(Received for publication, August 10, 1995; and in revised form, January 22, 1996)
From the
The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.
B-myb has been cloned by virtue of its homology with
the c-myb protooncogene (1) and its structure
indicates that it may be a transcriptional activator. However,
conflicting data have hampered a definitive evaluation of B-myb function(s). Some investigators have provided evidence of
transcriptional activation of promoters containing Myb-responsive
elements by B-myb(2, 3) , whereas others
showed that B-myb is unable to transactivate these promoters,
acting rather as a repressor of c-myb transcriptional
activity(4, 5) . A recent report has established that
B-myb transcriptional activity is highly dependent on the cell
type(6) , thus explaining the sharp discrepancies in
B-myb-mediated biological effects observed in different
systems. It has been clearly shown, however, that B-myb is
required for the proliferation of murine fibroblasts, human
glioblastoma, human leukemic cells lines, and human neuroblastoma cells (7, 8, 9, 10) and that deregulated
expression of B-myb induces abnormal cell growth and
activation of genes such as cyclin D1 and cdc2 in rodent
fibroblasts(7) . Consistent with the notion that B-myb may be a regulator of the cell cycle, B-myb expression
has been correlated with cell proliferation in different systems and is
induced at the G/S border of the cell
cycle(11, 12, 13, 14) . Furthermore,
ectopic expression the HPV16 E7 transforming protein induces
up-regulation of B-myb expression, whereas growth suppression
agents like TGF-
or the p53 protein cause down-regulation of
B-myb mRNA levels (15, 16, 17) . In
a recent study it was provided evidence that B-myb can bypass
p53-induced Waf1/Cip1-mediated G
arrest(8) .
p21Waf1/Cip1 can inhibit G
cyclin protein kinase and the
phosphorylation of pRb(
)(18) , suggesting that
B-myb may overcome retinoblastoma-associated functions.
The
retinoblastoma family comprises three members, pRb, p107, and p130,
which share structural and functional characteristics. These proteins
all induce growth arrest when overexpressed in certain cell lines and
are able to bind the viral transforming protein E1A and the E2F family
of transcription
factors(19, 20, 21, 22, 23, 24, 25) .
It is notable that p107, but not pRb, can inhibit DNA synthesis of C33A
cervical carcinoma cells(25) ; furthermore, E2F-1 can rescue
pRb-mediated growth arrest of SAOS2 osteosarcoma cells, whereas it is
ineffective in bypassing a p107 block (25, 26) .
Recent evidence indicates that p107 and pRb may associate in vivo with specific E2F proteins (we will use the term E2F to indicate
all the possible members of the family); pRb has been found associated
in cell extracts with E2F1, E2F2, and E2F3(27) , whereas p107
seems to bind specifically E2F4(19, 22) . However,
when E2F1 and p107 are transiently transfected an interaction can be
observed(19) , suggesting that weak binding between p107 and
E2F1 may also exist in vivo but can be detected only by
overexpression. There is mounting evidence that E2F and the associated
pRb family proteins are involved in cell cycle regulation of B-myb transcription (14, 15, 28) . Thus, a
conserved E2F site is located in the human and mouse B-myb promoters within a region specifying the multiple 5` termini of
the mRNA. It has been proposed that specific E2F-p107 and E2F-p130
complexes repress transcription in G/G
through
binding to this site; induction of B-myb transcription at the
G
/S boundary, which appears to arise predominantly by a
derepression mechanism, correlates with modification of these complexes
by cyclin A/Cdk2 association(14, 15) . Further
evidence that that p107 is responsible for transcription repression in
murine NIH 3T3 fibroblasts was provided by the finding that induction
of B-myb transcription by HPV E7 protein mutants correlated
with their ability to bind p107 rather than pRb(15) . These
findings indicate that B-myb may be a downstream target of
p107 and, as such, suggest a mechanism by which this protein may act as
growth suppressor. To provide further evidence for this hypothesis, we
have investigated the functional relationship among B-myb,
p107, and pRb in human cells.
