Examination of clinically-derived p210 BCR/ABL1 RhoGEF mutations in a murine bone marrow transplantation model of CML.

Expression of the p210 BCR/ABL1 fusion protein has been described in virtually all patients with chronic myelogenous leukemia (CML). Previous studies have identified a guanine nucleotide exchange factor (RhoGEF) domain within BCR that is retained in p210 BCR/ABL1. Missense mutations at residues T654 (T654K) and F547 (F547L) within this domain have been reported in a CML patient in blast crisis (BC). In this study, we have evaluated p210 BCR/ABL1 constructs that contain these substitutions in a murine bone marrow transplantation (BMT) model of CML. The mutants exhibit normal expression and tyrosine kinase activity but altered signaling. When examined in the BMT assay, mice that express the mutants exhibit earlier onset of disease but have significantly extended lifespans relative to mice that express unmodified p210 BCR/ABL1. While mice that express p210 BCR/ABL1 exhibit neutrophilia that progresses to a less differentiated phenotype at death, disease in the mutant mice is characterized by eosinophilia with no maturation arrest. This observation was confirmed in vitro using myeloid cells and was associated with enhanced p53 phosphorylation and G1/S arrest. These results suggest that residues within the RhoGEF domain of p210 BCR/ABL1 can influence disease progression.

Type III interferon is a critical regulator of innate antifungal immunity.

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus (Af) as a model to study antifungal immune responses, we found that depletion of CCR2+ monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2+ monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2+ monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2+ monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression.

Fasudil, a Clinically Used ROCK Inhibitor, Stabilizes Rod Photoreceptor Synapses after Retinal Detachment.

PURPOSE: Retinal detachment disrupts the rod-bipolar synapse in the outer plexiform layer by retraction of rod axons. We showed that breakage is due to RhoA activation whereas inhibition of Rho kinase (ROCK), using Y27632, reduces synaptic damage. We test whether the ROCK inhibitor fasudil, used for other clinical applications, can prevent synaptic injury after detachment. METHODS: Detachments were made in pigs by subretinal injection of balanced salt solution (BSS) or fasudil (1, 10 mM). In some animals, fasudil was injected intravitreally after BSS-induced detachment. After 2 to 4 hours, retinae were fixed for immunocytochemistry and confocal microscopy. Axon retraction was quantified by imaging synaptic vesicle label in the outer nuclear layer. Apoptosis was analyzed using propidium iodide staining. For biochemical analysis by Western blotting, retinal explants, detached from retinal pigmented epithelium, were cultured for 2 hours. RESULTS: Subretinal injection of fasudil (10 mM) reduced retraction of rod spherules by 51.3% compared to control detachments (n = 3 pigs, P = 0.002). Intravitreal injection of 10 mM fasudil, a more clinically feasible route of administration, also reduced retraction (28.7%, n = 5, P < 0.05). Controls had no photoreceptor degeneration at 2 hours, but by 4 hours apoptosis was evident. Fasudil 10 mM reduced pyknotic nuclei by 55.7% (n = 4, P < 0.001). Phosphorylation of cofilin and myosin light chain, downstream effectors of ROCK, was decreased with 30 µM fasudil (n = 8-10 explants, P < 0.05). CONCLUSIONS: Inhibition of ROCK signaling with fasudil reduced photoreceptor degeneration and preserved the rod-bipolar synapse after retinal detachment. TRANSLATIONAL RELEVANCE: These results support the possibility, previously tested with Y27632, that ROCK inhibition may attenuate synaptic damage in iatrogenic detachments.

Individualised Expert Feedback is Not Essential for Improving Basic Clinical Skills Performance in Novice Learners: A Randomized Trial.

OBJECTIVE: To determine whether unsupervised video feedback (UVF) is as effective as direct expert feedback (DEF) in improving clinical skills performance for medical students learning basic surgical skills-intravenous cannulation, catheterization, and suturing. BACKGROUND: Feedback is a vital component of the learning process, yet great variation persists in its quality, quantity, and methods of delivery. The use of video technology to assist in the provision of feedback has been adopted increasingly. METHODS: A prospective, blinded randomized trial comparing DEF, an expert reviewing students' performances with subsequent improvement suggestions, and UVF, students reviewing their own performance with an expert teaching video, was carried out. Medical students received an initial teaching lecture on intravenous cannulation, catheterization, and suturing and were then recorded performing the task. They subsequently received either DEF or UVF before reperforming the task. Students' recordings were additionally scored by 2 blinded experts using a validated proforma. RESULTS: A total of 71 medical students were recruited. Cannulation scores improved 4.3% with DEF and 9.5% with UVF (p = 0.044), catheterization scores improved 8.7% with DEF and 8.9% with UVF (p = 0.96), and suturing improved 15.6% with DEF and 13.2% with UVF (p = 0.54). Improvement from baseline scores was significant in all cases (p < 0.05). CONCLUSION: Video-assisted feedback allows a significant improvement in clinical skills for novices. No significant additional benefit was demonstrated from DEF, and a similar improvement can be obtained using a generic expert video and allowing students to review their own performance. This could have significant implications for the design and delivery of such training.

