Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) shows high frequency and mortality in Asian regions, including China. Previous analysis of genomic DNA of ESCC using comparative genomic hybridization indicated that amplification of the chromosome 5p regions is a common event in ESCC cell lines and patient cases of Hong Kong Chinese origin, and the results suggested that the genes located in the chromosome 5p regions may play crucial roles in the molecular pathogenesis of ESCC. Our previous studies on ESCC confirmed the tumorigenic and overexpression properties of a novel gene JS-1 located in chromosome 5p15.2 upstream to delta-catenin. In the present study, another novel gene JK-1 which is located at 5p15.1 downstream to delta-catenin was characterized for its roles in the pathogenesis of ESCC. Thirteen ESCC cell lines and 30 surgical specimens of esophageal tumors were studied for the overexpression of JK-1 using multiplex RT-PCR analysis. The transforming capacity of overexpression of JK-1 was also investigated by transfecting NIH 3T3 and HEK 293 cells with the expression vector cloned with JK-1, followed by the soft agar and foci formation assays. JK-1 was overexpressed in 9/13 (69%) of the ESCC cell lines and 9/30 (30%) of the ESCC patient cases. Both NIH 3T3 and HEK 293 cells acquired the properties of anchorage-dependent and -independent growth when JK-1 was overexpressed. Most significantly, subcutaneous sarcomas were formed in all (3/3) the athymic nude mice after NIH 3T3 cells overexpressing JK-1 were injected subcutaneously. Our results thus indicated that JK-1 is commonly overexpressed in ESCC and has a prominent capacity to transform normal cells. Our overall results thus provide the first evidence that the overexpression of JK-1 and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC.

Inhibitory effects of Gleditsia sinensis fruit extract on telomerase activity and oncogenic expression in human esophageal squamous cell carcinoma.

Previous studies have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) exhibited apoptotic properties in various solid and non-solid tumors in vitro. However, the inhibitory actions of GSE on oncogenic expression and telomerase activity in esophageal squamous cell carcinoma (ESCC) have not been studied before. In the present study, the anti-cancer effects of GSE were demonstrated in three ESCC cell lines (HKESC-1, HKESC-2 and SLMT-1) by MTS and anchorage-independent clongen-icity assays, expression studies on oncogenes at 11q13 (CCND1, INT2, FGF4 and EMS1) and real-time quantitative telomeric repeat amplification protocol assay to show the inhibitory effect of GSE on telomerase in ESCC. The means of MTS50 of GSE for the ESCC cell lines and non-tumor NIH 3T3 cells were 21 and 163 microg/ml respectively. The anchorage-independent clongenicity assay showed that SLMT-1 cells lost their colony-forming potential which was dose-dependent to GSE. Moreover, GSE demonstrated dose-dependent suppression on the expression of INT2, EMS1 and FGF4, and inhibition of telomerase activity in the ESCC cell lines. Our overall results thus provide the first evidence that the anti-cancer effects of GSE on ESCC involve the suppression of oncogenic expression and inhibition of telomerase activity. Our findings also offer a new opportunity for the future development of GSE as a novel anti-cancer agent for ESCC and possibly for other cancers.

Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

Observer variation in the diagnosis of follicular variant of papillary thyroid carcinoma.

The histopathologic diagnosis of follicular variant of papillary thyroid carcinoma (FVPCA) can be difficult. Recent reports have suggested that this neoplasm may be frequently overdiagnosed by pathologists. We examined the observer variation in the diagnosis of FVPCA in 87 tumors by 10 experienced thyroid pathologists. The criteria that the reviewers considered most helpful for making a diagnosis of FVPCA were also assessed. A concordant diagnosis of FVPCA was made by all 10 reviewers with a cumulative frequency of 39%. In this series, 24.1% of the patients had metastatic disease (n = 21). In the cases with metastatic disease, a diagnosis of FVPCA was made by all 10 reviewers with a cumulative frequency of 66.7%, and 7 of the reviewers made a diagnosis of FVPCA with a cumulative frequency of 100%. The most important criteria used to diagnose FVPCA included the presence of cytoplasmic invaginations into the nucleus (pseudo-inclusions), abundant nuclear grooves, and ground glass nuclei. These results suggest that although the diagnosis of FVPCA is variable even among experienced thyroid pathologists, most reviewers agreed on this diagnosis for patients with metastatic disease. The use of well-defined histopathologic features should improve the consistency in diagnosing FVPCA. Since most cases with metastatic disease had obvious invasion, caution should be used in making a diagnosis of FVPCA in the absence of the major histopathologic features or clear-cut invasive growth.

Establishment, characterization, karyotyping, and comparative genomic hybridization analysis of HKESC-2 and HKESC-3: two newly established human esophageal squamous cell carcinoma cell lines.

The establishment of esophageal cancer cell lines can facilitate the search for molecular mechanisms underlying its pathogenesis. Two novel human esophageal squamous cell carcinoma (ESCC) cell lines, HKESC-2 and HKESC-3, were established from a moderately differentiated ESCC of a 46-year-old Chinese woman and a well-differentiated ESCC of a 74-year-old Chinese man, both from Hong Kong. The pathological characteristics (morphological, immunohistochemical, and electron microscopic studies), tumorigenicity in nude mice, cytogenetic features, and DNA ploidy of the two cell lines were investigated. The two cell lines have been maintained in vitro for more than 17 months and passaged over 85 times for HKESC-2 and 58 times for HKESC-3. Both grew as monolayers, with a doubling time of 24 hours for HKESC-2 and 48 h for HKESC-3. Their squamous epithelial nature was authenticated by their strong immunopositivity with the anti-cytokeratin antibodies and the ultrastructural demonstration of tonofilaments and desmosomes. They are tumorigenic in nude mice and had DNA aneuploidy. G-banding cytogenetic analysis showed hyperdiploidy in HKESC-2 and near-tetraploidy in HKESC-3. Frequent breakpoints were noted at 1p22, 1p32, and 9q34 in HKESC-2 and at 1p31, 3p25, 3p14, 6q16, 6q21, 8p21, 9q34, 13q32, and 17q25 in HKESC-3. Comparative genomic hybridization analysis found that chromosomal gains were at 3q24-qter, 5q21-qter, 8q11-qter, 13q21-q31, 17q11-qter, 19, 22q22 for HKESC-2 and at 3q13-qter, 5p, 6p, 9q21-qter, 10q21-q22, 12q15-pter, 14q24-qter, 16, 17q24-qter, 20 for HKESC-3. Chromosomal losses were at 3p13-pter, 18q12-qter for HKESC-3. These two newly established cell lines will be useful tools in the study of the molecular pathogenesis and biological behavior of ESCC cells and for testing new therapeutic reagents for ESCC in the future.