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Department of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center [F. C., D. C., S. A. K., M. L.] and Section of Urology [M. G.], Program in Cellular and Molecular Biology [M. L.], University of Michigan Medical School, Ann Arbor, Michigan 48109-0396
Abstract
In this study, we explored what effect inhibitors of the 26S proteasome have on cell cycle distribution and induction of apoptosis in human skin fibroblasts and colon cancer cells differing in their p53 status. We found that proteasome inhibition resulted in nuclear accumulation of p53. This was surprising because it is thought that the degradation of p53 is mediated by cytoplasmic 26S proteasomes. Nuclear accumulation of p53 was accompanied by the induction of both p21WAF1 mRNA and protein as well as a decrease in cells entering S phase. Interestingly, cells with compromised p53 function showed a marked increase in the proportion of cells in the G2-M phase of the cell cycle and an attenuated induction of apoptosis after proteasome inhibition. Taken together, our results suggest that proteasome inhibition results in nuclear accumulation of p53 and a p53-stimulated induction of both G1 arrest and apoptosis.
Introduction
The ubiquitin-dependent protein degradation pathway involving the 26S proteasome plays an important role in the regulation of various cellular processes such as cell cycle progression, cell differentiation, signal transduction, stress responses, and apoptosis (1 , 2) . The orderly progression through the cell cycle is orchestrated by a tightly regulated ubiquitin-mediated proteolysis of both cell cycle inhibitors and cyclins (3) . Thus, inhibition of the proteasome-mediated pathway may influence cell cycle progression as well as many other cellular functions.
Under normal conditions, the p53 tumor suppressor protein is rapidly degraded by the 26S proteasome (4, 5, 6, 7) in a process mediated by MDM2 (8, 9, 10) and jun kinase (11) . It has been reported that nuclear export may be required for the efficient degradation of p53 (12, 13, 14) , suggesting that cytoplasmic but not nuclear proteasomes are responsible for the degradation of p53. After cellular stresses, such as exposure to DNA-damaging agents, the half-life of the p53 protein is significantly increased, and p53 accumulates in the nucleus of treated cells (15 , 16) . The mechanism by which the stability of p53 is increased after cellular stress is not fully understood, but it is likely that modifications of the p53 protein itself are involved (7) . Phosphorylation of Ser15, Ser20, and Ser37 has been suggested to result in attenuated interaction between p53 and the negative regulator MDM2 (17, 18, 19, 20) . Interference with components of the degradation pathway, such as phosphorylation of MDM2 (21) , could also result in increased stability of p53. Furthermore, blockage of nuclear export of MDM2 by the ARF protein (22) or by treatment with leptomycin B, an inhibitor of the CRM1-dependent nuclear export machinery (12, 13, 14) , leads to the accumulation of p53 in the nucleus.
Inhibition of the 26S proteasome results in the rapid accumulation of p53 (4 , 5 , 23) and of p53-inducible gene products such as p21WAF1, MDM2, and Bax (5 , 23, 24, 25) . In addition, certain cell types have been shown to undergo apoptosis after treatment with proteasome inhibitors (23 , 26 , 27) through a process that has been suggested to be p53 dependent (24) . Thus, accumulation of p53 by default by simply inhibiting its degradation appears to activate downstream events. However, the induction of p21WAF1, MDM2, and Bax by proteasome inhibition may not be entirely dependent on p53-mediated transactivation (18 , 25 , 28) . Because these proteins are normally subjected to regulation by proteasome-mediated degradation, it is possible that the accumulation observed was due to their increased half-lives in the absence of proteasome-mediated degradation (25 , 29) . Furthermore, recent studies have questioned the requirement of p53 in the induction of apoptosis resulting from inhibition of proteasome function (30 , 31) .
To address some of these controversies, we examined the role of p53 in the induction of p21WAF1, cell cycle arrest, and induction of apoptosis after proteasome inhibition using a panel of cell lines differing in their p53 status. We show that p53 actually accumulated in the cell nucleus rather than the cytoplasm after proteasome inhibition. Furthermore, proteasome inhibition resulted in the p53-dependent induction of p21WAF1, G1 arrest, and apoptosis, whereas a proteasome-mediated arrest in G2-M phase was revealed in cells with compromised p53 function.
