By
From the Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000
We have previously shown that transgenic mice expressing the oncoprotein v-Rel under the
control of a T cell-specific promoter develop T cell lymphomas. Tumor formation was correlated with the presence of p50/v-Rel and v-Rel/v-Rel nuclear B-binding activity. Since experimental evidence has led to the suggestion of a potential tumor suppressor activity for I
B
,
we have studied the role of I
B
in the transforming activity of v-Rel by overexpressing I
B
in v-rel transgenic mice. Overexpression of I
B
in v-rel transgenic mice resulted in an extended survival, and the development of cutaneous T cell lymphomas of CD8+CD4
phenotype. These phenotypic alterations were associated with a dramatic reduction of p50/v-Rel,
but not v-Rel/v-Rel nuclear DNA binding activity and an increased expression of the intercellular adhesion molecule 1. Our results indicate that v-Rel homodimers are active in transformation and that the capacity of v-Rel-containing complexes to escape the inhibitory effect
of I
B
may be a key element in its transforming capability.
The oncogene v-rel was originally identified as the
transforming component of the avian retrovirus reticuloendotheliosis virus T. v-Rel induces neoplastic disease
in birds, and compared to c-Rel, is missing 2 NH2-terminal
and 118 COOH-terminal amino acids, and has several internal changes (1). v-Rel is a member of the Rel/NF- The finding that v-Rel and the corresponding protooncogene c-Rel are members of the Rel/NF- The mechanism by which v-Rel induces oncogenic
transformation is not clear, and it was originally believed
that v-Rel could only transform avian cells (1). Recently, we demonstrated that v-Rel also has the capacity of
transforming mammalian cells in vivo. Transgenic mice expressing v-Rel in thymocytes develop T cell lymphomas
with very poor prognosis. In tumor cells from v-Rel transgenic mice, there are two major DNA-binding complexes
containing v-Rel homodimers and p50/v-Rel heterodimers.
However, when v-rel transgenic mice were crossed with
p50-deficient animals, T cell leukemia appeared at an earlier stage, suggesting that the v-Rel homodimer is the essential transforming complex (17).
In this report we address the question of whether overexpression of the inhibitory I Plasmid Construction and Generation of Transgenic Mice.
The generation of v-rel transgenic mice has been previously described
(17). A detailed description of the generation and characterization
of ikba transgenic mice will be reported elsewhere (Perez, P., unpublished observations). Screening of I Histopathology and Immunofluorescence.
Mice were killed and
tissues were immersion fixed in 10% buffered formalin. Tissues
were embedded in paraffin blocks and processed by routine
methods, sectioned at 5-10-µm thickness, stained with hematoxylin and eosin, and examined by light microscopy. Single cell suspensions from tumor-bearing spleens were prepared according to
standard procedures and spun onto slides as previously described (20). For immunofluorescence, the cytospins were incubated at 4°C overnight with anti-v-Rel or anti-I Flow Cytometric Analysis.
Single cell suspensions of spleens
bearing tumors were placed in RPMI 1640 medium supplemented with 10% FCS, 4 mM glutamine, 50 mM Western Blot Assays.
Cytoplasmic and nuclear fractions from
thymocytes were prepared as previously described (22). Aliquots
of cytoplasmic and nuclear extracts (20 µg) were boiled in Laemmli buffer and run overnight on a 12.5% acrylamide-bisacrylamide (200:1) gel at 12 mA. Western blotting procedures and antibodies have been previously described (23). The proteins were
transferred onto nitrocellulose membranes and transfer efficiency
assessed by Ponceau S staining. Purity of the nuclear extracts was
checked by incubating the membranes with an antiserum specific
for the cytosolic enzyme lactate dehydrogenase.
Cell Labeling, Lysis, and Immunoprecipitation.
Isolated thymocytes
were labeled for 2 h with 500 µCi/ml of [35S]methionine (1,000 Ci/mmol) in DMEM lacking methionine and containing 10%
heat inactivated and dialyzed FCS. The labeling medium was removed and the cells were washed with cold PBS and lysed on ice by adding radio immunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.5, 0.5% Nonidet P-40, 150 mM NaCl). Cell lysates
were first cleared with a preimmune serum (3 µl) for 3 h and
then immunoprecipitated with specific antisera as previously described (23).
Electrophoretic Mobility Shift Assay.
The palindromic Semiquantitative Reverse Transcription Analysis.
