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

Abstract

SCH58500 ( ACN53 ) is a replication -deficient, type 5 adenovirus ( Ad ) expressing human wild -type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first -line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53 -independent apoptosis, which might result in a``lowered threshold'' for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real -time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis -related genes, bax, bcl -2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3 ± 11 ± fold elevations of p21 expression were observed in tumor xenografts 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 -468 and SK -OV -3 cells, but not in PA -1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4 -fold in DU -145 and SK -OV -3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl -2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax / bcl -2 and bax / survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

Inefficient killing of quiescent human epithelial cells by replicating adenoviruses: potential implications for their use as oncolytic agents

“…11 Briefly, cells were harvested at various time points, pelleted, and frozen at À701C. DNA was prepared following lysis of the pellets with reagents from the QIAamp DNA mini kit (Qiagen, Valencia, CA).…”

Assessment of p53 gene transfer and biological activities in a clinical study of adenovirus-p53 gene therapy for recurrent ovarian cancer

“…26 The GAPDH housekeeping gene was used as an internal control to assess the quality of assay samples and to normalize results. No results were reported if GAPDH DNA or RNA was less than 1000 copies per PCR reaction.…”

“…Two types of standard curves were used to quantify gene expression in this study. cRNA 26 was used to quantify p53, p21/WAF1, bax, mdm-2, and GAPDH gene expression. A standard created from serially diluted RNA extracted from rAd-p53 SCH 58500-infected A549 cells was used to quantify caspase-3 and survivin expression.…”

“…This set of primers and probe do not recognize the endogenous p53 gene sequence, and therefore only amplify rAd-p53 SCH 58500. 26,27 Bioassay for adenovirus serum neutralizing factors HEK 293 cells were plated onto collagen-I-coated 96-well plates (BioCoat, Fort Washington, PA) and incubated at 37 o C in a 5% CO 2 incubator for 3 hours prior to sample addition. Sera for analysis were serially diluted two-fold All probes were labeled with FAM at the 5 0 end as the reporter and TAMRA at the 3 0 end as the quencher.…”

“…For this reason, prior to analyzing subject samples, we developed and validated quantitative PCR (QPCR) and quantitative RT-PCR (QRT-PCR) assays using various xenograft tumor models. 26 Since the presence of endogenous human p53 and p21 proteins causes interference in many protein-based assays, and because such methods are relatively insensitive for quantifying specific proteins, we also applied QRT-PCR assay as an alternative tool to quantify p53 gene expression at mRNA level, instead of as protein.…”