Rapid Analysis of Mitochondrial DNA Depletion by Fluorescence In Situ Hybridization and Immunocytochemistry : Potential Strategies for HIV Therapeutic Monitoring
Molecular Probes, Inc. (MSJ,BJH,DMH,GMB,JYA,SWS,WGC), and Institute of Molecular Biology, University of Oregon, Eugene, Oregon (KY,RAC)
Correspondence to: Michael S. Janes, Molecular Probes, Inc., 29851 Willow Creek Road, Eugene, Oregon 97402. E-mail: mike.janes{at}probes.com
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Summary |
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Key Words: mitochondria oxidative phosphorylation mitochondrial DNA HIV AIDS mitochondrial disease nucleoside reverse transcriptase inhibitor cytochrome c oxidase immunocytochemistry FISH
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Introduction |
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There is increasingly strong evidence that mitochondria originated as symbiotic bacteria. They retain a prokaryote-like DNA, which in all species encodes a fraction of the proteins of the organelle. In mammals, mtDNA is a 16-kb double-stranded circular DNA that encodes 13 different polypeptides, all involved in oxidative phosphorylation, along with two rRNAs and 22 tRNAs (Anderson et al. 1981). Unlike the nuclear genome, mtDNA is present in thousands of copies in mammalian cells, all of which are used in translation of gene products made within the organelle on bacterium-like ribosomes (Taanman 1999
). mtDNA lacks protective histones and has minimal repair mechanisms and, as a result, has a much higher spontaneous mutation rate than nuclear DNA (nDNA), which is significantly enhanced because the mitochondrion is a free radical-rich environment (Sastre et al. 2003
). Accumulation of mutations and deletions occurs throughout life and becomes physiologically relevant when there are sufficient copy numbers to alter oxidative phosphorylation (Schon et al. 1997
; Schapira 2002
). Protein synthesis in mitochondria involves several transcription factors, replication enzymes, DNA polymerases, and regulatory factors, all encoded by the nuclear genome and imported into the organelle (Taanman 1999
). Mutations in mtDNA occur that decrease the amount of mtDNA and result in concomitant mitochondrial dysfunction, leading to a set of diseases of variable phenotypes called mtDNA depletion syndromes (Taanman et al. 1997
; Elpeleg et al. 2002
; Carrozzo et al. 2003
).
The current strategy for managing mitochondrial toxicity associated with NRTI therapy requires temporarily removing patients from the treatment to recover mitochondrial function before the viral load becomes life threatening (Lewis 2003; Walker 2003
). This treatment regimen would be greatly aided by the availability of efficient and reliable methods for monitoring mtDNA depletion and by the development of less toxic NRTIs. Here we describe simple approaches for monitoring the levels of mtDNA that should be suitable for such clinical applications. Recent reports have demonstrated the utility of monitoring mtDNA abundance by real-time PCR in patients treated with NRTIs (Cote et al. 2002
; Petit et al. 2003
). A key observation in one of these reports is that measurable loss of mtDNA precedes measurable metabolic changes, such as hyperlactemia, associated with drug toxicity (Cote et al. 2002
). This means, in effect, that changes in mtDNA content may predict the onset of some side effects. However, cells contain thousands of copies of mtDNA and, because of variable segregation of the mtDNA, often only a proportion of the mitochondrial genomes within a cell carry a mutation. In fact, both heteroplasmy (intracellular variation) and mosaicism (intercellular variation) are characteristic of mtDNA mutations. Therefore, techniques that allow analysis of populations of cells on a single-cell basis are necessary for accurate determination of mitochondrial changes.
Here we report the use of mtDNA FISH and ICC to monitor mtDNA depletion in cultured fibroblasts treated with the NRTI ddC and compare the results obtained with these methods to those obtained by the population-based technique of conventional real-time PCR.
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Materials and Methods |
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Real-time PCR
Total DNA was extracted from cells using the Qiagen DNeasy kit per manufacturer's recommendations (Qiagen; Valencia, CA). The primers used to amplify the mtDNA were forward 5'-dTATTAGTGGCATCGCCTGCC-3' (19511970) and reverse 5'-dGACCCTCGTTTAGCCATTCATTC-3' (20712049). The carboxytetramethylrhodamine (TAMRA)- and carboxyfluorescein (FAM)-conjugated probe used was FAM 5'-dAAGGTAGGATAATCACTTGTTC-3' TAMRA (20122033). The total DNA quantity was corrected by simultaneous measurement of 28SrRNA using 5'-dAGAAATTCAATGAAGCGCGG-3' and 5'-dAGGGACAGTGGGAATCTCGTT-3' as primers and FAM 5'-dACGGCGGGAGTAAC-3' TAMRA as the probe (Yamagata et al. 2002). An initial 10-min denaturation at 95C was followed by 50 cycles of denaturation at 95C for 15 sec and annealing at 60C for 30 sec. A standard curve for real-time PCR was generated using serial dilutions of DNA obtained from human fibroblasts. The amount of mtDNA is expressed as relative quantity to nDNA using Ct values calculated by the sequence detector (Perkin Elmer; Boston, MA).
Fluorescence In Situ Hybridization
Six overlapping PCR products, averaging 3.3 kb in length, representing the entire human mtDNA were prepared. All primers were custom synthesized (Operon; Alameda, CA) and PCR reactions were performed using Amplitaq Gold PCR Master Mix (Applied Biosystems; Foster City, CA). PCR primers used for mtDNA FISH probe preparation were as follows: MtF1 5'-dTCCATGCATTTGGTATTTTCGTC-3'; MtR1 5'-dCGAAGGGTTGTAGTAGCCCGTAG-3'; MtF2 5'-dACCTTCAAATTCCTCCCTGTACG-3'; MtR2 5'-dTGATGGCCCCTAAGATAGAGGAG-3'; MtF3 5'-dGGCAACCTTCTAGGTAACGACCA-3'; MtR3 5'-dGAGGAGCG-TTATGGAGTGGAAGT-3'; MtF4 5'-dGTAAGCCTCTACCTGCACGACAA-3'; MtR4 5'-dGTGGATGCGACAATGGATTTTAC-3'; MtF5 5'-dGCTCACTCACCCACCACATTAAC-3'; MtR5 5'-dTGAAGGGCAAGATGAAGTGA-AAG-3'; MtF6 5'-dTGAGGGGCCACAGTAATTACAAA-3'; and MtR6 5'-dGAGTGGGAGGGGAAAATAATGTG-3'. Each PCR reaction was confirmed by agarose gel electrophoresis and the product was purified by QIAquick PCR Purification Kit (Qiagen). An equal amount of PCR product from each reaction was combined to represent the whole mtDNA. Ten Arabidopsis cDNA PCR products from the SpotReport cDNA Array Validation System (Stratagene; La Jolla, CA) were pooled and used as the template for labeled negative control probe generation. Pooled DNA for the mtDNA and the negative control probes was nick-translated and labeled with the Alexa Fluor 488 dye as instructed in the ARES Alexa Fluor 488 DNA Labeling Kit (Molecular Probes; Eugene, OR).
For microscopic analysis, MRC5 fibroblasts were grown to 5070% confluency on glass coverslips. In some instances, cells were incubated for 20 min at 37C in 300 nM MitoTracker Red dye (Molecular Probes) in culture medium to counterstain the mitochondria. Cells were then washed twice with PBS before fixation in 4% paraformaldehyde in complete medium for 20 min at 37C. After fixation, cells were washed in PBS and permeabilized in 0.1% Triton X-100 for 10 min at room temperature. Cells were again washed twice in PBS before treatment with 0.1 mg/ml DNase-free RNase (Roche Diagnostics; Indianapolis, IN) for 90 min at 37C. Meanwhile, microscope slides were pre-warmed to 74C on a slide warmer. The cells were then washed twice with PBS and excess liquid was blotted from the coverslips. To the pre-warmed slide, 15 µl of the mtDNA or negative control probe was added [0.5 ng/µl in 50% formamide, 10% dextran sulfate, 2 x saline sodium citrate (SSC) buffer]. Coverslips were mounted on the pre-warmed slides and incubated on the slide warmer for 5 min at 74C, allowing simultaneous denaturation of targets and probes. Coverslips were sealed to the slides with rubber cement and the probes were left to hybridize overnight at 37C in a dark humidified chamber. Coverslips were removed from the slides and washed once in 2 x SSC buffer at RT, followed by one wash in 2 x SSC and one in 0.5 x SSC each for 5 min at 40C. Samples were then rinsed in PBS, counterstained with 0.2 ng/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Molecular Probes) for 2 min, and mounted in ProLong antifade reagent (Molecular Probes). All samples prepared for microscopic evaluation were visualized with a Nikon Eclipse 800 fluorescence microscope (Nikon Instruments; Melville, NY) equipped with a Micromax 1300 YHS camera (Roper Scientific; Trenton, NJ). Images were acquired with MetaMorph software v4.6.9 (Universal Imaging; Downingtown, PA) and appropriate filter sets (Omega Optical; Brattleboro, VT).
For flow cytometric analysis, fibroblasts were labeled in suspension. The FISH protocol is the same as above, with the following exceptions. Cells were harvested by removing the culture medium from a nearly confluent culture dish, washing the cells once with pre-warmed (37C) PBS, releasing the cells by incubation in a pre-warmed solution of trypsin and EDTA (0.25% and 1 mM, respectively) for 5 min, and adding an equal volume of pre-warmed culture medium containing 10% FBS to quench the trypsin. Fibroblasts were collected by low-speed centrifugation, resuspended in complete medium containing 4% paraformaldehyde, and fixed for 20 min at 37C before a single PBS wash and cell count. Approximately two million cells per sample were resuspended, and both detergent and RNase treatments were carried out as above. Each sample for flow cytometry was hybridized with 50 µl of mtDNA or negative control probe. Simultaneous denaturation of the probes and targets took place in a water bath at 74C and hybridization took place in a 37C water bath protected from light. After labeling and posthybridization washing as described above, cells were resuspended in
0.3 ml of PBS for flow cytometric analysis. Samples prepared for flow cytometric analysis were not counterstained with MitoTracker Red dye or with DAPI. All samples prepared for flow cytometry were analyzed using an Elite flow cytometer equipped with EXPO 32 software v. 1.2, a 488-nm argon ion laser, a 633-nm HeNe laser, and bandpass filters at 525 ± 10 nm and 675 ± 10 nm with a 640-nm dichroic mirror (Beckman Coulter; Miami, FL).
Immunocytochemistry
For microscopic analysis, fibroblasts were grown to 50-70% confluency on glass coverslips. The coverslips were removed from culture medium and washed in PBS before fixation in complete medium containing 4% paraformaldehyde for 20 min at 37C. Coverslips were then washed with PBS and incubated in antigen retrieval buffer (0.1 M Tris-HCl at pH 9.5 containing 5% urea) for 20 min at
95C before permeabilization in 0.1% Triton X-100 for 10 min at RT. After a 30-min block in PBS containing 10% normal goat serum (blocking buffer), cells were incubated for 1 hr at RT with primary antibody cocktails consisting of mouse monoclonal 31HL anti-porin IgG2b at 5 µg/ml (Calbiochem; San Diego, CA) and mouse monoclonal 1D6 anti-OxPhos Complex IV subunit I (COX I) IgG2a at 2 µg/ml (Molecular Probes) diluted in blocking buffer. Coverslips were washed three times in blocking buffer and then incubated for 30 min at RT with secondary antibodies diluted in blocking buffer. Alexa Fluor 647 dyeconjugated goat anti-mouse IgG2b-specific IgG and Alexa Fluor 488 dyeconjugated goat anti-mouse IgG2a-specific IgG were used at 5 µg/ml to detect anti-porin and anti-COX-I primary antibodies, respectively. Isotype-specific secondary antibodies (Southern Biotech; Birmingham, AL) were conjugated to either Alexa Fluor 488 dye or Alexa Fluor 647 dye as described previously (Haugland 2000
). Negative ICC controls were perfomed by replacement of described specific primary antibodies with nonspecific mouse IgG2a and IgG2b mouse antibodies, exclusion of primary antibodies in the labeling scheme, or incubation of isotype secondary antibodies with mismatched isotypes of specific primary antibodies. Coverslips were washed three times in PBS before counterstaining with 0.2 ng/ml DAPI for 2 min at RT and mounting in ProLong antifade reagent.
For flow cytometric analysis, cells were harvested and fixed as described above for FISH methods. Approximately two million fixed cells per sample underwent antigen retrieval as described above, with vortexing every few minutes. Samples were washed once with PBS before being permeabilized with 0.1% Triton X-100 and blocked with PBS containing 30 µg goat IgG for 10 and 30 min at RT, respectively. Primary and secondary antibody incubations were for 60 and 30 min at RT, respectively, with a wash in between and after incubations using PBS containing 1% BSA. All antibodies were used at 2 µg per sample and ICC controls were performed as described above.
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Results |
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Discussion |
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mtDNA depletion can occur through several mechanisms and, when extensive, is detrimental to cellular respiration and ATP synthesis. Mutations in enzymes responsible for mtDNA transcription have been described that lead to mtDNA depletion syndromes (Taanman 1999). Most often, mtDNA depletion and subsequent mitochondrial dysfunction lead to undesirable side effects during drug therapies (Brinkman and Kakuda 2000
). For example, there is evidence of a myopathy induced by statin therapy for hypocholesterolemia that could be caused by loss of mtDNA (Fosslien 2001
; Phillips et al. 2002
). The use of NRTI treatment for HIV infection also leads to mtDNA depletion, with grave consequences for the patient. NRTIs have been the mainstay for treatment of HIV infections since the introduction of AZT in 1987. There are now 15 different anti-retroviral drugs that are FDA-approved for HIV treatment, six of which are NRTIs, three of which are non-nucleoside reverse transcriptase inhibitors, and six of which are protease inhibitors (Temesgen 2001
). Although mortality rates have decreased since the introduction of combination NRTI drug therapy, none of the current therapies can completely eradicate the virus and so long-term use of anti-viral drugs is needed to prevent viral regrowth.
