Tissue factor pathway inhibitor-2 (TFPI-2), a serine protease inhibitor abundant in the extra cellular matrix, is highly expressed in non-invasive cells but undetectable levels in highly invasive human glioma cells. The mechanisms responsible for its transcriptional regulation are not well elucidated. In this study, we made several deletion constructs from a 3.6 kb genomic fragment from Hs683 cells containing the 5'-¯anking region of the TFPI-2 gene, transiently transfected with these constructs into non-invasive (Hs683) and highly invasive (SNB19) human glioma cells, and assessed their expression by using a luciferase reporter gene. Three constructs showed high promoter activity (pTF5, 7670 to +1; pTF6, 7312 to +1; pTF2, 71511 to +1). Another construct, pTF8 (781 to +1), showed no activity. PTF9, a variant of pTF5 in which a further 231 bp fragment (7312 to 781) was deleted, from the [7670 to +1] pTF5 region, also showed no promoter activity. Hence, (7312 to 781) this region is essential for the transcription of TFPI-2 in glioma cells. Sequencing of this promoter region revealed that it has a high G+C content, contains potential SP1 and AP1 binding motifs, and lacks canonical TATA and CAAT boxes immediately upstream of the major transcriptional initiation site, although CAAT boxes were found about 73000 bp upstream of the transcription start site. We also found a strong repressor in the region between 7927 to ± 1181, upstream of the major transcriptional initiation site, followed by positive elements or enhancers between ± 1511 to 71181. These positive elements masked the silencer eect. Finally TFPI-2 was induced in Hs683 cells transfected with the pTF6 construct (7312 to +1) and stimulated with phorbol-12-myristate-13-acetate (PMA). We conclude that the 7312 to +1 region is critical for the minimal and inducible regulation of TFPI-2 in non-invasive (Hs683) and highly invasive (SNB19) human glioma cell lines. Oncogene (2002) 21, 921 ± 928.
The effects of intralesional chemotherapy with 7 different drugs on line 10 hepatoma grown in strain 2 guinea pigs were compared with the sensitivity of line 10 tumor cells in vitro, using a micro modification of the tumor stem assay with capillary tubes. A modified method was used to evaluate the in vitro dose-response curves. The correlation for in vivo/in vitro resistance was found to be 100% and for in vivo/in sensitivity it was 80%.
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