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Background Abl interactor 1 (Abi1) is a downstream target of Abl tyrosine kinases and a component of the WAVE regulatory complex (WRC) that plays an important role in regulating actin cytoskeleton remodeling and membrane receptor signaling. While studies using short hairpin RNA (shRNA) have suggested that Abi1 plays a critical role in Bcr-Abl-induced leukemogenesis, the mechanism involved is not clear. Methods In this study, we knocked out Abi1 expression in p185Bcr-Abl-transformed hematopoietic cells using CRISPR/Cas9-mediated gene editing technology. The effects of Abi1 deficiency on actin cytoskeleton remodeling, the Bcr-Abl signaling, IL-3 independent growth, and SDF-induced chemotaxis in these cells were examined by various in vitro assays. The leukemogenic activity of these cells was evaluated by a syngeneic mouse transplantation model. Results We show here that Abi1 deficiency reduced the IL3-independent growth and SDF-1α-mediated chemotaxis in p185Bcr-Abl-transformed hematopoietic cells and inhibited Bcr-Abl-induced abnormal actin remodeling. Depletion of Abi1 also impaired the Bcr-Abl signaling to the ERK and PI3 kinase/Akt pathways. Remarkably, the p185Bcr-Abl-transformed cells with Abi1 deficiency lost their ability to develop leukemia in syngeneic mice. Even though these cells developed drug tolerance in vitro after prolonged selection with imatinib as their parental cells, the imatinib-tolerant cells remain incapable of leukemogenesis in vivo. Conclusions Together, this study highlights an essential role of Abi1 in Bcr-Abl-induced leukemogenesis and provides a model system for dissecting the Abi1 signaling in Bcr-Abl-positive leukemia.

Tyrosine 177 and the Src homology 2 (SH2) domain play important roles in linking p185Bcr-Abl to downstream pathways critical for cell growth and survival. However, a mutant p185(Y177FR552L) (p185(YR)), in which tyrosine 177 and arginine 552 in the SH2 domain are mutated, is still capable of transforming hematopoietic cells in vitro. Transplant of these cells into syngeneic mice also leads to leukemogenesis, albeit with a phenotype distinct from that produced by wild-type p185Bcr-Abl (p185(wt))-transformed cells. Here we show that G-protein coupled receptor 34 (Gpr34) expression is markedly up-regulated in p185(YR)-transformed cells compared to those transformed by p185(wt). Knockdown of Gpr34 in p185(YR) cells is sufficient to suppress growth factor-independent proliferation and survival in vitro and attenuate leukemogenesis in vivo. The Erk and phosphatidylinositol 3-kinase/Akt pathways are activated in p185(YR) cells and the activation is dependent on Gpr34 expression. These studies identify Gpr34 as an alternative pathway that may mediate p185Bcr-Abl-induced transformation and leukemogenesis.

Coordinated actin remodeling is crucial for cell entry into mitosis. The WAVE regulatory complex is a key regulator of actin assembly, yet how the WAVE signaling is regulated to coordinate actin assembly with mitotic entry is not clear. Here, we have uncovered a novel mechanism that regulates the WAVE complex at the onset of mitosis. We found that the Bcr-Abl-stimulated F-actin assembly is abrogated during mitosis. This mitotic inhibition of F-actin assembly is accompanied by an attenuation of Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex. We identified serine 216 of Abi1 as a target of CDK1/cyclin B kinase that is phosphorylated in cells at the onset of mitosis. The Abi1 phosphorylated on serine 216 displayed greatly reduced tyrosine phosphorylation in the hematopoietic cells transformed by Bcr-Abl. Moreover, a phosphomimetic mutation of serine 216 to aspartic acid in Abi1 was sufficient to attenuate Bcr-Abl-induced tyrosine phosphorylation of the WAVE complex and F-actin assembly. Ectopic expression of Abi1 with serine 216 mutations interfered with cell cycle progression. Together, these data show that CDK1-mediated phosphorylation of serine 216 in Abi1 serves as a regulatory mechanism that may contribute to coordinated actin cytoskeleton remodeling during mitosis.

