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Cell Growth & Differentiation Vol. 11, 239-246, May 2000
© 2000 American Association for Cancer Research


Articles

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

Feng Chen, Daniel Chang, Meidee Goh, Sergey A. Klibanov and Mats Ljungman2

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. 1ACitation ), whereas cells irradiated with 30 J/m2 UVC light presented strong nuclear staining of p53 (Fig. 1BCitation ). Interestingly, lactacystin treatment for 6 h led to a strong nuclear accumulation of p53 (Fig. 1, C–FCitation ). At x100 magnification, using either fluorescence (Fig. 1DCitation ) or scanning confocal fluorescence microscopy (Fig. 1, E and FCitation ), 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.



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Fig. 1. Exposure of cells to lactacystin resulted in nuclear accumulation 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 µM lactacystin (C–F), followed by fixation and immunocytochemistry with anti-p53 antibodies and FITC-conjugated antimouse antibodies. A–C were photographed at x40 magnification, and D–F were photographed at x100 magnification. Images A–D were obtained using fluorescence microscopy and anti-p53 antibody 1801, whereas images E and F were captured digitally using scanning confocal microscopy and anti-p53 antibodies AB-1 and FL-393, respectively.

 


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Fig. 2. Induction of p21WAF1 accumulation by proteasome inhibition is attenuated in cells with compromised p53 function. Parental RKO cells and E6-expressing RKO-E6 cells were incubated with 30 µM LLnL or 10 µM lactacystin for 6 h before cells were collected, and the cellular levels of p53 (A) or p21WAF1 (B) were assessed by Western blot. Human fibroblasts from a normal (NF) or LFS (LFS) individual (C) or human colon carcinoma HT29 cells with mutant p53 (D) were treated with 10 µM lactacystin for 6 h before the cellular levels of p53 or p21WAF1 were assessed by Western blot. E, Northern blot of p21WAF1 mRNA (top) or ß-actin (bottom) expression in cells mock-treated or treated with 10 µM lactacystin for 6 h. The expression of ribosomal RNAs in these cells is shown in the left panel.

 
Accumulation of p21WAF1 by LLnL3 or Lactacystin Is Stimulated by Wild-Type p53.
To3 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 characterized 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 µM) or lactacystin (10 µM). These doses were chosen because they have been shown to be sufficient to inhibit proteasome activity 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 parental RKO cell line (Fig. 2ACitation ). 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 (Citation ). 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. 2BCitation ). 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. 2CCitation ). 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. 2DCitation ).

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. 2ECitation ). 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 BCitation ), 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. 2BCitation ). Using oneparameter flow cytometry of PI-stained RKO, RKO-E6, and mutant p53 RKO-M cells (Fig. 3ACitation ), 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. 3BCitation ; Table 1Citation ). 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)Citation . 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. 2BCitation ).



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Fig. 3. p53-mediated G1 arrest and p53-independent G2-M-phase arrest after proteasome inhibition. A, parental RKO, RKO-M, and RKO-E6 cells were incubated with 30 µM LLnL for 0, 6, or 24 h, followed by fixation, staining with PI, and flow cytometry. The position of G1, S phase, and G2-M phase in the flow diagrams is shown in the top left panel. B, RKO and RKO-E6 cells were treated with LLnL for 6 or 24 h or untreated (control) before BrdUrd was added to the media for 15 min to pulse-label cells actively synthesizing DNA. Cell cycle analysis was then performed using two-parameter flow cytometry. The amount of BrdUrd incorporation 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 regions represent the following: E, G1; F, S-phase DNA-containing cells not synthesizing DNA; G, G2-M; and H (the whole encircled region above the horizontal line), S-phase cells actively synthesizing DNA. C, HT29 cells treated with or without 10 µM lactacystin for 24 h before fixation, PI staining, and cell cycle analysis using flow cytometry.

 

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

 
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 (Table 1)Citation . 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)Citation . 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)Citation .

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. 3ACitation ). 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. 3CCitation ). 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)Citation . 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.



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Fig. 4. p53 stimulates LLnL-induced apoptosis in RKO cells. A, parental RKO and E6-expressing RKO-E6 cells were incubated with 30 µM LLnL for 48 h before apoptosis was assessed by measuring the percentage of cells with sub-G1 DNA content using PI staining and flow cytometry. B, quantification of multiple experiments show that a difference between the amount of apoptosis induced by the two cell lines was significant using the Student’s t test (P < 0.05). The values are the mean of two (control) or three (treated) different biological samples, with error bars showing the sample SD.

