Nuclear genes involved in mitochondria-to-nucleus communication in breast cancer cells
© Delsite et al; licensee BioMed Central Ltd. 2002
Received: 31 October 2002
Accepted: 12 November 2002
Published: 12 November 2002
The interaction of nuclear and mitochondrial genes is an essential feature in maintenance of normal cellular function. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, mitochondrial DNA (mtDNA) encodes 13 subunits and rest of the subunits are encoded by nuclear DNA. Mutations in mitochondrial genes encoding the 13 subunits have been reported in a variety of cancers. However, little is known about the nuclear response to impairment of mitochondrial function in human cells.
We isolated a Rho0 (devoid of mtDNA) derivative of a breast cancer cell line. Our study suggests that depletion of mtDNA results in oxidative stress, causing increased lipid peroxidation in breast cancer cells. Using a cDNA microarray we compared differences in the nuclear gene expression profile between a breast cancer cell line (parental Rho+) and its Rho0 derivative impaired in mitochondrial function. Expression of several nuclear genes involved in cell signaling, cell architecture, energy metabolism, cell growth, apoptosis including general transcription factor TFIIH, v-maf, AML1, was induced in Rho0 cells. Expression of several genes was also down regulated. These include phospholipase C, agouti related protein, PKC gamma, protein tyrosine phosphatase C, phosphodiestarase 1A (cell signaling), PIBF1, cytochrome p450, (metabolism) and cyclin dependent kinase inhibitor p19, and GAP43 (cell growth and differentiation).
Mitochondrial impairment in breast cancer cells results in altered expression of nuclear genes involved in signaling, cellular architecture, metabolism, cell growth and differentiation, and apoptosis. These genes may mediate the cross talk between mitochondria and the nucleus.
Mitochondria participate in numerous functions in the cell. In addition to producing energy, mitochondria are involved in intermediary metabolism, ion homeostasis, synthesis of lipids, amino acids, and nucleotides, active transport processes, cell motility, and cell proliferation [1–4]. Recent developments also demonstrate that mitochondria are key regulators of programmed cell death . Mitochondrial dysfunction is one of the most profound features of cancer cells. Several distinct differences between the mitochondria of normal cells and cancer cells have been observed at the microscopic, molecular, biochemical, metabolic and genetic levels . Microscopic study of oncocytic tumors revealed mitochondrial hyperplasia , and differential expression of mitochondrial cytochrome oxidase II in benign and malignant breast tissues has also been reported . Furthermore, mutations in mitochondrial DNA (mtDNA) are commonly found in a variety of cancers including the ovarian, thyroid, salivary, kidney, liver, lung, colon, gastric, brain bladder, head and neck, leukemia and breast cancers .
Mitochondrial DNA encodes two rRNA, 22 tRNA and 13 proteins . Each of the polypeptides form subunits of four respiratory enzyme complexes localized to the inner mitochondrial membrane . These subunits include seven subunits of respiratory enzyme complex I, one subunit of complex III, three subunits of complex IV, and two subunits of complex V . All other mitochondrial proteins, including those involved in the replication, transcription and translation of mtDNA, are encoded by nuclear genes and are targeted to the mitochondrion by a specific transport system . Although the mitochondrial and nuclear genomes are physically distinct, there is constant communication between the two genomes to carry out many of the mitochondrial functions. For example, in the yeast Saccharomyces cerevisiae, RTGproteins monitor the functional state of mitochondria and mediate the intergenomic communication between the mitochondria and the nucleus. In this organism, RTG regulates transcription of numerous genes involved in several pathways that help cells adapt to mitochondrial dysfunction . In addition, yeast cells shift their metabolic profile by altering expression of a number of genes that respond to depletion of the mitochondrial genome . As in the case of yeast, the depletion of mitochondrial DNA from human cell lines leading to the generation of cell lines devoid of mtDNA (denoted Rho0) provides an opportunity to study nuclear responses to impairment of mitochondrial function. In the present study we isolated a Rho0 derivative of a breast cancer cell line. Our results demonstrate that Rho0 derivative experience increased oxidative stress and has impaired mitochondrial function. Using cDNA microarray we compared differences in the nuclear gene expression profile between parental Rho+ breast cancer cell line and its Rho0 derivative. Our study suggests a variety of structural genes as well as genes involved in cellular signaling and transcriptional regulation are differentially regulated in Rho0 breast cancer cells.
