| 1. |
- Kågedal, Bertil, 1943-, et al.
(författare)
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How useful are housekeeping genes? Variable expression in melanoma metastases
- 2007
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Ingår i: Clinical Chemistry and Laboratory Medicine. - 1434-6621 .- 1437-4331. ; 45:11, s. 1481-1487
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Tidskriftsartikel (refereegranskat)abstract
- Background: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, beta(2)-glucuronidase and beta(2)-microglobulin, for normalization of expression data from melanoma metastases. Methods: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon alpha 2b. Results: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the metastases, whereas treatment did not seem to influence the expression. Conclusions: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.
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| 2. |
- Baryawno, Ninib, et al.
(författare)
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Tumor-growth-promoting cyclooxygenase-2 prostaglandin E2 pathway provides medulloblastoma therapeutic targets
- 2008
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Ingår i: Neuro-Oncology. - : Oxford University Press (OUP). - 1522-8517 .- 1523-5866. ; 10:5, s. 661-674
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Tidskriftsartikel (refereegranskat)abstract
- Prostaglandin E(2) (PGE(2)) has been shown to play important roles in several aspects of tumor development and progression. PGE(2) is synthesized from arachidonic acid by cyclooxygenases (COX) and prostaglandin E synthases (PGES) and mediates its biological activity through binding to the four prostanoid receptors EP(1) through EP(4). In this study, we show for the first time that medulloblastoma (MB), the most common malignant childhood brain tumor, expresses high levels of COX-2, microsomal prostaglandin E synthase-1, and EP(1) through EP(4) and secretes PGE(2). PGE(2) and the EP(2) receptor agonist butaprost stimulated MB cell proliferation. Treatment of MB cells with COX inhibitors suppressed PGE(2) production and induced caspase-dependent apoptosis. Similarly, specific COX-2 silencing by small interfering RNA inhibited MB cell growth. EP(1) and EP(3) receptor antagonists ONO-8713 and ONO-AE3-240, but not the EP(4) antagonists ONO-AE3-208 and AH 23848, inhibited tumor cell proliferation, indicating the significance of EP(1) and EP(3) but not EP(4) for MB growth. Administration of COX inhibitors at clinically achievable nontoxic concentrations significantly inhibited growth of established human MB xenografts. Apoptosis was increased, proliferation was reduced, and angiogenesis was inhibited in MBs treated with COX inhibitors. This study suggests that PGE(2) is important for MB growth and that therapies targeting the prostanoid metabolic pathway are potentially beneficial and should be tested in clinical settings for treatment of children with MB.
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| 3. |
- Berois, Nora, et al.
(författare)
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ppGalNAc-TI3 : A new molecular marker of bone marrow involvement in neuroblastoma
- 2006
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Ingår i: Clinical Chemistry. - : Oxford University Press (OUP). - 0009-9147 .- 1530-8561. ; 52:9, s. 1701-1712
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Tidskriftsartikel (refereegranskat)abstract
- Background: To identify new molecular markers of bone marrow dissemination in human neuroblastoma (NB), we studied the transcriptome profiles of malignant neuroblasts established from the human MYCN-amplified IGR-N-91 model. Methods: This experimental model includes human neuroblastoma cells derived from & subcutaneous stage 4 disease, myocardium (Myoc) and bone marrow (BM) metastatic cells. Results: Gene expression profiles obtained with Agilent oligo microarrays revealed a set of 107 differentially expressed genes in the metastatic neuroblasts. This set included up-regulated genes involved in chemoresistance, cell motility, neuronal structure/signaling, and the recently characterized GALNT13 gene encoding a glycosyltransferase that initiates mucin-type O-glycosylation. Because the glycosylation process is involved in the progression of primary tumor to metastatic disease, we investigated whether the most strongly upregulated gene, GALNT13, might be a marker of bone marrow involvement in stage 4 NB patients. Importantly, in the BM of healthy adults no GALNT13 transcript was detected with analysis by quantitative (n = 3) and nested reverse transcription-PCR (n = 4) assays. In contrast, GALNT13 transcripts were detected in 23/23 cytologically involved BM samples obtained at diagnosis of stage 4 NB patients and in 5/27 cytologically noninvolved BM samples obtained from patients with stage 1-4 and 4S and treated stage 4 NB. The quantitative measurements of tyrosine hydroxylase (TH), ganglioside D2 synthase, dopa decarboxylase, and GALNT13 transcript values were compared in the same NB patients, and the results showed that GALNT13 expression was most highly correlated to poor clinical outcome at diagnosis. Conclusion: We propose ppGalNAc-T13 as a new informative marker for the molecular diagnosis of BM involvement and the follow-up of minimal residual disease in NB patients. © 2006 American Association for Clinical Chemistry.
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| 4. |
- Björk, Per, et al.
(författare)
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Conjugated polythiophene probes target lysosome-related acidic vacuoles in cultured primary cells
- 2007
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Ingår i: Molecular and Cellular Probes. - : Elsevier BV. - 0890-8508. ; 21:5-6, s. 329-337
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Tidskriftsartikel (refereegranskat)abstract
- Conformation-sensitive optical probes for studying biological processes and structures are of great interest. The present work shows for the first time that conjugated polyelectrolyte (CPE) probes can be used for specific targeting of chromatin, nuclear and cytoplasmatic vesicles, and cytoskeletal components in a complex system of cultured cells. One of the probes could also be used for vital staining of live cells. When bound to different entities, the polythiophene derivative probes emitted light with different colors due to the unique spectral properties of these conformation sensitive probes. The physical pre-requisites for binding could also be exploited for characterization of the target. Unexpectedly, lysosome-related acidic vacuoles were targeted in cultured primary cells by both anionic, cationic, and zwitter-ionic polythiophene derivatives. Pre-treatment with Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, caused redistribution of the staining. The targeting of lysosome-related acidic vesicles could not be demonstrated in transformed cells (melanoma, neuroblastoma, and prostate cancer cell lines), indicating a difference in the localization, structure, accessibility, or quantity of the target in cultured normal cells as compared with the malignant cell lines. The chemical nature of the conjugated polyelectrolyte complex in the cytoplasmatic vacuoles remains elusive.
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| 5. |
- Hultdin, Johan, 1964-
(författare)
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Homocysteine in cardiovascular disease with special reference to longitudinal changes
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abnormalities in homocysteine metabolism have been suggested as risk factors for stroke and myocardial infarction. In retrospective studies, elevated levels of total plasma homocysteine (tHcy) and/or methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism have indicated an increase in risk. However, the fewer prospective studies have not been as conclusive. To further explore this, tHcy was studied in four prospective settings. The first was a prospective nested case-referent cohort within the Västerbotten Intervention Program (VIP) and WHO MONICA project on 312 ischemic and 60 haemorrhagic first-ever strokes. The aim was to study tHcy and its main genetic determinant MTHFR. Risk for haemorrhagic stroke increased exponentially through tHcy quartiles, independent of hypertension and BMI, and increased for MTHFR 677 CT and TT. MTHFR 1298A>C appeared to be protective. In multivariate models, after adjustment for tHcy, BMI and hypertension, both tHcy and MTHFR remained as independent predictors for hemorrhagic stroke. Neither tHcy, nor the two MTHFR polymorphisms were significant predictors for ischemic strokes. The second was a prospective long-term follow-up study within the VIP and MONICA cohorts to determine whether a first-ever myocardial infarction (AMI) causes increased levels of tHcy. Fifty cases developing AMI after the first screening participated in a second screening (mean follow-up 8.5 years) with 56 matched referents. Increase in tHcy did not differ between cases and referents. tHcy was related to AMI at follow-up, but not at baseline and no longer significant after adjusting for creatinine and albumin. The third was a method study to determine if cystatin C, creatinine, albumin and other lipoprotein risk markers of cardiovascular disease could be analysed in Stabilyte™ plasma stored at -80°C. It was found to be suitable for all analyses tested and using this tube would simplify sampling for epidemiological studies. The fourth study was a prospective longitudinal long-term study of 735 subjects (340 men and 395 women, age 25-64 at first screening), participating in two MONICA screenings nine years apart, who donated blood in Stabilyte™ tubes to study change over time in tHcy and its determinants. We confirmed the age dependency in a cross sectional setting. In contrast, if followed longitudinally over time, no change in tHcy or in the prevalence of hyperhomocysteinemia was found. Cystatin C and creatinine increased, and albumin decreased. In multivariate models baseline levels of albumin, creatinine, cystatin C, and to some extent hs-CRP, were predictors of tHcy at follow-up but gender differences were seen. Age was not a major determinant of change in tHcy over nine years. In conclusion, tHcy and MTHFR are risk factors for first-ever haemorrhagic, but not ischemic stroke in a prospective setting. A first myocardial infarction does not cause an increase in tHcy. No long-term changes were seen in tHcy over a nine-year period in neither men, nor in women.
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| 6. |
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| 7. |
- Johnsen, J I, et al.
(författare)
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Inhibitors of mammalian target of rapamycin downregulate MYCN protein expression and inhibit neuroblastoma growth in vitro and in vivo
- 2008
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 27:20, s. 2910-2922
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Tidskriftsartikel (refereegranskat)abstract
- Mammalian target of rapamycin (mTOR) has been shown to play an important function in cell proliferation, metabolism and tumorigenesis, and proteins that regulate signaling through mTOR are frequently altered in human cancers. In this study we investigated the phosphorylation status of key proteins in the PI3K/AKT/mTOR pathway and the effects of the mTOR inhibitors rapamycin and CCI-779 on neuroblastoma tumorigenesis. Significant expression of activated AKT and mTOR were detected in all primary neuroblastoma tissue samples investigated, but not in non-malignant adrenal medullas. mTOR inhibitors showed antiproliferative effects on neuroblastoma cells in vitro. Neuroblastoma cell lines expressing high levels of MYCN were significantly more sensitive to mTOR inhibitors compared to cell lines expressing low MYCN levels. Established neuroblastoma tumors treated with mTOR inhibitors in vivo showed increased apoptosis, decreased proliferation and inhibition of angiogenesis. Importantly, mTOR inhibitors induced downregulation of vascular endothelial growth factor A (VEGF-A) secretion, cyclin D1 and MYCN protein expression in vitro and in vivo. Our data suggest that mTOR inhibitors have therapeutic efficacy on aggressive MYCN amplified neuroblastomas. © 2008 Nature Publishing Group All rights reserved.
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| 8. |
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| 9. |
- Kågedal, Bertil, et al.
(författare)
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Failure of the PAXgene™ Blood RNA System to maintain mRNA stability in whole blood
- 2005
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Ingår i: Clinical Chemistry and Laboratory Medicine. - 1434-6621 .- 1437-4331. ; 43:11, s. 1190-1192
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Tidskriftsartikel (refereegranskat)abstract
- In multicentre studies of malignant and inflammatory diseases, whole blood, cell or tissue samples are often collected for analyses of gene expression to predict or monitor treatment effects. For correct analysis, sample stability during handling and transport is crucial. In developing the logistics for multicentre studies in malignant melanoma and inflammatory bowel disease, we found poor stability of a number of transcripts using the PAXgene™ Blood RNA System, which was advertised to maintain RNA stability for several days at room temperature. The results indicate that general statements on sample stability are not reliable and have to be verified for the specific transcripts of interest. © 2005 by Walter de Gruyter. Berlin.
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| 10. |
- Nezirevic Dernroth, Dzeneta, 1969-, et al.
(författare)
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Gas chromatography-mass spectrometry analysis of pheomelanin degradation products
- 2009
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673 .- 1873-3778. ; 1216:30, s. 5730-5739
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Tidskriftsartikel (refereegranskat)abstract
- Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylaianine and 3-amino4-hydroxyphenylaianine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.
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