Regulation of Fibroblast Growth Factor 2

Vol. 8, 1199-1210,

November

Regulation Melanoma

1997

Cell

of Fibroblast Growth Factor 2 Expression Cells by the c-MYB Proto-Oncoprotein’

Mark R. Miglarese,2 Ruth Halaban, and Neil W. Gibson3 Department of Cancer, Immunology and Infectious Deases,

Pfizer Groton, Connecticut 06340 [M. R. M., N. W. G.], and of Dermatology, Yale University School of Medicine, New

Central Research, Department Haven, Connecticut

06520

[B. H.]

Abstract Dysregulated expression of basic fibroblast growth factor [fibroblast growth factor 2 (FGF-2)] mediates autocrine growth of melanoma cells. The presence of a consensus Myb binding site in the human FGF-2 promoter prompted us to investigate whether this transcription factor could regulate FGF-2 expression in melanomas. We report that c-MYB mRNA is overexpressed in melanoma cell lines compared to normal melanocytes and that ectopic expression of murine c-Myb in SK-MEL-2 human melanoma cells resufted in increased expression of FGF-2 mRNA and FGF-2 protein. Furthermore, munne c-Myb transactivated a reporter plasmid containing the human FGF-2 promoter region in cotransfected SKMEL-2 human melanoma cells. Although a functional DNA-binding domain was required for transactivation, responsiveness to c-Myb was independent of the putative Myb binding site and mapped to two regions of the FGF-2 promoter that did not bind c-Myb In vltm. We suggest that c-MYB contributes to FGF-2-mediated autocrine growth of melanomas by indirectly regulating the FGF-2 promoter. Introduction FGF-24 is a member of a family of heparin-binding growth factors that play critical roles in development, wound healing, hematopoiesis, and tumorigenesis (1-6). FGFs mediate their

Received 5/5/97; revised 8/25/97; accepted 9/22/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-

cate this fact. in part by USPHS Grant CA44542 from the NIH (to A. H.). M. R. M. was supported as a Pfizer Post-Doctoral Research Scientist 2 To whom requests for repeints should be addressed. Present address: Department of Dermatology, Yale University Medical School, New Haven, CT 06520. Phone: (203) 785-4965; Fax: (203) 785-7234, E-mail: [email protected]. 3 Present address: Institute of Bone Joint Disorders and Cancer, Bayer Corporation, Pharmaceutical Division, West Haven, CT 06516. 4 The abbreviations used are: FGF. fibroblast growth factor bFGF, basic fibroblast growth factor DNAB, DNA binding; EMSA, electrophoretic mobility shift assay; mAb, monoclonal antibody; MBS, Myb binding site; MRE, Myb response element; RT-PCR, reverse transcription-PCR; TBE, I Supported

Tris-borate EDTA, HSF, heat shock factor; dHSE, distal heat shock element; CDK, cyclin-dependent kinase; YRIPA, yellow radioimmunoprecipitation

assay

buffer

MBS mut, mutated

Myb

binding

site.

Growth & Differentiation

in

effects in part by stimulating the catalytic activity of members of the FGF receptor tyrosine kinase family (7, 8). Activation of the FGF receptor stimulates intracellular signaling pathways involved in proliferation, differentiation, and migration (7, 914). FGF-2

is a potent

mitogenic

and

angiogenic

factor

that

is believed to play a role in the growth of several tumors, including melanomas, glioblastomas, hepatomas, leukemias, and cancers of the bladder, prostate, ovary, and breast (15-24). In fact, elevated levels of FGF-2 in the urine or serum of patients with several of these malignancies have been reported to correlate with poor prognosis (1 9, 25, 26). A role for FGF-2 in the growth of normal and transformed melanocytes has been firmly established. Normal melanocytes do not express FGF-2 but require an exogenous supply, most likely produced by basal keratinocytes and dermal fibroblasts (27-29). In contrast, melanoma cells produce FGF-2, which acts as an autocrine growth factor. This conclusion is supported by several lines of evidence: (a) melanoma cell proliferation in vitro is inhibited by internalized anti-FGF-2 antibodies (1 5), by antagonistic FGF-2 peptides spanning the receptor-binding domain (15), or by introduction

of

FGF-2 in murine

antisense

mRNA

oligodeoxynucleotides

(30); and (b) constitutive

melanocytes

results

directed

expression

in FGF-2-independent

against

of FGF-2 growth,

de-differentiation, and morphological transformation in culture (31). Despite the clear role for FGF-2 expression in the proliferation of melanoma cells defined by these observations, the mechanism by which FGF-2 expression is aberrantly activated in these tumors has not been elucidated. Several transcription factors have been reported to regulate FGF-2 promoter activity. In glioblastoma and hepatocellular carcinoma cells, FGF-2 promoter activity is stimulated by mutant p53, suggesting that mutations in the p53 gene may contribute to FGF-2-mediated autocrine growth (32). In addition, the product of the primary response gene, egr-1, has been shown to activate the FGF-2 promoter in human astrocytes, which exhibit FGF-2-mediated autocrine growth (33). Finally, the HOXB7 homeodomain transcription factor was reported to transactivate the FGF-2 promoter in HeLa cells (34). However, the regulation of FGF-2 promoter activity in melanoma cells has not been characterized. The TATA-less human FGF-2 promoter has at least two transcription initiation sites and contains several potential transcription factor binding sites, including a consensus MBS located at position +169 relative to the 5’ transcription initiation site (35, 36). The c-MYB proto-oncogene encodes a 75-kDa transcription factor that is expressed at high levels in immature normal and transformed hematopoietic cells, in which it plays important roles in both proliferation and differentiation (37). Expression of c-MYB mRNA has also been detected in several other normal and transformed cell types, including vascular smooth muscle cells (38), breast carcinomas (39), small cell lung carcinomas (40), colon carcinomas

1189

12

c-Myb

Regulation

of FGF-2

Expression

(41),

neuroblastomas

(42),

and

tion,

rearrangements

near

or

detected

been

that

in several

c-MYB

may

a role

of five

c-MYB

that

melanoma

oligodeoxynucleotides

growth of this

was

tested

of

that

in

growth

in

and in vivo (47). These results, in combination with the presence of a consensus MBS at position + 1 69 in the FGF-2

vitro

led us to speculate

promoter, in regulating

In this human both

FGF-2 report,

expression

we

c-Myb

and

transactivated

in transfected tional

DBD

was

melanoma required

and

elements

ing that c-Myb

the

0.

a func-

for transactivation,

FGF-2

binding to the FGF-2 promoter. plays a role in the FGF-2-mediated anomas

by contributing

c-Myb

without

dysregulated

a)

500

U)

c-MYB of mel-

expression

of

FGF-2.

B

Results Overexpression of FGF-2 Protein and c-MYB mRNA in Melanoma Cells and Transactivation of the FGF-2 Promoter by Munne c-Myb. As a first step toward examining a potential

role for c-MYB

examined expression man

FGF-2 each (Fig.

melanocytes

and

protein

and

melanoma

line

were

through

plasmid luciferase

+ 1 78 of the

stimulated

c-Myb

promoter-reporter cells

human

at position

promoterless

+169

luciferase

plasmid luciferase

increase

sequences

melanoma

including the

In contrast,

plasmid,

for

(see

melc-Myb

using

indicating

required

activity

FGF-2

SK-MEL-2

in experiments

were

in luciferase

then

of a murine

from

manner. reporter

actin

2,4). Fig. 2B shows

activity

activity

was

promoter,

(Fig.

in

amount of the that contains bp

in cotransfected

pGL2.BASIC

promoter

FGF-2

the

the

Fig. 1 . FGF-2 and c-MYB are co-overexpressed in human melanoma cell lines compared to normal melanocytes. A, quantitative ELISA showing several fold differences in the amount of FGF-2 immunoreactive protein in three melanoma cell lines compared to normal melanocytes. Each lysate

was prepared

disrupted

tioned

through

that

pared

luciferase

with FGF-2

the c-Myb promoter

activities

in SK-MEL-2

expression or a mutated

plasmid FGF-2

and

+169,

we com-

cells

cotransfected

either

the wild-type

promoter

cell cultures

and assayed

in

c-Myb-

Fig. 4A).

at position

growing

that

the

MBS

from exponentially

triplicate. The mean values are given, with error bars representing the SE. Inset, the results obtained for normal melanocytes and SK-MEL-2 melanoma cells on a smaller scale to better illustrate the 12-fold difference in the amount of FGF-2 detected in each lysate. B, RT-PCR analysis showing overexpression of c-MYB mRNA in melanoma cell lines compared to normal melanocytes (top panel) using p-actin as a control (bottom panel). Lane 1 , normal melanocytes; Lanes 2-4, melanoma cell lines as indicated; Lane 5, positive control PCR products using 50 amol of specific c-MYB or -actin DNA template.

c-Myb Does Not Function through the Putative MBS in the FGF-2 Promoter. To determine whether c-Myb functhe putative

c-MYB

transactivate

amounts

and a constant reporter plasmid

in a dose-dependent

did not stimulate

assay

human

various

I

!p

Both

melanocytes

could

SK-MEL-2 with

lines.

overexpressed

cotransfection

promoter.

C)

we

mRNA of normal hu-

to normal c-Myb

#{149}44

c-MYB

cell

were

murine

cotransfected

MBS

induced

expression,

melanoma

mRNA

whether

that

FGF-2

FGF-2

compared

1 , A and B). A transient

the putative

anoma

human

c-MYB

cell

c-Myb expression pGL2.bFGFpNB

-290

in regulating

steady-state FGF-2 protein and in exponentially growing cultures

used to determine the human FGF-2 cells

0

0

directly

We suggest that autocrine growth

to the

C .!

U.

Ui

In N.

suggest-

in vitro,

expression

0

1000

z

Ui

0

LL

the con-

U) a)

1500

protein

cells. Although

that did not bind

stimulates

2000

E

murine

FGF-2

-I-

2500

sensus MBS in the FGF-2 promoter did not mediate this response. Instead, responsiveness to c-Myb mapped to two sequence

=

0

overexpress

and stimulated

mRNA

3000

to normal

In addition,

promoter

FGF-2

3500

0.

cells. cells

protein.

a)

be involved

compared

melanoma

the FGF-2

SK-MEL-2

c-Myb

that

FGF-2

of endogenous

might

in melanoma

human

mRNA

expression

c-MYB

demonstrate

melanocytes, c-MYB

that

4000

C

antisense

melanoma

4500

notion,

expressed

and

inhibited

A

have

to speculation

deregulated

mRNA

lines

In addi-

locus

In support

c-MYB

cell

(43).

c-MYB

leading

in the

(44-46).

et a!. reported

Hijiya each

the

melanomas,

play

melanocytes

transformed

glioblastomas within

containing

putative

putative

stimulate

sion

did

at position

increase a

had

luciferase

c-Myb

cated

MBS

MBS

could

activity

Surprisingly,

3A).

on the

function through + 1 69 of the FGF-2 activity an inability

ability

in cotransfected

not

in luciferase reflect

(Fig.

no effect

putative

promoter

of c-Myb

of to

cells,

the

in response

mutation of c-Myb

(Fig.

to c-Myb to interact

indicating MBS

lo-

3B). The expreswith

the

Cell Growth

+55

1201

Activation of the FGF-2 Promoter by c-Myb Is Indirect. Because c-Myb did not seem to function through the putative MBS within the FGF-2 promoter region, we determined

A +1

& Differentiation

+178

which B

elements to c-Myb.

a c-Myb

expression

mids

the

+1

were

and

plas-

promoter.

FGF-2

region

of the FGF-2

transcription

no effect

the with

of reporter

of the

to +12

virtually

mediated

cotransfected

a series

one of the two

had

,

promoter

cells

portions

-290

contains

position

at

V

which

FGF-2

plasmid various

within

moter,

the

SK-MEL-2

containing

Deletions

0

within

response

pro-

start

on luciferase

sites

activity

0

compared ing

to that

that

the

generated

region

from

including

pGL2.bFGFpNB,

bp

+13

indicat-

through

+178

was

sufficient

to maintain maximal luciferase activity in the prosence of ectopic c-Myb (Fig. 4A). Cotransfection of the + 13/ + 1 78 promoter-reporter plasmid with either the c-Myb expression

Mgc.Myb

expression

plasmid

that

per dish

human FGF-2 promoter by c-Myb in SK-MEL-2 melanoma cells. A, schematic representation of the pGL2.bFGFpNB FGF-2 prornoter-luciferase reporter construct. The solid black rectangle marked LUC represents the firefly luciferase reporter gene, the open square represents the putative consensus MBS, and the thin black line represents the promoter elements. Arrows indicate the known transcription start sites, and numbers mark nucleotide position with respect to the most 5’ transcription start site (+1). B, transactivation of the FGF-2 promoter in melanoma cells by c-Myb. SK-MEL-2 melanoma cells were transiently cotransfected with increasing amounts of the pRMb3SV.wt expression plasmid (as indicated) plus 1 g per dish of pGL2.bFGFpNB. Data represent the mean fold activation of three independently transfected dishes over pR3SV empty vector control ± SE. Fig. 2.

Transactivation

of

the

putative MBS. We therefore ated with oligodeoxynucleotide wild-type

or mutated EMSAs

moter.

CMT3COS

a single

containing

that

lysates

MBS murine

bound

copy

whether

probes

putative

showed

cell

tested

c-Myb

containing from

the

c-Myb

of the

MBS

probe from

the

chicken mim-1A promoter (Fig. 3C, Lanes 1, 12, and 14-16). The presence of c-Myb in the major complex containing the mim-1A

probe

was

by a supershift

verified

induced

by a mAb

directed against c-Myb (Fig. 3C, Lane 3). Preincubation with a 100-fold molar excess of unlabeled mim-1A oligodeoxynucleotide ment

but

unlabeled

the

Lanes

13 and

senting

either

inhibited

specificity

of the

wild-type

the

1).

pete

Moreover,

or mutated

MBS

from

from

the

wild-type

or mutated

the

with

the

relative these

luciferase

activity

by c-Myb

putative

was

MBS

in the

FGF-2

promoter

and

indicated

from not

MBS located

that the specific

the FGF-2

mediated

at position

by an interaction

+169.

with

confirmed

highly

responsive

plasmid

resulted

and

+1 12 from

the + 1 3/+ 1 78 promoter-reporter

which

located

+50/+178).

removes

at position

activity

to

a

the

similar

gether,

these

data

through

+49

and

that

+129

that

at least +165)

region

were

required

luciferase

activity

in the

presence

murine

(+112/

for

site

of luciferase from

reporter

through

promoter

through start

generated

control

indicated

+13 plasmid

in a reduction

to

promoterless

re1 78 to

+ 13/+

transcription

+ 55, resulted

level

pGL2.BASIC

remaining

of bp +13/

in substantial

(Fig. 4A, compare Deletion of bp

activity

zX129-165

+ 1 78),

deletion from the

the

plasmid.

two

To-

elements

within

the

(+ 13 FGF-2

maintaining

maximal

of ectopically

expressed

c-Myb.

We next

used

EMSAs

elements

Similar

the results

to

CMT3COS

cell probe,

mim-1A

anti-c-Myb 2, 3, and

from

mouse

probe

FGF-2

(Fig.

4B).

in transfected

IgG (Fig.

control

empty

failed to induce a shift in the mobility (Fig. 4B, Lane 2). In contrast, lysates

fectants

these

c-Myb

with the positive was supershifted

but not with

Lysates

with

in Fig. 3, c-Myb

lysates interacted and the complex 7).

whether

associate

shown

antibody 14-1

to determine

could

control with the 4B, Lanes

vector

trans-

of the mimfrom control

1A

or

c-Myb-expressing CMT3COS cells failed to induce a shift in the mobility of radiolabeled probes corresponding to the + 13 +50,

+ 1 29

FGF-2

to

+ 1 65

and

+ 1 3 to

(Fig. 4B,

promoter

Lanes

Moreover, the interaction between beled mim-1A probe was effectively

+

178 regions

c-Myb and the radiolacompeted by a 100-fold

molar excess of unlabeled mim1A ohigodeoxynucleotide not by unlabeled ohigodeoxynucleotides derived +13 FGF-2

Lanes tion

to

+50,

+129

promoter

used

Together,

18-21). of the

to +165,

+ 1 3/+1

or +13

at the

same

these

78 region

of the

5, 6, 8, 9, 1 1 , and 12).

results

to +178 molar

regions ratios

indicated

of the FGF-2

but

from

the of the

(Fig.

4B,

that activa-

promoter

by c-

the

other

promoters

Thus,

we

stimulation

promoter-reporter

plasmid was

plasmid

of c-Myb for the mim-1A MBS was than that for the putative FGF-2 MBS.

data

control

promoter

Myb does not occur through a direct interaction with c-Myb. A Functional c-Myb DNA Binding Domain Is Required for Activation of the FGF-2 Promoter. The c-Myb DBD has been shown to be required for stimulation of at least two

affinity

greater

Together,

ohigodeoxynucleotides

empty

FGF-2

in luciferase

human

the FGF-2

or the of the

1 78 promoter-reporter

to

repro-

failed to comfor c-Myb binding to the mim-1A MBS probe (Fig. 3C, 15 and 16). These results indicated that c-Myb did not

significantly

tative

oligodeoxynucleotides

derived

MBS

FGF-2

interact

that

formation,

(Fig. 3C, compare

complexes with c-Myb (Fig. 3C, Lanes a 1 00-fold molar excess of unlabeled oh-

godeoxynucleotides

Lanes

ele-

did not form

promoter

putative

response

complex

interaction

In contrast,

14).

AMP

cyclic

oligodeoxynucleotide

confirming

4-1

not

+

ductions

promoter

transfected

control

high-affinity

the

region

to ectopic c-Myb (data not shown). However, +129 through +165 or +13 through +49

pro-

FGF-2

from

to a positive

associeither

plasmid

this

of pu-

compared

in the absence the abilities

of direct DNAB (48, 49). of wild-type c-Myb and a

1202

c-Myb

Regulation

of FGF-2

Expression

DNAB-defective

A +1

-290 MBS wt: F-fl

+55

+178

-_________

i+r

-i_____

stimulate reporter c-Myb

failed

-290

+1

+55

+178

Wi 85G c-Myb

MBS

luciferase

mut

(Fig. 5A), to

from the FGF-2 promoterto wild-type c-Myb, Wi 85G activity

from

both

promoter-reporter

FGF-2

the

plasmids

(Fig. 5B). The requirement for the c-Myb DBD was also demonstrated using a control promoter-reporter plasmid containing five copies of the mim-1A MBS, which specifically

r#{248} r

.

mutant,

to stimulate

and

wild-type

MBS mut:

c-Myb

luciferase activity plasmid. In contrast

B

binds

to c-Myb

with

Lanes

1-3 and

12-16

cell lysates ilar amounts

C 0

in transfected endogenous

the

see also

in lysates

from

blot

was

FGF-2

2 and

1 B showing

expressed

of

that simexpressed

Lanes

c-MYB

Fig. 3C,

analysis

3).

that

in SK-MEL-2 protein

was also

cells transfected with (Fig. 5C, Lane 1). These results mdic-Myb DBD is required for transacti-

vector

of the

5C, in Fig.

of endogenous

that a functional

vation

(Fig.

presented

mRNA

amount

control

cated

5B,

50). Western

cells

data

c-MYB

a small

empty

(Fig.

Ref.

SK-MEL-2 with

detected

affinity

and

with an anti-c-Myb antibody confirmed of wild-type and Wi 85G c-Myb were

Consistent cells,

high

SK-MEL-2

promoter

in

SK-MEL-2

cotransfected

cells.

Regulation of Endogenous FGF-2 Expression by cMyb. To determine whether c-Myb regulated endogenous FGF-2 expression, we transfected SK-MEL-2 cells with plas-

I

2

mids

encoding

Myb

protein

3

plasmid

expression

MBSWt

MBSmut

-

mlm.1A: CRE: MBSwI MBSmuI:

-+

+

--

I

-+

ciated empty

mim-1A

UI

I

anIl.Myb.

I

I

mid

+

FGF-2

expression

--+--

-

-

-

-

-+-

-

--+---+-

-

--+-

-

-

-

+-

-

-

-

-+

mid

or a dominant

interfering

in a 42%

increase

to cells transfected

in cell-asso-

with

a control

(Fig. 6A). In contrast, transfection with a plasMEnT resulted in a 41 % decrease in FGF-2

MEL-2

with

Myb

mRNA

mRNA (which

-:L:=.