The plasmid containing the mitochondrial DNA-specific probe, pTZ19/K5, was a generous gift from Dr. Michael King (Thomas Jefferson University, USA). The probe has bases 2578–4122 of the human mitochondrial genome corresponding to a portion of the NADH dehydrogenase I gene, subcloned into the pTZ19 plasmid. Chemical reagents were obtained from Sigma-Aldrich, Corp. (St. Louis, MO) unless otherwise indicated.
MDA-MB-435 cells were obtained from American Type Culture Collection, and maintained in Dulbecco's modified Eagle's medium/ Ham's F-12 (50:50 mix) (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (GIBCO/Invitrogen Life Technologies, Carlsbad, CA), 2 mM L-glutamine, penicillin, streptomycin, and 50 μg/ml uridine. For measurement of cell growth in glucose and galactose, a 50:50 mix glucose-free Dulbecco's modified Eagle's medium (Sigma-Aldrich) and glucose-free Ham's F-12 (Biofluids, Rockville, MD) was used. Either glucose or galactose was added to 3.15 g/L, and the medium was supplemented as described above. Cells were cultured in a water-humidified incubator at 37°C in 10 % CO2/90% air. Medium was replenished every 2 days.
Generation of ρ0 Cell Line
Cells were cultured in the routine growth medium containing 50 ng/ml ethidium bromide (0.22 μm-filtered) with regular replenishment of medium. After a minimum of 30 days culture, single cell clones were isolated by limiting dilution in 96 well culture clusters, in the presence of ethidium bromide. The mtDNA status of clones was determined by Southern blotting, after which Rho0 clones were maintained in medium without ethidium bromide. Cells were screened after a minimum of 30 population doublings in the absence of ethidium bromide to verify continued Rho0 status.
MtDNA Southern blotting
Total cellular DNA was isolated from washed exponentially growing cells extracted in 10 mM Tris-Cl, pH 7.5, 5 mM MgCl2, 320 mM sucrose, 1% Triton X-100, 0.1 mg/ml proteinase K, 0.58% sodium dodecyl sulfate at 37°C for 2 h. An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) (Fisher Scientific, Pittsburgh, PA) was added to the extracts, and DNA was isolated from the aqueous phases after phase separation by centrifugation. After a second round of phenol/chloroform/isoamyl alcohol extraction, DNA was cleaned with chloroform/isoamyl alcohol (25:1), twice with diethyl ether, and precipitated. Three μg of total DNA were digested with PvuII, which cuts a single site in the mitochondrial genome. Linearized DNA was electrophoresed on a 0.8% agarose gel in TAE containing ethidium bromide, and photographed. The DNA was denatured and transferred to Nytran SuPerCharge membrane (Schleicher & Schuell, Keene, NH) using TurboBlotter (Schleicher & Schuell, Keene, NH) rapid downward transfer with the alkaline transfer conditions recommended by the manufacturer. DNA was crosslinked by UV (1200 joules) and the membrane was prehybridized at 65°C for 4 h in 3X SSC, 0.25% milk powder, 0.25% SDS. Oligonucleotide probe was excised from pTZ19 (K5) by digestion with HindIII and EcoRI and isolated from low melting point agarose gel using β-agarase (New England Biolabs, Beverly, MA). Probe (25 ng) was labeled with 32P-deoxycytidine (New England Nuclear/ Perkin Elmer Life Sciences, Boston, MA) by RadPrime random primer labeling (Invitrogen) and unincorporated nucleotides were removed by Concert ™ PCR purification kit (In Vitrogen). After hybridization with labeled probe for a minimum of 12 h, the membrane was washed twice with 2X SSC, 0.25% milk powder, 0.25% SDS for 30 min, and twice for 30 min with 0.5X SSC, 0.25% milk powder, 0.25% SDS. All washes were performed at 65°C. Hybridization was visualized by autoradiography with X-ray film (Kodak) at -80°C.
