Optimization of Thymidine Kinase-Based Safety Switch for Neural Cell Therapy.

Cell therapies based on pluripotent stem cells (PSC), have opened new therapeutic strategies for neurodegenerative diseases. However, insufficiently differentiated PSC can lead to tumor formation. Ideally, safety switch therapies should selectively kill proliferative transplant cells while preserving post-mitotic neurons. In this study, we evaluated the potential of nucleoside analogs and thymidine kinase-based suicide genes. Among tested thymidine kinase variants, the humanized SR39 (SR39h) variant rendered cells most sensitive to suicide induction. Unexpectedly, post-mitotic neurons with ubiquitous SR39h expression were killed by ganciclovir, but were spared when SR39h was expressed under the control of the cell cycle-dependent Ki67 promoter. The efficacy of six different nucleoside analogs to induce cell death was then evaluated. Penciclovir (PCV) showed the most interesting properties with an efficiency comparable to ganciclovir (GCV), but low toxicity. We tested three nucleoside analogs in vivo: at concentrations of 40 mg/kg/day, PCV and GCV prevented tumor formation, while acyclovir (ACV) did not. In summary, SR39h under the control of a cell cycle-dependent promoter appears most efficient and selective as safety switch for neural transplants. In this setting, PCV and GCV are efficient inducers of cell death. Because of its low toxicity, PCV might become a preferred alternative to GCV.

Hypoxia theranostics of a human prostate cancer xenograft and the resulting effects on the tumor microenvironment.

Hypoxia is frequently observed in human prostate cancer, and is associated with chemoresistance, radioresistance, metastasis, and castrate-resistance. Our purpose in these studies was to perform hypoxia theranostics by combining in vivo hypoxia imaging and hypoxic cancer cell targeting in a human prostate cancer xenograft. This was achieved by engineering PC3 human prostate cancer cells to express luciferase as well as a prodrug enzyme, yeast cytosine deaminase, under control of hypoxic response elements (HREs). Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to HRE in the promoter region of target gene that results in their increased transcription. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation. HRE-driven luciferase expression allowed us to detect hypoxia in vivo to time the administration of the nontoxic prodrug 5-fluorocytosine that was converted by yeast cytosine deaminase, expressed under HRE regulation, to the chemotherapy agent 5-fluorouracil to target hypoxic cells. Conversion of 5-fluorocytosine to 5-fluorouracil was detected in vivo by 19F magnetic resonance spectroscopy. Morphological and immunohistochemical staining and molecular analyses were performed to characterize tumor microenvironment changes in cancer-associated fibroblasts, cell viability, collagen 1 fiber patterns, and HIF-1α. These studies expand our understanding of the effects of eliminating hypoxic cancer cells on the tumor microenvironment and in reducing stromal cell populations such as cancer-associated fibroblasts.

Issues facing employability assessment in total and permanent disability insurance claims: A rehabilitation perspective.

BACKGROUND: Expert employment information helps life insurers to decide total and permanent disability claims. The employability assessment model was developed a decade ago by rehabilitation counselors and has not been critically examined. OBJECTIVE: This exploratory descriptive study aims to elicit key issues of employability assessment within Australian life insurance. METHODS: Ten rehabilitation advisors with knowledge of employability assessment in the total and permanent disability sector participated in a single focus group. They each nominated issues of concern about employability assessment. These issues were collated then discussed within the group. Qualitative thematic analysis was used to identify key issues. RESULTS: The predominant theme was the forensic nature of employment evidence. There were three categories of key issues. First, assessment providers- their training, qualifications, experience, and role. Second, methodology- approaches that provide most realistic information about a claimant's likelihood of work. Third, policy definitions that illustrate reliance on legal interpretation of employability. CONCLUSIONS: To withstand legal scrutiny, the credentials of providers, assessment methodology, and quality of forensic reports are key issues which need to be addressed. This foundational study will contribute to broader research on this topic, with implications particularly for rehabilitation, life insurance, and claimants.

Delayed Progression of Lung Metastases Following Delivery of a Prodrug-activating Enzyme.

BACKGROUND: Chemotherapy is an effective option to treat recurrent or metastatic cancer but its debilitating side-effects limit the dose and time of exposure. Prodrugs that can be activated locally by an activating enzyme can minimize collateral damage from chemotherapy. We previously demonstrated the efficacy of a poly-L-lysine-based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that locally converted 5-fluorocytosine (5-FC) to the chemotherapeutic agent 5-fluorouracil in MDA-MB-231 primary tumor xenografts. MATERIALS AND METHODS: Here we used a more effective variant of bCD to target metastatic red fluorescence protein expressing MDA-MB-435 cells in the lungs. We used an intravenous injection of tumor cells and monitored tumor growth in the lungs for 5 weeks by which time metastatic nodules were detected with optical imaging. The animals were then treated with the bCD-nanoplex and 5-FC. RESULTS: We observed a significant decrease in metastatic burden with a single dose of the enzyme-nanoplex and two consecutive prodrug injections. CONCLUSION: These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the enzyme can be directly coupled to imaging reporters to time prodrug administration for the detection and treatment of aggressive metastatic cancer.

Creation of zebularine-resistant human cytidine deaminase mutants to enhance the chemoprotection of hematopoietic stem cells.

Human cytidine deaminase (hCDA) is a biomedically important enzyme able to inactivate cytidine nucleoside analogs such as the antileukemic agent cytosine arabinoside (AraC) and thereby limit antineoplastic efficacy. Potent inhibitors of hCDA have been developed, e.g. zebularine, that when administered in combination with AraC enhance antineoplastic activity. Tandem hematopoietic stem cell (HSC) transplantation and combination chemotherapy (zebularine and AraC) could exhibit robust antineoplastic potency, but AraC-based chemotherapy regimens lead to pronounced myelosuppression due to relatively low hCDA activity in HSCs, and this approach could exacerbate this effect. To circumvent the pronounced myelosuppression of zebularine and AraC combination therapy while maintaining antineoplastic potency, zebularine-resistant hCDA variants could be used to gene-modify HSCs prior to transplantation. To achieve this, our approach was to isolate hCDA variants through random mutagenesis in conjunction with selection for hCDA activity and resistance to zebularine in an Escherichia coli genetic complementation system. Here, we report the identification of nine novel variants from a pool of 1.6 × 106 transformants that conferred significant zebularine resistance relative to wild-type hCDA2. Several variants revealed significantly higher Ki values toward zebularine when compared with wild-type hCDA values and, as such, are candidates for further exploration for gene-modified HSC transplantation approaches.

Development of inhibitor-directed enzyme prodrug therapy (IDEPT) for prostate cancer.

Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.

Enzymes to die for: exploiting nucleotide metabolizing enzymes for cancer gene therapy.

Suicide gene therapy is an attractive strategy to selectively destroy cancer cells while minimizing unnecessary toxicity to normal cells. Since this idea was first introduced more than two decades ago, numerous studies have been conducted and significant developments have been made to further its application for mainstream cancer therapy. Major limitations of the suicide gene therapy strategy that have hindered its clinical application include inefficient directed delivery to cancer cells and the poor prodrug activation capacity of suicide enzymes. This review is focused on efforts that have been and are currently being pursued to improve the activity of individual suicide enzymes towards their respective prodrugs with particular attention to the application of nucleotide metabolizing enzymes in suicide cancer gene therapy. A number of protein engineering strategies have been employed and our discussion here will center on the use of mutagenesis approaches to create and evaluate nucleotide metabolizing enzymes with enhanced prodrug activation capacity and increased thermostability. Several of these studies have yielded clinically important enzyme variants that are relevant for cancer gene therapy applications because their utilization can serve to maximize cancer cell killing while minimizing the prodrug dose, thereby limiting undesirable side effects.

Evaluation of a UCMK/dCK fusion enzyme for gemcitabine-mediated cytotoxicity.

While gemcitabine (2'-2'-difluoro-2'-deoxycytidine, dFdC) displays wide-ranging antineoplastic activity as a single agent, variable response rates and poor intracellular metabolism often limit its clinical efficacy. In an effort to enhance dFdC cytotoxicity and help normalize response rates, we created a bifunctional fusion enzyme that combines the enzymatic activities of deoxycytidine kinase (dCK) and uridine/cytidine monophosphate kinase (UCMK) in a single polypeptide. Our goal was to evaluate whether the created fusion could induce beneficial, functional changes toward dFdC, expedite dFdC conversion to its active antimetabolites and consequently amplify cell dFdC sensitivity. While kinetic analyses revealed the UCMK/dCK fusion enzyme to possess both native activities, the fusion rendered cells sensitive to the cytotoxic effects of dFdC at the same level as dCK expression alone. These results suggest that increased wild-type UCMK expression does not provide a significant enhancement in dFdC-mediated cytotoxicity and may warrant the implementation of studies aimed at engineering UCMK variants with improved activity toward gemcitabine monophosphate.

Validation of an isocratic HPLC method to detect 2-fluoro-beta-alanine for the analysis of dihydropyrimidine dehydrogenase activity.

The efficacy of the chemotherapeutic drug 5'-fluorouracil is reduced by catabolism to 2'-fluoro-beta-alanine (FBAL), a three-step reaction in which dihydropyrimidine dehydrogenase (DPD) catalyzes the rate-limiting step. To study in vitro DPD activity, we developed and validated an isocratic, reverse-phase HPLC method to detect and quantify FBAL without using multiple columns or radiolabeled substrates. Pre-column derivatization of FBAL was performed using o-phthalaldehyde in the presence of two sulfur donors, ethanthiol or beta-mercaptoethanol, and the resulting products assayed. Calibration curves were linear over a range of 10-200 microg/ml and the method was successfully applied to the examination of DPD activity in cultured cells.

Development and validation of a rapid and sensitive HPLC method for the quantification of 5-fluorocytosine and its metabolites.

To study the intracellular metabolism of the prodrug 5-fluorocytosine (5FC), we developed a novel reverse-phase high-performance liquid chromatography method to simultaneously detect 5FC and its four major anabolic metabolites: 5-fluorouracil, 5-fluorouridine, 5-fluorouridine-monophosphate and 5-fluoro-2'deoxyuridine-5'-monophosphate. Separation of each compound was accomplished under isocratic conditions using a C(18) column and mobile phase of formic acid-water (1 : 99 v/v). The method was validated for both accuracy and reproducibility in cell culture media. Additionally, metabolites were assessed for stability at ambient temperatures and following freeze-thaw cycles. Calibration curves were linear over a range of 1-200 microg/mL. Limit of quantification for four of the five compounds was 1 microg/mL in cell culture media (RSD < 11%). This method was successfully used to monitor intracellular conversion of 5FC to its metabolic products over a 24h period.

Bacterial cytosine deaminase mutants created by molecular engineering show improved 5-fluorocytosine-mediated cell killing in vitro and in vivo.

Cytosine deaminase is used in combination with 5-fluorocytosine as an enzyme-prodrug combination for targeted genetic cancer treatment. This approach is limited by inefficient gene delivery and poor prodrug conversion activities. Previously, we reported individual point mutations within the substrate binding pocket of bacterial cytosine deaminase (bCD) that result in marginal improvements in the ability to sensitize cells to 5-fluorocytosine (5FC). Here, we describe an expanded random mutagenesis and selection experiment that yielded enzyme variants, which provide significant improvement in prodrug sensitization. Three of these mutants were evaluated using enzyme kinetic analyses and then assayed in three cancer cell lines for 5FC sensitization, bystander effects, and formation of 5-fluorouracil metabolites. All variants displayed 18- to 19-fold shifts in substrate preference toward 5FC, a significant reduction in IC(50) values and improved bystander effect compared with wild-type bCD. In a xenograft tumor model, the best enzyme mutant was shown to prevent tumor growth at much lower doses of 5FC than is observed when tumor cells express wild-type bCD. Crystallographic analyses of this construct show the basis for improved activity toward 5FC, and also how two different mutagenesis strategies yield closely related but mutually exclusive mutations that each result in a significant alteration of enzyme specificity.

Titration of variant HSV1-tk gene expression to determine the sensitivity of 18F-FHBG PET imaging in a prostate tumor.

UNLABELLED: Because of its high selectivity and specificity for the imaging reporter probe 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk) variant sr39tk is actively being studied as a PET reporter gene. We recently demonstrated the capability of using a prostate-specific transcriptional amplification PET reporter vector, AdTSTA-sr39tk, to target prostate cancer lymph node metastasis. However, one area that warrants further study is the examination of the sensitivity of PET by determining the minimum percentage of cells expressing the sr39tk transgene needed for detection. Addressing this question could determine the sensitivity of vector-mediated sr39tk PET in cancer-targeting strategies. METHODS: DU-145, PC-3, and CWR22Rv.1 prostate cancer cell lines (a total of 1 x 10(6) cells) were studied, of which 7%, 10%, 25%, 50%, or 70% were transduced with the lentiviral vector constitutively expressing HSV1-sr39tk-IRES-enhanced green fluorescent protein (EGFP). Cells were subcutaneously implanted into the left shoulder of severe combined immunodeficient mice and evaluated. Tumor cells comparably transduced with an EGFP control vector were implanted on the right shoulder. Mice were imaged using PET with (18)F-FHBG at 8, 15, and 22 d after tumor implant. On day 23, tumors were isolated and analyzed for sr39tk transgene expression by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting, immunohistochemistry, and flow cytometry for EGFP expression. RESULTS: Results showed a linear relationship between the level of sr39tk expression and the quantity of tracer accrual in DU-145, with the minimal value for PET detection at 10%. The magnitude of tracer retention in sr39tk-expressing cells was amplified over time as the tumor grew. Protein levels in the stepwise titration increased with the percentage of sr39tk-transduced cells. CONCLUSION: The stepwise titration of prostate cancer cells transduced with the lenti-CMV-sr39tk-IRES-EGFP determined the minimum number of sr39tk-expressing tumor cells necessary to be detected by PET using the (18)F-FHBG reporter probe. Furthermore, PET signal correlated well with traditional methods of protein evaluation such as flow cytometry, quantitative RT-PCR, Western blotting, and immunohistochemistry. Unlike the traditional methods, however, the use of PET is noninvasive and will be more advantageous in clinical situations.

Mutations at serine 37 in mouse guanylate kinase confer resistance to 6-thioguanine.

Guanylate kinase (GMK) is an essential nucleoside monophosphate kinase that catalyzes the phosphorylation of guanine-monophosphate (GMP) and dGMP to yield GDP and dGDP, respectively, important precursors for nucleotide synthesis. GMK is also responsible for the activation of 6-thioguanine (6-TG), a drug widely used as chemotherapeutic agent to treat leukemia. Several mechanisms of resistance to 6-TG have been reported but a subset of drug resistant cells cannot be explained by these mechanisms. We propose that mutations in GMK could result in drug resistance. Because cells require the presence of a functional GMK for viability, mutations that arise that lead to 6-TG resistance must retain activity toward GMP. We report three amino acid substitutions at serine 37 (S37) in mouse GMK that display activity toward GMP by conferring genetic complementation to a conditional GMK-deficient Escherichia coli and in enzyme assays. When 6-TG is included in complementation studies, cells expressing wild-type GMK are sensitive whereas all S37 mutants examined are able to effectively discriminate against 6-TG and display a drug resistance phenotype. Activity of the three S37 mutant enzymes toward clinically relevant concentrations of 6-TGMP is undetectable. Mutations in GMK, therefore, represent a previously undescribed mechanism for 6-TG resistance.

Molecular chemotherapy of pancreatic cancer using novel mutant bacterial cytosine deaminase gene.

The combination of molecular chemotherapy with radiation therapy has the potential to become a powerful approach for treatment of pancreatic cancer. We have developed an adenoviral vector (AdbCD-D314A) encoding a mutant bacterial cytosine deaminase (bCD) gene, which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil. The aim of this study was to investigate AdbCD-D314A/5-FC-mediated cytotoxicity in vitro and therapeutic efficacy in vivo alone and in combination with radiation against human pancreatic cancer cells and xenografts. AdbCD-D314A/5-FC-mediated cytotoxicity alone and in combination with radiation was analyzed using crystal violet inclusion and clonogenic survival assays. CD enzyme activity was determined by measuring conversion of [3H]5-FC to [3H]5-fluorouracil after adenoviral infection of pancreatic cancer cells in vitro and pancreatic tumor xenografts by TLC. S.c. pancreatic tumor xenografts were used to evaluate the therapeutic efficacy of AdbCD-D314A/5-FC molecular chemotherapy in combination with radiation therapy. AdbCD-D314A infection resulted in increased 5-FC-mediated pancreatic cancer cell killing that correlated with significantly enhanced CD enzyme activity compared with AdbCDwt encoding wild-type of bCD. Animal studies showed significant inhibition of growth of human pancreatic tumors treated with AdbCD-D314A/5-FC in comparison with AdbCDwt/5-FC. Also, a significantly greater inhibition of growth of Panc2.03 and MIA PaCA-2 tumor xenografts was produced by the combination of AdbCD-D314A/5-FC with radiation compared with either agent alone. The results indicate that the combination of AdbCD-D314A/5-FC molecular chemotherapy with radiation therapy significantly enhanced cytotoxicity of pancreatic cancer cells in vitro and increased therapeutic efficacy against human pancreatic tumor xenografts.

Yeast cytosine deaminase mutants with increased thermostability impart sensitivity to 5-fluorocytosine.

Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil, an inhibitor of DNA synthesis and RNA function. Over 150 studies of CD-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of CD are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study, we stabilized and extended the half-life of yeast CD (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated T(m) values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in stability, as well as a more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models.

Seeking to understand telephone support for dementia caregivers.

Caregivers of persons with dementia encounter particular challenges in their roles and often experience unmet needs for information and emotional support. This article describes a qualitative descriptive study designed to explore the intervention of telephone support for such caregivers. Data were collected from both caregivers and telephone support providers. Results revealed that telephone support met four specific needs of dementia caregivers: the need for (a) information and education, (b) referral and/or assistance required to navigate through the system, (c) emotional support, and (d) caregiver support that is convenient and hassle free. Caregivers' main experience with the intervention was the sense of companionship, whereas service providers experienced mixed feelings of helplessness and an opportunity to empower caregivers.

Computational thermostabilization of an enzyme.

Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10 degrees C increase in apparent melting temperature T(m) and a 30-fold increase in half-life at 50 degrees C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.

Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy.

Cytosine deaminase (CD) is currently being used as a suicide gene for cancer gene therapy. The premise of this therapy is the preferential deamination of 5-fluorocytosine (5FC) to 5-fluorouracil by cancer cells expressing cytosine deaminase. However, a lack of efficient gene transfer to tumors combined with inefficient 5FC turnover currently limits the clinical applications of this gene therapy approach. We have used random mutagenesis to create novel bacterial cytosine deaminases that demonstrate an increased preference for 5FC over cytosine. Among the 15 mutants isolated, one conferred sensitivity to Escherichia coli in a negative selection system at a concentration of 5FC that was 10-fold lower than a sublethal dose for wild-type CD. Evaluation of individual substitutions found in this double mutant (Q102R, D314G) demonstrated that the substitution at residue D314 was solely responsible for the observed increase in sensitivity to 5FC. Additional mutagenesis at D314 resulted in the identification of two more substitutions with the ability to confer enhanced 5FC sensitivity to E.coli. Structure determinations of the three CD variants in the presence and absence of a transition state 5FC analogue provide insights to the determinants of substrate binding specificity at the 5' position of the pyrimidine ring. CD mutant D314A is a promising candidate for further gene therapy studies.