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N J MacDonald

Publications and source records attributed to N J MacDonald.

At least 19 recordsLinked to original sources
PubMed2001

Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin.

The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as an Endostatin-binding protein. We show that recombinant human Endostatin binds tropomyosin in vitro and to tropomyosin-associated microfilaments in a variety of endothelial cell types. The most compelling evidence that tropomyosin modulates the activity of Endostatin was demonstrated when E37 blocked greater than 84% of the tumor-growth inhibitory activity of Endostatin in the B16-BL6 metastatic melanoma model. We conclude that the E37 peptide mimics the Endostatin-binding epitope of tropomyosin and blocks the antitumor activity of Endostatin by competing for Endostatin binding. We postulate that the Endostatin interaction with tropomyosin results in disruption of microfilament integrity leading to inhibition of cell motility, induction of apoptosis, and ultimately inhibition of tumor growth.

Actin Cytoskeleton↗
PubMed2000

Angiostatin and endostatin: endogenous inhibitors of tumor growth.

Considerable progress has been made in the understanding of the molecular structure and mechanistic aspects of Angiostatin and Endostatin, endogenous angiogenesis inhibitors that have been shown to regress tumors in murine models. The growing body of literature surrounding these molecules and on the efficacy of these proteins is in part due to the ability to generate these proteins in recombinant systems as well characterized molecules. Recombinant human Angiostatin and Endostatin are in Phase I trials, following the manufacture of clinical grade material at large scale. This review highlights the recent advances made on understanding the structure and function of Angiostatin and Endostatin.

Angiogenesis Inhibitors↗
PubMed1999

The tumor-suppressing activity of angiostatin protein resides within kringles 1 to 3.

Angiostatin protein, which comprises the first four kringle domains of plasminogen, is an endogenous inhibitor of angiogenesis that inhibits the growth of experimental primary and metastatic tumors. Truncation of Angiostatin K1-4 to K1-3 retained the activity of Angiostatin. We recombinantly expressed full-length human Angiostatin protein corresponding to the first four kringle domains of human plasminogen and a truncated form of the Angiostatin protein, kringles 1-3. Purified recombinant Angiostatin K1-3 and K1-4 proteins inhibited the formation of experimental B16-BL6 lung metastases by greater than 80% when administered at 30 nmol/kg/day. We demonstrate for the first time that Angiostatin protein, consisting of the first three kringle domains of human plasminogen, has in vivo biological activity in this assay indistinguishable from that of the full-length Angiostatin K1-4 protein and that the fourth kringle of plasminogen, when linked in sequence to K1-3, plays no direct role in the antitumor activity of Angiostatin.

Amino Acid Sequence↗
PubMed1998

Nm23 and tumour metastasis: basic and translational advances.

The nm23 genes were discovered on the basis of their reduced expression by highly metastatic cell lines. This trend was confirmed in cohorts of several types of human carcinomas and melanomas. Several transfection studies have demonstrated the suppressive effect of nm23 overexpression on the metastatic aggressiveness of melanoma and breast carcinoma cells in vivo. These transfection experiments have also demonstrated an effect of nm23 overexpression on cellular functions involved in the metastatic phenotype, such as cell motility, and point to a regulatory role for Nm23 proteins in cellular signalling pathways. Nm23 homologues from various species are also involved in normal tissue development and differentiation. Transfection of nm23-H1 into breast cancer cells provided a functional demonstration of the involvement of this gene in the differentiation of mammary epithelial cells. However, the molecular mechanism of these biological effects remains unknown. Several biochemical activities have been reported for Nm23, including NDP kinase activity, serine autophosphorylation and protein-histidine kinase activity. To define the possible significance of these biochemical activities, we carried out site-directed mutagenesis of the relevant codons of nm23-H1 cDNA and studied the effects upon transfection into MDA-MB-435 human breast carcinoma cells. We have also used Nm23 expression as a molecular marker to identify novel compounds that are active against the most aggressive tumour cells. This approach revealed that none of the standard agents currently in clinical use is preferentially active against the most aggressive tumour cells, and allowed us to identify new compounds that are preferentially inhibitory towards low-Nm23-expressing breast carcinoma and melanoma cell lines. This analysis also revealed a significant correlation between Nm23 levels and sensitivity of the tumour cells to alkylating agents. A functional implication of Nm23 proteins in this phenomenon was demonstrated after transfection of nm23 cDNAs into melanoma and breast and ovarian carcinoma cells.

