Human artificial episomal chromosomes for cloning large DNA fragments in human cells.

We have developed a human artificial episomal chromosome (HAEC) system, based on the latent replication origin of the large herpes Epstein-Barr virus, for the propagation and stable maintenance of DNA as circular minichromosomes in human cells. Individual HAECs carried human genomic inserts ranging from 60-330 kb and appeared genetically stable. An HAEC library of 1,500 independent clones carrying random human genomic fragments with average sizes of 150-200 kb was established and allowed recovery of the HAEC DNA. Our autologous HAEC system, with human DNA cloned directly in human cells, provides an important tool for functional study of large mammalian DNA regions and gene therapy.

PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling.

Wnt signalling regulates beta-catenin-dependent developmental processes through the Dishevelled protein (Dsh). Dsh regulates two distinct pathways, one mediated by beta-catenin and the other by Jun kinase (JNK). We have purified a Dsh-associated kinase from Drosophila that encodes a homologue of Caenorhabditis elegans PAR-1, a known determinant of polarity during asymmetric cell divisions. Treating cells with Wnt increases endogenous PAR-1 activity coincident with Dsh phosphorylation. PAR-1 potentiates Wnt activation of the beta-catenin pathway but blocks the JNK pathway. Suppressing endogenous PAR-1 function inhibits Wnt signalling through beta-catenin in mammalian cells, and Xenopus and Drosophila embryos. PAR-1 seems to be a positive regulator of the beta-catenin pathway and an inhibitor of the JNK pathway. These findings show that PAR-1, a regulator of polarity, is also a modulator of Wnt-beta-catenin signalling, indicating a link between two important developmental pathways.

Drosophila par-1 is required for oocyte differentiation and microtubule organization.

BACKGROUND: Drosophila oocyte determination involves a complex process by which a single cell within an interconnected cyst of 16 germline cells differentiates into an oocyte. This process requires the asymmetric accumulation of both specific messenger RNAs and proteins within the future oocyte as well as the proper organization of the microtubule cytoskeleton, which together with the fusome provides polarity within the developing germline cyst. RESULTS: In addition to its previously described late oogenic role in the establishment of anterior-posterior polarity and subsequent embryonic axis formation, the Drosophila par-1 gene is required very early in the germline for establishing cyst polarity and for oocyte specification. Germline clonal analyses, for which we used a protein null mutation, reveal that Drosophila par-1 (par-1) is required for the asymmetric accumulation of oocyte-specific factors as well as the proper organization of the microtubule cytoskeleton. Similarly, somatic clonal analyses indicate that par-1 is required for microtubule stabilization in follicle cells. The PAR-1 protein is localized to the fusome and ring canals within the developing germline cyst in direct contact with microtubules. Likewise, in the follicular epithelium, PAR-1 colocalizes with microtubules along the basolateral membrane. However, in either case PAR-1 localization is independent of microtubules. CONCLUSIONS: The Drosophila par-1 gene plays at least two essential roles during oogenesis; it is required early in the germline for organization of the microtubule cytoskeleton and subsequent oocyte determination, and it has a second, previously described role late in oogenesis in axis formation. In both cases, par-1 appears to exert its effects through the regulation of microtubule dynamics and/or stability, and this finding is consistent with the defined role of the mammalian PAR-1 homologs.

[Retrospective comparison of screening criteria for active surveillance for papillary thyroid microcarcinoma].

Objective: To investigate the rationality of management of active surveillance for papillary thyroid microcarcinoma (PTMC) and the main indications for active surveillance for PTMC. Methods: In this study, two criteria were used to evaluate patients with PTMC: low-risk PTMC conditions defined by Kuma hospital and Chinese Association of Thyroid Oncology (CATO) consensus on PTMC management of active surveillance. The patients had received surgical treatment. Clinicopathological characteristics and prognosis of the patients in different groups were compared. Results: A total of 778 patients were enrolled in the study, 565 (72.6%) of them met Kuma screening criteria and only 112 (14.4%) met CATO screening criteria. Kuma low-risk subgroup had lower incidence of cervical lymph node metastasis than Kuma high-risk PTMC subgroup(30.6% vs 47.9%, P<0.05). There were significant differences in multifocal lesions(6.3% vs 16.4%), extrathyroidal extension (1.8% vs 7.5%) and cervical lymph node metastasis(19.6% vs 38.0%) between low-risk and high-risk CATO PTMC subgroups. Patients in the CATO low-risk PTMC subgroup had lower recurrence and longer disease-free survival (DFS) than those in the CATO high-risk PTMC subgroup. But there was no significant difference in recurrence or DFS between Kuma low-risk and high-risk Kuma PTMC subgroups.The Chi-square test of Fisher's exact probabilities test was used to compare clinicopathological characteristics of patients between different groups.Rates of disease-free survival were calculated using the Kaplan-Meier method. Conclusion: CATO screening criteria is relatively strict and may be more suitable for Chinese patients with active surveillance for PTMC.

New clinical features of thyroid cancer in eastern China.

OBJECTIVE: Analyze recent clinical features of thyroid cancer in eastern China. METHODS: Investigation and comparison of clinical data of thyroid cancer patients from 1996 to 2006 from the Department of Head and Neck Surgery in the Cancer Hospital of Fudan University, Shanghai, China. RESULTS: The number of patients with thyroid cancer rose from 148 in the year 1996 to 572 in the year 2006, a 3.9-fold increase. Routine ultrasound survey during physical examination revealed four cases (2.7%) in 1996 and 285 cases (49.8%) in 2006. The sensitivity of the ultrasound survey in thyroid cancer diagnosis was 86.66% in 1996 and 88.20% in 2006 (P>0.05). Papillary carcinoma was most prevalent (87.8% in 1996 and 92.8% in 2006). An increasing proportion of small tumors was found. The incidence of microcarcinoma was 35.7% in 2006 in contrast with 20.3% in 1996 (P<0.01). Moreover, tumors with diameter from 1 to 2 cm were found in 38.5% patients in 2006 as opposed to 27.0% in 1996 (P<0.01). Extrathyroid extension was reported in 46 (31.1%) patients in 1996, but only in 39 (6.8%) in 2006 (P<0.01). Central cervical lymph node metastases were found in 98 (66.2%) patients in 1996, contrasting with 301 (52.6%) in 2006 (P<0.05). Thirty-seven (25.0%) patients had lateral cervical lymph node metastasis in 1996 compared with 117 (20.5%) in 2006 (P>0.05). Last, the proportion of stage I cancers in 2006 was higher than that in 1996. CONCLUSION: With the increasing incidence of thyroid cancer, cancer was discovered at an earlier stage. This is due to new clinical features of thyroid cancer, such as the decrease in tumor diameter, the lower rate of extrathyroid extension and of cervical lymph node metastasis. Routine ultrasound survey during physical examination has become the most common way to detect thyroid cancer. Increasing usage of diagnostic scrutiny, including the ultrasound survey, has most likely contributed to the increased incidence through detection of small thyroid cancers. Increased use of ultrasound to screen thyroid cancer in early stages should lead to better therapeutic outcome.

Structure determination of novel orthoborate NaMgBO(3): a promising birefringent crystal.

A novel orthoborate, NaMgBO(3), has been successfully synthesized by a standard solid-state reaction, and the crystal structure has been determined from powder X-ray diffraction data. It crystallizes in the monoclinic space group C2/c with lattice parameters a = 5.01313(6) Angstrom, b = 8.8007(1) Angstrom, c = 5.52831(7) Angstrom, and beta = 99.6962(6) degrees. Isolated [BO(3)](3-) anionic groups, which are the fundamental building units, are distributed perfectly parallel to each other. It is a typical structure of big birefringent crystals. The overall structure is similar to that of NaSrBO(3), which crystallizes in P2(1)/c. The correlation between these two structures is discussed. The Mg atoms in NaMgBO(3) are used instead of the position of Na in NaSrBO(3), while the Na atoms occupy the position of Sr. Then the directions of the BO(3) triangles are changed to balance the bond valence, which leads to a higher symmetry. They are the first pair of alkali-alkaline earth orthoborates with close space groups by exchange of the cation positions.

New steps in the Wnt/beta-catenin signal transduction pathway.

Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. Wnt family genes encode secreted glycoproteins that act as ligands for membrane receptors belonging to the Frizzled family of proteins. Wnt-1 originally was found as a proto-oncogene that was upregulated in tumors caused by the mouse mammary tumor virus. The Drosophila homologue of Wnt-1, wingless, is a segment polarity gene that regulates body patterning of the fly embryo. In Xenopus, the Wnt pathway regulates formation of the ventral-dorsal axis. Although Wnt proteins are expressed widely in mammals, the function of the Wnt signaling pathway in normal adult mammalian tissues is not understood. Downstream components of the Wnt pathway, APC (adenomatous polyposis coli) and beta-catenin, clearly are involved in human cancer. There are also several reports that Wnt ligands are highly expressed in tumors. Wnt stabilizes cytoplasmic beta-catenin and activates beta-catenin/Lef-1 (lymphoid enhancer factor), Tcf (T-cell factor)-dependent gene transcription. This regulation of cytosolic beta-catenin is mediated by glycogen synthase kinase-3 (GSK-3) activity but in neither case is the mechanism known. The mechanism by which Wnt inhibits GSK-3 is unknown. Recent studies have shown that some of the intracellular signaling molecules that mediate the Wnt pathway are in complexes, including Dishevelled (Dsh or Dvl), GSK-3beta, and APC protein. However, little is known about how Wnt or other upstream stimuli regulate these complexes to stabilize beta-catenin. We took a variety of approaches to identify new components of the Wnt pathway. Using an expression-cloning technique, we isolated casein kinase I (CKI)epsilon as a positive regulator of beta-catenin in the Wnt pathway. Overexpression of CKIepsilon mimics Wnt by stabilizing beta-catenin, thereby increasing expression of beta-catenin-dependent genes. Inhibition of endogenous CKIepsilon attenuated gene transcription stimulated by Wnt or by Dsh. CKIepsilon forms a complex with Axin and the other downstream components of the Wnt pathway. CKIepsilon is a positive regulator of the Wnt pathway and a possible functional link between upstream signals and the intracellular Axin signaling complex that regulates beta-catenin. In separate experiments, we have identified a Dishevelled-associated kinase (DAK) that binds to Dsh and regulates its functions. Dsh is required for two different pathways, the Wnt pathway and planar polarity pathway in Drosophila. DAK dramatically enhances the function of Dsh in the Wnt pathway and inhibits its function in the planar polarity pathway. This chapter will discuss these newly identified components of the Wnt pathway.

Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes.

The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functional self-replicating extrachromosomal DNA in target cells. Members of the herpesviral family are among the largest episomal double-stranded DNA viruses. As model systems of this family of endemic infectious agents, vectors derived from the human herpes 4 Epstein-Barr virus (EBV) were constructed which transferred up to 180 kb of DNA packaged as infectious virions. Such a transduction strategy was based on a non-oncogenic helper-dependent mini-EBV carrying minimal cis elements for latent replication and virus production. After exposure of human B lymphoma and lymphoblastoid cells to mini-EBVs transducing lacZ and human HPRT minigenes, stable cell transformants were selected which carried the delivered multimeric linear DNAs as circular episomes up to 160-180 kb in size. Following transduction of Lesch-Nyhan disease cells with a mini-EBV/HPRT, normal human HPRT function was restored in cells carrying large episomal HPRT minigenes. Direct visualization of the therapeutic mini-EBV by fluorescent in situ hybridization (FISH) on metaphase and interphase nuclei indicated that 99% (556/563) of the transduced mini-EBV DNA was episomal with an average copy number of one to two per nucleus. This system should allow the delivery of large genes in common diseases such as hemophilia A and codelivery of multiple genes in cells from polygenic diseases such as cancer. The extrachromosomal mini-EBV-based strategy offers an alternative to integrative or non-replicating gene therapy infectious vectors, which may be generally applicable to other herpesviruses characterized by different tropisms.

Cell autonomous regulation of multiple Dishevelled-dependent pathways by mammalian Nkd.

Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/beta-catenin signaling pathway, but its mechanism of action remains obscure [Zeng, W., Wharton, K. A., Jr., Mack, J. A., Wang, K., Gadbaw, M., et al. (2000) Nature (London) 403, 789--795]. Here we have cloned a cDNA encoding a mammalian homolog of Drosophila Nkd, mNkd, and demonstrated that mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. We showed that mNkd acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupted convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway.