HIF-1 Regulation of Chondrocyte Apoptosis: Induction of the Autophagic Pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

A role for c-FLIPL in the regulation of apoptosis,autophagy, and necroptosis in T lymphocytes

Caspase 8 plays a dual role in the survival of T lymphocytes. Although active caspase 8 mediates apoptosis upon death receptor signaling, the loss of caspase 8 activity leads to receptor-interacting protein (RIP)-1/RIP-3-dependent necrotic cell death (necroptosis) upon TCR activation. The anti-apoptotic protein c-FLIP (cellular caspase 8 (FLICE)-like inhibitory protein) suppresses death receptor-induced caspase 8 activation. Moreover, recent findings suggest that c-FLIP is also involved in inhibiting necroptosis and autophagy. It remains unclear whether c-FLIP protects primary T lymphocytes from necroptosis or regulates the threshold at which autophagy occurs. Here, we used a c-FLIP isoform-specific conditional deletion model to show that c-FLIP_L-deficient T cells underwent RIP-1-dependent necroptosis upon TCR stimulation. Interestingly, although previous studies have only described necroptosis in the absence of caspase 8 activity, we found that pro-apoptotic caspase 8 activity and apoptosis were also enhanced in c-FLIP_L-deficient T lymphocytes. Furthermore, c-FLIP_L-deficient T cells exhibited enhanced autophagy, which served a cytoprotective function. Together, these findings indicate that c-FLIP_L plays an important antinecroptotic role and is a key regulator of apoptosis, autophagy, and necroptosis in T lymphocytes.     

CFLAR/c-FLIPL

Necroptosis, a caspase-independent, receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)/RIPK3-dependent necrotic cell death, occurs in cells when apoptosis is blocked. A high level of macroautophagy (herein referred to as autophagy) is usually detected in necroptotic cells, although it is still controversial as to whether excessive autophagy leads to cell death or is cytoprotective. In a recently published paper, we show that the anti-apoptotic protein CFLAR (CASP8 and FADD-like apoptosis regulator) long isoform (CFLAR_L) plays a critical role in all three fundamental intracellular processes: autophagy, necroptosis, and apoptosis in T lymphocytes. CFLAR_L-deficient T cells suffer from severe cell death upon T cell receptor stimulation, in which both apoptosis and necroptosis are involved. Autophagy is enhanced in both naïve and activated CFLAR_L-deficient T cells and plays a cytoprotective function. Here, we summarize our findings and discuss the future direction in the study of the interplay of autophagy, apoptosis and necroptosis in T lymphocytes.     

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.     

Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound,Z18

Inhibitors of Bcl-X_L/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-X_L/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis. Unexpectedly, the inhibition of Beclin 1 and phosphatidylinositol 3-kinase have no obvious effect on Z18-induced autophagy in HeLa cells, implying that it is a non-canonical Beclin 1-independent autophagy. Meanwhile, the accumulation of autophagosomes is positively correlated with Z18-induced cell death and the full flux of autophagy is not necessary.     

Autophagy and CD4+ T lymphocyte destruction by HIV-1

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.     

Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.     

Anti‐cell death engineering of CHO cells: Co‐overexpression of Bcl‐2 for apoptosis inhibition,Beclin‐1 for autophagy induction

Genetic engineering approaches to inhibit cell death in Chinese hamster ovary (CHO) cell cultures have been limited primarily to anti‐apoptosis engineering. Recently, autophagy has received attention as a new anti‐cell death engineering target in addition to apoptosis. In order to achieve a more efficient protection of cells from the stressful culture conditions, the simultaneous targeting of anti‐apoptosis and pro‐autophagy in CHO cells (DG44) was attempted by co‐overexpressing an anti‐apoptotic protein, Bcl‐2, and a key regulator of autophagy pathway, Beclin‐1, respectively. Co‐overexpression of Bcl‐2 and Beclin‐1 exhibited a longer culture period as well as higher viability during serum‐free suspension culture, compared with the control (without co‐overexpression of Bcl‐2 and Beclin‐1) and Bcl‐2 overexpression only. In addition to the efficient inhibition of apoptosis by Bcl‐2 overexpression, Beclin‐1 overexpression successfully induced the increase in the autophagic marker protein, LC3‐II, and autophagosome formation with the decrease in mTOR activity. Co‐immunoprecipitation and qRT‐PCR experiments revealed that the enforced expression of Beclin‐1 increased Ulk1 expression and level of free‐Beclin‐1 that did not bind to the Bcl‐2 despite the Bcl‐2 overexpression. Under other stressful culture conditions such as treatment with sodium butyrate and hyperosmolality, co‐overexpression of Bcl‐2 and Beclin‐1 also protected the cells from cell death more efficiently than Bcl‐2 overexpression only, implying the potential of autophagy induction. Taken together, the data obtained here provide the evidence that pro‐autophagy engineering together with anti‐apoptosis engineering yields a synergistic effect and successfully enhances the anti‐cell death engineering of CHO cells. Biotechnol. Bioeng. 2013; 110: 2195–2207. © 2013 Wiley Periodicals, Inc.     

