Background: Gene-directed enzyme prodrug therapy (GDEPT) represents a technology to improve drug selectivity for cancer cells. It consists of delivery into tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. Major limitations of GDEPT that hinder its clinical application include inefficient delivery into cancer cells and poor prodrug activation by suicide enzymes. We tried to overcome these constraints through a combination of suicide gene therapy with immunomodulating therapy. Viral vectors dominate in present-day GDEPT clinical trials due to efficient transfection and production of therapeutic genes. However, safety concerns associated with severe immune and inflammatory responses as well as high cost of the production of therapeutic viruses can limit therapeutic use of virus-based therapeutics. We tried to overcome this problem by using a simple nonviral delivery system.

The kinetic parameters of reactions catalyzed by four guanyl-specific RNases TI, Pbl, Thl and Sa were studied comparatively using three types of substrates; guanosine-2',3'-cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the 'recognising' and catalytical parts of the active site) without changes in the catalytical parameters.Guanyl-specific ribonucleases from various fungal and bacterial species are subject to extensive studies whose purpose is to elucidate molecular mechanisms responsible for the specificity of their action in the cleavage of phosphodiester bonds in RNA. Gudnylic RNases are characterized by high similarity amongst their primary structures, although the taxonomic relationships between organisms producing them are quite different [I]. Comparison of tertiary structures of several microbial RNases suggests that the evolutionary-conserved parts form the active sites [2 -51. One may ask the question, how much this conservative structure of active sites in guanyl RNases is paralleled by the invariance of their kinetic characteristics, or to what extent the catalytic properties of guanylic RNases are affected by differences in their primary and tertiary structures at areas other than the evolutionary conservative regions. The main purpose of this work is to answer these questions. Catalytic properties were compared for the three RNases isolated from the microscopic fungi Aspergillus orysae (RNase Tl), Penicillium brevicompactum (RNase Pbl), Trichoderma harziunum (RNase Thl) and the prokaryotic KNase from Streptomyces uureofaciens (RNase Sa).

To determine the role of intramitochondrial protein synthesis (PS) and degradation (PD) in contractile activity-induced mitochondrial biogenesis, we evaluated rates of [(35)S]methionine incorporation into protein in isolated rat muscle subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. Rates of PS ranged from 47 to 125% greater (P < 0.05) in IMF compared with SS mitochondria. Intense, acute in situ contractile activity (10 Hz, 5 min) of fast-twitch gastrocnemius muscle resulted in a 50% decrease in PS (P < 0.05) in SS but not IMF mitochondria. Recovery, or continued contractile activity (55 min), reestablished PS in SS mitochondria. In contrast, PS was not affected in either SS or IMF mitochondria after prolonged (60-min) contractile activity in the presence or absence of a recovery period. PD was not influenced by 5 min of contractile activity in the presence or absence of recovery but was reduced after 60 min of contractions followed by recovery. Chronic stimulation (10 Hz, 3 h/day, 14 days) increased muscle cytochrome-c oxidase activity by 2.2-fold but reduced PS in IMF mitochondria by 29% (P < 0.05; n = 4). PS in SS mitochondria and PD in both subfractions were not changed by chronic stimulation. Thus acute contractile activity exerts differential effects on protein turnover in IMF and SS mitochondria, and it appears that intramitochondrial PS does not limit the extent of chronic contractile activity-induced mitochondrial biogenesis.

One of the main problems in oncology is the development of drugs that cause the death of cancer cells without damaging normal cells. Another key problem to be solved is to suppress the drug resistance of cancer cells. The third important issue is to provide effective penetration of drug molecules to cancer cells. TRAIL (TNFα-related apoptosis inducing ligand)/Apo2L is a highly selective anticancer agent. However, the recombinant TRAIL protein having high efficiency against cancer cells in vitro was not effective in clinical trials. Recently we have discovered an acquisition of TRAIL resistance by cancer cells in confluent cultures, which is apparently a manifestation of the general phenomenon of multicellular resistance. The aim of this study was to evaluate whether the anticancer effect of the recombinant protein TRAIL in vivo can be improved by the suppression of multicellular TRAIL-resistance using sorafenib and a tumor-penetrating peptide iRGD, c(CRGDKGPDC). The results testified a great increase in the resistance of human fibrosarcoma HT-1080 cells to izTRAIL both in confluent cultures and in spheroids. Sorafenib administered at nontoxic concentration effectively suppressed confluent- or spheroid-mediated TRAIL-resistance of HT-1080 cells in vitro. Sorafenib combined with iRGD significantly improved the anticancer effect of the recombinant protein izTRAIL in HT-1080 human fibrosarcoma grafts in BALB/c nude mice. Consistent with this finding, multicellular TRAIL-resistance may be a reason of inefficacy of izTRAIL alone in vivo. The anticancer effect of the recombinant protein izTRAIL in vivo may be improved in combination with sorafenib, an inhibitor of multicellular TRAIL resistance and iRGD, the tumor-penetrating peptide.