We have identified a novel human gene, designated C1orf10, using modified differential display PCR. The C1orf10 gene, which spans 5 kb in length, is composed of three exons. The deduced protein contains 495 amino acids with one transmembrane domain. The amino acid sequence of C1orf10 is characterized by the presence of a calcium-binding motif of about 90 amino acids at its N-terminal and a conserved consecutive repeat sequence of 60 amino acids that was identified previously only in bacterial ice nucleation proteins. In normal adult tissues, C1orf10 is highly expressed only in the esophagus and was undetectable in a total of 15 other tissues examined, suggesting its important role in esophageal cells. The expression of C1orf10 is either dramatically reduced or absent in esophageal cancer cell lines (3/3) as well as primary esophageal cancer tissues (35/37) compared with the corresponding normal esophageal mucosa. Using a radiation hybrid panel, C1orf10 was found to be located on chromosome 1q21. These findings suggest that expression of C1orf10 is unique to esophageal cells and that loss of its expression may play a role in the development of esophageal cancer.

The development and progression of human cancer are believed to be due to the alterations of multiple genes or/ and their protein products. For identifying the proteins associated with esophageal cancer, we analysed the protein profiles of 24 pairs of esophageal squamous cell carcinomas/matched adjacent normal epithelia. Micro-dissection of routinely unstained frozen sections was performed to purify cancerous and epithelial cells. The protein expression profiles were obtained by two-dimensional electrophoresis. Selected proteins dysregu-lated in tumors were identified by MALDI-TOF-MS. Three isoforms of annexin I were detected in normal esophageal mucosa and down-regulated in esophageal squamous cell carcinomas. RT

Migration inhibitory factor-related protein 14 (MRP14) is one of calcium-binding proteins, referred as S100A9. The heterodimeric molecule formed by MRP14 with its partner MRP8 (S100A8) is the major fatty acid carrier in neutrophils. The MRP8/14 complex has been also implicated in the intracellular transport of arachidonic acid and its precursors in keratinocytes. We show here the involvement of MRP14 in human esophageal cancer. In an initial study, mRNA differential display-reverse transcription polymerase chain reaction (DD-PCR) was performed with two esophageal carcinomas, one esophageal adenocarcinoma and matched normal adjacent mucosa. DD-PCR with the arbitrary primer OPA3 showed that one cDNA band was highly expressed in normal tissues, but disappeared or substantially decreased in tumor counterparts. It was later identified to be the 3'-end of migration inhibitory factor-related protein 14 (MRP14). Northern blotting, RT-PCR and Western blotting corroborated the down-regulation of MRP14 in 58/64 squamous cell carcinomas and 2/2 adenocarcinomas as compared with adjacent normal epithelia of the esophagus. MRP14 was undetectable in 3/3 esophageal-carcinoma cell lines. Immunochemistry demonstrated that expression of MRP14 was restricted to normal esophageal epithelia. No mutation was found in the genomic DNA of the MRP14 gene by PCR and directed DNA sequencing. Our finding suggested that the reduction of MRP14 expression is a frequent event in Chinese human esophageal cancer.

Esophageal cancer is the fourth most prevalent malignancy in China. So far, the genetic events involved in esophageal cancer remain largely unknown. To identify chromosomal alterations in this disease, comparative genomic hybridization was performed on 25 primary tumors of esophageal squamous cell carcinomas. Results exhibited nonrandom copy number changes in chromosome DNA, with higher incidence in gain than in loss. The average gains and losses per patient were 7.76 and 4, respectively. The most common gains were 3q (20/25), 1q (15/25), 8q (15/25), 20p (12/25), 20q (11/25), 5p (10/25), 15q (8/25), and 9q (8/25) with two minimal amplification loci mapped to chromosomal regions of 8q24 (2 cases) and 11q13 (7 cases). High-level amplification was observed at 3q (8 cases), 5p (4 cases), and 8q (4 cases). Losses at 3p (10/25), 13q (8/25), 18q (7/25), Xp (7/25), 4 (6/25), 9p (6/25), 14q (6/25), 18p (6/25), and 21q (6/25) were identified. Remarkably, ten cases showed both loss of the entire 3p and verrepresentation of almost the whole 3q. No significant differences in stage or grade of tumor were found for DNA copy number changes. The results provided candidate regions for potential oncogenes and tumor suppressor genes related to Chinese esophageal cancer, to which further molecular studies should be addressed.

The existence of unknown tumor suppressor gene (s) other than the APC gene has been hinted on 5q for esophageal squamous cell carcinoma (ESCC). In order to define minimal deletion intervals on 5q in ESCC and investigate the potential tumor suppressor gene(s), 9 microsatellite markers scattering the region from 5q22 to 5q35 were chosen for loss of heterozygosity (LOH) analysis in 50 primary ESCC from northern China. The results showed that six cases presented coexistence of LOH for three consecutive adjacent chosen markers, suggesting a minimal deletion region covering approximately 272 kb located on 5q23 from D5S1384 to D5S1505. It was a novel deletion region that was so far never reported in ESCC. Significant higher frequencies of LOH were observed in tumors with lower pathological grade at the locus D5S820 and with lymph node metastasis at the locus D5S408. The data suggested the possibility that one or more putative candidate tumor suppressor gene (s) on 5q23 might play an important role in the development and/or progression of ESCC.

Allelic loss on chromosome 13 occurs frequently in esophageal squamous cell carcinomas. To define minimal deletion intervals and find candidate tumor suppressor gene(s), we conducted a study of loss of heterozygosity (LOH) in 59 esophageal squamous cell carcinomas from northern China using a panel of ten microsatellite markers on chromosome 13q. The results showed that 12 of 59 (20%) cases presented allelic loss in three or more consecutive adjacent chosen markers, suggesting loss of larger fragments on chromosome 13q. Two minimal deletion regions of overlap were found: one was located on band 13q12.3 from markers D13S171 to D13S267, and the other on band 13q14.1-q14.3 from markers D13S263 to D13S168. The latter was a new deletion region that was never reported in esophageal squamous cell carcinomas. More frequent LOH was observed in higher pathological grade of esophageal cancer at loci D13S171, D13S263 and in later stage of esophageal cancer at D13S263. The data suggest the possibility that one or more unknown tumor suppressor gene(s) on 13q may play an important role in the development of esophageal squamous cell carcinomas.

p63 is expressed in basal cells of squamous epithelia and many kinds of tumors. To explore the penetrance of p63 in esophageal cancer, we analyzed p63 expression in squamous cell carcinomas, adjacent dysplasia and histologically normal mucosa of the esophagus by combination of immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that the Np63 mRNA was easily detectable in all malignant and histologically normal tissues, whereas TAp63 presented extremely low or no expression. The p63 protein was highly expressed in 50 of 51 tumor tissues without significant difference in gender, age, stage and grade. Ten of 11 dysplasia exhibited strong p63 staining in all abnormal cells. Interestingly, p63 expression was observed in 96% (45/47) histologically normal epithelia adjacent to the cancerous tissues but only in 47% (14/30) mucosa far from tumors. Most of the epithelia far from tumors showed weaker staining than that adjacent to the cancerous tissues. In all the histologically normal epithelia with p63 expression, irrespective of the distance from the tumors, immunohistochemical reaction was restricted to the basal and suprabasal cell layers. Our data suggested that of Np63 is the major isotype expressed in epithelia and tumors of the esophagus. Elevated expression of p63 is probably an early event in esophageal squamous cell carcinomas, which may play a significant role in the development of the disease.

Transglutaminase-3 (TGase-3) is an enzyme with the ability to catalyze the irreversible cross-linking of peptide-bound glutamine residues either with peptide-bound lysines or with primary amines. It has been implicated in the formation and assembly of the cornified cell envelope of the epidermis, hair follicle and perhaps other stratified squamous epithelia. We show here the involvement of TGase-3 in human esophageal cancer. In an initial study, mRNA differential display was performed with 3 pairs of esophageal cancer tissues and matched normal adjacent mucosa by a 10-mer arbitrary primer and mixed anchored primers (GT15N, N 5 A, C and G). Four differentially expressed cDNA bands were consistently observed in all 3 normal tissues but barely detected in their tumor counterparts. One of them was identified to be the 3* end of TGase-3. Northern blot and dot blot analyses of 14 samples confirmed the down-regulation of TGase-3 in malignant tissues compared with normal epithelia. RT-PCR revealed that TGase-3 expression was lost in 3 esophageal carcinoma cell lines and decreased in 35/38 tumors compared with adjacent normal mucosa. Taken together, 49/52 (94.2%) esophageal tumors presented down-regulation of the gene. Our data suggest that alteration of TGase-3 expression is a common event in the development of human esophageal cancer.

cDNA fragments that were differentially expressed between human oesophageal carcinomas and matched normal adjacent mucosa were isolated using an improved mRNA differential display technique. One of them was identified as the 3-untranslated region of SPRR3 and was homologous to the esophagin cDNA. Northern blot, dot blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that SPRR3 expression was lost in three cell lines of oesophageal carcinoma and was dramatically decreased in 54 out of 57 primary oesophageal carcinomas compared with adjacent normal mucosa. Esophagin has been shown to be down-regulated in western oesophageal carcinomas. The data suggest that esophagin is probably the protein product of the gene SPRR3 and that altered mRNA expression of SPRR3/esophagin is a frequent event in the development of Chinese oesophageal cancer.

To better understand the molecular events underlying the development of oesophageal cancer, we have isolated the genes dysregulated in primary oesophageal cancer tissues using a modified differential display polymerase chain reaction (DD-PCR). In the present study, a gene designated C15orf6 was identified. The C15orf6 gene, encompassing 25 kb, is composed of 11 exons with a mRNA of 1948 bp. Database searching showed that C15orf6 was 100% homologous to the Rh type C-glycoprotein (RhCG) with the same open reading frame, but 16 bp longer than RhCG at the 50-end. The gene was highly expressed in human oesophagus, cervix, oral cavity, skin and kidney, but undetectable in the other 14 adult normal tissues examined. Northern blot, RT-PCR and western blot analysis showed that RhCG/C15orf6 was frequently lost or dramatically reduced in primary oesophageal cancer tissues (30/34) compared with the corresponding normal oesophageal mucosa. Three oesophageal-cancer cell lines tested lacked RhCG/C15orf6 expression. Immunohistochemistry revealed that in normal oesophageal tissues, RhCG/C15orf6 was mainly expressed in the plasma membrane of the epithelial cells. In addition, Rh-associated glycoprotein (RhAG) expression was also commonly silenced in both oesophageal cancer cell lines (2/3) and primary oesophageal cancer tissues (11/13). To our knowledge, this is the first time that RhAG expression has been seen in oesophageal epithelium and extends the functional role of the RhAG protein beyond the erythrocyte. These data suggest that inactivation of RhCG/C15orf6 and RhAG occurs frequently during the development of human oesophageal cancer.