Isabella T. Tai

Alternative expression analysis by RNA sequencing

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription–PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

Reversal of Diabetes in BB Rats by Transplantation of Encapsulated Pancreatic Islets

Prolonged survival of pancreatic islet allografts implanted in diabetic BB rats was achieved by encapsulation of individual islets in a protective biocompatible alginate-polylysine-alginate membrane without immunosuppression. Intraperitoneal transplantation of the encapsulated islets reversed the diabetic state of the recipients within 3 days and maintained normoglycemia for 190 days. Normal body weight and urine volume were maintained during this period, and no cataracts were detected in the transplant recipients. In contrast, control rats receiving transplants of unencapsulated islets experienced normoglycemia for <2 wk. These results demonstrated that microencapsulation can protect allografted islets from both graft rejection and autoimmune destruction without immunosuppression in an animal model that mimics human insulin-dependent diabetes.

SPARC in cancer biology: Its role in cancer progression and potential for therapy

The ability to effectively target a tumor to achieve complete regression and cure is the ultimate goal that drives our need to better understand tumor biology. Recently, SPARC has generated considerable interest as a multi-faceted protein that belongs to a family of matricellular proteins. It functions not only to modulate cell-cell and cell-matrix interactions, but its de-adhesive and growth inhibitory properties in non-transformed cells have led to studies to assess its role in cancer. Its divergent actions reflect the complexity of this protein, because in certain types of cancers, such as melanomas and gliomas, SPARC is associated with a highly aggressive tumor phenotype, while in others, mainly ovarian, neuroblastomas and colorectal cancers, SPARC may function as a tumor suppressor. Recent studies have also demonstrated a role for SPARC in sensitizing therapy-resistant cancers. Here, the role of SPARC in cancer progression and its potential application in cancer therapy is discussed.

Genome-wide expression analysis of therapy-resistant tumors reveals SPARC as a novel target for cancer therapy

Overcoming resistance to chemotherapy and radiation therapy has been a difficult but important goal in the effort to cure cancer. We used gene-expression microarrays to identify differentially expressed genes involved in colorectal cancer resistance to chemotherapy and identified secreted protein, acidic and rich in cysteine (osteonectin) (SPARC) as a putative resistance-reversal gene by demonstrating low SPARC expression in refractory human MIP101 colon cancer cells. We were able to achieve restoration of their radiosensitivity and sensitivity to 5-fluorouracil and irinotecan by reexpression of SPARC in tumor xenografts. Moreover, treatment of mice with SPARC conferred increased sensitivity to chemotherapy and led to significant regression of xenografted tumors. The results show that modulation of SPARC expression affects colorectal cancer sensitivity to radiation and chemotherapy. SPARC-based gene or protein therapy may ameliorate the emergence of resistant clones and eradicate existing refractory clones and offers a novel approach to treating cancer.

Meta-analysis of Colorectal Cancer Gene Expression Profiling Studies Identifies Consistently Reported Candidate Biomarkers

Purpose: Elucidation of candidate colorectal cancer biomarkers often begins by comparing the expression profiles of cancerous and normal tissue by performing gene expression profiling. Although many such studies have been done, the resulting lists of differentially expressed genes tend to be inconsistent with each other, suggesting that there are some false positives and false negatives. One solution is to take the intersection of the lists from independent studies. However, often times, the statistical significance of the observed intersection are not assessed. Methods: Recently, we developed a meta-analysis method that ranked differentially expressed genes in thyroid cancer based on the intersection among studies, total sample sizes, average fold change, and direction of differential expression. We applied an improved version of the method to 25 independent colorectal cancer profiling studies that compared cancer versus normal, adenoma versus normal, and cancer versus adenoma to highlight genes that were consistently reported as differentially expressed at a statistically significant frequency. Results: We observed that some genes were consistently reported as differentially expressed with a statistically significant frequency (P < 0.05) in cancer versus normal and adenoma versus normal comparisons but not in the cancer versus adenoma comparison. Conclusion: Our meta-analysis method identified genes that were consistently reported as differentially expressed. A review of some of the candidates revealed genes described previously as having diagnostic and/or prognostic value as well as novel candidate biomarkers. The genes presented here will aid in the identification of highly sensitive and specific biomarkers in colorectal cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(3):543–52)

Prolonged Reversal of Diabetic State in NOD Mice by Xenografts of Microencapsulated Rat Islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25–0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 ± 1.4 to 4.0 ± 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for <10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.

Microencapsulation of recombinant cells: a new delivery system for gene therapy.

The therapeutic potential of somatic gene therapy has been extensively investigated in recent years, yet its slow progression into the clinical setting can be attributed to problems associated with the inability to achieve efficient gene transfers, to obtain sustained level of expression of the transfected gene, and the necessity to avoid immunorejection after transplantation. Here we report on an alternate strategy in gene therapy that overcomes all three problems by immunoisolating genetically modified cells in a biocompatible membrane, thereby introducing a system that can provide sustained delivery of the desired gene product. As a model, mouse fibroblasts transformed with the human growth hormone gene (Ltk‐GH) were encapsulated with an alginate‐poly‐L‐lysinealginate membrane. Long‐term in vitro studies showed that the encapsulation of the cells was physiologically compatible with growth and survival of the cells. Furthermore, there was a unique pattern of secretion of the human protein by the encapsulated cells: there was a phase of steady increase in the secretion of the human growth hormone by each cell, followed by a plateau phase. The most convincing evidence of the feasibility of this strategy was provided by the in vivo study: Balb‐c mice transplanted with encapsulated Ltk‐GH cells had detectable serum levels of human growth hormone (hGH) for the duration of the study (115 days). Moreover, encapsulated cells recovered from a recipient 1 year after the transplantation continued to secrete high levels of hGH in culture.—Tai, I. T., Sun, A. M. Microencapsulation of recombinant cells: a new delivery system for gene therapy. FASEB J. 7: 1061‐1069; 1993.

Xenografts of rat islets into diabetic mice : an evaluation of new smaller capsules

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.

A Novel Interaction between Procaspase 8 and SPARC Enhances Apoptosis and Potentiates Chemotherapy Sensitivity in Colorectal Cancers

Chemotherapy resistance accounts for the high mortality rates in patients with advanced cancers. We previously used a genomics approach to determine novel genes associated with this phenomenon and identified secreted protein acidic and rich in cysteine (SPARC) as a chemosensitizer capable of reversing therapy resistance in colorectal cancer cells by enhancing apoptosis in vitro and tumor regression in vivo. Here, we examined the mechanisms by which SPARC enhances apoptosis in the presence of chemotherapy. We show that SPARC potentiates apoptosis by augmenting the signaling cascade in a caspase-8-dependent manner, because apoptosis can be abolished by caspase 8 small interfering RNA in the presence of SPARC. This occurs independently of death receptor activation and leads to downstream involvement of Bid and subsequent apoptosis. Interestingly, this results from an interaction between SPARC and the N terminus of the procaspase-8 DED-containing domain. These exciting findings provide an initial map of the apoptosis signaling events mediated by SPARC and how this can ultimately result in the reversal of chemotherapy resistance and enhanced tumor regression. This signaling cascade can be exploited therapeutically and may have potential clinical implications for patients with advanced and therapy-refractory cancers.

Protective effects of methyl gallate on H2O2-induced apoptosis in PC12 cells.

Neurodegenerative disorders are a class of diseases that have been linked to apoptosis induced by elevated levels of reactive oxygen species (ROS). ROS activates the apoptotic cascade through mitochondrial dysfunction and damage to lipids, proteins and DNA. Recently, fruit and tea-derived polyphenols have been found to be beneficial in decreasing oxidative stress and increasing overall health. Further, polyphenols including epigallocatechin gallate (EGCG) have been reported to inhibit apoptotic signaling and increase neural cell survival. In an effort to better understand the beneficial properties associated with polyphenol consumption, the aim of this study was to explore the neuroprotective effects of EGCG, methyl gallate (MG), gallic acid (GA) and N-acetylcysteine (NAC) on H(2)O(2)-induced apoptosis in PC12 cells and elucidate potential protective mechanisms. Cell viability data demonstrates that MG and NAC pre-treatments significantly increase viability of H(2)O(2)-stressed cells, while pre-treatments with EGCG and GA exacerbates stress. Quantitation of apoptosis and mitochondrial membrane potential shows that MG pre-treatment prevents mitochondria depolarization, however does not inhibit apoptosis and is thus evidence that MG can inhibit mitochondria-mediated apoptosis. Subsequent analysis of DNA degradation and caspase activation reveals that MG inhibits activation of caspase 9 and has a partial inhibitory effect on DNA degradation. These findings confirm the involvement of both intrinsic and extrinsic apoptotic pathways in H(2)O(2)-induced apoptosis and suggest that MG may have potential therapeutic properties against mitochondria-mediated apoptosis.