High expression of Cks1 in human non-small cell lung carcinomas

Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers. Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1). There are no reports about the involvement of Cks1 in the pathogenesis of human cancer. Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR. The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples. In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas. These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.     

Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development

Retinal ganglion cells (RGCs) are the first cell type to differentiate during retinal histogenesis. It has been postulated that specified RGCs subsequently influence the number and fate of the remaining progenitors to produce the rest of the retinal cell types. However, several genetic knockout models have argued against this developmental role for RGCs. Although it is known that RGCs secrete cellular factors implicated in cell proliferation, survival, and differentiation, until now, limited publications have shown that reductions in the RGC number cause significant changes in these processes. In this study, we observed that Math5 and Brn3b double null mice exhibited over a 99% reduction in the number of RGCs during development. This severe reduction of RGCs is accompanied by a drastic loss in the number of all other retinal cell types that was never seen before. Unlike Brn3b null or Math5 null animals, mice null for both alleles lack an optic nerve and have severe retinal dysfunction. Results of this study support the hypothesis that RGCs play a pivotal role in the late phase of mammalian retina development.     

chokh/rx3 specifies the retinal pigment epithelium fate independently of eye morphogenesis

Despite the importance of the retinal pigment epithelium (RPE) for vision, the molecular processes involved in its specification are poorly understood. We identified two new mutant alleles for the zebrafish gene chokh (chk), which display a reduction or absence of the RPE. Unexpectedly, the neural retina (NR) in chk is specified and laminated, indicating that the regulatory network leading to NR development is largely independent of the RPE. Genetic mapping and molecular characterization revealed that chk encodes Rx3. Expression analyses show that otx2 and mitfb are not expressed in the prospective RPE of chk, indicating that the retinal homeobox gene rx3 acts upstream of the molecular network controlling RPE specification. Cellular transplantations demonstrate that rx3 function is autonomously required to specify the prospective RPE. Though rx2 is also absent in chk, neither rx2 nor rx1 is required for RPE development. Thus, our data provide the first indication that, in addition to controlling optic lobe evagination and proliferation, chk/rx3 also determines cellular fate.     

Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development

The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.     

Involvement of HMG-12 and CAR-1 in the cdc-48.1 expression of Caenorhabditis elegans

Caenorhabditis elegans possesses two p97/VCP/Cdc48p homologues, named CDC-48.1 (C06A1.1) and CDC-48.2 (C41C4.8), and their expression patterns and levels are differently regulated. To clarify the regulatory mechanisms of differential expression of two p97 proteins of C. elegans, we performed detailed deletion analysis of their promoter regions. We found that the promoter of cdc-48.1 contains two regions necessary for embryonic and for post-embryonic expression, while the promoter of cdc-48.2 contains the single region necessary for embryonic expression. In particular, two elements (Element A and Element B) and three conserved boxes (Box a, Box b and Box c) were essential for cdc-48.1 expression in embryos and at post-embryonic stages, respectively. By using South-Western blotting and MALDI-TOF MS analysis, we identified HMG-12 and CAR-1 as proteins that bind to Element A and Element B, respectively, from the embryonic nuclear extract. Importantly, we found the decreased expression of p97 in embryos prepared from hmg-12(RNAi) or car-1(RNAi) worms. These results indicate that both HMG-12 and CAR-1 play important roles in embryonic expression of cdc-48.1.     

Apoptosis related protein 3, an ATRA-upregulated membrane protein arrests the cell cycle at G1/S phase by decreasing the expression of cyclin D1

Human Apr3 was first cloned from HL-60 cells treated by ATRA. In this study, we further demonstrated that Apr3 could be obviously upregulated by ATRA in many other ATRA sensitive cells, suggesting a common role of Apr3 in ATRA effects. Indirect immunofluorescence assay indicates that Apr3 is a membrane protein, while its truncated form without the predicted transmembrane and intracellular domain, was likely a secreted one. Furthermore, FACS analysis showed that Apr3 overexpression could cause an obvious G1/S phase arrest which might be induced by dramatic reduction of cyclin D1 expression. Strikingly, the truncated Apr3 antagonized the negative role of Apr3 on cell cycle and cyclin D1. Taken together, our data suggest that Apr3 should play an important role in ATRA signal pathway and the predicted transmembrane and/or the intracellular domain mediates Apr3 membrane localization and is vital for the negative regulation on cell cycle and cyclin D1.     

Mutations in Nop60B,the Drosophila homolog of human dyskeratosis congenita 1, affect the maintenance of the germ-line stem cell lineage during spermatogenesis

Spermatogenesis in Drosophila is maintained by germ-line stem cells. These cells undergo self-renewing divisions and also generate daughter gonial cells, whose function is to amplify the germ cell pool. Gonial cells subsequently differentiate into spermatocytes that undergo meiosis and generate haploid gametes. To elucidate the circuitry that controls progression through spermatogenic stem cell lineages, we are identifying mutations that lead to either excess germ cells or germ cell loss. From a collection of male sterile mutants, we identified P-element-induced hypomorphic alleles of nop60B, a gene encoding a pseudouridine synthase. Although null mutations are lethal, our P element-induced alleles generate viable, but sterile flies, exhibiting severe testicular atrophy. Sterility is reversed by P-element excision, and the atrophy is rescued by a Nop60B transgene, confirming identity of the gene. Using cell-type-specific markers, we find that testicular atrophy is due to severe loss of germ cells, including stem cells, but much milder effects on the somatic cells, which are themselves maintained by a stem cell lineage. We show that Nop60B activity is required intrinsically for the maintenance of germ-line stem cells. The relationship of these phenotypes to the human syndrome Dyskeratosis congenita, caused by mutations in a Nop60B homolog, is discussed.     

