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1.
Gene Ther ; 17(9): 1105-16, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20428216

ABSTRACT

The anti-tumor efficacy of adoptively transferred T cells requires their in vivo persistence and memory polarization. It is unknown if human chimeric antigen receptor (CAR)-expressing T cells can also undergo memory polarization. We examined the functional status of CAR CD8(+) T cells, re-directed to Lewis Y antigen (LeY-T), throughout a period of ex vivo expansion. Immediately before culture CD8(+) T cells comprised a mixture of phenotypes including naive (CD45RA(+)/CCR7(+)/CD27(+)/CD28(+)/perforin-), central memory (CM, CD45RA(-)/CCR7(lo)/CD27(+)/CD28(+)/perforin(lo)), effector memory (EM, CD45RA(-)/CCR7(-)/CD27(+)/CD28(+)/perforin(mod)) and effector (Eff, CD45RA(+)/CCR7(-)/CD27(-)/CD28(-)/perforin(hi)) cells. After transduction and expansion culture of peripheral blood mononuclear cells from normal donors or multiple myeloma patients, CD8(+) LeY-T cells polarized to EM- and CM-like phenotype. CD8(+) LeY-T cells differed from starting CD8(+) CM and EM T cells in that CD27, but not CD28, was downregulated. In addition, CD8(+) LeY-T cells expressed high levels of perforin, similar to starting CD8(+) Eff. CD8(+) LeY-T cells also showed hallmarks of both memory and Eff function, underwent homeostatic proliferation in response to interleukin (IL)-15, and showed interferon (IFN)-γ production and cytotoxicity in response to Le-Y antigen on OVCAR-3 (human ovarian adenocarcinoma) cells. This study confirms CD8(+) LeY-T cells have a CM- and EM-like phenotype and heterogeneous function consistent with potential to persist in vivo after adoptive transfer.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cytotoxicity, Immunologic , Immunologic Memory , Receptors, Antigen/genetics , CD28 Antigens/immunology , Cell Proliferation , Humans , Interferon-gamma/metabolism , Leukocyte Common Antigens/immunology , Phenotype , Receptors, Antigen/immunology , Receptors, Antigen/metabolism , Recombinant Fusion Proteins/metabolism , Tumor Necrosis Factor Receptor Superfamily, Member 7/immunology
2.
Cell Death Differ ; 11(9): 1028-37, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15131592

ABSTRACT

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Adenosine Triphosphate/metabolism , Caspases/metabolism , Adenosine Triphosphatases/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cell Survival , Cytochromes c/metabolism , DNA Mutational Analysis , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Enzyme Activation , Green Fluorescent Proteins/metabolism , Humans , Hydrolysis , Hydroxamic Acids/pharmacology , Idarubicin/pharmacology , Lymphoma/drug therapy , Mitosis , Mutation , Retroviridae/genetics , Structure-Activity Relationship , Time Factors , Vincristine/pharmacology
3.
Cell Death Differ ; 9(11): 1266-72, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12404126

ABSTRACT

Previous studies by our laboratory have shown that the drug transporter protein P-glycoprotein, P-gp, can specifically inhibit Fas-induced caspase-3 activation and apoptosis. Importantly, inhibition of both caspase-3 activation and cell death could be reversed by pharmacological and antibody inhibitors of P-gp function. However, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remained unknown. We therefore sought to identify the point(s) within the death receptor pathway at which P-gp exerted its inhibitory effect and to determine whether the ATPase activity of P-gp was required. Structure-function analysis determined that ATP hydrolysis was necessary for P-gp to confer resistance to Fas-induced caspase activation and cell death. Importantly, although both FADD and caspase-8 were recruited to the Death Inducing Signal Complex (DISC) in wild-type P-gp expressing cells following Fas ligation, subsequent activation of caspase-8 at the DISC was inhibited. The ability of P-gp to inhibit caspase-8 activation was also ATP dependent. These studies demonstrate that P-gp inhibits Fas-induced caspase-8 activation but not formation of the DISC and that this activity of P-gp is dependent on ATP hydrolysis.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adaptor Proteins, Signal Transducing , Caspases/metabolism , Intracellular Signaling Peptides and Proteins , Receptors, Tumor Necrosis Factor/metabolism , fas Receptor/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Caspase 8 , Caspase 9 , Death Domain Receptor Signaling Adaptor Proteins , Fas-Associated Death Domain Protein , Genetic Vectors , Humans , Retroviridae , Transduction, Genetic
4.
Proc Natl Acad Sci U S A ; 98(19): 10833-8, 2001 Sep 11.
Article in English | MEDLINE | ID: mdl-11535817

