Role of p53 in Cell Cycle Regulation and Apoptosis by steepslope9876


									Vol. 11, 239 –246, May 2000                                                                                       Cell Growth & Differentiation   239

Role of p53 in Cell Cycle Regulation and Apoptosis following
Exposure to Proteasome Inhibitors1

Feng Chen, Daniel Chang, Meidee Goh,                                           Under normal conditions, the p53 tumor suppressor pro-
Sergey A. Klibanov, and Mats Ljungman2                                      tein is rapidly degraded by the 26S proteasome (4 –7) in a
Department of Radiation Oncology, Division of Cancer Biology,               process mediated by MDM2 (8 –10) and jun kinase (11). It has
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
                                                                            been reported that nuclear export may be required for the
Molecular Biology [M. L.], University of Michigan Medical School, Ann       efficient degradation of p53 (12–14), suggesting that cyto-
Arbor, Michigan 48109-0396                                                  plasmic 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
In this study, we explored what effect inhibitors of the
26S proteasome have on cell cycle distribution and                          of treated cells (15, 16). The mechanism by which the stability
induction of apoptosis in human skin fibroblasts and                        of p53 is increased after cellular stress is not fully under-
colon cancer cells differing in their p53 status. We                        stood, but it is likely that modifications of the p53 protein
found that proteasome inhibition resulted in nuclear                        itself are involved (7). Phosphorylation of Ser15, Ser20, and
accumulation of p53. This was surprising because it is                      Ser37 has been suggested to result in attenuated interaction
thought that the degradation of p53 is mediated by                          between p53 and the negative regulator MDM2 (17–20). In-
cytoplasmic 26S proteasomes. Nuclear accumulation                           terference with components of the degradation pathway,
of p53 was accompanied by the induction of both                             such as phosphorylation of MDM2 (21), could also result in
p21WAF1 mRNA and protein as well as a decrease in                           increased stability of p53. Furthermore, blockage of nuclear
cells entering S phase. Interestingly, cells with                           export of MDM2 by the ARF protein (22) or by treatment with
compromised p53 function showed a marked increase                           leptomycin B, an inhibitor of the CRM1-dependent nuclear
in the proportion of cells in the G2-M phase of the cell                    export machinery (12–14), leads to the accumulation of p53
cycle and an attenuated induction of apoptosis after                        in the nucleus.
proteasome inhibition. Taken together, our results                             Inhibition of the 26S proteasome results in the rapid
suggest that proteasome inhibition results in nuclear                       accumulation of p53 (4, 5, 23) and of p53-inducible gene
accumulation of p53 and a p53-stimulated induction of                       products such as p21WAF1, MDM2, and Bax (5, 23–25). In
both G1 arrest and apoptosis.                                               addition, certain cell types have been shown to undergo
                                                                            apoptosis after treatment with proteasome inhibitors (23,
Introduction                                                                26, 27) through a process that has been suggested to be
The ubiquitin-dependent protein degradation pathway in-                     p53 dependent (24). Thus, accumulation of p53 by default
volving the 26S proteasome plays an important role in the                   by simply inhibiting its degradation appears to activate
regulation of various cellular processes such as cell cycle                 downstream events. However, the induction of p21WAF1,
progression, cell differentiation, signal transduction, stress              MDM2, and Bax by proteasome inhibition may not be
responses, and apoptosis (1, 2). The orderly progression                    entirely dependent on p53-mediated transactivation (18,
through the cell cycle is orchestrated by a tightly regulated               25, 28). Because these proteins are normally subjected to
ubiquitin-mediated proteolysis of both cell cycle inhibitors                regulation by proteasome-mediated degradation, it is pos-
and cyclins (3). Thus, inhibition of the proteasome-mediated                sible that the accumulation observed was due to their
pathway may influence cell cycle progression as well as
                                                                            increased half-lives in the absence of proteasome-medi-
many other cellular functions.
                                                                            ated 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).
