Bcl-x Transcripts Research Articles

HnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the alternative splicing response to oxaliplatin-mediated DNA damage

Little is known about how RNA binding proteins cooperate to control splicing, and how stress pathways reconfigure these assemblies to alter splice site selection. We have shown previously that SRSF10 plays an important role in the Bcl-x splicing response to DNA damage elicited by oxaliplatin in 293 cells. Here, RNA affinity assays using a portion of the Bcl-x transcript required for this response led to the recovery of the SRSF10-interacting protein 14-3-3ε and the Sam68-interacting protein hnRNP A1. Although SRSF10, 14-3-3ε, hnRNP A1/A2 and Sam68 do not make major contributions to the regulation of Bcl-x splicing under normal growth conditions, upon DNA damage they become important to activate the 5′ splice site of pro-apoptotic Bcl-xS. Our results indicate that DNA damage reconfigures the binding and activity of several regulatory RNA binding proteins on the Bcl-x pre-mRNA. Moreover, SRSF10, hnRNP A1/A2 and Sam68 collaborate to drive the DNA damage-induced splicing response of several transcripts that produce components implicated in apoptosis, cell-cycle control and DNA repair. Our study reveals how the circuitry of splicing factors is rewired to produce partnerships that coordinate alternative splicing across processes crucial for cell fate.

Developmental consequences of alternative Bcl‐x splicing during preimplantation embryo development

Elevated cell death in human preimplantation embryos is one of the cellular events compromising pregnancy rates after assisted reproductive technology treatments. We therefore explored the molecular pathways regulating cell death at the blastocyst stage in human embryos cultured in vitro. Owing to limited availability of human embryos, these pathways were further characterized in mouse blastocysts. Gene expression studies revealed a positive correlation between the cell death index and the expression of Bcl-x transcript. Cell death activation in human blastocysts was accompanied by changes in Bcl-x splicing, favoring production of Bcl-xS, an activator of cell death. Expression of Bcl-xS was detected in a subset of human blastocysts that show particular clustering in dying and/or dead cells. Altering the Bcl-xL/Bcl-xS ratio in mouse embryos, in antisense experiments, confirmed that upregulation of Bcl-xS, with concomitant downregulation of Bcl-xL, compromised developmental potential and committed a subset of cells to undergoing cell death. This was accompanied by increased accumulation of reactive oxygen species levels without any impact on mtDNA content. In addition, altered Bcl-x splicing in favor of Bcl-xS was stimulated by culture in HTF medium or by addition of excessive glucose, leading to compromised embryo development. Thus, we conclude that inappropriate culture conditions affect Bcl-x isoform expression, contributing to compromised preimplantation embryo development.

The Oncogenic Kinase Bcr-Abl Directly Regulates Splicing of BclX through a Quaternary Complex Coordinated by Nck-Beta and Sam-68 Adapter Proteins.

