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Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion in a manner consistent with the inhibition of BMP signaling., Neural crest cells are both highly migratory and significant to vertebrate organogenesis. However, the signals that regulate neural crest cell migration remain unclear. In this study, we test the function of differential screening-selected gene aberrant in neuroblastoma (DAN), a bone morphogenetic protein (BMP) antagonist we detected by analysis of the chick cranial mesoderm. Our analysis shows that, before neural crest cell exit from the hindbrain, DAN is expressed in the mesoderm, and then it becomes absent along cell migratory pathways. Cranial neural crest and metastatic melanoma cells avoid DAN protein stripes in vitro. Addition of DAN reduces the speed of migrating cells in vivo and in vitro, respectively. In vivo loss of function of DAN results in enhanced neural crest cell migration by increasing speed and directionality. Computer model simulations support the hypothesis that DAN restrains cell migration by regulating cell speed. Collectively, our results identify DAN as a novel factor that inhibits uncontrolled neural crest and metastatic melanoma invasion and promotes collective migration in a manner consistent with the inhibition of BMP signaling.

Congenital secondary erythrocytosis is a rare disorder characterized by increased red blood cell production. An important cause involves defects in the oxygen sensing pathway, in particular the PHD2-VHL-HIF axis. Mutations in VHL are also associated with the von Hippel-Lindau tumor predisposition syndrome. The differences in phenotypic expression of VHL mutations are poorly understood. We report on three patients with erythrocytosis, from two unrelated families. All patients show exceptionally high erythropoietin (EPO) levels, and are homozygous for a novel missense mutation in VHL: c.162G>C p.(Met54Ile). The c.162G>C mutation is the most upstream homozygous VHL mutation described so far in patients with erythrocytosis. It abolishes the internal translational start codon, which directs expression of VHLp19, resulting in the production of only VHLp30. The exceptionally high EPO levels and the absence of VHL-associated tumors in the patients suggest that VHLp19 has a role for regulating EPO levels that VHLp30 does not have, whereas VHLp30 is really the tumor suppressor isoform.

Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protein (UV-DDB) has also been proposed as a damage recognition factor involved in repair of UV-photoproducts, especially CPD. Here, we show in human XP-E cells (UV-DDB deficient) that the incision complex formation at UV-induced lesions was severely diminished in locally damaged nuclear spots. Repair kinetics of CPD and 6-4PP in locally and globally UV-irradiated normal human and XP-E cells demonstrate that UV-DDB can mediate efficient targeting of XPC-hHR23B and other NER factors to 6-4PR The data is consistent with a mechanism in which UV-DDB forms a stable complex when bound to a 6-4PP, allowing subsequent repair proteins starting with XPC-hHR23B - to accumulate, and verify the lesion, resulting in efficient 6-4PP repair. These findings suggest that (i) UV-DDB accelerates repair of 6-4PP, and at later time points also CPD, (ii) the fraction of 6-4PP that can be bound by UV-DDB is limited due to its low cellular quantity and fast UV dependent degradation, and (iii) in the absence of UV-DDB a slow XPC-hHR23B dependent pathway is capable to repair 6-4PP, and to some extent also CPD. (c) 2005 Elsevier B.V. All rights reserved.

Polyglutamine diseases are characterized by neuronal intranuclear inclusions (NIIs) of expanded polyglutamine proteins, indicating the failure of protein degradation. UBB(+1), an aberrant form of ubiquitin, is a substrate and inhibitor of the proteasome, and was previously reported to accumulate in Alzheimer disease and other tauopathies. Here, we show accumulation of UBB(+1) in the NIIs and the cytoplasm of neurons in Huntington disease and spinocerebellar ataxia type-3, indicating inhibition of the proteasome by polyglutamine proteins in human brain. We found that UBB(+1) not only increased aggregate formation of expanded polyglutamines in neuronally differentiated cell lines, but also had a synergistic effect on apoptotic cell death due to expanded polyglutamine proteins. These findings implicate UBB(+1) as an aggravating factor in polyglutamine-induced neurodegeneration, and clearly identify an important role for the ubiquitin-proteasome system in polyglutamine diseases.

Cells derived from Fanconi anemia (FA) patients are hypersensitive for cross-linking agents, such as cisplatin, that are potent inducers of programmed cell death (PCD). Here, we studied cisplatin hypersensitivity in FA in relation to the mechanism of PCD in lymphoblastoid cells representing FA groups A and C. In FA cells, a low concentration of cisplatin caused chromatin condensation, phosphatidylserine (PS) externalization, and the expression of an 18-kDa variant of Bax, all indicators of apoptotic cell death, and the latter suggesting the involvement of a mitochondrial route. However, procaspases-3, -8, and -9, and PARP were not cleaved, although small increases in caspase activity could be detected. At a high concentration of cisplatin, both FA and corrected cells showed a robust cleavage of procaspases and PARP. DNA fragmentation was clearly visible under high cisplatin conditions and to some extent at a low concentration in FA-A cells, but not in the FA-C cell line regardless of the presence of functional FANCC, suggesting an unknown deficiency in these cells. We conclude that hypersensitivity in FA cells is associated with a mixture of necrotic and apoptotic features that is best described as apoptotic-like cell death, and that a defective FA pathway does not interfere with the proper activation of caspase-mediated cell death. (C) 2003 Elsevier Science (USA). All rights reserved.

1 The present study was performed to evaluate and compare the ability of human MDR1-, and rat Mdr1b- and Mdr2-P-glycoproteins to transport hydrophilic monoquaternary drugs. Transport studies were performed with plasma membrane vesicles isolated from MDR1-, Mdr1b-, or Mdr2-overexpressing insect cells. 2 As model substrates we used the N-methylated derivatives of the diastereomers quinidine and quinine, the monoquaternary compounds N-methylquinidine and N-methylquinine. Vincristine, an established MDR1- substrate, was used as a reference. 3 We observed ATP-dependent uptake of all drugs studied into MDR1- and Mdrlb-expressing vesicles. Mdr2 was not able to transport these compounds. MDR1- and Mdr1b-mediated transport was saturable, and could be inhibited by various drugs, including PSC-833. 4 For both MDR1- and Mdr1b the ratios (or clearance) of N-methylquinidine were greater than those determined for N-methylquinine. This stereoselective difference was also evident from differential inhibitory studies with the two isomers. 5 Comparison of normalized clearance indicated that human MDR1 was more effective in transporting the tested substrates than rat Mdr1b. 6 In conclusion, our results demonstrate that MDR1 and Mdrlb, but not Mdr2, are able to transport the monoquaternary model drugs; both MDR1- and Mdrlb display stereospecificity for these cations; and indicate human MDR1- is more efficient in transporting these cations than its rat orthologue Mdr1b.