Amino Acid Sequence↗
PubMed1997

Nm23/PuF does not directly stimulate transcription through the CT element in vivo.

Decreased levels of the nm23 gene product have been correlated with increased tumor metastatic potential in a variety of malignancies. At least a subset of the regulatory properties of Nm23 has been proposed to be due to transactivation of the human c-myc oncogene through binding to a homopyrimidine tract 140 base pairs upstream of the transcription start site (termed the CT element or the PuF site). Conventional transcription factors possess DNA binding and transactivation domains; Nm23 fusion proteins were used to address two questions. First, if provided with a well characterized DNA binding domain, does Nm23 possess a transactivation domain capable of stimulating transcription of an appropriate reporter? Second, if provided with a potent transactivation domain, is the DNA binding of Nm23 of sufficient specificity and affinity to direct the fusion protein to a CT-dependent reporter? Since reporter gene expression was not stimulated in either case, we conclude that Nm23 does not directly stimulate transcription through binding to the CT element and that its antimetastatic and other reported functions are likely due to other biochemical activities.

Animals↗
PubMed1997

Site-directed mutation of Nm23-H1. Mutations lacking motility suppressive capacity upon transfection are deficient in histidine-dependent protein phosphotransferase pathways in vitro.

We previously compared the structure and motility suppressive capacity of nm23-H1 by transfection of wild type and site-directed mutant forms into breast carcinoma cells. Wild type nm23-H1 and an nm23-H1(S44A) (serine 44 to alanine) mutant suppressed motility, whereas the nm23-H1(P96S), nm23-H1(S120G), and to a lesser extent, nm23-H1(S120A) mutant forms failed to do so. In the present study wild type and mutant recombinant Nm23-H1 proteins have been produced, purified, and assayed for phosphorylation and phosphotransfer activities. We report the first association of Nm23-H1 mutations lacking motility suppressive capacity with decreased in vitro activity in histidine-dependent protein phosphotransferase assays. Nm23-H1(P96S), a Drosophila developmental mutation homolog, exhibited normal autophosphorylation and nucleoside-diphosphate kinase (NDPK) characteristics but deficient phosphotransfer activity in three histidine protein kinase assays, using succinic thiokinase, Nm23-H2, and GST-Nm23-H1 as substrates. Nm23-H1(S120G), found in advanced human neuroblastomas, exhibited deficient activity in several histidine-dependent protein phosphotransfer reactions, including histidine autophosphorylation, downstream phosphorylation on serines, and slightly decreased histidine protein kinase activity; significant NDPK activity was observed. The Nm23-H1(S120A) mutant was deficient in only histidine-dependent serine autophosphorylation. Nm23-H1 and Nm23-H1(S44A) exhibited normal activity in all assays conducted. Based on this correlation, we hypothesize that a histidine-dependent protein phosphotransfer activity of Nm23-H1 may be responsible for its biological suppressive effects.

Dose-Response Relationship, Drug↗
PubMed1996

Site-directed mutagenesis of nm23-H1. Mutation of proline 96 or serine 120 abrogates its motility inhibitory activity upon transfection into human breast carcinoma cells.

We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway.

Alanine↗
PubMed1996

Increased sensitivity to cisplatin by nm23-transfected tumor cell lines.

We report a functional link between expression of the metastasis suppressor gene nm23 and cancer cell sensitivity to the alkylating agent cisplatin. Cisplatin was 2-15-fold more inhibitory to the growth in vitro of nm23 transfectants of the K-1735 TK murine melanoma, MDA-MB-435 human breast carcinoma, and OVCAR-3 human ovarian carcinoma cell lines as compared to matched control transfectants. Administration of a single dose of cisplatin i.v. after injection of control- or nm23-1-transfected K-1735 TK melanoma cells resulted in a more pronounced inhibition of pulmonary metastatic colonization by the nm23-1 transfectants. The mechanism of nm23-dependent sensitivity to cisplatin is unknown, but was correlated with increased formation of interstrand DNA cross-links in nm23-H1 transfected breast carcinoma cells. These data suggest that elevation of tumor cell nm23 expression may be considered as a potential therapeutic strategy in combination with cisplatin treatment.

Animals↗
PubMed1995

Expression of nm23 in cell lines derived from patients with metastatic renal cell carcinoma.

Reduced expression of nm23 has been associated with increased metastases and decreased survival in a variety of malignancies. In the present study, the expression of nm23 was examined by Northern and Western blot analyses in a series of cell lines derived from patients with metastatic renal cell carcinoma. Two of twelve (17%) informative cell lines derived from 9 patients had loss of heterozygosity at Nm23-H1. Twenty-two renal cancer cell lines derived from primary tumors, 5 cell lines derived from metastatic tumors and 4 short-term cultures of normal proximal renal tubular cells all expressed Nm23 mRNA in varying amounts. On average, the level of expression of Nm23 mRNA in short-term cultures of benign proximal renal tubular cells was found to be similar to the level seen in renal cancer cell lines. Twenty-eight cell lines derived from renal primary tumors and 8 cell lines derived from metastatic tumors expressed both the Nm23-H1 and Nm23-H2 proteins. High or low relative expression of nm23 at the mRNA or protein level did not correlate with survival. The absence of any anomalous pattern of expression of the nm23 genes and the lack of correlation of expression with survival suggests that nm23 does not play a central role in the progression of this tumor type.

Blotting, Northern↗
PubMed1993

A serine phosphorylation of Nm23, and not its nucleoside diphosphate kinase activity, correlates with suppression of tumor metastatic potential.

We describe a serine phosphorylation of the putative metastasis suppressor protein Nm23, and present evidence of its relevance to the signal transduction and tumor metastatic processes. Nm23 was previously demonstrated to exhibit nucleoside diphosphate kinase (NDPK) activity, which transfers a phosphate among nucleoside tri- and diphosphates via an Nm23-phospho-histidine intermediate. Recent data have dissociated the NDPK activity of Nm23 from its phenotypic effects; therefore we have asked whether Nm23 possesses additional biochemical functions. An acid-stable (nonhistidine) phosphorylation was identified on autophosphorylated purified recombinant Nm23 proteins and [32P]orthophosphate-labeled human breast carcinoma and murine melanoma Nm23. Phosphoamino acid analysis identified serine as the acid-stable phosphorylation and serine 44 as the major site of phosphorylation. The acid stable phosphorylation (serine) of Nm23 was inhibited by cAMP in vitro and forskolin in vivo, suggesting that this phosphorylation pathway is regulated in signal transduction. No effect of cAMP was observed on Nm23 NDPK activity. Once phosphorylated, Nm23-phosphoserine can release free phosphate in vitro. The biological relevance of the novel phosphorylation identified herein is suggested by the direct correlation of in vivo Nm23 acid-stable phosphorylation levels, but not Nm23 NDPK activity, with suppression of tumor metastatic potential among control and nm23-1 transfected murine melanoma cells.

Adenosine Triphosphate↗
PubMed1993

Nm23 and breast cancer metastasis.

The majority of breast cancer patients succumb to metastatic disease. We summarize published and recent research concerning the nm23 gene in breast cancer metastasis. In a murine developmental study, nm23 expression increased with the functional differentiation of the mammary gland in nulliparous and pregnant animals. In human breast cancer, five studies have now demonstrated a significant association between reduced nm23 expression, at the RNA or protein levels, and aggressive tumor behavior. Nm23-negative tumor cells have been observed in comedo ductal carcinoma in situ lesions in two independent studies, indicating that decreases in nm23 expression begin prior to actual histologically identifiable invasion. Transfection studies, in which human nm23-H1 cDNA was expressed in the metastatic human MDA-MB-435 breast carcinoma cell line, indicate that nm23-H1 suppresses in vivo metastatic potential by 50-90%. Finally, our data in melanoma and breast carcinoma transfection systems suggest that the biochemical mechanism of nm23 suppressive activity is likely not due to its nucleoside diphosphate kinase activity, association with GAP proteins, or secretion from cells.

Animals↗
PubMed1993

Molecular basis of tumour metastasis.

The metastatic process is complex, requiring invasion, haematogenous and/or lymphatic dissemination, angiogenesis, colonization of a distant site and avoidance of host immune responses. These phenotypic changes can be accomplished by a variety of molecular pathways. Recent evidence suggests that many of the genetic events required for metastatic competence can be coordinately regulated, perhaps by regulatory cascades controlling normal development and differentiation. In addition to alterations in cellular phenotypes, metastatic cells also exhibit genomic instability, the causes of which are presently unknown. The development of therapeutic strategies to counteract metastasis may be most promising if directed towards the final angiogenesis and colonization by tumour cells at the metastatic site. Such strategies must overcome the redundancy of molecular mechanisms.

Cell Adhesion↗
PubMed1991

The effect of renal impairment on the pharmacokinetics and metabolism of bopindolol.

The pharmacokinetics of the non-selective beta-adrenoceptor antagonist, bopindolol, have been studied in 18 hypertensive patients with varying degrees of renal impairment following single and multiple oral dosing. Bopindolol, which undergoes extensive hepatic metabolism, was found to accumulate in patients with chronic renal failure but the disposition in patients on regular haemodialysis did not differ significantly from patients with normal renal function. The mechanism underlying these changes in pharmacokinetics is not clear but suggests the presence of metabolic inhibitors in uraemic plasma which are removed by regular haemodialysis.

Adrenergic beta-Antagonists↗
PubMed1990

Different pathways mediate virus inducibility of the human IFN-alpha 1 and IFN-beta genes.

Multimerization of GAAANN generates sequences frequent in virus-inducible promoters. We distinguished different types of (GAAANN)4 sequences mediating virus inducibility. Type I (NN = GT, GC, CT, or CC) responds to IFNs and to IRF-1 and causes silencing. Type II (NN = TG) and type III (NN = CG) neither silence nor respond to IRF-1 or IFN. Type III mediates constitutive transcription and binds the constitutive IEFga factor, whereas type II binds the novel "TG protein". IFN-beta and IFN-alpha 1 promoters contain different response elements: The former has a type I-like sequence (PRDI) and an NF-kappa B-binding sequence (PRDII); the latter has a type II-like "TG sequence" and possibly additional elements but does not bind NF-kappa B. Type I, type II, and NF-kappa B elements represent three distinct terminal pathways mediating virus induction.

Animals↗
PubMed1990

Pharmacodynamic, pharmacokinetic and humoral effects of oral zabicipril, an angiotensin converting enzyme inhibitor in normotensive man.

1. Zabicipril, S9650, a new angiotensin converting enzyme inhibitor, was administered to salt-replete, normotensive males in single doses of up to 10 mg. 2. The safety, tolerance and dose-response relationship with regard to inhibition of plasma ACE activity were characterised initially in an open, pilot, dose-finding study in 12 subjects and further explored in a double-blind, parallel group, placebo controlled study in another 30 subjects. 3. The drug was generally well tolerated and produced no change in routine haematology or serum biochemistry tests. 4. Dose related (0.03 to 10 mg) inhibition of plasma converting-enzyme was observed, with 2.5 mg of zabicipril producing over 90% inhibition at 4 h and 60% at 24 h. 5. There were no significant changes in blood pressure or heart rate in normotensive subjects over the dose range studied. 6. A dose related rise in plasma renin activity and angiotensin I was observed. No dose related reduction in plasma aldosterone was observed. 7. These initial studies suggest that zabicipril is a well tolerated inhibitor of converting enzyme with near maximal inhibitory effect occurring at a dose of 2.5 mg. Further exploration of the dose range after single and multiple doses is indicated.

Adult↗
PubMed1989

A comparison of the effects of human alpha calcitonin gene-related peptide and glyceryl trinitrate on regional blood velocity in man.

1. Human alpha calcitonin gene-related peptide (h alpha CGRP) is a potent vasodilator which in doses up to 1.5 micrograms min-1 i.v. produces little or no fall in blood pressure in normal volunteers, but does cause a substantial tachycardia. 2. We have explored the underlying mechanism of this effect by comparing h alpha CGRP infused so as to maintain heart rate 25-30% above baseline with glyceryl trinitrate (GTN) in a dose sufficient to maintain a throbbing headache. 3. Ten normal volunteers were studied. In addition to blood pressure and heart rate, blood velocity and pulsatility index (PI) were determined from Doppler signals recorded from the internal and external carotid, renal and femoral arteries. 4. Following h alpha CGRP blood pressure (mean +/- s.d., mm Hg) did not significantly change: 120 +/- 10/70 +/- 7 before compared with 121 +/- 12/67 +/- 7 during h alpha CGRP infusion. Heart rate (mean +/- s.d., beats min-1) increased from 62 +/- 8 to 86 +/- 10 (P less than 0.0001). In contrast the blood pressure fell following GTN: 124 +/- 12/74 +/- 8 before compared with 111 +/- 13/62 +/- 6 following treatment (P less than 0.02). Heart rate did not change following GTN: 64 +/- 9 compared with 69 +/- 10. 5. GTN significantly increased PI (mean +/- s.d.) in the common carotid artery from 2.8 +/- 0.5 to 3.4 +/- 0.5 (P less than 0.003) while h alpha CGRP increased PI in the internal carotid from 1.3 +/- 0.2 to 2.1 +/- 0.4 (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