Results
Nuclear Accumulation of p53 in Cells Treated with Lactacystin.
Under nonstressed conditions, p53 is thought to be actively
translocated from the nucleus to the cytoplasm, where it is degraded by
the 26S proteasome (12, 13, 14)
. After drug-induced inhibition
of the proteasome, p53 would be expected to accumulate in the
cytoplasm, as suggested previously by transient transfection
experiments using p53-expressing vectors (32)
. To explore
the localization of p53 after proteasome inhibition, we treated diploid
human fibroblasts with the proteasome-specific inhibitor lactacystin,
followed by immunohistochemistry with various anti-p53 antibodies.
Using fluorescence microscopy it was found that untreated fibroblasts
showed only a faint staining (Fig. 1A
), whereas cells irradiated with 30 J/m2 UVC light presented
strong nuclear staining of p53 (Fig. 1B
). Interestingly,
lactacystin treatment for 6 h led to a strong nuclear accumulation
of p53 (Fig. 1, C–F
). At x100 magnification, using either
fluorescence (Fig. 1D
) or scanning confocal fluorescence
microscopy (Fig. 1, E and F
), it can be seen that
p53 appears to accumulate in hundreds of foci throughout the nucleus,
with some areas devoid of staining. Similar nuclear accumulation of p53
was observed with the specific 26S proteasome inhibitor MG132 (data not
shown). We conclude that although p53 is thought to be exported to
cytoplasmic proteasomes, inhibition of proteasome activity results in
nuclear accumulation of p53.
|
|
Here we show that the basal level of p53 was significantly lower in the
E6-expressing cells (RKO-E6) than in the parental RKO cell line (Fig. 2A
). LLnL and lactacystin induced p53 to high levels in the parental RKO
cell line, whereas the absolute p53 protein levels in the RKO-E6 cells
were not increased as much as in the parental RKO cell line (
). Because the untreated RKO-E6 cells had such a low basal
level of p53, the relative induction of p53 protein by proteasome
inhibition may rival that of the parental cells. However, this
quantification could not be done accurately due to the low p53 level in
the untreated RKO-E6 cells.
We next investigated the cellular levels of the p53-inducible
cyclin-dependent kinase inhibitor p21WAF1 and the ability
of LLnL to stimulate its expression. The basal level of
p21WAF1 was markedly lower in the RKO-E6 cells than in the
RKO cells (Fig. 2B
). Treatment of the cells with LLnL
strongly induced p21WAF1 expression in the parental RKO
cells, whereas we observed only a slight induction of
p21WAF1 in the E6-expressing cells. As for the accumulation
of p53, the relative induction of p21WAF1 by LLnL may be
similar between the two cell lines, whereas the absolute protein levels
were much higher in the parental RKO cells.
We then assessed the effect of lactacystin on the cellular levels of
p53 and p21WAF1 in primary human fibroblasts derived from a
normal individual (NFs) or from a Li-LFS patient lacking functional p53
expression. Lactacystin strongly induced p53 and p21WAF1 in
NF cells, whereas no expression was seen in the LFS cells either with
or without lactacystin (Fig. 2C
). The mutant p53
gene in the LFS cells has a frameshift mutation that leads to a
truncated p53 protein that is very unstable in the cells
(39)
. Finally, in a cell line overexpressing mutant p53
(HT29), which exhibits a significant baseline level of
p21WAF1 expression, lactacystin did not induce a
significant increase in p21WAF1 protein levels (Fig. 2D
).
To examine whether the accumulation of p21WAF1 by
proteasome inhibition was due to up-regulation of the
p21WAF1 gene, we performed Northern blots with
p21WAF1-specific probes (Fig. 2E
). Although
lactacystin caused a slight up-regulation of the
p21WAF1 gene as reported previously
(5)
, it clearly did not fully account for the dramatic
increase of the protein level of p21WAF1 observed in these
cells. Taken together, these results suggest that both protein
stabilization and, to some extent, transcriptional up-regulation are
responsible for the accumulation of p21WAF1 by proteasome
inhibitors.
Modest p53-dependent G1 Arrest after Proteasome
Inhibition.
