Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.     

Cancer stem cells in glioblastoma — molecular signaling and therapeutic targeting

Glioblastomas (GBMs) are highly lethal primary brain tumors. Despite current therapeutic advances in other solid cancers, the treatment of these malignant gliomas remains essentially palliative. GBMs are extremely resistant to conventional radiation and chemotherapies. We and others have demonstrated that a highly tumorigenic subpopulation of cancer cells called GBM stem cells (GSCs) promotes therapeutic resistance. We also found that GSCs stimulate tumor angiogenesis by expressing elevated levels of VEGF and contribute to tumor growth, which has been translated into a useful therapeutic strategy in the treatment of recurrent or progressive GBMs. Furthermore, stem cell-like cancer cells (cancer stem cells) have been shown to promote metastasis. Although GBMs rarely metastasize beyond the central nervous system, these highly infiltrative cancers often invade into normal brain tissues preventing surgical resection, and GSCs display an aggressive invasive phenotype. These studies suggest that targeting GSCs may effectively reduce tumor recurrence and significantly improve GBM treatment. Recent studies indicate that cancer stem cells share core signaling pathways with normal somatic or embryonic stem cells, but also display critical distinctions that provide important clues into useful therapeutic targets. In this review, we summarize the current understanding and advances in glioma stem cell research, and discuss potential targeting strategies for future development of anti-GSC therapies.     

Hypersensitivity of lymphoblastoid lines derived from ataxia telangiectasia patients to the induction of chromosomal aberrations by etoposide (VP-16)

Mammalian DNA topoisomerase II represents the cellular target of many antitumor drugs, such as epipodophyllotoxin VP-16 (etoposide). The mechanism by which VP-16 exerts its cytotoxic and antineoplastic actions has not yet been firmly established, although the unique correlation between sensitivity to ionizing radiation and to topoisomerase II inhibitors suggest the involvement of DNA double-strand breaks. In the present study we analyzed the chromosomal sensitivity of lymphoblastoid cell lines derived from ataxia telangiectasia (AT) patients to low concentrations of the drug. Our results indicate that AT derived cells are hypersensitive to the clastogenic activity of VP-16 either when the drug is present for the whole duration of the cell cycle or specifically in the G2 phase, confirming that the induction of DNA double strand breaks, to which AT cells seem typically sensitive, could have an important role in the biological activity of VP-16.     

NK4拮抗HGF促进肿瘤细胞凋亡的生物学效应

观察NK4通过拮抗肝细胞生长因子（HGF）诱导不同肿瘤细胞凋亡,研究其生物学作用及分子机制.以足叶乙甙（VP-16）诱导凋亡,分别或经HGF蛋白、NK4蛋白处理5种肿瘤细胞（B细胞淋巴瘤细胞系Raji、人急性粒细胞白血病细胞系HL-60、宫颈癌细胞系HeLa、前列腺癌细胞系PC-3、非小细胞肺癌细胞系A549）,采用流式细胞术（FCM）、吖啶橙 (AO) 染色法、苏木素 伊红（HE）染色法定量观察5种肿瘤细胞的凋亡情况,并进行相关分析. FCM发现,经VP-16处理5种肿瘤细胞凋亡率均显著高于对照组(P<0.001),而HGF+VP-16组凋亡率明显下降(P<0.01),HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.05). AO染色和HE染色结果也证实,5种肿瘤细胞经VP-16处理后凋亡率均显著增高 (P<0.001,P<0.001),而HGF+VP-16组细胞凋亡率均明显低于VP-16组(P<0.001,P<0.01), HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.001,P<0.05).此外,发现NK4+VP-16组、HGF+ NK4+VP-16组、VP-16组等3组间凋亡率无统计学差异(P>0.05). 以上结果提示,HGF蛋白可抵抗凋亡诱导剂VP-16的作用, 明显降低细胞凋亡;NK4通过竞争性抑制HGF从而促进肿瘤细胞的凋亡,具有潜在的肿瘤治疗价值.     

The Expression of Connexins and SOX2 Reflects the Plasticity of Glioma Stem-Like Cells

Glioblastoma (GBM) is the most malignant primary brain tumor, with an average survival rate of 15 months. GBM is highly refractory to therapy, and such unresponsiveness is due, primarily, but not exclusively, to the glioma stem-like cells (GSCs). This subpopulation express stem-like cell markers and is responsible for the heterogeneity of GBM, generating multiple differentiated cell phenotypes. However, how GBMs maintain the balance between stem and non-stem populations is still poorly understood. We investigated the GBM ability to interconvert between stem and non-stem states through the evaluation of the expression of specific stem cell markers as well as cell communication proteins. We evaluated the molecular and phenotypic characteristics of GSCs derived from differentiated GBM cell lines by comparing their stem-like cell properties and expression of connexins. We showed that non-GSCs as well as GSCs can undergo successive cycles of gain and loss of stem properties, demonstrating a bidirectional cellular plasticity model that is accompanied by changes on connexins expression. Our findings indicate that the interconversion between non-GSCs and GSCs can be modulated by extracellular factors culminating on differential expression of stem-like cell markers and cell-cell communication proteins. Ultimately, we observed that stem markers are mostly expressed on GBMs rather than on low-grade astrocytomas, suggesting that the presence of GSCs is a feature of high-grade gliomas. Together, our data demonstrate the utmost importance of the understanding of stem cell plasticity properties in a way to a step closer to new strategic approaches to potentially eliminate GSCs and, hopefully, prevent tumor recurrence.     

Autophagy interplay with apoptosis and cell cycle regulation in the growth inhibiting effect of resveratrol in glioma cells

Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.     

Heterogeneous phenotype of human glioblastoma: In vitro study

Glioblastomas (GBMs) are the most lethal primary brain tumours. Increasing evidence shows that brain tumours contain the population of stem cells, so‐called cancer stem cells (CSCs). Stem cell marker CD133 was reported to identify CSC population in GBM. Further studies have indicated that CD133 negative cells exhibiting similar properties and are able to initiate the tumour, self‐renew and undergo multilineage differentiation. GBM is a highly heterogeneous tumour and may contain different stem cell populations with different functional properties. We characterized five GBM cell lines, established from surgical samples, according to the marker expression, proliferation and differentiation potential. CD133 positive cell lines showed increased proliferation rate in neurosphere condition and marked differentiation potential towards neuronal lineages. Whereas two cell lines low‐expressing CD133 marker showed mesenchymal properties in vitro, that is high proliferation rate in serum condition and differentiation in mesenchymal cell types. Further, we compared therapy resistance capacity of GBM cell lines treated with hydroxyurea. Our results suggest that CSC concept is more complex than it was believed before, and CD133 could not define entire stem cell population within GBM. At least two different subtypes of GBM CSCs exist, which may have different biological characteristics and imply different therapeutic strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest

Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers.     

Rosuvastatin reduces microglia in the brain of wild type and ApoE knockout mice on a high cholesterol diet; implications for prevention of stroke and AD

IGFBP2 is overexpressed in the most common brain tumor, glioblastoma (GBM), and its expression is inversely correlated to GBM patient survival. Previous reports have demonstrated a role for IGFBP2 in glioma cell invasion and astrocytoma development. However, the function of IGFBP2 in the restricted, self-renewing, and tumorigenic GBM cell population comprised of tumor-initiating stem cells has yet to be determined. Herein we demonstrate that IGFBP2 is overexpressed within the stem cell compartment of GBMs and is integral for the clonal expansion and proliferative properties of glioma stem cells (GSCs). In addition, IGFBP2 inhibition reduced Akt-dependent GSC genotoxic and drug resistance. These results suggest that IGFBP2 is a selective malignant factor that may contribute significantly to GBM pathogenesis by enriching for GSCs and mediating their survival. Given the current dearth of selective molecular targets against GSCs, we anticipate our results to be of high therapeutic relevance in combating the rapid and lethal course of GBM.     

Lipid Peroxidation Plays an Important Role in Chemotherapeutic Effects of Temozolomide and the Development of Therapy Resistance in Human Glioblastoma

BACKGROUND: Glioblastoma (GBM) is the most malignant primary brain tumor. Relapse occurs regularly, and the clinical behavior seems to be due to a therapy-resistant subpopulation of glioma-initiating cells that belong to the group of cancer stem cells. Aldehyde dehydrogenase (ALDH) has been identified as a marker for this cell population, and we have shown previously that ALDH1A3-positive GBM cells are more resistant against temozolomide (TMZ) treatment. However, it is still unclear how ALDH expression mediates chemoresistance. MATERIALS AND METHODS: ALDH1A3 expression was analyzed in 112 specimens from primary and secondary surgical resections of 56 patients with GBM (WHO grade IV). All patients received combined adjuvant radiochemotherapy. For experimental analysis, CRISPR-Cas9–induced knockout cells from three established GBM cell lines (LN229, U87MG, T98G) and two glioma stem-like cell lines were investigated after TMZ treatment. RESULTS: ALDH1A3 knockout cells were more sensitive to TMZ, and oxidative stress seemed to be the molecular process where ALDH1A3 exerts its role in resistance against TMZ. Oxidative stress led to lipid peroxidation, yielding active aldehydes that were detoxified by ALDH enzymatic activity. During the metabolic process, autophagy was induced leading to downregulation of the enzyme, but ALDH1A3 is upregulated to even higher expression levels after finishing the TMZ therapy in vitro. Recurrent GBMs show significantly higher ALDH1A3 expression than the respective samples from the primary tumor, and patients suffering from GBM with high ALDH1A3 expression showed a shorter median survival time (12 months vs 21 months, P < .05). CONCLUSION: Oxidative stress is an important and clinically relevant component of TMZ-induced therapeutic effects. Cytotoxicity seems to be mediated by aldehydes resulting from lipid peroxidation, and ALDH1A3 is able to reduce the number of toxic aldehydes. Therefore, we present a molecular explanation of the role of ALDH1A3 in therapeutic resistance of human GBM cells.     

Cytomegalovirus pp71 Protein Is Expressed in Human Glioblastoma and Promotes Pro-Angiogenic Signaling by Activation of Stem Cell Factor

Glioblastoma multiforme (GBM) is a highly malignant primary central nervous system neoplasm characterized by tumor cell invasion, robust angiogenesis, and a mean survival of 15 months. Human cytomegalovirus (HCMV) infection is present in >90% of GBMs, although the role the virus plays in GBM pathogenesis is unclear. We report here that HCMV pp71, a viral protein previously shown to promote cell cycle progression, is present in a majority of human GBMs and is preferentially expressed in the CD133+, cancer stem-like cell population. Overexpression of pp71 in adult neural precursor cells resulted in potent induction of stem cell factor (SCF), an important pro-angiogenic factor in GBM. Using double immunofluorescence, we demonstrate in situ co-localization of pp71 and SCF in clinical GBM specimens. pp71 overexpression in both normal and transformed glial cells increased SCF secretion and this effect was specific, since siRNA mediated knockdown of pp71 or treatment with the antiviral drug cidofovir resulted in decreased expression and secretion of SCF by HCMV-infected cells. pp71- induced upregulation of SCF resulted in downstream activation of its putative endothelial cell receptor, c-kit, and angiogenesis as measured by increased capillary tube formation in vitro. We demonstrate that pp71 induces a pro-inflammatory response via activation of NFΚB signaling which drives SCF expression. Furthermore, we show that pp71 levels and NFKB activation are selectively augmented in the mesenchymal subtype of human GBMs, characterized by worst patient outcome, suggesting that HCMV pp71-induced paracrine signaling may contribute to the aggressive phenotype of this human malignancy.     

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma(GBM)is the most common and most aggressive primary brain tumor in adults.The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis.In this study,we performed an in-depth analysis of the DNA methylation landscape in GBMderived cancer stem cells(GSCs).Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls(a neural stem cell(NSC)line and normal brain tissue)identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls,primary GBMs,and their counterpart GSC lines,respectively.Interestingly,concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT,AJAP1 and PTPRN2.These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes.Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2,NEFM and PENK.Forced re-expression of SPINT2 reduced glioma cell proliferative capacity,anchorage independent growth,cell motility,and tumor sphere formation in vitro.The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.     

Elevated invasive potential of glioblastoma stem cells

Glioblastomas (GBMs) are the most lethal and common types of primary brain tumors. The hallmark of GBMs is their highly infiltrative nature. The cellular and molecular mechanisms underlying the aggressive cancer invasion in GBMs are poorly understood. GBM displays remarkable cellular heterogeneity and hierarchy containing self-renewing glioblastoma stem cells (GSCs). Whether GSCs are more invasive than non-stem tumor cells and contribute to the invasive phenotype in GBMs has not been determined. Here we provide experimental evidence supporting that GSCs derived from GBM surgical specimens or xenografts display greater invasive potential in vitro and in vivo than matched non-stem tumor cells. Furthermore, we identified several invasion-associated proteins that were differentially expressed in GSCs relative to non-stem tumor cells. One of such proteins is L1CAM, a cell surface molecule shown to be critical to maintain GSC tumorigenic potential in our previous study. Immunohistochemical staining showed that L1CAM is highly expressed in a population of cancer cells in the invasive fronts of primary GBMs. Collectively, these data demonstrate the invasive nature of GSCs, suggesting that disrupting GSCs through a specific target such as L1CAM may reduce GBM cancer invasion and tumor recurrence.     

Inhibition of Autophagy by Chloroquine Enhances the Antitumor Efficacy of Sorafenib in Glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive and common brain tumor in adults. Sorafenib, a multi-kinase inhibitor, has been shown to inhibit cell proliferation and induce apoptosis through inhibition of STAT3 signaling in glioblastoma cells and in intracranial gliomas. However, sorafenib also induces cell autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the therapeutic effect of sorafenib on glioblastoma is uncertain. Here, we combined sorafenib treatment in GBM cells (U373 and LN229) and tumors with the autophagy inhibitor chloroquine. We found that blockage of autophagy further inhibited cell proliferation and migration and induced cell apoptosis in vitro and in vivo. These findings suggest the possibility of combination treatment with sorafenib and autophagy inhibitors for GBM.     

