ICANCERRESEARCH
55, 1452—1457,
April1, 19951
Perspectives in Cancer Research
Transforming
Growth Factor fi and Cell Cycle Regulation'
Mark G. Alexandrow and Harold L. Moses2
Department of Cell Biology and The Vanderbilt Cancer Center, Nashville, TN 37232-2175
In many cases tumor cells develop when normal progenitor cells
lose control of signaling pathways that regulate responses to soluble
growth factors. These signaling pathways consist of the molecular
machinery that regulates cell cycle progression primarily during the
first gap phase (G1) of the cell cycle. Whereas growth factors such as
epidermal growth factor and platelet-derived growth factor stimulate
these signaling pathways (1), other soluble growth-regulatory factors
lead to inhibition of cell growth presumably through mechanisms that
target the same cell cycle-regulated events. Of key interest in the field
of growth-inhibitory peptides are the autocrine/paracrine effectors of
the type 13 transforming growth factors, TGF@31,3 TGF(32, and
TGF@33,the most commonly studied being TGF(31. TGF@1 has been
shown to be a potent inhibitor of the proliferation of most normal cell
types in culture as well as in vivo (2—5).Because many tumorigenic
cell lines have lost responsiveness to the negative growth-regulatory
effects of TGF@1 (6—8),it is anticipated that an understanding of the
molecular
mechanisms
by which TGFj31 arrests
cell growth
will also
provide information relevant to molecular events important in
neoplastic transformation.
The intracellular inhibitory effects produced by TGF@1 are initiated
following ligand binding to oligomeric complexes of high affinity
TGFI3 cell surface receptors (9, 10). The recent cloning of the three
types of high affinity TGFj3 cell surface receptors (1 1—13),designated
types I, II, and III, has helped to provide insights into the mechanisms
by which these receptors generate the cascade of inhibitory effects on
cells. The type I and type II receptors appear to interact with each
other directly (13); while functionally, the type I receptor requires the
type II receptor to bind specific ligands (14—16) and the type II
receptor requires the type I receptor in order to signal (17). The type
III TGFf3 receptor ((3-glycan) is proposed to be involved in regulating
access of ligand, particularly TGFI32, to the TGF(3 receptors but has
not been shown to be directly involved in generating the intracellular
signal transduction (1 1, 18—20).Both the type I and type II TGF(3
receptors contain a cytoplasmic region that shows homology to serine/
threonine kinases (10, 21, 22), and recent evidence suggests that
phosphorylation of the type I receptor by the kinase activity of the
type II receptor may initiate signaling from the heteromeric complex
(23). Because the structure and function of the TGF@3receptors have
TGF@31appears to be able to target factors that act early in G1 as well
as factors acting late in G1, it becomes difficult to determine with
certainty which of these putative targets are directly involved in
TGF@1-negative signaling and the resultant cell cycle arrest. There
fore, it is important to understand not only when in G@TGF@1 is
capable of inhibiting proliferation but also the probable point(s) in G1
at which the cells finally arrest in response to TGFj31. With these
kinetic concepts in perspective one can begin to delineate which
events are directly involved in the suppression of cell growth by
TGF@31and which events are simply consequential to the inability of
the cells to progress through G1 from the TGF@1 arrest point.
Cell CycleKineticsof TGF@31
Inhibition
Studies from several laboratories
have shown that TGF(31 can
inhibit the ability of cells to enter S phase when the inhibitory
peptide
is added to cultures at both early and late points during the prerepli
cative G@period (24, 27, 32, 33). While one of these reports demon
strates that a human keratinocyte cell line, HaCaT, loses sensitivity to
TGF@31approximately 6 h prior to the G1-S transition (33), the others
have shown that TGFf31 is able to inhibit mouse keratinocytes
(BALB/MK) and mink lung epithelial cells (MvlLu) from entering S
phase when added throughout the entire G@period up to the G1-S
transition. In addition, one report has shown that TGF@31may be more
potent at inhibiting the progression of MvlLu cells into S phase when
it is added to synchronized cultures in the latter hours of G@just prior
to the G1-S transition (32). This report also demonstrated that removal
of TGF@31from arrested cells followed by analysis of the kinetics of
entry into S phase showed that the cells might actually be arrested at
a point in G@approximately 1—2h prior to S phase (32). However,
since the latter TGFf31 release experiments were reliant upon the
effective removal of TGF@31from the cultures, which is difficult to
achieve (34), and since it has been reported that the half-life of TGFI31
effects may be a prolonged period of time rather than 1—2
h (35), we
hypothesize that the kinetic effects of TGF(31 on epithelial cells are
more complex than has been suggested previously. In retrospect,
when interpreting results of experiments relating TGFf31 and cell
cycle progression, it is important to distinguish between the ability of
TGF@31to simply inhibit proliferation when added to cultures in early
been the focus of several recent reviews (9, 10, 21), the reader is
or late G1 and the ability of TGF@31to cease cell cycle progression at
directed to these sources for further discussion of the mechanisms by
any particular point in G1 irrespective of the time that TGF@31was
which these receptors function in TGFI3 signal transduction.
added to the cultures.
Several biochemical targets of TGFf31-induced inhibitory signals
Because cells are sensitive to TGF(31 both early and late in G1, we
have been suggested (24—31).Among these are down-regulation of
hypothesize that TGF@31addition early in G1 may arrest cells through
transcription, decreased phosphorylation of target proteins, and mac
mechanisms that are different than those mechanisms by which
tivation of cell cycle-regulated enzymes. The cell cycle kinetics of the
TGF@31arrests cells when the inhibitory factor is added late in G1.
implicated targets of TGF@31suggests that each target normally func
Indeed, the proto-oncogene c-myc has been shown to be an immediate
tions temporally in a certain defined period of time during G1. Since
target ofTGF@1-induced signals (see below; Refs. 29—31)and kinetic
evidence would suggest that c-myc functions, at least in part, in the
Received 12/8/94; accepted 2/2/95.
early
half of G1 as a progression factor (36). Abrogation of c-nzyc
1 This
work
was
supported
by National
Cancer
Institute
Grants
CA42572
and
expression by TGF(31 early in G1 may actually lead to cell cycle arrest
CA48799. M. G. A. was supported in pail by NIH Training Grant DK07563 and United
States Army Breast Cancer Training Grant DAMD17-94-J-4024.
during this period of time. More specifically, progression of the cell
2 To
whom
requests
for
reprints
should
be addressed.
cycle may cease early in G1 due to the absence of c-myc-induced
3 The abbreviations
used are: TGF,
transforming
growth
factor; Cdk, cyclin-dependent
events required for G@progression. In the presence of TGF(31, some
kinase.
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Torn ANDCELLCYCLEREGULATION
01
events
may
occur
normally
and
appear
unaffected
by
to progress to the period in G1 when these cyclins normally become
expressed. In addition, it remains to be shown whether TGF@31treat
ment in late G1 produces the same suppressive effect on cyclin
expression as it does when TGF@31is present throughout the entire G@
period. Data showing down-regulation of cyclins A and/or E at the
transcriptional or post-transcriptional levels after TGF(31 treatment
late in G1 would suggest a more direct role for these cyclins in the
TGFf31 signaling pathway.
The product of the retinoblastoma susceptibility gene (pRB) has
been implicated as a target of TGF(31-induced negative signals and
the activity of pRB or other pRB-related proteins in cells has been
shown to be required for TGFf31 to efficiently suppress cell growth
(24, 27). Although the kinetics of the effects of TGF@1 appears to be
slow, it has been shown that MvlLu cells treated with TGFg31
contained more hypophosphorylated pRB than untreated cells (27).
Since hypophosphorylated pRB has been shown to be the growth
suppressive form of the protein (see Ref. 42 for a review), it has been
suggested that TGFf31-induced accumulation of hypophosphorylated
suppression
of the c-myc pathway, while other pathways may display inhibitory
effects caused by the loss of c-myc expression or simply by the
inability of the cells to progress to the latter part of G@.
The ability of TGF(31 to inhibit MvlLu and BALB/MK cells from
entering S phase when the inhibitory factor is added to cultures in late
G1 (24, 32) presents an interesting situation with important implica
tions for TGFf31 signaling. It has been shown that cells pass through
a restriction point in late G1 termed the R point after which time the
cells are committed to progress into S phase (37, 38). After the R point
at least some cell types can progress into S phase in the presence of
low doses of cycloheximide implying that they no longer require the
translation of a labile protein necessary for G1-S transition (38).
Additionally, in the last 1—2h of G@the cells no longer require the
presence of serum to enter S phase (37) nor is entry into S phase
affected by late G1 treatment of the cells with inhibitors of RNA
transcription (39, 40). This latter observation implies that the cells no
longer require de novo RNA synthesis in the last 2—3h of G1 for
progression into S phase. With these ideas in mind, the observed
ability of TGFI31 to potently inhibit cell cycle progression when
added to cultures in late G1, a time when cells no longer require
production of RNA and translation of certain protein factors, suggests
that TGF@31may be inhibiting initiation of S phase through post
transcriptional and likely post-translational mechanisms. In this re
pRB in the cells is one mechanism that prevents progression into S
phase (24, 27). It was hypothesized from these findings that TGF131
might be inactivating kinases which normally phosphorylate pRB. In
support
spect, potential targets of TGF(31 in late G1 are likely to be the Cdks
and factors which regulate these enzymes and their associated cyclins.
However, this model does not disqualify the hypothesis that TGFj31
addition early in G@is arresting cell growth mechanistically through
down-regulation of transcription of necessary progression factors such
as c-myc. Since cells require de novo RNA synthesis early in G@(39,
40), inhibition of any particular RNA species that is required for cell
cycle progression might be sufficient to cause cell cycle arrest at a
point in time in the early part of G@when activity of this transcribed
factor is necessary.
Cydins and Cdks as Targets of TGF@31Signals
Recent evidence has shown that certain G1 cyclins and cyclin
dependent kinases may be targets of the negative signaling pathways
induced by TGFfi1 (25, 26, 28, 33). Treatment of a human keratino
cyte line (HaCaT) with TGFf31 in the early part of G@results in the
lack of transcriptional induction of both cyclin A and cyclin E in late
G1 (33). The same effect of TGFj31 on cydlin A and cyclin E mRNA
expression has been shown in MvlLu cells (28); however, there is one
report showing that, although cyclin A mRNA is suppressed, TGFf31
treatment
addition,
cyclin E
treatment
of this hypothesis,
it has since been shown
that the activity
and rate of translation of two cell cycle-regulated kinases, Cdk2 and
Cdk4, respectively, are affected by TGF(31 treatment of MvlLu cells
fails to suppress cyclin E mRNA in these cells (41). In
BALBIMK cells also show that cyclin A mRNA, but not
mRNA, fails to increase in late G1 after early G1 TGF(31
(41). In the HaCaT cells, the down-regulation of cyclin A
and E mRNA is followed by a concomitant loss of the protein levels
of both cyclins (33), while in the MvlLu cells only cyclin A protein,
and not cyclin E protein, appears to be down-regulated following
TGF@31treatment (28). The findings described in these reports suggest
that there may be important differences between cell types in the
mechanisms by which TGF@31is able to inhibit proliferation.
Although it is apparent that there are inconsistent data from differ
ent cell types, the observations described above have led to the
hypothesis that TGF$31-induced prevention of cyclin expression may
be causally involved in blocking progression into S phase. It is likely
that loss of cyclin expression is one of the events which leads to cell
cycle arrest, but what is unknown about the effects of TGF@31on
cyclin expression is whether this inhibition is the cause of the growth
arrest or simply a consequence of TGF@31-induced failure of the cells
(25,
26).
