Abstract

     Manganese superoxide dismutase (MnSOD) is a primary antioxidant enzyme responsible for protecting the mitochondria from superoxide radical generated during aerobic respiration and pathological conditions. Increased expression of MnSODhas been shown to suppress cancer phenotypes in a large number of human and murine tumors. We have demonstrated that overexpression of MnSOD suppressed metastasis of highly invasive mouse fibrosarcoma (Fsa-II) cells. To explore the global change in gene expression patterns resulting from an increased cellular MnSOD level, we performed DNA microarray analysis of gene expression patterns in control Fsa-II NEO cells and Fsa-II SOD-H cells overexpressing the human MnSOD gene, previously shown to have reduced metastasis potential. Using Affymetrix mouse gene array and multidimensional analysis of genes expressed in the Fsa-II SOD-H over the Fsa-II NEO cells, we identified some genes as being up- or downregulated by MnSOD. Expressions of genes coding proteins known to promote angiogenesis and metastasis including VE-cadherin, MMP-1 and MMP-3 were decreased while i.e. expressions of lysyl oxidase and TIP30/CC3 putative cancer metastasis suppressor genes as well c-myc protooncogene were increased in Fsa-II SOD-H cells. Expressions of genes coding matrix proteins known to inhibit motility of tumor cells were increase, thus, consistent with the anti-metastatic effect of MnSOD. There was also increased llevel of the homolog to phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor. Biochemical analysis using chemicals affecting various superoxide metabolism pathways (i.e. metasuccinate and antimycin A) confirmed the role of superoxide/hydrogen peroxide in regulating expressions of genes revealed by microarray analysis. Taken together, these results are consistent with the hypothesis that regulation of redox status in the mitochondria can alter gene expression profile consistent with suppression of tumor metastasis.

Abbreviations: slg - signal log ratio, MnSOD manganese superoxide dismutase

Keywords: manganese superoxide dismutase, MnSOD, carcinogenesis, tumorigenesis, ROS