Abl interactor (Abi) 1 was first identified as the downstream target of Abl tyrosine kinases and was found to be dysregulated in leukemic cells expressing oncogenic Bcr-Abl and v-Abl. Although the accumulating evidence supports a role of Abi1 in actin cytoskeleton remodeling and growth factor/receptor signaling, it is not clear how it contributes to Bcr-Abl-induced leukemogenesis. We show here that Abi1 gene silencing by short hairpin RNA attenuated the Bcr-Abl-induced abnormal actin remodeling, membrane-type 1 metalloproteinase clustering and inhibited cell adhesion and migration on fibronectin-coated surfaces. Although the knock down of Abi1 expression did not affect growth factor-independent growth of Bcr-Abl-transformed Ba/F3 cells in vitro, it impeded competitive expansion of these cells in non obese diabetic (NOD)/ severe combined immuno-deficiency (SCID) mice. Remarkably, the knock down of Abi1 expression in Bcr-Abl-transformed Ba/F3 cells impaired the leukemogenic potential of these cells in NOD/SCID mice. Abi1 contributes to Bcr-Abl-induced leukemogenesis in part through Src family kinases, as the knock down of Abi1 expression attenuates Bcr-Abl-stimulated activation of Lyn. Together, these data provide for the first time the direct evidence that supports a critical role of Abi1 pathway in the pathogenesis of Bcr-Abl-induced leukemia.

MT1-MMP as a downstream target of BCR-ABL/ABL interactor 1 signaling: polarized distribution and involvement in BCR-ABL-stimulated leukemic cell migration

The hematopoietic cells from patients with Bcr-Abl-positive chronic myelogenous leukemia exhibit multiple abnormalities of cytoskeletal function. The molecular events leading to these abnormalities are not fully understood. Previously we showed that Bcr-Abl elicits ubiquitin-dependent degradation of Abl interactor proteins. Because recent studies have suggested a role of Abl interactor proteins in the pathway that regulates cytoskeletal function, we investigated whether mutations in Bcr-Abl that interfere with the signaling to Abl interactor proteins affect its leukemogenic activity. We report here that the Src homology 3 domain and C-terminal proline-rich sequences of Bcr-Abl are required for its binding to Abl interactor 2 as well as for the induction of Abl interactor 2 degradation. Although the deletion of these regions did not affect the ability of the mutant Bcr-Abl to transform hematopoietic cells to growth factor independence, it abrogated its ability to stimulate spontaneous cell migration on fibronectin-coated surfaces. Furthermore, the mutant Bcr-Abl, defective in binding to Abl interactor 2 and inducing its degradation, failed to induce chronic myelogenous leukemia-like disease in mouse. These results are consistent with a role of Abl interactor proteins in the regulation of cytoskeletal function as well as in the pathogenesis of Bcr-Abl-induced leukemogenesis.

The in vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development have been investigated in transgenic (Tg) mice (IGF-II/I Tg mice) in whom an IGF-II promoter-driven IGF-I transgene is highly expressed in cerebellum. Compared to normal littermates, the brains of IGF-II/I Tg mice exhibited overgrowth beginning from the second week of postnatal life. Among the brain regions examined, cerebellum exhibited the greatest increase in size, such that by 50 days of age cerebellar weight and DNA content were increased by 90% and 143%, respectively, compared to littermate controls. Morphological studies of adult IGF-II/I Tg mice showed that the total number of granule and Purkinje cells was increased by 82% and 20%, respectively, findings consistent with the increased cerebellar DNA content and indicating that the increased cerebellar weight was due in part to an increase in cell number. The thickness of the molecular layer also was increased in IGF-II/I Tg mice. During early postnatal development the number of external granular layer cells, as well as the number of BrdU labeled external granular cells, was increased. These data strongly indicate that IGF-I increases granule cell number by a mechanism that involves the stimulation of granule cell progenitor proliferation. Our findings also indicate that IGF-I influences the growth of Purkinje cells and possibly of other cell types in the cerebellum.

A protein has been identified that interacts specifically with both the Src homologous 3 (SH3) domain and carboxy-terminal sequences of the c-Abl tyrosine kinase. The cDNA encoding the Abl interactor protein (Abi-2), was isolated from a human lymphocyte library using the yeast two-hybrid system with the Abl SH3 domain as bait. Abi-2 binds to c-Abl in vitro and in vivo. Abi-2 is a novel protein that contains an SH3 domain and proline-rich sequences critical for binding to c-Abl. A basic region in the amino terminus of Abi-2 is homologous to the DNA-binding sequence of homeo-domain proteins. We show that Abi-2 is a substrate for the c-Abl tyrosine kinase. Expression of an Abi-2 mutant protein that lacks sequences required for binding to the Abl SH3 domain but retains binding to the Abl carboxyl terminus activates the transforming capacity of c-Abl. The properties of Abi-2 are consistent with a dual role as regulator and potential effector of the c-Abl protein and suggest that Abi-2 may function as a tumor suppressor in mammalian cells.

Three hemizygous transgenic (Tg) mouse lines were generated with a fusion gene composed of the mouse metallothionein promoter (mMT-I) and a full-length human insulin-like growth factor binding protein-1 (hIGFBP-1) complementary DNA that was truncated in its 3'-untranslated region. Despite high serum hIGFBP-1 levels (120-2570 micrograms/liter) before puberty in two of these lines, no significant alterations were observed in somatic growth, nor were marked alterations noted in fasting or random serum glucose or in the response of young adult Tg mice to ip glucose. The transgene was expressed in a number of tissues from each line, but liver was a significant site of transgene expression in only one line. Unexpectedly, liver hIGFBP-1 messenger RNA (mRNA) expression in this line was regulated in fashion similar to the native liver IGFBP-1 mRNA: 1) its abundance waned with advancing postnatal age and became minimal in early adult life, despite continuous zinc supplementation to stimulate its transcription; and 2) fasting increased its abundance 3- to 4.3-fold. The decline in transgene expression with aging was not due to a deletion, rearrangement, or a change in the methylation of liver transgene DNA. Transcriptional mechanisms also were not likely to account for the observed regulation of the transgene mRNA, because liver expression of the mMT-I gene, which shares identical genomic 5'-regulatory elements with the transgene, was not similarly altered by aging or fasting. Because cycloheximide (CHX) treatment of cultured rat H4IIE cells has been shown to prolong IGFBP-1 mRNA half-life while decreasing its transcription, Tg mice were treated with CHX to test the possibility that instability of the liver transgene mRNA influenced its abundance. After CHX and under conditions of chronic zinc supplementation, liver transgene mRNA abundance increased in parallel with that of the native IGFBP-1 mRNA. Although CHX is known to activate mMT-I transcription by mechanisms involving the 5'-regulatory regions contained in the transgene, CHX-induced transcription only in part accounted for the increase in liver transgene mRNA, because CHX induced an earlier and greater increase in liver transgene mRNA than in mMT-I mRNA. Taken together, these data indicate that both transgene and native IGFBP-1 liver mRNA are regulated by factors that alter mRNA stability. The finding that native liver IGFBP-1 mRNA abundance is influenced by transgene expression further supports the concept that both mRNAs share some common mechanisms of regulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Angiogenesis is essential for tumor growth and metastasis. VEGF has been shown to be a central player in this process. The biological activity of VEGF is mainly mediated by two tyrosine kinase receptors, VEGFR-1 and VEGFR-2. While increasing evidence suggests that VEGF/VEGFR-1 signaling is crucial for tumor angiogenesis, its molecular mechanism is not well understood. Here we show that VEGFR-1 knockdown dramatically inhibits tumor growth. This inhibition is associated with significant decrease of tumor VEGF levels and tumor angiogenesis as well as an increased tumor necrosis. Moreover, we demonstrate that VEGF in CRCC tumors is mainly produced by tumor stromal cells instead of the tumor cells themselves. It has been shown that macrophages constitute a significant part of tumor stromal cells and produce a large amount of VEGF. We therefore examined the macrophage infiltration in the xenograft tumors. Remarkably, VEGFR-1 knockdown attenuates the tumor macrophages infiltration. To understand the mechanism, we investigated the impact of VEGFR-1 knockdown on the expression of monocyte chemoattractant protein-1 (MCP-1), one of the main chemoattractants for macrophages. Significantly, VEGFR-1 knockdown inhibits MCP-1 expression of CRCC cells. Taken together, these data indicate that VEGF/VEGFR-1 signaling plays an essential role in initiating tumor angiogenesis by regulating MCP-1 expression, which in turn, attracts macrophages infiltration and VEGF production. Thus, these studies suggest that blockade of VEGFR-1 function may provide a tumor-specific, VEGF-based therapeutic strategy for treatment of CRCC.

