Pathologic Processing of Lung Cancer Resection Specimens After Neoadjuvant Therapy.

Neoadjuvant treatment of non-small cell lung cancer challenges the traditional processing of pathology specimens. Induction therapy before resection allows evaluation of the efficacy of neoadjuvant agents at the time of surgery. Many clinical trials use pathologic tumor response, measured as major pathologic response (MPR, ≤10% residual viable tumor [RVT]) or complete pathologic response (CPR, 0% RVT) as a surrogate of clinical efficacy. Consequently, accurate pathologic evaluation of RVT is crucial. However, pathologic assessment has not been uniform, which is particularly true for sampling of the primary tumor, which instead of the traditional processing, requires different tissue submission because the focus has shifted from tumor typing alone to RVT scoring. Using a simulation study, we analyzed the accuracy rates of %RVT, MPR, and CPR of 31 pretreated primary lung tumors using traditional grossing compared with the gold standard of submitting the entire residual primary tumor and identified the minimum number of tumor sections to be submitted to ensure the most accurate scoring of %RVT, MPR, and CPR. Accurate %RVT, MPR, and CPR calls were achieved in 52%, 87%, and 81% of cases, respectively, using the traditional grossing method. Accuracy rates of at least 90% for these parameters require either submission of all residual primary tumor or at least 20 tumor sections. Accurate %RVT, MPR, and CPR scores cannot be achieved with traditional tumor grossing. Submission of the entire primary tumor, up to a maximum of 20 sections, is required for the most accurate reads.

Prognostic significance of RNA-dependent protein kinase on non-small cell lung cancer patients.

PURPOSE: The role of RNA-dependent protein kinase (PKR) in antiviral defense mechanisms and in cellular differentiation, growth, and apoptosis is well known, but the role of PKR in human lung cancer remains poorly understood. To explore the role of PKR in human lung cancer, we evaluated the expression of PKR in tissue microarray (TMA) specimens from both non-small cell lung cancer (NSCLC) and normal human bronchial epithelium tissue. EXPERIMENTAL DESIGN: TMA samples (TMA-1) from 231 lung cancers were stained with PKR antibody and validated on TMA-2 from 224 lung cancers. Immunohistochemical expression score was quantified by three pathologists independently. Survival probability was computed by the Kaplan-Meier method. RESULTS: The NSCLC cells showed lower levels of PKR expression than normal bronchial epithelium cells did. We also found a significant association between lower levels of PKR expression and lymph node metastasis. We found that loss of PKR expression is correlated with a more aggressive behavior, and that a high PKR expression predicts a subgroup of patients with a favorable outcome. Univariate and multivariate Cox proportional hazards regression models showed that a lower level of PKR expression was significantly associated with shorter survival in NSCLC patients. We further validated and confirmed PKR to be a powerful prognostic factor in TMA-2 lung cancer (hazard ratio, 0.22; P < 0.0001). CONCLUSIONS: Our findings first indicate that PKR expression is an independent prognostic variable in NSCLC patients.

KLF4 inhibition of lung cancer cell invasion by suppression of SPARC expression.

Krüppel-Like Factor 4 (KLF4) functions as a tumor suppressor in some cancers, but its molecular mechanism is not clear. Our recent study also showed that the expression of KLF4 is dramatically reduced in primary lung cancer tissues. To investigate the possible role of KLF4 in lung cancer, we stably transfected KLF4 into cells from lung cancer cell lines H322 and A549 to determine the cells' invasion ability. Our results showed that ectopic expression of KLF4 extensively suppressed lung cancer cell invasion in Matrigel. This effect was independent of KLF4-mediated p21 up-regulation because ectopic expression of p21 had minimal effect on cell invasion. Our analysis of the expression of 12 genes associated with cell invasion in parental, vector-transfected, and KLF4-transfected cells showed that ectopic expression of KLF4 resulted in extensively repressed expression of secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix protein that plays a role in tumor development and metastasis. Knockdown of SPARC expression in H322 and A549 cells led to suppression of cell invasion, comparable to that observed in KLF4-transfected cells. Moreover, retrovirus-mediated restoration of SPARC expression in KLF4-transfected cells abrogated KLF4-induced anti-invasion activity. Together, our results indicate that KLF4 inhibits lung cancer cell invasion by suppressing SPARC gene expression.

