Stanislaw M. Mikulski

Inhibition of HIV-1 Production and Selective Degradation of Viral RNA by an Amphibian Ribonuclease

Ribonucleases appear to have physiologic roles in host defense against cancer, viruses, and other parasites. Previously it was shown that select ribonucleases added to cells concurrently with virions blocked human immunodeficiency virus, type I (HIV-1) infection of H9 cells. We now report that a ribonuclease homologous to RNase A, named onconase, inhibits virus replication in chronically HIV-1-infected human cells without killing the virally infected cell. Examining the mechanism of this inhibition shows that onconase enters the infected cells and degrades HIV-1 RNA without degrading ribosomal RNA or the three different cellular messenger RNAs analyzed. The homologous human pancreatic RNase lacks anti-viral activity. Comparing recombinant forms of onconase and a onconase-human RNase chimera shows that the N-terminal 9 amino acids and the pyroglutamyl residue of onconase are required for full anti-viral activity. Thus extracellular ribonucleases can enter cells, metabolize select RNAs, and inhibit HIV virion production within viable replicating cells.

Tumoricidal effects of onconase on various tumors.

The effects of Onconase (Onc) on the tumor growth in vitro and in vivo were examined. Because elevated tumor interstitial fluid pressure (TIFP) is one of the major causes of inadequate drug delivery into solid tumors, we tested if Onc could lower TIFP in solid tumors.

Effect of ONCONASE +/- tamoxifen on ASPC-1 human pancreatic tumors in nude mice.

To evaluate changes in tumor physiological parameters after treatment with ONCONASE (ONC), we used laser Doppler flowmetery for tumor blood flow (TBF) in legs of nude mice bearing AsPC-1 human pancreatic carcinoma. Tumor interstitial fluid pressure (TIFP) was measured using the wick-in-needle technique, while intratumoral pO2 utilized O2 sensitive needle electrodes. TBF was significantly increased by an i.p. injection of ONC, then returned to the untreated levels at 150 min post-treatment. Single and multiple intraperitoneal injections of ONC significantly reduced TIFP (post-treatment at 1-7 days). ONC significantly improved tumor oxygenation evidenced by an increase in median pO2 from 4.2 mm Hg to 8.2 mm Hg. Since ONC had a synergistic interaction with tamoxifen (TAMX) on the cytotoxic clonogenicity of AsPC-1 tumor cells in vitro, we evaluated the antitumoral effects in vivo by ONC +/- TAMX, using nude mice bearing AsPC-1 pancreatic tumor cells of ascite (intraperitoneally implanted) or solid (subcutaneously implanted to legs) tumors. ONC alone effectively retarded the tumor growth, but TAMX alone did not. ONC + TAMX was synergistically effective in inhibiting tumor growth.

Interferon enhances the activity of the anticancer ribonuclease, onconase.

Interferons (IFN) are biologic agents involved in the antiviral response and the inhibition of tumor growth. Biochemical pathways of IFN action include the double-stranded RNA-activated oligoadenylate synthetase, RNase L, and double-stranded RNA-dependent protein kinase (PKR). Extracellular ribonucleases, especially onconase, also display antiviral and antitumor properties and involve degradation of RNA. We find that IFN increases the anticancer activity of onconase. These two agents work synergistically, and the effect is seen at the level of translation probably because of the degradation of tRNA.

Human B-lymphocyte antigens expressed by lymphocytic and myelocytic leukemia cells: Lymphocyte-dependent antibody studies with rabbit antisera☆

Abstract Rabbit antisera raised to papain-solubilized antigens from malignant spleen cell membranes had high lymphocyte-dependent antibody activity against normal B lymphocytes but no significant activity against T lymphocytes from the same donor. Leukemia blast cells from acute lymphoblastic and acute myelogenous leukemia and cultured B lymphoblastoid cell lines were also positive but the T line, Molt 4, and PHA blast cells were negative. Twelve rabbits were immunized with spleen cell extracts from 4 different patients with advanced malignant lymphomas. All produced antisera showing high lymphocyte-dependent activity with titers ranging from 10 4 to 10 8 . Studies indicated that the sera were reacting against a common antigen present on B cells and certain leukemia cells. These conclusions are supported by earlier complement-dependent antibody studies which, in addition, strongly suggested that the antigen being detected was part of the Ia-like alloantigen found on B lymphocytes. Lymphocyte-dependent antibody effector function was blocked by the original antiserum but not by F(ab′) 2 fragments made from the same serum.