An inherited heteroplasmic mutation in mitochondrial gene COI in a patient with prostate cancer alters reactive oxygen, reactive nitrogen and proliferation

Abstract

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.

Figure 1

Detection of heteroplasmic point mutation of mitochondrial cytochrome oxidase subunit I (COI) mitochondrial gene from a single individual with prostate cancer. (a) Sequencing chromatograms of prostatic tissue and an Epstein-Barr transformed lymphoblast cell line show approximately equal levels of both the wild type (T) and mutant (C) DNA base. (b) Activity of cytochrome oxidase measured in isolated mitochondria prepared from the patient's heteroplasmic lymphoblasts (see Section 2 for details) compared to the average of two-lymphoblast lines from controls with only the wild type base at position 6124. (c) Flow cytometric analysis of DCF fluorescence in the patient's heteroplasmic lymphoblasts compared to the average of two-lymphoblast lines from controls with only the wild type base at position 6124 (gray bars). Cells were also analyzed for DCF fluorescence in the presence of FCCP (white bars). Error bars represent the standard deviation of 2–4 data points.

Figure 2

Cybrid cell lines with the T6124C mutation show increased proliferation. Proliferation was measured in 3 separate 6124WT clones and 3 separate 6124Mut clones using FluoReporter Blue Fluorometric dsDNA Quantitiation Kit (see Section 2). Symbols represent the following clones: ◊-6124WT3; ◯-6124WT5; □-6124WT6, ◆-6124Mut1; ⚫-6124Mut2; ■-6124Mut4. Error bars represent the standard error of the mean of triplicate data.

Figure 3

Peroxide and Nitric Oxide are elevated in 6124 mutant cybrid cells. (a) Peroxide levels are elevated in 6124Mut cell lines as measured by flow cytometric analysis of DCF fluorescence. 143B cybrids cell lines containing either the wild type base at position 6124WT or the mutation at position 6124Mut were analyzed. The average DCF fluorescence of five wild type and six mutant clonal cell lines are shown. Mutant cybrids produce significantly more peroxides (P ≤ 0.0001). Error bars represent the standard error of the mean of 10 data points (WT) and 12 data points (Mut). (b) Mitochondrial superoxide levels are decreased in 6124Mut cell lines compared to 6124WT (P ≤ 0.04). The average MitoSOX fluorescence of three wild type and three mutant clonal cell lines are shown. Error bars represent the standard error of the mean of 9 data points each WT and Mut. (c) NO levels are highly elevated in 6124Mut cells compared to 6124WT as measured by DAF-FM Fluorescence (P ≤ 0.00001). For comparison, 143B cybrids cell lines containing a separate patient mtDNA with either a mutation at position 8993 (8993Mut) or wild type 8993 (8993WT) is shown. Error bars represent the standard error of the mean of 9 data points each 6124WT, 6124Mut, and 3 data points each 8993WT and 8993Mut. (d) Hydroxyl radicals and peroxynitrite anions as measured by hydroxyphenylfluorescein (HPF) remain unchanged. Error bars represent the standard error of the mean of 9 data points each 6124WT and 6124Mut.

Figure 4

iNOS RNA is present in the cybrids cells. Reverse transcription followed by standard PCR was performed with iNOS specific primers. iNOS RNA was present in both 6124WT and 6124Mut cell lines. The breast cancer cell line BT474 is used as a robustly positive control for iNOS RNA. and 18S RNA was used as a quality control.

Figure 5

PARP cleavage is decreased in 6124Mut cybrids cells. (a) Western Blot analysis of uncleaved and cleaved PARP in 6124WT (left) and 6124Mut (right) cells. Figure is representative of three 6124WT and three 6124Mut clones. (b) Densitometric analysis of Western Blot results of three 6124WT and three 6124Mut clones using ImageJ software. Error bars represent the standard error of the mean of the three WT and three Mut clones.

Figure 6

6124Mut cell lines grow faster in nude mice. Growth curves of tumor xenografts in nude mice. Each line represents a cohort of 29-30 animals injected with the 6124WT cybrids cells (◊) or 6124Mut cybrids cells (■). Error bars denote the standard error of the mean for each cohort at each time. Data are representative of 3 individual experiments.