Structural mapping of post-translational modifications in human interleukin-24: role of N-linked glycosylation and disulfide bonds in secretion and activity.

Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications.

Putative tumor-suppressive function of Kruppel-like factor 4 in primary lung carcinoma.

PURPOSE: Krüppel-like factor 4 (KLF4) is a zinc-finger protein that plays important roles in stem cells and the development of gastric cancers. However, the role of KLF4 in primary lung cancer is unknown. The purpose of this study is to determine possible roles of KLF4 in lung cancer. EXPERIMENTAL DESIGN: The KLF4 expression in primary lung cancer tissues and case-matched normal lung tissues were determined by protein and mRNA analyses. The effects of KLF4 on cell proliferation, clonogenic formation, and cell cycle progression were determined in cultured lung cancer cells or bronchial epithelial cells after enforced KLF4 overexpression or small interfering RNA knockdown. The in vivo antitumor activity of KLF4 was evaluated by using stably transfected lung cancer cells and by adenovector-mediated gene delivery. The effect of KLF4 in regulating p21 and cyclin D1 was also evaluated. RESULTS: KLF4 protein and mRNA levels were dramatically decreased in most primary lung tumors compared with in case-matched normal lung tissues. Enforced expression of KLF4 resulted in marked inhibition of cell growth and clonogenic formation. The tumor-suppressive effect of KLF4 was associated with its role in up-regulating p21 and down-regulating cyclin D1, leading to cell cycle arrest at the G(1)-S checkpoint. Knockdown of KLF4 promoted cell growth in immortalized human bronchial epithelial cells. The enforced expression of KLF4 gene to lung cancer cells by ex vivo transfection or adenovector-mediated gene transfer suppressed tumor growth in vivo. CONCLUSIONS: Our results suggest that KLF4 plays an important role in suppressing the growth of lung carcinoma.

Double-stranded RNA-dependent protein kinase-dependent apoptosis induction by a novel small compound.

The interferon-induced, double-stranded RNA-dependent protein kinase (PKR) can play critical roles in inhibiting virus replication and inducing apoptosis. To develop new agents that may inhibit viral replication or induce apoptosis in cancer cells via the PKR signaling pathway, we screened a chemical library for compounds that have differential cytotoxic effects on wild-type [mouse embryonic fibroblast (MEF)/PKR(+/+)] and PKR-knockout [MEF/PKR(-/-)] mouse embryonic fibroblast cells. We identified a synthetic compound, BEPP [1H-benzimidazole1-ethanol,2,3-dihydro-2-imino-a-(phenoxymethyl)-3-(phenylmethyl)-,monohydrochloride], that induces a cytotoxic effect more effectively in MEF/PKR(+/+) cells than in MEF/PKR(-/-) cells. BEPP also relatively effectively inhibited the growth of a human lung cancer cell line overexpressing PKR, compared with other cancer cell lines. In sensitive cells, BEPP induced apoptosis with activation of caspase-3. Treatment with BEPP led to increased phosphorylation of PKR and eIF2alpha, increased expression of BAX, and decreased expression of Bcl-2. BEPP-induced apoptosis was PKR dependent and was blocked by the adenovector expressing the dominant-negative PKR. Furthermore, pretreatment of HeLa cells at a noncytotoxic dose of BEPP effectively inhibited Vaccinia virus replication. Together, our results suggest that BEPP and its analogs may induce PKR-dependent apoptosis and inhibition of viral replication and that they can be a potential anticancer or anti-virus agent.

Inhibition of RNA-dependent protein kinase (PKR) leads to cancer cell death and increases chemosensitivity.

RNA-dependent protein kinase is an interferon-induced, double-stranded (ds), RNA-activated serine/threonine protein kinase involved in the eukaryotic response to viral infection. While PKR also functions in cellular differentiation, growth control and apoptosis, its role in human cancer remains poorly understood. To explore a role for PKR in human cancer, we evaluated PKR expression and function in a series of cancer cell lines from different tumor types. We observed that PKR protein expression is high in various cancer cells and low in normal cells. Knockdown of PKR protein expression by PKR siRNA induced cell death, indicating a PKR-dependent survival pathway under normal growth conditions. Inhibition of PKR signaling using a dominant negative adenoviral PKR mutant (Ad-Delta6PKR) also induced cancer cell apoptosis via a mechanism that blocks activation of AKT-mediated survival while simultaneously inducing ER stress. ER stress-mediated apoptosis was evidenced by unregulated expression of phosphorylated JNK (p-JNK), phosphorylated cJun (p-cJun), and caspase-4 and was significantly reduced in cancer cells treated with JNK and caspase-4 inhibitors. We further demonstrated that inhibition of PKR signaling via either siRNA or Ad-Delta6PKR sensitizes cancer cells to etoposide or cisplatin-mediated cell death. Our results suggest a rationale to develop therapeutic strategies that target PKR signaling in human cancer cells.

Adenoviral endoplasmic reticulum-targeted mda-7/interleukin-24 vector enhances human cancer cell killing.

We developed several adenoviral vectors designed to target MDA-7 expression to different subcellular compartments [endoplasmic reticulum (ER), mitochondria, nucleus, and cytosol] and evaluated their ability to enhance apoptosis. Adenoviral ER-targeted mda-7/interleukin-24 vector (Ad-ER-mda7) selectively and effectively inhibited the growth and proliferation of lung (A549 and H1299) and esophageal (Seg1 and Bic1) cancer cells by enhancing cell killing. Both Ad-mda7 and Ad-ER-mda7 activated a novel pathway of ER stress-induced apoptosis characterized by unregulated expression of phosphorylated JNK, phosphorylated c-Jun, and phosphorylated RNA-dependent protein kinase. Caspase-4 activation mediated Ad-mda7- and Ad-ER-mda7-induced cell death. In addition, Ad-mda7- and Ad-ER-mda7-mediated growth inhibition correlated with activation of ER molecular markers RNA-dependent protein kinase and JNK both in vitro (in Ad-mda7- or Ad-ER-mda7-treated lung cancer cells) and in vivo. These findings suggest that vectors targeting the ER (Ad-ER-mda7) may be more effective in cancer gene therapy possibly through more effective induction or ER stress pathways.

Development of Ad-mda7/IL-24-resistant lung cancer cell lines.

Many cancers can become resistant to repeated administration of even the most effective therapeutic agents. In developing adenoviral mda-7/IL-24 (Ad-mda-7/IL-24) therapy for lung cancer, we have anticipated this potential clinical problem by attempting to identify the molecular mechanisms of Ad-mda7/IL-24 resistance in several Ad-mda7/IL-24-resistant lung cancer cell lines that we have developed. For the present study, we established four Admda7- resistant cell lines by repeated selection of resistant clones of parental Ad-mda7-sensitive A549 cells: two lines (A549R1 and A549R2) resistant to both adenoviral vector and the mda-7 gene and two (A549R3 and A549R4) resistant to the therapeutic mda-7 gene only. As shown by western blot analysis of several known anti-apoptotic proteins, parental A549 and resistant A549R3 cells expressed similar levels of AKT and phosphorylated AKT (p-AKT), whereas resistant A549R3 and A549R4 cells expressed higher levels of bcl-2 and lower levels of bcl-xL than did their parental cells. As shown by flow-cytometric analysis, treating resistant A549R3 and A549R4 cells with a combination of Ad-mda7 and 17-allyl-amino-17-demethoxygeldanamycin (17AAG) (50 nM) for 48 hours enhanced apoptosis. Together, these in vitro findings indicate that an antiapoptotic mechanism may underlie Ad-mda7 resistance and that such resistance can be overcome by addition of 17AAG. Further investigations along these lines are warranted.

