17 
process takes many months as the virus replicates through many cycles or the 
cells undergo further genetic changes. No evidence suggests the malignancies 
caused by these viruses result from a single hit event. 
Dr. Gottesman asked whether leukemia would develop if viral replication did 
not occur. rr. Mulligan replied that it is unlikely a proto-oncogene would be 
activated or inactivated without many cycles of replication. Dr. Joklik said 
replication is probably necessary for insertion to occur at the appropriate 
site in the appropriate cell. Not all cells in the animal are susceptible to 
tumor induction by the particular virus. 
[Rapporteur's Note: The ALV provirus integrates adjacent to the c -myc gene in 
several B cell lymphomas and next to c- erb proto-oncogenes in leukemic 
erythroblast cells, resulting in elevated levels of transcription from these 
genes.] 
Dr. LeRoy Walters of the Kennedy Institute and Chair of the RAC Working Group 
on Human Gene Therapy asked how many proto-oncogenes are in human cells. 
Dr. Mulligan said there are at least 30 proto-oncogenes in the human gencme 
and probably more. 
Dr. Anderson thought most retroviral insertions would not occur close enough 
to a proto-oncogene to activate it. The distance of the insertion site from 
a proto-oncogene and its regulatory signals is probably the primary factor 
affecting activation or inactivation of the proto-oncogene. 
Dr. Anderson said for human gene therapy the control regions of the retroviral 
vector will probably be eliminated; the vector sequences thus would not be 
able to override the chromosomal regulatory signals near the insertion site. 
Dr. Mulligan noted that even if such vectors were utilized, the transcriptional 
control signals used to promote expression of the inserted genes might 
activate adjacent cellular genes as well. 
Dr. Mulligan said in addition to insertion, additional events involving the 
deletion and/or rearrangement of viral sequences are parobably necessary 
for qene activation. 
[Rapporteur's Note: Several hut not all of the ALV- induced B cell lymphomas 
result from activation of the c- myc gene by premotor insertion: the ALV pro- 
viruses had integrated into seouences upstream of the oncogene such that myc 
transcripts were initiated from the promotor within the 3' LTR. In most other 
tumors examined, however, provirus integration occurs either in an orientation 
transcriptionally opposite to that of the putative oncoqene, or downstream of 
the gene within the 3' flanking sequences. In these cases, the provirus may be 
activating cellular gene expression by means of cis- acting transcriptional 
enhancer elanents in the LTR. 
[456] 