Semiquantitative
PCR was performed as described previously (17). The PCR conditions were established such that amplification of the cDNAs was
linearly dependent on the concentration of the corresponding
messenger RNAs (mRNAs). This was achieved by performing the reactions in the presence of [32P]dCTP; due to the greater
sensitivity of autoradiography as opposed to ethidium bromide
staining, a relatively low number of PCR cycles was sufficient to
detect the amplified product. In this way, the cDNA concentration remained the rate-limiting factor throughout the amplification procedure. Lymph node T cells derived from ikba, v-rel, and
v-rel/ikba transgenic mice were isolated on murine T cell enrichment columns (R&D Sys. Inc.), and total RNA was prepared and
quantified by absorbance at 260 nm. 10 µg of total RNA were used for cDNA synthesis using 1,000 U of reverse transcriptase (GIBCO BRL). After cDNA synthesis, each sample was diluted
five times and 5 µl were taken for amplification with 25 cycles by using Taq polymerase in the presence of [32P]dCTP and sequence-specific primers. Specific oligonucleotides were obtained
from Stratagene Corp. (La Jolla, CA). One-tenth of the PCR
products were separated on 8% native polyacrylamide gels run in
Tris base, boric acid, EDTA buffer, dried, and exposed to X-Omat
AR film (Kodak) at The transcriptional activity of Rel/NF- We recently generated transgenic mice specifically expressing v-Rel in thymocytes (17). These animals develop
T cell multicentric aggressive lymphomas. To study the effect of I The expression of v-Rel and I
The levels of I To examine whether the overexpression of I
The changes in the nuclear/cytoplasmic distribution of
v-Rel protein in the double transgenics were further confirmed by immunofluoresence (Fig. 2 B). v-Rel protein in
v-rel transgenic thymocytes is detected in both nucleus and
cytoplasm; in contrast, in v-rel/ikba double transgenic thymocytes, v-Rel is mainly detected in the cytoplasm (a and
b, respectively). In addition, the distribution of v-Rel and
I To investigate
whether the The p50/v-Rel DNA binding activity in nuclear extracts
from v-rel/ikba double transgenic thymocytes presents a
dramatic reduction compared to nuclear protein extracts
from v-rel transgenic thymocytes (compare lanes 1 and 4).
However, the v-Rel/v-Rel DNA-binding activity remains
almost unchanged (compare lanes 2 and 5). The amount of
nuclear protein extract added to the DNA binding reaction was controlled by measuring Oct 1 DNA-binding activity.
Oct 1 DNA-binding activity was comparable between the
different protein samples (data not shown).
These results indicate that v-Rel homodimers are significantly less sensitive than p50/v-Rel heterodimers to the inhibitory effect of I To investigate the
consequences of the changes in
The earliest manifestation of disease in
most of the v-rel/ikba double transgenic mice was the appearance of facial skin lesions that progressed slowly (Fig. 3
B). Light microscopy revealed cellular infiltration of the
dermis by large lymphocytes of irregular nuclei (Fig. 4, a-d).
Isolated foci of epidermal infiltration by tumor cells were
occasionally seen, but typical Pautrier's microabcesses and
massive epidermal infiltration were not observed (Fig. 4 e). The histologic appearance of this lymphoid infiltration resembles one seen in cutaneous T cell lymphomas (24).
In contrast to v-rel/ikba transgenic mice, dermal compromise was rarely seen in v-rel transgenic mice (17). Histopathology of v-rel/ikba transgenic animals revealed much less
severe tumoral cellular infiltration in other tissues besides
skin (17). For instance, in the lung of v-rel transgenic mice,
tumoral cellular infiltration around bronchi and alveoli was
severe enough to cause organ failure. In v-rel/ikba transgenic mice, lung compromise was much less severe with
only tumoral cell infiltration around the bronchi, but not in
the parenchyma (17; Fig. 4 e). The liver in v-rel transgenic mice was always enlarged and massively infiltrated by tumor cells that frequently plugged the vessels and produced
extensive areas of ischemia and tissue necrosis. In contrast,
in v-rel/ikba double transgenic mice, the liver was not enlarged, and lymphoid infiltration was seen in hepatic sinusoids and around the portal triad, but areas of necrosis were
not observed (Fig. 4 f ). The spleen in v-rel transgenic mice
was dramatically enlarged with complete distortion of the
normal architecture and massive areas of tissue necrosis. On
the other hand, in v-rel/ikba double transgenic mice, the
spleen was moderately enlarged and remnants of the normal splenic architecture with demarcation of white and red
pulp areas were always present (Fig. 4 g). Lymph nodes in
v-rel/ikba double transgenic mice were not severely compromised, even at very late stages of disease, as was the case
in v-rel transgenic mice (Fig. 4 h, and data not shown).
