Mitochondria damage checkpoint in apoptosis and genome stability

Authors

  • Keshav K. Singh

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    1. Department of Cancer Genetics, Cell and Virus Building, Room 247, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA
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*Tel.: +1-716-845-8017; fax: +1-716-845-1047, E-mail address: keshav.singh@roswellpark.org

Abstract

Mitochondria perform multiple cellular functions including energy production, cell proliferation and apoptosis. Studies described in this paper suggest a role for mitochondria in maintaining genomic stability. Genomic stability appears to be dependent on mitochondrial functions involved in maintenance of proper intracellular redox status, ATP-dependent transcription, DNA replication, DNA repair and DNA recombination. To further elucidate the role of mitochondria in genomic stability, I propose a mitochondria damage checkpoint (mitocheckpoint) that monitors and responds to damaged mitochondria. Mitocheckpoint can coordinate and maintain proper balance between apoptotic and anti-apoptotic signals. When mitochondria are damaged, mitocheckpoint can be activated to help cells repair damaged mitochondria, to restore normal mitochondrial function and avoid production of mitochondria-defective cells. If mitochondria are severely damaged, mitocheckpoint may not be able to repair the damage and protect cells. Such an event triggers apoptosis. If damage to mitochondria is continuous or persistent such as damage to mitochondrial DNA resulting in mutations, mitocheckpoint may fail which can lead to genomic instability and increased cell survival in yeast. In human it can cause cancer. In support of this proposal we provide evidence that mitochondrial genetic defects in both yeast and mammalian systems lead to impaired DNA repair, increased genomic instability and increased cell survival. This study reveals molecular genetic mechanisms underlying a role for mitochondria in carcinogenesis in humans.

1Introduction

Mitochondria play a central role in many cellular functions including energy production, respiration, heme synthesis, lipids synthesis, metabolism of amino acids, nucleotides, and iron, and maintenance of intracellular homeostasis of inorganic ions, cell motility, cell proliferation and apoptosis [1,2]. Mitochondria contain their own DNA (mtDNA) that amounts on average to about 15% of the DNA content of Saccharomyces cerevisiae. It means that a haploid cell contains about 50 copies of the 75-kb mitochondrial genome. The mtDNA occurs in small clusters called nucleoids or chondriolites. The number of mtDNA molecules in nucleoids varies in size and numbers in response to physiological conditions. All of the mtDNA molecules in S. cerevisiae are comprised of polydisperse linear tandem arrays of the genome. The linear molecules are accompanied by small amounts of circular forms [3]. While nuclear DNA encodes the majority of the mitochondrial proteins only a few of these proteins are encoded by mitochondrial DNA. A recent mitochondrial proteomic study in S. cerevisiae identified at least 750 mitochondrial proteins that perform mitochondrial function [4].

The last decade has witnessed an increased interest in mitochondria, not only because mitochondria were recognized to play a central role in apoptosis but also since mitochondrial genetic defects were found to be involved in the pathogenesis of a number of human diseases [2,5–9]. Human mitochondrial diseases are caused both by mutations in mtDNA and in nuclear DNA involved in mitochondrial function. In this paper we describe the importance of mitochondria in yeast apoptosis and genome stability.

2Mitochondria are the major site of oxidative stress in the cell

Mitochondria are the major source of endogenous reactive oxygen species (ROS) in the cell, because they carry the electron transport chain that during oxidative phoshorylation reduces oxygen to water by addition of electrons [1,10]. It is estimated that the endogenous production of ROS within human mitochondria is about 107 molecules/mitochondrion/day during normal oxidative phosphorylation [1,10]. Unlike nuclear DNA, human mtDNA contains no protective histones, and is continuously exposed to ROS generated during oxidative phosphorylation (it is estimated that up to 4% of the oxygen consumed by cells is converted to ROS under physiological conditions) [1]. ROS induce more persistent damage to mtDNA than to nuclear DNA [11]. ROS also produce more than 20 types of mutagenic base modifications in DNA [12]. These DNA lesions cause mutations in mtDNA that can lead to impairment of mitochondrial function [1,13]. Taken together, this makes clear that mtDNA is extremely susceptible to mutation by ROS-induced damage.

3Mitochondrial impairment leads to nuclear genome instability

Although the mitochondrial and nuclear genomes are physically distinct, there is a high degree of cross-talk and functional interdependence between the two genomes. Since mitochondria are the major site of ROS in the cell, and mtDNA is more frequently damaged, we were interested in identifying the consequences of mitochondrial dysfunction on nuclear genome stability. To address this question we isolated an S. cerevisiae clone that lacked mitochondrial genome entirely (ρ° strain). We also isolated a ρ strain containing a large deletion in mtDNA. Our studies conducted with these strains suggest that indeed mitochondrial dysfunction leads to nuclear genome instability [14]. We identified two pathways: a ROS-dependent and a ROS-independent pathway involved in mitochondria which leads to nuclear genome instability. Our studies also suggest that ROS-independent mitochondria-mediated nuclear genome instability was controlled by an error-prone DNA repair pathway [14]. However, the error-prone repair pathway was not involved in ROS-dependent nuclear mutator phenotype. We carried out similar studies in mammalian cells that suggest that mutations in nuclear DNA arise due to reduced DNA repair activity. Given that mitochondria are the major producer of ATP, it is also likely that mitochondrial dysfunction leads to the reduction in ATP level that may affect ATP-dependent pathways involved in transcription, DNA replication, DNA repair, and DNA recombination (see Table 1). Consistent with this notion a study recently reported chromosomal abnormalities in mouse fibroblast cells lacking mitochondrial MnSOD activity [15] and in response to mitochondrial inhibitors [16]. Together these studies suggest that mitochondrial dysfunction leads to nuclear genome instability.

Table 1.  Putative ATPases that may contribute to genomic instability in ρ° cells
Cellular functionGenes
TranscriptionDBP3, DBP6, DBP7, DBP8, DBP9, DBP10, and PRP2, PRP5
ReplicationCDC46, CDC47, DCD54, DNA2, HMI1, MCM2, MCM3, MCM6, ORC5, RRM3 and SGS1
Base excision repairUNG1 and CDC9, DNL4
Nucleotide excision repairRAD5, RAD7 RAD16, RAD18, RAD26
Mismatch repairMlh1, Msh2, Msh6, PMS1 Msh1
RecombinationRAD54, RDH54
Chromosome maintenanceSMC1, SMC2, SMC3 and SMC4

4Mitochondrial impairment leads to altered expression of antioxidant genes

In order to identify genes whose expression may contribute to increased mutagenesis in nuclear DNA we did a gene expression analysis using Affymetrix microarrays. Our results demonstrate that CTT1 and GPX1 antioxidant genes were down-regulated while GPX2 was up-regulated in ρ° cells. This indicates that an altered level of antioxidant status may contribute to nuclear DNA mutation. The list of genes and the fold changes in their expression in ρ° cells are described in Table 2.

Table 2.  Changes in antioxidant gene expression in ρ° cells
Gene nameFold changes
CTT1−5.9
GPX1−5.3
GPX27.9

5Mitochondrial impairment alters nucleotide metabolism

Although our studies suggest that reduced DNA repair and changes in expression of antioxidant genes may contribute to mutation in nuclear genome, it is likely that other factors are involved. Mitochondria are intimately involved in deoxyribose nucleoside triphosphate (dNTP) biosynthesis [17,18]. It is conceivable that mitochondrial impairment contributes to mutagenesis of the nuclear genome in part due to impaired nucleotide biosynthesis. In fact, it is well established that an imbalance in the dNTP pool is mutagenic to cells [19]. Studies demonstrate that a dNTP pool imbalance can induce nucleotide insertion, frame-shift mutation [19], sister chromatid exchange, recombination and double-strand break [20–22].

6Mitochondrial impairment alters ATP utilization

Mitochondria produce energy in the form of adenosine 5-triphosphate (ATP) in a process termed oxidative phosphorylation [23]. Under the condition of high proton-motive force, the F1F0-ATP synthase complex catalyzes the formation of ATP from adenosine 5-diphosphate (ADP) in the manner that is coupled to the transport of protons from the intermembrane space across the inner membrane to the matrix. A decrease in the proton-motive force as a result of oxygen deprivation to the cell, or because of the uncoupling of oxidative phosporylation, can cause the reversal of the action of F1F0 synthase, resulting in the hydrolysis of ATP to ADP and phosphate. This hydrolytic activity of F1F0-ATP synthase is regulated directly by the natural inhibitor protein called Inh1 in yeast [23,24]. In yeast, the inhibitory activity of Inh1 is enhanced by two stabilizing factors: STF1 and STF2 (stabilizing factors). Interestingly, microarray analysis showed that INH1, STF1 and STF2 are all down-regulated in ρ° cells (see Table 3). Our large-scale gene expression analysis suggests that down-regulation of genes involved in preventing the hydrolysis of ATP may allow essential ATP-dependent functions so that cellular functions are not compromised severely. However, this view may be simplistic because the level of RNA does not give a perfect image of enzyme activity.

Table 3.  Expression of genes involved in ATP hydrolysis in ρ° cells
Gene namesFold changes
  1. Total RNA was isolated from exponentially growing S. cerevisiae cells according to manufactures guide lines using RNeasy (QIAGEN). Total RNA (5 μg) was converted in double-stranded cDNA by GIBCO BRL's SuperScript Choice system for cDNA synthesis (Life Technologies) and a T7-(dT)24 oligomer provided by Research genetics (Huntsville, AL). Double-stranded cDNA was purified by phenol/chloroform extraction and ethanol precipitation. In vitro transcription was performed with T7 RNA polymerase following the instructions from BioArray high-yield RNA transcript Labeling kit from Enzo (Affymetrix). Gene array analysis was conducted using yeast gene array (from Affymetrix) as described [36].

INH1−7.2
STF1−2.2
STF2−4.3

7A mitochondria damage checkpoint (mitocheckpoint)

Checkpoint was defined by Hartwell and Weinert [37] as control mechanism that ensures the proper order of cellular events by arresting or delaying progression through the cell cycle in response to DNA damage [25]. Based on our comparative gene expression analysis between the wild-type yeast S. cerevisiae and the ρ° derivative cells, I propose that cells contain a mitochondria damage checkpoint (mitocheckpoint) that avoids production of cells defective in mitochondrial function. Mitocheckpoint monitors the functional state of mitochondria and responds accordingly when mitochondria are damaged or become dysfunctional. Mitocheckpoint can adjust the cell cycle response and gene expression to help repair-damaged mitochondria to restore normal mitochondrial function [7]. This hypothesis is consistent with (i) A study reporting cell cycle arrest of human cells in response to respiratory inhibitors [26]. Following low doses there was a significant increase in the proportion of cells in G1. However, exposure to higher doses of respiratory inhibitors caused G2-M arrest [26]. (ii) Existence of highly coordinated cross-talk between mitochondria and nucleus in yeast [27]. (iii) Mutations in CIT1 gene (encoding mitochondrial citrate synthase) of Podospora anserina show a defect in progression to meiosis, leading to developmental abnormalities. In this organism, citrate synthase appears to work as meiotic checkpoint [28]. And (iv) A link between nuclear DNA and mtDNA replication in Drosophila[29]. In Drosophila, a transcription factor DREF coordinates nuclear and mitochondrial DNA replication. DREF also controls the expression of genes encoding mitochondrial single-stranded DNA-binding protein, polymerase β and accessory subunit of polymerase γ involved in mtDNA replication [29]. DREF's role in controlling the expression of the DNA polymerase γ and β genes also establishes a common regulatory mechanism linking nuclear and mitochondrial DNA replication with repair. In S. cerevisiae Rtg1, Rtg2 and Rtg3 proteins monitor the functional state of mitochondria and coordinate mitochondria-to-nucleus signaling. Among RTG proteins Rtg2p contains an ATP-binding domain that may be the sensor of the ATP level in the cell [30]. RTG proteins are important candidates to serve as mitocheckpoint proteins. Several proteins involved in DNA replication in yeast are absolutely dependent on ATP for their function (see Table 1). The mitocheckpoint may regulate ATPases known to be involved in DNA repair and recombination. Examples of such ATPases are UNG1 and CDC9 (base excision) RAD5, RAD7 RAD16, RAD18, RAD26 (Nucleotide Excision repair), Mlh1, Msh2, Msh6, PMS1 (DNA mismatch repair in the nucleus), Msh1 (DNA mismatch repair in the mitochondria), RAD54, and RDH54 (recombination). In addition, mitocheckpoint can also regulate SMC (Structural Maintenance of Chromosome) proteins that contain ATPase activity and maintain chromosomal integrity ([30], see Table 1). The mitocheckpoint may coordinate transcription of genes by regulating ATP-dependent RNA helicases (see Table 1).

