Identification of genes overexpressed in head and neck squamous cell carcinoma using a combination of complementary DNA subtraction and microarray analysis.
OBJECTIVE
To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers and vaccine candidates.


METHODS
Molecular biological analysis of fresh-frozen head and neck squamous cell cancer (HNSCC).


METHODS
A subtractive library was made from two HNSCC and six normal tissues using a polymerase chain reaction (PCR)-based approach. Genes from this library were PCR amplified and placed on a microarray glass slide. RNA was prepared or obtained from 16 fresh-frozen HNSCC and 22 normal tissue sources. Fluorescent probes were made from the polyA+ RNA derived from the tumor and normal tissues. The probes were hybridized to the glass slides and excited by a tuneable laser. One hundred seven of the genes showing the highest differential fluorescence value between tumor and normal tissue were identified by sequence analysis.


RESULTS
Thirteen independent genes were found to be overexpressed in tumor tissues. Of these, nine were previously known: keratins K6 and K16, laminin-5, plakophilin-1, matrix metalloproteinase-2 (MMP), vascular endothelial growth factor, connexin 26, 14-3-3 sigma, and CaN19. The level of polyA+ RNA of these genes in the tumors was significantly different from the levels in normal tissue (P < .05). Four previously unidentified genes were also discovered to have increased expression in tumor tissue. Comparing the total tumor group (n = 16) to the normal group (n = 22), only one of these genes showed significant overexpression.


CONCLUSIONS
We report the identification of nine known genes that are significantly overexpressed in HNSCC as compared to normal tissue using subtractive and microarray technology. In addition, we present four previously unidentified genes that are overexpressed in a subset of tumors. These genes will be developed as tumor markers and vaccine candidates.
Villaret
D B
DB
Department of Otolaryngology-Head and Neck Surgery, University of Florida, Gainesville 32610, USA.
Wang
T
T
Dillon
D
D
Xu
J
J
Sivam
D
D
Cheever
M A
MA
Reed
S G
SG
D048948:14-3-3 Proteins; D000293:Adolescent; D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000235:genetics; D019496:Cancer Vaccines / Q000235:genetics; D002294:Carcinoma, Squamous Cell / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000235:genetics; D002630:Chemotactic Factors / Q000235:genetics; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics; D015342:DNA Probes; D018076:DNA, Complementary / Q000235:genetics; D016228:Endothelial Growth Factors / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D015723:Gene Library; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007633:Keratins / Q000235:genetics; D007834:Lasers; D008222:Lymphokines / Q000235:genetics; D020778:Matrix Metalloproteinase 2 / Q000235:genetics; D008875:Middle Aged; D008967:Molecular Biology; D051191:Plakophilins; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D009418:S100 Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D014446:Tyrosine 3-Monooxygenase; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000097794:Kalinin
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D019496:Cancer Vaccines; D015815:Cell Adhesion Molecules; D002630:Chemotactic Factors; D000072259:Connexin 26; D017630:Connexins; D015342:DNA Probes; D018076:DNA, Complementary; D016228:Endothelial Growth Factors; D007633:Keratins; D008222:Lymphokines; D020778:Matrix Metalloproteinase 2; D051191:Plakophilins; D011506:Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D009418:S100 Proteins; D014446:Tyrosine 3-Monooxygenase; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D000097794:Kalinin; C495335:PKP1 protein, human; C098997:S100A2 protein, human