Biomedical subjects

Ursula R Kees

Publications and source records attributed to Ursula R Kees.

At least 19 recordsLinked to original sources

PubMed2006

The role of MYCN in the failure of MYCN amplified neuroblastoma cell lines to G1 arrest after DNA damage.

We previously reported that 3 p53 wild type (wt) MYCN amplified (MNA) neuroblastoma cell lines failed to G1 arrest after DNA damage despite induction of p53, p21(WAF1) and MDM2. We hypothesised that this was due to high MYCN expression. p53 responses to DNA damage were examined in an additional 13 p53 wt neuroblastoma cell lines. MNA was significantly associated with a failure to G1 arrest after DNA damage (p < 0.001) and higher levels of apoptosis after irradiation (p < 0.05). p21(WAF1) and hypophosphorylated (hypo) RB accumulation post irradiation were significantly lower in cell lines that failed to G1 arrest (p < 0.05). Conditional MYCN expression in non-MNA SHEP Tet21N cells did not affect the G1 arrest after irradiation. MYCN knockdown using siRNA in 3 p53 wt MNA cell lines did not restore a G1 arrest after irradiation, but increased the baseline G1 population, p21(WAF1) and hypo RB expression. MYCN siRNA also caused a G1 arrest in a p53 mutant MNA cell line. This study is the first to determine that MNA correlates with a failure to G1 arrest and attenuated p21(WAF1) induction; however MYCN expression alone is not causally responsible.

Apoptosis↗

PubMed2006

Translating microarray data for diagnostic testing in childhood leukaemia.

BACKGROUND: Recent findings from microarray studies have raised the prospect of a standardized diagnostic gene expression platform to enhance accurate diagnosis and risk stratification in paediatric acute lymphoblastic leukaemia (ALL). However, the robustness as well as the format for such a diagnostic test remains to be determined. As a step towards clinical application of these findings, we have systematically analyzed a published ALL microarray data set using Robust Multi-array Analysis (RMA) and Random Forest (RF). METHODS: We examined published microarray data from 104 ALL patients specimens, that represent six different subgroups defined by cytogenetic features and immunophenotypes. Using the decision-tree based supervised learning algorithm Random Forest (RF), we determined a small set of genes for optimal subgroup distinction and subsequently validated their predictive power in an independent patient cohort. RESULTS: We achieved very high overall ALL subgroup prediction accuracies of about 98%, and were able to verify the robustness of these genes in an independent panel of 68 specimens obtained from a different institution and processed in a different laboratory. Our study established that the selection of discriminating genes is strongly dependent on the analysis method. This may have profound implications for clinical use, particularly when the classifier is reduced to a small set of genes. We have demonstrated that as few as 26 genes yield accurate class prediction and importantly, almost 70% of these genes have not been previously identified as essential for class distinction of the six ALL subgroups. CONCLUSION: Our finding supports the feasibility of qRT-PCR technology for standardized diagnostic testing in paediatric ALL and should, in conjunction with conventional cytogenetics lead to a more accurate classification of the disease. In addition, we have demonstrated that microarray findings from one study can be confirmed in an independent study, using an entirely independent patient cohort and with microarray experiments being performed by a different research team.

Cell Line, Tumor↗

PubMed2006

Is there a folate-related gene-environment interaction in the etiology of childhood acute lymphoblastic leukemia?

Acute lymphoblastic leukaemia (ALL) is the commonest childhood cancer in developed countries. Little is known about its causes, although its early age at diagnosis has focused interest on maternal and perinatal factors. We have previously observed a protective effect of maternal folate supplementation during pregnancy against ALL, and a number of studies have reported protective effects of some common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene. One study has suggested that the effect of MTHFR polymorphisms on risk of ALL may depend on folate status. This study aimed to look for evidence of an interaction between maternal folate supplementation and child's genotype among the cases from our previous study. Bone marrow specimens from 82 of 83 case children were available. DNA was extracted and genotyped for MTHFR C677T and A1298C using standard techniques. We used a case-only analysis to estimate the case-only odds ratio (COR) for MTHFR genotype and folate supplementation in association with ALL. None of the CORs indicated a significant departure from a multiplicative model. Adjustment for sex, age or genotype at the other locus had little effect on the results. Other studies of this gene and environment interaction in ALL and other cancers have produced contradictory results, perhaps because of varying definitions of folate exposure. Further research into the interaction of folate intake and genotype in causing ALL and other cancers is needed. We are specifically studying it in an Australian national case-control study of genetic and environmental causes of ALL.

