Hyaluronidase induces murine L929 fibrosarcoma cells resistant to tumor necrosis factor and fas cytotoxicity in the presence of actinomycin D

Actinomycin D (ActD) enhances the potency of tumor necrosis factor-α (TNF-α) in killing cancer cells. However, it is determined in this study that murine L929 fibrosarcoma cells, when pretreated with bovine testicular hyaluronidase for 12–24h, became resistant to the cytotoxic effect of TNF-α in the presence of DNA interacalators, such as ActD, doxorubicin, and daunorubicin. Monoclonal anti-Fas antibody-mediated apoptosis in the presence of ActD was also blocked in hyaluronidase-pretreated L929 cells. Hyaluronidase failed to up-or downregulate the expression of apoptosis regulatory proteins, including Bcl-2, Bcl-x_L, ICH-1, and TIAR, suggesting that these proteins were not involved in the hyaluronidase-induced resistance to TNF/ActD. A semisynthetic polysulfated hyaluronic acid (HA) inhibited the increased TNF/ActD resistance, whereas unmodified HA, dextran sulfate, and naturally polysulfated glycosaminoglycans had no effect. Evidence is provided here that the induced resistance is related to serum fetuin and a novel intracellular 35-kDa TNF-binding protein (intra TBP). Under serum-free conditions, L929 became refractory to TNF/ActD cytotoxicity and hyaluronidase reversed the resistance. Exogenous fetuin increased L929 cell spreading and proliferation, and restored hyaluronidase-induction of TNF/ActD resistance in these serum-starved cells. Hyaluronidase failed to reduce the expression of TNF-receptors and their binding of TNF-α. However, binding and Western-blotting analyses revealed that hyaluronidase downregulated the intra-TBP. Overall, these observations suggest that serum fetuin and intra TBP are involved in the hyaluronidase induction of TNF/ActD resistance.     

JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis

Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis. Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death. PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells. WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53. Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis. Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types. Also, JNK1 blocked WOX1 prevention of cell cycle progression. By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33). The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation. Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1. A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation. This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis. Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.     

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.     

TGF-beta-induced matrix proteins inhibit p42/44 MAPK and JNK activation and suppress TNF-mediated IkappaBalpha degradation and NF-kappaB nuclear translocation in L929 fibroblasts

The role of transforming growth factor beta1 (TGF-beta1)-induced extracellular matrix proteins in the modulation of cellular response to the cytotoxic effect of tumor necrosis factor (TNF) or Fas ligand was investigated. Murine L929 fibroblasts were prestimulated with or without TGF-beta1 for 1-24 h and the resulting extracellular protein matrices were prepared. Unstimulated control L929 cells were then cultured on these matrices. Compared to control matrix-stimulated L929 cells, the TGF-beta1 matrix-stimulated cells resisted TNF killing in the presence of actinomycin D (ActD), but became more susceptible to killing by anti-Fas antibodies/ActD. The induced TNF resistance is independent of the NF-kappaB antiapoptotic effect. For example, exposure of TGF-beta1 matrix-stimulated L929 cells to TNF failed to result in IkappaBalpha degradation and NF-kappaB nuclear translocation or activation. Also, control matrix stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) in L929 cells, whereas TGF-beta1 matrix suppressed the activation. Nonetheless, in response to TNF, JNK activation was restored in the TGF-beta1 matrix-stimulated cells. By metabolic labeling, ammonium sulfate precipitation and N-terminal amino acid microsequencing, TGF-beta1 was shown to induce a novel matrix protein of 46 kDa (p46) from L929 cells. Adsorption of p46 by peptide antibodies against its N-terminus removed the TGF-beta1 matrix protein-mediated protection against TNF/ActD cytotoxicity and its enhancement of anti-Fas/ActD killing, indicating that p46 is responsible for these effects. Immunostaining of L929 cells revealed that the antibodies were bound to a membrane protein of 100 kDa (p100). Thus, the matrix p46 is likely derived from the released membrane p100.     

Characterization of serum adhesive proteins that block tumor necrosis factor-mediated cell death

Previously we have shown that TGF-beta1 protects murine L929 fibroblasts from TNF/ActD-mediated cell death by inducing the expression of an extracellular matrix TNF-resistance triggering (TRT) protein. TRT promotes TNF-resistance via activation of tyrosine and serine/threonine kinases in L929 cells. To examine the presence of TRT activity in serum (designated STRT), human sera were diluted, treated with or without PMSF and subjected to sequential ammonium sulfate precipitation (ASP). Aliquots of the ASP protein fractions were coated onto 96-well plates, followed by thorough washing. When L929 cells were seeded and cultured on the wells coated with STRT proteins, these cells resisted killing by TNF, TNF/ActD, doxorubicin and serum deprivation, but not by anti-Fas/ActD, staurosporine and ActD. STRT activity was found at the 15% ASP fraction of untreated sera, but shifted to the 20% ASP fraction of PMSF-treated sera. Two likely STRT proteins of approximately 226 and 265 kDa were found in these fractions, compared to the corresponding nonfunctional ASP fractions. Functionally, STRT was inactivated by trypsin, but not by 5 M salt, various serine and/or cysteine protease inhibitors, and antibodies against fibronectin, vitronectin, C1q, histidine-rich glycoprotein, CD44, chondroitin sulfate and hyaluronic acid. STRT failed to alter the expression of proteins involved in apoptosis such as RIP, ICH-1L, BCL-X, TIAR and IkappaBalpha, and could not induce IkappaBalpha degradation. The induced TNF-resistance could be reversed by treatment of STRT-stimulated cells with testicular hyaluronidase, as well as with tyrosine kinase inhibitors tyrophostin, lavendustin A and AG-490 (a selective inhibitor of JAK2 kinase). However, the STRT function could not be blocked by the MEK kinase inhibitor PD98059 and the NF-kappaB inhibitors curcumin and a synthetic inhibitor peptide for NF-kappaB translocation. Together, our data suggest that tyrosine kinase activation is involved in the STRT-mediated resistance to TNF and TNF/ActD in L929 cells.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

Hyaluronidase induction of a WW domain-containing oxidoreductase that enhances tumor necrosis factor cytotoxicity

To determine how hyaluronidase increases certain cancer cell sensitivity to tumor necrosis factor (TNF) cytotoxicity, we report here the isolation and characterization of a hyaluronidase-induced murine WW domain-containing oxidoreductase (WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear localization sequence, and a C-terminal alcohol dehydrogenase (ADH) domain. WOX1 is mainly located in the mitochondria, and the mitochondrial targeting sequence was mapped within the ADH domain. Induction of mitochondrial permeability transition by TNF, staurosporine, and atractyloside resulted in WOX1 release from mitochondria and subsequent nuclear translocation. TNF-mediated WOX1 nuclear translocation occurred shortly after that of nuclear factor-kappaB nuclear translocation, whereas both were independent events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH domains as determined using stable cell transfectants. In parallel with this observation, WOX1 also enhanced TRADD (TNF receptor-associated death domain protein)-mediated cell death in transient expression experiments. Antisense expression of WOX1 raised TNF resistance in L929 cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its significant down-regulation of the apoptosis inhibitors Bcl-2 and Bcl-x(L) (>85%), but up-regulation of pro-apoptotic p53 ( approximately 200%) by the ADH domain. When overexpressed, the ADH domain mediated apoptosis, probably due to modulation of expression of these proteins. The WW domains failed to modulate the expression of these proteins, but sensitized COS-7 cells to TNF killing and mediated apoptosis in various cancer cells independently of caspases. Transient cotransfection of cells with both p53 and WOX1 induced apoptosis in a synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds to the proline-rich region of p53 via its WW domains. Blocking of WOX1 expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is a mitochondrial apoptogenic protein and an essential partner of p53 in cell death.     

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.     

Hyaluronidase enhancement of TNF-mediated cell death is reversed by TGF-beta 1

Bothhyaluronidase and transforming growth factor (TGF)-1 play asignificant role in the development of prostate cancer. In this study,the regulation of tumor necrosis factor (TNF)-mediated cell death byhyaluronidase and TGF-1 was investigated. Preexposure of L929fibroblasts, prostate LNCaP cells, and epithelial Mv 1 Lu cells tohyaluronidase for a minimum of 12 h resulted in significant enhancementof cell death by TNF. Phosphorylation of p42 and p44 mitogen-activatedprotein (MAP) kinases was found by stimulation of L929 cells withhyaluronidase for 30 min, indicating that the Raf/MAPkinase-extracellular signal-regulating protein kinase (MEK)/MAP kinase pathway was activated. However, blocking the activation of upstream MAP kinase kinase (MEK 1 and 2 kinase) byPD-98059 failed to inhibit the hyaluronidase-enhanced TNF killing ofcells, suggesting that hyaluronidase-mediated degradation of extracellular matrix and membrane components may elicit multiple signaling pathways. As a potent stimulator of extracellular matrix protein synthesis, TGF-1 blocked the hyaluronidase-enhanced death ofL929 and LNCaP cells mediated by TNF. TGF-1 activatedprotein-tyrosine kinases in L929 cells, in which the tyrosine kinaseinhibitors lavendustin A and tyrphostin blocked the activation as wellas the TGF-1 inhibition of hyaluronidase effects. Functionalantagonism was also observed between hyaluronidase and TGF-1 in cellgrowth regulation. For example, TGF-1-mediated suppression ofepithelial Mv 1 Lu cell growth was abolished by hyaluronidase. Overall,it is demonstrated in this study that hyaluronidase reciprocally antagonized TGF-1 in the modulation of cell proliferation and TNF-mediated death.

     

Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.     

c-Jun N-terminal kinase contributes to apoptotic synergy induced by tumor necrosis factor-related apoptosis-inducing ligand plus DNA damage in chemoresistant,p53 inactive mesothelioma cells

Apoptotic resistance of cancer cells may be overcome by the combination of treatments that activate the two major apoptotic pathways: (i) the death receptor pathway activated by death ligands and (ii) the DNA damage pathway activated by chemotherapy. We have previously shown that mesothelioma cells, resistant to most treatments, are sensitive to the combination of the death ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) plus chemotherapy. We investigated a possible role for c-Jun N-terminal kinase (JNK) in the synergistic effect, knowing that JNK can be activated separately by TRAIL and by DNA damage. We chose to study the M28 and REN human mesothelioma cell lines, which are p53-inactivated, to avoid an interaction between p53 and JNK. We showed that JNK was activated by TRAIL and by etoposide and that the activation was enhanced by the combination of the two treatments. We found this activation to be caspase-independent. To inhibit the JNK pathway, we used either dominant-negative constructs of JNK1 and JNK2 (compared with dominant-negative caspase 9) or a chemical inhibitor of the JNK pathway (SP600125). In cells treated with TRAIL plus etoposide, JNK inhibition increased cell survival and decreased apoptosis significantly. In transfected M28 cells, the effect of JNK inhibition was as great as that of the dominant-negative caspase 9 construct. We conclude that JNK contributes to the synergistic effect of TRAIL combined with DNA damage by mediating signals independent of p53 leading to apoptosis.     

Transforming growth factor beta 1 induces apoptosis through cleavage of BAD in a Smad3-dependent mechanism in FaO hepatoma cells

Transforming growth factor beta (TGF-beta) induces apoptosis in a variety of cells. We have previously shown that TGF-beta 1 rapidly induces apoptosis in the FaO rat hepatoma cell line. We have now studied the effect of TGF-beta 1 on the expression of different members of the Bcl-2 family in these cells. We observed no detectable changes in the steady-state levels of Bcl-2, Bcl-X(L), and Bax. However, TGF-beta 1 induced caspase-dependent cleavage of BAD at its N terminus to generate a 15-kDa truncated protein. Overexpression of the 15-kDa truncated BAD protein enhanced TGF-beta 1-induced apoptosis, whereas a mutant BAD resistant to caspase 3 cleavage blocked TGF-beta 1-induced apoptosis. Overexpression of Smad3 dramatically enhanced TGF-beta 1-induced cleavage of BAD and apoptosis, whereas antisense Smad3 blocked TGF-beta 1-induced apoptosis and BAD cleavage. These results suggest that TGF-beta 1 induces apoptosis through the cleavage of BAD in a Smad3-dependent mechanism.     

