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Departments of
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Biochemistry and
Laboratory Medicine and Pathology, Boston University Medical School, Boston MA 02118
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30–40% and 60–70% of cells
after 24 and 48 h, respectively (5). Thus, these
cells have been proposed as models for self-induced tolerance via
clonal deletion (1). Several years ago, we demonstrated
that BCR engagement of WEHI 231 cells resulted in a dramatic drop in
c-myc expression which preceded the induction of apoptosis
(6, 7, 8, 9). Furthermore, we noted that the rescue of WEHI 231
cells from anti-IgM-mediated killing that is mediated via CD40
ligand (CD40L) interaction with its receptor (10) was
accompanied by maintenance of elevated c-Myc expression
(11). We confirmed the ability of c-Myc to promote
survival of WEHI 231 cells by using c-myc stable
transfectants and microinjection analyses (5). In
particular, we showed that ectopic expression of c-Myc prevented
apoptosis induced by BCR engagement. Taken together, these results
indicate that the drop in c-myc expression plays a pivotal
role in the death of WEHI 231 cells upon BCR engagement. Although
c-myc overexpression in the absence of a critical
growth-promoting signal was originally shown to promote cell death
(12), a similar role for a decrease in c-myc in
promoting apoptosis has been reported in an ever-increasing number of
cell types and treatment conditions, e.g., in various leukemia,
lymphoid, melanoma, and breast cancer cells (12) as well
as in pre-B (13), murine erythroleukemia
(14), and immature T cells (15). Interestingly, apoptosis of WEHI 231 cells induced upon anti-IgM treatment is preceded by arrest of growth. This arrest was shown to occur in the late G1 phase immediately preceding entry into S phase, as judged by appearance of hypophosphorylated Rb and by FACS analysis of DNA content (8, 16). BCR engagement of WEHI 231 cells was shown by Scott and coworkers (17) to lead to the induction of p27Kip1 cyclin-dependent kinase (cdk) inhibitor, which promoted the cell cycle arrest. More recently, we demonstrated that anti-IgM treatment also results in p53-mediated induction of the cdk inhibitor p21WAF1/CIP1 and that inhibition of its expression provided WEHI 231 cells with partial protection from apoptosis (18). Interestingly, we found that inhibition of both p21 and p27 provided additional protection (18). Thus, in this paper we have evaluated the potential role of p27 in apoptosis of WEHI 231 cells. Our findings identify the p27Kip1 cdk inhibitor as a proapoptotic gene in WEHI 231 cells and show that its induction is a component of the c-Myc signaling pathway controlling cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WEHI 231 cells were maintained and treated with 1:1000 dilution of anti-µ heavy-chain Ab in the absence or presence of CD40L as previously described (11). For immunoblot analysis, whole cell extracts were prepared as described previously (5). Except where noted, for trypan blue analysis of cell viability, cultures were incubated with 0.2% trypan blue for 10–20 min and the percentage of cells excluding dye (viable cells) was determined by examination under phase contrast microscopy at x100. DNA fragmentation and Non-Radioactive Cell Proliferation (Promega, Madison, WI) assays were performed as described previously (5, 9).
Isolation of inducible p27 and c-myc antisense stable WEHI 231 cell transfectants
To construct an inducible p27 expression vector, a human p27 cDNA insert was cut from the vector pGlu-p27H (kindly provided Dr. Y. Xiong, University of North Carolina) with HindIII and BamHI, and was used to replace the chloramphenicol acetyltransferase (CAT) reporter gene in the pOPRSVICAT vector, generating a clone termed pOPRSVI-p27. Similarly, an expression vector for antisense c-myc RNA (pOPRSVI-as-c-myc) was prepared with the 1.8-kb HindIII and EcoRI insert, containing exon 2 and exon 3 of murine c-myc from the pRc-CMV-c-myc expression vector, which was blunt ended and subcloned in the reverse orientation into the NotI sites of pOPRSVICAT, replacing the CAT gene. Cells were electroporated as described previously (18), first with 30 µg p3'SS and stable transfectants selected with 350 µg/ml hygromycin (Boehringer Mannheim, Indianapolis, IN). Cultures were then electroporated with either 30 µg pOPRSVI-p27 or pOPRSVI-as-c-myc and were selected for stable transfectants with 350 µg/ml hygromycin plus 1.0 mg/ml G418 to isolate mixed populations of transfected cells.
