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Previous deletion analysis of the 5'-flanking region of human GnRH receptor (GnRHR) gene has revealed a powerful negative regulatory element (NRE) located between nucleotide -1017 and -771. In the present study, we demonstrated that this NRE could repress the homologous promoter, irrespective of its position and completely abolish the activity of a heterologous thymidine kinase promoter in an orientation-dependent manner. Progressive 3'-deletion analysis revealed that most of the silencing activity of the NRE resided in a putative octamer regulatory sequence (5'AAGCAAACT3'), which alone could repress the promoter activities by 69-90% in ovarian OVCAR-3, placental JEG-3, and gonadotrope-derived alphaT3-1 cells. Mutation of the AAAC residues of the octamer sequence completely removed its silencing activity. Interestingly, conversion of the octamer sequence into that of the rodent GnRHR promoter (5'AAGCAAAGT3') did not attenuate its silencing effect, indicating that the repressive role of the octamer sequence is evolutionarily conserved. EMSAs showed that common DNA-protein complexes of the same mobility were formed with nuclear extracts from the reproductive cells and gonadotropes, and a consensus octamer transcription factor-1 (Oct-1) binding sequence could dose dependently inhibit the complex formation. Antibody supershift and Southwestern blot assays confirmed that the protein binding to the octamer sequence was the ubiquitously expressed transcription factor Oct-1. Overexpression of Oct-1 augmented the silencing activity of the octamer sequence in alphaT3-1 cells. Taken together, our results clearly indicate a role of Oct-1 in the transcriptional repression of the human GnRHR gene.

GnRH has been implicated as an important local autocrine and paracrine factor in regulating ovarian function. However, to date, the transcriptional regulation of GnRH receptor (GnRHR) gene in human ovary remains poorly understood. Here we report the characterization of a new upstream promoter for the GnRHR gene in human granulosa-luteal cells. Using progressive deletion analysis, a region between nucleotide -1300 and -1018 (relative to the translation start site) was shown to exhibit the highest promoter activities in two immortalized human granulosa-luteal cell lines, SVOG-4o and SVOG-4m. Two putative CCAAT/enhancer binding protein (C/EBP) motifs and one GATA motif were identified within this region. Mutational studies showed that these three motifs cooperated synergistically to regulate GnRHR gene transcription in the granulosa cells but not in other cell types including human ovarian carcinoma OVCAR-3, human embryonic kidney-293 (HEK-293) and mouse pituitary gonadotrope-derived alphaT3-1 cells. Surprisingly, by competitive EMSAs, we found that an Oct-1 consensus sequence was able to inhibit protein complex formation with the distal C/EBP motif, suggesting a possible cross-talk between the Oct-1 transcription factor and this C/EBP motif. Taken together, our results strongly indicate a role of the C/EBP and GATA motifs in regulating GnRHR gene transcription in human granulosa-luteal cells and further suggest that tissue-specific expression of human GnRHR gene is mediated by differential promoter usage.

CONCLUSION:We established and characterized a colorectal cancer cell line, LST-R1 and LST-R1 has an obvious malignant tendency, which maybe partially attributed to the changes of the expression of some adhesion molecules, such as E-cadherin. It is also a versatile tool for exploring the original and progressive mechanisms of laterally spreading tumor and the early colon cancer genesis. INTRODUCTIONTwo routes have been described that can lead to colon carcinogenesis: one is the adenoma-carcinoma model, which is the main carcinogenetic pathway of prudent colorectal tumor and has been widely accepted in the west. However, an increasing number of superficial or flat colorectal tumors have been reported, particular in Japan [1,2] . The superficial tumors are considered to have a distinct clinicopathologic, genetic feature and behave a different carcinogenetic pathway, called de novo pathway [3][4][5][6] . Laterally spreading tumor (LST) is a unique subtype of superficial colorectal tumor, which was first reported by Kudo S in 1993 [7,8] and many names such as granular cluster type, nodule-aggregating tumor, creeping tumor, superficial spreading (epithelial) tumor, flower-bed-like lesion had been used. In 1998, it was defined as tumors originated from the colorectal mucous membranes and mainly extends laterally rather than vertically with its diameter greater than 1 cm ( Figure 1A) [9][10][11][12] . It has a tendency to affect the rectum, sigmoid colon, and caecum. In some studies, it has been reported that the carcinogenesis rate of LST is 8.64%-52.5% [13][14][15] . Abstract AIM: To study the molecular mechanism of laterally spreading tumor (LST), a cell line [Laterally Spreading Tumor-Rectum 1 (LST-R1)] was derived and the characteristics of this cell line were investigated. BASIC RESEARCH METHODS:A new cell line (LST-R1) originated from laterally spreading tumor was established. Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments. In vitro invasion assay, cDNA microarray and Western blotting were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer. RESULTS:Our study demonstrated that both epithelial special antigen (ESA) and cytokeratin-20 (CK20) were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability. cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells.