Simultaneous generation of multiple mitochondrial DNA mutations in human prostate tumors suggests mitochondrial hyper-mutagenesis

Junjian Z. Chen1,4, Neriman Gokden2, Graham F. Greene3, Bridgett Green1 and Fred F. Kadlubar1

1 Division of Molecular Epidemiology, National Center for Toxicological Research, Jefferson, AR 72079, USA
2 Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
3 Department of Urology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA

4 To whom correspondence should be addressed Email: jjchen{at}nctr.fda.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Multiple somatic mitochondrial DNA mutations are frequently reported in human tumors, but the process leading to homoplasmic transformation and accumulation of multiple mutations in the same tumor cell lineage remains a mystery. We address possible mechanisms responsible for the generation of multiple mitochondrial (mt)DNA mutations observed in a high frequency of prostate tumors using sensitive mutant-specific PCR coupled with laser capture microdissection. Analysis of prostate tumors with multiple mtDNA mutations in the control region indicates that the mutations are locally confined, that the multiple mutations exist on the same molecules and that more than one mtDNA mutant species co-exists in the same neoplastic lesion. These results suggest an unusually rapid process in mtDNA mutagenesis during tumor progression. On the basis of prostate tumor cell kinetics, we propose a unique process of mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, to account for a burst of multiple mtDNA mutations in human prostate tumors.

Abbreviations: D-loop, displacement loop; HV1, hyper-variable region 1; HV2, hyper-variable region 2; LCM, laser capture microdissection; mtDNA, mitochondrial DNA; PIN, prostatic intra-epithelial neoplasia; ROS, reactive oxygen species


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A mutator phenotype expressed early in tumor progression has been postulated as an underlying mechanism through which multiple genetic alterations are generated in the nuclear genome of a tumor cell lineage (1,2). However, it is not clear if an increased mutation rate also occurs in the cytoplasmic mitochondrial genome during tumor progression. The human mitochondrial genome is haploid, circular DNA that is semi-autonomously maintained in mitochondria, and exists in multiple copies in each cell. Because of its unique genetics and functional importance in cellular oxidative phosphorylation and apoptotic control, it has drawn increasing attention in cancer research (3,4). A high frequency of homoplasmic point mutations [i.e. the majority of the mitochondrial (mt)DNA copies contain the same mutation] is detected in many human tumors with incidence rates ranging from 70% in colon to 15% in prostate (511). Interestingly, multiple homoplasmic mutations in mtDNA appear to be common in human tumors. Seven or more mutations in the same tumor were reported in glioblastomas, ovarian cancer, prostate cancer as well as in hepatocellular carcinoma (12). Considering that new mutations probably begin as unique events in a single molecule among the many copies of mtDNA in each cell, how mtDNA mutation, especially multiple mutations expand and accumulate in the same cell or cell lineage over a clinically relevant time period remains a mystery. One interpretation is that the homoplasmic transformation of mtDNA mutations is driven by selection for the mutant phenotype, presumably that the mutations confer selective and/or replicating advantages (5,13). Another interpretation argues that homoplasmy of somatic mtDNA mutations was driven entirely by random processes (14). However, some exceptionally high number of mutational events and their frequently clustered distribution in the mitochondrial genome imply a ‘catastrophic’ process in mitochondrial mutagenesis in at least a subset of human tumors.

