et Biqphysica &a
ELSEVIER
BmchimicaetBiophysica.Acta
1287(1996) 103-120
’ Review
Cell cycle regulation by the retinoblastoma
proteins
family of growth inhibitory
Roderick L. Beijersbergen, RenC Bemards
*
Dkisiun of Mobcuhr Carcinogenesis, The NerherlandsCancer Instiwe. Plesmmdaan121. 101%CX Ansardam. The Nefherlonds
Received IO October 1995: accepted 24 January 1996
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.Summary...................
..... ........................... ....
. ..
.....
.
3. Gene exprewion and the cell cycle . . . . . . . .
.
4. Cl cyclins and thx Gl/S phase tranailion ......................
.
4.1. The restriction pant ...............................
4.2. D-type cyclins ..................................
.
4.3. CyclinE .....................................
.
4.4. CyclinA ....................................
5. Mitotic control by cyclin B .
....
....... .... ................
.
..................................
6. Regulationofcdks.
.
6.1. Regulation by phosphorylation .........................
.
6.2. Regulation hy specific inhibiton. ........................
2. lnlroduction
......
7. Substrates For cyclin-cdk complexes
. . . . . . .
. . . . . . . .
8. Targets of pRh and pRb rebued proteins: Regulation of the tranwiption
8.1. E2F complexes in the cell cycle
8.2. The E2F Family of transcription Factors .....................
8.3. E2FnSulated genes
8.4. Regulation of E2F activity.
Transcriptional repression mediated by E2F complexes
8.6. E2F and cell biology
8.7. E2C and apoptosis ...............................
8.8. @her large& of pRb Family members
8.5.
9. Functional differences
IO. Concluding remarks.
.
. . . . . . . . . . . . . . . . .
factor E2F
.
.
.........................
...............................
.
...........................
............
.
..............................
.
.
......................
between pRb Family members . . . . . . . . . . . . . . . . . . . . . . . . . .
.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Cwrcsponding author. Fax: +3l 20 5121954: e-mail: [email protected]
0304-419X/%/$I5.00
0 1996 Elsevier Science B.V. All rights reserved
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1. Summary
The retinoblastoma family of growth-inhibitory proteins
act by binding and inhibiting several proteins with
growth-stimulatory
activity, the most prominent of which
is the cellular transcription factor E2F. in higher organisms, progression through the cell division cycle is accom
panied by the cyclical activation of a number of protein
kinases, the cyclin-dependent
kinases. Phosphorylation of
retinoblastoma family proteins by these cyclin-dependent
kinases leads to release of the associated groath-stimulatory proteins which in turn mediate progression through
the cell division cycle.
2. Introduction
Prolifemtion of normal cells IS controlled by multiple
growth-regulatusy
pathways that act together to ensure
proper growth regulation. To evade these controls, tumor
cells have to acquire multiple genetic changes before they
display a fully transformed phenotype. Cells respond to a
variety of extracellular signals, including growth faciors,
mitogen antagonists and differentiation-inducing
factors.
Together, these factors dictate cellular behavior, including
the decision to grow, differentiate or commit suicide by
apoptosis. Cancer cells ignore many of these growth-regulatory signals due to mutations in genes that control either
the growth-promoting (proto-oncogenes) or growth-inhibitory pathways (tumor-suppressor
genes). Although these
mutations appear to affect different classes of genes, II has
become increasingly evident that they are both part of the
same regulatory system designed to maintain the integrity
of all tissues. The observation that proteins of both classes
often influence each other’s activity through direct interactions provides further proof of this intertwinement.
(G2) followed by rapid degradation at the end of mitosis
(M phase).
Apart from the cyclin genes, another group of genes,
collectively known as the immediate early response genes.
play a crucial role in the early phases of the cell cycle.
Growth factors bind to specific cell surface receptors
which trigger signaling cascades that ultimately result in
the transcription of immediate early response genes. The
immediate early mRNAs include c-f& c-iun and c-mvc
which appear within minutes followi;ng mitbgenic stimuiation. These mRNAs turn over rapidly and consequently
their encoded proteins appear only transiently. Immediate
early mRNAs even appear when protein synthesis is inhibited. indicating that their induction depends solely on the
post translational modification of pre-existing cellular factors. For c-mvc it has been shown that induction of its
mRNA is both necessary and sufficient for the transition
from quiescence to the &I and S phase of the cell cycle
lated in cancer.
The later transitions in the cell cycle are also marked by
the coordinate expression of yet other groups of genes that
are required for periodically occurring biochemical processes. For instance, at the GI/S transition genes required
for DNA synthesis have to be activated. This group of cell
cycle regulated genes include those for dihydrofolate reductase !DHFR), DNA polymerase a, the DNA polymerase a subunit PCNA and thvmidine kinase (TK) which
are all induced dt the GI/S
transition [7]. An important
aspect of cell cycle regulation is therefore the coordinated
eipiession of groups of genes that
together during the
specific phases of the cell cycle.
act
4. Cl cyclins and the Cl/S
3. Grne expression
phase transition
and the cell cycle
The major positive regulators of the cell division cycle
are a group of related proteins. the cyclins. first identified
by virtue of their cyclical appearance during the ceil cycle
of marine invertebrates [I]. Cyclins are the positive regulatory subunits of a class of related protein kinases, named
cyclin-dependent
kinases (cdks) [2]. The mammalian
genome encodes at least ten different cyclins and seven
cdks that can associate in at least IS dil? .-nt cyclin-cdk
complexes [3,4]. Together. these cyclin-cdk complexes are
the master regulators of the major ceil cycle transitions.
When cells emerge from quiescence (GO phase) and enter
the first phase of the cell cycle (GI), the expression of D
and E type cyclins is induced. At the onset of DNA
synthesis 6 phase) cyclin A is first detected followed by
cyclin B during the interval between S phase and mitosis
In the GI phase of the cell cycle growth-stimulatory
dnd growth-inhibitory
signals determine whether cells
progress through the cell cycle or whether they remain
quiescent. Later processes (DHA synthesis and mitosis) are
largely independent of extracellular signals but rather depend on intracellularly triggered controls. A critical moment in the cell cycle is the point after which the cell is
irreversibly committed to complete the division cycle: the
restriction point [8]. Beiore thic checkpoint growth factors
are required to progress throu,+ the tirst phases of the cell
cycle. The actual onset of i)NA synthesis, the GI to S
phase transition, follows one to 3 h after the restriction
point. Both transitions are marked by the appearance of
active kinase complexes of Gl cyclins and their associated
cdks, that phosphorylate key substrates essential for the
R.L Beijersbrrgen. R. Brmords/
Bidt~mica
execution of the different transitions [2,4]. The timing and
activation
-
of specific kinases indicates that the commir
rr Biophicu
expression
strongly implicated
ment to enter the cell cycle and the actual onset of S phase
mosome I lql3,
are separately controlled events.
parathyroid
[22-251.
4.2. D-type cylins
The
firstgroup
are stimulated
cyclins.
The
105
Acts 1287 f IYY6J 103-120
Deregulated
of
cyclin
in cancer. Cyclin
direct
the cell
role
progression through GI
of
cycle
are the D-type
the D-type
cyclins
in the
has been demonstrated in several
ways. First, the D-type
cyclins
are expressed and form
DI,
located at chro-
is over-expressed due to translocation
Cyclin DI amplitication
and squamous cell carcinomas
stimulatory
activity
cyclin
can cooperate
DI
transformation
in
B cell lymphomas
has been found in breast-,
291. Further evidence that cyclin
to enter
has also been
adenomas and centrocytic
gastric-, esophagealof cyclins that is expressed after cells
D
is provided
Dl
[26-
has strong growth-
by the observation
with
a
ms
oncogene
that
in the
of primary rat embryo tibroblasts and b:.q
rat kidney cells [30,31].
Furthermore, cyclin DI
can ca p
erate with c-rnyc to induce B cell lymphomas in transge nit
active kinase complexes in mid- alid late GI [4,9]. Second,
mice [32.33].
enforced expression of cyclin DI
mouse mammary tumor virus long terminal repea: causes
earlier in the cell cycle
accelerates entry into S phase [IO- 121. Third, the inactivation
of
cyclin
DI
through
plasmids or monoclonal
miz~oinjecrion
cyclin
DI
before the restriction
antisense
pre-
The inactivation
of
point blocks progression
into S phase, whereas inactivation
without
of
antibodies against cyclin DI
vents entry into S phase [lO,l3-151.
at later time points is
effect. This indicates that the point of cyclin
D
The
D-type
complexes
in the decision to pass the restriction
However,
induction of cyclin DI
alone is
D-type cyclins, cyclin Dl.
phenotype,
for cyclin Dl do not
although
some cell type specific abnormalities
the D-type
in
of MyoD
colony
[36,37].
Together
stimulating
factor
also inhibits the function
and therefore the differentiation
of myocytes into
these results
indicate
expression of cyclin D can dramatically
ence key ceb’ular processes such as proliferation
that
influ-
and differ-
entiation.