Autophagy is induced in CD4+ T cells and important for the growth factor-withdrawal cell death

Autophagy is a tightly regulated catabolic mechanism that degrades proteins and organelles. Autophagy mediates programmed cell death under certain conditions. To determine the role of autophagy in T cells, we examined, in mouse CD4+ T cells, conditions under which autophagy is induced and alterations of the cell fate when autophagy is blocked. We have found that resting naive CD4+ T cells do not contain detectable autophagosomes. Autophagy can be observed in activated CD4+ T cells upon TCR stimulation, cytokine culturing, and prolonged serum starvation. Induction of autophagy in T cells requires JNK and the class III PI3K. Autophagy is inhibited by caspases and mammalian target of rapamycin in T cells. Interestingly, more Th2 cells than Th1 cells undergo autophagy. Th2 cells become more resistant to growth factor-withdrawal cell death when autophagy is blocked using either chemical inhibitors 3-methyladenine, or by RNA interference knockdown of beclin 1 and Atg7. Therefore, autophagy is an important mechanism that controls homeostasis of CD4+ T cells.     

Vitamin D3 induces autophagy of human myeloid leukemia cells

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.     

The in vitro cleavage of the hAtg proteins by cell death proteases

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.     

Activation-induced cell death in human T cells is a suicidal process regulated by cell density but superantigen induces T cell fratricide

Repeated ligation of the TCR results in apoptosis (activation-induced cell death; AICD). Superantigens such as Staphylococcal enterotoxin B (SEB) are particularly efficient at inducing AICD in T cells. We investigated whether apoptosis in human T cell subsets was due to fratricide (killing of neighboring cells) or suicide (cell autonomous death). AICD of Th1, Th2, Tc1, and Tc2 effector cells was dramatically enhanced at low cell densities and could be observed in single cell microcultures. AICD was unaffected by adhesion molecules or neighboring cells undergoing AICD, confirming the predominance of a suicidal mechanism. However, SEB was able to induce fratricidal apoptosis of type 1, but not type 2 cells. Fratricide was also observed when unstimulated T cells were exposed to activated Tc1 effector cells. Thus, AICD is tightly regulated to allow clonal T cell expansion and memory cell generation, but superantigens may subvert this process by allowing T cell fratricide.     

The in vitro cleavage of the hAtg proteins by cell death proteases

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.     

DJ‐1 inhibits autophagy activity of prostate cancer cells by repressing JNK–Bcl2–Beclin1 signaling

The regulation of DJ‐1 on AR signaling plays an important role in the pathogenesis of prostate cancer (PCa). DJ‐1 could alter autophagy and regulate Beclin1‐involved autophagy response through JNK‐dependent pathway. JNK is known to mediate autophagy through Bcl2–Beclin1 complex. Therefore, this study aimed to investigate the significance of autophagy in DJ‐1‐modulated PCa cells. The current studies showed that DJ‐1 overexpression in LNCaP decreased LC3 transformation and autophagosome formation. However, DJ‐1 knockdown exerted the opposite effect. Moreover, DJ‐1 silencing inhibited survival and promoted death in LNCaP, which was recovered by autophagy inhibition with 3‐MA. In addition, DJ‐1 overexpression inhibited the phosphorylation of JNK and Bcl2, and the dissociation of Beclin1 and Bcl2; while the effect of silencing DJ‐1 was completely opposite. More important, JNK activated by anisimycin inhibited the proliferation and promoted death of DJ‐1‐overexpressed LNCaP while increasing LC3 transformation and LC3‐puncta formation, but these results were reversed by the decrease of Beclin1 (by spautin‐1). In contrast, when DJ‐1 was silenced, the death of LNCaP, LC3 transformation, and LC3‐puncta formation were inhibited by JNK inhibitor SP600125, which promoted cell proliferation. However, Bcl2 inhibition (by ABT737) reversed all the effects of SP600125. Our results suggested that DJ‐1 in PCa cells could promote the growth of PCa through autophagy inhibition, and JNK–Bcl2–Beclin1 signaling played an important role in it. The study provided new insights into the role of DJ‐1 in the development of PCa.     

