NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms.

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.     

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.     

Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.     

ZBTB34, a novel human BTB/POZ zinc finger protein, is a potential transcriptional repressor

Our work has identified a cancer-specific, cell surface and growth-related quinol oxidase with both NADH oxidase and protein disulfide-thiol interchange activities, a member of the ECTO-NOX protein family designated tNOX. We provide evidence for tNOX as an alternative drug target to COX-2 to explain the anticancer activity of COX inhibitors. Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib all specifically inhibited tNOX activity of HeLa (human cervical carcinoma) and BT-20 (human mammary carcinoma) cells (IC₅₀ in the nanomolar range) without effect on ECTO-NOX activities of non-cancer MCF-10A mammary epithelial cells. With cancer cells, rofecoxib was less effective and two NSAIDS selective for COX-1 were without effect in inhibiting NOX activity. The IC₅₀ for inhibition of tNOX activity of HeLa cells and the IC₅₀ for inhibition of growth of HeLa cells in culture were closely correlated. The findings provide evidence for a new drug target to account for anticancer effects of NSAIDS that occur independent of COX-2.     

Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface.

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tNOX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were resistant to heating and to protease digestion. The activity was purified 200- to 500-fold to provide apparently homogeneous gel bands for N-terminal sequencing using three different protocols. All three protocols involved heat (50 degrees C) and proteinase K treatment. Recovery of the total NADH oxidase activity was 86% and inhibition by capsaicin was 60 to 80%. After 450-fold purification, a 52-kDa component was obtained as a single gel band that retained the capsaicin-inhibited NADH oxidase activity. Amino acid composition and partial amino acid sequences were obtained. The partial amino acid sequences were used to generate peptide antisera. Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaicin-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa components on Western blots from different steps of the purification. The tNOX protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antibody to tNOX from sera of cancer patients. The results provide a direct sequence link between tNOX of the HeLa cell surface and the cloned tNOX representative of patient sera. The tNOX form from the surface of HeLa cells yielded N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities.     

Alternative splicing as the basis for specific localization of tNOX, a unique hydroquinone (NADH) oxidase, to the cancer cell surface

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera of healthy individuals. Full-length tNOX mRNA is present in both normal and tumor cells but appears not to be expressed in either. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. Four splice variants were found. Of these, the exon 4 minus and exon 5 minus forms present in cancer cell lines were absent in noncancer cell lines. In contrast to full-length tNOX cDNA, transfection of COS cells with tNOX exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the plasma membrane. The exon 4 minus form resulted in initiation of translation at a downstream M231 initiation site distinct from that of full-length mRNA. With replacement of M231 by site-directed mutagenesis, no translation of exon 4 minus cDNA or cell surface expression of 34 kDa mature tNOX was observed. The unprocessed molecular mass of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to that of the principal native tNOX form of the endoplasmic reticulum. Taken together, the molecular basis of cancer-cell-specific expression of 34 kDa tNOX appears to reside in the cancer-specific expression of exon 4 minus splice variant mRNA.     

RNA interference targeting tNOX attenuates cell migration via a mechanism that involves membrane association of Rac

tNOX, a tumor-associated NADH oxidase, is a growth-related protein present in transformed cells. In this study, we employed RNA interference (RNAi)-mediated down-regulation of tNOX protein expression to explore the role of tNOX in regulating cell growth in human cervical adenocarcinoma (HeLa) cells. In this first reported use of RNAi to decrease tNOX expression, we found that HeLa cell growth was significantly inhibited by shRNA-knockdown of tNOX. Furthermore, cell migration and membrane association of Rac were decreased concomitantly with the reduction in tNOX protein expression. These results indicate that shRNA targeting of tNOX inhibits the growth of cervical cancer cells, and reduces cell migration via a decrease in the membrane association of Rac. We propose that tNOX is a potential upstream mediator of Rho activation that plays a role in regulating cell proliferation, migration, and invasion.     

A site-directed mutagenesis analysis of tNOX functional domains

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.     

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 microM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 microM adriamycin with 1 and 10 microM adriamycin being less inhibitory than 0.1 microM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.     

