Complex tools and motor-to-mechanical transformations.

The ability to use complex tools is thought to depend on multifaceted motor-to-mechanical transformations within the left inferior parietal lobule (IPL), linked to cognitive control over compound actions. Here we show using neuroimaging that demanding transformations of finger movements into proper mechanical movements of functional parts of complex tools invoke significantly the right rather than left rostral IPL, and bilateral posterior-to-mid and left anterior intraparietal sulci. These findings emerged during the functional grasp and tool-use programming phase. The expected engagement of left IPL was partly revealed by traditional region-of-interest analyses, and further modeling/estimations at the hand-independent level. Thus, our results point to a special role of right IPL in supporting sensory-motor spatial mechanisms which enable an effective control of fingers in skillful handling of complex tools. The resulting motor-to-mechanical transformations involve dynamic hand-centered to target-centered reference frame conversions indispensable for efficient interactions with the environment.

SIAH ubiquitin ligases target the nonreceptor tyrosine kinase ACK1 for ubiquitinylation and proteasomal degradation.

Activated Cdc42-associated kinase 1 (ACK1) is a nonreceptor tyrosine kinase linked to cellular transformation. The aberrant regulation of ACK1 promotes tumor progression and metastasis. Therefore, ACK1 is regarded as a valid target in cancer therapy. Seven in absentia homolog (SIAH) ubiquitin ligases facilitate substrate ubiquitinylation that targets proteins to the proteasomal degradation pathway. Here we report that ACK1 and SIAH1 from Homo sapiens interact in a yeast two-hybrid screen. Protein-protein interaction studies and protein degradation analyses using deletion and point mutants of ACK1 verify that SIAH1 and the related SIAH2 interact with ACK1. The association between SIAHs and ACK1 depends on the integrity of a highly conserved SIAH-binding motif located in the far C-terminus of ACK1. Furthermore, we demonstrate that the interaction of ACK1 with SIAH1 and the induction of proteasomal degradation of ACK1 by SIAH1 are independent of ACK1's kinase activity. Chemical inhibitors blocking proteasomal activity corroborate that SIAH1 and SIAH2 destabilize the ACK1 protein by inducing its proteasomal turnover. This mechanism apparently differs from the lysosomal pathway targeting ACK1 after stimulation with the epidermal growth factor. Our data also show that ACK1, but not ACK1 mutants lacking the SIAH binding motif, has a discernable negative effect on SIAH levels. Additionally, knockdown approaches targeting the SIAH2 mRNA uncover specifically that the induction of SIAH2 expression, by hormonally-induced estrogen receptor (ER) activation, decreases the levels of ACK1 in luminal human breast cancer cells. Collectively, our data provide novel insights into the molecular mechanisms modulating ACK1 and they position SIAH ubiquitin ligases as negative regulators of ACK1 in transformed cells.

Ubiquitin conjugase UBCH8 targets active FMS-like tyrosine kinase 3 for proteasomal degradation.

The class III receptor tyrosine kinase FMS-like tyrosine kinase 3 (FLT3) regulates normal hematopoiesis and immunological functions. Nonetheless, constitutively active mutant FLT3 (FLT3-ITD) causally contributes to transformation and is associated with poor prognosis of acute myeloid leukemia (AML) patients. Histone deacetylase inhibitors (HDACi) can counteract deregulated gene expression profiles and decrease oncoprotein stability, which renders them candidate drugs for AML treatment. However, these drugs have pleiotropic effects and it is often unclear how they correct oncogenic transcriptomes and proteomes. We report here that treatment of AML cells with the HDACi LBH589 induces the ubiquitin-conjugating enzyme UBCH8 and degradation of FLT3-ITD. Gain- and loss-of-function approaches show that UBCH8 and the ubiquitin-ligase SIAH1 physically interact with and target FLT3-ITD for proteasomal degradation. These ubiquitinylating enzymes though have a significantly lesser effect on wild-type FLT3. Furthermore, physiological and pharmacological stimulation of FLT3 phosphorylation, inhibition of FLT3-ITD autophosphorylation and analysis of kinase-inactive FLT3-ITD revealed that tyrosine phosphorylation determines degradation of FLT3 and FLT3-ITD by the proteasome. These results provide novel insights into antileukemic activities of HDACi and position UBCH8, which have been implicated primarily in processes in the nucleus, as a previously unrecognized important modulator of FLT3-ITD stability and leukemic cell survival.

Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase.

Several lines of evidence point to an abnormality in the response of Fanconi anemia cells to reactive oxygen species. To investigate the potential pathologic consequences of an in vivo alteration of redox state in mice lacking one of the Fanconi anemia genes, animals were generated having combined deficiencies of the cytosolic Cu/Zn superoxide dismutase (Sod1) and Fanconi anemia complementation group C (Fancc) genes. Interestingly, hepatocytes of Fancc(-/-)Sod1(-/-) mice exhibited a zonal pattern of microvesicular steatosis, possibly as a result of oxidative stress-induced injury to hepatocyte membranes. Consistent with this idea, freshly explanted Fancc(-/-)Sod1(-/-) hepatocytes demonstrated increased spontaneous production of superoxide in vitro. The second phenotypic feature of Fancc(-/-) Sod1(-/-) mice was that of bone marrow hypocellularity accompanied by significant decreases in peripheral blood erythrocyte and leukocyte numbers as compared with wild-type controls. Although flow cytometry analysis with monoclonal antibodies against cell surface antigens revealed normal numbers of primitive hematopoietic progenitor populations in Fancc(-/-)Sod1(-/-) marrow, lineage-positive progenitor numbers were significantly reduced in these mice. Furthermore, the in vitro clonogenic growth of Fancc(-/-)Sod1(-/-) erythroid, myeloid, and early B-lymphoid colonies in semisolid media was profoundly compromised. These results suggested that the altered redox state likely present in Fancc(-/-) Sod1(-/-) hematopoietic progenitors was responsible for an impairment of cell proliferation or survival. (Blood. 2001;98:1003-1011)

Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1.

The Fanconi anemia group C protein (FANCC) plays an important role in hematopoiesis by ensuring the survival of hematopoietic progenitor cells through an unknown mechanism. We investigated the function of FANCC by identifying FANCC-binding proteins in hematopoietic cells. Here we show that glutathione S-transferase P1-1 (GSTP1) interacts with FANCC, and that overexpression of both proteins in a myeloid progenitor cell line prevents apoptosis following factor deprivation. FANCC increases GSTP1 activity after the induction of apoptosis. GSTP1 is an enzyme that catalyzes the detoxification of xenobiotics and by-products of oxidative stress, and it is frequently upregulated in neoplastic cells. Although FANCC lacks homology with conventional disulfide reductases, it functions by preventing the formation of inactivating disulfide bonds within GSTP1 during apoptosis. The prevention of protein oxidation by FANCC reveals a novel mechanism of enzyme regulation during apoptosis and has implications for the treatment of degenerative diseases with thiol reducing agents.

Targeting transgene expression to airway epithelia and submucosal glands, prominent sites of human CFTR expression.

Targeting therapeutic gene expression to disease-affected tissues is an essential component of effective and safe gene therapy. After birth, CFTR gene expression in human lungs is localized predominantly in the epithelial cells lining the upper airways, especially in the ducts and serous tubules of the submucosal glands. We have developed a K18 expression cassette, based on the DNA control elements of the human cytokeratin 18 gene. Temporal and spatial analyses of transgenic mice demonstrated that this expression cassette targets transgene expression to almost all cell types in which CFTR is expressed. Airway epithelium expression started as early as 11.5 days of gestational age and continued into the adulthood of the transgenic mice. In these adult mice, the pattern of the reporter expression strikingly matched that of the human cytokeratin 18 and human CFTR genes. The transgene expression was epithelium-specific and undetectable in connective tissue, muscle, bone, cartilage, blood, and endothelial cells. Significantly, high levels of expression were detected in tracheal submucosal glands. Together, these results suggest that our K18 expression cassette has a high potential for clinical application in gene therapy for patients with cystic fibrosis.

Isolation of a cDNA representing the Fanconi anemia complementation group E gene.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.

Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA.

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA.

The human homolog of insect-derived growth factor, CECR1, is a candidate gene for features of cat eye syndrome.

