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) 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 MDAMB-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.
Wen
S F
SF
Canji, San Diego, California 92121, USA.
Xie
L
L
McDonald
M
M
DiGiacomo
R
R
Chang
A
A
Gurnani
M
M
Shi
B
B
Liu
S
S
Indelicato
S R
SR
Hutchins
B
B
Nielsen
L L
LL
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis; D003131:Combined Modality Therapy; D005260:Female; D016158:Genes, p53 / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D051034:Inhibitor of Apoptosis Proteins; D051379:Mice; D016513:Mice, SCID; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009368:Neoplasm Transplantation; D010051:Ovarian Neoplasms / Q000628:therapy; D017239:Paclitaxel / Q000627:therapeutic use*; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000077022:Survivin; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*; D051028:bcl-2-Associated X Protein; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis
C494826:BAX protein, human; C107489:BIRC5 protein, human; C494827:Bax protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D000077022:Survivin; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D020741:rho GTP-Binding Proteins; D017239:Paclitaxel