Michael R. Roner

Reovirus reverse genetics: Incorporation of the CAT gene into the reovirus genome

We have modified the infectious reovirus RNA system so as to generate a reovirus reverse genetics system. The system consists of (i) the plus strands of nine wild-type reovirus genome segments; (ii) transcripts of the genetically modified cDNA form of the tenth genome segment; and (iii) a cell line transformed so as to express the protein normally encoded by the tenth genome segment. In the work described here, we have generated a serotype 3 reovirus into the S2 double-stranded RNA genome segment of which the CAT gene has been cloned. The virus is stable, replicates in cells that have been transformed (so as to express the S2 gene product, protein σ2), and expresses high levels of CAT activity. This technology can be extended to members of the orbivirus and rotavirus genera. This technology provides a powerful system for basic studies of double-stranded RNA virus replication; a nonpathogenic viral vector that replicates to high titers and could be used for clinical applications; and a system for providing nonselectable viral variants (the result of mutations, insertions, and deletions) that could be valuable for the construction of viral vaccine strains against human and animal pathogens.

Reovirus RNA is infectious

Conditions under which reovirus RNA is infectious have been worked out. In brief, single-stranded (plus-stranded, ss) and/or double-stranded (ds) RNA of reovirus serotype 3 (ST3 virus) is lipofected into L929 mouse fibroblasts together with a rabbit reticulocyte lysate in which ss or melted dsRNA has been translated. After 8 hr the cells are then infected with a helper virus, ST2 reovirus. Virus yields are harvested 24 or 48 hr later. Under these conditions virus that forms plaques by 5 days is produced, all of which is ST3 virus; ST2 virus forms plaques only after 12 days. No reassortants are present among the progeny. The virus yields are about 0.2 PFU/cell; immunofluorescence assays show that this progeny is derived from about 4% of the cells. Double-stranded RNA is 20 times as infectious as ssRNA; ds and ssRNA together yield 10 times as much infectious virus as dsRNA alone, the reason being that dsRNA greatly increases the infectiousness of ssRNA. All species of both ss and dsRNA are required for the operation of this additive effect. The primed rabbit reticulocyte lysate is not essential, but increases virus yields by 100-fold. Its activity is proportional to the time for which translation has proceeded; however, this activity is not due solely to newly synthesized proteins because destruction of the RNA following translation abolishes activity which cannot be restored by simple addition of more RNA. Translation of all species of RNA is essential. Whereas no reassortants are formed when ss and dsRNA of different genotypes are lipofected together, mixtures of dsRNAs of different genotypes do yield reassortants. The same is true for such mixtures of ssRNA. These findings will permit the introduction of new or altered genome segments into the reovirus genome. They open the way to the identification of encapsidation and assortment signals on reovirus genome segments, the characterization of functional domains on reovirus proteins, and the development of reovirus as an expression vector.

Organotin Polyethers as Biomaterials

Organotin polyethers are easily synthesized employing interfacial polymerization systems involving the reaction of hydroxyl-containing Lewis bases and organotin halides. A wide variety of organotin-containing polymeric products have been synthesized including those derived from natural and synthetic polymers such as lignin, xylan, cellulose, dextran, and poly(vinyl alcohol). Others have been synthesized employing known drug diols such as dicumarol, DES, and dienestrol and a wide variety of synthetic diols. Included in these materials are the first water soluble organotin polymers. The organotin polyethers exhibit a wide range of biological activities. Some selectively inhibit a number of unwanted bacteria, including Staph. MRSA, and unwanted yeasts such as Candida albicans. Some also inhibit a variety of viruses including those responsible for herpes infections and smallpox. Others show good inhibition of a wide variety of cancer cell lines including cell lines associated with ovarian, colon, lung, prostrate, pancreatic and breast cancer. The synthesis, structural characterization, and biological characterization of these materials is described in this review.

Structural Consideration in Designing Organotin Polyethers to Arrest the Growth of Breast Cancer Cells In Vitro

The ability to inhibit cancer is inherent in organotin materials yet the structural relationships that regulate/direct this activity remains unknown. We measured antitumor activity using a matched pair of cell lines MDA-MB-231 cells that are estrogen-independent, estrogen receptor negative and MCF-7 cells, a cell line that is estrogen receptor (ER) positive. Those polyethers that contained a O-phenyl unit were able to significantly inhibit the non-estrogen sensitive cell line but were much less effective against the estrogen sensitive cell line; that is, the human breast cancer cell line MDA-MB-231 showed better test results for polymers derived from diols containing the O-phenyl moiety than the breast cancer cell line MCF-7, a well-characterized estrogen receptor positive control cell line. Those polyethers that did not contain the O-phenyl unit inhibited both cell lines approximately the same. The differential activity of the O-phenyl-containing polyethers is likely due to the estrogen-sensitive cells combining with some of the organotin polyethers minimizing their ability to inhibit cell growth.

Antiviral Activity of Metal-Containing Polymers—Organotin and Cisplatin-Like Polymers

Polymers containing platinum and to a lesser extent tin, have repeatedly demonstrated antitumor activity in vitro and in vivo against a variety of cell and tumor types. The mechanisms responsible for the antitumor activity include inducing a delay in cell proliferation and sister chromatid exchanges blocking tumor growth. As most DNA and some RNA viruses require, and even induce, infected cells to initiate DNA replication and subsequent cell division, compounds with antitumor activity will very likely also possess antiviral activity. This article examines the use of metal-containing polymers as a novel class of antivirals.

Ability of Group IVB metallocene polyethers containing dienestrol to arrest the growth of selected cancer cell lines

BackgroundMonomeric Group IVB (Ti, Zr and Hf) metallocenes represent a new class of antitumor compounds. There is literature on the general biological activities of some organotin compounds. Unfortunately, there is little information with respect to the molecular level activity of these organotin compounds. We recently started focusing on the anti-cancer activity of organotin polymers that we had made for other purposes and as part of our platinum anti-cancer effort.MethodsFor this study, we synthesized a new series of metallocene-containing compounds coupling the metallocene unit with dienestrol, a synthetic, nonsteroidal estrogen. This is part of our effort to couple known moieties that offer antitumor activity with biologically active units hoping to increase the biological activity of the combination. The materials were confirmed to be polymeric using light scattering photometry and the structural repeat unit was verified employing matrix assisted laser desorption ionization mass spectrometry and infrared spectroscopy results.ResultsThe polymers demonstrated the ability to suppress the growth of a series of tumor cell lines originating from breast, colon, prostrate, and lung cancers at concentrations generally lower than those required for inhibition of cell growth by the commonly used antitumor drug cisplatin.ConclusionThese drugs show great promise in vitro against a number of cancer cell lines and due to their polymeric nature will most likely be less toxic than currently used metal-containing drugs such as cisplatin. These drugs also offer several addition positive aspects. First, the reactants are commercially available so that additional synthetic steps are not needed. Second, synthesis of the polymer is rapid, occurring within about 15 seconds. Third, the interfacial synthetic system is already industrially employed in the synthesis of aromatic nylons and polycarbonates. Thus, the ability to synthesize large amounts of the drugs is straight forward.

Molecular recognition in the assembly of the segmented reovirus genome.

Publisher Summary To lay the groundwork for examining molecular recognition interactions in reovirus genome segment assortment and reassortment, this chapter reviews the basic features of reovirus multiplication cycle, the nature of the genome segment sets of the three reovirus serotypes, and the nature of reovirus defective interfering (DI) particles that are genome segment size reassortants. When cells are infected with two reoviruses that belong to different serotypes, reassortants that contain n genome segments of one parent and 10– n of the other are formed at frequencies close to those expected on the basis of random reassortment— that is, there is little linkage between any pair or combination of genome segments. It has also been found that the introduction of heterologous, or modified homologous, genome segments into the reovirus genome requires the presence of acceptance signals on certain genome segments of recipient genomes. The finding that established genome segment reassortment analysis as a very powerful technique involved the demonstration that the double stranded RNA genome segments and proteins of the three reovirus serotypes possess readily distinguishable electrophoretic migration rates.

Influence of DMSO on the inhibition of various cancer cells by water-soluble organotin polyethers

The effect of solvent on the ability to inhibit a group of cell lines was studied employing a series of water-soluble dibutyltin polyethers derived from various hydroxyl-terminated poly(ethylene glycols). Molecular weight as a function of time was essentially unchanged for a period of 30 weeks for the polyethers. Variations in test media can affect biological activities on the cell level as well as at the molecular level. The current study involves the evaluation of the cellular effects when dimethyl sulfoxide (DMSO) is initially employed to dissolve the polymers compared with using water alone to dissolve the polymer. Even though caution was taken to employ DMSO at concentrations where cell interactions are not found for the control, differences were found between samples exposed to DMSO and those not exposed to DMSO. The general inhibition fingerprint is similar for both conditions but the particular EC50 results can vary 10-fold. Thus, the presence of DMSO does have some effect on the particular biolog...

Control of Prostate Cancer Using Organotin Polymers

The current efforts described in this paper are aimed at developing compounds that inhibit the growth of prostate cancer and to identify structure/property relationships that will further assist us towards this goal. The growth of prostate cancer is affected employing a wide range of organotin condensation polymers. The EC50 values are primarily dependant on the nature of the Lewis base but the CI50 is dependent on both the nature of the Lewis base and Lewis acid. EC50 values down to the picogram/mL range are found. A number of products exhibit CI50 values greater than two. For polymers derived from hydroquinone and hydroquinone derivatives ability to inhibit cancer cell growth decreases as the bulk of substituents increases.

Effect of Electrical Conductivity Through the Bulk Doping of the Product of Titanocene Dichloride and 2-Nitro-1,4-phenylenediamine

The condensation polymer derived from reaction between titanocene dichloride and 2-nitro-1,4-phenylenediamine was doped by mixing the polymer with different amounts of iodine. This bulk doping of the titanocene polyamine resulted in an increase in bulk conductivity from 10 to over 1,000 fold. Conductivity increased to a doping level of about 10 to 15% iodine. Conductivity decreased as the sample discs were heated returning to pre-doped levels after the samples were heated for eight minutes. It is believed that this decrease in conductivity is due to the surface evaporation of iodine as the samples were heated. MALDI MS and IR results are consistent with the formation of C-I compounds for doped materials.