From the
The B- myb gene belongs to a family of transcription
factors that also includes A- myb and c- myb. B- myb is expressed in many cell types including human neuroblastoma
cells. Here we demonstrate that B- myb expression is
down-regulated during retinoic acid-induced neural and glial
differentiation of neuroblastoma cells. This modulation is an early
event, is maintained at late times of induction, and is in part
regulated at the transcriptional level. Constitutive expression of
B- myb prevents retinoic acid-induced neural differentiation as
reflected by morphological features and the expression of (or lack of)
biochemical markers associated with the undifferentiated phenotype.
Furthermore, the expression of antisense B- myb transcripts
does not allow the rescue of viable cells, suggesting an important role
for B- myb in the survival of neuroblastoma cells. These
results indicate that B- myb plays a functional role in the
differentiative potential of neuroblastoma cells, raising the
possibility that this gene is one of the nuclear regulators in the
cascade of events leading to cellular differentiation.
Many transcription factors that normally play a physiological
role during development or in adult life can become activated
oncogenically upon overexpression or mutation. Several such genes have
been characterized with respect to structure, function, and mechanism
of action
(1) .
The Myb family of transcription factors
includes A- myb, B- myb, and c- myb (2) , which are each located on a different chromosome
(3, 4) . Each member of the Myb family is characterized
by the presence of DNA-binding and transactivation domains.
B- myb, located on the long arm of chromosome X (Xq13)
(3) , has a DNA-binding domain consisting of three tandemly
repeated segments of 51-52 amino acids located near the NH
c- myb is expressed at high levels in
hematopoietic cells, where it plays a major role in the regulation of
both normal and malignant hemopoiesis in vitro (7, 8) and in vivo (9) . Several leukemic cell
lines express c- myb at high levels that decline during
terminal differentiation
(10) . In addition, constitutive
expression of c- myb blocks Me
The tissue distribution of B- myb expression is more widespread than that of c- myb and is
detectable in stomach, lung, colon, and thyroid carcinoma and
neuroblastoma cell lines
(2) . Overexpression of B- myb reduces the growth factor requirements of Balb/c 3T3 fibroblasts
(17) and bypasses, in part, the p53 growth-suppressive effects
in human glioblastoma T98G cells
(18) . In addition, antisense
B- myb oligonucleotides inhibit the proliferation of human
leukemic cell lines
(19) . Although B- myb expression,
like that of c- myb, correlates with proliferation
(17, 20, 0) , transcription regulation by
B- myb appears to be distinct from that of c- myb (21, 22) . Furthermore, c- myb and
B- myb appear to recognize the same DNA sequence, but with
different affinities
(23) . Although B- myb appears to
be involved in cellular proliferation, recent work of Foos et al. (24) demonstrating that chicken B- myb inhibits
c- myb transactivation of the MIM-1 gene promoter
(25) raises the possibility that B- myb can act, at
least in some instances, as a competitor of c- myb.
Despite
the wealth of data on B- myb structure and B- myb function as relates to cell proliferation, no evidence exists for
its possible involvement in differentiative processes. In this paper,
we have investigated the expression and regulation of the B- myb gene during neural and glial differentiative pathways in a
neuroblastoma differentiation model.
Neuroblastoma is a malignant
childhood tumor with a very undifferentiated appearance and is thought
to arise from embryonal neural crest tissue that may be arrested at an
early stage of differentiation
(26) . The mechanisms
contributing to the onset and progression of neuroblastomas are largely
unknown, but nonrandom chromosomal changes suggest the involvement of
genetic alterations
(27) . The N- myc oncogene is
frequently expressed in advanced neuroblastomas and cell lines
(28) and undergoes early transcriptional down-regulation when
cells are induced to differentiate with retinoic acid (RA)
Like N- myc, c- myb is
down-modulated in neuroblastoma upon induction of differentiation
(30) , and we recently showed that down-regulation of c- myb expression is associated with inhibition of neuroblastoma and
neuroepithelioma cell proliferation
(31) . Thus, to determine
the role, if any, of B- myb in the growth and differentiation
of neuroblastoma cells, we examined the effects of ablating B- myb expression on the survival of neuroblastoma cell lines and the
effects of constitutive expression of B- myb on neural
differentiation of neuroblastoma cells.
For cell
differentiation experiments, cells were seeded at an initial density of
5
LAN-5 cells were transfected by the calcium
phosphate precipitation technique as described
(31) or by
Lipofectin according to the manufacturer's instructions
(Boehringer Mannheim). Briefly, cells were seeded at a density of 1.5
Nuclei for
run-on transcription were prepared as described
(37) , with at
least 2
Polymerase chain reaction
analyses were carried out as described
(38) . Amplification
products were separated on standard agarose gels and transferred to
nylon membranes. Filters were prehybridized in 5
Rates of growth under normal serum conditions were
comparable in B- myb transfectants, pRc/CMV controls, and LAN-5
parental cells (data not shown), suggesting that B- myb overexpression does not confer a proliferative advantage to
neuroblastoma cells. Nevertheless, it should be pointed out that LAN-5
cells express the endogenous B- myb mRNA at high levels.
Morphological analysis of B- myb transfectants and controls
induced to differentiate by 6-day treatment with RA revealed the
expected differentiation of LAN-5 cells toward a prevalently neural
phenotype, with most cells showing long neuritic processes and few
cells with a flat morphology (Fig. 3 F). The pRc/CMV
transfectants behaved similarly (Fig. 3 E). By contrast,
most of the B- myb transfectants showed cell body enlargement
with a flat morphology without neuritic processes (Fig. 3 D).
To determine whether the morphology of the RA-treated B- myb transfectants correlated with a different biochemical phenotype,
these cells were tested for the presence of biochemical markers of
differentiation (Fig. 4). Vimentin and neurofilaments, two
components of the cytoskeleton that are typical markers of
differentiation in neuroblastoma cells
(43, 44) , were
initially examined. Vimentin was highly expressed in controls and
uninduced B- myb transfectants and remained at high levels in
RA-treated B- myb transfectants, but was dramatically reduced
in RA-treated pRc/CMV transfectants and LAN-5 parental cells
(Fig. 4, top panel). Neurofilaments were absent
in all uninduced cells under normal growth conditions (data not shown)
and were expressed in pRc/CMV controls and LAN-5 cells after RA
induction, but not in the B- myb transfectants (Fig. 4,
bottom panel). This pattern of intermediate filament
expression, together with the morphological data, indicates that
RA-treated pRc/CMV transfectants and LAN-5 cells undergo the expected
pattern of neural differentiation. By contrast, the high level of
vimentin expression and the lack of neurofilaments suggest that neural
differentiation is inhibited in RA-treated B- myb transfectants.
The induction of differentiation and the commitment to enter
a particular pathway are associated with well defined changes in the
regulation of gene expression
(10) . c- myb, the most
extensively studied member of the Myb family, is transcriptionally
down-regulated during differentiation of hematopoietic
(48) and
neuroblastoma
(30) cells. The functional significance of this
modulation is suggested by the reported inhibition of
Me
In
neuroblastoma, c- myb down-regulation is an early event
(30) . We have previously demonstrated that c- myb down-regulation inhibits the proliferation of neuroblastoma and
neuroepithelioma cells
(31) . We now find that B- myb is
expressed in several neuroblastoma cell lines and that B- myb mRNA is down-regulated long before any detectable morphological
change when these cells are induced to differentiate along neural and
Schwann pathways. Such down-regulation appears to be regulated
primarily at the transcriptional level. These findings might be
explained in at least three ways. (i) B- myb is involved in the
control of proliferative mechanisms that must be down-regulated before
differentiation can occur; (ii) B- myb regulates the expression
of genes that inhibit differentiation; or (iii) B- myb modulation is a passive event affecting a bystander gene. The last
possibility seems unlikely in light of the finding that transfections
carried out using an expression vector in which a region of the
B- myb gene was cloned in the antisense orientation resulted in
a substantially lower number of transfected clones as compared with
control transfections (). These data are in agreement with
previous studies indicating that B- myb down-regulation
inhibits the proliferation of fibroblasts
(17) and
hematopoietic
(19) cells. Thus, it appears that B- myb expression is important for the survival of neuroblastoma cells,
although the mechanism by which B- myb affects this process
remains unclear.
Neuroblastoma cells can differentiate in vivo and in vitro toward neural, Schwann, and melanocytic
phenotypes, recapitulating the multipotential of neural crest cells
from which neuroblastomas derive
(41) . Although many
neuroblastoma cell lines show a certain degree of heterogeneity in
terms of neurotransmitter expression
(33) and differentiative
potential
(49) , each cell line has a prevalent behavior in
response to differentiation inducers
(41) . LAN-5 cells
differentiate to a prevalent neural phenotype by induction with RA.