Autophagy delays apoptotic death in breast cancer cells following DNA damage

Early signaling in camptothecin-treated MCF-7 cells followed an intrinsic pathway, but death was delayed and late events exhibited few hallmarks of apoptosis. BH3-only proteins, such as Noxa, Puma and BimEL, were activated and localized to mitochondrial sites within 24 h following drug exposure. However, caspase activity was low and death was unaffected by caspase inhibition. Transmission electron micrographs showed the presence of large vacuoles in drug-treated cells. An autophagic survival response has been attributed to MCF-7 cells following nutrient starvation or exposure to tamoxifen. Here, we show that autophagy also plays an important role in the delayed DNA damage response. Confocal microscopy revealed colocalization of mitochondria with large autophagic vacuoles and inhibitors of autophagy increased mitochondrial depolarization and caspase-9 activity, and accelerated cell death. Furthermore, downregulation of autophagy proteins, Beclin 1 and Atg7, unmasked a caspase-dependent, apoptotic response to DNA damage. We propose that a post-mitochondrial caspase cascade is delayed as a result of early disposal of damaged mitochondria within autophagosomes. Our data also suggest that the use of autophagy as a means of delaying apoptosis or prolonging survival may be characteristic of noninvasive breast tumor cells. These studies underscore a potential role for autophagy inhibitors in combination with conventional chemotherapeutic drugs in early breast cancer therapy.     

BECN1 and BIM interactions with MCL-1 determine fludarabine resistance in leukemic B cells

The purine analog fludarabine (Fd) is an essential therapeutic for chronic lymphocytic leukemia (CLL). Innate or acquired resistance to Fd is a significant clinical problem and is largely mediated by increased expression of BCL-2 family members. The antiapoptotic BCL-2 family proteins inhibit both apoptosis and autophagy, therefore, downregulation of antiapoptotic BCL-2 family proteins and enhanced autophagy must coexist in cells dying in response to an apoptosis inducing therapeutic. However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. Here, we examined the role of the BCL-2 family in regulating cell death and autophagy in leukemic cell lines and their derivative isogenic Fd-resistant (FdR) cells. MCL-1 degradation following Fd treatment freed the proapoptotic effectors BIM and BECN1, thus leading to cell death-associated autophagy in Fd-sensitive cells. However, in FdR cells, low BIM expression and BECN1 sequestration by MCL-1 prevented cell death. Consistently, in sensitive cells inhibition of apoptosis using siBIM and of both the early-phase autophagy nucleation steps by siBECN1, shATG7 or 3-methyladenine and the late-phase autophagy by shLAMP2, significantly reduced Fd-induced cell death. Paradoxically, FdR cells were addicted to basal autophagy, which was dependent on AMP-activated protein kinase (AMPK) but not BECN1. Moreover, in FdR cells, inhibition of autophagy by shLAMP2, but not siBECN1, enhanced cell death. The BH3-mimetic obatoclax released BIM and BECN1 from MCL-1 in Fd-sensitive and BECN1 from MCL-1 in FdR cells, and was effective at killing both Fd-sensitive and - resistant leukemic cells, including primary CLL cells. Therefore, a differential regulation of autophagy through BECN1 and AMPK signaling in Fd-sensitive and - resistant cells determines the different possible outcomes of autophagy inhibition. These findings suggest effective means to overcome Fd resistance by induction of BIM-dependent apoptosis and activation of BECN1-dependent autophagy.     

Autophagy as the main means of cytotoxicity by glucocorticoids in hematological malignancies

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. We have recently shown that dexamethasone induces autophagy in lymphoid leukemia cells and in this particular setting this cell death modality is a prerequisite for the efficient killing of the leukemic cells by dexamethasone.¹ Hence, inhibition of autophagy by siRNA-mediated silencing of Beclin 1, as well as chemical inhibition of type III PtdIns3K, inhibits apoptosis, demonstrating an important role of autophagy in dexamethasone-induced cell death. In this brief report, we review these findings and introduce the Multiple Myeloma cells as a novel system to study autophagy in response to dexamethasone.     

Tumor cells can evade dependence on autophagy through adaptation

The autophagy-lysosome and the proteasome constitute the two major intracellular degradation systems. Suppression of the proteasome promotes autophagy for compensation and simultaneous inhibition of autophagy can selectively increase apoptosis in transformed cells, but not in untransformed or normal cells. Transformed cells are thus more dependent on autophagy for survival. However, it is unclear whether long-term autophagy inhibition/insufficiency would affect such dependency. To address this question, we transformed wild-type and autophagy-deficient cells lacking a key autophagy-related gene Atg5 with activated Ras. We found that such transformation did not make the autophagy-deficient tumor cells more susceptible to proteasome inhibitors than the wild type tumor cells, although the transformed cells were in general more sensitive to proteasome inhibition. We then compared the effect of acute versus constitutive knock-down of a key autophagy initiating molecule, Beclin 1, in an already transformed cancer cell line. In a wild-type U251 glioblastoma cell line (autophagy intact), increased sensitivity to proteasome inhibition was induced immediately after the knock-down of Beclin 1 expression with a specific siRNA (acute autophagy deficiency). On the other hand, when the tumor cell line was selected over a long period to achieve constitutive knock-down of Beclin 1, its sensitivity to proteasome inhibitors was no higher than that of the wild-type tumor cells. These results suggest that long-term autophagy deficiency either before or after oncogenic transformation can render the tumor cell survival independent of the autophagic activity, and the response to chemotherapy is no longer affected by the manipulation of the autophagy status.