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Loss of chromosome 13q regions in esophageal squamous cell carcinoma (ESCC) is a frequent event. Monochromosome transfer approaches provide direct functional evidence for tumor suppression by chromosome 13 in SLMT-1, an ESCC cell line, and identify critical regions at 13q12.3, 13q14.11, and 13q14.3. Differential gene expression profiles of three tumor-suppressing microcell hybrids (MCH) and their tumorigenic parental SLMT-1 cell line were revealed by competitive hybridization using 19k cDNA oligonucleolide microarrays. Nine candidate 13q14 tumor-suppressor genes (TSG), including RB1, showed down-regulation in SLMT-1, compared with NE1, an immortalized normal esophageal epithelial cell line; their average gene expression was restored in MCHs compared with SLMT-1. Reverse transcription-PCR validated gene expression levels in MCHs and a panel of ESCC cell lines. Results suggest that the tumor-suppressing effect is not attributed to RB1, but instead likely involves thrombospondin type I domain-containing 1 (THSD1), a novel candidate TSG mapping to 13q14. Quantitative reverse transcription-PCR detected down-regulation of THSD1 expression in 100\% of ESCC and other cancer cell lines. Mechanisms for THSD1 silencing in ESCC involved loss of heterozygosity and promoter hypermethylation, as analyzed by methylation-specific PCR and clonal bisulfite sequencing. Transfection of wild-type THSD1 into SLMT-1 resulted in significant reduction of colony-forming ability, hence providing functional evidence for its growth-suppressive activity. These findings suggest that THSD1 is a good candidate TSG.

In up to 87\% of nasopharyngeal carcinoma (NPC) clinical tumor specimens, there was either down-regulation or loss of TSLC1 gene expression. Using a tissue microarray and immunohistochemical staining, the frequency of down-regulated or loss of expression of TSLC1 in metastatic lymph node TNPC was 83\% and the frequency of loss of expression of TSLC1 was 35\%, which was significantly higher than that in primary NPC (12\%). To examine the possible growth-suppressive activity of TSLC1 in NPC, three NPC cell lines, HONE1, HNE1, and CNE2, were transfected with the wild-type TSLC1 gene cloned into the pCR3.1 expression vector; a reduction of colony formation ability was observed for all three cell lines. A tetracycline-inducible expression vector, pETE-Bsd, was also used to obtain stable transfectants of TSLC1 There was a dramatic difference between colony formation ability in the presence or absence of doxycycline when the gene is shut off or expressed, respectively, with the tetracycline-inducible system. Tumorigenicity assay results show that the activation of TSLC1 suppresses tumor formation in nude mice and functional inactivation of this gene is observed in all the tumors derived from tumorigenic transfectants. Further studies indicate that expression of TSLC1 inhibits HONE1 cell growth in vitro by arresting cells in G(0)-G(1) phase in normal culture conditions, whereas in the absence of serum, TSLC1 induced apoptosis. These findings suggest that TSLC1 is a tumor suppressor gene in NPC, which is significantly associated with lymph node metastases.

Esophageal squamous cell carcinoma (ESCC) is the eighth most common cancer worldwide. Geographically, its incidence is unevenly distributed throughout the world and the current prognosis for this cancer is poor. Genetic alterations of tumor suppressor genes (TSGs) are central to tumor development. In the present study, the role of a known TSG was studied in esophageal cancer. p16INK4a, a G1-specific cell cycle regulatory gene located on chromosome 9p21, shows frequent loss of heterozygosity (LOH) in many human malignant tumors. It has been found to have a high alteration frequency including promoter hypermethylation, deletion, and mutation among the ESCC cell lines and primary tumors, which is suggestive that p16INK4a is playing a critical role in tumor growth through the p16INK4a/pRb pathway in ESCC. To elucidate the genetic status and role of p16INK4a in three ESCC cell lines, HKESC-2, T-Tn6/TSIS2V, and SLMT-1S9, a series of p16INK4a molecular analyses including polymerase chain reaction (PCR), methylation-specific PCR (MSP), reverse transcription-PCR (RT-PCR), Western blot, and transfection assays were performed. The p16INK4a gene was inactivated by promoter hypermethylation and deletion in all three cell lines. No p16INK4a RNA or protein expression was detected in the ESCC cell lines. HKESC-2 and T-Tn6/TSIS2V were transfected with a full-length p16INK4a cDNA. The growth of p16INK4a transfected cells was significantly reduced in both cell lines, illustrating the functional significance of the gene in cell cycle progression. These studies should help us better understand the key role of p16INK4a in ESCC development.