When transfected with a B- myb cDNA, LAN-5 cells overexpressed
B- myb. Unlike the parental cell line and control
transfectants, B- myb transfectants did not show decreased
B- myb expression after treatment with RA. The continuous
B- myb expression is due to the expression of the exogenous
gene driven by the cytomegalovirus promoter that is not affected by the
RA treatment. By contrast, RA appeared to enhance B- myb expression in the B- myb transfectants both at the
messenger and protein levels (Fig. 2 B; data not shown).
The product of the B- myb construct appears to be functional as
nuclear extracts from B- myb transfectants retain the ability
to interact with a B- myb-specific binding site even after RA
induction (Fig. 2 C).
The constitutive expression of
B- myb results in the inhibition of neural differentiation as
reflected by the enlargement of B- myb transfectant cells, the
absence of outgrowing neurites, the maintenance of high level vimentin
expression, the lack of neurofilament production, and the moderate
increase in collagen type IV expression after 6 days of RA treatment.
Such an increase in collagen type IV, which is typical of Schwann-like
differentiation, is the only marker of other known differentiative
pathways found in RA-treated B- myb transfectants. Thus, it is
possible that B- myb-transfected neuroblastoma cells begin a
differentiative pathway that is distinct from neural differentiation,
but resembles a Schwann-like differentiation. Consistent with this
hypothesis, we found only a slight down-modulation of B- myb in
SK-N-SH cells differentiating toward a Schwann-like phenotype.
Nevertheless, a decrease in B- myb expression under a certain
threshold might be required for complete schwannian differentiation.
Recently, the mechanism of action of RA on the differentiation of
neuroblastoma cells has been analyzed in detail
(50) . RA
reportedly induces expression of the trkB receptor, which, in turn,
renders neuroblastoma cells sensitive to the differentiative action of
brain-derived nerve growth factor
(50) . However, the nuclear
effectors in the signal transduction cascade promoted by the activation
of the trkB receptor remain unknown. Based on the results described
here, it is tempting to suggest that B- myb is one of the
nuclear targets of the differentiation-promoting signal transduced by
the trkB receptor. A complete understanding of the process by which a
differentiative signal is transmitted to the nucleus is particularly
important for the development of effective therapies that may take
advantage of the differentiative potential of neuroblastoma cells
(51) .
We thank Dr. Robert E. Lewis for the gift of the
antibody to B-Myb and Adelma Di Stefano for technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus
(5) . This region is highly homologous to the
corresponding domains of A- myb and c- myb (2) .
Deletion of the second and third repeats completely abolishes the DNA
binding-dependent transactivating ability of B- myb (5) . A transcriptional activation domain containing a
cluster of acidic amino acids and different from the homologous domain
of c- myb (6) is located downstream of the DNA-binding
domain of B- myb (5) . Unlike c- myb, B- myb does not have a negative regulatory domain, and it does not show
negative autoregulatory activity; however, like c- myb, it has
multiple nuclear localization signals located near the COOH terminus
(5) .
SO-induced
differentiation of Friend murine erythroleukemia cells
(11) .
c- myb also appears to play an important role in the
proliferation of arterial smooth cells in vivo (12) .
Detectable amounts of c- myb are also found in many tumors of
different embryonic origin such as small cell lung carcinoma
(13) , colon carcinoma
(14, 15) , and
neuroblastoma
(16) .
(
)(29) .
Cloning of Antisense and Sense B-myb Vectors
The
EcoRI- ScaI region (714 base pairs) spanning
nucleotides 941-1655 of the human B- myb cDNA
(12) was
cloned into plasmid pRc/CMV (Invitrogen, San Diego, CA) in the
antisense or sense orientation as confirmed by restriction analysis.
The full-length cDNA sequence was subcloned in the sense orientation
from plasmid pSV-B-myb
(17) into vector pRc/CMV.
Cell Culture and Transfection
The neuroblastoma
cell line LAN-5
(32) was grown in RPMI 1640 medium (Sigma)
supplemented with 15% fetal bovine serum (Sigma). The neuroblastoma
cell line SK-N-SH
(33) was grown in minimal essential medium
(Life Technologies, Inc.) with 10% fetal bovine serum.
