EYA4 is inactivated biallelically at a high frequency in sporadic lung cancer and is associated with familial lung cancer risk.

In an effort to identify novel biallelically inactivated tumor suppressor genes (TSGs) in sporadic invasive and preinvasive non-small-cell lung cancer (NSCLC) genomes, we applied a comprehensive integrated multiple 'omics' approach to investigate patient-matched, paired NSCLC tumor and non-malignant parenchymal tissues. By surveying lung tumor genomes for genes concomitantly inactivated within individual tumors by multiple mechanisms, and by the frequency of disruption in tumors across multiple cohorts, we have identified a putative lung cancer TSG, Eyes Absent 4 (EYA4). EYA4 is frequently and concomitantly deleted, hypermethylated and underexpressed in multiple independent lung tumor data sets, in both major NSCLC subtypes and in the earliest stages of lung cancer. We found that decreased EYA4 expression is not only associated with poor survival in sporadic lung cancers but also that EYA4 single-nucleotide polymorphisms are associated with increased familial cancer risk, consistent with EYA4s proximity to the previously reported lung cancer susceptibility locus on 6q. Functionally, we found that EYA4 displays TSG-like properties with a role in modulating apoptosis and DNA repair. Cross-examination of EYA4 expression across multiple tumor types suggests a cell-type-specific tumorigenic role for EYA4, consistent with a tumor suppressor function in cancers of epithelial origin. This work shows a clear role for EYA4 as a putative TSG in NSCLC.

Functions of the von Hippel-Lindau tumour suppressor protein.

Von Hippel-Lindau disease (VHL) is caused by germline mutations in the VHL tumour suppressor gene. Tumour development in this setting is due to loss or inactivation of the remaining wild-type VHL allele. The VHL gene product (pVHL) resides primarily in the cytoplasm. A frequently mutated region of pVHL can bind to complexes containing elongin B, elongin C and Cul2. Loss of pVHL leads to an inappropriate accumulation of hypoxia-inducible mRNAs, such as the mRNA encoding vascular endothelial growth factor (VEGF), under normoxic conditions. This finding is most likely to account for the hypervascular nature of VHL-associated neoplasms. Current studies are focussed on understanding if and how binding to elongins and Cul2 is linked to the ability of pVHL to regulate hypoxia-inducible mRNAs. In this regard, it is perhaps noteworthy that elongin C and Cul2 are homologous to yeast proteins Skp1 and Cdc53. These latter proteins participate in the formation of complexes that target certain proteins for ubiquitination.

The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix.

Fibronectin coimmunoprecipitated with wild-type von Hippel-Lindau protein (pVHL) but not tumor-derived pVHL mutants. Immunofluorescence and biochemical fractionation experiments showed that fibronectin colocalized with a fraction of pVHL associated with the endoplasmic reticulum, and cold competition experiments suggested that complexes between fibronectin and pVHL exist in intact cells. Assembly of an extracellular fibronectin matrix by VHL-/- renal carcinoma cells, as determined by immunofluorescence and ELISA assays, was grossly defective compared with VHL+/+ renal carcinoma cells. Reintroduction of wildtype, but not mutant, pVHL into VHL-/- renal carcinoma cells partially corrected this defect. Finally, extracellular fibronectin matrix assembly by VHL-/- mouse embryos and mouse embryo fibroblasts (MEFs), unlike their VHL+/+ counterparts, was grossly impaired. These data support a direct role of pVHL in fibronectin matrix assembly.

The von Hippel-Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes affected individuals to the human VHL cancer syndrome and is associated with sporadic renal cell carcinomas (RCC) and brain hemangioblastomas. VHL-negative 786-0 RCC cells are tumorigenic in nude mice which is suppressed by the reintroduction of VHL. Remarkably, this occurs without affecting the growth rate and cell cycle profile of these cells in culture. The 786-0 cell line, like many cancer cells, fails to exit the cell cycle upon serum withdrawal. Here, it is shown that reintroduction of the wild-type VHL gene restores the ability of VHL-negative RCC cancer cells to exit the cell cycle and enter G0/quiescence in low serum. Both VHL-positive and VHL-negative RCC cells exit the cell cycle by contact inhibition. The cyclin-dependent kinase inhibitor, p27, accumulates upon serum withdrawal, only in the presence of VHL, as a result of the stabilization of the protein. We propose that the loss of wild-type VHL gene results in a specific cellular defect in serum-dependent growth control, which may initiate tumor formation. This is corrected by the reintroduction of wild-type VHL, implicating VHL as the first tumor suppressor involved in the regulation of cell cycle exit, which is consistent with its gatekeeper function in the kidney.

