Heterozygous Cell Lines Research Articles

Abstract 4731: Resequencing of the MYC-335 enhancer in the colorectal cancer associated 8q24 region

Abstract Genome-wide association studies have identified multiple different regions at 8q24 associated with risk of colorectal, prostate, breast and bladder cancers. It has been suggested that the cancer predisposition could be mediated through altered MYC expression. One of the regions, marked by SNP rs6983267, has been consistently shown to confer susceptibility to colorectal and prostate cancers. In our previous work, we aimed to understand the biological basis of the colorectal cancer (CRC) predisposition associated with the G allele of rs6983267. By utilizing a computer program, Enhancer Element Locator, we showed that rs6983267 affects a TCF4 binding site in an evolutionary conserved enhancer element (MYC-335). Enhancer activity of this element was confirmed in vitro and in vivo. The G allele of rs6983267 was shown to enhance the activity of the MYC-335, and in the presence of active Wnt signaling potentially lead to increased expression of a target gene, possibly MYC. To examine the contribution of other variants in the MYC-335 to CRC predisposition, we determined the genetic variation within MYC-335 in different ethnic groups by resequencing the 1271-bp genomic fragment in Caucasian, African and African American samples. This effort identified eight variants which showed population-specific allele frequencies. One variant, a 2-bp deletion, affected a putative transcription factor binding site and was present only in individuals with African origin. The deletion affected GA nucleotides at the fifth and sixth position of the putative binding sequence for transcriptional repressor GFI1 (CAGAGATTGC). The identified GFI1 binding sequence is evolutionary highly conserved and the deletion is predicted to cause a 5.5-fold lower binding affinity. Both the SNP rs6983267 and the GA deletion affect transcription factor binding sites and are differentially distributed in different ethnic populations, which suggests that both alleles are functional and under selective pressure from environmental exposure. We hypothesized that the deletion could contribute to CRC predisposition by abolishing the binding of the repressor, which in turn would lead to enhanced transcriptional activity. Here, we show preliminary results that GFI1 has preferential binding affinity for the wild type allele in the heterozygous cell line HeLa. Also, we investigated the role of the deletion in CRC predisposition in African American individuals. The 2-bp deletion identified here might be a novel population-specific colorectal cancer risk variant. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4731.

Multiplexed transposon-mediated stable gene transfer in human cells.

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes.

Stable Expression of Brain Sodium Channels in Human Cells by Multiplexed Transposon-Mediated Gene Transfer

Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to co-express multiple transgenes or multi-protein complexes. We harnessed the highly efficient, nonviral and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated ∼60% (three transposons) or ∼30% (four transposons) co-expression of all delivered transgenes despite selection of a single marker transposon. We validated multiplexed piggyBac transposon delivery by co-expressing large transgenes encoding a multi-subunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Co-transfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We concluded that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells and this technology may be valuable for applications requiring concurrent expression of multi-protein complexes.

Distinct Expression and Localization of the Ku Autoantigens in Patients with Chronic Lymphocytic Leukaemia (CLL).

