The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine.

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.

Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Transgenic mice expressing the Sh ble bleomycin resistance gene are protected against bleomycin-induced pulmonary fibrosis.

Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fibrotic response of the lung is a major dose-limiting side effect. Here, we explore the possibility of a direct inhibition of lung tissue injury by in vivo expression of the actinomycetes BLM resistance protein Sh ble. Transgenic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3 ES cells. The protein was detected at high level in lungs, spleen, and kidney. We then assessed its ability to modulate the BLM-induced fibrotic response in the transgenic mice in comparison with C57BL/6 and 129/Sv parental mice. Cumulative doses of 300, 400, or 500 mg/kg BLM were administered either by i.p. or s.c. repeated injections in the different strains. Transgenic mice were shown to be clearly less sensitive to BLM toxicity, as assessed by lung histology. The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mice. These observations are consistent with the hypothesis that a resistance gene specifically expressed in lungs may prevent the BLM-induced inflammation.

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

CL307-24, a new antibiotic complex from Saccharopolyspora aurantiaca sp. nov. II. Physico-chemical and biological properties.

CL307-24I, the main component of the CL307-24 complex produced by Saccharopolyspora aurantiaca sp. nov., was found to be a potent inhibitor of yeast mitochondrial ATPase. CL307-24I displayed a high degree of activity towards some coryneform bacteria and also has been shown to possess an insecticidal activity. Its biological and physico-chemical properties clearly distinguish it from previously known ATPase inhibitors.

Genetic improvements of an industrial strain of Aspergillus flavus for urate oxidase production.

Urate oxidase, an enzyme used in human therapy, is currently produced industrially by a strain of Aspergillus flavus. Two strategies of strain improvement were tested in order to obtain higher yields of urate oxidase. The first one, based on a classical mutation-selection protocol, led to the isolation of a mutant strain that overproduced uricase two-fold as compared to the industrial strain. The second one consisted in the construction of transformed strains that had integrated multiple copies of a urate oxidase-expression vector. A twenty-fold improvement in urate oxidase was obtained by this method.

A screening method for antifungal substances using Saccharomyces cerevisiae strains resistant to polyene macrolides.

Strains of Saccharomyces cerevisiae FL200 capable of growing on a solid medium containing a mixture of polyene macrolide antibiotics (nystatin, 40 micrograms/ml, amphotericin B, 40 micrograms/ml, pimaricin, 150 micrograms/ml and RP9971 antibiotic, 10 micrograms/ml) have been isolated after successive selection steps. The mutant strains, PR13 and PRC24, are 10 to 100 times more resistant than the polyene macrolide antibiotics. When 4% Tween 80 is added to the medium, resistance to these antifungal drugs is further increased. In addition, strain PRC24, derived from strain PR13, is resistant to a non-polyene macrolide antifungal antibiotic, cycloheximide. In contrast, PR13 and PRC24 are both highly susceptible to a large range of compounds, including non-polyenic antifungal, antitumor and antibacterial agents. These particular characteristics make these strains useful for the rapid detection of antifungal compounds of the polyene macrolide and cycloheximide types, as well as for the recognition of antimitotic substances.

Phleomycin resistance as a dominant selectable marker for plant cell transformation.

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.