Regulation of hypoxia-inducible mRNAs by the von Hippel-Lindau tumor suppressor protein requires binding to complexes containing elongins B/C and Cul2.

The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.

Expression of a continuous open reading frame encoding subunits 1 and 2 of cytochrome c oxidase in the mitochondrial DNA of Acanthamoeba castellanii.

We have investigated the expression of a continuous open reading frame (ORF) present in the mitochondrial genome of Acanthamoeba castellanii and specifying the two largest subunits (COX1 and COX2) of the cytochrome c oxidase complex. Northern hybridization and primer extension analysis demonstrated that this ORF (cox1/2, 873 codons) is transcribed as part of a 4.7 kb RNA that also includes the upstream small subunit rRNA sequence. Between the cox1 and cox2 portions of the transcript, RNA sequence exactly matches gene sequence, excluding the possibility that a standard cox1 termination codon is created by post-transcriptional RNA processing or editing. Western analysis revealed an A. castellanii COX2 protein with a mobility matching that of mature COX2 from yeast (Saccharomyces cerevisiae) mitochondria. These observations indicate that although A. castellanii COX1 and COX2 are apparently translated from the same ORF, they do not exist in mature form as a COX1-COX2 "fusion" protein. Whereas translation of COX2 could potentially be initiated from an internal AUG codon in the cox1/2 ORF, COX1 must be generated either through an unusual translation termination mechanism acting between the cox1 and cox2 coding regions of the cox1/2 mRNA, or by co-translational or post-translational proteolytic processing of a translation product whose synthesis continues into the cox2 coding region. Because the cox2 nucleotide sequence predicts a COX2 protein considerably larger than that observed by Western analysis, A. castellanii COX2 may undergo additional post-translational processing to its final form.

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion).

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non-Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site-2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba-like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.

The mitochondrial DNA of the amoeboid protozoon, Acanthamoeba castellanii: complete sequence, gene content and genome organization.

In phylogenetic trees based on comparison of nuclear small subunit rRNA sequences, Acanthamoeba castellanii (an amoeboid protozoon) is positioned near the base of the radiation leading to the animals, fungi and plants. However, the specific affiliation of this protist with the major multicellular lineages of eukaryotes is currently uncertain. To further explore the evolutionary position of A. castellanii, we have determined the complete primary sequence of its mitochondrial genome. We find that the circular mtDNA (41,591 bp; 70.6% A+T) encodes two rRNAs (small subunit and large subunit), 16 tRNAs and 33 proteins (17 subunits of the respiratory chain and 16 ribosomal proteins). As well, this genome contains eight open reading frames (ORFs) larger than 60 codons and of undefined function. Two of these ORFs (orf124 and orf142) have homologs in other mtDNAs ("orf25" and "orfB", respectively), three are unique to A. castellanii mtDNA (orf83, orf115 and orf349), and three are intronic ORFs. Among notable features of A. castellanii mtDNA are the following: (1) Genes and ORFs are all encoded on the same strand and are tightly packed, with only 6.8% of the total sequence not having an evident coding function and intergenic spacer sequences ranging from only 1 to 616 bp (average 64 bp). Ten pairs of protein-coding genes overlap by up to 38 bp and two subunits of cytochrome oxidase (COX1 and COX2) are specified by a single continuous ORF. (2) Only three introns, all group I and each containing a free-standing ORF, are present; these are localized in the 3'-half of the large subunit rRNA gene. (3) The genome encodes fewer than the minimal number of tRNA species required to support mitochondrial protein synthesis, suggesting that additional tRNAs are imported from the cytosol into A. castellanii mitochondria. Of the 16 tRNAs specified by A. castellanii mtDNA (one with an 8-nucleotide anticodon loop), 13 have been shown or are predicted to undergo a novel form of RNA editing within the acceptor stem. (4) A modified genetic code is used in which UGA specifies tryptophan. (5) Repeated sequences and obvious small sequence motifs that might represent regulatory elements are absent. In overall size, gene content and organizational pattern, A. castellanii mtDNA most closely resembles the mtDNA of the chlorophycean alga Prototheca wickerhamii (55,326 bp; 74.2% A+T), but is quite different in these respects from the mtDNA of Chlamydomonas reinhardtii (15,758 bp; 54.8% A+T), another chlorophycean alga, as well from characterized animal and fungal mitochondrial genomes.(ABSTRACT TRUNCATED AT 250 WORDS)

The ribosomal RNA gene region in Acanthamoeba castellanii mitochondrial DNA. A case of evolutionary transfer of introns between mitochondria and plastids?

