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Amy Hartman

Dr. Bert Ely

Biol 303

1 November 2012

Effect of Mitochondrial DNA Mutations on Tumorigenesis Colorectal Cancer and Barrett’s Metaplasia

As humans start leading longer lives, scientists and doctors are faced with a new problem:

addressing the multitude of debilitating diseases that develop over multiple years. The studies discussed

in this paper focused on two of these diseases: colorectal cancer and Barrett’s Esophagus. Colorectal

cancer, or colon cancer, targets over a hundred thousand people every year in the United States alone;

it is affected by many different factors, including age, genetics (family history), and lifestyle. This

particular cancer is the third most diagnosed cancer and the second leading cause of cancer-related

death in the United States. Like most types of cancer, there are distinctions between the different forms

of the disease. Early on, benign polyps form along the digestive tract (generally along the descending

portion, or the sigmoid colon.) The transition between the early stages to the advanced stages can take

up to ten years. As the transition occurs, these polyps gradually morph into malignant tumors and these

tumors start to spread to neighboring healthy tissue, infecting and destroying other parts of the body. At

this point, the prognosis for the patient becomes very grim, resulting in over fifty thousand deaths in the

United States every year (PubMed Health 2012). Diseases like colon cancer that present such a risk to

many individuals often warrant further study. Another example meriting further study is Barrett’s

metaplasia. Barrett’s metaplasia, or Barrett’s esophagus, is a disease where the cells lining the

esophagus are mutated into cells that are more like those lining the intestine. (This is where the term

“Barrett’s metaplasia” comes from; metaplasia describes when one mature cell type is replaced by

another.) Normally, Barrett’s metaplasia is caused by gastroesphageal reflux disease (GERD), which can

be described as chronic acid reflux. Barrett’s metaplasia patients are at higher risk of adenocarcinoma, a

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cancer of the esophagus (Anand and Weinstein 2012). In order to prevent the development of such

debilitating diseases, scientists are looking into several different types of genetic causes for affected

tissue for diseases like colorectal cancer and Barrett’s Metaplasia. Both of the experiments pursue a

certain type of genetic mutation, not in the nuclear DNA, but in the mitochondrial DNA.

Ask any eager freshman biology student the importance of the mitochondria and he or she will

automatically describe how these organelles provide almost all of the cell’s energy. However, these

students may not immediately consider the major role the mitochondria’s genome. Inside the

mitochondria, as ATP Synthesis occurs, a potentially harmful by-product is produced: reactive oxygen

species (ROS), or oxygen-containing chemicals with an unpaired electron. ROS is especially prevalent in

“chronic inflammatory tissue.” Why exactly is the presence of ROS such an issue that it warrants the

interest of many research teams? The mitochondrial DNA (mtDNA), which exists separately from the

nuclear DNA, is a target of ROS. ROS overproduction can hinder the function of mtDNA and cause

several types of genetic mutations including large deletions, minisatellite instability, and changes in copy

number (Ishikawa et al 2008). (Unlike nuclear DNA, mtDNA lacks many of the repair mechanisms that

protect against mutations.) Large deletions occur when thousands of nucleotides are erased from the

genome during replication. One of the most common large deletions in mtDNA is that of 4,977 base

pairs (bp); this large deletion includes genes that are involved in oxidative phosphorylation (ATPases 8

and 6). (Lim et al, 2012). Another type of mutation includes the changes in copy number, which can be

measured using minisatellite markers, like 16189 polyC, 303 polyC, and 514 (CA) repeat (Lee et al 2012,

Lim et al 2012). These minisatellite markers are short repeating sequences of nucleotides. In order to

determine minisatellite instability, the regions were sequenced and the researchers noted any

mutations. The copy number in a genome is the number of times a certain active gene is repeated. Both

research teams were very interested in these mtDNA mutations, and followed a similar procedure to

compare “normal” mtDNA with affected mtDNA (Lee et al 2012, Lim et al 2012).

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The procedures for the two experiments were extremely similar, though the hypotheses and

conclusions were interpreted and applied differently. First, the teams gathered samples from both

normal tissue and affected tissue from each individual in the study. The mtDNA from each sample was

sequenced and analyzed at the three minisatellite markers listed above (16189 polyC, 303 polyC, and

514(CA) repeat). Using an established method for quantification PCR to determine the concentration of

plasmid DNA, both research teams calculated the mtDNA copy number of CYTB and β-actin genes. (The

copy number was calculated as a ratio of CYTP to β-actin genes.) Additionally, a PCR was run with the

samples to separate the samples by size and determine the percentage of the 4,977 bp deletion. In

order to test for the presence and possible effect of ROS on mtDNA mutations, the hydrogen peroxide

content in the affected tissue samples were also determined. (Hydrogen peroxide is a form of ROS.) (Lee

et al 2012, Lim et al 2012)

To understand why the results from the colorectal cancer tissue and the Barrett’s Metaplasia

tissue are similar, the significance of the results must be explained. Lee et al (2012) and Lim et al (2012)

were examining the mitochondria in mutated cells – whether the cells were mutated cancerous cells or

mutated tissue in the esophagus. In this respect, a very general hypothesis can be formed: mutations in

the mtDNA create mutations in the corresponding cell. These mutations may present in a multitude of

ways. Both sets of results showed an increase in large deletion of the affected mtDNA, an increase in

copy number of the affected mtDNA, and an increase of hydrogen peroxide concentration in the

affected cells (Lee et al 2012, Lim et al 2012).