Early developmental expression of a normally tumor-associated and drug-inhibited cell surface-located NADH oxidase (ENOX2) in non-cancer cells

Full length mRNA to a drug-inhibited cell surface NADH oxidase, tNOX or ENOX2, is present in both non-cancer and cancer cells but is translated only in cancer cells as alternatively spliced variants. ENOX2 is a growth-related protein of the external plasma membrane surface that is shed into the circulation and is inhibited by a series of quinone site inhibitors with anticancer activity. To test the possibility that ENOX2 expression might be important to early stages of non-cancer cell development, the expression of the protein was monitored in chicken embryos during their development. Polyclonal antisera to a 34 kDa human serum form of ENOX2 cross-immunoreactive with the drug-responsive NADH oxidase of chicken hepatoma cells was used. The protein was identified based on drug-responsive enzymatic activities and analyses by western blots. The drug-responsive activity was associated with plasma membranes and sera of early chicken embryos and with chicken hepatoma plasma membranes but was absent from plasma membranes prepared from livers or from sera of normal adult chickens and from late embryo stages. The findings suggest that ENOX2 may fulfill some functions essential to the growth of early embryos which are lost in late embryo stages and absent from normal adult cells but which then reappear in cancer.     

NAD+/NADH and/or CoQ/CoQH2 ratios from plasma membrane electron transport may determine ceramide and sphingosine-1-phosphate levels accompanying G1 arrest and apoptosis

To elucidate possible biochemical links between growth arrest from antiproliferative chemotherapeutic agents and apoptosis, our work has focused on agents (EGCg, capsaicin, cis platinum, adriamycin, anti-tumor sulfonylureas, phenoxodiol) that target tNOX. tNOX is a cancer-specific cell surface NADH oxidase (ECTO-NOX protein), that functions in cancer cells as the terminal oxidase for plasma membrane electron transport. When tNOX is active, coenzyme Q(10) (ubiquinone) of the plasma membrane is oxidized and NADH is oxidized at the cytosolic surface of the plasma membrane. However, when tNOX is inhibited and plasma membrane electron transport is diminished, both reduced coenzyme Q(10) (ubiquinol) and NADH would be expected to accumulate. To relate inhibition of plasma membrane redox to increased ceramide levels and arrest of cell proliferation in G(1) and apoptosis, we show that neutral sphingomyelinase, a major contributor to plasma membrane ceramide, is inhibited by reduced glutathione and ubiquinone. Ubiquinol is without effect or stimulates. In contrast, sphingosine kinase, which generates anti-apoptotic sphingosine-1-phosphate, is stimulated by ubiquinone but inhibited by ubiquinol and NADH. Thus, the quinone and pyridine nucleotide products of plasma membrane redox, ubiquinone and ubiquinol, as well as NAD(+) and NADH, may directly modulate in a reciprocal manner two key plasma membrane enzymes, sphingomyelinase and sphingosine kinase, potentially leading to G(1) arrest (increase in ceramide) and apoptosis (loss of sphingosine-1-phosphate). As such, the findings provide potential links between coenzyme Q(10)-mediated plasma membrane electron transport and the anticancer action of several clinically-relevant anticancer agents.     

tNOX is both necessary and sufficient as a cellular target for the anticancer actions of capsaicin and the green tea catechin (-)-epigallocatechin-3-gallate

Capsaicin and the principal green tea catechin, (-)-epigallocatechin-3-gallate (EGCg), target tNOX, a tumor (cancer)-specific surface hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ECTO-NOX protein). Accordingly vector-forced over expression of tNOX in MCF-10A mammary epithelia or COS cells that lack tNOX or in COS cells that underexpress tNOX enhanced the susceptibility of growth and apoptosis to both EGCg and capsaicin. Additionally, the tNOX-transfected MCF-10A cells proliferated in Matrigel, a measure of invasiveness. In contrast, oligomeric antisense tNOX DNA abrogated growth inhibition by EGCg and capsaicin and reduced anchorage-dependent growth of HeLa (human cervical carcinoma) cells that naturally overexpress tNOX. The findings show cell surface expression of tNOX as both necessary and sufficient for the cellular anticancer activities attributed to both EGCg and capsaicin.     

Reciprocal Relationship Between Cytosolic NADH and ENOX2 Inhibition Triggers Sphingolipid‐Induced Apoptosis in HeLa Cells

ENOX2 (tNOX), a tumor‐associated cell surface ubiquinol (NADH) oxidase, functions as an alternative terminal oxidase for plasma membrane electron transport. Ubiquitous in all cancer cell lines studied thus far, ENOX2 expression correlates with the abnormal growth and division associated with the malignant phenotype. ENOX2 has been proposed as the cellular target for various quinone site inhibitors that demonstrate anticancer activity such as the green tea constituent epigallocatechin‐3‐gallate (EGCg) and the isoflavene phenoxodiol (PXD). Here we present a possible mechanism that explains how these substances result in apoptosis in cancer cells by ENOX2‐mediated alterations of cytosolic amounts of NAD⁺ and NADH. When ENOX2 is inhibited, plasma membrane electron transport is diminished, and cytosolic NADH accumulates. We show in HeLa cells that NADH levels modulate the activities of two pivotal enzymes of sphingolipid metabolism: sphingosine kinase 1 (SK1) and neutral sphingomyelinase (nSMase). Their respective products sphingosine 1‐phosphate (S1P) and ceramide (Cer) are key determinants of cell fate. S1P promotes cell survival and Cer promotes apoptosis. Using plasma membranes isolated from cervical adenocarcinoma (HeLa) cells as well as purified proteins of both bacterial and human origin, we demonstrate that NADH inhibits SK1 and stimulates nSMase, while NAD⁺ inhibits nSMase and has no effect on SK1. Additionally, intact HeLa cells treated with ENOX2 inhibitors exhibit an increase in Cer and a decrease in S1P. Treatments that stimulate cytosolic NADH production potentiate the antiproliferative effects of ENOX2 inhibitors while those that attenuate NADH production or stimulate plasma membrane electron transport confer a survival advantage. J. Cell. Biochem. 110: 1504–1511, 2010. © 2010 Wiley‐Liss, Inc.     

Differential response of the NADH oxidase of plasma membranes of rat liver and hepatoma and HeLa cells to thiol reagents

NADH oxidase activity of plasma membranes from rat hepatoma and HeLa cells responded to thiol reagents in a manner different from that of plasma membranes of liver. Specifically, the NADH oxidase activity of plasma membranes of HeLa cells was inhibited by submicromolar concentrations of the thiol reagentsp-chloromercuribenzoate (PCMB),N-ethylmaleimide (NEM), or 5,5-dithiobis-(2-nitrophenylbenzoic acid) (DTNB), whereas that of the rat liver plasma membranes was unaffected or stimulated over a wide range of concentrations extending into the millimolar range. With some hepatoma preparations, the NADH oxidase activity of hepatoma plasma membranes was stimulated rather than inhibited by PCMB, whereas with all preparations of hepatoma plasma membranes, NEM and DTNB stimulated the activity. In contrast, NADH oxidase activity of rat liver plasma membrane was largely unaffected over the same range of PCMB concentrations that either stimulated or inhibited with rat hepatoma or HeLa cell plasma membranes. Dithiothreitol and glutathione stimulated NADH oxidase activity of plasma membranes of rat liver and hepatoma but inhibited that of HeLa plasma membranes. The findings demonstrate a difference between the NADH oxidase activity of normal rat liver plasma membranes of rat hepatoma and HeLa cell plasma membranes in addition to the differential response to growth factors and hormones reported previously (Brunoet al., 1992). Results are consistent with a structural modification of a NADH oxidase activity involving thiol groups present in plasma membranes of rat hepatoma and HeLa cells but absent or inaccessible with plasma membranes of rat liver.     

Cancer isoform of a tumor-associated cell surface NADH oxidase (tNOX) has properties of a prion.

We have described a drug-responsive form of a cell surface NADH oxidase (hydroquinone oxidase) of cancer cells (tNOX) that exhibits unusual characteristics including resistance to proteases, resistance to cyanogen bromide digestion, and an ability to form amyloid filaments closely resembling those of spongiform encephalopathies and all of which are characteristics of PrP(sc) (PrP(res)), the presumed infective and proteinase K resistant particle of the scrapie prion. The tNOX protein from the HeLa cell surface copurified with authentic glyceraldehyde-3-phosphate dehydrogenase (muscle form) (GAPDH). Surprisingly, the tNOX-associated muscle GAPDH also was proteinase K resistant. In this paper, we show that combination of authentic rabbit muscle GAPDH with tNOX renders the GAPDH resistant to proteinase K digestion. This property, that of converting the normal form of a protein into a likeness of itself, is one of the defining characteristics of the group of proteins designated as prions.     

Membrane topology of the murine fatty acid transport protein 1

The murine fatty acid transport protein (FATP1) was identified in an expression cloning screen for proteins that facilitate transport of fatty acids across the plasma membranes of mammalian cells. Hydropathy analysis of this protein suggests a model in which FATP1 has multiple membrane-spanning domains. To test this model, we inserted a hemagglutinin epitope tag at the amino terminus or a FLAG tag at the carboxyl terminus of the FATP1 cDNA and expressed these constructs in NIH 3T3 cells. Both tagged constructs produce proteins of the expected molecular masses and are functional in fatty acid import assays. Indirect immunofluorescence studies with selective permeabilization conditions and protease protection studies of sealed membrane vesicles from cells expressing epitope-tagged FATP1 were performed. These experiments show that the extreme amino terminus of tagged FATP1 is oriented toward the extracellular space, whereas the carboxyl terminus faces the cytosol. Additionally, enhanced green fluorescent protein fusion constructs containing predicted membrane-associated or soluble portions of FATP1 were expressed in Cos7 cells and analyzed by immunofluorescence and subcellular fractionation. These experiments demonstrate that amino acids 1-51, 52-100, and 101-190 contain signals for integral association with the membrane, whereas residues 258-313 and 314-475 are only peripherally membrane-associated. Amino acid residues 191-257 and 476-646 do not direct membrane association and likely face the cytosol. Taken together, these data support a model of FATP1 as a polytopic membrane protein with at least one transmembrane and multiple membrane-associated domains. This study provides the first experimental evidence for topology of a member of the family of plasma membrane fatty acid transport proteins.     

Stress-induced down-regulation of tumor-associated NADH oxidase during apoptosis in transformed cells

Tumor-associated NADH oxidase (tNOX) is a growth-related protein expressed in transformed cells. tNOX knockdown using RNA interference leads to a significant reduction in HeLa cell proliferation and migration, indicating an important role for tNOX in growth regulation and the cancer phenotype. Here, we show that tNOX is down-regulated during apoptosis in HCT116 cells. Treatment with diverse stresses induced a dose- and time-dependent decrease in tNOX expression that was concurrent with apoptosis. Moreover, shRNA-mediated tNOX knockdown rendered cells susceptible to apoptosis, whereas re-expression of tNOX partially recovered cell proliferation. Our results indicate that tNOX is suppressed during apoptosis and demonstrate that tNOX down-regulation sensitizes cells to stress-induced growth reduction, suggesting that tNOX is required for transformed cell growth.     

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane.

NADH oxidase activity (electron transfer from NADH to molecular oxygen) of plasma membranes purified from rat liver was characterized by a cyanide-insensitive rate of 1 to 5 nmol/min per mg protein. The activity was stimulated by growth factors (diferric transferrin and epidermal growth factor) and hormones (insulin and pituitary extract) 2- to 3-fold. In contrast, NADH oxidase was inhibited up to 80% by several agents known to inhibit growth or induce differentiation (retinoic acid, calcitriol, and the monosialoganglioside, GM3). The growth factor-responsive NADH oxidase of isolated plasma membranes was not inhibited by common inhibitors of oxidoreductases of endoplasmic reticulum or mitochondria. As well, NADH oxidase of the plasma membrane was stimulated by concentrations of detergents which strongly inhibited mitochondrial NADH oxidases and by lysolipids or fatty acids. Growth factor-responsive NADH oxidase, however, was inhibited greater than 90% by chloroquine and quinone analogues. Addition of coenzyme Q10 stimulated the activity and partially reversed the analogue inhibition. The pH optimum for NADH oxidase was 7.0 both in the absence and presence of growth factors. The Km for NADH was 5 microM and was increased in the presence of growth factors. The stoichiometry of the electron transfer reaction from NADH to oxygen was 2 to 1, indicating a 2 electron transfer. NADH oxidase was separated from NADH-ferricyanide reductase, also present at the plasma membrane, by ion exchange chromatography. Taken together, the evidence suggests that NADH oxidase of the plasma membrane is a unique oxidoreductase and may be important to the regulation of cell growth.     

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.     

Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell proliferation and migration in cancer cells

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. Consistent with this function, tNOX knockdown by RNA interference leads to a significant reduction in cell proliferation and migration in HeLa cells, whereas tNOX overexpression confers an aggressive phenotype. Here, for the first time, we report that tNOX is phosphorylated by protein kinase Cδ (PKCδ) both in vitro and in vivo. Replacement of serine-504 with alanine significantly reduces phosphorylation by PKCδ. Co-immunoprecipitation experiments reveal an interaction between tNOX and PKCδ. Moreover, whereas overexpression of wild-type tNOX in NIH3T3 cells increases cell proliferation and migration, overexpression of the S504A tNOX mutant leads to diminished cell proliferation and migration, reflecting reduced stability of the unphosphorylatable tNOX mutant protein. Collectively, these results suggest that phosphorylation of serine-504 by PKCδ modulates the biological function of tNOX.