Summary BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph 1 )-positive human leukemias. Sequences within the first exon of BCR are required to activate the transforming potential of BCR-ABL. The SH2/SH3 domain-containing GRB-2 protein links tyrosine kinases to Ras signaling. We demonstrate that BCR-ABL exists in a complex with GRB-2 in vivo. Binding of GRB-2 to BCR-ABL is mediated by the direct interaction of the GRB-2 SH2 domain with a phosphorylated tyrosine, Y177, within the BCR first exon. The BCR-ABL-GRB-2 interaction is required for activation of the Ras signaling pathway. Mutation of Y177 to phenylalanine (Y177F) abolishes GRB-2 binding and abrogates BCR-ABL-induced Ras activation. The BCR-ABL (Y177F) mutant is unable to transform primary bone marrow cultures and is impaired in its ability to transform Rat 1 fibroblasts. These findings implicate activation of Ras function as an important component in BCR-ABL-mediated transformation and demonstrate that GRB-2 not only functions in normal development and mitogenesis but also plays a role in oncogenesis.

FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells, exhibit a marked mitogenic response to insulin-like growth factors (IGFs) when they are exposed to TSH. Because IGF binding proteins (IGFBPs) are important modulators of IGF actions, we investigated the capacity of FRTL-5 cells to synthesize IGFBPs. We found that FRTL-5 cell conditioned media contained a single band of approximately 31 kilodaltons on ligand blot analysis. This band represents IGFBP-5 because: 1) it can be immunostained with a specific antibody raised against human IGFBP-5; 2) by Northern analysis, total RNA from FRTL-5 cells contains a major 6-kilobase transcript when hybridized with a cDNA for rat IGFBP-5; and 3) no transcripts were observed when Northern blots of FRTL-5 cells were hybridized with complementary DNAs for rat IGFBP-1, -2, -3, -4 or -6. Conditioned media IGFBP-5 increased in response to IGF-I, IGF-II, and insulin in a dose-dependent fashion, compared with unstimulated FRTL-5 cells. At maximally effective concentrations IGF-I was 3.5- and 6-fold more potent than IGF-II and insulin, respectively. The addition of a monoclonal antibody (Sm 1.2) to IGF-I completely inhibited IGF-I stimulation of the IGFBP-5 6-kilobase transcript and the appearance of IGFBP-5 in FRTL-5 conditioned media. Stimulation of IGFBP-5 synthesis by the IGFs and insulin appeared to be regulated at the messenger RNA (mRNA) level, because each stimulated similar increases in both IGFBP-5 mRNA and media protein at maximally effective concentrations. TSH, on the other hand, inhibited basal levels of IGFBP-5 mRNA and attenuated the increase in IGFBP-5 mRNA stimulated by IGF-I. FRTL-5 cells provide a relatively uncomplicated model to study the regulation and action of IGFBP-5 and the mechanisms by which IGFs interact with this binding protein.

The expression of p185(Bcr-Abl) in Ba/F3 cells inhibits the chemotactic response of these cells to SDF1alpha. A mutant p185(Bcr-Abl) with deletion of amino acids from 176 to 426 (p185(Delta176-426)) is deficient in suppressing SDF1alpha-stimulated chemotaxis. Comparison of the gene expression profiles among parental Ba/F3 cells and cells transformed by p185(Bcr-Abl) and p185(Delta176-426) reveals that class II phosphoinositide 3-kinase gamma (PI3KC2gamma) expression is markedly down-regulated by p185(Bcr-Abl) but not p185(Delta176-426). Furthermore, knockdown of PI3KC2gamma expression in p185(Delta176-426) cells is sufficient to suppress SDF1alpha-stimulated chemotaxis and to promote infiltration of these cells into the liver. Together, these studies suggest that inhibition of PI3KC2gamma expression may represent a mechanism by which Bcr-Abl suppresses SDF1alpha-induced chemotaxis and induces abnormal homing of leukemic cells.

In a prior report we presented evidence that insulin-like growth factor-I (IGF-I) can act in an autocrine fashion by demonstrating that FRTL-5 cells transfected with hIGF-IA fusion genes express and secrete biologically active IGF-I that renders the stimulation of DNA synthesis in FRTL-5 cells independent of their requirement for exogenous IGFs or insulin. To determine if IGF-I's autocrine actions require secretion or can be mediated by interactions with intracellular receptors, we have created a new line of FRTL-5 cells that express a mutant IGF-IA precursor containing the endoplasmic reticulum retention amino acid sequence, Lys-Asp-Glu-Leu (KDEL), at its carboxyl terminus. The mutant IGF-IA/KDEL precursor expressed by stably transfected FRTL-5 cells was shown to be retained intracellularly and to have biological activity comparable with mature IGF-I, as judged by the activity of partially purified IGF-IA/KDEL in wild type FRTL-5 cells. Expression of IGF-IA/KDEL in FRTL-5 cells, however, neither augmented TSH-stimulated DNA synthesis nor stimulated IGF-binding protein-5 expression, as does IGF-IA expression in transfected FRTL-5 cells and the addition of exogenous IGF-I to wild type FRTL-5 cells. IGF-IA/KDEL expression, however, desensitized FRTL-5 cells to the actions of exogenous IGF-I despite having only minimal effects on cell surface type I receptor number, suggesting that intracellular IGF-I is capable of significant biological actions. The failure of IGF-IA/KDEL to replicate the actions of secreted IGF-I, taken together with the findings that a monoclonal antibody against IGF-I blocked IGF-I's actions in IGF-I-secreting transfected FRTL-5 cells, provides evidence that IGF-I secretion and interaction with cell surface type I IGF receptors is the dominant mechanism of IGF-I's autocrine actions.

Abl interactor 1 (Abi1) is a key regulator of actin polymerization/depolymerization. The involvement of Abi1 in the development of abnormal cytoskeletal functions of cancer cells has recently been reported. It remains unclear, however, how Abi1 exerts its effects in tumor cells and whether it contributes to tumor progression in vivo. We report here a novel function for Abi1 in the regulation of invadopodia formation and Src-inhibitor of differentiation protein 1 (Id1)-matrix metalloproteinase (MMP)-9 pathway in MDA-MB-231 human breast cancer cells. Abi1 is found in the invadopodia of MDA-MB-231 cells. Epigenetic silencing of the Abi1 gene by short hairpin RNA in MDA-MB-231 cells impaired the formation of invadopodia and resulted in downregulation of the Src activation and Id1/MMP-9 expression. The decreased invadopodia formation and MMP-9 expression correlate with a reduction in the ability of these cells to degrade extracellular matrix. Remarkably, the knockdown of Abi1 expression inhibited tumor cell proliferation and migration in vitro and slowed tumor growth in vivo. Taken together, these results indicate that the Abi1 signaling plays a critical role in breast cancer progression and suggest that this pathway may serve as a therapeutic target for the treatment of human breast cancer.

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as β1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.

Oncogenic forms of the Abl and Src tyrosine kinases trigger the destruction of the Abi proteins, a family of Abl-interacting proteins that antagonize the oncogenic potential of Abl after overexpression in fibroblasts. The destruction of the Abi proteins requires tyrosine kinase activity and is dependent on the ubiquitin–proteasome pathway. We show that degradation of the Abi proteins occurs through a Ras-independent pathway. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr–Abl-positive leukemias. These findings suggest that loss of Abi proteins may be a component in the progression of Bcr–Abl-positive leukemias and identify a novel pathway linking activated nonreceptor protein tyrosine kinases to the destruction of specific target proteins through the ubiquitin–proteasome pathway.