As discussed above, it has been reported that, although TGF(31
prevents induction of cyclin E mRNA in some MvlLu strains, there
is no effect of TGF(31 on the total levels of cyclin E protein produced
in the cells (28). One of the cyclin-dependent kinases which associates
with the cyclin E protein, Cdk2, was also found to be unaffected in
expression at any level by TGF@31in these MvlLu cells (25, 28), but
the normal association of Cdk2 and cyclin E in late G1 did not occur
and the late-G1 enzymatic activity of cyclin E/Cdk2 was inhibited in
cells treated with TGFf31 in the early part of G@(25, 28). In addition,
Cdk2 appeared to be dephosphorylated on threonine-160 (25), phos
phorylation of which is presumably required for enzymatic activity of
the cyclin E/Cdk2 complex (43). However, because of the design of
these experiments, it still remains to be shown whether treatment with
TGFf31 in late G1 results in an immediate inactivation of Cdk2
activity. Acute inactivation of Cdk2 in late G1 might be sufficient to
explain the ability of TGFf31 to block entry into S phase since active
Cdk2
complexes
with
both
cyclin
E and cyclin
required for the G1-S transition and progression
A are presumably
through S phase,
respectively.
Cdk4 has been postulated to be another target of TGF@31in that it,
like c-myc, has been shown to be rapidly down-regulated in expres
sion by TGFJ31 treatment of MvlLu cells (26). This regulation of
Cdk4 expression by TGFj31 appears to be at the level of translation of
the protein and the effects on Cdk4 production occur after treatment
with TGFf31 throughout the entire prereplicative G1 period (26).
Further evidence supporting that Cdk4 may indeed be an important
target of TGF@1-induced negative signals was derived from experi
ments showing that ectopically expressed Cdk4, but not Cdk2, could
block the ability of TGFf31 to inhibit the proliferation of MvlLu cells
(26). However, the importance of Cdk4 regulation by TGF@31in the
growth-inhibitory pathway requires further analysis. Although inhibi
tion of Cdk4 translation by TGFf31 may be one of the mechanisms by
which TGFf31 arrests cell growth or holds cells in an arrested state, it
is not sufficient to explain the mechanism by which TGFf31 can
inhibit entry into S phase when TGFf31 is added in late G1. The
induction of the D-type cyclins, the only known regulatory cyclin
subunits for Cdk4, occurs in mid-G1 and presumably leads to activa
tion of the catalytic activity of Cdk4 during this period of time (44).
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TGFB AND CELL CYCLE REGULATION
In addition, it has recently been shown that Cdk4 activity peaks by the
time cells reach late G1 (44). For these reasons, the ability of TGF@1
to inhibit the translation of Cdk4 after cyclin D/Cdk4 has already
become active may only be serving to prevent further translation and
activation of Cdk4 in arrested cells. In this manner, inhibition of Cdk4
translation may not actually cause the growth arrest but may instead
comprise a mechanism by which TGFf31 blocks further cell cycle
stimulation.
Mediation
of TGF@1 Effects by Cdk Inhibitors
The interest in Cdks and cyclins as targets of negative regulatory
signals that are initiated by factors such as TGFf31 has led to the
fortuitous discovery of several small proteins which appear to nega
tively regulate Cdk activity in vitro and in vivo (45—51).The hypoth
esis which directed research efforts at cloning and purifying these Cdk
inhibitors was that inhibitory growth factors, such as TGF@31,or DNA
damage-inducible factors, such as the p53 tumor suppressor protein,
might be inactivating Cdks mechanistically through activation of
latent Cdk inhibitors or through increased expression of the inhibitors.
The first of these Cdk inhibitors to receive attention was the
p21Waf1@@@
gene
product.
Cloned
by
virtue
of
its
ability
to
interact
with and inhibit Cdk2 as well as increase in expression in response to
p53 induction, p2lWa@@C@
appearsto be able to inhibit all cyclin
dependent kinases, including Cdc2, and has been shown to be a
downstream transcriptional target of p.53 (45—47,52). Although a
direct role for p21 in the TGF@1 signaling pathway has not been
definitively shown, initial studies suggest that p21 may participate in
TGF@31-induced cell cycle arrest in certain cell types.4 Two other Cdk
inhibitors, p16INK4A and p15INK4B, which have recently been identi
fled as the products of the multiple tumor suppressor 1 (MTSJ) and 2
(MTS2) genes, respectively, appear to selectively inhibit Cdk4 and/or
Cdk6 (51, 53, 54). The possibility that Cdk4 may be a target of
TGF@1 signaling, as discussed above, suggests that p16INK4A or
pl5tN@@4B
may also play a role in TGFf31-induced cell cycle arrest.
Additional evidence for the involvment of pl5I@4B in TGFf31 sig
naling derives from data showing that TGF@31treatment of HaCaT
cells leads to an increased abundance of p15INK4BmRNA and and an
increased association of the inhibitor with Cdk4 and Cdk6 within a
few hours of exposure of the cells to TGF@31(54).
Another potential mediator of TGFf31-induced inhibitory pathways
is the p27K@@@
protein (48—50).Two groups have reported that this
protein is able to inhibit the formation of active cyclin E/Cdk2
complexes (28, 48), and one group showed evidence that it might also
be able to inactivate cyclin E/Cdk2 complexes that are already enzy
matically active (28). The p27'@'I―
protein appears to be present in
asynchronous cells and in cells that are in G0, and it has been
suggested that this inhibitor protein may be present in a latent state
throughout the cell cycle (28, 48). Both contact-inhibited and TGFf31inhibited MvlLu cells show increased inhibitory activity of p27@'1
toward Cdk2 and the inhibitory activity of p27K@@)l
increases the
longer the cells are in the growth-arrested state (48).
