Figure 3.

Detection of RB expression after LPD-RB administration. (A) Detection by microdissection and PCR analysis of the RB transgene in metastatic cells from lung tissue of mice treated with either LPD-RB (lanes 2–5) or LPD-RBH209 (H209, lanes 6 and 7) but not in those from mice exposed to LPD-Luc (lane 1). The 114-bp and 194-bp PCR fragments are diagnostic for RB cDNA and the Rb gene, respectively (20). Lane 6, DNA size markers (Amresco, Euclid, OH). (B) Immunoprecipitation and immunoblot analysis of RB expression in lungs of mice treated with LPD-RB (lanes 1–4), LPD-RBH209 (lanes 5 and 6), or in those of mice exposed to LPD-Luc (lanes 7 and 8). Immunoprecipitation (IP) was performed with C-15 antiserum, which recognizes both human RB (110 kDa) and mouse Rb (105 kDa), or mAb 11D7, which is specific for human RB protein, and immunoblot analysis was performed with mAB 245 (Upper). Detection of p84, a nuclear matrix protein (57), was assessed as a control for gel loading (Lower). (C) Detection of Rb and RB (Upper, 161 bp) and β-actin (Lower, 306 bp) mRNAs in the lung (lane 1), thyroid C cells (lane 2), and lung metastases treated with LPD-RB (lane 3), LPD-RBH209 (lane 6), LPD-Luc (lane 4), or LPD-lacZ (lane 7). Lanes 5 and 8, reverse transcriptase (RT)–PCRs without RT. (A and C) Polyacrylamide gel stained with silver. (D) Relative amounts of Rb cDNA (mean ± SD) in the lung (n = 8), C cells (CC, n = 5), and metastatic cells treated with either LPD-RB (RB, n = 6) or LPD-RBH209 (H209, n = 4) after densitometric readings of PCR products in exponential phase of amplification and subsequent β-actin normalization for internal errors in loading and amplification. Two-tailed Student’s t test P values are <0.0001, <0.0001, 0.7430, and 0.6135 for lung vs. CC, lung vs. RB, CC vs. RB, and CC vs. H209, respectively. All experiments were performed in triplicate and repeated at least twice. In all experiments, mice were treated as described in the legend to Fig. 2 G–K.