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Advances in Experimental Medicine and Biology 1038
Hongzhi Sun
Xiangdong Wang Editors
Mitochondrial
DNA and
Diseases
Advances in Experimental Medicine
and Biology
Volume 1038
Editorial Board
IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel
ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research, Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
NIMA REZAEI, Tehran University of Medical Sciences Children’s Medical
Center, Children’s Medical Center Hospital, Tehran, Iran
More information about this series at http://www.springer.com/series/5584
Hongzhi Sun • Xiangdong Wang
Editors
Mitochondrial DNA
and Diseases
Editors
Hongzhi Sun Xiangdong Wang
Liaoning Province Key Laboratory of Zhongshan Hospital Institute of Clinical
Cancer Metabolomics Science
Jinzhou Hospital of Jinzhou Medical Fudan University, Shanghai Medical
University College
Jinzhou, Liaoning, China Shanghai, China
ISSN 0065-2598 ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-981-10-6673-3 ISBN 978-981-10-6674-0 (eBook)
https://doi.org/10.1007/978-981-10-6674-0
Library of Congress Control Number: 2017958161
© Springer Nature Singapore Pte Ltd. 2017

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.
Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
Contents
1 How Far Can Mitochondrial DNA Drive the Disease?�� 1

Hongzhi Sun, Weibin Shi, and Xiangdong Wang
2 Mitochondrial DNA in Lung Cancer �� 9
Fangming Liu, David E. Sanin, and Xiangdong Wang
3 Approach, Application, and Bioethics of mtDNA Sequencing
in Cancer�� 23
Mengjia Qian, Claudio Spada, and Xiangdong Wang
4 Roles of Mitochondrial DNA Signaling in Immune Responses �� 39
Lingyan Wang, Michael N. Liebmen, and Xiangdong Wang
5 Mitochondrial DNA in Telocytes�� 55
Dongli Song, Dragos Cretoiu, and Xiangdong Wang
6 Roles of Mitochondrial DNA in Energy Metabolism�� 71
Jiapei Lv, Madhav Bhatia, and Xiangdong Wang
7 The Role of DNA Repair in Maintaining Mitochondrial
DNA Stability�� 85
Linlin Zhang, Aurelio Reyes, and Xiangdong Wang
8 Is Mitochondrial Cell Fragility a Cell Weakness?�� 107
William Wang, Jiayuan Hou, Zhenghua Zhu, and Hao Fang
9 Mitochondrial DNA Methylation and Related Disease�� 117
Danyan Gao, Bijun Zhu, Hongzhi Sun, and Xiangdong Wang
10 Beyond Deubiquitylation: USP30-Mediated Regulation
of Mitochondrial Homeostasis�� 133
Jiayun Hou, Mohmmad Eldeeb, and Xiangdong Wang
11 Metabolic Regulation in Mitochondria and Drug Resistance�� 149
Yue Pan, Min Cao, Jianzhou Liu, Qing Yang, Xiaoyu Miao,
Vay Liang W. Go, Paul W. N. Lee, and Gary Guishan Xiao
v
vi Contents
12 Mitochondrial Lon Protease and Cancer�� 173

Bin Lu
13 Regulatory Roles of Mitochondrial Ribosome in Lung
Diseases and Single Cell Biology�� 183
Linlin Zhang, William Wang, Bijun Zhu, and Xiangdong Wang
14 Epithelial Mitochondrial Dysfunction in Lung Disease �� 201
Linlin Zhang, William Wang, Bijun Zhu, and Xiangdong Wang
15 Significance of Mitochondria DNA Mutations in Diseases�� 219
Zhenhua Zhu and Xiangdong Wang
About the Editors
Hongzhi Sun, MD, PhD, is a famous surgical specialist

in China and the director of the First Affiliated Hospital
of Liaoning Medical University. He is the chairman of
Surgical Biomaterials Specialized Committee, the vice
chairman of Tumor Cell Biology Specialized Committee
and Surgical Biological Cell Technology Specialized
Committee in Liaoning Cell Biology Society, a member
of the Breast Sciences Section of Liaoning Medical
Association, the executive director of Liaoning
Anticancer Association, the associate editor of the inter-
national journal Molecular and Cellular Therapies, the
editorial board member of Chinese Journal of Tissue Engineering Research and
Clinical Rehabilitation and Liaoning Medical University newspaper. He hosted
seven fund projects and published more than ten scientific papers with the impact
factor over 20. His major research is focused on the mechanism of tumorigenesis
and progression, invasion and metastasis, and the basic and clinical research of
biotherapy of tumor.
Xiangdong Wang, MD, PhD, is a distinguished pro-

fessor of medicine, director of Shanghai Institute of
Clinical Bioinformatics, executive director of Clinical
Science Institute of Fudan University Zhongshan
Hospital, director of Fudan University Center of Clinical
Bioinformatics, deputy director of Shanghai Respiratory
Research Institute, and visiting professor of King’s
College London. His main research is focused on clini-
cal bioinformatics, disease-specific biomarkers, lung
chronic diseases, cancer immunology, and molecular
and cellular therapies. He is the author of more than 200
scientific publications with the impact factor about 900, citation number about
6918, h-index 46, i10-index 181, and cited journal impact factor about 7000.
vii
Chapter 1
How Far Can Mitochondrial DNA Drive
the Disease?
Hongzhi Sun, Weibin Shi, and Xiangdong Wang
Abstract Mitochondria are one of the dominant drivers for producing cellular
energy to meet a large number of biological functions, of which the mitochondrial
DNA (mtDNA) is the control center of energetic driving force and the dominant
driver of mitochondrial molecular diversification. mtDNA transcription generates
the necessary RNAs to regulate the extent and nature of mtRNA post-transcriptional
modifications and the activity of nucleus-encoded enzymes. With a special focus on
mtDNA, the current volume aims to overview the biology and structures of mtDNA,
regulatory roles of mtDNA in lung diseases, or involvement of mtDNA in metabo-
lism. We explore the significance of mtDNA sequencing, methylation, stability, and
mutation in the pathogenesis of the diseases. Molecular mechanisms by which
mtDNA contribute to the regulation of mitochondrial homeostasis and drug resis-
tance are also discussed. We also point out the importance of mitochondrial ribo-
some, single cell biology, and gene editing in the understanding of the development
of mitochondrial dysfunction in lung disease.
Keywords Mitochondria • mtDNA • Diseases • Energy • Metabolism
H. Sun (*)
Liaoning Province Key Laboratory of Cancer Metabolomics, Jinzhou Hospital of Jinzhou
Medical University, Jinzhou, Liaoning, China
e-mail: [email protected]
W. Shi (*)
Medical Examination Center of Zhongshan Hospital, Fudan University, Shanghai, China
X. Wang (*)
Zhongshan Hospital Institute of Clinical Science, Fudan University,
Shanghai Medical College, Shanghai, China
© Springer Nature Singapore Pte Ltd. 2017 1

H. Sun, X. Wang (eds.), Mitochondrial DNA and Diseases, Advances in
Experimental Medicine and Biology 1038,
https://doi.org/10.1007/978-981-10-6674-0_1
2 H. Sun et al.
1 Why We Focus on mtDNA
Mitochondria are the dominant driver of cellular energy for a large number of bio-
logical functions, while mitochondrial DNA (mtDNA) is the control center of ener-
getic driving force and the dominant driver of mitochondrial molecular diversification.
mtDNA can act as the part of genome to transcript genetic messages to maintain
cell’s own function, as the genetic copy for other cells which have the capacity of the
sensibility to mtDNA and as the toxicant to “poison” owners intra- or extramitochon-
drially. Macrophages have strong uptake and weak digestion capacities of tumor
mtDNA, while dendritic cells have strong sensitivity and digestion capacities [1].
Both cells belong to the family of native immunity but have different senses to
mtDNA, due to the variation of integrin-associated protein-signal regulatory protein-α
axis function between cells. Elements of mtDNA or elements associated with mtDNA
function are considered as diagnostic biomarkers or therapeutic targets. With the
development of biotechnology, mapping mtDNA genomes can provide more details
of the genetic information for the understanding molecular mechanisms mtDNA is
involved in. For example, a new strategy of PacBio full-length transcriptome sequenc-
ing was applied for the first full-length human mitochondrial transcriptome, and two
novel long non-coding RNAs of ATP-binding cassette permease MDL1 and MDL1AS
were discovered in human mitochondrial DNA from animal mitochondrial genomes
[2]. mtDNA epigenetics is also a new merging area to be explored. mtDNA methyla-
tion and histone modifications were detected and suspected to regulate the mitochon-
drial unfolded protein response, although the exact mechanisms are not unclear yet.
The oxidative phosphorylation system and RNA granules ribosome are the major
players in the posttranscriptional processes of mitochondrial gene expression,
mtDNA perturbation, and the coordination of mitochondrial and cytosolic translation
[3]. mtDNA transcription generates the necessary RNAs to regulate the extent and
nature of mtRNA posttranscriptional modifications and the activity of nucleus-
encoded enzymes. With a special focus on mtDNA, the current volume aims to over-
view the biology and structures of mtDNA, regulatory roles of mtDNA in lung
diseases, or involvement of mtDNA in metabolism. We explore the significance of
mtDNA sequencing, methylation, stability, and mutation in the pathogenesis of the
diseases. Molecular mechanisms by mtDNA that contribute to the regulation of mito-
chondrial homeostasis and drug resistance are also discussed. We also point out the
importance of mitochondrial ribosome, single-cell biology, and gene editing in the
understanding of the development of mitochondrial dysfunction in lung disease.
2 hy We Start with the Relationship Between mtDNA

