Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange.

Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 microM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4-20 microM cholesterol with the exception of PG at above 8 microM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO-CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 microM NAO provides an inverse linear fluorescence response at 0.2-10 microM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 microM and the limit of quantification is 0.6 microM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 microM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.

Killing of sarcoma cells by proapoptotic Bcl-X(S): role of the BH3 domain and regulation by Bcl-X(L).

Kaposi's sarcoma (KS) is the most common tumor affecting AIDS patients with over 20% of these patients afflicted by this disease. Previous studies have demonstrated that KS tumor cells predominantly express the prosurvival protein Bcl-X(L) compared with Bcl-2. In the current study, we have used an adenoviral vector that expresses Bcl-X(S), a functional inhibitor of Bcl-X(L), to study the significance of Bcl-X(L) expression in the KS cell line (SLK) or KS primary cultures. The results demonstrate that 75% to 80% of SLK or KS primary cells were killed by the Bcl-X(S) containing adenovirus whereas KS cells infected with control adenovirus showed no significant cell death or growth inhibition. Overexpression of Bcl-X(L), but not Bcl-2, in SLK cells attenuated apoptosis induced by adenovirus Bcl-X(S). Immunoprecipitation experiments revealed that adenoviral Bcl-X(S) associated with Bcl-X(L), but not with Bcl-2. Mutational analysis showed that the alpha 2 helical region of Bcl-X(S) containing the BH3 motif was critical for killing activity and interaction with Bcl-X(L). These results suggest that Bcl-X(S) is a direct killer and Bcl-X(L) may act by interacting with and sequestering Bcl-X(S.) These studies also suggest that targeting Bcl-X(L) may be of therapeutic benefit for the treatment of tumors that are characterized by inappropriate expression of Bcl-X(L).

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells.

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.

Specific and rapid induction of the proapoptotic protein Hrk after growth factor withdrawal in hematopoietic progenitor cells.

Hrk is a newly described proapoptotic member of the Bcl-2 family that is mainly expressed in hematopoietic tissues and cultured neurons. In this study we have examined the expression and activity of Hrk in hematopoietic progenitors. To address these issues, we used 3 growth factor-dependent murine hematopoietic cell lines, HCD-57, FDCP-Mix, and FL5.12. The expression of Hrk was undetectable in cells cultured with growth factors, but it was rapidly up-regulated on growth factor withdrawal. In contrast, the expression of Bcl-x(L) decreased and that of proapoptotic Bax, Bad, and Bak was unchanged or down-regulated after removal of growth factors. This pattern of expression correlated with the induction of apoptosis. Hrk was also up-regulated in human cell lines and in bone marrow-derived CD34(+) cells cultured in the absence of growth factors. In addition, the levels of Hrk were up-regulated after treatment with the chemotherapeutic drug etoposide. Expression of prosurvival Bcl-x(L) or Bcl-2 proteins blocked the induction of Hrk. Hrk was induced in FDCP-Mix cells treated with ionomicin in the presence of IL-3, suggesting that cytosolic calcium may regulate the expression of this proapoptotic protein. Furthermore, ectopic expression of Hrk induced cell death of hematopoietic progenitors in the presence of IL-3. Thus, Hrk is specifically and rapidly induced in hematopoietic progenitors after growth factor deprivation or treatment with chemotherapeutic drugs, and this may be sufficient to induce apoptosis in these cells. (Blood. 2000;95:2742-2747)

ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells.