Because the RKO and RKO-E6 cells showed a differential expression of
p53 and p21WAF1 after exposure to LLnL (Fig. 2, A and B
), these cells were used to explore the
role of p53 in cell cycle regulation after exposure to LLnL. The RKO
and RKO-E6 cell lines were incubated with LLnL for 6 or 24 h,
followed by a 15-min BrdUrd incubation to specifically label nascent
DNA synthesis. Cells were then fixed and stained for DNA content, and
anti-BrdUrd antibodies and secondary antibodies conjugated with FITC
were used to identify cells synthesizing DNA at the time of labeling.
Using two-parameter flow cytometry, the cell cycle effects of LLnL
incubation were then analyzed.
It was expected that LLnL would induce a G1 arrest in the
parental RKO cells because this treatment resulted in a significant
accumulation of p21WAF1 (Fig. 2B
). Using
oneparameter flow cytometry of PI-stained RKO, RKO-E6,
and mutant p53 RKO-M cells (Fig. 3A
), no obvious G1 arrest was observed in the cell lines tested.
However, when using two-parameter flow cytometry of cells pulse-labeled
with BrdUrd, we observed a marked difference between parental RKO and
RKO-E6 cells in the G1-S-phase compartment of the cell
cycle (Fig. 3B
; Table 1
). Whereas the RKO-E6 cells were stimulated to enter S phase after 6 h of treatment, the RKO cells were not. In fact, 24 h of LLnL
treatment led to a visible decline in RKO cells occupying the S-phase
compartment. At this time the number of parental RKO cells synthesizing
DNA had dropped from 45% to 19% (Table 1)
. Thus, compared with the
RKO-E6 cells, the parental RKO cells appeared to be blocked from
entering the S phase, perhaps as a result of the strong
induction of p21WAF1 in these cells (Fig. 2B
).
|
|
. Thus, not only were
fewer parental RKO cells entering S phase, the cells that entered did
synthesize DNA less efficiently. By multiplying the mean FITC signal
with the number of BrdUrd-positive cells, we obtained an estimation of
the relative amount of DNA synthesis in the different cell populations.
LLnL did not affect DNA synthesis negatively during the first 6 h
of incubation in either cell line (Table 1)
. However, after 24 h
of LLnL incubation, the DNA synthesis in parental RKO cells was reduced
to 26% of the level found in untreated control cells. In contrast, the
LLnL-treated RKO-E6 cells synthesized DNA at a rate comparable with
that of untreated control cells (Table 1)
.
Proteasome Inhibition Results in a G2-M-phase Arrest in
p53-compromised Cells.
Whereas LLnL treatment led to a modest G1 arrest in the
wild-type p53-expressing RKO cells, the RKO-M and RKO-E6 cells showed a
significant accumulation in the G2-M phase of the cell
cycle (Fig. 3A
). The percentages of cells in
G2-M phase increased from 15% in untreated RKO-E6 cells to
27% and 49% after 6 and 24 h of drug treatment, respectively.
During the same time period, the population of cells in G1
was severely diminished.
Using HT29 colon cancer cells, which harbor mutant p53, we also
observed an accumulation of cells in the G2-M phase of the
cell cycle after proteasome inhibition. Treatment with 10
µM lactacystin increased the percentage of cells in
G2-M phase from 9% in untreated cells to 27% in treated
cells (Fig. 3C
). We conclude that LLnL and lactacystin
induce an accumulation of p53-deficient cells in the G2-M
phase of the cell cycle, whereas cells with wild-type p53 do not reveal
a G2-M phase arrest, presumably because these cells arrest
in G1 instead.
Wild-Type p53 Sensitizes Cells to Proteasome Inhibitor-induced
Apoptosis.
Previous studies have shown that treatment of human and mouse cancer
cells with proteasome inhibitors induces apoptosis (26
, 27 , 30
, 31)
. Furthermore, induction of apoptosis by proteasome
inhibitors appears to be p53 dependent in some cell types
(24)
but not in others (30
, 31)
and may be
selective for transformed cells (31)
. Here we investigated
the effect of LLnL on the induction of apoptosis in the parental RKO
cells and compared it with the effect on the E6-expressing cells. Cells
were incubated for 48 h in the presence of 30 µM
LLnL before both floating and attached cells were collected, fixed, and
stained with PI. Flow cytometric analysis of the percentage of cells
containing sub-G1 DNA content as a measure of apoptosis
revealed that LLnL induced apoptosis in both cell types (Fig. 4)
. However, the induction of apoptosis was significantly higher
(P < 0.05) in the parental RKO cells (58%) as
compared with the RKO-E6 cells (38%). Thus, induction of apoptosis by
LLnL in these human colon cancer cells did not require wild-type p53
function. However, wild-type p53 significantly enhanced LLnL-induced
apoptosis.
|
). These results are in agreement with the preferential induction of
apoptosis in the wild-type p53-expressing RKO cells after LLnL
treatment.
|
), supporting the findings obtained in the RKO-E6
cells after LLnL treatment (Fig. 3A
). Discussion
In this study, we investigated the role of p53 in mediating cellular responses after inhibition of the 26S proteasomes. We found that proteasome inhibition resulted in a significant accumulation of p53 in the nucleus of treated cells. This finding was surprising, considering that p53 is thought to be degraded primarily in the cytoplasm and not in the cell nucleus (12, 13, 14) . One hypothesis for the nuclear accumulation of p53 after proteasome inhibition is that p53 proteins targeted for degradation by cytoplasmic proteasomes may be "recycled" and recruited back into the nucleus. Increased concentration of nuclear p53 would favor p53 tetramerization, which would block nuclear export (14) and further augment nuclear accumulation. Alternatively, p53 may, under normal conditions, be degraded by nuclear proteasomes. However, this scenario is less plausible because p53 has been shown to accumulate in the cell nucleus after overexpression of wild-type p53 (40) or inhibition of the nuclear export machinery with leptomycin B (12, 13, 14) . Finally, it is possible that the nuclear accumulation of p53 by proteasome inhibition is due to a direct or an indirect inhibition of a proteasome-mediated step involved in nuclear export. Additional studies are needed to elucidate the true mechanism by which proteasome inhibition leads to nuclear accumulation of p53.
Both lactacystin and LLnL strongly induced the accumulation of
wild-type p53 in the parental RKO cell line and in NFs. In contrast,
only a marginal increase or no increase in the absolute p53 protein
level was observed in the E6expressing cells, the LFS cells, or
the HT29 cells containing mutant p53. Furthermore, induction of
p21WAF1 by proteasome inhibition was seen only in the cell
cultures in which p53 accumulated, suggesting that the induction of
p21WAF1 may have been, at least in part, p53 dependent.
This finding is similar to those in studies in which forced expression
of exogenous p53 in cells (41)
or treatment of cells with
the nuclear export inhibitor leptomycin B (12
, 28) resulted in induction of p21WAF1 expression. However, the
induction of p21WAF1 after proteasome inhibition could only
partially be explained by an up-regulation of the
p21WAF1 gene (Fig. 2E
). Thus, the
accumulation of p21WAF1 protein cannot be entirely
explained by p53-mediated transactivation but may also result from
increased stability of the p21WAF1 protein in the absence
of proteasome degradation (5)
. Although p53 isolated from
proteasome inhibitor-treated cells has been reported to be fully
capable of binding to oligonucleotides containing
p21WAF1 promoter sequences (18)
,
some studies suggest that these p53 proteins are fairly poor in
transactivating either endogenous (18)
or exogenous
p21WAF1 promoters (28)
. The limited
transactivation activity of p53 after proteasome inhibition may result
from conformational misfolding of nascent p53 in the presence of
proteasome inhibitors (32)
. Alternatively, the relatively
low transactivation activity of drug-accumulated p53 may be due to the
lack of protein modifications, such as phosphorylation or acetylation
(19
, 42) , which are thought to stimulate the
transactivation function of p53 (43)
.
Because the stability of p21WAF1 is increased after
proteasome inhibition, it is expected that p21WAF1 levels
should increase, even in cells with compromised p53 function. However,
the rather small increase of p21WAF1 protein levels in
LLnL-treated RKO-E6 cells and the absence of such an increase in the
LFS cells may have been related to the rather low basal expression of
this protein due to the absence of functional p53. However, our result
using the p53 mutant cell line HT29 shows that the detectable basal
level of p21WAF1 did not increase after incubation with
lactacystin (Fig. 2D
). This would argue against protein
stabilization as the major mechanism of p21WAF1 induction
after proteasome inhibition. The marginal induction of p53 expression
after treatment with proteasome inhibitors in the E6-expressing cells
is in disagreement with a previous study in which E6-transfected normal
fibroblasts were shown to readily accumulate p53 after incubation with
the proteasome inhibitor MG132 (5)
. The reason for this
discrepancy is not clear, but it may be that the RKO-E6 cell line used
in our study expressed the E6 protein to a higher level than the
E6-transfected fibroblasts. Whereas Maki et al.
(5)
readily detected p53 in the untreated E6-expressing
fibroblasts used in their study, no p53 protein expression was detected
in the RKO-E6 cells (Fig. 2A
; Refs. 37
and 38
).
Whereas wild-type p53-expressing RKO cells tended to arrest in
G1 after treatment with LLnL, RKO-E6 cells appeared to be
stimulated to enter S phase (Fig. 2B
; Table 1
). This finding
that LLnL blocked cells from entering S phase in wild-type p53 cells is
in agreement with a previous study (44)
. The most likely
explanation for this G1 arrest is that the accumulation of
the cyclin-dependent kinase inhibitor p21WAF1 after LLnL
treatment blocked the G1 to S-phase transition of these
cells. The mechanism responsible for the increased S-phase entry of the
RKO-E6 cells after LLnL treatment is not clear, but it may involve
accumulation of some cellular component(s) driving the progression of
cells from G1 into S phase. Such components may include
cyclin D1, cyclin E, and/or the transcription factor E2F1, which are
normally subjected to degradation by the ubiquitin-proteasome pathway
(3
, 25 , 45
, 46)
.
A profound arrest in the G2-M phase of the cell cycle was
found for cells with compromised p53 status but not in the wild-type
p53-expressing cells after treatment with either LLnL or lactacystin
(Figs. 3
and 5)
. One explanation for these results is that inhibition
of proteasome activity causes a G2-M-phase arrest that is
only revealed in cells lacking wild-type p53. Because the entry of
wild-type p53-expressing cells into S phase was reduced after drug
treatment, a smaller percentage of cells would be expected to reach the
G2-M-phase compartment of the cell cycle. The mechanism for
the G2-M-phase arrest in the p53-compromised cells after
proteasome inhibition may be due to the inability of the cells to exit
mitosis. The exit of the M phase requires proteasome activity to
degrade the targets of the anaphase promoting complex such as cyclins A
and B (3)
. An alternative hypothesis for the lack of
G2-M-phase arrest in the wild-type p53-expressing cells
could be that the exit from the G2-M phase is stimulated by
wild-type p53 expression. This hypothesis is supported by findings that
p53 may play a role in the exit from DNA damage-induced G2
arrest (47)
.
The role of p53 in the induction of apoptosis by proteasome inhibitors
is controversial (24
, 30
, 31)
. Proteasome inhibitors have
been shown to induce apoptosis in a number of human cell lines
(26
, 27
, 30
, 31)
. However, the induction of apoptosis in
sympathetic neurons and thymocyte cultures has been shown to be
inhibited by proteasome inhibitors (48
, 49)
. Our results
suggest that wild-type p53 function is not required for the induction
of apoptosis by LLnL or lactacystin in the cells used in this study
(Figs. 4
and 5)
. However, wild-type p53 function clearly stimulated the
induction of apoptosis after proteasome inhibition. It has been shown
that certain apoptosis-promoting proteins such as Bax are subjected to
proteasome-mediated degradation (25)
. It is thus possible
that inhibition of proteasome-mediated degradation of Bax together with
increased expression of Bax in the presence of high levels of p53 may
explain the stronger induction of apoptosis in the wild-type
p53-expressing cells. It is also possible that the potential
p53-stimulated exit from the G2-M phase of the cell cycle
may enhance apoptosis, as has been suggested previously
(47)
.
In conclusion, we have found that proteasome inhibition leads to nuclear accumulation of p53, a p53-stimulated induction of p21WAF1, and cell cycle arrest in the G1 phase of the cell cycle. Furthermore, cells with compromised p53 function accumulated in the G2-M phase of the cell cycle and were somewhat protected against the induction of apoptosis by proteasome inhibition. Because proteasome inhibitors are potentially useful as antitumor agents (50 , 51) , the true mechanisms of how these agents induce apoptosis and how p53 modifies these events warrants further exploration.