The mechanism by which p27'@ inhibits Cdk2 is not completely
understood. Although Cdk2 has been shown to be in a dephosphoryl
ated state on threonine-160 in TGF@1-arrested cells (25), which could
implicate a role for a protein phosphatase in the inactivation of Cdk2,
it appears that p27'@ is not a phosphatase and is thus unlikely to be
related to the recently cloned dual specificity phosphatase Cdii shown
to interact with Cdc2, Cdk2, and Cdk3 (48, 55). It is also unlikely that
p27K@@inactivates the kinase, Cdk-activating kinase, that phospho
rylates Cdk2 on threonine-160 (48); however, the data would suggest
4 J. M.
Nigro
and
H.
L.
Moses,
unpublished
data.
that p27@1 is able to block, perhaps by a simple steric hindrance,
the ability of Cdk-activating kinase to phosphorylate and activate
Cdk2 (48).
Studies of the p27'@―@1
inhibitor have also shown that the cyclin
D2/Cdk4 complex is able to bind the inhibitor directly in vitro (48).
Binding of p27'@ to the cyclin D2/Cdk4 complex appears to seques
ter the inhibitor away from the cyclin E/Cdk2 complexes and enables
this latter complex to become active (48). Although these experiments
were all performed in vitro, they would suggest that the cyclin
D2/Cdk4 complex may be acting upstream of the cyclin E/Cdk2
complex by modulating the levels of free p27@'1 which can interact
with and inhibit the cyclin E/Cdk2 complex.
It is clear that these Cdk inhibitors are important for cell cycle
progression and there is the potential that one or more of these
inhibitors may play an important role in the mechanisms leading to
TGFf31-induced cell cycle arrest. However, because it still remains to
be shown whether TGFf31 treatment of cells actually results in an
immediate activation or induction of any of the Cdk inhibitors, it is
possible that these inhibitors may simply be increasing in activity as
a consequence of the state of being growth arrested by TGF@31or
contact inhibition and may, in reality, not be directly responsible for
the cell cycle arrest. Another plausible hypothesis is that these inhib
itors could be acting to hold TGF@31-arrested cells in an inhibited state
through suppression of the activities of one or more of the Cdk
enzyme complexes. The latter hypothesis could also suggest that, after
the growth-inhibitory signals have been removed, the Cdks might
remain inactivated until upstream cell cycle-regulated events have
occurred properly. Considering each of these possibilities, it is evident
that further research is necessary to clarify the roles these Cdk
inhibitors play in the cell cycle and the response to inhibitory factors
such as TGF@31.
Role of c-myc
TGFf31 treatment of several different cell lines results in the rapid
down-regulation of c-myc RNA and protein levels, and in at least one
case this effect has been shown to occur at the level of transcriptional
initiation (29—31,56—60). Because c-myc expression is required for
cell growth, and more specifically entry into S phase (29, 30, 61), it
has been suggested that one mechanism by which TGF@31inhibits cell
cycle progression is through suppression of the c-myc gene product.
Additional support for this hypothesis comes from recent studies
which showed that in at least two different cell types overexpression
of c-Myc protein can block the ability of TGFf31 to inhibit entry into
S phase (62, 63). However, a model which incorporates the function
of c-myc as a target of TGFf31 is probably more complex than initially
suggested.
Although the actual function of c-myc in the cell cycle remains
elusive, it is believed that c-myc function is required in the early half
of G@because c-myc RNA and protein levels peak during this period
of time (36). With this idea it can be suggested that treatment of cells
with TGFf31 in the early part of G@and subsequent down-regulation
of c-myc would likely prevent cell cycle progression toward the latter
part of G1 due to the absence of events that are normally regulated by
c-myc and required for cell growth. In support of this hypothesis are
reports that c-myc may, at least indirectly, regulate the expression of
G1 cyclins including cyclin E, cyclin A, and cyclin Dl (64—66).In
this way it is likely that a consequence of the failure of cells to
progress to the latter part of G1 when TGF@1 is added early would be
the absence of induction of cyclins A and/or E, an observation
recently shown in three different cell types (28, 33, 41). The potential
ability of c-myc to normally down-regulate cyclin Dl expression (65)
presents another hypothesis. Since overexpression of cyclin Dl has
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TGFB AND CELL CYCLE REGULATION
been shown to block some cell types from entering S phase (65, 67),
loss of c-myc expression due to TGF@31would likely allow endoge
noun cyclin Dl to be expressed at higher than normal levels which
might be sufficient to block progression into S phase. Directly related
to the above ideas is the prediction that early G@ treatment with
TGF@1 might also be able to prevent activation of the cyclin E/Cdk2
complex or other Cdk complexes in late G1 due indirectly to the
effects of TGF@1 on c-myc and cyclin expression.
Signals
The involvementof c-myc in the mechanismsby which TGF(31
inhibits entry into S phase when the inhibitory factor is added in late
G1 is at present unclear. Although controversial, there is evidence
suggesting that the c-Myc protein may have a function in late G1 and
perhaps at the G1-S transition (30, 68, 69). The early G@induction is
not followed by a reduction to basal or below basal levels as is seen
with other immediate early genes such as c-fos, but rather is followed
by a suprabasal level of expression throughout G@and into S phase
(70, 71). The necessity of this suprabasal expression in the latter part
Fig. 1. Schematic representation
of the putative nuclear targets of TGF(31-induced
inhibitory signals showing a potential common pathway in which these factors may act.
of G@was demonstratedusing antisense oligonucleotidesdirected Seetextfor discussion.
against c-myc mRNA to inhibit the translation of c-Myc protein in late
G1, the result of which was failure of the cells to enter S phase (30).
In addition, the c-Myc protein has been found in complexes contain
lug DNA polymerase a and other enzymes required for DNA repli
cation (68), and studies in Xenopus laevis embryogenesis have sug
gested that c-myc may be involved in controlling the rate of DNA
replication in the dividing blastocyst (69). Furthermore, these latter
studies suggested that the function of c-myc in these early cell divi
sions does not require the putative transcriptional regulatory function
of c-myc. Although initial data from our laboratory indicate that
overexpression of c-Myc protein in late G1 coincident with TGF@31
treatment in late G@is incapable ofblocking the induced growth arrest
(63), the involvement of c-myc in the late G1 effects of TGF@31
requires further analysis. TGF@31could target the endogenous c-Myc
protein late in G@at the post-translation level through altered phos
phorylation of c-Myc or through effects on proteins complexed with
function of cyclins and Cdks suggests another interesting hypothesis.
