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J Stables

Publications and source records attributed to J Stables.

12 recordsLinked to original sources
PubMed2001

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Animals↗
PubMed2001

Indazolylamino quinazolines and pyridopyrimidines as inhibitors of the EGFr and C-erbB-2.

Described herein is the design and synthesis of indazolylaminopyridopyrimidines and quinazolines as inhibitors of the class 1 tyrosine kinase receptor family. Data is presented for N(4)-(1-benzyl-1H-indazol-5-yl)-N(6),N(6)-dimethylpyrido[3,4-d]pyrimidine-4,6-diamine 3B. This compound inhibited EGFr and c-erbB-2 enzymes selectively over other kinases. It inhibited the proliferation of a range of tumour cell lines in vitro and the growth of BT474 xenografts in SCID mice.

Animals↗
PubMed1997

A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor.

Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the co-factor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ETA, angiotensin ATII, thyrotropin-releasing hormone (TRH), and neurokinin NK1 receptors, all of which interact pre-dominantly with the G alpha q-like phosphoinositidase-linked G-proteins. A substantially greater luminescence was obtained with mitochondrially targeted apoaequorin compared to cytoplasmically expressed apoaequorin. To generate a system amenable for the study of agonist activity at virtually any G-protein-coupled receptor the alpha subunit of the receptor promiscuous G-protein G alpha 16 was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing G alpha 16, but not in its absence, agonists at a series of receptors which normally interact with either G alpha s or G alpha i were now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A1 adenosine receptor, this response was clearly a result of activation of G alpha 16 and not a consequence of the release of the G alpha i-associated beta/gamma complex, as the luminescent response was unaffected by pertussis toxin treatment of the cells, whereas agonist-mediated inhibition of adenylyl cyclase activity was attenuated. These studies describe the use of coexpressed apoaequorin as a reporter for G-protein-coupled receptor-mediated calcium signaling. Furthermore, coexpression of G alpha 16 and apoaequorin provides a basis for a generic mammalian cell microplate assay for the assessment of agonist action at virtually any G-protein-coupled receptor, including orphan receptors for which the physiological signal transduction mechanism may be unknown.

Adenosine-5'-(N-ethylcarboxamide)↗
PubMed1996

Recognition of CD52 allelic gene products by CAMPATH-1H antibodies.

Cloning of the CD52 from a B-lymphocyte tumour cDNA library revealed two closely related sequences differing only at two amino acids C-terminal to the proposed point of glycosylphosphatidylinositol (GPI)-linkage. When transfected into CHO cells only one of these sequences gave high-level expression of the antigen recognized by the prototypic anti-CD52 antibody CAMPATH-1 whereas in JURKAT cells good expression levels were obtained with both sequences. Fusion of the sequence from the second sequence to DNA encoding the extracellular domain of CD4 indicated that this sequence was capable of directing GPI linkage. The possible implications for the function of CD52 and serotherapy with anti-CD52 antibodies are discussed.

Alemtuzumab↗
PubMed1996

Bicistronic vector for the creation of stable mammalian cell lines that predisposes all antibiotic-resistant cells to express recombinant protein.

We have developed an improved vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pCIN, both the recombinant cDNA and the neomycin phosphotransferase selection marker are transcribed from a single promoter element. To facilitate translation of the second open reading frame, the encephalomyocarditis virus internal ribosome entry site has been inserted into the expression cassette immediately before the start codon of this sequence. We report the use of this vector to generate stable cell lines expressing the human 5-HT1Da serotonin receptor and show that following transfection and clonal selection, all ten cell lines characterized express similar and high levels of receptor (1.5-11.9 pmol receptor/mg protein). Use of pCIN should permit the rapid and efficient production of stable mammalian cell lines for the characterization of recombinant protein, as this vector appears to predispose all transfected cells to express such protein.

Animals↗
PubMed1994

Effect of purine synthesis inhibition on WiDr spheroids in vitro or on WiDr or colon 38 tumors in vivo. Complete growth inhibition but not regression.