The double-stranded RNA-activated protein kinase mediates radiation resistance in mouse embryo fibroblasts through nuclear factor kappaB and Akt activation.

PURPOSE: Activation of the double-stranded RNA-activated protein kinase (PKR) leads to the induction of various pathways including the down-regulation of translation through phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF-2alpha). There have been no reports to date about the role of PKR in radiation sensitivity. EXPERIMENTAL DESIGN: A clonogenic survival assay was used to investigate the sensitivity of PKR mouse embryo fibroblasts (MEF) to radiation therapy. 2-Aminopurine (2-AP), a chemical inhibitor of PKR, was used to inhibit PKR activation. Nuclear factor-kappaB (NF-kappaB) activation was assessed by electrophoretic mobility shift assay (EMSA). Expression of PKR and downstream targets was examined by Western blot analysis and immunofluorescence. RESULTS: Ionizing radiation leads to dose- and time-dependent increases in PKR expression and function that contributes to increased cellular radiation resistance as shown by clonogenic survival and terminal nucleotidyl transferase-mediated nick end labeling (TUNEL) apoptosis assays. Specific inhibition of PKR with the chemical inhibitor 2-AP restores radiation sensitivity. Plasmid transfection of the PKR wild-type (wt) gene into PKR(-/-) MEFs leads to increased radiation resistance. The protective effect of PKR to radiation may be mediated in part through NF-kappaB and Akt because both NF-kappaB and Akt are activated after ionizing radiation in PKR+/+ but not PKR-/- cells. CONCLUSIONS: We suggest a novel role for PKR as a mediator of radiation resistance modulated in part through the protective effects of NF-kappaB and Akt activation. The modification of PKR activity may be a novel strategy in the future to overcome radiation resistance.

Role for the double-stranded RNA-activated protein kinase PKR in Ad-TNF-alpha gene therapy in esophageal cancer.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha) is a cytokine with direct antitumor activity. Clinical trials with TNF-alpha have been limited because of the severe side effects of systemic administration. Gene therapy with an adenoviral vector allows delivery of high local doses of TNF-alpha. Activation of protein kinase R (PKR) has been implicated as a general transducer of apoptosis in response to a variety of different stimuli including TNF-alpha. We, therefore, evaluated the role of PKR in Ad-TNF-alpha-induced apoptosis in esophageal cancer cells. METHODS: A tetracycline-responsive adenoviral vector was used to transfect the TNF-alpha gene (Ad-TNF-alpha) into human esophageal cancer cell lines Bic1, Seg1 and TT, as well as in transformed PKR(+/+) and PKR(-/-) early-passage mouse embryo fibroblasts. Ad-luciferase, Ad-Bak, and mock infection with phosphate buffered saline solution were used as controls. Gene expression was determined by Western blot analysis. Apoptosis was detected by propidium iodide staining and fluorescence-activated cell sorter analysis. RESULTS: Overexpression of TNF-alpha in the lysate was evident in all cell lines treated with Ad-TNF-alpha. Treatment with Ad-TNF-alpha was associated with PKR upregulation and induction of apoptosis. Inhibition of TNF-alpha expression by tetracycline resulted in downregulation of PKR and decreased apoptosis. Transduction of PKR(+/+) and PKR(-/-) mouse embryo fibroblasts with Ad-TNF-alpha demonstrated that Ad-TNF-alpha-induced apoptosis was mediated in part through a PKR-dependent process. CONCLUSIONS: These results suggest that Ad-TNF-alpha-mediated apoptosis in esophageal cancer cell lines is dependent in part on PKR upregulation. Strategies to enhance PKR upregulation may allow increased Ad-TNF-alpha antitumoral activity in the treatment of esophageal cancer.

Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR.