Received 9/29/99; revised 2/16/00; accepted 4/5/00.                            To address some of these controversies, we examined the
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
                                                                            role of p53 in the induction of p21WAF1, cell cycle arrest, and
advertisement in accordance with 18 U.S.C. Section 1734 solely to indi-     induction of apoptosis after proteasome inhibition using a
cate this fact.                                                             panel of cell lines differing in their p53 status. We show that
  Supported by Seed Grant 836 from the Michigan Memorial-Phoenix
Project, by a grant from the University of Michigan Comprehensive Cancer    p53 actually accumulated in the cell nucleus rather than the
Center’s Institutional Grant from the American Cancer Society, by NIH       cytoplasm after proteasome inhibition. Furthermore, protea-
Grant CA82376-01, and by start-up funds supplied by the Department of
Radiation Oncology, University of Michigan.                                 some inhibition resulted in the p53-dependent induction of
  To whom requests for reprints should be addressed, at the Department      p21WAF1, G1 arrest, and apoptosis, whereas a proteasome-
of Radiation Oncology, Division of Cancer Biology, University of Michigan
Comprehensive Cancer Center, 4306 CCGC, 1500 East Medical Center            mediated arrest in G2-M phase was revealed in cells with
Drive, Ann Arbor, MI 48109-0396. E-mail:                compromised p53 function.
240   p53 and Proteasome Inhibitors

                                                                                   though p53 is thought to be exported to cytoplasmic pro-
                                                                                   teasomes, inhibition of proteasome activity results in nuclear
                                                                                   accumulation of p53.
                                                                                      Accumulation of p21WAF1 by LLnL3 or Lactacystin Is
                                                                                   Stimulated by Wild-Type p53. To examine whether the
                                                                                   drug-induced accumulation of p53 resulted in the induction
                                                                                   of p21WAF1 expression, a panel of isogenic cell lines differing
                                                                                   in their functional p53 status was used. First we character-
                                                                                   ized the RKO parental and human papillomavirus 16 E6-
                                                                                   transfected RKO cell lines in terms of their ability to induce
                                                                                   cellular accumulation of p53 and/or p21WAF1 after 6 h of
                                                                                   incubation with the proteasome inhibitors LLnL (30 M) or
                                                                                   lactacystin (10 M). These doses were chosen because they
                                                                                   have been shown to be sufficient to inhibit proteasome ac-
                                                                                   tivity in cells (33, 34). The E6 protein interacts with the cellular
                                                                                   protein E6AP to target the p53 protein for ubiquitylation and
                                                                                   subsequent proteasome-mediated degradation (35, 36). It
                                                                                   has been shown that the E6-mediated degradation in these
                                                                                   cells overrides the p53 stabilization normally seen after
                                                                                   exposure to ionizing radiation, UV light, or actinomycin D
                                                                                   (37, 38).
                                                                                      Here we show that the basal level of p53 was significantly
                                                                                   lower in the E6-expressing cells (RKO-E6) than in the paren-
                                                                                   tal RKO cell line (Fig. 2A). LLnL and lactacystin induced p53
                                                                                   to high levels in the parental RKO cell line, whereas the
      Fig. 1. Exposure of cells to lactacystin resulted in nuclear accumulation    absolute p53 protein levels in the RKO-E6 cells were not
      of p53. Diploid human fibroblasts were mock-treated (A), irradiated with
      30 J/m2 UVC and incubated for 16 h (B) or 6 h with 10 M lactacystin (C–F),
                                                                                   increased as much as in the parental RKO cell line (Fig. 2A).
      followed by fixation and immunocytochemistry with anti-p53 antibodies        Because the untreated RKO-E6 cells had such a low basal
      and FITC-conjugated antimouse antibodies. A–C were photographed at           level of p53, the relative induction of p53 protein by protea-
        40 magnification, and D–F were photographed at 100 magnification.
      Images A–D were obtained using fluorescence microscopy and anti-p53          some inhibition may rival that of the parental cells. However,
      antibody 1801, whereas images E and F were captured digitally using          this quantification could not be done accurately due to the
      scanning confocal microscopy and anti-p53 antibodies AB-1 and FL-393,        low p53 level in the untreated RKO-E6 cells.