Abstract 2171Poster Board II-148Chronic Myelogenous Leukemia is the prototype of myeloproliferative disorder characterized by a reciprocal chromosomal translocation, involving the chromosomes 9 and 22 —t(9;22)-. The molecular consequence of this translocation is the generation of the Bcr-Abl oncogene that encodes the chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. Its expression in hematopoietic cells induces uncontrolled and growth factor independent cellular proliferation, alteration in cell-cell and cell-matrix adhesion, resistance to apoptosis which altogether are leukemogenesis landmarks.Although it is well established that Bcr-Abl-expressing leukemic cells are highly resistant to apoptotic cell death induced by chemotherapeutic drugs and a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway/s triggered by this oncogene has not been fully understood.The numerous experimental evidences collected in the last years highlight the crucial role played by alternative splicing in the control of apoptosis. Several pre-mRNAs for cell death factors are alternatively spliced, yielding isoforms with opposing functions during programmed cell death. A clear example is Bcl-x transcript which is alternatively spliced to produce the antiapoptotic Bcl-x(L) or the proapoptotic Bcl-x(S). Identical features are shared by another member of the Bcl-2 family, the myeloid cell leukemia-1 (MCL-1) gene which encodes two alternative splicing variants MCL-1S and MCL-1L displaying pro- and anti-apoptotic effects, respectively.CML-derived cell lines overexpress the antiapoptotic protein Bcl-x(L) and their erythroid differentiation is inhibited by Bcl-x(L).The data so far collected indicate that there is an extensive cross-talk among BCL-2 family members by virtue of their protein-protein interactions and the ratio of pro-apoptotic to anti-apoptotic proteins has been shown to be a major detereminant of the cell propensity to undergo apoptosis.Furthermore, it is well established that accelerated and blastic phases of the disease are characterized by deregulated WT1 expression. WT1/KTS- gene encodes for a transcription factor but the WT1/KTS+ isoform has been reported to localize into nuclear speckles, the major sub-nuclear structures enriched pre-messenger RNA and splicing factors.Based on the above premise we started investigating the possibility of an active involvement of Bcr-Abl as candidate regulator of splicing events affecting Bcl-x pre-mRNA.By means of an interactomic approach, based on proteomic strategy using GST-Pull Down assay with an array of SH2 containing proteins, we attempted to gain insight into the role played by adapter molecules and Bcr-Abl in splicing assembling machinery. The data presented aims to demonstrate the presence of quaternary complex involving the SH2-SH3 containing adapter protein Nck-beta, the oncogenic tyrosine kinase Bcr-Abl, the RNA binding protein Sam68, the spliceosome ribonucleprotein hnRNPA1 and WT1. The experimental evidences we have collected support the hypothesis of an Imatinib-dependent interaction occurring between Nck-beta and Bcr-Abl. Furthermore, Pull Down experiments indicate an intermolecular interaction between Nck-beta, Sam68, and hnRNPA1 supporting the idea of a novel complex Bcr-Abl/Nck-beta/Sam68/hnRNPA1/WT1. Biochemical analysis carried-out by Pull-Down experiments has been further corroborated by immunofluorescence staining. RNA Pull Down assay suggest that the quaternary complex Nck-beta/Sam68/hnRNPA1/Bcr-Abl/WT1 might modulates splicing process of Bcl-x gene, whose function has been recently described as crucial in myeloproliferative disorders. Astoningshly, the data collected so far indicates that other mRNAs are pulled-down together with the quaternary complex. Taken together these results represent the first experimental evidences showing an interaction between the oncogene Bcr-Abl and Sam-68 leading to speculate a novel putative role played by Bcr-Abl in the intriguing and complex pre-mRNA splicing scenario. Disclosures:No relevant conflicts of interest to declare.

SAHA induces apoptosis in hepatoma cells and synergistically interacts with the proteasome inhibitor Bortezomib

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.

Molecular requirements for doxorubicin-mediated death in murine oocytes

We previously published evidence that oocytes exposed to doxorubicin (DXR), a widely used chemotherapeutic agent, rapidly undergo morphological and biochemical changes via discrete effector signaling pathways consistent with the occurrence of apoptosis. In this report, we elucidated the molecular requirements for actions of this drug in oocytes. Our results indicate that within 1 h of exposure DXR causes rapid DNA damage, and commits the oocyte to cytoplasmic fragmentation by the fourth hour, followed by delayed oocyte activation and execution of cytoplasmic fragmentation. Inhibitors that interfere with oocyte activation consistently rescue cytoplasmic fragmentation, but fail to suppress DNA damage. There was evidence of depletion of Bax, Caspase-2, MA-3 and Bcl-x transcripts, suggesting that modulations by DXR caused recruitment of these maternal transcripts into the translation process. Furthermore, sphingolipids such as sphingosine-1-phosphate and ceramide modulate DXR actions by, respectively, altering its intracellular trafficking, or by sustaining the drug's contact with DNA.

The Minimal Epo Receptor Allele, EpoR-H, Regulates ERK1,2, (But Not JNKs or p70S6K), and Induces Pim-1, SOCS3 and Oncostatin-M (But Not BcL-XL) in Primary Erythroblasts.

In studies of core Epo receptor signals that are necessary-and-sufficient for erythroblast development, we have shown that an EpoR-PY343-Stat5 axis is required for efficient erythropoiesis during anemia. This involved analyses of knocked-in truncated Epo receptor alleles that retain only a PY343 Stat5 binding site (EpoR-H allele), or lack all cytoplasmic PY motifs (EpoR-HM allele). Presently we have analyzed core signaling capacities of these receptor forms (and the wt-EpoR) in purified developmentally-staged marrow- derived erythroblasts. Analyses of JNKs, p70s6K and ERKs revealed that JNK and p70s6K activation is mediated by non-essential C-terminal EpoR domains. ERKs, in contrast, remained coupled to EpoR-H and EpoR-HM forms. EpoR-H/PY343/Stat5 response genes that might support EpoR-H's wild-type activity next were analyzed. Despite evidence for Stat5 binding at a Bcl-x intron-1 element, no significant Epo-induction of Bcl-x transcripts was detected for any EpoR allele. In contrast, Pim-1, oncostatin-M and SOCS-3 were induced multi-fold in wt-EpoR and EpoR-H, but not EpoR-HM erythroblasts (and for EpoR-HM, no activation of Stat-5, -1 or-3 was detectable). In addition, receptor cross-talk analyses revealed that Kit selectively modulates EpoR-PY343-Stat5- dependent SOCS-3 and oncostatin-M transcription, and enhances Pim-1 expression several-fold via apparent post-transcriptional mechanisms. Epo action therefore is proposed to depend primarily on the regulation of ERKs, Pim-1 and possibly oncostatin-M (but not JNKs, p70S6K or Bcl-x).

Heterogeneous Nuclear Ribonucleoprotein F/H Proteins Modulate the Alternative Splicing of the Apoptotic Mediator Bcl-x

Bcl-x is a member of the Bcl-2 family of proteins that are key regulators of apoptosis. The Bcl-x pre-mRNA is alternatively spliced to yield Bcl-x(S) and Bcl-x(L), two isoforms that have been associated, respectively, with the promotion and the prevention of apoptosis. We have investigated some of the elements and factors involved in the production of these two splice variants. Deletion mutagenesis using a human Bcl-x minigene identifies two regions in exon 2 that modulate Bcl-x 5'-splice site selection in human HeLa cells. One region (B3) is located upstream of the Bcl-x(L) 5'-splice site and enforces Bcl-x(L) production in cells and splicing extracts. The other region (B2) is located immediately downstream of the 5'-splice site of Bcl-x(S) and favors Bcl-x(S) production in vivo and in vitro. A 30-nucleotide G-rich element (B2G) is responsible for the activity of the B2 element. We show that recombinant heterogeneous nuclear ribonucleoprotein (hnRNP) F and H proteins bind to B2G, and mutating the G stretches abolishes binding. Moreover, the addition of hnRNP F to a HeLa extract improved the production of the Bcl-x(S) variant in a manner that was dependent on the integrity of the G stretches in B2G. Consistent with the in vitro results, small interfering RNA-mediated RNA interference targeting hnRNP F and H decreased the Bcl-x(S)/Bcl-x(L) ratio of plasmid-derived and endogenously produced Bcl-x transcripts. Our results document a positive role for the hnRNP F/H proteins in the production of the proapoptotic regulator Bcl-x(S.).

Transcription of Bcl-x In erythroid progenitor cells

Abstract Bcl-x has been shown to be essential for survival and maturation of late erythroid progenitors and for survival of cells in the developing brain. During the late phases of erythroid differentiation there is a large increase in the Bcl-x protein and its mRNA in mouse and human erythroblasts. In mouse erythroblasts and other hematopoietic cells, the main transcription initiation sites form a cluster at positions −662, −653 and −643 relative to the ATG initiation codon. This cluster of sites accounts for about 70% of all transcripts in these cells and another 15 to 20% appear to be initiated at position −112. Human erythroblasts exhibit a doublet of transcription start sites at −660 and −654 relative to the initiation codon that is homologous to the main cluster in the mouse. In contrast to hematopoietic tissues, a large fraction of bcl-x transcripts isolated from mouse brain originates from start sites far upstream of those used in erythroblasts. To determine which sequences upstream of the coding region of the gene are important for regulation of transcription, plasmid constructs were made that contain various portions of this region of bcl-x (from −1 to −1211) placed just upstream of the firefly luciferase gene. These plasmids were used in transient transfection assays of bcl-x promoter function in the mouse erythroid cell line HCD57, a line that is dependent on erythropoietin, EPO, for viability. A DNA sequence between positions −196 and −96 was found to be essential for transcription, and this sequence was sufficient for transcription at nearly the maximum rate observed for any of the plasmids. Additional bcl-x sequence between −196 and −998 had little effect on the rate of transcription. However, plasmids containing the sequence between −998 and −1211 exhibited a significant reduction of transcription rate, indicating that there is a negative regulatory element in that region. Transient transfection experiments did not show evidence for localized regulatory sequences within the −1 to −1211 region that modulate transcription specifically in response to EPO.

Bcl-x and Bax regulate mouse primordial germ cell survival and apoptosis during embryogenesis.