Abstract 3239Poster Board III-176 BackgroundThe Ku autoantigen is a heterodimer composed of a 70kDa subunit (Ku70) and an 80kDa subunit (Ku86), which is critical for repairing DNA double-strand breaks (DSB) by non-homologous end joining and for telomere maintenance. Knockdown of either one or both Ku components results in rapid shortening of telomere ends and genomic instability (Fattah et al., DNA Repair 2008; Faure et al., DNA Repair 2008). We and others have demonstrated significantly shorter telomeres in patients with chronic lymphocytic leukemia (CLL) (Röth A et al, Brit. J. Haematol. 2008). In addition, in over 80% of patients with CLL cytogenetic aberrations are found (Döhner H, NEJM, 2000). Based on these observations we hypothesized that differences in Ku protein expression or function could be involved in the pathophysiology of CLL cells. Our aim was to assess the localization and expression levels of the Ku components in leukemic cells of patients with CLL Patients and MethodsSo far, we examined the localization and expression of the Ku-proteins in 20 patients with CLL (14 with mutated IgVH genes, 6 with unmutated IgVH genes), and in peripheral blood lymphocytes of 4 healthy individuals. The localization of the Ku proteins was assessed by immunological staining and fluorescence microscopy. Expression levels of the Ku components in whole cells as well as in the cytoplasmic and nuclear fractions were assessed by Western blotting. DNA sequencing was done by cycle sequencing and capillary electrophoresis. Telomere length measurements were performed by automated multicolor Flow-FISH. ResultsLymphocytes of healthy individuals with a normal telomere length showed an expression of Ku70 and Ku86 in the nucleus without any cytoplasmic expression. Interestingly, in leukemic cells from CLL patient samples, Ku86 was not detectable in the nucleus, but was abundantly found in the cytoplasm, while Ku70 was found in approximately equal amounts in the nucleus and cytoplasm. The expression levels of both Ku proteins were comparable in lymphocytes from CLL patients and from healthy controls. Sequence analysis of the Ku70 and Ku86 DNA revealed no mutations. All leukemic cells of CLL patients had short telomeres below the 50th percentile of healthy individuals with significantly shorter telomeres in patient samples with unmutated IgVH (mean: 2.8kb ± 0.03kb) than with mutated IgVH (mean: 6.15kb ± 2.92kb). In contrast, no difference in the expression pattern of Ku70 and Ku86 was found between these two prognostic subgroups of CLL patients. ConclusionFor the first time we can demonstrate clear differences in the expression and localization of the Ku proteins in leukemic cells of patients with CLL with short telomeres compared to lymphocytes of healthy individuals with telomeres in the normal range. The high cytoplasmic expression of both Ku proteins in the leukemic cells of CLL patients and the lack of nuclear Ku86 protein suggested the presence of a genetic alteration. Although we could not find any mutations in the DNA sequence of the Ku proteins, the delocalization could point to changes in the Ku proteins (i.e. by post-translational modifications) and/or in the transport system of the Ku proteins between the nucleus and the cytoplasm. Deficient nuclear expression of Ku86 and/or Ku70 in CLL cells might lead to shortening of telomeres and genomic instability as has been seen in heterozygous Ku86 deficient human cell lines. Since there was no difference in the expression and localization of the Ku proteins in CLL cells with mutated and unmutated IgVH it is unlikely that the absolute telomere length, which differs highly between the two subgroups of CLL patients, is directly related to the level of the nuclear Ku protein expression. Further molecular and functional studies of the Ku proteins are ongoing. DisclosuresNo relevant conflicts of interest to declare.

Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect

Mutations in the gene encoding phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate "heterozygous" and "homozygous" states in patients. Mutants in the N-terminus, C-terminus, and kinase domain were generated and cloned into a two-gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild-type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (DeltaPsi(m)) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in DeltaPsi(m), and increased time-dependent cell loss when subjected to oxidative stress compared to the wild-type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and DeltaPsi(m) decrease than those expressing one copy of the mutant. In time-dependent experiments, there was a significant difference between "homozygous," "heterozygous," and wild-type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild-type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss.

Neutrophil gelatinase-associated lipocalin suppresses cyst growth by Pkd1 null cells in vitro and in vivo

Cyst growth in patients with autosomal dominant polycystic kidney disease is thought to be due to increased tubular cell proliferation. One model to explain this altered proliferation suggests that the polycystin proteins PC1 and PC2 localize to apical cilia and serve as an integral part of the flow-sensing pathway thus modulating the proliferative response. We measured proliferation and apoptosis in proximal tubule derived cell lines lacking PC1. These cells showed increased rates of proliferation, a decreased rate of apoptosis, compared to control heterozygous cell lines, and spontaneously formed cysts rather than tubules in an in vitro tubulogenesis assay. Addition of neutrophil gelatinase associated lipocalin (NGAL), a small secreted protein that binds diverse ligands, to the cells lacking PC1 inhibited proliferation and increased apoptosis leading to slower cyst growth in vitro. Sustained over-expression at low level of NGAL by an adenoviral delivery system suppressed cyst enlargement without improving renal function in the Pkd1 mutant mice. Our studies show that renal epithelial cells lacking PC1 have an inherent tendency to hyper-proliferate forming cysts in vitro independent of a flow stimulus. The potential benefit of attenuating cyst growth with NGAL remains to be determined.

Impact of EGFR Gene Polymorphisms on Anticancer Drug Cytotoxicity In Vitro

The epidermal growth factor receptor (EGFR) plays a major role in cell proliferation of epithelial tissues, and its alterations frequently contribute to oncogenesis. Several common polymorphisms of the EGFR gene have been described, at the level of both coding and regulatory sequences. Some of these polymorphisms are associated with alterations of EGFR expression and/or activity and may have an impact on the activity of anticancer agents. This study aims to analyze the relationships between specific EGFR functional polymorphisms and anticancer drug activity. We investigated, in the panel of 60 human tumor cell lines established by the National Cancer Institute (NCI-60), whether the EGFR polymorphisms -216G>T, -191C>A, Arg521Lys (R521K), Val592Ala (V592A), and Cys624Phe (C624F), and the intron 1 (CA)n repeat were associated with EGFR gene expression and the in vitro cytotoxicity of anticancer agents using data extracted from the NCI database. We also looked for mutations of exons 18-21, known to enhance the activity of tyrosine kinase inhibitors, and the deletion of exons 2-7, associated to the oncogenesis of glioblastomas. In the NCI-60 cell lines, only two mutations were observed, both in exon 19, in a leukemia and melanoma cell line. These mutations have not been described previously in clinical samples and their functional role is uncertain. The allele frequencies of the -216G>T, -191C>A, and R521K single nucleotide polymorphisms (SNPs) in the NCI-60 panel were 33%, 8.5%, and 27%, respectively; the V592A and C624F SNPs were not found in any NCI-60 cell line. The intron 1 CA repeat was highly variable in the cell lines; 32 cell lines having a total number of repeats below 35, and 27 having a total number of repeats above 35. The heterozygous and variant homozygous cell lines for the -216G>T SNP presented a significantly higher expression of the EGFR gene than the homozygous wild-type lines. In contrast, there was no association between the -191C>A or R521K SNPs and EGFR gene expression. No association could be detected between the number of CA repeats in intron 1 and the expression of EGFR. The cell lines having at least one variant T allele at the -216G>T SNP were more sensitive to erlotinib and less sensitive to geldanamycin, topoisomerase I and II inhibitors, and alkylating agents than those without a variant allele. No relationship was detected between anticancer drug sensitivity and the -191C>A SNP. The R521K SNP was associated to lower sensitivity to alkylating agents. The number of CA repeats was associated with significant differences in anticancer drug activity: a high total number of CA repeats (>35 per diploid genome) was associated to increased sensitivity to alkylating agents and topoisomerase I and II inhibitors. We provide evidence in this work that EGFR polymorphisms are associated with significant differences in the in vitro cytotoxicity of several types of DNA-interfering agents. Studies attempting a clinical validation of these clues are warranted.

Haplotype‐specific expression of the human PDGFRA gene correlates with the risk of glioblastomas

Aberrant expression of the platelet-derived growth factor alpha-receptor (PDGFRA) gene has been associated with various diseases, including neural tube defects and gliomas. We have previously identified 5 distinct haplotypes for the PDGFRA promoter region, designated H1, H2alpha, H2beta, H2gamma and H2delta. Of these haplotypes H1 and H2alpha are the most common, whereby H1 drives low and H2alpha high transcriptional activity in transient transfection assays. Here we have investigated the role of these PDGFRA promoter haplotypes in gliomagenesis at both the genetic and cellular level. In a case-control study on 71 glioblastoma patients, we observed a clear underrepresentation of H1 alleles, with pH1 = 0.141 in patients and pH1 = 0.211 in a combined Western European control group (n = 998, p < 0.05). Furthermore, in 3 out of 4 available H1/H2alpha heterozygous human glioblastoma cell lines, H1-derived mRNA levels were more than 10-fold lower than from H2alpha, resulting at least in part from haplotype-specific epigenetic differences such as DNA methylation and histone acetylation. Together, these results indicate that PDGFRA promoter haplotypes may predispose to gliomas. We propose a model in which PDGFRA is upregulated in a haplotype-specific manner during neural stem cell differentiation, which affects the pool size of cells that can later undergo gliomagenesis.

Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Gene targeting by homologous recombination is a powerful and widely used technique for introduction of specific gene mutations (frequently a gene inactivation) in transgenic animals. The basic method detailed in this unit uses sequences homologous to the endogenous gene flanking the mutation. While methods using bacterial artificial chromosomes (BACs) and recombineering may be used, in most cases simpler bacterial plasmid clones with several kb of homology are sufficient. This protocol details the strategic factors in designing the constructs for selection and screening for homologous recombination.

Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

Highly Active Antiretroviral Therapy Does Not Affect Mitochondrial β-Oxidation of Fatty Acids: An in Vitro Study in Fibroblasts

Preeclampsia is a multifactorial pregnancy-specific disease. In some cases, severe preeclampsia and related disorders of acute fatty liver of pregnancy and hemolysis, elevated liver enzymes, low platelets syndrome are associated with inherited defects in mitochondrial beta-oxidation of fatty acids, especially a deficiency of long-chain 3-hydroxyacyl coenzyme A dehydrogenase (LCHAD). Recently, an unexplained increase in the incidence of preeclampsia has been documented in human immunodeficiency virus (HIV)-infected pregnant women on treatment with highly active antiretroviral therapy (HAART). We performed this study to determine if antiretroviral drugs affect mitochondrial beta-oxidation fatty acids in vitro. Two normal and 1 heterozygous LCHAD-deficient cell lines were exposed to up to 5 times the therapeutic concentrations of the following antiretroviral drugs: nevirapine, didanosine, lamivudine, and a combination of nelfinavir, zidovudine, and lamivudine. One homozygous LCHAD-deficient cell line served as the positive control. After exposure of the fibroblasts to these drugs for periods ranging from 2 to 10 days, accumulations of even-chain 3-hydroxy fatty acids (3-OH-C6 to 3-OH-C18) in the culture media were measured by stable-isotope dilution gas chromatography/mass spectrometry. Compared to the respective unexposed fibroblasts, there was no significant build-up of 3-hydroxy fatty acids in the culture media of normal or heterozygous LCHAD-deficient fibroblasts exposed to antiretroviral drugs. Our results show that the commonly used antiretroviral drugs do not adversely affect fatty acid oxidation in fibroblasts. Therefore, an altered fatty acid oxidation may not be the mechanism for the reported increased risk of preeclampsia in HIV-infected pregnant women on HAART.

Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

Haplotype-specific expression of the N-terminal exons 2 and 3 at the human MAPT locus

The microtubule-associated protein tau ( MAPT) H1 haplotype shows a strong association to the sporadic neurodegenerative diseases, progressive supranuclear palsy and corticobasal degeneration. The functional biological mechanisms behind the genetic association have started to emerge with differences recently shown in haplotype splicing of the neuropathologically relevant exon 10. Here we investigate the hypothesis that expression of the alternatively spliced N-terminal exons also differs between the two MAPT haplotypes. We performed allele-specific gene expression analysis on a H1/H2 heterozygous human neuronal cell line model and 14 H1/H2 heterozygous human post-mortem brain tissues from two brain regions. In both cell culture and post-mortem brain tissue, we show that the protective MAPT H2 haplotype significantly expresses two-fold more 2N (exons 2+3+) MAPT transcripts than the disease-associated H1 haplotype. We suggest that inclusion of exon 3 in MAPT transcripts may contribute to protecting H2 carries from neurodegeneration.