W
and Lung Diseases
Lung diseases include acute and chronic lung injury and failure, infections, intersti-
tial inflammation, vascular pathology, allergic immune disorders, and cancer. The
incidence of lung diseases is one of the highest ones in the world, with a continuous
increase. For example, the incidence and mortality of lung cancers become the top
one in all kinds of cancers, and chronic lung diseases become the leading one in all
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1 How Far Can Mitochondrial DNA Drive the Disease? 3
kinds of chronic diseases, especially in the developing countries with air pollution
[4–6]. Even though, lung diseases are relatively ignored, gain less attentions than
they should have, or are underestimated, due to a large compensatory capacity the
lung has, less incidence of sudden death, and wide distribution of clinical category.
Lu et al. collects experimental and clinical evidence to show the potential links
between mtDNA and lung cancer and demonstrates the fact that alterations of
mtDNA copy number and sequence mutations may contribute to carcinogenesis and
development of lung cancer [7]. One of the exciting points from the chapter is that
variations of mtDNA sequencing, mutation, and epigenetics are suggested to be
potential biomarkers to monitor severity, duration, stage, response to therapy, and
prognosis in patients with lung cancer. The significance of mtDNA mutations and
altered copy numbers in lung cancer and clinical relevance was comprehensively
overviewed [8–10], although a number of issues should be considered before
mtDNA changes can be an effective strategies for lung cancer diagnosis and therapy.
One of the most challenges to develop mtDNA-dependent biomarkers is to ensure
the disease specificity and sensitivity, since mitochondria are the power and metabo-
lism driver of the cells and have the function and responsibility more generally.
Qian et al. bring a critical question how we can properly identify disease-specific
values from mtDNA sequencing, mutations, molecular interactions and networks, sig-
naling pathways, and heteroplasmy in mitochondrial genome [11]. From practical
points, this chapter presents what the proper and optimal way to prepare the library
preparation, sequencing, data analysis, and professional interpretations and call the spe-
cial attentions to recognize advantages and disadvantages of mtDNA. One of the critical
issues the chapter delivers is how and why we should choose the most appropriate
mtDNA sequencing in the real application, and we should seriously take care of bioeth-
ics in multi-aspects. Another important issue the chapter bring on the table at the first
time is the urgent need to evaluate the potential cytotoxicity and genotoxicity of gene
editing, especially the long-term effects, before mtDNA editing marches into clinic.
3 How mtDNA Works in the Disease
It is easier to understand the importance of mtDNA in mitochondrial function, cel-

lular metabolism, and organ function, while it is hard to describe how mtDNA
works and regulates the element interaction intra- or extramitochondrially. The
mechanism by which mtDNA regulates and controls the occurrence of the disease
is more complex than we can image. Of those, the epigenetic mechanisms are
recently overviewed and believed to be important in bidirectional mitonuclear com-
munication between mitochondria and the nuclear epigenome represents [12]. It
seems that the mitochondria can generate a number of the intermediate or terminal
metabolites to mediate or participate in the processes of acetylation or methylation
to regulate and alter nDNA epigenetic phenotypes. The modified epigenetic mes-
sages in the nuclear are transferred into the mitochondria to mediate mtDNA gene
expression of mitochondria-produced proteins or mtDNA acetylation or methyla-
tion. The mitochondrial proteins can participate and regulate the production of bio-
energy, oxygenation, and metabolism in mitochondria, which in turn can feedback
4 H. Sun et al.
the signaling to the nuclear or mitochondria. It is possible or necessary for nDNA

and mtDNA to have such crosstalk and interactions.
Wang et al. furthermore explore the intercommunication and interaction of the
molecules within mitochondria during immune responses or the development of
immune-related diseases [13]. This chapter collects scientific evidence to show the
roles of mtDNA in immune responses and lead an initial discussion on the main func-
tion and mechanisms of mtDNA in immune responses. mtDNA per se can interact
with native immune cells and influence their capacities, dependently upon the sensi-
tivity of cells. mtDNA can also contribute to the immune response by regulating
mtDNA-dependent TLR9 signaling pathways, autophagy pathways, or cGAS-STING
signaling pathways. The chapter also uncovers the potentials of mtDNA-oriented
therapeutic targets and mtDNA gene editing in immune diseases and inflammation,
although it is still at the early stage of the discovery and development.
Huo et al. describe potential mechanisms by which mutations or sequence aberra-
tions of nDNA or mtDNA occur in the disease and specially focus on USP30. USP30
is a mitochondrial deubiquitinase to contribute to mitochondrial fusion by mediating
the deubiquitination of ubiquitylated forms of mitofusins [14]. USP30 can regulate
the removal of Ub-chains from Lys 6 and Lys 11 on mitochondria-derived proteins
and of the ubiquitin chains added by Parkin, a cytoplasmic E3 ubiquitin-ligase.
Mitochondrial fission and mitophagy as well as mtDNA genetic alterations can be
regulated and controlled by autophagy-related genes, mitochondrial-quality control
pathways, or USP30-triggered mitochondrial dynamic signaling.
4 Whether mtDNA Can Influence Cell Metabolism
Lv et al. demonstrate the fact that metabolic disorders, oxidative stress, or inflamma-
tion can alter mtDNA, resulting in the occurrence of mitochondrial dysfunction [15].
It is questioned how mtDNA control and influence energy metabolism, signaling
pathways, and cell death. The intra- or extracellular factors can induce DNA single-
or double-strain breaks and provoke DNA-dependent protein kinase, e.g., aging [16].
The activated DNA-dependent protein kinase can phosphorylate threonines 5 and 7
of HSP90α and downregulate the activity of AMP-activated protein kinase, leading to
the dysfunction of mitochondrial biogenesis, energy metabolism, and physical fit-
ness. Drugs and toxicants could alter mitochondrial motility, structure, and cell injury
[17–19], probably through mtDNA-associated mitochondrial dysfunction. Intra- or
extracellular toxicants could induce overproductions of oxidative stress, dysfunction
of mitochondrial respiration, alterations of mitochondrial membrane potential, and
apoptosis through the downregulation of intracellular adenosine triphosphate activi-
ties and the down-activation of mitochondrial enzymes, e.g., mitochondrial respira-
tory enzymes, NADH dehydrogenase, cytochrome c oxidase, and oxidative
stress-sensitive aconitase. It is critical that toxins-induced compromises and fragmen-
tation of nDNA and mtDNA can deliver “wrong” messages to activate the apoptosis
signal pathways, e.g., apoptosis-initiator caspase-9 and apoptosis-effector caspase-3/
caspase-7. This is one of complex mechanisms to reach mtDNA-associated or mito-
chondria-dependent cytotoxicity and metabolic disorders. Song et al. at first time
describe potential mechanisms of mtDNA in telocytes and foresee the importance to

