Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) express ing human wild-type p53 rumor suppressor. It is currently undergoing clinic al trials as a cancer therapeutic. Many SCH58500 clinical trials incorporat e an arm comparing traditional chemotherapy against chemotherapy combined w ith SCH58500. Paclitaxel was chosen for combination therapy in the preclini cal study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, an d its activation of p53-independent apoptosis, which might result in a "low ered threshold" for tumor cell death. SCID mice bearing human tumor xenogra fts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, w ith or without paclitaxel. Real-time quantitative reverse transcriptase pol ymerase chain reaction assays were developed and validated to quantitate ex pression of p53, the p53 downstream effector gene p21, and the apoptosis-re lated genes, bax, bcl-2, and survivin. Protein expression was confirmed usi ng immunohistochemical assays for p53 and p21. Only tumors injected with SC H58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of pal expression were observed in tumor xe nografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU -145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDA-MB-46 8 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel a lone had no effect on p21 mRNA levels in most tumors. However, paclitaxel s uppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 t umors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin a fter SCH58500 treatment with or without paclitaxel between tumor types, alt hough there were consistent responses within individual tumor lines. The mR NA ratios for bax/bcl-2 and bax/survivin were also not informative across t umor types. OF the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful fo r confirming SCH58500 activity in human tumor biopsies.