Not only may TGFf31-induced inhibitory signals directly inhibit
events which are required for the G1-S transit but they may also block
opposing stimulatory signals. There is evidence that members of the
E2F family of transcription factors may regulate the expression of
c-myc during G@(72—74),and data also suggest that the functions of
the E2F factors may be regulated by pRB or other related proteins
(Ref. 75 and references therein). Since pRB has been shown to be a
target of TGF@31-induced inhibitory signals (24, 27) and these effects
on pRB are postulated to be derived from negative effects of TGF@1
on Cdk activity toward pRB (25, 26), it is possible that c-myc is
regulated through a feedback mechanism (Fig. 1). In this manner, we
hypothesize that the potential ability of c-myc to stimulate cyclin and
Cdk activity
might allow for positive
feedback
to c-myc through
pRB
and E2F factors. Abrogation of Cdk activity by TGF(31 would then be
in G1 may not suffice for altered biochemical regulation of the predicted not only to inhibit late G1 progression but also to sustain
cells in a growth-inhibited state by preventing further stimulation of
endogenous c-Myc protein by TGF(31.
late G1 events by c-myc.
It is evident that the mechanisms by which TGF/31 arrests cell
AMOdelforTGFfi1Inhibition
growth are complex and may likely include effects on multiple path
The complex cell cycle kinetics of the postulated targets of TGF(31- ways. Current studies suggest that TGF@31may be targeting proteins
induced negative signals indicates that the mechanisms by which
which are in a common signaling pathway (Fig. 1). With this in mind,
TGF@1 inhibits cell growth may be through effects on multiple
it becomes important that one determine how potential targets are
independent and synergistic pathways that are required for cell cycle
affected by TGFI31 and whether these targets are causally involved in
progression. We predict that members of the cyclin-dependent kinase
arresting growth or whether the effects on these targets are simply the
family will be the ultimate targets of growth-inhibitory signals gen
consequence of TGF@31-inducedfailure to progress through the cell
crated by [email protected], negative regulation of the Cdks
cycle. Overall, the study of the mechanisms of TGF@1-induced
may likely be shown to occur through activation, or an increase in growth arrest has produced new avenues of investigation and has
the expression, of Cdk inhibitors such as p16tN@C4@@@,
p15INK4B, helped to explain how cells control G@progression. It is clear, how
p2lWdl/QP1,
or p27'°― (see above and Fig. 1).
ever, that the dynamic inhibitory effects of TGFj31 on cell cycle
Alternatively, we propose that the negative effects on Cdk activity
progression are only partly understood and will require further
could be derived from inhibition of the expression of genes in G1 analysis before a complete story can be told.
which arc required to coordinate the events leading to activation of the
References
Cdks. An example of this type of control by TGF@1 is its effects on
1. Heldin, C-H., and Westermark, B. Growth factors: mechanism of action and relation
the expression of c-myc. As discussed above, several reports suggest
to oncogenes. Cell, 37: 9—20,1984.
that c-myc may regulate, at least indirectly, the expression of cyclin E,
2. Moses, H. L., Yang, E. Y., and Pietenpol, J. A. TGF-@ stimulation and inhibition of
cyclin A, and cyclin Dl (64—66). In this manner, c-myc might
cell proliferation: new mechanistic insights. Cell, 63: 245—247,1990.
3. Moses, H. L. The biological actions of transforming growth factor @3.
In: V. Sara, K.
indirectly be regulating Cdk activity in the latter half of G@through its
Hall, and H. Low (eds.), Growth Factors from Genes to Clinical Application, pp.
effects on the regulatory cyclin subunits. As a consequence of TGF(31
141—155.
New York: Raven Press, 1990.
c-Myc.In this situation,simpleoverexpressionof c-Mycproteinlate
treatment, suppressed levels of active Cdks would be observed due to
@
4. Silberstein, G. B., and Daniel, C. W. Reversible inhibition ofmammary gland growth
by transforming growth factor-a. Science (Washington DC), 237: 291—293,1987.
the lack of appropriate expression of c-myc-sensitive cyclins.
5. Russell, W. E., Coffey, R. J., Jr., Ouellette, A. J., and Moses, H. L Type
The ability ofTGF@31to rapidly down-regulate c-myc expression in
transforming growth factor reversibly inhibits the early proliferative response to
partial hepatectomy in the rat. Proc. Natl. Acad. Sci. USA, 85: 5126—5130, 1988.
late G@ together with the possibility that c-myc may regulate the
1455
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
@
Torn
CELL CYCLE REGULATION
34. Sporn,M.B.,andRoberts,A. B.Transforming
growthfactor-a.Multipleactionsand
6. Shipley, G. D., Pittelkow, M. R., willie, J. J., Jr., Scott, R. E., and Moses, H. L
@
@
Reversible inhibition of normal human prokeratinocyte proliferation by type
transforming growth factor-growth inhibitor in serum-free medium. Cancer Rca., 46:
2068—2071, 1986.
7. Knabble, C., Lippman, M. E., Wakefield, L M., Flanders, K. C., Kasid, A., Derynck,
R., and Dickson, R. B. Evidence that transforming growth factor-a is a hormonally
regulated negative growth factor in human breast cancer cells. Cell, 48: 417—428,
1987.
8. Masui, T., Wakefield, L M., Lechner, J. F., LaVeck, M. A., Spom, M. B., and Harris,
C. C. Type p transforminggrowthfactoris the primarydifferentiation-inducing
serum factor for normal human bronchial epithelial cells. Proc. Nail. Aced. Sci. USA,
83: 2438—2442,1986.