Thymic atrophy was a constant observation in both v-rel
and v-rel/ikba transgenic mice (data not shown). These histopathological studies demonstrate that v-rel/ikba double transgenic mice developed lymphoma, but the magnitude
of the visceral compromise was much less than in v-rel
transgenic mice of the same age. These results indicate that
I
To understand the changes in the
clinical expression of v-rel/ikba double transgenics compared to v-rel transgenic mice, we analyzed lymphoid cell
populations by flow cytometry (Fig. 5). Flow cytometric
analysis of lymphoid cells in v-rel transgenic mice have
been previously described (17). Analyses of thymuses and
spleens from young v-rel/ikba double transgenic animals
(3-8 wk) did not reveal any alterations when compared to
ikba transgenic littermates (Fig. 5 A, a-d, and data not
shown). ikba transgenic littermates were used as a control,
since no differences in lymphoid populations have been detected in ikba transgenic mice when compared to wild-type
animals (Perez, P., unpublished results and data not shown).
Analysis of spleens from moribund and euthanized v-rel/ ikba double transgenic animals showed an expanded T cell
population characterized by intermediate and low levels of
the TCR
To understand why higher
levels of I
To correlate the changes in the clinical expression and the cutaneous infiltration observed in v-rel/ikba double transgenic mice, we examined the level of expression of In this work we have demonstrated that overexpression
of the Rel/NF- Previous studies have
suggested a potential tumor supresser activity for I In this work, we have demonstrated that although v-rel/
ikba double transgenic thymocytes expressed I The fact that I When nuclear The lymphomatous infiltration observed in v-rel/ikba transgenic mice resembles that seen in T cell cutaneous lymphomas: the Sezary syndrome, mycosis fungoides, or other related disorders (35). Our observation that v-rel/ikba double
transgenic mice developed cutaneous T cell lymphomas is
particularly interesting in light of the observation that chromosomal translocations associated with structural alterations
of the Rel/NF-B
family of eukaryotic transcription factors, which includes
c-Rel, RelA (p65), RelB, NF-
B1 (p50/p105), NF-
B2
(p52/p100), and the Drosophila proteins Dorsal, dorsal-related immunity factor, and Relish (6).
B transcription factor family led to the suggestion that transformation
resulted from v-Rel-induced changes in gene expression
(1). Rel/NF-
B proteins are related through an ~300-
amino acid NH2-terminal region known as the Rel homology domain (RHD)1, which contains sequences important
for dimerization, DNA binding, inhibitor binding, and nuclear localization. The activity of Rel/NF-
B complexes is
modulated by their interaction with the I
B family of inhibitors, which contain ankyrin repeats. In unstimulated cells, Rel/NF-
B dimers remain in the cytoplasm as inactive complexes through association with I
B molecules
that mask their nuclear localization signals. A wide variety
of stimuli result in the rapid phosphorylation and degradation of I
B molecules and nuclear translocation of Rel/
NF-
B complexes (6).
B
protein, which has been
suggested to have tumor suppresser activity (18), can reverse the transforming activity of v-Rel in v-rel transgenic
mice. Overexpression of I
B
extended the survival of
v-rel transgenic mice and reduced the severity of lymphomas. Surprisingly, I
B
overexpression resulted in a change
in the clinical expression of the disease with an expansion
of CD8+CD4
T cells in peripheral tissues. These T cell
changes were associated with increased levels in the expression of the intercellular adhesion molecule 1 (ICAM-1),
increased dermotropism and the development of cutaneous
lymphoma. T cells from v-rel/ikba double transgenic mice
presented a dramatic reduction of p50/v-Rel but not of
v-Rel/v-Rel nuclear DNA-binding activity. Our results
indicate that v-Rel homodimers are active in transformation and that v-Rel containing complexes have an intrinsic
capability to escape the inhibitory effect of I
B
. We postulate that variations in the clinical expression of related
lymphoid malignancies may reflect subtle changes in the
nuclear composition and interplay among different Rel/
NF-
B and I
B molecules.
B
transgenic mice was
performed as described (19), and line 1 was selected for its high
level of expression as determined by immunoblot analysis using a
mouse monoclonal I
B
antibody. To generate double v-rel/ikba transgenic mice, ikba transgenic mice were bred to homozygocity and crossed with heterozygote v-rel transgenic animals. The mice obtained from these intercrosses were screened by PCR using a pair of specific v-rel oligonucleotides: 5
-TTTCTCACCAACCTCCGATTCACTG-3
and 5
-ATCTCTGCAGCCTTTTCCAACTAGA-3
.
B
polyclonal antibodies. Anti-v-Rel and anti-I
B
antibodies were visualized with a
donkey anti-rabbit immunoglobulin conjugated with Texas red
(Amersham Corp. Arlington Heights, IL). For ICAM-1 immunofluorescence analysis, lymph node T cells were isolated on murine T cell enrichment columns (R&D Sys. Inc., Minneapolis,
MN) according to manufacturer's recommendation from v-rel
and v-rel/ikba transgenic mice. T cells were spun onto slides and
incubated at 4°C overnight with anti-ICAM-1 FITC-labeled monoclonal antibody (PharMingen, San Diego, CA).
-mercaptoethanol, 50 U/ml penicillin, and 50 mg/ml streptomycin. Flow
cytometry was performed with a flow cytometer and cell sorter (Epics Profile II; Coulter Corp., Hialeah, FL). Single cell suspensions from ikba and v-rel/ikba transgenic thymi and spleens were
prepared and analyzed for surface expression of CD4 (clone
H129.19), CD8 (clone 7D4), TCR-
/
(clone H57-597), B220
(clone RA3-6B2), HSA (clone J11d), Mac-1 (clone M17/70HL),
and Gr-1 (clone RB6-8C5) as previously described (21). Monoclonal antibodies were obtained from GIBCO BRL (Gaithersburg, MD) and PharMingen.
B site
used for these assays has been previously described (23). Nuclear
extracts (3 µg) were incubated with 20,000 cpm 32P-labeled
probe, 3 µg poly (dI/dC) in buffer containing 20 mM Hepes, pH
7.9, 60 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM
PMSF, and 17% glycerol in 25 ml final volume for 15 min on ice.
Protein loading was checked by Oct 1 DNA-binding activity. Complexes were separated on 5.5% native polyacrylamide gels
run in 0.25 × Tris base, boric acid, EDTA buffer, dried, and exposed to X-Omat AR film (Kodak, Rochester, NY) at
70°C.
70°C.
Generation of v-rel Transgenic Mice Overexpressing IB
in
the Thymus.
B
family of proteins is regulated, in part, by their association
with inhibitory molecules (I
Bs) that sequester them as inactive complexes in the cytoplasm. I
B
is the predominant inhibitory protein in most cell types (5, 13).
B
on the transforming capability of v-Rel, we
produced double transgenic v-rel/ikba mice by crossing
transgenic mice expressing v-Rel with transgenic mice
overexpressing I
B
. Both transgenes are under the control of the mouse T cell-specific lck proximal promoter.
B
in thymocytes from
the double transgenic mice was assessed by immunoprecipitation (Fig. 1 A). These analyses demonstrate the association of v-Rel complexes with I
B
(Fig. 1, lanes 1 and 3)
and show that there is an excess of I
B
protein that is not
associated with v-Rel (Fig. 1, lane 4). In addition, these
data show that under these conditions, I
B
, another I
B
inhibitory protein, is not associated with v-Rel, suggesting
that this molecule does not play a major role in the inactivation of v-Rel complexes (Fig. 1 A, lane 2).
Fig. 1.
v-Rel/IB
complexes (A) and I
B
protein expression (B) in v-rel/ikba double
transgenic thymocytes. To generate v-rel/ikba double transgenic
mice, ikba homozygous transgenic mice were crossed with v-rel
heterozygous transgenic mice.
Both transgenes were under the
control of the lck promoter.
Screening of double transgenic mice was done by PCR analysis
using v-rel-specific oligonucleotides. (A) Immunoprecipitations
were performed as previously
described (31). [35S]methionine-labeled thymocytes were lysed
under nondenaturing conditions,
and total protein extract was immunoprecipitated with anti-v-Rel
antibodies. The immunoprecipitate was denatured to dissociate
the v-Rel-containing immune
complex and then reprecipitated, first with I
B
and subsequently
with I
B
and v-Rel antibodies (left). The supernatant of the total protein extract treated with anti-v-Rel antibody was subsequently reprecipitated with anti-I
B
and anti-I
B
antibodies (right). (B) The levels of
I
B
protein expression were analyzed by Western blot in total protein
extracts from control (w.t.), ikba transgenic, v-rel transgenic and v-rel/ikba double transgenic thymocytes.
[View Larger Version of this Image (28K GIF file)]
B
protein expression in v-rel/ikba double transgenic mice was determined by Western blot with
whole protein extract prepared from transgenic thymocytes
and compared with the levels of I
B
protein expression in
wild-type, ikba transgenic, and v-rel transgenic thymocytes
(Fig. 1 B). The highest levels of I
B
protein expression
were detected in v-rel/ikba double transgenic thymocytes
(lane 4). This represents almost two times the amount of
I
B
present in ikba transgenic (lane 2) and v-rel transgenic
thymocytes (lane 3). As described previously, there is an increase in the levels of I
B
in v-rel transgenic thymocytes when compared with control thymocytes (lane 1) due to I
B
protein stabilization by v-Rel-containing complexes (17).
B
Overexpression Changes v-Rel Nucleus/cytoplasm Distribution.
