The goal of this proposal is to develop a method which will increase the resistance of hematopoietic cells to agents known to be leukemogenic. Alkylating agents, in particular, nitrosoureas, cause secondary leukemias in humans and animals because of their ability to create mutagenic DNA damage. It appears that hematopoietic cells are susceptible to nitrosoureas because they contain low levels of a DNA repair enzyme which normally protects cells from the DNA damage caused by these agents. The repair enzyme, 06 alkylguanine-DNA alkyltransferase (alkyltransferase), removes 06 alkylguanine adducts which are formed by nitrosoureas and, if left unrepaired, cause mutations during DNA replication. This proposal will use molecular cloning techniques to introduce the bacterial alkyltransferase gene, ada, into hematopoietic cells and then use assays of hematopoietic stem cells and bone marrow transplantation to test the ability of ada to be expressed in hematopoietic precursors. Because high levels of ada can also confer drug resistance, these studies will evaluate a potentially new drug resistance gene that may be useful in future gene transfer experiments.
The specific aims of this proposal are: (1) Introduce ada as a chimeric gene consisting of a mammalian promoter ligated to the coding sequence of the E. coli alkyltransferase gene into a replication defective retroviral vector. This vector will be transfected into cells which can produced helper free retroviral particles containing the ada gene. (2) Use retroviral vectors containing ada to infect mouse bone marrow cells, resulting in efficient transfer of the alkyltransferase gene into hematopoietic precursors. Ada expression and nitrosourea resistance will be studied in these cells in vitro. (3) Bone marrow cells infected with defective retroviral particles containing ada will be used to transplant mice. The ability of bone marrow cells to express ada and become resistant to nitrosoureas in vivo will be determined. Finally, we will determine whether these animals are protected from the development of leukemia following nitrosourea exposure. These studies will provide evidence about the etiology of secondary leukemias.
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