Microinjection analysis
A pOPRSVI-p27 construct was also prepared with reverse
orientation of the p27 cDNA insert using the pOPRSVICAT as above,
yielding expression of antisense p27 RNA (termed pOPRSVI-as-p27). For
microinjection analysis, WEHI 231 cells were allowed to attach to
tissue culture plastic in the presence of culture medium containing
0.4% FBS and supplemented with 20 mM HEPES (pH 7.3). After 30 min of
incubation at 37°C, all cells in duplicate circled areas
(150–300/sample) were microinjected using a Narishige (Tokyo,
Japan) micromanipulator, as previously described (19) with
the indicated mammalian expression plasmid or control DNA samples,
adjusted to 130 mM KCl and 10 mM sodium phosphate buffer (pH 7.3)
before microinjection. Where indicated, vector DNA was microinjected in
the presence of Ab or control BSA protein in the absence or presence of
cognate peptide. After microinjection, cloning rings were placed over
the microinjected areas and the medium was replaced with 10% FBS/DMEM.
After 30 min of incubation at 37°C, cells were removed by gentle
trituration, transferred to multiwell plates, and incubated at 37°C
in the absence or presence of anti-IgM, as indicated. To measure
cell death, one-tenth volume trypan blue solution (0.04% final) was
added to the wells, and the cells were incubated for another 15 min;
the percentage of trypan blue positive cells was determined by phase
contrast microscopy.
Protein and RNA blot analysis
For analysis of levels of p27, c-Myc, p21, and p53 proteins, whole cell extracts were prepared as described previously (5). Immunoblotting was performed as previously described (11) using a p27 Ab (Transduction Laboratories, Lexington, KY), affinity-purified c-Myc-specific Ab (50-23) (kindly provided by S. Hann, Vanderbilt University), p21 Ab (sc-397; Santa Cruz Biotechnology, Santa Cruz, CA), and p53 Ab (Pab 421; Oncogene Science, Cambridge, MA). Cytoplasmic RNA was isolated and analyzed as previously described (11) using either the HindIII-BamHI fragment of the pGlu-p27H or the c-myc vector (5) as probe.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently Scott and coworkers (17) noted an increase
in p27 protein levels after 24 h of anti-IgM treatment of WEHI
231 cells. We first sought to determine whether CD40L-mediated rescue
of WEHI 231 cell apoptosis in response to anti-IgM treatment
(10, 11) ablates the increase in p27 expression.
Immunoblotting for p27 expression was performed on total cellular
proteins extracted from exponentially growing WEHI 231 cells treated
with anti-IgM alone or costimulated in the presence of CD40L for up
to 24 h. As a control to verify the effectiveness of the
anti-IgM treatment, c-Myc protein levels were similarly analyzed.
Treatment with anti-IgM alone for 24 h led to the expected
induction in p27 protein level and drop in c-Myc (Fig. 1
). In contrast, in cells cotreated with
CD40L plus anti-IgM, an initial drop occurred in the level of p27
protein below baseline values, after which there was a return by
24 h to essentially the level seen in untreated cells. CD40L plus
anti-IgM caused an initial increase in c-Myc levels that was
maintained for 12 h, after which there was a return to essentially
baseline values by 24 h, which is consistent with our previous
observations (11). Treatment with CD40L alone had little
effect on overall levels of either protein (data not shown). Thus, the
induction of cdk inhibitor p27 levels is ablated during
CD40L-induced rescue of WEHI 231 cells from receptor-mediated
apoptosis. Interestingly, the changes in p27 expression varied
inversely with the pattern of c-Myc expression.