)&

3 4

5

of the putative

6

7

MBS

8

91011

in the FGF-2

1213141516

promoter

does

not

transactivation. A, schematic representations (MBS wt) and pGL2.MBS mut (MBS mut) reporter

of the pGL2.bFGFpNB plasmids. Conventions are similar to those described in Fig. 2A. U, putative MBS; U, mutated putative MBS. B, transactivation of the wildtype and mutated FGF-2 promoters by c-Myb. Luciferase activity in 5KMEL-2 cells transiently cotransfected with 8 pg per dish of the pRMb3SV.wt expression plasmid and 1 g per dish of either the pGL2.bFGFpNB (MBS wt)or pGL2.MBSmut(MBS mut) promoter-reporter plasmids. Histograms show the mean fold activation ± SE by c-Myb of wt FGF-2 promoter (E or FGF-2 promoter with mutated MBS () over pR3SV control of three independent experiments, each performed in triplicate. C,

with a control

transcriptional

plasmids

in FGF-2

expression

cells by RT-PCR.

MEnT

engrailed

expression

changes

(Fig. 6B, Lanes encoding

to cells transfected the

plas-

repressor

DBD (Fig. 6A). To determine whether the in FGF-2 expression in response to trans-

a c-Myb expression mRNA expression

iY,

c-Myb-mediated

EnT,

lacking the c-Myb observed changes fection

1 2

compared

encoding

FGF-2

Disruption

resulted

compared

vector encoding

responding

abrogate

c-Myb

+-

C

Fig. 3.

murine

consisting of the murine c-Myb DBD fused to the Drosophila engrailed transcriptional repressor (51). Transient transfection of SK-MEL-2 cells with the c-Myb

Experiment mim-1A

either

(MEnT)

were

mRNA

in transiently

Transfection

plasmid compared

resulted to the

1 and 2), whereas substantially

by cor-

we analyzed

transfected

of SK-MEL-2 in increased empty vector

transfection

reduced

reflected

levels,

with

the amount

5K-

cells with FGF-2 control a plasmid of FGF-2

compared to transfection with the EnT or MEnTAl lacks the entire MEnT coding sequence) control plas-

DNAB analysis of c-Myb with the consensus mim-1A MBS or the putative MBS from the FGF-2 promoter. Lysates from CMT3COS cells transfected with the pRMb3SVori+.wt were incubated with radiolabeled oligodeoxynucleotides and analyzed by EMSA. The c-Myb-DNA complex (solid arrowhead) was supershifted (a) in the presence of an anti-c-Myb antibody (Lane 3). The open arrowhead indicates a specific interaction not supershifted by the anti-c-Myb antibody. The radiolabeled probes indicated at the top were: mim- 1A, positive control probe encoding a consensus MBS from the chicken mim-1 promoter; MBS wt, the wt putative MBS from the FGF-2 promoter, and MBS mut, the mutated MBS from FGF-2 promoter. Additional components of the reaction are listed on the left, and their presence or absence is indicated by + or - , respectively. They include the anti-c-Myb antibody (anti-Myb) or various unlabeled competitor oligodeoxynucleotides (100-fold molar excess). CRE, negative control oligodeoxynucleotide encoding the cAMP response element derived from the mouse c-fos promoter.

Cell Growth

& Differentiation

1203

A Fig. 4. Responsiveness to c-Myb maps to two regions of the FGF-2 promoter that do not directly bind c-Myb. A, relative activities of FGF-2 promoter deletions in the presence ofectopic c-Myb. SK-MEL-2 cells were tran-

siently cotransfected

+1 +55 rr

with 8 g.tg of pcD3.myb

1+

I

plus 1 g of each individual FGF-2 promoter-reporter plasmid per dish. Regions of the FGF-2 promoter in each reporter construct are shown schematically to the left of their corresponding bars. Numbers delineate the nucleotide position numbers for the 5’ and 3’

.267/+178

r$’

.25V+178

I+l$’

I

I

-71/+178

I +13/+178

h--I

I-I

II.,,

#{149}

M29-165

I +50/+178 I-f

boundaries, except for M29-165, which indicates deletion of bp + 1 29 through + 165

+112/+178

4

PGU.BASIC

from the + 13/+1 78 promoter construct. pGL2.BASIC is a prornoterless reporter plasmid. Data are plotted as mean luciferase activity ± SE relative to the pGL2.bFGFpNB promoter-reporter plasmid (-2901+178). Each bar represents 8-25 individually trans-

fected dishes in at least 3 independent

-290/+178

,

I

1+ f$’

II

0

0.2

0.4

0.6

0.8

1.0

B

ex-

competitor

‘.

mOmlA

Normal

3-5).

diated

in part

4,

i/S..?ii

‘7::

&,

.5

. C

.4

I

.4

FP-

A-L

k

FP-

FP-

w.

melanocytes

2

were

3

4

used

as

for FGF-2

by changes

$

+131+178

je

mRNA expression (Fig. 6B, Lane 6). Thus, the abilities of c-Myb and MEnT to modulate FGF-2 expression in transfected SK-MEL-2 cells seems to be mecontrol

0

1’

1

a negative

+1291+165

‘/d

oligodeoxynucleoti-

(Fig. 6B, Lanes

+131+50

.

des, including AP-2, the consensus binding site for the AP-2 transcription factor, is mdicated above each lane. The c-Myb-DNA complex (solid arrowhead) was supershifted (*) by an anti-c-Myb antibody (Lane 16). The open arrowhead indicates a specific interaction not supershifted by the anti-c-Myb antibody. FP, free probe.

mids

1.6

cells

transfected with either pR3SVori (Vector) or pRMb3SVori+.wt (c-Myb) were incubated with radiolabeled oligodeoxynucleotides and analyzed by EMSA. The radiolabeled probes indicated at the top are: mim-1A, positive control probe; + 13/+50, upstream c-Mybresponsive region; + 1291+ 165, downstream c-Myb-responsive region; and + 13/ +178, minimal FGF-2 promoter region fully responsive to c-Myb. The addition of supershifting antibody(anti-Myb), control antibody (mouse IgG), or 100-fold molar excess of

unlabeled

1.4

actIvIty

mim.1A

periments. B, DNAB analysis of c-Myb and c-Myb-responsive regions of the FGF-2 promoter compared to the mim-1A positive

control probe. Lysates from CMT3COS

1.2

Relative luciforasa

in steady-state

amounts

5

-

6

7

8

.iM

-FP

I 9

10 11 12

Myb

to

13

modulate

FGF-2

1415

1617

protein

18

and

19

23

mRNA

24

expression

in

SK-MEL-2 cells and the ability of endogenous c-Myb ectopic murine c-Myb to regulate FGF-2 promoter-driven luciferase activity in cotransfection assays.

or

of FGF-2

mRNA. To confirm

protein function

that

and mRNA through

the

MEnT-mediated

expression

the FGF-2

decrease

was reflected

promoter

region,

in FGF-2

by an ability SK-MEL-2

to

cells

were cotransfected with the MEnT expression plasmid and FGF-2 promoter-reporter plasmid. Fig. 6C shows that luciferase activity was drastically reduced by cotransfection of the FGF-2 promoter-reporter plasmid with the MEnT ex-

the

pression

plasmid

EnT or MEnThl

compared

control

to cotransfection

plasmids.

with

either

the

Furthermore, cotransfection of SK-MEL-2 cells with the FGF-2 promoter-reporter plasmid and both the c-Myb and MEnT expression plasmids showed that MEnT completely abrogated the increase in luciferase activity mediated by ectopically expressed murine c-Myb (Fig. 6D). Together, these results revealed a positive correlation between the ability of ectopically expressed c-

Discussion We

have

demonstrated

differentially

that

expressed

anoma

lines. Relative cell line examined

FGF-2.

We have

noma

rine fected

suIted and

cell

c-Myb

also

stimulated

c-MYB

in normal

mRNA

and

melanocytes

to normal melanocytes, overexpressed c-MYB

shown

that

luciferase

ectopically activity

FGF-2 and

each melmRNA and

expressed from

are mela-

mu-

a cotrans-

FGF-2 promoter-containing reporter plasmid and rein increased expression of endogenous FGF-2 mRNA protein

in SK-MEL-2

melanoma

cells.

These

results

ar-

gue that c-MYB plays an important role in contributing to the aberrant expression of FGF-2 and the ensuing unchecked proliferation characteristic of melanoma cells. Whereas the importance of dysregulated FGF-2 expression

to melanoma

cell

proliferation

is well

established,

the

1204

c-Myb

Regulation

of FGF-2

Expression

A DNAB

TA

NR

role of c-MYB

in melanomas

ied.

studies

Previous

expressed

I I111

I

I Wild-type

I I1*1

I !+

I

W185G

G

in melanoma

and

mRNA

B

(47). to

which

c-MYB

mRNA

nomas

locus

cells

seems

type

and

to

were

used c-MYB

associated

with

the relative

Regulation

of

target

In contrast, the

FGF-2

C

FGF-2

kQa

83 62 47.5

2

3

mim-1

(55), and bcl-2 hsp

match

flanking

binding (59). its effect by binding

ments

within c-Myb

the FGF-2 and

a profound

because from

that c-Myb DNA ele-

interactions

the FGF-2

in vitro,

that

a functional

c-Myb DBD

did not bind

was

required

to FGF-2

by c-Myb

with

other

transcription

DBD. Indeed, increasing evidence that the Myb DBD domain is important for mediating protein interactions with other transcription factors For

indicates protein(48, 60-

its

example,

transactivation

Kanei-lshii of the

interaction

in

that it functions indirectly of other factors that bind to

or by interacting

promoter via

be-

promoter

by EMSAs.

our observation

the FGF-2

can have

It does not seem to novel non-MBS

derived

in the 5’-(C/

has been reported

it

MBS

promoter,

probes

detected

not

physical

has

70 promoter

the putative MBS to the consensus,

because

on c-Myb

tween

62).

(50),

(56, 57).

by c-Myb

human

(58). However,

factors Fig. 5. Requirement for a functional c-Myb DBD for transactivation of the FGF-2 promoter. A, schematic representations of the wild-type and W185G c-Myb proteins. The three triangles represent the three repeats of the DBD (DNAB). Solid bars represent the transactivation domain, and gray bars represent the negative regulatory (NR) domain. The single amino acid substitution (glycine for tryptophan) in the third repeat ofthe DBD is indicated at the bottom (W-+G). B, transactivation of the FGF-2 promoter by wild-type and W185G c-Myb. Luciferase activity in SK-MEL-2 cells transiently cotransfected with 8 Lg per dish of pRMb3SV.wt (black bars) or pRMb3SV.W185G (gray bats) expression plasmids and 1 g per dish of either the pGL2.bFGFpNB (MBS wt), pGL.2.MBSmut (MBS mut), or the ptk5xMRE.luc (5x mim- 1A) positive control promoter-reporter plasmids (as indicated). The data represent the mean fold activation over pR3SV empty vector of three independently transfected cultures. C, Western analysis of wild-type and mutant Wi 85G c-Myb proteins. SK-MEL-2 cells were transiently transfected with either pR3SV (Vector, Lane 1), pRMb3SV.wt (wt, Lane 2), or pRMb3SV.W1 85G (W185G, Lane 3), and equal amounts of cellular proteins were subjected to Western blotting with anti-c-Myb mAb. The arrowhead indicates Myb proteins. The positions of prestained molecular mass standards are marked on the left in kDa.

both

for the majority

transactivation

cotransfection assays, suggesting either by inducing the expression

I

via

in

MBS-de-

including

the core

promoter -

occur

described

to date,

is a perfect

sequences

Despite

-

additional of FGF-2

can

that

were

that

mechanisms.

T)AAC(GTF)GN(A/CIr)-3’

mediates

I 75

c-Myb

for the

unexpected, promoter

effect

It is

EBV BMRF1 promoter (48, 49). We report that the promoter belongs to this latter group. This result was

somewhat

1

suggesting

(54), CD34

demonstrated

the notion

of endogenous c-MYB cell line did not correlate

has been identified

MBS-independent

clearly

supports

to the expression by

ex-

cell cultures

role in melanomas.

of FGF-2,

(52), Ick (53), CD4

c-myc

pheno-

because

melanoma

-independent

genes

in mela-

in melanoma

transformed

observation

transcription

and

near or within

proliferation, and

contribute cell lines.

transactivation

pendent

of Myb

and

the

by

melanoma

implicated mRNA

an important

amount

factors

MBS-dependent

ileporter

This

play

melanoma

been

with

in

c-MYB

mechanism

in these

been

cellular

growth

cell line exam-

The

that the relative amount detected in each melanoma

different

5xmim-1A

melanoma

was c-MYB

that

rearrangements

melanocyte

in this study.

cell-specific

MBSmut

cell

of c-MYB

be

might

melanoma demonstrated

previously

simply

growing

that

notable mRNA

although

Expression

not

antisense

overexpressed

stud-

mRNA

and that

in each

was

extensively

c-MYB

melanocytes.

have

(44-46).

ponentially

MBSWt

have

normal

is not clear,

c-MYB

with

We

was overexpressed compared

cell lines

that

inhibited

in vivo

med

the

shown

cell lines

oligodeoxynucleotides vitro

has not been

have

et

recently

a!.

hsp

70 promoter

between

the c-Myb

is

showed

that

mediated

and HSF3

by

DBDs

a

and

binding of HSF3-c-Myb-containing complexes to the dHSE within the hsp 70 promoter (60). A similar mechanism may be operating

at the

an association vator.

Interestingly,

FGF-2

promoter,

activity

FGF-2

promoter

between

c-Myb

the which

HSF3

+ 1 3 to was

in cotransfection

to the hsp 70 dHSE coactivate

in which

and +49

required

assays,

the

shows

region for

promoter

of

coactithe

human

maximal

luciferase

sequence

similarity

(Fig. 7A). It is conceivable

the FGF-2

DBD mediates

a transcriptional

through

that c-Myb the

and

+ 1 3/+49

Cell Growth

A

Fig. 6. Endogenous FGF-2 expression in SK-MEL-2 cells is regulated by c-Myb. A, relative amounts of FGF-2 in lysates from SK-MEL-2 cells transiently transfected with pcD3.myb (cMyb) or pSCDMS/MEnT (MEnT) normalized to empty vector controls [pcDNA3.1(+) for pcD3.myb and pSCD/EnT for pSCDMS/MEnT]. B, RT-PCR analysis of FGF-2 (top) and 3-actin (bottom) mRNA expression in SK-MEL-2 cells transfected with pcDNA3.1(+) (Vector, Lane 1), pcD3.myb (c-Myb, Lane 2), pSCDMS/ MEnT (MEnT, Lane 3), pSCD/EnT (EnT, Lane 4) or pSCDMS/MEnTl (MEnThJ, Lane 5), and in normal necnatal human foreskin melanocytes (Melanocytes, Lane 6). C, repression of luciferase activity from the FGF-2

promoter-reporter

by dominant

C 0 U) U)

0.

1205

B 1.6

SK-MEL-2 I

1.4

transfectants I

,

1.2

A,b

w

01 U.

.,

1.0

I

C, U.

0.8

C

I

.

0.6

0.4

123456 c-Myb

MEnT

C

U

S

FGF-2

actin

0

inter-

fering Myb. Luciferase activity in 5KMEL-2 cells transiently cotransfected with pGL2.bFGFpNB (reporter) and either pSCDMS/MEnThl (MEn T+I), pSCD/EnT (En?), or pSCDMS/MEnT (MEnT). Data represent mean counts registered during a 15-s interval minus background ± SE of two independently transfected dishes. 0, inhibition ofectopic c-Myb-mediated stimulated luciferase activity from the FGF-2 promoter-reporter plasmid by dominant interfering Myb. Luciferase activity in SK-MEL-2 cells transiently cotransfected with indicated amounts ofpGL2.bFGFpNB (reporter), pcDNA3.1(+) (vector), pcD3.myb (cMyb), pSCD/EnT (En?), and pSCDMS/ MEnT (MEnT) per dish. Data represent the mean fold activation ± SE of three independently transfected dishes over empty vectors control (first bar on the left).

& Differentiation

D 150

12

125

10

100

8

T

75 .

0

-J

50

4

25

2

0

0

reporter: EnT: MEnTAl:

MEnT:

-

reporter: vector:

10

-

c-Myb:

-

10

10

-

-

l9

1 2 8

EnT: MEnT:

per dish

1 2 8

8

-

-

8

Mg per dish

The

A

+ 1 29/+165

also required FGF-2 HSP

+131+49:

I

70 dHSE:

1111111 III

assays,

I 1111

GTGAATCCCAGAAGACTCTGGAGAGTTC

contains

sponse

FGF-2

the +1 29/+165:

G

with

ICCGCGG

ICCCGGCCCAGI1GCGGACGG’1C

HOXB7 Binding Site

mRNA

FGF-2

may

stimulates

HOXB7.

and

modulated + 1 3/+49

that

FGF-2

by heat region

shock

functions

expression

in melanomas

or stress. at the

level

The

notion

can

that

of transcription

supported by the fact that it lies within the nontranscribed region of the FGF-2 promoter in the highly c-Myb-responsive + 1 3/+

1 78 promoter-reporter

plasmid.

be

the

a novel

is

we cannot

role

expression

interfering

likely

a reflection using

mRNA

this

Myb

study).

of the

Thus, switch

FGF-2

mRNA response

modulated

the

of prothat

or the MEnT

c-Myb

cthat

it

stability

role as a HOXB7 protein

and

to normal

of murine

endogenous FGF-2 protein changes in FGF-2 expression obtained

c-MYB compared

rule out the possibility

in regulating

to its defined

Indeed,

activity in the presence 1 65 region of the FGF-2

the

is transcribed,

nant

promoter + 1 29/+

cooperate

in proliferating

a transformation-associated

FGF-2

plays

Ectopic

34 and

(Ref.

may

activity.

levels

whereas

re-

to specu-

to transactivate

promoter

in melanomas

melanocytes

represent

moter

HOXB7

at similar

was

HOXB7

c-MYB

melanomas,

Because

addition element

with

FGF-2

is expressed and

proliferating

MYB

interact

are overexpressed

which

in cotransfection

characterized

Alternatively,

to stimulate

melanocytes Fig. 7. The +13/+49 and +129/+165 regions ofthe human FGF-2 promoter required by c-Myb contain potential binding sites for other transcription factors. A, alignment of the + 1 31+49 region of the human FGF-2 promoter with the c-Myb-HSF3-responsive dHSE from the human hsp7O promoter. Vertical lines indicate sequence identity. B, location of the HOXB7 binding site and response element within the + 1291+ 1 65 region of the human FGF-2 promoter. Open rectangle, probe used by Care et al. (34) to demonstrate HOXB7 binding activity in nuclear extracts from melanomas cells; shaded box, core HOXB7 binding site.

might

promoter,

activity

Ref. 34). It is interesting

7B;

promoter.

HOXB7

HOXB7

previously

(Fig.

c-MYB

FGF-2

of the FGF-2 luciferase

the

element

late that

B

region

for maximal

in

element.

domi-

expression

of

and mRNA. The moderate in these experiments were

low transfection

the calcium-phosphate

efficiency

routinely

precipitation

method

1

c-Myb

Regulation

of FGF-2 Expression

(63). MEnT also repressed luciferase activity generated from the FGF-2 promoter-reporter plasmid in cotransfected 5KMEL-2 cells in the absence of ectopic c-Myb, indicating that endogenous c-MYB functions through the FGF-2 promoter region. These data, along with our deletion analysis of the FGF-2 promoter, are consistent with c-MYB regulating FGF-2 expression at the level of transcription and clearly support the notion that FGF-2 is a physiologically relevant target for regulation by c-MYB in melanomas. It will be interesting to determine whether c-MYB regulates FGF-2 expression in other cell types, including hematopoietic tumors in which (64,

c-MYB

and

FGF-2

are

known

to be coexpressed

65).