Introduction

     Cells of aerobic organisms are armed in enzymatic systems protecting them from oxidative stress caused by reactive oxygen species (ROS) [1]. The crucial role in detoxicating ROS play superoxide dismutases (SODs) catalysing conversion of superoxide to hydrogen peroxide and molecular oxygen:
2O₂^• – + 2H⁺ ® H₂O₂ + O₂
Eucaryotic cells have two metal-containing SOD isoenzymes - mitochondrial MnSOD and cytosolic Cu/ZnSOD [5]. Superoxide is produced by complex I and complex III of the respiratory chain, flavoenzymes, e.g. xanthine oxidase, lipoxygenase and cyclooxygenase[2]. Hydrogen peroxide is removed by catalases (CAT) and glutathione peroxidases (GPx) [1].
     Manganese superoxide dismutase (MnSOD) was proposed as a tumor suppressor [3, 11]. It was shown, that it's overexpression suppresses metastasis of mouse fibrocarcoma [5], radiation-induced neoplastic transformation of mouse C3H 10T1/2 cells [4], malignant phenotype of human breast cancer MCF-7 cells [6], human glioma cells [9] and tumor malignancy of human oral squamous carcinoma SCC-25 cells [7] . The increase of MnSOD activity can also reverse part of the malignant phenotype in SV40-transformed human lung fibroblasts [8] and correlation between the loss of the transformed phenotype and an increase in superoxide dismutase activity in a revertant subclone of sarcoma virus-infected rat kidney (NRK) cells was reported [16]. Human chromosome 6 region where MnSOD gene has been maped is frequently lost in SV40-transformed (TF) human fibroblasts [3] and melanoma cells [17] and it is correlated with decrease in MnSOD activity. Moreover stable transfection of MnSOD cDNA into melanoma cells exerts a biological effect similar to observed after introduction of an entire human chromosome 6 [17]. Also mutations in the proximal region of MnSOD promoter were identified in several human tumor cell lines with reduced MnSOD activity [18]. These mutations lead to increased binding site for AP-2, which downregulates transcription of the human MnSOD gene via its interaction with Sp1 [143]. Previously was shown, that a mouse fibrosarcoma cell line (Fsa-II) exhibiting low endogenous levels of MnSOD stably transfected with MnSOD cDNA were less malignant than control cells [5].
     Diminished amounts of MnSOD have been found in many tumors [10, 11, 14], however MnSOD level is decreased only in 10-14% of human breast cancer tumors [13] and increased in gastric cancer [12]. Moreover MnSOD overexpression resulted in a 50% increase in hamster cheek pouch carcinoma HCPC-1 cell invasiveness [15].
     Mechanism of MnSOD role as tumor suppressor is not well established. As possible way of MnSOD action was proposed changing of gene expression profile by affecting transcription factors action. Overexpression of MnSOD in mouse fibrosarcoma cells FSaII really leads to decrease of activity of two transcription factors - AP-1 and CREB [24, 25]. MnSOD protects also these cells against apoptosis and, what might contribute to carcinogenesis inhibition, promotes a differentiation program what involves NFkappaB and Raf1/MEK/ERK pathways [26, 27]. Activation of NF-kappaB and AP-1 transcription factors and suppresion of apoptosis by MnSOD was observed also in MCF-7 cells [29]. MnSOD overexpression in these cells upregulates MET, GADD153, CD9, a-catenin and plakoglobin and downregulates VEGFR-1, TNF-a converting enzyme and interleukin-1b. Involved in DNA damage response GADD153 gene shows 33 fold increase of expression [28]. Activated is also the matrix metalloproteinase-2 what involves reactive oxygen species [19]. Matrix metalloproteinases are well known enzymes involved in tumor invasion by affecting the extracellular matrix [20, 21, 22, 23, 59]. All these changes except differentiation should rather increase survival of tumor cells and promote metastasis and the mechanism of MnSOD action remained still unsolved. Previous studies revealed also activation of MnSOD promoter by TPA [142] known to be able to promote not malignant skin tumors (papilloma) converting later into malignant tumors (carcinoma). This is also consistent with data showing, that the NF-kB present in the second intron of MnSOD gene is essential for high-level induction of MnSOD by cytokines [144]. Tumor promotion may be however independent on MnSOD activation even this enzyme inhibits late steps of carcinogenesuis (malignancy).
     Because previous data in general pointed at least at changes in transcription factors activity in cells overexpressing MnSOD, we decided to solve this problem comparing gene expression patterns in control and MnSOD stably transfected FSa cells using microarray technique. It allowed us to screan the transcription of about 12000 genes, what gave a real chance to explain the mechanism of carcinogenesis inhibition by MnSOD. We also tryed to mimic these changes by affecting reactive oxygen species metabolism using specific agents (mostly inhibitors) affecting ROSs metabolism.

Materials and Methods

Cell culture and treatment

     We performed our experiments on murine fibrosarcoma cells Fsa-II transfected with pSV2-NEO plasmid (control cell line Fsa-II NEO) and the same cell line cotransfected with the MnSOD expressing plasmid pHbAPR-1 (clone revealing high MnSOD expression - Fsa-II SOD-H) [5]. The cells were maintained in McCoy's medium 5a supplemented with 10% fetal bovine serum, 250 mg/ml penicillin, 250 mg/ml streptomycin and 500 mg/ml of geneticyn (G418) in a humidified atmosphere containing 5% CO₂ [145].
     Cells were treated for 24 hours with 500 mM N^G-amino-L-arginine (L-NAA.HCl) (Sigma), 500 mM DETA NONOate (Sigma), 100 mM ebselen (Alexis), 10 mM mercaptosuccinate (MS) (Sigma-Aldrich), 50 mM 3-amino-1,2,4-triazole (3-AT) (Calbiochem) and 10mM antimycin A (Sigma). Ebselen, 3-AT, and antimycin A stocks were DMSO solutions, other chemicals were diluted in the water.


