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Lars Bolund

Publications and source records attributed to Lars Bolund.

18 recordsLinked to original sources
PubMed2025

Spatial-Temporal Diversity of Extrachromosomal DNA Shapes Urothelial Carcinoma Evolution and Tumor-Immune Microenvironment.

Extrachromosomal DNA (ecDNA) presents a promising target for cancer therapy; however, its spatial-temporal diversity and influence on tumor evolution and the immune microenvironment remain largely unclear. We apply computational methods to analyze ecDNA from whole-genome sequencing data of 595 urothelial carcinoma (UC) patients. We demonstrate that ecDNA drives clonal evolution through structural rearrangements during malignant transformation and recurrence of UC. This supports a model wherein tumors evolve via the selective expansion of ecDNA-bearing cells. Through multi-regional sampling of tumors, we demonstrate that ecDNA contributes to the evolution of multifocality and increased intratumoral heterogeneity. EcDNA is present in 36% of UC tumors and correlates with an immunosuppressive phenotype and poor prognosis. Single-cell RNA sequencing analyses reveal that ecDNA+ malignant cells exhibit diminished expression of major histocompatibility complex class I molecules, enabling them to evade T-cell immunity. Finally, we show that sequencing of urinary sediment-derived DNA has excellent specificity in detecting ecDNA.

Journal Article↗
PubMed2003

Integrating proteomic and functional genomic technologies in discovery-driven translational breast cancer research.

The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.

Academies and Institutes↗
PubMed2003

Expression of short-chain acyl-CoA dehydrogenase (SCAD) proteins in the liver of SCAD deficient mice after hydrodynamic gene transfer.

Hydrodynamic administration of naked DNA was investigated as a method for in vivo expression of variant proteins involved in metabolic diseases, using short-chain acyl-CoA dehydrogenase (SCAD) deficient mice (BALB/cByJ) as a model. Human SCAD wild-type (WT) and two disease-associated SCAD variant proteins (R147W and G185S) were expressed in mouse liver by means of single injections of SCAD cDNA under the control of a ubiquitin promoter. SCAD expression was detected two days after injection. The activity decreased after the first week but continued to be detectable for at least 31 days after injection. Analysis of SCAD WT, R147W, and G185S proteins in liver cells showed that all three SCAD proteins were processed to the mature protein in mitochondria. Concomitantly, the SCAD activity in BALB/cByJ mice injected with SCAD WT, G185S, and R147W cDNA was 30, 39, and 13%, respectively, of the level in normal mice. A tendency to a reduction in the level of butyrylcarnitine in blood was observed although only approximately 5% of the liver cells expressed the SCAD protein. Thus, hydrodynamic gene transfer allows for functional testing of SCAD variant proteins in vivo.

Animals↗
PubMed2003

Twelve single nucleotide polymorphisms on chromosome 19q13.2-13.3: linkage disequilibria and associations with basal cell carcinoma in Danish psoriatic patients.

The genetic susceptibility to basal cell carcinoma (BCC) among Danish psoriatic patients was investigated in association studies with 12 single nucleotide polymorphisms on chromosome 19q13.2-3. The results show a significant association between BCC and the A-allele of a polymorphism in ERCCI exon4 (Odds ratio 12;95% Confidence Interval 1.17-124; p(chi2, two-side) = 0.019) and to a lesser extent with XPD exon6 (p = 0.06). This is in accordance with recent studies of a different group of BCC cases (Rockenbauer et al. (in press) Carcinogenesis; Yin et al. (manuscript submitted for publication). Cancer Epidemiol. Biomarkers Prev), which places two highly influential markers between these two genes. The analysis also confirmed that considerable linkage disequilibrium exists between SNPs both within genes and between genes in this region. The combined studies suggest that genetic variation in nucleotide excision repair is of importance for the development of BCC.