We previously reported that adenoviral-mediated overexpression of the melanoma differentiation-associated gene-7 (Ad-mda7; approved gene symbol IL24) leads to the rapid induction of PKR and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. To evaluate the mechanism by which Ad-mda7 activates PKR, we studied the interaction between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, intracellular and extracellular MDA-7 protein was generated, leading to dose- and time-dependent PKR induction. Purified MDA-7 protein administered extracellularly did not induce PKR or apoptosis, suggesting that Ad-mda7-mediated PKR activation and apoptosis were not dependent on extracellular MDA-7 protein. Following Ad-mda7 transduction, RT-PCR demonstrated no increase in PKR mRNA levels despite increased levels of PKR protein, suggesting posttranscriptional regulation of PKR by MDA-7. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 protein physically interacts with PKR. Transduction of PKR+/+ and PKR-/- transformed MEFs with Ad-mda7 demonstrated phosphorylated MDA-7 and PKR proteins in the lysates of PKR+/+ but not PKR-/- cells. These findings identify the first binding partner for MDA-7 and suggest that direct interaction between PKR and MDA-7 may be important for PKR activation and apoptosis induction, possibly through MDA-7 phosphorylation or activation of other downstream targets.

Genetically targeted cancer therapy: tumor destruction by PKR activation.

The is a double-stranded RNA-activated protein kinase (PKR) has been largely investigated for its key role in viral host defense. Although best characterized by its function in mediating the antiviral and antiproliferative effects of interferon (IFN), PKR is also implicated in transcriptional regulation, cell differentiation, signal transduction, and tumor suppression. However, recent findings identifying PKR as an important effector of apoptosis have led to an increased interest in PKR modulation as an antitumor strategy. PKR can either be up-regulated through direct induction by the transcription factor E2F-1, or it can be activated through direct protein-protein interactions with the melanoma differentiation-associated gene-7 (MDA7, IL-24). Additionally, the intracellular formation of double-stranded RNA by transfection with antisense RNA complementary to tumor-specific RNA sequences can induce PKR activation and apoptosis selective to these tumor cells. The growing application of viral vector-based gene therapies and oncolytic, replicating viruses that must elude viral defense in order to be effective, has also drawn attention to PKR. Oncolytic viruses, like the attenuated herpes simplex virus R3616, the vesicular stomatitis virus, or reovirus, specifically replicate in tumor cells only because the viral host defense in the permissive cells is suppressed. In this article we review the role of PKR as an effector of apoptosis and a target for tumor treatment strategies and discuss the potential of PKR-modifying agents to treat patients with cancer. Targeted gene therapy against cancer can be approached by activation of PKR with the down-regulation of protein synthesis and induction of apoptosis, or by suppression of PKR with the propagation of oncolytic virus. Since the PKR pathway can be modified by many routes, antitumor therapies combining oncolytic virus, gene therapies, and chemotherapy with PKR modifiers are likely to emerge in the near future as therapeutic options in the treatment of patients with cancer.

The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells.

mda-7/IL-24 (HGMW-approved symbol IL24) is a tumor suppressor gene whose expression is lost during tumor progression. Gene transfer using adenoviral mda-7/IL-24 (Ad-mda7) exhibits minimal toxicity on normal cells while inducing potent apoptosis in a variety of cancer cell lines. Ad-mda7-transduced cells express high levels of MDA-7 protein intracellularly and also secrete a soluble form of MDA-7 protein. In this study, we sought to determine whether the intracellular or secreted MDA-7 protein was responsible for anti-tumor activity in H1299 lung tumor cells. Ad-mda7 transduction of lung tumor cells increased expression of stress-related proteins, including BiP, GADD34, PP2A, caspases 7 and 12, and XBP-1, consistent with activation of the UPR pathway, a key sensor of endoplasmic reticulum (ER)-mediated stress. Blocking secretion of MDA-7 did not inhibit apoptosis, demonstrating that intracellular MDA-7 was responsible for cytotoxicity. Consistent with this result, when applied directly to lung cancer cells, soluble MDA-7 protein exhibited minimal cytotoxic effect. We then generated mda-7 expression constructs using vectors that target the expressed protein to various subcellular compartments, including cytoplasm, nucleus, and ER. Only full-length and ER-targeted MDA-7 elicited cell death in tumor cells. Thus in lung cancer cells, Ad-mda7 activates the UPR stress pathway and induces apoptosis via intracellular MDA-7 expression in the secretory pathway.