4.3. Cvclin E
uniikely that any one is essential for cell cycle progression.
Consistent with this, mice nullizygous
a dramatic
role
D2
and D3, which are expressed in a cell-lineqe
specitic
manner. Because there are several D-type cyclins it is
manifest
an important
into S phase indicating
that more proteins are involved in this process [ 121.
There are three different
also play
32D myeloid cells that ectopically
in the presence of granulocyte
myotubes
D-cdk
under the control of the
[35]. The expression of cyclin DI
deregulatrd
not sufficient to progress from Gl
cyclins
cellular differentiation.
action is before the onset of S phase. Taken together, these
point [9,16,17].
cyclin Dl
express cyclin D2 or D3 are prevented from differentiation
data provide strong evidence for a critical
role of cyclin
Finally,
mammary hyperplasia and mammary carcinomas [34].
they do have
[18]. The genes for
cyclins are induced when cells are mitogeni-
After cyclin DI induction, but well before the onset of
S phase. cyclin E is transcriptionally induced and forms an
active complex with cdk2. Cyclin E expression reaches its
maximum near the G I /S
odically
boundary and is expressed peri-
during the following
cell cycles [38,39].
Several
lines of evidence support a role for cyclin E in the onset of
DNA
synthesis. Entry into S phase of mammalian cells is
tally stimulated as part of the delayed early response. The
blocked by the inhibition
half life of the D-type cyciins is very short CT,,? < 25 mitt)
antibody microinjection
and the withdrawal
negative cdk2 mutant [&l-43],
whereas overexpression of
cyclin E shortens the GI interval in mammalian cells
of the cell
cyclins.
stimulating
of growth factors during the GI phase
cycle result
When
is a raptd
macrophages
factor
I, cyclin
decrease of D-type
are deprived
from
colony
D expression is immediately
of cyclin E and cdk2, either by
or by the expression of a dominant
[I 1,121. Like cyclin Dl,
cyclin E expression only moder-
ately accelerates entry into S phase. However,
when both
reduced. As a result, these cells can no longer pass the
cyclin D and cyclin E are over-expressed simultaneously a
restriction point [19]. These data have led to the idea that
D-type cyclins act as growth factor sensors. A prediction
further
ir&.&ig
from this would be tlrn deregulated expression of cyclin D
processes during
would
contribute
to tumorigenesis
by making
cells less
decrease in the time spent in Gl is observed,
thz; D and E-type cyclins regulate different
the Gl
to S phase transition [44]. This
was also suggesied by the fact that cyclin E, but not cyclin
dependent on growth factors. Indeed, Resnizky et al. have
DI.
shown
product of the retinoblastoma
that overexpression
of
cyclin
D
reduces serum
requirement for cell cycle entry [ 121. The
cyclin Dl transcription can be stimulated
finding that
tbmugh in-
creased expression of c-myc, provides a possible comrection between the immediate
control [20,21].
early genes and cell cycle
A role for cyclin DI
also suggested by the finding
in cell proliferation
that
loss of
function in mice leads to reduced proliferative
retina and breast epithelium
[l8].
cyclin
is
DI
capacity of
is essential for S phase entry in cells lacking
(see below and [43&t]).
the
tumor-suppressor gene, pRb
Also. in lower organisms, cyclin
E is required for the initiation of S phase since Drosophila
embryos in which cyclin E is homozygously deleted arrest
in the GI
normally
cyclin
G I /S
phase of
cyclin
cell
cycle
17. the moment
when
E expression is induced 1451. Although
E seems to regulate an important
aspect of the
transition. no strong evidence is present that deregu-
lated cyclin E expression contributes to human cancer.
IO6
R.L. Beijersburgm.R. Bemards/Wuchimun et Biophuica Acta 1287 (19!%~103-120
4.4. Cyclin A
Cyclin A is induced shortly after cyclin E and binds and
activates cdk2 in S phase and cdc2 in G2 and M phase.
Although cyclin A has been implicated in the control of
mitosis, it appears that cyclin A is also involved in the
regulation of S phase entry. Inactivation of cyclin A by
antibody injection blocks entry into S phase [41,46]. Furthermore, the enforced expression of cyclin A early in Gl,
results in the acceleration of S phase entry and the over-expression of cyclin A in asynchronous cells causes a decrease of the number of cells in GI [47]. Together these
results suggest a role for cyclin A in the regulation of S
phase entry. Consistent with this is the observation that
cyclin A deregulation is implicated in transformation. In
one hepatoma, the integration of hepatitis B virus resulted
in tb formation of a chimeric cyclin A protein that lacks
the cyclio destruction box. As a result, the half life of the
ch:.metic protein was prolonged significantly, causing a net
increase in cyclin A protein levels in the vitally infected
cells ]48]. One observation suggests that cyclin A expression is stimulated by signals from cell surface adhesion
receptors and mediates cell growth control. A stable cell
line of NRK tibroblasts expressing ectopic cyclin A is able
to grow in suspension, whereas the parental cell line is
anchorage dependent (491. This could indicate that cyclin
A synthesis is involved in mediating the anchorage independent growth properties of transformed cells.
5. Mitotic control by cyclin i?
Mitosis is regulated by cyclin B in association with
cdc2 (reviewed in [50,51]). Cyclin B is synthesized in S
phase and accumulates with cdc2 towards M phase and is
rapidly degraded during mitosis. The exit from mitosis
depends on the abrupt ubiquitin-mediated
degradation of
cyclin B. In normal cells, unduplicated DNA or DNA
damage prevents the activation of the cyclin B-cdc2 complex with the result that cells arrest in G2. In tumor cells
this checkpoint is often defective. causing cells to become
anaploid by entering M phzse regardless of the replication
state of the DNA [52]. The role of cyclin B in mitotic
control was also illustrated in Drosophila cells that ucdergo endo-reduplication,
continuous DNA synthesis without intervening mitosis. These cells do not contain mitotic
cyclin B, thereby skipping M phase and as a consequence
progress into Gl with double DNA content [53].
6. Regulation of cdks
As was mentioned above, the activation of a cdk requires the association with a regulatory subunit, the cyclin.
The cell cycle-dependent expression of these cyclins is one
mechanism by which cdk activity is controlled. However,
it has become clear that additional
control cdk activity.
mechanisms
exist that
6.1. Regrrlatitin by phosphor?;lation
First, the cdk component needs to be phosphorylated to
acquire full activation of its kinase activity. The targets of
this phosphotylation
are the residues that block the protein-substrate binding site in cdks. The phosphoylation
of
threonine I60 in cdk2 or I61 in cdc2 makes the catalytic
pocket accessible for the protein substrate [54]. The Cdk
Activating Kinase (CAK) responsible for phosphorylation
of these thmonine residues is the MOl5-cdk7
kinase,
which in turn requires cyclin H for activity [55,56]. CAK
can phosphorylate cyclincdk2.
cyclin-cdc2 and cyclin D2cdk4 complexes. In contrast to cdc2. cdk2 can be phosphorylated by CAK in the absence of bound cyilin. This could
represent a cdk2-specific activation pathway, allowing the
formation of phosphorylated inactive monomeric cdk2 kinases. The relevance of this observation under physiological conditions is still unclear. The identif~ca%n of
CAK as a cyclin-cdk complex which can function as an
activator of other cyclin-kinase complexes. suggests that
cyclin-cdk cascades may reflect an important regulatory
pathway
in cell cycle control. In addition to CAK
phosphorylation, cdks are regulated by a second phospho
rylation event mediated by the wee-l/&k-l
related protein kinases 1511. The cdc2 kinase is inactive in S phase
due to the phosphorylations on Tyr-I5 and Thr-14. Phosphorylation of cdc2 on Thr-I61 stabilizes the interaction
with cyclin B and is essential for the activation of cdc2
kinase activity. The cdc25C phosphatase dephosphorylates
Tyr- I5 and Thr-I4 at the end of G2, thereby activating
cdd kinase activity [50].
6.2. Regttlatiotz by specific inhibitors
Recently it has been shown that a family of cyclin
dependent kinase inhibitors (cdkfs) plays a major role in
the negative regulation of cyclin-cdk activity (for reviews
see [57-591). These GI cyclin inhibitors are involved in
the arrest in GI of cells in response to anti-proliferative
signals. This arrest enables cells to enter processes such as
terminal differentiation. cellular senescence, or to repair
DNA damage. The cdkls can be subdivided into two
categories. The first class is a group of broadly-acting
inhibitors that associate with a complex containing a cyclin, a cdk and the prolifenting
cell nuclear antigen
WCNA) [60.61]. The first cdkl of this class that was
identified is p2l “Pt. A molecule of ~21“~’ binds to and
inhibits a wide variety of cyclin-cdk complexes including
cyclin D-cdk4, cyclin E-cdk2, cyclin A-cdk2 [62-661.
However, p2l <‘PI is present in most cyclin-cdk complexes
in normal cycling cells. Significantiy,
active cyclin-cdk
complexes can be immunoprecipitated
with p2I”r’ antibodies. indicating that p2I”P’ can be present in active
R.L
Eeijersbergen.
