Search

Aims: To compare the natural history of sporadic optic glioma with those associated with neurofibromatosis type 1 (NF1 ). Methods: Optic glioma cases were identified using both the Manchester Children's [...]

The effect of expression of an O6-benzylguanine (O6-beG)-resistant mutant (hATPA/GA) of human O6-alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant in the presence of the ATase inactivator O6-beG (3. 5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O6-beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O6-beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animals (P = 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O6-beG-resistant protection against the toxicity and clastogenicity of a number of O6-alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents. [ABSTRACT FROM AUTHOR]

Ionising radiation induces the expression of a number of radiation-responsive genes and there is current interest in exploiting this to regulate the expression of exogenous therapeutic genes in gene therapy strategies for cancer. However, the radiation-responsive promoters used in these approaches are often associated with low and transient levels of therapeutic gene expression. We describe here a novel radiation-triggered molecular switching device based on promoter elements from the radiation-responsive Egr-1 gene and the cre-LoxP site-specific recombination system of the P1 bacteriophage. Using this system, a single, minimally toxic dose of radiation induced cre-mediated excision of a Iox-P flanked stop cassette in a silenced expression vector and this resulted in amplified levels of CMV-promoter-driven expression of the exogenous tumour-sensitising gene, HSVtk. This strategy could be used in combination with targeted delivery and tumour-specific promoters to elicit the tumour targeted and prolonged expression of a variety of tumour sensitising genes and provide an unprecedented level of control and tumour selectivity. [ABSTRACT FROM AUTHOR]

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future. [ABSTRACT FROM AUTHOR]

Retroviral transduction was used to introduce cDNAs encoding two mutants of human O6-alkylguanine-DNA alkyltransferase (hAT), one of which (hATPA) is 16 times more resistant to O6-benzylguanine (O6-beG), and the other (hATPA/GA) which is almost totally refractory to inactivation relative to the wild-type protein, into K562 human erythroleukaemic cells. A colony-forming assay was used to demonstrate significant protection (P < 0.001) against mitozolomide or temozolomide toxicity in k562 clones expressing either hat mutant, as determined from an in vitro assay of activity. however, protection against these agents was reduced in hatpa expressing cells in the presence of 1 μm o6-beG and was lost in the presence of 20 μM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 μM O6-beG (P < 0.001). using primary human cord blood-derived cd34+ haemopoietic cells in which PCR analy-sis indicated that up to 70% of progenitors were trans- duced with retroviral constructs harbouring hATPA/GA, we observed significant protection of the granulocyte–macrophage colony-forming cells against mitozolomide (P < 0.05) and temozolomide (p < 0.001) induced toxicity in the presence of o6-beG. These findings indicate that retrovirus-mediated expression of hATPA/GA in primitive primary human haemopoietic cells is possible and does provide O6-beG-resistant protection for these cells. Using this strategy in patients may simultaneously permit attenuated myelosuppression and increased sensitivity of tumour cells to the effects of O6-alkylating agent chemotherapy. These data, taken together with the study reported by Chinnasamy et al in the accompanying article in this issue showing reduced toxicity and clastogenicity in murine haemopoietic progenitors, make a compelling case to test this strategy clinically. [ABSTRACT FROM AUTHOR]

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O6-benzylguanine (O6-beG) insensitive, double mutant form of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA). In animals reconstituted with the transduced bone marrow, about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte–macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive for hATPA/GA by immunocytochemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P = 0.001) and GM-CFC (P < 0.024) against the toxicity of the antitumour methylating agent, temozolomide, given in combination with o6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P < 0.003). this study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an o6-alkylating agent in the presence of O6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34+ haemopoietic progenitors to combined O6-beG/O6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue may be combined with O6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index. [ABSTRACT FROM AUTHOR]

The uptake of recombinant α-(l)-iduronidase into glial and neuronal cells, produced by retrovirally transduced NIH3T3 fibroblasts, was studied. We demonstrate that: (1) neuronal and glial cells take up α-(l)-iduronidase released into the medium by retrovirally transduced fibroblasts expressing high levels of α-(l)-iduronidase; (2) both glial and neuronal cells express the cation independent mannose-6-phosphate receptor responsible for lysosomal enzyme uptake; and (3) uptake of the lysosomal enzyme can be blocked by excess free mannose-6-phosphate, but not glucose-6-phosphate. Thus, various brain cells take up α-(l)-iduronidase, possibly through a cation independent mannose-6-phosphate receptor mediated pathway, and this uptake is higher in actively dividing or immature brain cells. Consequently, (1) neuronal metabolism ought to be capable of cross correction by enzyme provided by genetically engineered and transplanted cells provided by bone marrow transplantation (BMT); (2) that BMT could have a more beneficial effect on neurological function if performed as early as possible; and (3) given that the uptake mechanism of glial cells has a higher capacity, it might be easier to target diseases like the leukodystrophies in which lysosomal enzymes are needed in glial cells, compared to diseases where lysosomal enzymes ought to be delivered into neurons. [ABSTRACT FROM AUTHOR]

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

Background and Objective: As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production., Design and Methods: To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test)., Results: An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants., Interpretation and Conclusions: A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

Many of the problems with current anti-tumour therapies stem from a lack of specificity for tumour as opposed to normal tissues. To address the problem of collateral toxicity during anti-tumour chemotherapy we have been developing a gene therapy approach to protect normal tissues from the toxic and potentially mutagenic effects of chemotherapeutic agents. As a paradigm for this we have been examining the potential of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (ATase) to confer genetic chemoprotection to the bone marrow. By transfer and expression of a mutant form of this protein, which is resistant to inactivation by the tumour sensitising agent O6-benzylguanine (O6-beG), we have been able to demonstrate protection of murine bone marrow in vitro from the cytotoxic and clastogenic effects of O6-beG in combination with the anti-tumour agent temozolomide. This protection is seen in multiple lineages, including erythroid and granulocyte/macrophage progenitors, as well as more primitive cells. Importantly, significant protection of the platelet lineage is also seen, with faster recovery of platelets. The multi-lineage protection seen has encouraged us to take this approach forward to clinical trial in the near future.

Retroviral gene transfer was used to achieve expression in mouse bone marrow of a mutant form of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (hATPA/GA), which exhibits resistance to inactivation by O6-benzylguanine (O6-beG). After reconstitution of mice with transduced bone marrow, approximately 50% of the bipotent granulocyte-macrophage colony-forming cell (GM-CFC) and multipotent spleen colony-forming unit (CFU-S) hemopoietic populations showed expression of the transgene; this expression was associated with resistance to either mitozolomide or to a combination of O6-beG and mitozolomide, relative to mock-transduced controls. Thus, at a dose of mitozolomide in vivo that allowed only 70% and 62% survival of mock-transduced GM-CFC and CFU-S, respectively, the hATPA/GA CFC were totally resistant to the same dose of mitozolomide (P < .05 and .001, respectively). In the presence of O6-beG, the toxicity of mitozolomide was greatly potentiated. Only 24% and 18%, respectively, of mock-transduced GM-CFC and CFU-S survived combination treatment, whereas 45% (P < .05) and 37% (P < .01) of GM-CFC and CFU-S, respectively, from hATPA/GA mice survived the same combination of doses. Furthermore, as a result of transgene expression, the number of micronucleated polychromatic erythrocytes induced by mitozolomide was significantly reduced (P < .05) by 40% relative to mock-transduced controls, indicating the potential of this approach to reduce the frequency of mutation associated with chemotherapy exposure. The protection against the toxic and clastogenic effects of mitozolomide in both primitive and more mature hemopoietic cells suggests that the severe myelosuppression that halted further clinical investigation of this drug could be substantially ameliorated by the exogenous expression of O6-alkylguanine-DNA alkyltransferase. Therefore, these data raise the prospect for the reinvestigation of mitozolomide and other proscribed drugs in the context of genetically protected hemopoiesis.