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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2004 as doi:10.1096/fj.04-1813fje. |
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* Dipartimento di Biologia,
Dipartimento Medicina Sperimentale,
Cattedra di Medicina del Lavoro, Universita’ di Roma "Tor Vergata," Rome, Italy; and
DABAC, Universita’ della Tuscia, Viterbo, Italy
1 Correspondence: Dipartimento di Biologia Universita’ di Roma "Tor Vergata", via Ricerca Scientifica, Rome 100133, Italy. E-mail: ghibelli{at}uniroma2.it
SPECIFIC AIMS
In many cell systems, pharmacological glutathione depletion with GSH neosynthesis inhibitor buthionine sulfoximine (BSO) leads to cell death per se and highly sensitizes target tumor cells to apoptosis induced by standard chemotherapeutic agents. For these reasons, BSO is widely used in clinical practice as a chemosensitizing agent.
We studied cell systems (U937 and HepG2 human tumor cells) in which BSO is neither apoptogenic nor chemosensitizing. Upon BSO treatment, apoptotic signaling is allowed to begin in these cells, since early apoptotic events such as Bax translocation and cytochrome c release occur as an early response, but the signal is blocked at the caspase activation level. These cells are able to adapt to increasing oxidizing conditions by developing a survival pathway, which may involve production (up-regulation?) of a molecule (protein?) that is able to stop and abort an ongoing apoptotic signaling. We performed the present study with the aim of understanding the mechanism through which U937 and HepG2 adapt to GSH depletion and abort apoptosis, focusing on possible up-regulation of the main antiapoptotic protein, Bcl-2.
PRINCIPAL FINDINGS
1. GSH depletion up-regulates Bcl-2 in U937 cells
To analyze the mechanism through which U937 adapts to BSO, we explored the possibility of an up-regulation of the antiapoptotic protein Bcl-2. We measured intracellular levels of Bcl-2 upon BSO treatment by Western blot analysis with anti-Bcl-2 monoclonal antibodies. Figure 1
A shows an increase in Bcl-2 levels at 20 h of treatment with BSO of about 2-fold. Flow cytometric analysis with anti-Bcl-2 monoclonal antibodies in U937 cells +/– BSO confirmed the results. When GSH depletion was sped up by pretreatment with the thiol alkylating agent diethylmaleate (DEM), reaching total depletion within 2 h (as opposed to 20 h of BSO); Bcl-2 up-regulation was also anticipated, since it was detectable at 3 h post-DEM and peaked at 6 h, reaching a 3.2-fold increase with respect to the steady state.
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To investigate the mechanism of BSO-dependent Bcl-2 up-regulation, we analyzed mRNA levels. We found that the increase in Bcl-2 protein synthesis is paralleled by increased mRNA concentration, as shown by specific RT-PCR on control vs. BSO-treated U937 (Fig. 1B
). Quantification of the bands corresponding to amplified Bcl-2 sequence, normalized for actin mRNA, revealed an increase in Bcl-2 mRNA to an extent of 
4-fold (i.e., higher with respect to the protein increase).
We wondered whether Bcl-2 mRNA up-regulation was due to transcriptional regulation and analyzed the possible involvement of NF-
B, one of the key transcriptional regulators in oxidative stress-induced cell response. We observed that NF-B activity, measured by electrophoretic mobility shift assay (EMSA) performed on nuclear extracts of U937 cells, was slightly activated by BSO. Inhibition of NF-B activation with phenylethylester of caffeic acid (CAPE) during BSO treatment strongly reduced Bcl-2 up-regulation due to BSO. Studies with more specific means of NF-B inhibition will clarify whether NF-B is actually involved in Bcl-2 up-regulation.
2. BSO-induced Bcl-2 up-regulation occurs in spite of increased breakdown
It has often been reported that GSH depletion decreases the half-life of Bcl-2, leading to a decrease in Bcl-2 concentration in the presence of BSO. We found instead an increase in Bcl-2 levels, due to increased synthesis. To determine whether increasing Bcl-2 levels in U937 cells was actually the result of an equilibrium between increased synthesis ("in") and increased breakdown ("out"), we monitored the rate of Bcl-2 consumption in control vs. BSO-treated cells. With the purpose of eliminating the "in", we evaluated the effect of protein synthesis inhibition on Bcl-2 protein intracellular levels in control vs. BSO-treated cells. We could not make use of standard protein synthesis inhibitors such as cycloheximide or puromycin, since they induced apoptosis in U937 cells starting from 6–7 h incubation. We selected amino acid starvation by incubating cells in medium deprived of methionine and cysteine (met-). This treatment is more physiological, and is essentially non toxic for U937 up to 24 h. Figure 1C
shows that protein synthesis inhibition for 18 h did not substantially alter Bcl-2 levels in control cells, suggesting a rather stable protein. Instead, Bcl-2 levels drastically decreased in BSO-treated cells, indicating that under these conditions Bcl-2 is more labile. Thus, we may conclude that in U937 treated with BSO, the increase in Bcl-2 levels is the algebraic sum between increased synthesis and increased breakdown.