R. Remxds/Riochimica
cyclin-cdk complexes. Expression of ~21”~’ is induced
when quiescent tibroblasts and T lymphocyu:s are mitogenitally stimulated [66-691. This probable Indicates that
low concentrations of p21c’p’ are synthesized in growthstimulated cells to facilitate the assembly of active cyclincdk complexes, whereas higher concentrations of ~21”~’
are inhibitory.
P2l “P’ expression is in part under the control of wild
type p53 [65]. DNA damage results in the increase of ~53
protein levels that in turn induce p2P’P synthesis. This
increase in ~21”~ levels results in a further binding of
P2’ “p’ to cyclin-cdk complexes. This inhibits cyclin-cdii
kinase activity, thereby allowing cells time to repair DNA
damage before proceeding into S phase. p2l”r expression
is also increased IO- to 20-fold in senescing libroblasts,
coincident with their loss of proliferative capacity [66]. In
these cells an accumulation of inactive cyclin E-cdk2 is
observed presumably as the result of p21c’p’ activation
1701.
In addition to a role in cell cycle control and senescence, it is likely that ~21-“P’ is also involved in regulation
of differentiation.
Thus, induction of p2l”r
has been
observed in cultured hematopoetic cell lines undergoing
differentiation and in differentiating
myoblasts [?I -731.
However, mice lacking a functional ~21”~’ gene do not
manifest major haematopoetic or muscle abnormalities [74].
This may indicate that ~21 “P’ is redundant with other
members of this gene family. At the same time, these data
argue against a unique and essential role for ~21”~’ in the
assembly of active cyclin-cdk complexes.
Other members of the p2l”r
family of cdkls include
p27 krr’ and ~57”~‘. Both appear to have a similar substrate specificity as p21c@ but probably allow the cell to
respond to different growth-regulatory
signals. Thus.
p27 “P’ is lost from cyclin D-cdk4 complexes following
stimulation of T ceils with IL-2. whereas inducers of
CAMP increase p27*P’ levels in macrophages [69.75].
A second group of cdk!s is more restricted in its ability
to inhibit cdk activity. ~16’~~” was the first member of
this family of cdkIs that now consists of at least four
members, including, apart from ~16’~~‘“, ~15’~~~“. ~18,
and p19 [76-SO]. These cdkIs act as competitive inhibitors
of D-type cyclins by forming specific complexes with the
D-type cyclin partners cdk4 and cdk6 [76,79,80]. When
overexpressed in pRb positive cells, all four members of
this family can cause a Gl arrest, indicating that they are
potent inhibitors of cell cycle progression. Consistent with
this, loss of ~16’~~~~ has been observed in a variety of
human cancers and germ line mutations in ~16’~~” have
been found in familial melanoma [S I-831. In normal cells
these inhibitors probably also act to mediate growth-inhibitory signals to the cell cycle machinery. Thus, treatment of
human keratinocytes with TGFP leads to a rapid induction
of pl5’NK4b, causing the ceils to arrest in GI. whereas in a
different cell system, Ewen et al. found that TGFP induces
a Gl arrest by reducing the expression of cdk4 [80,84].
er Ri?phyxca
Arm
1287 (1996)
103-120
107
The enforced expression of cdk4 is able to overcome a
TGFPinduced
cell cycle block 1841. This indicates that a
TGFPinduced
growth arrest depends on the efficient hthibition of D-type cyclin-associated kinase activity.
3.
Susubstrates
for cyclin-cdkcomplexes
The direct involvement of the sequentially activated
cyclic-cdk complexes in cell cycle progression supports a
model in which each successive cyclin-cdk complex phosphorylates a unique set of substrates that is essential for
each transition. Thus, S phase cyclin-cdk complexes phosphotylate and activz:e proteins essential fo: ?“lA symhesis, whereas M phase cyclin-cdk complexes phosphorylate
protrins involved in mitosis and cytokinesis. ,%e targets of
the D-type cyclins would then control the proliferation-oiffere-itiation switch.
/,lthough many substrates for cyclin-cdk complexes
have been identified in vitro, relatively few proteins are
known whose phosphorylation
is relevant to cell cycle
progression. The best examples come from the study of
cyclin B-cdc2 in mitotic events. For example, phosphorylation of lamins by cyclin B-cdc2 plays a major role in the
disassembly of the karyoskeletal system. Similarly, chromosome condensation, occurring in M phase, is also accompanied by extensive phosphorylation of histone HI on
cdk sites (reviewed in [85]).
Recently, major attention has been focused on the phosphorylation of growth regulators in the GI phase of the
cell cycle. A key substrate for Gl cyclin-cdk complexes is
the product of the retinoblastoma gene, pRb. Over the past
decade it has become increasingly clear that pRb is a
negative growth regulator that acts in the Gl phase of the
cell cyc!e. In man, inactivation of one allele of RB predisposes to retinoblastoma. In these tumors the second allele
is also inactivated, indicating that the loss of RB is an
essential step in tumorigenesis.
Somatic mutations that
inactivate RB are also found in other human tumors such
as osteosarcomas,
indicating that loss of RB also contributes to other types of human cancer [86]. Reintroduction of a wild type RB gene in certain RB negative tumor
cc!1 lines causes a reversion of the transformed phenotype,
supporting the role of pRb in regulation of cell proliferation [87,88]. pRb acts as a negative regulator in the Gl
phase of the ceil cycle because over-expression of pRb
arrests most cells in Gl and the introduction of pRb after
the restriction point is without effect [89,90]. Adenovirus
and SV40 can induce quiescent cells to enter S phase, most
likely to enforce the expression of host cellular genes
required for viral DNA replication. The adenovirus EIA,
HPV E7 and SV40 huge T proteins bind to pRb and
thereby inactivate pRb’s ability to restrain cell division
[91-931. For all the viral oncoproteins it was demonstrated
that the region involved in the interaction with pRb is also
requited for their transforming activity [94].
108
pRb t; phosphorylated in a cell cycle-dependent
[95-971.
manner
in GO and early GI phases of the cell cycle pRb
IS found in the hypophosphorylated
gression through GI,
rylations
resulting
state. During
pRb undergoes additional
in a hyperphosphorylated
the prophospho-
form
that
cdk complexes are primarily
rylation.
responsible for pRb phospho-
there are indications
also contribute
to pRb
that other cyclin-cdks
phosphorylation.
When
it causes an arrest in the Gl
can
pRb
over-expressed in the osteosarcoma cr!l line SAOS-2
is
cells,
phase of the cell cycle.
persists through S, G2 and most of M phase. Several lines
Ectopic expression of either cyclin E or cyclin A rescues
of evidence indicate that the growthsuppressive
the
pRb is regulated by phosphorylation.
ryla:ion
of pRb
growth.
whereas
is stimulated
phosphorylation
by signals that favor
inhibitory
signals
Moreover,
the transforming
of
the hypophosphorylated
SV40
binds
and thereby
Furthermore,
binds
prevent
inactivates
proteins.
among
factor E2F. pRb phosphorylation
to viral
proteins
of pRb,
cellular
proteins
thereby allowing
(see below).
All
form of
which
the
or binding
cell cycle progression
state can prevent
the cell cycle.
prefer-
results in the release of these
these data suggest that pRb
pophosphotylated
T
form is active in growth
the hypophosphorylated
several cellular
the
form of pRb. suggesting
that only the hypophosphorylated
transcription
cell
growth
antigen
pRb
First, the phospho-
of pRb.
entially
inhibition.
activity of
In mid GI,
pRb
in its hy-
progression
through
is phosphorylated
and
cell
arrest
[99,107.108].
and
causes
Although
pRb-mediated
phosphorylation
cyclin
A
of
growth arrest. it seems unlikely
A-associated
kinase activity
phosphorylation
pRb
is able to release the
is responsible
of pRb in mid GI.
that cyclin
for the early
Rather it would seem
possible that cyclin A-cdk2 contrilouies to subsequent additional phosphorylation
of pRb in the S and G2 phases ot
the cell cycle.
In contrast. cyclin E expression increases at the time of
pRb phosphorylation
cell line C33A.