The mitocheckpoint must coordinate and maintain a proper balance between apoptotic and anti-apoptotic signals. Thus mitochondria must regulate mechanisms that promote cell survival. Our studies show that a mitochondrial genetic defect causes high frequency of mutations in the nuclear genome and promotes cell survival when exposed to DNA-damaging agents such as adriamycin (see Fig. 1, Table 4 and [14]). Similar to studies in yeast, we also find that a variety of mammalian ρ° cells are resistant to apoptosis induced by oxidative agents [31]. Altogether our studies provide evidence that mitochondrial dysfunction leads to evasion of apoptosis, increased cell survival and genomic instability in both yeast and mammalian cells [14,31,32]. Fig. 2 summarizes our studies in a model where mitocheckpoint monitors damage to the mitochondria and responds to dysfunctional state of mitochondria. When mitochondria are damaged, mitocheckpoint is activated which helps cells repair the damage and restore normal mitochondrial function. If the mitochondria are severely damaged, mitocheckpoint may not be able to protect the cells. Such an event will trigger apoptosis [33–35]. If damage to mitochondria is continuous or persistent (such as mutations in mtDNA) mitocheckpoint system can fail which can lead to genomic instability (mutator phenotype, chromosomal aneuploidy, loss of heterozygosity), and increased cell survival and cancer in humans.

Figure 1.

Cell survival in response to DNA-damaging agent. Both wild-type (circles) and ρ° (squares) cells were grown to log phase in YPD as described [14]. Cells were centrifuged, resuspended in sterilized distilled water containing various concentrations of adriamycin. Appropriate dilutions were made and cells were plated on YPD medium. Colonies were counted after three to four days.

Table 4.  Frequency of mutations in nuclear genome (Frequency of CanR mutant × 10−7)
Adriamycin (μg ml−1)Untreated control20 (μg ml−1)40 (μg ml−1)
  1. Both wild-type and ρ° cells were grown to log phase as described [14]. Cells were centrifuged, resuspended in sterilized distilled water containing various concentrations of adriamycin. Appropriate dilutions were made and cells were plated on YPD and SD medium containing canavanine as described [14]. The CAN1 gene of S. cerevisiae encodes a transmembrane amino acid transporter that renders the cell sensitive to a lethal arginine analogue, canavanine. Any inactivating mutation in this gene results in a canavanine-resistant phenotype (CAN1R). Thus, the frequency of canavanine-resistant colonies measures spontaneous nuclear mutational events. Canavanine-resistant colonies were counted after five days.

Wild type5.06.49.7
ρ°7.111.5195.3
Figure 2.

Integrating apoptosis, genomic instability and cell survival. A model which integrates a role for mitocheckpoint in apoptosis, genomic stability, and cell survival. For details see Section 7 in the text.

It is interesting to note how much of our current understanding of genetic and biochemical activities of the mitochondria is owed to the unique ability of the humble brewer's yeast S. cerevisiae to survive without respiration [3]. The S. cerevisiae genome was the first eukaryotic genome that was sequenced. A comprehensive approach to the deletion of and expression of all Open Reading Frames has been performed [4]. In addition to availability of deletion mutants of all genes, sophisticated biochemical and genetic analysis tools are also available to perform functional genomics and genome-wide gene expression. In summary, yeast will continue to serve as an excellent model to understand the underlying genetic mechanisms involved in apoptosis and genome instability relevant to human carcinogenesis.

Acknowledgements

This research was supported by a grant from the National Institutes of Health RO1-097714. I thank Drs. Lene Rasmussen and Anna Rasmussen for their contribution to the data presented in Table 4 and Dr. Kylie Keshav for critical reading of this manuscript.

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