| 1. |
- Adolfsson, Jörgen
(författare)
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Expression and role of the cytokine tyrosine kinase receptor flt3 in early hematopoiesis
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mature blood cells are crucial for life, but they have a short lifetime and thus have to be replaced. These mature blood cells are produced by lineage restricted progenitors, which themselves are generated by rare multipotent hematopoietic stem cells (HSCs) in a highly dynamic process called hematopoiesis. In addition to the ability of HSCs to generate all blood cell lineages, they possess the unique property of self-renewal, a process in which a HSC, during a cell division generate at least one daughter cell identical to the parental cell. The maintenance and regulation of HSCs and earliest stages of lineage development are partly controlled by soluble and membrane bound regulators called cytokines. The focus in this thesis has been on the cytokine tyrosine kinase receptor flt3 and its ligand and their role in regulating HSCs and the earliest progenitors in adult murine bone marrow (BM). Earlier studies had implicated a role for flt3 and its ligand in the maintenance of HSCs. When studying mice with targeted deletion of the flt3 ligand (FL), we failed to find any role of flt3 and its ligand in HSC regulation. However, the generation of the common lymphoid progenitor (CLP) and earliest stages of B- and T cell development were severely affected, but not myeloid progenitors or later stages of lymphoid development. Based on flt3 expression, we subfractionated the Lin(-)Sca-1(+)c-kit(+) (LSK) stem cell pool in mouse bone marrow. In contrast to LSKflt3- cells, which sustained multilineage long-term reconstitution, LSKflt3+ cells generated only short-term lymphoid dominated reconstitution when injected into lethally ablated recipients. We also demonstrated the hierarchical relationship at the earliest stages of hematopoiesis, in that LSKflt3- HSCs generate LSKflt3+ cells, which generate the CLP but not the LSKflt3- cells. In the classical model of the hematopoietic hierarchy, the first lineage commitment step of HSCs results in a strict separation into distinct lymphoid and myeloid pathways, generating the recently identified CLP and the common myeloid progenitor (CMP). However, in sharp contrast to LSKflt3-, cells which could generate all blood cell lineages, LSK cells expressing flt3 could not generate megakaryocytes or erythroid cells. Thus, these finding together with the above mentioned relationship between the LSKflt3-, LSKflt3+ and CLP do not support the classical hematopoietic hierarchy model depicting the first lineage commitment generating a strict separation of lymphoid and myeloid pathways. The LSK population contains all LT-HSC activity. However, this population is not homogenous neither in phenotype or function and it has been proposed to contain at least two populations, one with long-term reconstitution (LTR) potential and one with short-term reconstitution potential. Based on the expression of CD34 and flt3 within the adult LSK stem cell pool we were able to subfractionate short-term hematopoietic stem cells (ST-HSC) from the LT-HSCs. In contrast to the LSKCD34-flt3- and LSKCD34+flt3+ cells, the LSKCD34+flt3- is highly enriched for CFU-S activity and capable of rescuing lethally irradiated recipients, fulfilling the criteria of ST-HSCs.
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| 2. |
- Agarwal, Prasoon
(författare)
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Regulation of Gene Expression in Multiple Myeloma Cells and Normal Fibroblasts : Integrative Bioinformatic and Experimental Approaches
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The work presented in this thesis applies integrative genomic and experimental approaches to investigate mechanisms involved in regulation of gene expression in the context of disease and normal cell biology.In papers I and II, we have explored the role of epigenetic regulation of gene expression in multiple myeloma (MM). By using a bioinformatic approach we identified the Polycomb repressive complex 2 (PRC2) to be a common denominator for the underexpressed gene signature in MM. By using inhibitors of the PRC2 we showed an activation of the genes silenced by H3K27me3 and a reduction in the tumor load and increased overall survival in the in vivo 5TMM model. Using ChIP-sequencing we defined the distribution of H3K27me3 and H3K4me3 marks in MM patients cells. In an integrated bioinformatic approach, the H3K27me3-associated genes significantly correlated to under-expression in patients with less favorable survival. Thus, our data indicates the presence of a common under-expressed gene profile and provides a rationale for implementing new therapies focusing on epigenetic alterations in MM.In paper III we address the existence of a small cell population in MM presenting with differential tumorigenic properties in the 5T33MM murine model. We report that the predominant population of CD138+ cells had higher engraftment potential, higher clonogenic growth, whereas the CD138- MM cells presented with less mature phenotype and higher drug resistance. Our findings suggest that while designing treatment regimes for MM, both the cellpopulations must be targeted.In paper IV we have studied the general mechanism of differential gene expression regulation by CGGBP1 in response to growth signals in normal human fibroblasts. We found that CGGBP1 binding affects global gene expression by RNA Polymerase II. This is mediated by Alu RNAdependentinhibition of RNA Polymerase II. In presence of growth signals CGGBP1 is retained in the nuclei and exhibits enhanced Alu binding thus inhibiting RNA Polymerase III binding on Alus. Hence we suggest a mechanism by which CGGBP1 orchestrates Alu RNA-mediated regulation of RNA Polymerase II. This thesis provides new insights for using integrative bioinformatic approaches to decipher gene expression regulation mechanisms in MM and in normal cells.
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| 3. |
- Agnarsdóttir, Margrét, 1970-
(författare)
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Biomarker Discovery in Cutaneous Malignant Melanoma : A Study Based on Tissue Microarrays and Immunohistochemistry
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The incidence of cutaneous malignant melanoma has increased dramatically in Caucasians the last few decades, an increase that is partly explained by altered sun exposure habits. For the individual patient, with a localized disease, the tumor thickness of the excised lesion is the most important prognostic factor. However, there is a need to identify characteristics that can place patients in certain risk groups. In this study, the protein expression of multiple proteins in malignant melanoma tumors was studied, with the aim of identifying potential new candidate biomarkers. Representative samples from melanoma tissues were assembled in a tissue microarray format and protein expression was detected using immunohistochemistry. Multiple cohorts were used and for a subset of proteins the expression was also analyzed in melanocytes in normal skin and in benign nevi. The immunohistochemical staining was evaluated manually and for part of the proteins also with an automated algorithm. The protein expression of STX7 was described for the first time in tumors of the melanocytic lineage. Stronger expression of STX7 and SOX10 was seen in superficial spreading melanomas compared with nodular malignant melanomas. An inverse relationship between STX7 expression and T-stage was seen and between SOX10 expression and T-stage and Ki-67, respectively. In a population-based cohort the expression of MITF was analyzed and found to be associated with prognosis. Twenty-one potential biomarkers were analyzed using bioinformatics tools and a protein signature was identified which had a prognostic value independent of T-stage. The protein driving this signature was RBM3, a protein not previously described in malignant melanoma. Other markers included in the signature were MITF, SOX10 and Ki-67. In conclusion, the protein expression of numerous potential biomarkers was extensively studied and a new prognostic protein panel was identified which can be of value for risk stratification.
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| 4. |
- Ahlford, Annika, 1980-
(författare)
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Applications of Four-Colour Fluorescent Primer Extension Technology for SNP Analysis and Discovery
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Studies on genetic variation can reveal effects on traits and disease, both in humans and in model organisms. Good technology for the analysis of DNA sequence variations is critical. Currently the development towards assays for large-scale and parallel DNA sequencing and genotyping is progressing rapidly. Single base primer extension (SBE) is a robust reaction principle based on four-colour fluorescent terminating nucleotides to interrogate all four DNA nucleotides in a single reaction. In this thesis, SBE methods were applied to the analysis and discovery of single nucleotide polymorphism (SNP) in the model organism Drosophila melanogaster and in humans. The tag-array minisequencing system in a microarray format is convenient for intermediate sized genotyping projects. The system is scalable and flexible to adapt to specialized and novel applications. In Study I of the thesis a tool was established to automate quality control of clustered genotype data. By calculating “Silhouette scores”, the SNP genotype assignment can be evaluated by a single numeric measure. Silhouette scores were then applied in Study I to compare the performance of four DNA polymerases and in Study III to evaluate freeze-dried reagents in the tag-array minisequencing system. The characteristics of the tag-array minisequencing system makes it suitable for inexpensive genome-wide gene mapping in the fruit fly. In Study II a high-resolution SNP map, and 293 genotyping assays, were established across the X, 2nd and 3rd chromosomes to distinguish commonly used Drosophila strains. A database of the SNP markers and a program for automatic allele calling and identification of map positions of mutants was also developed. The utility of the system was demonstrated by rapid mapping of 14 genes that disrupt embryonic muscle patterning. In Study III the tag-array minisequencing system was adapted to a lab-on-a-chip format for diagnostic testing for mutations in the TP53 gene. Freeze-drying was evaluated for storing reagents, including thermo-sensitive enzymes, on the microchip to reduce the complexity of the integrated test. Correct genotyping results were obtained using freeze-dried reagents in each reaction step of the genotyping protocol, both in test tubes and in single polymer test chambers. The results showed the potential of the approach to be implemented in fully integrated systems. The four-colour chemistry of SBE has been developed further to allow massively parallel sequencing (MPS) of short DNA fragments as in the Genome Analyzer system (Solexa/Illumina). In Study IV MPS was used to compare Nimblegen arrays and the SureSelect solution-based system for targeted enrichment of 56 continuous human candidate-gene regions totalling 3.1 Mb in size. Both methods detected known SNPs and discovered novel SNPs in the target regions, demonstrating the feasibility for complexity reduction of sequencing libraries by hybridization methods.
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| 5. |
- Ahmed, Meftun
(författare)
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Oscillatory Ca2+ signaling in glucose-stimulated murine pancreatic β-cells : Modulation by amino acids, glucagon, caffeine and ryanodine
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.
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| 6. |
- Akkuratov, Evgeny E.
(författare)
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The Biophysics of Na+,K+-ATPase in neuronal health and disease
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete. In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid-onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions. In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.
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| 7. |
- Al-Behadili, Ali
(författare)
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Molecular insights into primer removal during mtDNA replication
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mitochondria are vital for cell survival, and the primary producers of ATP, the energy currency used for various metabolic processes. Mitochondria are unique from other cellular compartments because they have their own genomes of circular small double-stranded DNA (mtDNA) of approximately 16.6 kbp in size. The mtDNA is highly compact, containing no introns and little non-coding DNA. MtDNA has two non-coding regions: one large region known as the control region or the non-coding region that contains the promoters for transcription (LSP and HSP) and the origin of replication of the H strand (OriH), and a smaller region containing the origin of replication for the L-strand (OriL). MtDNA is replicated by a set of replication factors distinct from those needed for DNA replication in the nucleus. A fundamental step in mtDNA replication is the processing of the RNA primers needed for replication initiation.In this thesis, we could demonstrate that Ribonuclease H1 (RNase H1) is essential for the process of replication initiation at OriH. We couldalso elucidate the role of RNase H1 during primer removal and ligation at the mitochondrial origin of light-strand DNA synthesis (OriL) andexplain the pathogenic consequences of disease-causing mutations in RNase H1.These findings have taken the field of mitochondrial DNA transcription and replication forward and generated knowledge to build further research.In the last project, we studied EXOG, a mitochondrial exonuclease. We demonstrated that EXOG could interplay with RNase H1 and other mitochondrial nucleases in vitro and identified a possible pathway for EXOG to function in.
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| 8. |
- Alaei-Mahabadi, Babak
(författare)
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Comprehensive analysis of structural genomic alterations in cancer : Computational approaches for identifying cancer driver events
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transformation of a normal cell into a cancer cell involves the accumulation of somatic DNA alterations that confer growth and survival advantages. These genomic alterations can be different in terms of pattern and size, comprising single nucleotide variants (SNVs), small insertions or deletions (indels), structural variations (SVs) or foreign DNA insertions such as viral DNA. Cancer genomes typically harbor numerous such changes, of which only small fractions are driver events that are positively selected for during the evolution of the tumor. High throughput sequencing has enabled systematic mapping of somatic DNA alterations across thousands of tumor genomes. Mutations in particular have been thoroughly explored in this type of data, and this has implicated many new genes in tumor development. However, our knowledge remains more limited when it comes to the contribution of SVs to cancer. In the present thesis, we made use of publicly available cancer genomics data to gain further insight into the role of structural genomic alterations in tumor development. Viruses cause 10-15% of all human cancers through multiple mechanisms, one of which is structural genomic changes due to viral DNA being integrated into the human genome. Thus, in the first study, we performed an unbiased screen for viral genomic integrations into cancer genomes. We developed a computational pipeline using RNA-Seq data from ~4500 tumors across 19 different cancer types to detect viral integrations. We found that recurrent events typically involved known cancer genes, and were associated with altered gene expression. SVs can lead to copy number amplification of specific cancer driver genes, as well as the formation of fusion oncogenes, but their importance in cancer beyond these types of events is underexplored. We mapped SVs to the human genome using whole genome sequencing data from 600 tumors across 18 different cancer types and investigated the global relationship between SVs and mRNA changes. We found that such events often contribute to altered gene expression in human tumors, but we were not able to detect novel recurrent driver events. To increase the cohort size, we used a larger but lower resolution and more limited dataset, comprising of microarray based DNA copy number profiles from ~10,000 tumors across 32 cancer types, with the aim of identifying recurrent SV driver events in tumors. Specifically, we investigated SVs predicted to result in promoter substitution events, a known mechanism for gene activation in cancer, and found several recurrent activating events with potential cancer driver roles. Notable among our findings in all the studies were human papillomavirus integrations in RAD51B and ERBB2 and gene fusions involving NFE2L2, TIAM2 and SCARB1, all being known cancer genes. Taken together, massive amounts of genomic and transcriptomic sequencing data allowed us to comprehensively map viral integrations and structural variations in cancer, which led to the identification of several genes with potential roles in tumor development.
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| 9. |
- Alajbegovic, Azra
(författare)
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Transcriptional and post-transcriptional regulation of vascular smooth muscle cell phenotype - Implications for vascular disease states
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- As the world population is pushing toward 8 billion, cardiovascular diseases (CVD) remain the leading cause of death worldwide, representing 30% of all global deaths. A large body of work has recognized that smooth muscle cells (SMCs) surrounding the blood vessels play a prominent role in the development and progression of cardiovascular diseases. SMCs are highly specialized cells with the main function to maintain vascular tension and thereby regulate blood pressure and blood flow. SMCs retain remarkable plasticity. In response to changes in external cues, SMCs can modulate their phenotype from a highly mature contractile phenotype to a synthetic, proliferative phenotype. Although beneficial during key physiological processes such as wound healing, phenotypic modulation can contribute to the development and progression of several vascular disease states. Despite extensive studies on the transcriptional programs that define smooth muscle phenotype, the endogenous regulators that control smooth muscle specificity are still far from understood. The aim of this thesis was to gain further insight into the transcriptional and post-transcriptional regulation of gene expression that occurs during disease development and how these changes affect the function of the vascular wall.The work in the following papers has identified previously unknown mechanisms by which small non-coding RNAs (miRNAs), actin polymerization and transcriptional regulators MRTFA and GATA6 can contribute to the changes in vascular smooth muscle observed in vascular disease states. In summary, we show that actin polymerization and MRTFA regulate a profile of miRNAs that are downregulated in patients with mildly dilated aorta. Moreover, we demonstrate a novel role for MRTFA in lipid accumulation and foam cell formation. We further demonstrate the importance of miRNA-143 and miRNA-145 for vascular function and for adaptation to hypertension. Lastly, we show that GATA6 regulates migration of SMCs. A deeper understanding into the underlying molecular mechanisms is crucial in order to develop new efficient therapeutic approaches against cardiovascular disease states.
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| 10. |
- Albertsson, Per, 1964
(författare)
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Matrix metalloproteinases in natural killer cells. Expression of MMPs, IL-2 activation and killer cell interactions with Matrigel® and model tumours
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: IL-2 activated natural killer (A-NK) cells can recognize malignant cells and exert tumouricidal activities by multiple mechanisms that require close contact with the target cells. Upon adoptive transfer, some A-NK cells accumulate in tumours by migrating from the vascular bed to a position inside the malignant tissue, forming close contacts with target cells. This process requires that naturally occurring barriers are passed, i.e. the endothelial lining with the underlying basement membrane and the adjacent extracellular matrix, ECM. Thus, tumour infiltration by A-NK cells is believed to depend in part on proteolytic matrix degradation, in analogy with malignant cell invasion during formation of metastasis. This focuses on expression, release, and possible functions of ECM degrading proteases in NK cells. Specific aims: to explore the repertoire of matrix metalloproteinases (MMPs) in NK/A-NK cells of various species; to examine whether MMPs are utilised in the process of basement membrane transmigration; to describe the morphology of A-NK cell infiltration in ECM and in model tumours as related to proteolytic mechanismsMaterials & Methods: Gelatin zymography, Western blotting and reverse transcriptase (RT-) PCR were used to detect and identify the various MMPs in NK/A-NK cell membranes and culture supernatants. Transmigration assays using Matrigel (reconstituted basement membrane ECM) covered invasion-chambers. A-NK-to-matrix and A-NK-to-tumour cell interactions were investigated by means of light and fluorescence microscopy, and electron microscopy. Model B16 melanoma tumours formed in vitro and growing intraperitoneally were used.Results: Expression and release of multiple MMPs were demonstrated in NK cells from different species. In rat MMP-2 and MMP-9 were found, in mice MMP-2, MMP-9, MMP-11, MMP-13, MT1-MMP, MT2-MMP and the specific inhibitors TIMP-1 and TIMP-2. In human NK cells MMP-2, MMP-8, MMP-9 and MT1-MMP as well as TIMP-1 were detected. A-NK cell transmigration through a basement membrane material was inhibited to about 50% (rat cells) or 90% (murine cells) by the MMP inhibitor batimastat (BB94). Human A-NK cell migrated across the membranes in a seemingly IL-2-dependent manner. Microscopy demonstrated that infiltrating A-NK cells disintegrate B16 tumour cell aggregates in vitro, which was not related to cytolysis. Further, an A-NK cell age-dependent alteration of Matrigel was detected. Short-term cultured cells caused a general digestion of the matrix material which contrasted to the effect of older A-NK cells that created large excavations around the cells with little alteration on the remaining Matrigel. A-NK cells infiltrated, but did not disintegrate, intraperitoneal tumours.Conclusions: NK/A-NK cells express and release a broad array of MMPs including their inhibitors. Further, MMPs are involved in A-NK cell basement membrane transmigration. Disintegration of tumour cell aggregates in vitro and digestion of Matrigel are interpreted as proteolytic effects. The additional matrix-dilating effect may be explained as a release of proteoglycans. The present studies confirm that intravital tumours largely resist derangement of the tumour structure in spite of A-NK cell infiltration.
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