Notch3 overexpression causes arrest of cell cycle progression by inducing Cdh1 expression in human breast cancer cells

Uncontrolled cell proliferation, genomic instability and cancer are closely related to the abnormal activation of the cell cycle. Therefore, blocking the cell cycle of cancer cells has become one of the key goals for treating malignancies. Unfortunately, the factors affecting cell cycle progression remain largely unknown. In this study, we have explored the effects of Notch3 on the cell cycle in breast cancer cell lines by 3 methods: overexpressing the intra-cellular domain of Notch3 (N3ICD), knocking-down Notch3 by RNA interference, and using X-ray radiation exposure. The results revealed that overexpression of Notch3 arrested the cell cycle at the G0/G1 phase, and inhibited the proliferation and colony-formation rate in the breast cancer cell line, MDA-MB-231. Furthermore, overexpressing N3ICD upregulated Cdh1 expression and resulted in p27^Kip accumulation by accelerating Skp2 degradation. Conversely, silencing of Notch3 in the breast cancer cell line, MCF-7, caused a decrease in expression levels of Cdh1 and p27^Kip at both the protein and mRNA levels, while the expression of Skp2 only increased at the protein level. Correspondingly, there was an increase in the percentage of cells in the G0/G1 phase and an elevated proliferative ability and colony-formation rate, which may be caused by alterations of the Cdh1/Skp2/p27 axis. These results were also supported by exposing MDA-MB-231 cells or MCF-7 treated with siN3 to X-irradiation at various doses. Overall, our data showed that overexpression of N3ICD upregulated the expression of Cdh1 and caused p27^Kip accumulation by accelerating Skp2 degradation, which in turn led to cell cycle arrest at the G0/G1 phase, in the context of proliferating breast cancer cell lines. These findings help to illuminate the precision therapy targeted to cell cycle progression, required for cancer treatment.     

Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27(KIP1) protein expression

Helicobacter pylori, the main cause of chronic gastritis, plays a central role in the etiology of peptic ulcer disease and gastric cancer. In vitro studies have shown that H. pylori increases gastric epithelial cell turnover, thus increasing the risk for the development of neoplastic clones. The mechanisms by which H. pylori promotes perturbation of cell proliferation are not yet elucidated. To investigate whether products released by H. pylori in culture media interfere with cell cycle progression of human gastric epithelial cells, four cell lines (MKN 28, MKN 7, MKN 74, and AGS) were incubated in the presence of H. pylori broth culture filtrate. Cell cycle analysis showed that a H. pylori-released factor(s) significantly inhibited the G1- to S-phase progression of MKN 28 and MKN 7 cell lines, with a reversible, nonlethal mechanism, independent of the expression of VacA, CagA, and/or urease. The cell cycle inhibition occurred concomitantly with an increase in p27(KIP1) protein levels, a reduction in Rb protein phosphorylation on serine residues 807-811, and a significant decrease in cyclin E-associated cdk2 activity. In contrast, the cell cycle progression of MKN 74 and AGS cell lines was not affected by the H. pylori-released factor(s). In normal human fibroblasts, G1-phase cell accumulation was concomitant with the reduction in Rb protein phosphorylation; that, however, appeared to be dependent on p21(WAF1/CIP1) rather than on p27(KIP1) protein. A preliminary characterization showed that the molecular mass of the partially purified cell cycle inhibitory factor(s) was approximately 40 kDa. These results suggest that H. pylori releases a soluble factor(s) that may affect cell cycle progression of gastric epithelial cells through elevated levels of cdk inhibitor p27(KIP1). This factor(s) might act in vivo on noncolonized distant cells, the most proliferating cells of human gastric mucosa.     

C. elegans RBX-2-CUL-5- and RBX-1-CUL-2-based complexes are redundant for oogenesis and activation of the MAP kinase MPK-1

Cul5-based complex is a member of ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) ubiquitin ligase family. The cellular function of the Cul5-based complex is poorly understood. In this study, we found that oocyte septum formation and egg production did not occur in either cul-5- or rbx-2-depleted cul-2 homozygotes, although control cul-2 homozygotes laid approximately 50 eggs. These phenotypes are reminiscent of those caused by the MAP kinase mpk-1 depletion. In fact, activation of MPK-1 was significantly inhibited in cul-5-depleted cul-2 mutant and cul-2-depleted cul-5 mutant. Yeast two-hybrid analysis and RNAi-knockdown experiments suggest that oocyte maturation from pachytene exit and MPK-1 activation are redundantly controlled by the RBX-2-CUL-5- and RBX-1-CUL-2-based complexes.