Nm23-transfected MDA-mB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: A31P nuclear magnetic resonance study in vivo and in vitro

Bhujwalla, Zaver M.; Aboagye, Eric O.; Gillies, Robert J.; Chacko, V. P.; Mendola, Charmaine E.; Backer, Joseph M.

doi:10.1002/(sici)1522-2594(199905)41:5<897::aid-mrm7>3.0.co;2-t

Cited by 84 publications

(19 citation statements)

References 18 publications

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“…), although Nm23 expression does not represent an independent prognostic or predictive factor (4). Ten transfection experiments have shown that nm23-transfected, metastatically competent cell lines are 40 -98% less metastatic in vivo than control transfectants (5)(6)(7)(8)(9)(10)(11)(12)(13)(14). For nm23-H1, the most studied member, overexpression in human MDA-MB-435 breast carcinoma cells reduced colonization in soft agar, both unstimulated and transforming growth factor-␤-stimulated (6), and invasion/motility to a variety of chemoattractants (15)(16)(17).…”

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confidence: 99%

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

Hartsough¹,

Morrison²,

Salerno³

et al. 2002

Journal of Biological Chemistry

181

151

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The metastasis-suppressive activity of Nm23-H1 was previously correlated with its in vitro histidine protein kinase activity, but physiological substrates have not been identified. We hypothesized that proteins that interact with histidine kinases throughout evolution may represent partners for Nm23-H1 and focused on the interaction of Arabidopsis "two-component" histidine kinase ERS with CTR1. A mammalian homolog of CTR1 was previously reported to be c-Raf; we now report that CTR1 also exhibits homology to the kinase suppressor of Ras (KSR), a scaffold protein for the mitogen-activated protein kinase (MAPK) cascade. Nm23-H1 co-immunoprecipitated KSR from lysates of transiently transfected 293T cells and at endogenous protein expression levels in MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1 phosphorylated KSR in vitro. Phosphoamino acid analysis identified serine as the major target, and two peaks of Nm23-H1 phosphorylation were identified upon high performance liquid chromatography analysis of KSR tryptic peptides. Using site-directed mutagenesis, we found that Nm23-H1 phosphorylated KSR serine 392, a 14-3-3-binding site, as well as serine 434 when serine 392 was mutated. Phosphorylated MAPK but not total MAPK levels were reduced in an nm23-H1 transfectant of MDA-MB-435 cells. The data identify a complex in vitro histidine-to-serine protein kinase pathway, which may contribute to signal transduction and metastasis.Metastasis suppressor genes are credentialed by their ability to suppress metastatic potential in vivo upon injection of a transfected tumor cell line, without a concomitant reduction in primary tumor size (reviewed in Ref. 1). The nm23 gene family was described by its reduced expression in highly metastatic murine melanoma cell lines, as compared with related, tumorigenic but less metastatic cell lines (2) and consists of eight family members (reviewed in Ref.3). In a recent review, 18 of 24 studies found a significant relationship between decreased Nm23 expression in primary human breast carcinomas and an aspect of aggressive clinical course (patient survival, lymph node metastasis, etc.), although Nm23 expression does not represent an independent prognostic or predictive factor (4). Ten transfection experiments have shown that nm23-transfected, metastatically competent cell lines are 40 -98% less metastatic in vivo than control transfectants (5-14). For nm23-H1, the most studied member, overexpression in human MDA-MB-435 breast carcinoma cells reduced colonization in soft agar, both unstimulated and transforming growth factor-␤-stimulated (6), and invasion/motility to a variety of chemoattractants (15-17). Nm23-H1 breast carcinoma transfectants exhibited morphological (ascinus formation) and biosynthetic aspects of differentiation in three-dimensional culture (18), and this finding is supported by similar studies in neural cells (19 -25).Despite extensive work, the biochemical mechanism of action whereby Nm23-H1 suppresses the metastatic potential of cancer cells is unk...

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confidence: 99%

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

Hartsough¹,

Morrison²,

Salerno³

et al. 2002

Journal of Biological Chemistry

181

151

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“…NMR spectroscopy has long been known to be important tool for exploring Plp metabolism (3)(4)(5)(6). Novel NMR developments, such as high-resolution magic angle spinning (HRMAS) probes, have provided improved spectra of cultured cells (7) and normal (8,9) and cancer (10 -12) tissues.…”

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confidence: 99%

Quantitative HRMAS proton total correlation spectroscopy applied to cultured melanoma cells treated by chloroethyl nitrosourea: Demonstration of phospholipid metabolism alterations

Morvan

Demidem

Papon

et al. 2003

Magnetic Resonance in Med

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The ability to follow phospholipid (Plp) metabolism is of paramount importance in many circumstances in which cell survival and cell proliferation are of concern-for example in neurological disorders and cancer (1,2). NMR spectroscopy has long been known to be important tool for exploring Plp metabolism (3-6). Novel NMR developments, such as high-resolution magic angle spinning (HRMAS) probes, have provided improved spectra of cultured cells (7) and normal (8,9) and cancer (10 -12) tissues. Recently, using HRMAS together with proton total correlation spectroscopy (TOCSY), we showed that a mousebearing B16 melanoma tumor responded to chloroethyl nitrosourea (CENU) treatment in vivo by altering its Plp metabolism (12).The first purpose of the present study was to demonstrate on cultured B16 melanoma cells in vitro that HRMAS proton TOCSY cross-peak signal variations of Plp derivatives reflected concentration variations. The second purpose was to determine the Plp metabolism response of cultured B16 melanoma cells to CENU treatment in vitro, as a model for Plp metabolism alterations previously observed in vivo.The Plp derivatives that were simultaneously observed using proton TOCSY were choline (Cho), phosphocholine (PC), cytidyl-diphosphate-choline (CDP-Cho), ethanolamine (Eth), phosphoethanolamine (PE), CDP-ethanolamine (CDP-Eth) (all of which are derivatives of the phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) biosynthetic pathways), and glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) (derivatives of PtdCho and PtdEth hydrolysis) (1-3).As regards the most expressed Plp derivatives in cultured B16 melanoma cells, HRMAS proton TOCSY signals revealed linearity with 1D saturation recovery signals, the NMR spectroscopy reference for quantifying concentrations. Therefore, HRMAS proton TOCSY was used to quantify concentration changes of water-soluble Plp derivatives in CENU-treated cultured melanoma cells. The response of cultured B16 melanoma cells to CENU treatment involved a transient accumulation of GPC and GPE, a down-regulation of PC and increase of CDP-Eth during cell proliferation inhibition, and a dramatic and irreversible rise of PE during and after cell proliferation inhibition. These data are discussed in relation to mouse-bearing B16 melanoma tumor response to CENU in vivo, and to Plp metabolism enzymatic involvement. METHODS ChemicalsNЈ-[2-chloroethyl]-N[2-(methylsulfonyl)ethyl]-NЈ-nitrosourea (cystemustine) is a CENU antineoplastic agent (13) that has been proposed for the treatment of human malignant melanoma and glioma. Cystemustine (Orphachem, Clermont-Ferrand, France) was supplied as a 5-mM solution in 0.9% NaCl. D 2 O (SDS, Peypin, France) was used to lock the spectrometer. Cell CulturesTransplantable B16 melanoma cells originating from C57BL6/6J Ico mice (ICIG, Villejuif, France) were adapted

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“…For example, expression of wild-type NM23 metastatis suppressor gene in human breast cancer cells, leading to a decrease in metastatic potential, was associated with a fall in the phosphomonoester/ phosphodiester ratio (38). Up-regulation of mutant RAS in NIH3T3 fibroblasts correlated with increased phosphocholine content that was reversed on exposure to RAS signaling inhibitors (39).…”

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confidence: 99%

Changes in choline metabolism as potential biomarkers of phospholipase Cγ1 inhibition in human prostate cancer cells

Beloueche‐Babari

Peak

Jackson

et al. 2009

Molecular Cancer Therapeutics

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Phosphoinositide-specific phospholipase Cγ1 (PLCγ1) is activated downstream of many receptor tyrosine kinases to promote cell motility. Inhibition of this protein is being explored as a therapeutic strategy for blocking cancer cell invasion and metastasis. The clinical development of such cytostatic therapies requires the implementation of pharmacodynamic biomarkers of target modulation. In this study, we use magnetic resonance spectroscopy to explore metabolic biomarkers of PLCγ1 down-regulation in PC3LN3 prostate cancer cells. We show that inhibition of PLCγ1 via an inducible short hairpin RNA system causes a reduction in phosphocholine levels by up to 50% relative to the control as detected by 1 H and 31 P magnetic resonance spectroscopy analyses. This correlated with a rounded-up morphology and reduced cell migration. Interestingly, the fall in phosphocholine levels was not recorded in cells with constitutive PLCγ1 knockdown where the rounded-up phenotype was no longer apparent. This study reveals alterations in metabolism that accompany the cellular effects of PLCγ1 knockdown and highlights phosphocholine as a potential pharmacodynamic biomarker for monitoring the action of inhibitors targeting

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Nm23-transfected MDA-mB-435 human breast carcinoma cells form tumors with altered phospholipid metabolism and pH: A31P nuclear magnetic resonance study in vivo and in vitro

Cited by 84 publications

References 18 publications

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

Nm23-H1 Metastasis Suppressor Phosphorylation of Kinase Suppressor of Ras via a Histidine Protein Kinase Pathway

Quantitative HRMAS proton total correlation spectroscopy applied to cultured melanoma cells treated by chloroethyl nitrosourea: Demonstration of phospholipid metabolism alterations

Changes in choline metabolism as potential biomarkers of phospholipase Cγ1 inhibition in human prostate cancer cells

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