and used for production of high-titer virus stock (the titers ranged from 5-9 X lO^/ml) To confirm titers 
and test the relative infectivity of human breast cancer cell lines we have infected MCF-7 cells with 
amphotropic viral stocks from producer cells which produce the following viruses: 1) XM6-Antisense c- 
fos; 2) XM6-Sense c-myc (a control vector which does not express antisense RNA; 3) XM6-Antisense c- 
myc; 4) XM6-Rb; 5) XM6-CAT. We standardized the viral stocks such that each T25 flask containing 1 
million cells received 3 million viral particles. Forty-eight hours following infection we counted the 
number of cells in triplicate, as presented in Appendix C: Figure 2. This preliminary data suggests that 
antisense inhibition of c-myc or introduction of Rb results in growth inhibition in infected MCF-7 breast 
cancer cells. These experiments were performed on MCF-7 cells cultured in T25 flasks. The sample 
marked sense c-myc was infected with an XM6-sense myc retrovirus which expresses a control gene 
containing 750 base pairs from the 5' region of the c-myc gene, and serves as a control for the antisense c- 
myc retroviral vector. 
C. Toxicity studies of viral stocks in the peritoneal cavity 
To test for toxic effects or potential carcinogenic effects of injection of retroviral vector into 
mesothelial-lined spaces, we have infused e nude mice with 1 milliliter of retroviral vector XM6: anti fos 
and 3 other nude mice with 1 milliliter of XM6:antimyc and observed the mice for 6 weeks at the date of 
this submission. None of these mice have developed tumors, rashes, clear allergic responses, or evident 
pathology. Autopsies will be performed on these mice in a few weekks to determine the extent of viral 
infection and to determine whether retroviral vector has infected the gonadal region. 
D. In vivo infection of MCF-7 cells injected into nude mice 
Infection of cultured MCF-7 cells with XM6:antifos produced no significant decrement in cell 
proliferation rate, but produced a marked slowing of tumor formation when these ex vivo infected cells 
were injected into estrogen-treated nude mice. Examination of the cultured cells and the tumors revealed 
that although only 30-60% of the cells had been infected with the retroviral vector as determine by 
Southern blot (Appendix C; Figure 3), there was a marked decrease in tumor formation. These results are 
presented in Appendix C; Table I and Figure 4. Histologic examination of these tumors showed that the 
tumors derived from cells infected with XM6antifos were predominantly encapsulated and the majority did 
not invade tissue structures (compared with control tumors infected with XM6:sensefos which usually 
invading skeletal muscle or subcutaneous lymphatics). These tumors also showed frequent apoptosis and 
induction of differentiation which was most evident when sections were stained with alcian blue. By 
contrast, the control tumors were larger, showed less apoptosis, and were extremely undifferentiated. 
Taken together, the Southern blot and histologic findings suggests a "bystander" phenomenon: in which 
infected cells signal other tumor cells to die and/or differentiate. We are presently studying the mechanism 
responsible for this phenomenon but do not presently know whether it involves cell-cell contact, soluble 
factors, and/or other explanations. 
Infection of cultured MCF-7 cells with XM6:antimyc produced a moderate decrease in cell 
proliferation rate, but completely prevented tumor formation when these ex vivo infected cells were 
injected into estrogen-treated nude mice. This occurred even though only 30-60% of the cells had been 
infected with the retroviral vector as shown by Southern blot (Appendix C; Figure 3), suggesting a 
bystander phenomenon to prevent tumor formation by the remaining cells. However, because no tumors 
developed in this treatment group there was no way to determine whether induction of either apoptosis or 
differentiation occurred. These results are presented in Appendix C; Table II and Figure 5. 
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Recombinant DNA Research, Volume 20 
