Arthur Zelent

The effect of antisense inhibition of urokinase receptor in human squamous cell carcinoma on malignancy

Concomitant expression of urokinase type plasminogen activator (uPA) and its surface receptor (uPAR) has been shown to correlate strongly with a more invasive tumor cell phenotype. A highly malignant human epidermoid carcinoma cell line (HEp3) was transfected with a vector capable of expressing an antisense transcript complementary to 300 bases of the 5′ end of uPAR, including the ATG codon. Six stably transfected antisense (AS‐2, 3, 5, 9, 10, 12) and eight control clones were characterized. All clones produced high levels of uPA activity. Examination of collagenase production and doubling time showed that all of the clones tested produced similar activities. The antisense clones showed a 20‐74% reduction in the uPAR sites; the uPAR mRNA level was also reduced. A test of the invasive ability of all clones in a modified chorioallantoic membrane (CAM) showed that invasiveness of the antisense‐inhibited clones was directly proportional to the density of surface uPAR. The AS‐2 clone, which expressed the lowest number of uPARs showed a significantly reduced level of invasion. The invasiveness of additional AS‐inhibited clones was also reduced. Seven control and four AS‐inhibited clones were tested for tumorigenicity on CAMs of chick embryos. Inoculation of control cells produced large tumors, while the As clones were non‐tumorigenic. AS‐2 did not produce tumors even if kept in vivo for up to 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of protein-binding sites in the hepatitis B virus enhancer and core promoter domains

We have investigated the role of liver-specific trans-acting factor(s) in the regulation of hepatitis B virus (HBV) gene expression. A recorder plasmid (pEcoAluCAT; HBV nucleotides 1 through 1878) was constructed containing the HBV enhancer and the promoter region of the pregenomic RNA, which was ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene. Upon transfecting this plasmid into various cell lines, the CAT gene was expressed only in cells of liver origin. Moreover, competition cotransfections with pEcoAluCAT and plasmids containing HBV enhancer sequences in human hepatoblastoma-derived HepG2 cells indicated the presence of titratable trans-acting factor(s) in these cells. Gel mobility shift assays using HBV enhancer and core promoter domains confirmed the existence of sequence-specific DNA-binding proteins in liver cell nuclear extract which bound to these regions. These binding sites encompass 17- and 12-nucleotide palindromes in the HBV enhancer and core promoter domains, respectively, when mapped by the methylation interference assay.