Cytochrome oxidase immunoblotting
Exponentially growing cells were washed with ice-cold PBS, and collected in AG Buffer(0.3 M PMSF,0.9% NaCl,0.1% Triton X,1 mM EDTA,0.5% NP40,50 mM Na2HPO4), PMSF (100 μg/ml), leupeptin(2 μg/ml), and aprotinin (2 μg/ml). Extracts were homogenized for 20 strokes on ice and protein concentration was estimated using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were separated by SDS-PAGE on 12% polyacrylamide gels under denaturing conditions. Proteins were transferred to Immobilon-P nylon membrane by electrophoresis, and protein transfer visualized by staining with Ponceau S and destaining with deionized water. The membrane was blocked in 5% milk in TBST overnight at 4°C, washed with TBST and hybridized with mouse anti-cytochrome oxidase II (Molecular Probes Inc., Eugene, OR) 1:1000 in 3% milk/TBST for 1 h at room temperature. After washing, membranes were hybridized with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:5000 in 3% milk/TBST for 1 h at room temperature, washed, and incubated with ECL chemiluminescence solution. Antibody binding was visualized by exposure of membrane to X-ray film and subsequent processing. Membranes were stripped at 50°C in, washed extensively with TBST, blocked and hybridized as described above using a mouse anti-actin primary antibody at 1:1000 and HRP-conjugated goat anti-mouse IgG secondary antiserum as described above.
Cell growth in glucose or galactose
The Rho+ and Rho0 cells were plated in 24 well tissue culture clusters (50,000 cells/well) in growth medium containing 3.15 g/L glucose. After overnight culture, cells were washed three times with PBS, and growth medium containing either glucose or galactose (3.15 g/L) was added back to the cells. MTT at a final concentration of 1 μg/ml was added to triplicate wells of both cell lines grown in both carbon sources immediately (t = 0) and at 24, 48, and 96 h following medium change. MTT was also added to one well containing each medium only, as a control. After 4 h incubation with MTT, medium was aspirated, the converted MTT-formazan extracted with DMSO, NaCl, glycine, pH 10.5, and the absorbance at 553 nm was measured by spectrophotometer.
Measurement of lipid peroxidation
Thiobarbituric acid reactivity was measured by a modification of Ohkawa et al.,  as described by Bowie et al. . Subconfluent cultures were collected by trypsinization, washed with ice cold PBS and lysed by three freeze/thaw cycles in sterile deionized water. Protein concentration of samples was estimated using Bio-Rad Protein Assay Reagent compared with BSA standards. 1',1',3',3'-tetramethoxypropane (TMP) was diluted to 10 mM in 20 mM Tris-Cl, pH 7.5, and serially diluted for standards. Equal volumes (200 μl) of lysate or TMP standards were mixed with 800 μl of solution containing 4 mg/ml 2-thiobarbituric acid (TBA), 0.5% SDS, and 9.4% glacial acetic acid, and heated at 95°C for 1 h. Samples were cooled to room temperature, centrifuged for 10 min at 16,000 × g, and the absorbances of the supernatants were read at 532 nm. Concentrations of TBA-reactive substances were estimated by comparing values to TMP standard curve.
Total RNA was extracted from exponentially growing cells with Trizol. Precipitated RNA was resuspended in DEPC-treated deionized water, and concentration was estimated by absorbance at 260 nm. Labeled cDNA was prepared by RT-PCR according to the Array-Advantage UA protocol from Ambion (Ambion Inc., Austin, TX). Briefly, 5 μg experimental RNAs and 1 μl alignment control RNA were denatured at 70°C for 10 min and allowed to anneal to oligodT by cooling to room temperature for 5 min. M-MLV reverse transcriptase (400 units), [α-33P] dATP, dCTP, dGTP, dTTP and 2 μl 10X RT buffer were added to a final reaction volume of 20 μl, and RT reaction proceeded for 2 h at 42°C. Unincorporated nucleotides were removed by passing reaction through NucAway™ Spin Column and radiolabel incorporation of the reaction determined by liquid scintillation counting. Human LifeGrid Array membranes (Incyte Genomics, Palo Alto, CA), which contain 8000 cloned genes spotted in two positions on each filter, were used for analysis. Membranes were blocked with heat denatured herring sperm DNA and prehybridized for 1 h with 15 ml ULTRArray Hybridization Buffer at 68°C. Labeled cDNA from each RT reaction was heat denatured at 95°C for 5 min, added to the prehybridization buffer, and hybridized to an array for 16 h at 68°C. Membranes were washed twice 30 min each with 2X SSC, 0.5% SDS, followed by 0.5X SSC, 0.5% SDS, at 60°C. Membranes were exposed to phosphorimager screen for 3 hr, and imaged as .gel files using a phosphorimaging scanner with TyphoonScan (Molecular Dynamics, Amersham Biosciences, Piscataway, NJ) software. Spot intensities were measured using ArrayVision software (Imaging Research, Inc., St. Catharines, Ontario, Canada), and the values. Genes corresponding to differentially expressed RNAs were identified by Incyte Genomics clone identification numbers. Membranes were stripped and reconstituted according to the manufacturer's instructions, and hybridizations repeated using freshly labeled cDNAs. In repeats, each cDNA was hybridized to the opposite membrane from that to which it had been previously hybridized. Only those genes that were consistently differentially expressed in 3 hybridizations were selected for analysis.
Trizol reagent (In Vitrogen) was used to isolate RNA from 106 cultured cells according to the manufacturer's instuction. One and half micrograms of total RNA was reverse transcribed using Superscript II Rnase H- reverse transcriptase (In Vitrogen). Two microlitres of the reverse transcribed products was used in the PCR reactions. Primers were made for a twenty of the differentially expressed genes and for G3PDH a housekeeper gene. Twenty five microlitres of the PCR reactions contained 20 mM Tris-HCL, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 200 μM dNTP and 10 picomoles of each primer and one unit of Taq DNA polymerase (Invitrogen). Each gene was amplified by PCR using various cycle numbers ranging from 30 to 55 cycles to ascertain its linear amplification. In general the PCR profile consisted of an initial denaturation at 94°C for 5 minutes and a variable number of cycles of denaturation at 94°C for 45 sec, annealing at 58°C for 1 min and extension for 2 min at 72°C with a final extension at 72°C for 10 min. To aid comparison five microliters of the PCR products of the differentially regulated genes were mixed with an equal amount of the housekeeper gene products and electrophoresed on a 1% agarose gel and visualized after staining with ethidium bromide (0.5 μg/ml) under ultra violet.
Generation of Rho0 breast cancer cell line
Increased lipid peroxidation in Rho0 cells
Genes Up-regulated in Rho0 cells (≥ 3 fold)
v-maf protein G
General transcription factor IIH polypeptide 3
IFN consensus sequence binding protein 1
Runt-related transcription factor 1(AML-1Oncogene)
Slit(Drosophila) homolog 3
Phospholipase C, epsilon
HLA DR beta 5
Tubulin specific chaperone D
Collagen Type IV
Gap Junction Protein 43
Arylacetamide deacytylase (esterase)
UDP-galactose:N-acetylglucosamine beta-1, 4-galactosyltransferase I
Cysteine dioxygenase type I
Cell growth and differentiation
Cyclin-dependent kinase inhibitor P19
Microtubule-associated protein 4
CDC28 protein kinase
TNF superfamily member 6, (Fas Ligans)
Ectodermal dysplasia 1, anhidrotic.