Three lines of transgenic (Tg) mice carrying a fusion gene linking the mouse metallothionein-I promoter to a cDNA encoding human insulin-like growth factor binding protein-1 (hIGFBP-1) were found to express the transgene in brain. As judged by comparing Tg brain weights to those of non-transgenic littermates, adult hemizygotic Tg mice of each line exhibited brain growth retardation (16.2%, 14.4% and 8.1% reductions in weight, respectively in each line). In two lines, total brain DNA and protein content were decreased. Further analysis indicated that the brain growth retardation was manifested in the second week of postnatal life. Given that the insulin-like growth factors (IGFs) stimulate cell proliferation and/or survival in neural cultures and that hIGFBP-1, when present in a molar excess, inhibits IGF interactions with their cell surface receptors, the brain growth retardation in hIGFBP-1 Tg mice likely results from hIGFBP-1 inhibition of IGF-stimulated growth-promoting actions. These hIGFBP-1 Tg mice should prove useful in defining IGF actions during postnatal brain maturation.

Although there is much evidence that insulin-like growth factor-I (IGF-I) is delivered to its target tissues via the circulation from distal sites of synthesis, many other observations suggest that it is synthesized in or near its target tissues and acts by autocrine and/or paracrine modalities. Studies of the mechanisms of such local actions, however, have been problematic, because in vivo studies of a single tissue are technically difficult and confounded by many variables, whereas in vitro studies of autocrine/paracrine actions have been limited by low levels of IGF-I expression and/or lack of dramatic or clearly defined responses to IGF-I. We, therefore, set about to create IGF-I expression in FRTL-5 cells, a diploid nontransformed line of rat thyroid follicular cells that have been extensively studied as a model of TSH action. The modest increase in thymidine incorporation stimulated by TSH in wild type FRTL-5 cells is markedly increased in the presence of exogenous IGF-I. By transfecting these cells with a chimeric IGF-IA gene, driven either by the mouse metallothionein-1 or IGF-II 5' genomic regulatory regions, we were able to generate stable cell lines that synthesize and secrete mature IGF-I. This was demonstrated by RIA, by Northern analysis, and by polyacrylamide gel electrophoresis characterization of the radiolabeled intracellular and extracellular products that reacted with an IGF-I antibody. The mitogenic responses to TSH in IGF-I-expressing transfected FRTL-5 cells were indistinguishable from those stimulated by TSH and IGF-I in wild type or control-transfected cells (FRTL-5 cells stably transfected with a similar transgene that does not encode IGF-I). Basal DNA synthesis was higher and the peak of thymidine incorporation was earlier in IGF-I-expressing transfected FRTL-5 cells than in wild type or control cells (18-24 h vs. 30-36 h). The concentrations of TSH that maximally stimulate the incorporation of thymidine were not altered by IGF-I expression, and transfected cells did not appear to be transformed, as judged by their inability to form colonies in soft agar. TSH-stimulated DNA synthesis was blocked in IGF-I-expressing FRTL-5 cell by a monoclonal antibody to IGF (Sm 1.2). Thus, secretion of IGF-I appears to be required for the autocrine effects observed. These IGF-I-expressing FRTL-5 cell lines provide a model in vitro system to study the intracellular processing of IGF-I and the mechanisms by which IGF-I acts in an autocrine manner.

Actin polymerization is a fundamental cellular process that controls cytoskeletal functions such as adhesion, migration, and homing. This dynamic cellular process is spatiotemporally regulated in normal cells but its dysregulation is often observed in tumor cells and is believed to contribute to tumor metastasis. Hematopoietic cells isolated from Bcr-Abl positive leukemia patients exhibit multiple abnormalities in cytoskeletal function. The mechanism associated with these abnormalities is not completely understood. Previously, we showed that the expression of Bcr-Abl in hematopoietic cells induces tyrosine phosphorylation of Ableson interactor (Abi) 1 and ubiquitin dependent degradation of Abi 2. The dysregulation of Abi 1 and Abi 2 requires the SH3 domain and C-terminal proline-rich sequences of Bcr-Abl. Deletion of these sequences in Bcr-Abl not only abrogates Abi-1 tyrosine phosphorylation and Abi-2 degradation but also impairs leukemogenic activity of Bcr-Abl. Notably, the mutant Bcr-Abl with deletion of these sequences failed to induce splenomegaly in a bone marrow transplant mouse model. Because Abi proteins have been shown to function as a key regulator of actin polymerization, we examined the F-actin organization in Ba/F3 cells transformed by wild type Bcr-Abl (p185wt) and a mutant Bcr-Abl defective in signaling to Abi (p185ΔSH3ΔC). Actin polymerization is increased in Ba/F3 cells transformed by either p185wt or p185ΔSH3ΔC, as compared to control Ba/F3 cells. However, the pattern of F-actin organization in these cells appears different. The expression of p185wt induces a localized abnormal F-actin structure which is not observed in control Ba/F3 cells and the Ba/F3 cells transformed by p185ΔSH3ΔC. We have characterized this abnormal F-actin structure and show that it co-localizes with Bcr-Abl. Investigation is now underway to determine whether the Abi/WAVE pathway is involved in Bcr-Abl-induced abnormal F-actin structure. These studies may provide insight into the mechanism by which Bcr-Abl induces cytoskeletal abnormalities in CML cells.

Bcr-Abl elicits the ubiquitin-dependent degradation of Abl interactor (Abi) proteins, a family of proteins that antagonize the oncogenic potential of Abl. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr-Abl-positive leukemias. To determine the role of Abi degradation in Bcr-Abl-induced leukemogenesis, we have mapped the sequences in Bcr-Abl that are required for inducing Abi degradation. The deletion of C-terminal proline-rich sequences (p185 Bcr-AblΔ924-1018 ) severely impairs Bcr-Abl-induced Abi degradation. The double deletions of the SH3 domain and C-terminal proline-rich sequences (p185 Bcr-AblΔ924-1018 ) completely abolish the degradation of Abi proteins. We then compared the leukemogenic activity of these mutant forms of Bcr-Abl to that of the wild type Bcr-Abl in a murine bone marrow transduction/transplantation model. Like wild type P185 Bcr-Abl , both p185 Bcr-AblΔ924-1018 and p185 Bcr-AblΔSH3Δ924-1018 transform mouse bone marrow cells with similar potency, as judged by factor-independent growth in an agar colony assay. The mutant forms of Bcr-Abl, however, showed different leukemogenic activity in mice. Mice reconstituted with wild type P185 Bcr-Abl transduced marrow cells developed chronic myeloid leukemia (CML)-like disease and died in 5–7 weeks after transplantation. The peripheral white blood cell (WBC) count of these mice was significantly elevated and the mice had enlarged spleens. Mice reconstituted with p185 Bcr-AblΔ918-1018 transduced marrow cells also died in 5–7 weeks. The majority of these mice, however, did not show the elevated WBC count or the spenomegaly, suggesting that they died of a disease other than CML. Furthermore, mice reconstituted with p185 Bcr-AblΔSH3Δ924-1018 transduced marrow cells survived much longer post-transplantation without the elevated WBC count. These results are consistent with the hypothesis that Abi degradation is involved in the development of Bcr-Abl positive CML. Studies are now in progress to define the pathology of diseases caused by mutant forms of Bcr-Abl.

Background Polycystic Kidney Disease (PKD) kidneys exhibit increased extracellular matrix (ECM) collagen expression and metalloproteinases (MMPs) activity. We investigated the role of these increases on cystic disease progression in PKD kidneys. Methods We examined the role of type I collagen (collagen I) and membrane bound type 1 MMP (MT1-MMP) on cyst development using both in vitro 3 dimensional (3D) collagen gel culture and in vivo PCK rat model of PKD. Results We found that collagen concentration is critical in controlling the morphogenesis of MDCK cells cultured in 3D gels. MDCK cells did not form 3D structures at collagen I concentrations lower than 1 mg/ml but began forming tubules when the concentration reaches 1 mg/ml. Significantly, these cells began to form cyst when collagen I concentration reached to 1.2 mg/ml, and the ratios of cyst to tubule structures increased as the collagen I concentration increased. These cells exclusively formed cyst structures at a collagen I concentration of 1.8 mg/ml or higher. Overexpression of MT1-MMP in MDCK cells significantly induced cyst growth in 3D collagen gel culture. Conversely, inhibition of MMPs activity with doxycycline, a FDA approved pan-MMPs inhibitor, dramatically slowed cyst growth. More importantly, the treatment of PCK rats with doxycycline significantly decreased renal tubule cell proliferation and markedly inhibited the cystic disease progression. Conclusions Our data suggest that increased collagen expression and MMP activity in PKD kidneys may induce cyst formation and expansion. Our findings also suggest that MMPs may serve as a therapeutic target for the treatment of human PKD.