tech: Mitochondrial mutations in cancer

[Guest guest]

Hi All

The below pdf-available paper

identified in a Science Editor's

Choice is available and suggests

that mitochondrial DNA mutations (mtDNAs) may

be involved in cancer of the prostate.

The pdf was corrupted, unfortunately.

Although I tend not to be so impressed

by in vitro cancer cell growth data, it

was noted that, for the cybrids derived from

the mtDNA mutants-prostate cancer cells,

tumors showed " more than a 7-fold

increase. " The data of Fig. 2 was

impressive.

For definitions, there are:

cybrid: A cell with cytoplasm from two

different cells as a result of cell hybridization.

hetero-: Combining form from the

Greek heteros meaning different.

plastic: Having the power to give

form or fashion to a mass of matter

The Table 2 data is included to demonstrate

precisely how great the difference was in

frequency of the mtDNA mutations in prostate

cancer.

It is especially gratifying to note that CR

seems to greatly diminish cytochrome

oxidase, of which subunit I was analyzed in

the current report, in an earlier report:

http://tinyurl.com/5kwgs

Now, here is the current article.

Science 28 January 2005: 483

Editors' Choice: Highlights of the recent literature

MEDICINE: Mitochondria and Cancer

Human tumors often contain mutations in mitochondrial DNA

(mtDNA). Whether these mutations are causally involved in

tumorigenesis and the mechanisms by which they might contribute are

pressing questions that remain unanswered. One hypothesis suggests

that tumor-associated mtDNA mutations lead to increased production of

reactive oxygen species (ROS), a by-product of mitochondrial

oxidative phosphorylation, which can stimulate cell proliferation.

Data from a new study of mtDNA in human prostate tumors are

consistent with this hypothesis. Petros et al. identified mutations

in two mitochondrial genes encoding proteins involved in oxidative

phosphorylation: cytochrome oxidase subunit I and ATP6. Notably, when

mtDNA containing an ATP6 mutation close to the site of the tumor-

associated mutation was introduced into prostate cancer cells, the

cells generated significantly more ROS in comparison with wild-type

controls and grew at a much faster rate in mice, supporting the

notion that such mutations play a causal role in tumorigenesis. -- PAK

Proc. Natl. Acad. Sci. U.S.A. 102, 719 (2005).

mtDNA mutations increase tumorigenicity in prostate cancer.

Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, Lim

S, Issa MM,

Flanders WD, Hosseini SH, Marshall FF, Wallace DC.

Mutations in the mtDNA have been found to fulfill all of the

criteria expected

for pathogenic mutations causing prostate cancer. Focusing on the

cytochrome

oxidase subunit I (COI) gene, we found that 11-12% of all prostate

cancer

patients harbored COI mutations that altered conserved amino acids

(mean

conservation index = 83%), whereas <2% of no-cancer controls and 7.8%

of the

general population had COI mutations, the latter altering less

conserved amino

acids (conservation index = 71%). Four conserved prostate cancer COI

mutations

were found in multiple independent patients on different mtDNA

backgrounds.

Three other tumors contained heteroplasmic COI mutations, one of

which created a

stop codon. This latter tumor also contained a germ-line ATP6

mutation. Thus,

both germ-line and somatic mtDNA mutations contribute to prostate

cancer. Many

tumors have been found to produce increased reactive oxygen species

(ROS), and

mtDNA mutations that inhibit oxidative phosphorylation can increase

ROS

production and thus contribute to tumorigenicity. To determine

whether mutant

tumors had increased ROS and tumor growth rates, we introduced the

pathogenic

mtDNA ATP6 T8993G mutation into the PC3 prostate cancer cell line

through cybrid

transfer and tested for tumor growth in nude mice. The resulting

mutant (T8993G)

cybrids were found to generate tumors that were 7 times larger than

the

wild-type (T8993T) cybrids, whereas the wild-type cybrids barely grew

in the

mice. The mutant tumors also generated significantly more ROS.

Therefore, mtDNA

mutations do play an important role in the etiology of prostate

cancer.

PMID: 15647368 [PubMed - in process]

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Abbreviations: CI, conservation index; CO, cytochrome oxidase

subunit; ETC, electron transport chain; LCM, laser capture

microdissection; nDNA, nuclear DNA; np, nucleotide pair; OXPHOS,

oxidative phosphorylation; ROS, reactive oxygen species; SDH,

succinate dehydrogenase.

There is increasing evidence that mitochondrial gene mutations

are associated with various cancers, but their pathophysiological

significance remains unclear. The first clear demonstration that

mtDNA mutations in cancer could have functional significance came

with the report of a middle-aged woman with a renal adenocarcinoma

that was heteroplasmic (mixture of mutant and normal mtDNAs) for a

deletion of 294-nucleotide pairs (nps) in the mtDNA oxidative

phosphorylation (OXPHOS) gene ND1 (1). Subsequently, a variety of

mtDNA coding region and control region mutations have been reported

in colon cancer cells (2), prostate cancer (3–5), and a variety of

other solid tumors (6).

Mutations in nuclear DNA (nDNA)-encoded mitochondrial genes have

also been linked to cancer. Mutations in the nDNA-encoded succinate

dehydrogenase (SDH) B, SDHC, and SDHD subunits of OXPHOS complex II

have been linked to paragangliomas (7–10). However, mutations in

SDHA, the succinate-binding subunit, have been linked to Leigh

Syndrome (11), not paraganglioma, demonstrating that transformation

due to complex II mutants is not simply the result of energy

deficiency. An alternative mitochondrial contribution to

tumorigenicity could be increased reactive oxygen species (ROS)

production. This possibility is supported by the observation that the

mev-1 mutation in the SDHC gene of Caenorhabditis elegans markedly

increases ROS production (12, 13), and increased ROS production has

been proposed to be an important factor in tumor formation in

association with inactivation of p16ink4a and p53 (14).

ROS are generated as a toxic by-product of mitochondrial OXPHOS.

OXPHOS is composed of the electron transport chain (ETC),

encompassing complexes I, II, III, and IV and the H+-transporting ATP

synthase, complex V. Reducing equivalents (electrons) from dietary

calories are passed down the ETC, where they ultimately reduce O2

(four electrons) to generate H2O. The energy that is released is used

to pump protons out across the mitochondrial inner membrane through

complexes I, III, and IV to create an electrochemical gradient (P =

+ µH+). P is then used as a source of potential energy by complex V

to condense ADP and phosphate to generate ATP, and ATP is exchanged

across the mitochondrial inner membrane for spent cytosolic ADP by

the adenine nucleotide translocators (15).

When calories are plentiful, the ETC becomes more reduced and

electrons from complexes I and III can be donated directly to O2 to

generate superoxide anion , the first of the ROS. is converted to

H2O2 by mitochondrial manganese superoxide dismutase, and H2O2 can be

converted to water by glutathione peroxidase or catalase. H2O2 can

also acquire an additional electron from a reduced transition metal

to generate the highly reactive hydroxyl radical OH. H2O2, which is

semistable, can also diffuse out of the mitochondrion and into the

cytosol and the nucleus, where it can act (15).

At high levels ROS are toxic, but at low levels they are

mitogenic, presumably interacting with various nuclear regulatory

factors (AP-I, NF-B APE/ref-1) (16), regulatory kinases (Src kinase,

protein kinase C, mitogen-activated protein kinase), receptor

tyrosine kinases (17), protein-tyrosine phosphatases (18), and

angiogenic factors (19, 20). Consistent with mitochondrial ROS being

important in tumor formation, mitochondrial manganese superoxide

dismutase is reduced in many types of tumors including prostate

cancer, mutations in the mitochondrial manganese superoxide dismutase

gene promoter have been observed in a number of tumors, and

transformation of certain tumors with the mitochondrial manganese

superoxide dismutase cDNA can reverse the malignant phenotype (17, 21–

23).

Mitochondrial OXPHOS is assembled from multiple polypeptides, some

encoded by the mtDNA and others by the nDNA. In addition to the 12S

and 16S rRNAs and 22 tRNAs for mitochondrial protein synthesis, the

mtDNA encodes for 13 polypeptides, 7 (ND1, ND2, ND3, ND4L, ND4, ND5,

and ND6) of 46 polypeptides of complex I, 1 (cytochrome of 11

polypeptides of complex III, 3 [cytochrome oxidase subunit (CO) I,

II, and III] of 13 polypeptides of complex IV, and two (ATP6 and

ATP8) of 16 polypeptides of complex V. COI is the main catalytic

subunit of cytochrome c oxidase (complex IV), and ATP6 is central to

the proton channel of the ATP synthase (complex V) (15).

The inhibition of OXPHOS that increases ROS production has been

confirmed in mice in which the heart/muscle isoform gene of the

adenine nucleotide translocator gene (Ant1) was inactivated. This

inactivation resulted in the hyperpolarization of P, increased ROS

generation, and elevated mtDNA damage (24, 25).

If mitochondrial dysfunction and ROS production contribute to

cancer, then this finding would elicit two hypotheses. First, cancers

should harbor both germ-line and somatic mtDNA mutations, which

should partially inhibit OXPHOS and, thus, increase ROS production.

Second, mtDNA mutations that increase ROS production should stimulate

tumor growth. We have tested both of these predictions on prostate

cancer and confirmed their validity. Therefore, this article supports

the conclusion that mitochondrial defects contribute to the etiology

of cancer.

Materials and Methods

Patient Materials. All patient studies were implemented under

Emory University Institutional Review Board approved protocols.

Histologically confirmed prostate cancer samples were selected from

our collection of radical prostatectomies, institutional tissue

resources, and microdissected samples prepared between 1995 and 2002.

The " no-cancer " control group was assembled from subjects who had

undergone prostate biopsy and had been found to be free of prostate

cancer. These individuals were all at least 50 years old and had a <4

ng/ml prostate-specific antigen.

...

Results

Identification of the mtDNA Variants in LCM-Isolated Prostate

Cancer Epithelium. To determine whether mtDNA mutations were

associated with the prostate cancer, we used LCM to isolate prostate

cancer epithelial cells from several prostate tumors and sequenced

segments of their mtDNAs. This experiment revealed a variety of

potentially pathogenic mtDNA mutations. As a example, for tumor 18,

the entire mtDNA was sequenced in a series of overlapping segments.

This finding revealed 38 base substitutions relative to the Cambridge

Reference Sequence (MITOMAP), including 31 previously reported

polymorphisms and 7 previously uncharacterized mutations; the latter

including 1 ribosomal RNA mutation and 3 missense mutations. The

three new amino acid substitution mutations included a chain

termination mutation in COI (G5949A) and two missense mutations, one

in cytb (A14769G) and the other in ATP6 (C8932T).

The G5949A mutation introduced a stop codon into COI at amino acid

16 (G16X) (Fig. 1A). To determine the origin of this mutation, we

developed a restriction fragment length polymorphism test for the

mutation and tested the cancerous and normal epithelium from this

prostate. This experiment revealed that the cancerous epithelial

cells of tumor 18 were homoplasmic mutant, whereas the adjacent

normal epithelial cells were homoplasmic wild-type (Fig. 1B). Hence,

this mutation must have arisen during the genesis of the cancer cell

and then segregated to a pure mutant in the malignant cells.

To determine whether the G16X mutation actually eliminated the COI

protein from the prostate cancer cells, we performed

immunohistochemistry on tumor sections (Fig. 1C). The normal

epithelial cells proved to be strongly positive for COI, whereas the

adjacent cancer cells were completely negative.

The cytb A14769G mutation in this patient altered an amino acid

(N8S) with a relatively low interspecific amino acid conservation

index (CI) of 20.5%, indicating that this variant probably had

limited effect on the cellular physiology. By contrast, the ATP6

C8932T mutation altered an amino acid (P136S) with a CI of 64%, which

could be functionally significant. Therefore, both germ-line and

somatic mtDNA mutations may have contributed to the formation of

tumor 18.

COI Mutations in Prostate Cancer. The loss of the mtDNA-encoded

COI subunit in tumor 18 is consistent with proteomic surveys of LCM-

isolated prostate cancer epithelia, which revealed that the ratio of

nDNA-encoded complex IV subunits (COX IV, Vb, and VIc) to mtDNA-

encoded subunits (COI and COII) was increased in most prostate tumors

(32, 33). Hence, deficiency of mtDNA COI, COII, and COIII subunits

might be a common feature of prostate cancer.

To determine whether COI was in fact deficient, we sequenced the

COI genes from multiple prostate cancer tumors and controls. We chose

to study only the COI genes because this permitted us to survey a

large number of tumor and control samples, thus making statistical

evaluation feasible. Moreover, COI mutations have been observed in

the mtDNAs in colon cancer cell lines (2), colonic crypt cells (34),

and sideroblastic anemia patients (35, 36). However, polymorphisms

and pathogenic mutations in COI are relatively uncommon (MITOMAP)

(26, 37).

DNA was extracted and the COI gene sequenced from prostatectomy

specimens or peripheral blood cells taken from 260 European and

African American patients with pathologically confirmed prostate

cancer and from the lymphocytes of 54 " no-cancer " (prostate cancer

negative) controls. COI missense mutations (Table 1) were found in

12% of the prostate cancer patients but in only 1.9% of the no-cancer

controls, a significant increase in frequency (P = 0.023) (Table 2).

Furthermore, in a population sample of 1,019 European and African

mtDNA sequences, 7.8% had COI mutations, which was also significantly

lower than that of cancerous prostates (P = 0.015) (Table 2). Because

COI missense polymorphisms are more common in African mtDNAs of

macrohaplogroup L than in the rest of the world (37), we also

analyzed only patients and controls of European ancestry. From this

group, COI missense mutations were found in 11% of the prostate

cancer specimens, 0% of no-cancer controls (P = 0.016), and 6.5% in a

population sample of 898 Europeans (P = 0.025) (Table 2). Thus, COI

mutations are significantly increased in prostate cancer over the no-

cancer controls and the general population.

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Table 2. Frequency of COI mutations in prostate cancer patients,

controls, and the general population

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n COI mutant Frequency, % P

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Cancer 260 31 11.9ab

EA 180 19 10.6cd

AA 80 12 15.0

No cancer 54 1 1.9a 0.023

EA 46 0 0c 0.016

AA 8 1 12.5 0.674

Population 1338 104 7.8

EA + AA 1019 79 7.8b 0.015

EA 898 58 6.5d 0.025

AA 121 21 17.4 0.432

Non(EA + AA) 319 25 7.8

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Variants 7389 and 7146, defining L0 and L0L1, respectively, were not

considered. EA, European ancestry; AA, African ancestry. Frequencies

with the same superscript were compared, and the P value (from

Fisher's exact test) is represented in the right column.

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The interspecific conservation of the altered COI amino acids in

prostate cancer was also significantly higher than that in the

general population. The average CI for the prostate cancer mutations

was 83 ± 25%, whereas that for a general population sample of 1,338

mtDNA sequences was 71 ± 35 (P = 0.029). The CI of the prostate

cancer COI mutations was comparable with the CI observed for global

human mtDNA " adaptive mutations " (85 ± 9%) and far above the CI of

global " neutral polymorphisms " (23 ± 15%) (26). Thus, the prostate

cancer COI mutations must be functionally significant.

Three of the prostate cancer COI mutations had the characteristics

of new somatic pathogenic mutations, being heteroplasmic and changing

highly conserved amino acids. The first of these mutations was the

tumor 18 chain-termination mutation, G5949A (G16X). The second was a

T6124C mutation (M74T) with a CI of 95% that was heteroplasmic in

both the prostate tissue and blood cells. The third mutant was C6924T

(A341S), which had a CI of 100% and was primarily a mutant in the

prostate tissue but wild-type in blood (Table 1).

Four other prostate cancer COI mutations were found in more than

one patient and in each case was associated with prostate cancer. The

T6253C mutation (CI = 69%) was found in three independent cases, all

on haplogroup H, the most common European haplogroup. The C6340T

mutation (CI = 79%) was observed in two patients on two different

haplogroup backgrounds (H and N). The G6261A mutation (CI = 97%) was

observed in six patients on four different haplogroups (J, T, L1, and

N). Finally, the A6663G mutation (CI = 95%) was observed in five

patients on two different haplogroups (L2 and unclassified) (Table

1).

Because the T6253C, C6340T, G6261A, and A6663G mutations were

homoplasmic in these patient's lymphocytes, they must have arisen in

the female germ-line. The importance of germ-line mutations in

prostate cancer was supported by the observation that none of the

European descent no-cancer control men had COI mutations, yet 6.5% of

the European population had COI mutations and 11% of the European

prostate cancer patient men had COI mutations. Because the frequency

of COI mutations is significantly different between the noncancer

controls and the general population (P = 0.05) and between the

general population and the prostate cancer-positive men (P = 0.016),

it follows that men that harbor germ-line COI mutations must have a

substantially increased risk of developing prostate cancer.

Therefore, both somatic and germ-line COI mutations are associated

with prostate cancer, and COI mutations must be a significant risk

factor for prostate cancer.

We have exhaustively surveyed only COI mutations in this study.

However, it is likely that additional mtDNA polypeptide mutations can

contribute to the etiology of prostate cancer (5). This possibility

is supported by the ATP6 C8932T (P136S) observed in tumor 18 as well

as the presence of two previously uncharacterized missense mutations

in the complete mtDNA sequence of the PC3 tumor cell line, which is

haplogroup U5, and contained a np T11120C (ND4/F121L; CI = 12.8%)

variant of unlikely functional significance and a np C13802T

(ND5/T489M; CI = 62.5%) variant that could be functionally relevant.

Additional mtDNA missense mutations were found in other tumors

analyzed from LCM material (data not shown).

Cybrid Studies Reveal That mtDNA Mutations Enhance Cancer Cell

Growth. To investigate the functional importance of mtDNA mutations

for prostate cancer, we chose to model the tumor 18 germ-line ATP6

C8932T (P136S) mutation by using the well characterized pathogenic

ATP6 np 8993G L156R mutation (38). The ATP6 np 8993G mutation is just

20 amino acids away from the P136S mutation and is known to cause a

70% inhibition in ADP-stimulated respiration (30) and an increase in

mitochondrial ROS production (39).

We introduced mtDNAs harboring the ATP6 mutant T8993G or wild-type

T8993T base into a prostate cancer cell line through

transmitochondrial cybrids. The mitochondrial donor cells were

derived from the same heteroplasmic patient (30) and were homoplasmic

for either the T8993G or T8993T mtDNA. These cells were enucleated

and the cytoplasts fused to PC3 cells that had been cured of their

resident mtDNAs by using rhodamine 6-G (29). Six PC3 cybrids that

were homoplasmic for the mutant mtDNA (T8993G) [PC3(mtDNA T8993G)

nos. 2, 5, 8, 20, 21, and 26] and four cybrids that were homoplasmic

wild-type (T8993T) [PC3(mtDNA T8993T) WT nos. 1, 3, 5, and 10] were

isolated and studied further.

The PC3(mtDNA ATP6 T8993T) and PC3(mtDNA ATP6 T8993G) cybrids were

injected s.c. into nude mice in four separate experiments conducted

on both early passage (at about passage 6) and late-passage (at about

passage 25) cultures. The results from all four experiments

encompassing multiple trials for both the T8993G versus the T8993T

clones were combined for each time point and the values averaged and

plotted (Fig. 2). These experiments revealed that the average tumor

volume of the mutant PC3(mtDNA T8993G) cybrids was significantly

higher than that of the wild-type PC3(mtDNA T8993T) cybrids at every

time point (P < 0.026 by Mann–Whitney test) (Fig. 2). Indeed, the PC3

(mtDNA T8993T) wild-type cybrids barely grew at all in the mice. By

day 110, the average tumor volume of the PC3(mtDNA T8993T) wild-type

cybrids was 0.11 ml, whereas that of the PC3(mtDNA T8993G) mutant

cybrids was 0.78 ml, more than a 7-fold increase. Moreover, this

result is an underestimate of the differential growth rate of the PC3

(mtDNA T8993G) tumors because mice with rapidly growing tumors had to

be euthanized throughout the various experiments. This decision

removed the fastest growing PC3(mtDNA T8993G) tumors from the later

average tumor-size calculations, resulting in an uneven mutant cybrid

curve with a reduced average slope (Fig. 2). Hence, the tumor growth

rate of the PC3 prostate cancer cell nucleus was enhanced by the

introduction of an ATP6 mutation known to reduce ATP synthase

activity and increase mitochondrial ROS production (30, 39).

To confirm that the mutant PC3(mtDNA T8993G) cybrids generated

more ROS, we tested the tumor cells for superoxide anion production

by staining tumor sections with dihydroethidium (Fig. 3). The

nonfluorescent dihydroethidium is oxidized to fluorescent ethidium

by . The average fluorescence pixel density of the PC3(mtDNA T8993G)

mutant tumor cells was 71.2 ± 9.2 (n = 3; MT5, MT8, and MT20),

whereas that of the PC3(mtDNA T8993T) wild-type tumor cells was 46.7

± 4.2 (n = 2, WT3 and WT10). Thus, there was significantly more ROS

produced by the PC3(mtDNA T8993G) mutant tumors (P = 0.013 by t

test). Therefore, prostate cancer cells that harbor mtDNA mutations,

which increase ROS production, show increased tumor growth.

Discussion

The current study provides convincing evidence that mtDNA

mutations play an important role in the etiology of prostate cancer.

Prostate cancers have a significantly increased frequency of

functionally important COI mutations, and the introduction into

prostate cancer cells of a mtDNA mutation, which inhibits OXPHOS and

increases ROS production, increased their in vivo growth.

The COI mutations that we identified in prostate cancer fulfilled

all of the criteria expected for mtDNA mutations that cause this

disease. The COI mutations were significantly more frequent in

prostate cancer patients than in no-cancer controls or in the general

population. The COI mutations altered significantly more conserved

amino acids, and they included both new heteroplasmic somatic and

recurrent homoplasmic germ-line mutations. Hence, mtDNA COI mutations

appear to be a causal factor in the etiology of prostate cancer.

Germline COI mutations were also found to be an important risk

factor for developing prostate cancer. COI missense mutations were

common in the general population (7.8%), yet virtually absent (<2%)

in cancer-negative controls. Thus, most men harboring COI missense

mutations must move into the prostate cancer category by late middle

age. The association between germ-line COI mtDNA mutations and

prostate cancer risk might also explain why African American men are

more prone to prostate cancer than European American men (40).

Overall, COI variants are relatively common in African mtDNA (17.4%,

Table 2), in part due to certain African mtDNA lineages harboring

ancient COI protein polymorphisms (e.g., the np 7389 and 7146

variants in African lineages L0 and L0L1) (26). Therefore, these

ancient COI protein polymorphisms may be contributing to an increased

predisposition to prostate cancer in African American men today.

However, a much larger study will be required to test the statistical

validity of this proposition.

Given that highly conserved COI mtDNA missense variants (CI = 70%)

are so common in the general population (7.8%), we wonder why COI

mutations aren't more commonly found in neuromuscular disease. One

possibility is that the human cell has the capacity to partially

compensate for complex IV defects by changing the expression of the

COX subunits. Mice lacking liver adenine nucleotide translocators

were found to selectively upregulate complex IV by 2-fold (41).

Hence, the biochemical effects of partial complex IV defects might be

ameliorated by altered complex IV gene expression. This result would

be consistent with the observation that prostate cancers have

increased levels of nDNA-encoded to mtDNA-encode complex IV subunits

(32, 33). Even so, the COI mutations would inhibit the ETC, and this

result could chronically increase mitochondrial ROS production and

stimulate cell proliferation (15, 17, 23).

If prostate cancer is the most common clinical consequence of COI

mutations, then this finding may explain why COI mutations are so

common in the general population. Prostate cancer kills middle aged

or older males, but the mtDNA is exclusively maternally inherited.

Hence, deleterious COI mutations that cause prostate cancer would

have minimal effect on the genetic fitness of the mutant mtDNA.

Mutations in the mtDNA that inhibit the ETC and increase ROS

production could act as both tumor promoters and tumor initiators.

The fact that mtDNA mutations which increase ROS production can be

potent tumor promoters was demonstrated by our introduction of the

pathogenic mtDNA ATP6 T8993G mutation (30, 39) into PC3 cells and

showing a dramatic increased tumor growth rate in association with

increased cellular ROS production. Moreover, the lack of tumor growth

observed for the PC3(mtDNA T8993T) wild-type cybrids might also

support this conclusion because it is well established that PC3 cells

readily form tumors in nude mice. Because the PC3 mtDNA was found to

harbor a conserved ND5 np C13802T (T489M) mutation, it is possible

that removal of this mutation reduced the tumorigenic potential of

the PC3 cells.

Whether mtDNA mutations might also serve as tumor initiators was

suggested by tumor 18, which harbored both a germ-line ATP6 P136S and

a somatic COI G16X mutation. Because the germ-line ATP6 mutation must

have preceded that COI G16X mutation, it is possible that ROS

generated as a result of the ATP6 P136S mutation could have damaged

to the mtDNA and caused the COI G16X mutation.

These observations also indicate that mtDNA variants could have

accounted for earlier somatic cell genetic observations that the

cybrid transfer of chloramphenicol resistant (CAPR) mtDNAs from a

nontumorigenic Chinese hamster cell line into a tumorigenic cell line

suppressed tumorigenesis (42), whereas the reciprocal transfer had no

effect. Similarly, the transfer of CAPR mtDNAs from the near euploid

HT1080 cells into the aneuploid HeLa cells suppressed growth, but the

reciprocal transfer caused no change (43).

Our demonstration that partial defects in OXPHOS, which increase

ROS, can contribute to cancer now provides an explanation for the

observation of Otto Warburg >70 years ago that solid tumors have a

high rate of " aerobic-glycolysis " (44). Mutations that inhibit OXPHOS

would not only make more ROS, they would oxidize less pyruvate and

NADH. The pyruvate and NADH would be converted to lactate by lactate

dehydrogenase, resulting in excessive lactate production during

aerobic respiration, aerobic-glycolysis, a physiological state that

has been documented for cells harboring the ATP6 T8993G mutation

(45). If mitochondrial ROS production is essential for solid tumor

promotion, then aerobic-glycolysis should be a common feature of

solid tumors, which Warburg noted.

In conclusion, this study has revealed that mtDNA mutations are

not only associated with a predisposition to neuromuscular disease

but also a predisposition to cancer. Therefore, we can now add cancer

to the list of mitochondrial diseases.

Cheers, Al Pater.