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).

Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9.

Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.

bcl-X(S)-induced cell death in 3T3 cells does not require or induce caspase activation.

Using a tetracycline-regulated expression system, we have shown that expression of bcl-X(s) is sufficient to induce acute cell death in 3T3 cells, and that the manner in which these cells die is both morphologically and biochemically different from Fas/CD95-induced apoptosis. bcl-X(s) expression causes loss of the inner mitochondrial membrane potential (deltapsim) but does not induce caspase activation. Loss of viability, as determined by mitochondrial function and ethidium bromide exclusion, was not inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of a dominant negative caspase 9 (9DN). However, zVAD-fmk was efficacious in inhibiting cell death triggered by an activating anti-Fas/CD95 antibody. In addition, bcl-X(s) does not possess the 5th and 6th alpha-helices (thought to be the membrane-spanning domains in bcl-2, bcl-X(L), and bax) and, therefore, should not be able to form membrane channels, thus eliminating this possible mechanism of action. The finding that bcl-X(s) kills 3T3 cells without caspase activation, along with the absence of membrane spanning domains in bcl-X(s), may, therefore, represent a novel cell death pathway for the pro-death bcl-2 family members.

Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis.

Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associated with and activated procaspase-9, but failed to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis.

Identification of regulatory and catalytic domains in the apoptosis nuclease DFF40/CAD.

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290-345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1-83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian and Drosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway.

Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation.

Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9. Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells. The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1. Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1. Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo. Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells. The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats. Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells. Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9. Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1. These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Caspases: the proteases of the apoptotic pathway.

Apoptosis, a morphologically defined form of physiological cell death, is implemented by a death machinery whose executionary arm is a family of cysteine proteases called caspases. These death proteases are part of a proteolytic caspase cascade that is activated by diverse apoptotic stimuli from outside and inside of the cell. The cell death machinery is evolutionarily conserved and composed of caspases and their regulatory components that include activators and repressors. These key components of the death machinery are linked to signaling pathways that are activated by either ligation of death receptors expressed at the cell surface or intracellular death signals. Caspases are normally present in the cell as proenzymes that require limited proteolysis for activation of enzymatic activity. Recent studies suggest that the basic mechanism of caspase activation is conserved in evolution. Binding of initiator caspase precursors to activator molecules appears to promote procaspase oligomerization and autoactivation. Enzymatic activation of initiator caspases leads to proteolytic activation of downstream (effector) caspases and cleavage of a number of vital proteins, resulting in the orderly demise and removal of the cell.

A1 demonstrates restricted tissue distribution during embryonic development and functions to protect against cell death.

Members of the bcl-2 gene family are essential regulators of cell survival in a wide range of biological processes. A1, a member of the family, is known to be expressed in certain adult tissues. However, the precise tissue distribution and function of A1 remains poorly understood. We show here that A1 is expressed in multiple tissues during murine embryonic development. In the embryo, A1 was detected first at embryonic day 11.5 in liver, brain, and limbs. At day 13.5 of gestation, A1 expression was observed in the central nervous system, liver, perichondrium, and digital zones of developing limbs in a pattern different from that of bcl-X. In the central nervous system of 15.5-day embryos, A1 was expressed at high levels in the ventricular zone and cortical plate of brain cortex. Significantly, the interdigital zones of limbs and the intermediate region of the developing brain cortex, two sites associated with extensive cell death, were devoid of A1 and bcl-X. The expression of A1 was retained in many adult tissues. To assess the ability of A1 to modulate cell death, stable transfectants expressing different amounts of A1 protein were generated in K562 cells. Expression of A1 was associated with retardation of apoptotic cell death induced by actinomycin D and cycloheximide as well as by okadaic acid. Confocal microscopy showed that the A1 protein was localized to the cytoplasm in a pattern similar to that of Bcl-2. These results demonstrate that the expression of A1 is wider than previously reported in adult tissues. Furthermore, its distribution in multiple tissues of the embryo suggests that A1 plays a role in the regulation of physiological cell death during embryonic development.

A recombinant bcl-x s adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.

We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.

Fluid replacement after dehydration: influence of beverage carbonation and carbohydrate content.

This investigation evaluated the effects of beverage carbonation and carbohydrate (CHO) content on fluid replacement following exercise/thermal dehydration. On four occasions separated by at least 7 days, eight healthy men cycled at 50% of VO2max in a hot environmental chamber (40 degrees C, 40% relative humidity) until a weight loss of 4.12 +/- 0.22% was attained. In the subsequent four hours, subjects ingested one of four solutions at 15-min intervals. The total volume ingested equalled that lost during dehydration. The solutions were administered in randomized order and varied in their carbonation and carbohydrate (CHO) content: 1. CK: carbonated 10% glucose-fructose solution, 2. NCK: non-carbonated 10% glucose-fructose solution, 3. CNK: carbonated non-caloric solution, and 4. NCNK: non-carbonated non-caloric solution. Plasma volume changes, total plasma protein concentration, plasma osmolality, and the plasma glucose concentration were determined at rest before and after dehydration, and at 30, 90, 150, and 240 min of recovery. Plasma volume changes and the plasma protein concentration were not different (p greater than 0.05) between treatments. Values for the plasma glucose concentration and the change in plasma osmolality were significantly elevated when CHO beverages were ingested when compared with non-CHO beverage ingestion. Five-min cycling bouts were performed at 70% of VO2max before and after dehydration and at 60, 120, 180, and 240 min of rehydration. The respiratory exchange ratio was elevated in both of the CHO treatments when compared with both of the non-CHO treatments at 60, 120, 180 and 240 min of rehydration.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of protein synthesis inhibition on the transcription and transcript stability of Dictyostelium prespore genes.