Figure 1: B-myb can partially rescue 107-induced block of DNA synthesis. A, exponentially growing SAOS2 cells were transfected with control vector, CMV-Bmyb, or with a combination of increasing amounts of CMV-p107 plus (+) or minus(-) a fixed amount of CMV-Bmyb (10 µg). 10 µg of a CMV vector producing a B-myb mutant, lacking the DNA-binding domain(8) , was also tested and is indicated as +/mut. A representative experiment is shown. B, quantitation of the percentage of cells reentering S phase in p107 + B-myb transfected cells as compared with pRb+ B-myb transfected cells. Cells transfected with p107 or pRb plus a CMV-empty vector provided the base-line values of S phase rescue. Columns represent the mean of three experiments, and the S.D. is indicated by error bars.
The possibility that B-myb might neutralize p107 by protein-protein interaction was explored in vivo by co-transfecting B-myb and p107 expression vectors in SAOS2 cells, followed by immunoprecipitation with p107 antibody and Western blot with a B-myb-specific antibody. No association was found between B-myb and p107, although we cannot exclude the possibility that a weak or unstable interaction went undetected due to the limits of this technique (data not shown).
Figure 2: Basal activity and transactivation of the human B-myb promoter by E2F is inhibited by co-transfection with retinoblastoma family members in SAOS2 cells. Extracts were made from cells 36 h following transfection, and luciferase assays were performed as indicated under ``Materials and Methods.'' Data are expressed as -fold activation over the control. The mean of three independent experiments ± S.D. is shown. A, pGLHB-myb (5 µg) was cotransfected with 1 µg of CMV-E2F1 or CMV empty vector (promoter plus empty vector was used to determine the basal promoter activity) with or without CMVp107 or CMVpRb (5 µg each). B, pGLHB-myb (5 µg) was cotransfected with 1 µg of CMV-E2F4 + 1 µg of CMV-DP1 or CMV empty vector with or without CMVp107 or CMVpRb (5 µg each). C, pGLHB-myb (5 µg) was cotransfected with CMVp107 or CMVpRb (5 µg) or CMV empty vector.
Figure 3: Expression levels of p107 and pRb proteins in SAOS2 cells following transient transfection with CMV-driven p107, pRb, and control vector. The cells were collected 36 h after transfections, and equal amounts of cells were directly lysed in SDS sample buffer and subjected to polyacrylamide gel electrophoresis. After blotting the filter was stained with Poinceau Red to check for equal loading of the lanes and incubated with XZ-37 antibody. Lane 1, CMV; lane 2, p107; lane 3, pRb.
Figure 4: Cell cycle profiles of the human glioblastoma GM-47 cell line (A T98G subline) and the derivative GM-B-myb cell line at various time points after serum stimulation. A, quantitative analysis; B, graphic display of the data shown in A.
We also co-transfected T98G cells with pCMV-B-myb and the pCMV CD20 plasmids followed by cell cycle analysis after
48 h. Enforced expression of B-myb in asynchronously growing
cells reduced the percentage of cells in G, while the
percentage of cells in S phase was increased (Table 1). Although
the extent of S phase induction in this experiment does not appear to
be dramatic, we should consider that at the time of transfection these
cells are actively proliferating and that the degree of stimulation is
comparable with that obtained by ectopic expression of E2F4 in SAOS2
cells under similar experimental conditions(19) .
Recent evidence indicates that the different products of the
retinoblastoma family participate in cell cycle control, performing
similar yet distinct functions as the cell progresses from G to mitosis. A model has been developed that shows, depending on
the phases of the cell cycle, different retinoblastoma family members
in association with E2F. The complexity is even greater if we take into
account that five different E2F genes have been cloned, whose encoded
products can interact with two and maybe more DP proteins (27) . It has been shown that several growth-regulated genes
contain an E2F site located in close proximity to the transcription
start site (31) . Some, but not all, of these promoters are
activated by E2F1 (14, 30) and it is thought that
interaction of E2F with retinoblastoma family members can repress
E2F-induced transactivation, switching the E2F site from a positive to
a negative element(28, 32) .
