Cellular protection from H2O2 toxicity by Fv-Hsp70: protection via catalase and gamma-glutamyl-cysteine synthase.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 µM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.

Combining intracellular antibodies to restore function of mutated p53 in cancer.

TP53 is a tumor suppressor gene that is mutated in 50% of cancers, and its function is tightly regulated by the E3 ligase, Mdm2. Both p53 and Mdm2 are localized in the cell nucleus, a site that is impervious to therapeutic regulation by most antibodies. We identified a cell-penetrating lupus monoclonal anti-DNA antibody, mAb 3E10, that targets the nucleus, and we engineered mAb 3E10 to function as an intranuclear transport system to deliver therapeutic antibodies into the nucleus as bispecific single chain Fv (scFv) fragments. Bispecific scFvs composed of 3E10 include PAb421 (3E10-PAb421) that binds p53 and restores the function of mutated p53, and 3G5 (3E10-3G5) that binds Mdm2 and prevents destruction of p53 by Mdm2. We documented the therapeutic efficacy of these bispecific scFvs separately in previous studies. In this study, we show that combination therapy with 3E10-PAb421 and 3E10-3G5 augments growth inhibition of cells with p53 mutations compared to the effect of either antibody alone. By contrast, no enhanced response was observed in cells with wild-type p53 or in cells homozygous null for p53.

Gene expression and splicing alterations analyzed by high throughput RNA sequencing of chronic lymphocytic leukemia specimens.

BACKGROUND: To determine differentially expressed and spliced RNA transcripts in chronic lymphocytic leukemia specimens a high throughput RNA-sequencing (HTS RNA-seq) analysis was performed. METHODS: Ten CLL specimens and five normal peripheral blood CD19+ B cells were analyzed by HTS RNA-seq. The library preparation was performed with Illumina TrueSeq RNA kit and analyzed by Illumina HiSeq 2000 sequencing system. RESULTS: An average of 48.5 million reads for B cells, and 50.6 million reads for CLL specimens were obtained with 10396 and 10448 assembled transcripts for normal B cells and primary CLL specimens respectively. With the Cuffdiff analysis, 2091 differentially expressed genes (DEG) between B cells and CLL specimens based on FPKM (fragments per kilobase of transcript per million reads and false discovery rate, FDR q < 0.05, fold change >2) were identified. Expression of selected DEGs (n = 32) with up regulated and down regulated expression in CLL from RNA-seq data were also analyzed by qRT-PCR in a test cohort of CLL specimens. Even though there was a variation in fold expression of DEG genes between RNA-seq and qRT-PCR; more than 90 % of analyzed genes were validated by qRT-PCR analysis. Analysis of RNA-seq data for splicing alterations in CLL and B cells was performed by Multivariate Analysis of Transcript Splicing (MATS analysis). Skipped exon was the most frequent splicing alteration in CLL specimens with 128 significant events (P-value <0.05, minimum inclusion level difference >0.1). CONCLUSION: The RNA-seq analysis of CLL specimens identifies novel DEG and alternatively spliced genes that are potential prognostic markers and therapeutic targets. High level of validation by qRT-PCR for a number of DEG genes supports the accuracy of this analysis. Global comparison of transcriptomes of B cells, IGVH non-mutated CLL (U-CLL) and mutated CLL specimens (M-CLL) with multidimensional scaling analysis was able to segregate CLL and B cell transcriptomes but the M-CLL and U-CLL transcriptomes were indistinguishable. The analysis of HTS RNA-seq data to identify alternative splicing events and other genetic abnormalities specific to CLL is an added advantage of RNA-seq that is not feasible with other genome wide analysis.

DNA-dependent targeting of cell nuclei by a lupus autoantibody.

A nuclear-penetrating lupus anti-DNA autoantibody, 3E10, has been found to inhibit DNA repair and selectively kill certain cancer cells that are highly vulnerable to DNA damage. In addition, a 3E10 single chain variable fragment (scFv) has been developed for use as a delivery vehicle to carry therapeutic cargo proteins into cell nuclei. A greater understanding of the mechanism by which 3E10 penetrates cell nuclei is needed to help determine the scope of its potential therapeutic applications. Here we show that the presence of extracellular DNA significantly enhances the nuclear uptake of 3E10 scFv. In addition, we find that 3E10 scFv preferentially localizes into tumor cell nuclei in vivo, likely due to increased DNA in the local environment released from ischemic and necrotic regions of tumor. These data provide insight into the mechanism of nuclear penetration by 3E10 and demonstrate the potential for use of 3E10 in therapeutic approaches to diseases ranging from malignancy to ischemic conditions such as stroke.

Amplification of B cell receptor-Erk signaling by Rasgrf-1 overexpression in chronic lymphocytic leukemia.

Rasgrf-1 is a guanine exchange factor (GEF) that catalyzes the exchange of GDP for GTP. In a RNA microarray analysis of chronic lymphocytic leukemia (CLL) specimens (n = 5), this gene was found to be overexpressed in CLL as compared to normal peripheral blood mononuclear cell (PBMC) CD19 + B cells (n = 3). CLL specimens (n = 29) expressed Rasgrf-1 RNA at levels 5-300-fold higher as compared to normal B cells. CLL specimens expressed a 75 kDa isoform that was smaller than the expected full-length protein (140 kDa) and the truncated variant had higher GEF activity. Knockdown of Rasgrf-1 in CLL specimens inhibited active GTP-bound Ras and the Ras/Erk/mitogen-activated protein kinase (MAPK) pathway. Rasgrf-1 was phosphorylated and activated by B cell receptor (BCR) signaling that increased its GEF function, and this phosphorylation was blocked by Src and Bruton's tyrosine kinase (BTK) inhibitors. Rasgrf-1 is a novel GEF protein that has a role in BCR signaling and its overexpression further activates the Ras/Erk/MAPK pathway in CLL specimens.

Effect of epigenetic histone modifications on E-cadherin splicing and expression in lung cancer.

We have identified an alternatively spliced, non-functional aberrant E-cadherin transcript that lacks exon 11 and is over expressed in malignant cells as compared to the normal non-malignant cells. This increase in the aberrant transcript is a mechanism of loss of E-cadherin gene expression as it is rapidly degraded by the nonsense mediated decay pathway. To study the mechanism of this gene missplicing we analyzed the role of histone epigenetic modifications in lung cancer cell lines. The treatment of low E-cadherin lung cancer cell lines with histone deacetylase inhibitor (HDACi, MS-275) resulted in the preferential expression of the correctly spliced transcripts in the low E-cadherin expressing cell lines only. Chromatin immunoprecipitation (ChIP) assays revealed that the histone hypoacetylation levels correlate with aberrant exon 11 splicing as there is more aberrant splicing in cell lines with E-cadherin promoter hypoacetylation. Inactivation of histone deacetylases (HDAC) 1, 2 and 3 resulted in an increase in E-cadherin expression and an increase in the ratio of the correctly spliced E-cadherin transcript. As transcription of the gene is closely linked to splicing, we considered the possibility that change in E-cadherin transcription correlates with splicing. The Zeb1 epithelial-mesenchymal transformation (EMT) inducer silences E-cadherin expression and could also alter the splicing of this exon. Inhibition of the E-cadherin promoter transcription with Zeb1 expression increases aberrant splicing and the reverse is observed when Zeb1 is knocked down. The role of HDAC inhibitors was also studied in vivo in a immunodeficient mouse xenograft model. Exposure of mice to HDACi resulted in growth inhibition, increase in E-cadherin expression, alteration of aberrant splicing and the reversal of EMT in mouse tumors. The findings support the modulation of E-cadherin exon 11 inclusion or exclusion by histone epigenetic modifications as they change the overall chromatin structure. The results provide an interesting link between epigenetic alterations in cancer cells and gene splicing in addition to their effect on gene silencing.

E-cadherin gene re-expression in chronic lymphocytic leukemia cells by HDAC inhibitors.

BACKGROUND: The tumor suppressor gene E-cadherin gene is frequently silenced in chronic lymphocytic leukemia (CLL) cells and results in wnt-pathway activation. We analyzed the role of histone epigenetic modifications in E-cadherin gene silencing. METHODS: CLL specimens were treated with histone deacetylase inhibitor (HDACi) MS-275 and analyzed for E-cadherin expression with western blot and RT-PCR analysis. The downstream effects of HDACi treated leukemic cells were studied by analyzing the effect on wnt-pathway signaling. HDACi induced alterations in E-cadherin splicing were investigated by transcript specific real time PCR analysis. RESULTS: Treatment of CLL specimens with histone deacetylase inhibitors (HDACi) treatment resulted in an increase of the E-cadherin RNA transcript (5 to 119 fold increase, n=10) in eight out of ten CLL specimens indicating that this gene is down regulated by histone hypoacetylation in a majority of CLL specimens. The E-cadherin re-expression in CLL specimens was noted by western blot analysis as well. Besides epigenetic silencing another mechanism of E-cadherin inactivation is aberrant exon 11 splicing resulting in an alternatively spliced transcript that lacks exon 11 and is degraded by the non-sense mediated decay (NMD) pathway. Our chromatin immunoprecipitation experiments show that HDACi increased the acetylation of histones H3 and H4 in the E-cadherin promoter region. This also affected the E-cadherin exon 11 splicing pattern as HDACi treated CLL specimens preferentially expressed the correctly spliced transcript and not the exon 11 skipped aberrant transcript. The re-expressed E- cadherin binds to ß-catenin with inhibition of the active wnt-beta-catenin pathway in these cells. This resulted in a down regulation of two wnt target genes, LEF and cyclinD1 and the wnt pathway reporter. CONCLUSION: The E-cadherin gene is epigenetically modified and hypoacetylated in CLL leukemic cells. Treatment of CLL specimens with HDACi MS-275 activates transcription from this silent gene with expression of more correctly spliced E-cadherin transcripts as compared to the aberrant exon11 skipped transcripts that in turn inhibits the wnt signaling pathway. The data highlights the role of epigenetic modifications in altering gene splicing patterns.