Germline thymidylate synthase deficiency impacts nucleotide metabolism and causes dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone-marrow-failure disorder characterized by a triad of mucocutaneous features that include abnormal skin pigmentation, nail dystrophy, and oral leucoplakia. Despite the identification of several genetic variants that cause DC, a significant proportion of probands remain without a molecular diagnosis. In a cohort of eight independent DC-affected families, we have identified a remarkable series of heterozygous germline variants in the gene encoding thymidylate synthase (TYMS). Although the inheritance appeared to be autosomal recessive, one parent in each family had a wild-type TYMS coding sequence. Targeted genomic sequencing identified a specific haplotype and rare variants in the naturally occurring TYMS antisense regulator ENOSF1 (enolase super family 1) inherited from the other parent. Lymphoblastoid cells from affected probands have severe TYMS deficiency, altered cellular deoxyribonucleotide triphosphate pools, and hypersensitivity to the TYMS-specific inhibitor 5-fluorouracil. These defects in the nucleotide metabolism pathway resulted in genotoxic stress, defective transcription, and abnormal telomere maintenance. Gene-rescue studies in cells from affected probands revealed that post-transcriptional epistatic silencing of TYMS is occurring via elevated ENOSF1. These cell and molecular abnormalities generated by the combination of germline digenic variants at the TYMS-ENOSF1 locus represent a unique pathogenetic pathway for DC causation in these affected individuals, whereas the parents who are carriers of either of these variants in a singular fashion remain unaffected.

Thymidine-Dependent Staphylococcus aureus Small-Colony Variants Are Induced by Trimethoprim-Sulfamethoxazole (SXT) and Have Increased Fitness during SXT Challenge.

Trimethoprim-sulfamethoxazole (SXT) is a possible alternative for the treatment of community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) due to the susceptibility of most MRSA strains to the drug. However, after long-term treatment with SXT, thymidine-dependent (TD) SXT-resistant small-colony variants (SCVs) emerge. In TD-SCVs, mutations of thymidylate synthase ([TS] thyA) occur. Until now, it has never been systematically investigated that SXT is triggering the induction and/or selection of TD-SCVs. In our study, we performed induction, reversion, and competition experiments in vitro and in vivo using a chronic mouse pneumonia model to determine the impact of SXT on the emergence of TD-SCVs. SCVs were characterized by light and transmission electron microscopy (TEM) and auxotrophism testing. Short-term exposure of S. aureus to SXT induced the TD-SCV phenotype in S. aureus SH1000, while selection of TD-SCVs with thyA mutations occurred after long-term exposure. In reversion experiments with clinical and laboratory TD-SCVs, all revertants carried compensating mutations at the initially identified mutation site. Competition experiments in vitro and in vivo revealed a survival and growth advantage of the ΔthyA mutant under low-thymidine availability and SXT exposure although this advantage was less profound in vivo. Our results show that SXT induces the TD-SCV phenotype after short-term exposure, while long-term exposure selects for thyA mutations, which provide an advantage for TD-SCVs under specified conditions. Thus, our results further an understanding of the dynamic processes occurring during SXT exposure with induction and selection of S. aureus TD-SCVs.

Identification of thymidylate synthase as a potential therapeutic target for lung cancer.

BACKGROUND: Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is an important chemotherapeutic target for malignant tumours including lung cancer. Although inhibition of TS has an antiproliferative effect in cancer cells, the precise mechanism of this effect has remained unclear. METHODS: We examined the effects of TS inhibition with an RNA interference-based approach. The effect of TS depletion on the growth of lung cancer cells was examined using colorimetric assay and flow cytometry. RESULTS: Measurement of the enzymatic activity of TS in 30 human lung cancer cell lines revealed that such activity differs among tumour histotypes. Almost complete elimination of TS activity by RNA interference resulted in inhibition of cell proliferation in all tested cell lines, suggestive of a pivotal role for TS in cell proliferation independent of the original level of enzyme activity. The antiproliferative effect of TS depletion was accompanied by arrest of cells in S phase of the cell cycle and the induction of caspase-dependent apoptosis as well as by changes in the expression levels of cyclin E and c-Myc. Moreover, TS depletion induced downregulation of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP), and it seemed to activate the mitochondrial pathway of apoptosis. CONCLUSION: Our data provide insight into the biological relevance of TS as well as a basis for clinical development of TS-targeted therapy for lung cancer.

Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans.

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.

Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10.

Genetically modified Lactococcus lactis secreting interleukin 10 provides a therapeutic approach for inflammatory bowel disease. However, the release of such genetically modified organisms through clinical use raises safety concerns. In an effort to address this problem, we replaced the thymidylate synthase gene thyA of L. lactis with a synthetic human IL10 gene. This thyA- hIL10+ L. lactis strain produced human IL-10 (hIL-10), and when deprived of thymidine or thymine, its viability dropped by several orders of magnitude, essentially preventing its accumulation in the environment. The biological containment system and the bacterium's capacity to secrete hIL-10 were validated in vivo in pigs. Our approach is a promising one for transgene containment because, in the unlikely event that the engineered L. lactis strain acquired an intact thyA gene from a donor such as L. lactis subsp. cremoris, the transgene would be eliminated from the genome.

Chromosome breakage induced by thymidylate stress in thymidylate synthase-negative mutants of mouse FM3A cells.

In thymidylate synthase-negative mutants of mouse FM3A cells, thymidine starvation rapidly decreased mitotic activity and resulted in cell death (thymineless death). When the thymidine starvation was reversed by an addition of thymidine, mitotic activity was recovered, but the majority of mitotic cells exhibited extensive chromosome aberrations, including chromatid breaks, chromatid exchanges, and pulverizations. Autoradiographic examination revealed that chromosome instability was induced only in cells arrested in the S phase during thymidine starvation. Furthermore, the most sensitive sites to the chromosome-damaging effect appeared to be sites which had replicated just prior to thymidine starvation. During thymidine starvation, cells at other stages in the cell cycle were accumulated at the G1-S boundary, and they were insensitive to the chromosome-damaging effect. Thymidine starvation was also found to be recombinagenic. Complete removal from the medium of a thymidine analogue, 5-bromo-2'-deoxyuridine, resulted in a dramatic increase in the frequency of sister chromatid exchanges. These results support the view that thymidine starvation in mammalian cells results in thymineless death via induction of DNA double-strand breaks, leading to chromosome fragmentation as well as rearrangements in the cells synthesizing DNA.

Cell cycle control processes determine cytostasis or cytotoxicity in thymineless death of colon cancer cells.

Thymidylate synthase (TS) is a target of critical importance to the survival of human colon cancer cells since, upon inhibition, cells subsequently undergo thymineless death induced by dTTP deficiency. Using genetically marked mutants deficient in TS (TS-) and a derived population (Thy4) that is resistant to commitment to thymineless death, resistance was conferred by the ability of cells to arrest at a point either in late G1 or at the onset of S induced by dThd deprivation. Thus, Thy4 cells initially synchronized in G0 by leucine deprivation and released in the absence of dThd remained viable at 5 days, demonstrated delayed onset of nucleosomal ladder formation, and retained clonogenic potential (cytostatic response). In contrast, TS- and asynchronous Thy4 cells lost 50% clonogenic potential in 65 h and > 90% in 5 days (cytotoxic response). [3H]DNA precursor studies indicated failure of synchronized Thy4 but not TS- cells to progress through S, with arrest of Thy4 close to the G1/S boundary. Cell cycle control processes including: (a) the locus of dThd deprivation in G1; and (b) a potential checkpoint close to the G1/S border, may dictate whether consequences of dThd or dTTP restriction become cytostatic or cytotoxic.

Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization.

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.

Acute and delayed apoptosis induced by thymidine deprivation correlates with expression of p53 and p53-regulated genes in colon carcinoma cells.

The endogenous expression of p53 and p53-regulated genes has been examined in a thymidylate synthase-deficient colon carcinoma cell line (TS-) and a derived mutant clone (Thy4) that exhibit acute or delayed apoptotic responses, respectively, when released from G0 synchrony under conditions of dThd starvation. These cell clones demonstrate heterozygosity in p53, thereby expressing one wt allele and one with an A-->C point mutation at codon 240. Following release from G0, upregulated expression of both alleles occurred. During apoptosis in TS-, a wtp53 phenotype was expressed and in Thy4 during cytostasis, a mp53 phenotype was manifested, as determined from the ratios of wtp53/mp53 proteins, transactivation of p50-2 (a wtp53-responsive CAT reporter construct) and the endogenous expression of MDM2. Neither cytotoxicity nor cytostasis correlated with expression of p21Waf1/Cip1 Thy4 cells sustained accumulation of high levels of Bax in a wtp53-independent and dThd-independent manner and survival was associated with upregulated expression of Bcl-2. In contrast, Bax expression decreased in TS- during apoptosis, except in a highly resistant subpopulation that retained high levels of Bax. Data suggest that resistant cells (Thy4) can sustain high Bax expression and that Bcl-2 is upregulated in response to an apoptotic stimulus due to the absence of negative regulation by wtp53.

