Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.
The mitochondrion contains the only functional DNA outside the eukaryotic cell nucleus and mutations in this genome have been linked to a vast array of human disease including pediatric neurologic disease, degenerative muscular disease, and blindness in the pediatric population and more recently to chronic diseases of adults including diabetes, Alzheimer’s dementia, Parkinson’s disease, cardiovascular disease, and cancer [
Prostate cancer is one example of an adult disease tightly linked to ageing for which there is evidence linking both inherited and somatic mtDNA mutations to disease [
While multiple independent investigators have confirmed mtDNA mutations in prostate cancer, there is little understanding of the cell biologic and biochemical consequences of specific prostate cancer-associated mtDNA mutations. We investigated the mtDNA from a patient with prostate cancer and found a heteroplasmic missense mutation in the mitochondrial COI gene that impairs the oxidation of cytochrome c (respiratory complex IV inhibition) and increases the generation of both reactive oxygen species (ROS) and reactive nitrogen species (RNS).
The mtDNA region encompassing COI was amplified using a forward primer starting at nucleotide position (np) 5772 (5′ AGG TTT GAA GCT GCT TCT TC 3′) and a reverse primer ending at np 7600 (5′ CGC TGC ATG TGC CAT TAA GA 3′). The template was denatured at 95°C for 7 min and primers extended for 35 cycles of 94°C for 1 min, then 55°C for 1 min, and 72°C for 1 min. Both strands of the COI polymerase chain reaction (PCR) product were cycle-sequenced using the slip primers in the forward direction starting at np 6080 (5′ TCT ACA ACG TTA TCG TCA CA 3′) and at np 6930 (5′ TGC AGT GCT CTG AGC CCT AG 3′) and in the reverse direction starting at np 6340 (5′ CTA GGT GTA AGG AGA AGA TG 3′) and at np 7150 (5′ GAT TTA CGC CGA TGA ATA TG 3′). The templates were denatured at 96°C and primers extended in the presence of “Big Dye Terminators” for 25 cycles of 96°C for 10 sec, then 55°C for 5 sec, and 60°C for 4 min. The reactions were chilled to 4°C, and the excess dye terminators removed by Centri-Sep Columns. The trace files were determined using an Applied Biosystems (ABI) Prism 3100 genetic analyzer, analyzed using Sequencher gene analysis software v 4.1 (Gene Codes, Ann Arbor, MI), and interpreted within the context of MITOMAP (
LCM was carried out by the Winship Cancer Institute Pathology Core using the Molecular Machines & Industries, Inc. Cell cut laser microdissector to isolate pure populations of prostatic epithelium (malignant and benign) and stromal cells. DNA was purified from LCM of frozen tissue using PicoPure DNA Extraction Kit (Arcturus, Mt View, CA) according to the recommended protocol.
Lymphocytes were isolated from whole blood by centrifugation and diluted with phosphate buffered saline (PBS). Red cells were lysed by the addition of H2O. After 20 s, osmolarity was restored with 10x concentrated PIPES (piperazine-
Mitochondria were isolated by cell fractionation and centrifugation. All procedures were carried out on ice. Cells were pelleted and washed in cold PBS and resuspended in 10 volumes of isolation buffer (250 mM sucrose, 10 mM HEPES, 1 mM EDTA, pH 7.35). Cell lysis was performed with 5 passes of a hand held Dounce homogenizer and centrifuged at 500 g (4°C) for 5 min. The supernatant was reserved and the pellet was resuspended in isolation buffer, rehomogenized, and centrifuged. This process was repeated a third time and supernatants combined and centrifuged 12,000 g at 4°C for 10 min to pellet mitochondria which were then washed in isolation buffer and repelleted three times. Following the final wash, mitochondria were resuspended in reaction buffer (1 mL/mL of original cell pellet), and frozen until assayed.
Cytochrome c oxidase activity of isolated mitochondrial preparations was measured by dual wavelength spectrophotometry (550/540 nm) as previously described [
Mitochondrial donors (patient lymphoblasts) were enucleated by short term incubation with Actinomycin D, which was subsequently removed from the culture medium by centrifugation and washing. These cytoplasts were then rescued by polyethylene glycol (PEG)-induced fusion with 143B rho zero cells. Fusions are monitored by phase contrast microscopy and isolated by ring cloning. Clones were expanded and genotyped to assure that the donor mtDNA had been incorporated (sequencing) and that a single nucleus was present (Karyotyping).
Peroxides Assay: Lymphoblasts were grown in suspension in flasks. When cells were in growth phase, 1 × 106 cells per sample were removed, pelleted, and resuspended in 2
Cell were plated in 6 well plates, 18,000 cells per well in triplicate for each time point and grown in RPMI 1640 (Mediatech) containing 15% Fetal Bovine Serum. Cells were harvested after 1, 2, 3, or 4 days and cell number was determined using FluoReporter Blue Fluorometric dsDNA Quantitation Kit (Life Technologies) according to manufacturer’s protocol.
In order to determine which isoform of Nitric Oxide Synthase (NOS) is present in our cybrid cell lines we performed RT-RCR on RNA from the cybrid cell lies and a positive control. cDNA was obtained from the RNA of 6124WT, 6124Mut, and BT474 (positive control) by reverse transcription using Advantage RT for PCR from Clontech (a Takara Bio Company, Mountain View, CA) according to protocol. The presence of NOS1, NOS2, and NOS3 were examined. Using the Amplitaq Gold Kit (Life Technologies), reactions were as follows: cDNA, 1XBuffer, 1.5 mM MgCl2, 0.2 mM each dNTPs, 150 nM each forward and reverse primers for NOS1 (Forward 5′ CGA CAC CAC TAG CAC TTA CCA G 3′; Reverse 5′ CAG ACT CGG AAG TCG TGC TTG 3′), NOS2 (Forward 5′ TCG GCT GCA GAA TCC TTC ATG A 3′; Reverse 5′ CAT TGT CTT GCG CAT CAG CAT AC 3′) or NOS3 (Forward 5′ GAA GCA CCT GGA GAA TGA GCA G 3′; Reverse 5′ CTT CAC TCG CTT CGC CAT CAC 3′) and 5 units of Taq for a final volume of 100 uL. Primers were designed to cross two introns and amplify all major isoforms of NOS1. The PCR reaction included an initial cycle of 95°C for 10 min followed by 40 cycles of 95°C for 30 sec and 61°C for 30 sec, 72°C for 1 min. A 3% agarose gel was run after PCR. The presence of NOS1, NOS2, and NOS3 were indicated by the presence of a 261 nt band, 288 nt or a 274 nt band, respectively.
Whole-cell extracts were obtained by lysing cells with lysis buffer containing 50 mmol/L, Tris Base, 5 mmol/L EGTA, 150 mmol/L NaCl, and 1% Triton X-100 (pH 7.4). One tablet of protease inhibitor (Roche Applied Science, Indianapolis, IN) was dissolved in 7 mL of lysis buffer. Total protein 30
Male nu/nu mice, 6–8-weeks old, were purchased from Charles River Laboratories (Wilmington, MA) and housed in ventilated cages under sterile conditions. For surgical manipulation, mice were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride, Xylazine, and Acepromazine.
Mice were injected subcutaneously in the neck with 3 separate 6124WT clones for a total of 29 mice and 3 separate 6124Mut clones for a total of 30 mice.
A 60-year old patient underwent radical prostatectomy after presenting to his primary care physician with an elevated-serum-prostate-specific antigen (PSA = 5.5 ng/mL) that prompted a prostate biopsy revealing a Gleason 6 prostatic adenocarcinoma. Histopathologic examination of the radical prostatectomy specimen revealed a Gleason’s score 6 (moderately differentiated-grade G2) conventional (acinar) prostatic adenocarcinoma involving ~5% of the left lobe of the prostate with negative surgical margins (pathologic stage pT2a). Follow-up of over 7 years with history, physical examinations and serum PSA determinations found the patient to be without evidence of prostate cancer recurrence. There was no family history of prostate cancer and the patient was otherwise healthy. DNA from the prostate tissue was the first specimen to be sequenced from this patient and this demonstrated a heteroplasmic point mutation at nucleotide position (np) 6124 of the mitochondrial genome. The sequencing chromatogram demonstrated approximately equal proportions of the wild type base (T) and mutant base (C) (Figure
Patient mitochondrial DNA mutations. Patient peripheral blood, lymphoblast, and cybrid mtDNA was sequenced in its entirety. All changes from rCRS are shown as is the region in which the change occurs and the amino acid alteration when applicable. AI (allelic index) is the measure of the frequency of the mutation when compared to mtDB—Human Mitochondrial Genome Database [
ΔrCRS | Amino acid | Region | AI |
---|---|---|---|
A73G | noncoding | D-loop | 83.4 |
A263G | noncoding | D-loop | 99.7 |
309insC | noncoding | D-loop | |
311insC | noncoding | D-loop | |
523delA | noncoding | D-loop | |
524delC | noncoding | D-loop | |
G709A | noncoding | 12S rRNA | 16.4 |
A750G | noncoding | 12S rRNA | 99.2 |
A1438G | noncoding | 12S rRNA | 96.9 |
G1888A | noncoding | 16s rRNA | 5.3 |
A2706G | noncoding | 16s rRNA | 80.5 |
T4216C | Tyr → His | ND1 | 9.0 |
A4769G | Met | ND2 | 99.0 |
A4917G | Asn → Asp | ND2 | 4.8 |
| |||
T6124 T&C | Met → Thr | COI | unique |
