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: firstname.lastname@example.org.
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 ?
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.
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www.pnas.org?cgi?doi?10.1073?pnas.231467698 Malstrom et al.