Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents.

Cell cycle progression and apoptosis are controlled by regulatory proteins, including p53, of which functional alterations are linked to carcinogenesis. Recently, malignant mesothelioma (MM), a primary tumour related to asbestos exposure, alternatively to post therapeutic radiations, has proven to be an important problem in oncogenesis. The p53 protein does not seem mutated or deleted in MM but a possible inactivation by binding to other proteins [mdm2; SV40 large T antigen (Tag)] has been suggested. The present work investigated cell cycle regulation in normal rat pleural mesothelial cells (RPMC) and in RPMC expressing Tag (RPMC-TSV40), under exposure to asbestos and radiations. In RPMC, these agents induced activation of cell cycle checkpoints located at G1/S and G2/M and/or mitosis but a lack of control at G1/S was found in RPMC-TSV40. A loss of G2/M control may account for the formation of micronuclei observed after exposure of RPMC-TSV40 to radiations. In RPMC-TSV40 the enhancement of abnormal mitoses and apoptosis after asbestos exposure, in comparison with RPMC, suggests a loss of mitotic control and a p53-independent mechanism of apoptosis. Thus Tag expression in mesothelial cells might have both adverse and beneficial effects by impairing the control of DNA integrity and enhancing apoptosis respectively.

Novel clinical strategies for the treatment of pancreatic carcinoma.

Pancreatic carcinoma ranks as the eighth most frequent type of solid tumour arising worldwide yet it represents the fourth most frequent cause of death. This discrepancy reflects the current lack of effective treatment available for the pancreatic cancer patient and highlights the urgent need for new therapeutic principles in this area. The last five years have seen an increasing number of novel approaches both in the pre-clinical area as well as in clinical trials for pancreatic cancer treatments. This review summarizes these new developments and attempts to rationalize the possibilities available for the patient at the beginning of the new millennium.

Construction of retroviral vectors for targeted delivery and expression of therapeutic genes.

Current gene therapy protocols take an ex vivo approach in which cells are removed from a patient, genetically modified and then reimplanted. However this kind of approach is both cumbersome and costly, requiring high tech facilities and is limited to cell types that can be easily cultured. The in vivo delivery of genes by retroviral vectors will greatly facilitate gene therapy protocols of the future. However before in vivo gene therapy becomes a reality a number of problems must be overcome. Ideally therapeutic genes should be delivered only to the relevant cell type and/or expressed in this cell type. Strategies are described that (I) limit therapeutic gene delivery, using pseudotyping or vectors based on retroviruses that show a restricted infection spectrum or (II) limit the expression of transferred genes by inclusion of tissue specific promoters or cis acting regulatory elements. The combination of some of these strategies should permit the construction of novel retroviral vectors that provide safe and targeted in vivo gene transfer.

Development of retroviral vectors as safe, targeted gene delivery systems.

The transfer of genes of potential therapeutic benefit is presently being attempted in the clinic to treat a number of genetic and virally induced diseases. Many of these protocols use retroviral vectors derived from murine leukemia retroviruses as gene delivery systems. Although these viral delivery systems are well suited for this purpose, a number of their characteristics, some of which are discussed here, are still troublesome. Future retroviral vectors will incorporate nonretroviral features and will be tailored to desired needs for specific uses. These vectors will be safer, more efficient, and targeted in their delivery. Further, expression of the therapeutic genes carried will be limited to the specific target cell type. Some of the recent advances that have been made towards this goal are reviewed here.

Inducible expression of p21WAF-1/CIP-1/SDI-1 from a promoter conversion retroviral vector.

Constitutive, high-level expression of the potentially therapeutic WAF-1/CIP-1/SDI-1 gene is incompatible with cell growth. A promoter conversion retroviral vector carrying the WAF-1/CIP-1/SDI-1 gene under the transcriptional control of the glucocorticoid inducible promoter of mouse mammary tumor virus was used to infect human bladder carcinoma or feline kidney cells. Reduced cell growth due to a greater proportion of cells being in the G0/G1 phase of the cell cycle was observed when WAF-1/CIP-1/SDI-1 expression was activated by addition of glucocorticoid hormone. This system demonstrates the potential long-term therapeutic use of WAF-1/CIP-1/SDI-1 delivered by retroviral vectors for inhibiting the growth of rapidly proliferating cells. Moreover, the conditional expression of genes such as WAF-1/CIP-1/SDI-1 from such retroviral vectors may facilitate analysis of their function.

A mammary-specific promoter directs expression of growth hormone not only to the mammary gland, but also to Bergman glia cells in transgenic mice.

The whey acidic protein (WAP) promoter has been previously used to target the expression of heterologous genes to the mammary glands of transgenic mice. To direct the expression of human GH (hGH) to mouse mammary glands, hGH-coding sequences have been coupled to WAP promoter sequences (WAP-hGH). Female transgenic mice carrying the WAP-hGH constructs show expression of hGH in the mammary gland, demonstrating the functionality of the transgenes. However, when other organs from these transgenic mice were examined, high level expression of hGH was unexpectedly observed in the brains of all male and female mice. Using in situ hybridization or immunohistochemistry, hGH expression from the transgene was seen to occur specifically in Bergman glia cells. In contrast, mice carrying hGH-coding sequences linked to the metallothionein promoter do not express hGH in these cells. Neither the endogenous WAP gene nor at least three other transgenes in which heterologous genes have been placed under the transcriptional control of the WAP promoter are expressed in the brain. Thus, we propose that the combination of the WAP promoter and the hGH structural gene results in a novel tissue specificity in the Bergman glia.

Changes in selected subpopulations of lymphocytes in dogs infected with Babesia canis treated with imidocarb.

OBJECTIVE: The purpose of this study was to track changes in selected subpopulations of lymphocytes in the blood of dogs infected with Babesia (B.) canis and treated with imidocarb. MATERIAL AND METHODS: The study included 16 dogs divided into two groups. The first group (n = 6) consisted of healthy control animals. Dogs of the se- cond group (n = 10) were infected with B. canis and after establishment of the diagnosis each animal received a single dose of imido- carb (5 mg/kg). Flow cytometry was used to enumerate several immune cell phenotypes. RESULTS: It was concluded that the invasion of B. canis contributes to the decreased percentage of CD3+, CD4+, CD8+, CD21+ lymphocytes in the blood of infected animals. The decreased level of tested subpopulations of lymphocytes in group 2 persisted for the entire 12-day period of the test. After the administration of imidocarb, each tested lymphocyte fraction in the blood of the dogs with babesiosis increased, but did not reach physiological values. CONCLUSION: The presented results indicate that the resolution of clinical signs associated with babesiosis may be related to the stimulation and intensity of cellular immunity, dependent on the CD4+ T cells profile. After administration of imidocarb, the parasitemia is cleared which allows the recovery of the lymphocyte populations.

Targeting of retroviral vectors for gene therapy.

Retroviral vectors are one of the most promising vehicles for the delivery of therapeutic genes in human gene therapy protocols. Retroviral-mediated gene transfer currently being used in human clinical trials is based upon ex vivo transduction of target cells. The ability to target the delivery and expression of therapeutic genes in vivo using retroviral vectors is a prerequisite for widespread and routine use in the clinic and will be of great importance for the safe and successful treatment of certain genetic disorders as well as tumors and viral infections. A number of approaches have been taken to develop retroviral vectors that are able to target particular cell types both at the level of the transduction event and at the level of expression. Using various combinations of the restrictive features reviewed in this article, it should be possible to achieve definitive targeting of genes transduced by retroviral vectors.

Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.

Continuous and sustained in vivo production of monoclonal antibodies by engineered cells might render long-term antibody-based treatments cost-effective, avoid side effects associated with infusion of massive doses of antibody, and circumvent possible antiidiotypic responses against the therapeutic agent. The FrCasE retrovirus induces a lethal neurodegeneration on infection of newborn mice. We report here that implantation of cellulose sulfate capsules containing cells secreting an ectopic monoclonal antibody neutralizing FrCasE can prevent animals from developing the disease. All treated mice showed reduced or undetectable viremia in addition to a lack of the histopathological lesions characteristic of FrCasE infection. This work paves the way for a novel gene/cell antibody-based immunotherapy of a variety of severe viral and nonviral diseases.

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype.

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.

Cell targeting by murine retroviral vectors.

Gene therapy involves the transfer of new genetic material to cells of individuals with the aim of conferring a therapeutical benefit. Theoretically, gene therapy can be used to treat a variety of life-threatening disorders, such as inherited genetic diseases, cancer, chronic viral infections as well as a number of other severe diseases which are not treatable at present. To this aim, both efficient gene delivery techniques and controlled gene expression systems are required. Engineered murine retroviruses are the most widely used vectors for stable clinical gene transfer because of their ability to integrate--along with the transgenes they are engineered to carry--into the genome of infected cells. However, this technology still suffers from a number of drawbacks and limitations. Particularly, the ability to target cells of therapeutic interest together with the controlled expression of transferred genes would improve both the efficiency and the safety of retroviral vectors. Such improvements would additionally allow the development of new animal models of human diseases as well as enable more fundamental investigations in the fields of oncogenesis and developmental biology.

Necrotic, rather than apoptotic, cell death caused by cytochrome P450-activated ifosfamide.

Feline kidney cells were transfected with a vector overexpressing cytochrome P450 2B1 (CYP2B1). Transfected cells acquired a new specific biochemical activity, which could be demonstrated by a rapid CYP2B1 detection assay and showed selective sensitivity to the antitumorigenic prodrug ifosfamide (IFO). Further, the cell-killing effect was also mediated on nonmodified cells like feline kidney cells, mouse lymphoma, and human pancreatic cells in the vicinity of the CYP2B1-expressing cells due to the diffusible nature of the activated IFO metabolites. One of these, phosphoramide mustard, causes interstrand DNA cross-linking and it has been thought that the inability to repair this damage results in apoptosis. Surprisingly, our results clearly demonstrate a necrotic mechanism of IFO-induced cell death. This may have important implications for the activation of the immune system during CYP2B1/IFO suicide gene therapy of cancer.

Combined chemotherapy of murine mammary tumors by local activation of the prodrugs ifosfamide and 5-fluorocytosine.

The success of chemotherapeutic intervention is limited because the necessary high local drug doses cannot be achieved without systemic toxicity. Application of suicide genes (SGs) and direct conversion of prodrugs (PDs) to toxic metabolites in situ by SGs may enhance the efficacy of chemotherapy. To evaluate this strategy in two murine breast cancer models, TS/A and GR, we injected cellulose sulfate capsules harboring cat kidney cells expressing the SGs cytosine deaminase and cytochrome P450 2B1 (CYP2B1) intratumorally. The PDs 5-fluorocytosine and ifosfamide were administered in 3-day intervals. The effect of in situ chemotherapy with each PD alone and the combination was analyzed over a period of 100 days. The results reveal that for TS/A tumors, the antitumoral effect mediated by CYP2B1 is more efficient than that of cytosine deaminase, whereas for GR tumors, both systems worked equally well. Furthermore, we find additive toxicity using both SG/PD systems for both TS/A and GR tumors.

A transient assay for gene expression studies in B lymphocytes and its use for superantigen assays.

Superantigen assays are used to determine the stimulation of classes of T cells in response to the presence of a superantigen. This requires that antigen-presenting cells, such as B-lymphocytes, are co-cultured with syngeneic T cells over a four-day period. This prerequisite rules out conventional transient transfection procedures, since the cells must remain metabolically active for around five days. Consequently, stably transfected B cells have previously been used to test recombinant DNA constructs for superantigen activity. Here we present a protocol for the transient transfection of B cells that results in metabolically viable cells. Mouse mammary tumor virus (MMTV) encodes an endogenous superantigen. Using this transient transfection procedure, we show that a number of MMTV-based constructs give rise to functional superantigen activity. The ability to transiently transfect lymphocytes and maintain their viability should greatly facilitate studies of genes encoding products, such as superantigens, that can only be indirectly assayed on a second cell type as a result of expression in the recipient cell.

A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.

The endogenous mouse mammary tumour virus locus Mtv-8 contains a defective envelope gene.

The Mtv-8 associated provirus, GR40, is not expressed in vivo. However, upon transfection into rat XC cells, the two MMTV specific mRNAs of 35S and 24S are transcribed. Further, the level of these transcripts is augmented when the transfected cells are grown in the presence of dexamethasone (Ponta et al., 1983). No virus can be detected in the medium of the transfected cells. Intracellular protein analysis of these transfected cells shows that although apparently authentic gag proteins are synthesized, a novel protein of 68 kDa is the only env related protein detectable. The sequence of the env gene of GR40 was determined and the predicted amino acid sequence of the env protein obtained. A premature termination codon is present 68 amino acids before the COOH-terminus of the known MMTV env precursor Pr73env. This would result in an env protein of about 68 kDa, in agreement with the size of the env protein found in GR40 transfected cells. The lack of the carboxy terminus may be responsible for the non-processing of the aberrant env precursor protein, p68.

Mapping of a mouse mammary tumor virus integration site by retroviral LTR--arbitrary polymerase chain reaction.

The de novo integration of retroviral genomes within the mammalian genome is believed to contribute to the tumorigenic process. Integration may result in the disruption or inappropriate transcription of key regulatory genes. We describe the application of an arbitrarily primed PCR method for the mapping and cloning of genomic integration sites of the mouse mammary tumor virus (MMTV). We have amplified DNA sequences between a selected retroviral MMTV-LTR and random sites in the 3' flanking DNA. Using this technique we were able to visualize several proviral integration sites present in a MMTV-induced mammary tumor derived cell line that were absent from the germ line. Cloning and sequencing of the PCR product corresponding to one site established its identification as an unique 3' flanking sequence.

Characterization of a human cell clone expressing cytochrome P450 for safe use in human somatic cell therapy.

We have previously demonstrated the therapeutic effect and efficacy of implantation of cells genetically modified to express cytochrome P450 2B1 in a nude mouse tumor model. The cells are encapsulated in polymerized cellulose sulphate and injected into preformed tumors. Upon administration of ifosfamide, the P450 enzyme converts the ifosfamide into antitumorigenic toxic metabolites at the site required, thereby significantly reducing tumor burden. Feline kidney epithelial cells were chosen for these studies, because they are easy to culture and can readily be transfected. However, these cells are not suitable for eventual use in human patients, since they are known to express endogenous retroviruses that are able to infect mammalian cells. They thus represent a safety risk. Here we describe the establishment of a human cell line that has been genetically modified to express the same cytochrome P450 construct and their characterization. The usefulness of mitomycin C treatment, both to protect the cells from the toxic metabolites that they produce and to incapacitate these cells from replicating, should they escape from the capsules, has also been investigated.

Development of cellulose sulfate-based polyelectrolyte complex microcapsules for medical applications.

Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.

Injection of encapsulated cells producing an ifosfamide-activating cytochrome P450 for targeted chemotherapy to pancreatic tumors.

The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low-dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.