Clinical responses for anticancer agents are based upon tumor regression. We have investigated the potential of glycineamide ribonucleotide transformylase (GAR TFase) inhibitors to produce regressions in multiple preclinical models of colon carcinoma. The growth of multicellular tumor spheroids of WiDr human colon carcinoma was inhibited by the GAR TFase inhibitors 5-deazaacyclotetrahydrofolate (5-DACTHF), its 2'-fluoro, 3'-fluoro, 10-deaza, and 10-thia analogs as well as 5,10-dideazatetrahydrofolate, but none of the compounds caused spheroid regressions. By contrast, complete spheroid disruption was observed with exposure to etoposide, m-AMSA (amsacrine), piritrexim, or 2-desamino-2-methyl-10-propargyl-5,8-dideazafolate (DMPDDF). Light microscopy of the spheroids treated with either 5-DACTHF or DMPDDF suggested that the reason for the difference is extensive cell kill throughout the spheroid in the presence of DMPDDF compared with little or no kill, over that found in controls, with 5-DACTHF. Treatment of spheroids with 5-DACTHF in the presence of 1 microM hypoxanthine resulted in no significant reversal of growth inhibition; 50% reversal required 10 microM hypoxanthine. The spheroid studies were extended to in vivo studies examining the effects of 5-DACTHF on established WiDr and colon 38 tumors. The results showed that, in contrast to melphalan, which produced cures and tumor regressions, 5-DACTHF produced reversible growth inhibition with no significant regression of tumors. The results predict that clinical response, typically measured by tumor regression, may be rare following single agent therapy with inhibitors of de novo purine biosynthesis.

Acyltransferases↗
PubMed1992

Ontogeny and characterization of [125I]Bolton Hunter-eledoisin binding sites in rat spinal cord by quantitative autoradiography.

The distribution and characteristics of [125I]Bolton Hunter-eledoisin binding sites in rat lumbar spinal cord were studied during postnatal development by in vitro receptor autoradiography. At three, six and 10 days of age, specific [125I]eledoisin binding was distributed throughout the dorsal and ventral horns of the spinal cord. In contrast, from day 24 onwards, specific binding of [125I]eledoisin was confined to superficial layers of the dorsal horn, with negligible amounts of specific binding in the ventral horn. [125I]Eledoisin binding to neonatal (three day) and adult (eight to 12 weeks) spinal cord sections was characterized using tachykinin agonists. In both dorsal and ventral horns of neonatal spinal cord, the rank order of potency of agonists indicated that the majority (64%) of specific [125I]eledoisin binding was to neurokinin-3 binding sites. The identity of the non-neurokinin-3 sites labelled by [125I]eledoisin remains to be determined. In adult rat spinal cord, [125I]eledoisin appeared to bind exclusively to neurokinin-3 binding sites. These results suggest that major changes take place in the localization of neurokinin-3 receptors during postnatal ontogeny of the rat spinal cord. These changes may reflect an important role for tachykinins in neuronal plasticity of the developing spinal cord.

Animals↗
PubMedDate not supplied

Development of a dual glow-signal firefly and Renilla luciferase assay reagent for the analysis of G-protein coupled receptor signalling.

Several reporter gene assays have been described where gene transcription is activated as a consequence of a specific signal transduction event, such as activation of adenylyl cyclase (1.2). Reporter genes typically consist of specific responsive elements placed upstream of a minimal promoter, which together control the expression of a readily detectable reporter protein, such as luciferase. We have developed a dual glow-signal firefly and Renilla luciferase assay, which allows the simultaneous measurement of two reporter genes in the same well of a 96-well plate. In this report we demonstrate the use of this assay for the simultaneous analysis of agonist activity at two G-protein coupled receptors which signal through activation of the G-protein alpha sub-unit, G alpha S. Chinese hamster ovary (CHO) cells stably transfected with a cAMP responsive firefly luciferase reporter were further transfected with the human Vasopressin V2 receptor. Similarly, CHO cells stably transfected with a cAMP responsive Renilla luciferase reporter were further transfected with the human beta 2-adrenoceptor. The two cell lines were mixed in individual wells of a 96-well plate and a number of compounds were screened to determine their activity at both receptors. Stimulation with vasopressin and beta 2-adrenoceptor agonists resulted in the activation of the firefly and Renilla luciferases respectively. Stimulation with forskolin, which directly stimulates adenylyl cyclase, caused the activation of both reporter genes, and stimulation with a range of further compounds with no activity at either receptor did not generate a reporter response. The dual luciferase assay allows the simultaneous screening of two receptors in a 96-well format resulting in significant time and cost savings.

Animals↗