1 Department of Pediatrics, 2 Department of Urology, 3 Research Institute for Frontier Questions of Medicine and Biotechnology and 4 Institute of Pathology, Paracelsus Private Medical University Salzburg, Muellner Hauptstr. 48, A-5020 Salzburg, Austria
5 To whom correspondence should be addressed Email: b.kofler{at}lks.at
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Abstract |
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Abbreviations: COX, cytochrome c oxidase; m/r ratio, relative ratio of mitochondrial DNA to 18S rDNA; OXPHOS, oxidative phosphorylation
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Introduction |
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Studies by Warburg over five decades ago demonstrated that the vast majority of human and animal tumours display a high rate of glycolysis under aerobic conditions (4). Human solid tumours endure profound hypoxia, which indicates that adaptation to hypoxic conditions is a crucial step in tumour progression. The anaerobic use of glucose as an energy source through glycolysis is therefore a feature common to several solid tumours, in turn leading to a lesser dependence on the mitochondria for oxidative phosphorylation (5,6).
There is increasing evidence that mitochondrial mutations and/or functional abnormalities are associated with various neoplasms, although it is not clear whether mitochondrial lesions are contributing factors in carcinogenesis or whether they simply arise as part of secondary effects in cancer development (710). In many solid tumours the changes of the energy metabolism are associated with a reduction of the mitochondrial DNA content and the activity of enzymes of oxidative phosphorylation (OXPHOS) and are frequently related to the aggressiveness of the tumours including hepatocellular and renal carcinoma (1113).
To elucidate if there is a link between malignancy and changes of the mitochondrial energy metabolism in renal carcinomas we examined nephrectomy specimens from 37 patients. In each case, tissue removed from the corresponding healthy part of the kidney served as matched control for the renal tumour tissue. For each set of paired specimens we investigated the relationship of the activity of OXPHOS and Krebs cycle enzymes and the amount of mitochondrial DNA in correlation to cell proliferation, ploidy, tumour grade and histological type of renal carcinoma.
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Materials and methods |
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Quantitative Southern blot analysis
The relative amount of mitochondrial DNA was determined by Southern blot analysis by correlating the mitochondrial to the 18S nuclear DNA signal. Total cellular DNA was isolated from frozen renal tissues according to standard procedures. Southern blot analysis was carried out as described previously (14). The same mixture of mitochondrial and nuclear probes was used in each experiment. In order to determine the intensity of the signals, densitometry on autoradiographs was performed. The relative amount of mitochondrial DNA was expressed as the ratio of the signal of the mitochondrial probe to that of the nuclear probe (m/r ratio).
Ploidy
Fresh renal carcinoma and control tissues were touched onto poly-L-lysine slides, Feulgen stained and DNA cytometry was performed with the ACAS DNA system (Ahrens ACAS, Bargteheide, Germany) (15,16). To determine ploidy, 200 renal cells of each specimen were measured. Leukocytes and granulocytes were used as reference cells.
Immunohistochemistry
Immunofluorescence with a monoclonal antibody directed against human mitochondrial porin (A-21317; Molecular Probes, Eugene, OR) and cytochrome c oxidase (COX) subunit I (A-6403; Molecular Probes) was performed according to the manufacturer's instructions. In the present study we used formalin-fixed, paraffin-embedded material (2 µm) from renal carcinomas and the corresponding control tissue. After deparaffinization in xylene and graded alcohol, treatment with 0.01 M sodium citrate for 15 min at 95°C followed. The slides were incubated for 2 h with the primary antibody (porin, diluted 1:30 in PBS; COX subunit I, diluted 1:30 in PBS). After washing the sections three times for 5 min in PBS, slides were incubated for 30 min with a FITC-labelled goat anti-mouse secondary antibody (AB 124 F; Chemicon, Temecula, CA) diluted 1:100 in PBS.
Sample preparation for enzyme measurements and western blot analysis
Renal tumour and control tissues (20100 mg) were homogenized with a tissue disintegrator (Ultraturrax, IKA, Staufen, Germany) in extraction buffer (20 mM TrisHCl, pH 7.6, 250 mM sucrose, 40 mM KCl, 2 mM EGTA) and finally homogenized with a motor-driven Teflon-glass homogenizer (Potter S, Braun, Melsungen, Germany). The homogenate was centrifuged at 600 g for 10 min at 4°C. The supernatant (600 g homogenate) containing the mitochondrial fraction was used for measurement of enzyme activities and western blot analysis.
