Biology Reference
In-Depth Information
differentially spliced overlapping transcripts, referred to as 13S and
12S RNA, respectively. Whereas the 13S-derived protein (E1A-
13S) is required for viral replication, the 12S product (E1A-12S)
encodes all functions necessary for immortalization.
In general, E1A-immortalized cells are not completely trans-
formed in that they grow slowly and not to high densities, are
anchorage dependent, and usually are not tumorigenic. Instead, full
manifestation of the transformed phenotype requires expression of
a cooperating second oncogene (reviewed in refs.
2
,
3
). Such coop-
erating oncogenes include cellular genes like activated ras. However,
within the context of the adenoviral genome, the role of this second
oncogenic determinant has been traditionally ascribed to E1B. This
transcription unit encodes two major proteins, E1B-55 kDa and
E1B-19 kDa, which are structurally unrelated but independently
cooperate with E1A proteins in the complete oncogenic transfor-
mation of primary cells in culture. This function is, at least in part,
a result of their ability to inhibit E1A-induced p53-dependent and
p53-independent apoptotic cell death (reviewed in ref.
4
). It appears
that much of the oncogenic activity of E1B-55 kDa is related to its
interaction with the cellular tumor suppressor protein p53, although
p53-independent mechanisms are now known to exist. The E1B-
19 kDa shares structural and functional similarities with the cellular
anti-apoptotic Bcl-2 protein.
Over the past 20 years we and others have shown that, besides
E1A and E1B, human Ads contain an additional third oncogenic
region, E4 (reviewed in ref.
5
). Specifi cally, the protein products of
three open reading frames within this transcription unit—E4orf1,
E4orf3, and E4orf6—have been implicated in oncogenic transfor-
mation. Work by Javier and colleagues suggests that transforma-
tion by E4orf1 differs considerably from the E1-encoded functions
and is mediated through a novel mechanism that involves interac-
tions with multiple PDZ-domain-containing proteins [
6
,
7
]. In
contrast, the oncogenic activities of E4orf3 and E4orf6 resemble
those of the E1B oncoproteins. They can individually cooperate
with E1A to completely transform primary rodent cells in vitro.
Moreover, they enhance transformation mediated by the E1A and
E1B oncogenes. The transforming properties of E4orf6 involve
two distinct mechanisms, one of which is linked to degradation of
the p53 protein in combination with E1B-55 kDa [
8
]. The molec-
ular basis of transformation by E4orf3 is not known, but it appears
not to be linked to this protein's ability to interact with cellular
promyelocytic leukemia bodies (ref.
9
and unpublished results).
The transforming activities of adenoviral E1- and E4-encoded
oncogenes can be determined by DNA transfection of primary
rodent cells. For this purpose, we routinely use primary kidney
epithelial cells prepared from neonatal rats. These cells are
commonly referred to as baby rat kidney (BRK) cells. They provide
at least two advantages over other cell culture systems, including