| |||
C7028T | Ala | COI | 81.3 |
8270-8278del | noncoding | NC7 | |
G8697A | Met | ATPase6 | 4.7 |
G8854A | Ala → Thr | ATPase6 | 0.1 |
A8860G | Thr → Ala | ATPase6 | 99.8 |
T10463C | noncoding | tRNA Arg | 4.7 |
A11251G | Leu | ND4 | 8.7 |
G11719A | Gly | ND4 | 77.7 |
A11812G | Leu | ND4 | 3.3 |
G13368A | Gly | ND5 | 4.9 |
A14233G | Asp | ND6 | 3.4 |
C14766T | Ile → Thr | Cytb | 77.4 |
G14905A | Met | Cytb | 5.1 |
A15326G | Thr → Ala | Cytb | 99.4 |
C15452A | Leu → Ile | Cytb | 8.7 |
A15607G | Lys | Cytb | 5.5 |
G15928A | noncoding | tRNA Thr | 4.9 |
T16126C | noncoding | D-loop | 8.9 |
T16189C | noncoding | D-loop | 28.0 |
C16278T | noncoding | D-loop | 7.7 |
C16294T | noncoding | D-loop | 5.7 |
C16296T | noncoding | D-loop | 2.4 |
T16519C | noncoding | D-loop | 59.7 |
Detection of heteroplasmic point mutation of mitochondrial cytochrome oxidase subunit I (COI) mitochondrial gene from a single individual with prostate cancer. (a) Sequencing chromatograms of prostatic tissue and an Epstein-Barr transformed lymphoblast cell line show approximately equal levels of both the wild type (T) and mutant (C) DNA base. (b) Activity of cytochrome oxidase measured in isolated mitochondria prepared from the patient’s heteroplasmic lymphoblasts (see Section
In order to study the biochemical consequences of this mutation we studied reactive oxygen production and enzymatic activity of respiratory complex (RC) IV (cytochrome oxidoreductase). The COI polypeptide forms the catalytic core of this enzyme. Cytochrome oxidation was measured in mitochondria isolated from the patient’s lymphoblast cell line and compared to other lymphoblast cell lines from two unrelated, individuals’ lymphoblast lines, sequence proven to be wild type at the COI locus (Figure
Mutations in the COI polypeptide that lead to decreased RCIV activity could potentially lead to increased reactive oxygen [
In order to eliminate the potential confounding effects of the nuclear genome we made cytoplasmic hybrids (cybrids) that combined either pure mutant or pure wild type genomes from this patient with a stable nuclear background (143 B cells). The resultant pair of cybrids thus have the exact same nucleus (different from the patient), and the exact same mitochondrial DNA sequence except for the single base mutation at n.p. 6124. MtDNA genotypes were sequence verified in cybrids.
Three cybrids 6124 wild type clones and three 6124 mutant clones were analyzed for proliferation in culture. All three mutant 6124 cybrid clones grew faster than the three wild type clones with an average doubling time of
Cybrid cell lines with the T6124C mutation show increased proliferation. Proliferation was measured in 3 separate 6124WT clones and 3 separate 6124Mut clones using FluoReporter Blue Fluorometric dsDNA Quantitiation Kit (see Section
We then compared multiple wild type and mutant clones for reactive oxygen species (ROS) and reactive nitrogen species generation using flow cytometry and ROS and RNS sensitive fluorescent dyes. In order to study the cellular peroxide levels in cells that were either entirely mutant or entirely wild type at this base, cybrid cell lines were made and multiple clones assayed by DCF fluorescence. Overall, 6 individual mutant clones showed a consistent increase in peroxide levels when compared to 5 individual wildtype clones (data not shown). When averaging the relative level of fluorescence in all wildtype clones compared to mutant clones, the mutation is associated with a statistically significant (
Peroxide and Nitric Oxide are elevated in 6124 mutant cybrid cells. (a) Peroxide levels are elevated in 6124Mut cell lines as measured by flow cytometric analysis of DCF fluorescence. 143B cybrids cell lines containing either the wild type base at position 6124WT or the mutation at position 6124Mut were analyzed. The average DCF fluorescence of five wild type and six mutant clonal cell lines are shown. Mutant cybrids produce significantly more peroxides (
In order to determine the possible source of the increased NO, we harvested RNA from the 6124WT and 6124Mut cybrids cell lines, followed by reverse transcription and PCR. There were no detectable levels of NOS1 or NOS3 observed (data not shown). However, NOS2 was demonstrated to be in both WT and Mut cell lines (Figure
iNOS RNA is present in the cybrids cells. Reverse transcription followed by standard PCR was performed with iNOS specific primers. iNOS RNA was present in both 6124WT and 6124Mut cell lines. The breast cancer cell line BT474 is used as a robustly positive control for iNOS RNA. and 18S RNA was used as a quality control.