explore telocyte mitochondrial function and mtDNA regulations in this volume [20].
Pan et al. extend the discussion from mtDNA-regulated mitochondrial function
to mitochondria-dependent metabolism in cancer and mitochondria-mediated meta-
bolic regulation in cancer drug resistance [21]. This chapter systemically overviews
the historical development of mitochondrial metabolism and characteristic pheno-
types of mitochondria-dependent metabolism from various aspects, e.g., glycolysis
and the Warburg effect, pentose phosphate pathway, glutamine reductive carboxyl-
ation lipid, and amino acid metabolism. Pan et al. combine their own with published
knowledge and experience to propose a new therapeutic strategy for different cate-
gories of cancer on the basis of the principle of mitochondrial metabolism. Firstly,
it is critical to select precision methodologies to carry out the personalized diagno-
sis tools by measuring mutations of nDNA and mtDNA, mass of mtDNA and mito-
chondria, quality of mitochondrial OXPHOS, or levels of mitochondrial metabolites
and RO. Secondly, novel therapeutics have been developed and optimized, e.g.,
drug repositioning, specific pathways of inhibitors/activators of genetic engineering
techniques, and mitochondria-targeting drug resistance. Finally, the mitochondria-
targeting delivery systems for cancer should be designed and validated to meet the
clinical needs, including targeting peptides, lipophilic cations, nanoparticles, nano-
vesicles, or nano-liposomes. The chapter clearly points out the future development
of mitochondria-based therapies and explore the potential of clinical application.
Lu emphasizes the importance of mitochondrial ATP-dependent Lon protease on
the pathogenesis of diseases, especially in cancer, as the potential biomarker for
cancer diagnosis and novel target for drug discovery and development [22]. nDNA
can deliver the messages for the encoding ATP-dependent Lon proteases in the
mitochondria in various cells, which are the microenvironment for evolution and
quality control of proteins by the signaling pathways and mediating factors. The
balance of mitochondrial genome (mtDNA) and folding of mitochondria proteins
can be maintained by the selective degradation of misfolded, oxidized, and short-
lived regulatory proteins within mitochondria. A large number of intra- or extracel-
lular factors, e.g., ROS, inflammatory mediators, or kinases, can influence the
expressions and activities of ATP-dependent Lon proteases, leading mtDNA muta-
tions, mitochondrial dysfunction, or cell injury. Disordered activities of ATP-
dependent Lon proteases also change the sensitivity and susceptibility of cells to
drugs and increase drug resistance and tolerance [23–27]. Mitochondrial ATP-
dependent Lon protease was suggested as a potential biomarker for cancer diagno-
sis and therapeutic target for drug discovery and development, even though the
mechanism by proteases that interact with mtDNA remains unclear.
5 hether mtDNA Mutations and Genetic Changes Make

W
the Sense
Zhang et al. describe that the mitochondria are sensitive to both endogenous and
exogenous factors and become structural alterations of mitochondria and mtDNA
[28]. One of the most important mechanisms is mtDNA repair pathways to maintain
6 H. Sun et al.
the mtDNA integrity and mitochondrial injury as well as the occurrence of diseases.
The base excision repair pathway may contribute to mtDNA mutations and genetic
changes and can be an alternative to treat related diseases. Potential mechanisms
include a large number of enzymes, e.g., DNA glycosylases, polymerase γ, ligase,
AP endonuclease, and mtDNA repair pathways, e.g., SP-BER, LP-BER, mtDNA
degradation, direct DNA repair, nucleotide excision repair, mismatch repair, DNA
strand break repair, or nonhomologous or microhomology-mediated end joining.
Zhang et al. also discuss about roles of mitochondrial ribosomes, as oxidative phos-
phorylation, in the production of ATP during the development of diseases [29]. A
particular example of epithelial mitochondria and mtDNA is illustrated to under-
stand the relationship among mtDNA, mitochondria, cell, organ, and diseases [30].
Gao et al. arise another important roles of mtDNA epigenetics, since there is a
little discussion on the methylation of mtDNA, and question whether mtDNA meth-
ylation occurs in biology and pathology [31]. More evidence suggests that mtDNA
methylation plays an important role in mitochondrial gene regulation and the devel-
opment of diseases and is affected by a large number of intra- or extracellular fac-
tors. mtDNA methylation is proposed as a useful biomarker of disease diagnosis,
although those studies on mtDNA methylation are just initial. There are great needs
to develop methodologies to detect mtDNA methylation-orientated signal pathways,
interaction, and communications among regions of mtDNA sequences and between
mtDNA and nDNA. It is also perspective to consider that mtDNA heterogeneity
epigenetics should be investigated by measuring single-cell DNA sequencing, com-
prehensive characterizations of mtDNA, and bidirectional effects between mtDNA
and 3D genome, instability, and gene editing. It would be more help to combine the
single-cell biology with CRIPRS to mtDNA function [32–35]. Wang and Fang over-
view the sensitivity and vulnerability of mitochondria to drugs and drug candidates
or compromised effects of drugs on mitochondrial function and provide the compre-
hensive understanding regarding the mechanisms underlying mitochondrial dys-
function to uncover therapeutic targets in multiple pathologies [36].
Acknowledgments The work was supported by Zhongshan Distinguished Professor Grant

(XDW), the National Nature Science Foundation of China (91230204, 81270099, 81320108001,
81270131, 81300010), the Shanghai Committee of Science and Technology (12JC1402200,
12431900207, 11410708600, 14431905100), Operation funding of Shanghai Institute of Clinical
Bioinformatics, Ministry of Education for Academic Special Science and Research Foundation for
PhD Education (20130071110043), and National Key Research and Development Program
(2016YFC0902400, 2017YFSF090207).
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drug resistance. Adv Exp Med Biol. 2017;1038:8
22. Lu B. Mitochondrial Lon protease and cancer. Adv Exp Med Biol. 2017;1038:9
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acquired tamoxifen resistance. Cell Biol Toxicol. 2016;32(6):563–81. PMID: 27585693
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Biol. 2017;1038:14
Weibin Shi, who graduated from Shanghai Jiao Tong University,

is now working as the supervising technologist and director of the
Health Control Center in Zhong Shan Hospital. Since joining
Zhong Shan Hospital in 1989, he has been working for nearly 30
years. And during the period, he has acquired Radiology
Technology Qualification (Intermediate). His publication record
includes three research papers in core journals. He is a member of
the committee of Shanghai Medical Association, Shanghai Health
Administration Association, and Chinese Medical Association
Shanghai branch.
Chapter 2
Mitochondrial DNA in Lung Cancer
Fangming Liu, David E. Sanin, and Xiangdong Wang
Abstract Mitochondrial DNA (mtDNA) variations are increasingly discovered

and expected to be potential biomarkers to monitor severity, duration, stage,
response to therapy, and prognosis in patients with lung cancer. The present article
illustrates alterations of mtDNA in lung cancer, including alterations of mtDNA
copy number and sequence mutations, as well as their possible mechanisms for
carcinogenesis and development of lung cancer. The clear and comprehensive rela-
tionships between mtDNA variations and lung cancer are to be further confirmed to
benefit effective strategies for lung cancer diagnosis and therapy.
Keywords Mitochondria • DNA • Mutation • Lung cancer • Diagnosis
1 Introduction
Lung cancer is an increasingly severe disease, accounting for 30% of all cancer-
related deaths [1], and has the highest occurrence and mortality among malignan-
cies. This will continue to increase if there are still no effective measures and
therapies against lung cancer. Although the mechanism of lung cancer carcinogen-
esis remains unclear, tumor development is associated with mitochondrial DNA
(mtDNA) mutations and alterations in mitochondrial genomic function [2, 3].
mtDNA mutations are found in various kinds of tumors, such as lung cancer [1, 4],
bladder cancer [4], breast cancer [5], thyroid cancer [6], and prostate cancer [7],
since mtDNA homogeneity mutation has been identified in colorectal cancer [8].
F. Liu
Zhongshan Hospital Clinical Science Institute, Fudan University, Shanghai, China
D.E. Sanin
Department of Immunometabolism, Max Planck Institute of Immunobiology
and Epigenetics, Freiburg im Breisgau, Germany
X. Wang (*)
Zhongshan Hospital Institute of Clinical Science, Fudan University,
Shanghai Medical College, Shanghai, China
© Springer Nature Singapore Pte Ltd. 2017 9