ESTs, Weakly similar to AAB47496 NG5 [H. sapiens]
Uncharacterized Bone marrow protein, BM033
Genes Down regulated in Rho0 (≥ 3 fold)
Dachshund (Drosophila) Homolog
LIM binding domain 2
Phospholipase A2 Group IB
Phospholipase C, epsilon
Lipopolysaccharide response-68 protein
Agouti related protein
Protein Tyrosine Phosphatase, Receptor Type c, Polypeptide-Associated Protein
Neurotensin Receptor I
Phosphodiesterase 1A, calmodulin dependent
Adaptor related protein complex 4 mu 1 subunit
Carboxypeptidase B2 (plasma)
Cytochrome P 450 subfamily VIIB, polypeptide 1
Protective protein for beta-galactosidase
Cell growth and differentiation
Cyclin-dependent kinase inhibitor P19
Intracellular Protein degradation
Huntingtin interacting protein B
This paper reports on a comprehensive analysis of the nuclear gene expression in response to the absence of mtDNA in breast cancer cells. We demonstrate that expression of a number of nuclear genes is altered in response to the absence of mtDNA in breast cancer cells. These genes ranged from transcription factors to genes that are involved in metabolism, cell architecture and signaling. Our results are consistent with previous studies in yeast and mammalian cells that reported altered pattern of nuclear gene expression due to elimination of mtDNA [15–17].
We conducted comparative microarray analysis on Rho+ MDA-435 cell line and its Rho0 derivative. Rho0 cells have been used extensively to study the role of mitochondria in a variety of cellular processes that are affected by mitochondrial dysfunction (16, 17, 18). Our results showed that 35 genes are up regulated (> 3 fold) and 22 are down regulated (>3 fold) in Rho0 breast cancer cells. We chose 10 genes randomly to confirm the results of microarray analysis with RT-PCR (Figure 5). Among genes that are up regulated in Rho0 cells p19 (INK4d), a cyclin dependent kinase inhibitor, is focally expressed during fetal development and plays a role in terminal differentiation . Members of the group of INK proteins are involved in arresting cells in G1 phase of the cell cycle. Conceivably, p19 may be involved in the observed slow growth rate of the Rho0. A number of genes that influence gene transcription are differentially expressed after mitochondrial impairment. The Maf oncoprotein, a basic leucine zipper-bearing transcriptional activator that recognizes the Maf recognition element (MARE) DNA sequence (20) is noticeably up regulated in the Rho0 cell line. c-MAF is highly expressed in developing skeletal tissues, cells where mitochondria play a central role in their functioning [20, 21]. The transcription factors TFIIH and AML1 were also up regulated in the Rho0 cell lines. TFIIH is associated with the RNA polymerase II transcription complex, which is involved in transcription and transcription-mediated DNA repair . The transcription factor AML1 is frequently found translocated in leukemic cells . Conceivably, altered expression of these transcriptional regulators in response to mitochondrial impairment could directly or indirectly lead to the changes in expression of other genes seen in Rho0 cells.
Phosphoinositide-specific phospholipase C (PLC) ε was among the signaling molecules that were up regulated in the Rho0 cell line. PLC is a critical signaling enzyme that hydrolyzes membrane phospholipids to generate inositol trisphosphate (IP3), which binds to IP3 receptors and increases intracellular Ca2+. The ε isoform of PLC is a novel effector of ras[25, 26]. Another signaling molecule of significance that is up regulated in the Rho0 cells is the Slit (Drosophila) homolog 3. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function . Slit3 gene product is the least evolutionarily conserved of the vertebrate Slit genes. The fact that Slit3, but not Slit2, is predominantly localized within the mitochondria makes its up regulation in the Rho0 cells an intriguing phenomenon .