Soluble Urokinase Receptor Is Released Selectively by Glioblastoma Cells That Express Epidermal Growth Factor Receptor Variant III and Promotes Tumor Cell Migration and Invasion

Genomic heterogeneity is characteristic of glioblastoma (GBM). In many GBMs, the EGF receptor gene (EGFR) is amplified and may be truncated to generate a constitutively active form of the receptor called EGFRvIII. EGFR gene amplification and EGFRvIII are associated with GBM progression, even when only a small fraction of the tumor cells express EGFRvIII. In this study, we show that EGFRvIII-positive GBM cells express significantly increased levels of cellular urokinase receptor (uPAR) and release increased amounts of soluble uPAR (suPAR). When mice were xenografted with human EGFRvIII-expressing GBM cells, tumor-derived suPAR was detected in the plasma, and the level was significantly increased compared with that detected in plasma samples from control mice xenografted with EGFRvIII-negative GBM cells. suPAR also was increased in plasma from patients with EGFRvIII-positive GBMs. Purified suPAR was biologically active when added to cultures of EGFRvIII-negative GBM cells, activating cell signaling and promoting cell migration and invasion. suPAR did not significantly stimulate cell signaling or migration of EGFRvIII-positive cells, probably because cell signaling was already substantially activated in these cells. The activities of suPAR were replicated by conditioned medium (CM) from EGFRvIII-positive GBM cells. When the CM was preincubated with uPAR-neutralizing antibody or when uPAR gene expression was silenced in cells used to prepare CM, the activity of the CM was significantly attenuated. These results suggest that suPAR may function as an important paracrine signaling factor in EGFRvIII-positive GBMs, inducing an aggressive phenotype in tumor cells that are EGFRvIII-negative.     

GL3, a Novel 4β-Anilino-4′-O-Demethyl-4-Desoxypodophyllotoxin Analog,Traps Topoisomerase II Cleavage Complexes and Exerts Anticancer Activities

A novel VP-16 derivative, 4β-[N -(4?-acetyloxyl-phenyl-1?-carbonyl)-4″-aminoanilino]-4′-O-demethyl-4-desoxypodophyllotoxin (GL3), displayed a wide range of cytotoxicity in a panel of human tumor cell lines, with half-maximal inhibitory concentration (IC₅₀) values ranging from 0.82 to 4.88 µM, much less than that of VP-16 (4.18–39.43 µM). Importantly, GL3 induces more significant apoptosis and cell cycle arrest than VP-16. The molecular and cellular machinery studies showed that GL3 functions as a topoisomerase II (Top 2) poison through direct binding to the enzyme, and the advanced cell-killing activities of GL3 were ascribed to its potent effects on trapping Top 2-DNA cleavage complex, Moreover, GL3-triggered DNA double-strand breaks and apoptotic cell death were in a Top 2-dependent manner, because the catalytic inhibitor aclarubicin attenuated these biologic consequences caused by Top 2 poisoning in GL3-treated cells. Taken together, among a series of 4β-anilino-4′-O-demethyl-4-desoxypodophyllotoxin analog, GL3 stood out by its improved anticancer activity and well-defined Top 2 poisoning mechanisms, which merited the potential value of GL3 as an anticancer lead compound/drug candidate deserving further development.     

Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma

Glioblastomas (GBMs) are considered to be one of the deadliest human cancers, characterized by a high proliferative rate, aggressive invasiveness and insensitivity to radio- and chemotherapy, as well as a short patient survival period. Moreover, GBMs are among the most vascularized and invasive cancers in humans. Angiogenesis in GBMs is correlated with the grade of malignancy and is inversely correlated with patient survival. One of the first steps in tumor invasions is migration. GBM cells have the ability to infiltrate and disrupt physical barriers such as basement membranes, extracellular matrix and cell junctions. The invasion process includes the overexpression of several members of a super-family of zinc-based proteinases, the Metzincin, in particular a sub-group, metalloproteinases. Another interesting aspect is that, inside the GBM tissue, there are up to 30% of microglia or macrophages. However, little is known about the immune performance and interactions of the microglia with GBMs. These singular properties of GBMs will be described here. A sub-population of cells with stem-like properties may be the source of tumors since, apparently, GBM stem cells (GSCs) are highly resistant to current cancer treatments. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not eliminate GSCs, which survive to regenerate new tumors. Finally, GBM patient prognostic has shown little improvement in decades. In this context, we will discuss how the membrane-acting toxins called cytolysins can be a potential new tool for GBM treatment.     

Apoptosis-resistant mitochondria in T cells selected for resistance to Fas signaling

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.