RB activity alters checkpoint response and chemosensitivity in lung cancer lines.

BACKGROUND

The retinoblastoma tumor suppressor (RB) is a key regulator of cell cycle progression and is functionally inactivated in the majority of human non-small cell lung cancers (NSCLC). The specific influence of RB on therapeutic response in NSCLC remains elusive.

MATERIALS AND METHODS

We investigated the consequence of reintroduction of RB on checkpoint response and chemosensitivity in NSCLC cell lines. RB introduction into RB-proficient (NCI-H1299) and -deficient (H1734, H2172) NSCLC cells was achieved by adenoviral infection. RB/E2F target gene expression was determined by immunoblot analysis. Cell cycle response and viability after chemotherapeutic exposure were assessed by flow cytometry and MTT viability assay.

RESULTS

RB reconstitution in RB-deficient lines restored regulation of topoIIalpha, thymidylate synthase, and cyclin A. Similarly, RB overexpression in RB-proficient cells caused further regulation of some RB/E2F target genes including thymidylate synthase and topoIIalpha. In addition, RB overexpression resulted in restoration of the G1 arrest mechanism. Exposure of RB-proficient cells to cisplatin, etoposide, or 5-fluorouracil elicited arrest in various phases of the cell cycle while lines deficient for RB exhibited different checkpoint responses. However, introduction of RB restored ability to arrest following chemotherapeutic exposure. Chemotherapeutic challenge resulted in varying effects on cellular viability independent of RB status, yet restoration of RB activity conferred partial chemoresistance.

CONCLUSIONS

These results demonstrate that RB reconstitution into RB-deficient NSCLC lines establishes regulation of certain RB/E2F target genes and restores G1 arrest mechanisms. Furthermore, introduction of RB enhances the G1 checkpoint response to chemotherapeutics and decreases chemosensitivity. Knowledge of RB-dependent chemosensitivity may ultimately contribute to individualized therapy based on molecular characterization of tumors.