B
in v-rel transgenic mice alters the nuclear-cytoplasmic distribution of v-Rel, we performed Western blot analyses using thymocyte extracts from wild-type, v-rel transgenic,
and v-rel/ikba double transgenic mice (Fig. 2 A). In v-rel
transgenic thymocytes, a significant amount of v-Rel,
~30% of the total protein, was localized in the nucleus
(lanes 3 and 4). In contrast, in v-rel/ikba double transgenic thymocytes, >90% of the v-Rel protein is retained in the
cytoplasm, and only a small amount translocates to the nucleus (lanes 5 and 6). The blot containing lanes 5 and 6 was
overexposed to detect the min amounts of v-Rel protein in
the nucleus. Incubation of the same membrane with antiserum specific for the cytosolic enzyme lactic dehydrogenase
demonstrated no cross-contamination of nuclear and cytoplasmic extracts (data not shown).
Fig. 2.
Change of nucleus/cytoplasm distribution of v-Rel in v-rel/
ikba double transgenic thymocytes. (A) Western blot analysis. Cytoplasmic (C) and nuclear (N) fractions from thymocytes and Western blot assays were performed as previously described (31). (B) Immunofluoresence. Thymocytes from 6-wk-old v-rel (a) and v-rel/ikba (b) transgenic
mice were spun onto slides, incubated with anti-v-Rel antibodies, and
visualized with donkey anti-rabbit Texas red-labeled antibodies. T cells
from spleen-bearing tumors of v-rel/ikba double transgenic mice were
spun onto slides, incubated with anti-v-Rel (c) or anti-IB
(d) antibodies, and visualized with donkey anti-rabbit Texas red-labeled antibodies.
(C) Decreased p50/v-Rel
B-binding activity in v-rel/ikba double transgenic thymocytes. Electrophoretic mobility shift assays were performed as
previously described (23) using nuclear protein extracts from v-rel and
v-rel/ikba double transgenic thymocytes. Lanes 1 and 4 were treated with
preimmune sera (p.i.), lanes 2 and 5 were treated with anti-p50 antibody
(
-p50), and lanes 3 and 6 were treated with anti-v-Rel antibody (
-v-Rel).
[View Larger Version of this Image (23K GIF file)]
B
proteins in transformed T cells from v-rel/ikba double transgenic mice is also mainly in the cytoplasm (c and d, respectively).
B
Overexpression Decreases p50/v-Rel Heterodimer, but
Not v-Rel Homodimer DNA-binding Activity.
B-binding activity was altered in thymocytes
of v-rel/ikba double transgenic mice compared to v-rel
transgenic mice, thymocyte protein extracts were analyzed by electrophoretic mobility shift assays (EMSA) using a palindromic
B site (Fig. 2 C).
B
. The reduced affinity of v-Rel for
I
B
may be one of the key molecular basis for v-Rel
transformation.
B
Overexpression Extends Survival and Changes the
Clinical Expression of v-rel Transgenic Mice.
B binding activity in
v-rel/ikba double transgenic mice, a colony of these animals
was bred and their health status monitored over time in a
nongerm-free environment (Fig. 3). The v-rel/ikba double
transgenic mice presented a longer survival and appeared
completely healthy for a longer period of time when compared with v-rel transgenic mice. The mortality curve demonstrates that high levels of I
B
extended the survival of
v-rel transgenic mice (75% of v-rel transgenic mice succumbed before 25 wk of age, whereas 100% of the v-rel/ ikba double transgenic mice were unaffected at that time).
Young v-rel/ikba double transgenic mice, between 3 and
10 wk old, appeared normal as assessed by habits, weight,
posture, and histopathology. However, all v-rel/ikba double
transgenic mice developed skin lesion in face, ears, tail, and
feet after 15 wk of age. These lesions, characterized by loss
of hair, thickening of the skin, and exfoliative plaques (Fig.
3 B) progressed slowly with no major compromise in the
health status of the mice. Evaluation for a longer period of
time revealed that v-rel/ikba double transgenic were susceptible to bacterial infections and that they succumbed due to secondary infections by pathogens that colonize the
skin; antibiotic prophylactic treatment extended their survival for an additional 2-4 wk (data not shown). Autopsies
of v-rel/ikba double transgenic mice did not reveal massive
tumoral cell burden in lung, liver, lymph nodes, and
spleen, indicating that in contrast to v-rel transgenic mice,
massive tumor cellular infiltration was not the primary reason for organ failure in the double trangenic animals (17).
Fig. 3.
Overexpression of IB
increases survival of v-rel transgenic
mice and changes the clinical expression. (A) Mortality curve. Deaths in
v-rel/ikba transgenic animals occurred at later times and were the result of
secondary opportunistic infection according to pathologic analysis and
microbiology. Autopsy, and histologic examination of v-rel/ikba transgenic mice revealed a less severe lymphomatous infiltrate than in v-rel
transgenic mice. n = 29 for v-rel 35, and n = 13 for v-rel/ikba. (B) T-cell
cutaneous lymphoma in v-rel/ikba double transgenic mice. All v-rel/ikba transgenic mice developed skin lesions characterized by thickening and
exfoliative plaques. Arrow indicates a v-rel/ikba transgenic mouse; the
control mouse is an ikba transgenic littermate. (The lesions were never
observed in v-rel transgenic mice.)
[View Larger Versions of these Images (83 + 10K GIF file)]
B
overexpression does not prevent tumor formation,
but does delay the appearance of tumors, the progression of
the disease, and decreases the severity of the lymphomatous
state observed in v-rel transgenic mice. In addition, these
results indicate that I
B
overexpression induces transcriptional changes in v-Rel-transformed T cells that resulted in
their increased tropism for the skin.
Fig. 4.
Tumor pathology in
v-rel/ikba transgenic mice. Tissue
blocks were fixed in 10% neutral
buffered formalin and processed
for paraffin embedding. 5-µm
sections were stained with hematoxylin and eosin. Sections from
skin (a, b, c, and d), lung (e), liver ( f ), spleen (g), and lymph node
(h) are shown. Arrow heads indicate sites of lymphoid infiltration. Photomicrographs were taken at ×100 (a, b, e, and f ),
×250 (c and d), ×12.5 (g and h).
E, epidermis; D, dermis; PA,
paracortical area; PT, portal triad;
RP, red pulp; WP, white pulp.
[View Larger Version of this Image (125K GIF file)]
and
chains (Fig. 5 B, a and b). In addition, a
striking difference between v-rel transgenic and v-rel/ikba
double transgenic mice was a significant increase in CD8+
single positive T cells in v-rel/ikba transgenic mice, a population of cells that was not always increased in v-rel transgenic mice. These results indicate that I
B
overexpression
in v-rel transgenic mice alters the developmental profile of
the v-rel transformed T cells, allowing the T cells to reach a
more mature phenotype. Only a moderate increase of T
cells coexpressing markers associated with an immature
phenotype (HSA) was detected in the double transgenic (Fig. 5 B, e and f ), in contrast to the dramatic changes that we previously observed in v-rel transgenic mice. An increased population of granulocytes as a result of infection
was detected in v-rel/ikba double transgenic mice (Fig. 5 B,
g and h).
Fig. 5.
Flow cytometric
analysis in v-rel/ikba transgenic
mice. (A) Flow cytometric analysis
of CD4 and CD8 surface markers in thymocytes from 6-wk-old
ikba (a and c) and v-rel/ikba (b and
d) transgenic mice. (B) Flow cytometric analysis of surface markers
in splenocytes from 6-mo-old
ikba (a, c, e, and g) and v-rel/ikba
(b, d, f, and h) transgenic mice.
Flow cytometry was performed
using a flow cytometer (Epics
Profile II) and cell sorter (Coulter
Corp.). Single cell suspensions
from thymocytes and splenocytes
were prepared and analyzed for
surface markers expression of
CD4, CD8, HSA, TCR-/
,
B220, Mac-1, and Gr-1 as previously described (8).
[View Larger Version of this Image (27K GIF file)]
B
than
c-Rel-containing Complexes.
B
are required to reduce nuclear translocation
of v-Rel and p50/v-Rel
B-binding activity in transgenic
thymocytes, we compared the inhibitory effect of I
B
on
B binding activity in nuclear protein extract derived from
v-rel transgenic (Fig. 6 A) and
c-rel transgenic thymocytes.
