Research article
Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

The proteasome inhibitor MG-132 sensitizes PC-3 prostate cancer cells to ionizing radiation by a DNA-PK-independent mechanism

  • Frank Pajonk1Email author,
  • Arndt van Ophoven2,
  • Christian Weissenberger3 and
  • William H McBride1
BMC Cancer20055:76

https://doi.org/10.1186/1471-2407-5-76

©  Pajonk et al; licensee BioMed Central Ltd. 2005

Received: 23 March 2005

Accepted: 07 July 2005

Published: 07 July 2005

Open Peer Review reports

Abstract

Background

By modulating the expression levels of specific signal transduction molecules, the 26S proteasome plays a central role in determining cell cycle progression or arrest and cell survival or death in response to stress stimuli, including ionizing radiation. Inhibition of proteasome function by specific drugs results in cell cycle arrest, apoptosis and radiosensitization of many cancer cell lines. This study investigates whether there is also a concomitant increase in cellular radiosensitivity if proteasome inhibition occurs only transiently before radiation. Further, since proteasome inhibition has been shown to activate caspase-3, which is involved in apoptosis, and caspase-3 can cleave DNA-PKcs, which is involved in DNA-double strand repair, the hypothesis was tested that caspase-3 activation was essential for both apoptosis and radiosensitization following proteasome inhibition.

Methods

Prostate carcinoma PC-3 cells were treated with the reversible proteasome inhibitor MG-132. Cell cycle distribution, apoptosis, caspase-3 activity, DNA-PKcs protein levels and DNA-PK activity were monitored. Radiosensitivity was assessed using a clonogenic assay.

Results

Inhibition of proteasome function caused cell cycle arrest and apoptosis but this did not involve early activation of caspase-3. Short-time inhibition of proteasome function also caused radiosensitization but this did not involve a decrease in DNA-PKcs protein levels or DNA-PK activity.

Conclusion

We conclude that caspase-dependent cleavage of DNA-PKcs during apoptosis does not contribute to the radiosensitizing effects of MG-132.

Background

One of the most challenging ongoing efforts in radiotherapy is the search for agents that target tumor-specific characteristics to cause radiosensitization without increasing normal tissue complications. The malignant phenotype is normally associated with acquisition of mutations in genes encoding signal transduction molecules that control cell proliferation and/or cell death. Numerous experimental studies have shown, that the expression of mutated oncogenes and tumor suppressor genes in normal cells alters their intrinsic cellular radiosensitivity and clinical studies have, in some cases, indicated that such mutations influence disease free survival after chemotherapy and radiation therapy [1–4]. These observations suggest that the pathways leading to cell cycle arrest and cell death following exposure to ionizing irradiation are linked to DNA damage and repair. Our knowledge of the molecular circuitry that is involved is still however far from complete.

One of the processes that controls expression of short-lived cell cycle and cell death regulators and transcription factors, such as cyclin A, B and E, p21 and p27, p53, cJun, cFos, and nuclear factor κB (NF-κB), is the proteolytic degradation through the 26S proteasome [5–7]. Indeed, the initial response to many stress signals such as radiation-induced DNA damage [8], hypoxia/reperfusion [9], and exposure to heat [10, 11] or cytokines [12] appears to involve rapid alterations in 26S proteasome activity resulting in cell cycle arrest and/or apoptosis.

The 26S proteasome is a 2 MDa proteolytic complex that has 3 different cleavage activities [13]. Its function is highly regulated [14–18]. Highly specific and potent inhibitors of the 26S proteasome like the reversible inhibitor MG-132 or lactacystein, which acts irreversibly, have been shown to induce apoptosis in many tumor cell lines [19–27] and in SV-40-transformed but not normal human fibroblasts [27]. Apoptosis is regulated by the ubiquitin/proteasome system at various levels and inhibition of proteasome function may induce [21] or prevent apoptosis [28]. A possible association between apoptosis and radiosensitization exist through caspase activation and subsequent proteolytic destruction of DNA repair enzymes. Caspase activation has been reported to follow proteasome inhibition [24, 29, 30] and is known to degrade the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), a key DNA repair enzyme of non-homologous end-joining (NHEJ) of DNA double strand breaks [31, 32].

Still, the exact mechanisms of how proteasome inhibition causes apoptosis are not fully understood but the fact, that malignant cells are much more sensitive to the death-promoting aspects of proteasome inhibition than normal cells [33] makes the ubiquitin/26S proteasome system a target for cancer therapy. With Velcade (formerly known as bortezomib, PS-341) a first proteasome inhibitor has become available for clinical use [34]. The excellent results achieved in patients with multiple myeloma suggest the use of Velcade in solid cancers, especially in combination with classical chemotherapies or radiation therapy. So far, several Phase I/II studies have used Velcade as mono-therapy in advanced chemotherapy refractory solid cancers [35–40] but knowledge of in-vivo effects of combined modality approaches using Velcade is limited. It is known that proteasome inhibition can sensitize cells to chemotherapy [41] and ionizing radiation [26, 42] and therefore combination studies will be launched in the near future.

However, a recent in-vitro report demonstrated that Velcade treatment of A549 lung cancer might enhance or attenuate cisplatin toxicity, depending on the sequence of application of both drugs [28]. These findings underline the need for a better understanding of the mechanisms of action of these new compounds as they may interfere with standard therapies.

In this study, we investigated a possible link between induction of the apoptotic death program and radiosensitization following proteasome inhibition.

Methods

Cell culture

Cultures of PC-3 prostate carcinoma cells American Type Culture Collection (ATCC, Manassas) cells were grown in 75 cm2 flasks (Falcon) at 37°C in a humidified atmosphere at 5 % CO2. PC-3 cells were cultured in DMEM medium (Cell Concepts, Umkirch Germany) supplemented with 10 % heat-inactivated fetal calf serum (FCS, Sigma, St. Louis, MO) and 1 % penicillin/streptomycin (Sigma).