Early Embryonic Lethality of Mice Lacking the Essential Protein SNEV

SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development. Mutant preimplantation embryos initiated blastocyst formation but died shortly thereafter. Outgrowth of SNEV-null blastocysts showed a lack of proliferation of cells of the inner cell mass, which subsequently underwent cell death. While SNEV-heterozygous mice showed no overt phenotype, heterozygous mouse embryonic fibroblast cell lines with reduced SNEV levels displayed a decreased proliferative potential in vitro. Our experiments demonstrate that the SNEV protein is essential, functionally nonredundant, and indispensable for mouse development.

Aberrant Expression of X-Linked Genes RbAp46, Rsk4, and Cldn2 in Breast Cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.

DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-29 cells.

Functional loss of both alleles of the breast cancer susceptibility gene, BRCA2, facilitates tumorigenesis. However, the direct effects of BRCA2 heterozygosity remain unclear. Here, BRCA2 heterozygosity was mimicked in HT-29 colon cells by reducing levels of BRCA2 through stable RNA interference. No difference in RAD51 subcellular localization and focus formation was observed between control and mimicked heterozygous cell lines. DNA repair ability, as measured by colony survival following mitomycin C treatment and ultraviolet radiation exposure, was also unaffected by reduced levels of BRCA2. Interestingly, the growth rate of the mimicked BRCA2 heterozygous cell line was significantly lower than that of control cells. Increased expression of p53 in the mimicked heterozygous cells was observed, perhaps in response to BRCA2 deficiency. Levels of p27 were also found to be slightly increased in cells with reduced BRCA2, perhaps contributing to the slower growth rate. Overall, these results suggest that tumors are unlikely to arise directly from BRCA2 heterozygous cells without other genetic events such as loss of the wild-type BRCA2 allele and/or loss of p53 function or other cell cycle inhibitors.

Haplotype-specific expression of exon 10 at the human MAPT locus

Neurofibrillary tangles composed of exon 10+ microtubule associated protein tau (MAPT) deposits are the characteristic feature of the neurodegenerative diseases progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). PSP, CBD and more recently Alzheimer's disease and Parkinson's disease, are associated with the MAPT H1 haplotype, but the relationship between genotype and disease remains unclear. Here, we investigate the hypothesis that H1 expresses more exon 10+ MAPT mRNA compared to the other haplotype, H2, leading to a greater susceptibility to neurodegeneration in H1 carriers. We performed allele-specific gene expression on two H1/H2 heterozygous human neuronal cell lines, and 14 H1/H2 heterozygous control individual post-mortem brain tissue from two brain regions. In both tissue culture and post-mortem brain tissue, we show that the MAPT H1 haplotype expresses significantly more exon 10+ MAPT mRNA than H2. In post-mortem brain tissue, we show that the total level of MAPT expression from H1 and H2 is not significantly different, but that the H1 chromosome expresses up to 1.43-fold more exon 10+ MAPT mRNA than H2 in the globus pallidus, a brain region highly affected by tauopathy (maximum exon 10+ MAPT H1:H2 transcript ratio=1.425, SD=0.205, P<0.0001), and up to 1.29-fold more exon 10+ MAPT mRNA than H2 in the frontal cortex (maximum exon 10+ MAPT H1:H2 transcript ratio=1.291, SD=0.315, P=0.006). These data may explain the increased susceptibility of H1 carriers to neurodegeneration and suggest a potential mechanism between MAPT genetic variability and the pathogenesis of neurodegenerative disease.

The BCL2 major breakpoint region (mbr) regulates gene expression

BCL2 expression is finely tuned by a variety of environmental and endogenous stimuli and regulated at both transcriptional and post-transcriptional levels. Our previous investigations demonstrated that the BCL2 major breakpoint region (mbr) in the 3'-UTR upregulates reporter gene expression, which implies that this region possessed intrinsic regulatory function. However, the effect of the mbr on BCL2 expression, and the underlying regulatory mechanisms, remain to be elucidated. To assess the direct effect of the mbr on the transcriptional activity of the BCL2 gene, we employed targeted homologous recombination to establish a mbr(+)/mbr(-) heterozygous Nalm-6 cell line and then compared the transcriptional activity and apoptotic effect on transcription between the wild type and targeted alleles. We found that deletion of the mbr significantly decreased the transcriptional activity of the corresponding allele in the mbr(+)/mbr(-) cell. The BCL2 allele deleted of the mbr had a slower response to apoptotic stimuli than did the wild type allele. The regulatory function of the mbr was mediated through SATB1. Overexpression of SATB1 increased BCL2 expression, while knockdown of SATB1 with RNAi decreased BCL2 expression. Our results clearly indicated that the mbr could positively regulate BCL2 gene expression and this regulatory function was closely related to SATB1.