Alanine↗

PubMed2006

Increased frequency of aberrations in the p53/MDM2/p14(ARF) pathway in neuroblastoma cell lines established at relapse.

p53 mutations have been reported in cell lines derived from relapsed neuroblastoma tumors. We hypothesize that functional inactivation of p53 by mutation or other mechanisms is common in relapsed neuroblastoma and can contribute to chemoresistance. Our aim was to determine the frequency of p53 mutations, p14(ARF) methylation, or deletion and MDM2 amplification in 23 neuroblastoma cell lines (6 derived at diagnosis and 17 derived at relapse). One cell line was p53 mutant (BE2c) and two cell lines were deleted for p14(ARF) (LAN-6 and SHEP). Two cell lines were methylated for p14(ARF) (GIMEN and PER-108), one of which had low levels of p14(ARF) mRNA expression which increased following demethylation with 5-aza-2/deoxycytidine treatment (GIMEN), and four cell lines were confirmed to be MDM2-amplified. All these cell lines were derived from neuroblastomas at relapse. Inactivation of the p53 pathway was observed in 9 out of 17 neuroblastoma cell lines (53%) established at relapse and in none of the cell lines established from pretreatment tumors. If these data are confirmed in neuroblastoma tumors, this suggests that p53-independent therapy and reactivation of inactive p53 approaches would be useful in the management of relapsed neuroblastoma.

Cell Line, Tumor↗

PubMed2005

Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR -- how well do they correlate?

BACKGROUND: The use of microarray technology to assess gene expression levels is now widespread in biology. The validation of microarray results using independent mRNA quantitation techniques remains a desirable element of any microarray experiment. To facilitate the comparison of microarray expression data between laboratories it is essential that validation methodologies be critically examined. We have assessed the correlation between expression scores obtained for 48 human genes using oligonucleotide microarrays and the expression levels for the same genes measured by quantitative real-time RT-PCR (qRT-PCR). RESULTS: Correlations with qRT-PCR data were obtained using microarray data that were processed using robust multi-array analysis (RMA) and the MAS 5.0 algorithm. Our results indicate that when identical transcripts are targeted by the two methods, correlations between qRT-PCR and microarray data are generally strong (r = 0.89). However, we observed poor correlations between qRT-PCR and RMA or MAS 5.0 normalized microarray data for 13% or 16% of genes, respectively. CONCLUSION: These results highlight the complementarity of oligonucleotide microarray and qRT-PCR technologies for validation of gene expression measurements, while emphasizing the continuing requirement for caution in interpreting gene expression data.

Algorithms↗

PubMed2005

Genomic deletions in cell lines derived from primitive neuroectodermal tumors of the central nervous system.

Extensive genomic deletions affecting a variety of chromosomes are a common finding in primitive neuroectodermal tumors of the central nervous system (CNS-PNETs), implicating the loss of multiple tumor suppressor genes in the pathogenesis of these tumors. We have used representational difference analysis, microsatellite mapping, and quantitative polymerase chain reaction to identify and verify the presence of genomic deletions on a number of chromosomes in CNS-PNET cell lines. This systematic approach has confirmed the importance of deletions at 10q, 16q, and 17p in PNET pathology and has revealed other regions of deletion not commonly described (e.g., Xq, 1p, 7p, and 13q). These data highlight the prevalence of hemizygous loss in CNS-PNET cells, suggesting that haploinsufficiency affecting multiple tumor suppressor genes may play a fundamental role in CNS-PNET pathogenesis. The identification of specific genes and signaling pathways that are compromised in CNS-PNET cells is crucial for development of more efficacious and less invasive treatments, as are urgently needed.

Cell Line, Tumor↗

PubMed2005

Detection of hemizygous deletions in genomic DNA from leukaemia specimens for the diagnosis of patients.

Hemizygous deletions in genomic DNA appear to play an important role in tumorigenesis. The loss or inactivation of tumour suppressor genes (TSGs) is of critical importance in most malignancies, and has been shown to affect response to therapy. Here, we report a quantitative real-time polymerase chain reaction (qPCR) designed to detect two TSGs at the CDKN2A locus, p16(INK4A) and p14(ARF) that allows the detection of hemizygous deletions. Testing by qPCR of 18 bone marrow specimens from paediatric acute lymphoblastic leukaemia (ALL) patients at diagnosis revealed nine to be GG, six to be GD and three to be DD for exon 2 of p14(ARF)/p16(INK4A), concordant with Southern blotting analysis. A panel of 13 ALL cell lines was investigated for deletions at the CDKN2A locus and one of the lines, typed as GD for all exons, was further assessed by fluorescence in situ hybridisation, confirming the qPCR findings. The expression levels of p16(INK4A) and p14(ARF) were measured in all cell lines and these quantitative reverse transcriptase PCR results also agreed with the typing by qPCR. The qPCR method described is suitable for detection of hemizygous loss in primary patient material and the accuracy of the method was verified by three independent techniques.