Human prostatic carcinogenesis is a multistage process with a long latency in the development of a life-threatening prostate cancer (15,16). The slow pathogenesis of prostate malignancy is characterized by a unique bio-energetic transformation from low oxygen consumption in the benign epithelial cell to high oxygen consumption in the malignant cell (17), and by early inactivation of the {pi}-class glutathione S-transferase gene (GSTP1) that functions to inactivate cellular oxidants and electrophiles (18,19). It has been suggested that prostatic carcinoma cells may be susceptible to chronic oxidative stresses and prone to suffer oxidative genomic damage (20,21). Because mitochondria are the major source of endogenous reactive oxygen species (ROS), mtDNA and its maintenance machinery for DNA replication and repair may be particularly susceptible to oxidative damage. We reported extensive somatic mitochondrial mutation in human prostate cancer using the approach of laser capture microdissection (LCM) (22). Surprisingly, a high proportion of cancer cases had multiple mtDNA mutations in a 1.1 kb control region from the same neoplastic lesion. Three or more mutations were detected in six of 29 neoplastic lesions analyzed. To address possible mechanisms responsible for the generation of multiple mtDNA mutations, we developed a sensitive mutant-specific PCR coupled with LCM to characterize both the association of multiple mutations (more than mutational events) identified from three prostate tumors and their topographic distribution within each neoplastic lesion. Our new findings of local homoplasmy, linkage of multiple mtDNA mutations and the existence of more than one mutant species suggest an unusually rapid process in mtDNA mutagenesis during tumor progression. On the basis of prostate tumor cell kinetics, we propose that a process of mitochondrial hyper-mutagenesis, probably mediated by cellular oxidative stress, leads to a burst of multiple mtDNA mutations in human prostate tumors.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor specimens, microdissection and DNA extraction
Tumor specimens and methods for both LCM-based microdissection and DNA extraction were the same as described previously (22). However, this study focused on several cases of prostatectomy specimens that harbored more than three mtDNA mutations in the same lesion. Cases 1, 4 and 6, each containing five or more mutations in the displacement (D)-loop region, were chosen for the characterization of both the association of multiple mutations identified from each case and their distribution in serial tissue sections. The histology of the tumor focus and its microdissection from Case 4 were illustrated in Figure 1(A–C).



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Fig. 1. Histology of the tumor focus and its microdissection from Case 4. (A) The histology of the tumor focus analyzed for Case 4 was shown in a 5 µm tissue section stained with H&E. The majority of tumor cells in the lesion were dissected for mutation analysis. The small rectangle indicates an area enlarged in (B and C). (B) The enlarged area of tumor cells before microdissection. (C) A portion of microdissected cells using the PixCell II LCM system (Arcturus).

 
Allele/mutant-specific PCR
Mutant-specific PCR reactions were designed to determine the specificity and linkage of the mutations detected in Cases 1, 4 and 6. A mutant-specific reaction consisted of a mutant-specific downstream primer and an upstream primer containing wild-type sequence. Each mutant-specific primer was designed to contain a mutant-specific 3'-terminal base and a 3'-penultimate base mismatched to both the mutant and wild-type alleles. The cancer focus of Case 1 contained eight heteroplasmic substitution mutations that were distributed in the hyper-variable (HV)1-containing fragment. A mutant-specific primer for two closely spaced substitutions at positions 16311 and 16304, AS-MR16304 (5'-ATGGCTTTATGTGCTATGAG-3') and its upstream primer MF-16055 (5'-GAAGCAGATTTGGGTACCAC-3') were used to amplify a 248 bp mutant fragment flanking the six remaining mutations. MF and MR designates mtDNA forward and reverse primers, respectively; the number specifies the nucleotide position of a primer's 3' terminus according to the revised Cambridge reference sequence (23); the underlined bases were mutant-specific, while the italic base was mismatched to both the mutant and wild-type alleles. The cancer cell of Case 4 contained 10 near-homoplasmic substitutions that distributed equally in the HV1- and HV2-containing fragments. A mutant-specific PCR based on the mutation at position 16111 was designed to amplify a 464 bp mutant fragment flanking four substitutions in the HV1-containing segment using primers AS-MF16111 (5'-TTTCGTACATTACTGCCACT-3') and MR6 (5'-TTAATAGGGTGATAGACCTG-3'). Another mutant-specific PCR based on the mutation at position 73 was designed to amplify a 471 bp mutant fragment flanking the four other substitution mutations in the HV2-containing segment using primers AS-MF73 (5'-ATTTTCGTCTGGCGGGCG-3') and MR545 (5'-TTGGGGTTTGGTTGGTTC-3'). The prostatic intra-epithelial neoplasia lesion of Case 6 contained five heteroplasmic mutations that were all distributed in the HV2-containing fragment including the same mutation at position 73 as in Case 4. The same mutant-specific PCR for position 73 was used for analysis of Case 6.

