consideration is the functional role of proto-oncogenes and tumor suppressor genes in the regulation and 
suppression of cell growth. Multiple studies have indicated that inhibition of proto-oncogenes can inhibit 
cell growth even in cells lacking mutations in these oncogenes, suggesting that inhibition of proto- 
oncogenes can have effects in cells which go beyond a mere "genetic repair" concept [22-30]. 
Studying how mutations in specific genes produce cancers in humans will produce therapeutic 
implications beyond “genetic repair” of specific patient mutations. Both oncogenes and tumor suppressor 
genes appear to fall into certain groups or pathways. It may well be possible to alter the phenotypic 
consequences of numerous specific genetic mutations by manipulating certain signal transduction 
pathways. An example from the dominant oncogene field: CML is associated with (and presumably 
caused at least in part by) a chromosomal translocation of the bcr and c-abl genes producing an activated 
abl tyrosine kinase; and Burkitt’s lymphoma is associated with a chromosomal translocation of 
immunoglobulin genes with the nuclear c-myc proto-oncogene. Studies employing dominant-negative myc 
genes [20] and/or antisense c-myc [19] indicate that inhibition of c-myc expression will inhibit both types 
of tumor cells. The Burkitt’s lymphoma cells are inhibited by direct inhibition of the c-myc oncogene, and 
the CML ceils are inhibited because the abl tyrosine kinase apparently requires c-myc expression for its 
effects [22]. An example from the tumor suppressor gene field: the p53 tumor suppressor gene complexes 
with the murine double minute 2 gene(mdm-2) and many tumor cells contain either p53 deletions (or 
mutations) or mdm-2 amplification but generally not both. Overexpression of mdm-2 inhibits p53- 
mediated transactivation suggesting that altered expression of these genes might influence the growth of 
cells which contain mutations in either of these genes [3 1]. 
Gene therapy approaches to dominant oncogenes and tumor suppressor genes must be different: 
dominant oncogenes must be inhibited and tumor suppressor genes must be replaced. Both of these 
approaches are technically feasible. Dominant oncogenes may be inhibited by either 1) antisense methods 
which inhibit specific gene expression by producing the complementary RNA and blocking translation of 
the oncogene mRNA through hybridization or mRNA destruction; or 2) overexpression of a dominant- 
negative mutant which competes with the mutant activated oncogenes. The Holt laboratory was the first to 
demonstrate that antisense methods could inhibit dominant oncogenes and inhibit the growth of cells [22]. 
Evidence from homologous recombination studies and the realization that c-fos and c-myc are members of 
gene families has resulted in a more complex theory, suggesting that these proto-oncogenes have key roles 
in growth and differentiation which have specialized or generalized functions (redundant functions [25,32, 
33]. Both antisense methods and dominant negative mutants have suggested an important role for the c- 
myc oncogene during cell growth [19,20]. Additional studies have suggested an interaction between the 
proto-oncogene c-myc and the tumor suppressor gene Rb suggesting that interactions may occur between 
oncogene and tumor suppressor gene which must be considered in evaluation of studies of the type 
proposed [34-36]. 
III. MMTV and Breast Specific Gene Expression 
Retroviruses have evolved as effective agents which can introduce DNA into specific cells and 
employ molecular mimicry to cause cancer in animals. The Mouse Mammary Tumor Virus (MMTV) 
causes a transmissible breast cancer in certain strains of mice as a consequence of efficient transcription in 
breast cells and activation of oncogenes. The transcriptional unit which produces efficient transcription of 
the MMTV genome in breast cells is the MMTV long terminal repeat (LTR) which is a steroid-responsive 
promoter [1 1, 12]. the steroid-responsive nature and breast specificity of the MMTV promoter have been 
exploited in expression vectors to produce regulated expression in cultured cells and in transgenic mice 
[13-17]. The specific cis-elements within the MMTV promoter which are responsible for breast-targeted 
expression have not been definitively identified, but studies in transgenic mice have shown that certain 
truncations of the MMTV promoter show promiscuous expression in diverse tissues (16). For these 
reasons, the breast- targeted retroviral vectors employed in this protocol employ the entire 5' MMTV LTR 
region. 
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