BACKGROUND: Inactivation of retinoblastoma (RB) tumor suppressor function occurs 
frequently in lung cancer. Short-hairpin RNA can be constructed to target 
specific sequences and efficiently knock down protein expression. We developed a 
short-hairpin RNA approach to specifically target Rb in lung cancer cells to 
determine the influence of RB knockdown on proliferation.
METHODS: NCI-H520 human lung cancer cells (wild-type Rb) were transfected with 
pMSCVpuro-Rb3C, a plasmid containing a short-hairpin sequence targeted to human 
Rb. Transfectants harboring the construct were selected with puromycin. Loss of 
RB expression in selected cell populations was determined by immunoblotting. 
Proliferating cells were counted to establish growth rates. 
Retinoblastoma-proficient and RB-deficient tumor growth was monitored in nude 
mice.
RESULTS: Transfection with pMSCVpuro-Rb3C dramatically diminished RB expression 
and led to aberrant expression of RB-regulated genes. Cells harboring 
pMSCVpuro-Rb3C grew at an increased rate compared with control cells: 480.6 +/- 
37.7 versus 159.4 +/- 36.2 (relative cell count at 12 days). Tumor growth in 
nude mice also increased with RB knockdown compared with control mice: 135.2 +/- 
73.6 mm3 versus 40.0 +/- 17.0 mm3 (tumor volume at 10 days).
CONCLUSIONS: Inhibition of RB expression is efficiently achieved in lung cancer 
cells with short-hairpin RNA. Genetic targets of RB are deregulated with RB 
knockdown. Retinoblastoma depletion increases growth in vitro and in murine 
xenografts. These studies indicate that even in the context of an established 
tumor cell line, RB limits tumorigenic proliferation. Additionally, this model 
will serve as an ideal system to evaluate the role of RB activity on therapeutic 
response.