Ischemia induces apoptosis as well as necrosis of cardiac myocytes. We recently reported the cloning of a cDNA that encodes an apoptotic inhibitor, ARC, that is expressed predominantly in cardiac and skeletal muscle. In the present study, we examined the ability of ARC to protect rat embryonic heart-derived H9c2 cells from apoptosis induced by hypoxia, a component of ischemia. We found that H9c2 cells express ARC and that exposure to hypoxia substantially reduces ARC expression while inducing apoptosis. Transfected H9c2 cells in which cytosolic ARC protein levels remain elevated during hypoxia were significantly more resistant to hypoxia-induced apoptosis than parental H9c2 cells or H9c2 cells transfected with a control vector. Loss of endogenous ARC in the cytosol of H9c2 cells was associated with translocation of ARC from the cytosol to intracellular membranes, release of cytochrome c from the mitochondria, activation of caspase-3, poly(ADP-ribose)polymerase (PARP) cleavage, and DNA fragmentation. All of these events were inhibited in H9c2 cells overexpressing ARC when compared with control cells. In contrast, caspase inhibitors prevented PARP cleavage but not cytochrome c release, suggesting that exogenously expressed ARC acts upstream of caspase activation in this model of apoptosis. These results demonstrate that ARC can protect heart myogenic H9c2 cells from hypoxia-induced apoptosis, and that ARC prevents cytochrome c release by acting upstream of caspase activation, perhaps at the mitochondrial level.

Erythropoietin can induce the expression of bcl-x(L) through Stat5 in erythropoietin-dependent progenitor cell lines.

Erythropoietin (Epo) initiates its cellular response by binding to the Epo receptor, which triggers the activation of signal transducer and activator of transcription (Stat) 5 protein. Cell culture studies of erythroid progenitors have suggested that Epo functions as a survival factor by repressing apoptosis at least in part through Bcl-x(L), an anti-apoptotic protein of the Bcl-2 family. In this report, we examine whether Stat5 can induce transactivation of the bcl-x gene in response to Epo. Two Epo-responsive progenitor cell lines, HCD-57 and Bcl-2-transfected Ba/F3-Epo receptor (Ba/F3-EpoR-Bcl-2), were used in this study. After Epo stimulation, we observed a correlation between expression of bcl-x(L) and activation of Stat5 as assessed by the expression of oncostatin M, a direct target of Stat5, and the phosphorylation and nuclear translocation of Stat5. Moreover, a Stat binding element in the bcl-x promoter was found to be active in response to Epo, a finding that was further confirmed because mutagenesis of this sequence motif abrogated its promoter activity and overexpression of a dominant negative Stat5 protein blocked transactivation. When DNA-protein binding analyses were performed, we found that Stat5, not Stat1 or Stat3, was the protein bound to the bcl-x promoter in response to Epo. These data suggest that Epo-dependent activation of Stat5 is a transcriptional pathway that can be used by Epo-responsive progenitor cells to induce the expression of bcl-x(L) and consequently to inhibit apoptosis.

Mtd, a novel Bcl-2 family member activates apoptosis in the absence of heterodimerization with Bcl-2 and Bcl-XL.

We have identified and characterized Mtd, a novel regulator of apoptosis. Sequence analysis revealed that Mtd is a member of the Bcl-2 family of proteins containing conserved BH1, BH2, BH3, and BH4 regions and a carboxyl-terminal hydrophobic domain. In adult tissues, Mtd mRNA was predominantly detected in the brain, liver, and lymphoid tissues, while in the embryo Mtd mRNA was detected in the liver, thymus, lung, and intestinal epithelium. Expression of Mtd promoted the death of primary sensory neurons, 293T cells and HeLa cells, indicating that Mtd is a proapoptotic protein. Unlike all other known death agonists of the Bcl-2 family, Mtd did not bind significantly to the survival-promoting proteins Bcl-2 or Bcl-XL. Furthermore, apoptosis induced by Mtd was not inhibited by Bcl-2 or Bcl-XL. A Mtd mutant with glutamine substitutions of highly conserved amino acids in the BH3 domain retained its ability to promote apoptosis, further indicating that Mtd does not promote apoptosis by heterodimerizing with Bcl-2 or Bcl-XL. Mtd-induced apoptosis was not blocked by broad range synthetic caspase inhibitors z-VAD-fmk or a viral protein CrmA. Mtd is the first example of a naturally occurring Bcl-2 family member that can activate apoptosis independently of heterodimerization with survival-promoting Bcl-2 and Bcl-XL.

Genomic organization, promoter region analysis, and chromosome localization of the mouse bcl-x gene.

The bcl-x gene, a bcl-2 family member, is highly regulated during lymphoid development, and its expression modulates apoptosis in lymphoid and other cell populations. Several forms of bcl-x mRNAs with different biologic functions have been described in rodents and humans. In this study, we have determined the organization and promoter region of the mouse bcl-x gene in an effort to understand the molecular basis for the different bcl-x mRNA species identified in tissues. We show that mouse bcl-x maps to the distal mouse chromosome 2 at approximately 89 cM, and exhibits a three-exon structure with an untranslated first exon and a facultative first intron. The coding region of bcl-xL is generated by the juncture of exons II and III through a splicing reaction, whereas bcl-xS is generated by an alternatively utilized donor splice site located within exon II. Analysis of multiple cDNAs and primer extension experiments revealed major transcription initiation sites in brain and thymus within a GC-rich region, with multiple Sp1-binding motifs located upstream of exon I. Another promoter was mapped to a 57-bp region localized upstream of the translation initiation codon by transfection of reporter constructs into FL5.12 and K562 cell lines. The remarkable similarity between the genomic regions of bcl-2 and bcl-x suggests that these genes have evolved from a common ancestral gene or through gene duplication.

Molecular cloning and characterization of P113, a mouse SNF2/SWI2-related transcription factor.

A full-length cDNA encoding a 113-kDa transcription factor, named P113, was cloned from mouse preadipocyte line 30A5. P113 binds to a 7-bp consensus TNF-response element and a 30-bp fragment from mouse PAI-1 promoter (-88/-59). Sequence analysis indicates that the P113 is highly homologous to HIP116/HLTF (human) and RUSH-1alpha (rabbit). The sequence homology and the fact that P113 contains seven motifs conserved in many DNA-dependent helicases/ATPases indicate that it is a new member of the SNF2/SWI2 protein family. A cysteine-rich motif, called RING finger, was found close to the C-terminus of P113. The expression pattern of P113 mRNA in rat tissues is significantly different from that of HLTF in human tissues. Affinity-purified P113 has an ATPase activity that is activated by DNA in a sequence-specific manner. Using Northern blot analysis and the PAI-1 promoter/luciferase system, we demonstrated that P113 is a transcription factor that activates the transcription of the PAI-1 gene in 30A5 cells.

Regulation of acetyl coenzyme-A carboxylase gene in a transgenic animal model.

The gene for acetyl-CoA carboxylase, the rate-limiting enzyme in the biosynthesis of long-chain fatty acids, contains two promoters; promoter I (PI) and promoter II (PII) which are responsible for generation of class I and class II ACC mRNAs, respectively. Class I ACC mRNAs are present in adipose tissue, but only a trace was found in the liver under normal physiological conditions. However, class I mRNAs were induced under stimulated lipogenic conditions. To investigate how PI is regulated in vivo, we generated transgenic mice containing a reporter gene under the control of PI. In transgenic mice, PI is generally inactive and a small amount of PI activity was found only in the adipose tissues of female animals. Stimulated lipogenic conditions activated PI about 17-fold over normal conditions and again only in white adipose tissues of female animals.

Retroviral vector-mediated transfer and expression of human tissue plasminogen activator gene in human endothelial and vascular smooth muscle cells.

PURPOSE: Enhancement of the fibrinolytic activity of vascular cells by tissue plasminogen activator (tPA) gene transfer has considerable clinical potential. However, it is unknown whether greater constitutive expression of the tPA gene might increase plasminogen activator inhibitor-1 (PAI-1) secretion, which could negate expected increases in fibrinolytic activity that accompany greater tPA protein production. The objective of this investigation was to determine whether transduction of human endothelial cells (EC) and vascular smooth muscle cells (SMC) with a retroviral vector containing the human tPA gene would increase tPA production and what effect this would have on endogenous PAI-1 secretion and subsequent fibrinolytic activity. METHODS: Cultivated human EC and SMC either were transduced with a murine leukemia retroviral vector (MFG) containing the human tPA gene and, in the case of controls, the lacZ gene, or they were exposed to media alone. On days 14 and 28 after transduction, supernatent tPA antigen and PAI-1 antigen levels were measured by ELISA, and supernatent tPA activity was quantitated with a spectrolyse tPA/PAI assay. RESULTS: Southern and Northern blot analyses documented integration and transcription of the tPA gene in both EC and SMC. Greater tPA antigen production occurred in MFG-tPA-transduced EC and SMC compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). The tPA activity increased in transduced human saphenous vein EC (up to 5.1-fold) and human iliac artery EC (up to 4.7-fold), but no increased tPA activity occurred in transduced SMC, compared with nontransduced or MFG-lac Z-transduced cells (p < 0.05). PAI-1 antigen was unchanged in transduced SMC but decreased in MFG-tPA-transduced EC (p < 0.05). PAI-1 mRNA was unchanged in the transduced EC and SMC compared with nontransduced cells, suggesting that posttranslational events may have caused the changes in EC PAI-1. CONCLUSIONS: This investigation demonstrated that MFG-mediated tPA gene transfer into human EC resulted in a significant increase in tPA activity. Enhancement of adult human EC fibrinolytic activity by transfer of the human tPA gene has not been previously reported and represents a necessary finding in the development of this gene therapy technology for the prevention of thrombotic complications of vascular disease.

Transplantation of lac-Z-transduced microvascular endothelial cells into the skeletal muscle capillary bed of the rat hindlimb occurs independent of the duration of femoral artery occlusion after injection of cells.

The skeletal muscle capillary bed may be an ideal recipient site for transplantation of genetically modified autologous endothelial cells and thus provide a basis for a technique of somatic gene therapy that would be applicable to a variety of acquired and inherited human diseases. The purpose of this study was to test the hypothesis that adhesion of lac-Z-transduced microvascular endothelial cells (MVEC) in the skeletal muscle capillary bed in vivo is dependent on the duration of arterial occlusion after injection of the transduced MVEC. MVEC derived from the abdominal fat pad of syngeneic rats (Wistar F-455) were transfected with the BAG vector, a replication-incompetent retroviral vector containing the lac-Z gene for beta-galactosidase and the Tn5 gene for selection of the transduced cells by the neomycin analogue, G418. lac-Z-transduced MVEC were radiolabeled with 125I-PKH-95, and, after the femoral artery was occluded for 10 min, these cells (1 to 2 x 10(6)) were injected intraarterially into the rat hindlimb. In the experimental groups the femoral artery clamp was removed at 0, 60, or 120 min after injection. A control group without pre- or postinjection femoral arterial occlusion was also studied. Adhesion of MVEC in the skeletal muscle capillary bed (mean percentage of injected 125I activity) was determined in groups of 4 rats at 1 day, 1 week, and 1 month after injection. Adhesion of the transduced MVEC did not increase as the duration of femoral artery occlusion after injection was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythropoietin does not cross the placenta into the fetus.

Erythropoiesis in the fetus is controlled by erythropoietin (Ep). To determine the role of maternal Ep in this process, we used catheterized preparations of sheep and monkey fetuses to assess the ability of Ep administered to the mother to cross the placental barrier into the fetus. Ep was injected into pregnant sheep (3,600 IU/sheep) or monkeys (800-2,000 IU/animal) as a single intravenous dose, or into sheep in intravenous doses of 2,000 IU once every 12 h for a total of 4 injections. Maternal and fetal blood samples for Ep and reticulocyte determinations were obtained before and at intervals after Ep injections. The administration of Ep resulted in significant increases in maternal circulating Ep levels in sheep and monkeys. Despite the presence of high levels of maternal Ep, however, no increase in fetal plasma Ep levels was detected. The administration of Ep to the mother caused significant increases in reticulocyte production in the mother but not the fetus; injection of Ep directly to the fetus stimulated fetal erythropoiesis. These results demonstrate that Ep does not cross the placenta into the fetus even under conditions of chronically elevated maternal Ep levels, and suggest that red cell production in the fetus is regulated by Ep produced from sites within the fetus.

Adhesion and incorporation of lacZ-transduced endothelial cells into the intact capillary wall in the rat.

Use of the capillary bed of skeletal muscle as an in vivo recipient site to transplant autologous endothelial cells that have undergone gene transfer ex vivo has considerable potential as a technique of somatic gene therapy. Here we document a previously unrecognized capacity of endothelial cells to adhere and incorporate spontaneously into confluent endothelial cell monolayers in vitro and in vivo. This spontaneous adhesion and incorporation of endothelial cells enabled us to seed lacZ-transduced endothelial cells into the wall of skeletal muscle capillaries of the hindlimb of the rat. Certain transduced endothelial cells became incorporated within the capillary wall, whereas others remained within the capillary lumen where they formed focal, electron-dense, contacts with host endothelium. lacZ expression in the capillary bed was documented for up to 1 month after transplantation. Use of the intact capillary bed of skeletal muscle as an in vivo recipient site for transduced, autologous endothelial cells holds promise as a strategy for somatic gene therapy to treat various genetic and acquired human diseases.

High-level expression of recombinant human tPA in cultivated canine endothelial cells under varying conditions of retroviral gene transfer.

Successful genetic transduction of endothelial cells (EC) provides a theoretic means of increasing luminal secretion of tissue-type plasminogen activator (tPA) and lessening arterial and venous thrombotic processes. To identify the duration and number of retroviral exposures for an optimal tPA expression, enzymatically derived adult canine jugular venous EC were subjected to different exposure regimens using an amphotropic murine retroviral vector, MFG, containing the human tPA gene. Human tPA antigen secretion and its functional activity were determined at 2 days (subconfluent cells) and 14 days (confluent cells) after retroviral exposure. High-level secretion of human tPA was detected among transduced EC in all experimental groups. No secretion of human tPA occurred in control EC exposed to media alone. At 2 days after transduction, no significant differences in tPA secretion rates occurred among the different exposure regimens. At 14 days, the 12-hour X two-exposure group exhibited higher tPA secretion rates than all other exposure regimens (analysis of variance, p < 0.05). All exposure groups at 14 days exhibited significantly higher tPA secretion compared with those at 2 days (analysis of variance, p < 0.05). The presence of retroviral sequences in the genome of transduced EC was confirmed by Southern blot analysis. At 14 days, increased EC numbers were observed in vector-exposed wells compared with controls. Human tPA functional activity paralleled tPA antigen secretion. Genetically modified canine EC are capable of high levels of constitutive expression of human tPA after relatively short exposures to a retroviral vector containing the reporter gene. Increased cell number of tPA-transduced EC in culture suggests that tPA also may have other biologically important effects. These results support the efficacy of MFG-tPA gene transfer as a means of genetically modifying EC fibrinolytic activity and establishes the potential of this technology in vivo.

Retroviral vector-mediated transfer of the bacterial neomycin resistance gene into fetal and adult sheep and human hematopoietic progenitors in vitro.

We compared the efficiency of retroviral vector (N2)-mediated transfer of the bacterial neomycin resistance gene (NeoR) into adult and fetal hematopoietic progenitors of sheep and humans by assessing their ability to form colonies in the presence of lethal doses of the neomycin analogue G418 in vitro. Fetal cells from both sheep and humans exhibited a higher degree of NeoR transfer than adult cells. The overall level of NeoR expression was significantly higher for sheep than human cells. The transfer/expression of NeoR into adult human bone marrow hematopoietic progenitors was not affected by the presence or absence of T cells and monocyte/macrophages. The efficiency of NeoR transfer into both adult and fetal human cells, however, was improved when transduction was carried out in the presence of recombinant human interleukin-3 and granulocyte-macrophage colony-stimulating factor. These results demonstrate the greater efficiency of NeoR gene transfer into fetal hematopoietic progenitors, which may provide a basis for the relatively higher efficiency of the in utero approach to gene therapy.