Recent studies have indicated that mtDNA depletion can lead to many of the severe side effects that occur during drug therapy. The best-documented and most important example is the now widely described NRTI-induced mtDNA depletion present in a significant proportion of patients being treated with these drugs for HIV infection (Lewis 2003; Walker 2003
). Mitochondrial toxicity associated with long-term use of NRTIs may manifest itself in a variety of forms, including peripheral neuropathy, myopathy, lipidopathy, lactic acidosis, and hepatic dysfunction (Chariot et al. 1999
; Brinkman and Kakuda 2000
; Carr and Cooper 2000
). NRTIs competitively inhibit HIV reverse transcriptase as they are incorporated into the newly synthesized DNA strand, but because the NRTIs lack the 3'-hydroxyl group needed for further chain elongation, the growing viral DNA chain is terminated. Although most cellular replicative DNA polymerases are not significantly inhibited by NRTIs, mtDNA polymerase
, the sole polymerase responsible for replicating the mitochondrial genome, is an exception. mtDNA polymerase
shows relatively high incorporation rates of NRTIs into mtDNA as well as low excision rates (Kukhanova et al. 1998
; Feng et al. 2001
; Lim and Copeland 2001
). The resulting inhibition of the mtDNA polymerase
leads to mtDNA depletion and its pathological consequences (Brinkman and Kakuda 2000
; Walker 2003
).
The current strategy for managing side effects arising from mitochondrial toxicity associated with NRTI treatment is the temporary removal of HIV-affected patients from therapy to allow for new mtDNA replication before a deleterious increase in viral load ensues (Moyle 2000). Deciding when to remove a patient from the therapy and when to reinstate NRTI treatment is subjective until measurable criteria can be established. In this study we have demonstrated two approaches, mtDNA FISH and ICC, for measuring mtDNA levels that should be useful in this regard. Both methods are advantageous in that they require very little live cell/tissue manipulation and are amenable to analysis of populations of cells on a single-cell basis by flow cytometry. In contrast to real-time PCR, both FISH and ICC do not require DNA isolation, and the antibody-based approach is particularly expeditious relative to FISH and real-time PCR.
We have described microscopic observations of samples prepared for FISH and ICC that provide a simple qualitative assessment of mtDNA depletion suitable for even the most basic clinical situations. Furthermore, microscopy revealed clear mosaicism by 3 PDs in ddC, a clinically significant observation not evident from real-time PCR analysis in which subpopulations are lost in an average of the whole population. Inasmuch as this additional information reflects compromised oxidative phosphorylation and subsequent side effects, the use of monoclonal antibodies should prove quite valuable in therapeutic monitoring during management of pathologies such as HIV infection. A variety of different cell and tissue types, including leukocytes and adipose tissue, are affected by NRTI therapy and represent potential targets for immunological detection of mtDNA depletion in the context of HIV therapeutic monitoring (Cote et al. 2002; Cossarizza 2003
; Hellerstein 2003
).
The FISH method follows the loss of mtDNA copies directly, but the protocol requires 24 hr and multiple steps to perform. The loss of COX I is an indirect measure of mtDNA depletion because the levels of subunit observed are determined by the transcription and translation rates from available mtDNA copies and the rate of polypeptide loss as previously assembled cytochrome c oxidase complexes are degraded. Not surprisingly, the loss of COX I trails the loss of mtDNA copies but, in effect, both the ICC and FISH methods can be used to establish a standard curve for mtDNA depletion (Figure 5, inset graph). As demonstrated here, one advantage of the ICC approach is that it can be completed and the data analyzed within 4 hr. Moreover, this method is readily adaptable to a 96-well plate format for simultaneously screening multiple patient samples. Such a high-throughput format should aid in the development of less toxic NRTIs and in drug evaluation where mitochondrial toxicity is of concern. Although all three methods were effective in monitoring changes in mtDNA content, the ICC approach was considerably faster, less expensive, and is easily amenable to high-throughput screening. Importantly, ICC data were confirmed by direct detection of mtDNA content by real-time PCR and FISH analysis. This illustrates the utility of antibodies directed against mtDNA-encoded proteins as surrogate markers for mtDNA in the context of NRTI screening and as a front-line method to monitor mtDNA toxicity.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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