If we allowed cells to recover normal GSH level (i.e., by washing out BSO), we expected that the lower Bcl-2 protein consumption of a recovered normal redox environment would lead to a further increase in Bcl-2 protein levels, since these cells have high Bcl-2 mRNA levels due to the 24 h incubation in BSO. We performed wash out experiments in which BSO was removed at 24 h, and cells were replated for recovery in fresh medium. As expected, we found that upon this treatment, Bcl-2 levels continued to increase, peaking at 24 h recovery, and only returning to normal levels at 5 days recovery (Fig. 1D
).
3. BSO-dependent Bcl-2 up-regulation is linked to the ability to survive to BSO
We began to explore the possible role of Bcl-2 up-regulation in the survival of U937 to BSO treatment. We approached this question by utilizing two strategies.
First, we analyzed which effects may exert the inhibition of Bcl-2 up-regulation in the survival to BSO on U937 cells. We used the possibility of frustrating BSO-dependent Bcl-2 up-regulation as a tool for investigating the role of Bcl-2 overexpression in BSO resistance. We exploited the two instances where we inhibited BSO-dependent Bcl-2 up-regulation, namely protein synthesis inhibition (medium deprived of cysteine and methionine) and CAPE. Figure 2
A shows that CAPE and amino acid starvation, by themselves essentially nonapoptogenic on U937, were able to sensitize cells to BSO, thus supporting the hypothesis that Bcl-2 up-regulation actually plays a role in the resistance to BSO.
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In the second strategy, we analyzed a panel of tumor cell lines with the purpose of comparing their sensitivity to the apoptogenic action of BSO and their ability to up-regulate Bcl-2.
The human B cell Burkitt lymphoma cell line BL41 is induced to apoptosis by 1 mM BSO. Three additional human tumor cell lines, primary human glioblastoma HGB1, hepatoma HepG2, and monocytic U937 are resistant to this treatment (Fig. 2B
). Analysis of Bcl-2 levels at 24 h of BSO treatment, revealed that cells that survived BSO are the same that up-regulate Bcl-2 in response to BSO. BL41 cells, unable to survive BSO, accordingly failed to up-regulate Bcl-2 protein (Fig. 2C
).
4. BSO-dependent Bcl-2 up-regulation correlates to lack of chemosensitization by BSO
To investigate whether BSO-dependent Bcl-2 up-regulation may also affect BSO ability to sensitize tumor cells to apoptogenic treatments, we analyzed the effect of GSH depletion on chemoresistance in the same cell lines. BL41 cells were sensitized to apoptosis induced by puromycin, according to canonical chemosensitizing protocols. U937 and HGB1 cells were not sensitized. HGB1 cells were even less sensitive to apoptosis (Fig. 2D
). These were the cells in which Bcl-2 up-regulation reached the highest level ( 3-fold). In these cells, BSO exerted the paradoxical role of increasing chemoresistance.
CONCLUSIONS AND SIGNIFICANCE
Bcl-2 protein and GSH are two different types of molecules that share the function of tutoring cell survival. Accordingly, decrease in either Bcl-2 or GSH level favors cell death. Links between GSH and Bcl-2 appear quite often, concerning compensation of functions and coordinate regulation. Thus, modulation of Bcl-2 protein levels as an adaptive response to oxidative stress should conceivably be an important regulatory mechanism for cell survival in conditions of sudden environmental changes.
The finding that different cell types react differently to BSO, and that the ability of trans-activating Bcl-2 is linked to survival, give a possible explanation for the varying susceptibility to BSO treatment cited in many published studies. The apparent incongruence of an opposite response to BSO in terms of Bcl-2 concentration, increase vs. decrease, found in the literature is probably due to the overlapping of two independent phenomena, increased breakdown vs. increased neosynthesis. The net concentration of Bcl-2 upon BSO treatment will thus be the algebraic sum of two independent phenomena with the oxidative death signal contrasted rather than potentiated (Fig. 3
).
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The survival and increased resistance to apoptosis of some tumor cells treated with BSO is strictly related to the clinical exploitation of BSO as a chemoadjuvant in antitumor therapies. In the last decade, the deciphering of the apoptotic signaling led to a breakthrough in the designing of antitumor therapies that are mainly focused on the development of proapoptotic treatments that discriminate between normal and tumor cells.
The theoretical basis for the use of BSO as a chemosensitizer stems from the general findings that cells deprived of GSH are less prone to: 1) extrude xenobiotics (via glutathione-S-transferase), thus bypassing the problem of multidrug resistance; and 2) resist apoptosis due to reduced antioxidant ability and reduced levels of the antiapoptotic protein Bcl-2. Most cells respond to GSH depletion by down-regulating Bcl-2. This is probably due to an increased breakdown of Bcl-2 protein in an oxidizing environment.
These data recommend a warning against the indiscriminate use of BSO as a chemoadjuvant and recommend a careful evaluation of the tumor cells before using BSO in chemotherapy since it may increase resistance in some tumors (e.g., glioblastoma).
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1813fje;
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