Nelfinavir and lenalidomide/dexamethasone in patients with lenalidomide-refractory multiple myeloma. A phase I/II Trial (SAKK 39/10).

The antiretroviral agent nelfinavir has antimyeloma activity and can overcome resistance to bortezomib. Our phase I/II trial investigated whether adding nelfinavir to lenalidomide-dexamethasone can overcome lenalidomide resistance in lenalidomide-refractory multiple myeloma (MM). Twenty-nine patients were included (high-risk cytogenetic aberrations 31%; ≥2 prior therapy lines 93%; lenalidomide-bortezomib double-refractory 34%). Twenty-four patients (83%) had prior bortezomib and 10 (34%) were lenalidomide-bortezomib double-refractory. They received four cycles of nelfinavir 2500 mg/day with standard-dose lenalidomide (25 mg days 1-21) and dexamethasone (40/20 mg days 1, 8, 15, 22). Minor response or better was achieved in 16 patients (55%; 95% CI 36-74%), including 40% of those who were lenalidomide-bortezomib double-refractory, and partial response or better in nine patients (31%; 95% CI 15-51%). Median progression-free survival was 3.4 (95% CI 2.0-4.9) months and median overall survival 21.6 (13.0-50.1) months. Lenalidomide-related pneumonitis, pneumonia, and neutropenic fever occurred, but there were no unexpected adverse events. Peripheral blood mononuclear cells showed a 45% (95% CI 40-51%) reduction in total proteasome activity from baseline and significant induction of unfolded protein response and autophagy. Thus, nelfinavir-lenalidomide-dexamethasone is an active oral combination in lenalidomide-refractory MM.

NSAID treatment with meloxicam enhances peripheral stem cell mobilization in myeloma.

Chemotherapy with G-CSF is used to mobilize peripheral stem cells in multiple myeloma (MM) patients, with plerixafor as a rescue strategy for poorly mobilizing patients. Preclinical studies suggested that the nonsteroidal anti-inflammatory drug meloxicam enhances the mobilization of CD34+ cells. In this single-center study, we evaluated whether adding meloxicam to chemotherapy/G-CSF mobilization increases peripheral hematopoietic CD34+ cell levels and reduces the need of using plerixafor. We prospectively compared two consecutive cohorts of MM patients in first remission mobilized with G-CSF and non-myelosuppressive chemotherapy with vinorelbine or gemcitabine. The second cohort additionally received oral meloxicam. The cohorts comprised 84 patients without meloxicam (-M) and 66 patients with meloxicam (+M). Meloxicam was well tolerated and associated with similar hematologic engraftment after transplantation and equal survival rates. However, the meloxicam group had higher CD34+ cell levels on day 8 of the mobilization procedure (53 200 versus 35 600 CD34+ cells/mL; P=0.007), and fewer patients needed >1 collection day (+M: 6 (9%) patients versus -M: 16 (19%) patients; P=0.04). This resulted in reduced plerixafor administrations (+M: 7 (11%) patients versus -M: 18 (21%) patients; P=0.03) and less costs. Our data suggest that meloxicam enhances the mobilization of hematopoietic CD34+ blood cells in MM patients.

Safety and immunogenicity of varicella-zoster virus vaccine in pediatric liver and intestine transplant recipients.

Primary varicella-zoster virus (VZV) infections following organ transplantation may cause significant morbidity. We examined the safety and immunogenicity of Varivax after transplantation as a potential prophylactic tool. Pediatric liver and intestine transplant recipients without history of chickenpox received one dose of Varivax. VZV humoral and cellular immunity were assessed before and > or =12 weeks after vaccination. Adverse events (AE) and management of exposure to wild type VZV were monitored. Sixteen VZV-naïve subjects, 13-76 months of age, at 257-2045 days after transplantation were immunized. Five children developed mild local AE of short duration. Four subjects developed fever and four developed non-injection site rashes, three of whom received acyclovir. Liver enzymes did not increase during the month after vaccination. Eighty-seven percent and 86% of children developed humoral and cellular immunity, respectively. There were five reported exposures to varicella in four children, none of which resulted in chickenpox. One subject received VZV-immunoglobulin and another subject with liver enzyme elevations after exposure received acyclovir; all remained asymptomatic. Varivax was safe and immunogenic in pediatric liver and intestine transplant recipients. Larger studies are needed to establish the efficacy and role of varicella vaccination after transplantation.

Tara, a novel F-actin binding protein, associates with the Trio guanine nucleotide exchange factor and regulates actin cytoskeletal organization.

Reorganization of the actin cytoskeleton is essential to numerous cellular processes including cell locomotion and cytokinesis. This actin remodeling is regulated in part by Rho family GTPases. Previous studies implicated Trio, a Dbl-homology guanine nucleotide exchange factor with two exchange factor domains, in regulating actin cytoskeleton reorganization, cell motility and cell growth via activation of Rho GTPases. Trio is essential for mouse embryonic development and Trio-deficiency is associated with abnormal skeletal muscle and neural tissue development. Furthermore, genetic analyses in Caenorhabditis elegans and Drosophila demonstrate a role for trio-like genes in cell migration and axon guidance. Herein we characterize a novel Trio-binding protein, Tara, that is comprised of an N-terminal pleckstrin homology domain and a C-terminal coiled-coil region. Trio and Tara associate as assessed by the yeast interaction-trap assays and mammalian co-immunoprecipitation studies. Ectopically expressed Tara localizes to F-actin in a periodic pattern that is highly similar to the pattern of myosin II. Furthermore, a direct interaction between Tara and F-actin is indicated by in vitro binding studies. Cells that transiently or stably overexpress Tara display an extensively flattened cell morphology with enhanced stress fibers and cortical F-actin. Tara expression does not alter the ability of the cell to attach or to initially spread, but rather increases cell spreading following these initial events. Tara stabilizes F-actin structures as indicated by the relative resistance of Tara-expressing cells to the F-actin destabilizer Latrunculin B. We propose that Tara regulates actin cytoskeletal organization by directly binding and stabilizing F-actin, and that the localized formation of Tara and Trio complexes functions to coordinate actin remodeling.

Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos.

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation (trio(-/-)). Trio function is essential during late embryonic development as genotype analysis indicated that trio(-/-) embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the trio(-/-) embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal trio(-/-) skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of trio(-/-) secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. trio(-/-) embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues.

The trio guanine nucleotide exchange factor is a RhoA target. Binding of RhoA to the trio immunoglobulin-like domain.

Trio is a complex protein containing two guanine nucleotide exchange factor domains each with associated pleckstrin homology domains, a serine/threonine kinase domain, two SH3 domains, an immunoglobulin-like domain, and spectrin-like repeats. Trio was originally identified as a LAR tyrosine phosphatase-binding protein and is involved in actin remodeling, cell migration, and cell growth. Herein we provide evidence that Trio not only activates RhoA but is also a RhoA target. The RhoA-binding site was mapped to the Trio immunoglobulin-like domain. RhoA isoprenylation is necessary for the RhoA-Trio interaction, because mutation of the RhoA carboxyl-terminal cysteine residue blocked binding. The existence of an intramolecular functional link between RhoA activation and RhoA binding is suggested by the finding that Trio exchange activity enhanced RhoA binding to Trio. Furthermore, immunofluorescence studies of HeLa cells showed that although ectopically expressed Trio was evenly distributed within the cell, co-expression of Trio with RhoA resulted in relocalization of Trio into punctate structures. Relocalization was not observed with Trio constructs lacking the immunoglobulin-like domain, indicating that RhoA acts to regulate Trio localization via binding to the immunoglobulin-like domain. We propose that Trio-mediated RhoA activation and subsequent RhoA-mediated relocalization of Trio functions to modulate and coordinate Trio signaling.

Trio amino-terminal guanine nucleotide exchange factor domain expression promotes actin cytoskeleton reorganization, cell migration and anchorage-independent cell growth.

Rho family GTPases regulate diverse cellular processes, including extracellular signal-mediated actin cytoskeleton reorganization and cell growth. The functions of GTPases are positively regulated by guanine nucleotide exchange factors, which promote the exchange of GDP for GTP. Trio is a complex protein possessing two guanine nucleotide exchange factor domains, each with adjacent pleckstrin homology and SH3 domains, a protein serine/threonine kinase domain with an adjacent immunoglobulin-like domain and multiple spectrin-like domains. To assess the functional role of the two Trio guanine nucleotide exchange factor domains, NIH 3T3 cell lines stably expressing the individual guanine nucleotide exchange factor domains were established and characterized. Expression of the amino-terminal guanine nucleotide exchange factor domain results in prominent membrane ruffling, whereas cells expressing the carboxy-terminal guanine nucleotide exchange factor domain have lamellae that terminate in miniruffles. Moreover, cells expressing the amino-terminal guanine nucleotide exchange factor domain display more rapid cell spreading, haptotactic cell migration and anchorage-independent growth, suggesting that Trio regulates both cell motility and cell growth. Expression of full-length Trio in COS cells also alters actin cytoskeleton organization, as well as the distribution of focal contact sites. These findings support a role for Trio as a multifunctional protein that integrates and amplifies signals involved in coordinating actin remodeling, which is necessary for cell migration and growth.

Filling the gut activates paradoxical sleep in suckling rats.

Recent studies with rat pups suggest that suckling and sleeping are coordinated through milk-related events in the gut. Our experiments revealed that suckling rats respond to milk in the upper gastrointestinal tract by displaying more paradoxical sleep (PS) as the volume increases to 4% of the pup's body weight. Conversely, gastric loads larger than 4% reduced PS as a function of the volume. We also discovered that filling the stomach with warm non-nutritive paraffin is as effective as an equivalent volume of warm milk for enhancing PS. Although the temperature of the gut load did not appear to play a major role in the amount of PS displayed, increasing ambient temperature from 22 degrees C to 32 degrees C increased PS significantly. Moreover, a gut load of milk (4% body weight) was more effective than the same volume of water or no load for enhancing PS. Gut loads that stay in the stomach and warm ambient temperature appear to work in an additive manner to enhance PS. The electrophysiological data together with the stomach volume data and behavioral observations of nipple attachment revealed that milk-related stimuli along the gastrointestinal tract, especially gastric distension, alter sleep patterns in predictable ways that permit us to distinguish postingestive satiety from a deprivation state and nimiety in suckling rats.

The multidomain protein Trio binds the LAR transmembrane tyrosine phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanine nucleotide exchange factor domains.

rho-like GTP binding proteins play an essential role in regulating cell growth and actin polymerization. These molecular switches are positively regulated by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP. Using the interaction-trap assay to identify candidate proteins that bind the cytoplasmic region of the LAR transmembrane protein tyrosine phosphatase (PT-Pase), we isolated a cDNA encoding a 2861-amino acid protein termed Trio that contains three enzyme domains: two functional GEF domains and a protein serine/threonine kinase (PSK) domain. One of the Trio GEF domains (Trio GEF-D1) has rac-specific GEF activity, while the other Trio GEF domain (Trio GEF-D2) has rho-specific activity. The C-terminal PSK domain is adjacent to an Ig-like domain and is most similar to calcium/calmodulin-dependent kinases, such as smooth muscle myosin light chain kinase which similarly contains associated Ig-like domains. Near the N terminus, Trio has four spectrin-like repeats that may play a role in intracellular targeting. Northern blot analysis indicates that Trio has a broad tissue distribution. Trio appears to be phosphorylated only on serine residues, suggesting that Trio is not a LAR substrate, but rather that it forms a complex with LAR. As the LAR PTPase localizes to the ends of focal adhesions, we propose that LAR and the Trio GEF/PSK may orchestrate cell-matrix and cytoskeletal rearrangements necessary for cell migration.

RNA polymerase II C-terminal domain required for enhancer-driven transcription.