Adenoviral melanoma differentiation-associated gene 7 induces apoptosis in lung cancer cells through mitochondrial permeability transition-independent cytochrome c release.

OBJECTIVE: Melanoma differentiation-associated gene 7 is a novel tumor suppressor gene that induces apoptosis in lung cancer cells when delivered by adenoviral gene transfer as Ad-mda7. The mechanisms of action are not well defined but may involve release of cytochrome c from the mitochondria with subsequent caspase activation. METHODS: The lung cancer cell lines A549 and H1299 were transduced with Ad-mda7, adenovirus containing the gene for p53 (Ad-p53), and control adenoviral luciferase vectors. Staurosporine was used as a positive control to induce cytochrome c release through mitochondrial permeability transition-dependent pores, whereas cyclosporine (INN: ciclosporin) was used to specifically inhibit these mitochondrial permeability transition-dependent pores. Apoptosis was evaluated with fluorescence-activated cell sorting analysis of subdiploid populations and mitochondrial membrane potential changes with tetramethylrhodamine ethylester perchlorate. RESULTS: Melanoma differentiation-associated gene 7, transduced by Ad-mda7 into H1299 and A549 lung cancer cells, resulted in sharp increases in cytosolic cytochrome c levels followed by induction of apoptosis and cellular death. The release of cytochrome c from the mitochondria occurred without changes in the mitochondrial membrane potential. Unlike staurosporine treatment, transduction with Ad-p53 and Ad-mda7 caused releases of cytochrome c and apoptosis that were not blocked by cyclosporine, suggesting a mitochondrial permeability transition pore-independent pathway. CONCLUSIONS: Ad-mda7 induces apoptosis in lung cancer cells through mitochondrial cytochrome c release in a process that is not dependent on mitochondrial membrane potential changes and occurs through mitochondrial permeability transition-independent pores. This unique mechanism of action may allow treatment of patients with lung cancer resistant to mitochondrial permeability transition-dependent cell death processes.

The mitochondrial apoptosis-inducing factor plays a role in E2F-1-induced apoptosis in human colon cancer cells.

BACKGROUND: Overexpression of the transcription factor E2F-1 provokes apoptosis in cancer cells; the mechanism, however, is not completely understood. We sought to evaluate E2F-1 gene therapy in human colon cancer and to investigate the apoptotic pathway involved. METHODS: Adenoviral vectors were used to transfect the E2F-1 gene (Ad5E2F-1) or the control gene luciferase (Ad5Luc) into four human colon carcinoma cell lines. Apoptosis was confirmed by flow cytometry and poly (ADP-ribose) polymerase cleavage. Expression of apoptotic factors was determined with Western blot analysis. Inhibitory assays were used to determine the involvement of caspases in the apoptotic pathway. RESULTS: Overexpression of E2F-1 was evident in all cells treated with Ad5E2F-1; upregulation of Bcl-2, and activation of caspases were noted. The apoptosis-inducing factor in the cytosolic fraction was markedly upregulated after Ad5E2F-1 treatment. E2F-1 overexpression inhibited proliferation and induced significant apoptosis in all cell lines (P <.005). This apoptotic response could be only partially blocked by caspase inhibitors. CONCLUSIONS: These findings demonstrate that E2F-1 induces apoptosis and inhibits proliferation in human colon cancer cell lines. The marked upregulation of apoptosis-inducing factor and the fact that E2F-1-induced apoptosis is incompletely blocked by caspase inhibitors suggest a caspase-independent pathway of E2F-1-mediated apoptosis, reported here for the first time.

Role for the double-stranded RNA activated protein kinase PKR in E2F-1-induced apoptosis.

The transcription factor E2F-1 induces cell cycle progression at the G1/S checkpoint, and deregulation of E2F-1 provokes apoptosis in a wide variety of malignant cells. To date only p14(ARF) and p73, a p53 homologue, have been identified as E2F-1-inducible genes capable of mediating an apoptotic response. Here we show that adenovirus-mediated E2F-1 overexpression in cancer cells induces expression and autophosphorylation of the double-stranded RNA-dependent protein kinase PKR leading to phosphorylation of its downstream target, the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha) and to apoptotic cell death. This PKR-dependent apoptosis occurs in cell lines with mutated p53 and in cell lines with mutated p53 and p73, and is significantly reduced by the chemical inhibition of PKR activation. Further, PKR(-/-) mouse embryo fibroblasts, but not PKR(+/+) mouse embryo fibroblasts, demonstrate significant resistance to E2F-1-induced apoptosis. We conclude that an important pathway of E2F-1-mediated apoptosis is dependent on PKR activation and does not require p53 or p73.

Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR).

Adenoviral-mediated overexpression of the melanoma differentiation-associated gene 7 (Ad-mda7) induces apoptosis in a wide range of cancer cells, although themechanism is not well understood. We report that Ad-mda7 induces andactivates the double-stranded RNA-dependent protein kinase (PKR), which leads to phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha) and the induction of apoptosis in lung cancer cells. Treatment with 2-aminopurine (2-AP), a serine/threonine kinase inhibitor, inhibits PKR activation, eIF2alpha phosphorylation, and apoptosis induction by Ad-mda7. Additionally, PKR null but not wild-type fibroblasts are resistant to Ad-mda7-induced apoptosis. These results suggest that the activation of PKR and its downstream targets may be a critical pathway for Ad-mda7-mediated apoptosis.

Bax-mediated Ca2+ mobilization promotes cytochrome c release during apoptosis.

Previous studies have demonstrated that Ca(2+) is released from the endoplasmic reticulum (ER) in some models of apoptosis, but the mechanisms involved and the functional significance remain obscure. We confirmed that apoptosis induced by some (but not all) proapoptotic stimuli was associated with caspase-independent, BCL-2-sensitive emptying of the ER Ca(2+) pool in human PC-3 prostate cancer cells. This mobilization of ER Ca(2+) was associated with a concomitant increase in mitochondrial Ca(2+) levels, and neither ER Ca(2+) mobilization nor mitochondrial Ca(2+) uptake occurred in Bax-null DU-145 cells. Importantly, restoration of DU-145 Bax expression via adenoviral gene transfer restored ER Ca(2+) release and mitochondrial Ca(2+) uptake and dramatically accelerated the kinetics of staurosporine-induced cytochrome c release, demonstrating a requirement for Bax expression in this model system. In addition, an inhibitor of the mitochondrial Ca(2+) uniporter (RU-360) attenuated mitochondrial Ca(2+) uptake, cytochrome c release, and DNA fragmentation, directly implicating the mitochondrial Ca(2+) changes in cell death. Together, our data demonstrate that Bax-mediated alterations in ER and mitochondrial Ca(2+) levels serve as important upstream signals for cytochrome c release in some examples of apoptosis.

Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.

Alterations in intracellular Ca(2+) homeostasis and cytochrome c release from mitochondria have been implicated in the regulation of apoptosis, but the relationship between these events remains unclear. Here we report that enforced expression of either Bax or Bak via adenoviral gene delivery results in the accumulation of the proteins in the endoplasmic reticulum (ER) and mitochondria, resulting in early caspase-independent BCL-2-sensitive release of the ER Ca(2+) pool and subsequent Ca(2+) accumulation in mitochondria. The inhibition of ER-to-mitochondrial Ca(2+) transport with a specific inhibitor of mitochondrial Ca(2+) uptake attenuates cytochrome c release and downstream biochemical events associated with apoptosis. Bax and Bak also directly sensitize mitochondria to cytochrome c release induced by immediate emptying of ER Ca(2+) pool. Our results demonstrate that the effects of the "multidomain" proapoptotic BCL-2 family members Bak and Bax involve direct effects on the endoplasmic reticular Ca(2+) pool with subsequent sensitization of mitochondria to calcium-mediated fluxes and cytochrome c release. These effects modulate the kinetics of cytochrome c release and apoptosis.