10
cells/cm
and grown for 16 h before
the medium was replaced with fresh medium containing 5 µ
M all- trans-retinoic acid (Sigma). Medium with RA was then
replaced every 3 days.
10
cells/dish and 18 h later exposed to a mixture
of 10 µg of plasmid DNA and Lipofectin reagent for an additional 18
h. Cells were washed and grown in normal medium for 24 h. G418 (Sigma)
was then added at a concentration of 400 µg/ml to the medium, and
transfectant clones were isolated 3 weeks later.
RNA Analysis
Total RNA was prepared by acid
guanidinium/thiocyanate/phenol/chloroform extraction
(34) ,
separated on a formaldehyde-agarose gel using standard procedures
(35) , and transferred to nylon membranes (Stratagene, La Jolla,
CA). Filters were hybridized with specific probes using the high
stringency protocol suggested by the manufacturer. The
EcoRI- ScaI fragment derived from plasmid pSV-B-myb
containing the full-length cDNA for human B- myb (17) and the 2.1-kilobase XhoI insert of clone
HFA-1 containing the cDNA for human
-actin
(36) were
used as molecular probes in Northern blot analyses.
10
cells as starting material. Run-on
transcription was carried out with 1
10
nuclei in
the presence of 100 µCi of [
-
P]UTP
(DuPont NEN, Bad Homburg, Germany). Five µg of each linearized
plasmid was denatured and blocked on a nitrocellulose membrane
(Schleicher & Schuell, Dassel, Germany) using a slot blot
apparatus. Plasmids used in this experiment were as follows: pSV-B-myb
(17) , a Bluescript plasmid containing the complete cDNA for
human c- myb (subcloned in our laboratory); pNB-19-21
containing a fragment spanning the second exon of the human N- myc gene (kindly obtained from Dr. F. Alt, Columbia University);
-actin plasmid HF
A-1
(36) ; and the pVC vector used as
negative control. Filters were hybridized for 36 h at 65 °C in 10
m
M TES, pH 7.4, 0.2% SDS, 10 m
M EDTA, 300 m
M NaCl, and 5
10
cpm/ml nuclear RNA from the
run-on transcriptions. After hybridization, filters were washed with
several changes of 2
SSC (1
SSC = 0.15
M NaCl, 0.015
M trisodium citrate) for 2 h at 65 °C,
incubated at 37 °C in 2
SSC with 10 µg/ml RNase A
(Sigma) for 30 min, washed again with 2
SSC for 1 h at 37
°C, air-dried, and exposed to x-ray film.
Polymerase Chain Reaction
Primers used to confirm
integration of the sense and antisense B- myb inserts in
transfected cells were as follows: CMV-1,
5`-AATGGGAGTTTGTTTTGGCACCAA-3`, corresponding to nucleotides
699-722 of the pRc/CMV vector; SP6-1,
5`-GCACAGTCGAGGCTGATCAGCGAG-3`, complementary to nucleotides
1020-1043 of the pRc/CMV vector; and B-myb-1,
5`-TGGCATTGCTGGTCAGTGCGGTTA-3`, complementary to nucleotides
313-336 of the human B- myb cDNA sequence
(2) .
The oligonucleotide T7-1 (5`-AATACGACTCACTATAGGGAGACC-3`, corresponding
to nucleotides 865-888 in the pRc/CMV vector) was used as a
specific probe for hybridizations.
SSC, 1% SDS
for 2 h at 47 °C. Hybridizations were carried out in 5
SSC,
1% SDS, 100 mg/ml sonicated salmon sperm DNA, and 10
cpm of
5`-end-labeled probe (T7-1) for 16 h at 47 °C. After washing in 2
SSC, 0.5% SDS for 15 min at room temperature and in the same
buffer for 15 min at 50 °C, filters were exposed in an x-ray
cassette with an intensifier screen for 1-4 h.
In Vitro Translation
Plasmid pRc/CMV-B-myb
containing the full-length sequence of the human B- myb cDNA
was linearized with XbaI restriction endonuclease (Promega).
In vitro transcription using T7 RNA polymerase (Promega) was
carried out according to the manufacturer's instructions, and 1
µg of the B- myb mRNA produced in vitro was
translated in a rabbit reticulocyte system (Boehringer Mannheim) in the
presence of [S]methionine (DuPont NEN).