The RNA polymerase II carboxy-terminal domain (CTD) consists of tandem repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The CTD may participate in activated transcription through interaction with a high-molecular-weight mediator complex. Such a role would be consistent with observations that some genes are preferentially sensitive to CTD mutations. Here we investigate the function of the mouse RNA polymerase CTD in enhancer-driven transcription. Transcription by alpha-amanitin-resistant CTD-deletion mutants was tested by transient transfection of tissue culture cells in the presence of alpha-amanitin in order to inhibit endogenous RNA polymerase II. Removal of most of the CTD abolishes transcriptional activation by all enhancers tested, whereas transcription from promoters driven by Sp1, a factor that typically activates housekeeping genes from positions proximal to the initiation sites, is not affected. These findings show that the CTD is essential in mediating 'enhancer'-type activation of mammalian transcription.

[Smoking cessation at work exemplified by "action blanca casa", a project for smoking cessation in the D. Swarovski & Co., Wattens, firm]. / Rauchertherapie im Betrieb am Beispiel der "Aktion blanca casa", einem Projekt für das Nichtrauchen im Unternehmen D. Swarovski & Co., Wattens.

Recent studies about the efficacy of smoking cessation programs in the workplace have brought up to common difficulties. Only long-term-programs show remarkable effects on smoking habits of the workers. Presenting ongoing long-term strategies for open discussion can improve the design of more powerful cessation programs in the future. Our program as an important part of modern "human resources management" is demonstrated and the results after 18 months are discussed.

Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

A minimal transcription activation domain consisting of a specific array of aspartic acid and leucine residues.

Transcriptional activation by the herpesvirus protein VP16 (= Vmw65, alpha TIF) is mediated by its C-terminal acidic activation domain. Using GAL4 fusion proteins, we have previously shown that a construct containing two tandem copies of a short eleven amino acid fragment derived from the VP16 domain (DALDDFDLDML, residues 437-447) activates transcription in mammalian cells with an efficiency comparable to a GAL4 fusion with the full VP16 activation domain (residues 413-490). Here we have mutagenized this eleven amino acid core sequence and find that a mutant sequence with little inherent activity can cooperate with a wildtype sequence to yield almost full activity. Moreover, greater activity is observed when the wildtype sequence is positioned at the distal, rather than the proximal, end of the fusion protein, indicating that the distal position facilitates contacts to the transcription apparatus. We have also further reduced the eleven amino acid activating sequence to shorter sequence motifs. Two copies of eight and seven amino acids (DALDDFDL and DDFDLDL, respectively), or four copies of the sequence motif DDFDL are required to reach the activation potential of two eleven amino acid motifs. Four copies of the sequence DDLDL still activate transcription strongly (up to two-thirds of DDFDL), indicating that an aromatic residue is not an essential feature of this type of activation domain. However, repetitions of DDL or DL do not yield activity. Thus the minimal requirement for transcriptional activation is the presence of a sequence of some fifteen to twenty amino acids consisting of a specific array of aspartic acid and leucine residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue-Specific Expression of as-1 in Transgenic Tobacco.

When integrated as a transgene in one or a few copies, the -90 35S promoter of cauliflower mosaic virus confers expression in roots with little or no expression in cotyledons and leaves. The responsible cis element, activation sequence-1 (as-1), can bind to the nuclear factor ASF-1 as well as to the transcription factor TGA1a. Here, we show that microinjection of 104 molecules of TGA1a per cotyledon cell activated transgenes containing as-1-linked promoters. Transgenes with promoters linked to the octopine synthase (ocs) element, which also binds TGA1a, responded similarly. The acidic, N-terminal segment of TGA1a is important for transcription activation in vivo because a deletion mutant without the first 80 amino acids was inactive. Finally, we show that the -90 35S-[beta]-glucuronidase (GUS) fusion gene conferred GUS expression in cotyledon cells when injected at 50,000 copies per cell. Collectively, these results provide support for the hypothesis that the undetectable expression of the as-1-linked transgene in cotyledon cells is most likely a result of its inability to compete for a limiting amount of its cognate transcription factor(s), presumably TGA1a or related proteins.

Transcriptional activation modulated by homopolymeric glutamine and proline stretches.

Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.

Functional differences between mammalian transcription activation domains at the yeast GAL1 promoter.