9. Zhan, 0., Bae, I., Kastan, M. B., and Fomace, A. J., Jr. The p53-dependent y-ray
response of GADD45. Cancer Res., 54: 2755—2760,
1994.
10. Hori, M., Kamijo, R., Takeda, K., and Nagumo, M. Downregulation of c-myc
expression by tumor necrosis factor-a in combination with transforming growth
factor-@3or interferon-'y with concomitant inhibition of proliferation in human cell
R. A. Expressioncloningandcharacterization
of theTGF-f3typeIII receptor.Cell,
cloning of the TGF-(3type II receptor, a functional transmembrane serine/threonine
kinase. Cell, 68: 775—785,
1992.
13. Franzen, P., Ten Dijke, P., Ichijo, H., Yamashita, H., Schulz, P., Heldin, C-H., and
Miyazono, K. Cloning of a TGF@type I receptor that forms a heteromeric complex
with the TGF@3type II receptor. Cell, 75: 1—20,
1993.
14. Ebner, R., Chen, R-H., Lawler, S., Zioncheck, T., and Derynck, R. Determination of
type I receptor specificity by the type II receptors for TGF-@or activin. Science
(Washington DC), 262:900-902,1993.
15. Ten Dijke, P., Yamashita, H., Ichijo, H., Franzén,P., Laiho, M., Miyazono, K., and
Heldin, C-H. Characterization of type I receptors for transforming growth factor-a
and activin. Science (Washington DC), 264: 101—104,1994.
16. Wrana, J. L, Attisano, L, Cárcamo,J., Zentella, A., Doody, J., Laiho, M., Wang,
X-F.,andMassagué,
J. TGF@signalsthrougha heteromericproteinkinasereceptor
complex. Cell, 71: 1003—1014,
1992.
18. Lopez-Casillas, F., Cheifetz, S., Doody, J., Andres, J. L, Lane, W. S., and Massagué,
J. Structureandexpressionof themembraneproteoglycan@-glycan,
a componentof
the TGF43 receptor system. Cell, 67: 785—795,1991.
19. López-Casillas, F., Wrana, J. L, and Massagué,J. Betaglycan presents ligand to the
TGF@signaling receptor. Cell, 73: 1435—1444,
1993.
20. Moustakas, A., Un, H. Y., Hems, Y. I., Plamondon, J., O'Connor-McCourt, M. D.,
and Lodish, H. F. The transforming growth factor @3
receptors types I, II, and III form
of ligand. J. Biol. Chem., 268:
22215—22218,
1993.
serum requirement. Exp. Cell Rca., 116: 103—113,
1978.
38. Rossow, P. W., Riddle, V. 0. H., and Pardee, A. B. Synthesis of labile, serum
dependent protein in early G@controls animal cell growth. Proc. Nail. Acak Sci.
USA, 76: 4446—4450,1979.
39. Wells, D. J., Stoddard, L S., Getz, M. J., and Moses, H. L a-Amanitin and
5-fluorouridine inhibition of serum-stimulated DNA synthesis in quiescent AItR-2B
mouseembryocells.J. Cell.PhysioL,100: 199—214,
1979.
40. Olson,J. E.,Winston,J. T.,Whitlock,J. A.,andPledger,W.J. Cellcycle-dependent
333-342, 1993.
41. Safterwhite, D. J., Mitre, M. E., Gorska, A. E., and Moses, H. L Inhibition of cell
42. Nevins, J. R. E2F: A link between the Rb tumor suppressor protein and viral
oncoproteins. Science (Washington DC), 258: 424—429,1992.
43. Gu, Y., Rosenblatt, J., and Morgan, D. 0. Cell cycle regulation of CDK2 activity by
phosphorylation of Thri6O and Tyri5. EMBO J., 11: 3995—4005,1992.
44. Matsushime,H.,Quelle,D. E., Shurtleff,S. A., Shibuya,M.,Sherr,C. J., andKato,
J-Y.D-typecyclin-dependent
kinaseactivityin mammaliancells.Mol.Cell.Biol.,
14: 2066—2076,
1994.
45. Xiong, Y., Hannon,G. J., Zhang,H., Casso, D., Kobayashi, R., and Beach, D. p21 ii
a universal inhibitor of cyclin kinases. Nature (Lond.), 366: 701—704,1993.
46. El-Deiry,W.S.,Tokino,T.,Velculescu,V.E.,Levy,D.B.,Parsons,R.,Trets@
J. M.,
Lin, D., Mercer,W. E., Kinzler,K. W., and Vogelstein,B. WAFJ,a potential
mediator ofp53 tumor suppression. Cell, 75: 817—825,1993.
47. Gu, Y., Turck, C. W., and Morgan, D. 0. Inhibition of CDK2 activity in vivo by an
associated 20K regulatory subunit. Nature (Lond.), 366: 707—710,1993.
49. Polyak, K., Lee, M-H., Erdjument-Bromage, IL, Koff, k, ROberts,J. M., Tempst, P.,
and Massagué,
J. aonh@gof p27Kipl,a cyclin-dependent
kinaseinhibitorand a
potential mediator of extracellular antimitogenic signals. Cell, 78: 59—66, 1994.
50. Toyoshima,H.,andHunter,T.pZ7,a novelinhibitorof G1cyclin-cdkproteinklnaae
activity, is related to p21. Cell, 78: 67—74,1994.
51. Serrano, M., Hannon, 0. J., and Beach, D. A new regulatory motif in call-cycle
control causing specific inhibition ofcydlin D/CDK4. Nature (Lond.), 366: 704-707,
1993.
52. El-Deiry, W. S., Harper, J. W., O'Connor, P. M., Velculescu, V. B., Canman, C. B..
21. Kingsley, D. M. The TGF-@3superfamily: new members, new receptors, and new
Jackman,J., Pietenpol,J. A., Burrell,M., Hill, D. E., Wang,Y., Wiman,K. 0.,
genetic tests of function in different organisms. Genes Dcv., 8: 133—146,
1994.