H. Sun, X. Wang (eds.), Mitochondrial DNA and Diseases, Advances in
Experimental Medicine and Biology 1038,
https://doi.org/10.1007/978-981-10-6674-0_2
10 F. Liu et al.
Characterizations of mtDNA genes vary among lung cancer subtypes, stages, and
severity [9, 10]. The present article aims at overviewing the understanding of
mtDNA biology, the significance of mtDNA mutations and altered copy numbers in
lung cancer, the potential of mtDNA-associated biomarkers, and the comparison
between mtDNA and nDNA biomarkers.
2 mtDNA Biology
mtDNAs are double-stranded closed circular DNA with 16,596 base pairs, composed
of coding regions without introns and noncoding region. The coding region contains
13 polypeptides, 22 transfer RNAs, and 2 ribosomal RNAs [11]. The noncoding
region (also called D-loop region) is responsible for mtDNA replication and tran-
scription [12, 13], which has significant regulatory elements, such as L-strand pro-
moter, H-strand promoter, and H-strand replication origin. In coding regions, almost
every base plays a role in gene assembly [14]. As a result, once mutations occur,
DNA sequence will probably be altered and followed by the alteration of coding
proteins and mitochondrial dysfunction, as an important cause of human pathology.
Compared with nuclear DNA (nDNA), mtDNAs have higher mutation rates, and
there are hundreds of thousands of mtDNA copies per cell. Mitochondrion is a
major source to generate adenosine triphosphate (ATP) for cells through oxidative
phosphorylation [15], during which reactive oxygen species (ROS) are generated as
by-products and lead to DNA damage. mtDNAs are in close physical proximity to
the electron transport chain and superoxide anions generated in oxidative phos-
phorylation and easier to acquire oxidative damage [11] (Fig. 2.1). mtDNAs contain
Fig. 2.1 An illustration of mtDNA distribution in mitochondria. mtDNAs are double-stranded

closed circular DNA; they are in close physical proximity to the electron transport chain and reac-
tive oxygen species produced in oxidative phosphorylation. It’s more likely for mtDNAs to acquire
oxidative damage
2 Mitochondrial DNA in Lung Cancer 11
naked and lacked protective histones, more susceptible to the damage of carcino-
gens. Once mtDNA is compromised, mtDNA is hardly self-repaired due to the lack
of DNA repair system and excision repair capacity in the mitochondria [14]. There
are a large number of intra- and extracellular factors to contribute to the DNA dam-
age of base pair mismatch and high mutation rates of mtDNAs, responsible for the
development of mitochondrial dysfunction and cell susceptibility [15].
High mutations and copies of mtDNA as distinct features are involved in the
carcinogenesis and are expected to be potential biomarkers for cancer detection and
monitoring. The frequency of sequence mutations may increase during mtDNA
copying and sequencing. The higher the number of mtDNA copies, the more sus-
ceptible mtDNAs are for mutation. The small size and closed circular structure of
mtDNAs are more resistant to the DNA damages [11]. The appearance of the same
mutation in mtDNAs and tumors indicates the clear relationship between [4], since
it is questioned whether the gene mutation is from mtDNA to tumor tissue or
reversely, genetically regulated from the source, and represent the targeting similar-
ity between. It seems clear that mtDNAs can impact tumor cell growth and sensitiv-
ity to treatments [4, 16, 17].
3 The Significance of mtDNA Mutations
mtDNA may undergo mutations, e.g., insertion, deletion, or point mutations, in

response to carcinogenetic factors [18, 19]. The mutation rate of mtDNA is much
higher than that of nDNA due to its sensitivity to cancerogens. There are an amount
of mutations in mitochondrial D-loop in lung cancer [19, 20]. mtDNA noncoding
region is important in the transcription and duplication of mtDNA. D-loop is
1124 bp in size and a replicate origin where heavy and light chains of mtDNA are
located [18]. There are two high-variable regions HVI and HVII located between
16,024–16,383 and 57–372 in D-loop, respectively. Alterations of D-loop region are
possibly associated with mitochondrial dysfunction and mutation of nuclear
genome. Single nucleotide polymorphisms of minor alleles of nucleotides 235A/G
and 324A/G in D-loop region can be a risk factor of lung cancers. Of single nucleo-
tide polymorphisms, the minor alleles 151C/T, 200A/G, 524C/CA, and 16274G/A
are associated with the induction of squamous cell carcinoma, while the mutation of
the minor allele 16,298 T/C may trigger the occurrence of small cell lung cancer [1].
In another sequencing of mtDNA, 8 of 27 primary lung tumors had mutations in the
D-loop region, including a C-to-G transversion in nucleotide 16,114 and deletions/
insertions in polyinosinic acid-polycytidylic acid [19]. In addition, 12 SCLC and 16
NSCLC cell lines of lung tumors have high frequency of homopolymeric C tract or
single nucleotide polymorphisms in the D-loop region [18].
Mutations in mtDNA coding region are correlated with clinical characteristics in
patients with lung cancer. Of those,36 mutations were detected in mtDNA coding
region and 19 missense mtDNA mutations in 9 mtDNA coding region, including
COI, COII, COIII, ATPase 6, ND2, ND3, ND4, ND5, and Cyt b [3]. These alterations
of mtDNA may play an important role in tumorigenesis and metastasis of lung can-
12 F. Liu et al.
Fig. 2.2 Mutations in

different regions of
mtDNA. mtDNAs are
composed of 16 coding
and noncoding regions
without introns. Several
mutations of mtDNA in
lung cancer were showed
in this figure (insertion,
deletion, or point
mutations), according to
the literature. The
noncoding region D-loop is
the “hot spot” of mutations
cer, as shown in Fig. 2.2. Mitochondrial DNA is vulnerable to alterations. When a

mutation takes place, the altered mtDNAs can coexist with wild-type mtDNA in a
heteroplasmy state [21, 22] . During cell replication, mtDNAs were divided into
daughter cells randomly, and the percentage of variant mtDNAs in daughter cells is
uncertain. After lots of passages of those heteroplasmic cells, the wild-type or muta-
tion mtDNAs will transform into a predominant rate called homoplasmy. The het-
eroplasmic mutations result in highly variable inheritance and biological impact
[22] due to the random distribution of genetic materials (Fig. 2.3). Thus, there are
still several challenges to identify the association between mtDNA mutations and
clinical phenotypes.
mtDNA mutations may contribute to carcinogenesis by decreasing cellular
energy capacities, although the exact mechanism remains unclear. The mutation rate
of mtDNA at a certain proportion can reduce the cellular energy capacity and cause
the lack of cell and tissue bioenergetics to maintain normal functions. Or it is pos-
sible that tumors are induced by increasing mitochondrial oxidative stress [23]. The
mitochondria are the primary source of endogenous ROS, including unpaired elec-
trons and oxygen free radical. mtDNA encodes 13 polypeptides involved in electron
transport chain, vulnerable to mutagens due to the lack of introns and protective
histones. It is also possible that mtDNA mutations altered proteins and the function
of respiratory chain is damaged [24]. The increased amount of electrons could result
in more free radical production and interact with genome as tumor initiators, muta-
genizing proto-oncogenes into oncogenes [25]. mtDNA mutations induce carci-
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Fig. 2.3 The random genetic mutations of mtDNA. The altered mtDNAs can coexist with wild-
type mtDNA in a heteroplasmy state. During cell replication, mtDNAs were divided into daughter
cells randomly. After lots of passages of those heteroplasmic cells, the wild-type or mutation mtD-
NAs will transform into a predominant rate called homoplasmy that causes highly variable inheri-
tance and biological impact
noma through modulating apoptosis controlled by electrochemical gradients. The

reaction through mitochondrial electron transport chain can reduce electrochemical
gradients, opening the mitochondrial permeability transition pore [22] and releasing
cell death-promoting factors to destruct cytoplasm and chromatin. In cancer cells
with mtDNA mutations, the electrochemical gradients increase more than in wild-
type cells [26], possibly making carcinoma cells resistant to apoptosis (Fig. 2.4).
4 Altered Copy Number of mtDNAs
Each human cell contains an average of 100–500 mitochondria of which each has
two to ten copies of mtDNAs [27]. The normal combination and operation of the
respiratory chain require a complete and functional mitochondrial genome. mtDNA
function depends not only on the integrity of mtDNAs’ molecular structure but also
on the copy number of mtDNAs in the cell. In addition to somatic mtDNA muta-
tions including point mutation, deletion, and insertion, alterations of mtDNA copy
number vary among human cancers. For instance, the copy number of mtDNAs
14 F. Liu et al.
Fig. 2.4 Hypotheses about mtDNA mutations contributing to carcinogenesis. Proteins of electron
transport chain are altered by mtDNA mutation, and the function of respiratory chain is damaged.
The increased amount of electrons could result in more free radical production. In addition, mito-
chondria control cell apoptosis by electrochemical gradient (△Ψ), produced by mitochondrial
electron transport chain. Loss of △Ψ results in opening of the mitochondrial permeability transi-
tion pore (mtPTP). In cancer cells with mtDNA mutations, the △Ψ may increase, and mtPTP is
closed; thus, the release of cell death-promoting factors was held back. By this way, mtDNA muta-
tions possibly make carcinoma cells resistant to apoptosis
increased in prostate cancer cells [28] or in saliva of head and neck cancer [29] or
serum levels of cell-free mtDNAs in testicular cancer [30]. Those data show that
mtDNAs are altered not only in cancer cells but also in other body fluids. On the
other hand, the contents of mtDNA reduced in 82% of cancerous breast tissues [31]
and in hepatocellular carcinoma patients associated with poorer prognosis and
shorter 5-year overall survival rates [32]. It indicates that the regulation of mtDNA
copy number is out of control, with cancer site specificity [2, 33].
Alterations of mtDNA content also exist in patients with lung cancer. Lee et al.
[34] found increased mtDNA content in about 50% of patients with lung cancers,
while decreased mtDNA content was found in about 23%. Multiple factors influ-
ence alterations of mtDNA content, e.g., cancer type, DNA damage type, or sample
size. The reduction of mtDNA copy number seems more common in lung cancer.
For example, the copy number of mtDNAs is reduced in lung carcinoma tissues [35]
and in advanced stages of the disease correlated with a shorter survival time [36].
There was no correlation between mtDNA content and gender, age, or smoking
status [11] [36, 37]. Lee et al. reported that mtDNA contents initially increased with
pack-years of smoking and then declined to levels below nonsmokers [38].
Different changes of mtDNA content in cancer may be caused by diverse mecha-

nisms. D-loop region has regulatory elements containing promoters and replication
origin and controls mtDNA replication and transcription. Once mutations occurred
in D-loop region especially in regulatory elements, the replication and transcription
rate of mtDNAs would be changed. The mutation in D-loop region was about 40%
hepatocellular cancer, while decreased mtDNA content was more than 70% cancer
with D-loop region mutation [34]. It is possible that the mutation in D-loop region
decreases mtDNA content or a point mutation in coding region reduces mtDNA
copy number. The introduction of mtDNA with mutant A3243G into mtDNA-
deficient teratocarcinoma cells results in the depletion of mtDNA content [39].
Another possibility is that mtDNA contents are altered through ROS-mediated
pathway. High levels of ROS would lead to mtDNA damage (oxidative damage),
and 8-hydroxy-2-deoxyguanosine (8-oxo-G) as an indicator of oxidative damage
increased in lung tissues of heavy smokers [27]. The 8-oxoguanine-DNA glycosyl-
ase 1 protein and polymerase γ are two key enzymes to repair 8-oxo-G damage in
mtDNAs [40, 41]. Devitalized 8-oxoguanine-DNA glycosylase 1 and deficient
polymerase γ lead to accumulation of mtDNA mutations responsible for changes of
mtDNA content. The deficiency of repair system probably is responsible for the
decrease of mtDNA content in ROS-mediated damage. ROS-mediated damage can
also be a compensatory mechanism by which the process of mtDNA content to copy
increases an oxidative stress [27]. When it comes to oxidative damage, the combina-
tion of mtDNA mutations and compensatory mechanism should be considered to
estimate the changes of mtDNA copy number (Fig. 2.5).
Fig. 2.5 Possible mechanisms of altered copy number of mtDNAs. When mutations occurred in
regulatory elements in D-loop region, the replication and transcription rate of mtDNAs would be
changed, decreasing mtDNA copy number. Another possibility is that high levels of ROS would
lead to production of 8-hydroxy-2-deoxyguanosine (8-oxo-G) as an indicator of oxidative damage.
Meanwhile, devitalized 8-oxoguanine-DNA glycosylase 1 and deficient polymerase γ lead to a
failure of repairing 8-oxo-G damage in mtDNAs, which is responsible for changes of mtDNA
content. ROS-mediated damage can also be a compensatory mechanism to estimate changes of
mtDNA copy number
16 F. Liu et al.
Loss of p53 protein is another reason to reduce mtDNA content through increased
accumulation of mtDNA damage [42], since p53 protein regulates the response to
DNA damage and can maintain the stability of mtDNAs [43]. In coping with
mtDNA damage, p53 protein can move to mitochondria and interact with poly-
merase γ to strengthen the expression of polymerase γ to repair oxidative damage
[42]. The alteration of mtDNA copy number in lung cancer is subject to the mtDNA
mutations and oxidative damage. Although there are several causes to interpret the
changes of mtDNA content, a clear and comprehensive explanation for increased or
decreased mtDNA content of each case remains unknown. More research is required
to determine the exact relationship between mtDNA content and lung cancer
development.
5 otential Value of mtDNA as Biomarkers for Lung Cancer

P
Diagnosis
Although nuclear genome detection plays a significant role in cancer screening, the
biological role of mtDNA alteration in tumor formation has drawn more and more
attention in recent years. The alteration of mtDNA content or sequence mutations
has evitable relationship with oncogenesis and becomes potential biomarkers for
certain types of cancers [4, 44]. It is widely accepted that mtDNA is more vulnera-
ble to mutagens by genotoxin, oxidative press, or other factors compared with
nuclear genome [45, 46]. mtDNA has potential implications for lung cancer diagno-
sis, since mtDNA is easier to be detected and has a high copy number or mtDNA
mutations have strong resistance to damage. The 16,596 base pairs make up 16 gene
regions, arranging closely on the double-strand loop, which should be easier to be
characterized with current advances of methodologies. There are several thousand
copies of mtDNA in a cell [47], as compared to two copies of nuclear genome. Less
samples were required for DNA extraction to evaluate mtDNA. For precious clini-
cal samples, mtDNA content is usually enough for sequencing and other detection
methods. On the other hand, mtDNA sequence alterations are more stable and can
be detected with high repeatability.
Abnormal expression of mtDNA encoding proteins can also cause mitochondrial
dysfunction and cell damage, due to the lack of introns and accumulation of muta-
tions in coding regions. Changes of downstream proteins and clinical phenotypes
can also imply the corresponding mtDNA mutations. Due to the high heterogeneity
of mtDNA mutations, it is a challenge to identify lung cancer in an early stage.
Sanger sequencing is a common type of mitochondrial sequencing technique, but as
to heterogeneity detection, the next generation of sequencing will be more competi-
tive. In addition, there are quantitative PCR techniques, array chips [48], and other
measurement techniques, which can also achieve good detection results.
It is necessary for further study to identify and validate the function of mtDNA
mutations in initiation and progression of lung cancer in order to be helpful to
develop lung cancer detection and treatment strategies. It is also a challenge to
Fig. 2.6 Potential value of mtDNA as biomarkers. mtDNA has a prospect of becoming biomark-
ers to develop lung cancer detection and treatment strategies, to monitor disease severity and stage,
to monitor the transit of chronic diseases especially chronic obstructive pulmonary disease to lung
cancer, and to describe alterations of cell functions induced by mtDNA mutations. In addition, it
will be a power if mtDNA alterations can act as a biomarker to measure the cell behavior in drug
pharmacology
apply mtDNA changes for clinical application as biomarkers, especially to monitor

the transit of chronic obstructive pulmonary disease to lung cancer [49–51], to inte-
grate clinical phenotype mtDNA mutations for dynamic monitoring of disease
severity and stage [52–54], and to describe alterations of cell functions induced by
mtDNA mutations [55–58]. Mitochondrion is one of the most important organelles
to regulate and control drug pharmacological profiles. It will be a power if mtDNA
alterations can act as a biomarker to measure the cell behavior in drug metabolism
[59–61], pharmacokinetics [62–64], and sensitivity to drug toxicity [65–68]
(Fig. 2.6).
6 Conclusion
Alterations of mtDNA possibly are related to carcinogenesis of lung cancer, as well

as their mechanisms for promoting the development of lung cancer, including alter-
ation of mtDNA copy number and sequence mutations. Although the definite mech-
anism by which mtDNA is altered is to be further confirmed, it will be helpful in
developing diagnostic and therapeutic strategies for lung cancer.
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selityksen: emme voineet, kun oli niin kuuma, Musta oli laiska ja me
väsyneitä…
Ja hyvällä omallatunnolla me makasimme, haistelimme kanervan

kukkia ja söimme katajanmarjoja.
Vaan sitten. Eräällä kertaa, kun me kahden miehen (poikasia me

sentään olimmekin) astelimme hiekkakuorman perässä ja Musta
melkein joka askeleella seisahtui, päällään huitastakseen
elementissään surisevia paarmoja kupeiltaan, tuli "Kapteeni"
vastaamme ratsastaen Valkosella ratsullaan, itsellään päällään
valkonen takki, — koko mies tavallisuuden mukaan jäykkänä,
ylpeänä ja tuimana. Kun hänet huomasimme, yritimme reipastua,
suoristaa velttoja ruumiitamme ja yleensä näyttäytyä miehullisilta.
Kun hän saapui lähelle, nostimme säädyllisesti lakkia. Hän sanoi:
"Kenenkä sianpaimenia te olette?"
Jaska katsoi minuun, minä Jaskaan, suumme vääntyivät hymyyn

