Phosphorylation/inactivation of RB is typically required for cell cycle pro
gression. However, we have identified a tumor cell line, C33A, which progre
sses through the cell cycle in the presence of an active allele of RB (PSM-
RB), To determine how C33A cells evade RB-mediated arrest, we compared RB s
ignaling to downstream effecters in this resistant cell line to that of the
RB-sensitive SAOS-2 cell line. Although introduction of PSM-RB repressed E
2F-mediated transcription in both C33A and SAOS-2 cells, PSM-RB failed to r
epress Cyclin A promoter activity in C33A, Ectopic expression of PSM-RB in
SAOS-2 cells resulted in a decrease in both Cyclin A and Cdk2 protein level
s without affecting Cyclin E or Cdk4, In contrast, over-expression of PSM-R
B in C33A cells did not alter endogenous Cyclin A, Cyclin E, or Cdk2 protei
n levels or impact Cdk2 kinase activity, indicating that signaling from RB
to downstream targets is abrogated in this cell line. The importance of Cdk
2 activity was demonstrated by p27Kip1, which attenuated Cdk2 activity and
inhibited cell cycle progression in C33A cells, Since RB signaling to Cdk2
is disrupted in these tumor cells, we coexpressed two proteins that coopera
te with RB transcriptional repression, AHR and BRG-1, in an attempt to corr
ect this signaling dysfunction, Coexpression of AHR/BRG-1 with PSM-RB atten
uated Cyclin A and Cdk2 expression as well as Cdk2-associated kinase activi
ty, resulting in cell cycle inhibition of C33A cells. Importantly, ectopic
expression of Cyclin A was able to reverse the arrest mediated by co-expres
sion of AHR/BRG-1 with PSM-RB, These results indicate that down-regulation
of Cdk2 activity is requisite for RB-mediated cell cycle arrest. Thus, this
study reveals a new mechanism through which tumor cells evade anti-prolife
rative signals, and provides insight into how RB-signaling is mediated.