Mercer, W. E., Kastan, M. B., Kahn, K. W., Elledge, S. J., Kinzlcr, K. W., and
Vogelstein, B. WAFJ/CIPI is induCed in p53-mediated 0, arrest and apoptosis.
Cancer Res., 54: 1169—1174,1994.
22. Bartkova, J., Lukas, J., Strauss, M., and Bartek, J. Cell cycle-related variation and
tissue-restricted expression of human cyclin Dl protein. J. Pathol., 172: 237—245,
1994.
23. Wrana,J. L, Auisano,L, Wieser,R., Ventura,F., andMassagué,
J. Mechanismof
53. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, 0., Harshman, K., Tavtigian, S. V.,
Stockert, E., Day, R. S., III, Johnson, B. E., and Skolnick, M. H. Acell cycle regulator
potentiallyinvolvedingenesisof manytumortypes.Science(WashingtonDC),264:
activation of the TGF-(3receptor. Nature (Land.), 370: 341—347,
1994.
24. Pietenpol, J. A., Stein, R. W., Moran, E., Yaciuk, P., Schlegel, R., Lyons, R. M.,
Pittelkow, M. R., MUnger,K., Howley, P. M., and Moses, H. L TGF-Iii inhibition
of c-myc transcription and growth in keratinocytes is abrogated by viral transforming
436—440,1994.
54. Hannon, 6. J., and Beach, D. pJ5tNK4B is a potential effector ofTGF-@-induced
cell
cycle arrest. Nature (Land.), 371: 257-261, 1994.
55. Gyuris, J., Golemis, E., Chertkov, H., and Brent, R. Cdii, a human 01 and S phase
proteins with pan binding domains. Cell, 61: 777—785,
1990.
25. Koff, A., Ohtsuki,M., Polyak,K., Roberts,J. M., and Massagué,
J. Negative
regulation of G@in mammalian cells: inhibition of cyclin E-dependent kinase by
[email protected] (Washington DC), 260: 536—539,1993.
26. Ewen, M. E., Sluss, H. K., Whitehouse, L L, and Livingston, D. M. TGFI3 inhibition
protein phosphatase that associates with Cdk2. Cell, 75: 791—803,1993.
56. Takehara, K., LeRoy, E. C., and Grotendorst, 0. R. TGF-@inhibition of endothdlial
cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory
(competence) gene expression. Cell, 49: 415-422, 1987.
57. Fernandez Pol, J. A., Hamilton, P. D., and Kios, D. J. Transcriptional
of Cdk4 synthesis is linked to cell cycle arrest. Cell, 74: 1009—1020,
1993.
27. Laiho, M., DeCaprio, J. A., Ludlow, J. W., Livingston, D. M., and Massagué,J.
Growth inhibition by TGF-f3 linked to suppression of retinoblastoma protein phos
phorylation. Cell, 62: 175—185,1990.
28. Slingerland,J. M.,Hengst,L, Pan,C-H.,Alexander,D.,Stampfer,M.R.,andReed,
S. I. A novelinhibitorof cyclin-Cdkactivitydetectedin transforminggrowthfactor
a-arrested epithelial cells. Mol. Cell. Biol., 14: 3683—3694,
1994.
29. Pietenpol, J. A., Holt, J. T., Stein, R. W., and Moses, H. L Transforming growth
factor @3-1
suppression of c-myc gene transcription: role in inhibition of keratinocyte
proliferation. Proc. NatI. Acad. Sci. USA, 87: 3758—3762,1990.
regulation of
p1,and
trilodothyronine
in
MDA-468
cells.
J.
Biol.
Chem.,
264:
4151-4156
1989.
proto-oncogene expression by epidermal growth factor, transforrming growth factor
58. Mulder, K. M., Levine, A. E., Hernandez, X., McKnight, M. K., Brattain, D. B., and
Brattain, M. 0. Modulation ofc-myc by transforming growth factor-fl in human colon
carcinoma cells. Biochem. Biophys. Rca. Commun., 150: 711-716, 1988.
59. Ito,N.,Kawata,S.,Tamura,S.,Takaishi,K.,Saitoh,R.,andTarui,S. Modulationof
c-myc expression by transforming growth factor
1 in human hepatoma cell lines.
Jpn. J. Cancer Rca., 81: 216—219,1990.
60. Ruegemer,3.J., Ho,S. N.,Augustine,J. A., Schlager,J. W.,Bell,M. P., McKean,
30. Munger, K., Pietenpol, J. A., Pittelkow, M. R., Holt, J. T., and Moses, H. L.
Transforming growth factor @31
regulation of c-myc expression, pRB phosphoryl
D. J., and Abraham, R. T. Regulatory effects oftransforming growth factor-a on IL-2-
ation, and cell cycle progression in keratinocytes. Cell Growth & Differ., 3:
291—298,
1992.
31. Coffey, R. J., Jr., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E., and
Moses, H. L Selective inhibition of growth-related gene expression in murine
61. Gal, X., Rizzo, M., Valpreda, S., and Baserga, R. Regulation of c-myc mRNA levels
keratinocytes by transforming growth factor 13.Mol. Cell. Biol., 8: 3088—3093,1988.
32. Howe, P. H., Draetta, G., and Leaf, E. B. Transforming growth factor @iinhibition
of p34cdc2 phosphorylation and histone Hi kinase activity is associated with G1/S@
Biol. Cell, 5: 375—388,1994.
37. Yen, A., and Pardec, A. B. Exponential 3T3 cells escape in mid-G1 from their high
Koff, A. p27@C@P1,
A cyclin-Cdk inhibitor, links transforming growth factor-a and
contact inhibition to cell cycle arrest. Genes Dcv., 8: 9—22,1994.
lymphocytes in the 0, phase. Scand. J. Immunol., 39: 499—504,1994.
@
genes and identification of a subset regulated by conditional myc expression. Mol.