Although the mechanism by which c-MYB mRNA expression is up-regulated in melanoma cell lines is not understood, lesions in the cell cycle regulatory machinery in melanomas might be a contributing factor. A common lesion in familial melanomas is the inactivation of the INK4A/CDKN2 tumor suppressor gene, which encodes the p1 6 cell cycle inhibitor (66, 67). The p16 protein can inhibit CDK4 or CDK6 activity by either disrupting their association with cychin Dl or by forming a ternary p1 6-CDK-cyclin Dl complex (68-72). The inhibition of CDK4 and CDK6 activity leads to the accumulation of the hypophosphorylated form of the retinoblastoma susceptibility gene product (pRB), which in turn associates with transcription factors from the E2F family, leading to the down-regulation of E2F target genes and subsequent cell cycle arrest (73-75). Inactivation of!NK4NCDKN2 is considered to contribute to tumorigenesis by causing the accumulation of hyperphosphorylated pRB and release of active E2Fs that then stimulate the expression of several genes involved in DNA synthesis and cell cycle progression, including c-MYB (76, 77). We thus hypothesize that the absence of functional p1 6 might result in the overexpression of c-MYB, which in turn would activate FGF-2 expression and thereby stimulate the uncontrolled autocrine growth typical of melanomas. Materials

and

Methods

Cell LInes and Culture Conditions. SK-MEL-2 and A-375 (from American CMT3COS African Rekosh (University

cDMEM

[DMEM

The

human

melanoma

cell

lines

Type Culture Collection) and the kidney cell line [a gift from Dr. David

green monkey of Virginia, Charlottesville, supplemented with 1 g/liter

VA)] were maintained in o-glucose and 1 10 mg/liter and 10% fetal bovine serum (Life

sodium pyruvate, 2 mM glutamine, Technologies, Inc.)]. The YUGEN8 human melanoma cell line was isolated from a brain metastasis (78) and maintained in Ham’s F-b medium supplemented with 2 mM glutamine and 10% fetal bovine serum (all from Life Technologies, Inc.). Normal human melanocytes derived from necnatal foreskins were cultured in medium consisting of Ham’s F-i 0 medium supplemented with 2 mM glutammne, 100 units/mI penicillin G (Life Technologies, Inc.), 100 g/ml streptomycin sulfate (Life Technologies, Inc.), 2.5% newborn calf serum, 2% FCS, 2.5% catt serum (all sara from Life Technologies, Inc.), 85 flM 12-O-tetradecanoylphorbol-13-acetate, 100 n 3-isobutyl-i-methylxanthine, 10 n choleratoxin, 1 sodium orthovanadate, and 100 nM N6, 2’-O-dibutyryladenosmne 3’,5’-cyclic monophosphate (all from Sigma). Cell cultures were maintained in a humidified incubator at 37#{176}C in 5% CO2. Plasmids. The murine c-Myb expression vectors pRMb3SV.wt and pRMb3SVori+.wt and the empty control vectors pR3SV and pR3SVori that lack c-Myb coding sequences have been described previously (79, 80). The pcD3.myb murine c-Myb expression plasmid was constructed by ligating the 2.3-kb Hindlll-BgIll fragment containing the full-length murine

c-Mj, cDNA from pRMb3SV.wt into pcDNA3.1(+) (Invitrogen). The pRMb3SV.W185G plasmid encodes a full-length murine c-Myb protein

containing a single amino acid substitution at position 185 in the DBD (W to G) that results in defective sequence-specific DNAB activity (80). The pSCDMS/MEnT and pSCD/EnT plasmids [kindly provided by Dr. Kathleen Weston (Institute of Cancer Research, Chester Beatty Laboratories, London, United Kingdom)] encode a dominant interfering Myb fusion protein consisting of a portion of the c-Myb DBD fused amino-terminally to the Drosophila engrailed transcriptional repressor, and the engrailed protein without the c-Myb DBD, respectively (51). The empty control vector, pSCDMS/MEnTM, was generated by digestion of pSCDMS/MEnT with Xbal to release the fusion cDNA insert and religating the vector to itself.

The pGL2.bFGFpNB reporter plasmid contains a portion of the human FGF-2 promoter cloned upstream of the firefly luciferase reporter gene and was generated by ligating a 468-bp Nhel-BamHl fragment (-290 to +178) isolated from pF2.1CAT [a kind gift of Dr. Robert Florkiewicz(Pnzm Pharmaceuticals,

pGL2.MBSmut +171

to +173

La Jolla, CA)] into pGL2.BASIC (Promega). The reporter plasmid contains a 3-bp substitution at positions of the FGF-2 promoter (ACG to TGC) that disrupts the

putative

MBS and creates a unique Sphl site. pGL2.MBSmut

structed

by oligodeoxynucleotide-directed

Sites II kit (Promega).

mutagenesis

Briefly, the 0.5-kb SacI-Hindlll

was con-

using

the Aftered

fragment

containing

the FGF-2 promoter was excised from pGL2.bFGFpNB and ligated into pALTER-i (Promega). Mutations were introduced by following the manufacturer’s instructions (Promega), and the mutated fragment was ligated back into pGL2.BASIC. Mutant plasmids were initially identified by SphI digestion and then confirmed by DNA sequencing on an Applied BioSystems 373 automated sequencer. Nested deletions from the 5’ end of the FGF-2 promoter were generated from pGL2.bFGFpNB using the EraseA-Base system (Promega). Internal deletion ofthe +129 to +165 region of the FGF-2 promoter was performed by digestion with SacIl and religation of the promoter-reporter plasmid containing bp +13 through +178 of the FGF-2 promoter. The ptk5xMRE.Iuc plasmid (81) contains a minimal herpes virus thymidine kinase promoter into which five copies of the highaffinity chicken mim-1A MRE have been inserted. Quantitation of FGF-2. The Quantikine human basic FGF immunoassay (R&D Systems) was used to quantitate FGF-2 in total cell lysates by following the manufacturer’s instructions, with minor modifications. Briefly, total cell lysates were prepared in YRIPA lysis buffer [12 m NaHPO4, 4 mM NaH2PO4, 2 m EDTA, 1 .5 m EGTA, 150 m NaCI, 20 mM NaF, 0.1 % SDS, 1 % sodium deoxycholate, 1 % NP4O, 10 g/ml leupeptin, 10 g/ml pepstatin, 100 g/ml Pefabloc, 1 mg/mI aprotinin, 0.01 unit/mI a2-macroglobulin (Boehringer Mannheim), 1 m phenylmethylsulfonyl fluoride, and 1 mi sodium orthovanadate(Sigma)], as described previously (81). Protein content was determined by BCA protein assay (Pierce), and each lysate was diluted in YRIPA to yield 1 mg protein/mI. Some lysates (SK-MEL-2, A-375, and YUGEN8) were further diluted to maintain linearity of the FGF-2 immunoassay (determined to be 5-320 pg of recombinant FGF-2/m. Each lysate was analyzed in triplicate, and the data are reported as either the mean picograms of FGF-2 per milligrams of cellular protein or as the relative amount of FGF-2 detected in lysates from c-Myb or MEnT transfectants normalized to the amount of FGF-2 detected in lysates from corresponding control vector transfectants. Error bars represent SE. RT-PR. Total cellular RNA was prepared from exponentially growing

normal neonatal

human

melanocytes

and human

melanoma

cell lines

using the RNeasy kit with QlAshredders according to the manufacturer’s instructions (Qiagen, Inc., Chatsworth, CA). RNA samples were quantitated spectrophotometrically, and their integrity was assessed visually by electrophoresis through a 0.8% denaturing agarose gel (2.2 M formaldehyde) with subsequent ethidium bromide staining. The relative amounts of 3-actin mRNA per microgram of total RNA in each sample were determined to be equivalent by conventional RNA blotting using a (3-actin

cDNA probe. One g of total cellular RNA from each sample was treated with 0.1 unit/ph DNase l(amplification grade; LifeTechnologies, lnc.)for 15 mm at 27#{176}C. DNase I was inactivated by the addition of EDTA to a final concentration of 2.5 mM followed by heating to 65#{176}C for 15 mm. Firststrand cDNA was synthesized using the Advantage RT-for-PCR kit (Clontech). Each reaction contained 5X reaction buffer [1 x buffer

3

mM MgClJ,

random

1 pii ohigo-dT18(for hexamers (for FGF-2

1 g of DNase I-treated total RNA, 5 p1 of = 50 mM Tris-HCI (pH 8.3), 75 m KCI, and c-Myb and (3-actin experiments) and (3-actin experiments), 0.5

or 1 w mu each

Cell Growth

triphosphate, 2 units of recombinant RNase inhibitor, and 200 units of Moloney murine leukemia virus reverse transcriptase in a final volume of 20 .tl. Samples were incubated for 1 h at 42#{176}C followed by 5 mm at 94#{176}C and diluted to 100 p1 with diethylpyrocarbonate-treated H20. PCR-mediated amplification of c-MYB, FGF-2, and /3-actin cDNAs was carried out using the Advantage KlenTaq Polymerase Mix from Clondeoxynucleotide

tech

according to the manufacturer’s instructions. Reactions were set up in 0.5-mI thin-walled reaction tubes (GeneAmp; Perkin-Elmer Corp.) and contained 10 gd of individual cDNA synthesis reaction; 5 .d of lOx reaction buffer [1 x buffer = 40 mM Tricine-KOH (pH 9.2 at 25#{176}C), 15 m.i potassium acetate, 3.5 mM magnesium acetate, and 75 g/ml BSA]; 0.2 mM each deoxynucleotide triphosphate; 0.4 individual forward and reverse primers for c-MYB (Clontech), FGF-2 (82), or f3-actin (Clontech);

and 1 d ofAdvantage

KlenTaq Polymerase

Mix in a total volume of 50 tl.

The primers used were as follows: human c-MYB forward primer, 5’-AAUAAATACGGTCCCCTG4AGATG-3’; human c-MYB reverse primer, 5’-CAGGTACTGCTACAAGGCTGCAAGG-3’; human FGF-2 forward primer, 5’-GGAGTGTGTGCTAACCG1TACCTGGCTATG-3’; human

FGF-2 reverse primer, 5’-TCAGCTClTAGCAGACAUGGAAGAAAAAG3’; human (3-actin forward primer, 5’-ATCTGGCACCACACCUCTACMTGAGCTGCG-3’; and human (3-actin reverse primer, 5’-CGTCATACTCCTGCTrGCTGATCCACATCTGC-3. Positive control reactions for c-MYB and (3-actin included 50 amol of specific PCR product as a template instead of cDNA(Clontech). Negative control reactions included 10 d of mock cDNA synthesis reactions (no reverse transcriptase) from RNA preparations and yielded no detectable amplification products. The c-MYB primers were designed to amplify a 390-bp fragment of the human c-MYB cDNA (83), whereas the FGF-2 primers amplified a 243-bp fragment of the human FGF-2 cDNA (84). The f3-actin primers were designed to amplify a 838-bp fragment (bp 2941 131) of the human (3-actin cDNA (85). Amplifications were carried out in

a PTC-i00 programmable thermal controller (M. J. Research, Inc.) using the following cycling conditions: 30 cycles of94#{176}C for 1 mm; 62#{176}C for45 s; and 72#{176}C for 45 s followed by 1 cycle of 72#{176}C for 7 mm and incubation at 4#{176}C until sample recovery. Ten pJ from each c-MYB and FGF-2 amphification and 5 d from each (3-actin amplification were fractionated by electrophoresis through a 2% agarose gel (Seakem GTG; FMC Bioproducts) containing 0.2 gImI ethidium bromide in 1 x Tris-acetate EDTA buffer[40 mM Tris-acetate, 10 m EDTA, and 20 m glacial acetic acid (pH 8.4)]. The gel was photographed under UV illumination using Polaroid 55

mega)

were

for empty

vector

the SE EMSAS.