Cat eye syndrome (CES) is a developmental disorder with multiple organ involvement, associated with the duplication of a 2-Mb region of 22q11.2. Using exon trapping and genomic sequence analysis, we have isolated and characterized a gene, CECR1, that maps to this critical region. The protein encoded by CECR1 is similar to previously identified novel growth factors: IDGF from Sarcophaga peregrina (flesh fly) and MDGF from Aplysia californica (sea hare). The CECR1 gene is alternatively spliced and expressed in numerous tissues, with most abundant expression in human adult heart, lung, lymphoblasts, and placenta as well as fetal lung, liver, and kidney. In situ hybridization of a human embryo shows specific expression in the outflow tract and atrium of the developing heart, the VII/VIII cranial nerve ganglion, and the notochord. The location of this gene in the CES critical region and its embryonic expression suggest that the overexpression of CECR1 may be responsible for at least some features of CES, particularly the heart defects.

Hematopoietic compartment of Fanconi anemia group C null mice contains fewer lineage-negative CD34+ primitive hematopoietic cells and shows reduced reconstruction ability.

Fanconi anemia (FA) is a complex recessive genetic disease that causes bone marrow failure in children. The mechanism by which the gene for FA group C (Fancc) impinges on the normal hematopoietic program is unknown. Here we demonstrate that the bone marrow from Fancc-/- mice have reduced ability for primary and secondary long-term reconstitution of myeloablated recipients compared to wild-type or heterozygous mice, indicating that the Fancc gene product is required for the maintenance of normal numbers of hematopoietic stem cells. Long-term and secondary transplant studies suggested that there also were qualitative changes in their developmental potential. Consistent with the reduction in reconstitution, flow cytometric analysis of the primitive subfractions of hematopoietic cells obtained from the bone marrow of Fancc -/- mice demonstrated that they contained 40 to 70% fewer lineage-negative (Lin-)Thy1.2-/lowScal(+) c-Kit(+)CD34+ cells compared to controls. In contrast, the number of Lin Thy1.2-/ lowScal(+)c-Kit CD34(-)cells was comparable to that of wild-type mice. The differential behavior of Lin(-)Thy1.2-/lowScal+c-Kit+CD34+ and Lin(-)Thy1.2-/lowScal(+)c-Kit CD34 subfractions also was observed in mice treated with the DNA cross-linking agent mitomycin C(MMC). Fancc-/- mice treated with MMC had an 92% reduction of CD34 cells as compared to Fancc+/+ mice. The number of CD34 cells only was reduced about 20%. These results suggest that the Fancc gene may act at a stage of primitive hematopoietic cell development identified by CD34 expression.

Drug sensitivity spectra in Fanconi anemia lymphoblastoid cell lines of defined complementation groups.

Fanconi anemia (FA) is one of several genetic diseases with characteristic cellular hypersensitivity to DNA crosslinking agents which suggest that FA proteins may function as part of DNA repair processes. At the clinical level, FA is characterized by bone marrow failure that affects children at an early age. The clinical phenotype is heterogeneous and includes various congenital malformations as well as cancer predisposition. FA patients are distributed into eight complementation groups suggesting a complex molecular pathway. Three of the eight possible FA genes have been cloned, although their function(s) have not been identified. FA cells are highly sensitive to DNA crosslinking agents (mitomycin C (MMC) and diepoxybutane), with some variability between cell lines. Sensitivity to monofunctional alkylating agents has been reported in some cases, although these studies were performed with genetically unclassified FA cells. To further analyse and characterize the newly identified FA complementation groups, we tested their sensitivity to UV radiation, monofunctional and bifunctional alkylating agents and to the X-ray mimetic drug bleomycin. We found that FA complementation groups D to H show increased sensitivity to the X-ray mimetic drug bleomycin. Furthermore, the single known FA-H cell line shows increased sensitivity to ethylethane sulfonate (EMS), methylmethane sulfonate (MMS) in addition to the characteristic sensitivity to crosslinking agents, suggesting a broader spectrum of drug sensitivities in FA cells.

Loss of FancC function results in decreased hematopoietic stem cell repopulating ability.