Acanthamoeba castellanii, an amoeboid protozoan, occupies an intriguing position in phylogenetic trees based on nuclear rRNA sequences, branching together with or near (as an outgroup to) green algae and land plants. To gain insight into the organization, expression and evolutionary affiliations of the mtDNA of this non-photosynthetic protist, we determined the sequence of a 7778 base-pair region containing the single-copy large subunit (LSU) and small subunit (SSU) rRNA genes (rnl and rns, respectively) of the approximately 40 kilobase-pair A. castellanii mitochondrial genome. We also sequenced the 5'- and 3'-terminal portions of the corresponding LSU and SSU rRNAs. In A. castellanii mtDNA, rnl is flanked both upstream and downstream by a cluster of five tRNA genes, with rns and then cox1 (the cytochrome oxidase subunit 1 gene) following immediately further downstream. These genes are all in the same transcriptional orientation and are separated by only short non-coding spacers. Although rnl and rns are organized in a novel way in A. castellanii mtDNA, their SSU and LSU rRNA products are strikingly similar to their eubacterial homologs in primary sequence, secondary structure and post-transcriptional modification. In these characteristics, the A. castellanii mitochondrial rRNAs much more closely resemble their counterparts in land plants than do the corresponding mitochondrial rRNAs in the green alga, Chlamydomonas reinhardtii. Although no intervening sequences have so far been found in the mitochondrial rnl of angiosperms (flowering plants), A. castellanii mitochondrial rnl contains three group I introns, all located within highly conserved regions in the 3'-half of the gene and each possessing a free-standing open reading frame (ORF). The insertion site of one of these introns is identical to that of the single group I intron in the chloroplast rnl of C. reinhardtii, and sequence comparison reveals that these two introns (one mitochondrial, the other chloroplast) are structurally homologous both within the core region and within the ORFs they encode. These observations are indicative of intron movement between mitochondria and chloroplasts, either intracellularly in a photosynthetic, remote common ancestor of A. castellanii and C. reinhardtii or, more recently, as a result of an intercellular exchange of genetic information.

Editing of transfer RNAs in Acanthamoeba castellanii mitochondria.

With the discovery of RNA editing, a process whereby the primary sequence of RNA is altered after transcription, traditional concepts of genetic information transfer had to be revised. The known RNA editing systems act mainly on messenger RNAs, introducing sequence changes that alter their coding properties. An editing system that acts on transfer RNAs is described here. In the mitochondria of Acanthamoeba castellanii, an amoeboid protozoan, certain transfer RNAs differ in sequence from the genes that encode them. The changes consist of single-nucleotide conversions (U to A, U to G, and A to G) that appear to arise posttranscriptionally, are localized in the acceptor stem, and have the effect of correcting mismatched base pairs. Editing thus restores the base pairing expected of a normal transfer RNA in this region.

Rhythms of cell proliferation in the hindlimb epidermis of control and thyroxine-treated Rana pipiens tadpoles.

Labeling and mitotic index rhythms were studied in premetamorphic tadpoles under an LD 12:12 with the light phase beginning at 0800 hr. In a 72-hr experiment, control labeling and mitotic index curves showed a peak in the light and a peak in the dark with labeling index rhythms of 12.4, 17.7, and 23.6 hr and a 21.4-hr mitotic index rhythm. Thyroxine (T4) treatment resulted in a marked elevation of labeling index by 24 hr and of mitotic index by 48 hr, obscured the control bimodal pattern of peaks, and altered the rhythms. During the first 3 days of T4 treatment, a labeling index rhythm of 22 hr and a mitotic index rhythm of 37.5 hr occurred. However, additional work demonstrated that the dominant control rhythms of labeling and mitotic indices returned in the T4-treated during Days 4 and 5. The same pattern of change in labeling index occurred during Day 3 of T4 treatment when hormone administration began at different times in the diurnal phase of the light-dark cycle. The findings suggest that cell proliferation rhythms can be temporarily disturbed by an exogenous T4 stimulus without apparent reference to the phase of the circadian rhythm.