Werner's syndrome (WS) 1 is a rare autosomal recessive disorder characterized by premature aging and an early onset of age-related diseases including arteriosclerosis, malignant neoplasms, melituria, and cataract (1). Somatic cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening (2, 3). WS cells have subtle defects in DNA replication, resulting in a reduced frequency of firing of replication origins (4). In addition, a large number of reports have shown that many cellular events including DNA repair, transcription, and apoptosis are affected in WS cells (5-7). The gene responsible for WS encodes a protein (WRN) that is a member of the RecQ family of DNA helicases (8). Most of the WS mutations that have been identified are nonsense or frameshift mutations, resulting in the truncation of WRN (9, 10). The clinical features and cellular phenotypes of most WS patients seem to be due to an absolute lack of WRN in the nucleus because the nuclear localization signal of WRN resides in its C-terminal end (11).The RecQ family includes Escherichia coli RecQ, S. cerevisiae Sgs1, Shizosaccharomyces pombe Rqh1, and five human RecQ helicases, namely DNA helicase Q1/RecQL (RecQL1), WRN, Bloom's syndrome gene product (BLM), Rhusmund-Thomson's syndrome gene product (RecQL4), and RecQL5 (12-19). Rhusmund-Thomson's syndrome also shows some features of the premature aging phenotype, and Bloom's syndrome is characterized by a predisposition to various malignant neoplasms. In S. cerevisiae, mutations in the SGS1 gene caused premature aging and hyper-recombination phenotypes (20, 21). The sgs1 mutants showed higher sensitivity to MMS and hydroxyurea (22)(23)(24)(25)37). Thus, sgs1 mutants exhibit some of the phenotypes of WS.WRN has been shown to have DNA helicase and exonuclease activity (26 -29). Recent studies (30 -32) have revealed that WRN interacts with replication protein A, PCNA, DNA topoisomerase I, and DNA polymerase ␦, indicating the involvement of WRN in some aspects of DNA replication. WRN also interacts with the p53 and Ku 70/86 heterodimer, suggesting that WRN is involved in apoptosis and the repair of DNA double strand breaks (7,(33)(34)(35). Despite these observations, it is not clear how the dysfunction of WRN is related to the observed phenotypes of WS cells. To obtain further insight into the process in which WRN is involved, we performed a two-hybrid screening using mouse WRN (mWRN) as bait and identified three interacting proteins: Ubc9, SUMO-1 (small ubiquitinrelated modifier-1), and a novel protein, WHIP (Werner Helicase Interacting Protein), which is conserved from E. coli to human (36). Here we report that mWRN physically interacts with mWHIP, and the yeast homologue of WRN, Sgs1, genetically interacts with yWHIP. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The yeast strains and plasmids for two-hybrid screening were described previously (36).

No abstract

The Saccharomyces cerevisiae gene WHIP/ MGS1 encodes a protein related to the subunits of Replication Factor C (RFC). We found that the RFC-like motifs in Whip/Mgs1 are essential for its function. Furthermore, by screening for synthetic dosage lethality, we have shown that overexpression of MGS1 causes lethality in combination with mutations in genes that encode replication proteins such as DNA polymerase delta, RFC, PCNA and RPA. Moreover, loss of MGS1 function interferes with the ability of multicopy PCNA to suppress the replication defect of the rfc5-1 mutant. At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, which bear mutations in the smaller subunits of DNA polymerase delta, and at semipermissive and non-permissive temperatures mgs1delta partially alleviates the growth defects of the pol31 mutant. We also report that the growth defect and HU sensitivity of the pol31 mutant are suppressed by mms2delta and rad18delta mutations. We suggest that Mgs1 interacts with the DNA replication machinery to modulate the function of DNA polymerase delta during replication or replication-associated repair, and influences the choice of the pathway employed for replication fork reactivation. Possible roles of Mgs1, DNA polymerase delta, Rad18 and Mms2 in replication and replication fork restart are discussed.

BackgroundIn breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression?MethodsThroughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment.ResultsMCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells.ConclusionsMCF-7-14 cells are a novel model for breast cancer metastasis without requiring constitutive EMT and are categorized as a "metastable phenotype", which can be distinguished from both epithelial and mesenchymal cells. The alterations and characteristics of MCF-7-14 cells, especially nuclear β-catenin and CD44 upregulation, may characterize invasive cell populations in breast cancer.

No abstract