Alleles↗

PubMed2005

The gene expression signature of relapse in paediatric acute lymphoblastic leukaemia: implications for mechanisms of therapy failure.

Despite significant improvements in the treatment of childhood acute lymphoblastic leukaemia (ALL), the prognosis for relapsing patients remains poor. The aim of this study was to generate a transcriptional profile of relapsed ALL to increase our understanding of the mechanisms involved in therapy failure. RNA was extracted from 11 pairs of cryopreserved pre-B ALL bone marrow specimens taken from the same patients at diagnosis and relapse, and analysed using HG-U133A microarrays. Relapse specimens overexpressed genes that are involved with cell growth and proliferation, in keeping with their aggressive phenotype. When tested in 72 independent specimens of pre-B ALL and T-ALL, the identified genes could successfully differentiate between diagnosis and relapse in either lineage, indicating the existence of relapse mechanisms common to both. These genes have functions relevant for oncogenesis, drug resistance and metastasis, but are not related to classical multidrug-resistance pathways. Increased expression of the top-ranked gene (BSG) at diagnosis was significantly associated with adverse outcome. Several chromosomal loci, including 19p13, were identified as potential hotspots for aberrant gene expression in relapsed ALL. Our results provide evidence for a link between drug resistance and the microenvironment that has previously only been considered in the context of solid tumour biology.

Adolescent↗

PubMed2005

Gene expression levels in small specimens from patients detected using oligonucleotide arrays.

Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.

Child↗

PubMed2004

Deletion of one copy of the p16INK4A tumor suppressor gene is implicated as a predisposing factor in pediatric leukemia.

The p16INK4A tumor suppressor gene is frequently disrupted by mutation or deletion in a wide range of cancer types, ranging from leukemia to cancers of the bladder, skin, lung, liver, and spleen. We have previously shown that deletion of at least one copy of the p16INK4A gene is associated with an increased risk of relapse in pediatric leukemia. Our data suggest that hemizygous p16INK4A deletion may be constitutional, conferring susceptibility to leukemia. Confirmation of this association is worthy of a larger study. Data from primary leukemia specimens are also presented here which examined the possibility that the remaining allele of the gene was inactivated by another mechanism such as mutation or was silenced by methylation. These possibilities were formally excluded in a case of hemizygous loss of the p16INK4A gene in leukemia, establishing that in this case the p16INK4A deletion was either semidominant or fully haploinsufficient for relapse susceptibility in this disease. Implementation of high throughput methods such as those used here for detecting hemizygous loss of tumor suppressor genes will become increasingly important for molecular diagnosis of cancer. This is particularly true for the emerging class of tumor suppressor genes where deletion of one allele is sufficient to confer cancer susceptibility or poor prognosis with standard treatment.

Adolescent↗

PubMed2004

The functional mapping of long-range transcription control elements of the HOX11 proto-oncogene.

Mapping of transcriptional control elements normally depends on the generation of a series of deletion mutants. The consequences of particular deletions are then functionally assessed by their ability to alter gene expression. The information derived from such investigations provides a general regulatory profile of the gene of interest, as well as generating a focus for future experiments. Due to the limitations of conventional DNA cloning methods, it has previously not been possible to use such an approach to rapidly assess the role of long-range regulatory elements that frequently lie further than 20 kb away from the coding region. In order to identify regulatory elements of the proto-oncogene HOX11 that may be mutated in a subset of childhood T-cell acute lymphoblastic leukaemia specimens, we generated nested deletions from a P1 artificial chromosome (PAC). This clone contained 95 kilobases (kb) of the HOX11 locus at 10q24; including 63 kb of 5' regulatory DNA. The deletion series was produced by the use of a recombination based cloning system and clones were subsequently transfected into mammalian cells. We have identified several long-range regulatory elements that mediate transcriptional control of HOX11. This approach is simple, rapid, and inexpensive. Furthermore, it generates multiple deletion clones in a single experiment. This novel approach opens up a new avenue for investigating long-range transcription control. Additionally, by allowing analysis of these elements in the natural context of large integrants the approach does not require the use of artificial extrachromosomal elements. This methodology can be applied to any gene cloned into a PAC or BAC vector and could also be useful in identifying appropriately sized deletion mutants for functional testing in transgenic models.