Adult↗
PubMed2003

Functional testing of keratin 14 mutant proteins associated with the three major subtypes of epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominantly inherited skin disorders characterized by the development of intra-epidermal skin blisters on mild mechanical trauma. The three major clinical subtypes (Weber-Cockayne, Koebner and Dowling-Meara) are all caused by mutations in either the keratin 5 (KRT5) or keratin 14 (KRT14) gene. Previously, we identified three novel KRT14 missense mutations in Danish EBS patients associated with the three different forms of EBS (1). The identified KRT14 mutations represent the full spectrum of the classical EBS subtypes. In the present study we investigated these mutations in a cellular expression system in order to analyse their effects on the keratin cytoskeleton. KRT14 expression vectors were constructed by fusing the nucleotide sequence encoding the FLAG reporter peptide to the 3' end of the KRT14 cDNA sequences. The expression vectors were transiently transfected into normal human primary keratinocytes (NHK), HaCaT or HeLa cells in order to analyze the ability of the mutant K14 proteins to integrate into the existing endogenous keratin filament network (KFN). No effect on the keratin cytoskeleton was observed upon transfection of NHK with the various K14 constructs neither with nor without a subsequently induced heat-stress. In contrast, all constructs, including wild-type K14, caused collapse of the endogenous KFN in a small fraction of the transfected HeLa and HaCaT cells. However, overexpression of the mutation associated with the most severe form of the disease, EBS Dowling-Meara, resulted in a higher number of transfected HaCaT cells with KFN collapse (P < 0.001). Thus, although a background KFN perturbance was observed upon transfection with the wild-type K14 construct, the mutant protein associated with the most severe form of EBS worsened the KFN perturbation significantly compared with the mutant proteins associated with the milder forms of the disease and the normal K14 protein. This shows that the clinical severity of disease-associated mutations identified in patients can be tested using this expression system, although it can not at present be used to discriminate between the milder forms. Assessment of the endogenous K14 protein expression in NHK and HaCaT cells indicated that the higher level of endogenous keratin expression in NHK might make these cells more resistant to perturbation of the keratin cytoskeleton by overexpressed K14 protein than HaCaT cells.

Base Sequence↗
PubMed2003

Epidermolysis bullosa simplex keratinocytes with extended lifespan established by ectopic expression of telomerase.

As part of a strategy to develop somatic gene therapy of epidermolysis bullosa simplex (EBS) we have established patient keratinocytes with expanded lifespan by ectopic expression of the human telomerase gene (hTert). The presence of an active telomerase enzyme was demonstrated by the telomerase repeat amplification protocol (TRAP). The hTert(+) cells have a normal karyotype and the cells have, until now, undergone more than 80 population doublings (PDs) after hTert retroviral transduction while control cells exhibited senescence-associated proliferation arrest after 8 PDs. In organotypic culture the hTert(+) cells are capable of forming a stratified epidermis illustrating their preserved ability to differentiate.

Cell Line, Transformed↗
PubMed2003

Platelet derived growth factor (PDGF) responsive epidermis formed from human keratinocytes transduced with the PDGF beta receptor gene.

Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes are not known to express any member of the platelet-derived growth factor receptor family. In order to study if epidermis may be genetically transformed to a platelet-derived growth factor sensitive compartment we aimed to introduce the gene encoding human platelet-derived growth factor receptor beta (PDGF beta R) into epidermal keratinocytes using a retrovirus-derived vector. Successful gene transfer to primary cells was confirmed by immunofluorescence staining, southern blotting, and ligand-induced receptor autophosphorylation. By culturing a mixture of PDGF beta R-transduced and unmodified keratinocytes at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue cultured for 6 days and examined by immunofluorescence microscopy was shown to contain PDGF beta R-expressing keratinocytes distributed in all layers of living epidermis. By continued tissue culture in serum-containing medium, the epidermis became increasingly cornified although receptor-positive cells were still observed within the viable basal compartment. Stimulation of PDGF beta R-transduced epidermis with recombinant platelet-derived growth factor BB had a mitogenic effect as reflected by an increased frequency of Ki-67 positive keratinocytes. The study demonstrates that transgene expression of human PDGF beta R can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation of such gene-modified skin equivalent enhances cell proliferation. In perspective, viral PDGF beta R gene transfer to keratinocytes may be a useful approach in studies of receptor tyrosine kinase mediated skin repair and epithelialization.

3T3 Cells↗
PubMed2003

Production of retroviral vectors in primary human keratinocytes after DNA-mediated gene transfer leads to prolonged gene expression.

Prolonged stability and controlled expression of gene constructs transferred directly to human skin improve the possibility of using this tissue in somatic gene therapy. We aim to develop a simple transfection method resulting in retroviral mediated gene transfer to keratinocyte stem cells in situ. We here show that after DNA-mediated gene transfer into primary human keratinocytes it is possible to achieve production of retroviral vectors, leading to the transduction of co-cultured keratinocytes and prolonged reporter gene expression. The method is a first step in a strategy to generate retroviral producer cells in situ in the skin furthermore the method can be used for rapid analysis of the possible effects of transgenes in cultured human keratinocytes without preparatory retroviral vector production in packaging cell lines.

Cell Culture Techniques↗
PubMed2003

A population threshold for functional polymorphisms.

We sequenced 114 genes (for DNA repair, cell cycle arrest, apoptosis, and detoxification)in a mixed human population and observed a sudden increase in the number of functional polymorphisms below a minor allele frequency of approximately 6%. Functionality is assessed by considering the ratio in the number of nonsynonymous single nucletide polymorphisms (SNPs)to the number of synonymous or intron SNPs. This ratio is steady from below 1% in frequency-that regime traditionally associated with rare Mendelian diseases-all the way up to about 6% in frequency, after which it falls precipitously. We consider possible explanations for this threshold effect. There are four candidates as follows: (1). deleterious variants that have yet to be purified from the population, (2). balancing selection, in which a selective advantage accrues to the heterozygotes, (3). population-specific functional polymorphisms, and (4). adaptive variants that are accumulating in the population as a response to the dramatic environmental changes of the last 7000 approximately 17000 years.

Animals↗
PubMed2002

Mechanisms underlying targeted gene correction using chimeric RNA/DNA and single-stranded DNA oligonucleotides.

Chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides have been developed for site-specific correction of episomal and chromosomal target genes. The gene repair approach relies on specific hybridization of the oligonucleotides to the target gene generating a mismatch with the targeted point mutation. Restored gene function is anticipated to occur through activation of endogenous repair systems that recognize the created mismatch. We present an overview of the gene correction results obtained in several target genes by employing various oligonucleotide designs and a discussion of the possible mechanisms underlying the gene correction techniques. Experimental data suggest that modified single-stranded oligonucleotides form intermediate three-stranded heteroduplexes involving the human RecA homologue, hRad51, whereas chimeric RNA/DNA oligonucleotides may participate in three or four-stranded intermediate structures. Protein factors such as hRad52, hRad54, hRPA, and p53 may modulate the heteroduplex formation and participate in the activation of the endogenous mismatch repair and/or nucleotide excision repair pathway(s). The efficiency of the gene correction process may furthermore be influenced by the differential recognition of mismatches by repair enzymes and possible sequence context effects.

Adenosine Triphosphatases↗
PubMed2002

Genomic structure of the human mitochondrial chaperonin genes: HSP60 and HSP10 are localised head to head on chromosome 2 separated by a bidirectional promoter.

Although the mitochondrial chaperonin Hsp60 and its co-chaperonin Hsp10 have received great attention in the last decade, and it has been proposed that mutations and variations in these genes may be implicated in genetic diseases, the genome structure of the human HSP60 and HSP10 genes (also known as HSPD1 and HSPE1, respectively) has not been firmly established. The picture has been confused by the presence of many pseudogenes of both HSP60 and HSP10 and the long surviving assumption that the HSP60 gene is intron-less. An earlier report on the partial sequence of the human HSP60 gene and the presence of introns has largely been overlooked. We present the full sequence of the human HSP60 and HSP10 genes. The two genes are linked head to head comprising approximately 17 kb and consist of 12 and 4 exons, respectively. The first exon of the human HSP60 gene is non-coding and the first exon of the human HSP10 gene ends with the start codon. Analysis of human and mouse expressed sequence tag sequences in GenBank indicates that alternative splicing occurs resulting in HSP60 gene transcripts with different exon-1 sequences. By sequencing of the exons, the exon/intron boundaries and the region between the two genes in 10 Danish individuals (five couples), nine nucleotide variations and one intronic deletion have been detected that, by subsequent typing of one child from each couple, have been assigned to five haplotypes. The human HSP60 gene has been localised, by radiation hybrid mapping, between markers AFMA121YH1 and WI-10756 on chromosome 2. This location and the position of two homologous fragments in the Human Genome Assembly are consistent with cytogenetic position 2q33.1. Using a luciferase-reporter assay, we demonstrate that the region between the two genes functions as a bi-directional promoter. The transcriptional activity of the promoter fragment in the HSP60 direction is approximately twice that in the HSP10 direction under normal growth conditions and, upon heat-shock, promoter activity in either direction increased by a factor of approximately 12. One of the nucleotide variations detected is localised in a putative SP1-transcription-factor-binding site in the bidirectional promoter region and analysis of the transcriptional activity of the promoter fragment with this variation has shown that it does not affect transcription levels both with and without heat-shock.

3' Untranslated Regions↗
PubMed2002

Genomic organization, transcript variants and comparative analysis of the human nucleoporin 155 (NUP155) gene.