[109]. Also, in the cervical carcinoma
transfected pRb is readily phosphorylated
in the absence of
apparent
activity [ 105,107]. Although
in Gl
cyclin
D-associated
kinase
premature cyclin E expression
results in a earlier entry into S phase, this is not
accompanied
by the earlier onset of pRb phosphorylation
[44]. It is possible that under normal conditions, phospho-
thereby inactivated as brake on the cell cycle. The lack of
rylation of pRb by D-type cyclins must occur before it can
pRb or inactivation
function as a substmte for cyclin E-cdk2 complexes.
constraint
by viral proteins will remove the pRb
on cell cycle contrji
with the consequence of
Accumulating
evidence
of pRb is controlled
indicn:es that phosphorylation
by cyclin-cdk
are cdk consensus
sites [98]. Because of the timing of pRb phosphorylation.
seems likely that Gi
phosphorylations.
cyclin-cdk
activity
it
complexes mediate these
The connection
kinase
and
between D-type cyclinpRb
phosphorylation
is
strengthened by a large body of evidence. Ftrst. the timing
of pRb phosphorylation
from pRb.
were identified
complexes. The sites in
the pRb protein that are phosphorylated
associated
Apart
two plb-related
proteins. ~107
and
~130. share many features with pRb. Both ~107 and ~130
deregulated cell proliferation.
coincides with the appearance of
as pro:eins that bind to the region of EIA
required for transformation
with
pRb
[I IO-I
small family
teins.
and share significant homology
131, indicating
of structurally
indeed.
like pRb,
that pRb
belongs
and functionally
both ~107
and ~130
stable complexes with the cellular transcription
(see below and [I 14-l
~130
that
~107
can form
factor E2F
171). The finding that both ~107 and
are bound by viral transforming
possibility
to a
related pro-
and
proteins raised the
130 are also endowed
with
active cyclin Dl-cdk4 complexes. Also, when pRb protein
was incubated with lysates of insect St9 cells engineered
growth-inhibitory
activity. Indeed. when transiently transfected. both proteins are able to induce a GI arrest in
to express both cyclins and cdks. pRb could be phospho-
certain cell types [I 18-1201.
rylatcd by cyclin DI (and D2 or D3) together with cdk4 or
pISO. not shared by pRb.
cyclin
within the region required for viral protein binding. called
D2
or
Furthermore.
D3
in combination
cdk2
[99 1001.
premature induction of D-type cyclin expres-
sion after serum stimulation
phorylation
with
in tibroblasts results in phos-
of pRb at an earlier time point [12&t].
Inter-
the spacer, with
cyclin
E-cdk2
which
or cyclin
One idiosyncrasy of ~107 and
is that they contain a domain
they can form
A-cdk2
complexes
[I 13.121.122].
cyclin E-cdk2 and cyclin A-cdk2
with
Although
are able to phosphorylate
estingly. cells that lack a functional pRb do not appear to
require cyclin D-associated kinase activity to enter S phasr
~107 in vitro, it is unlikely that these cyclin-cdk
suggesting that pRb is a critical target of cyclin D-associ-
First, the presence of stable higher order complexes be-
ated kinase activity
[15.101].
tween ~107,
the .:dk4 inhibitors.
~16’“~‘”
Significantly.
the ability
of
E2F
acnvity
and cyclin
indicates
cycle arrest is also dependent on the presence of a func-
E2F-~107
tional pRb [77.102-1041.
are unable to rescue a pl07-induced
DI
The interplay between pRb and
is also apparent from studies with tumor cells
that cyclin
A or cyclin
complexes. Furthermore,
[1071. Rather
inactivation
act to bind and inactiva’e
cyclin
correlates with a decrease in
D expression. suggesting strongly that cyclin D is
only required in pRb positive cells [15.105.106].
compelling
Although
evidence has been obtained that D-type cyclin-
of ~107
complexes, in a similar
cyclin
or cyclin
E-cdk2
E do not disrupt
the
both cyclin A and E
growth arrest, suggest-
ing that these cyclins are not involved
lacking pRb function. Loss of pRb function. either through
mutation or viral inactivation.
complexes
of ~107 and ~130.
A-cdk2
to induce a cell
cyclin
and ~18.
control the growth-inhibitory
in the functional
it seems that ~107 may
A and cyclin
fashion as the ~21””
E kinase
family
of
cdkls. Indeed comparison of the structure of ~107 and
p21ctPt reveals a short region of homology between the
two proteins that is responsible for the cyclin-cdk
tion. Consistent with a p21 “p’-like
al. have shown that overexpression of ~107
pRb phosphorylation
interac-
E-cdk2 complexes [
role for ~107. Zhu et
mediated by cyclin
can inhibit
E or cyclin
1141.
Whether
cyclin E-cdk2 can con-
tribute to the subsequent phosphorylation
of ~130 later in
the cell cycle remains unclear.
A
complexes [123]. Thus, ~107 can cause a Gl arrest by one
of two mechanisms. One involving the binding and inactivation of cyclin-cdk
involving
complexes
and another mechanism
tbe binding and inactivating
promoting factors like E2F [ 124).
Apart
from
Compelling
being
able to bind
~107
can interact
spacer element,
cyclins
with
through
D-type
the
cyclins
through the pocket structure. Indeed, Li et al. have demonstrated that pi07
and a cyclin
is associated with D-type cyclins in vivo
D-cdk4
complex
is able to phosphotylate
~107 in an in vitro kinase assay [I 131. ~107 is hypophosphorylated in the GO and early GI
Phosphorylation
8. Targets of pRb and pRb related proteins: Regulation
of the transcription factor E2F
of cellular growth-
phase of the cell cycle.
1.coincident
of p 107 occurs in mid G
with
evidence has been obtained that pRb regu-
lates the activity of transcription
factors that in turn regu-
late the expression of division-promoting
lines of evidence indicate
factor\
that the cellular
Several
transcription
factor E2F is an important target for pRb-mediated
control.
First,
the interaction
growth
between pRb and E2F
dependent on the pocket region of pRb. Mutations
found
in human
domain
tumors
frequently
and abolish the interaction
involve
is
in pRb
the pocket
with E2F [127-1291.
the appearance of cyclin D expression [125]. in contrast to
Second. the binding of viral proteins to pRb prevents the
pRb, hypophosphorylated
interaction between pRb and E2F [I 301. The activation
~107 reappears at the beginning
of
of S phase. This may be explained by the observation that
the viral E2 promoter region, which carries two E2F DNA
~107 abundance strongly increases at the G 1 /S
binding sites. depends on the interaction between EIA
Most
likely.
the newly synthesized ~107 canno: be efli-
ciently phospholylated
ated
kinase
by the declining
activity,
pophosphorylated
photylation
causing
~107
of pi07
expression of cyclin Dl
the reappearance
of ~107
vivo cyclin D-associated
~107 phospholylation.
phosphorylation
of
can be mirruckcd
in combination
sion of a kinase-inactive
phosphorylation
cyclin DI-associ-
in early S phase [I 14,125].
in mid GI
by cyclin A or E in combination
mediated G
transition.
appearance of free E2F that can activate the E2 promoter.
hy-
Phosby the
uith cdkd. but not
with cdk2. Overexpres-
mutant
of cdk4
abolishes
in viva. both indicating
the
that in
kinase activity is responsible for
The functional
significance
of this
was illustrated by the finding that a p107-
I arrest could
cyclin DI-cdk4,
& overcome by co-expression of
but not by the overexpression of cyclin E
plb-related
phosphorylation.
phosphorylation
~130
is
In quiescent
pophosphorylated
Third,
E2F associates only with the hypophosphorylated.
growth-inhibitory,
species of
pRb
state.
With
also
regulated
cells ~130
through
is in the hy-
progression
through
G
I,
of ~130 is induced. The moment at which
[95-97.1281.
Finally,
E2F binding sites are present in a number of genes that are
regu’ated in a ceil cycle-dependent
manner and the pres-
ence of these E2F sites contributes to the cell cycle-regulated expression of genes such as c-myc, b-myb.
kinase. dihydrofolate
E2F itself [ I3
reductase, DNA
1,132].
Together
in which hypophospborylated
thymidine
polymerase
a and
these data suggest a model
pRb inhibits the transcrip-
tion factor E2F tbrokgh direct binding, thereby preventing
the expression of genes whose products mediate cell cycle
progression.
The
phosphorylation
complexes abolishes pRb-mediated
or cyclin A [107,125].
The
and
pRb. The binding of pRb by viral proteins results in the
of pRb by cyclin-cdk
inhibition
of E2F indi-
cating the functional interplay between the cell cycle clock
and cell cycle-regulated
gene expression.
function,
mutation.
either
phosphorylation
through
viral
Loss of
inactivation
pRb
or
results in the loss of control of the E2F
~130 is phosphoryiated coincides with the phosphorylation
transcription
of ~107 [126]. Thus, efficient
~107 and ~130 are also found in complex with E2F. These
accompanied
family
entry into the cell cycle is
by the phosphorylation
members. The
of
moment uf pi30
cyclin
associated
inactivated by ryclin
kinase
activity.
D-cdk4-mediated
the activity
That
of
~130
is
phosphorylation
is
substantiated by the finding that a pl30-induced
arrest can be rescued by cyclin DI-cdk4.
that the p I30 protein can be efticiently
three pRb
phosphorylation
indicates that it is too mediated through
D-type
all
interactions
factor. Apart from pRb, the related proteins
are also disrupted
phosphorylation.
upon viral
infection
suggesting a more complicated
and
network
in cell cycle control by pocket proteins and EZF.
cell cycle
Wolf et al. show
phosphorylated
by
cdk2 in vitro [I 191. However the timing of the phosphorylation of ~130 during the cell cycle suggests an earlier
time point for the onset of ~130 phosphorylation.
