Recommendations for the evaluation of specimen stability for flow cytometric testing during drug development.

The objective of this manuscript is to present an approach for evaluating specimen stability for flow cytometric methods used during drug development. While this approach specifically addresses stability assessment for assays to be used in clinical trials with centralized testing facilities, the concepts can be applied to any stability assessment for flow cytometric methods. The proposed approach is implemented during assay development and optimization, and includes suggestions for designing a stability assessment plan, data evaluation and acceptance criteria. Given that no single solution will be applicable in all scenarios, this manuscript offers the reader a roadmap for stability assessment and is intended to guide the investigator during both the method development phase and in the experimental design of the validation plan.

Signalling cross-talk between hepatocyte nuclear factor 4alpha and growth-hormone-activated STAT5b.

In the present study, we have characterized signalling cross-talk between STAT5b (signal transducer and activator of transcription 5b) and HNF4alpha (hepatocyte nuclear factor 4alpha), two major regulators of sex-dependent gene expression in the liver. In a HepG2 liver cell model, HNF4alpha strongly inhibited beta-casein and ntcp (Na+/taurocholate cotransporting polypeptide) promoter activity stimulated by GH (growth hormone)-activated STAT5b, but had no effect on interferon-gamma-stimulated STAT1 transcriptional activity. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4alpha towards the ApoCIII (apolipoprotein CIII) promoter. The inhibitory effect of HNF4alpha on STAT5b transcription was associated with the inhibition of GH-stimulated STAT5b tyrosine phosphorylation and nuclear translocation. The short-chain fatty acid, butyrate, reversed STAT5b transcriptional inhibition by HNF4alpha, but did not reverse the inhibition of STAT5b tyrosine phosphorylation. HNF4alpha inhibition of STAT5b tyrosine phosphorylation was not reversed by pervanadate or by dominant-negative phosphotyrosine phosphatase 1B, suggesting that it does not result from an increase in STAT5b dephosphorylation. Rather, HNF4alpha blocked GH-stimulated tyrosine phosphorylation of JAK2 (Janus kinase 2), a STAT5b tyrosine kinase. Thus STAT5b and HNF4alpha exhibit bi-directional cross-talk that may augment HNF4alpha-dependent gene transcription while inhibiting STAT5b transcriptional activity via the inhibitory effects of HNF4alpha on JAK2 phosphorylation, which leads to inhibition of STAT5b signalling initiated by the GH receptor at the cell surface.

Role of hepatocyte nuclear factors in transcriptional regulation of male-specific CYP2A2.

Cytochrome P450 2A2 (CYP2A2) is an adult male-specific rat liver steroid hydroxylase whose sex-dependent expression is regulated at the transcriptional level by sexually dimorphic pituitary growth hormone (GH) secretory patterns. In contrast to CYP2C11 and other male-specific, plasma GH pulse-inducible liver genes, CYP2A2 is highly expressed in hypophysectomized rat liver, despite the absence of GH stimulation. CYP2A2 promoter fragments 0.9-6.2 kb long exhibited unusually high basal promoter activity when transfected into the liver cell line HepG2. A further approximately 2.5-fold increase in activity was obtained by cotransfection of hepatocyte nuclear factor (HNF) 3gamma or HNF4alpha. CYP2A2 promoter activity was inhibited approximately 85% by transfection of HNF3beta or HNF6, both of which are more highly expressed in female than male liver and can strongly trans-activate the female-specific CYP2C12 promoter. The male GH pulse-activated transcription factor STAT5b had no effect on CYP2A2 promoter activity, either alone or in combination with HNF3gamma and HNF4alpha, consistent with the GH pulse-independence of CYP2A2 expression. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4alpha toward two other male-specific liver target genes, Cyp2d9 and CYP8B1. Furthermore, STAT5b in combination with the HNF4alpha coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha strongly enhanced the transcriptional activity of HNF4alpha toward CYP8B1 but not toward CYP2A2. These findings support the hypothesis that sex-dependent HNFs contribute to the sexually dimorphic expression of CYP2A2 and other liver CYPs and highlight the ability of STAT5b to act in concert with HNF4alpha to regulate select male-specific liver CYP genes.

Sexual dimorphism of rat liver nuclear proteins: regulatory role of growth hormone.