We have fused representatives of three structurally and functionally distinct classes of mammalian transcription activation domains for RNA polymerase II to the yeast GAL4 DNA binding domain. All fusion proteins were stable when expressed in yeast and were tested for their ability to activate transcription from various positions in the yeast GAL1 promoter. Activation domains functional from remote as well as TATA-proximal positions in mammalian cells, e.g. the acidic-type domain of VP16, also stimulate transcription in yeast from various promoter positions. Proline-rich domains, as e.g. in AP-2 and CTF/NF1, with considerable promoter activity and low enhancer activity in mammalian cells stimulate transcription in yeast only from a position close to the TATA box. The glutamine-rich domains of Oct1, Oct2 and Sp1, which activate transcription in mammalian cells from close to the TATA box in response to a remote enhancer, are inactive in the yeast GAL1 promoter. This finding might reflect some basic difference between the organization of yeast and mammalian promoters.

Different potential of cellular and viral activators of transcription revealed in oocytes and early embryos of Xenopus laevis.

Many protein domains for transcriptional activation also function when fused to a heterologous DNA binding domain. In mammalian/HeLa cells, we have previously characterized the activation domains of several transcription factors using GAL4 fusion proteins. Here we have tested their transcriptional activity in oocytes and developing embryos of the clawed toad Xenopus laevis. We find that the "acidic" C-terminal domain of the herpesvirus VP16 (= Vmw65) activator, which is active from yeast to man, is also very active in the two Xenopus systems. The constitutive nature of this viral domain may have evolved to be refractory to cellular defense mechanisms. By contrast, activation domains from cellular eukaryotic transcription factors (TFE3, ITF2, MTF-1) are differentially active in oocytes and early embryos. This indicates that their activity can be regulated by protein modification and/or availability of specific coactivators. We have also compared VP16 induced enhancement of transcription from remote and promoter-proximal positions. In both oocytes and late blastula embryos, activation from a promoter-proximal position was more than 50 fold, while only a moderate stimulation (3-8 fold) was observed from remote positions. This may mean that frog oocyte and early embryos are not yet fully geared for gene control by remote enhancers, i.e. respond predominantly to close-by regulatory sequences. The fact that cellular enhancers are naturally located at various distances from the responsive promoters may thus be exploited by multicellular organisms for differential gene control at early and late stages of development.

C-terminal domain (CTD) of RNA-polymerase II and N-terminal segment of the human TATA binding protein (TBP) can mediate remote and proximal transcriptional activation, respectively.

Activation domains of mammalian transcription factors can be subdivided into at least two functional classes. One, exemplified by the glutamine-rich activation domains of Oct and Sp1 factors, mediates transcriptional activation only from a proximal promoter position, and in response to an enhancer. The other, exemplified by the 'acidic' domain of the viral activator VP16, has the ability to activate from remote enhancer as well as from proximal promoter positions. Here we report that two proteins of the basal transcription apparatus also contain activation domains whose stimulatory effect can be detected in fusion proteins containing the GAL4 DNA binding domain. The human TATA-binding protein (TBP) contains at its N-terminus a domain with typical 'promoter' activity. We propose that the TBP N-terminal region acts as an auxiliary activation domain which reinforces the activity of other promoter-bound factors. The largest subunit of RNA polymerase II contains at its C-terminus a conserved heptad repeat structure (CTD). Both natural and synthetic CTD consensus repeats fused to GAL4 can activate transcription from remote positions like a typical enhancer-active domain. Accordingly we propose that the RNA polymerase II large subunit contains a 'portable' domain for transcriptional activation which may synergize with the activation domains of enhancer-bound transcription factors.

Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions.

We reported previously that the lymphocyte-derived octamer transcription factor 2A (Oct-2A or OTF-2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the 'octamer' site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. 'Proximal' activation domains, exemplified by glutamine-rich domains of Oct-1, Oct-2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. 'General' activation domains, derived from VP16, GAL4, p65 (NF-chi B), TFE3, ITF-1 and ITF-2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline-rich activation domains of AP-2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine-rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.