Microarray analysis

     Total RNA was isolated using Invitrogen TRIzol and cleaned up using Quiagen RNeasy Mini Kit. Final RNA concentration was about 2 mg/ml. Next steps of Affymetrix GeneChip sample and array processing were performed as in Affymetrix GeneChip Expression Analysis Technical Manual in microarray core facility equipped with a GeneChip Fluidics Station 400, a GeneChip Hybridization Oven, a Gene Array Scanner and the Affymetrix Microarray Suite (MAS) workstation. Target was hybridized to the Murine Genome U74Av2 Array (MG-U74Av2) belonging to Affymetrix GeneChip Murine Genome U74v2 Set. This array represents all sequences (about 6000) in the Mouse UniGene database (Build 74) that have been functionally characterized and additionally about 6000 other EST clusters.
     According to Affymetrix guidelines for determining the most robust changes of genes expression, we analyzed data using three metrics calculated by GeneChip Analysis Suite: detection, change and signal log ratio. Detection is the qualitative measure telling if a particular transcript is present (P) or absent (A), change is a qualitative measure of it's increase (I) or decrease (D) and signal log ratio is the quantitative measure of gene expression levels difference:

signal log ratio = log2 fold change fold change = 2signal log ratio	}	for increases and
signal log ratio = log2(-fold change) fold change = (-1)×2-signal log ratio	}	for decreases

     As in Affymetrix "Data analysis fundamentals manual", probe sets were sorted for robust increases by eliminating pairs called in the experimental sample "absent" (A), subsequent selecting these of them called "increase" (I) and eliminating sets with a signal log ratio < 1 (fold change < 2). To determine robust decreases eliminated were pairs called "absent" in the baseline sample, selected ones called "decrease" (D) and eliminated sets with signal log ratio > -1 (fold change > -2).


RT PCR

     Total RNA was isolated using TRIZol reagent (Invitrogen). The first strand cDNA was synthetized using using 2 mg of total RNA as a template and SuperScript II RNAse H^- reverse transcriptase (Invitrogen). Reaction was primed using oligo(dT)_12-18 primers (Invitrogen). After termination of the reverse transcription RNA template was removed from the cDNA:RNA hybrid by digestion with RNAse H (Invitrogen). For PCR reaction Taq DNA Polymerase (Invitrogen) was used. All of above procedures were performed according to manufacturer protocols.
     PCR reaction conditionas were as follows: denaturation at 94°C, 2 min, 28 cycles of PCR (denaturing at 94°C, 1 min, annealing, 1 min, extension at 72°C, 1 min) and final extension at 72°C, 10 min. Primers sequences (5'®3'), annealing temperatures and product sizes were as follows: mouse and human b-actin: cac act gtg ccc atc tac ga, gta ctt gcg ctc agg agg ag (53.0°C, 531 bp), human MnSOD: ccc tgg aac ctc aca tca ac, cct tgc agt gga tcc tga tt (51.2°C, 406 bp), mouse lysyl oxidase (LOX): tca tct gcc tga aag cac ac, ggt tgg gga cag tcg ttt ta (51.3°C, 424 bp), mouse MMP3: tgg aga tgc tca ctt tga cg, gat gga aga gat ggc caa aa (49.9°C, 449 bp), mouse homolog to phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor, gb=AK014199: agg aac cag aca cca aca gg, ttg gca tgg gta ggt aga gg (52.5°C, 566 bp). Mouse VE cadherin primers vere designed to amplify DNA sequence in the fragment shared by it's published mRNA (gb=X83930) and gene sequences (gb=X83678): tgg aga ttc acg agc agt tg, gat cca ggt tgc aat gag gt (50.1°C, 497 bp).


Northern blottig and hybrydization

     30 mg of total RNA isolated as above were separated in denaturing agarose gel (Sigma, for routine use) containing 2.2 M formaldehyde [146]. RNA was transferred to nylon membrane (Nytran SuPerCharge, Schleicher & Schuell) by capillary transfer using Nytran SuPerCharge Turbo Blotter rapid downward transfer kit (Schleicher & Schuell) following manufacturer protocols and ultraviolet cross-linked.
     DNA probes for particular transcripts were prepared using RT PCR products as templates and PCR reaction described above, suspected to electroforesis and isolated from the gel using Quiagen Gel Extraction Kit. About 20 ng of a probe was radiolabeled with [a-³²P] dCTP (Company?) using Amersham Rediprime^TMII system and purified on the Amersham ProbeQuantTM G-50 Micro Column. Membrane was prehybridized, hybridized in QuickHybâ Rapid Hybridization Solution (Stratagene) and washed as in manufacturer protocol. Autoradiography was performed using Amersham Hyperfilm MP.


