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John C Reed

Publications and source records attributed to John C Reed.

At least 127 records · Page 7Linked to original sources
PubMed2003

Apoptotic-regulatory and complement-protecting protein expression in chronic lymphocytic leukemia: relationship to in vivo rituximab resistance.

PURPOSE: Rituximab has clinical activity in patients with chronic lymphocytic leukemia (CLL) and has a variety of proposed mechanisms, including apoptosis, complement-dependent cell lysis (CDC), and antibody-dependent cellular cytotoxicity (ADCC). Here we examine pretreatment biologic features that promote resistance to apoptosis and CDC in CLL patients and correlate it with clinical outcome to rituximab-based therapy. PATIENTS AND METHODS: Pretreatment samples from 21 CLL patients treated on a prospective, single-agent rituximab trial were examined for quantitative expression of apoptotic and CDC regulatory proteins, and the level of expression of these proteins was correlated with clinical outcome. RESULTS: Of the 21 patents for whom samples were available, 10 attained a partial response and 11 failed to respond to rituximab therapy. The mean pretreatment expression of Bcl-2, Mcl-1, XIAP, and the ratio of Bcl-2/Bax were higher but not statistically increased in nonresponding patients versus those responding to treatment. In contrast, the pretreatment Mcl-1/Bax ratio was significantly elevated (0.82 +/- 0.28 v 0.39 +/- 0.29, P <.016) in nonresponding patients compared with patients responding to rituximab therapy. Although pretreatment expression of CD55 and CD59 was not associated with response to rituximab therapy, significantly higher levels of CD59 were observed in the CLL cells that were not cleared from the blood at completion of therapy than the level observed at baseline levels (P =.02). CONCLUSION: These data indicate that baseline expression of the Mcl-1/Bax ratio, but not CD55 and CD59, predict for clinical response to rituximab therapy in CLL patients. Further study of disrupted apoptosis in CLL as a potential mechanism of resistance to rituximab appears warranted.

Antibodies, Monoclonal↗
PubMed2003

A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.

Animals↗
PubMed2003

Depsipeptide (FR901228) induces histone acetylation and inhibition of histone deacetylase in chronic lymphocytic leukemia cells concurrent with activation of caspase 8-mediated apoptosis and down-regulation of c-FLIP protein.

Depsipeptide is in clinical trials for chronic lymphocytic leukemia (CLL) on the basis of earlier observations demonstrating selective in vitro activity in CLL. We sought to determine the relationship of histone H3 and H4 acetylation, inhibition of histone deacetylase, and apoptosis observed in CLL cells to justify a pharmacodynamic end point in these clinical trials. We demonstrate that in vitro depsipeptide induces histone H3 and H4 acetylation and histone deacetylase enzyme inhibition at concentrations corresponding to the LC50 (concentration producing 50% cell death) for cultured CLL cells (0.038 microM depsipeptide). The changes in histone acetylation are lysine specific, involving H4 K5, H4 K12, and H3 K9, and to a lesser extent H4 K8, but not H4 K16 or H3 K14. Depsipeptide-induced apoptosis is caspase dependent, selectively involving the tumor necrosis factor (TNF) receptor (extrinsic pathway) initiating caspase 8 and effector caspase 3. Activation of caspase 8 was accompanied by the down-regulation of cellular FLICE-inhibitory protein (c-FLIP, I-FLICE) without evidence of Fas (CD95) up-regulation. Changes in other apoptotic proteins, including Bcl-2, Bax, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), were not observed. Our results demonstrate a relationship between target enzyme inhibition of histone deacetylase, histone H3 and H4 acetylation, and apoptosis involving the TNF-receptor pathway of apoptosis that is not used by other therapeutic agents in CLL. These data suggest use of histone H3 and H4 acetylation, inhibition of histone deacetylase, and down-regulation of FLIP as pharmacodynamic end points for further evaluation of this drug in patients.

Acetylation↗
PubMed2003

Functional blocks in caspase activation pathways are common in leukemia and predict patient response to induction chemotherapy.