Mixtures with known mutant fractions were reconstructed at positions 16111, 16304 and 73 using amplified mtDNA molecules of known sequence. Titration experiments were performed to establish conditions for each mutant-specific PCR reaction. The mutant-specific amplification was carried out in a 50 µl reaction that consisted of 1–5 fmol of template DNA amplified using the Expand High Fidelity PCR system (Roche Molecular Biochemicals, Indianapolis, IN), 1x Stoffel buffer (10 mM Tris–HCl, 10 mM KCl, pH 8.3), 2 mM MgCl2, 40 µM of each dNTP, 2.5 pmol of mutant-specific primer, 2.0 pmol of upstream primer and 2.5 U of Stoffel fragment (Applied Biosystems, Forst City, CA). A step-down PCR protocol was used for amplification at position 16304, which consisted of 95°C for 2 min, 1 cycle; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 8 cycles with a 0.5°C reduction per cycle; 94°C for 30 s, 54°C for 30 s, 72°C for 30 s, 22–24 cycles; and a final extension at 72°C for 5 min. The same procedure but with annealing temperatures at 62–58°C and 56–52°C were used for mutant-specific amplification at positions 16111 and 73, respectively. Mutant-specific fragments were visualized in a 1.8% agarose gel with ethidium bromide staining.

PCR amplification of the control region and a cytochrome b fragment
Two overlapping fragments that covered the entire sequence of the control region were PCR amplified as described previously (22). PCR primers MF15264 (5'-GGCTACTCAGTAGACAGTCC-3') and MR15980 (5'-TCTTAGCTTTGGGTGCTAATGG-3') were used to amplify a 715 bp adjacent fragment containing the tRNA-Thr gene and part of the cytochrome b gene. The same PCR protocol used for the D-loop fragments was used for the amplification of the cytochrome b fragment.

Sequencing of mtDNA
PCR products were fractionated with 1.2–1.6% agarose gels followed by purification with the QIAEXII gel extraction kit (Qiagen, Valencia, CA). Cycle sequencing was performed using Thermosequenase (USB, Cleveland, OH) and IRDye 800 terminator mix kit (Li-cor, Lincoln, NE). Sequences were resolved on a polyacrylamide gel mounted on a NEN Global IR2 sequencer (Li-cor) according to the manufacturer's recommendations. The two D-loop fragments were sequenced as described previously. The purified cytochrome b fragment was sequenced using nested primers MF15277 (5'-AGTCCCACCCTCACACGA-3') and MR15967 (5'-GTGCTAATGGTGGAGTTAAAGAC-3'). The mutant-specific PCR fragments were sequenced using internal primers MF16089 (5'-ACCCATCAACAACCGCTATG-3'), MR16391 (5'-AGGATGGTGGTCAAGGGAC-3') and MR521 (5'-TGGGGTTAGCAGCGGTGT-3'), respectively.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specificity and linkage of multiple mtDNA mutations
Whether multiple mutations detected in a neoplastic lesion are distributed randomly among different copies of mtDNA molecules or linked together on the same molecule reflects very different mutagenic processes. A strategy of mutant-specific amplification was devised to determine if multiple mutations detected in the same prostate lesion were both associated on the same molecule and specific to the diseased focus at high sensitivity (Figure 2A). It was based on the assumption that mutant-specific amplification anchored by a flanking mutation would selectively amplify a mutant fragment containing downstream mutations if all the mutations were linked on the same molecule. Three mutant-specific primers based on mutations at positions 16111, 16304 and 73 were developed to analyze 23 mutations previously detected in Cases 1, 4 and 6. Titration experiments established conditions for each mutant-specific reaction that allowed reproducible detection of a mutant fraction of three anchor mutations at a sensitivity of 10-3–10-4. Under such conditions, mutant-specific fragments with expected sizes were amplified only from diseased cells but not from corresponding benign cells in all three cases (Figure 2B). This result was confirmed by analyzing independent DNA templates amplified from the same genomic DNA from Cases 4 and 6. Further sequence analysis of amplified mutant-specific fragments demonstrated the presence of all other mutations in each fragment as predicted (Figure 2C). These results suggest that the multiple mutations detected previously in Cases 1, 4 and 6 not only were specific to the neoplastic cell, but also were linked on the same mtDNA molecule in each focus regardless of their state of existence (i.e. homoplasmy versus heteroplasmy) in each analyzed cell population.