Enzyme measurements
The following enzyme activities were determined: citrate synthase (17), COX (18), complex II (19) with modifications (20) and isocitrate dehydrogenase (21) with the following modifications to the assay buffer: 50 mM HEPES pH 7.6, 2 mM MnCl2, 4 mM DL-isocitrate and 0.1 mM NADP (nicotinamide adenine dinucleotidephosphate). Oligomycin and aurovertin-sensitive ATPase activity of complex V was determined using buffer conditions described by Rustin et al. (19), but by sonifying the whole reaction mixture for 10 s with an ultra-sonifier (Bio cell disruptor 250, Branson, Vienna, Austria) at the lowest energy output (22). The concentration of oligomycin was 3 µM and of aurovertin B 60 µM. All spectrophotometric measurements (Uvicon 922, Kontron, Milano, Italy) were performed at 37°C.
Western blot analysis
After separation of the 600 g homogenate (18 µg protein/lane) on 520% polyacrylamide gradient gels, western blot analysis was performed according to Berger et al. (20).
The following antibodies were used: mouse monoclonal antibodies against COX subunit I (A-6403; Molecular Probes; 1:250); COX subunit IV (A-6431; Molecular Probes; 1:1500); porin (A-21317; Molecular Probes; 1:250) alkaline phosphatase-conjugated rabbit anti-mouse immunoglobulins (Dako, Golstrup, Denmark; 1:5000). Images were analysed by densitometry with image analysis software (Molecular Analyser, Bio-Rad, Hercules, CA). Coomassie staining of the acrylamide gel was performed, to show equal protein loading.
Determination of proliferation index
Immunostaining on paraffin-embedded sections (2 µm) for Ki-67 was carried out with the antibody clone MIB-I (Dako Cytomation, Carpinteria, CA) according to the manufacturer's instruction. The Ki-67 labelling indices were determined by light microscopy with a 40x objective. All nuclei with a visible staining reaction were registered as positive regardless of staining intensity or quality (granular or diffuse). In each section at least 800 nuclei were counted and the Ki-67 labelling indices was defined as the percentage of positively stained nuclei.
Statistical analysis
Significance of the differences among renal carcinoma and control tissues was examined by the Wilcoxon t-test. Comparison of relative enzyme activity and mitochondrial DNA levels to biological features was analysed with the MannWhitney or the KruskalWallis test using PrismTM 3.03 software (GraphPad Software Inc., San Diego, CA). A P-value of <0.05 was considered statistically significant.
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Results |
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The inter-assay variability of the quantitative Southern blot analysis ranged from 5 to 20%.
Analysis of the content of mitochondria in renal carcinoma
In order to determine if the observed low mitochondrial DNA content in renal carcinoma is due to a reduction of the amount of mitochondria per cell, 15 renal carcinoma tissue sections were analysed by immunofluorescence with an antibody directed against mitochondria. The sections showed intense mitochondrial staining in the control and variable intensity in renal carcinoma tissues (Figure 2). In contrast, immunofluorescence using an antibody directed against COX subunit I revealed an intense signal only in control kidney tissues (Figure 2).
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Thirty-four of thirty-seven renal carcinoma tissues displayed a 210-fold reduction of the enzyme activities of complex II, COX, complex V and isocitrate dehydrogenase, while citrate synthase was less affected (Figure 1 and Table I).
However, one case of grade 3 conventional renal carcinoma showed close to normal activities of COX and complex II and a 3-fold higher activity of the Krebs cycle enzymes citrate synthase and isocitrate dehydrogenase together with a 1.7-fold increase of mitochondrial DNA. Another grade 3 conventional renal carcinoma tissue also displayed a 23-fold induction of citrate synthase and isocitrate dehydrogenase accompanied by mitochondrial DNA reduction and low COX and complex II, which was in the range of all other renal carcinomas investigated. The third case was a grade 1 tumour with normal values compared with renal control tissue, except for a 1.5-fold increase in isocitrate dehydrogenase and complex V enzyme activity.
Since these three cases did not follow the trend of the other 34 carcinoma specimens, we used the median instead of the mean values for all of our calculations.
The collecting duct carcinoma showed a severe reduction in the lower range of all analysed enzyme activities (Table I).
The comparison of aurovertin- and oligomycin-sensitive ATPase activity in 10 low grade conventional renal carcinomas (grade 1 and 2) showed that the relative decrease of the ATPase activity of complex V in the tumour tissues compared with the matched control tissues was in the same range (oligomycin 83 ± 13% decrease; aurovertin B 77 ± 20% decrease). Oligomycin- as well as aurovertin-sensitive ATPase activities were lower in tumour samples compared with normal kidney in all samples investigated.
Correlation of enzyme activities to tumour type and biological features
In order to evaluate a possible correlation of the mitochondrial energy metabolism to the proliferative activity of the tumour, the median proliferation index was calculated by Ki-67 staining. The index was 2.0 (range 068, n = 37) in renal carcinoma and 0.0 (range 07.7, n = 13) in controls. No significant correlation of the proliferation index of the tumours to OXPHOS enzyme activity and mitochondrial DNA content could be detected (Figure 4A and D).