Materials and Methods
Cell Culture and Chemicals.
Three isogenic human colon cancer cell lines, the parental cell line
RKO, the mutant p53-expressing cell line RKO-M, and the human
papillomavirus 16 E6-expressing cell line RKO-E6, were generously given
to us by Dr. Albert Fornace, Jr., (National Cancer Institute, NIH,
Bethesda, MD) via Drs. Ted Lawrence and Mary Davis (University of
Michigan). These cell lines have been described in detail previously
(37
, 52
, 53)
. The human colon carcinoma cell line HT29
(mutant p53) and a normal diploid skin fibroblast cell strain were
generously provided to us by Drs. Ted Lawrence and Mary Davis
(University of Michigan). Finally, the spontaneously immortalized LFS
fibroblast cell strain MDAH041, which harbors a single mutant copy of
the p53 gene (p53 -/mt) was a generous gift from Michael
Tainsky (Wayne State University, Detroit, MI). Cells were grown
as monolayers in MEM (Life Technologies, Inc.) supplemented with 10%
fetal bovine serum, 1 x vitamins, 1 x amino acids, and
1 x antibiotics. Subconfluent cells seeded 2 days before the
experiments were treated with LLnL (Sigma), which is a reversible
inhibitor of the chymotryptic site on the proteasome (33)
,
and the irreversible proteasome inhibitor lactacystin (Ref. 33
;
Oncogene Research Products). Both drugs were dissolved in DMSO.
Immunocytochemistry.
After incubation with lactacystin or UV irradiation, cells grown on
coverslips were fixed in 100% methanol and stored at -20°C for
1 h. The coverslips were then rinsed in PBS, followed by the
addition of 100 µl of undiluted supernatant from hybridoma cells
expressing mouse anti-p53 mAb 1801 (a gift from Dr. Jiayuh Lin,
University of Michigan). After a 1-h incubation, the antibody solution
was aspirated, and the coverslips were incubated on a rocker platform
three times for 5 min with PBSBT. The coverslips were then incubated
for 1 h in the dark with 100 µl of a secondary FITC-conjugated
antimouse IgG antibody (Sigma; 1:1000 dilution in PBSBT), followed by
aspiration and incubation three times for 5 min in PBSBT. The
coverslips were then mounted on microscope slides in one drop of
Vectashield (Vector Laboratories, Inc.) and viewed and photographed
using a fluorescence microscope (Nikon Eclipse E600) or a confocal
microscope (MRC 600; BioRad/Nikon).
Western Blot.
Western blots were performed as described previously (54)
using 12% SDS-PAGE for analysis of p53 and 15% SDS-PAGE for analysis
of p21WAF1. For the detection of p53 and
p21WAF1 proteins, anti-p53 [Ab-2 (Oncogene Research
Products) and FL-393 (Santa Cruz Biotechnology, Inc.)] and
anti-p21WAF1 (Oncogene Research Products) as well as
horseradish peroxidase-conjugated secondary antibodies (Sigma) were
used together with enhanced chemiluminescence (SuperSignal CL-HRP
Substrate System; Pierce) and X-ray film. The protein content of each
sample was determined by the use of a UV spectrophotometer and equal
protein loading, and transfer was confirmed by staining the nylon
membranes with Coomassie Blue after the completion of the film
exposure.
Northern Blot.
Total RNA was isolated from untreated NFs and NFs treated with 10
µM lactacystin using Trizol (Life Technologies, Inc.).
Equal amounts of RNA (10 µg/lane) were loaded on a 1.2%
agarose/formaldehyde gel, and after electrophoresis, the RNA was
transferred onto Nytran nylon membranes (Schleicher & Schuell). Blots
were hybridized with [
-32P]dCTP (Amersham Pharmacia
Biotech) radiolabelled cDNA probes p21WAF1 and ß-actin.
The cDNA probes for p21WAF1 and ß-actin were generated
using reverse transcription-PCR and isolated through agarose gel
extraction.
BrdUrd Incubation, Cell Fixation, and Flow Cytometry.