                                                                                      We next investigated the cellular levels of the p53-induc-
                                                                                   ible cyclin-dependent kinase inhibitor p21WAF1 and the ability
                                                                                   of LLnL to stimulate its expression. The basal level of
      Results                                                                      p21WAF1 was markedly lower in the RKO-E6 cells than in the
      Nuclear Accumulation of p53 in Cells Treated with Lac-                       RKO cells (Fig. 2B). Treatment of the cells with LLnL strongly
      tacystin. Under nonstressed conditions, p53 is thought to                    induced p21WAF1 expression in the parental RKO cells,
      be actively translocated from the nucleus to the cytoplasm,                  whereas we observed only a slight induction of p21WAF1 in
      where it is degraded by the 26S proteasome (12–14). After                    the E6-expressing cells. As for the accumulation of p53, the
      drug-induced inhibition of the proteasome, p53 would be                      relative induction of p21WAF1 by LLnL may be similar be-
      expected to accumulate in the cytoplasm, as suggested                        tween the two cell lines, whereas the absolute protein levels
      previously by transient transfection experiments using p53-                  were much higher in the parental RKO cells.
      expressing vectors (32). To explore the localization of p53                     We then assessed the effect of lactacystin on the cellular
      after proteasome inhibition, we treated diploid human fibro-                 levels of p53 and p21WAF1 in primary human fibroblasts
      blasts with the proteasome-specific inhibitor lactacystin, fol-              derived from a normal individual (NFs) or from a Li-LFS
      lowed by immunohistochemistry with various anti-p53 anti-                    patient lacking functional p53 expression. Lactacystin
      bodies. Using fluorescence microscopy it was found that                      strongly induced p53 and p21WAF1 in NF cells, whereas no
      untreated fibroblasts showed only a faint staining (Fig. 1A),                expression was seen in the LFS cells either with or without
      whereas cells irradiated with 30 J/m2 UVC light presented                    lactacystin (Fig. 2C). The mutant p53 gene in the LFS cells
      strong nuclear staining of p53 (Fig. 1B). Interestingly, lacta-              has a frameshift mutation that leads to a truncated p53
      cystin treatment for 6 h led to a strong nuclear accumulation                protein that is very unstable in the cells (39). Finally, in a cell
      of p53 (Fig. 1, C–F). At 100 magnification, using either                     line overexpressing mutant p53 (HT29), which exhibits a
      fluorescence (Fig. 1D) or scanning confocal fluorescence                     significant baseline level of p21WAF1 expression, lactacystin
      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 accumu-                    The abbreviations used are: LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-
                                                                                   norleucinal; BrdUrd, bromodeoxyuridine; PI, propidium iodide; LFS, Li-
      lation of p53 was observed with the specific 26S proteasome                  Fraumeni syndrome; NF, normal fibroblast; PBSBT, 5 g of bovine albumin
      inhibitor MG132 (data not shown). We conclude that al-                       and 500 l Tween 20/liter PBS.
                                                                                                                         Cell Growth & Differentiation   241

                                                                                  protein level of p21WAF1 observed in these cells. Taken to-
                                                                                  gether, these results suggest that both protein stabilization
                                                                                  and, to some extent, transcriptional up-regulation are re-
                                                                                  sponsible for the accumulation of p21WAF1 by proteasome
                                                                                     Modest p53-dependent G1 Arrest after Proteasome In-
                                                                                  hibition. 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 sig-
                                                                                  nificant accumulation of p21WAF1 (Fig. 2B). Using one-
                                                                                  parameter 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, com-
                                                                                  pared with the RKO-E6 cells, the parental RKO cells ap-
                                                                                  peared to be blocked from entering the S phase, perhaps
                                                                                  as a result of the strong induction of p21WAF1 in these cells
                                                                                  (Fig. 2B).