The rationale of the
present study is based on experiments that demonstrated the presence of
cell cycle-regulated complexes containing distinct retinoblastoma
family members bound to the E2F site of the human and mouse B-myb promoters. In particular, it was shown that p107 containing
complexes present during G and S phase on the murine
B-myb promoter are disrupted by the E7 oncoprotein, resulting
in the activation of B-myb transcription(15) . These
experiments suggested that p107 might be specifically required to
modulate growth-regulated activity of the B-myb promoter and
that p107-induced growth arrest may be due, at least in part, to
repression of B-myb transcription. B-myb is required
for the proliferation of several human and murine cell lines, it is
broadly expressed, and its overexpression induces altered growth
characteristics of murine Balb/c 3T3 cells(7) . We have
therefore investigated the possibility that B-myb and p107 may
be functionally linked. B-myb overexpression induces S phase
in cells that have been blocked by p107 (Fig. 1A); this
effect seems to be specific because B-myb cannot rescue the
block induced by pRb (Fig. 1B). To test directly the
hypothesis that p107 may be involved in the control of B-myb transcription, we analyzed the activity of the human B-myb promoter in the presence of E2F in combination with p107 or pRb.
The human B-myb promoter is activated during the
G
/S transition of the cell cycle, whereas it is silent
during G
and early G
(14) . We found
that p107 was more effective than pRb in the inhibition of
E2F1-mediated activation of the B-myb promoter (Fig. 2A). This effect is even more impressive if we
take into account that pRb is expressed at higher level than p107 in
transiently transfected SAOS2 cells (Fig. 3). On the other hand,
both p107 and pRb appeared to possess similar activity in the
inhibition of E2F4-induced transactivation, which was very weak with
respect to that of E2F1 (Fig. 2B). Based on these
experiments and previous data, we can hypothesize that E2F1 and/or
other G
/S-induced E2Fs are involved in the activation of
the B-myb gene during S phase, whereas E2F4, which is thought
to be associated with the G
/G
transition of the
cell cycle(33) , might rather be involved in the repression of
B-myb expression during G
and early G
.
The evidence provided in this study strongly suggests that p107
might be required to modulate E2F-mediated activation of the B-myb promoter. There is no indication to date of a direct interaction
between p107 and E2F1 in vivo, although these proteins do
associate when overexpressed(19) . Our finding that p107 was
more efficient than pRb in the inhibition of E2F1-mediated
transactivation of a physiological promoter (B-myb) might be
the consequence of enforced expression of the retinoblastoma family
members; alternatively, it would suggest that the weak interaction
between p107 and E2F1 is functionally relevant. An important finding in
this study was that B-myb promotes S phase in a human cell
line, thus defining a cause/effect relationship between B-myb expression and DNA synthesis. In fact, B-myb overexpression induced lengthening of the S phase and thus massive
accumulation of glioblastoma cells in this phase after the addition of
serum (Fig. 4); B-myb was not able to override the
G arrest imposed by serum deprivation, indicating that its
function can be exerted exclusively during S phase. At least some
B-myb functions appear to be cell type-dependent;
overexpression of B-myb did not induce enhanced proliferation
of SAOS2 cells, nor of other murine cell lines(34) . Indeed, in
a recent report it was shown that B-myb transcriptional
activity is cell type-dependent and requires a specific
co-factor(6) .
Three different classes of growth suppressor
agents (p107, p53, and TGF-) seem to target expression levels of
the B-myb gene ( Fig. 2and Refs. 8, 13, and 16).
Conversely, overexpression of B-myb appears to overcome the
block induced by both p53 and p107 ( (8) and Fig. 1).
This is suggestive of a possible involvement of B-myb in human
tumorigenesis. The mechanism(s) underlying the positive action of
B-myb on cell cycle progression probably rests in the
activation of genes required for DNA synthesis. We previously showed
that B-myb induces up-regulation of genes involved in cell
cycle control such as cyclin D1 and cdc2 in murine Balb/c 3T3
cells(7) . Cyclin D1 is required for cell proliferation and,
when overexpressed, shortens the G
phase of the cell
cycle(35, 36) , whereas cdc2 is required for
DNA synthesis and mitosis(37, 38) . The identification
of B-myb molecular targets would represent the next step
needed in understanding its role in the regulation of the cell cycle.