An exegesis of IAPs: salvation and surprises from BIR motifs.

The BIR (baculovirus IAP repeat) motif is a conserved sequence of approximately 70 amino acids that was identified originally in the 'inhibitor of apoptosis' (IAP) family of proteins. BIR-containing proteins (BIRPs) are found in viruses, yeast and metazoans. Recent genetic analysis of a nematode BIRP demonstrated an essential role in cytokinesis instead of apoptosis. It is likely that BIRs originated in eukaryotes to serve a role in cytokinesis and/or mitotic spindle function during cell division and that, with gene duplication, the more recent adaptation of some BIRPs to the regulation of apoptosis was possible. IAPs interact with a variety of proteins, including members of the caspase protease family. This article discusses current research on the structure and function of the BIR motifs and how it could provide insight into the function of BIRPs in cell division.

Freezing tolerance in an adult insect.

The adult carabid beetle Pterostichus brevicornis tolerates freezing under natural conditions. Laboratory tests confirm that winter beetles tolerate temperatures below -35 degrees C, whereas summer beetles die if frozen at -6.6 degrees C. Winter beetles can be cooled to about -10 degrees C before freezing, and they thaw near -3.5 degrees C. Summer beetles thaw at -0.7 degrees C. To avoid freezing damage even in winter beetles, cooling rates must be near 20 degrees C per hour or less.

Prevention of apoptosis by a baculovirus gene during infection of insect cells.

Programmed cell death is an active process of self destruction that is important in both the development and maintenance of multicellular animals. The molecular mechanisms controlling activation or suppression of programmed cell death are largely unknown. Apoptosis, a morphologically and biochemically defined type of programmed cell death commonly seen in vertebrates, was found to be initiated during baculovirus replication in insect cells. A specific viral gene product, p35, was identified as being responsible for blocking the apoptotic response. Identification of the function of this gene will allow further definition of the molecular pathways involved in the regulation of programmed cell death and may identify the role of apoptosis in invertebrate viral defense systems.

A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase.

The predicted amino acid sequence of a newly identified gene of the insect baculovirus Autographa californica nuclear polyhedrosis virus was similar to several uridine 5'-diphosphate (UDP)-glucuronosyl transferases and at least one UDP-glucosyl transferase. Genetic and biochemical studies confirmed that this gene encodes an ecdysteroid UDP-glucosyl transferase (egt). This enzyme catalyzes the transfer of glucose from UDP-glucose to ecdysteroids, which are insect molting hormones. Expression of the egt gene allowed the virus to interfere with normal insect development so that molting was blocked in infected larvae of fall armyworm (Spodoptera frugiperda).

Bacterial, viral, and fungal insecticides.

Microorganisms that are pathogenic to insects provide a wealth of biological material that can be exploited by humans to control insect pests. Innovative applications of a few such entomopathogens are found throughout the world, but widespread commercial production of microbial insecticides awaits further studies of the biology, ecology, and pathogenicity of the agents. Genetic engineering techniques may be used to increase the virulence of these microorganisms, as well as to make them more tolerant of physical and chemical conditions and perhaps to broaden their host ranges. The use of microbial insecticides could decrease our dependence on chemical pesticides.

Baculoviruses: high-level expression in insect cells.

Baculoviruses continue to serve as workhorse vectors for the high-level expression of eukaryotic genes in insect cells; however, numerous researchers are also finding novel uses for these vectors by taking advantage of the unique nature of the viruses and their host cells. Concurrently, the technology involved in constructing and utilizing these vectors is being improved so that the time and effort required to construct expression vectors are reduced.

Caenorhabditis elegans CED-4 stimulates CED-3 processing and CED-3-induced apoptosis.