MG-132 treatment

MG-132 (Calbiochem, San Diego, CA) was dissolved in DMSO (10 mM) and small aliquots (30 μl) were stored at -20°C. Velcade (Janssen-Cilag, Neuss, Germany) was solubilized in water at a concentration of 1 mg/ml and stored in aliquots of 50 μl at -80°C. Three hours before irradiation growth medium was replaced by medium containing MG-132 (50 μM, 0.5% DMSO) or Velcade (100 nM in water). Control cells were subjected to DMSO treatment alone (0.5 %). In clonogenic assays, cells were incubated at 37°C for 3 hours, washed with DMEM, trypsinized, counted, diluted and irradiated at room temperature.

Irradiation

Exponentially growing PC-3 cells were trypsinized, counted, and diluted. The cell suspensions were immediately irradiated at room temperature using a 137Cs-laboratory irradiator (JLShephard, Mark I) at a dose rate of 5.80 Gy/min and a Gammacell 40 137Cs-laboratory irradiator at a dose rate of 0.83 Gy/min respectively.

Clonogenic survival

Colony-forming assays with PC-3 cells were performed immediately after irradiation by plating an appropriate number of cells into culture dishes, in triplicate. Before plating, the viability of the cells was assessed during counting by a dye exclusion test with tryphan blue. After 14 days, colonies were fixed with methanol, stained with crystal violet, and were counted if they consisted of more than 50 cells. The fraction of cells surviving irradiation was normalized to the surviving fraction of the corresponding control and survival values and curves were fitted to the data using a linear-quadratic model.

Flow cytometry

For analysis of cell cycle distribution, 1 × 105 cells were trypsinized, washed in PBS and fixed with ice-cold ethanol (70 %). After RNAse treatment (1 mg/ml), cells were permeabilized with Triton X-100 and stained with propidium iodide (0.1 mg/ml). To determine the cell cycle distribution, DNA content was measured using a flow cytometer (FACScan, Becton Dickinson). TUNEL-Assay was carried out as described previously [43].

DNA-PK activity assay

In order to assess DNA-PK activity, DNA-PK-dependent phosphorylation of a biotinylated p53-derived peptide (Glu-Pro-Pro-Leu-Ser-Gln-Glu-Ala-Phe-Ala-Asp-Leu-Trp-Lys-Lys. Promega, Madison, WI; [44]) was measured in the presence of [32P-γ]-ATP. Drug-treated or control cells were washed in low salt buffer (10 mM HEPES, 25 mM KCl, 10 mM NaCl, 1.1 mM MgCl2, 0.1 mM ethylenediaminetetraacetic acid [EDTA], 0.5 mM phenylmethanesulfonyl fluoride [PMSF], pH 7.2), pelleted and lysed by one freezing/thawing cycle as described in [45]. After centrifugation at 10,000 × g for 5 minutes at 4°C, the supernatant was collected and used as cell extract. Protein content was determined using the Micro-BCA protocol (Pierce) with bovine serum albumin (Sigma) as standard. 10 μg protein were incubated for 30 minutes at 30°C in DNA-PK reaction buffer (50 mM HEPES (KOH, pH 7.5), 100 mM KCl, 10 mM MgCl2, 0.2 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid [EGTA], 0.1 mM EDTA, 1 mM DTT), 0.025 mM ATP, 0.5 μCi [32P-γ]-ATP, bovine serum albumin [BSA] 0.1 mg/ml, and with human p53 oligopeptide as substrate in the presence or absence of activated calf thymus DNA to measure DNA-DSB-dependent phosphorylation of p53. The final volume was 25 μl. The reaction was stopped by addition of 25 μl 30 % acetic acid. 10 μl was spotted on Whatman P81 membranes in duplicates and washed four times with 15 % acetic acid.

Membranes were placed on a phosphor imager screen for 2 hours. The screen was read on a phosphor imager (Storm 860, Molecular Dynamics) and the activity measured using the ImageQuant software package (Molecular Dynamics). Activity in the absence of activated DNA was assumed to be unspecific and thus subtracted from corresponding measurements in presence of activated DNA.

Caspase-3 activity assay

For assessment of caspase-3 activity, cells were plated into culture dishes 24 hours before drug treatment. After drug treatment, cells were dislodged mechanically and washed twice in PBS. Caspase-3 activity was assessed as described by Enari et al. [46] with minor modifications: After five cycles of freezing and thawing in extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol [DTT], 20 μM cytochalasin B, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 50 μg/ml antipain, 10 μg/ml chymopain) lysates were centrifuged at 10,000 × g for 12 minutes (4°C). The supernatant was immediately frozen in liquid nitrogen and stored at -80°C. Protein concentrations were determined using the Micro-BCA protocol (Pierce) with bovine serum albumin (Sigma) as standard. 36 μg protein were diluted in ICE standard buffer (100 mM HEPES-KOH, pH 7.5, 10 % sucrose, 0.1 % CHAPS, 10 mM DTT, 0.1 mg/ml ovalbumin) containing the fluorogenic caspase-3 substrate DEVD-7-amido-4-methylcoumarin (DEVD-AMC, 1 μM) and incubated for 30 minutes at 30°C. Fluorescence was measured using a fluorescence plate reader (Tecon, excitation 380 nm, emission 460 nm).

Immunoblotting

Cells were washed with PBS and lysed in TOTEX buffer (20 mM HEPES [pH 7.9], 0.35 mM NaCl, 20 % glycerol, 1 % Nonidet P-40, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 50 μM PMSF and 90 trypsin inhibitor units [TIU's]/ml aprotinin) for 30 minutes on ice. The lysate was centrifuged at 12,000 × g for 5 minutes and the supernatant transferred to fresh tubes. Protein concentrations were determined using the Micro-BCA protocol (Pierce, Rockford, IL) with bovine serum albumin as standard. 100 μg or 50 μg of protein were subjected to SDS gel electrophoresis (0.1 % Sodium dodecyl sulfate [SDS]/6 % polyacrylamide) and blotted to PVDF membranes. Equity of protein loading was confirmed by Ponceau staining of the PVDF membranes. After blocking with 5 % skim milk in PBS, membranes were incubated with a mouse polyclonal antibody against human DNA-PKcs (Santa Cruz Biotechnologies). A secondary horseradish-peroxidase-conjugated antibody and the ECL Plus System (Amersham) were used for visualization. Fluorescence was measured using a Typhoon 9410 Phosphorimager (Blue fluorescence, 600 V, Amersham).

Results

MG-132 induces apoptosis and sensitizes PC-3 cells to ionizing radiation

To investigate if transient proteasome inhibition by proteasome inhibition affects PC-3 cells induction of apoptosis and long-term clonogenic survival was examined following MG-132 treatment. Cells were exposed to 50 μM of MG-132 for 3 hours, washed twice, trypsinized and plated into tissue culture dishes in triplicates. Clonogenicity, as assessed by colony counts after 2 weeks incubation, was decreased by MG-132 treatment of PC-3 cells from 65 ± 5.1 % in dimethyl sulfoxide (DMSO)-treated versus 35 ± 2.1 in MG-132-treated cells. Decreased clonogenicity was due to apoptosis, as assessed morphologically. 3 hours exposure of PC-3 cells to MG-132 at 25 μM and 50 μM concentrations increased the TUNEL-positive (terminal deoxynucleotidyl transferase-mediated nick end labeling) population after 24 hours of incubation from initially 8 % to 67 % and 73 % respectively (Fig. 1). Short-term proteasome inhibition for 3 hours was therefore effective in causing apoptosis in a proportion of cells, but a considerable number remained clonogenically viable.
Figure 1

MG-132 induces apoptosis in PC-3 cells. Representative (n = 3) FACS analysis of PC-3 prostate cancer cells incubated with 0, 25 and 50 μM concentrations of MG-132 for 24 h. TUNEL-staining indicated an increase of apoptotic cells from 7.6 % in control cells to 66.5 % and 72.8 % in MG-132 treated cells.

Clinical treatment with proteasome inhibitors might also be expected to spare a proportion of clonogenic tumor cells and it would be an advantage if these were sensitized to some other form of therapy. To test whether they were sensitized to radiation, PC-3 cells were exposed to short-term treatment with MG-132. After 3 hours, cells were washed, irradiated and plated in a clonogenic assay, as described previously [43]. Inhibition of proteasome function by MG-132 sensitized the surviving clonogenic PC-3 cells to the effects of ionizing radiation as shown by the left-shift of the survival curve (Fig. 2).
Figure 2

MG-132 sensitizes human cancer cell lines to ionizing radiation. PC-3 prostate cancer cells were incubated with MG-132 (50 μM) for 3 hours, washed twice, irradiated and plated into culture dishes. After 14 days the colonies were fixed with ethanol, stained with crystal violet, and counted. MG-132 treatment sensitized PC-3 cells to ionizing radiation. Radiobiological parameters obtained from a linear-quadratic fit (LQ-fit): PC-3 control α = 0.3, β = 0.043, α/β = 7.6; PC-3 MG-132 α = 0.3, β = 0.07, α/β = 4.3. Data shows means (± standard deviation) from 3 experiments (each plated in triplicates).

Effect of 26S proteasome inhibition on radiation-induced cell cycle arrest and apoptosis

Having validated PC-3 cells as a model for radiosensitization by proteasome inhibitors, we first excluded that the observed radiosensitizing effect was a result of changes in cell cycle distribution. Inhibition of 26S proteasome function is known to block cellular transition from G1- to S-phase and from late S- to G2/M-phase, as well as S phase transition [47]. This is considered to be due to alterations in expression of molecules such as p53, p21WAF/CIP1, pRB, p27, and cyclins A, B, and E. Similar effects can be seen following exposure of cells to ionizing radiation, which can cause arrest at the G1 and G2/M checkpoints, as well as S-phase delay, and apoptosis in certain cancer cell lines [48]. In cells with mutated p53, like PC-3 prostate cancer cells, the radiation-induced G1 checkpoint delay and the pro-apoptotic response are often abrogated, but the cells will arrest in G2/M (reviewed in [49]).

The effect of the combined treatment of proteasome inhibitors and ionizing irradiation on cell cycle arrest was tested using PC-3 cells treated continuously with MG-132 (50 μM) from 3 hours prior up to 24 hours after irradiation. This treatment blocks proteasome activity almost completely [50]. Flow cytometric analyses indicated that radiation induced G2/M but not G1 arrest, as would be expected in the p53-null PC-3 cells (Fig. 3). MG-132 treatment caused p53-independent cell cycle arrest in all phases and, as a result, the combination of both treatments did not lead to a radiation-induced G2/M arrest (Fig. 3).
Figure 3

The effect of proteasome inhibition by MG-132 on cell cycle progression and radiation-induced cell cycle arrest and apoptosis. Cell cycle distribution of PC-3 prostate cancer cells assessed by flow cytometry after staining with propidium iodide. Cells were pre-incubated with MG-132 (50 μM) for 3 hours, irradiated, and incubated for additional 24 hours in the presence of MG-132. MG-132 prevented cell cycle progression and, consequently, dose-dependent radiation-induced G2/M-arrest. Additionally, MG-132 treatment increased the number of cells in late S-phase.

The effect of MG-132 treatment on caspase-3 and DNA-PK activities

Since short-term proteasome inhibition caused both apoptosis and radiosensitization in PC-3 cells, this model can be used to investigate the relationship between these phenomena and to explore the pathways that might interconnect them. We considered the hypothesis that both involve caspase-3 activation. Proteasome inhibition has been reported to induce apoptosis that is mediated by caspase activation [20]. Also, the catalytic subunit of the DNA-PK complex, DNA-PKcs, which is required for NHEJ repair pathway of DNA double strand breaks, is a known substrate of caspase-3 [51]. Its destruction could result in radiosensitization.