oxynucleotide used

6 x 1O cells/i00-mm

washed once with PBS (137 m NaCI, 2.7 m KCI, 4.3 mp+iNa2HPO4#{149}H20, and 1 .4 mM KH2PO), incubated in growth media for an additional 24 h, and harvested. M cotransfectionsforluciferase assays were performed in duplicate or triplicate and included the indicated amounts of expression and reporter plasmids per dish. Where appropriate, the total amount of

DNA per transfection

was made

equal

by the addition

of pR3SV,

pcDNA3.1(+), pSCDMS/MEnThI, tions for FGF-2 immunoassays 8 pg/dish of either pcDNA3.i(+),

or pSCD/EnT control vectors. Transfecwere performed in duplicate and included pcD3.myb, pSCDMS/MEnT, or pSCD/ EnT. Transfections for anti-c-Myb immunoblots included 25 .&g of mdividual expression plasmid (pR3SV, pRMb3SV.wt, and pRMb3SV.W185G) per dish. Transfections for the preparation of total cellular RNA included 25 g of individual expression plasmid [pcDNA3.1(+), pcD3.myb, pSCDMS/MEnT, pSCDMS/MEnTM, or pSCD/EnT] per dish. Cell lysates were prepared, and luciferase assays were performed using the luciferase assay kit (Promega) according to the manufacturer’s instructions. Briefly, transfected cells were washed once with PBS and incubated in 250 p1 of Cell Culture Lysis Reagent (Promega) for 10 mm at 27#{176}C. Cells were harvested by scraping with a rubber policeman and transferred to microcentrifuge Twenty

AJ

tubes on ice. Lysates were kept on ice of each lysate and 100 .d of luciferase

throughout the assay. Assay

Reagent

(Pro-

(pR3SV

96-well

was measured using In some experiments,

microtiter a DynaTech luciferase

a liquid scintillation counter as described is defined by the mean relative light units

or pcD3.myb)

Total cell lysates

containing

as a positive

from

transfectants.

transfected

Error bars represent

cells express-

CMT3COS

a single

control

MBS

for c-Myb

from binding.

the mim-1A

MRE (79) was

Double-stranded

oligode-

oxynucleotides containing the wild-type (MBS wt) or a mutated (MBS mut) putative MBS from the human FGF-2 promoter spanning positions +162 through +183 were used. In addition, ohigodeoxynucleotide probes ocrresponding to the +13 through +50 and +129 through +165 regions of the human FGF-2 promoter were used. The +13 through +178 fragment of the human FGF-2 promoter was excised from the + 1 3/+ 178 promoterreporter plasmid by digestion with Smal and HindlIl (New England Biolabs), treated with calf intestinal alkaline phosphatase (New England Biolabs), and purified by the phenol-freeze method as described previously (87). Double-stranded ohigodeoxynucleotides contained either blunt ends or 4-bp 5’ overhangs and were radiolabeled to a specific activity of at least 1 .1 x io cpm/ng using T4 polynucleotide kinase (New England Biolabs) with [‘-P]ATP(DuPont New England Nuclear 6000 CVmmoI) or Klenow fragment (New England Biolabs) with [cr-32P]dCTP (DuPont New England Nuclean The sequences 166-bp +13/+178

6000

Ci/mmol),

+50, +165,

respectively.

of the upper strands probe)

cattaTMCGGlTttttcga-3’; ggtcga-3’; MBS mut,

are as follows: MBS

wt,

of the probes mim-1A

MAE,

(except for the 5’-tcgatcgace-

5’-tcgagcggftgCAACGGGAtcccg-

5’-tcgagcggttgCAtgcGGAtcccgggtcga-3’; 5’-gaggccggccccagaaaacccgagcgagtagggggcggc-3’;

and

+

+13/ 129/

5’-gcccggcgggtgccagattagcggacggtgcccgcgg-3’.

Unincorporated nucleotides were removed from the reactions using Quick Spin Sephadex G-25 columns (Boehringer Mannheim) or Probe

Quant G-50

assays and FGF-2 immunoassays)or

on an opaque

ing wild-type murine c-Myb were prepared, and EMSAS were performed as described previously (79). The sequences of the upper strands of the probes (except for the 166-bp +13/+178 probe) are shown below with consensus MBS shown in uppercase. A double-stranded oligode-

turers’

dish (for anti-c-Myb immunoblots and preparation of total cellular RNA) and transfected 16-20 h later. After overnight incubation, the cells were

wells

minus background for pRMb3SV.wt, pRMb3SV.W185G, or pcD3.myb transfectants divided by the mean relative light units minus background

TranslentTransfectlens tions into SK-MEL-2 precipitation method

(forluciferase

to individual

and luciferase activity plate luminometer.

activity was measured using previously (79). Fold activation

film. and Luciferase Assays. Transient transfeccells were performed using the calcium-phosphate (86) as deSCribed previously (81). The SK-MEL-2 human melanoma cell line was chosen for these studies, because it was the most readily transfected using the CaPO4 precipitation method, and because ft expressed the lowest amount of endogenous FGF-2 among the melanoma cellhines tested. Cells were plated at 2 x 1O cells/60-mm dish

added

plate (DynaTech), ML2250 microtiter

& Differentiation

microspin instructions.

columns (Pharmacia) according to the manufacEMSA reactions contained 1 p1 of transfected

cell lysate, 2 pJ of lOx buffer [1 x buffer = 10 m Tris-HCI (pH 7.9), 50 mM NaCI, 1 mM EDTA, 0.05% nonfat dry milk, 5% glycerol, and 0.01 % saturated bromphenol blue], 2 .&l of 100 m DII (Boehringer CMT3COS

Mannheim),

1 sI of 1 mg/mI poly(deoxyinosinic-deoxycytidylic acid) (Sigand 1 d (approximately 0.1 ng or at least 10,000 cpm) of P-labeled probe and were incubated at 27#{176}C for 45 mm. For competition assays, cell extracts were preincubated with unlabeled competitor oligodeoxynucleotide for 30 mm at 27#{176}C, at which time labeled probe was added to the reaction for an additional 15 mm. Double-stranded ohigodeoxynucleotides containing a cyclic AMP response element or a

ma), 13

;.tJ of H20,

consensus AP-2 binding site were used as a nonspecific competitors. For supershifts, cell extract was preincubated with 20 g of anti-c-Myb type I mAb (UBI) for 30 mm at 4#{176}C. Reactions were loaded onto 4 or 6% polyacrylamide gels and run at 100 V in 0.25x TBE buffer at 4#{176}C or 0.5x TBE bufferat 27#{176}C [0.25x ThE = 12.5 mp,i Tris-HCI(pH 8.5), 12.5 m boric acid, and 0.25 mM EDTA]. Gels were dried onto Whatman 3M paper and

film (Kodak) or Reflection film (DuPont New England at -80#{176}C with an intensifying screen for 4-16 h. Immunoblot Detection of c-Myb. Transiently transfected SK-MEL-2 cells were harvested at 48 h posttransfection, lysates were prepared in YRIPA buffer, and protain content was quantitated as described above. exposed Nuclear;

Fifty-six

to XAR-5 NEF-486)

of each lysate were fractionated by 4-20% gradient SDS(BIO-Rad Ready Gels) and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell) using a semidry transfer cell (Bio-Rad TransBlot SD). Nitrocellulose membranes were blocked in TBS [50 mM Tris-HCI (pH 8) and 150 m.i NaCI] plus 5% nonfat dry milk (Bio-Rad blotting grade blocker) for 1 h at room temperature followed by detection of c-Myb by enhanced chemiluminescence as described previously (79).

PAGE

1207

12

c-MybRegulation

of FGF-2

Expression

Acknowledgments We thank Dr. Timothy P. Bender(University for the murine c-Myb expression plasmids,

ofvirginia,

Charlottesville,

VA) Dr. Kathleen Weston (Institute for Cancer Research, Chester Beatty Laboratories, London, United Kingdom) for the pSCDMS/MEnT and pSCD/EnT plasmids, and Dr. Robert Florkiewicz (Prizm Pharmaceuticals, La Jolla, CA) for the parental

pF2.1CAT

FGF-2 promoter

clone. We thank Dr. Alison Richardson

(Uni-

versity

of Virginia, Charlottesville, VA) for sharing technical expertise for EMSAS and Yevette Clancy (Pfizer Central Research, Groton, CT) for sequencing the FGF-2 promoter clones. We also thank Drs. Jean Beebe and Kevin Coleman (Pfizer Central Research, Groton, CT), Dr. Rick Moran (Medical College of Virginia, Richmond, VA), and Dr. Yoen Smicun (Yale University, New Haven, CT) for helpful discussions.

18. Ogasawara, S., Yano, H., lemura, A., Hisaka, T., and Kojiro, M. Expressions of basic fibroblast growth factor and its receptors and their relationship to proliferation of human hepatocellular carcinoma cell lines. Hepatology, 24: 198-205, 1996. 19. Nguyen, M., Watanabe, H., Budson, A. E., Richie, J. P., Hayes, D. F., and Folkman, J. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J. NatI. Cancer Inst., 86: 356-361 , 1994. 20. Menzel, T., Rahman, Z., Calleja, E., White, K., Wilson, E. L, Wieder, R., and Gabrilove, J. Elevated intracellular level of basic fibroblast growth factor correlates with stage of chronic lymphocytic leukemia and is associated with resistance to fludarabine. Blood, 87: 1056-1063, 1996. 21

.

O’Brien,

T., Cranston,

D., Fuggle,

S., Bicknell,

R., and

References

Two mechanisms of basic fibroblast growth factor-induced in bladder cancer. Cancer Res., 57: 136-140, 1997.

1 . Yamaguchi, T. P., and Rossant, J. Fibroblast growth factors in mammalian development. Curr. Opin. Genet. Dcv., 5: 485-491 , 1995.

22. Story, M. T. Regulation of prostate growth by fibroblast tore. World J. Urol., 13: 297-305, 1995.

2. Brew, E. C., Mitchell, M. B., and Harken, A. H. Fibroblast growth factors in operative wound healing. J. Am. CoIl. Surg., 180: 499-504, 1995.

23. Crickard,

3. Allouche, (FGF-2) 4.

M., and Bikfalvi,

in hematopoiesis.

A. The role of fibroblast

Prog.

Growth

Factor

R. E., and Maciag, T. Molecular

Friesel,

fibroblast

growth factor signal transduction.

Res.,

growth

6: 35-48,

factor-2 1995.

K., Gross, J. L, Crickard,

A. L

growth fac-

M., Lele, S., Herblin,

W. F., and Eidsvoog, K. Basic fibroblast growth factor and receptor expression in human ovarian cancer. Gynecol. Oncol., 55: 277-284, 1994. 24. el Yazidi,

mechanisms of angiogenesis: FASEB J., 9: 919-925, 1995.

U., Yoonessi,

Harris,

angiogenesis

fibroblast

I., and

Boilly-Marer,

Y. Production

of acidic

growth factor by the hormone-independent

line MDA-MB-231

.

Anticancer

Res.,

15: 783-790,

and

basic

breast cancer cell 1995.

5. Johnson, D. E., and Williams, L T. Structural and functional diversity in the FGF receptor multigene family. Adv. Cancer Res., 60: 1-41 , 1993.

25. Nguyen, M., Watanabe, H., Budson, A. E., Richie, J. P., and Folkman, J. Elevated levels of the angiogenic peptide basic fibroblast growth factor

6. Basilico,

C., and Moscatelli,

oncogenes.

Adv.

in urine 1993.

Cancer

Res.,

7. Mohammadi,

M., Dikic,

Schlessinger,

J. Identification

fibroblast receptor 1996.

I., Sorokin,

A., Burgess,

factors

W. H., Jaye,

of six novel autophosphorylation

and

cancer

patients.

J. NatI.

activity.

J. Signal transduction

Cell, 61: 203-212,

by receptors

with

M. Broadly expressed to activators of Ras.

1 1 . Wang, J. K., Gao, G., and Goldfarb, M. Fibroblast growth factor receptors have different signaling and mitogenic potentials, Mel. Cell. Biol., 14: 181-188, 1994. 12. Hall, H., Williams, E. J., Moore, S. E., Walsh, F. S., Prochiantz, A., and Doherty, P. Inhibition of Fgf-stimulated phosphatidylinositol hydrolysis, and neurite outgrowth by a cell membrane-permeable phosphopeptide. Curr. Biol., 6: 580-587, 1996. Itoh,

N., Mima, T., and Mikawa, T. Loss of fibroblast growth factor receptors is necessary for terminal differentiation of embryonic limb muscle. Development (Camb.), 122: 291-300, 1996. 14. DeVore, D. L, Horvitz, H. A., and Stem, M. J. An FGF receptor signaling pathway is required for the normal cell migrations of the sex myoblasts in C. elegans hermaphrodites. Cell, 83: 61 1-620, 1995. 15. Halaban, A., Kwon, as an autocrine growth 177-186, 1988.

B. S., Ghosh, S., Delli Bovi, P., and Baird, factor for human melanomas. Oncogene

16. Takahashi, J. A., Fukumoto, M., Igarashi, Hatanaka, M. Correlation of basic fibroblast

levels with the degree of malignancy J. Neurosurg.,

76: 792-798,

A. bFGF Res., 3:

K., Oda, V., Kikuchi, H., and growth factor expression

and vascularity

85: 241-242,

evaluation 2675-2677,

1990.

10. Wang, J. K., Xu, H., Li, H. C., and Goldfarb, SNT-like proteins link FGF receptor stimulation Oncogene, 13: 721-729, 1996.

Inst.,

26. Sliutz, G., Tempfer, C., Obermalr, A., Dadak, C., and Kainz, C. Serum

27.

of basic 1995.

Halaban,

FGF in breast

A., Ghosh,

cancer

S., and

Baird,

growth factor for human melanocytes. kinase

Cancer

M., and

9. Garfinkel, S., Hu, x., Prudovsky, I. A., McMahon, G. A., Kapnik, E. M., McDowell, S. D., and Maciag, T. Fgf-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothehial cells and correlate with the inability to signal tyrosmne phosphorylation of fibroblast growth factor receptor-i substrates. J. Cell Biol., 134: 783-791, 1996.

13.

of bladder

sites on

growth factor receptor 1 and elucidation of their importance in activation and signal transduction. Mol. Cell. Biol., 16: 977-989,

8. Ullrich, A., and Schlessinger, tyrosine

D. The FGF family of growth 59: 1 15-165, 1992.

in human gliomas.

1992.

17. Maret, A., Galy, B., Arnaud, E., Bayard, F., and Prats, H. Inhibition of fibroblast growth factor 2 expression by antusense RNA induced a loss of the transformed phenotype in a human hepatoma cell line. Cancer Res., 55: 5075-5079, 1995.

patients. A. bFGF

Anticancer

Res.,

is the putative

15:

natural

In Vitro Cell. Dev. Bid., 23: 47-52,

1987.

28. Halaban, A., Langdon, R., Birchall, N., Cuono, C., Baird, A., Scott, G., Moellmann, G., and McGuire, J. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J. Cell Biol., 107: 1611-1619,

1988.

29. Scott, G., Stoler, M., Sarkar, S., and Halaban, A. Localization fibroblast growth ization. J. Invest.

factor mRNA in melanocytic lesions Dermatol., 96: 318-322, 1991.

of basic

by in situ

hybrid-

30. Becker, D., Meier, C. B., and Herlyn, M. Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor. EMBO J., 8: 368-3691, 1989. 31 . Dotto, G. P., Moellmann, G., Ghosh, S., Edwards, M., and Halaban, R. Transformation of murine melanocytes by basic fibroblast growth factor

cDNA and oncogenes and selective suppression notype in a reconstituted cutaneous environment. 3128, 1989. 32.

Ueba,

T., Nosaka,

Vogelstein, regulation

blastoma

of basic

fibroblast

and hepatocellular

91: 9009-9013, 33.

T., Takahashi,

B., Oda, Y., Kikuchi,

of the transformed J. Cell Bid.,

J. A., Shibata,

F., Florkiewicz,

H., and Hatanaka,

growth

factor

carcinoma

gene

phe-

109:3115R. 1,

M. Transcriptional

by p53 in human

glio-

cells. Proc. NatI. Acad. Sci. USA,

1994.

Biesiada,

E., Razandi, M., and Levin, E. A. Egr-1 activates basic fibroblast growth factor transcription. Mechanistic implications for astrocyte proliferation. J. Biol. Chem., 271: 18576-18581 , 1996. 34. Care, Parmiani,

A., Silvani, G., Peschle,

A., Meccia, E., Matha, G., Stoppacciaro, A., C., and Colombo, M. P. HOXB7 constitutively activates basic fibroblast growth factor in melanomas. Mol. Cell. Bid., 16: 4842-4851, 1996. 35. Bost, production

L M., and Hjelmeland, of bFGF transcripts.

L M. Cell density regulates Growth Factors, 9: 195-203,

36. Shibata, F., Baird, A., and Florkiewicz, tion of the human basic fibroblast growth Factors, 4: 277-287, 1991.

differential 1993.

A. Z. Functional characterizafactor gene promoter. Growth

Cell Growth

37.

Graf,

T. Myb:

ferentiation

a transcriptional

in hematopoietic

activator

linking

proliferation

and dif-

cells. Curr. Opin. Genet. Dev., 2: 249-55,

1992.

267: 4625-4630,

D., Badiani,

mutant causes apeptosis

38. Brown, K. E., Kindy, M. S., and Sonenshein, G. E. Expression of the c-Myb proto-oncogene in bovine vascular smooth muscle cells. J. Biol. Chem.,

57. Taylor,

1992.

& Differentiation

P., and Weston, K. A dominant interfering Myb in T cells. Genes Dcv., 10: 2732-2744, 1996.

58. Deng, 0. L, lshii, S., and Sarai, A. Binding site analysis of c-Myb: screening of potential binding sites by using the mutation matrix derived from systematic binding affinity measurements. Nucleic Acids Res., 24: 766-774, 1996.

39. Guenn, M., Sheng, Z. M., Andrieu, N., and Riou, G. Strong association between c-Myb and oestrogen-receptor expression in human breast cancer. Oncogene, 5: 131-135, 1990.

59.

40. Griffin, C. A., and Baylin, S. B. Expression of the c-Myb human small cell lung carcinoma. Cancer Res., 45: 272-275,

60. Kanei-lshii, C., Tanikawa, J., Nakai, A., Morimoto, A. I., and lshii, Activation of heat shock transcription factor 3 by c-myb in the absence cellular stress. Science (Washington DC), 277: 246-248, 1997.

oncogene 1985.

in

41 . Alitalo, K., Wmnqvist, A., Un, C. C., dela Chapelle, A., Schwab, M., and Bishop, J. M. Aberrant expression of an amplified c-Myb oncogene in two cell lines from a colon carcinoma. Proc. NatI. Aced. Sci. USA, 81: 45344538, 1984. 42. Thiele,

C. J., Cohen,

P. S., and

Israel,

M. A. Regulation

expression in human neuroblastoma cells during retinoic differentiation. Mol. Cell. Biol., 8: 1677-1683, 1988. 43. Wefter,

C., Henn,

W., Theisinger,

B., Fischer,

H., Zang,

N. The cellular myb oncogene

is amplified,

rearranged

human

Cancer

52: 57-62,

glioblastoma

cell lines.

Left.,

of c-myb acid-induced

K. D., and Blin, and activated in 1990.

44. Trent, J. M., Thompson, F. H., and Meyskens, F. L, Jr. Identification of a recurring translocation site involving chromosome 6 in human malignant melanoma. Cancer Res., 49: 420-423, 1989. 45. Dasgupta, P., Linnenbach, A. J., Reddy, E. P. Molecular cloning of the 6 in cutaneous malignant melanoma: locus and translocation of a segment 1201-1205, 1989. 46.

Linnenbach,

A. J., Huebner,

K., Reddy,

A. H., Nowell, P. C., and Koprowski, protooncogene and deletion kinase C in human melanoma 74-78, 1988.

Giaccia, A. J., Stamato, T. D., and breakpoint region on chromosome evidence for deletion in the c-myb of chromosome 12. Oncogene, 4: E. P., Herlyn,

H. Structural

within the gene cell lines. Proc.

alteration

M., Parmiter,

in the MYB

encoding a-type protein NatI. Aced. Sci. USA, 85:

47. Hijiya, N., Zhang, J., Ratajczak, M. Z., Kant, J. A., DeRiel, K., Herlyn, M., Zon, G., and Gewirtz, A. M. Biologic and therapeutic significance of

MYB expression in human melanoma. 4499-4503, 1994.

Proc. NatI. Aced. Sci. USA, 91:

48. Kanei-lshii, C., Yasukawa, T., Morimoto, A. I., and lshii, S. c-Mybinduced trans-activation mediated by heat shock elements without soquence-specific DNA binding of c-Myb. J. Biol. Chem., 269: 1576815775, 1994. 49.

Kenney,

S. C., Holley-Guthrie, E., Quinlivan, E. B., Gutsch, D., Zhang, T., Giot, J. F., and Sergeant, A. The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLFI transactivator in lymphoid cells. Mol. Cell. Biol., 12: 136-146, 1992.

Q., Bender,

50. Ness, S. A., Marknell, A., and Graf, T. The v-myb oncogene product binds to and activates the promyelocyte-specific mim-i gene. Cell, 59: 1115-1125,

1989.

51 . Badiani, P., Corbella, P., Kioussis, D., Marvel, J., and Weston, K. Dominant interfering alleles define a role for c-Myb in T-cell development. Genes Dcv., 8: 770-782, 1994.

52. Evans, J. L, Moore, T. L, Kuehl, W. M., Bender, T., and Ting, J. P. Functionalanalysis ofc-Myb protein in T-lymphocytic celllines shows that it trans-activates the c-myc promoter. Mel. Cell. Biol., 10: 5747-5752, 1990. 53. McCracken, S., Leung, S., Bosselut, R., Ghysdael, J., and Miyamoto, N. G. Myb and Ets related transcription factors are required for activity of the human Ick type I promoter. Oncogene, 9: 3609-3615, 1994. 54. Siu, G., Wurster, A. L, Upsick, J. S., and Hedrick, S. M. Expression of the CD4 gene requires a Myb transcription factor. Mel. Cell. Biol., 12: 1592-1604, 1992. 55. Melotti, P., Ku, D. H., and Calabretta, B. Regulation of the expression of the hematopoietic stem cell antigen CD34: role of c-myb. J. Exp. Med., 179: 1023-1028, 1994. 56.

Frampton,

up-regulates Dev.,

J., Ramqvist,

T., and Graf, T. v-Myb

bcl-2 and suppresses

10: 2720-2731,

1996.

apoptosis

of E26 leukemia

in myeloid

virus

cells. Genes

12

Howe,

specific

K. M., and Watson,

recognition

3913-3919,

A. J. Nucleotide

of DNA by c-myb

preferences

protein.

in sequence-

Nucleic Acids Res., 19:

1991. S. of

61 . Dash, A. B., Omco, F. C., and Ness, S. A. The EVES motif mediates both intermolecular and intramolecular regulation of c-Myb. Genes Dcv., 10: 1858-1869, 1996. 62.

Mink,

S., Kerber,

U., and

Klempnauer,

and v-Myb is required for synergistic Cell.

Biol.,

63.

FeIgner,

16: 1316-1325,

K. H. Interaction

activation

of C/EBPf3

of the mim-1 gene. Mol.

1996.

P. L, Gadek,

T. A., Holm,

M., Roman,

A., Chan,

H. W., Wenz,

M., Northrop, J. P., Ringold, G. M., and Danielson, M. Upofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. NatI. Acad. Sci. USA, 64. Gu,

84: 7413-7417,

Constitutive human

1987.

X. F., Bikfalvi,

A., Chen,

V. Z., Caen,

and selective expression

leukaemia

cell lines.

Han, 1 C. growth factor in

J. P., and

of basic fibroblast

Eur. J. Haematol.,

55: 189-194,

1995.

65. Allouche, M., Bayard, F., Clamens, S., Fillola, G., Sic, P., and Amalric, F. Expression of basic fibroblast growth factor (bFG9 and FGF-receptors in human leukemic cells. Leukemia (Baltimore), 9: 77-86, 1995. 66. W.,

Kamb, A., Shattuck-Eidens, Hussey, C., Tran, T., Miki,

D., Eeles, R., Uu, Q., Gruis, N. A., Ding, Y., Weaver-Feidhaus, J., McClure, M.,

Aitken, J. F., Anderson, D. E., Bergman, W., Frants, A., Goldgar, D. E., Green, A., MacLennan, R., Martin, N. G., Meyer, L J., Youl, P., Zone, J. J., Skolnick,

M. H., and

Connon-Albright,

L A. Analysis

(CDKN2) as a candidate for the chromosome locus. Nat. Genet., 8: 23-26, 1994. 67.

FitzGerald,

M. G., Harkin,

D. P., Silva-Arrieta,

Lucchina, L C., Unsal, H., O’Neill,

of the

9p melanoma

p16

gene

susceptibility

S., MacDonald,

D. J.,

E., Koh, J., Finkelstein,

D. M.,

Isselbacher, K. J., Sober, A. J., and Haber, D. A. Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population. Proc. NatI. Aced. Sci. USA, 93: 8541-8545, 1996. 68.

Aprelikova,

0., Xiong,

V., and Liu, E. T. Beth

cyclin-dependent

kinase (CDK) inhibitors

cyclin-dependent 270: 18195-18197,

kinases 1995.

69.

Parry,

D., Bates,

by the CDK-activating

S., Mann,

p16 and p21 families

D. J., and

kinase. Peters,

D-Cdk complexes in Rb-negative cells correlates p16INK4/MTS1 tumour suppressor gene product. 511,

of

block the phosphorylation J. Biol.

of

Chem.,

G. Lack with high

EMBO

of cyclin levels of J., 14: 503-

1995.

70. Guan, K. L, Jenkins, C. W., Li, Y., Nichols, M. A., Wu, X., O’Keefe, C. L, Matera, A. G., and Xiong, Y. Growth suppression by p18, a pl6lNK4/ MTS1 - and p14INK4B/MTS2-related CDK6 inhibitor, correlates with wildtype pRb function, Genes Dcv., 8: 2939-2952, 1994. 71 . Serrano, M., Hannon, G. J., and Beach, D. A new regulatory cell-cycle control causing specific inhibition of cyclin D/CDK4. (Lond.), 366: 704-707, 1993.

motif in Nature

72. Hirai, H., Reussel, M. F., Kate, J. Y., Ashmun, R. A, and Sherr, C. J. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6. Mel. Cell. Biol., 15: 2672-2681, 1995.

73. Weintraub,

S. J., Chow, K. N., Lue, A. X., Zhang, S. H., He, S., and

Dean, D. C. Mechanism of active blastoma protein. Nature (Lond.), 74. Sherr, dependent

transcriptional 375: 812-815,

repression 1995.

C. J., and Roberts, J. M. Inhibitors of mammalian kinases. Genes Dcv., 9: 1 149-1 163, 1995.

75. Nevins, J. A. E2F: alink viral oncoproteins. Science

between the Rb tumorsuppresser (Washington DC), 258: 424-429,

by the retinoG1 cyclinprotein 1992.

and

1210

c-Myb

76.

Regulation

DeGregeri,

J.,

of FGF-2

Expression

Kowalik,

T., and

targets

for

by the E2F1 transcription factor include DNA synthesisG1-S-regulatory genes. Mel. Cell. Biol., 15: 4215-4224, 1995.

Nevins,

J. A. Cellular

and

activation

77. Sala, A., Nicolaides, N. C., Engelhard, A., Bellon, T., Lawe, D. C., Arnold, A., Grana, X., Giordano, A., and Calabretta, B. Correlation between E2F-1 requirement in the S phase and E2F-1 transactivatien of cell cycle-related genes in human cells. Cancer Roe., 54: 1402-1406, 1994. 78. Halaban, A., Ghosh, S., Duray, P., Kirkweed, J. M., and Lamer, A. B. Human melanocytes cultured from nevi and melanemas. J. Invest. Dermatol., 87: 95-101 , 1986. 79. Miglarese, M. R., Richardson, A. F., Aziz, N., and Bender, T. P. Differential regulation of c-Myb-induced transcription activation by a phesphorylation site in the negative regulatory domain. J. Biol. Chem., 271: 22697-22705, 1996. 80. Cuddihy, A. E., Brents, L A., Aziz, N., Bender, T. P., and Kuehl, W. M. Only the DNA binding and transactivation domains of c-Myb are required to block terminal differentiation of murine erythroleukemia cells. Mel. Cell. Biol., 13: 3505-3513, 1993. 81

.

Aziz,

N.,

Miglarese,

M.

R., Hendrickson,

A. C.,

Sturgill, T. W., Hunt, D. F., and Bender, T. P. Modulation transcription activation ulatory domain. Proc.

Shabanowitz,

Brogi,

E., Winkles,

J. A., Underwood,

A., Clinton,

S. K., Alberts,

Libby, P. Distinct pattems their receptors in human

of expression of fibroblast growth atheroma and nonatherosclerotic

sociation

with

plaque

J. Clin. Invest., 92: 2408-2418,

i993.

83.

of acidic

Majello,

microvessels

B., Kenyon,

L C., and

Dalla-Favera,

nucleotide

sequence

of

tooncogene: genomic

FGF

locus.

Proc.

cDNA

NatI. Acad. Sci. USA, 83:

and

and As-

macrophages.

R. Human and

G. F.,

factors arteries.

c-myb

organization

9636-9640,

proof the

1986.

A., Mergia, A., Friedman, J., Gespodarowicz, D., and Fiddes, J. C. Human basic fibroblast growth factor: nucleetide sequence and genomic organization. EMBO J., 5: 2523-2528, 1986. 84. Abraham,

J. A., Whang, J. L, Tumole,

85. Ponte, P., Ng, S. Y., Engel, J., Gunning, P., and Kedes, L Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human

(3-actin

cDNA.

Nucleic

Acids

Res.,

12: 1687-1696,

1984.

86. Graham, F. L and Van der Eb, A. J. A new technique for the assay infectivity of human adenovirus 5 DNA. Virology, 52: 456-467, 1973.

of

J.,

of c-Myb-induced

by a phesphorylation site near the negative NatI. Aced. Sci. USA, 92: 6429-6433, 1995.

82.

reg-

87. Bewsey, K. E., Johnson, M. E., Huff, J. P. Rapid isolation cation of DNA from agarose gels: the phenol-freeze-fracture Biotechniques,

10: 724-725,

1991.

and purifimethod.