The in vivo accumulation of several prespore transcripts of Dictyostelium discoideum has previously been shown to depend upon concomitant protein synthesis (Ratner, D.I., Pentz, W.H. and Pelletier, D.A. (1989) Biochim. Biophys. Acta 1008, 71-78). Measurements of in vivo mRNA decay and nuclear run-on transcription assays have now been used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. The translational inhibitors cycloheximide and pactamycin stabilized existing prespore transcripts, despite their effect upon mRNA accumulation. Transcriptional assays, performed at intervals throughout the developmental cycle, demonstrated that temporal changes in the abundance of several cell-specific transcripts correlated closely with changes in their rates of synthesis. Finally, blocking protein synthesis strongly inhibited the transcription of the prespore genes examined. These results imply that one or more developmentally regulated, labile proteins are needed for the activation of prespore gene transcription.

Muscle glycogenolysis during differing intensities of weight-resistance exercise.

Skeletal muscle glycogen metabolism was investigated in eight male subjects during and after six sets of 70% one repetition maximum (1 RM, I-70) and 35% 1 RM (I-35) intensity weight-resistance leg extension exercise. Total force application to the machine lever arm was determined via a strain gauge and computer interfaced system and was equated between trials. Compared with the I-70 trial, the I-35 trial was characterized by almost double the repetitions (13 +/- 1 vs. 6 +/- 0) and half the peak concentric torque for each repetition (12.4 +/- 0.5 vs. 24.2 +/- 1.0 Nm). After the sixth set, muscle glycogen degradation was similar between I-70 and I-35 trials (47.0 +/- 6.6 and 46.6 +/- 6.0 mmol/kg wet wt, respectively), as was muscle lactate accumulation (13.8 +/- 0.7 and 16.7 +/- 4.2 mmol/kg wet wt, respectively). After 2 h of passive recovery without caloric intake, muscle glycogen increased by 22.2 +/- 6.8 and 14.2 +/- 2.5 mmol/kg wet wt in the I-70 and I-35 trials, respectively. Optical absorbance measurement of periodic acid-Schiff-stained muscle sections after the 2 h of recovery revealed larger absorbance increases in fast-twitch than in slow-twitch fibers (0.119 +/- 0.024 and 0.055 +/- 0.024, P = 0.02). Data indicated that when external work was constant, the absolute amount of muscle glycogenolysis was the same regardless of the intensity of resistance exercise. Nevertheless the rate of glycogenolysis during the I-70 trial was approximately double that of the I-35 trial.

Developmental protein synthesis is required for the transcription of Dictyostelium prespore genes.

It has been established previously that the maintenance of expression of prespore-specific genes of Dictyostelium discoideum is prevented by the translational inhibitor cycloheximide. The drug had no effect upon the level of transcripts of the other genes examined, prestalk-specific or cell type-nonspecific. However, the interpretation of this result is open to question, because of possible nonspecific effects of cycloheximide. We have now characterized the cellular specificity and temporal profiles of mRNA accumulation of additional Dictyostelium cDNA clones, and have examined other inhibitors of in vivo protein synthesis. Four structurally and mechanistically distinct translational inhibitors each prevented the reaccumulation of prespore transcripts in cyclic AMP-primed, disaggregated amoebae. These results establish the importance of developmental protein synthesis in the accumulation of prespore gene transcripts. Nuclear run-on transcription assays were used to learn whether protein synthesis is required primarily for mRNA synthesis or transcript stability. A transcriptional time course first demonstrated that the abundance of these cell-specific transcripts during development mirrors their rates of synthesis. Significantly, the protein synthesis requirement of the prespore genes examined also occurs at the level of mRNA transcription, implying the existence of one or more developmentally regulated transcriptional activators.

Identification of murine complement receptor type 2.

A rabbit antiserum reactive with the human complement component C3d/Epstein-Barr virus receptor (complement receptor type 2, CR2) immunoprecipitates a Mr 155,000 murine B-cell surface antigen. The apparent molecular weight and cellular distribution of this murine antigen are similar to those of human CR2. Cells expressing the murine protein bind sheep erythrocytes coated with antibody and murine C1-C3d but do not bind Epstein-Barr virus at all. The monospecific antiserum to human CR2 together with goat F(ab')2 anti-rabbit IgG blocks attachment of the C3d-coated erythrocytes to receptor-bearing murine B lymphocytes. To further characterize murine CR2, a lambda gt11 library from the murine late pre-B-cell line 70Z/3 was screened with human CR2 cDNA. A partial cDNA clone of 3.5 kilobases with 79% amino acid sequence identity to human CR2 in the unique intracytoplasmic region and 63% identity to the sixth human CR2 repeat was obtained. Blot hybridization with the murine cDNA clone identified an RNA species of approximately 4.7 kilobases, similar in size to human CR2 mRNA, from a murine B-cell line but not from a murine T-cell line.