 93, 14569–14573.
28. Jones, R. G., Parsons, M., Bonnard, M., Chan, V. S., Yeh, W. C., Woodgett,
J. R. & Ohashi, P. A. (2000) J. Exp. Med. 191, 1721–1734.
29. Parsons, M. J., Jones, R. G., Tsao, M. S., Odermatt, B., Ohashi, P. S. &
Woodgett, J. R. (2001) J. Immunol. 167, 42–48.
30. Aoki, M., Batista, O., Bellacosa, A., Tsichlis, P. N. & Vogt, P. K. (1998) Proc.
Natl. Acad. Sci. USA 95, 14950–14955.
31. Muise-Helmericks, R. C., Grimes, H. L., Bellacosa, A., Malstrom, S. E.,
Tsichlis, P. N. & Rosen, N. (1998) J. Biol. Chem. 273, 29864–29872.
32. Diehl, J. A., Cheng, M., Roussel, M. F. & Sherr, C. J. (1998) Genes Dev. 12,
33. Ahmed, N. N., Grimes, H. L., Bellacosa, A., Chan, T. O. & Tsichlis, P. N. (1997)
Proc. Natl. Acad. Sci. USA 94, 3627–3632.
34. Brennan, P., Babbage, J. W., Burgering, B. M., Groner, B., Reif, K. & Cantrell,
D. A. (1997) Immunity 7, 679–689.
Cell 85, 829–839.
36. Knoblich, J. A., Sauer, K., Jones, L., Richardson, H., Saint, R. & Lehner, C. F.
(1994) Cell 77, 107–120.
37. Casaccia-Bonnefil, P., Tikoo, R., Kiyokawa, H., Friedrich, V., Jr., Chao, M. V.
& Koff, A. (1997) Genes Dev. 11, 2335–2346.
39. Tuttle, R. L., Gill, N. S., Pugh, W., Lee, J. P., Koeberlein, B., Furth, E. E.,
Polonsky, K. S., Naji, A. & Birnbaum, M. J. (2001) Nat. Med. 7, 1133–1137.
www.pnas.org?cgi?doi?10.1073?pnas.231467698 Malstrom et al.