The invisible killer of research?

Environmental stress may change the biological outcomes via signaling pathways

Ever wonder why the results of cell culture experiments are sometimes difficult to replicate? A recent study in the journal Molecular & Cellular Proteomics may shed some lights on the secrets behind the complex biological mechanisms.

Pedro Cutillas

Protein phosphorylation, a common type of post-translational modification, plays critical roles in regulating signaling pathways involved in disease progression, including cancers. With the advancement of mass-spectrometry technology, phosphoproteomics now enables scientists to map in depth the delicate changes of protein biomarkers and allows them to interrogate important research questions. However, a team led by Pedro Casado and Pedro Cutillas at Barts Cancer Institute in London discovered that cells are extremely susceptible to environmental stress stimuli, including temperature, and consequently elicit numerous protein modifications that may lead to errant data interpretation.
The team tested the MCF7 breast cancer cell line in room temperature and on ice and then monitored the changes by mass spectrometry. Nearly 1.5 percent of 3,500 phosphorylation sites measured were changed after leaving the cells in room temperature for 15 minutes. In addition, while the effects were delayed compared with those in room temperature, maintaining cells on ice did not prevent the cells from responding to metabolic stress. The researchers found that ambient conditions at room temperature stimulated catabolic pathways involving AMPK and GSK3beta and inactivated anabolic pathways regulated by AKT, ERK and mTOR.
Autophagy also was induced by the environmental stress in this experiment. Cutillas’ team used immunofluorescence microscopy to measure two common autophagic markers, WIPI2 and LC3. After two hours of exposure to room-temperature conditions, the number and size of the markers increased dramatically. The protein assay also confirmed the increased phosphorylation of numerous other autophagy markers.
This study raises an interesting question not frequently asked by the scientific community: Is it a real biology response, or is it a response caused by sample retrieval? The discoveries made by the Cutillas group provide another angle from which to interpret research data more carefully.

Shirley TanShirley H. Tan ( is a postdoctoral research fellow at Johns Hopkins School of Medicine.