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To determine whether apoptosis resulting from the drop in
c-Myc might be mediated via a p27-signaling pathway, a microinjection
strategy was employed, as described previously (19). WEHI
231 cells were microinjected in duplicate with
pOPRSVI-as-c-myc, a vector expressing c-myc
antisense transcripts, in the absence or presence of an increasing dose
of pOPRSVI-as-p27, a plasmid yielding expression of p27 transcripts in
an antisense orientation. Alternatively, to control for nonspecific
effects of the antisense p27 plasmid backbone, cells were microinjected
with the antisense c-myc vector and the parent vector of the
as-p27 construct (pOPRSVICAT). Furthermore, as additional controls,
cells were microinjected with a pBluescript vector DNA or were not
microinjected at all. After 20 h, cultures were analyzed for cell
viability via trypan blue staining or for apoptosis using propidium
iodide staining. Antisense c-myc vector induced death in
33.2 ± 8.0% of cells (Fig. 2A). The presence of condensed
chromatin upon staining with propidium iodide confirmed death was due
to apoptosis (data not shown; see below). Upon comicroinjection of
pOPRSVI-as-p27, a significant dose-dependent decrease in the extent of
cell death was noted with only 10.9 ± 4.1% dead cells detected
at the highest amount (p < 0.05) (Fig. 2A). In contrast, comicroinjection of a similar quantity of
the pOPRSVICAT parental DNA had essentially no effect. As expected,
only low levels of dead cells were seen in nonmicroinjected cells
(2.5 ± 1.3%) or in cells microinjected with pBluescript DNA
(4.0 ± 2.4%). Furthermore, similar protection was observed upon
comicroinjection of an affinity-purified anti-p27 Ab, which could
be ablated with the cognate peptide (Fig. 2B). Thus,
inhibition of the induction of p27 provides significant protection
against antisense c-Myc-induced apoptosis, indicating a functional role
for the p27 gene in control of survival of WEHI 231 cells mediated by
the drop in c-Myc.
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To directly investigate the potential role of p27 protein in
mediating signals leading to apoptosis, the isopropyl
ß-D-thiogalactopyranoside (IPTG)-inducible pOPRSVI
LacSwitch two-vector system was employed (18). Cells were
transfected sequentially with the p3'SS eukaryotic vector carrying the
lacI gene expressing the lac repressor and then with
pOPRSVI-p27 DNA encoding the full-length p27 protein under the control
of the lac repressor. Expression of the lac repressor was confirmed
by immunoblotting of cell extracts (data not shown). Immunoblotting of
total proteins extracted at 24 h after 20 mM IPTG treatment
confirmed a 2.0-fold induction of p27 protein in the stable pOPRSVI-p27
transfectants, which is consistent with the 1.9-fold increase seen
following anti-IgM treatment (Fig. 1
), but not in the control p3'SS
cells (Fig. 3A). Treatment of
pOPRSVI-p27 transfectants with IPTG led to a significant increase in
cell death, as judged by DNA ladder fragmentation assays (Fig. 3B) and quantified using trypan blue exclusion assays (Fig. 3C). Induction of p27 resulted in death of 30% of cells
in the mixed population of WEHI 231 pOPRSVI-p27 transfectants. As
expected, IPTG had little effect on the survival of cells containing
only the p3'SS vector expressing the lac repressor (Fig. 3, and data
not shown). Thus, induction of p27 is sufficient to promote apoptosis
of WEHI 231 cells.
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To confirm the role of the drop in c-Myc expression on p27
expression and apoptosis of WEHI 231 cells, a stable population of
cells containing the IPTG-inducible antisense c-myc
expression vector pOPRSVI-as-c-myc was similarly isolated.
RNA and proteins were prepared 0, 14, and 24 h after addition of
20 mM IPTG. These were used in Northern blot (Fig. 4A) and immunoblot analyses
(Fig. 4B) and confirmed the ability of the antisense RNA to
effectively inhibit c-myc RNA and protein expression,
respectively. We next evaluated the effects of IPTG stimulation on
levels of p27 and cell death. An increase in p27 levels was detected
within 14 h of IPTG treatment and was even more pronounced at
24 h (Fig. 4C). In contrast, no increase was noted in
the levels of the p21 cdk inhibitor (Fig. 4D). Thus, the
drop in c-myc expression is sufficient to lead to a
selective increase in p27 protein levels. We next assessed the effects
of the IPTG treatment on cell survival using DNA fragmentation to
verify apoptosis and trypan blue staining to quantify the extent of
cell death. The pOPRSVI-as-c-myc and control p3'SS cells
were incubated in the absence or presence of IPTG for 24 h and DNA
was assessed for fragmentation (Fig. 5A). Inhibition of
c-myc expression in WEHI 231 cells resulted in extensive DNA
laddering, whereas no effect was seen in the control cells. As judged
by trypan blue staining, IPTG induction of c-myc antisense
transcripts led to a time-dependent loss in cell viability (Fig. 5B). By 14 h, 25% of cells were trypan blue
positive and the numbers increased to >40% within 40 h.
