No abstract

Abstract. The PML protein was first identified as part of a fusion product with the retinoic acid receptor (RARa), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RARa hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.T HE eukaryotic nucleus is highly organized into discrete domains which spatially separate different biochemical processes. A variety of metabolic activities such as DNA replication, ribosome assembly, transcription, and pre-mRNA processing localize to distinct subnuclear compartments. The most conspicuous example is the nucleolus in which rRNAs are assembled into ribosomal subunits (reviewed by Scheer and Weisenberger, 1994). DNA replication sites were also shown to concentrate within discrete regions containing the proliferating cell nuclear antigen (PCNA; 1 Bravo and MacdonaldBravo, 1987) as well as DNA methyltransferase (Leonhardt et al., 1992). In addition, the small nuclear ribonucleoprotein particles (snRNPs), which are the major subunits of spliceosomes, were similarly found to localize to T. Carvalho and J.-S. Seeler should be considered as first authors.

How melanoma acquire a metastatic phenotype is a key issue. One possible mechanism is that metastasis is driven by microenvironment-induced switching between noninvasive and invasive states. However, whether switching is a reversible or hierarchical process is not known and is difficult to assess by comparison of primary and metastatic tumors. We address this issue in a model of melanoma metastasis using a novel intravital imaging method for melanosomes combined with a reporter construct in which the Brn-2 promoter drives green fluorescent protein (GFP) expression. A subpopulation of cells containing little or no pigment and high levels of Brn2::GFP expression are motile in the primary tumor and enter the vasculature. Significantly, the less differentiated state of motile and intravasated cells is not maintained at secondary sites, implying switching between states as melanoma cells metastasize. We show that melanoma cells can switch in both directions between high-and low-pigment states. However, switching from Brn2::GFP high to low was greatly favored over the reverse direction. Microarray analysis of high-and lowpigment populations revealed that transforming growth factor (TGF)B2 was up-regulated in the poorly pigmented cells. Furthermore, TGFB signaling induced hypopigmentation and increased cell motility. Thus, a subset of less differentiated cells exits the primary tumor but subsequently give rise to metastases that include a range of more differentiated and pigment-producing cells. These data show reversible phenotype switching during melanoma metastasis. [Cancer Res 2009;69(20):7969-77]

We have cloned and characterized a novel human serine/ threonine protein kinase gene from chromosome 12p13.3 encoding 2382 amino acids. Remarkably, the catalytic domain sequence contains a cysteine in place of a lysine residue conserved in subdomain II of most kinases. The same amino acid alteration was recently described for rat WNK1 (with no K=lysine) in which another nearby lysine residue was shown to confer kinase activity to the protein. Rat WNK1 is 85% identical to a splice variant lacking exons 11 and 12 of the described human kinase which we have called human WNK1. The WNK1 catalytic domain has closest homology with human PAK2, MEKK3, and Raf-1. Three additional, partial human protein kinase sequences, WNK2, WNK3 and WNK4, are also reported here with catalytic domains that are 95% homologous to WNK1. These genes di er both in chromosomal location and tissue-speci®c expression. Moreover, we have identi®ed in the database a total of 18 WNK-related genes, all exclusively from multicellular organisms, which share a WNK kinase sequence signature within subdomains I and II of the catalytic domain. We suggest that they constitute a novel subfamily of protein kinases that evolved together with cell adhesion and tissue-formation. Oncogene (2001) 20, 5562 ± 5569.

The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.

Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that ampli®cation by RT ± PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identi®ed in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel eector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.

cis-diamminedichloroplatinum(II) or cisplatin is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin cross-links to DNA, forming intra- and interstrand adducts, which bend and unwind the duplex and attract high-mobility-group domain and other proteins. Presumably due to a shielding effect caused by these proteins, the cisplatin-modified DNA is poorly repaired. The resulting DNA damage triggers cell-cycle arrest and apoptosis. Although it is still debatable whether the clinical success of cisplatin relies primarily on its ability to trigger apoptosis, at least two distinct pathways have been proposed to contribute to cisplatin-induced apoptosis in vitro. One involves the tumour-suppressor protein p53, the other is mediated by the p53-related protein p73. Coupling cisplatin damage to apoptosis requires mismatch repair activity, and recent observations further suggest involvement of the homologous recombinatorial repair system. At present it is generally accepted that abortive attempts to repair the DNA lesions play a key role in the cytotoxicity of the drug, and loss of the mismatch repair activity is known to cause cisplatin resistance, a major problem in antineoplastic therapy. Clearly, a better understanding of the signalling networks involved in cisplatin toxicity should provide a rational basis for the development of new therapeutic strategies.

Rac1 is a member of the Rho family of small GTPases, which control signaling pathways that regulate actin cytoskeletal dynamics and gene transcription. Rac1 is activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. In addition, Rho-GDP dissociation inhibitors (Rho-GDIs) can inhibit Rac1 by sequestering it in the cytoplasm. We have found previously that colorectal tumors express an alternatively spliced variant, Rac1b, containing 19 additional amino acids following the switch II region. Here we characterized the regulation and downstream signaling of Rac1b. Although little Rac1b protein is expressed in cells, the amount of activated Rac1b protein often exceeds that of activated Rac1, suggesting that Rac1b contributes significantly to the downstream signaling of Rac in cells. The regulation of both Rac1 and Rac1b activities is dependent on guanine nucleotide exchange factors and GTPase-activating proteins, but the difference in their activation is mainly determined by the inability of Rac1b to interact with Rho-GDI. As a consequence, most Rac1b remains bound to the plasma membrane and is not sequestered by Rho-GDI in the cytoplasm. Unlike Rac1, activated Rac1b is unable to induce lamellipodia formation and is unable to bind and activate p21-activated protein kinase nor activate the downstream protein kinase JNK. However, both Rac1 and Rac1b are able to activate NFB to the same extent. These data suggest that alternative splicing of Rac1 leads to a highly active Rac variant that differs in regulation and downstream signaling.