Deborah R. Wilson

Autoregulation of the human C/EBP alpha gene by stimulation of upstream stimulatory factor binding.

The human C/EBP alpha gene promoter shares significant sequence homology with that of the mouse but has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP alpha to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately threefold (R. J. Christy, K. H. Kaestner, D. E. Geiman, and M. D. Lane, Proc. Natl. Acad. Sci. USA 88:2593-2597, 1991; K. Legraverend, P. Antonson, P. Flodby, and K. G. Xanthapoulos, Nucleic Acids Res. 21:1735-1742, 1993). Unlike its murine counterpart, the human C/EBP alpha gene promoter does not contain a cis element that binds the C/EBP alpha protein. Neither C/EBP alpha nor C/EBP beta (NF-Il-6) binds the human C/EBP alpha promoter within 437 bp. However, cotransfection studies show that C/EBP alpha stimulates transcription of a reporter gene driven by 437 bp of the C/EBP alpha promoter. Our studies show that the human C/EBP alpha protein stimulates USF to bind to a USF consensus element within C/EBP alpha promoter and activates it by two- to threefold. We propose that the human gene employs the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP alpha promoter was abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP beta following transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a trans-acting factor that is expressed in all cell types. Thus, direct binding of the C/EBP alpha protein to the promoter of the C/EBP alpha gene is not required for autoregulation.

Demonstration of Epstein-Barr virus in primary central nervous system lymphomas by the polymerase chain reaction and in situ hybridization☆

Primary lymphomas of the central nervous system (CNS) account for 0.3% to 1.5% of all intracranial neoplasms. Several reports have noted a coincidence between this neoplasm and serologic evidence of Epstein-Barr virus (EBV) infection, but in only a few instances has the EBV genome been demonstrated in these tumors. To further evaluate the frequency of this occurrence, we analyzed primary CNS lymphomas using nucleic acid hybridization methods and the polymerase chain reaction (PCR). In situ hybridization was used in selected cases. Sequences of EBV were found in two of nine cases by PCR and in situ hybridization. Southern blot hybridization of genomic DNA from these samples was negative for EBV. Both tumors arose in patients with conditions shown to produce secondary immunodeficiency, namely, chronic alcohol abuse and diabetes mellitus. We conclude that the association of EBV and CNS lymphoma is not restricted to patients with severe primary immune deficiency, and that PCR can be applied successfully to paraffin-embedded tissue for the detection of low-abundance viral sequences.

500. Local and systemic inhibition of lung tumor growth after liposome mediated mda-7/IL-24 gene delivery

The melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, anti-angiogenic and cytokine properties. In vitro adenovirus-mediated gene transfer of the mda-7/IL-24 gene (Ad-mda7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda7 to lung tumor xenografts results in growth suppression via induction of apoptosis and anti-angiogenic mechanisms. Although these results are encouraging, one limitation of this approach is its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is liposomal gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) liposomes effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated liposome-mediated delivery of the mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P = 0.001) and metastatic lung tumors (P = 0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 complex (P = 0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer and (2) anti-tumor effects of mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.