Also the
existence of higher order complexes containing E2F, ~130
and cyclin
unlikely
E/cdkZ.
that ~130
as was found
is functionally
for
~107,
inactivated
makes it
by cyclin
Rg. I The E?F family of’ tranrcnplionfactor\ and their pocketprotein
piInner\.
Fig. 2. Model for the regulauonof EZF mediatedwnsacwawm and reprewon in the ccl: cycle.The hypopho@orylated>pecierof ~130. pi07 and pRb
in white.phosphorylaled
~130 ~107 and pRb are indicatedin black. The anount of E?F and EZF-packer protein complexes are a relleclion of
are depicted
the pho\pborylalion
~IIU\
of their respective pocket protem partner and the exprewon
levels of the different E2h
and pocket proteins. l%e complex
iomationof EZF-J-p130 and E?FJ-pi07 and EZF-I-pRb we depicted.In S phase.cyclin A-cdk2 phorphorylates
DPI and DP-2. as indicatedin black.
reducingthe DNA bindingaffinily of the EZF-DP comp!r* The lowerpanelrepre.snt\Ihe activityof the differentE2h. The plO7/pl30
E2h resemble
the activity asroc~ted
with E2F-4
and EZF-5.
wherea
pRb-EZF\
are comprom~ng
EZF-I.
EZF-2
and E2F-3
ill
8.1. E2F complexes in the cell cycle
lasts hypophosphorylated
pi07
is observed in GO cells, it
does not result in complex formation
The complexes between E2F and pRb family
teins are subject to cell cycle regulation
GI/S
phase transition,
(Fig.
of pro-
2). At the
an increase in free E2F
is ob-
served, whereas quiescent cells contain predominantly
in complex with pocket proteins [I 17,133,134].
E2F
However,
with E2F. It is likely
that the ~130 out-competes ~107 in early GI for binding to
E2F indicating that ~130 has a higher affinity for EZF-4.
late Gl
~107 expression is strongly
synthesized hypophosphorylated
clin D-associated
kinast
In
induced and newly
~107 reappears when cy-
activity
declines. This results in
most cycling cells contain these higher order complexes as
the emergence of E2F-~107
w&l. In general. E2F in complex with pRb is found in the
pression of ~130 does not increase or in some cell types
GI
even decreases in late GI.
As a result of this, E2F-~130
complexes
in S phase [I 19.1261.
phase of the cell cycle. Although
pRb takes place before *he Gl
EZF-pRb
phosphorylation
of
to S phase transition,
the
complex persists well into S and G2 phases of
the cell cycle [I 171. This probably
pearance of free E2F at the GI/S
ily due to the dissociation
indicates that the aptransition is not primar-
of pre-existing
EZF-pRb
com-
plexes but rather the result of the new synthesis of E2F
together,
are not found
the formation
complexes.
of the differeut
largely depends on the availability
In contrast, ex-
E2F
Taken
complexes
and relative affinity
of
the different components.
The changes in the multi-protein
complexes do not only
occur during the cell cycle, but also following
the induc-
[ 135.1361. This newly synthesized E2F is no longer bound
tion of differentiation
by the phosphorylated
complexes undergo dramatic changes during embryo carci-
the generation
complexes.
‘free’ pRb. This mechanism allows
of free EPF in the presence of EZF-pRb
Mouse
tibroblasts
that
are
homozygously
deleted for the RB gene, appear not to differ
in their
noma
cell
or senescence. For instance,
differentiation:
The
declines and the amount
wcket
proportion
of E2F
of
complexes
proteins increases [ I38,139].
E2F
free
E2F
containing
Similar changes occur
compositicn of higher order E2F complexes in the GO state
during
of the cell cycle [I 141. This in&
shown that free E2F is present in Xenopus oocytes and
z,.es that pRb is not a
Xewpus
development.
major component of higher order E2F complexes in quies-
early embryos [ 1401. E2F-pRb
cent mouse fibroblasts.
in tl,e mid-blastula
In mos: quiescent cells. such as
Philpot
and Friend
have
complexes are tint detected
phase and become more prominent at
fibroblasts and T lymphocytes, E2F forms a complex with
later stages of development. In mammals, E2F complexes
~130
can be modulated
[I II,!
17.121,137].
complexes containing
However,
E2Fp
in certain cell types,
107 and E2F-pRb
can also be
by a variety of signals, including
tokines [141]. Stimulation
of Burkitt’s
cy-
lymphoma cells and
observed in GO cells [I 141. That some cell types differ in
myeloblastic cells with either interlcukin-6
the presence of GO E2F complexes could reflect a differ-
or /3 gave an immediate reduction of E2F DNA
ence in their capacity to become quiescent. The E2F-~130
complexes. This decrease was correlated with a decrease in
complexes
c-mnw expression and the induction
disappear
when fibroblasts
are stimulated
to
enter the cell cycle [I 14.1 IO]. In mouse tibroblasts,
disappearance of the E2F-~130
phosphorylation
pl30
contains
cyclin-cdk
E2F-~130
vitro
cyclin
complex coincides with the
of ~130 by cyclin D-cdk4
a spacer element
[126]. Because
that can interact
with
complexes. higher order complexes containing
and cyclin-cdk
reconstitution
pl30-E2F
the
complexes are also observed. In
experiments
have demonstrated
that
complexes can associate with cyclin E-cdk2 and
A-cdk2.
The E2F-~130
cyclin
E-cdk2
have been observed in fibroblasts in the !ate Gl
1141.
the cell cycle [
The complexes
complexes
phase of
and E2F
also show a
complex pattern of appearance during the cell cycle. In late
GI,
DNA
binding
complexes
in these complexes,
instead E2F
binding
activity
that cytokines regulate DNA
lational
by which cytokines re-
is undear,
but the restoration of
in cell extracts by EDTA
modifications.
suggests
binding activity by post-trans-
Finally,
upon muscle differentia-
tion, multiple changes in E2F complexes occur I! <2]. The
p130-E2F
complex
is only present in fully differel.tiated
myotubes. The formation
of pl30-E2F
occur in a differeniL&on-dcfc&;c
other higher
detected. Thus, the formation
complex:? did not
~ryoh!z~r cell line
order E2F
complexes
al-
were readily
of the p130-F2F
complex
seems a necessary event in the onset of differentiation.
entry into mitosis the cyclin
A component
8.2. The E2F family
of transcriprion factor.~
is
found associated with ~107, cyclin A and cdk2 [I 17.1211.
Upon
E2F binding
arrest,
event in cy-
have been observed that
contain E2F, ~107, cyclin E and cdk2. In S phase cyclin E
is no longer found
tokine signaling. The mechanism
duce E2F DNA
(Y
binding
of a growth
suggesting that it may reflect an important
though
between ~107
or interferon
is de-
The
DNA
binding
complex
named
E2F
is a het-
erodimeric complex consisting of an E2F component and a
graded and at the beginning of the subsequent GI phase
E2F is again found in complex with ~107 alone. When
the E2F and DP component to DNA
quiescent cells are stimulated to enter the cell cycle, the
quently. E2F site-dependent transactivation by E2F and DP
pl07-E2F
complex only becomes apparent in late Gl
and
dimerization
partner, the DP component.
The bindina
of
is synergistic. Conse-
proteins is also highly interdependent [143-1461.
The in-
persists in S phase [ 1341. The late G I complex consists of
teraction with the DP component is also essential for the
E2F-~107
high affinity
and cyclin E-cdk2.
Although
in mouse fibrob-
interaction of E2F with pRb and pi07
[146-
112
1481. The
E2F
component
DP-2
is encoded by at least five
I through
different genes, E?.Fnent two different
E2F-5.
For the DP compo-
genes have been isolated,
[147,149-1541.
Ai1 the different
DP-I
E2Fs
and
are struc-
cycle. Vairo
et al. have shown that the predominant
present in unstimulated
pressed throughout
T cells is E2F-4.
E2F
E2F-4
is ex-
the ceil cycle of re-stimulated
quies-
cent tibrobiasts and human keratinocytes [120,153].
This in
turally related and have several regions that share a high
contrast to the pRb interacting E2F-I
degree of
very low or absent in quiescent ceils and is induced only
binding
homology.
domain,
These
the DP
transactivation/pocket
regions
include
dimerization
two DP proteins share limited
the DNA
domain
protein binding
and the
region 11351. The
homology
with the E2Fs
except for the region that corresponds to the DNA
and dimLrization
Although
F.ote;n
interaction
domains,
only EZF-L.
EZF-2
and E2F-3
pRb in viva [I51 .l52].
under pRb
pocket
control
[120.135,147,153,158].
DP-I
and DP-2
activating
specificity
for
Of the five known E2Fs
E2F-4 and E2F-5
~107
and/or
All
appear not to be
~130
the
I)
(Fig.
E2Fs can interact
with
both
in viva. and each complex is capable of
transcription of reporter genes that have an E2F
consensus DNA
binding sites in their promotors. This also
suggests that the pocket binding
serum
stimulation
activation
specificity
is not detcr-
The regulation of EZF.pocket
cyclins
and the targeting
transforming
of gene expression during the cell cycle. Indeed, the list of
promoters containing E2F binding sites includes genes that
b-m&
EZF-I
proteins for cell cycle-regulated biochemical processes such
as DNA
polymerase
(Y. thymidine
kinase and dihydrofolate
dependent
is not found
in
Drosophila.
At
Many of these
genes are induced in quiescent cells following
:his
in mammals
synthetase, thymidine
reductase (DHFR).
‘rhe
DPs
cdc2.
and cyclin A, as well as genes encoding
and
E2Fs
by viral
encode ceil cycle regulators such as c-mnxc. N-r?z~.
subunit
of the multiple
protein complexes by Gi
of these complexes
proteins Indicates that E2F is a key regulator
virus infection. concomitant
compiexily
The
tions of the E2F family of transcription factors.
mined by the DP component but is mediated by the E’!F
[152].The
[135,136,150].
strongly argues for specific func-
8.3. E2F regulated genes
are found associated with
but rather tound associated with
proteins
after
in their pocket
they display
pocket protein binding [151,157].
h
strictly-timed
binding
region of these proteins [152.155,156].
the E2Fs are highly homologous
S-10
whose expression is
ability
of
these viruses to activate
on their
DNA
tumor
with the induction of S phase.
ability
to bind
these genes is
and
inactivate
the
moment only a single homologue of F2F. DP and the RB
retinobiastoma
gene
the important role of E2F in the regulation of these genes.
have been
comm.).
identified
Significantly.
Drosophikc
indicating
[159.160]
all E2F
DNA
Dyson,
binding
pers.
activity
in
can be accounted for by these three proteins.
that it is unlikely
bers exist in Dro.wphi/o.
that additional
The functional
the Dro.wp/ti/a
E2F homologue
by disruption
of the single dE2F
homozygous
induce
(N.
dE2F
for null mutations
DNA
family
significance
was demunstrated
of dE2F
can no longer
17. when maternally
provided dE2F is no longer present. Mutant
replication.
This
transcription
embryos also
of genes essential for
suggests that dE2F.
essential for the Gl
of
gene [ 1611. Embryos
synthesis after cycle
lack the coordinated
mem-
in most ceils.
is
to S phase transition.
E2Fs
in higher organisms is still unclear. One possibility is that,
although
they can all recognize the same E2F consensus
sequence. they differ
As a result.
subtly in DNA
they may control
Recent analyses have indicated that the E2F binding sites
are critical for the growth-regulated
moters [
binding
different
specificity.
sets of genes.
164.166
!681.
The DHFR
inverted and overlapping
tion initiation
activity of these propromoter contains two
E2F sites located at the transcrip-
site. Transcription
gene promoter
from a truncated DHFR
increases more than IO-fold
phase transition. which was lost following
E2F sites [169].
Furthermore,
EZF sites upstream of a heterologous
strong transcriptional
iXlFR
for
the ceil
cycle-regulated
expression
One puzzling
aspect of thr different
EZF sites is that their transcriptional
genes containing
activation
with
EZF-pi07
complexes
[162-1641.
Further-
more, infection of rat fibrobiasts with a recombinant adenovirus that mediates expression o!’ E2F-I.
leads to tran-
does not
occur at the came time in the cell cycle. c-m_vc and N-nzyc
are immediate early genes, whose expression is induced
within
entially
of the
gene.
genes encoding products involved in DNA
preter-
causes a
boundary [170].
and b-ttt!b
E2F sites that interact
of the DHFR-
promoter
increase at the GI/S
Consistent with this, the promoters of the thymidine kinase
genes contain
at the Gi/S
mutation of the
introduction
Together these data indicate that the E2F sites are essential
elements
The relevance of the existence of many different
protein and its relatives. This emphasizes
minutes after serum stimulation.
first expressed at the G
I/Stransition.
depend on E2F. additional
In contrast, the
replication
are
Since both appear to
mechanisms must exist to oc-
scriptionul activation of only a subset of EZF-site contain-
count for the distinct expression patterns. One hypothesis
to explain this difference is that other transcription factors
ing promoters 11651. In addition.
act in concert
appearance
of the EZF-pocket
cell cycle indicates
different
the specific
protein
that the various
pattern of
complexes
E2R
points in the cell cycle. Moreover.
in the
are active at
the different
E2Fs show a unique pattern of expression during the cell
with
E2F
to mediate
promoter
activity.
Elements that contain binding sites for Spl and CCAATbox binding factors have been implicated to cooperate with
E2F in growth-regulated
transcription [I 36.1X].
the existence of different
E2Fs.
that disp’ay
However.
a distinct
113
pattern of expression and complex
formation
with their
that E2F is subject to negative regulation by phosphoryla-
inhibitory pocket proteins in the cell cycle, may also allow
tion. In S phase, the E&I-DP-1
differential
quarternary
regulation of promoters that contain E2F sites.
cdk-2
8.4. Regulation
of E2F actiriu
heterodimer is found in a
complex with cyclin A and cdk2. Cyclin
complexes
can directly
interact
DP-I
dimer with cyclin A-cdk2
tion
of
DP-I
[177,181.182].
with
A-
the amino
in vivo [181]. The association of E2F-I-
terminus of E2F-I
and
loss
leads to ihe phosphoryla-
of
DNA
The phosphorylation
binding
of DP-I,
affinity
which can be
observed in late S phase when cyclin A expression peaks,
can only occur when DP-I
region of E2F-I
A-cdk2
is bound to E2F-1
involved
in the interaction
tlSl].
contrast, the N-terminal
in the plO7-
The
with cyclin
is conserved between the pRb-interacting
E2Fs. In
cyclin A binding domain is absent
and pl30-interacting
E2F-4
and E2F-5,
sug-
gesting mat these E2F subtypes escape this type of negative controol. This is also suggested by the observation that
in S phase DNA
binding complexes containing
E2F and
~107 and cyclin A are readily observed. Downregulation
of E2F
explair
activity
by cyclin
A-cdk2
during
S phase may
why many of the genes that are transcriptionally
induced
at the GI/S
transition
decrease in expression
during S phase. Recent data by Resnitzky et al. cast doubt
on this model.
They
cyclin A enhan,es
show that enforced
Recently.
yet another
of E2F activity [47].
level of E2F-I
been described. The proto-oncogene
the activation
domain
of E2F-I,
activity of the EZF-I-DP-I
8.5.
Trunscripfiorml
Whereas
expression of
5 phase entry rather than inhibit it by
the presumed down modulation
regulation
MDM2
thereby stimulating
the
complex [183].
repwssio~~ mediared
roost E2F
has
interacts with
sites in cellular
by E2F
com-
promoters act as
positive elements that confer cell cycle-regulated
activa-
tion. in some cases E2F sites have been shown to act
primarily as negative elements. For instance. the b-myb
promoter
is repressed in GO and GI
E2F-~107
complex to an upstream E2F site. Mutation
this E2F site was sufficient
by binding
of an
to relieve transcriptional
of
re-
pression in GO, resulting in a promoter with constitutively
high activity that was equal to the G I /S
levels seen with
the wild type promoter [ 1621. This indicates that pl07-E2F
complexes can act as active transcripttonal repressors in
GO and early Gl.
The disruption
of these complexes in
mid to late Gl
leads to de-repression and activation
other regulatory
elements in the same promoter.
by
Similar
mechanisms have been observed for the promoter of the
insulin-like
E2F-I
growth
genes [ 136, I7
factor-l
(IGF-I),
I.I72.I84,I85].
cdc-2.
c-m.w
and
Consistent with this.
Weintraub et al. showed that a synthetic promoter construct in which E2F sites were placed upstream of a strong
promoter, the E2F sites act as negative elements [186]. The
transcriptional
repression
is most likely
caused by the
recruitment of pRb to the promoter because co-expression
of EIA
(that disrupts EZF-pRb
complexes)
relieves this
R.L Br&rsbergen.
114
repression.
Furthermore,
the E2F
negative elements in pRb-‘E2F-pRb
When
R. Bemards/
sites did
Biochimira et Biophwica Acta 12X7 f I996J 103-120
not act as
cells, suggesting that only
complexes were active repressor complexes [ 1861.
fused to a DNA
binding
domain of GAL
4, both
pRb or ~107 repress transcription of promoters that contain
GAL
4 DNA
binding
sites, indicating
repression is a universal
property
that transcriptional
of the r:tinoblastoma
W?intraub
e! a!. have suggested a mechanism
repression by pRb [ 1891. They
suggest that pRb is recruited
to promoters through
where pRb binds and inactivates neighboring
factors.