PAPR (poly ADP ribose polymerase) cleavage was analyzed to determine if changes in cellular proliferation were in part due to changes in apoptosis. Western blot analysis of PARP cleavage demonstrates that PARP cleavage was reduced in 6124Mut cybrids compared to 6124WT (Figure
PARP cleavage is decreased in 6124Mut cybrids cells. (a) Western Blot analysis of uncleaved and cleaved PARP in 6124WT (left) and 6124Mut (right) cells. Figure is representative of three 6124WT and three 6124Mut clones. (b) Densitometric analysis of Western Blot results of three 6124WT and three 6124Mut clones using ImageJ software. Error bars represent the standard error of the mean of the three WT and three Mut clones.
6124Mut cybrid cell lines grew faster
6124Mut cell lines grow faster in nude mice. Growth curves of tumor xenografts in nude mice. Each line represents a cohort of 29-30 animals injected with the 6124WT cybrids cells (
The mitochondrially encoded COI gene was first implicated in cancer biology in 1998 when a somatically acquired chain termination mutation was reported in colon cancer by the Vogelstein group at Johns Hopkins [
The biochemical analysis of mtDNA mutations requires that viable cells are obtained from patients with such mutations and that potentially confounding nuclear events are controlled. This is made possible by the combination of capturing clinically relevant mutations in patient lymphoblast cell lines and the subsequent formation of cybrids with a common nuclear background. The cybrid formation process has the further advantage of allowing the mutant base to be studied separately from the wild type base. This paper documents that a prostate cancer-associated COI mutation affects the normal functioning of respiratory complex (RC) IV (cytochrome oxidase) in at least two distinct ways: decreasing the rate of mitochondrial cytochrome c oxidation and increasing the rate of ROS and NO generation in intact cells. It therefore seems likely that both of these effects are a direct consequence of the mutation. As Figure
The possible tumorigenic effects of increases in ROS are well known and include (at least) an increased rate of DNA mutations and ROS-induced promitogenic signaling [
The possible mechanism by which a decreased efficiency of oxidative phosphorylation is related to malignant transformation is less obvious. It is probably relevant to the so called “Warburg Effect” wherein tumor cells exhibit defective oxidative phosphorylation and increased glycolysis as a primary means of ATP generation [
The possible tumorigenic effects of increases in RNS are also well known. Nitric oxide (NO) is generated enzymatically by synthases (NOS), which oxidize L-arginine to L-citrulline. The inducible form, iNOS, is present in a variety of cell types. Over the past 20 years iNOS expression has been associated with various human tumors including: breast, brain, lung, prostate, colorectal, and melanoma [
We have demonstrated that the T6124C (M74T) mutation was inherited in a heteroplasmic state by a patient that developed prostate cancer and that the mutation not only causes increased ROS and nitric oxide but also induces increased cellular proliferation, decreased apoptosis, and increased
Mitochondrial DNA from a prostate cancer patient with an inherited-heteroplasmic-mtDNA mutation in COI, the catalytic core of mitochondrial respiratory complex IV was studied. In the laboratory, this mutation was found to simultaneously decrease the activity of the respiratory complex as measured by the rate of cytochrome c oxidation and to increase the rate of mitochondrial reactive oxygen generation. Other mutation-induced biochemical changes included increased generation of nitric oxide. Cells harboring the mutation proliferated faster
This work is supported by NIH Grant nos. CA98912 (Petros), CA96994 (Petros), and NS21328 (Wallace). It is also supported by a VA MERIT award (Petros). The authors wish to thank Mr. Larry Williams (The Breckenridge Group) for his personal support of this research and the Evans County Cares Foundation. In addition, cytochrome oxidase activity assays performed by Leslie Costello, Ph.D. (University of Maryland). Technical help was also provided by Amanda Parrish Ph.D. and Carina Fenandez-Golarz M.D.