c-rel transgenic animals express a truncated version of the
mouse c-Rel protein (
c-Rel) under the control of the lck
promoter, and, like the v-Rel oncogenic protein, lacks part
of the COOH-terminal transcriptional activation domain
keeping the RHD intact. The generation of
c-rel transgenic mice has been previously described (17). Increasing
levels of purified baculovirus expressed I
B
protein were
added to identical amounts of nuclear protein extracts from
c-rel transgenic thymocytes (Fig. 6 A a) and v-rel transgenic thymocytes (Fig. 6 A b) before starting the
B-binding reaction. The total amount of protein extract used in
the reaction was tested by Oct 1 DNA-binding activity and
the identification of the complexes by antibody supershifts
(data not shown). In the absence of added I
B
, similar
amounts of total
B binding activities were detected in
c-rel transgenic (Fig. 6 A a, lane 1) and in v-rel transgenic thymocytes (Fig. 6 A b, lane 1). However, when increased
amounts of I
B
protein were added to the binding reaction, the binding of p50/
c-Rel was dramatically reduced
(Fig. 6 A a, lanes 2 and 3), whereas the binding of v-Rel
containing complexes was slightly affected (Fig. 6 A b, lanes
2 and 3). At 100 ng of I
B
, the binding of v-Rel-containing complexes is significantly reduced (Fig. 6 A b, lane
4), and only when the highest amounts of I
B
were used (1 µg) and when endogenous p50/p50
B binding also began to be affected (Fig. 6 A a, lane 5), was the binding of
v-Rel-containing complexes completely reduced (Fig. 6 A
b, lane 5). These results demonstrate that the binding of
v-Rel-containing complexes is more resistant than the
binding of complexes containing the cellular homologue
c-Rel to the inhibitory effect of I
B
, and offer an attractive explanation for the molecular mechanisms involved in the transforming activity of v-Rel. This is in agreement
with previous in vitro studies that demonstrated a reduced
I
B
inhibition of DNA binding by the oncogenic v-Rel
protein when compared to the nononcogenic c-Rel protein (28).
Fig. 6.
Reduced IB
inhibition of v-Rel DNA binding
activity (A) and increased levels
of ICAM-1 in v-rel/ikba double
transgenic T cells (B and C). (A)
Equal amounts of nuclear protein extracts from
c-rel (a) or
v-rel (b) transgenic thymocytes
were incubated in the presence
of increased amounts of I
B
protein and analyzed by EMSA
using a palindromic
B-binding
site. (B) Total RNA was prepared from ikba transgenic, v-rel
transgenic or v-rel/ikba double
transgenic lymph node-derived
cells. After cDNA synthesis, one-tenth of the reaction mixture was amplified in the presence of 0.1 µCi of [32P]dCTP using Taq polymerase and sequence-specific primers. One-fifth of the amplified products were separated on 6% nondenaturing polyacrilamide gels. Gels were dried and exposed to x-ray film. (C) Purified T cells from lymph
nodes of v-rel transgenic (a) and v-rel/ikba (b) double transgenic mice were spun onto slides and incubated with anti-ICAM-1 FITC-labeled antibody.
[View Larger Version of this Image (27K GIF file)]
B responsive genes encoding adhesion molecules that may be involved in the increased tropism of v-rel/ikba double transgenic T cells for the skin (22, 24, 29, 30). We analyzed
mRNA expression levels of ICAM-1 by reverse transcription PCR in ikba, v-rel, and v-rel/ikba transgenic T cells
(Fig. 6 B). cDNA was prepared from total mRNA extracted from single cell suspension from ikba normal lymph
nodes and from v-rel and v-rel/ikba tumor-bearing lymph
nodes. The cDNA was amplified with 25 PCR cycles in
the presence of [32P]dCTP and the appropriate pairs of specific oligonucleotides. The same amount of total mRNA
was present in all the samples, as demonstrated by equal
amounts of
-actin PCR products (Fig. 6 B). By comparing the amount of PCR products detected in ikba (lane 1),
v-rel (lane 2), and v-rel/ikba lymph node-derived T cells
(lane 3), it is evident that there is a dramatic increase in the
expression of ICAM-1 in v-rel/ikba transgenic T cells. No
major changes in the expression of vascular cell adhesion
molecule 1 and dorsal midline-globulin-restricted axonal
surface protein were detected between v-rel and v-rel/ikba-
transformed T cells (Fig. 6 B and data not shown). Increased
levels of ICAM-1 expression in v-rel/ikba transgenic T cells
were also detected on cytospin preparation by indirect
immunofluorescence using anti-ICAM-1 FITC-labeled antibody (Fig. 6 C). The difference in the level of ICAM-1 expression between v-rel and v-rel/ikba may explain the increased tropism for the skin of v-rel/ikba-transformed T cells.
B inhibitory protein, I
B
, delays the development of T cell lymphomas in a transgenic mouse
model that expresses the oncogenic v-Rel protein under
the control of the lck T cell-specific promoter (17). Previous studies indicate that v-Rel transformation requires its
translocation to the nucleus and binding to a set of specific
Rel/NF-
B responsive genes (1).
B
in v-Rel Transformation.
B
(4).
I
B
-deficient mice displayed elevated levels of nuclear
Rel/NF-
B activity in hemopoietic tissues. Unfortunately, these animals do not survive long enough for evaluation of
appearance of lymphoid malignancies (31, 32).
B
protein
at very high levels, there was still some nuclear v-Rel protein in the double transgenic cells, suggesting an intrinsic
ability of v-Rel to escape the inhibitory effect of I
B
.