Fanconi anemia (FA) is a complex genetic disorder characterized by progressive bone marrow (BM) aplasia, chromosomal instability, and acquisition of malignancies, particularly myeloid leukemia. We used a murine model containing a disruption of the murine homologue of FANCC (FancC) to evaluate short- and long-term multilineage repopulating ability of FancC -/- cells in vivo. Competitive repopulation assays were conducted where "test" FancC -/- or FancC +/+ BM cells (expressing CD45.2) were cotransplanted with congenic competitor cells (expressing CD45.1) into irradiated mice. In two independent experiments, we determined that FancC -/- BM cells have a profound decrease in short-term, as well as long-term, multilineage repopulating ability. To determine quantitatively the relative production of progeny cells by each test cell population, we calculated test cell contribution to chimerism as compared with 1 x 10(5) competitor cells. We determined that FancC -/- cells have a 7-fold to 12-fold decrease in repopulating ability compared with FancC +/+ cells. These data indicate that loss of FancC function results in reduced in vivo repopulating ability of pluripotential hematopoietic stem cells, which may play a role in the development of the BM failure in FA patients. This model system provides a powerful tool for evaluation of experimental therapeutics on hematopoietic stem cell function.

Characterization of regions functional in the nuclear localization of the Fanconi anemia group A protein.

Fanconi anemia (FA) is an autosomal recessive disease characterized by a variety of congenital abnormalities. Cells from FA patients show chromosomal instability and are hypersensitive to DNA cross-linking agents, though the basic cellular defect in FA is not known. The FANCA gene encodes a protein with an Mr of 162 kDa and with unknown function. The cellular localization of the FANCA protein has been controversial, and has been shown in different reports to be exclusively cytoplasmic and predominantly nuclear. In the present study, we further confirm that FANCA localizes primarily to the nucleus. Fusions of FANCA with the green fluorescent protein (GFP) showed a strong nuclear signal and a weak cytoplasmic signal in several cell types. Confocal laser microscopy confirmed that FANCA is evenly distributed throughout the nucleus. We also examined regions in FANCA that participate in its nuclear import. FANCA contains two bipartite nuclear localization signal (NLS) motifs at the extreme N-terminus. Deletion of amino acids N-terminal to the NLS motifs had no effect on the nuclear localization of FANCA or on its ability to correct mitomycin C sensitivity in an FA-A cell line, while deletion of both motifs impeded but did not prevent nuclear import. Deletions of 75, 90 and 150 residues from the N-terminus yielded a mixture of cells with only a cytoplasmic signal, and with both a nuclear and cytoplasmic signal. Deletion of the N-terminal 250 amino acids was required to block nuclear localization completely. Fusion of GFP to the N-terminal 250 amino acids showed a localization pattern similar to FANCA. Mutant forms of FANCA with deletions of the C-terminal 70 or 260 residues localized to the cytoplasm, although the C-terminal 260 amino acids alone lacked NLS activity. The results show that nuclear localization of FANCA involves several functional regions.

The Fanconi anaemia group G gene FANCG is identical with XRCC9.

Fanconi anemia (FA) is an autosomal recessive disease with diverse clinical symptoms including developmental anomalies, bone marrow failure and early occurrence of malignancies. In addition to spontaneous chromosome instability, FA cells exhibit cell cycle disturbances and hypersensitivity to cross-linking agents. Eight complementation groups (A-H) have been distinguished, each group possibly representing a distinct FA gene. The genes mutated in patients of complementation groups A (FANCA; refs 4,5) and C (FANCC; ref. 6) have been identified, and FANCD has been mapped to chromosome band 3p22-26 (ref. 7). An additional FA gene has recently been mapped to chromosome 9p (ref. 8). Here we report the identification of the gene mutated in group G, FANCG, on the basis of complementation of an FA-G cell line and the presence of pathogenic mutations in four FA-G patients. We identified the gene as human XRCC9, a gene which has been shown to complement the MMC-sensitive Chinese hamster mutant UV40, and is suspected to be involved in DNA post-replication repair or cell cycle checkpoint control. The gene is localized to chromosome band 9p13 (ref. 9), corresponding with a known localization of an FA gene.

Is Fanconi anemia caused by a defect in the processing of DNA damage?