Individual clones were isolated by limiting dilution and two of these,
pYA2 and pYA7, were subjected to a similar analysis. Even higher levels
of cell death were observed after IPTG induction of c-myc
antisense RNA in pYA2 and pYA7 cells (Fig. 6). Furthermore, extensive cell death was
confirmed using the Non-Radioactive Cell Proliferation assay, i.e.,
51% of pYA2 cells lost viability after 15 h of IPTG treatment.
Thus, consistent with the microinjection analysis, decreased c-Myc
expression in WEHI 231 cells leads to increased levels of p27 and
to apoptosis.
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As noted above, the drop in c-Myc, which resulted in increased p27
expression, did not cause any detectable increase in the levels of the
p53-regulated p21 protein. To determine whether the inhibition of the
p53 pathway affects signaling via c-Myc and p27, we used a WEHI 231
cell transfectant expressing a temperature-sensitive dominant-negative
p53 protein, termed p53#11. This clone was isolated following
cotransfection of WEHI 231 cells with the p53-expression plasmid
pLTRp53cGVal135 and a pSV2neo plasmid, for selection, as described
previously (18). In these cells, growth at 38.5°C
results in expression of a dominant-negative form of p53 protein, which
is unable to signal activation of p21 protein after BCR engagement
(18). WEHI 231 p53#11 or control WEHI 231 cells
transfected with the pSV2neo plasmid alone were treated with
anti-IgM for 0, 2, 4, 8, 10, 12, or 24 h (Fig. 7). Inhibition of p53 signaling failed to
ablate either the drop in c-Myc or the increase in expression of p27
protein. As expected, the p53#11 line contains extremely high levels of
p53 protein, which fail to show the normal changes following BCR
engagement (Fig. 7). As expected, the normal increase in p21 protein
was totally ablated in the p53#11 line incubated with anti-IgM at
38.5°C (Ref. 18 , and data not shown). Thus, BCR-mediated
changes in c-Myc and p27 levels appear independent of p53
signaling.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously we demonstrated that anti-IgM treatment of WEHI 231
cells results in two signaling pathways that contribute approximately
equally in promoting cell death. One pathway is mediated via an
increase in expression of p53 and p21 (18), and the other
involves a decline in the constitutive NF-
B/Rel activity that leads
directly to the drop in c-Myc level (5, 9, 19). Taken
together with the results presented in this paper, we propose that this
latter pathway leading to cell death can now be extended as follows:
anti-IgM 
decreased NF-B/Rel decreased c-Myc
increased p27 G1 arrest apoptosis.
Furthermore, the results suggest that signaling via regulation of p27
levels represents another mechanism for the observed effects of the Rel
family of transcription factors in promoting cell proliferation and
cell survival (20).
Although not universal, several studies have recently shown that induction of p27 promotes apoptosis in several cell types. For example, adenoviral-mediated induction of p27 led to increased cell death of human carcinoma, melanoma, lung fibroblast, breast cancer, and HeLa cells (21, 22). Furthermore, induction of apoptosis of skin keratinocytes upon treatment with TGF-ß1 was found to be accompanied by a drop in level of c-myc and by induction of p27 expression (23). Thus, these findings suggest that enhanced cell survival may also contribute to the large mouse phenotype of the p27 knockout animals (24); however, it should be noted that cells isolated from p27-/- mice displayed increased apoptosis upon growth factor deprivation (25).
The observation that apoptosis of WEHI 231 cells is induced upon
inhibition of c-Myc expression confirms and extends previous work from
several groups. We have shown that ectopic expression of
c-myc protects WEHI 231 cells from anti-IgM-mediated
apoptosis and that inhibition of c-Myc function upon expression of Mad1
leads to apoptosis (5). Scott and coworkers
(26) and Krieg and coworkers (27) have found
that receptor-mediated apoptosis of WEHI 231 cells can be ablated upon
treatment with CpG-containing oligonucleotides, which induce expression
of NF-B/Rel and lead to maintenance of c-Myc. TGF-ß1-mediated
apoptosis of WEHI 231 or CH33 B cells, which occurs after a drop in
c-myc expression, can similarly be rescued by ectopic c-Myc
expression (28). In contrast, however, Hagiyama et al.