Cell Line, Tumor↗

PubMed2004

Regions syntenic to human 17q are gained in mouse and rat neuroblastoma.

Gain of chromosome arm 17q is the most frequent chromosomal change in human neuroblastoma and is a powerful predictor of adverse outcome of disease. This suggests that the region of gain includes a gene or genes critical for tumor pathogenesis. Analyses of breakpoint positions have revealed that the shortest region of gain (SRG) extends from MPO (17q23.1) to 17qter. Because this encompasses >300 genes, it precludes the identification of candidate genes from human breakpoint data alone. However, mouse chromosome 11, which is syntenic to human chromosome 17, is gained in up to 30% of neuroblastoma tumors developed in a murine MYCN transgenic model of this disease. To confirm that this key genetic change indicates the involvement of a molecular pathway conserved between mouse and man and is not occurring coincidentally in the transgenic model, we used fluorescence in situ hybridization to analyze sporadic cases of both mouse and rat neuroblastoma. Our results confirmed the presence of chromosome 11 gain in all three of the mouse cell lines we analyzed, with the SRG extending from Stat5b (101.6 Mb) to tel. In addition, the rat neuroblastoma cell line harbors an extra copy of distal chromosome 10, extending from 92.8 to 109.3 Mb, which is also syntenic to human 17q. Comparison of the regions gained in all three species has excluded 4.2 Mb from the previously defined region of 17q gain in humans as a likely location of the candidate gene or genes, and strongly suggests that the molecular etiology of neuroblastoma is similar in all three species.

Animals↗

PubMed2004

A microarray model system identifies potential new target genes of the proto-oncogene HOX11.

HOX11 is a homeobox gene originally identified at a chromosomal breakpoint in T-cell acute lymphoblastic leukemia (T-ALL). It is one of the most frequently deregulated genes in T-ALL, although the precise role of HOX11 in leukemogenesis as well as in normal development remains obscure. To gain more insight into the functional role of HOX11, we utilized a microarray model system to characterize the gene expression network that it directs. Using one of our T-ALL cell lines that had been stably transfected to express HOX11 and high-density oligonucleotide HG-U95A arrays, we identified a large number of differentially expressed genes in response to the enforced expression of HOX11. We focused on examining genes found to be up-regulated according to the microarray analysis and selected three putative target genes, NFKB2, SMARCD3, and NR4A3, for further investigation. We could not only confirm the up-regulation of NR4A3 by an independent method in all clones expressing HOX11, but luciferase reporter assays demonstrated that the effect that HOX11 exerted on the proximal promoter of NR4A3 was dependent on the presence of an intact homeodomain, providing support for the idea that HOX11 manifests its regulatory function via its action as a transcription factor.

Cell Line, Tumor↗

PubMed2004

Gene expression signatures in lymphoid tumours.

Lymphoid tumours comprise the acute and chronic leukaemias, the broad spectrum of lymphomas, including Hodgkin's disease, and multiple myeloma. The subdivision of the acute leukaemias according to the proliferating type of white blood cells has had a major impact on the care of these patients. More recently, specific chromosomal translocations have been used to identify patients who may benefit from more intensive therapies. The novel high-throughput genomic technologies, such as microarrays, provide new avenues for the molecular diagnosis of the haematological malignancies. Rapid advances in genome sequencing and gene expression profiling provide unprecedented opportunities to identify specific genes involved in complex biological processes, including tumorigenesis. The features of microarray technology and the variety of experimental approaches to elucidate lymphoid malignancies are discussed. Microarray technology has the potential to lead to more accurate prognostic assessment for patients and is expected to ultimately allow the clinician to select therapies optimally suited to each patient.

Antineoplastic Agents↗

PubMed2003

Specific alternative HOX11 transcripts are expressed in paediatric neural tumours and T-cell acute lymphoblastic leukaemia.