Dr. Sang Woo Lim and his team’s (2012) objective was to analyze the mtDNA instability for

colorectal cancer tissues and normal tissue at a control region, two coding regions, and mtDNA

minisatellites. The mitochondrial minisatellite instability (mtMSI) was determined by performing a gene

scan analysis and examining the patterns of affected and corresponding normal mtDNA. (See figure 1 for

illustrated results.) Additionally, the mtDNA copy number was reported as 3,998 ± 1,975.9 and 3,329

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±1,315 for the colorectal cancer tissue and the normal tissue, respectively (Lim et al 2012). Though Lim

et al (2012) calculated the p-value to be 0.048, the degree that the two values overlapped should be

noted. (See figure 2.)Graphically, the two values are very similar, and the standard deviation values

showed a large degree of overlap. On the other hand, the degree of difference in the mtDNA large

deletion between the two samples appeared to be much more significant, and the calculated p-value is

0.03. The colorectal cancer tissue had a much lower level of mtDNA large deletion than in normal tissue.

(See figure 2.) Lim et al (2012) offered several possible explanations for the connection between the

increase in mtDNA copy, the decrease in mtDNA large deletion, and the elevated ROS (measured as

hydrogen peroxide content) in the colorectal cancer tissue. It was possible that the large deletion (which

deleted several ATPases) prevented overproduction of ROS, and the lack of this large deletion may cause

a cycle of ROS overproduction and mtDNA mutation (Lim et al 2012). The results of this experiment

supported the hypothesis that mtDNA mutations are linked to the development of tumors, but the

authors agreed that the sample size was too limited to provide enough evidence. These general

conclusions – that mtDNA mutations are linked to cancerous tissue development – are not limited to

only colorectal cancer tissue. The authors cite many other experiments involving Barrett’s esophagus,

breast cancer, thyroid cancer, and (nonmelanoma) skin cancer that all concluded that the mtDNA large

deletion was significantly decreased in the affected tissue, which suggests that this 4,977 bp deletion

may be a factor in the development of advanced stages of cancer.

Lee et al (2012) also connected mtDNA with carcinogenesis. Lee et al (2012) hypothesized that

higher levels of reactive oxygen species (ROS) produced higher levels of mutation in mtDNA. Though the

hypotheses for the two experiments varied, the results and conclusions were very similar. This study

indicated that the copy number of the two genes studied was significantly increased in the affected

tissue. (See figure 4.) However, the values reported (4.75±12.14 and 1.22±0.78) were much more

precise than those of Lim et al (2012). Lee et al (2012) also reported similar results for the mtDNA large

Figure 2: (a) mtDNA copy number and (b) large deletion in colorectal cancer tissue. Source: Lim et al 2012.

Figure 1: Minisatellite instability in colorectal cancer tissue at (a) 16184polyC, (b) 303polyC, and (c) 514(CA) repeat. Source: Lim et al 2012

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deletion of 4,977 bp (see figure 5). The microsatellite instability results were very similar among the two

experiments, especially for 514(CA) repeats (See figure 3). These combined results – from two different

types of tissues – support the hypothesis that mtDNA takes a contributing role in cell metastasis or

mutation. These results also suggest that the mutations do not necessarily indicate cancerous tissue, but

rather a much broader scope of diseases.

Figure 3: Microsatellite instability for Barrett’s Esophagus at A) 303 polyC, B) 16189 polyC and C) 514(CA) repeats. Note the similarities between C) and c) of figure 1. Source: Lee et al 2012.

Figure 4: mtDNA copy number for normal tissue and Barrett’s Esophagus tissue. Note the similarities to part a) of figure 2. Source: Lee et al 2012.

Figure 5: mtDNA large deletion for normal tissue and Barrett’s Esophagus tissue. Note the similarities to part b) of figure 2. Source: Lee et al 2012.

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It turns out that Lee et al (2012) and Lim et al (2012) were not the first scientists to ask questions about

the role of mtDNA mutations in metastasis. Ishikawa et al (2008) also questioned if and how the mtDNA

mutations could contribute to tumor growth. However, Ishikawa et al (2008) mainly pursued the

implications of ROS overproduction, concluding like Lim et al (2012), the somatic mutations create an

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environment that is conducive to ROS overproduction. In turn, the overabundance of ROS increases the

likelihood of metastasis (Ishikawa et al 2008, Lim et al 2012). An obvious next step would be to

determine just how significant mtDNA mutations are in cell metastasis and tumorigenesis. Additionally,

how much can these conclusions be generalized? Are they applicable to all types of cancer and

metastasis, or do other outside factors inhibit or augment the productivity of mtDNA mutations? Future

studies may answer these questions and bring humans closer to curing these diseases.

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Literature Cited

Anand B, Weinstein WM. 2012. Barrett’s Esophagus. Available from:

http://www.medicinenet.com/barretts_esophagus/article.htm

Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A et al. 2008. ROS-generating

mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320: 661-664.

Available from: http://www.sciencemag.org.pallas2.tcl.sc.edu/content/320/5876/661.full.pdf

Lee S, Han M-J, Lee K-S, Back S-C, Hwang D et al. 2012. Frequent occurrence of mitochondrial DNA

mutations in Barrett’s Metaplasia without the presence of dysplasia. PLoS ONE 7(5): e37571.

Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358277/pdf/pone.0037571.pdf

Lim SW, Rim HR, Kim HY, Huh JW, Kim YJ et al. 2012. High-frequency minisatellite instability of the

mitochondrial genome in colorectal cancer tissue associated with clinicopathological values.

Cancer131:1332-1341. Available from: http://onlinelibrary.wiley.com/doi/10.1002/ijc.27375/pdf

PubMed Health. 2012. Colon cancer. Available

from: http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001308/