(29) have recently presented evidence for overexpression
of a c-Myc/estrogen receptor (ER) fusion protein promoting apoptosis
upon anti-IgM treatment. It is unlikely that a simply
physiologically relevant increase in c-Myc expression can result in
apoptosis because cotreatment with CD40L and anti-IgM or TGF-ß1
induces a very large increase in expression of c-Myc but protects WEHI
231 cells from apoptosis (11, 28). However, levels of
induction with ER vectors are often extremely high, and unfortunately
no measurements of the functional c-Myc protein expressed before or
after anti-IgM treatment were reported by Hagiyama et al.
(29). Differences in WEHI 231 cell lines used in these
studies are also apparent because in Hagiyama et al.’s
(29) experiments, essentially no cell death was detected
even after 24 h of incubation with F(ab')2
fragments, whereas extensive cell death is normally detected within
24 h of treatment with either F(ab')2
(11) or anti-IgM (4, 5, 11). Lastly, one
cautionary note needs to be raised about the use of the Myc/ER vector
because several groups have now reported evidence that some of the
apparent functions of the Myc/ER are due to the ER moiety alone
(12, 30). Thus, experiments to verify the absence of any
effect on apoptosis in WEHI 231 cells of a control vector expressing ER
alone are needed.
To date, several posttranscriptional sites of regulation of p27 activity have been observed, e.g., at the levels of protein stability (31, 32) and protein sequestration (33). Furthermore, increased p27 steady-state mRNA levels were demonstrated upon dexamethasone-induced arrest of normal human blood lymphocyte proliferation (34). Analysis of the sequence of the TATA-less p27-promoter DNA sequence (35) has revealed the presence of a putative initiator-like element at the start site: CCAGACC (where +1 is underlined), as defined by Lo and Smale (36). Interestingly, transcription of many genes containing initiator elements can be repressed by c-Myc (37). Thus, the findings presented in this paper suggest the intriguing possibility that p27 is a novel c-Myc target gene within a signaling pathway induced by BCR engagement, and that derepression of its expression leads to growth arrest and apoptosis. Work is in progress to test this hypothesis.
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Acknowledgments |
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Footnotes |
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2 M.W., R.E.B., and J.S. have contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Gail E. Sonenshein, Department of Biochemistry, Boston University Medical School, 715 Albany Street, Boston, MA 02118. E-mail address:
4 Abbreviations used in this paper: BCR, B cell receptor; CAT, chloramphenicol acetyltransferase; CD40L, CD40 ligand; cdk, cyclin-dependent kinase; IPTG, isopropyl ß-D-thiogalactopyranoside; ER, estrogen receptor.
Received for publication August 24, 1999. Accepted for publication October 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/Rel and c-myc expression during CD40 ligand rescue of WEHI 231 early B cells from receptor-mediated apoptosis through modulation of IB proteins. J. Immunol. 157:81.[Abstract]
B in the survival of hematopoietic cells mediated by interleukin 3 and the oncogenic TEL/platelet-derived growth factor receptor ß fusion protein. Proc. Natl. Acad. Sci. USA 95:8081.B/c-myc-dependent survival pathway is targeted by corticosteroids in immature thymocytes. J. Immunol. 162:314.B/Rel induces apoptosis of murine B cells. EMBO J. 15:4682.[Medline]
B transcription factors and the control of apoptosis. Semin. Cancer Biol. 8:113.[Medline]
B/Rel activity inducing apoptosis of B cells: transcriptional activation of IB
. Immunity 5:31.[Medline]
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) and p3'SS-transfected (
) WEHI
231 cells were treated, in duplicate, with 20 mM IPTG for 0, 14, 24, or
40 h, and induction of cell death was determined by trypan blue
staining. The statistical significance was obtained using the Student
t test (p < 0.05), and the data are
plotted as the mean percentage of cells stained positive for trypan
blue ± SD.
) or 20 mM IPTG (