Translation products were separated by electrophoresis on a 10%
SDS-polyacrylamide gel. The gel was dried and exposed in an x-ray
cassette for 2 h.
Mobility Shift Assays
Nuclear extracts from
B- myb transfectants, pRc/CMV transfectants, and LAN-5 cells
treated for 6 days with RA (5 µ
M) were prepared as
described
(39) . The protein content of each extract was
determined using the protein assay kit II (Bio-Rad). One strand of the
myb-specific oligonucleotide MBS-1
(5`-AGAATGTGTGTCAGTTAGGGTGTAGAG-3`) was labeled with
[-
P]ATP and polynucleotide kinase before
annealing to the complementary strand. Mobility shift assays were
performed with 6 µg of each nuclear extract or 4 µl of
programmed reticulocyte lysate in 40 m
M KCl, 15 m
M HEPES, pH 7.9, 1 m
M EDTA, 0.5 m
M dithiothreitol,
5% glycerol, 1 m
M MgCl
, and 0.2 µg/µl
poly[d(I-C)] (Boehringer Mannheim) in the presence of 0.01
pmol of double-stranded
P-labeled MBS-1 at room
temperature for 20 min. Where appropriate, a 100-fold molar excess of
the unlabeled double-stranded MBS-1 or YY1
(5`-CCGAGCCCGCTTCAAAATGGAGACCCTC-3`) oligomer was added to the mixtures
as a specific or nonspecific competitor, respectively. Reactions were
loaded on a native polyacrylamide gel (5%) run on 0.5
Tris
borate/EDTA (1
Tris borate/EDTA = 0.090
M Tris
borate, 0.002
M EDTA) for 4 h at 140 V at 4 °C. The gel
was dried and exposed to x-ray film.
Immunoprecipitation
LAN-5, B- myb transfectants, and pRc/CMV transfectants were metabolically
labeled with 100 µCi/ml [S]methionine (1000
Ci/mmol) for 3 h. Extracts were prepared from 1
10
labeled cells in 50 m
M Tris-HCl, pH 8.0, 150 m
M NaCl, 5 m
M EDTA, 1% Nonidet P-40, 1 m
M phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 1
µg/ml leupeptin on ice. Immunoprecipitations were carried out
essentially as described
(40) using a specific anti-B-Myb
polyclonal antibody (gift of Dr. Robert E. Lewis, University of
Nebraska, Omaha, NB) raised in rabbits by immunization with a
glutathione S-transferase fusion protein containing amino
acids 553-611 of the human B-Myb protein.
Immunocytochemistry
Cells were seeded at a density
of 5 10
cells/cm
in Labtek chamber
slides (NUNC, Naperville, IL) and grown under normal conditions or
treated with RA for the appropriate time. After washing in
phosphate-buffered saline (137 m
M NaCl, 2.7 m
M KCl,
4.3 m
M Na
HPO
7H
O, 1.4
m
M KH
PO
), cells were fixed in
methanol/acetone (25:75) for 20 min at -20 °C and incubated
with the appropriately diluted primary monoclonal antibody for 1 h at
room temperature. Cells were then extensively washed with
phosphate-buffered saline and incubated for 1 h at room temperature
with fluoresceinated anti-mouse IgG (Sigma) diluted 1:40. The
monoclonal antibodies and relative dilutions used were as follows:
anti-vimentin (Sigma), 1:40; anti-glial fibrillary acidic protein
(Sigma), 1:400; anti-laminin (Sigma), 1:2000; anti-neurofilament (160
kDa; Sigma), 1:40; anti-collagen type IV (Sigma), 1:500; and anti-S-100
(Boehringer Mannheim), 1:500.
B-myb Is Transcriptionally Down-regulated during Neural
Differentiation of Neuroblastoma Cells
B- myb mRNA
levels in several neuroblastoma cell lines were determined by Northern
blot analysis. Transcripts were detected in all cases (Fig. 1, A and C; data not shown). Since neuroblastoma cells can
differentiate toward a neural, Schwann, or melanocytic phenotype
depending on the cell line and the inducer
(41) , we
investigated whether B- myb mRNA levels might be modulated by
differentiative processes. LAN-5 and SK-N-SH cells were treated with
RA, which induces predominant neural
(42) and Schwann-like
(41) phenotypes, respectively. Total RNA was extracted at
different times after treatment, separated on a formaldehyde-agarose
gel, and transferred to a nylon filter. Filters were hybridized with a
B- myb-specific probe, washed, and rehybridized with a probe
for -actin to control the integrity and the amount of each RNA
loaded on the gel. After densitometric reading of the autoradiograms
and normalization for
-actin mRNA levels, the amount of B- myb mRNA, in arbitrary units, was plotted against the time of RA
induction. After RA treatment, B- myb mRNA levels decreased
sharply in LAN-5 cells (Fig. 1, A and B), and a
moderate, but reproducible decrease was detected in SK-N-SH cells
( C and D); B- myb down-regulation occurred as
early as 6 h after the beginning of the RA treatment and was maintained
at later times in both cell lines (Fig. 1, A-D).
To determine whether the down-regulation of B- myb mRNA during
RA treatment was due to a transcriptional mechanism, nuclei from
untreated and RA-treated (for 6 days) LAN-5 cells were isolated, and
in vitro transcription in the presence of
[
P]UTP was carried out. The signal intensity for
B- myb in filters hybridized with the labeled RNA from
untreated LAN-5 cells was >3-fold greater than that detected in
filters hybridized with RNA from RA-treated cells (data not shown),
demonstrating that the rate of B- myb transcription decreases
during RA-induced differentiation. As expected from previous studies
(29, 30) , transcription of N- myc and c- myb was also down-regulated (data not shown).
Figure 1:
Detection of B- myb mRNA in
neuroblastoma cell lines at different times after RA induction. LAN-5
( A) and SK-N-SH ( C) cells were uninduced ( lane 1) or were induced with RA for 6 h ( lane 2), 1 day ( lane 3), 5 days ( lane 4), 7 days ( lane 5), or 10 days
( lane 6). Filters were also hybridized with a
-actin probe. Also shown is the densitometric analysis of
B- myb RNA levels in LAN-5 ( B) and SK-N-SH
( D) cells during RA treatment (mean of two experiments in each
panel).
B-myb Expression Is Required for Neuroblastoma Cell
Survival
A B- myb fragment (714 base pairs) spanning a
region of minimum homology to the other members of the Myb family was
cloned in the polylinker region of the pRc/CMV plasmid in the sense or
antisense orientation under the control of the cytomegalovirus
promoter. Parallel transfections using sense and antisense B- myb plasmids were carried out in LAN-5 cells. After 21 days of
selection in the presence of the antibiotic G418, plates were fixed and
stained, and the number of resistant clones was counted. In four
independent experiments (Table I), the number of resistant clones
obtained in the antisense B- myb transfections was
significantly lower than that obtained with the sense B- myb transfections, with an average reduction of 79% in the four
experiments. Southern blot and polymerase chain reaction analyses of
the genomic DNA of the residual antisense B- myb transfectants
after antibiotic selection demonstrated rearrangements in the antisense
B- myb insert that prevented expression of the antisense
B- myb transcript (data not shown), suggesting a
counterselection of cells expressing antisense B- myb RNA. The
marked reduction in the colony-forming ability of antisense
B- myb-transfected cells is in good agreement with the results
of similar experiments demonstrating that antisense B- myb RNA
or DNA inhibits the proliferation of mouse fibroblasts
(17) and
human hematopoietic cell lines
(19) . Thus, our findings
indicate that B- myb is required for the survival of
neuroblastoma cells.
Constitutive Expression of B-myb Affects the
Differentiative Potential of Neuroblastoma Cells
A full-length
B- myb cDNA was cloned in the polylinker region of vector
pRc/CMV downstream of the cytomegalovirus promoter. The molecular
weight of the protein product from the cloned cDNA was verified by
in vitro translation (Fig. 2 A).
B- myb-transfected LAN-5 cells were grown for 21 days in
antibiotic selection medium, and a pool of 60-70 resistant clones
was expanded. Pooled transfectants were used in order to maintain the
heterogeneity of the parental cell line. Integration of the B- myb construct was monitored by polymerase chain reaction and Southern
blot analyses (data not shown). Overexpression of the B- myb transcript in the transfectant clones was analyzed by Northern
blot analysis before and after RA treatment for 6 days (Fig.