Tile
promoter-localized
like Elf-l,
effect on VP-16.
difference
SP-I
PU-I
pRb
PU-I
and c-Myc
like
localized
pRb
but not to the insensitive
factors
VP-16,
SP-I
and CTF
transactivators
by pRb prevents their interaction
transcriptional
machinery [ 1891.
significancl:
trans-repression
remains
a mechanism
(1891.
transrepression
unknown.
to silence
might
E2F-~130
be required
In differentiated
complex is E2F-~130.
complex
with the
protein-mediated
Obviously,
it would
S phase-specific
gene
8.6. E2F and
cell
for
the induction
cells the predominant
of
E2F
It is therefore well possible that the
in these cells
suppress proliferation-associated
functions
to actively
genes.
biolop
a central
position
E2F
target
genes is involved
[ 1941. The role of DP-I
of rat embryo
in a regulatory
these
ras oncogene in the
tibroblasts,
seem to depend on the intern&on
an EZF-independent
in the
and DP-2
is still unclear. Although
this does not
with E2F. This suggests
effector function
for the DP proteins
in cell growth control [195].
the binding
of pRb
and plb-related
network
that controls
The observation
that E2Fs are
proteins
~107
and
~130. That the major consequence of this binding, release
of E2F transcription
was shown
factors, is essential in S phase entry
by the co-expression
of
an E2F
dominant
negative mutant. This mutant was able to block El A-induced cell cycle progression, indicating
is essential for El A-induced
that activation
of
cell cycle progression
[196]. The hypothesis that members of the pocket protein
family can induce a cell arrest by inhibition
funher
substantiated
by the observation
of E2Fs was
that pocket pro-
tein-induced cell cycle blocks could be overcome by overexpression of their E2F partners.
cycle block could
E2F-I
The
plb-induced
cell
be rescueo by the overexpression
of
and to a much lesserextend by E2F-4 [107,120.191].
In contrast. a p IO7- or p I30-mediated
growth arrest could
efficiently
be rescued by overtixpression of E2F-4 and not
by E2F-I
[I 18.1201. The observation
and cyclin E are transcriptionally
The results discussed above pl;ice the different E2Fs in
growth and diiferentiatton.
transformation
E2F
of pocket
expression in other periods of the cell cycle. In addition.
differentiation.
of
observed transformation
The expression of the viral El A protein in quiescent
for this
in vitro data suggest that the binding of these
provide
the activation
is re-
ability. This suggests that
cells can induce S phase entry. This effect is mediated by
transcription
in vivo
exhibits increased transformation
a chimetic
domain
of herpes virus VP16
to repress
Furthermore.
The
Furthermore,
domain
but has no
One explanation
that promotor
E2F.
transcription
is able
and c-Myc,
and CTF.
is the observation
binds to Elf-l,
by the activation
proteins cooperate with an activated
for the active transcriptional
transactivators
[147,194].
E2F- I protein in which the transactivation
proteins in transformation
protein family [ 187.1881.
Recerlly
mt embryo iibroblasts
placed
gest a potential inactivation
ted transcriptional
activation
indicate that the different
that both cyclin A
induced by E2F-I.
sug-
of pRb through EZF-l-media(1651. Together these results
E2Fs are involved in cell cycle
under the control of the retinoblastoma family of growthinhibitory proteins suggests that the inactivation of E2Fs
control and that the different pocket proteins regulate cell
plays an important role in the cell cycle arrest imposed on
members of the E2F rranscription factor family.
cells by the pocket
proteins. That
E2F
indeed plays an
important role in the control of the transition from GI to S
cycle progression via distinct pathways involving
8.7. E2F md apoptoxiv
Overexpression
is suggested by several observations. Ectopic expression of
E2F-I
can prevent cells from entering quiescence and can
induce S phase entry in quiescent fibroblasts
sence of serum [ 190,191].
ability
of E2F-I
The ability
in the ab-
These effects depend on the
to bind DNA
and activate transcription.
to stimulate cell cycle progression is not lim-
ited to the pRb-interacting
E2Fs.
together with its dimerization
Introduction
partner DP-I
of E2F-4
in the osteosar-
coma cell line SAOS-2, reduces the number of cells in G I,
indicating that E2F-4 activation stimulates cell cycle pro-
plete a cell cycle
[
different
cell types, indicating
not tolerated
these cells do not com-
undergo
programmed
cell
unpublished
that high levels of E2F are
in most cells (R.
Kerkhoven
data). One explanation
and R.L.B.
for the induction
of
apoptosis by El!F is that E2F stimulates the expression of
proteins that are able to induce apoptosis. For example an
moqt members of the E2F gene
E2F
2, 3, 4 and DP-I
apoptosis [2Ol].
proteins. are
can induce S phase entry in
but rather
death l97-2001.
Also the generation of stable cell lines
that overexpress E2Fs has been unsuccessful in several
family,
E2F-I.
of E2F-I
the absence of serum. However.
gression [ 1471. Moreover.
including
different
target
such as c-myc
has been shown
to induce
It is also possible that the untimely entry
able to transform cells. Deregulated expression of E2F- I. 2
into S phase is responsible for apoptosis after the induction
or 3 can lead to transformation
of
cell line [192,193].
E2F-4
of a rat embryo fibrobiast
whereas the oveicrptession
together with activated
of ZF.!
ras can transform
or
primary
E2F
abptuais
in
low
serum.
provides
rn
The
ability
erp!anatioli
apoptosis by the functional
of
E2F
to induce
for the indac;ioe
inactivation
of
of pRb, either by
115
viral inactivation or mutation. White et al. have shown that
EIA
expression
12021.
can induce
Furthermore,
apoptosis
in primary
cells
the loss of both pRb alleles in mice
causes degeneration
p53 in the E2F-induced
MDM2
and UBF
have been re-
ported that seem to play different roles in the pRb-mediated cell cycle arrest. The nuclear tyrosine kinase c-Abl
is
rylated form binds to the catalytic domain of c-Abl thereby
Recent data have implicated
apoptosis. EZF-l-mediated
BRG-I,
found associated with pRb in vivo. Only the hypophospho-
of a number of tissues, most likely
due to apoptosis [203-2051.
proteins c-Abl,
apopto-
inactivating
the
kinase
activity
of
c-Abl.
Although
sis is suppressed by co-expression of pRb or a transdomi-
phosphorylation
nant negative mutant of ~53 [198,200].
Abl, its binding to pRb is not mediated by the E2F binding
demonstrated
that ~53 is important
results from loss of pRb function.
in the apoptosis that
In the absence of ~53,
loss of pRb results in uncontrolled
inactivation
It has also been
proliferation
and the
of one RR allele in a p53 nullizygous
mouse
of pRb regulates the interaction
with c-
domain of pRb. Welch and Wang have suggested that the
assembly of multiprotein
complexes containing
both E2F,
pRb and c-Abl are essential to control of the activity of a
number
of
genes
involved
in cell
cycle
proliferation
to-
[213,21/r].
pRb also associates with
BRGI
gether, this could indicate that the loss of pRb results in
[215.216].
Both BRCil
share extensive se-
the activation of E2F that causes p53-dependent apoptosis.
quence similarity
Although
activator of homeotic gene expression and the yeast tran-
gives rise to a higher tumor incidence [206].
E2F-I
mediated apoptosis appears to be p53-de-
pendent, the inability
to generate stable E2F overexpreps-
ing cell lines that lack ~53 function,
pendent and p53-independent
of pRb
8.8. Orher targets
Apart
from
Taken
the E2Fs.
indicates that p53-de-
family
Drosophila
activator SNF2/SW12.
to specific
DNA
show that BRGI
member.?
in chromatin
in cell cycle regula-
block the pRb-BRG-I
is abolished
Interestingly.
ATF-2,
Elf-l
PU- I, UBF, BRG- I, MDM2
is a member of the ETS
MyoD,
and c-Abl[207-2
family
IO].
of transcription
does not bind
seems to function
structure. Dunaief
by
et al.
in pocket mutant
interactions and BRG-I
with pRb in the formation
Elf-l,
an
associates with the hypophosphorylated
regulators
including
Brahms.
pRb from human tumor cell lines 12151. Viral oncoproteins
tion. pRb influences the activities of other transcriptional
by direct interaction.
and hBRhI1
gene
Brahms
sequences but
form of pRb and binding
ihe pocket proteins reguhne a
number of other pathways involved
scriptional
inducing alterations
pathways are involved.
and hBRMl
to the
cooperates
of flat, growth-arrested
cells.
the cervical carcinoma cell line C33A has no
detectable level of BIG-I
to a plb-induced
expression and is not sensitive
cell cycle arrest, suggesting that BRG-I
factors that regulates gene expression during T cell devel-
and pRb may cooperate to induce growth arrest [107]. This
opment. The mechanism by which pRb regulates Elf-l
is also substantiated by the finding that the BRG-I
analogous to E2F: hypophosphorylated
pRb interacts with
the transcriptional
activation domain of Elf-l
inactivating
in resting T-cells.