RhoA Signaling and Synaptic Damage Occur Within Hours in a Live Pig Model of CNS Injury, Retinal Detachment.

PURPOSE: The RhoA pathway is activated after retinal injury. However, the time of onset and consequences of activation are unknown in vivo. Based on in vitro studies we focused on a period 2 hours after retinal detachment, in pig, an animal whose retina is holangiotic and contains cones. METHODS: Under anesthesia, retinal detachments were created by subretinal injection of a balanced salt solution. Two hours later, animals were sacrificed and enucleated for GTPase activity assays and quantitative Western blot and confocal microscopy analyses. RESULTS: RhoA activity with detachment was increased 1.5-fold compared to that in normal eyes or in eyes that had undergone vitrectomy only. Increased phosphorylation of myosin light chain, a RhoA effector, also occurred. By 2 hours, rod cells had retracted their terminals toward their cell bodies, disrupting the photoreceptor-to-bipolar synapse and producing significant numbers of spherules with SV2 immunolabel in the outer nuclear layer of the retina. In eyes with detachment, distant retina that remained attached also showed significant increases in RhoA activity and synaptic disjunction. Increases in RAC1 activity and glial fibrillary acidic protein (GFAP) were not specific for detachment, and sprouting of bipolar dendrites, reported for longer detachments, was not seen. The RhoA kinase inhibitor Y27632 significantly reduced axonal retraction by rod cells. CONCLUSIONS: Activation of the RhoA pathway occurs quickly after injury and promotes synaptic damage that can be controlled by RhoA kinase inhibition. We suggest that retinal detachment joins the list of central nervous system injuries, such as stroke and spinal cord injury, that should be considered for rapid therapeutic intervention.

The influence of academic discourses on medical students' identification with the discipline of family medicine.

PURPOSE: To understand the influence of academic discourses about family medicine on medical students' professional identity construction during undergraduate training. METHOD: The authors used a multiple case study research design involving international medical schools, one each from Canada, France, Spain, and the United Kingdom (UK). The authors completed the fieldwork between 2007 and 2009 by conducting 18 focus groups (with 132 students) and 67 semistructured interviews with educators and by gathering pertinent institutional documents. They carried out discursive thematic analyses of the verbatim transcripts and then performed within- and cross-case analyses. RESULTS: The most striking finding was the diverging responses between those at the UK school and those at the other schools. In the UK case, family medicine was recognized as a prestigious academic discipline; students and faculty praised the knowledge and skills of family physicians, and students more often indicated their intent to pursue family medicine. In the other cases, family medicine was not well regarded by students or faculty. This was expressed overtly or through a paradoxical academic discourse that stressed the importance of family medicine to the health care system while decrying its lack of innovative technology and the large workload-to-income ratio. Students at these schools were less likely to consider family medicine. CONCLUSIONS: These results stress the influence of academic discourses on medical students' ability to identify with the practice of family medicine. Educators must consider processes of professional identity formation during undergraduate medical training as they develop and reform medical education.

Regulation of vesicle transport and cell motility by Golgi-localized Dbs.

DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration.

Ubiquitin-mediated interaction of p210 BCR/ABL with ß-catenin supports disease progression in a murine model for chronic myelogenous leukemia.

We have identified a ubiquitin-binding domain within the NH2-terminal sequences of p210 BCR/ABL and determined that the binding site co-localizes with the binding site for ß-catenin. The domain does not support the auto- or trans-kinase activity of p210 BCR/ABL or its ability to interact with GRB2 and activate ERK1/2 signaling. Expression of p210 BCR/ABL, but not a ß-catenin-binding mutant, in hematopoietic cells is associated with the accumulation of p-ß-catenin (Tyr654) and increased TCF/LEF-mediated transcription. In a bone marrow transplantation model, the interaction between ß-catenin and p-ß-catenin (Tyr654) is detectable in mice transplanted with p210 BCR/ABL, but not the mutant. Whereas mice transplanted with p210 BCR/ABL exhibit myeloid disease with expansion of monocytes and neutrophils, mice transplanted with the mutant predominantly exhibit expansion of neutrophils, polycythemia, and increased lifespan. The increased disease latency is associated with expansion of megakaryocyte-erythrocyte progenitors, a decrease in common myeloid progenitors, and reduced ß-catenin signaling in the bone marrow of the diseased mice. These observations support a model in which p210 BCR/ABL may influence lineage-specific leukemic expansion by directly binding and phosphorylating ß-catenin and altering its transcriptional activity. They further suggest that the interaction may play a role in chronic phase disease progression.

