Novel mRNA isoforms and mutations of uridine monophosphate synthetase and 5-fluorouracil resistance in colorectal cancer.

The drug fluorouracil (5-FU) is a widely used antimetabolite chemotherapy in the treatment of colorectal cancer. The gene uridine monophosphate synthetase (UMPS) is thought to be primarily responsible for conversion of 5-FU to active anticancer metabolites in tumor cells. Mutation or aberrant expression of UMPS may contribute to 5-FU resistance during treatment. We undertook a characterization of UMPS mRNA isoform expression and sequence variation in 5-FU-resistant cell lines and drug-naive or -exposed primary and metastatic tumors. We observed reciprocal differential expression of two UMPS isoforms in a colorectal cancer cell line with acquired 5-FU resistance relative to the 5-FU-sensitive cell line from which it was derived. A novel isoform arising as a consequence of exon skipping was increased in abundance in resistant cells. The underlying mechanism responsible for this shift in isoform expression was determined to be a heterozygous splice site mutation acquired in the resistant cell line. We developed sequencing and expression assays to specifically detect alternative UMPS isoforms and used these to determine that UMPS was recurrently disrupted by mutations and aberrant splicing in additional 5-FU-resistant colorectal cancer cell lines and colorectal tumors. The observed mutations, aberrant splicing and downregulation of UMPS represent novel mechanisms for acquired 5-FU resistance in colorectal cancer.

SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2'deoxycytidine to increase SPARC expression and improve therapy response.

Poor clinical outcomes in cancer can often be attributed to inadequate response to chemotherapy. Strategies to overcome either primary or acquired chemoresistance may ultimately impact on patients' survival favourably. We previously showed that lower levels of SPARC were associated with therapy-refractory colorectal cancers (CRC), and that upregulating its expression enhances chemo-sensitivity resulting in greater tumour regression in vivo. Here, we examined aberrant hypermethylation of the SPARC promoter as a potential mechanism for repressing SPARC in CRCs and whether restoration of its expression with a demethylating agent 5-Aza-2'deoxycytidine (5-Aza) could enhance chemosensitivity. Initially, the methylation status of the SPARC promoter from primary human CRCs were assessed following isolation of genomic DNA from laser capture microdissected specimens by direct DNA sequencing. MIP101, RKO, HCT 116, and HT-29 CRC cell lines were also used to evaluate the effect of 5-Aza on: SPARC promoter methylation, SPARC expression, the interaction between DNMT1 and the SPARC promoter (ChIP assay), cell viability, apoptosis, and cell proliferation. Our results revealed global hypermethylation of the SPARC promoter in CRCs, and identified specific CpG sites that were consistently methylated in CRCs but not in normal colon. We also demonstrate that SPARC repression in CRC cell lines could be reversed following exposure to 5-Aza, which resulted in increased SPARC expression, leading to a significant reduction in cell viability (by an additional 39% in RKO cells) and greater apoptosis (an additional 18% in RKO cells), when combined with 5-FU in vitro (in comparison to 5-FU alone). Our exciting findings suggest potential diagnostic markers of CRCs based on specific methylated CpG sites. Moreover, the results reveal the therapeutic utility of employing demethylating agents to improve response through augmentation of SPARC expression.

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-lysine-alginate 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.

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.

Free versus microencapsulated pancreatic islet xenografts producing amelioration of streptozotocin toxicity.

This study examines the effect of pancreatic islet transplants on the streptozotocin(STZ)-associated toxicity in diabetic animals. Mice with STZ-induced diabetes were implanted with microencapsulated or free rat islets. The effectiveness of the transplant was evaluated in terms of: (A) blood glucose monitoring, (B) determination of subset levels of the helper and cytotoxic T-lymphocytes, and (C) STZ-associated mortality. The experimental results demonstrate that the transplanted islets can quickly restore normoglycemia. The restoration of normal blood glucose levels is accompanied by a significant increase in proportions of helper and cytotoxic T-cells. There was no mortality in the transplant recipients as a result of the STZ administrations, whereas a significant mortality was observed in the control group of mice. No significant differences between the encapsulated and free islet transplant recipients were observed.

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 less than 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.

Xenotransplantation of microencapsulated fetal rat islets.

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.

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 less than 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.