Cells were pulse-labeled with 30 mM BrdUrd (Sigma) at
37°C for 15 min as described previously (38)
. The plates
were then rinsed with PBS, and then the cells were collected by
scraping them with a rubber policeman in 2 ml of PBS. After
centrifugation, the cells were fixed by adding ice-cold 70% ethanol
dropwise under vortexing. Staining of BrdUrd-containing DNA was then
performed as described previously (38
, 55)
. The cell
samples were analyzed for BrdUrd content (FITC) and DNA content (PI)
using two-parameter flow cytometry (Coulter Elite ESP Cell Sorter).
Quantification of Apoptosis.
Cells were treated with 30 µM LLnL for 48 h or 10
µM lactacystin for 24 h before cells were harvested
and fixed, and the DNA was stained by PI as described previously
(54
, 56) . Samples were then analyzed using flow cytometry
and cells with sub-G1 DNA content were scored as apoptotic.
Acknowledgments
We thank Dr. Al Fornace and Dr. Ted Lawrence for their kind gifts of the RKO and HT29 cell lines and Michael Tainsky for kindly supplying us with the Li-Fraumeni cell strains used in this study. We also thank the members of the Flow Cytometry Core at the University of Michigan for excellent technical assistance and Dr. Bruce McKay for valuable input into this study.
Footnotes
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 indicate this fact.
1 Supported by Seed Grant 836 from the Michigan
Memorial-Phoenix Project, by a grant from the University of Michigan
Comprehensive Cancer Center’s Institutional Grant from the American
Cancer Society, by NIH Grant CA82376-01, and by start-up funds supplied
by the Department of Radiation Oncology, University of Michigan. 
2 To whom requests for reprints should be
addressed, at the Department of Radiation Oncology, Division of Cancer
Biology, University of Michigan Comprehensive Cancer Center, 4306 CCGC,
1500 East Medical Center Drive, Ann Arbor, MI 48109-0396. E-mail: ljungman{at}umich.edu
3 The abbreviations used are: LLnL,
N-acetyl-L-leucinyl-L-leucinyl-Lnorleucinal;
BrdUrd, bromodeoxyuridine; PI, propidium iodide; LFS, Li-Fraumeni
syndrome; NF, normal fibroblast; PBSBT, 5 g of bovine albumin and
500 µl Tween 20/liter PBS.
Received for publication 9/29/99. Revision received 2/16/00. Accepted for publication 4/ 5/00.
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 L. Chen, L. Smith, Z. Wang, and J. B. Smith Preservation of Caspase-3 Subunits from Degradation Contributes to Apoptosis Evoked by Lactacystin: Any Single Lysine or Lysine Pair of the Small Subunit Is Sufficient for Ubiquitination Mol. Pharmacol., August 1, 2003; 64(2): 334 - 345. [Abstract] [Full Text] [PDF]
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 S. Yanamadala and M. Ljungman Potential Role of MLH1 in the Induction of p53 and Apoptosis by Blocking Transcription on Damaged DNA Templates Mol. Cancer Res., August 1, 2003; 1(10): 747 - 754. [Abstract] [Full Text] [PDF]
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 A. Chauchereau, L. Amazit, M. Quesne, A. Guiochon-Mantel, and E. Milgrom Sumoylation of the Progesterone Receptor and of the Steroid Receptor Coactivator SRC-1 J. Biol. Chem., March 28, 2003; 278(14): 12335 - 12343. [Abstract] [Full Text] [PDF]
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M. Leverkus, M. R. Sprick, T. Wachter, T. Mengling, B. Baumann, E. Serfling, E.-B. Brocker, M. Goebeler, M. Neumann, and H. Walczak Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation Mol. Cell. Biol., February 1, 2003; 23(3): 777 - 790. [Abstract] [Full Text]
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 S. Klibanov, H. O'Hagan, and M Ljungman Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress J. Cell Sci., January 5, 2001; 114(10): 1867 - 1873. [Abstract] [PDF]
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J. S. Isaacs, S.'i. Saito, and L. M. Neckers Requirement for HDM2 Activity in the Rapid Degradation of p53 in Neuroblastoma J. Biol. Chem., May 18, 2001; 276(21): 18497 - 18506. [Abstract] [Full Text] [PDF]
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