Inhibition of Escherichia coli DNA polymerase-I by the anti-cancer drug cis-diaminedichloroplatinum(II): what roles do polymerases play in cis-platin-induced cytotoxicity?

Activities of Escherichia coli DNA polymerase-I were examined in the presence of the anti-tumor drug cis-diaminedichloroplatinum(II) and its inactive geometric isomer trans-diaminedichloroplatinum(II). The trans-isomer did not inhibit the enzyme activity. The anti-tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis-diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective preincubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05-3.0 mM. A mechanism that deals with the formation of an initial platinum-polymerase-I complex with a binding constant > 10(5) M(-1) followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.     

Glioma IL13Rα2 Is Associated with Mesenchymal Signature Gene Expression and Poor Patient Prognosis

A major challenge for successful immunotherapy against glioma is the identification and characterization of validated targets. We have taken a bioinformatics approach towards understanding the biological context of IL-13 receptor α2 (IL13Rα2) expression in brain tumors, and its functional significance for patient survival. Querying multiple gene expression databases, we show that IL13Rα2 expression increases with glioma malignancy grade, and expression for high-grade tumors is bimodal, with approximately 58% of WHO grade IV gliomas over-expressing this receptor. By several measures, IL13Rα2 expression in patient samples and low-passage primary glioma lines most consistently correlates with the expression of signature genes defining mesenchymal subclass tumors and negatively correlates with proneural signature genes as defined by two studies. Positive associations were also noted with proliferative signature genes, whereas no consistent associations were found with either classical or neural signature genes. Probing the potential functional consequences of this mesenchymal association through IPA analysis suggests that IL13Rα2 expression is associated with activation of proinflammatory and immune pathways characteristic of mesenchymal subclass tumors. In addition, survival analyses indicate that IL13Rα2 over-expression is associated with poor patient prognosis, a single gene correlation ranking IL13Rα2 in the top ~1% of total gene expression probes with regard to survival association with WHO IV gliomas. This study better defines the functional consequences of IL13Rα2 expression by demonstrating association with mesenchymal signature gene expression and poor patient prognosis. It thus highlights the utility of IL13Rα2 as a therapeutic target, and helps define patient populations most likely to respond to immunotherapy in present and future clinical trials.     

Autogenous growth factor production by reticuloendotheliosis virus-transformed hematopoietic cells

Reticuloendotheliosis virus strain T (REV-T)-transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo-producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum-free medium conditioned by the growth of REV-T-transformed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV-T-transformed cells under conditions where they normally failed to proliferate.     

Opiate receptors in mice: genetic differences.

The concentration of opiate receptors in the brains of mice was determined by means of a naloxone-binding assay. The strains of mice used in these experiments were C57BL/6By, BALB/cBy, their reciprocal F₁ hybrids, and 7 recombinant-inbred strains derived by inbreeding from the F₂ generation. These strains could be divided into 3 groups on the basis of the number of opiate receptors: high (CXBH); low (CXBK); and intermediate (all the other strains). The difference in stereospecific binding of naloxone reflects a difference in the total number of receptor sites rather than in the affinity for the drug. The recombinantinbred strains also differ in their analgesic response to morphine, as previously determined by the tail-flick assay. The differences in the number of opiate receptors are not enough to account for the genetic difference in analgesic responsiveness. Both these parameters appear to be under different genetic control, and at least 2 genetic determinants may be involved in regulating the level of opiate receptors.     

Inhalation toxicity of methylisocyanate: assessment of germ cell mutagenicity and reproductive effects in rats.

Adult male Wistar rats were exposed to methylisocyanate (MIC, 3.2 mg/l, single inhalation exposure for 8 min under static condition) or ethyl methanesulphonate (EMS, 150 mg/kg, single ip dose) for the assessment of germ cell mutagenicity and reproductive effects. Sequential matings of treated males with normal females on days 1-7, 8-14 and 15-21 post-exposure did not indicate any induction of dominant lethal mutation (increased frequency of preimplantation losses and early fetal deaths) by MIC but it was significantly induced by EMS as compared to respective controls. Males, necropsied after 21 days of exposure, showed no effect of MIC on epididymal sperm density and morphology. EMS also had no effect on sperm density but it significantly induced morphological abnormalities in sperm as compared to untreated controls. There was an acute and transitional reduction in reproductive performance (10-21%) of MIC-exposed males during days 1-14 post-exposure followed by recovery to the normal level during days 15-21 post-exposure. The progeny of MIC-exposed males was also normal in terms of litter size, litter weight, neonatal survival and body weight gain in litters up to 10 days post-partem. It is concluded with the evidence at hand that the observed failure of MIC to cause germ cell mutagenicity is related to its poor biodistribution to the target site(s) and a transient reduction in the reproductive performance of MIC-exposed males is a result of general stress and disconsolate copulation.     

Molecular cloning of Clostridium difficile toxin A gene fragment in lambda gt11

Toxin A of Clostridium difficile has been purified and monospecific antiserum produced. A reliable procedure for isolation and restriction of C. difficile chromosomal DNA was developed which allowed for the construction of a genomic library in lambda gt11. Approx. 35,000 plaques were screened using anti-toxin A which resulted in the identification of one stable positive clone, lambda cd19. Verification of the immunological identity of the isolated toxin A gene fragment in lambda cd19 was determined by affinity purifying toxin A antibodies specific for lambda cd19 gene product, and using these selected antibodies to probe a Western blot of purified toxin A. The insert in lambda cd19 was demonstrated to be a 0.3 kb fragment by restriction digestion, and by hybridization of the clone to a chromosomal digest of C. difficile. The peptide coded for by the toxin A gene fragment in lambda cd19 was not cytotoxic for 3T3 mammalian tissue culture cells.