Bivariate analysis of the p53 pathway to evaluate Ad-p53 gene therapy efficacy.
BACKGROUND
Gene therapy of human tumors with adenovirus vectors presents a clinical research challenge and a potential opportunity in cancer therapy. One of the research challenges is that endpoints like tumor reduction, time to recurrence, and survival do not provide information about whether a potential therapeutic infects the targeted cells or whether the transferred gene functions or induces a cellular response. Therefore, a flow cytometric approach was developed for a wildtype, p53 encoding adenoviral vector (Ad-p53) that provides (1) the relative level of p53 transferred by p53 immunoreactivity, (2) mdm2 immunoreactivity as an assay of p53 activity, and (3) estimates of the percentage of infected cells by dual parameter analysis (p53 versus mdm2).


METHODS
Three prostate cancer cell lines (PC-3, LNCaP, DU 145) that are null, wild-type, and mutant for p53, respectively, and two ovarian cancer cell lines (PA1, MDAH 2774) that are wild-type and mutant for p53, respectively, were tested for immunoreactivity and lack of cross-reactivity with the monoclonal antibodies, DO-7 (anti-p53) and IF2 (anti-mdm2). Optimal dual staining conditions for a flow cytometric assay employing saturating levels of antibody were developed and tested by infection of PC-3, PA1, and MDAH 2774 with Ad-p53 or a control virus, Ad-luc. Dual staining with DO-7 and propidium iodide was used to determine any biological effect of the transferred gene.


RESULTS
Neither DO-7 nor IF2 showed appreciable cross-reactions by Western blot analysis of representative prostate or ovarian cell lines. By flow cytometric titration, DO-7 appears to be a high avidity antibody (saturation staining of 10(6) DU 145 cells with 0.5ug) whereas IF2 appears less so (optimum signal to noise ratio at 1ug/10(6) cells). Infection with Ad-p53 was detected at 6 to 48 hours post infection as a uniform relative increase in p53 levels over background p53 levels. Coincident increases in mdm2 immunoreactivity were also detected. DNA content measurements of PA1 and MDAH 2774 cells indicated that G1 arrest and/or apoptosis occurred subsequent to Ad-p53 infection. p53 and mdm2 levels and DNA content distributions for Ad-luc infected cells were equivalent to uninfected cells.


CONCLUSIONS
A flow cytometric approach to measure the efficacy of an Ad-p53 gene therapy vector was developed that detects not only the gene transferred but also the activity of the transferred gene product.
Jacobberger
J W
JW
Cancer Research Center, Case Western Reserve University, Cleveland, Ohio.
Sramkoski
R M
RM
Zhang
D
D
Zumstein
L A
LA
Doerksen
L D
LD
Merritt
J A
JA
Wright
S A
SA
Shults
K E
KE
D000256:Adenoviridae / Q000235:genetics*; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002469:Cell Separation; D003429:Cross Reactions; D004273:DNA, Neoplasm / Q000032:analysis; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D018014:Gene Transfer Techniques; D016158:Genes, p53; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009687:Nuclear Proteins; D010051:Ovarian Neoplasms / Q000737:chemistry / Q000235:genetics / Q000628:therapy*; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000235:genetics / Q000628:therapy*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D051736:Proto-Oncogene Proteins c-mdm2; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000032:analysis*
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2