MG-132 (50 μM) was maintained in the growth medium of PC-3 cells for various time periods. DNA-PKcs protein levels were monitored by western blotting (Fig. 4). Expression of the intact 460 kDa (upper arrow) protein did not change after 3 (lane 1) and 6 hours (lane 2) but slightly decreased after 24 hours of incubation (lane 3) compared to DMSO-treated control cells (lane 7). At this time-point the specific 160 kDa caspase-3-dependent degradation product of DNA-PKcs appeared (lane 3, lower arrow) which coincided with occurrence of morphological signs of apoptosis. Comparable results were obtained using Velcade (100 nM) another specific proteasome inhibitor (lane 4,5 and 6, Fig. 4).
Figure 4

Degradation of DNA-PKcs by caspase-3 is a late event in apoptosis induced by proteasome inhibition in PC-3 cells. Representative (n = 3) western blot of lysates from PC-3 prostate cancer cells after 3, 6 and 24 hours incubation with MG-132 (50 μM) or Velcade (100 nM) using a specific antibody against human DNA-PKcs. The antibody recognizes the intact 460 kDa protein as well as the specific 160 kDa fragment observed after caspase-3 dependent cleavage of DNA-PKcs. MG-132 caused no significant change of the DNA-PKcs protein levels over a period of 6 hours. Detection of a caspase-3-cleavage specific 160 kDa fragment coincided with apoptosis at 24 hours, but did not occur during the first 6 hours.

Because the level of DNA-PKcs might not reflect the total kinase activity of the enzyme complex, functional activity was assessed by phosphorylation of a p53 protein fragment in the presence of DNA double strand breaks. This was unchanged over a period of 5 hours after MG-132 treatment, which is the period over which most DNA repair would be expected to take place (Fig 5). In order to exclude a possible trivial explanation that MG-132 might directly interfere with DNA-PK activity, lysates of PC-3 cells were incubated with MG-132 and studied for p53 phosphorylation. The drug did not affect DNA-PK activity directly (Fig. 6). Since caspase-3 activity has been reported to be increased by proteasome inhibition in the MOe7 cell line [20], caspase-3 activity was measured using a fluorogenic substrate in PC-3 cells at various time points after short-term (3 hours) treatment of PC-3 cells with MG-132. We found a substantial drop in caspase-3-like activity as early as 15' minutes after the end of MG-132 incubation if compared to untreated control cells. Caspase-3-like cleavage activity slowly recovered about 3 hours after the end of MG-132 incubation (Fig. 7), but was never increased above baseline. Radiation (30 Gy) with or without MG-132 pre-treatment also failed to activate caspase-3-like activity during the first 3 hours.
Figure 5

MG-132 does not decrease DNA-PK activity. PC-3 cells were incubated for 3 hours with MG-132 (50 μM). Cells were washed and incubated at 37°C. At indicated times, cells were lysed and DNA-PK activity was measured by phosphorylation of a human p53 derived oligopeptide in the presence of DNA DSB's. Proteasome inhibition did not cause any significant decrease of DNA-PK activity (n = 2, data expressed as mean ± standard error mean).

Figure 6

MG-132 does not interfere with DNA-PK directly. Lysates of untreated PC-3 cells were incubated with MG-132. There was no decrease in DNA-PK activity, excluding any direct interaction with the drug (n = 3, data expressed as mean ± standard error mean).

Figure 7

MG-132 treatment reduces caspase-3-like activity in PC-3 cells. PC-3 cells were pre-incubated with MG-132 (50 μM) for 3 hours, washed, irradiated, and incubated at 37°C. 15 minutes, 1, and 3 hours later, cells were lysed and total cellular protein was assayed for caspase-3-like activity, measuring the release of AMC from the fluorogenic caspase-3 substrate DEVD-AMC. Irradiation with 30 Gy initially decreased constitutive caspase-3 activity in PC-3 cells at 15 minutes followed by a slight increase at 1 and 3 hours after irradiation. In contrast, proteasome inhibition with MG-132 completely inhibited caspase-3 activity at 15 minutes. Activity recovered slowly reaching about 30% of baseline levels after 3 hours. This was not altered by combination of MG-132 treatment with ionizing radiation (n = 3, data expressed as mean ± standard error mean).

Discussion

Proteasome inhibitors like MG-132 sensitize cancer cells to ionizing radiation. In this study, we investigated interference of proteasome inhibition with NHEJ by caspase-3-dependent cleavage of DNA-PKcs as a possible underlying mechanism.

Treatment with MG-132 caused cell cycle arrest in un-irradiated and irradiated cells, induced apoptosis, decreased clonogenicity, and sensitized the surviving cells to ionizing radiation. This was in accordance with previous reports [19–21, 47, 52–55].

Drug-induced changes in cell cycle redistribution were most unlikely responsible for this effect because even after 24 hours MG-132 treatment mainly increased the number of cells in late S-phase, which has been shown to coincide with greatest radioresistance [56].

The relationship between pro-apoptotic pathways and radiosensitization is controversial, as is the importance of apoptosis in cell death following exposure to clinically relevant doses of ionizing radiation. In general, the majority of cells in solid carcinomas do not enter the apoptotic death pathway [57]. Instead, they undergo several cell divisions until they die or finally survive. Thus, the fate of cells in solid carcinomas after irradiation may be determined more by their ability to repair DNA damage caused by ionizing radiation than by initiation of apoptosis [58]. However, common molecular pathways may link these two phenomena. So far, any link between survival pathways and molecules involved in DNA repair has been elusive but cannot be excluded.

The most important DNA lesions occurring after exposure of cells to ionizing radiation that determines death or survival of a cancer cell are the double strand breaks. A process called non-homologous end joining (NHEJ) repairs these lesions in eukaryote cells. Concerted concession of NHEJ requires the activity of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), which is a known substrate of caspase-3. In this study we therefore focused on the possibility that proteasome inhibition by MG-132 activates caspase-3, as has been reported previously [24, 30]. This could mediate apoptosis and cause degradation of DNA-PKcs, the catalytic subunit of DNA-PK [20], resulting in radiosensitization. Reduction of DNA-PK activity following inactivation or mutation of DNA-PKcs is known to enhance radiosensitivity by decreasing repair of DNA-DSB's [59–61]. Although DSB repair is critical for cell survival after exposure to ionizing radiation [62], it is not clear whether this is a rate-limiting step dependent on the level of expression of DNA-PKcs. For example, DNA-PKcs level has been reported not to correlate with radiosensitivity of gliomas [63] and normal fibroblasts [64]. In any event, we were not able to detect meaningful changes in DNA-PK activity following MG-132 drug treatment. Degradation that did occur, appeared late at 24 hours and was probably an effect rather than a cause of the apoptotic process. According to the kinetics of the DSB-repair process described previously [65], any event interfering with the repair of DNA-DSB's has to take place during the initial 6 hours after irradiation. Consistent with the late cleavage of DNA-PKcs, we were not able to detect early activation of caspase-3 following treatment of PC-3 cells with MG-132. In fact, we observed a substantial drop in DEVD-AMC cleavage activity, which might be explained by the observation, that proteasome activity is necessary to activate caspase-3 at least in some cells [66, 67]. Our observations are in accordance with data from Hideshima and coworkers who also could not detect changes in DNA-PKcs levels or caspase-3 activation during the initial 6 hours of Valcade-treatment in multiple myeloma cells [68]. Additionally, Wu and coworkers excluded the involvement of caspases in apoptosis of MO7e cells following proteasome inhibition, as caspase inhibitors failed to prevent DNA fragmentation [20] and it is therefore possible that proteasome inhibition induces caspase-independent apoptosis as described for cells, defective in the ubiquitin pathway [69].

Taken together, we conclude that although proteasome inhibition induces apoptosis in most cancer cells, sensitization of PC-3 cells to ionizing radiation occurs through mechanism that does not involve cleavage of DNA-PKcs.

Declarations

Acknowledgements

FP was supported by grants from the German Research Foundation Pa 723-2-1 and Pa 723/3-1. WM was supported by a grant from the National Cancer Institute, Department of Health and Human Services PHS grant CA-87887.

Authors’ Affiliations

(1)
Department of Radiation Oncology, David Geffen School of Medicine at UCLA
(2)
Department of Urology, University Hospital Münster
(3)
Department of Radiation Oncology, University Hospital Freiburg