Restricted germ cell loss through apoptosis is initiated in the fetal gonad around embryonic day 13.5 (E13.5) as part of normal germ cell development. The mechanism of this germ cell attrition is unknown. We show that Bcl-x plays a crucial role in maintaining the survival of mouse germ cells during gonadogenesis. A bcl-x hypomorphic mouse was generated through the introduction of a neomycin (neo) gene into the promoter of the bcl-x gene by homologous recombination. Mice that contained two copies of the hypomorphic allele had severe reproductive defects attributed to compromised germ cell development. Males with two mutant alleles lacked spermatogonia and were sterile; females showed a severely reduced population of primordial and primary follicles and exhibited greatly impaired fertility. Primordial germ cells (PGCs) in bcl-x hypomorph mice migrated to the genital ridge by E12.5 but were depleted by E15.5, a time when Bcl-x and Bax were present. Two additional bcl-x transcripts were identified in fetal germ cells more than 300 bp upstream of previously reported start sites. Insertion of a neo cassette led to a down-regulation of the bcl-x gene at E12.5 in the hypomorph. Bax was detected by immunohistochemistry in germ cells from bcl-x hypomorph and control testes at E12.5 and E13.5. Bcl-x function was restored, and animals of both genders were fertile after removal of the neo selection cassette using Cre-mediated recombination. Alternatively, the loss of Bcl-x function in the hypomorph was corrected by the deletion of both copies of the bax gene, resulting in a restoration of germ cell survival. These findings demonstrate that the balance of Bcl-x and Bax control PGC survival and apoptosis.

Costimulatory function of CD28 in avian gammadelta T cells is evolutionarily conserved.

CD28 costimulatory signals are required for T-cell proliferation and lymphokine production. In this work, the functional conservation of CD28 was studied in avian gammadelta T cells. The avian CD28 molecule is expressed on all alphabeta T cells and is capable of giving a costimulatory signal. Most peripheral gammadelta T cells are CD28 negative; however, we identified a CD28-positive gammadelta T-cell subset from peripheral blood comprising about 12% of gammadelta T cells. The peripheral CD28+ gammadelta T-cell subset included all CD8+ gammadelta T cells known to be a responding subset during activation. After polyclonal activation, the frequency of CD28+ gammadelta T cells was increased and the activation also up-regulated CD5, CD25 and major histocompatibility complex (MHC) class II molecules. These changes were detected after both polyclonal and antigen-specific T-cell activation. In addition, we also showed that CD28 can give a costimulatory signal to gammadelta T cells and that this signal leads to up-regulation of IL-2 and bcl-x transcripts. These results indicate that the function of CD28 is evolutionarily conserved and can already be detected in avian gammadelta T cells.

Identification of a Human cDNA Encoding a Novel Bcl-x Isoform

Alternative splicing has been shown to generate two isoforms of the apoptosis regulator bcl-x, Bcl-xLand Bcl-xS, in humans. Here we describe the identification and characterization of a third splice variant of the human bcl-x gene. It differs from previously described bcl-x transcripts in two respects: (1) a novel facultative intron is spliced out at the 5′ untranslated region and (2) the open reading frame arises from a continuous genomic sequence extending over the splice donor sites utilized by the bcl-xLand bcl-xStranscripts. Since the resulting molecule has an organisation homologous to mouse and rat Bcl-xβwe suggest calling this novel protein human Bcl-xβ. Northern blot analysis revealed that bcl-xβmRNA is expressed in numerous cell lines. Like Bcl-xL, h-Bcl-xβbinds to the pro-apoptotic protein Bax, suggesting a functional activity in vivo.

Interferon-γ Modulates a p53-independent Apoptotic Pathway and Apoptosis-related Gene Expression

Interferon (IFN)-gamma increases the sensitivity of tumor cell lines, many of which are p53 mutants, to tumor necrosis factor-alpha-mediated and anti-Fas antibody-mediated cell death. To better understand the mechanism of IFN-gamma action in modulating the cell death response independently of p53 function, we analyzed the death of the human colon adenocarcinoma cell line, HT-29, following treatment with IFN-gamma and various cytotoxic agents. Here we show that IFN-gamma modulates cell death by sensitizing the cells to killing by numerous pro-apoptotic stimuli but not pro-necrotic stimuli. Furthermore, we show that select genes from several important apoptosis-related gene families are induced by IFN-gamma, including the apoptosis-signaling receptors CD95 (Fas/APO-1) and TNFR 1 and interleukin-1beta-converting enzyme (Ice) family members Ice, CPP32 (Yama, apopain), ICErel-II (TX, Ich-2), Mch-3 (ICE-LAP3, CMH-1), Mch-4, and Mch-5 (MACH, FLICE). Of the bcl-2 family members, IFN-gamma directly induced bak but notably not bax, which is activated by p53. The IFN-responsive transcriptional activator interferon regulatory factor-1 was also strongly induced and translocated into the nucleus following IFN-gamma treatment. We propose that IFN-gamma modulates a p53-independent apoptotic pathway by both directly and indirectly inducing select apoptosis-related genes.

Progestins Prevent Apoptosis in a Rat Endometrial Cell Line and Increase the Ratio of bcl-XL tobcl-XS

Endometrial cell proliferation and cell death are regulated by ovarian hormones. The fall of ovarian progesterone in late secretory phase, or the artificial withdrawal of ovarian hormones during early pregnancy, are followed by programmed cell death of uterine epithelial cells. Aspects of this cell-specific response have been reproduced in a newly established rat endometrial cell line which expresses functional progesterone receptor. At low concentrations of serum and in the absence of glucocorticoids, these cells were dependent on progestins for survival. Removal of progesterone or addition of the antiprogestins RU38486 or ZK98299 led to a substantial increase of apoptotic cells indicated by the accumulation of internucleosomally degraded DNA. The hormonal control of cell proliferation and cell death correlated with the overall quantity and distribution of the different bcl-X transcripts. Progesterone administration not only increased total bcl-X mRNA level but also shifted the quantitative ratio between the different mRNA isoforms in favor for the apoptosis inhibiting form, bcl-XL, compared with the apoptosis promoting form, bcl-XS. These effects were rapid and could not be prevented by inhibitors of protein synthesis. As the low level of bcl-2 and bax mRNA was not influenced by progesterone treatment, the observed changes in total amount of bcl-X transcripts and spliced isoforms could represent the mechanism by which progesterone controls cell death in epithelial cells of the endometrium.

The Expression of Bcl-x Is Downregulated During Differentiation of Human Hematopoietic Progenitor Cells Along the Granulocyte But Not the Monocyte/Macrophage Lineage

AbstractExpression of the apoptosis inhibitory protein Bcl-x was studied in CD34+ hematopoietic precursor cells and in the promyelocytic leukemia cell line HL-60. The enriched population of CD34+ cells (more than 95%) was cultured in the presence of stem cell factor, interleukin-3 (IL-3), IL-6, and either granulocyte colony-stimulating factor or macrophage colony-stimulating factor to achieve granulocyte or monocyte/macrophage differentiation, respectively. The expression of Bcl-x increased in the early stages of both differentiation pathways. However, by day 21 of culture mature granulocytes were Bcl-x–negative, whereas monocytes/macrophages either maintained or increased the expression of Bcl-x. The pattern of Bcl-x expression in the differentiated CD34+ cells was similar to that observed in HL-60 cells differentiated along the granulocyte lineage (induced by incubation with retinoic acid), or along the monocyte/macrophage lineage (induced by incubation with phorbol diester). The bcl-x transcript predominant in HL-60 and CD34+ cells differentiated into monocytes/macrophages was bcl-xL . Although little is yet known regarding the functional significance of Bcl-x within the granulomonocytic compartment, marked changes in the pattern of its expression, as observed during granulomonocytic differentiation of HL-60 and CD34+ cells, are likely to alter the life span of mature granulocytes and monocytes/macrophages.

Primitive Human Hematopoietic Precursors Express Bcl-x But Not Bcl-2

Bcl-2 and its homologue, bcl-xL, encode membrane-associated proteins that suppress programmed cell death of hematopoietic cell lines after growth factor withdrawal, and are expressed in hematopoietic precursor cells. To better understand the maintenance of long-term survival in the hematopoietic stem cell population, we evaluated the expression patterns of Bcl-2 and Bcl-x in primitive hematopoietic precursor populations. Hematopoietic precursor cells expressing CD34 (CD34+) and lacking maturation-linked surface antigens (lin-) were isolated from adult human bone marrow using two-color immunofluorescence cell sorting and fractionated on the basis of forward light scatter characteristics into blast-sized and small to medium lymphocyte-sized cell populations. Bcl-2 expression was shown in 78% to 90% of CD34+ lin- blast-sized cells versus less than 10% of small to medium lymphocyte-sized CD34+ lin- cells by immunohistochemical analysis. Small to medium lymphocyte-sized CD34+ lin- cells were further enriched for primitive precursors by selecting cells that lacked expression of CD38 (CD34+ lin- CD38-). In parallel experiments, only 1% to 4% of CD34+ lin- CD38- cells expressed Bcl-2, whereas 45% to 56% of these cells generated colony-forming cells. In contrast, > or = 94% of cells in all bone marrow subpopulations studied expressed Bcl-x protein. Both alternatively spliced bcl-x transcripts, bcl-xL and bcl-xs, were present. Our data show that the most primitive hematopoietic precursors express Bcl-x but not Bcl-2. Thus, the functional bcl-2 homologue, bcl-xL, may be essential for the long-term survival of the hematopoietic stem cell population.

Primitive human hematopoietic precursors express Bcl-x but not Bcl-2

Bcl-2 and its homologue, bcl-xL, encode membrane-associated proteins that suppress programmed cell death of hematopoietic cell lines after growth factor withdrawal, and are expressed in hematopoietic precursor cells. To better understand the maintenance of long-term survival in the hematopoietic stem cell population, we evaluated the expression patterns of Bcl-2 and Bcl-x in primitive hematopoietic precursor populations. Hematopoietic precursor cells expressing CD34 (CD34+) and lacking maturation-linked surface antigens (lin-) were isolated from adult human bone marrow using two-color immunofluorescence cell sorting and fractionated on the basis of forward light scatter characteristics into blast-sized and small to medium lymphocyte-sized cell populations. Bcl-2 expression was shown in 78% to 90% of CD34+ lin- blast-sized cells versus less than 10% of small to medium lymphocyte-sized CD34+ lin- cells by immunohistochemical analysis. Small to medium lymphocyte- sized CD34+ lin- cells were further enriched for primitive precursors by selecting cells that lacked expression of CD38 (CD34+ lin- CD38-). In parallel experiments, only 1% to 4% of CD34+ lin- CD38- cells expressed Bcl-2, whereas 45% to 56% of these cells generated colony- forming cells. In contrast, > or = 94% of cells in all bone marrow subpopulations studied expressed Bcl-x protein. Both alternatively spliced bcl-x transcripts, bcl-xL and bcl-xs, were present. Our data show that the most primitive hematopoietic precursors express Bcl-x but not Bcl-2. Thus, the functional bcl-2 homologue, bcl-xL, may be essential for the long-term survival of the hematopoietic stem cell population.

Bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria.

Most examples of cell death in animals are controlled by a genetic program that is activated within the dying cell. The apoptotic process is further regulated by a set of genes that act as repressors of cell death. Of these, bcl-2 is expressed in a variety of embryonic and postnatal tissues which suggests a critical role for bcl-2 in organogenesis and tissue homeostasis. Surprisingly, mutant mice with targeted disruption of bcl-2 appear normal at birth and complete maturation of lymphoid tissues before succumbing to fulminant lymphopenia and polycystic renal disease by 2-5 weeks of age. This suggests that there may be genes other than bcl-2 that can regulate apoptosis during development. To begin to investigate this possibility, we have cloned and characterized the murine bcl-x gene, whose human counterpart displays striking homology to bcl-2. The predicted murine bcl-xL gene product exhibits a high level of amino acid identity (97%) to its human counterpart. Just like Bcl-2, the murine bcl-xL gene product can act as a dominant inhibitor of cell death upon growth factor withdrawal. In addition, the bulk of the bcl-xL product localizes to the periphery of mitochondria as assessed by a bcl-xL-tag expression system, suggesting that both Bcl-2 and Bcl-xL proteins prevent cell death by a similar mechanism. bcl-xL is the most abundant bcl-x mRNA species expressed in embryonic and adult tissues. The levels of bcl-xL mRNA appear higher than those of bcl-2 during embryonal development and in several adult organs including bone marrow, brain, kidney and thymus. In addition to bcl-xL, we have identified another form of bcl-x mRNA, bcl-x beta, that results from an unspliced bcl-x transcript. bcl-x beta mRNA is expressed in various embryonic and postnatal tissues. Surprisingly, the expression of bcl-xS (a negative regulator of programmed cell death) was undetectable by a sensitive S1-nuclease assay and polymerase chain reaction analysis of mouse tissues. Based on its tissue and developmental patterns of expression, it appears that bcl-x may play an important role in the regulation of cell death during development and tissue homeostasis.