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dc.contributor.authorHickson, Ian
dc.contributor.authorFairbairn, Leslie J
dc.contributor.authorChinnasamy, Nachimuthu
dc.contributor.authorLashford, Linda S
dc.contributor.authorThatcher, Nick
dc.contributor.authorMargison, Geoffrey P
dc.contributor.authorDexter, T Michael
dc.contributor.authorRafferty, Joseph A
dc.date.accessioned2010-02-12T16:16:54Z
dc.date.available2010-02-12T16:16:54Z
dc.date.issued1998-06
dc.identifier.citationChemoprotective gene transfer I: transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro. 1998, 5 (6):835-41 Gene Ther.en
dc.identifier.issn0969-7128
dc.identifier.pmid9747464
dc.identifier.doi10.1038/sj.gt.3300698
dc.identifier.urihttp://hdl.handle.net/10541/92054
dc.description.abstractRetroviral 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 microM O6-beG and was lost in the presence of 20 microM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 microM O6-beG (P < 0.001). Using primary human cord blood-derived CD34+ haemopoietic cells in which PCR analysis indicated that up to 70% of progenitors were transduced 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.
dc.language.isoenen
dc.subjectHaematopoietic Stem Cellsen
dc.subject.meshAntigens, CD34
dc.subject.meshAntineoplastic Agents, Alkylating
dc.subject.meshCell Line
dc.subject.meshCell Survival
dc.subject.meshDacarbazine
dc.subject.meshGene Transfer Techniques
dc.subject.meshGenetic Vectors
dc.subject.meshGuanine
dc.subject.meshHematopoietic Stem Cells
dc.subject.meshHumans
dc.subject.meshNitrogen Mustard Compounds
dc.subject.meshO(6)-Methylguanine-DNA Methyltransferase
dc.subject.meshPolymerase Chain Reaction
dc.subject.meshRetroviridae
dc.titleChemoprotective gene transfer I: transduction of human haemopoietic progenitors with O6-benzylguanine-resistant O6-alkylguanine-DNA alkyltransferase attenuates the toxic effects of O6-alkylating agents in vitro.en
dc.typeArticleen
dc.contributor.departmentCRC Section of Haemopoietic Cell, Paterson Institute for Cancer Research, Manchester, UK.en
dc.identifier.journalGene Therapyen
html.description.abstractRetroviral 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 microM O6-beG and was lost in the presence of 20 microM O6-beG while cells expressing hATPA/GA retained protection even in the presence of 20 microM O6-beG (P < 0.001). Using primary human cord blood-derived CD34+ haemopoietic cells in which PCR analysis indicated that up to 70% of progenitors were transduced 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.


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