References

  1. Caffo O, Doglioni C, Veronese S, Bonzanini M, Marchetti A, Buttitta F, Fina P, Leek R, Morelli L, Palma PD, Harris AL, Barbareschi M: Prognostic value of p21(WAF1) and p53 expression in breast carcinoma: an immunohistochemical study in 261 patients with long-term follow-up. Clin Cancer Res. 1996, 2 (9): 1591-1599.PubMedGoogle Scholar
  2. Bonin SR, Pajak TF, Russell AH, Coia LR, Paris KJ, Flam MS, Sauter ER: Overexpression of p53 protein and outcome of patients treated with chemoradiation for carcinoma of the anal canal: a report of randomized trial RTOG 87-04. Radiation Therapy Oncology Group. Cancer. 1999, 85 (6): 1226-1233. 10.1002/(SICI)1097-0142(19990315)85:6<1226::AID-CNCR3>3.0.CO;2-B.View ArticlePubMedGoogle Scholar
  3. Hayakawa K, Mitsuhashi N, Hasegawa M, Saito Y, Sakurai H, Ohno T, Maebayashi K, Ebara T, Hayakawa KY, Niibe H: The prognostic significance of immunohistochemically detected p53 protein expression in non-small cell lung cancer treated with radiation therapy. Anticancer Res. 1998, 18 (5B): 3685-3688.PubMedGoogle Scholar
  4. Pomp J, Davelaar J, Blom J, van Krimpen C, Zwinderman A, Quint W, Immerzeel J: Radiotherapy for oesophagus carcinoma: the impact of p53 on treatment outcome. Radiother Oncol. 1998, 46 (2): 179-184. 10.1016/S0167-8140(97)00163-1.View ArticlePubMedGoogle Scholar
  5. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL: Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell. 1994, 78 (5): 761-771. 10.1016/S0092-8674(94)90462-6.View ArticlePubMedGoogle Scholar
  6. Rolfe M, Chiu MI, Pagano M: The ubiquitin-mediated proteolytic pathway as a therapeutic area. J Mol Med. 1997, 75 (1): 5-17. 10.1007/s001090050081.View ArticlePubMedGoogle Scholar
  7. Weissman AM: Regulating protein degradation by ubiquitination. Immunol Today. 1997, 18 (4): 189-198. 10.1016/S0167-5699(97)84666-X.View ArticlePubMedGoogle Scholar
  8. Pajonk F, McBride WH: Ionizing radiation affects 26s proteasome function and associated molecular responses, even at low doses. Radiother Oncol. 2001, 59 (2): 203-212. 10.1016/S0167-8140(01)00311-5.View ArticlePubMedGoogle Scholar
  9. Stempien-Otero A, Karsan A, Cornejo CJ, Xiang H, Eunson T, Morrison RS, Kay M, Winn R, Harlan J: Mechanisms of hypoxia-induced endothelial cell death. Role of p53 in apoptosis. J Biol Chem. 1999, 274 (12): 8039-8045. 10.1074/jbc.274.12.8039.View ArticlePubMedGoogle Scholar
  10. Kueckelkorn U, Knuehl C, Boes-Fabian B, Drung I, Kloetzel PM: The effect of heat shock on 20S/26S proteasomes. Biol Chem. 2000, 381 (9-10): 1017-1023. 10.1515/BC.2000.125.Google Scholar
  11. Pajonk F, van Ophoven A, McBride WH: Hyperthermia-induced proteasome inhibition and loss of androgen receptor expression in human prostate cancer cells. Cancer Res. 2005, 65 (11): 4836-4843.View ArticlePubMedGoogle Scholar
  12. Glockzin S, von Knethen A, Scheffner M, Brune B: Activation of the cell death program by nitric oxide involves inhibition of the proteasome. J Biol Chem. 1999, 274 (28): 19581-19586. 10.1074/jbc.274.28.19581.View ArticlePubMedGoogle Scholar
  13. Hasselgren PO, Fischer JE: The ubiquitin-proteasome pathway: review of a novel intracellular mechanism of muscle protein breakdown during sepsis and other catabolic conditions. Ann Surg. 1997, 225 (3): 307-316. 10.1097/00000658-199703000-00011.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Tanaka K: Molecular biology of proteasomes. Mol Biol Rep. 1995, 21 (1): 21-26. 10.1007/BF00990966.View ArticlePubMedGoogle Scholar
  15. Stohwasser R, Kloetzel PM: Cytokine induced changes in proteasome subunit composition are concentration dependent. Biol Chem. 1996, 377 (9): 571-577.PubMedGoogle Scholar
  16. Nandi D, Woodward E, Ginsburg DB, Monaco JJ: Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits. EMBO J. 1997, 16 (17): 5363-5375. 10.1093/emboj/16.17.5363.View ArticlePubMedPubMed CentralGoogle Scholar
  17. Schmidt M, Kloetzel PM: Biogenesis of eukaryotic 20S proteasomes: the complex maturation pathway of a complex enzyme. FASEB J. 1997, 11 (14): 1235-1243.PubMedGoogle Scholar
  18. Kloetzel PM, Soza A, Stohwasser R: The role of the proteasome system and the proteasome activator PA28 complex in the cellular immune response. Biol Chem. 1999, 380 (3): 293-297. 10.1515/BC.1999.040.View ArticlePubMedGoogle Scholar
  19. Drexler HC: Activation of the cell death program by inhibition of proteasome function. Proc Natl Acad Sci U S A. 1997, 94 (3): 855-860. 10.1073/pnas.94.3.855.View ArticlePubMedPubMed CentralGoogle Scholar
  20. Wu LW, Reid S, Ritchie A, Broxmeyer HE, Donner DB: The proteasome regulates caspase-dependent and caspase-independent protease cascades during apoptosis of MO7e hematopoietic progenitor cells. Blood Cells Mol Dis. 1999, 25 (1): 20-29. 10.1006/bcmd.1999.0223.View ArticlePubMedGoogle Scholar
  21. Herrmann JL, Briones FJ, Brisbay S, Logothetis CJ, McDonnell TJ: Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. Oncogene. 1998, 17 (22): 2889-2899. 10.1038/sj.onc.1202221.View ArticlePubMedGoogle Scholar
  22. McDade TP, Perugini RA, Vittimberga FJJ, Callery MP: Ubiquitin-proteasome inhibition enhances apoptosis of human pancreatic cancer cells. Surgery. 1999, 126 (2): 371-377. 10.1067/msy.1999.98785.View ArticlePubMedGoogle Scholar
  23. Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott PJ: Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res. 1999, 59 (11): 2615-2622.PubMedGoogle Scholar