                                                                                     The mean FITC signal of the BrdUrd-positive cells, which
                                                                                  is an indication of the rate of BrdUrd incorporation and thus
                                                                                  the rate of DNA synthesis, was lower for the parental RKO
                                                                                  cells than for the RKO-E6 cells after 24 h of LLnL incubation
Fig. 2. Induction of p21WAF1 accumulation by proteasome inhibition is             (Table 1). Thus, not only were fewer parental RKO cells
attenuated in cells with compromised p53 function. Parental RKO cells
and E6-expressing RKO-E6 cells were incubated with 30 M LLnL or 10                entering S phase, the cells that entered did synthesize DNA
  M lactacystin for 6 h before cells were collected, and the cellular levels of   less efficiently. By multiplying the mean FITC signal with the
p53 (A) or p21WAF1 (B) were assessed by Western blot. Human fibroblasts
                                                                                  number of BrdUrd-positive cells, we obtained an estimation
from a normal (NF) or LFS (LFS) individual (C) or human colon carcinoma
HT29 cells with mutant p53 (D) were treated with 10 M lactacystin for 6 h         of the relative amount of DNA synthesis in the different cell
before the cellular levels of p53 or p21WAF1 were assessed by Western             populations. LLnL did not affect DNA synthesis negatively
blot. E, Northern blot of p21WAF1 mRNA (top) or -actin (bottom) expres-
sion in cells mock-treated or treated with 10 M lactacystin for 6 h. The          during the first 6 h of incubation in either cell line (Table 1).
expression of ribosomal RNAs in these cells is shown in the left panel.           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
did not induce a significant increase in p21WAF1 protein levels                   cells synthesized DNA at a rate comparable with that of
(Fig. 2D).                                                                        untreated control cells (Table 1).
   To examine whether the accumulation of p21WAF1 by pro-                            Proteasome Inhibition Results in a G2-M-phase Arrest
teasome inhibition was due to up-regulation of the p21WAF1                        in p53-compromised Cells. Whereas LLnL treatment led to
gene, we performed Northern blots with p21WAF1-specific                           a modest G1 arrest in the wild-type p53-expressing RKO
probes (Fig. 2E). Although lactacystin caused a slight up-                        cells, the RKO-M and RKO-E6 cells showed a significant
regulation of the p21WAF1 gene as reported previously (5), it                     accumulation in the G2-M phase of the cell cycle (Fig. 3A).
clearly did not fully account for the dramatic increase of the                    The percentages of cells in G2-M phase increased from 15%
242   p53 and Proteasome Inhibitors

                                                                                     Table 1 The relative DNA synthesis is reduced in a p53-dependent
                                                                                     manner after LLnL treatment

                                                                                                             % BrdUrd-incorporating   Mean    Relative DNA
                                                                                          Cells   Time (h)
                                                                                                                    cellsa            FITCa    synthesisb
                                                                                         RKO         0                45               36         100
                                                                                                     6                52               41         132
                                                                                                    24                19               22          26
                                                                                         RKO-E6      0                37               63         100
                                                                                                     6                64               63         173
                                                                                                    24                43               64         118
                                                                                      Data taken from window H in Fig. 3B.
                                                                                      Relative DNA synthesis was calculated by multiplying the mean FITC
                                                                                     value by the number (percentage) of cells in the H window and then
                                                                                     expressing the results relative to untreated control cells.

                                                                                     of the cell cycle after proteasome inhibition. Treatment with
                                                                                     10 M 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 ac-
                                                                                     cumulation 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 Inhibi-
                                                                                     tor-induced Apoptosis. Previous studies have shown that
                                                                                     treatment of human and mouse cancer cells with protea-
                                                                                     some inhibitors induces apoptosis (26, 27, 30, 31). Further-
                                                                                     more, induction of apoptosis by proteasome inhibitors ap-
                                                                                     pears 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 M LLnL before both floating
                                                                                     and attached cells were collected, fixed, and stained with PI.
                                                                                     Flow cytometric analysis of the percentage of cells contain-
                                                                                     ing sub-G1 DNA content as a measure of apoptosis revealed
                                                                                     that LLnL induced apoptosis in both cell types (Fig. 4). How-
                                                                                     ever, the induction of apoptosis was significantly higher (P
                                                                                     0.05) in the parental RKO cells (58%) as compared with the
      Fig. 3. p53-mediated G1 arrest and p53-independent G2-M-phase arrest           RKO-E6 cells (38%). Thus, induction of apoptosis by LLnL in
      after proteasome inhibition. A, parental RKO, RKO-M, and RKO-E6 cells          these human colon cancer cells did not require wild-type p53
      were incubated with 30 M LLnL for 0, 6, or 24 h, followed by fixation,
      staining with PI, and flow cytometry. The position of G1, S phase, and         function. However, wild-type p53 significantly enhanced
      G2-M phase in the flow diagrams is shown in the top left panel. B, RKO         LLnL-induced apoptosis.