 
We next repeated the experiments using lactacystin instead of LLnL to more specifically inhibit 26S proteasome activity. We found that 10 µM lactacystin induced a more severe induction of apoptosis in the RKO cells than was observed after incubation with 30 µM LLnL. At 24 h after adding lactacystin, 84% of the RKO cells were already scored as apoptotic, whereas 55% of the RKO-E6 cells had undergone apoptosis (Fig. 5ACitation ). These results are in agreement with the preferential induction of apoptosis in the wild-type p53-expressing RKO cells after LLnL treatment.



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Fig. 5. Lactacystin treatment resulted in accumulation of p53-compromised cells in the G2-M phase and a slightly higher percentage of wild-type p53-expressing cells undergoing apoptosis compared with p53-compromised cells. A, RKO and RKO-E6 cells were treated with 10 µM lactacystin for 24 h before both attached and floating cells were collected, fixed, and stained with PI. Flow cytometry analysis revealed massive apoptosis in both cell lines and accumulation of nonapoptotic cells in the G2-M phase of the cell cycle in the RKO-E6 cells. B, normal human skin fibroblasts (NF) or a cell line derived from a LFS patient (LFS) was treated with 10 µM lactacystin. After 24 h of incubation, detached and attached cells were collected, fixed, and stained with PI. The percentages of cells with a sub-G1 DNA are indicated.

 
Finally, we explored the induction of apoptosis in NF and LFS cells treated with 10 µM lactacystin. Again, the NF cells with wild-type p53 exhibited a slightly higher induction of apoptosis than the LFS cells, although this difference was not dramatic. Furthermore, whereas no significant cell cycle perturbation was observed in the nonapoptotic population of NF cells, a clear accumulation of cells in the G2-M phases was observed in the LFS cell population (Fig. 5BCitation ), supporting the findings obtained in the RKO-E6 cells after LLnL treatment (Fig. 3ACitation ).

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. 2ECitation ). 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. 2DCitation ). 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. 2ACitation ; 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. 2BCitation ; Table 1Citation ). 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. 3Citation and 5)Citation . 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. 4Citation and 5)Citation . 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 [{alpha}-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. Back

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 Back

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. Back

Received for publication 9/29/99. Revision received 2/16/00. Accepted for publication 4/ 5/00.

References

  1. Varshavsky A. The ubiquitin system. Trends Biochem. Sci., 22: 383-387, 1997.[Medline]
  2. Hilt W., Wolf D. H. Proteasomes: destruction as a programme. Trends Biochem. Sci., 21: 96-102, 1996.[Medline]
  3. Koepp D., Harper J., Elledge S. How the cyclin became a cyclin: regulated proteolysis in the cell cycle. Cell, 97: 431-434, 1999.[Medline]
  4. Chowdary D., Dermody J., Jha K., Ozer H. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway. Mol. Cell. Biol., 14: 1997-2003, 1994.[Abstract/Free Full Text]
  5. Maki C. G., Huibregtse J. M., Howley P. M. In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Res., 56: 2649-2654, 1996.[Abstract/Free Full Text]
  6. Brown J., Pagano M. Mechanism of p53 degradation. Biochim. Biophys. Acta, 1332: 1-6, 1997.
  7. Ljungman M. Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress. Neoplasia, 2: 208-225, 2000.[Medline]
  8. Haupt Y., Maya R., Kazaz A., Oren M. Mdm2 promotes the rapid degradation of p53. Nature (Lond.), 387: 296-299, 1997.[Medline]
  9. Kubbutat M. H. G., Jones S. N., Vousden K. H. Regulation of p53 stability by Mdm2. Nature (Lond.), 387: 299-303, 1997.[Medline]
  10. Honda R., Tanaka H., Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett., 420: 25-27, 1997.[Medline]
  11. Fuchs S. Y., Adler V., Buschmann T., Yin Z. M., Wu X. W., Jones S. N., Ronai Z. JNK targets p53 ubiquitination and degradation in nonstressed cells. Genes Dev., 12: 2658-2663, 1998.[Abstract/Free Full Text]
  12. Freedman D. A., Levine A. J. Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol. Cell. Biol., 18: 7288-7293, 1998.[Abstract/Free Full Text]
  13. Tao W. K., Levine A. J. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc. Natl. Acad. Sci. USA, 96: 3077-3080, 1999.[Abstract/Free Full Text]
  14. Stommel J. M., Marchenko N. D., Jimenez G. S., Moll U. M., Hope T. J., Wahl G. M. A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J., 18: 1660-1672, 1999.[Abstract]
  15. Maltzman W., Czyzyk L. UV irradiation stimulates levels of p53 cellular antigen in nontransformed mouse cells. Mol. Cell. Biol., 4: 1689-1694, 1984.[Abstract/Free Full Text]
  16. Kastan M., Onyekwere O., Sidransky D., Vogelstein B., Craig R. Participation of p53 protein in the cellular response to DNA damage. Cancer Res., 51: 6304-6311, 1991.[Abstract/Free Full Text]
  17. Shieh S. Y., Ikeda M., Taya Y., Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell, 91: 325-334, 1997.[Medline]
  18. Siliciano J. D., Canman C. E., Taya Y., Sakaguchi K., Appella E., Kastan M. B. DNA damage induces phosphorylation of the amino terminus of p53. Genes Dev., 11: 3471-3481, 1997.[Abstract/Free Full Text]
  19. Shieh S. Y., Taya Y., Prives C. DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization. EMBO J., 18: 1815-1823, 1999.[Abstract]
  20. Unger T., Juven-Gershon T., Moallem E., Berger M., Sionov R. V., Lozano G., Oren M., Haupt Y. Critical role for Ser20 of human p53 in the negative regulation of p53 by Mdm2. EMBO J., 18: 1805-1814, 1999.[Abstract]
  21. Mayo L. D., Turchi J. J., Berberich S. J. Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53. Cancer Res., 57: 5013-5016, 1997.[Abstract/Free Full Text]
  22. Zhang Y. P., Xiong Y. Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol. Cell, 3: 579-591, 1999.[Medline]
  23. Ljungman M., Zhang F. F., Chen F., Rainbow A. J., McKay B. C. Inhibition of RNA polymerase II as a trigger for the p53 response. Oncogene, 18: 583-592, 1999.[Medline]
  24. Gazos Lopes U., Erhardt P., Yao R., Cooper G. p53-dependent induction of apoptosis by proteosome inhibitors. J. Biol. Chem., 272: 12893-12896, 1997.[Abstract/Free Full Text]
  25. Chang Y. C., Lee Y. S., Tejima T., Tanaka K., Omura S., Heintz N. H., Mitsui Y., Magae J. mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Differ., 9: 79-84, 1998.[Abstract]
  26. Shinohara K., Tomioka M., Nakano H., Tone S., Ito H., Kawashima S. Apoptosis induction resulting from proteasome inhibition. Biochem. J., 317: 385-388, 1996.
  27. Drexler H. C. A. Activation of the cell death program by inhibition of proteasome function. Proc. Natl. Acad. Sci. USA, 94: 855-860, 1997.[Abstract/Free Full Text]
  28. Lain S., Midgley C., Sparks A., Lane E. B., Lane D. P. An inhibitor of nuclear export activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs. Exp. Cell. Res., 248: 457-472, 1999.[Medline]
  29. Maki C. G., Howley P. M. Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation. Mol. Cell. Biol., 17: 355-363, 1997.[Abstract/Free Full Text]
  30. Herrmann J. L., Briones F., Jr., Brisbay S., Logothetis C. J., McDonnell T. J. Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. Oncogene, 17: 2889-2899, 1998.[Medline]
  31. An B., Goldfarb R., Siman R., Dou P. Novel dipeptidyl proteasome inhibitors overcome Bcl-2 protective function and selectively accumulate the cyclin-dependent kinase inhibitor p27 and induce apoptosis in transformed, but not normal, human fibroblasts. Cell Death Differ., 5: 1062-1075, 1998.[Medline]
