Downregulation of RAD17 in head and neck cancer.
BACKGROUND
DNA repair genes play a critical role in maintaining genome stability and have been implicated in tumorigenesis. Head and neck squamous cell carcinoma (HNSCC) often shows chromosomal instability. We examined the expression of human RAD17, a DNA damage cell cycle checkpoint gene, in primary head and neck cancer tissue.


METHODS
Significance analysis of microarrays was applied to expression array results examining more than 12,000 genes in 7 samples of primary HNSCC and 6 samples of normal control oral epithelial tissue. Additional confirmation was performed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) in these samples and western blot with an additional 12 primary HNSCC and 7 normal samples, followed by loss of heterozygosity (LOH) analysis and quantitative PCR at the RAD17 locus.


RESULTS
Multiple checkpoint and DNA repair genes were downregulated in primary head and neck tumor tissue compared with normal control epithelial tissue, including hRAD17. Its Z-score and fold change were -2.5 and 0.39, respectively. The results of normalized, quantitative RT-PCR showed decreased expression of hRAD17 mRNA in tumor tissue (mean value 0.2166) when compared with normal tissue (mean value 0.3957, p < .05). Western blot demonstrated undetectable expression of hRAD17 protein in primary tumor tissue (0/12), while there was strong expression of hRAD17 protein in normal oral mucosal tissue (6/7). To determine possible mechanisms of inactivation, the hRAD17 locus at 5q13 was analyzed using microsatellite markers, showing 70% LOH in 30 primary HNSCCs. Quantitative PCR showed that RAD17 DNA copy number was decreased in the majority of head and neck tumor tissue samples.


CONCLUSIONS
Loss of hRAD17 expression occurs frequently in HNSCC, is often due to genomic deletion, and may facilitate genomic instability in HNSCC.
Zhao
Ming
M
Department of Otolaryngology-Head and Neck Surgery, Head and Neck Cancer Research Division, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
Begum
Shahnaz
S
Ha
Patrick K
PK
Westra
William
W
Califano
Joseph
J
D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D006258:Head and Neck Neoplasms / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019656:Loss of Heterozygosity; D018895:Microsatellite Repeats; D040081:Protein Array Analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018797:Cell Cycle Proteins; D012333:RNA, Messenger; C113575:Rad17 protein, human