Many genes are expressed in mammalian liver in a sexually dimorphic manner. DNA microarray analysis has shown that growth hormone (GH) and its sex-dependent pattern of pituitary secretion play a major role in establishing the sexually dimorphic patterns of liver gene expression. However, GH may exert effects on protein post-translational modification and nuclear localization that are not reflected at the mRNA level. To investigate these potential effects of GH, we used two-dimensional gel electrophoresis followed by LC-MS/MS to: 1) identify rat liver nuclear proteins whose abundance or state of post-translational modification displays sex-dependent differences; and 2) determine the role of the plasma GH profile in establishing these differences. Nuclear extracts prepared from livers of individual male (n=9) and female (n=5) adult rats, and from males given GH by continuous infusion for 7 days to feminize liver gene expression (n=5 rats), were resolved by two-dimensional electrophoresis. Image analysis of SYPRO Ruby-stained gels revealed 165 sexually dimorphic protein spots that differ in normalized volume between male and female groups by >1.5-fold at p<0.05. Sixty of these proteins exhibited female-like changes in spot abundance following continuous GH treatment. Comparison of male and GH-treated male groups revealed 130 proteins that displayed >1.5-fold differences in abundance, with 60 of these GH-responsive spots being sexually dimorphic. Thus, GH plays an important role in establishing the sex-dependent differences in liver nuclear protein content. Twenty-eight of the sexually dimorphic and/or GH-regulated protein spots were identified by LC-MS/MS. Proteins identified include regucalcin, nuclear factor 45, and heterogeneous nuclear ribonucleoproteins A3, D-like, and K, in addition to proteins such as GST, normally associated with cytosolic extracts but also reported to be localized in the nucleus.

Role of hepatocyte nuclear factors in growth hormone-regulated, sexually dimorphic expression of liver cytochromes P450.

The liver is a sexually dimorphic organ in many species, including humans. In rodent models, dramatic sex differences characterize the expression of numerous plasma proteins, receptors and other signaling molecules, and enzymes of steroid and foreign compound metabolism, including members of the cytochrome P450 (CYP) superfamily. The sexual dimorphism of liver gene expression is dictated by the temporal pattern of plasma growth hormone (GH) stimulation, which is intermittent and highly pulsatile in males and more frequent in females. Many liver-specific genes, including CYP genes, are regulated by the coordinated action of multiple hepatic nuclear factors (HNFs) through a complex transcriptional hierarchy. These HNFs are proposed to collaborate with the GH pulse-activated latent cytoplasmic transcription factor STAT5b to regulate the sex-dependent expression of liver CYPs. This hypothesis is supported by the finding that certain HNFs are regulated by GH and exhibit a differential responsiveness to the sex-specific pattern of GH secretion. In particular, recent studies of an HNF4alpha-deficient mouse model demonstrate an essential role for this nuclear receptor in regulating several liver-enriched transcription factors and sexually dimorphic CYPs in liver in vivo. Further studies on the mechanisms by which HNF4alpha and other liver factors respond to GH may expand our understanding of the mechanisms by which GH, via the coordinated action of HNFs and STAT5b, regulate sexually dimorphic liver gene expression.

Sexually dimorphic P450 gene expression in liver-specific hepatocyte nuclear factor 4alpha-deficient mice.

Hepatocyte nuclear factor (HNF) 4alpha is a liver-enriched nuclear receptor that plays a critical role in regulating the expression of numerous hepatic genes, including members of the cytochrome P450 (CYP) superfamily, several of which are expressed in a sex-dependent manner. Presently, we use a liver-specific Hnf4alpha-deficient mouse model to investigate the role of HNF4alpha in regulating liver-enriched transcription factors and sexually dimorphic Cyps in liver in vivo. Real-time PCR analysis of RNA isolated from livers of wild-type and Hnf4alpha-deficient mice revealed the following: 1) HNF4alpha exerts both positive regulation (Hnfalpha, C/ebpalpha, and C/ebpbeta) and negative regulation (Hnf3alpha and the HNF4alpha coactivator Pgc-1alpha) on liver transcription factor expression; 2) a strong dependence on HNF4alpha characterizes several male-predominant Cyps (2d9 and 8b1), female-predominant Cyps (2b10, 2b13, 3a41, and 3a44) and Cyps, whose expression is sex independent (3a11, 3a25); 3) HNF4alpha confers a unique, positive regulation of two male-expressed genes (Cyp4a12 and GSTpi) and a negative regulation of several female-predominant genes (Cyp2a4, Cyp2b9, Hnf3beta, and Hnf6), both of which are manifest in male but not female mouse liver. These trends were confirmed at the protein level by Western blot analysis using antibodies raised to Cyp2a, Cyp2b, and Cyp3a family members. Thus, HNF4alpha is an essential player in the complex regulatory network of liver-enriched transcription factors and the sexually dimorphic mouse Cyp genes that they regulate. HNF4alpha is proposed to contribute to the sex specificity of liver gene expression by positively regulating a subset of male-specific Cyp genes while concomitantly inhibiting the expression of certain female-specific Cyps and liver transcription factors, by mechanisms that are operative in male, but not female, mouse liver.