48. Polyak,K., Kato,J., Solomon,M.J., Sherr,C. J., Massagué,
J., ROberts,J. M.,and
17. Kato, J., Kohgo, Y., Kondo, H., Sasaki, K., and Niitsu, Y. Antisense oligodeoxynucle
otides for IL-2, c-myc and transferrin receptor synchronize mitogen-activated
in the presence
36. Tavtigian,S.V.,Zabludoff,S. D.,andWold,B.J. aoning ofmid-G1serumresponse
growth by TGF@1 is associated with inhibition of B-myb and cyclin A in both
Balb/MK and MviLu cells. Cell Growth & Differ., 5: 789-799, 1994.
67: 797—805,1991.
12. Lin, H. Y., Wang, X-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. Expression
complexes
Transforming growth factor type can act as a potent competence factor for AKR-2B
cells Exp. Cell Res., 172: 293-303, 1987.
gene expression in V point-arrested BALB/c3T3 cells. J. Cell. PhysioL, 154:
lines. J. Interferon Res., 14: 49—55,1994.
11. Wang, X-F., Lin, H. Y., Ng-Eaton, E., Downward, J., Lodish, H. F., and Weinberg,
hetero-oligomeric
potential clinical applications. JAMA, 262: 938—941,1989.
35. Goustin,A. S., Nuttall,0. A., Leof,E. B., Ranganathan,0., and Moses,H. L
phase growth arrest. Mol. Cell. Biol., 11: 1185—1194,1991.
33. Geng, Y., and Weinberg, R. A. Transforming growth factor
by insulin or platelet-poor plasma. Oncogene Res., 5: 111—120,
1990.
62. Selvakumaran, M., Un, H-K., Sjin, R. T. T., Reed, J. C., Liebermann, D. A., and
Hoffman, B. The novel primary response gene MyD118 and the proto-oncogenes
myb, myc, and bcl-2 modulate transforming growth factor p1-induced apoptosis of
myeloid leukemia cells. MoL Cell. BioL, 14: 2352—2360,
1994.
63. Alexandrow, M. 0., Kawabata, M., Aakre, M. E., and Moses, H. L Overexpreasion
of the c-Myc oncoprotein blocks the growth-inhibitory response, but is required for
the mitogeniceffects,of transforminggrowthfactortype @i.
Proc.Nail.Aced.Sal.
effects on expression
of G, cyclins and cyclin-dependent protein kinases. Proc. Nail. Acad. Sal. USA, 90:
10315—10319,1993.
andIL-4-dependent
T cellcycleprogression.J. Immunol,144: 1767—1776,
1990.
USA, in press, 1995.
64. Jansen-Durr,P.,Meichle,A.,Steiner,P.,Pagano,M.,Finke,K.,Botz@
J.,Wessbecber,
1456
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1995 American Association for Cancer Research.
TGFB AND CELL CYCLE REGULATION
J., Draetta,G., and Eilers,M. Differentialmodulationof cyclingeneexpressionby
MYC. Proc. Natl. Acad. Sci. USA, 90: 3685—3689,
1993.
65. Philipp, A., Schneider, A., Vasrik, I., Finke, K., Xiong, Y., Beach, D., Alitalo, K., and
Eilers, M. Repression of cyclin Dl: a novel function of MYC. Mol. Cell. Biol., 14:
4032—4043,1994.
66. Shibuya,H., Yoneyama,M.,Ninomiya-Tsuji,
J., Matsumoto,K., andTaniguchi,T.
IL-2 and EGF receptors stimulate the hematopoietic cell cycle via different signaling
pathways: demonstration of a novel role for c-myc. Cell, 70: 57—67,1992.
67. Quelle, D. E., Ashmun, R. A., Shurtleff, S. A., Kato, J., Bar-Sagi, D., Roussel, M. F.,
and Sherr, C. J. Overexpression of mouse D-type cydlins accelerates G, phase in
70. Greenberg, M. E., and Ziff, E. B. Stimulation of3T3 cells induces transcription of the
c-fos proto-oncogene.Nature (Lond.), 311: 433—438,1984.
71. Kelly, K., Cochran, B. H., Stiles, C. D., and Leder, P. Cell-specific regulation of the
c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell, 35:
603-610, 1983.
72. Thalmeier, K., Synovzik, H., Mertz, R., Winnacker, E-L., and Lipp, M. Nuclear factor
E2F mediates basic transcription and trans-activation by Ela of the human MYC
promoter. Genes Dcv., 3: 527—536,1989.
73. Hiebert, S. W., Lipp, M., and Nevins, J. R. E1A-dependent trans-activation of the
human MYC promoter is mediated by the E2F factor. Proc. NatI. Acad. Sci. USA, 86:
rodent fibroblasts. Genes Dcv., 7: 1559—1571,1993.
3594—3598,
1989.
68. Studzinski,G. P., Shankavaram,U. T., Moore,D. C., andReddy,P. V. Association
of c-myc protein with enzymes of DNA replication in high molecular weight fractions
from mammalian cells. J. Cell. Physiol., 147: 412—419,1991.
69. Gusse, M., Ghysdael, J., Evan, G., Soussi, T., and Mechali, M. Translocation of a
74. Roussel, M. F., Davis, J. N., Cleveland, J. L., Ghysdael, J., and Hiebert, S. W. Dual
control of myc expression through a single DNA binding site targeted by ets family
proteins and E2F-i. Oncogene, 9: 405—415, 1994.
75. Helm, K., Harlow, E., and Fattaey, A. Inhibition of E2F-1 transactivation by direct
store of matemal cytoplasmic c-myc protein into nuclei during early development.
Mol.Cell.Biol.,9: 5395—5403,
1989.
binding of the retinoblastoma
protein. Mol. Cell. Biol., 13: 6501—6508, 1993.
1457
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Transforming Growth Factor β and Cell Cycle Regulation
Mark G. Alexandrow and Harold L. Moses
Cancer Res 1995;55:1452-1457.
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