TABLE 2.

Loss of IκBα-mediated inhibition of nuclear import and DNA binding correlates with increased transformation

Rel proteina	Leptomycin B sensitivityb	IκBα-mediated displacementc	Transformationd (%)	Establishment of cell linese (%)
c-Rel-546	2	1	6	<1
c-Rel-546-SP	58	25	23	65
v-Rel-YL	23	1	29	65
v-Rel	92	125	100	90

^aSNV-derived expression vectors encoding the indicated Rel proteins were constructed (Fig. 1). 

^bCEF were transfected with retroviral expression vectors that encoded the indicated Rel protein and wild-type IκBα. Leptomycin B was added to the culture medium at a concentration of 5 nM 30 min prior to fixation of the CEF for indirect immunofluorescence. Values are percentages of cells that displayed predominantly nuclear staining relative to the total number of cells that displayed staining for the transfected Rel proteins. For each treatment, a total of 200 cells that were positive for expression of the respective Rel proteins were examined. 

^cCOS-1 cells were singly transfected with CMV-derived expression vectors encoding the indicated Rel proteins. Equivalent amounts of cell lysates were incubated with a ³²P-labeled oligonucleotide containing a palindromic κB site for 10 min at room temperature. The relative amounts of each of the Rel proteins that remained bound to the ³²P-labeled oligonucleotide following incubation with increasing amounts of baculovirus-purified IκBα were determined by solution UV cross-linking. Values are fold excesses of IκBα required to displace the indicated Rel proteins from DNA, relative to the amount of IκBα required to displace the c-Rel-546 protein from the c-Rel-546–DNA complex. 

^dAvian lymphoid cells were infected with viruses encoding the indicated Rel proteins. The ability of avian lymphoid cells infected with viruses encoding the indicated Rel proteins to form colonies in soft agar was determined. The relative efficiency of transformation by the indicated Rel proteins is derived from the mean of six independent experiments, in which the total number of colonies obtained with each virus within a single experiment was normalized to the total number of colonies obtained with virus encoding the wild-type v-Rel protein (set at 100%). The actual numbers of colonies that formed in soft agar for the six independent experiments were as follows: for c-Rel-546, 1, 2, 13, 7, 9, and 0; for c-Rel-546-SP, 3, 5, 47, 8, 67, and 0; for v-Rel-YL, 11, 26, 17, 45, 14, and 3; for v-Rel, 59, 45, 130, 99, 85, and 15. 

^eTransformed cell lines could not be established following infection of avian lymphoid cells with virus encoding the c-Rel-546 protein. In contrast, cell lines could be established from more than 65% of colonies obtained following infection with virus encoding c-Rel-546-SP or v-Rel-YL, and from more than 90% of colonies obtained following infection with virus encoding the wild-type v-Rel protein.