Laboratory variability does not preclude identification of biological functions impacted by hydroxyurea.
The multi-lab International Life Sciences Institute (ILSI) project on the application of genomics to risk assessment offered the unique opportunity to investigate the influence of variability within and between laboratories on identifying biologically relevant gene expression changes. We assessed the gene expression profiles of mouse lymphoma L5178Y cells treated with hydroxyurea (HU) in three independent studies from two different laboratories, Sanofi Aventis and Procter and Gamble. Cells were dosed for 4 hr and harvested immediately at the end of the treatment or after a 20-hr recovery period. Cytotoxicity and genotoxicity were evaluated by standard assays. Statistical analysis of these data revealed that, while gene expression responses to HU treatment were markedly different at 4 hr vs. 24 hr, there was otherwise a consistent pattern of dose-response across the three studies. Therefore, the studies were merged and each time point was evaluated separately. At 4 hr, we identified 173 (P < 0.0001) dose-responsive genes with a common trend in all three studies. These were mainly associated with the cell cycle, DNA repair and DNA metabolism, and in particular, the intra-S and G2/M phase checkpoints. At 24 hr, we identified 434 dose-responsive genes common across studies. These genes were involved in lymphocyte-specific activities and the activation of apoptosis via the caspase cascade. Our results show that despite inter-laboratory variability, combining the three studies in a single statistical analysis identifies more significantly-modulated genes than in any of the individual studies, due to improved statistical sensitivity. The genes identified in our study provide information that is relevant to HU biology.
Müller
Arne
A
Drug Safety Evaluation, sanofi aventis, Vitry-Sur-Seine, France.
Boitier
Eric
E
Hu
Ting
T
Carr
Gregory J
GJ
Le Fèvre
Anne-Céline
AC
Marchandeau
Jean-Pierre
JP
Flor
Manoli
M
Jefferson
Felicia
F
Aardema
Marilyn J
MJ
Thybaud
Véronique
V
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D019411:Clinical Laboratory Techniques / Q000592:standards* / Q000706:statistics & numerical data; D004305:Dose-Response Relationship, Drug; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006918:Hydroxyurea / Q000633:toxicity*; D007940:Leukemia L5178; D051379:Mice; D008954:Models, Biological; D009152:Mutagenicity Tests; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D006918:Hydroxyurea