The cell surface marker CD34 marks mouse hair follicle bulge cells, which have attributes of stem cells, including quiescence and multipotency. Using a CD34 knockout (KO) mouse, we tested the hypothesis that CD34 may participate in tumor development in mice because hair follicle stem cells are thought to be a major target of carcinogens in the two-stage model of mouse skin carcinogenesis. Following initiation with 200 nmol 7,12-dimethylbenz(a)anthracene (DMBA), mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. Under these conditions, CD34KO mice failed to develop papillomas. Increasing the initiating dose of DMBA to 400 nmol resulted in tumor development in the CD34KO mice, albeit with an increased latency and lower tumor yield compared with the wild-type (WT) strain. DNA adduct analysis of keratinocytes from DMBA-initiated CD34KO mice revealed that DMBA was metabolically activated into carcinogenic diol epoxides at both 200 and 400 nmol. Chronic exposure to TPA revealed that CD34KO skin developed and sustained epidermal hyperplasia. However, CD34KO hair follicles typically remained in telogen rather than transitioning into anagen growth, confirmed by retention of bromodeoxyuridine-labeled bulge stem cells within the hair follicle. Unique localization of the hair follicle progenitor cell marker MTS24 was found in interfollicular basal cells in TPA-treated WT mice, whereas staining remained restricted to the hair follicles of CD34KO mice, suggesting that progenitor cells migrate into epidermis differently between strains. These data show that CD34 is required for TPA-induced hair follicle stem cell activation and tumor formation in mice. [Cancer Res 2007;67(9):4173-81]

Mammalian mitochondrial DNA (mtDNA) is replicated by the heterotrimeric Pol γ comprised of a single catalytic subunit, encoded by Polg, and a homodimeric accessory subunit encoded by the Polg2 gene. While the catalytic subunit has been shown to be essential for embryo development, genetic data regarding the accessory subunit are lacking in mammalian systems. Here, we describe the generation of heterozygous (Polg2(+/-)) and homozygous (Polg2(-/-)) knockout (KO) mice. Polg2(+/-) mice are haplosufficient and develop normally with no discernable difference in mitochondrial function through 2 years of age. In contrast, the Polg2(-/-) is embryonic lethal at day 8.0-8.5 p.c. with concomitant loss of mtDNA and mtDNA gene products. Electron microscopy shows severe ultra-structural defects and loss of organized cristae in mitochondria of the Polg2(-/-) embryos as well as an increase in lipid accumulation compared with both wild-type (WT) and Polg2(+/-) embryos. Our data indicate that Polg2 function is critical to mammalian embryogenesis and mtDNA replication, and that a single copy of Polg2 is sufficient to sustain life.

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg 2؉ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.Chronic disruption of mitochondrial function can result in a broad array of neuromuscular degenerative disorders known as mitochondrial diseases, including progressive external ophthalmoplegia (PEO), 2 Alpers syndrome, parkinsonism, and several complex ataxia neuropathy syndromes (1-3). A heritable form of PEO was first mapped to a chromosomal interval near position 10q24 in a Finnish pedigree (4). One molecular hallmark of PEO was the age-dependent accumulation of mtDNA deletions in somatic tissues of affected individuals, which suggested that this locus was needed for replication or maintenance of mtDNA (4). In 2001, autosomal dominant PEO with mtDNA deletions was shown to co-segregate with 11 different missense mutations in the C10orf2 gene (5), which encoded a protein containing predicted amino acid sequences homologous to portions of the bacteriophage T7 gene product 4 and other superfamily 4 DNA helicases (5, 6). Since that time, numerous reports have identified 23 additional missense mutations in C10orf2 associated with heritable mitochondrial diseases such as adPEO, hepatocerebral mtDNA depletion syndrome (MDS), and infantile-onset spinocerebellar ataxia (IOSCA) (7, 8) (see Fig. 1).Several lines of evidence more directly link C10orf2 function to maintenance of mtDNA. The C10orf2 gene product dynamically colocalizes with mtDNA in nucleoprotein structures known as mitochondrial nucleoids (5, 9), and knocking down expression of C10orf2 by RNAi results in the rapid decrease in mtDNA copy number in cultured human osteosarcoma (143B) cells (10). Overexpression of catalytic mutants and dominant disease variants of the mtDNA helicase in cultured human or Schneider cells results in stalled mtDNA replication or depletion of mtDNA (11-13), which emulates the...

The Elk subfamily of the Eag K+ channel gene family is represented in mammals by three genes that are highly conserved between humans and rodents. Here we report the distribution and functional properties of a member of the human Elk K+ channel gene family, KCNH8. Quantitative RT-PCR analysis of mRNA expression patterns showed that KCNH8, along with the other Elk family genes, KCNH3 and KCNH4, are primarily expressed in the human nervous system. KCNH8 was expressed at high levels, and the distribution showed substantial overlap with KCNH3. In Xenopus oocytes, KCNH8 gives rise to slowly activating, voltage-dependent K+ currents that open at hyperpolarized potentials (half-maximal activation at -62 mV). Coexpression of KCNH8 with dominant-negative KCNH8, KCNH3, and KCNH4 subunits led to suppression of the KCNH8 currents, suggesting that Elk channels can form heteromultimers. Similar experiments imply that KCNH8 subunits are not able to form heteromultimers with Eag, Erg, or Kv family K+ channels.