ja — vastaukseksi huitasin raipalla Mustaa lanteille. Vaan Valkonen
pelästyi, hypähti syrjään, kapteeni kirosi ja hankkiusi alas astumaan
meille selkään antaaksensa. Me emme ruvenneet odottelemaan,
vaan varustausimme suuren ojan ja aidan yli nevalle ja tunsimme
jaloissamme erinomaista kepeyttä. Hän todellakin astui alas
hevosensa selästä, mutta meitä jo alkoi naurattaa, sillä neva oli
meidän tietämme. Hänen närilleen nähtävästi kävivät asianhaarat,
sillä hän kiroili yhtämyötään, kuten jylhä tapansa oli todessa ja
leikissä. Minä olin luonnoltani pelkurimpi kuin Jaska ja minun täytyy
tunnustaa, että hiukan hätkähdin, kun Jaska tempasi
seipäänkappaleen, täräytti sillä aitaan ja huusi:
"No tule koettamaan!"
Tätä Kapteenia pidettiin yleensä koiramaisena herrana, kaikki

vihasivat häntä ja hän vihasi muita. Ja miten ollakaan: yleinen
mielipide tempasi minutkin helmaansa aivan huomaamattani, monta
kärsittyä haukkumista ja pakoretkeä vilahti mielessäni: koettelin,
oliko puukko tupessani tallella. Vaan asia ei jatkunut aijottuun
suuntaan, hän näetten, nousi rähisten ja uhkaillen ratsulleen ja ajoi
pois.
Se nyt oli aivan varmaa, että meidän aikomuksemme oli alusta

loppuun pääasiassa taistella säärillämme, sillä tuo uhka oli vaan
pieni välinäytös. Mutta nyt me aloimme, aluksi leikillä, kerskata, että
hän oli pelännyt, kunnes jo uskoimme, että niin todellakin oli
tapahtunut. Eikä meitä kuumuuskaan enää aivan paljon rasittanut.
*****
Alkoi jyrähdellä ukkonen, taivaalle ilmestyi mustanpuhuvia

pilvenlonkareita, lyhyet, vinhakat tuulenpuuskaukset lennättivät
ilmaan kytömultaa, tien tomua ja muuta törkyä. Meille tuli kiire. Vielä
kaksi kuormaa ja sitten kotiin. Mustakin oli virkistynyt, — olimme
kiireissämme yksistä neuvoin.
Vaan viimeistä kuormaa viedessämme leimahti ja jyrähti ukkonen

hirvittävästi. Musta lyyhistyi, minun käteni kohosivat vaistomaisesti
taivasta kohti ja Jaska oli valkoinen kuin päivänpolttama luu. Nyt
alkoivat salamat leimahdella aivan katkeamatta, jyrinä jatkui
loppumatta ja raekuuro, kivisateen tapainen, syöksi alas.
Lähellä oli lato, jossa oli vähän heiniä. Me aloimme kiidättää sinne,
minkä kintuista lähti.
"Mutta Musta?" älähdin minä Jaskalle, joka edellä juoksi. Ja me

palasimme, laskimme hevosen valjaista ja veimme mukanamme
latoon. Se turvausi meihin kuin lapsi. Ja vaikka salamat leimusivat,
taivas jyrisi ja rakeet pieksivät pelottavalla kopinalla ladon seiniä ja
sinkoilivat ympärillämme, emme saattaneet olla Mustan päätä
syleilemättä ja turpaa suutelematta… Meitä oli siinä kolme
ajattelevaa olentoa, jotka yhteinen vaara ja pelko liitti lähemmäksi
toisiamme kuin milloinkaan ennen. Minua niin lämmitti Mustan
avuton ja pelokas katse.
Ladossa oli hiukan heiniä. Aloimme kaivaa sinne koloja

itsellemme, että olisi mihin peittäytyä, kun rakeet alkoivat yhä tulla
suurempina ja sinkoilivat seinien hataroista raoista meihin
koskettaen kipeästi.
Yht'äkkiä ilmestyi Kapteeni Valkosineen ladon ovelle. Samassa

leimusivat tuliset kielet ja valaisivat tumman ilman kamalalla
hehkuvalolla. Jyräys, joka tuntui maan repeämältä, seurasi perässä
pitkänä, venyvänä, hirmuisena. Me parkasimme kauhusta, Musta
lyyhistyi melkein maahan, kapteenin Valkonen hypähti vapisten
syrjään…
"Tpruu! — Älkää pelätkö lapset." Ääni millä tuo lausuttiin,

kummastutti meitä ja rauhoittikin. Hän koetti saada Valkosta latoon,
vaan se ei tullut. Täytyi johdattaa suojan puolelle latoa ja sitoa kiinni
seinään.
Hän oli kauheasti märkänä ja vakavana, ei kiroillut ollenkaan, —

niin kuin olin kuullut hänen tekevän ukonilmoilla.
Ja miten olikaan. Viiden minuutin kuluttua makasimme heinissä

kylki kylkeä vasten, kaikin: Kapteeni, Jaska ja minä. Omituinen,
selittämätön, turvallisuutta etsivä tunne veti meitä toisiimme. Riidasta
ei virketty sanaakaan. Kapteeni oli tavallisissa oloissa juro, nyt hän
meille jutteli lämmöllä, melkeinpä innostuneena. Minusta tuntui, että
hän piti meitä jonkullaisina tovereina tuossa yhteisessä rajuilman
pakopaikassa. Luulenpa vielä, ettei otaksumani ollut edes erehdys.
Rajuilman loputtua lähdimme ja hän sanoi meille hyvästit aivan

kuin kaltaisillensa.
Me emme kertoneetkaan enää tappeluyritystä siinä valossa kuin

olimme aikoneet. Me emme siitä edes puhuneet mitään. Vaan
toistemme mieliala selvisi siitä, että toinen sanoi ensin:
"Hän on oikein sopiva mies." Toinen siihen innokkaasti yhtyi ja
sitten me yhdessä ylistimme häntä, ystäväämme, jolla oli niin paljon
vihamiehiä.
Monta viikkoa pysyi tämä ystävyys mielessämme elämämme

suurimpana muistona. Vaan sitten se haihtui, kun ei enää satuttu
rajuilmalla milloinkaan yhteiseen turvapaikkaan.
Ja syksynpuolella puhutteli hän meitä uudelleen sianpaimeniksi,

pelotteli ja kiusasi samoin kuin muitakin poikia. Hyvät muistot
hälvenivät meistä, me liityimme muihin, jotka hänelle kiusaa tekivät.
Hän oli taas yksin koko kylässä, kaikki pitivät häntä ilkeänä, raakana
kylän herrana.
Kylässä ei ollut ensinkään muita herroja, harvoin niitä kävikin, hän

edusti niitä kaikkia alusta loppuun.
Ja kyläläiset vihasivat vaistomaisesti kaikkia herroja Kapteenin

persoonassa.
Mutta minun mielestäni ei ole milloinkaan täysin haihtunut

"ystävyytemme" rajuilman aikana. Olen vasta jälestäpäin käsittänyt,
että se oli hänen luonnollinen, pilaantumaton itsensä, joka silloin,
rajuilman aikana pilkisti esiin kuin auringon säde, kätkeytyäkseen
taasen, kun piti esiintyä "kylänherrana," mökkiläisten ja talollisten
kauhuna… Kuinkas muuten, vanha sotaherra!
Aasi ja hevonen.
Satu.
Karhu kerran katseli hevosen virmaa, kaunista juoksua, ravisti
mietteissään päätänsä ja virkkoi:
"Ei se kelpaa, että kotieläimet tuolla tavalla juoksevat, voi mennä

jalka poikki, pää halki, tahi saattaisi joku hidasliikkeisempi elukka
jalkoihinkin joutua. Ja sitäpaitsi on se liian nopeata eteenpäin
menoa."
"Setä, pane hänelle aasi holhoojaksi", kehoitti kettu, joka

tavallisuuden mukaan oli karhulla adjutanttina.
"Se saattaisi olla tarpeeksi vakavaluontoinen siihen virkaan."
"Olisipa hyvinkin", kehui kettu, jonka kieli roikkui suusta lerpallaan

siinä kun se syrjittäin, lurjusmaisesti katseli setänsä totista naamaa.
Karhu tuijotti nyrpeän näköisenä suoraan eteensä ja virkkoi:
"En viitsi sitä asiaa niin paljon ajatella. Se nyt on vaan estettävä
tuo hevosen tavaton juoksu ja kopea käytös. Sellaista en minä
kärsi!"
"Saanko minä toimittaa asian, setä?"
"Saat."
Kettu juoksi aasin luokse:
"Tomppeli!" huusi tälle korvaan.
Aasi kohotti hiukan, hyvin laiskasti päätänsä rehukasasta, suu