Defects in apoptosis mechanisms contribute to chemoresistance in malignancy. However, correlations of apoptosis-regulating proteins with clinical outcome in cancer patients are variable, presumably reflecting the difficulty of using static tests of gene expression in a scenario influenced by a dynamic interplay of multiple pro- and antiapoptotic molecules. Therefore, we assessed the functional integrity of apoptosis pathways in intact primary leukemia cells and correlated the functional status of these pathways with clinical outcome. Active apoptogenic proteins were introduced into primary leukemia cells by electroporation followed by measurement of active caspases by flow cytometric techniques. Cytochrome c was introduced to activate the intrinsic (mitochondrial) pathway, whereas caspase-8 was introduced to activate the extrinsic (death receptor) pathway. In a series of 24 patients with acute myeloid leukemia, 79% had a block in at least one pathway, indicating that defects in caspase activation mechanisms are common in patients with leukemia. Simultaneous blocks in both pathways correlated with chemoresistant disease (92% of patients with chemoresistant disease versus 33% of patients with chemosensitive disease; P = 0.005) and decreased overall patient survival (35% versus 89% 1-year survival; P = 0.02). Simultaneous blockage of the intrinsic and extrinsic pathways could be explained by a defect located at a point of convergence of the two pathways, probably related to overexpression of endogenous inhibitors of the effector-caspases, rather than decreased levels of these proteases. This study supports the importance of apoptosis pathways in determining response to chemotherapy and suggests that functional defects in caspase activation are prognostic in patients with leukemia.

Adult↗
PubMed2003

Peptides targeting caspase inhibitors.

Here we report on the identification of peptides targeting the X-inhibitor of apoptosis protein (XIAP). XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. IAPs are often overexpressed in cancers and leukemias and are associated with an unfavorable clinical prognosis. We have selected peptides from a phage library by using recombinant full-length human XIAP or a fragment containing only the baculovirus IAP repeat 2 (BIR2) domain. A consensus motif, C(D/E/P)(W/F/Y)-acid/basic-XC, was recovered from two independent screenings by using different libraries. Phage-displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. The interaction was specific as it could be blocked by the cognate synthetic peptides in a dose-dependent manner. Phage displaying the XIAP-binding motif CEFESC bound to the BIR2 domain of XIAP with an estimated dissociation constant of 1.8 nm as determined by surface plasmon resonance. Protein-protein interaction assays revealed that caspase-3 and caspase-7 (but not caspase-8) blocked the binding of the CEFESC phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. In fact, the sequence EFES is homologous to a loop unique to the executioner caspase-3 and caspase-7 that are targeted by XIAP. Finally, we demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings (PFKQ) can induce programmed cell death in leukemia cells. Peptides interacting with XIAP could serve as prototypes for the design of low molecular weight modulators of apoptosis.

Amino Acid Motifs↗
PubMed2003

Bcl-2 and apoptosis in chronic lymphocytic leukemia.

The Bcl-2 family of pro- and antiapoptotic proteins are key regulators of the apoptosis cascade and the mitochondrial-mediated pathway of caspase activation. Of this family, Bcl-2 was the first identified and remains the best characterized. Aberrant expression of Bcl-2 is common in chronic lymphocytic leukemia (CLL) and is associated with poor response to chemotherapy and decreased overall survival. Bcl-2 is an attractive target for novel therapeutic agents. Antisense oligonucleotides directed against Bcl-2 are effective in vitro and are being evaluated in clinical trials in CLL. Small molecule Bcl-2 inhibitors are in preclinical development and should be ready for clinical evaluation in the next few years. Strategies that induce apoptosis and bypass Bcl-2 may also be therapeutically useful in CLL. Thus, over the next decade, one can envision incorporating measurements of apoptotic proteins such as Bcl-2 into the risk assessment and treatment algorithms for individual patients. In addition, we anticipate that in the next decade, rationally designed therapies targeting specific molecular defects in the malignant CLL lymphocytes will be introduced into the clinic.

Apoptosis↗
PubMed2003

Temporal alterations in cellular Bax:Bcl-2 ratio following traumatic brain injury in the rat.

Cell death/survival following CNS injury may be a result of alterations in the intracellular ratio of death and survival factors. Using immunohistochemistry, Western analysis and in situ hybridization, the expression of the anti-cell death protein, Bcl-2, and the pro-cell death protein, Bax, was evaluated following lateral fluid-percussion (FP) brain injury of moderate severity (2.3-2.6 atm) in adult male Sprague-Dawley rats. By 2 h post-injury, a marked reduction of cellular Bcl-2-immunoreactivity (IR) and a mild decrease in cellular Bax IR were observed in the temporal and occipital cortices, and in the hippocampal CA3 ipsilateral to the site of impact. These decreases in Bcl-2 and Bax IR appeared to precede the overt cell loss in these regions that was evident at 24 h. Immunoblot analysis supported the immunohistochemical data, with a modest but significant reduction in the intensities of both the Bcl-2 and Bax protein bands at 2 h (p < 0.05 compared to sham levels). However, the Bax:Bcl-2 ratio increased significantly at 2 h (2.28 +/- 0.13) and remained elevated up to 7 days (2.05 +/- 0.13) post-injury compared to sham-injured control tissue (1.62 +/- 0.10, p < 0.05). Furthermore, cortical, but not hippocampal, levels of Bax protein increased by 25% (p < 0.05 compared to sham-injured controls) at 24 h post-injury, and returned to control levels by 7 days. In situ hybridization analysis of Bax mRNA revealed increased cellular grain density in the injured cortex (p < 0.05 compared to sham-injured brains), but not in the CA3 region of the injured hippocampus. No injury-induced changes in the expression of Bcl-2 mRNA were observed in any brain region. Taken together, these data suggest that the association between regional post-traumatic cell death and alterations in the cellular ratio of Bcl-2 and Bax may be, in part, due to alterations in mRNA and/or protein expression of the Bcl-2 family of proteins.

Animals↗
PubMed2003

Comparative analysis of apoptosis and inflammation genes of mice and humans.

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFkappaB induction. Proteins involved in apoptosis and NFkappaB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFkappaB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.

Animals↗
PubMed2003

BAG1 over-expression in brain protects against stroke.

The co-chaperone BAG1 binds and regulates 70 kDa heat shock proteins (Hsp70/Hsc70) and exhibits cytoprotective activity in cell culture models. Recently, we observed that BAG1 expression is induced during neuronal differentiation in the developing brain. However, the in vivo effects of BAG1 during development and after maturation of the central nervous system have never been examined. We generated transgenic mice over-expressing BAG1 in neurons. While brain development was essentially normal, cultured cortical neurons from transgenic animals exhibited resistance to glutamate-induced, apoptotic neuronal death. Moreover, in an in vivo stroke model involving transient middle cerebral artery occlusion, BAG1 transgenic mice demonstrated decreased mortality and substantially reduced infarct volumes compared to wild-type littermates. Interestingly, brain tissue from BAG1 transgenic mice contained higher levels of neuroprotective Hsp70/Hsc70 protein but not mRNA, suggesting a potential mechanism whereby BAG1 exerts its anti-apoptotic effects. In summary, BAG1 displays potent neuroprotective activity in vivo against stroke, and therefore represents an interesting target for developing new therapeutic strategies including gene therapy and small-molecule drugs for reducing brain injury during cerebral ischemia and neurodegenerative diseases.

Animals↗
PubMed2003

ZIP kinase triggers apoptosis from nuclear PML oncogenic domains.

PML oncogenic domains (PODs), also referred to as nuclear dot 10 bodies, Kreb's bodies, or nuclear bodies, represent nuclear structures implicated in the regulation of a variety of cellular processes, including transcription, tumor suppression, and apoptosis. ZIP kinase (ZIPK) is a proapoptotic protein kinase with homology to DAP kinase, a protein kinase implicated in apoptosis. We show here that ZIPK is present in PODs, where it colocalizes with and binds to proapoptotic protein Daxx. Arsenic trioxide (As(2)O(3)) and gamma interferon (IFN-gamma), which accentuate POD formation, increased the association of ZIPK with PODs. In contrast, the kinase-inactive ZIPK resides in nuclei with a diffuse pattern and significantly prevents the association of Daxx with PODs, implying that ZIPK recruits Daxx to PODs via its catalytic activity. ZIPK also binds and phosphorylates proapoptotic protein Par-4. Association of ZIPK with Daxx was enhanced by coexpression of Par-4. Activation of caspases and induction of apoptosis were also observed in cells overexpressing these proteins. Conversely, small-interfering RNA-mediated reduction of ZIPK, Daxx, or Par-4 expression decreased activation of caspase and apoptosis induced by As(2)O(3) and IFN-gamma. These results suggest that ZIPK, in collaboration with Daxx and Par-4, mediates a novel nuclear pathway for apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Pediatric acute lymphoblastic leukemia.

The outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically with current therapy resulting in an event free survival exceeding 75% for most patients. However significant challenges remain including developing better methods to predict which patients can be cured with less toxic treatment and which ones will benefit from augmented therapy. In addition, 25% of patients fail therapy and novel treatments that are focused on undermining specifically the leukemic process are needed urgently. In Section I, Dr. Carroll reviews current approaches to risk classification and proposes a system that incorporates well-established clinical parameters, genetic lesions of the blast as well as early response parameters. He then provides an overview of emerging technologies in genomics and proteomics and how they might lead to more rational, biologically based classification systems. In Section II, Drs. Mary Relling and Stella Davies describe emerging findings that relate to host features that influence outcome, the role of inherited germline variation. They highlight technical breakthroughs in assessing germline differences among patients. Polymorphisms of drug metabolizing genes have been shown to influence toxicity and the best example is the gene thiopurine methyltransferase (TPMT) a key enzyme in the metabolism of 6-mercaptopurine. Polymorphisms are associated with decreased activity that is also associated with increased toxicity. The role of polymorphisms in other genes whose products play an important role in drug metabolism as well as cytokine genes are discussed. In Sections III and IV, Drs. James Downing and Cheryl Willman review their findings using gene expression profiling to classify ALL. Both authors outline challenges in applying this methodology to analysis of clinical samples. Dr. Willman describes her laboratory's examination of infant leukemia and precursor B-ALL where unsupervised approaches have led to the identification of inherent biologic groups not predicted by conventional morphologic, immunophenotypic and cytogenetic variables. Dr. Downing describes his results from a pediatric ALL expression database using over 327 diagnostic samples, with 80% of the dataset consisting of samples from patients treated on a single institutional protocol. Seven distinct leukemia subtypes were identified representing known leukemia subtypes including: BCR-ABL, E2A-PBX1, TEL-AML1, rearrangements in the MLL gene, hyperdiploid karyotype (i.e., > 50 chromosomes), and T-ALL as well as a new leukemia subtype. A subset of genes have been identified whose expression appears to be predictive of outcome but independent verification is needed before this type of analysis can be integrated into treatment assignment. Chemotherapeutic agents kill cancer cells by activating apoptosis, or programmed cell death. In Section V, Dr. John Reed describes major apoptotic pathways and the specific role of key proteins in this response. The expression level of some of these proteins, such as BCL2, BAX, and caspase 3, has been shown to be predictive of ultimate outcome in hematopoietic tumors. New therapeutic approaches that modulate the apoptotic pathway are now available and Dr. Reed highlights those that may be applicable to the treatment of childhood ALL.

Child↗
PubMed2002

The PAAD/PYRIN-family protein ASC is a dual regulator of a conserved step in nuclear factor kappaB activation pathways.

Apoptosis-associated speck-like protein containing a Caspase recruitment domain (ASC) belongs to a large family of proteins that contain a Pyrin, AIM, ASC, and death domain-like (PAAD) domain (also known as PYRIN, DAPIN, Pyk). Recent data have suggested that ASC functions as an adaptor protein linking various PAAD-family proteins to pathways involved in nuclear factor (NF)-kappaB and pro-Caspase-1 activation. We present evidence here that the role of ASC in modulating NF-kappaB activation pathways is much broader than previously suspected, as it can either inhibit or activate NF-kappaB, depending on cellular context. While coexpression of ASC with certain PAAD-family proteins such as Pyrin and Cryopyrin increases NF-kappaB activity, ASC has an inhibitory influence on NF-kappaB activation by various proinflammatory stimuli, including tumor necrosis factor (TNF)alpha, interleukin 1beta, and lipopolysaccharide (LPS). Elevations in ASC protein levels or of the PAAD domain of ASC suppressed activation of IkappaB kinases in cells exposed to pro-inflammatory stimuli. Conversely, reducing endogenous levels of ASC using siRNA enhanced TNF- and LPS-induced degradation of the IKK substrate, IkappaBalpha. Our findings suggest that ASC modulates diverse NF-kappaB induction pathways by acting upon the IKK complex, implying a broad role for this and similar proteins containing PAAD domains in regulation of inflammatory responses.

Binding Sites↗
PubMed2002

Nod1, a CARD protein, enhances pro-interleukin-1beta processing through the interaction with pro-caspase-1.

The production of bioactive interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, is mediated by activated caspase-1. One of the known molecular mechanisms underlying pro-caspase-1 processing and activation involves interaction between the caspase recruit domains (CARDs) of caspase-1 and a serine/threonine kinase RIP2. While the association of Nod1 with both caspase-1 and RIP2 is already known, the consequences of these interactions are poorly understood. Because Nod1 also binds to RIP2, we hypothesized that Nod1 plays a role in pro-caspase-1 activation and IL-1beta processing. We show here that Nod1 binds to both RIP2 and caspase-1 by CARD interactions. Nod1 enhances pro-caspase-1 oligomerization and pro-caspase-1 processing. Nod1 enhances caspase-1-induced IL-1beta secretion, as well as lipopolysaccharide (LPS)-induced IL-1beta secretion in transfected cells. Moreover, HT1080 cells stably transfected with Nod1 showed higher LPS-induced IL-1beta secretion than non-transfected cells, suggesting a role of Nod1 in LPS-induced responses. Our data indicate that Nod1 can regulate IL-1beta secretion, implying that Nod1 may play a role in inflammatory responses to bacterial LPS.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The bioenergetic signature of cancer: a marker of tumor progression.

Mitochondrial H+-ATP synthase is required for cellular energy provision and for efficient execution of apoptosis. Almost one century ago, Otto Warburg proposed the hypothesis that mitochondrial function might be impaired in cancer cells. However, his hypothesis was never demonstrated in human carcinomas. In this study, we have analyzed the expression of the beta-catalytic subunit of the H+-ATP synthase (beta-F1-ATPase) of mitochondria in carcinomas of the human liver, kidney, and colon. We show that carcinogenesis in the liver involves a depletion of the cellular mitochondrial content, as revealed by reduced content of mitochondrial markers, whereas in kidney and colon carcinomas, it involves a selective repression of the expression of the beta-F1-ATPase concurrent with an increase in the expression of the glycolytic glyceraldehyde-3-phosphate dehydrogenase. Both mechanisms limit mitochondrial cellular activity in cancer, strongly supporting Warburg's hypothesis, and suggest a mechanism for the resistance and compromised apoptotic potential of tumor cells. Furthermore, we show that the metabolic state of the cell, as defined by a bioenergetic mitochondrial index relative to the cellular glycolytic potential, provides a signature of carcinogenesis of prognostic value in assessing the progression of colorectal carcinomas.

Animals↗
PubMed2002

Structural analysis of Siah1 and its interactions with Siah-interacting protein (SIP).

Seven in absentia homologue (Siah) family proteins bind ubiquitin-conjugating enzymes and target proteins for proteasome-mediated degradation. Recently we identified a novel Siah-interacting protein (SIP) that is a Sgt1-related molecule that provides a physical link between Siah family proteins and the Skp1-Cullin-F-box ubiquitin ligase component Skp1. In the present study, a structure-based approach was used to identify interacting residues in Siah that are required for association with SIP. In Siah1 a large concave surface is formed across the dimer interface. Analysis of the electrostatic surface potential of the Siah1 dimer reveals that the beta-sheet concavity is predominately electronegative, suggesting that the protein-protein interactions between Siah1 and SIP are mediated by ionic contacts. The structural prediction was confirmed by site-directed mutagenesis of these electronegative residues, resulting in loss of binding of Siah1 to SIP in vitro and in cells. The results also provide a structural basis for understanding the mechanism by which Siah family proteins interact with partner proteins such as SIP.