ABSTRACT

Many chemotherapeutic agents induce mitochondrial-membrane disruption to initiate apoptosis. However, the upstream events leading to drug-induced mitochondrial perturbation have remained poorly defined. We have used a variety of physiological and pharmacological inhibitors of distinct apoptotic pathways to analyze the manner by which suberoylanilide hydroxamic acid (SAHA), a chemotherapeutic agent and histone deacetylase inhibitor, induces cell death. We demonstrate that SAHA initiates cell death by inducing mitochondria-mediated death pathways characterized by cytochrome c release and the production of reactive oxygen species, and does not require the activation of key caspases such as caspase-8 or -3. We provide evidence that mitochondrial disruption is achieved by means of the cleavage of the BH3-only proapoptotic Bcl-2 family member Bid. SAHA-induced Bid cleavage was not blocked by caspase inhibitors or the overexpression of Bcl-2 but did require the transcriptional regulatory activity of SAHA. These data provide evidence of a mechanism of cell death mediated by transcriptional events that result in the cleavage of Bid, disruption of the mitochondrial membrane, and production of reactive oxygen species to induce cell death.


Subject(s)
Antineoplastic Agents/metabolism , Apoptosis , Carrier Proteins/metabolism , Enzyme Inhibitors/metabolism , Histone Deacetylase Inhibitors , Hydroxamic Acids/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Antineoplastic Agents/pharmacology , BH3 Interacting Domain Death Agonist Protein , Carrier Proteins/genetics , Caspase 10 , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolism , Cytochrome c Group/metabolism , Enzyme Inhibitors/pharmacology , Gene Expression , Humans , Hydroxamic Acids/pharmacology , Proto-Oncogene Proteins c-bcl-2/genetics , Transcription, Genetic , Tumor Cells, Cultured , Vorinostat
5.
J Biol Chem ; 276(20): 16667-73, 2001 May 18.
Article in English | MEDLINE | ID: mdl-11278745

ABSTRACT

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance (MDR). In addition to its ability to efflux toxins, P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We therefore hypothesized that P-gp may have additional functions in addition to its role in effluxing xenotoxins that could provide protection to tumor cells against a host response. There have been a number of contradictory reports concerning the role of P-gp in regulating complement activation. Given the disparate results obtained by different laboratories and our published results demonstrating that P-gp does not affect cell death induced by another membranolytic protein, perforin, we decided to assess the role of P-gp in regulating cell lysis induced by a number of different pore-forming proteins. Testing a variety of different P-gp-expressing MDR cell lines produced following exposure of cells to chemotherapeutic agents or by retroviral gene transduction in the complete absence of any drug selection, we found no difference in sensitivity of P-gp(+ve) or P-gp(-ve) cells to the pore-forming proteins complement, perforin, or pneumolysin. Based on these results, we conclude that P-gp does not affect cell lysis induced by pore-forming proteins.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Cell Survival/physiology , Membrane Glycoproteins/physiology , Antibodies/pharmacology , Antibodies, Monoclonal/pharmacology , Antigens, CD/immunology , Antigens, CD/physiology , Antigens, Differentiation, B-Lymphocyte/physiology , Cell Survival/drug effects , Doxorubicin/toxicity , Drug Resistance, Multiple , Humans , K562 Cells , Kinetics , Leukemia, T-Cell , Membrane Glycoproteins/pharmacology , Perforin , Pore Forming Cytotoxic Proteins , Receptors, IgG/physiology , Receptors, Transferrin , Recombinant Proteins/metabolism , Rubidium/pharmacokinetics , Transfection , Tumor Cells, Cultured , Vincristine/toxicity
6.
Biochem Biophys Res Commun ; 276(1): 231-7, 2000 Sep 16.
Article in English | MEDLINE | ID: mdl-11006111

ABSTRACT

P-glycoprotein (P-gp) is an ATP-dependent drug pump that confers multidrug resistance. In addition to its ability to efflux toxins P-gp can also inhibit apoptosis induced by a wide array of cell death stimuli that rely on activation of intracellular caspases for full function. We have previously demonstrated that stimuli including drugs such as hexamethylene bisacetamide (HMBA), the cytotoxic lymphocyte granule protein granzyme B, and pore-forming proteins such as perforin, kill P-gp positive cells in a caspase-independent manner. We therefore hypothesised that drugs that are not effluxed by P-gp and which induce cell death in the absence of caspase activation could induce death of P-gp expressing cells. Staurosporine has been previously shown to kill cells in the absence of caspase activation. Consistent with our hypothesis, we demonstrate here that staurosporine can equivalently kill P-gp(+ve) and P-gp(-ve) tumor cell lines in a caspase-independent manner.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Apoptosis/drug effects , Apoptosis/genetics , Enzyme Inhibitors/pharmacology , Staurosporine/pharmacology , Humans , Protein Kinase C/antagonists & inhibitors , Tumor Cells, Cultured
7.
Genome Res ; 10(6): 832-8, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10854414

ABSTRACT

We have previously localized the core centromere protein-binding domain of a 10q25.2-derived neocentromere to an 80-kb genomic region. Detailed analysis has indicated that the 80-kb neocentromere (NC) DNA has a similar overall organization to the corresponding region on a normal chromosome 10 (HC) DNA, derived from a genetically unrelated CEPH individual. Here we report sequencing of the HC DNA and its comparison to the NC sequence. Single-base differences were observed at a maximum rate of 4.6 per kb; however, no deletions, insertions, or other structural rearrangements were detected. To investigate whether the observed changes, or subsets of these, might be de novo mutations involved in neocentromerization (i.e., in committing a region of a chromosome to neocentromere formation), the progenitor DNA (PnC) from which the NC DNA descended, was cloned and sequenced. Direct comparison of the PnC and NC sequences revealed 100% identity, suggesting that the differences between NC and HC DNA are single nucleotide polymorphisms (SNPs) and that formation of the 10q25.2 NC did not involve a change in DNA sequence in the core centromere protein-binding NC region. This is the first study in which a cloned NC DNA has been compared directly with its inactive progenitor DNA at the primary sequence level. The results form the basis for future sequence comparison outside the core protein-binding domain, and provide direct support for the involvement of an epigenetic mechanism in neocentromerization.


Subject(s)
Base Sequence/genetics , Centromere/genetics , Chromosomes, Human, Pair 10/genetics , Alleles , Cell Line , Chromosomes, Human, Pair 10/chemistry , Cloning, Molecular , Gene Expression Regulation/genetics , Humans , Kinetochores , Male , Molecular Sequence Data , Reproducibility of Results , Sequence Analysis, DNA
8.
Leuk Lymphoma ; 38(1-2): 1-11, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10811443

ABSTRACT

P-glycoprotein (P-gp) is an energy dependent drug pump responsible for multidrug resistance (MDR) in human cancers. While it is irrefutable that P-gp can efflux xenobiotics out of cells, the biological function of P-gp in multicellular organisms has yet to be firmly established. The question of what, if anything, P-gp does when not effluxing drugs has been raised by recent reports indicating that P-gp may regulate apoptosis, chloride channel activity, cholesterol metabolism and immune cell function. There is now a lively debate regarding the possible role of P-gp in regulating cell differentiation, proliferation and survival.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1 , Apoptosis , Cell Death , Humans , Neoplasms/metabolism , Neoplasms/pathology
9.
Genomics ; 47(3): 399-404, 1998 Feb 01.
Article in English | MEDLINE | ID: mdl-9480754

ABSTRACT

The transformation-associated recombination (TAR) procedure allows rapid, site-directed cloning of specific human chromosomal regions as yeast artificial chromosomes (YACs). The procedure requires knowledge of only a single, relatively small genomic sequence that resides adjacent to the chromosomal region of interest. We applied this approach to the cloning of the neocentromere DNA of a marker chromosome that we have previously shown to have originated through the activation of a latent centromere at human chromosome 10q25. Using a unique 1.4-kb DNA fragment as a "hook" in TAR experiments, we achieved single-step isolation of the critical neocentromere DNA region as two stable, 110- and 80-kb circular YACs. For obtaining large quantities of highly purified DNA, these YACs were retrofitted with the yeast-bacteria-mammalian-cells shuttle vector BRV1, electroporated into Escherichia coli DH10B, and isolated as bacterial artificial chromosomes (BACs). Extensive characterization of these YACs and BACs by PCR and restriction analyses revealed that they are identical to the corresponding regions of the normal chromosome 10 and provided further support for the formation of the neocentromere within the marker chromosome through epigenetic activation.


Subject(s)
Centromere/genetics , Chromosomes, Human, Pair 10/genetics , Cloning, Molecular , DNA/genetics , Saccharomyces cerevisiae/genetics , Transformation, Genetic , Chromosome Mapping , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Bacterial , DNA/analysis , Genetic Vectors , Humans
11.
Nat Genet ; 16(2): 144-53, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9171825

ABSTRACT

We recently described a human marker chromosome containing a functional neo-centromere that binds anti-centromere antibodies, but is devoid of centromeric alpha-satellite repeats and derived from a hitherto non-centromeric region of chromosome 10q25. Chromosome walking using cloned single-copy DNA from this region enabled us to identify the antibody-binding domain of this centromere. Extensive restriction mapping indicates that this domain has an identical genomic organization to the corresponding normal chromosomal region, suggesting a mechanism for the origin of this centromere through the activation of a latent centromere that exists within 10q25.


Subject(s)
Centromere , Chromosomes, Human, Pair 10 , DNA, Satellite/genetics , Binding Sites, Antibody , Chromosomes, Artificial, Yeast , DNA, Satellite/metabolism , Genetic Markers , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence
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