Proteins upregulated by mild and severe hypoxia in squamous cell carcinomas in vitro identified by proteomics.
BACKGROUND
Solid malignant tumours are characterised by an inadequate vascular system, which can give rise to micro-regional hypoxic areas. As the negative impact of tumour hypoxia is believed largely to depend on dynamic changes in gene expression, it is important to identify the genes regulated by hypoxia to further enlighten the biology behind the cellular response to hypoxia. Previous studies have demonstrated that hypoxia has an impact not only on the gene transcription, but also on gene-specific mRNA translation. Therefore, proteomics is a suitable approach to understand the complexity of gene regulation under hypoxia at protein level. In this in vitro study we have studied the proteome of cells under intermediate hypoxia (1% O2) and anoxia and compared these to normoxic (21% O2) cells to identify proteins upregulated by mild and severe hypoxia.


METHODS
A human cervix cancer cell line (SiHa) and a human head and neck cancer cell line (FaDu(DD)) were used. Total cell lysate from hypoxic and normoxic cells was separated by 2-dimensional gel electrophoresis, and images were analysed using Quantity One software. Proteins from significant spots (difference in intensity by more than a factor 2) were identified by Liquid chromatography-mass spectrometry (LC-MS/MS). In order to confirm the hypoxic regulation of the identified proteins, immunoblotting and qPCR were employed when possible.


RESULTS
All together 32 spots were found to be upregulated in the hypoxic gels. Of these, 11 different proteins were successfully identified and largely confirmed by Western blotting and qPCR. Amongst these proteins are protein disulfide isomerase family A, member 6 (PDIA6) and dynein light chain roadblock-type 1 (DynLRB1). Both 2D gels and Western blots revealed that PDAI6 exhibited a cell line specific pattern; in FaDu(DD) there was upregulation at 1% and further upregulated at 0% compared to atmospheric air, whereas there was no upregulation in SiHa cells. DynLRB1 was found to be upregulated in FaDu(DD) at both 1% and 0% oxygen.


CONCLUSIONS
The upregulated proteins observed in this study are involved in different cellular processes, as regulators of both cell metabolism and stress response, and in cell migration and cell division. All of which may contribute to cell survival and adaptation during oxygen starvation.
Sørensen
Brita Singers
BS
Department of Experimental Clinical Oncology, Aarhus University Hospital, Denmark. bsin@oncology.dk
Horsman
Michael R
MR
Vorum
Henrik
H
Honoré
Bent
B
Overgaard
Jens
J
Alsner
Jan
J
D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000503:physiopathology; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000503:physiopathology; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011336:Probability; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D012016:Reference Values; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000378:metabolism; D015854:Up-Regulation; D002583:Uterine Cervical Neoplasms / Q000235:genetics* / Q000503:physiopathology
D009363:Neoplasm Proteins; D020543:Proteome; D010100:Oxygen