Pilot Projects

Project Title: Mitochondrial Proliferation, Oxidative Stress, and Cancer

Principal Investigator: Eric Schon, Ph.D. (Department of Neurology)

Year: 2005
Award Amount: $25,000

Summary. Mitochondria are the location of a number of vitally important processes; foremost among these is the production of energy in the form of ATP via the respiratory chain/oxidative phosphorylation system. Pathogenic mutations in mitochondrial DNA (mtDNA) cause a variety of disorders, particularly in systems with high oxidative energy requirements, such as brain and muscle. Many mitochondrial diseases are characterized by respiratory chain deficiency and lactic acidosis. Mitochondria proliferate in tissues of patients, seen in muscle as red or purple blotches in "ragged-red fibers" (RRFs). The RRF's contain the vast majority of mutated mtDNAs. Importantly, it is not the presence of the mutation per se, but rather the inexorable and unregulated proliferation of mitochondria containing mutated mtDNAs that is devastating to the patient. Resulting defects of respiratory chain complexes have been shown to increase reactive oxygen species (ROS) and therefore oxidative stress, which also contributes to pathogenesis. The genes that control this unregulated proliferation are currently unknown. This Pilot Project aims to isolate a gene that controls the unregulated proliferation of the mutated mtDNAs in cells in order to halt proliferation and resulting disease. We propose to identify one such gene, which we believe straddles a translocation breakpoint in a critical 1-Mb region at chromosome locus 11q13.3 that we defined in three independent oncocytomas, benign tumors characterized by massive cytoplasmic mitochondrial proliferation.

Results. In 2004, after publishing the hypothesis that CCND1 might be involved in the regulation of mitochondrial proliferation, two groups demonstrated that CCND1 is indeed an inhibitor of mitochondrial proliferation. Therefore, immunohistochemistry for CCND1, COXI (mtDNA-encoded respiratory chain subunit of complex IV), SDHC (nDNA-encoded respiratory chain subunit of complex II) and porin (nDNA-encoded mitochondrial outer membrane protein) was performed on paraffin-embedded tissue from several oncocytoma patients. Using the proliferation markers COX, SDH and porin, the massive mitochondrial proliferation in the oncocytomas was confirmed. Interestingly, no expression for SDHC was found in the case with a duplication, suggesting that the telomeric duplication breakpoint interrupts SDHD (one of the four subunits of succinate dehydrogenase), which is located ~42 mB away from CCND1. CCND1 was not expressed in ragged-red fibers, implying that CCND1 expression is not necessary for mitochondrial proliferation to occur in muscle. In two of the three oncocytomas cases (t(4;11) and duplication), approximately 20% of cells expressed CCND1 (in their nuclei); in the third case, (t(9;11)), whose breakpoint is much further from CCND1 (~1 Mb) and is closer to MRPL21 (encoding a mitochondrial ribosomal protein), nearly 40% of nuclei were CCND1-positive. In contrast, essentially 100% of cell nuclei from control renal cell carcinoma cells (without mitochondrial proliferation) were CCND1-positive. As with those observed in RRF, these results imply that CCND1 expression is unrelated to mitochondrial proliferation, and that the breakpoints may indeed be disrupting a "proliferation-specific" DNA element.

The breakpoint in one case has been fine mapped to a 160-kb interval between MRPL21 and TPCN2. Bacterial artificial chromosomes BACs covering the gene-rich region between MRPL21 and TPCN2 have been prepared and characterized, providing a good chance of characterizing the breakpoint right to the gene level. If this is not possible, long PCR probes from the area of interest will help to get right to the gene level. Inverse PCR will give the exact junction sequence. The fine mapping and characterizing the breakpoints at the nucleotide level should be finished in about 6 months. The characterization of the mitochondrial proliferation gene is dependant upon finding a gene interrupted by one of the breakpoints.

Outcomes/Publications. Muscular Dystrophy Association (USA), Mattie Stepanek Neuromuscular Disease Research Fellowship awarded to Stefanie Zanssen, MD, PhD, a postdoctoral fellow working with Dr. Schon on this pilot; 1/1/2006-12/31/2007; "Genetic Control of Mitochondrial Proliferation"; $60,000/year.