  24. Zhang XM, Lin H, Chen C, Chen BD: Inhibition of ubiquitin-proteasome pathway activates a caspase-3-like protease and induces Bcl-2 cleavage in human M-07e leukaemic cells. Biochem J. 1999, 340 (Pt 1): 127-133. 10.1042/0264-6021:3400127.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Kitagawa H, Tani E, Ikemoto H, Ozaki I, Nakano A, Omura S: Proteasome inhibitors induce mitochondria-independent apoptosis in human glioma cells. FEBS Lett. 1999, 443 (2): 181-186. 10.1016/S0014-5793(98)01709-8.View ArticlePubMedGoogle Scholar
  26. Pajonk F, Pajonk K, McBride WH: Apoptosis and radiosensitization of hodgkin cells by proteasome inhibition. Int J Radiat Oncol Biol Phys. 2000, 47 (4): 1025-1032. 10.1016/S0360-3016(00)00516-2.View ArticlePubMedGoogle Scholar
  27. An B, Goldfarb RH, Siman R, Dou QP: 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. 1998, 5 (12): 1062-1075. 10.1038/sj.cdd.4400436.View ArticlePubMedGoogle Scholar
  28. Mortenson MM, Schlieman MG, Virudachalam S, Bold RJ: Effects of the proteasome inhibitor bortezomib alone and in combination with chemotherapy in the A549 non-small-cell lung cancer cell line. Cancer Chemother Pharmacol. 2004, 54 (4): 343-53. 10.1007/s00280-004-0811-4.View ArticlePubMedGoogle Scholar
  29. Pasquini LA, Besio Moreno M, Adamo AM, Pasquini JM, Soto EF: Lactacystin, a specific inhibitor of the proteasome, induces apoptosis and activates caspase-3 in cultured cerebellar granule cells. J Neurosci Res. 2000, 59 (5): 601-611. 10.1002/(SICI)1097-4547(20000301)59:5<601::AID-JNR3>3.0.CO;2-1.View ArticlePubMedGoogle Scholar
  30. Qiu JH, Asai A, Chi S, Saito N, Hamada H, Kirino T: Proteasome inhibitors induce cytochrome c-caspase-3-like protease- mediated apoptosis in cultured cortical neurons. J Neurosci. 2000, 20 (1): 259-265.PubMedGoogle Scholar
  31. Han Z, Malik N, Carter T, Reeves WH, Wyche JH, Hendrickson EA: DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease. J Biol Chem. 1996, 271 (40): 25035-25040. 10.1074/jbc.271.40.25035.View ArticlePubMedGoogle Scholar
  32. Song Q, Burrows SR, Smith G, Lees-Miller SP, Kumar S, Chan DW, Trapani JA, Alnemri E, Litwack G, Lu H, Moss DJ, Jackson S, Lavin MF: Interleukin-1 beta-converting enzyme-like protease cleaves DNA- dependent protein kinase in cytotoxic T cell killing. J Exp Med. 1996, 184 (2): 619-626. 10.1084/jem.184.2.619.View ArticlePubMedGoogle Scholar
  33. Masdehors P, Omura S, Merle-Beral H, Mentz F, Cosset JM, Dumont J, Magdelenat H, Delic J: Increased sensitivity of CLL-derived lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Br J Haematol. 1999, 105 (3): 752-757. 10.1046/j.1365-2141.1999.01388.x.View ArticlePubMedGoogle Scholar
  34. Kane RC, Bross PF, Farrell AT, Pazdur R: Velcade: U.S. FDA approval for the treatment of multiple myeloma progressing on prior therapy. Oncologist. 2003, 8 (6): 508-513. 10.1634/theoncologist.8-6-508.View ArticlePubMedGoogle Scholar
  35. Maki RG, Kraft AS, Scheu K, Yamada J, Wadler S, Antonescu CR, Wright JJ, Schwartz GK: A multicenter Phase II study of bortezomib in recurrent or metastatic sarcomas. Cancer. 2005, 103 (7): 1431-1438. 10.1002/cncr.20968.View ArticlePubMedGoogle Scholar
  36. Price N, Dreicer R: Phase I/II trial of bortezomib plus docetaxel in patients with advanced androgen-independent prostate cancer. Clin Prostate Cancer. 2004, 3 (3): 141-143.View ArticlePubMedGoogle Scholar
  37. Papandreou CN, Daliani DD, Nix D, Yang H, Madden T, Wang X, Pien CS, Millikan RE, Tu SM, Pagliaro L, Kim J, Adams J, Elliott P, Esseltine D, Petrusich A, Dieringer P, Perez C, Logothetis CJ, Chatta G: Phase I trial of the proteasome inhibitor bortezomib in patients with advanced solid tumors with observations in androgen-independent prostate cancer . J Clin Oncol. 2004, 22: 2108-21. 10.1200/JCO.2004.02.106.View ArticlePubMedGoogle Scholar
  38. Blaney SM, Bernstein M, Neville K, Ginsberg J, Kitchen B, Horton T, Berg SL, Krailo M, Adamson PC: Phase I study of the proteasome inhibitor bortezomib in pediatric patients with refractory solid tumors: a Children's Oncology Group study (ADVL0015). J Clin Oncol. 2004, 22 (23): 4804-4809. 10.1200/JCO.2004.12.185.View ArticlePubMedGoogle Scholar
  39. Kondagunta GV, Drucker B, Schwartz L, Bacik J, Marion S, Russo P, Mazumdar M, Motzer RJ: Phase II trial of bortezomib for patients with advanced renal cell carcinoma. J Clin Oncol. 2004, 22 (18): 3720-3725. 10.1200/JCO.2004.10.155.View ArticlePubMedGoogle Scholar
  40. Shah MH, Young D, Kindler HL, Webb I, Kleiber B, Wright J, Grever M: Phase II study of the proteasome inhibitor bortezomib (PS-341) in patients with metastatic neuroendocrine tumors. Clin Cancer Res. 2004, 10 (18 Pt 1): 6111-6118.View ArticlePubMedGoogle Scholar
  41. Hideshima T, Richardson P, Chauhan D, Palombella VJ, Elliott PJ, Adams J, Anderson KC: The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001, 61 (7): 3071-3076.PubMedGoogle Scholar
  42. Russo SM, Tepper JE, Baldwin ASJ, Liu R, Adams J, Elliott P, Cusack JCJ: Enhancement of radiosensitivity by proteasome inhibition: implications for a role of NF-kappaB. Int J Radiat Oncol Biol Phys. 2001, 50 (1): 183-193. 10.1016/S0360-3016(01)01446-8.View ArticlePubMedGoogle Scholar
  43. Pajonk F, Pajonk K, McBride W: Apoptosis and radiosensitization of Hodgkin’s cells by proteasome inhibition. Int J Radiat Oncol Biol Phys. 2000, 47 (4): 1025-1032. 10.1016/S0360-3016(00)00516-2.View ArticlePubMedGoogle Scholar
  44. Lees-Miller SP, Sakaguchi K, Ullrich SJ, Appella E, Anderson CW: Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992, 12 (11): 5041-5049.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Ihara M, Suwa A, Komatsu K, Shimasaki T, Okaichi K, Hendrickson EA, Okumura Y: Heat sensitivity of double-stranded DNA-dependent protein kinase (DNA- PK) activity. Int J Radiat Biol. 1999, 75 (2): 253-258. 10.1080/095530099140717.View ArticlePubMedGoogle Scholar
  46. Enari M, Talanian RV, Wong WW, Nagata S: Sequential activation of ICE-like and CPP32-like proteases during Fas- mediated apoptosis. Nature. 1996, 380 (6576): 723-726. 10.1038/380723a0.View ArticlePubMedGoogle Scholar
  47. Machiels BM, Henfling ME, Gerards WL, Broers JL, Bloemendal H, Ramaekers FC, Schutte B: Detailed analysis of cell cycle kinetics upon proteasome inhibition. Cytometry. 1997, 28 (3): 243-252. 10.1002/(SICI)1097-0320(19970701)28:3<243::AID-CYTO9>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
  48. Maity A, McKenna WG, Muschel RJ: The molecular basis for cell cycle delays following ionizing radiation: a review. Radiother Oncol. 1994, 31 (1): 1-13. 10.1016/0167-8140(94)90408-1.View ArticlePubMedGoogle Scholar
  49. Hwang A, Muschel RJ: Radiation and the G2 phase of the cell cycle. Radiat Res. 1998, 150 (5 Suppl): S52-9.View ArticlePubMedGoogle Scholar
  50. Lee DH, Goldberg AL: Selective inhibitors of the proteasome-dependent and vacuolar pathways of protein degradation in Saccharomyces cerevisiae. J Biol Chem. 1996, 271 (44): 27280-27284. 10.1074/jbc.271.44.27280.View ArticlePubMedGoogle Scholar
  51. Teraoka H, Yumoto Y, Watanabe F, Tsukada K, Suwa A, Enari M, Nagata S: CPP32/Yama/apopain cleaves the catalytic component of DNA-dependent protein kinase in the holoenzyme. FEBS Lett. 1996, 393 (1): 1-6. 10.1016/0014-5793(96)00842-3.View ArticlePubMedGoogle Scholar
  52. Dietrich C, Bartsch T, Schanz F, Oesch F, Wieser RJ: 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 U S A. 1996, 93 (20): 10815-10819. 10.1073/pnas.93.20.10815.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Hashemolhosseini S, Nagamine Y, Morley SJ, Desrivieres S, Mercep L, Ferrari S: Rapamycin inhibition of the G1 to S transition is mediated by effects on cyclin D1 mRNA and protein stability. J Biol Chem. 1998, 273 (23): 14424-14429. 10.1074/jbc.273.23.14424.View ArticlePubMedGoogle Scholar
  54. Rao S, Porter DC, Chen X, Herliczek T, Lowe M, Keyomarsi K: Lovastatin-mediated G1 arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase. Proc Natl Acad Sci U S A. 1999, 96 (14): 7797-7802. 10.1073/pnas.96.14.7797.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Delic J, Masdehors P, Omura S, Cosset JM, Dumont J, Binet JL, Magdelenat H: The proteasome inhibitor lactacystin induces apoptosis and sensitizes chemo- and radioresistant human chronic lymphocytic leukaemia lymphocytes to TNF-alpha-initiated apoptosis [see comments]. Br J Cancer. 1998, 77 (7): 1103-1107.View ArticlePubMedPubMed CentralGoogle Scholar
  56. Sinclair WK, Morton RA: X-ray sensitivity during the cell generation cycle of cultured Chinese hamster cells. Radiat Res. 1966, 29 (3): 450-474.View ArticlePubMedGoogle Scholar
  57. Meyn RE, Stephens LC, Milas L: Programmed cell death and radioresistance. Cancer Metastasis Rev. 1996, 15 (1): 119-131. 10.1007/BF00049491.View ArticlePubMedGoogle Scholar
  58. Weichselbaum RR, Little JB: The differential response of human tumours to fractionated radiation may be due to a post-irradiation repair process. Br J Cancer. 1982, 46 (4): 532-537.View ArticlePubMedPubMed CentralGoogle Scholar
  59. Rosenzweig KE, Youmell MB, Palayoor ST, Price BD: Radiosensitization of human tumor cells by the phosphatidylinositol3- kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G2-M delay. Clin Cancer Res. 1997, 3 (7): 1149-1156.PubMedGoogle Scholar
  60. Chernikova SB, Wells RL, Elkind MM: Wortmannin sensitizes mammalian cells to radiation by inhibiting the DNA-dependent protein kinase-mediated rejoining of double-strand breaks. Radiat Res. 1999, 151 (2): 159-166.View ArticlePubMedGoogle Scholar
  61. Hsieh CL, Arlett CF, Lieber MR: V(D)J recombination in ataxia telangiectasia, Bloom's syndrome, and a DNA ligase I-associated immunodeficiency disorder. J Biol Chem. 1993, 268 (27): 20105-20109.PubMedGoogle Scholar
  62. Jeggo PA: Identification of genes involved in repair of DNA double-strand breaks in mammalian cells. Radiat Res. 1998, 150 (5 Suppl): S80-91.View ArticlePubMedGoogle Scholar
  63. Allalunis-Turner MJ, Lintott LG, Barron GM, Day RS, Lees-Miller SP: Lack of correlation between DNA-dependent protein kinase activity and tumor cell radiosensitivity. Cancer Res. 1995, 55 (22): 5200-5202.PubMedGoogle Scholar
  64. Dikomey E, Borgmann K, Brammer I, Kasten-Pisula U: Molecular mechanisms of individual radiosensitivity studied in normal diploid human fibroblasts. Toxicology. 2003, 193 (1-2): 125-135. 10.1016/S0300-483X(03)00293-2.View ArticlePubMedGoogle Scholar
  65. Olive PL, Banath JP, MacPhail HS: Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines. Cancer Res. 1994, 54 (14): 3939-3946.PubMedGoogle Scholar
  66. Grimm LM, Goldberg AL, Poirier GG, Schwartz LM, Osborne BA: Proteasomes play an essential role in thymocyte apoptosis. Embo J. 1996, 15 (15): 3835-3844.PubMedPubMed CentralGoogle Scholar
  67. Sadoul R, Fernandez PA, Quiquerez AL, Martinou I, Maki M, Schroter M, Becherer JD, Irmler M, Tschopp J, Martinou JC: Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. Embo J. 1996, 15 (15): 3845-3852.PubMedPubMed CentralGoogle Scholar
  68. Hideshima T, Bergsagel PL, Kuehl WM, Anderson KC: Advances in biology of multiple myeloma: clinical applications. Blood. 2004, 104 (3): 607-618. 10.1182/blood-2004-01-0037.View ArticlePubMedGoogle Scholar
  69. Monney L, Otter I, Olivier R, Ozer HL, Haas AL, Omura S, Borner C: Defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by Bcl-2. J Biol Chem. 1998, 273 (11): 6121-6131. 10.1074/jbc.273.11.6121.View ArticlePubMedGoogle Scholar

    70. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/5/76/prepub

Copyright

© Pajonk et al; licensee BioMed Central Ltd. 2005

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement