Establishment and characterization of a canine soft tissue sarcoma patient-derived xenograft model.

Spontaneously occurring soft tissue sarcoma (STS) is relatively common in canine cancer patients. Because of the similarities to human disease, canine STSs are a valuable and readily available resource for the study of new therapeutics. In this study, a canine patient-derived xenograft (PDX) model, CDX-STS2, was established. The CDX-STS2 model was engrafted and expanded for systemic administration studies with chemotherapeutic agents commonly used to treat STS, including doxorubicin, docetaxel and gemcitabine. Immunohistochemistry for drug-specific biomarkers and tumour growth measurement revealed tumour sensitivity to doxorubicin and docetaxel, whereas gemcitabine had no effect on tumour growth. Although many human PDX tumour models have been established, relatively few canine PDX models have been reported to date. CDX-STS2 represents a new STS PDX research model of canine origin that will be useful in bridging preclinical research with clinical studies of STS in pet dogs.

Selective inhibition of low affinity IgE receptor (CD23) processing: P1' bicyclomethyl substituents.

Using a variety of alpha-hydroxy hydroxamic acid derivatives, the size and shape of the S1' pocket for the CD23 processing metalloprotease has been explored. It has been demonstrated that a P1' 2-naphthylmethyl group occupies most of the available space and gives excellent selectivity against fibroblast collagenase (matrix metalloproteinase-1, MMP-1) and other MMPs.

The intranuclear organization of normal, hemizygous and excision-deficient rRNA genes during developmental amplification in Tetrahymena thermophila.

In the ciliated protozoan, Tetrahymena thermophila, the diploid germinal micronucleus contains two allelic copies of the gene for ribosomal RNA (rDNA). During genesis of new somatic macronuclei the germline rDNA gene is excised by developmentally programmed chromosome breakage and preferentially amplified to approximately 9, 000 copies. We have studied this process by fluorescence in situ hybridization. We find that initially rDNA amplification is restricted to two separate and highly confined regions of the nucleus. Analysis of nuclei that are hemizygous for the rDNA locus reveals that each focus of hybridization is derived from a single allele of the rDNA. As rDNA amplification progresses these two foci of hybridization disperse and spread throughout the macronucleus, eventually forming approximately 100-500 new nucleoli. These events are correlated with morphologically distinct developmental stages. We investigated the amplification of the C3 allele of the rDNA that confers a replication advantage over the B allele during vegetative propagation, and find no evidence for preferential amplification of the C3 early in rDNA maturation. We also show that the rmm 11 rDNA mutant allele, which is defective for developmentally programmed rDNA excision, can be amplified during the two-foci stage in mutant homozygotes and heterozygotes, but fails to amplify further and disperse into multiple nucleoli. These data indicate that amplification of the rmm 11 allele is not delayed during the initial rounds of amplification, and suggest that efficient excision is not required for this amplification to occur. We propose that rDNA amplification is a two-step process. First, the two rDNA alleles are independently amplified, while allelic copies remain closely associated. Later, copies of the rDNA disperse and are further amplified, presumably because rDNA excision has occurred, generating fully mature rDNA minichromosomes that are able to replicate to high copy number.

Effects of the transcription inhibitor actinomycin D on postzygotic development of Tetrahymena thermophila conjugants.

During Tetrahymena thermophila conjugation, new somatic macronuclei develop from a common zygotic nucleus derived from meiotic products of the germline, and the old parental somatic nucleus is destroyed. The transcription inhibitor actinomycin D disrupts many events of postzygotic conjugation (cycloheximide causes indistinguishable effects). Early treatment causes a block of all postzygotic development, suggesting a transcription requirement for conjugants to pass a checkpoint, allowing entry into postzygotic development. Thereafter, pair separation, resorption of the old macronucleus, and elimination of one of the new micronuclei are blocked if actinomycin D is added at least 1.5 hr before each of these events normally occurs. Treatment just before DNA rearrangements in the developing macronuclei (anlagen) causes aberrant anlage DNA loss, suggesting that this DNA loss may be caused by inhibition of gene expression involved in genome rearrangements. DNA loss, and correlated lethality, appear to require previous gene expression, since actinomycin D added earlier causes cells to arrest in development without anlage DNA loss, and these conjugants can (at some frequency) complete conjugation and make viable progeny once actinomycin D is removed. The old macronucleus already had been inactivated before most actinomycin D treatments were initiated, indicating that the various induced defects we observed are the result of inhibition of postzygotic gene expression, presumably in anlagen. The defects induced by actinomycin D are similar to defects previously observed in conjugants harboring nullisomic germline deficiencies but proficient old macronuclei.

Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development.

Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies. Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis). Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die. Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration. Some deficiencies cause aberrant anlage DNA loss. Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes. The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed. Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage. The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate.

Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena.

During conjugation in the ciliated protozoan Tetrahymena, new macronuclei differentiate from germinal zygotic micronuclei while parental (old) macronuclei are eliminated in two stages, condensation or pycnosis coincident with cessation of transcription followed by resorption. We show that pycnosis is accompanied by degradation of old macronuclear DNA into oligonucleosome-sized fragments, a hallmark of programmed cell death, or apoptosis, in a variety of eukaryotic systems. As expected, oligonucleosome formation does not occur in the new micro- and macronuclei, confirming the coordination of different developmental fates for different nuclei in a common cytoplasm. NULLI 3 conjugants have wild-type old macronuclei but lack chromosome 3 germinally and hence in the new macronucleus. In NULLI 3 conjugants, old macronuclear pycnosis and oligonucleosome fragmentation occur normally but the resorption step fails, and the pycnotic old macronucleus is retained, demonstrating that the two steps are genetically separable and thus distinct and implying that genes on chromosome 3 in the new macronucleus are required for the resorption step. Comparison of whole cell polypeptides synthesized during stages of macronuclear development in both wild-type and NULLI 3 crosses reveal similar profiles. However, a polypeptide (apparent M(r) of 53 kDa) synthesized during old macronuclear elimination is not observed in NULLI 3 conjugants; its role, if any, in elimination of the old macronucleus is unknown. The results show that the old macronucleus is selectively destroyed by a mechanism which is remarkably similar to apoptosis in other eukaryotes and that the zygotic genome is required for the resorption step.

The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol.

Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed.

Influence of Mg2+ and pH on n.m.r. spectra and radioligand binding of inositol 1,4,5-trisphosphate.

We and others have shown that the binding of Ins(1,4,5)P3 to its receptor is pH-sensitive and can be inhibited by Mg2+. In the present study we have used 1H- and 31P-n.m.r. spectroscopy to study whether these effects results from increased ionization of Ins(1,4,5)P3 and a direct interaction with Mg2+ respectively. Under near-physiological conditions of ionic strength (100 mM-KCl), three ionizable groups were observed. The pH titration curve of the 1-phosphate was monophasic, with a pKa of 6.3. The titration curves of the 4- and 5-phosphates were biphasic, suggesting that these groups interact; the pKa values for the 4-phosphate determined by 31P-n.m.r. were 5.7 and 7.8, and for the 5-phosphate they were 5.3 and 7.9. 1H- and 31P-n.m.r. measurements suggest that Mg2+ binds weakly to Ins(1,4,5)P3 at physiological pH. Mg2+ non-competitively inhibited binding of Ins(1,4,5)P3 to its receptor in rat cerebellum and bovine adrenal cortex. Inhibition curves for rat cerebellum at pH 7.1 and 8.5, and also for bovine adrenal cortex at pH 8.5, appeared to be monophasic, with IC50 values (concn. of displacer giving 50% inhibition of specific binding) of 214 microM, 572 microM and 9.1 mM respectively. Scatchard analysis revealed that Mg2+ inhibited binding of Ins(1,4,5)P3 to bovine adrenal cortex at pH 8.5 in a non-competitive manner. Our results suggest that the previously reported pH-sensitivity of the binding of Ins(1,4,5)P3 may be caused by ionization of the phosphate groups in positions 4 and 5, and that the ability of Mg2+ to inhibit the binding of Ins(1,4,5)P3 is not mediated by direct chelation but through a site located on, or close to, the Ins(1,4,5)P3 receptor. Inhibition by Mg2+ is pH-sensitive and can vary at least 10-fold between tissues, suggesting possible receptor heterogeneity. Mg2+ may exert an important regulatory control on the release of Ca2+ by Ins(1,4,5)P3.

Purine derivatives as competitive inhibitors of human erythrocyte membrane phosphatidylinositol 4-kinase.

The possibility of deriving a potent, cell-penetrating inhibitor of human erythrocyte PI 4-kinase, competitive with respect to ATP, has been investigated in a series of purine derivatives and analogues. The purine nucleus is not essential for binding to the ATP site but offers the advantage of synthetic accessibility to its derivatives. The optimum substitution pattern in purine was found to be an electron-releasing substituent in the 6-position (e.g. amino, as in adenine, 1) and a compact, lipophilic group in either the 8-position or, preferably, the 9-position, suggesting the importance of the N-1 lone pair and hydrophobic contributions of the 8- and 9-substituents to binding. The most potent inhibitor synthesized was 9-cyclohexyladenine (54), which has an apparent Ki value of 3.7 microM.

Total synthesis of the four stereoisomers of dihexadecanoyl phosphatidylinositol and the substrate stereospecificity of human erythrocyte membrane phosphatidylinositol 4-kinase.

A new and convenient method for the preparation of the four stereoisomers of dihexadecanoyl phosphatidylinositol has been developed. An enantiomeric pair of acid-labile, pentaprotected myo-inositol building blocks was synthesized in high yield and coupled with chiral phenyl dihexadecanoylglyceryl phosphates to give the fully protected phosphatidylinositols. These were subsequently deprotected by hydrogenolysis and self-hydrolysis in aqueous ethanol to give the desired pure products. Comparison of these compounds as potential substrates for a partially purified phosphatidylinositol 4-kinase (EC 2.7.1.67) derived from human erythrocyte membranes revealed that the chirality of the inositol ring is crucial for efficient phosphorylation, whereas the chirality of the glycerol moiety is relatively unimportant. Moreover, the similarity in phosphorylation rates of the naturally occurring mammalian phospholipid, I, and its synthetic stereochemical counterpart, compound 10a, suggests that the enzyme is relatively tolerant to changes in fatty acid composition.