Characterization of an apoptosis inhibitory domain at the C-termini of FE65-like protein

TR2(L) is a 56-amino-acid polypeptide that has been shown to block TNF cytotoxicity. FE65-like (FE65L) proteins possess this conserved TR2(L) sequence at their C-termini, whereas variations in the sequences are found in the FE65 proteins. To further analyze the antiapoptotic function of TR2(L), here we utilized an isolated murine partial FE65L cDNA that encodes an N-terminal phosphotyrosine-binding domain (PTB) and the conserved C-terminal TR2(L) sequence. When L929 cells were stably transfected with the FE65L cDNA or its 3' end TR2(L) DNA sequence, these cells became resistant to TNF killing. Replacement of the N-terminal PTB domain with GFP failed to abolish the FE65L-mediated TNF resistance. Ablation of the C-terminal TR2(L) sequence through frame-shift mutation resulted in a complete loss of the FE65L function against TNF. Various protein kinase inhibitors, including lavendustin A, tyrphostin, H7, and staurosporine, which may affect the PTB domain function, could not abolish the FE65L-mediated TNF resistance. A prolonged exposure of L929 cells to these inhibitors for 24 h resulted in cell death, whereas FE65L significantly blocked the cell death. Polyclonal antibodies were generated against a synthetic peptide and shown to interact with a 38-kDa FE65L in L929 cells. Hyaluronidase downregulates the expression of FE65L gene and protein in L929 cells, and this correlates with its enhancement of TNF killing of these cells. Together, our data indicate that the TR2(L) amino acid sequence is an apoptosis-inhibitory domain commonly present in the FE65 and FE65-like family proteins.     

Modes of L929 cell death induced by TNF-alpha and other cytotoxic agents.

Recent studies have variably reported that tumour necrosis factor alpha (TNF-alpha) induces either necrosis or apoptosis in L929 cells. This study was undertaken to better characterize the mode of death induced in L929 cells by this agent. We determined the effects of exposure to TNF-alpha and other cytotoxic agents on cell size and morphology, cell membrane permeability, exposure of phosphatidylserine at the cell surface, nuclear morphology and fragmentation of DNA. Our results suggest that L929 cells treated with TNF-alpha alone show nuclear changes and a pattern of DNA fragmentation that are atypical of apoptosis. In contrast, our results demonstrate that, when augmented with actinomycin D, TNF-alpha induces classical apoptosis in L929 cells. We also provide the first report that, in L929 cells, staurosporine induces classical apoptosis and colchicine induces a form of apoptosis lacking internucleosomal DNA fragmentation. Previous studies of TNF-alpha-induced death in L929 cells relied on measurements of only one or two parameters to define the mode of death. Overall, our results suggest that in future cellular or biochemical studies of the effects of TNF-alpha on L929 cells it will be prudent to characterize the mode of death in each case using a multi-parameter approach, as done here.     

Cardiotoxin III-induced apoptosis is mediated by Ca2+-dependent caspase-12 activation in K562 cells

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. When K562 cells were treated with CTX III, cytosolic calcium concentration was rapidly and persistently increased. This CTX III-induced cell death was partially reversed by pretreatment with BAPTA/AM (20 microM), a chelator of intracellular Ca2+. Moreover, CTX III-induced apoptotic signals, such as caspase-12 and c-Jun N-terminal kinase (JNK) activation, were induced in a time-dependent manner and inhibited by BAPTA/AM. In contrast, the neutral protease micro-calpain, a key enzyme in endoplasmic reticulum (ER) stress-related apoptosis via caspase-12 activation, was unchanged during apoptosis. Taken together, our findings suggest CTX III-induced apoptosis is triggered by Ca2+ influx, then activated caspase-12 and JNK through micro-calpain-independent cascade, and consequently caused apoptosis.     

Glutathione transferases and development of new principles to overcome drug resistance

Chemoresistance is a multifactorial phenomenon and many studies clearly show that a coordinated expression of efflux transporter proteins and phase II conjugating enzymes in tumor cells is linked to the development of the multidrug resistance phenotype. In particular, the overexpression of glutathione S-transferases and efflux pumps in tumors may reduce the reactivity of various anticancer drugs. In recent years it has become evident that glutathione S-transferases are also involved in the control of apoptosis through the inhibition of the JNK signaling pathway. As such, the glutathione S-transferase superfamily has become the focus of extensive pharmaceutical research in attempt to generate more efficient anticancer agents. Here we present an overview of the GST inhibitors and the GST-activated pro-drugs utilized to date to overcome drug resistance.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.