Regulation of breast cancer cell motility by T-cell lymphoma invasion and metastasis-inducing protein.

INTRODUCTION: T-cell lymphoma invasion and metastasis-inducing protein (Tiam1) is a Ras-related C3 botulinum toxin substrate (Rac)-specific guanine nucleotide exchange factor that was isolated based on its ability to induce a metastatic phenotype. In polarized migrating keratinocytes, Tiam1 is found at the leading edge where it cooperates with the Protease-activated receptor 1 (Par1) complex to establish front-rear polarity. Although a positive correlation has been observed between Tiam1 expression and tumor grade in a variety of human malignancies, including breast, its role in breast cancer cells has not yet been examined. METHODS: Tiam1 expression and Rac activity were examined in a panel of human breast cancer cell lines which exhibit different degrees of cell motility. The contribution of Tiam1 to cell motility was directly examined using transwell motility, and wound healing assays. RESULTS: Although we observe a striking, positive correlation between Tiam1 expression and cell motility in the panel of breast cancer cell lines, we do not observe a correlation between Tiam1 expression and overall levels of Rac activity. Consistent with this, small interfering ribonucleic acid (siRNA)-mediated suppression of Tiam1 expression limits the motility of cell lines in which Tiam1 expression is high (MDA-MB-231 and MDA-MB-453), but does not substantially alter the overall levels of activated Rac. Tiam1 overexpression is also not sufficient to increase the motility of more poorly motile cells (T-47D) or increase Rac activity. Immunofluorescence and cellular fractionations indicate that Tiam1 is found predominantly in the Golgi of breast cancer cells, and in the latter case Tiam1 was shown to co-fractionate with a limited pool of Rac1. Consistent with this Golgi localization, Tiam1 supports cell motility and Golgi reorientation in response to serum in a wound healing assay using MDA-MB-231and MDA-MB-435S cells. CONCLUSIONS: Tiam1 expression correlates with cell motility in human breast cancer cells, and is required to support the motile phenotype. Localization of endogenous Tiam1 to the Golgi, and its demonstrated role in Golgi reorientation, suggest that it may support motility through a mechanism that is discrete from its known function in leading edge dynamics.

The rho-specific guanine nucleotide exchange factor Dbs regulates breast cancer cell migration.

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) that regulates neurotrophin-3-induced cell migration in Schwann cells. Here we report that Dbs regulates cell motility in tumor-derived, human breast epithelial cells through activation of Cdc42 and Rac1. Cdc42 and Rac1 are activated in T47D cells that stably express onco- or proto-Dbs, and activation is dependent upon growth of the cells on collagen I. Transient suppression of expression of Cdc42 or Rac1 by small interfering RNAs attenuates Dbs-enhanced motility. Both onco- and proto-Dbs-enhanced motility correlates with an increase in tyrosine phosphorylation of focal adhesion kinase on Tyr-397 and p130(Cas) on Tyr-410 and an increase in the abundance of the Crk.p130(Cas) complex. Suppression of expression of Cdc42 or its effector, Ack1, reduces tyrosine phosphorylation of focal adhesion kinase and p130(Cas) and disrupts the Crk.p130(Cas) complex. We further determined that suppression of expression of Cdc42, Ack1, p130(Cas), or Crk reduces Rac1 activation and cell motility in Dbs-expressing cells to a level comparable with that in vector cells. Therefore, a cascade of activation of Cdc42 and Rac1 by Dbs through the Cdc42 effector Ack1 and the Crk.p130(Cas) complex is established. Suppression of the expression of endogenous Dbs reduces cell motility in both T47D cells and MDA-MB-231 cells, which correlates with the down-regulation of Cdc42 activity. This suggests that Dbs activates Cdc42 in these two human breast cancer cell lines and that the normal function of Dbs may be required to support cell movement.

ROCK I-mediated activation of NF-kappaB by RhoB.

RhoB is a short-lived protein whose expression is increased by a variety of extra-cellular stimuli including UV irradiation, epidermal growth factor (EGF) and transforming growth factor beta (TGF-beta). Whereas most Rho proteins are modified by the covalent attachment of a geranylgeranyl group, RhoB is unique in that it can exist in either a geranylgeranylated (RhoB-GG) or a farnesylated (RhoB-F) form. Although each form is proposed to have different cellular functions, the signaling events that underlie these differences are poorly understood. Here we show that RhoB can activate NF-kappaB signaling in multiple cell types. Whereas RhoB-F is a potent activator of NF-kappaB, much weaker activation is observed for RhoB-GG, RhoA, and RhoC. NF-kappaB activation by RhoB is not associated with increased nuclear translocation of RelA/p65, but rather, by modification of the RelA/p65 transactivation domain. Activation of NF-kappaB by RhoB is dependent upon ROCK I but not PRK I. Thus, ROCK I cooperates with RhoB to activate NF-kappaB, and suppression of ROCK I activity by genetic or pharmacological inhibitors blocks NF-kappaB activation. Suppression of RhoB activity by dominant-inhibitory mutants, or siRNA, blocks NF-kappaB activation by Bcr, and TSG101, but not by TNFalpha or oncogenic Ras. Collectively, these observations suggest the existence of an endosome-associated pathway for NF-kappaB activation that is preferentially regulated by the farnesylated form of RhoB.

Protein kinase A-independent cAMP stimulation of progesterone in a luteal cell model is tyrosine kinase dependent but phosphatidylinositol-3-kinase and mitogen-activated protein kinase independent.

Comprehensive understanding of the cellular mechanisms utilized by luteal cells in response to extracellular hormonal signals resulting in the normal synthesis and secretion of their steroid and peptide products has yet to be achieved. Previous studies have established that cAMP functions as a second messenger in mediating gonadotropin stimulated luteal progesterone secretion. Classically, increased intracellular concentrations of cAMP result in activation of protein kinase A (PKA), which in turn phosphorylates gene regulatory transcription factors. Recent studies demonstrate that non-PKA mediated actions of cAMP exist, yet the mechanisms are not well understood. In addition to gonadotropic hormones, such growth factors as insulin, insulin-like growth factor 1, and epidermal growth factor have been shown to modulate luteal steroid hormone synthesis and steroidogenic enzyme expression as either independent effects or via amplification or modulation of the action of gonadotropic hormones or cAMP. Thus, mechanisms independent of cAMP and also downstream to cAMP that do not involve PKA are likely to be important in steroidogenesis in mammalian cells. The present studies were performed to help define the cellular mediators involved in cAMP-stimulated progesterone expression. Our data demonstrate that, in an in vitro steroidogenic cell model, 1) cAMP-stimulated progesterone occurs in a manner that is independent of PKA, 2) neither phosphatidylinositol-3-kinase nor mitogen-activated protein kinase are involved in PKA-independent cAMP-stimulated progesterone production, 3) tyrosine kinase activity does mediate cAMP-stimulated progesterone production, and 4) cAMP directly activates the Ras protein. These data suggest novel mediators of cAMP-stimulated progesterone production.

Ccpg1, a novel scaffold protein that regulates the activity of the Rho guanine nucleotide exchange factor Dbs.

Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member.

Bcr interacts with components of the endosomal sorting complex required for transport-I and is required for epidermal growth factor receptor turnover.

Virtually all patients with chronic myelogenous leukemia (CML) express an aberrant protein (p210 Bcr-Abl) that contains NH2-terminal sequences from Bcr fused to COOH-terminal sequences from Abl. In a yeast two-hybrid screen, we have identified TSG101 as a binding partner for Bcr. Because TSG101 is a subunit of the mammalian endosomal sorting complex required for transport (ESCRT), which regulates protein sorting during endosomal trafficking, this association suggests that Bcr may have a related cellular function. The docking site for TSG101 has been mapped to the COOH terminus of Bcr, indicating that this interaction may be disrupted in CML. Overexpression studies with full-length TSG101 and Bcr reveal that this interaction can be recapitulated in mammalian cells. The association can also be observed between natively expressed proteins in a panel of hematopoietic and nonhematopoietic cell lines, where a second subunit of the ESCRT complex, vacuolar sorting protein 28 (Vps28), was also found to interact with Bcr. Both Bcr and TSG101 exhibit a punctate cytoplasmic distribution and seem to colocalize in HeLa cells, which would be consistent with an in vivo association. Bacterially purified Bcr and TSG101 also bind, suggesting that the interaction is direct and is not dependent on ubiquitination. Disruption of the endosomal pathway with an ATPase-defective Vps4 mutant results in the cellular redistribution of Bcr, and suppression of Bcr in HeLa cells by small interfering RNA impairs epidermal growth factor receptor turnover. Taken together, these observations suggest that Bcr is a component of the mammalian ESCRT complexes and plays an important role in cellular trafficking of growth factor receptors.

Transformation by the Rho-specific guanine nucleotide exchange factor Dbs requires ROCK I-mediated phosphorylation of myosin light chain.

Dbs was identified in a cDNA-based expression screen for sequences that can cause malignant growth when expressed in murine fibroblasts. In previous studies we have shown that Dbs is a Rho-specific guanine nucleotide exchange factor that can activate RhoA and/or Cdc42 in a cell-specific manner. In this current study we have used a combination of genetic and pharmacological approaches to examine the relative contributions of RhoA x PRK and RhoA x ROCK signaling to Dbs transformation. Our analysis indicates that ROCK is activated in Dbs-transformed cells and that Dbs transformation is dependent upon ROCK I activity. In contrast, there appears to be no requirement for PRK activation in Dbs transformation. Dbs transformation is also associated with increased phosphorylation of myosin light chain and stress fiber formation, both of which occur in a ROCK-dependent manner. Suppression of myosin light chain expression by small interfering RNAs impairs Dbs focus formation, thus establishing a direct link between actinomyosin contraction and Rho-specific guanine nucleotide exchange factor transformation.

XGef mediates early CPEB phosphorylation during Xenopus oocyte meiotic maturation.

Polyadenylation-induced translation is an important regulatory mechanism during metazoan development. During Xenopus oocyte meiotic progression, polyadenylation-induced translation is regulated by CPEB, which is activated by phosphorylation. XGef, a guanine exchange factor, is a CPEB-interacting protein involved in the early steps of progesterone-stimulated oocyte maturation. We find that XGef influences early oocyte maturation by directly influencing CPEB function. XGef and CPEB interact during oogenesis and oocyte maturation and are present in a c-mos messenger ribonucleoprotein (mRNP). Both proteins also interact directly in vitro. XGef overexpression increases the level of CPEB phosphorylated early during oocyte maturation, and this directly correlates with increased Mos protein accumulation and acceleration of meiotic resumption. To exert this effect, XGef must retain guanine exchange activity and the interaction with CPEB. Overexpression of a guanine exchange deficient version of XGef, which interacts with CPEB, does not enhance early CPEB phosphorylation. Overexpression of a version of XGef that has significantly reduced interaction with CPEB, but retains guanine exchange activity, decreases early CPEB phosphorylation and delays oocyte maturation. Injection of XGef antibodies into oocytes blocks progesterone-induced oocyte maturation and early CPEB phosphorylation. These findings indicate that XGef is involved in early CPEB activation and implicate GTPase signaling in this process.

The Sec14 homology domain regulates the cellular distribution and transforming activity of the Rho-specific guanine nucleotide exchange factor Dbs.

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose overexpression cause deregulated growth in murine fibroblasts. Dbs contains multiple recognizable motifs including a centrally located Rho-specific guanine nucleotide exchange factor domain, a COOH-terminal Src homology 3 domain, two spectrin-like repeats, and a recently identified NH(2)-terminal Sec14 homology domain. The transforming potential of Dbs is substantially activated by the removal of inhibitory sequences that lie outside of the core catalytic sequences, and in this current study we mapped this inhibition to the Sec14 domain. Surprisingly removal of the NH(2) terminus did not alter the catalytic activity of Dbs in vivo but rather altered its subcellular distribution. Whereas full-length Dbs was distributed primarily in a perinuclear structure that coincides with a marker for the Golgi apparatus, removal of the Sec14 domain was associated with translocation of Dbs to the cell periphery where it accumulated within membrane ruffles and lamellipodia. However, translocation of Dbs and the concomitant changes in the actin cytoskeleton were not sufficient to fully activate Dbs transformation. The Sec14 domain also forms intramolecular contacts with the pleckstrin homology domain, and these contacts must also be relieved to achieve full transforming activity. Collectively these observations suggest that the Sec14 domain regulates Dbs transformation through at least two distinct mechanisms, neither of which appears to directly influence the in vivo exchange activity of the protein.

Bone marrow stroma influences transforming growth factor-beta production in breast cancer cells to regulate c-myc activation of the preprotachykinin-I gene in breast cancer cells.

Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-beta (TGF-beta) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-beta bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-beta in each other. In cocultures, PPT-I and TGF-beta expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-beta and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5' flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-beta, and transient tranfections all showed that c-myc is required for TGF-beta-mediated induction of PPT-I in BCCs. TGF-beta was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-beta is important to the regulation of PPT-I in the early events of bone invasion by BCCs.