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Discussion |
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Correlation of low respiratory chain content with tumour aggressiveness in renal carcinoma has been reported recently (11). In this previous study, enzyme activities of the mitochondrial energy metabolism of high (3 + 4) and low (1 + 2) grade conventional renal carcinomas were decreased to a comparable extent as in our study with the exception of complex V activity. Simonnet et al. reported that complex V activities, measured enzymatically as aurovertin-sensitive ATPase, were reduced in high grade but normal in low grade conventional renal carcinomas. This is in contrast with our results where we found comparable median low activity of complex V in low grade (25% of control kidney) and high grade (19% of control kidney) conventional renal carcinomas by measurement of oligomycin-sensitive ATPase activity, which determines the activity of assembled FoF1-ATPase, the form of the enzyme necessary in the energy metabolism. In comparison, measurements of aurovertin-sensitive ATPase in 10 cases of low grade conventional renal carcinomas also revealed a reduction of aurovertin-sensitive ATPase in all cases. In the former study (11), the level of complex V protein determined by 2D gel electrophoresis was found to be reduced in low grade (20% of control kidney) and in high grade (27% of control kidney) renal carcinomas. However, the same study showed in low grade renal carcinomas that the mean activity of aurovertin-sensitive ATPase was even slightly elevated compared with control kidney. This result was due to highly elevated ratios in two of ten patients. A similar discrepancy was found in the previous study for papillary renal carcinomas where unchanged activities of aurovertin-sensitive ATPase were found along with a reduction of complex V protein (34% of control kidney). In our study, where we also included five papillary renal carcinomas, the median activity of complex V was significantly reduced (36% of control kidney).
Our results and the data from 2D analysis of the previous study, question the quantification of complex V enzyme activity reported previously (11). The measurement of complex V activity is problematic because the enzyme activity is measured by the use of helper enzymes (pyruvate kinase and lactate dehydrogenase), which must get in the vicinity of ATPase, which is enclosed in the two mitochondrial membranes. To circumvent any assay variations in our study, sonification of the whole assay mixture was performed to bring the enzymes evenly in contact to achieve a significant better standardization of the measurement of complex V activity (data not shown). Features of progression of renal carcinoma such as the mitotic index, metastasis, tumour size and ploidy were not found to be related to the extent of alterations of mitochondrial energy metabolism and the reduction of mitochondrial DNA levels. Although sarcomatoid renal carcinomas have a poor prognosis, the two unclassified carcinomas with sarcomatoid alterations displayed even less reduced enzyme activity compared with the other renal carcinoma tissues investigated. As we could not find a clear distinction between carcinoma types and aggressiveness, it seems unlikely that alterations of the mitochondrial energy metabolism are strongly associated with the outcome of patients with renal carcinomas.
Since in our Southern blot analysis relative mitochondrial DNA levels are correlated to nuclear DNA, aneuploidy of tumour cells would falsify the relative mitochondrial DNA content per cell. The tumour tissues had only a slightly higher overall ploidy compared with controls, which does not significantly influence the m/r ratio in the renal carcinoma tissues investigated and therefore no correction of m/r data regarding ploidy has been carried out.
Down-regulation of the mitochondrial DNA content and the enzyme activities of the mitochondrial energy metabolism seem to be a co-ordinated process. This is evident by the fact that COX, which is partially encoded by the mitochondrial DNA, is diminished to the same extent as complex II, an enzyme of the respiratory chain, which is entirely encoded by the nuclear genome. In contrast, mutations in the deoxyguanosine kinase gene lead to a mitochondrial DNA depletion (24), which results in enzymatic defects of enzymes that contain mitochondrial-encoded subunits but normal activities of the other enzymes-encoded exclusively by the nuclear genome. Interestingly, the down-regulation of citrate synthase is less pronounced than the decrease of other enzymes measured. This might be due to additional functions of the Krebs cycle enzymes for example in the amino acid metabolism. This should be considered when using citrate synthase as a marker for mitochondrial energy metabolism.
In agreement with an earlier report (25) where the amount of mitochondria was examined by electron microscopy, no obvious reduction of the mitochondrial marker enzyme porin was found in western blot analysis but a clear decrease of the mitochondrial and nuclear-encoded COX subunits comparing renal carcinoma with control kidney (Figure 3). Other studies, however, found variable or decreased content of mitochondria in renal carcinoma tissue (2628).
In conclusion, we found no correlation of any of the enzymes of the mitochondrial energy metabolism investigated with carcinoma type and aggressiveness. These findings indicate that mitochondrial alterations occur early in the progression process of cancer. In general, low mitochondrial activity rather seems to be an adaptation to environmental conditions of solid tumours, which have to endure hypoxia during their development. Low mitochondrial activity leads to lower oxidative stress under hypoxic conditions and might therefore represent an advantage for carcinoma progression.
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Acknowledgments |
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References |
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