Nucleoporin 155 (Nup155) is a major component of the nuclear pore complex (NPC) involved in cellular nucleo-cytoplasmic transport. We have acquired the complete sequence and interpreted the genomic organization of the Nup155 orthologos from human (Homo sapiens) and pufferfish (Fugu rubripes), which are approximately 80 and 8 kb in length, respectively. The human gene is ubiquitously expressed in many tissues analyzed and has two major transcript variants, resulted from an alternative usage of the 5' cryptic or consensus splice donor in intron 1 and two polyadenylation signals. We have also cloned DNA complementary to RNAs of the Nup155 orthologs from Fugu and mouse. Comparative analysis of the Nup155 orthologs in many species, including H. sapiens, Mus musculus, Rattus norvegicus, F. rubripes, Arabidopsis thaliana, Drosophila melanogaster, and Saccharomyces cerevisiae, has revealed two paralogs in S. cerevisiae but only a single gene with increasing number of introns in more complex organisms. The amino acid sequences of the Nup155 orthologos are highly conserved in the evolution of eukaryotes. Different gene orders in the human and Fugu genomic regions harboring the Nup155 orthologs advocate cautious interpretation of synteny in comparative genomic analysis even within the vertebrate lineage.

3T3 Cells↗
PubMed2002

Keratoacanthomas frequently show chromosomal aberrations as assessed by comparative genomic hybridization.

Keratoacanthomas are commonly occurring benign skin lesions localized to sun-exposed areas. They typically develop rapidly and may show cellular atypia and infiltration like cutaneous squamous cell carcinomas, but they finally regress spontaneously. This benign lesion shows a high degree of genetic instability as assessed by comparative genomic hybridization, with 35.7% (25 of 70) of the analyzed lesions harboring chromosomal aberrations. The same frequency of genetic imbalance was found in lesions from immunosuppressed organ transplant recipients (36.4%, 20 of 55) and in patients with keratoacanthomas without immunosuppression (33.3%, five of 15), indicating a common pathway in both situations. Recurrent aberrations, given as a fraction of lesions with aberrations, were gains on 8q (20.0%), 1p and 9q (each 16.0%), and deletions on 3p (20.0%), 9p (20.0%), 19p (20.0%), and 19q (16.0%). Many of the most frequently appearing aberrations in keratoacanthomas were not detected in any of the 10 squamous cell carcinomas analyzed, whereas some aberrations were shared by both types of lesions. Aberrations were found in early and late stages of keratoacanthoma development, indicating a role for genetic instability in the progression as well as involution of keratoacanthomas. There were no significant correlations between cytologic atypia and genetic imbalance, or between degree of infiltration and genetic aberrations, although there was a trend for keratoacanthomas with severe atypia to have aberrations. Thus malignant phenotypic development does not appear to be driven by the detected genetic aberrations. More detailed studies of chromosomal areas with recurrent aberrations are needed for the localization of putative genes that determine the biologic behavior of keratoacanthomas, and that may distinguish them from squamous cell carcinomas.

Carcinoma, Squamous Cell↗
PubMed2002

Association of chromosome 19q13.2-3 haplotypes with basal cell carcinoma: tentative delineation of an involved region using data for single nucleotide polymorphisms in two cohorts.

We have previously used single nucleotide polymorphisms to detect an association of basal cell carcinoma (BCC) in Caucasian Americans and Danes with the genome region 19q13.2-3, which contains several genes involved in the nucleotide excision repair of DNA. In this exploratory paper we have extended the data and used them in a chromosomal scan. The results indicate the presence of a gene variation modulating the risk of developing BSS in a submegabase region including and surrounding the gene RAI. Specifically, persons that are homozygous for the haplotype RAI intron 1(A) RAI exon 6(A) appear at increased risk for BCC. In addition, we have looked for possible synergisms between all pairs of markers. We find that a marker in GLTSCR1, presumably separated from RAI by several million bases, supplements the most significant marker in RAI in separating cases from controls, which may suggest the presence of an independent, risk-modulating variation in this second gene region.

Adult↗
PubMed2002

Multiple single nucleotide polymorphisms on human chromosome 19q13.2-3 associate with risk of Basal cell carcinoma.

In this paper, we present evidence that alleles of several polymorphisms in the chromosomal region 19q13.2-3, encompassing the genes RAI and XPD, are associated with occurrence of basal cell carcinoma in Caucasian Americans. The association of one of these, RAI-intron1, is sufficiently strong to make mass significance unlikely (P = 0.004, chi(2)). We interpret our combined data to indicate that a specific haplotype partly defined by the alleles of three single nucleotide polymorphisms, RAI intron1(G), RAI exon6(T), and XPD exon 6(C), is associated with a protective gene variant in a region spanning from XPD to ERCC1.

Alleles↗