One feature of the Rho0 cell line is the differential expression of certain genes that are involved directly or indirectly in metabolism of fatty acids or signaling (Figure 5 and Table 1 and 2). Among those that are up regulated are apolipoprotein D (ApoD) and arylacetamide deacetylase. While ApoD may have a similar function as the other apoproteins, it does not share a similar protein structure to the other family members, and does not bind cholesterol with high affinity . Hence, it may have a unique effect via binding of different ligands. ApoD may be involved in the binding of steroids or fatty acids, Apo D interacts with Ob-Rb, but not Ob-Ra, in hypothalamic neurons in vivo. In the central nervous system it was found that Apo D may be activated through its interaction with a leptin-stimulated Ob-Rb and may bind a specific ligand in hypothalamic neurons, where it exerts signaling functions . Arylacetamide deacetylase may also function as a lipase during the process of lipoprotein secretion [31, 32]. Other genes involved in fatty acid signaling were down regulated in Rho0 cells. These include phospholipase A2, a protein closely involved with arachidonic acid metabolism and also involved with cell signaling [33–35], that was found to be maximally down regulated in Rho0 cells. Phospholipase A2 also plays a role in the translocation of PKC-mediated by fatty acids. Another gene with functions closely related to the arachidonic acid metabolism that is down regulated in the Rho0 cells is the progesterone-induced blocking factor 1 (PIBF1). PIBF1 is known to act on the phospholipase A2 enzyme, and so interferes with arachidonic acid metabolism, inducing a Th2 biased immune response . PIBF1 exerts an anti-abortive effect by controlling NK activity . A gene related to lipid signaling that encodes agouti-related protein was also down regulated in the Rho0 cells. Agouti is a paracrine-signaling factor that acts to block melanocortin action [38, 39]. Disruption of melanocortin signaling with antagonist administration leads to an increase in feeding and eventually to obesity. Such blockage has been observed in both lipopolysaccharide-induced animals and also during illness . The data exhibiting down regulation of the agouti gene is further strengthened by the down regulation of the LPS reponse-68 protein in the Rho0 cells.
The ATP synthase 5A1 subunit gene that is directly involved in mitochondrial function was up regulated in the Rho0 cell lines. ATP synthase is a multimeric complex composed of at least 16 different polypeptides, two of which are mitochondrially encoded, and the remainder encoded by nuclear genes . Certain leukemic cells show an increased production of the ATP5A1 subunit . Interestingly, a down regulated gene CYP7B1 encoding an oxysterol 7 alpha-hydroxylase (Cytochrome P450 subfamily VII BI) is involved in many metabolic processes including bile acid synthesis and neurosteroid metabolism .
Mitochondria control Ca++ homeostasis . Protein kinase C γ, neurotensin receptor, and neuromodulin (GAP-43) are among the genes that are involved in calmodulin regulation or related to calmodulin function [43–45] which are down regulated in Rho0 cells. Neuromodulin is also a substrate for phosphorylation by protein kinase C  which may reflect an influence of mitochondrial function on multiple members of a signal transduction pathway. Another interesting feature in the expression profile of cells lacking mtDNA is the low expression of many genes that play a role in the development and maintenance of the nervous system [47, 48]. Of particular interest is the gene that encodes neurotensin receptor I, a short peptide receptor for neurotensin that exerts neuromodulatory functions in the central nervous system and endocrine/paracrine actions in the periphery . Neuromodulin is also involved in the growth and regeneration of axons, and in the elongation of axons as an axonal transport membrane protein . Synuclein is expressed in the nervous tissues and mutations in this gene are associated with rare familial cases of early-onset Parkinson's disease . Gamma synuclein is also involved in tumorigenesis . PKC-g mutation has been shown to lead to neurodegeneration . Dachshund, a putative transcription factor, plays a role in retinal development . Huntingtin-interacting protein-2 is involved in the neurodegenerative Huntington disease .
In summary, our data provides an overview of the genes that are involved in the nuclear and mitochondrial cross talk and respond to oxidative stress manifested due to impaired mitochondrial function. The identification of a large number of genes whose expression is influenced by mitochondrial function provides a foundation for future investigation of the pathways affected by the impairment of mitochondria in pathological conditions. An understanding about the role of the proteins in the maintenance of proper mitochondrial function and in oxidative stress response is critical for understanding the role of mitochondria in carcinogenesis.
Research in our laboratory was supported by grants from National Institutes of Health RO1-097714, Elsa U. Pardee Foundation and a pilot project from CA 88843 (to KKS). Dr. Robert Delsite was supported in part by National Institutes of Health training grant T32 CA0936 from the National Cancer Institute and a minority supplemental grant from National Institute of Environmental Health. The authors wish to thank Daniel Bornman for his excellent technical assistance.
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