Biomedical subjects

J Lundeberg

Publications and source records attributed to J Lundeberg.

At least 19 recordsLinked to original sources

PubMed2006

Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis.

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.

Apoptosis↗

PubMed2005

Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms.

We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and downregulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

Acetylcysteine↗

PubMed2005

PTCH codon 1315 polymorphism and risk for nonmelanoma skin cancer.

BACKGROUND: The PTCH tumour suppressor gene is involved in the development of nearly all basal cell carcinomas (BCCs) of the skin and a fraction of squamous cell carcinomas (SCCs). A nonconservative Pro/Leu nucleotide polymorphism within PTCH exon 23 at codon 1315 was recently reported to be potentially important for the development of breast epithelial cell cancers. Objectives Accordingly, the status of PTCH codon 1315 was analysed for a possible association with the development of nonmelanoma skin cancers (NMSCs) in a pilot study. Because skin cancer risk is affected by specific population-dependent phenotypes such as skin and hair colour, codon 1315 was also analysed for normal allele frequency variation in human populations having differing extents of eumelanin vs. phaeomelanin. METHODS: The single nucleotide polymorphism in codon 1315 of the human PTCH gene was analysed in genomic DNA from six different populations comprising 472 blood samples and from 170 patients in four different categories with NMSC. Polymerase chain reaction and pyrosequencing were used to determine the allele frequencies. Allelic loss was furthermore determined in tumours following microdissection. RESULTS: The Pro/Pro genotype frequency ranged from 30% to 65% between populations, with a significant trend for a reduced frequency of the Pro/Pro genotype in populations having lighter pigmentation (P = 0.020). Pro/Pro frequency showed an increasing trend with increasing tumour case severity (P = 0.027). In 260 samples from 180 Swedish patients with NMSC and a control group of 96 healthy ethnically matched volunteers, no statistically significant pairwise differences between groups were detected in the PTCH codon 1315 allelic distribution, neither was a difference seen for multiple or early onset cases of BCC in the Swedish population. In Swedish patients with single tumours, allelic loss (loss of heterozygosity) was observed in 20 of 30 (67%) patients with BCC and four of 22 (18%) patients with SCC, with no preference in the allele lost. In contrast, the Pro/Pro genotype was frequent in seven U.S. patients having multiple independent BCCs. One of these patients was heterozygous, enabling allelic loss studies. Of 20 independent tumours, 11 had lost an allele; 10 of the 11 had lost Leu, suggesting nonrandom loss that favoured retention of Pro (P = 0.0059). CONCLUSIONS: Our results indicate an association between the eumelanin-to-phaeomelanin shift and a shift from the Pro/Pro genotype to Leu-containing genotypes. Failure to lose Pro during the shift to phaeomelanin may be associated with an increased population risk for BCC and increased individual risk for multiple BCC. During development of a tumour, the effect of Pro may be magnified by loss of the Leu allele.

Basal Cell Carcinoma↗

PubMed2005

Expressed sequence tags from the midgut and an epithelial cell line of Chironomus tentans: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

Animals↗

PubMed2005

ESTs from brain and testis of White Leghorn and red junglefowl: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

We report the generation, assembly and annotation of expressed sequence tags (ESTs) from four chicken cDNA libraries, constructed from brain and testis tissue dissected from red junglefowl and White Leghorn. 21,285 5'-end ESTs were generated and assembled into 2,813 contigs and 9,737 singletons, giving 12,549 tentative unique transcripts. The transcripts were annotated using BLAST by matching to known chicken genes or to putative homologues in other species using the major gene/protein databases. The results for these similarity searches are available on www.sbc.su.se/~arve/chicken. 4,129 (32.9%) of the transcripts remained without a significant match to gene/protein databases, a proportion of unmatched transcripts similar to earlier non-mammalian EST studies. To estimate how many of these transcripts may represent novel genes, they were studied for the presence of coding sequence. It was shown that most of the unique chicken transcripts do not contain coding parts of genes, but it was estimated that at least 400 of the transcripts contain coding sequence, indicating that 3.2% of avian genes belong to previously unknown gene families. Further BLAST search against dbEST left 1,649 (13.1%) of the transcripts unmatched to any library. The number of completely unmatched transcripts containing coding sequence was estimated at 180, giving a measure of the number of putative novel chicken genes identified in this study. 84.3% of the identified transcripts were found only in testis tissue, which has been poorly studied in earlier chicken EST studies. Large differences in expression levels were found between the brain and testis libraries for a large number of transcripts, and among the 525 most frequently represented transcripts, there were at least 20 transcripts with significant difference in expression levels between red junglefowl and White Leghorn.

Animals↗

PubMed2004

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs.

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.

Animals↗

PubMed2002

Monitoring of representational difference analysis subtraction procedures by global microarrays.

Various approaches to the study of differential gene expression are applied to compare cell lines and tissue samples in a wide range of biological contexts. The compromise between focusing on only the important genes in certain cellular processes and achieving a complete picture is critical for the selection of strategy. We demonstrate how global microarray technology can be used for the exploration of the differentially expressed genes extracted through representational difference analysis (RDA). The subtraction of ubiquitous gene fragments from the two samples was demonstrated using cDNA microarrays including more than 32 000 spotted, PCR-amplified human clones. Hybridizations indicated the expression of 9100 of the microarray elements in a macrophage/foam cell atherosclerosis model system, of which many were removed during the RDA process. The stepwise subtraction procedure was demonstrated to yield an efficient enrichment of gene fragments overrepresented in either sample (18% in the representations, 86% after the first subtraction, and 88% after the second subtraction), many of which were impossible to detect in the starting material. Interestingly, the method allowed for the observation of the differential expression of several members of the low-abundant nuclear receptor gene family. We also observed a certain background level in the difference products of nondifferentially expressed gene fragments, warranting a verification strategy for selected candidate genes. The differential expression of several genes was verified by real-time PCR.

Cell Line↗

PubMed2001

Genotyping by apyrase-mediated allele-specific extension.

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.

Alleles↗

PubMed2001

A transcriptional roadmap to wood formation.

The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

Cell Wall↗

PubMed2001

PATCHED and p53 gene alterations in sporadic and hereditary basal cell cancer.

It is widely accepted that disruption of the hedgehog-patched pathway is a key event in development of basal cell cancer. In addition to patched gene alterations, p53 gene mutations are also frequent in basal cell cancer. We determined loss of heterozygosity in the patched and p53 loci as well as sequencing the p53 gene in tumors both from sporadic and hereditary cases. A total of 70 microdissected samples from tumor and adjacent skin were subjected to PCR followed by fragment analysis and DNA sequencing. We found allelic loss in the patched locus in 6/8 sporadic basal cell cancer and 17/19 hereditary tumors. All sporadic and 7/20 hereditary tumors showed p53 gene mutations. Loss of heterozygosity in the p53 locus was rare in both groups. The p53 mutations detected in hereditary tumors included rare single nucleotide deletions and unusual double-base substitutions compared to the typical ultraviolet light induced missense mutations found in sporadic tumors. Careful microdissection of individual tumors revealed genetically linked subclones with different p53 and/or patched genotype providing an insight on time sequence of genetic events. The high frequency and co-existence of genetic alterations in the patched and p53 genes suggest that both these genes are important in the development of basal cell cancer.

Adult↗

PubMed2001

Monitoring of the subtraction process in solid-phase representational difference analysis: characterization of a candidate drug.

In this study, we have applied and evaluated a modified cDNA representational difference analysis (RDA) protocol based on magnetic bead technology to study the molecular effects of a candidate drug (N,N'-diacetyl-L-cystine, DiNAC) in a model for atherosclerosis. Alterations in a gene expression profile induced by DiNAC were investigated in a human monocytic cell line (THP-1) differentiated into macrophage-like cells by lipopolysaccharide and further exposed to DiNAC. Three rounds of subtraction have been performed and the difference products from the second and third rounds have been characterized in detail by analysis of over 1000 gene sequences. Two protocols for analysis of the subtraction products have been evaluated, a shotgun approach and size selection of both distinct fragments and band-patterned smear. We demonstrate that in order to obtain a representative view of the most abundant gene fragments, the shotgun procedure is preferred. The obtained sequences were analyzed against the UniGene and Expressed Gene Anatomy Database (EGAD) databases and the results were visualized and analyzed with the ExProView software enabling rapid pair-wise comparison and identification of individual genes or functional groups of genes with altered expression levels. The identified differentially expressed gene sequences were comprised of both genes with known involvement in atherosclerosis or cholesterol biosynthesis and genes previously not implicated in these processes. The applicability of a solid-phase shotgun RDA protocol, combined with virtual chip monitoring, results in new starting points for characterization of novel candidate drugs.

Arteriosclerosis↗

PubMed2001

Cloning and characterization of spliced fusion transcript variants of synovial sarcoma: SYT/SSX4, SYT/SSX4v, and SYT/SSX2v. Possible regulatory role of the fusion gene product in wild type SYT expression.

The synovial sarcoma translocation t(X;18)(p11.2; q11.2) results in the fusion of the SYT gene on chromosome 18 to exon 5 of either SSX1 or SSX2 genes on chromosome X. We recently reported that the SSX4 gene is also involved in such a translocation. In the present investigation we cloned and sequenced the full-length cDNA of SYT/SSX1, SYT/SSX2 and SYT/SSX4 from synovial sarcoma tissues. We isolated a novel fusion transcript type variant involving the fusion of SYT with exon 6 of the SSX4 gene (SYT/SSX4v). The SYT/SSX4 and SYT/SSX2 open reading frame also differed from previously reported SYT/SSX sequences by an in-frame addition of 93bp exon located in the junction between exon 7 and 8 of the SYT. This exon is identical to that reported for the murine SYT but has not been previously found in the human transcript. Two SYT transcripts, with and without the 93 bp exon, were co-expressed in mouse NIH3T3 cells, human malignant cells and human testis tissue, but not in human normal fibroblasts. Stable transfection of an SYT/SSX4 expression vector into human and murine cell lines correlated with a down-regulation of SYT transcripts. This was also observed in a synovial sarcoma tumor expressing SYT/SSX4. This suggests that the SYT/SSX fusion gene may regulate SYT expression from the normal allele and as such alter the normal function of SYT.