Results

Microarray Analysis

     Microarray analysis revealed, that MnSOD transfection resulted in robust expression decreases (signal log ratio £ -1, fold decrease £ -2) of many functionally characterized and other genes homological respectively to 50 and 41 array probes (oligonucleotides on the surface of the probe array). Overexpression of this enzyme caused also robust increases of levels (signal log ratio ³ 1, fold increase ³ 2) of known and unknown transcripts homological respectively to 43 and 31 probes.
     Among sequences representing not characterized transcripts coresponding to probes which revealed robust decreases we identified one (gb=AI853217) as practically identical with fragment of 3'-terminus of M.musculus VE-cadherin gene (gb=X83678) but not homological to any part of published M.musculus mRNA for VE-cadherin (gb=X83930). Sequence comparison of VE-cadherin transcript and gene sequences revealed overlapping of 5'-part of gene and 3'-part of mRNA. It suggests, that VE cadherin transcript contains probably much longer 3'-UTR than in published mRNA sequence, including fragment homological to the probe from the array. And that VE cadherin gene has much longer 5'-UTR than in published sequence.
     Another not characterized sequence (gb=AI882309) coresponding to other probe was identified as mouse homolog to phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor (gb=AK014199). However our analysis has shown, that aminoacid sequence of this hypothetical protein is almost identical with human protein described as weakly similar to glutathione peroxidase 2 (gb=AK027683, gb=CL683) and that it is difficult to say, which glutathione peroxidase is most similar to this protein.
     All robust changes of functionally characterized transcripts, putative VE-cadherin, phospholipid peroxidase and some of minor changes which seem to be important for carcinogenesis process are summarized in table 1.


RT PCR verification of microarray analysis

     To assure microarray comparison results, we performed additionally RT PCR analysis for some of robust changes (Picture 1). Using this method we confirmed very significant decreases of vascular endothelial cadherin (VE cadherin) and matrix metalloproteinase 3 (MMP3) as well increases of lysyl oxidase (LOX) and homolog to M.musculus phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor (gb=AK014199) expressions. We also detected expected very severe overexpression of human MnSOD in FsaII cell line transfected with this gene. b actin was used as a control.


Molecules affecting ROS metabolism pathways change gene expression levels

     We treated cells with many ROS metabolism inhibitors and examined transcripts levels of genes activated (LOX and homolog to M.musculus phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor, gb=AK014199) and inhibited (VE cadherin) by MnSOD overexpression using Northern hybridization (Picture 3). These results confirmed changes revealed by microarray analysis an have shown, that ROS may contribute to gene expression regulation.


