Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro.

R115777 [(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. In vitro, using isolated human farnesyl protein transferase, R115777 competitively inhibited the farnesylation of lamin B and K-RasB peptide substrates, with IC50s of 0.86 nM and 7.9 nM, respectively. In a panel of 53 human tumor cell lines tested for growth inhibition, approximately 75% were found to be sensitive to R115777. The majority of sensitive cell lines had a wild-type ras gene. Tumor cell lines bearing H-ras or N-ras mutations were among the most sensitive of the cell lines tested, with responses observed at nanomolar concentrations of R115777. Tumor cell lines bearing mutant K-ras genes required higher concentrations for inhibition of cell growth, with 50% of the cell lines resistant to R115777 up to concentrations of 500 nM. Inhibition of H-Ras, N-Ras, and lamin B protein processing was observed at concentrations of R115777 that inhibited cell proliferation. However, inhibition of K-RasB protein-processing could not be detected. Oral administration b.i.d. of R115777 to nude mice bearing s.c. tumors at doses ranging from 6.25-100 mg/kg inhibited the growth of tumors bearing mutant H-ras, mutant K-ras, and wild-type ras genes. Histological evaluations revealed heterogeneity in tumor responses to R115777. In LoVo human colon tumors, treatment with R115777 produced a prominent antiangiogenic response. In CAPAN-2 human pancreatic tumors, an antiproilferative response predominated, whereas in C32 human melanoma, marked induction of apoptosis was observed. The heterogeneity of histological changes associated with antitumor effects suggested that R115777, and possibly farnesyl protein transferase inhibitors as a class, alter processes of transformation related to tumor-host interactions in addition to inhibiting tumor-cell proliferation.

Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: assay design and optimization.

BACKGROUND: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. METHODS: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. RESULTS: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. CONCLUSION: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.

DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format.

BACKGROUND: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. METHODS: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. RESULTS: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum. CONCLUSION: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.

Protein synthesis in the liver of Bufo marinus: cost and contribution to oxygen consumption.

While many estimates of the contribution of protein synthesis to metabolic rate exist for a variety of animals, most rely on theoretical costs of protein synthesis. The limitations of this approach are that theoretical costs depend upon variable estimates of ATP cost per peptide bond. In addition, they do not take into account the fact that there are protein-specific pre- and post-translational costs. By inhibiting, protein synthesis with cycloheximide and measuring the resultant decrease in oxygen consumption, we have measured the actual cost of protein synthesis and its contribution to metabolic rate in an in vitro system of tissue slices from Bufo marinus. Such measurements exist for endotherms, but there are few such measurements for ectotherms, and none have been done previously for amphibians. The cost of protein synthesis in liver slices from B. marinus was 7.32+/-1.19 mmol O2 x g(protein)(-1) (x +/- SE, n = 48) and protein synthesis accounted for 12% of the total metabolic rate of this tissue. This cost is comparable to values measured for other ectotherms although the contribution of protein synthesis to metabolic rate is at the lower end of the range of estimates for other ectotherms.

The role of protein synthesis during metabolic depression in the Australian desert frog Neobatrachus centralis.

Little is known about the role of energy consuming processes during metabolic depression. We have shown that aestivation in the Australian desert frog Neobatrachus centralis is accompanied by an in vivo metabolic depression of 77%. Using an in vitro liver slice preparation, we have measured an in vitro metabolic depression in liver of 55%, with a concomitant 67% decrease in the rate of protein synthesis. The decrease in protein synthesis accounts for 52% of the metabolic depression of the tissue, but only 4.9% of the metabolic depression of the whole animal. No in vitro metabolic depression or decrease in protein synthesis during aestivation was measured in muscle, but a decrease in the low rate of protein synthesis in muscle in vivo could not, in any case, account for more than 3% of the metabolic depression of the whole animal. The liver, although not a quantitatively important tissue in terms of metabolic depression in vivo, offers the opportunity to characterise the regulation of protein synthesis in a system in which metabolic depression is not confounded by changes in ambient temperature and PO2.

Effects of urea on M4-lactate dehydrogenase from elasmobranchs and urea-accumulating Australian desert frogs.

We measured the effect of urea on M4-lactate dehydrogenase (M4-LDH) from elasmobranchs and Australian desert frogs (urea accumulators) and from two animals that do not accumulate urea, the axolotl and the rabbit. An analysis of the effect of urea on the Kd(NADH), V, V/K(m(prr)) and V/K(m(NADH)) shows that in all cases the major effect of urea was on the binding of pyruvate, which fits with data in the literature that show that urea acts as a competitive inhibitor of LDH. The characteristics of the elasmobranch enzymes are consistent with a proposed adaptation model, but the situation for the enzymes from the aestivating frogs is equivocal. Urea (400 mM) had less effect on the K(m(prr)) of M4-LDH from the urea accumulators than it did on the non-accumulators, suggesting a general adaptation and that the enzyme produced by the aestivating frogs (urea accumulators) is kinetically different from that of non-aestivating frogs (non-accumulators). A new approach is used to characterize the overall pattern of adaptation to urea. The pattern is similar in an enzyme from an elasmobranch and an aestivating frog despite the temporary presence of urea in the latter and the phylogenetic difference between these animals.

Effectors of metabolic depression in an estivating pulmonate snail (Helix aspersa): whole animal and in vitro tissue studies.

We have examined metabolic depression in the land snail (Helix aspersa) during estivation, and have developed a tissue model of metabolic depression using an in vitro mantle preparation. The metabolic rate of H. aspersa is depressed by 84% in vivo within 4 weeks of onset of estivation, and this metabolic depression is accompanied by a decrease in haemolymph PO2 and pH, and an increase in haemolymph PCO2. The in vitro mantle preparation has a stable O2 consumption and energy charge, and an energy charge similar to that of mantle in vivo. The in vitro mantle is an O2-conforming tissue, with VO2 varying curvilinearly with PO2. Consequently, we have developed a mathematical method of calculating tissue VO2 at any PO2. These calculations show that under appropriate incubation conditions of pH and PO2, the mantle from estivating animals shows a stable in vitro metabolic depression of 48% compared to mantle from control snails. The extrinsic effects of PO2 and pH account for 70% of the total in vitro metabolic depression of mantle tissue; intrinsic effectors contribute a further 30%.

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog, Neobatrachus wilsmorei.

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6-12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na+ and K+ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na+ and K+ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K+ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K+ did not increase above that which can be accounted for by dehydration, indicating that K+ gradients were maintained.