Amino Acid Sequence↗

PubMed2001

Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection.

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

Base Sequence↗

PubMed2001

Persistent p53 mutations in single cells from normal human skin.

Epidermal clones of p53-mutated keratinocytes are abundant in chronically sun-exposed skin and may play an important role in early development of skin cancer. Advanced laser capture microdissection enables genetic analysis of targeted cells from tissue sections without contamination from neighboring cells. In this study p53 gene mutations were characterized in single cells from normal, chronically sun-exposed skin. Biopsies were obtained from skin subjected to daily summer sun and skin totally protected from the sun by blue denim fabric. Using laser capture microdissection, 172 single-cell samples were retrieved from four biopsies and analyzed using single-cell polymerase chain reaction and direct DNA sequencing. A total of 14 different mutations were identified in 26 of 99 keratinocytes from which the p53 gene could be amplified. Mutations displayed a typical UV signature and were detected in both scattered keratinocytes and in a small cluster of p53-immunoreactive keratinocytes. This minute epidermal p53 clone had a diameter of 10 to 15 basal cells. Two missense mutations were found in all layers of epidermis within the p53 clone. The presented data show that p53 mutations are common in normal skin and that a clone of keratinocytes with a mutated p53 gene prevailed despite 2 months of total protection from ultraviolet light.

Adult↗

PubMed2001

cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol.

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 microg of total RNA and 1-2 microg of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 microg of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell layers with a fresh weight of approximately 0.5 mg.

DNA, Complementary↗

PubMed2001

HPV-related cancer susceptibility and p53 codon 72 polymorphism.

Conflicting results regarding the association of a polymorphism at codon 72 of the p53 tumour suppressor gene and susceptibility to develop human papilloma virus (HPV)-associated cervical cancer have been published over the last year, implicating differences in ethnic background, sample origin, sample size and/or detection assay. The material for this study was collected in the identical geographical region as for 2 previous reports with contradictory results regarding the association of codon 72 genotype with squamous cell cancer (SCC). We have used an alternative detection assay, based on pyrosequencing technology, that interrogates the variable position by the accuracy of DNA polymerase. In addition to cervical clinical specimens from SCC, HPV16- and HPV18-infected adenocarcinoma cases as well as cervical intraepithelial neoplasia (CIN) were investigated. No significant association was found between p53 codon 72 genotype and the risk to develop adenocarcinoma, SCC or CIN in the Swedish population.

Adenocarcinoma↗

PubMed2001

Screening and scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) by pyrosequencing.

The increasing interest in the discovery and characterization of single nucleotide polymorphisms (SNPs) emphasis the need for high-throughput and cost effective scoring methods. Pyrosequencing is a novel method for screening SNPs. In this study we examine breed specific SNPs in the pig melanocortin 1 receptor gene (MC1R), some causing coat color phenotypes. A total of fifteen pigs representing eight breeds and crosses were analyzed by pyrosequencing. In addition to nine previously known SNPs, we also detected one new missense mutation by pyrosequencing. We here show that the SNPs were readily scored using standard reaction conditions. Insertions as well as substitutions were unambiguously detected and all genotypes were resolved in terms of homo- and heterozygozity.

Animals↗

PubMed2001

Monitoring resistance to human immunodeficiency virus type 1 protease inhibitors by pyrosequencing.

The emergence of drug-resistant viral variants is the inevitable consequence of incomplete suppression of human immunodeficiency virus type 1 (HIV-1) replication during treatment with antiretroviral drugs. Sequencing to determine the resistance profiles of these variants has become increasingly important in the clinical management of HIV-1 patients, both in the initial design of a therapeutic plan and in selecting a salvage regimen. Here we have developed a pyrosequencing assay for the rapid characterization of resistance to HIV-1 protease inhibitors (PIs). Twelve pyrosequencing primers were designed and were evaluated on the MN strain and on viral DNA from peripheral blood mononuclear cells from eight untreated HIV-1-infected individuals. The method had a limit of detection of 20 to 25% for minor sequence variants. Pattern recognition (i.e., comparing actual sequence data with expected wild-type and mutant sequence patterns) simplified the identification of minor sequence variants. This real-time pyrosequencing method was applied in a longitudinal study monitoring the development of PI resistance in plasma samples obtained from four patients over a 2 1/2-year period. Pyrosequencing identified eight primary PI resistance mutations as well as several secondary mutations. This sequencing approach allows parallel analysis of 96 reactions in 1 h, facilitating the monitoring of drug resistance in eight patients simultaneously and, in combination with viral load analysis, should be a useful tool in the future to monitor HIV-1 during therapy.

Acquired Immunodeficiency Syndrome↗