BACKGROUND: Programmed cell death or apoptosis is a key feature of normal development, tissue homeostasis and disease progression in metazoans. Genetic studies in the nematode C. elegans have identified three key genes involved in apoptosis, ced-3, ced-4 and ced-9. Expression of ced-3 and ced-4 is required for the induction of cell death, whereas expression of ced-9 is necessary to inhibit cell death. The precise mechanism by which these genes influence the life or death decision of a cell is not known. In this study, we have expressed the genes in an insect cell line to explore their role in the apoptotic pathway. RESULTS: Co-expression of ced-4 with ced-3 in insect cells stimulated both the induction and the level of CED-3-mediated apoptosis. Stimulation of CED-3-dependent apoptosis by CED-4 was accompanied by accelerated processing of CED-3, which was dependent on the presence of a wild-type CED-3 prodomain and a conserved lysine residue within a putative ATP/GTP-binding motif of CED-4. Co-expression of ced-9 with ced-4 and ced-3 inhibited the ability of CED-4 to stimulate CED-3 processing and CED-3-dependent apoptosis. Although a temperature-sensitive CED-9 mutant was unable to block CED-4 activity and failed to associate with CED-4, a deletion mutant of CED-9 lacking the carboxy-terminal hydrophobic domain could associate with CED-4 and block CED-4 activity. CONCLUSIONS: Our results establish a role for CED-4 in the processing of CED-3 and the stimulation of CED-3-induced apoptosis. Furthermore, we show that CED-9 achieves its anti-apoptotic effect by associating with CED-4 and blocking the ability of CED-4 to process CED-3.

Control of programmed cell death by the baculovirus genes p35 and iap.

The SF-21 insect cell line undergoes rapid and widespread apoptosis when treated with actinomycin D or when infected with a mutant of the baculovirus Autographa californica nuclear polyhedrosis virus lacking a p35 gene or a functionally active iap (inhibitor of apoptosis) gene. Here we provide evidence that the basis for the induction of apoptosis by these two different stimuli is the cessation of RNA synthesis. We also show that expression of either p35 or two different functional iap homologs blocks apoptosis independently of other viral genes, indicating that these gene products act directly on the cellular apoptotic pathway. The iap genes encode a C3HC4 (or RING) finger motif found in a number of transcriptional regulatory proteins, as well as two additional Cys/His motifs (baculovirus iap repeats). We show that specific amino acids within both the C3HC4 finger and the N-terminal baculovirus iap repeat are critical for anti-apoptosis function. Overexpression of either mammalian bcl-2 or adenovirus E1B-19K, genes which block apoptosis when overexpressed in a number of mammalian cells, does not block actinomycin D-induced apoptosis in SF-21 cells.

Characterization of reaper- and FADD-induced apoptosis in a lepidopteran cell line.

Expression of the reaper gene (rpr) correlates with the initiation of apoptosis in Drosophila melanogaster. Transient expression of rpr in the lepidopteran SF-21 cell line induced apoptosis displaying nuclear condensation and fragmentation, oligonucleosomal ladder formation, cell surface blebbing, and apoptotic body formation. Inhibitors of ICE-family proteases p35 and crmA, as well as members of the iap class of genes, Op-iap and D-iap2, but not bcl-2 family members, blocked rpr-induced apoptosis. Mutational analysis of rpr provided no support for the proposed sequence similarity of Reaper and death domain proteins. Mutations in the N-terminal region of Reaper, which displays sequence similarity to Hid and Grim, other Drosophila gene products correlated with the initiation of apoptosis, suggested that these residues might be functionally important. The mammalian cDNA encoding FADD (Fas-associating protein with a death domain) also induced cell death in SF-21 cells, but death progressed more slowly and with features which distinguished it from rpr-induced apoptosis. Several bcl-2 family members delayed or blocked FADD-induced cellular death. Thus, apoptosis initiated by Reaper progressed by a faster path which appeared to differ from that of FADD-induced apoptosis.

Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM.

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183-10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.

Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.

Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.

The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.

Bidirectional transcription from a solo long terminal repeat of the retrotransposon TED: symmetrical RNA start sites.

A single copy of the retrotransposon TED was found integrated within the DNA genome of the insect baculovirus, Autographa californica nuclear polyhedrosis virus. After excision of the element from the viral genome, a single long terminal repeat (LTR) remained behind. We have examined the effect of this solo TED LTR on the local pattern of viral transcription. Most prominent was the transcription of two sets of abundant RNAs; both originated within the LTR but extended in opposite directions into flanking viral genes. By promoting symmetric transcription of adjacent genes, the solo LTR has the capacity to activate or repress gene expression in two directions. Primer extension analysis demonstrated that the divergent LTR transcripts were initiated near the same point within a 22-base-pair sequence having hyphenated twofold symmetry. Analogous symmetries at the initiation sites of other retrotransposon LTRs, including copia and Ty, suggested that these sequences serve to establish the precise start for transcription.

Anti- and pro-apoptotic activities of baculovirus and Drosophila IAPs in an insect cell line.

The anti-apoptotic activities of two baculovirus IAPs, OpIAP and CpIAP, were directly compared with that of two Drosophila IAPs, DIAP1 and DIAP2, in the same insect cell line, SF-21 cells. Like OpIAP and CpIAP, DIAP1 inhibited actinomycin D-induced apoptosis and apoptosis induced by Doom. Removal of the RING finger of DIAP1 reduced but did not eliminate its anti-apoptotic activity. DIAP2 was unable to inhibit actinomycin-D induced apoptosis but was able to partially inhibit Doom-induced apoptosis. The baculoviral BIR and RING finger regions, when separated, were unable to block apoptosis induced by actinomycin D or Doom. Instead, the BIR regions of OpIAP and CpIAP as well as the RING finger regions of CpIAP and DIAP1 induced apoptosis. Thus, there were significant differences in the manner in which the different domains of the viral and cellular homologues of IAPs interacted with the components of the pathways regulating apoptosis in SF-21 cells.

Anti-apoptotic genes of baculoviruses.

Baculoviruses possess two different classes of genes with anti-apoptptic activity: p35 and iap. The p35 gene product (P35) is able to block apoptosis induced by a variety of stimuli in phylogenetically diverse organisms. P35 has recently been shown to be capable of inhibiting the ICE/ced-3 family of cysteine proteases, a family of enzymes which are implicated in cell death and which exhibit specificity for cleavage at aspartate residues. The products of the iap genes are a distinct class of proteins containing a carboxyl ring finger and tandem duplications of a unique motif known as the BIR motif. Homologues of the baculovirus iap genes have been identified in the human genome. Both classes of baculovirus anti-apoptotic genes will continue to be important tools in defining the pathways involved in apoptosis. Since our demonstration in 1991 that a baculovirus prevents host cells from undergoing apoptosis by expressing a gene known as p35(Clem et al., 1991), the study of baculovirus-induced apoptosis and the anti-apoptotic genes they possess has led to discoveries with far-reaching implications for viral pathogenesis, human disease, and the study of cell death. It is now known that a variety of eukaryotic viruses encode genes which allow them to control cellular apoptosis. Understanding the mechanism(s) by which these viral gene products act provides fundamental insights into the pathways regulating apoptosis. In this review, we discuss the inhibition of apoptosis by baculoviruses, concentrating mainly on the nature and mechanism of action of the two classes of baculovirus genes, p35 and iap, which are able to control apoptosis in a diversity ofeukaryotes.

Phase I study of paclitaxel given by seven-week continuous infusion concurrent with radiation therapy for locally advanced squamous cell carcinoma of the head and neck.

PURPOSE: Paclitaxel is one of the most active agents for squamous cell carcinoma of the head and neck (SCCHN) and an in vitro radiosensitizer. The dose-response relationship for paclitaxel may depend more on exposure duration than on peak concentration. This National Cancer Institute-sponsored phase I trial was designed to determine the feasibility of combining continuous-infusion (CI) paclitaxel with concurrent radiation therapy (RT). PATIENTS AND METHODS: Patients with previously untreated stage IVA/B SCCHN were eligible. Primary end points were determination of the maximum-tolerated dose, dose-limiting toxicity, and pharmacokinetics for paclitaxel given by CI (24 hours a day, 7 days a week for 7 weeks) during RT (70 Gy/7 weeks). RESULTS: Twenty-seven patients were enrolled and assessable for toxicity. Nineteen of the patients who completed > or = 70 Gy were assessable for response. Grade 3 skin and mucosal acute reactions occurred at 10.5 mg/m(2)/d, but uninterrupted treatment was possible in five of six patients. At 17 mg/m(2)/d, skin toxicity required a 2-week treatment break for all three patients. The mean paclitaxel serum concentration at dose levels > or = 6.5 mg/m(2)/d exceeded that reported to achieve in vitro radiosensitization. Initial locoregional control was achieved in 14 (58%) of 24 of patients treated to 70 Gy, and control persisted in nine (38%). CONCLUSION: CI paclitaxel with concurrent RT is a feasible and tolerable regimen for patients with advanced SCCHN and good performance status. Preliminary response and survival data are encouraging and suggest that further study is indicated. The recommended phase II dose of paclitaxel by CI is 10.5 mg/m(2)/d with RT for SCCHN.

Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene.

A series of recombinant baculoviruses containing linker-substituted polyhedrin promoters attached to a reporter gene encoding chloramphenicol acetyl transferase (CAT) were constructed and tested for expression of the gene. The major determinant for promoter activity was narrowed to within eight nucleotides, TAAGTATT, at the start point of polyhedrin mRNA transcription. Mutations within TAAGTATT blocked initiation of transcription from this site and resulted in a 2000-fold decrease in CAT activity. Linker mutations from 12 to 22 bases upstream from the TAAGTATT sequence increased the steady-state levels of RNAs initiated within TAAGTATT and increased CAT expression by up to 50%. Mutations downstream from TAAGTATT and within the region specifying the untranslated RNA leader diminished transcriptional initiation at TAAGTATT and decreased CAT activity two- to 20-fold. The half-lives of CAT RNAs were not noticeably affected by mutations in the untranslated RNA leader region and thus RNA turn-over was not responsible for the reduced levels of these CAT RNAs. Nuclear run-on analysis of two mutant viruses showed that these mutations decrease the rate of transcriptional initiation. Transcriptional initiation thus appears to be the major means of polyhedrin gene regulation. The data define promoter-related roles for TAAGTATT and the sequences specifying the untranslated mRNA leader in transcriptional initiation. A model by which the viral-induced RNA polymerase distinguishes late and very late initiation sites is proposed.

Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol.

Estrogen receptors in cytoplasmic extracts of breast tumors from more than 40 patients were separately analyzed by gel filtration and/or ultracentrifugation under diverse conditions. Resultant patterns are presented for specimens from 11 women with infiltrating duct carcinoma and are representative of results obtained in all samples of sufficient size and receptor content (approximately 40 fmol/mg cytosol protein) for accurate determination of hydrodynamic parameters. Estradiol-binding components of intracellular origin were distinguished from the serum contaminant, sex hormone-binding globulin by their high affinity for diethylstilbestrol and negligible affinity for 5 alpha-dihydrotestosterone. The predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-L-leucyl-DL-arginine aldehydes (leupeptin). At least three discrete forms of the intracellular receptors were detected. The smallest labeled complex, the mero-receptor, with a sedimentation coefficient of about 3S and a Stokes radius of about 24 A, was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (approximately 5S) and Stokes radius (approximately 34A) were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 nM leupeptin was analyzed in hypertonic buffer. The largest receptor form (10.5S, 71A) was predominant in cytosol prepared with 50 mM leupeptin and analyzed in hypotonic buffer. In this small series of patients, there was no obvious correlation between the molecular form of the receptors and the clinical status or eventual responsiveness to endocrine therapy. Preliminary studies of endogenous proteolytic enzymes in breast tumor cytosol that may be involved in mero-receptor formation included assays of plasminogen activators (EC 3.4.21.-) by fibrinolytic and spectrofluorometric techniques. The detection of high concentrations of plasminogen activators in several tumor cytosols and the inhibition of this activity by leupeptin, which stabilizes the large receptor forms in this and other systems, are consistent with a possible role of these enzymes in receptor cleavage.

PIP: The multiple molecular forms of steroid hormone receptors in human breast tissue were characterized and are reported. 40 patients provided cytoplasmic extracts for assay by either gel filtration or ultracentrifugation under diverse conditions. In general, the predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-Dl-arginine aldehydes (leupeptin). 3 discrete forms of intracellular receptors were detected. The mero-receptor, the smallest labeled complex, had a sedimentation coefficient of 3S and a Stokes radius of 24 angstroms; it was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (5S) and Stokes radius (34 angstroms), were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 mM leupeptin was analyzed in hypertonic buffer. There was no obvious correlation between the molecular form and clinical status or eventual responsiveness to endocrine therapy.

Human breast tumor estrogen receptor: effects of molybdate and electrophoretic analyses.

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.

Twenty-four-hour mean plasma testosterone concentration declines with age in normal premenopausal women.

The 24-h mean plasma concentration of total testosterone (T) was measured in 33 healthy, regularly cycling, nonobese women between 21 and 51 yr of age. Percent free T was measured in 17 of them. Plasma dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were measured in 24 of them, and the DHEA-to-T and DHEAS-to-T ratios were calculated. It was found that the concentration of total T showed a steep decline with age; the regression equation was: T (nanomoles per L) = 37.8 x age-1.12 (r = -0.54; P < 0.003). According to this equation, the expected T concentration of a woman of 40 would be 0.61 nmol/L, about half that of a woman of 21 (1.3 nmol/L). The percent free T did not vary significantly with age, so free T concentration likewise showed a steep decline with age. The DHEA-to-T and DHEAS-to-T ratios were both age invariant, clearly because the levels of DHEA and DHEAS also decline steeply with age, as previously reported.