Genetic hybridization at the unlinked thy and str loci of Streptococcus.

The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci.

The fas signaling pathway is functional in colon carcinoma cells and induces apoptosis.

Fas is expressed in colonic epithelial cells and is also expressed in colon carcinomas, although its functional significance in the regulation of apoptosis in cells outside of the immune system remains unknown. In this study, we determined the role of Fas signaling on cellular growth of cultured colon carcinoma cells and demonstrated apoptosis induced by a cytotoxic anti-Fas monoclonal antibody (CH-11) in cells of the GC3/c1 lineage (GC3/c1, TS-, Thy4) but not in HCT116 or CaCo2 cells. Growth inhibition was detected at concentrations of CH-11 as low as 1 ng/ml, and clonogenic survival studies yielded IC50 values of 3-26 ng/ml. Cytotoxicity was inhibited by ZB4, a monoclonal antibody inhibitory to Fas signaling. In addition, the survival factor Bcl-2, which has demonstrated inconsistent protective effects against Fas signaling in other systems, was inhibitory to Fas-induced apoptosis in colon carcinoma cells after adenoviral transduction. Fas was expressed at the highest levels in TS- and Thy4 cells, which were the most sensitive cell lines to Fas-induced apoptosis. FAP-1, a protein tyrosine phosphatase that interacts with the cytosolic negative regulatory domain of Fas, was expressed in each cell line but did not correlate with sensitivity to Fas-mediated apoptosis. These data have therefore identified a functional Fas pathway in colon carcinoma cells when Fas is expressed at high levels. Hence, the role of Fas signaling in the regulation of apoptosis in colon carcinoma cells and its role in influencing the response to treatment with chemotherapeutic agents should be further explored.

Cellular pharmacology and in vivo activity of a new anticancer agent, ZD9331: a water-soluble, nonpolyglutamatable, quinazoline-based inhibitor of thymidylate synthase.

ZD9331 is a drug that was developed from a potent class of water-soluble, C7-methyl-substituted, quinazoline-based inhibitors of thymidylate synthase (TS) that are transported into cells via a saturable, carrier-mediated system (reduced folate carrier, or RFC) but are not substrates for folylpolyglutamate synthetase. ZD9331 is the gamma-tetrazole analogue of 2-desamino-2, 7-dimethyl-N10-propargyl-2'fluoro-5,8-dideaza folate (ZM214888), with a TS Ki of approximately 0.4 nM. ZD9331 exhibits potent growth inhibitory and cytotoxic activity; e.g., IC50 for the inhibition of human W1L2 lymphoblastoid cell line was 7 nM. The addition of thymidine to the culture medium increased the IC50 in W1L2 cells >10, 000-fold, demonstrating the high specificity of the drug for TS. ZD9331 is transported into cells predominantly via the RFC. Accordingly, it competes with methotrexate (MTX) and folinic acid for cellular uptake and has reduced activity against two cell lines with low expression of the RFC (L1210:1565 and CEM/MTX). In addition, a cell line with acquired resistance to ZD9331 displays reduced uptake of both ZD9331 and MTX. A mouse cell line (L1210:RD1694), with acquired resistance to ZD1694 due to reduced folylpolyglutamate synthetase activity, was not significantly cross-resistant to ZD9331. The flux through TS, as measured by 3H release from 5-[3H]deoxyuridine, was rapidly inhibited when cells were incubated with ZD9331. However, because ZD9331 cannot form polyglutamates, TS activity recovered rapidly once cells were placed in drug-free medium. The minimum curative dose of ZD9331 in the i.m. L5178Y TK-/- tumor model was approximately 3 mg/kg when given by 24-h continuous infusion, and it was 25-50 mg/kg when given by a single i.p. or i.v. injection. ZD9331 had antitumor activity against the L5178Y TK+/- tumor when administered by 7-day continuous infusion; growth delays of more than 5 days (and some cures) were seen at doses of 25-50 mg/kg/day. At higher doses, significant weight loss (gastrointestinal toxicity) and myelosuppression (neutropenia and thrombocytopenia) were observed, suggesting that these may be dose-limiting toxicities in the Phase I clinical studies.

Thymidylate synthetase-positive and -negative murine mammary FM3A carcinoma cells as a useful system for detecting thymidylate synthetase inhibitors.

The murine mammary FM3A/O and the thymidylate (dTMP) synthetase-deficient FM3A/TS- carcinoma cell lines can be considered as a novel and useful test system for the detection of nucleoside analogues which are directly aimed at the thymidylate synthetase. These compounds should be inhibitory for FM3A/O but not for FM3A/TS- cells, and their inhibitory effects on FM3A/O cell growth should be readily reversed by exogenous dThd within the concentration range of 5-20 microM.

Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line.

Following mutagenesis of the human colorectal tumor cell line HCT C with ethyl methanesulfonate, clonal sublines were isolated that survived on medium toxic to cells expressing thymidylate synthase (TS). The subline exhibiting the lowest TS activity, designated as C18, was characterized. Extracts from C18 cells were mixed with extracts from parental C cells to determine whether the TS-deficient phenotype is trans-acting. No effect was observed on the activity of TS in parental extracts. The levels of functional TS in C18 cells were analyzed by the binding of the mechanism-based inhibitor 5-fluoro-2'-deoxyuridylate (FdUMP) under conditions that allowed for the detection of 10 fmol of TS. Only a low level of FdUMP-TS complexes was detected in C18 extracts. The level of TS expression in C18 cells was similar to that in parental C cells, as indicated by immunoblot and RNA analyses. DNA sequence analysis of TS cDNA from C18 cells revealed the existence of a point mutation (C-->T) at nucleotide 647 that predicts the replacement of Ser216 by a leucine residue. That the C18 cell line was homozygous for this mutation was indicated by restriction fragment-length polymorphism analysis and by primer extension analysis. To provide additional evidence that substitution of Ser216 by a leucine residue created a defective protein, a TS-deficient bacterial strain was transformed with an expression vector containing the mutated human TS cDNA. The transformed strain exhibited thymidine auxotrophy, indicating that the mutant TS (Leu216) is nonfunctional.

Caffeine enhances fragile (X) expression in somatic cell hybrids.

Yunis and Soreng recently demonstrated enhanced expression of common fragile sites as and of fra(X) when 2.2 mM caffeine is added to FUdR treated lymphocyte cultures 6 hours before harvest. We failed to replicate this finding for fra(X) expression in lymphocytes. However, we do find a consistent increase in expression levels in somatic cell hybrids between a Chinese hamster cell line and 3 unrelated individuals with the fra(X) mutation when caffeine is present for the last 2 hours before harvest. This was particularly true for "low-expressing" cell lines, in which a 4-6 fold increase was observed. Using a thymidylate synthase deficient hybrid which could be blocked in the S phase by thymidine deprivation, we found that caffeine significantly reduced the recovery time from thymidine release to mitosis. This produced the highest level of fra(X) expression (48%) only one hour after release from thymidine deprivation. These results show that caffeine does enhance fra(X) expression in at least some cell types. The effect is probably indirect, inhibiting the mitotic delay usually associated with DNA damage.

Molecular cloning of genomic DNA segments partially coding for human thymidylate synthase from the mouse cell transformant.

Mouse cells deficient in the enzyme thymidylate synthase [TS; EC 2.1.1.45] were serially transformed with human DNA to yield primary and secondary transformants which produced human TS [Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., & Seno, T. (1983) J. Biol. Chem. 258, 48-53]. Southern blot hybridization of their genomic DNA showed that six secondary transformants examined contained in common a 5.5 kb EcoRI fragment hybridized with a human Alu sequence. From the secondary transformant genomic library constructed with phage lambda Charon 4A, two recombinant phage clones carrying Alu sequences were isolated. Restriction endonuclease mapping revealed that the insert DNAs of the two phage clones overlapped and covered a region of 19 kb in total. Within this region at least six Alu sequences were located. A 2.0 kb DNA fragment, prepared from an EcoRI fragment subcloned in plasmid pBR322 and free of Alu sequences, hybridized to a single band on RNA blots of primary and secondary transformant poly(A)+ RNA, but not to RNA of mouse wild-type and recipient cell lines. The relative amount of the presumed human TS mRNA was linearly correlated with the relative activity of human TS in various types of mouse transformant cells. These results indicate that these two phage clones contain genomic DNA sequences encoding human TS.

Incorporation of 5-substituted pyrimidine nucleoside analogues into DNA of a thymidylate synthetase-deficient murine FM3A carcinoma cell line.

Various 5-substituted 2'-deoxyuridine (dUrd), 2'-deoxycytidine (dCyd), 1-beta-D-arabinofuranosyluracil (araU) and 1-beta-D-arabinofuranosylcytosine (araC) analogues have been investigated for their stimulatory effect on the growth of a thymidylate (dTMP) synthetase-deficient murine mammary carcinoma cell line (FM3A/TS-) that is auxotrophic for thymidine (dThd). Such stimulatory effect may be considered as indicative for the incorporation of the nucleoside analogue into host cell DNA. Based on this premise, several dUrd analogues were found to be incorporated into FM3A/TS- cell DNA (in decreasing order of incorporation): 5-bromo-dUrd greater than 5-chloro-dUrd greater than 5-(3-hydroxy-1-propynyl)-dUrd greater greater than 5-(1-pentynyl)-dUrd approximately 5-(1-propynyl)-dUrd approximately 5-iodo-dUrd greater than 5-(5-carboxy-1-hexenyl)-dUrd greater than 5-(3,3-dimethyl-1-butynyl)-dUrd greater than 5-ethyl-dUrd greater than 5-(5-chloro-1-pentynyl)-dUrd greater than 5-ethynyl-dUrd approximately 5 vinyl-dUrd greater than 5-phenylethynyl-dUrd greater than 5-(5-cyano-1-pentenyl)-dUrd greater than 5-(1-propenyl)-dUrd greater than 5-(1-hexynyl)-dUrd greater than 5-(5-hexyn-1-enyl)-dUrd. Among the 5-substituted dCyd analogues, 5-methyl-dCyd, 5-chloro-dCyd, 5-bromo-dCyd and 5-iodo-dCyd were also found to stimulate cell growth, and are therefore assumed to be incorporated into FM3A/TS- cell DNA. Since the stimulatory effects of these compounds on FM3A/TS- cell proliferation were suppressed in the presence of a Cyd deaminase inhibitor (tetrahydrouridine) or dCMP deaminase inhibitor (2'-deoxytetrahydrouridine), it is surmised that the dCyd analogues are first deaminated to the corresponding dUrd analogues before they are incorporated into DNA. None of the 5-substituted araU or araC analogues tested were able to sustain the growth of FM3A/TS- cells. It is postulated, therefore, that these araU or araC analogues are not incorporated to any appreciable extent into the DNA of FM3A/TS- cells, or, if they are incorporated, prevent cell growth. Thus, the dTMP synthetase-deficient FM3A/TS- cell line represents a unique system to distinguish those pyrimidine nucleoside analogues that are able to sustain cell growth and, therefore, assumed to be incorporated into the host cell DNA from those pyrimidine nucleoside analogues that are not.

Two-tiered biological containment strategy for Lactococcus lactis-based vaccine or immunotherapy vectors.

The concept of biological containment was developed as a strategy to prevent environmental dissemination of engineered live vaccine or drug delivery vehicles. A mutation in the gene encoding thymidylate synthase (thyA), a key enzyme in the pyrimidine biosynthetic pathway, has previously been shown to limit growth of L. lactis vectors under restrictive conditions. We hypothesized that further mutations in the pyrimidine biosynthetic pathway might enhance the stability and safety of live L. lactis vectors. We show that a double mutation in the genes encoding ThyA and CTP synthase (PyrG) in L. lactis confers double auxotrophy for both thymidine and cytidine. However, the combination of two mutations failed to enhance the biological containment phenotype of the engineered strain. In the absence of thymine/thymidine, the thyA mutant exhibited a strong bactericidal phenotype. However, creation of the double mutant caused the loss of this phenotype, though survival in the mouse GI tract was enhanced. The implications for biological containment of live L. lactis based delivery vectors are discussed.