Fanconi anemia (FA) is an autosomal genetic disease characterized by a complex array of developmental disorders, a high predisposition to bone marrow failure and to acute myelogenous leukemia. The chromosomal instability and the hypersensitivity to DNA cross-linking agents led to its classification with the DNA repair disorders. This review aimed at establishing whether it is still appropriate to consider 1/approximately FA within a DNA repair framework taking into account the recently discovered genetic heterogeneity characteristics of the defect (eight complementation groups). We discuss the possibility that the FA proteins interact to form a complex which may control different functions, including the processing of specific DNA lesions. Such a complex may act as a sensor to initiate protective systems as well as transcription of specific genes specifying, among others proteins, growth factors. Such steps may be organized as a linear cascade or more likely under the form of a web network.

Specific activation of Smad1 signaling pathways by the BMP7 type I receptor, ALK2.

BMP7 and activin are members of the transforming growth factor beta superfamily. Here we characterize endogenous activin and BMP7 signaling pathways in P19 embryonic carcinoma cells. We show that BMP7 and activin bind to the same type II receptors, ActRII and IIB, but recruit distinct type I receptors into heteromeric receptor complexes. The major BMP7 type I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRIB). BMP7 and activin elicited distinct biological responses and activated different Smad pathways. BMP7 stimulated phosphorylation of endogenous Smad1 and 5, formation of complexes with Smad4 and induced the promoter for the homeobox gene, Tlx2. In contrast, activin induced phosphorylation of Smad2, association with Smad4, and induction of the activin response element from the Xenopus Mix.2 gene. Biochemical analysis revealed that constitutively active ALK2 associated with and phosphorylated Smad1 on the COOH-terminal SSXS motif, and also regulated Smad5 and Smad8 phosphorylation. Activated ALK2 also induced the Tlx2 promoter in the absence of BMP7. Furthermore, we show that ALK1 (TSRI), an orphan receptor that is closely related to ALK2 also mediates Smad1 signaling. Thus, ALK1 and ALK2 induce Smad1-dependent pathways and ALK2 functions to mediate BMP7 but not activin signaling.

Association of the TLX-2 homeodomain and 14-3-3eta signaling proteins.

Homeodomain proteins play important roles in various developmental processes, and their functions are modulated by polypeptide cofactors. Here we report that both in vitro and in vivo, 14-3-3eta is associated with the TLX-2 homeodomain transcription factor that is required for mouse embryogenesis. Expression of 14-3-3eta shifts the predominant localization of TLX-2 in COS cells from the cytoplasm to the nucleus. Tlx-2 and 14-3-3eta are expressed in the developing peripheral nervous system with spatially and temporally overlapping patterns, and they are also coexpressed in PC12 cells. Increased expression of either gene by transfection considerably inhibited nerve growth factor-induced neurite outgrowth of PC12 cells, and cotransfection of both genes led to a synergistic effect of suppression. These findings define 14-3-3eta as a functional modulator of the TLX-2 homeodomain transcription factor and suggest that the in vivo function of TLX-2 in neural differentiation is likely regulated by signaling mediated by 14-3-3eta.

Development and characterization of immortalized fibroblastoid cell lines from an FA(C) mouse model.

Fanconi anemia (FA) is an autosomal recessive disorder, characterised by multiple congenital malformations, bone marrow failure and a predisposition to developing malignancies, especially leukemia. FA cells show increased levels of spontaneous chromosomal aberrations and a hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB). There are at least eight complementation groups involved in FA, and the genes for two of these groups, FA(A) and FA(C), have been isolated and cloned. Mouse models for FA(C) have been developed by replacing exon 8 or exon 9 of Fac with the neo gene. Mice homozygous for Fac mutations show reduced fertility and hypersensitivity to induction of chromosomal aberrations by MMC and DEB. To facilitate the study of cellular defects in vitro, transformed mouse fibroblast cell lines were established. Cell-killing experiments and cytogenetic analyses were performed on these cells following treatment with MMC and DEB. Fac-/- showed significant hypersensitivity to MMC and DEB as compared with Fac+/+ and +/- for both cellular phenotypes. This is consistent with results obtained from similar studies on human fibroblasts and lymphoblastoid cell lines. Therefore, these isogenic transformed mouse fibroblasts provide as in vitro model for further investigation of the hypersensitivity of Fanconi anemia cells to DNA cross-linking agents.