This observation was correlated with an increased resistance of v-Rel DNA-binding activity to the inhibitory effect of I
B
when compared to a truncated version of c-Rel
that lacks the transcriptional activation domain. The reduced affinity of v-Rel for I
B
may be associated with
some of the internal mutations present in the Rel homology domain of v-Rel (1). It is noteworthy to mention
that transgenic mice overexpressing either RelA or RelB in
T cells under the control of the lck did not develop lymphomas. In the case of rela transgenic mice, the absence of
tumor formation was correlated with the fact that there was
no increased DNA-binding activity in T cells, because RelA protein was efficiently retained in the cytoplasm by
I
B
(19). However, relb transgenic thymocytes presented
a dramatic increase in DNA-binding activity due to the fact
that RelB complexes were not efficiently retained in the
cytoplasm because of their low affinity for I
B
(33), indicating that increased nuclear levels of Rel/NF-
B activity
appear to be insufficient for transformation. Therefore, together with the low affinity for I
B
, it appears that v-Rel
possesses additional and unique properties that render a potent transforming potential in mammalian cells.
B
overexpression extended the survival
but did not prevent tumor formation in v-rel transgenic
mice indicates that it would be impossible to achieve in
vivo the levels of I
B
required to compensate for the reduced affinity of v-Rel for I
B
to prevent v-Rel transformation.
B-binding activity was assessed by EMSA
in v-rel/ikba double transgenic thymocytes, the most striking observation was the dramatic reduction in the p50/v-Rel
DNA-binding activity with almost no change in v-Rel/
v-Rel DNA binding activity. The reduction in the p50/
v-Rel DNA binding correlated with the changes observed
in the clinical expression of v-rel/ikba double transgenic mice, and supports our previous indications that v-Rel may
be active as a p50/v-Rel heterodimer and as a homodimer
(17), with the p50/v-Rel heterodimeric form being more
susceptible to I
B
inhibition. Because of the changes in
the clinical expression and the differences observed in p50/
v-Rel and v-Rel/v-Rel DNA-binding activity in v-rel/
ikba double transgenic T cells, it is possible to speculate that
v-Rel either as a heterodimeric complex with p50 or as a
homodimeric complex may target different sets of
B-regulated genes. An example of this differential regulation is
the observation of increased levels of ICAM-1 in T cells
derived from v-rel/ikba double transgenic mice, but not in
T cells derived from v-rel transgenic mice. Interestingly,
strong induction of ICAM-1 has been observed in human
lymphoproliferative disorders (29, 34), and ICAM-1 overexpression in v-rel/ikba double transgenic mice may be responsible for the increased tropism of T cells for the skin
observed in these animals (25, 22).
B Activity in Cutaneous T Cell Lymphomas.
B family of proteins have been documented in several cases of T cell cutaneous lymphomas in
human patients (26, 35). In particular, rearrangement
and altered expression of the NFKB-2 gene have been
identified in the HUT 78 human cutaneous T cell leukemia line (39, 40). In addition, structural alterations of the
NFKB-2 gene have also been identified very commonly
(14%) in neoplasms derived from mature T cells such as
mycosis fungoides and Sezary syndrome (24, 38). These
structural alterations may contribute to lymphomagenesis by determining a constitutive activation of the NF-
B system and, in particular, of NFKB-2 target genes (36, 37).
This data, in addition to our observations, strongly supports
the notion that structural alterations of other Rel/NF-
B
family members may play a role in lymphomagenesis. Furthermore, our results indicate that variations in the clinical
expression of lymphoid malignancies such us mycosis fungoides and Sezary syndrome may be based on subtle
changes in the nuclear composition and interplay among the different Rel/NF-
B and I
B molecules. In this sense,
v-rel and v-rel/ikba transgenic mice constitute a potential
model to study the molecular mechanisms involved in the
generation of lymphoid malignancies.
Address correspondence to Rodrigo Bravo, Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000. Phone: 609-252-5744; FAX: 609-252-3307; E-mail: Bravo#m#_Rodrigo.PRILVMS1{at}msmail.bms.com
Received for publication 11 April 1997.
1Abbreviations used in this paper: EMSA, electrophoretic mobility shift assay; ICAM-1, intercellular adhesion molecule 1; mRNA, messenger RNA; RHD, Rel homology domain.We would like to thank C. Raventos-Suarez and K. Class for FACS® analysis; K. Dorfman and L. Chen for technical assistance; S. Lira, M. Swerdel, and P. Zalamea for the generation of the transgenic mice, and all the staff in Veterinary Sciences of Bristol-Myers Squibb for their excellent support; C. Gelinas for anti- v-Rel antibodies, Roger M. Perlmutter for the mouse lck promoter, and Dimitris Kioussis for the human CD2 LCR sequences; and Janet Cheng for her valuable comments on this manuscript.
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