Calcium-Binding Proteins↗
PubMed2002

The triterpenoid CDDO induces apoptosis in refractory CLL B cells.

Chronic lymphocytic leukemia (CLL) cells develop chemo-resistance over time. Most anticancer agents function through induction of apoptosis, and therefore resistance against these agents is likely to be caused by selection for CLL cells with defects in the particular apoptosis pathway that is triggered by these drugs. Anticancer agents that function through alternative apoptotic pathways might therefore be useful in treating chemo-resistant CLL. Triterpenoids represent a class of naturally occurring and synthetic compounds with demonstrated antitumor activity. We examined the effects of CDDO (triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid) on CLL B cells in vitro. CDDO induced apoptosis in a dose-dependent manner in all (n = 30) CLL samples tested, including previously untreated and chemo-resistant CLL specimens. CDDO induced rapid proteolytic processing of caspase-8, but not caspase-9, in CLL B cells, suggesting activation of a mitochondria-independent pathway. CDDO-induced apoptosis of CLL B cells was blocked by cytokine response modifier A (CrmA), a suppressor of caspase-8, but not by X-linked inhibitor of apoptosis protein-baculovirus IAP repeat-3 (XIAP-BIR3), a fragment of XIAP, which selectively inhibits caspase-9. Examination of CDDO effects on expression of several apoptosis-relevant genes demonstrated significant reductions in the levels of caspase-8 homolog Fas-ligand interleukin-1-converting enzyme (FLICE)-inhibitory protein (c-FLIP), an endogenous antagonist of caspase-8. However, reductions of FLIP achieved by FLIP antisense oligonucleotides were insufficient for triggering apoptosis, indicating that CDDO has other targets in CLL B cells besides FLIP. These data suggest that the synthetic triterpenoid CDDO should be further explored as a possible therapeutic agent for treatment of chemo-resistant CLL.

Antineoplastic Agents↗
PubMed2002

Protection of CLL B cells by a follicular dendritic cell line is dependent on induction of Mcl-1.

Chronic lymphocytic leukemia (CLL) B cells have defects in apoptosis pathways and therefore accumulate in vivo. However, when removed from the patient and cultured in vitro, these malignant cells rapidly undergo apoptosis. Recent studies suggest that leukemia cell survival is influenced by interactions with nonleukemia cells in the microenvironment of lymph nodes, marrow, and other tissues. To model such cell-cell interactions in vitro, we cultured freshly isolated CLL B cells with a follicular dendritic cell line, HK. CLL B cells cocultured with HK cells were protected from apoptosis, either spontaneous or induced by treatment with anticancer drugs. Protection against spontaneous apoptosis could also be induced by coculturing the CLL B cells with normal dendritic cells (DCs) or with a CD40-ligand (CD154)-expressing fibroblast cell line. Examination of the expression of several apoptosis-regulatory proteins revealed that coculture with HK cells or DCs induced up-regulation of the antiapoptotic Bcl-2 family protein Mcl-1 in CLL B cells, whereas CD40 ligation increased expression of Bcl-X(L). Cell-cell contact was required for HK-induced protection, and introducing neutralizing antibodies against various adhesion molecules showed that CD44 was involved in HK-mediated survival, whereas CD40, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were not. Anti-CD44 antibodies also blocked Mcl-1 induction by HK cells. Mcl-1 antisense oligonucleotides reduced leukemia cell expression of Mcl-1, and significantly suppressed HK-induced protection against apoptosis, whereas control oligonucleotides had no effect. Thus, HK cells protect CLL B cells against apoptosis, at least in part through a CD44-dependent mechanism involving up-regulation of Mcl-1, and this mechanism is distinct from that achieved by CD40 ligation. Consequently, the particular antiapoptotic proteins important for CLL survival may vary depending on the microenvironment.

Apoptosis↗