biAb Mediated Restoration of the Linkage between Dystroglycan and Laminin-211 as a Therapeutic Approach for α-Dystroglycanopathies.

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.

Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1ß and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1ß and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases.

Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.

BACKGROUND: Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. RESULTS: The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. CONCLUSIONS: This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.

Phage display of functional human TNF-alpha converting enzyme catalytic domain: a rapid method for the production of stabilized proteolytic proteins for assay development and high-throughput screening.

The catalytic domain of human tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) was expressed in a phage display system to determine whether stable and active enzyme could be made for high-throughput screening (HTS). This would address many issues around screening of proteases in this class. The phage-displayed TACE catalytic domain (PDT) properly cleaved the fusion protein of glutathione S-transferase (GST)-pro-TNF-alpha to generate the mature TNF-alpha in vitro. To determine the utility of the PDT in HTS, the authors further demonstrated that PDT was able to generate a strong reproducible fluorescence signal by cleaving a fluorogenic TNF-alpha-specific peptide in vitro. More important, the catalytic activity of the PDT was inhibited by a broad-spectrum matrix metalloprotease (MMP) inhibitor but not by an MMP-I specific inhibitor, illustrating the potential utility of PDT for HTS. The PDT was also compared with baculovirus-expressed TACE (BET) in these assays to establish the relative efficacy of PDT. Both PDT and BET showed a similar specific cleavage profile against the defined substrates. Activity of the BET, however, was stable at 4 degrees C for less than 24 h. In contrast, the PDT exhibited remarkable stability, losing very little activity even after 2 years at 4 degrees C. On the basis of these results, the authors concluded that the phage display system might be a useful tool for expressing proteins that have stability issues related to auto-proteolytic activity. Furthermore, the ease and low cost of large-scale production of phage should make it suitable for assay development and HTS.

Bisphenol-A and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells.

Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17beta-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ERalpha-HA) human breast cancer cells derived from the MCF-7 cell line. The ERalpha-HA cells (stably over-expressing ERalpha) were exposed to E2 (10(-8)M) or BPA (10(-6)M), for 3h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ERalpha-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4-12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott-Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ERalpha expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth.

1B50-1, a mAb raised against recurrent tumor cells, targets liver tumor-initiating cells by binding to the calcium channel α2δ1 subunit.

The identification and targeted therapy of cells involved in hepatocellular carcinoma (HCC) recurrence remain challenging. Here, we generated a monoclonal antibody against recurrent HCC, 1B50-1, that bound the isoform 5 of the α2δ1 subunit of voltage-gated calcium channels and identified a subset of tumor-initiating cells (TICs) with stem cell-like properties. A surgical margin with cells detected by 1B50-1 predicted rapid recurrence. Furthermore, 1B50-1 had a therapeutic effect on HCC engraftments by eliminating TICs. Finally, α2δ1 knockdown reduced self-renewal and tumor formation capacities and induced apoptosis of TICs, whereas its overexpression led to enhanced sphere formation, which is regulated by calcium influx. Thus, α2δ1 is a functional liver TIC marker, and its inhibitors may serve as potential anti-HCC drugs.

Human T helper (Th) cell lineage commitment is not directly linked to the secretion of IFN-gamma or IL-4: characterization of Th cells isolated by FACS based on IFN-gamma and IL-4 secretion.

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.

Gene expression in lung adenocarcinomas of smokers and nonsmokers.

Adenocarcinoma (AC) has become the most frequent type of lung cancer in men and women, and is the major form of lung cancer in nonsmokers. Our goal in this paper was to determine if AC in smokers and nonsmokers represents the same genetic disease. We compared gene expression profiles in resected samples of nonmalignant lung tissue and tumor tissue in six never-smokers with AC and in six smokers with AC, who were matched for clinical staging and histologic criteria of cell differentiation. Results were analyzed using a variety of bioinformatic tools. Four times as many genes changed expression in the transition from noninvolved lung to tumor in nonsmokers as in smokers, suggesting that AC in nonsmokers evolves locally, whereas AC in smokers evolves in a field of genetically altered tissue. There were some similarities in gene expression in smokers and nonsmokers, but many differences, suggesting different pathways of cell transformation and tumor formation. Gene expression in the noninvolved lungs of smokers differed from that of nonsmokers, and multidimensional scaling showed that noninvolved lungs of smokers groups with tumors rather than noninvolved lungs of nonsmokers. In addition, expression of a number of genes correlated with smoking intensity. Our findings, although limited by small sample size, suggest that additional studies comparing noninvolved to tumor tissue may identify pathogenetic mechanisms and therapeutic targets that differ in AC of smokers and nonsmokers.