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Fig. 2. Specificity and linkage of multiple mtDNA mutations using mutant-specific PCR. (A) A strategy of mutant-specific amplification. The upper portion of the diagram shows map positions of segments of the 1.1 kb control region that were amplified to yield two overlapping fragments (empty block arrows) and for three mutant-specific segments (filled block arrows). The vertical lines with a solid dot at positions 16111, 16304 and 73 indicate anchor mutations used for each mutant-specific primer. The lower portion of the diagram shows mutant specific segments with expected sizes from Cases 1, 4 and 6. The vertical arrows indicate positions of downstream mutations on each mutant-specific segment. (B) Mutant-specific fragments detected only in neoplastic cells. The labels M, C, P and B stand for molecular marker, cancer focus, PIN lesion and benign epithelial cells, respectively. The subscript number 1 indicates amplified DNA template from the original tissue section in each case, and the superscript primer represents a confirmative analysis in Cases 4 and 6. Reconstructed mixtures with known mutant fractions (0.1 and 0%) were included in each mutant-specific reaction as controls. (C) Fluoroimages showing the presence of multiple linked mutations in a mutant-specific fragment amplified from Case 1. As indicated by arrows, heteroplasmic mutations with an estimated mutant fraction of 70% were presented in the cancer template (C1). These same mutations were present in homoplasmy in the mutant-specific template (MS-C1). Note that genetic instability also occurred at the 12-cytosine tract as evidenced by the presence of shutter bands in sequence ladders downstream of the tract in both mutant-specific and cancer templates.

 
The multiple mtDNA mutations observed in neoplastic lesions were not limited to the non-coding sequence. A 715-bp fragment containing the tRNA-Thr gene and partial cytochrome b gene that was adjacent to the control region as amplified from the same genomic DNA from Cases 1, 4 and 6, and used for sequence analysis. Three substitution mutations in the coding sequence of the cytochrome b gene were identified in the tumor cell from Case 4. Two of the mutations (G15535A and T15877C) were homoplasmic and the third one (A15795G) was heteroplasmic with an estimated mutant fraction of 60%. Interestingly, both homoplasmic substitutions occurred at the third coding base causing synonymous mutations, while the heteroplasmic substitution resulted in an amino acid change (Ile -> Thr) at codon 350. No DNA sequence change was observed in the cytochrome b fragment in Cases 1 and 6.

Localization of multiple mutations and co-existence of additional mutant species
The spatial/topographic distribution of the mutant mtDNA molecules containing multiple mutations was analyzed in each neoplastic lesion and its serial tissue sections. The D-loop fragments were first amplified from microdissected cells from each serial section, and then used for mutant-specific PCR and direct sequencing. Among tumor DNA templates amplified from serial sections 2, 3 and 10 of Case 4, a mutant-specific fragment was amplified only at position 73 from section 2 that was adjacent to the initial tumor cells analyzed (Figure 3A). No mutant fragment was generated from two other tumor templates and four corresponding benign tissues from this case. Meanwhile, no mutant-specific fragment was generated at positions 73 and 16304 from the neoplastic cell of two serial sections in Cases 1 and 6, respectively. The failure to detect known mutations in a serial section of the same neoplastic lesion is unexpected, but it suggests local confinement of the mutants in the lesions where they were initially detected. Direct sequence analysis of the D-loop fragments revealed no mutations in serial sections from Cases 1 and 6. Surprisingly, multiple novel mutations were detected in tumor cells from three serial sections of Case 4, including three new mutations and a shared mutation in serial section 2, and a new mutation each in sections 3 and 10 (Figure 3B). The shared mutation at position 73 explained the generation of a mutant-specific fragment in serial section 2 of this case. The multiple mutant species detected in the same tumor focus could not be attributed to experimental errors because no mutation was observed in four corresponding benign controls from the same case. Interestingly, all the newly detected mutations were distributed in the HV2-containing fragment and located in close approximation to the previously detected mutations (Figure 3C), suggesting strong structural and/or conformational effects of the D-loop sequence on mtDNA mutagenesis.