Discussion

     As we predicted, MnSOD overexpression altered transcription of multiple genes. Majority of these changes are supposed to cause angiogenesis, migration and invasion inhibition (Scheme ).
     The most robust decrease we detected is transcription inhibition of the vascular endothelial cadherin VE cadherin (slg=-5.7) a protein localized at the intercellular junctions [34] implicated in many aspects of angiogenesis like homotypic endothelial cell adhesion [34], survival [35] and contact inhibition [33] required for normal organ functions [32]. Despite its name, VE cadherin is expressed not only by endothelial cells, but also by aggressive human melanoma cells, where is needed for vasculogenic networks formation [36]. Mice deficient in VE-cadherin or expressing truncated VE-cadherin die in midgestation from severe vascular defects as endothelial cells apoptosis and disrupted survival signaling pathways [35]. Moreover VE cadherin is involved in fibrin or collagen-induced capillary tube formation in vitro [31]. It was shown also, that monoclonal antibodies to VE cadherin are potent inhibitors of angiogenesis, tumor growth and metastasis and some of them disrupt also existing adherent junctions increasing vascular permeability [30, 40, 41]. Similarly reduction of the VE-cadherin gene transcription by TNF-a is associated with induction of vascular permeability [39]. It was shown, that a highly conserved region present in the juxtamembrane domain of VE-cadherin is responsible for binding p120 catenin (p120ctn, d-catenin), what plays an important role in cellular growth [37]. Moreover VE-cadherin clustering may trigger intracellular signals via small GTPases as Rac, which exert changes in actin cytoskeleton organization and cell shape [38].
     Other example proteins downregulated in Fsa-II SODH cells which could contribute to possible angiogenesis inhibition are uridine phosphorylase UPase (slr=-1.9) [112], interleukin-17 (IL-17) receptor (slr=-1.7), [117]. angiopoietin-2 (slr=-0.9) [130, 131] and endothelial protein C receptor EPCR (slr=-1.0).
     Fibrosarcoma cells ransfection with MnSOD upregulated also transcription of many genes coding proteins of the extracellular matrix (ECM), involved in ECM modifications or binding cells to it. We speculate, that ECM protein networks can trape tumor cells, increase their attachment and thus make difficult cell migration needed for invasion and subsequently also for angiogenesis and metastasis. Likewise downregulation of proteases digesting ECM and upregulation of their inhibitors should decrease invasiveness of cells. Moreover some of above listed types of molecules directly or indirectly can trigger or inhibit signal transduction pathways regulating apoptosis, growth or motility of tumor cells.
     Most significant among these changes (and all increases) is overexpression of lysyl oxidase LOX (slr=+5.9) - a copper-dependent amine oxidase that initiates the covalent cross-linking of extracellular matrix proteins - collagens and elastin by catalyzing oxidative deamination of the epsilon-amino group in certain lysine and hydroxylysine residues [42, 43]. LOX was shown as downregulated in many tumor cells and may be induced along with the reversion process [45, 46, 47, 48, 49, 50]. It occurs mainly in cells transformed by ras or ras-dependent oncogenes and therefore is called ras recision gene (rrg) since was identified as a tumor-suppressor gene [45]. However, LOX is upregulated in highly invasive human breast cancer cells MDA-MB-231 in comparison with less invasive MCF-7 cells and inhibition of it's expression or activity decreases metastatic properties of these cells [44]. Fibrosarcoma cells transfected with MnSOD weakly overexpress also N-acetylglucosamine-6-O-sulfotransferase (slr=+1.7) [90] and UDP-Gal-bGlcNAc b 1,3-galactosyltranferase-III (slr=+0.9) [108] involved in glycoproteins modyfying.
     The ECM Leu-rich proteglycan biglycan very significantly overexpressed by transfected fibrosarcoma cells (slr=+5.3) has been shown to inhibit growth of pancreatic cancer cells by arresting them in G1 and may be part of a host defense mechanism induced by transforming growth factor-b (TGF-b) TGF-b1 aimed at slowing down tumor progression [76]. Moreover TGF-b induction of BGN expression requires activation of MKK6-p38 MAPK signaling downstream of tumor suppressor gene product Smad [78, 77]. MnSOD transfected cells overexpress also many other extracellular matrix proteins including collagen VI a3(VI) chain (slr=+3.2), decorin (slr=+1.1) [79], procollagen type Va2 (slr=+1.6) [91]. Weakly increased extracellular matrix proteins - transforming growth factor-b inducible matrix protein P68 big-h3 (slr=+1.0) [96, 97], biliary glycoprotein BGP (slr=+1.1) [100] and a member of transmembrane-4 superfamily (TM4SF or tetraspannins) TM4SF6 (slr=+1.1) [106] are suggested to be involved in metastasis inhibition. Moreover MnSOD upregulated Oncostatin M (OM) receptor b OSMRb (slr=+1.1) expression, which ligand - IL-6 cytokine family member oncostatin M (OM) was reported to be able to suppress growth and induce the production of ECM components [95]. Among upregulated molecules related to ECM are worth to be mentioned also membrane associated protein e-sarcoglycan (slr=+1.3) [99], fibrillin 1 (slr=+1.5) [92, 93] and matrix Gla protein MGP (slr=+2.4) [83, 84?, 85]. Some of them as CD44 (slr=-1.1) [124], galectin-9 (slr=-1.1) [125] and PSGL-1 (slr=-1.2) [120] have been reported to be involved in tumor cells function.
     So significant inrease in production of extracellular and membrane associated proteins requires development of secretory machinery. In accordance with these expectations our cells overexpressed at least two proteins involved in secretion process aquaporin AQP1 (slr=+5.7) [80] and secretogranin II SgII (slr=+2.8) [81].
     MnSOD overexpression not only upregulated transcription of genes coding ECM proteins, but also diminished transcripts levels of three matrix metalloproteinases digesting ECM - MMP-3/Stromelysin-1 (slr=-4.3), MMP-1/Collagenase-1 (slr=-2.1) and ADAM-8 (slr=-1.1) and one of protease inhibitors - TFPI-2 (slr=+1.1). We hypothesize, that these changes should result in the inhibition of the ability of tumor cells to ECM degradation and as consequence tissue invasion by tumor cells as well as tissue remodeling needed for angiogenesis, tumor growth and finally metastasis. Matrix metalloproteinases (MMPs) are a family of over 20 zincinc-dependent neutral endopeptidases that cleave the various components of the extracellular matrix (ECM) as collagens, proteoglycans or gelatin. They allow not only mentioned above tissue remodeling, but also release from ECM and other proteins fragments with biological activities, including other MMPs. All above taken together facilitates not only normal tissue remodeling, but also pathological destruction and tumor cells growth, invasion, metastasis and angiogenesis [for review see 20, 21, 22, 23, 59]. MMP3 and/or MMP1 can also release or activate growth factors including the basic fibroblast growth factor (FGF) [60] insulin-like growth factor IGF [62, 63], transforming growth factor b1 (TGF-b1) [61], EGF [64] and interleukin-1-b [70]. Moreover MMP-3 facilitates cell migration by cleaving an ectodomain of a cell-cell adhesion and signal-transducing molecule E-cadherin disrupting adhesion junctions and releasing it's soluble fragment, what timulates cell migration and invasion in a paracrine way [65]. In addition, chondrocytic MMP-3 generates an unidentified macrophage chemoattractant that stimulates proteolytically active macrophages infiltration of the herniated disc and subsequent proteoglycan degradation [66]. MMP-3 cleaves also osteopontin - inducible inhibitor of ectopic vascular calcification [83]. The MMP-1 or MMP-3 overexpression in mouse skin cause skin abnormalities, hyperplasia and increase susceptibility or promote mammary carcinogenesis [67, 68, 69]. MMP-3 activation correlates also with squamous cell carcinoma (SCC) invasiveness in vitro [75]. Similarly, overexpression of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) can inhibit angiogenesis and associated tumor growth in a murine tumor model [74]. The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression [72]. Moreover, MMPs including MMP-1 and MMP-3 can be used as markers to predict tumor recurrence and risk of death in several cancer types [71] and the promoter polymorphism of the MMP-3 or MMP-1 gene enhances susceptibility to several cancers including the breast cancer [73]. Slightly downregulated in MnSOD transfected cells ADAM-8 belongs to the family of membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM [123] and may also contribute to invasiveness and metastasis. Weakly overexpressed human tissue factor pathway inhibitor-2 (TFPI-2) is a serine protease inhibitor repressing invasion by HT-1080 fibrosarcoma, LNCaP prostate cancer, JAR choriocarcinoma, C-32 amelanotic melanoma and human glioma cells in vitro [101, 102, 103, 104, 105] and by JAR choriocarcinoma cells in vivo [103].
     MnSOD affected transcription of few genes coding proteins taking part in signal transduction. We observed i.e. upregulation of the cellular retinol (vitamin A) binding protein 1 CRBP1 (slr=+2.3) inhibitor of the PI3K/Akt survival pathway and suppressor of anchorage-independent growth [87]. Down-regulation of CRBP1 has been shown in breast cancer cell lines and tumors, what was associated with common in tumors and cancer cell lines hypermethylation [88].
     So large number and wide range of genes, which transcription was affected in Fsa-II cells after transfection with MnSOD, and some of these changes point at switch in differentiation programme as a most general cause of decrease of our cells malignancy. One of proteins known to be involved in development and differentiation process is very strongly downregulated in our cells keratin 18 (slr=-3.6). This protein belongs to acidic type I keratins (K9 to K20), which together with basic type II keratins (K1 to K8) and four other categories of proteins (types III-VI) are the largest family of cytoskeletal molecules and form the intermediate filaments (IF). The expression patterns of keratins are tissue- and differentiation-specific [132]. Keratins K18 (endoB in mice) and K8 (Endo A in mice) are useful markers for human carcinomas arising from simple epithelia [134]. Keratins functions are not only structural. K18 may suppress TNF and Fas mediated apoptosis. It's overexpression increases also L fibroblasts migration and invasion. It is postulated also involvement of K18 (and K8) in TGF-b signaling [132]. Other proteins implicated in differentiation, downregulated in Fsa-II SOD-H cells are glypican-4 (slr=-2.3) [110, 111] and N-myc downregulated gene Ndr1 (slr=-1.1) [128]. Involvement of MnSOD in differentiation regulation was postulated also earlier [141].
     It is worth to tell also about several other genes potentially involved in carcinogenesis inhibition, which transription was affected by MnSOD overexpression. Downregulated in our cells ornithine decarboxylase ODC (slr=-1.1) gene was reported as a transcriptional target of c-Myc in association with induction of cell proliferation and transformation, but not with induction of apoptosis [126]. ODC is a key enzyme in biosynthesis of polyamines, which increased levels are required for growth, differentiation, and transformation of cells and its expression is upregulated in cancer cells and coincides with expression of oncogenes [127]. Changes in MRP (slr=-1.0) [129], protein S (slr=+2.9) [82], the CC chemokine eotaxin CCL11 (slr=+1.4) [94] and Friend-virus-susceptibility-1 Fv1 (slr=-1.9) [115, 116] genes expression may be also linked to decreased tumorigenicity of Fsa-II SOD-H cells.
     MnSOD overexpression not only affected transcription factors activity, what was reported earlier, but also their expression levels. It concerns many proteins related to them too. These changes probably directly resulted in the affection of gene expression profile after transfection with MnSOD and subsequent decrease of malignant properties of fibrosarcoma cells. Most important seems to be here downregulation of myelocytomatosis oncogene c-myc (slr=-1.3). Constitutive expression of the proto-oncogene c-myc contributes to progression of a wide range of human and animal tumors. Myc executes its multiple activities mostly through positive and negative transcriptional regulation of the target genes, mostly regulating cell growth and proliferation. Some observations however suggest that Myc also affects later stages of tumorigenesis, most notably angiogenesis [119]. MnSOD decreased also transcription of Pvt 1 gene (slr=-1.1) located 260 kb downstream of c-myc on mouse chromosome 15. Its juxtaposition onto the immunoglobulin light chain gene, similarly as c-myc-activating translocation that juxtapose c-myc onto the immunoglobulin heavy chain, is the principal lesion in many B cell malignancies including Burkitt's Lymphoma (BL), AIDs-NHL, mouse plasmacytoma (Pct) and possibly multiple myeloma (MM) [109]. It is very interesting, that two different chromosomal translocations (Txs) produce indistinguishable disease phenotypes. Moreover Pvt 1 and c-myc genes are both amplificated and present in the same amplicons forming double minute (DM) chromosomes found in many tumors, i.e. in acute myeloid and nonlymphatic leukemias [135, 136]. We noted also upregulation of c-myc coding region determinant binding protein CRD-BP (slr=+2.5) protecting c-myc mRNA from endonucleolytic attack during translation, what occurs probably when ribosomes reach a translation pause site [119]. It suggests indirectly inhibition of c-myc translation in addition to transcription in fibrosarcoma cells overexpressing MnSOD. At c-myc activity decrease points also downregulation of its transcriptional target ornithine decarboxylase ODC (slr=-1.1), what was mentioned earlier.
     Moreover MnSOD transfections upregulated expression of transcriptional cofactor TIP30/CC3 (slr=+1.2), which specifically enhances HIV-1 Tat-activated transcription [52, 51, 52, 57]. TIP30/CC3 was shown to be associated with metastasis suppression for small cell lung carcinoma (SCLC) and a mouse melanoma B16 in vivo [53, 54, 55]. The mechanism is not fully understood, but in SCLC cells likely is due at least in part to the induction of apoptosis, which involves disruption of the mitochondrial membrane potential [56, 57]. TIP30/CC3 inhibits also angiogenic properties of tumor cells in vitro and induced changes of several angiogenic modulators as angiopoietin 1, slightly overexpressed in our cells angiopoietin 2 (slr=+0.9), BAI1, VEGF and SPARC [54].
     MnSOD upregulated also expression of well known tumor suppressors retinoblastoma family member p107 (slr=+1.0). It is inhibitor of E2F4 transcription factor acting predominantly in the S phase of the cell cycle, but surprisingly causing G1 arrest in some of cells. p107 can recruit also CDK2 containing complexes to promoters and also inhibit CDK function [107]. MnSOD downregulated also transcription of other nuclear proteins which can be partially responsible for carcinogenesis inhibition by MnSOD: a mouse homolog of zinc finger gene OZF (slr=-1.6) amplified and overexpressed in human pancreatic carcinomas [118], the cell typerestricted ankyrin repeat protein CARP (slr=-1.2) possibly inducing angiogenesis [122, 121] as well npdcf-1 (slr=-3.0) and Slfn2 (slr=-2.4) involved in discussed earlier differentiation process [137, 138].
     The possible explanation of all these changes in transcripts levels is that increased expression of MnSOD in Fsa-II SOD-H cells catalyzing transformation of oxygen superoxide to hydrogen peroxide:
2O₂^• – + 2H⁺ ® H₂O₂ + O₂
affected concentration of an unidentified oxygen derivative which in some way takes part in genes regulation, what is perhaps dependent on oxygen availability in the cell environment. It could be for example part of mechanism regulating for example tissue remodeling needed for angiogenesis. To localyse and identify this hypothetical molecule we were trying to exert similar effect as MnSOD transfection (Pictures 2 and 3).
     Because MnSOD overactivity supposed to decrease concentration of superoxide [2], we treated cells with respiratory chain complex III inhibitor antimycin A, well known agent increasing superoxide production [2,139]. As we expected, it diminished transcription levels of genes induced by MnSOD in SOD-H cells, but surprisingly exerted similar effect on these genes which were expressed only in NEO cells. However this change was less significant. We speculate, that ATP production decrease caused by antimycin A and perhaps increased cell death [19], may be responsible for not specific decrease transcripts levels in both cell lines. But this effect probably was overlapped by some kind of specific action possibly caused by increased superoxide level and it could explain, why expressions of MnSOD inducible genes were decreased more then inhibitable ones.
     Since MnSOD substrate superoxide reacts with nitric oxide (NO) producing peroxynitrite (OONO-) and thus concentrations of these molecules seem to be dependent on superoxide availability and MnSOD acivity, we examined, if NO known as very important factor in redox control of cellular function [140], is able to affect gene expression. To check this possibility we treated cells with endothelial and inducible NO synthases (eNOS/NOS III, iNOS/NOS II) inhibitor NG-amino-L-arginine (L-NAA) and with NO donor DETA NONOate, but these chemicals did not cause any significant changes in genes expressions levels. Treatment with ebselen which is scavenger of oxidants, specifically peroxynitrite had no effect on transcript levels too.
     The other possibility of affecting gene expression by changes of superoxide concentration could be acting through the hydrogen peroxide, what is direct product of superoxide detoxication reaction catalysed by MnSOD. Because hydrogen peroxide may be further neutralysed by catalase (CAT) and peroxidases (GPx) [1], we treated cells with catalase inhibitor 3-amino-1,2,4-triazole (3-AT), peroxidases inhibitor mercaptosuccinate (MS) and both these chemicals simultaneously. After MS treatment we detected very significant specific decrease expressions of genes induced by MnSOD without change those ones expressed only in NEO cells. It suggests, that this hypothetical molecule triggering/inhibitting transcription may be among intermediates of peroxidation reactions. Simultaneous treatment of cells with 3-AT didn't cause such effect like MS alone because catalase inhibition probably redirected more hydrogen peroxide towards peroxidation reaction and MS effect in the presence of 3-AT was not so strong because of much higher availability of peroxidases substrate (hydrogen peroxide). There is very abundant variety of peroxidation intermediates including glutathione and products of its reaction with proteins. Trying to guess which one is responsible for MnSOD action we subjected cells to some of them: 4-Hydroxy-2-noneal (HNE), HpETE, HpODE and arachidonic acid) and to glutathione metabolism inhibitors: 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine), D,L - buthionine sulfoximine (BSO), but without significant effects (data not shown).
     It is worth to mentione, that transfection of Fsa-II cells with MnSOD induced expression of the homolog to phospholipid hydroperoxide glutathione peroxidase, mitochondrial precursor (AK014199). We speculate, that this putative enzyme might be part of the inducible mechanism scavenging hydrogen peroxide produced by overactivated MnSOD.
     Taking together these data supported our speculations, that MnSOD overexpression inhibited tumorigenicity of Fsa cells affecting their malignant properties rather then proliferation or apoptosis.