Chromosomal aberrations in squamous cell carcinoma and solar keratoses revealed by comparative genomic hybridization

OBJECTIVE: To identify chromosomal copy numbers of frequent genetic aberrations within squamous cell carcinomas (SCCs) and solar keratoses (SKs), and provide further evidence to support or challenge current dogma concerning the relationship between these lesions. DESIGN: Retrospective analysis of genetic aberrations in DNA from SK and SCC biopsy specimens by comparative genomic hybridization. SETTING: University-based research laboratory in Queensland, Australia. PATIENTS: Twenty-two biopsy specimens from patients with diagnosed SKs (n = 7), cutaneous SCCs (n = 10), or adjoining lesions (n = 5). MAIN OUTCOME MEASURES: Identification of frequent genetic aberrations both specific to SK and SCC and shared by these lesions to investigate their clonal relationship. RESULTS: Shared genomic imbalances were identified in SK and SCC. Frequent gains were located at chromosome arms 3q, 17q, 4p, 14q, Xq, 5p, 9q, 8q, 17p, and 20q, whereas shared regional losses were observed at 9p, 3p, 13q, 17p, 11p, 8q, and 18p. Significant loss of 18q was observed only in SCC lesions. CONCLUSIONS: Our results demonstrate that numerous chromosomal aberrations are shared by the 2 lesions, suggesting a clonal relationship between SK and SCC. Additionally, the genomic loss of 18q may be a significant event in SK progression to SCC. Finally, the type and frequency of aberrations suggests a common mode of tumorigenesis in SCC-derived tumors.

Regional chromosomal assignment of human renin gene to 1q12→qter and use in linkage studies in Charcot-Marie-Tooth disease

The gene for renin, previously mapped to human chromosome 1, was further localized to 1q12 → qter using human-mouse somatic cell hybrid DNAs. The renin DNA probe used (λ HR5) could detect a HindIII restriction fragment length polymorphism. When used in studies of 12 informative families, no linkage could be found between the renin and Charcot-Marie-Tooth disease. Furthermore, an association of any renin allele with hypertension was not apparent.

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Chromosomal genotoxicity of nitrobenzene and benzonitrile

In order to investigate the chromosomal genotoxicity of nitrobenzene and benzonitrile, we studied the induction of micronuclei (MN) by these test compounds in V79 cells, as well as effects on the formation and stability of microtubules and on motor protein functions. No cytotoxicity was seen in V79 cell cultures in terms of Neutral red uptake after 18 h treatment with up to 1 mM nitrobenzene or 1 mM benzonitrile. Subsequently, a concentration range up to 100 μM was used in the experiments on induction of MN. Both test compounds exhibit a weak, but definitely positive test result compared to the solvent (DMSO) control. Minimal effect concentrations of nitrobenzene and benzonitrile appeared as low as 0.01 μM, and no-effect-concentrations were between 0.001 and 0.005 μM. Clearly enhanced MN rates were found at 0.1 μM and higher. Both, nitrobenzene and benzonitrile, induced mostly kinetochor (CREST)-positive micronuclei, thus characterising the chromosomal effects as aneugenic. In cell-free assays, a slight effect on tubulin assembly was observed at 1 mM nitrobenzene without addition of DMSO. Higher concentrations (5 mM) led to secondary effects. In presence of 1% DMSO, nitrobenzene exerted no detectable effect on tubulin assembly up to the solubility limit in water of about 15 mM. For benzonitrile in presence of DMSO, a clear dose-response of inhibition of tubulin assembly at 37°C was seen above the no-effect-concentration of 2 mM, with an IC50 of 13 mM and protein denaturation starting above a level of about 20 mM. The nature of the effects of nitrobenzene and benzonitrile on the association of tubulin to form microtubules was confirmed by electron microscopy. Treatment by either 5 mM nitrobenzene or 13 mM benzonitrile plus 1% DMSO left the microtubular structure intact whereas 5 mM nitrobenzene, in absence of DMSO, led to irregular cluster formations. The experiments demonstrate that both nitrobenzene and benzonitrile, in millimolar concentration ranges, may lead to interference with tubulin assembly in a cell-free system. The functionality of the tubulin-kinesin motor protein system was assessed using the microtubule gliding assay. Nitrobenzene affected the gliding velocity in a concentration-dependent manner, starting at about 7.5 μM and reaching complete inhibition of motility at 30 μM, whereas benzonitrile up to 200 μM did not affect the kinesin-driven gliding velocity. The micronucleus assay data demonstrate a chromosomal endpoint of genotoxicity of nitrobenzene and benzonitrile. Aneugenic effects of both compounds occur at remarkably low concentrations, with lowest-effect-concentrations being 0.1 μM. This points to the relevance of interactions with the cellular spindle apparatus.

Chromosomal rearrangements, phenotypic variation and modularity: A case study from a contact zone between house mouse Robertsonian races in Central Italy

The Western European house mouse, Mus musculus domesticus, is well-known for the high frequency of Robertsonian fusions that have rapidly produced more than 50 karyotipic races, making it an ideal model for studying the mechanisms of chromosomal speciation. The mouse mandible is one of the traits studied most intensively to investigate the effect of Robertsonian fusions on phenotypic variation within and between populations. This complex bone structure has also been widely used to study the level of integration between different morphogenetic units. Here, with the aim of testing the effect of different karyotypic assets on the morphology of the mouse mandible and on its level of modularity, we performed morphometric analyses of mice from a contact area between two highly metacentric races in Central Italy. We found no difference in size, while the mandible shape was found to be different between the two Robertsonian races, even after accounting for the genetic relationships among individuals and geographic proximity. Our results support the existence of two modules that indicate a certain degree of evolutionary independence, but no difference in the strength of modularity between chromosomal races. Moreover, the ascending ramus showed more pronounced interpopulation/race phenotypic differences than the alveolar region, an effect that could be associated to their different polygenic architecture. This study suggests that chromosomal rearrangements play a role in the house mouse phenotypic divergence, and that the two modules of the mouse mandible are differentially affected by environmental factors and genetic makeup.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Queer

The word “queer” is a slippery one; its etymology is uncertain, and academic and popular usage attributes conflicting meanings to the word. By the mid-nineteenth century, “queer” was used as a pejorative term for a (male) homosexual. This negative connotation continues when it becomes a term for homophobic abuse. In recent years, “queer” has taken on additional uses: as an all encompassing term for culturally marginalised sexualities – gay, lesbian, trans, bi, and intersex (“GLBTI”) – and as a theoretical strategy which deconstructs binary oppositions that govern identity formation. Tracing its history, the Oxford English Dictionary notes that the earliest references to “queer” may have appeared in the sixteenth century. These early examples of queer carried negative connotations such as “vulgar,” “bad,” “worthless,” “strange,” or “odd” and such associations continued until the mid-twentieth century. The early nineteenth century, and perhaps earlier, employed “queer” as a verb, meaning to “to put out of order,” “to spoil”, “to interfere with”. The adjectival form also began to emerge during this time to refer to a person’s condition as being “not normal,” “out of sorts” or to cause a person “to feel queer” meaning “to disconcert, perturb, unsettle.” According to Eve Sedgwick (1993), “the word ‘queer’ itself means across – it comes from the Indo-European root – twerkw, which also yields the German quer (traverse), Latin torquere (to twist), English athwart . . . it is relational and strange.” Despite the gaps in the lineage and changes in usage, meaning and grammatical form, “queer” as a political and theoretical strategy has benefited from its diverse origins. It refuses to settle comfortably into a single classification, preferring instead to traverse several categories that would otherwise attempt to stabilise notions of chromosomal sex, gender and sexuality.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.