olkia täynnä.
"Lurjus!" toisti kettu. Aasi muljautti silmiään, laski pitkät korvansa,
jos mahdollista, vielä enemmän lerpalleen ja jatkoi syömistään. Kettu
purasi sitä vähän takakoipeen koetellakseen arastaako tuo. Veti se
pois säärensä maltillisesti, ilman potkimatta.
Kettu vetäysi sivulle, purskahti nauramaan ja tuumi itsekseen:
"Mitkä verrattomat tomppelin-lahjat! Siihen virkaan erinomainen

kuoma."
Aasi kohotti korviaan 45° asteen kulmaan:
"Mihinkä virkaan?" kysyi.
"Ähäh", ilkkui kettu. "Kyllä kuulee sillä korvalla. Oikein kelpo

aines!"
"Mikä aines?" kyseli aasi, jonka uteliaisuus oli herännyt.
Kettu ei viitsinyt edes vastata, se kun niin sydämestään halveksi

tuota ainesta. Itsekseen vaan jupisi:
"Tuollainen nälkiintynyt tomppeli, jolla ei ole mitään kunnianhimoa,

ei mitään muita vaatimuksia kuin vatsan… Sitä saa haukkua, pilkata,
jopa hutkiakin ilman että suuttuu… Kerrassa oiva kätyri!"
Ja kettu teki aasista hevosen juoksun tarkastajan; tämä otti viran

iloiten vastaan.
Miten aasi virkaansa toimitti, ei enää kuulu tämän kertomuksen

piiriin. Vaan lopputulos on ollut se, että nykyisellä hetkellä ei enään
koko avarassa maailmassa liene yhtään aasia, joka vielä olisi
virassaan, hevosten juoksun tarkastajana. He, näettekös, eivät
kyenneet estämään hevosten juoksua. Päät pystössä, kaulat
sankarillisesti kaarella, jouhet upeasti tuulessa heiluen, — sillä lailla
kulkevat nämät uljaat eläimet. Hevosen vauhti lisääntyy vuosi
vuodelta ja maine kasvaa. Sitä vastoin aasin maine keskittyy yhä
enempi — pitkin korviin.
Jesus ja sunnuntaimetsästäjä.
Eräänä sunnuntaipäivänä käveli Jesus vaatimattomaksi

suomalaiseksi talonpojaksi pukeutuneena muutamata Suomen
maantietä. Aurinko heloitti kuultavalta taivaslaelta ja paahtoi
maantien hiekan niin kuumaksi, että matkamiehen jalkoja poltti ja
kuumuus alkoi painostaa ruumista. Hän istahti maantien viereen
ruohikolle levähtämään. Hiljakkoin oli satanut, maantien vieressä
oleva metsä tuoksui hurmaten, laulurastaat, tiaiset, pajupiiparit,
närhit ja sirkut lauloivat metsässä tuhatsävelistä, loppumatonta
virttänsä. Maantien toisella puolella levisi avara timotee-niitty, jota
hauskasti ja elähyttävästi kirjavoi puna-apilan muhkeat, pehmoiset
kukkaset. Lukematon määrä hyttysiä ja metsäkärpäsiä hyräili ja
surisi matkamiehen pään ympärillä ja ruohikossa hänen vierellänsä
samoilivat ahkerat muurahaiset ristinrastin, osuivatpa joskus
kaahimaan paljaille jaloille, housuille ja takinhelmoillekin. Muuan
sirkka sirkutti etäämmällä, mutta aivan tuossa vieressä oli pari
toukkaa kiertynyt puolukkavarvun ympäri. Jesus katseli ja kuunteli.
Kaikkialla niin ihana, suloinen rauha. Hänen suunsa vetäysi
nauttivaan hymyyn, sitten pani hän kätensä ristiin, otti pois lakin
päästänsä ja — kiitti Isäänsä ihmisten puolesta. Hän oli juuri
lopettanut, kun metsästä alkoi kuulua jänistä ajavan metsäkoiran
ulina ja haukahteleminen. Kohtapa syöksikin jänes ohitse ja koira
perässä. Samassa kuului pamaus, — Jesus tunsi jalkansa puutuvan
samassa kuin jänes kiljahtaen otti pitkän loikkauksen ja tupertui
henkitoreissa mättäitten väliin. Hämmästyneenä tästä äkkinäisestä
tapauksesta, ei Jesus aluksi pannut mitään huomiota jalkaansa,
vaan tuijotti säälien jänestä, jota nyt takaa ajava koira raateli. Kohta
ilmestyi metsästä aukealle myöskin nuori metsästäjä sesettäen ja
miellytellen koiraansa. Mutta kun koira näytti jänestä repivän, huusi
metsästäjä täyttä kurkkua: "Anna perhanan elukka sen olla!" Jesus
säpsähti sitä ääntä. Nousi ja kävi ampujan luokse. Jalasta, johon
pyssystä oli useita hauleja lentänyt, vuosi verta.
"Hyvää päivää", tervehti Jesus ystävällisesti.
Sunnuntaimetsästäjä, joka paraillaan purkasi jäneksen sisälmyksiä

koiralleen, kääntyi, myöskin säpsähtäen, ja näki nyt edessään
köyhän puoleisesti puetun miehen, joka säälien katseli ei ainoastaan
jänestä vaan häntäkin. Tervehdykseen ei hän vastannut mitään.
Jesus aikoi istahtaa muutamalle kivelle siihen lähelle, mutta

metsästäjä, joka katsoi itsensä arvokkaammaksi tässä joukossa,
virkkoi käskijän äänellä: "Enhän minä ole pyytänyt sinua seuraani?"
Jesus punastui korvia myöten. Mitään vastaamatta lähti hän ontuen,
omaa tuskaansa valittamatta kävelemään pitkin tietä, johon
jokaiseen jälkeen jäi verinen pilkku…
Ylistysvirsi valon vastustajain kunniaksi.

Teidän ylistykseksenne, te valon vakaiset vastustajat, ei ole
luullakseni vielä koskaan kiitosvirsiä kirjoitettu, sillä kirjoitustaitoinen
maailma ei voi teitä ylistää syystä, että te vihaatte sitä. Mutta te
olette kuitenkin maailman pylväitä tänään ja vielä huomennakin, sillä
teidän käytettävinänne ovat kaikki väkivallan periaatteet ja sota-
aseet. Teidän mielestänne on yksilön persoona asetettava kaiken
yhteishyvän yläpuolelle, oma etu elämän korkeimmaksi
tarkoitusperäksi, käsivarren voima ja väkivallan kauhu kaiken
inhimillisen edistyksen johtavaksi periaatteeksi. Te siunaatte sitä
aikaa, jolloin kenenkään katse ei ulottunut oman kaalimaan laitoja
ulommaksi, jolloin orjat raatoivat palkatta teidän työnne ja te saitte
vaan laiskoina partojanne hoitaa ja "voida hyvin". Te nykyiset valon
vastustajat ette kyllä ole itse noita riemuaikoja nähneet, mutta te
uskotte niiden olleen onnellisia. Sillä kun kirjain oli ihme itse
isännillekin, kun kirjoittaa ei osanneet muut kun pappi ja ruununvouti,
— ei kukaan kirjoittanut sanomalehtiin eikä kukaan niitä lukenut.
Rauhassa saitte te silloin toteuttaa periaatteitanne elämän
korkeimmasta hyvästä. Te kokositte, niinkuin muinoin Joosef,
aittoihinne viljaa, riistaa ja rahaa pahaa päivän varalle. Mutta muiden
pahat päivät eivät teidän sydämiänne koske, sillä teillä ovat vaan
omat päivänne. Ja vaikka teidän ympärillänne sadottain
pitäjäläisiänne nääntyy nälkään ja viluun, vaikka kipu ja kärsimykset
kaataisivat kokonaisia kyliä, ja vaikka niiden sielut valon ja
opastuksen puutteessa kuihtuisivat ja näivettyisivät niiden irvikuvien
kaltaisiksi [tarkoitetaan noita inhoittavia kuvia, joita kaikkialla tapaa
piirreltyinä huoneitten seiniin], joita he piirtelevät seinillensä, — se ei
koske teidän sydämihinne. "Sillä kussa on teidän tavaranne, siellä
on myös teidän sydämenne." Mutta kun jossain joku nääntyvän
näyttää, te niitä perimään riennätte jo ennen kuin riutuva henki vielä
on ehtinyt kituvan kuoren heittää. Sillä teillä on melkein jokaisessa
kuolinpesässä saatavia ja velkakirjoja, joiden alkuperä on usein
hämärä, mutta lainvoima aina pätevä. Siten te lisäilette aarteitanne,
tulojanne ja talojanne, liittämällä omiin sarkoihinne kuolevain maita,
hävittäen kiinnekirjat ja perintöoikeudet laillisilta perillisiltä, joista
teette mieronkiertäjäjoukon, joilla ei ole kontua eikä "kotia mihin
päänsä kallistaisivat."
Jos sitten joku uskaltaa teidän toimianne moittia ja edes ihmetellä

teidän suuntanne armotonta tylyyttä, jos torpparien akat ja velallisten
lesket asiasta itkussa-silmin toistensa korviin kuiskuttelevat, te
hymyillen mainitte siinä kärsimyksestä, jota tekonne synnyttävät, sillä
teidän ilonne on ostettu köyhäin kärsimyksillä. Mutta kun parku liian
suureksi paisuu, kun sen soraäänet alkavat teidän korviinne tunkea
ja häiritä teidän nautintoanne, silloin te julmassa vihassanne
"panette kätenne akkain suun päälle". Silloin näiden huulet ajettuvat
ja kangistuvat, puhe ei enää suju suulla. Mutta he viittailevat ja
puhuvat käsillänsä. Ne viittaukset kyllä usein ovat ilkeämmät kuin se
sana jonka he sanoisivat, jos puhua saisivat, mutta te ette usko
viittausten levenevän eikä niitä kaikkein ymmärtävän.
Ja niin olette te valtanne kukkuloilla te valon väkevät vastustajat,

te luulette että maailmassa on rauha, että köyhälistön ja velallistenne
ja lampuotienne mökeissä kaikuvat ylistysvirret teidän viisaista
toimenpiteistänne ja kohoilevat ihastuneet ihmetyshuudahdukset
teidän rikkauksianne katsellessa.
Jos minulla olisi runoilijan taito ja osaisin vertauksilla puhua, niin

minä sanoisin, että teidän rikkautenne ja teidän valtanne
muodostavat suuren, pääsemättömän vuoren. Mutta jos minä olisin
korppi ja osaisin ennustaa, niin sanoisin, että sillä aikaa kuin
aarrevarastonne kasvaa, sen ilmattomassa sisustassa levenee
mätä. Te kapuatte yhä korkeammalle vuorenne huippua kohti sen
loistavaa pintaa, sen leveyttä ja korkeutta ihaellen, koskaan
tutkimatta sen sisuksia ja yrittämättäkään poistaa siellä levenevää
mätää. Ken lähelle uskaltaa tulla ja heikkoja puolianne paljastaa, sen
palkatut kätyrinne ryöstävät ja vievät vuoristonne sokkeloihin, johon
on monta menneitä, vaan harvoja takaisin palanneita. Ja siellä, siellä
vallitsee uni, nälkä ja ilman puute luonnonlain rajattomalla voimalla.
Mutta eräänä päivänä mätä puhkaisee reijän valtanne loistavaan

vuoreen, vapaa ilma tunkeutuu sinne sisään, uneksivat eläjät
aukovat silmiänsä, oikovat jäseniänsä ja kiinnittävät katseensa sinne
missä on reikä, josta ilmaa ja elämätä virtaa ja josta Jumalan vapaa
aurinko loistaa. Vuosituhansia vangittuina olleet voimat murtavat
itsensä vapaiksi ja te kukistutte kuin juurettomat puut. Te tulette
näkemään sen ikuisen keväimen, jolloin valon voimat murtavat kaikki
luonnottomat esteet, jolloin naisten kielet tulevat laulamaan
sorrettujen vapautuksen ihanoita ylistysvirsiä ja anteeksi-annon ja
veljeyden sointuvat sävel-luomat sulattavat teidänkin sydämenne ja
osoittavat teillekin oikean tien elämän ikuiseen onneen. Silloin on se
suuri huomen, jolloin väkivallan voima on vaan kauhea muisto ja
ihmiskunta on ottanut omiintuntoihinsa kirjoitetun Jumalan lain
ainoaksi ohjaajakseen, jolloin joukkojen murha on yhtä suuri synti
kun miestappokin, ja jolloin henkinen murha on suurempi rikos kuin
ruumiillinen. Silloin tekevät ihmiset maailman rauhan sen periaatteen
vuoksi, että murha on raaka synti ja että ketään ei haluta murhata.
Kukaan ei anasta keltään mitään, ei oikeuden varjolla enempää kuin
suoranaisella vääryydelläkään. Silloin on valo valtias maan. Silloin
hallitsevat ne voimat, jotka nyt ovat lapsina, joiden kasvamista kaikin
voimin koetetaan ehkäistä, että pysyisivät pieninä, eivätkä koskaan
kasvaisi suuriksi. Mutta samaten kuin Galilein maapallo "pyörii
kuitenkin", samaten kasvavat nämät vesat, sillä niitä kasvattavat
luonnonvoimat ja luomisen lait, ja niiden käyttäjänä on Jumala.
Sen pituinen on ylistysvirsi teidän kunniaksenne te valon nykyiset

ja vastaiset vastustajat, joiden joukko on niin suuri ja valta niin
mahtava ja vastustamaton.
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Cellular and Molecular Basis of Mitochondrial Inheritance

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Species conservation in managed habitats the myth of a

Growing Without Schooling The Complete Collection Volume 3

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1 How Far Can Mitochondrial DNA Drive the Disease?������������������������ 1

12 Mitochondrial Lon Protease and Cancer���������������������������������������������� 173

Hongzhi Sun, MD, PhD, is a famous surgical specialist

Xiangdong Wang, MD, PhD, is a distinguished pro-

Hongzhi Sun, Weibin Shi, and Xiangdong Wang

Keywords Mitochondria • mtDNA • Diseases • Energy • Metabolism

© Springer Nature Singapore Pte Ltd. 2017 1

1 Why We Focus on mtDNA

2  hy We Start with the Relationship Between mtDNA

3 How mtDNA Works in the Disease

It is easier to understand the importance of mtDNA in mitochondrial function, cel-

the signaling to the nuclear or mitochondria. It is possible or necessary for nDNA

4 Whether mtDNA Can Influence Cell Metabolism

describe potential mechanisms of mtDNA in telocytes and foresee the importance to

5  hether mtDNA Mutations and Genetic Changes Make

Acknowledgments The work was supported by Zhongshan Distinguished Professor Grant

Weibin Shi, who graduated from Shanghai Jiao Tong University,

Fangming Liu, David E. Sanin, and Xiangdong Wang

Abstract Mitochondrial DNA (mtDNA) variations are increasingly discovered

Keywords Mitochondria • DNA • Mutation • Lung cancer • Diagnosis

© Springer Nature Singapore Pte Ltd. 2017 9

Fig. 2.1 An illustration of mtDNA distribution in mitochondria. mtDNAs are double-stranded

3 The Significance of mtDNA Mutations

mtDNA may undergo mutations, e.g., insertion, deletion, or point mutations, in

Fig. 2.2 Mutations in

cer, as shown in Fig. 2.2. Mitochondrial DNA is vulnerable to alterations. When a

noma through modulating apoptosis controlled by electrochemical gradients. The

4 Altered Copy Number of mtDNAs

Different changes of mtDNA content in cancer may be caused by diverse mecha-

5  otential Value of mtDNA as Biomarkers for Lung Cancer

apply mtDNA changes for clinical application as biomarkers, especially to monitor

Alterations of mtDNA possibly are related to carcinogenesis of lung cancer, as well

Heillä oli kuitenkin ollut jonkunverran hauskaa. Eivät kiirehtineet

Ja kun oli tutkimuksensa perinpohjin suorittanut, istui hän

"Jos torppari haluaa torppansa lunastaa palstatilaksi, on hänellä

Kun kaikki oli valmiina, lähti kartanonomistaja kahden

Niin tuli tämä lähetystö myöskin Pekan mökille.

Kun toinen kartanonomistajan apulaisista oli Pekalle selittänyt

Kun kaikki oli selitetty, kysyi joku Pekalta:

Pekka astui kartanon omistajan eteen, kopeloi tämän käden

Vaimokin tuli kiittämään. Hänkin sanoi ymmärtäneensä. Vaan vielä

"Anna kättä kartanon herralle ja sano 'Jumala siunatkoon teitä'."

Poika peräytyi vierastaen, mutta isä ei hellittänyt. Herra läheni

"Annatko sinä kättä? Mikä poikanne nimi on?"

1 How Far Can Mitochondrial DNA Drive the Disease?�� 1

12 Mitochondrial Lon Protease and Cancer�� 173

1 Why We Focus on mtDNA

2 hy We Start with the Relationship Between mtDNA

3 How mtDNA Works in the Disease

4 Whether mtDNA Can Influence Cell Metabolism

5 hether mtDNA Mutations and Genetic Changes Make

3 The Significance of mtDNA Mutations

4 Altered Copy Number of mtDNAs

5 otential Value of mtDNA as Biomarkers for Lung Cancer