Elf-l
results in phosphorylation
is
Activation
and thereby
of T cells
of pRb and activation
of tran-
logue hBRM
SAOS-2
cells (2151. The interaction of pRb and hBRMl
involved in the stimulation
scription mediated by Elf- I. In addition IO the repression
mediated transcription,
pRb-mediated
it is possible that pRb activates genes that
of transcription.
pointing to hBRMl
transcriptional
Recently, an interaction
is
The presence
of both proteins upregulates the glucocorticoid
of transcription,
are involved in suppression of cell growth. Indeed positive
homo-
cooperates with pRb in flat cell induction in
receptor-
as a target for
activation [206].
between pRb and MDM2
has
regulation of transcription by pRb has been reported in the
case of the ATF-2 transcription factor, which mediates rhe
also been described [202]. The cellular oncoprotein MDM2
activation
cenzn
TGFP
of the TGFp2
promoter
proteins induce a Gi
by pRb
[207].
The
arrest in many cell types, so
was originally
identified
tumors. MDM2
that the activation of expression of these factors provides a
that MDM2
means to constrain celi proliferation.
Although a direct
interaction between ATF-2
and pRb has been demon-
pRb growth-inhibitory
strated in vitro, the cxaci orecirr(&~~ tr
ATF-2
dependent transcription
tional activation
control
This
G,<i~
pZ
is still unclear. Transcrip-
can also be mediated
element (RCE).
rrh;&
element
through
tbp PRb
is present in the
promoters of TGFp I, and c-Jun. The pRb-specific
on RCEs is probably
mediated by SPI.
The SPI
effect
protein
as a protein that is amplified
in
also binds to and inhibits transacti-
vation by the ~53 tumor suppressor gene. Xiao et al. show
interacts with pRb and, as with ~53. inhibits
function [2lO].
MDM2
C-terminus of pRb. but binding of MDM2
the interaction
with E2F, thus providing
nation for the observed rescue by MDM2
binds to the
appears to block
a possible explaof pRb growth
suppressive activity. On the other hand. Martin et al. have
demonstrated
that MDM2
can also directly
activate E2F- I [ 1831. Together
MDM2
bind to and
these results indicate that
can stimulate E2F mediated transactivation
via two
can bind to the RCE element and pRb can enhance both
ways. first by releasing pRb and second through the activa-
the DNA
binding
tion of E2F itself.
[211.212].
This activation
and transactivational
sequestering of an inhibitor
activity
of
SPI
is most likely mediated by the
of SPI by pRb, named SPI-I.
The interaction between pRb and the RNA
transcription
factor UBF
polymerase
gives another dimension
role of pRb in the regulation
Db!A
growing cells require the ongoing synthesis of ribosomal
as!!
ta
Recently,
activa!e transcription 17 I?]
interactions
between
pRb
and the cellular
RNA.
Cavanaugh
of transcription.
I
to the
which in the absence of pRb prevents SPI from binding to
Actively
et al. show that the pocket of pRb is
116
required for the interaction between pRb and UBF
in cell
that ~107 and ~130 share the ability to interact with E2F-4
extracts [217].
by the
and cyclin A or E-cdk complexes.
addition
The
activity
of
UBF
is inhibited
of pRb in vitro. The nature of this interaction
would suggest a general role for pRb in the modulation
of
Several
important
lines of evidence
in growth
The myogenic helix-loop-helix
protein MyoD
promotes
a functional
pRb
Furthermore,
cyclin
arrest. The
members of
the retinoblastoma
gene
family appear to play an essential role in the withdrawal
of
the cell cycle and phenotypic
an
between myoD
differentiation.
and pRb
Although
has been observed
the mechanism by which myoD induces differentia-
tion is still unclear. Recent data indicate that myoD may
is more
and ~130.
For
of all three
members of the pRb family. requires only the presence of
skeletal muscle specific gene expression and induces a cell
cycle
interaction
that pRb
than ~107
instance, ~16, which prevents phosphorylation
gene expression.
[208],
indicate
inhibition
to induce
a GI
D-associated
arrest [77,102-104).
kinase activity
is only
required for cell cycle progression in pRb positive cells. At
first glance, this may seem surprising, as the downstream
:argets or ~107 and ~130 (E2F-4
growth-promoting
in addition
and c-Myc)
activity [147.219].
to pRb,
contribute
have strong
That ~107 and ~130,
to cell cycle
control
is
induce terminal cell cycle arrest by increasing the expres-
substantiated by several observations. First, all three pocket
sion of the cdk inhibitor
proteins are phosphotylated
remains
induce
p2l [73]. As a result of this pRb
in the hypophospiiorylated
a GO arrest. Consistent
myogenesis, inactivation
state and is able to
with
a role for pRb
in
of pRb bv mutation or binding to
D-type cyclin-associated
forming
and inactivated at mid Gl
by
kinase activity. Second, the trans-
proteins of several DNA
tumor viruses bind to
and inactivate all three members of the pRb family. This
viral proteins interferes with myogenesir. In contrast, myo-
may indicate that for the efficient induction of S phase, all
genie differentiation
three family
m pRb-delicient
mice seems normal.
One explanation for this apparent discrepancy is the observation that muscle differentiation
‘-
in pRb_
lates with the increased expression of ~107
ever, the phenotype of these differentiated
different
from their wild
cells corre[218].
How-
muscle cells is
type counterparts
because they
members have to be inactivated.
Consistent
with this view, efftcient induction of a cell cycle block by
X-ray
irradiation
family
requires the presence of all three pRb
members [222].
The
loss of pocket
function
is
tolerated by most cell types with respect to their capacity
to complete
cell
division.
It
is therefore
unlikely
that
are able to re-enter the cell cycle after serum stimu;.uion.
pocket proteins are an integral component of the cell cycle
Apparently,
clock. Rather, it would seem more likely that the primary
induction
Apart
~107 is not able to fully replace pRb in the
of myocyte differentiation.
from the interaction
have shown that ~107
with E2F-4.
forms a complex
oncoprotein (219.2201.
tion domain of c-Myc
we and others
with the c-Myc
~107 interacts with the transactivaresulting in the inhibition
mediated tnnsactivation.
This interaction
interest because of the interaction
gene and a growth-inhibitory
communication
role of pRb family proteins is in the regulation of differen-
of c-Myc-
is of particular
between a proto-onco-
protein could reflect a direct
between proteins that regulate cell growth
and dtfferentiation.
tants of
the
Burkitt’s
lymphoma.
Furthermore.
c-rrryc proto
sion, providing
naturally
oncogene
ihat
occurring
are found
escape the pl07-mediated
in
suppres-
inactivation
of both RB alleles in mice leads to embryonic
lethality
between day I3 and I5 of gestation as a result of defects in
erythropoiesis
and neural development
other hand, many important
[204,205].
differentiation
tion decisions can be taken in the developing
the absence of
pRb.
It
is well
possible
On the
and proliferaembryo in
that
tn these
pRb-‘-
embryo’s ~107 and ~130 can compensate for loss
of pRb.
As was discussed above, in differentiating
oblasts, ~107 can indeed partially
tor in differentiation.
my-
replace pRb as a cofac-
Conversely,
loss of ~107
or ~130
for the increased
may be compensated by pRb. That this is indeed the case
growth rate of these cells [220]. II will be worthwhile to
investigate whether similar mutations. that allow the es-
is supported by the recent finding that mice that carry only
cape from
a possible explanation
mu-
tiation. In agreement with this, the functional
pocket protein
inhibition.
ore also present in
E2F genes in human tumors.
have reduced body
pRb-“+ and p 107-/abnormalilies
9. Functional
differences between pRb family members
Although pRb, ~107 and ~130 all have strong growth
suppressive activity, only pRb has been found mutated in
human cancer. Furthermore.
only loss of one allele of Rtl
in mice leads to predisposition to cancer ([203-205.22
D. Cobrinik.
~107
personal communication).
and ~130
are functionally
I].
It is possible that
redundant and that only
(M-H
10. Concluding
All
weight and viability, whereas both
mice show no major developmental
Lee. personal communicationl.
remarks
of our knowledge
concerning
action of the retinoblastoma
family
the mechanisn.
of
of growth-inhibitory
proteins stems from the last decade [223]. Although
many,
if not most. of the basics of pRb action are now understood
in some detail.
particular,
several issues remain to be clarified.
In
the recent finding that the fruit fly carries only
loss of both genes in the same cell causes growth deregula-
one EZF-like
tion. In support of this redundancy argument
mammalian
is the fact
RB allele and two inactivated ~107 alleles
one functional
gene and one pRb-like
gene. whereas the
genome carry at least 5 E2F genes and en-
R.L. Beijersbergen. R. Bema&/
Biochimlca er Bioplyica
codes three retinoblastoma family members is intriguing. It
most likely indicates that for proper differentiarion and
growth control of the multitude of specialized cell types in
the mammalian
body, the network of growth stimulatory
and growth-inhibitory
proteins had to be expanded. hrdeed.
the initial studies using mice that carry targeted disruptions
of these genes seems to support the notion that the three
pRb family members differ subtly in their role in differentiation and proliferation. At the same time this would
suggest that the five E2Fs also differ with respect to their
ability
to control proliferation
probably
and differentiatton.
it will
be a lot less than ten years before we get the
answers to these questions.
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