p53-dependent stimulation of redox-related genes in the lymphoid organs of gamma-irradiated--mice identification of Haeme-oxygenase 1 as a direct p53 target gene.

Recent data showed that p53 stimulates the expression of genes encoding not only pro- but also antioxidant enzymes. It was suggested that antioxidant genes could be induced under physiologic levels of stress while the prooxidant ones respond to higher level of stress. Results presented in this article illustrate an additional degree of complexity. We show that the expression of Haeme-oxygenase 1 (HO-1), a stress-inducible gene that codes for an enzyme having antioxidant properties, is stimulated in a p53-dependent manner in the thymus and spleen of irradiated mice. We prove that HO-1 is a direct p53 target gene by showing that the p53RE identified within human and mouse genes is specifically bound by p53. The threshold of irradiation dose required to induce a significant response of HO-1 in the lymphoid organs of the irradiated mice is higher than that for Waf1/p21 that encodes an universal inhibitor of cell cycle. Moreover, induction of HO-1 occurs later than that of Waf1/p21. Finally, the higher stimulation of HO-1 is reached when Waf1/p21 stimulation starts to decrease.

Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists.

A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means for correcting for serum matrix effects and allows residual drug levels or anti-drug neutralizing antibodies to be quantified even in serum samples with a relatively high degree of cytotoxicity.

One-step assay for quantification of neutralizing antibodies to biopharmaceuticals.

Assessment of immunogenicity is an important part of biopharmaceutical drug safety evaluation and a prerequisite for the development of less immunogenic and safer biopharmaceuticals since anti-drug antibodies can impair the activity and compromise the safety of biopharmaceuticals. Although regulatory authorities recommend cell-based assays for detection of neutralizing antibodies (NAbs), such assays are difficult to standardize, and ill adapted to high-throughput analysis. These limitations have been overcome by the development of a unique one-step cell-based assay that allows both drug activity and drug NAbs to be quantified rapidly and with a high degree of precision simply be adding reporter cells to a sample. The reporter cells have been engineered to express firefly luciferase (FL) under the control of a drug-responsive promoter, and to express the drug of interest, the production of which is normalized relative to the expression of Renilla luciferase (RL) transcribed from a common doxycycline-inducible promoter. Residual drug levels present in a sample are first quantified by determination of FL expression, autocrine drug synthesis is then induced, and NAb activity is quantified from the difference in the ratio of FL/RL expression in the presence or absence of the sample. Since assay results are normalized relative to the expression of an internal standard, results are independent of cell number or differences in cell viability thus affording a high degree of assay precision and reducing serum matrix effects to a minimum. This unique assay platform is ideally suited for high-throughput analysis, is applicable to most biopharmaceuticals, and will facilitate standardization and comparison of immunogenicity data. The performance of the one-step assay is illustrated for interferon alpha2 (IFNalpha2) used widely to treated chronic hepatitis C (HCV) infection and neoplastic disease.

GAAP-1: a transcriptional activator of p53 and IRF-1 possesses pro-apoptotic activity.

The mechanisms that regulate the transcription of the tumour suppressor genes p53 and IRF-1 are poorly understood. We have characterized a 68-kDa transcription factor, GAAP-1 (gatekeeper of apoptosis activating proteins), encoded by an alternative splice product of the PRDII-BF1 gene, that recognizes a novel regulatory element within the p53 and IRF-1 promoters. Transfection of U937 cells with GAAP-1 activates p53 and IRF-1 expression and leads to apoptosis, whereas over-expression of GAAP-1 in K562 cells that lack p53 and IRF-1 induces cell differentiation. Alterations in the 6p24 locus containing the GAAP-1 gene are frequent in acute myelogenous leukemia (AML), and AML-derived cell lines display reduced GAAP-1 mRNA levels. Together, these results suggest that GAAP-1 acts as a gatekeeper at a critical point in the tumour suppressor gene pathway.