THY-1 induction is associated with up-regulation of fibronectin and thrombospondin-1 in human ovarian cancer.

We recently identified THY-1 as a putative tumor suppressor gene for human ovarian cancer. To understand the carcinogenic role of THY-1, and its downstream effects on cancer cells, a THY-1 inducible system was established in the human ovarian cancer cell line SKOV-3 based on the tetracycline (tet) regulating system. To establish an inducible system for Thy-1 expression, two plasmids, pUHD172-1neo and pTEP4mThy-1, which are neomycin and hygromycin resistant were co-transfected into the ovarian carcinoma cell line, SKOV-3. The inducibility of Thy-1 expression in the SKOV-3 cell line by doxycycline (dox) was determined by northern blot analysis, immunocytochemistry, and flow cytometry. A time course study revealed that Thy-1 expression is induced 3 hours post dox exposure. Expression was reversible such that 12 hours post the removal of dox almost no Thy-1 could be detected. Furthermore, 2 genes, Fibronectin (FN) and Thrombospondin (TSP-1) involved in cellular differentiation and the regulation of tumor angiogenesis, respectively, were found to be up-regulated upon THY-1 induction. In contrast, the gene SPARC was found to be independent of Thy-1 expression. This study supports the hypothesis that THY-1 plays a critical role in regulating downstream genes associated with the regulation of ovarian tumor growth and cellular differentiation.

The role of the THY1 gene in human ovarian cancer suppression based on transfection studies.

In our recent studies, the expression of the THY1 gene encoding a 25-28 kDa glycoprotein located at 11q23-q24, was found to be associated with complete tumor suppression of the ovarian cancer cell line SKOV-3 after the transfer of chromosome 11. These studies raised the possibility that THY1 maybe a candidate tumor suppressor gene for ovarian cancer. To investigate this, the complete cDNA sequence for THY1 was cloned and transfected into SKOV-3 ovarian cancer cells. The expression of THY1 in the transfectants was confirmed by Northern blot analysis, immunocytochemistry, and flow cytometry. Both SKOV-3-THY1 and SKOV-3-null cells were inoculated subcutaneously into severe combined immunodeficiency (SCID) mice to determine in vivo tumorigenicity. THY1 transfectants formed tumors, but overall tumor growth rate and tumor size was significantly reduced compared with their null counterparts. To further correlate THY1 expression with tumorigenicity, the THY1 antisense was transfected into the nontumorigenic clone, 11(C)9-8, which resulted in restoration of tumorigenicity. These data indicate that THY1 expression alone cannot suppress tumorigenicity; however, abrogation of THY1 expression from nontumorigenic cells can restore tumorigenesis. Taken together, the data suggest that THY1 is necessary but not sufficient to suppress ovarian tumorigenicity. Therefore, THY1 can be designated as a putative tumor suppressor gene for human ovarian cancer.

Suppression of HIV-1 viral replication and cellular pathogenesis by a novel p38/JNK kinase inhibitor.

OBJECTIVE: To analyze a novel compound, which inhibits serine-threonine protein kinase p38, for its possible bioactivity against HIV-1 infection. METHODS: Proteins involved in cellular signal transduction pathways represent a novel class of host therapeutic targets for infectious diseases. In this regard the serine/threonine kinase p38 MAPK, a member of the mitogen-activated protein (MAP) kinase superfamily of signal transduction molecules may play an important role in HIV-1 infection. We analyzed the ability of this compound (RWJ67657) to inhibit HIV replication in primary T cells and monocytes. Cellular expression of phospho-p38MAPK was studied by Western blot analysis. Blockade of HIV infection induced apoptosis was measured by Annexin V staining. RESULTS: p38 inhibitor RWJ67657 was effective in inhibiting HIV-1 replication in both T-cell and monocyte cell lines, irrespective of the coreceptor used by the virus for entry into the cell. Importantly, both reverse transcriptase and protease resistant escape mutant viruses were effectively suppressed by RWJ67657. In addition, the tested compounds block HIV-induced T-cell apoptosis, a critical means of T-cell depletion linked to AIDS progression. CONCLUSION: Several steps in the HIV-1 virus life cycle appear to depend on cellular activation, including activation of the p38 pathway. Without activation virus replication is thought to be blocked due to incomplete reverse transcription and a lack of proviral DNA integration. The data collectively illustrate that inhibition of the p38 pathway can affect HIV-1 replication. Interruption of HIV infection by p38 inhibitors underscores the value of exploring antiviral drugs that target host cellular proteins.

THY1 expression is associated with tumor suppression of human ovarian cancer.

Microcell-mediated transfer of chromosome 11 into the human ovarian cancer cell line SKOV-3 results in suppression of tumorigenicity in severe combined immunodeficiency (SCID) mice. To identify the differentially expressed transcripts associated with suppression of tumorigenicity, cDNA populations from the slow-growing tumorigenic clone 11(H)8-3, tumorigenic clone 11(H)8-4, and parental SKOV-3 cells were subtracted from the nontumorigenic clones, 11(H)7-2 and 11(C)9-8. The subtracted cDNA populations were either cloned, sequenced and searched in GenBank, or analyzed by gene discovery array screening. A cDNA transcript corresponding to the THY1 gene located at chromosome 11q23 approximately q24 was found to be exclusively expressed in the two nontumorigenic cell clones. In contrast, THY1 expression was not detected in SKOV-3, the tumorigenic hybrid clones, or six other tumorigenic ovarian cancer cell lines. Further analysis using immunocytochemistry and quantitative flow cytometry with a Thy-1-specific antibody confirmed the exclusive expression of THY1 at the protein level in the two nontumorigenic clones. Several cell growth and differentiation-related genes, including thrombospondin 1 (THBS1), SPARC [secreted protein, acidic, cysteine-rich (osteonectin)], and fibronectin (FN1) were also found to be upregulated in the nontumorigenic clones; however, these were expressed in the slow-growing tumorigenic clones as well. Expression of these genes was not observed in the parental SKOV-3 cell line and therefore must be regulated by a gene or genes on chromosome 11. Our results suggest that THY1 is a putative tumor suppressor gene for ovarian cancer and that THBS1, SPARC, and FN1 are genes associated with the regulation of in vivo tumor growth rate.

Comparison of endothelin-1-mediated tissue tension and calcium mobilization effects in isolated rabbit corpus cavernosum.

OBJECTIVES: To directly compare and contrast the effects of endothelin-1 (ET-1) and adrenoreceptor agonists norepinephrine and phenylephrine on eliciting calcium influx in primary rabbit corpus cavernosum cells and their ability to elicit tissue contractions. The potent vasoconstrictor peptide ET-1 and the alpha-adrenoreceptor agonists are important modulators of smooth muscle tone in the penile corpus cavernosum. However, the mechanisms involved in maintaining smooth muscle tone and contraction are not clearly understood. METHODS: Intracellular calcium mobilization was measured in cultured corpus cavernosum smooth muscle cells using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader. Tissue tension studies on rabbit corpus cavernosum were conducted using organ chambers. RESULTS: ET-1 at concentrations as low as 10 nM was sufficient to induce a transient increase of intracellular calcium in these cells. In contrast, concentrations of 1 mM and greater of norepinephrine and phenylephrine were required to elicit comparable calcium fluxes in cavernosum cells. Tissue bath studies indicated that ET-1 is a potent stimulator of corpus cavernosum smooth muscle contraction, with concentrations as low as 10 nM sufficient to initiate contraction. CONCLUSIONS: The potency of ET-1 in producing contraction on tissue strips and inducing calcium flux suggests that ET-1 might be an important mediator for modulating and maintaining corpus cavernosum smooth muscle tone. Therefore, additional exploration of the role of endothelins and their receptors in the tumescence and detumescence states of the penis would be extremely valuable.