Cystic fibrosis transmembrane conductance regulator trafficking is mediated by the COPI coat in epithelial cells

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted beta-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. Beta-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of beta-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the alpha-, beta-, and gamma-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.     

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for beter understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G₁ phase, cyclins C, D and E, or in S, G₂ and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G₁ phase, cyclin A in late G₁/early S phase and cyclin B1 in G₂. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones—oestrogens and progestins—and growth factors—insulin-like investigated for their effects on G₁ cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G₁ arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies

Key message

An algae-based vaccine model against atherosclerosis was developed with positive findings in terms of antigen yield and immunogenicity in mouse.

Abstract

Several immunotherapies against atherosclerosis have been evaluated at the preclinical level thus far, with some of them currently under evaluation in clinical trials. In particular, the p210 epitope from ApoB100 is known to elicit atheroprotective responses. Considering that Chlamydomonas reinhardtii is an attractive host for the production and delivery of subunit vaccines, in this study a chimeric protein consisting of the B subunit of the cholera toxin and the p210 epitope from ApoB100 (CTB:p210) has been expressed in C. reinhardtii chloroplast as an attempt to establish an oral vaccine candidate against atherosclerosis. The Chlamydomonas-made CTB:p210 protein was successfully expressed at levels of up to 60 µg per g of fresh weight biomass. The antigenic activity of the CTB and the p210 moiety was preserved in the CTB:p210 chimera. Moreover the algae-made CTB:p210 showed an immunogenic activity, when orally administered to BALB/c mice, as evidenced the presence of anti-p210 serum antibodies in mice treated with the algae-derived CTB:p210. The antibody response lasts for at least 80 days after the last boost. This experimental model is proposed as a convenient tool in the development of low cost atherosclerosis vaccines of easy compliance and friendly delivery. Further studies will determine the therapeutic potential of this algae-made vaccine in atherosclerosis animal models.

     

Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations

Campylobacter isolates from diverse samples within broiler production and processing environments were typed by using flaA short variable region DNA sequence analysis. Sixteen flocks from four different farms representing two broiler producers in Arkansas and California were analyzed. Fourteen of the flocks (87.5%) were Campylobacter-positive; two remained negative throughout the 6-week rearing period. In general, multiple clones were present within a flock. Additionally, clones found within a flock were also present on the final product, although the diversity of Campylobacter spp. on the final product appeared to be reduced relative to that observed within the flock. Comparison of clones between flocks on the same farm revealed that some clones of Campylobacter persisted in multiple flocks. Furthermore, some clones were identified across the two farms that were under the same management. In two sampling periods, environmental isolates were positive for Campylobacter prior to flock shedding. Environmental samples associated with five additional flocks were positive for Campylobacter concomitantly with recovery of Campylobacter from the birds. Analysis of the environmental isolates that were positive prior to flock shedding demonstrated that in some instances the environmental isolates possessed genotypes identical to those of isolates originating from the flock, while in other cases the environmental isolates possessed genotypes that were distantly related to isolates obtained from the flock. Analyses of environmental isolates that tested positive concurrently with the positive isolates from the flocks demonstrated varied results; in some instances the environmental isolates possessed genotypes identical to those of isolates originating from the flock, while in other cases the environmental isolates possessed genotypes that were distantly related to isolates obtained from the flock. These data suggest that the external environment may contribute to Campylobacter contamination during poultry production and processing. However, environmental contamination with Campylobacter does not appear to be the sole contributing factor.     

Increased proliferation and altered growth factor dependence of human mammary epithelial cells overexpressing the Gab2 docking protein

The docking protein Gab2 is a proto-oncogene product that is overexpressed in primary breast cancers. To determine the functional consequences of Gab2 overexpression, we utilized the immortalized human mammary epithelial cell line MCF-10A. In monolayer culture, expression of Gab2 at levels comparable with those detected in human breast cancer cells accelerated epidermal growth factor (EGF)-induced cell cycle progression and was associated with increased basal Stat5 tyrosine phosphorylation and enhanced and/or more sustained EGF-induced Erk and Akt activation. Three-dimensional Matrigel culture of MCF-10A cells resulted in the formation of polarized, growth-arrested acini with hollow lumina. Under these conditions, Gab2 increased cell proliferation during morphogenesis, leading to significantly larger acini, an effect dependent on Gab2 binding to Grb2 and Shp2 and enhanced by recruitment of the p85 subunit of phosphatidylinositol 3-kinase. Pharmacological inhibition of MEK revealed that, in addition to direct activation of phosphatidylinositol 3-kinase, increased Erk signaling also contributed to Gab2-mediated enhancement of acinar size. In addition, Gab2 overcame the proliferative suppression that normally occurs in late stage cultures and conferred independence of the morphogenetic program from exogenous EGF. Finally, higher levels of Gab2 expression led to the formation of large disorganized structures with defective luminal clearance. These findings support a role for Gab2 in mammary tumorigenesis.     

The PDZ-binding motif of MCC is phosphorylated at position -1 and controls lamellipodia formation in colon epithelial cells

In this study, we describe a new post-translational modification at position -1 of the PDZ-binding motif in the mutated in colorectal cancer (MCC) protein and its role in lamellipodia formation. Serine 828 at position -1 of this motif is phosphorylated, which is predicted to increase MCC binding affinity with the polarity protein Scrib. We show that endogenous MCC localizes at the active migratory edge of cells, where it interacts with Scrib and the non-muscle motor protein Myosin-IIB. Expression of MCC harboring a phosphomimetic mutation MCC-S828D strongly impaired lamellipodia formation and resulted in accumulation of Myosin-IIB in the membrane cortex fraction. We propose that MCC regulates lamellipodia formation by binding to Scrib and its downstream partner Myosin-IIB in a multiprotein complex. Importantly, we propose that the function of this complex is under the regulation of a newly described phosphorylation of the PDZ-binding motif at position -1.     

Chromosome-breakage genomic instability and chromothripsis in breast cancer

Background

Chromosomal breakage followed by faulty DNA repair leads to gene amplifications and deletions in cancers. However, the mere assessment of the extent of genomic changes, amplifications and deletions may reduce the complexity of genomic data observed by array comparative genomic hybridization (array CGH). We present here a novel approach to array CGH data analysis, which focuses on putative breakpoints responsible for rearrangements within the genome.

Results

We performed array comparative genomic hybridization in 29 primary tumors from high risk patients with breast cancer. The specimens were flow sorted according to ploidy to increase tumor cell purity prior to array CGH. We describe the number of chromosomal breaks as well as the patterns of breaks on individual chromosomes in each tumor. There were differences in chromosomal breakage patterns between the 3 clinical subtypes of breast cancers, although the highest density of breaks occurred at chromosome 17 in all subtypes, suggesting a particular proclivity of this chromosome for breaks. We also observed chromothripsis affecting various chromosomes in 41% of high risk breast cancers.

Conclusions

Our results provide a new insight into the genomic complexity of breast cancer. Genomic instability dependent on chromosomal breakage events is not stochastic, targeting some chromosomes clearly more than others. We report a much higher percentage of chromothripsis than described previously in other cancers and this suggests that massive genomic rearrangements occurring in a single catastrophic event may shape many breast cancer genomes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-579) contains supplementary material, which is available to authorized users.