RB activity affects checkpoint control and chemosensitivity in lung cancer lines

Abstract

Body. Our hypothesis was that restoration of retinoblastoma (RB) activity alters cell-cycle checkpoint control and chemosensitivity in non-small-cell lung cancer (NSCLC) lines. Methods. Adenoviral constructs were created containing wild-type RB, RBΔcdk (a constitutively active phosphorylation-site mutant), and an empty control. NSCLC lines NCI-H520 (wild-type RB), NCI-H596 (RB null), NCI-H1734 (RB null), and NCI-H2172 (mutant RB) were infected. Protein levels were determined by immunoblotting. Cell-cycle distribution was measured using flow cytometry. Chemosensitivity was tested by MTT viability assay. Results. NSCLC lines with disrupted RB expressed elevated levels of thymidylate synthase (TS) and Topoisomerase IIα (TopoIIα). Reintroduction of RB activity into H596, H1734, and H2172 down regulated TS and TopoIIα, as well as cyclin A. However, RB and RBΔcdk could not be overexpressed after adenoviral introduction into H520, and no alteration of TS, TopoIIα, or cyclin A levels were seen. Adenovirally mediated RB and RBΔcdk expression in H596, H1734, and H2172 led to a G1 arrest, while H520 exhibited no change in cell-cycle distribution. The cell-cycle checkpoint responses to chemotherapeutic treatments varied between cell lines. In H520, exposure to cisplatin, VP-16, or 5-FU resulted in a G1 arrest. But S, G2/M, or limited G1 arrests were observed in lines deficient for RB activity. Reintroduction of RB activity established a G1 arrest with chemotherapeutic exposure. MTT assays showed normal viability in all NSCLC lines after 5-FU treatment, but cisplatin and VP-16 reduced viability in all lines. Restoration of RB activity conferred partial chemoresistance in H596, H1734, and H2172. Conclusions. RB reconstitution into NSCLC lines with disrupted RB re-establishes normal expression of targets of RB-mediated repression and confers a G1 arrest. The G1 checkpoint response to chemotherapy is restored by RB reintroduction. Restoration of RB activity in RB-deficient NSCLC lines decreases chemosensitivity.