HOX11 is a proto-oncogene, which is silent in normal mature T-cells, while being aberrantly activated in T-cell acute lymphoblastic leukaemia (T-ALL) by translocations t(10;14)(q24;q11) or t(7;10)(q35;q24). Although many oncogenes are expressed in alternative forms in cancer, thus far, only one form of the human HOX11 transcript has been reported. We describe here the identification of three alternative transcripts of the HOX11 proto-oncogene, expressed in primary T-ALL specimens. Using rapid amplification of cDNA ends (RACE) and targeted RT-PCR, we have sequenced 23 individual cDNA clones characterising these novel transcripts. Northern hybridisation identified particular novel exons expressed in T-ALL, which are not expressed in normal T-cells. To date, aberrant expression of HOX11 has only been associated with leukaemia. Our survey of a range of neuroblastoma and primitive neuroectodermal tumour (PNET) cell lines demonstrated the expression of these novel HOX11 transcripts in tumours of neural origin, while their expression was not detected in normal brain tissues. Strikingly, the dominant transcript in these neural tumour cell lines is more than 1 kb larger than the dominant transcript in T-ALL. These observations, combined with sequence data from several EST clones derived from medulloblastoma cDNA libraries, support a new hypothesis that HOX11 may also function as a neural oncogene or brain tumour marker.

Alternative Splicing↗

PubMed2003

The diphtheria toxin/urokinase fusion protein (DTAT) is selectively toxic to CD87 expressing leukemic cells.

Diphtheria fusion proteins are a novel class of agents for the treatment of chemotherapy resistant acute myelogenous leukemia (AML). We prepared diphtheria toxin/urokinase fusion protein (DTAT) composed of the amino terminal fragment of the urokinase-type plasminogen activator (uPA) fused to the catalytic and translocation domains of diphtheria toxin (DT) and assessed its activity on leukemic cell lines. The number of uPA receptors (uPAR or CD87) was measured using a phycoerythrin conjugated monoclonal antibody to CD87 and flow cytometry. Seven of 23 cell lines (30%) showed CD87 expression (> or =5000 receptors/cell). DTAT cytotoxicity (IC(50)< or =30pM) was observed in all seven of these samples and none of the 16 samples with low or absent CD87 expression. There was a significant correlation between DTAT sensitivity and CD87 density (P=0.0007). These results show that specific CD87 binding is one factor important in the sensitivity of patient's leukemic blasts to DTAT and demonstrate for the first time that the CD87/uPAR can be used as a target for fusion protein therapy of AML.

Acute Disease↗

PubMed2003

Gene expression profiles in a panel of childhood leukemia cell lines mirror critical features of the disease.

The development of new drugs against cancer requires established cell lines. They are needed for in vitro studies to identify candidate drugs and in xenograft models to measure drug efficacy in vivo. Specific criteria need to be fulfilled by cell lines used in the evaluation of potential novel therapeutic agents. It is imperative that they display the features of the particular cancer under investigation. Given the documented heterogeneity of cancers, relevant subtypes need to be represented. In this study, we have examined these aspects for pediatric acute lymphoblastic leukemia. A panel of 13 leukemia cell lines recently established in our laboratory was analyzed. We used cDNA microarrays to define the gene expression profiles and compared the data with immunophenotyping and cytogenetic analyses. The expression profiles obtained showed excellent concordance with corresponding protein levels. Importantly, the panel of lines displayed the critical genetic features identified in clinically important acute lymphoblastic leukemia subtypes in childhood leukemia patients.

Child↗

PubMed2002

Expression and purification of the human homeodomain oncoprotein HOX11.

HOX11 is a transcription factor belonging to the homeodomain family that is essential for spleen development during embryogenesis. It is also tumorigenic, being associated with T-cell acute lymphoblastic leukemia in children. In order to understand the functional role of HOX11 in both normal development and malignancy, protein-DNA and protein-protein interaction studies involving this factor are required. Such investigations would be facilitated by the availability of significant amounts of purified HOX11 protein. However, expression of full-length HOX11 in bacteria has been reported to be problematic owing to fusion protein instability. Here, we report the purification of human HOX11 expressed in Escherichia coli as a soluble and functional glutathione S-transferase (GST) fusion protein. In addition, a mutant version of HOX11 was produced (HOX11 Delta H3) which lacked the DNA-recognition helix (helix 3) of the homeodomain. Through a single purification procedure using glutathione-Sepharose, 2mg of the recombinant proteins were obtained per liter of bacterial culture. Notably, recombinant GST-HOX11 fusion proteins had a markedly higher stability when purified at low temperature (4 degrees C). Purification to near-homogeneity was achieved as judged by SDS-PAGE and the purified proteins were recognized by anti-HOX11 antibodies. The biological activity of the recombinant protein was verified by the specific binding of GST-HOX11, but not GST-HOX11 Delta H3, to DNA containing consensus HOX11 recognition sites.

DNA-Binding Proteins↗