  32. Magae J., Illenye S., Tejima T., Chang Y. C., Mitsui Y., Tanaka K., Omura S., Heintz N. H. Transcriptional squelching by ectopic expression of E2F-1 and p53 is alleviated by proteasome inhibitors MG-132 and lactacystin. Oncogene, 15: 759-769, 1997.[Medline]
  33. Rock K., Gramm C., Rothstein L., Clark K., Stein R., Dick L., Hwang D., Goldberg A. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell, 78: 761-771, 1994.[Medline]
  34. Fenteany G., Standaert R., Lane W., Choi S., Corey E., Schreiber S. Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. Science (Washington DC), 268: 726-731, 1995.[Abstract/Free Full Text]
  35. Scheffner M., Werness B., Huibregtse J., Levine A., Howley P. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell, 63: 1129-1136, 1990.[Medline]
  36. Scheffner M., Huibregtse J. M., Howley P. M. Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc. Natl. Acad. Sci. USA, 91: 8797-8801, 1994.[Abstract/Free Full Text]
  37. Smith M. L., Chen I. T., Zhan Q. M., Oconnor P. M., Fornace A. J. Involvement of the p53 tumor suppressor in repair of u.v.-type DNA damage. Oncogene, 10: 1053-1059, 1995.[Medline]
  38. Chang D., Chen F., Zhang F. F., McKay B. C., Ljungman M. Dose-dependent effects of DNA-damaging agents on p53-mediated cell cycle arrest. Cell Growth Differ., 10: 155-162, 1999.[Abstract/Free Full Text]
  39. Loignon M., Fetni R., Gordon A. J. E., Drobetsky E. A. A p53-independent pathway for induction of p21waf1cip1 and concomitant G1 arrest in UV-irradiated human skin fibroblasts. Cancer Res., 57: 3390-3394, 1997.[Abstract/Free Full Text]
  40. Liang S. H., Hong D., Clarke M. F. Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein. J. Biol. Chem., 273: 19817-19821, 1998.[Abstract/Free Full Text]
  41. El-Deiry W., Tokino T., Velculescu V., Levy D., Parsons R., Trent J., Lin D., Mercer W., Kinzler K., Vogelstein B. WAF1, a potential mediater of p53 tumor suppression. Cell, 75: 817-825, 1993.[Medline]
  42. Sakaguchi K., Herrera J. E., Saito S., Miki T., Bustin M., Vassilev A., Anderson C. W., Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev., 12: 2831-2841, 1998.[Abstract/Free Full Text]
  43. Lambert P. F., Kashanchi F., Radonovich M. F., Shiekhattar R., Brady J. N. Phosphorylation of p53 serine 15 increases interaction with CBP. J. Biol. Chem., 273: 33048-33053, 1998.[Abstract/Free Full Text]
  44. Dietrich C., Bartsch T., Schanz F., Oesch F., Wieser R. J. p53-dependent cell cycle arrest induced by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal in platelet-derived growth factor-stimulated human fibroblasts. Proc. Natl. Acad. Sci. USA, 93: 10815-10819, 1996.[Abstract/Free Full Text]
  45. Diehl J., Zindy F., Sherr C. Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteosome pathway. Genes Dev., 11: 957-972, 1997.[Abstract/Free Full Text]
  46. Hofmann F., Martelli F., Livingston D., Wang Z. The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteosome pathway. Genes Dev., 10: 2949-2959, 1996.[Abstract/Free Full Text]
  47. Guillouf C., Rosselli F., Krishnaraju K., Moustacchi E., Hoffman B., Liebermann D. A. p53 involvement in control of G2 exit of the cell cycle: role in DNA damage-induced apoptosis. Oncogene, 10: 2263-2270, 1995.[Medline]
  48. Grimm L. M., Goldberg A. L., Poirier G. G., Schwartz L. M., Osborne B. A. Proteasomes play an essential role in thymocyte apoptosis. EMBO J., 15: 3835-3844, 1996.[Medline]
  49. Sadoul R., Fernandez P. A., Quiquerez A. L., Martinou I., Maki M., Schroter M., Becherer J. D., Irmler M., Tschopp J., Martinou J. C. Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J., 15: 3845-3852, 1996.[Medline]
  50. Adams J., Palombella V., Sausville E., Johnson J., Destree A., Lazarus D., Maas J., Pien C., Prakash S., Elliott P. Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res., 59: 2615-2622, 1999.[Abstract/Free Full Text]
  51. Meng L., Kwok B., Sin N., Crews C. Eponemycin exerts its antitumor effect through the inhibition of proteasome function. Cancer Res., 59: 2798-2801, 1999.[Abstract/Free Full Text]
  52. Kuerbitz S., Plunkett B., Walsh V., Kastan M. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc. Natl. Acad. Sci. USA, 89: 7491-7495, 1992.[Abstract/Free Full Text]
  53. Kessis T., Slebos R., Nelson W., Kastan M., Plunkett B., Han S., Lorincz A., Hedrick L., Cho K. Human papillomavirus 16 E6 expression disrupts p53-mediated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA, 90: 3988-3992, 1993.[Abstract/Free Full Text]
  54. Ljungman M., Zhang F. Blockage of RNA polymerase as a possible trigger for uv light-induced apoptosis. Oncogene, 13: 823-831, 1996.[Medline]
  55. Hoy C., Seamer L., Schimke R. Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram. Cytometry, 10: 718-725, 1989.[Medline]
  56. Chung D. H., Zhang F. F., Chen F., McLaughlin W. P., Ljungman M. Butyrate attenuates BCLXL expression in human fibroblasts and acts in synergy with ionizing radiation to induce apoptosis. Radiat. Res., 149: 187-194, 1998.[Medline]



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