2 B, lanes 1 and 2). In comparison
with transfectants obtained with the pRc/CMV vector (Fig. 2 B,
lanes 3 and 4) and with the LAN-5 parental
cells ( lanes 5 and 6), B- myb mRNA
levels were readily down-regulated in control cells, whereas B- myb transfectants expressed B- myb mRNA at high levels, which
were even increased after RA induction.
Figure 2:
A,
in vitro translation of RNA obtained from the transcription of
plasmid pRc/CMV-B-myb. Control and BMV are
reticulocyte lysate without RNA and stimulated with brome mosaic virus
RNA, respectively. Numbers on the right indicate the molecular mass
markers in kilodaltons. B, B- myb mRNA detection in
B- myb transfectants ( lanes 1 and
2), pRc/CMV transfectants ( lanes 3 and
4), and LAN-5 cells ( lanes 5 and 6)
under normal growth conditions ( lanes 1, 3,
and 5) or after 6 days of RA treatment ( lanes 2, 4, and 6). Filters were stripped of
the previous probe and hybridized with -actin. C,
mobility shift assays performed with nuclear extracts from B- myb transfectants RA-treated for 6 days ( lanes 1-3),
pRc/CMV transfectants RA-treated for 6 days ( lanes 4-6),
and B- myb synthesized in rabbit reticulocyte lysate
( RRL; lanes 7 and 8). The
myb-specific
P-labeled oligonucleotide used in
this experiment was MBS-1. Lanes 2 and 5 contain unlabeled nonspecific competitor YY1. Lanes 3, 6, and 8 contain unlabeled specific
competitor MBS-1.
Mobility shift assays
performed on nuclear extracts from B- myb and pRc/CMV
transfectants before and after 6-day RA treatment (5 µ
M)
revealed a specific band in B- myb transfectants
(Fig. 2 C, lanes 1 and 2), but
not in RA-treated pRc/CMV transfectants ( lanes 4 and
5). The B- myb-specific band had a shift pattern
similar to that obtained with a B- myb-programmed reticulocyte
lysate (Fig. 2 C, lane 7). This result
suggests that the protein product of the transfected B- myb construct retains the DNA binding activity, which is still
detectable after 6 days of RA induction when endogenous B- myb is down-regulated. Finally, B-Myb protein was immunoprecipitated
in B- myb transfectants and controls using a specific rabbit
polyclonal antibody to the human B-Myb protein (data not shown).
Consistent with the RNA levels, B-Myb protein in B- myb transfectants was more abundant after RA treatment (data not
shown).
Figure 3:
Morphology of B- myb transfectants
( A and D), pRc/CMV transfectants ( B and
E), and LAN-5 cells ( C and F) under normal
growth conditions ( A-C) and after 6 days of treatment
with RA ( D-F). Original magnification was
200.
Figure 4:
Top panel, indirect immunofluorescence
detection of vimentin. Shown are B- myb transfectants ( A and D), pRc/CMV transfectants ( B and
E), and LAN-5 cells ( C and F) under normal
growth conditions ( A-C) and after 6 days of treatment
with RA ( D-F). Bottom panel, indirect
immunofluorescence detection of neurofilament (160 kDa). Shown are
B- myb transfectants ( A), pRc/CMV transfectants
( B), and LAN-5 cells ( C) after 6 days of treatment
with RA.
To determine whether RA-treated B- myb transfectants were induced to enter other differentiative
pathways, several components of the extracellular matrix were also
investigated: laminin, fibronectin, and collagen type IV, which are
produced by neuroblastoma cells and can be modulated during
differentiation processes
(45) ; and glial fibrillary acidic
protein, which has been described in human brain tumors
(46) .
Finally, the S-100 protein, which is expressed in 50% of glial
tumors, was also tested
(47) . Most of the markers analyzed did
not change after RA induction in B- myb transfectants and in
controls (). The only exception was collagen type IV, a
marker of Schwann differentiation, which was increased in SK-N-SH cells
(). This marker was moderately increased in B- myb transfectants after RA treatment, but not in pRc/CMV transfectants
or in LAN-5 cells. Overall, the phenotypic data indicate the
maintenance of many undifferentiated features in B- myb transfectants treated with RA, although they do not rule out the
onset of a different, yet not completely defined, differentiative
pathway.
SO-induced differentiation in mouse erythroleukemia cells
constitutively expressing c- myb (11) .
Table: Clonogenic assay using an antisense B-myb
vector in LAN-5 cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.