      and RKO-E6 cells were treated with LLnL for 6 or 24 h or untreated                We next repeated the experiments using lactacystin in-
      (control) before BrdUrd was added to the media for 15 min to pulse-label
      cells actively synthesizing DNA. Cell cycle analysis was then performed        stead of LLnL to more specifically inhibit 26S proteasome
      using two-parameter flow cytometry. The amount of BrdUrd incorporation         activity. We found that 10 M lactacystin induced a more
      per cell is expressed on the Y axis (LOG FITC), whereas the DNA content
      per cell is expressed on the X axis (PI-DNA). The different encircled
                                                                                     severe induction of apoptosis in the RKO cells than was
      regions represent the following: E, G1; F, S-phase DNA-containing cells        observed after incubation with 30 M LLnL. At 24 h after
      not synthesizing DNA; G, G2-M; and H (the whole encircled region above         adding lactacystin, 84% of the RKO cells were already
      the horizontal line), S-phase cells actively synthesizing DNA. C, HT29 cells
      treated with or without 10 M lactacystin for 24 h before fixation, PI          scored as apoptotic, whereas 55% of the RKO-E6 cells had
      staining, and cell cycle analysis using flow cytometry.                        undergone apoptosis (Fig. 5A). These results are in agree-
                                                                                     ment with the preferential induction of apoptosis in the wild-
                                                                                     type p53-expressing RKO cells after LLnL treatment.
      in untreated RKO-E6 cells to 27% and 49% after 6 and 24 h                         Finally, we explored the induction of apoptosis in NF and
      of drug treatment, respectively. During the same time period,                  LFS cells treated with 10 M lactacystin. Again, the NF cells
      the population of cells in G1 was severely diminished.                         with wild-type p53 exhibited a slightly higher induction of
         Using HT29 colon cancer cells, which harbor mutant p53,                     apoptosis than the LFS cells, although this difference was
      we also observed an accumulation of cells in the G2-M phase                    not dramatic. Furthermore, whereas no significant cell cycle
                                                                                                                           Cell Growth & Differentiation    243

                                                                             Fig. 5. Lactacystin treatment resulted in accumulation of p53-compro-
                                                                             mised cells in the G2-M phase and a slightly higher percentage of wild-
Fig. 4. p53 stimulates LLnL-induced apoptosis in RKO cells. A, parental      type p53-expressing cells undergoing apoptosis compared with p53-
RKO and E6-expressing RKO-E6 cells were incubated with 30 M LLnL for         compromised cells. A, RKO and RKO-E6 cells were treated with 10 M
48 h before apoptosis was assessed by measuring the percentage of cells      lactacystin for 24 h before both attached and floating cells were collected,
with sub-G1 DNA content using PI staining and flow cytometry. B, quan-       fixed, and stained with PI. Flow cytometry analysis revealed massive
tification of multiple experiments show that a difference between the        apoptosis in both cell lines and accumulation of nonapoptotic cells in the
amount of apoptosis induced by the two cell lines was significant using      G2-M phase of the cell cycle in the RKO-E6 cells. B, normal human skin
the Student’s t test (P 0.05). The values are the mean of two (control) or   fibroblasts (NF) or a cell line derived from a LFS patient (LFS) was treated
three (treated) different biological samples, with error bars showing the    with 10 M lactacystin. After 24 h of incubation, detached and attached
sample SD.                                                                   cells were collected, fixed, and stained with PI. The percentages of cells
                                                                             with a sub-G1 DNA are indicated.

perturbation was observed in the nonapoptotic population of                  However, this scenario is less plausible because p53 has
NF cells, a clear accumulation of cells in the G2-M phases                   been shown to accumulate in the cell nucleus after overex-
was observed in the LFS cell population (Fig. 5B), supporting                pression of wild-type p53 (40) or inhibition of the nuclear
the findings obtained in the RKO-E6 cells after LLnL treat-                  export machinery with leptomycin B (12–14). Finally, it is
ment (Fig. 3A).                                                              possible that the nuclear accumulation of p53 by protea-
                                                                             some inhibition is due to a direct or an indirect inhibition of a
Discussion                                                                   proteasome-mediated step involved in nuclear export. Ad-
In this study, we investigated the role of p53 in mediating                  ditional studies are needed to elucidate the true mechanism
cellular responses after inhibition of the 26S proteasomes.                  by which proteasome inhibition leads to nuclear accumula-
We found that proteasome inhibition resulted in a significant                tion of p53.