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Fig. 3. Localization of multiple mutations and co-existence of additional mutant species. (A) Detection of mutant-specific fragments in serial tissue sections. The labels are the same as in Figure 1B and the subscript number indicates serial sections analyzed in each case. (B) Fluoroimages showing different mtDNA mutations detected in a 20 bp sequence in tumor cells isolated from serial tissue sections of Case 4. As indicated by arrows, two known mutations (T73C, C94T) were detected in tumor cells from C1, a shared mutation (T73C) in C2, and a new mutation (G79A) in C10. Note that the sequence ladders were generated using infrared dye-labeled reverse primer, IR-MR266. (C) The nature and distribution of novel mutations detected in three serial tumor sections from Case 4. The horizontal line stands for the HV2-containing fragment used for sequence analysis. Arrows above the line indicate mutations detected previously in the original tissue section (1) and arrows below the line represent new mutations detected in serial tumor sections 2, 3 and 10.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The purpose of this study was to address possible mechanisms that were responsible for the generation of a high frequency of multiple mtDNA mutations observed in human prostate tumors using sensitive mutant-specific PCR coupled with laser capture microdissection. We demonstrated that multiple mutations identified previously in three prostate tumors were specific to neoplastic lesions and were linked on the same mutant molecule in each microdissected cell population. Further topographic analysis using serial tissue sections showed local confinement of the mutants containing multiple mutations in each neoplastic lesion and revealed additional mutant species with novel mutations in one of the cancer foci analyzed. Our new findings of locally confined, multiple linked mtDNA mutations and co-existence of more than one mutant species in the same neoplastic lesion suggest not only active mitochondrial mutagenesis in prostatic neoplastic lesions, but also an accelerated process for the burst of multiple mtDNA mutations.

The frequency of homoplasmic mtDNA mutations is determined by the mutation rate and the segregating unit (mtDNA copy number) in tumor cells. The high frequency of locally confined multiple mtDNA mutations cannot be sufficiently explained by random processes through increased turnover of tumor progenitor cells. The observed frequency (6/29) of three or more mutations in prostate tumors is 17.2- and 3.8-fold higher than that expected after 300 and 600 generations, respectively, according to a simulation analysis based on unbiased mtDNA replication and sorting during cell division (14). On the basis of prostate tumor cell kinetics, a tumor progenitor cell with an estimated mean doubling time of 479 days requires 39.4 years (or 30 population doublings) to reach a clinically detectable mass of 1 cm3 (24,25). This slow rate of tumor growth can be converted into an average of 276 cell generations for the clonal outgrowth of a localized prostate tumor with an annual turnover of seven generations. The local confinement of multiple mutations and the existence of independent mutant species in close approximation as detected in prostate tumors suggest a recent origin for the mutations during tumor progression, and cannot be explained by pre-existing mutations in normal or tumor progenitor cells. This is because the late scenario would lead to ‘global’ homoplasmy due to clonal expansion of a tumor progenitor cell, rather than ‘local’ homoplasmy or heteroplasmy within a neoplastic lesion, although both types of mutations are reported. Paradoxically, the occurrence of five or more mtDNA mutations was not expected within the first 300 generations and would require a clinically unrealistic number of generations (>1000) for their random genesis. This apparent discrepancy could not be simply accounted for by a higher mutation rate in the D-loop region because the multiple mutations were not restricted to the non-coding sequence in prostate tumors. An exceptionally high number of multiple homoplasmic mutations distributed throughout the mitochondrial genome were also reported in many tumor types (810,12). The random process essentially implies a sequential accumulation of individual mtDNA mutations in a tumor cell lineage over time. In contrast, the locally confined multiple mtDNA mutations detected in tumor cells appear to be generated simultaneously on the same mtDNA molecule in a unique process of mitochondrial hyper-mutagenesis, a phenomenon reminiscent of transient mutators in bacteria (26).

The proposed mitochondrial hyper-mutagenesis probably involves error-prone mtDNA replication and/or repair, and fluctuation of mtDNA copy number in tumor cells. The error-prone process mediated by an array of by-pass polymerases was reported in both bacterial and mammalian cells, and frequently associated with cellular adaptive response (27). It is less probable that a by-pass polymerase plays a role in mitochondrial mutagenesis in human tumors because the human mtDNA polymerase {gamma} (pol {gamma}) is so far the only polymerase identified in mitochondria and is involved in both mtDNA replication and repair (28). We postulate that an integrated action by increased oxidative stress in mitochondria may be responsible for the burst of multiple mutational events observed in patches of tumor cells. Because the mitochondrial respiratory chain is a major source of ROS that are responsible for oxidative modification of biomolecules, we envision the expression of error-prone mtDNA replication/repair as mediated by defective proteins or protein assemblies caused by oxidative modification in mitochondria. Since modified proteins exist only temporarily, the error-prone process is likely to persist transiently. This postulation is supported by recent findings that pol {gamma} was detected as a major oxidized mitochondrial matrix protein, with a detectable decline in polymerase activity (29). It is conceivable that a functional but modified pol {gamma} may also compromise its replication fidelity, leading to transient error-prone mtDNA synthesis and repair. Moreover, the partitioning and assembly of nuclear-encoded proteins, especially limiting polypeptide chains, in multiple mitochondria within a cell are susceptible to oxidative damage. One of the consequences of oxidative insult is altered mitochondrial membrane potential ({Delta}{Psi}m) that serves as one of the major driving forces for the vectorial transport of polypeptide chains (30). An impaired import mechanism could lead to unbalanced partitioning of nuclear-encoded proteins and faulty assembly of a functional complex involved in mtDNA replication and repair, another potential source for transient hyper-mutagenesis. Because a feed-forward cascade in ROS generation in tumor cells can lead to a mutagenic tumor-microenvironment (31), the transient hyper-mutagenesis could be an inherent property associated with mtDNA mutagenesis and responsible for the majority of multiple mtDNA mutations observed in tumor cells. It is important to point out that the copy number of mtDNA may fluctuate in tumor cells, a process that may lead to the ‘bottleneck’ effect responsible for rapid homoplasmic transformation of somatic mutations (4,7). The effective unit in mtDNA segregation may be substantially reduced in a tumor cell lineage that sustains persistent oxidative DNA damage. Therefore, it is possible that transient expression of mitochondrial hyper-mutagenesis may be coupled with severe depletion in mtDNA content in a tumor cell. The coupling of these two processes constitutes a catastrophic mode of hyper-mutagenesis (Figure 4A and B). Under such a condition, transient hyper-mutagenesis probably confers immediate replicating advantage to a severely damaged cell, leading to cell survival. Alternatively, it is also possible that the transient hyper-mutagenesis constantly occurs in a subset of mitochondria in a cell. This spontaneous mode of hyper-mutagenesis may provide a baseline of pre-existing multiple mutations in tumor cells. In either case, the homoplasmic transformation of newly arisen multiple mutations is probably accelerated through the fluctuation of mtDNA content and biased mtDNA replication of mutant molecules in a tumor cell lineage (Figure 4C). Although the discussion on the ROS-mediated mitochondrial hyper-mutagenesis is speculative in nature, it offers new perspectives in understanding a possible mutator phenotype in mitochondria and the role of tumor-microenvironment in mitochondrial mutagenesis. Further experimental evidence is needed for its proof. Besides, somatic mutation in tumor cells may share a common mechanism with germ-line mutations in human populations (22,32,33). The possibility for simultaneous generation of multiple mutational events in mtDNA may raise questions on both the use of a molecular clock in phylogenetic analysis and the forensic identification employing mtDNA sequence polymorphism, especially in the D-loop sequence.



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Fig. 4. A catastrophic mode of transient hyper-mutagenesis in tumor mitochondria. Differential susceptibility of individual tumor cells to increased cellular oxidative insults could lead to the coupling of two biological processes that trigger a catastrophic mode of hyper-mutagenesis in mitochondria. (A) The initial process involves substantial mtDNA depletion in a subset of tumor cells as consequences of persistent mtDNA damage and compromised repair capacity caused by oxidative damage. The damaged tumor cell could either undergo apoptosis as triggered by severe mtDNA damage (30) or survive the damage by entering temporary growth arrest (34). (B) Under such conditions, a process of error-prone mtDNA replication and/or repair probably mediated by oxidized proteins or faulty protein assemblies in mitochondria would confer immediate replicating advantage to the damaged cell, an ‘SOS’-like response in mitochondria. Multiple mtDNA mutations could be generated simultaneously in a single round of mtDNA replication as a consequence of the error-prone process. (C) A final step involves recovery of the mtDNA content of the survived cell and subsequent homoplasmic transformation of the multiple mutations through either a replicating advantage or a stochastic process, leading to the formation of locally confined clonal cells containing the same multiple mtDNA mutations.

 

    Acknowledgments
 
We thank Brett Thorn for computer simulation, Hilary Coller for providing raw simulation data (14), Gerald Holmquist and Lili Liu for useful discussion. We also thank Robert Heflich and Carrie Valentine for their comments on an earlier version of the manuscript. This work was supported by a protocol from FDA (E07113.01) to J.Z.C.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 11, 2003; revised June 2, 2003; accepted June 14, 2003.