Characterization of the breast cancer associated ATM 7271T&gt;G (V2424G) mutation by gene expression profiling

Mutations in ATM are responsible for the autosomal recessive disorder ataxia telangiectasia. Heterozygous mutations in ATM have been associated with an elevated risk of breast cancer. We previously reported one breast cancer family in which ATM 7271T>G (V2424G) segregated with disease, and apparently acted in a dominant negative manner. We now report the screening of 782 multiple-case breast cancer families that identified two additional index cases with ATM 7271T>G. Phylogenetic sequence analysis showed that V2424 is a highly conserved residue, and that the 2424G variant is likely to interfere with function. To elucidate the consequences of this mutation, we expression profiled wild-type, heterozygous, and homozygous lymphoblastoid cell lines (LCLs) from Scottish and Australian families using an oligonucleotide microarray. Cluster analysis revealed 77 genes that were differentially expressed in homozygous and heterozygous V2424G cells (compared to wild-type) and 11 genes differentially expressed in the homozygous cells. We also evaluated the profiles of LCLs after exposure to ionizing radiation (IR) and identified 77 genes that were differentially expressed in wild-type cells, but not in homozygous or heterozygous V2424G cells. We validated the expression differences by RT-PCR in additional heterozygous V2424G LCLs from another breast cancer family. We found no consistent cytotoxicity or abrogation of ATM kinase activity after IR in seven heterozygous V2424G LCLs, compared to wild-type LCLs, but did find an increase in the number of chromosomal aberrations. These data suggest that the V2424G missense mutation acts largely as a dominant negative in terms of the associated expression profiles.

Relationships Between Genetic Polymorphisms and Anticancer Drug Cytotoxicity Vis-à-Vis the NCI-60 Panel

The National Cancer Institute (NCI)-60 panel consists of 60 human tumor cell lines initially established for screening thousands of molecules for antiproliferative activity. It has been powerful for deciphering the relationships between anticancer drug cytotoxicity and cell molecular characteristics. We tested its potential interest for establishing relationships between the polymorphism of genes involved in drug metabolism and transport or in DNA repair, and drug cytotoxicity extracted from NCI databases. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques, three frequent single nucleotide polymorphisms (SNPs) were analyzed: Lys751Gln in the Xeroderma pigmentosum complementation group D (XPD, ERCC2) gene, Asp1104His in the Xeroderma pigmentosum complementation group G (XPG, ERCC5) gene and Ile105Val in the glutathione S-transferase P1 (GSTP1) gene. The allelic frequencies of the variants were 33% for ERCC2, 23% for ERCC5 and 39% for GSTP1. The ERCC2 polymorphism appeared to be a strong determinant of the in vitro cytotoxicity of most anticancer agents, with lower half maximal inhibitory concentration (IC50) values in variant homozygous lines than in common homozygous or heterozygous cell lines. Unexpectedly, the cytotoxicity of taxanes appeared markedly dependent upon the ERCC2 genotype, with threefold lower mean IC50 values in variant homozygous cell lines. The ERCC5 genotype appeared to be important only for taxanes, with fourfold higher IC50 values in variant homozygous cell lines. The GSTP1 polymorphism was related to the cytotoxicity of several drug classes, especially topoisomerase inhibitors, antimetabolites and N7 alkylating agents. The NCI-60 panel is capable of providing clues and tracks for the establishment of clinically useful relationships between a given genotype and the cytotoxicity of an anticancer agent.