RESUMO
BACKGROUND: The use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22-55 years and > 55 years) and gender. METHODS: Eleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays. RESULTS: Gene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with approximately 2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF), 95% of all samples profiled fell within 1.5-2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers. CONCLUSION: This study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated expression in a subject with iron deficiency anemia and another subject being treated for lung cancer.
RESUMO
Interferon-alpha with its antiproliferative activity is widely used for the treatment of viral infections and tumor therapy such as melanoma. Naturally occurring resistance to recombinant interferon alpha-2a (IFN-alpha) and severe side effects limit the therapeutic efficacy. Understanding of the molecular mechanisms involved in unresponsiveness may therefore lead to the development of novel formulations that overcome resistance. Here, we have applied oligonucleotide DNA microarrays with probe sets for about 11,400 human transcripts to study the expression of interferon-alpha inducible genes in a sensitive and resistant melanoma cell line over multiple time points and two interferon formulations. We identified two major groups of genes with termed interferon primary response genes (IPRGs) or interferon secondary response genes (ISRGs). IPRGs are upregulated early after interferon stimulation in both the sensitive and the resistant line and they contain IREs in the noncoding regulatory region. In contrast, ISRG expression occurs preferentially in the sensitive line ME15 at late time points, and this group of genes lacks typically IREs. In addition to these two major interferon response gene classes, we identified a relatively small number of genes with complex kinetic expression modes. In addition, we show for the first time that regular and pegylated recombinant interferons are equally potent activators of interferon (IFN) gene expression. Finally, we propose that the ISRGs are activated downstream of the primary response genes by a molecule or pathway, which awaits identification, and interferon inducible gene expression is thus more complicated than previously thought.