accumulation of p53 in the nucleus of treated cells. This                       Both lactacystin and LLnL strongly induced the accumu-
finding was surprising, considering that p53 is thought to be                lation of wild-type p53 in the parental RKO cell line and in
degraded primarily in the cytoplasm and not in the cell nu-                  NFs. In contrast, only a marginal increase or no increase in
cleus (12–14). One hypothesis for the nuclear accumulation                   the absolute p53 protein level was observed in the E6-
of p53 after proteasome inhibition is that p53 proteins tar-                 expressing cells, the LFS cells, or the HT29 cells containing
geted for degradation by cytoplasmic proteasomes may be                      mutant p53. Furthermore, induction of p21WAF1 by protea-
“recycled” and recruited back into the nucleus. Increased                    some inhibition was seen only in the cell cultures in which
concentration of nuclear p53 would favor p53 tetrameriza-                    p53 accumulated, suggesting that the induction of p21WAF1
tion, which would block nuclear export (14) and further aug-                 may have been, at least in part, p53 dependent. This finding
ment nuclear accumulation. Alternatively, p53 may, under                     is similar to those in studies in which forced expression of
normal conditions, be degraded by nuclear proteasomes.                       exogenous p53 in cells (41) or treatment of cells with the
244   p53 and Proteasome Inhibitors

      nuclear export inhibitor leptomycin B (12, 28) resulted in          normally subjected to degradation by the ubiquitin-protea-
      induction of p21WAF1 expression. However, the induction of          some pathway (3, 25, 45, 46).
      p21WAF1 after proteasome inhibition could only partially be             A profound arrest in the G2-M phase of the cell cycle was
      explained by an up-regulation of the p21WAF1 gene (Fig. 2E).        found for cells with compromised p53 status but not in the
      Thus, the accumulation of p21WAF1 protein cannot be entirely        wild-type p53-expressing cells after treatment with either
      explained by p53-mediated transactivation but may also re-          LLnL or lactacystin (Figs. 3 and 5). One explanation for these
      sult from increased stability of the p21WAF1 protein in the         results is that inhibition of proteasome activity causes a
      absence of proteasome degradation (5). Although p53 iso-            G2-M-phase arrest that is only revealed in cells lacking wild-
      lated from proteasome inhibitor-treated cells has been re-          type p53. Because the entry of wild-type p53-expressing
      ported to be fully capable of binding to oligonucleotides           cells into S phase was reduced after drug treatment, a
      containing p21WAF1 promoter sequences (18), some studies            smaller percentage of cells would be expected to reach the
      suggest that these p53 proteins are fairly poor in transacti-       G2-M-phase compartment of the cell cycle. The mechanism
      vating either endogenous (18) or exogenous p21WAF1 pro-             for the G2-M-phase arrest in the p53-compromised cells
      moters (28). The limited transactivation activity of p53 after      after proteasome inhibition may be due to the inability of the
      proteasome inhibition may result from conformational mis-           cells to exit mitosis. The exit of the M phase requires pro-
      folding of nascent p53 in the presence of proteasome inhib-         teasome activity to degrade the targets of the anaphase
      itors (32). Alternatively, the relatively low transactivation ac-   promoting complex such as cyclins A and B (3). An alterna-
      tivity of drug-accumulated p53 may be due to the lack of            tive hypothesis for the lack of G2-M-phase arrest in the
      protein modifications, such as phosphorylation or acetylation       wild-type p53-expressing cells could be that the exit from the
      (19, 42), which are thought to stimulate the transactivation        G2-M phase is stimulated by wild-type p53 expression. This
      function of p53 (43).                                               hypothesis is supported by findings that p53 may play a role
         Because the stability of p21WAF1 is increased after protea-      in the exit from DNA damage-induced G2 arrest (47).
      some inhibition, it is expected that p21WAF1 levels should              The role of p53 in the induction of apoptosis by protea-
      increase, even in cells with compromised p53 function. How-         some inhibitors is controversial (24, 30, 31). Proteasome
      ever, the rather small increase of p21WAF1 protein levels in        inhibitors have been shown to induce apoptosis in a number
      LLnL-treated RKO-E6 cells and the absence of such an                of human cell lines (26, 27, 30, 31). However, the induction of
      increase in the LFS cells may have been related to the rather       apoptosis in sympathetic neurons and thymocyte cultures
      low basal expression of this protein due to the absence of          has been shown to be inhibited by proteasome inhibitors (48,
      functional p53. However, our result using the p53 mutant cell       49). Our results suggest that wild-type p53 function is not
      line HT29 shows that the detectable basal level of p21WAF1          required for the induction of apoptosis by LLnL or lactacystin
      did not increase after incubation with lactacystin (Fig. 2D).       in the cells used in this study (Figs. 4 and 5). However,
      This would argue against protein stabilization as the major         wild-type p53 function clearly stimulated the induction of
      mechanism of p21WAF1 induction after proteasome inhibi-             apoptosis after proteasome inhibition. It has been shown that
      tion. The marginal induction of p53 expression after treat-         certain apoptosis-promoting proteins such as Bax are sub-
      ment with proteasome inhibitors in the E6-expressing cells is       jected to proteasome-mediated degradation (25). It is thus
      in disagreement with a previous study in which E6-trans-            possible that inhibition of proteasome-mediated degradation
      fected normal fibroblasts were shown to readily accumulate          of Bax together with increased expression of Bax in the
      p53 after incubation with the proteasome inhibitor MG132            presence of high levels of p53 may explain the stronger
      (5). The reason for this discrepancy is not clear, but it may be    induction of apoptosis in the wild-type p53-expressing cells.
      that the RKO-E6 cell line used in our study expressed the E6        It is also possible that the potential p53-stimulated exit from
      protein to a higher level than the E6-transfected fibroblasts.      the G2-M phase of the cell cycle may enhance apoptosis, as
      Whereas Maki et al. (5) readily detected p53 in the untreated       has been suggested previously (47).
      E6-expressing fibroblasts used in their study, no p53 protein           In conclusion, we have found that proteasome inhibition
      expression was detected in the RKO-E6 cells (Fig. 2A; Refs.         leads to nuclear accumulation of p53, a p53-stimulated in-
      37 and 38).                                                         duction of p21WAF1, and cell cycle arrest in the G1 phase of
         Whereas wild-type p53-expressing RKO cells tended to             the cell cycle. Furthermore, cells with compromised p53
      arrest in G1 after treatment with LLnL, RKO-E6 cells ap-            function accumulated in the G2-M phase of the cell cycle and
      peared to be stimulated to enter S phase (Fig. 2B; Table 1).        were somewhat protected against the induction of apoptosis
      This finding that LLnL blocked cells from entering S phase in       by proteasome inhibition. Because proteasome inhibitors are
      wild-type p53 cells is in agreement with a previous study (44).     potentially useful as antitumor agents (50, 51), the true mech-
      The most likely explanation for this G1 arrest is that the          anisms of how these agents induce apoptosis and how p53
      accumulation of the cyclin-dependent kinase inhibitor               modifies these events warrants further exploration.
      p21WAF1 after LLnL treatment blocked the G1 to S-phase
      transition of these cells. The mechanism responsible for the        Materials and Methods
      increased S-phase entry of the RKO-E6 cells after LLnL              Cell Culture and Chemicals. Three isogenic human colon cancer cell
      treatment is not clear, but it may involve accumulation of          lines, the parental cell line RKO, the mutant p53-expressing cell line
                                                                          RKO-M, and the human papillomavirus 16 E6-expressing cell line RKO-
      some cellular component(s) driving the progression of cells         E6, were generously given to us by Dr. Albert Fornace, Jr., (National
      from G1 into S phase. Such components may include cyclin            Cancer Institute, NIH, Bethesda, MD) via Drs. Ted Lawrence and Mary
      D1, cyclin E, and/or the transcription factor E2F1, which are       Davis (University of Michigan). These cell lines have been described in
                                                                                                                             Cell Growth & Differentiation      245

detail previously (37, 52, 53). The human colon carcinoma cell line HT29      References
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