| 1. |
- Hashemi, Jamileh, et al.
(författare)
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Molecular Characterization of Acquired Tolerance of Tumor Cells to Picropodophyllin (PPP)
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:3, s. e14757-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Picropodophyllin (PPP) is a promising novel anti-neoplastic agent that efficiently kills tumor cells in vitro and causes tumor regression and increased survival in vivo. We have previously reported that PPP treatment induced moderate tolerance in two out of 10 cell lines only, and here report the acquired genomic and expression alterations associated with PPP selection over 1.5 years of treatment. Methodology/Principal Findings: Copy number alterations monitored using metaphase and array-based comparative genomic hybridization analyses revealed largely overlapping alterations in parental and maximally tolerant cells. Gain/amplification of the MYC and PVT1 loci in 8q24.21 were verified on the chromosome level. Abnormalities observed in connection to PPP treatment included regular gains and losses, as well as homozygous losses in 10q24.1-q24.2 and 12p12.3-p13.2 in one of the lines and amplification at 5q11.2 in the other. Abnormalities observed in both tolerant derivatives include amplification/gain of 5q11.2, gain of 11q12.1-q14.3 and gain of 13q33.3-qter. Using Nexus software analysis we combined the array-CGH data with data from gene expression profilings and identified genes that were altered in both inputs. A subset of genes identified as downregulated (ALDH1A3, ANXA1, TLR4 and RAB5A) or upregulated (COX6A1, NFIX, ME1, MAPK and TAP2) were validated by siRNA in the tolerant or parental cells to alter sensitivity to PPP and confirmed to alter sensitivity to PPP in further cell lines. Conclusions: Long-term PPP selection lead to altered gene expression in PPP tolerant cells with increase as well as decrease of genes involved in cell death such as PTEN and BCL2. In addition, acquired genomic copy number alterations were observed that were often reflected by altered mRNA expression levels for genes in the same regions.
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| 2. |
- Larsson, Mårten, et al.
(författare)
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Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins
- 2003
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 373:2, s. 381-391
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Tidskriftsartikel (refereegranskat)abstract
- Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
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| 3. |
- Aftab, Obaid, 1984-, et al.
(författare)
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Detection of cell aggregation and altered cell viability by automated label-free video microscopy : A promising alternative to endpoint viability assays in high throughput screening
- 2015
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 20:3, s. 372-381
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Tidskriftsartikel (refereegranskat)abstract
- Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.
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| 4. |
- Aftab, Obaid, 1984-, et al.
(författare)
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Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
- 2014
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10:1, s. 57-69
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
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| 5. |
- Aftab, Obaid, 1984-, et al.
(författare)
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Label free high throughput screening for apoptosis inducing chemicals using time-lapse microscopy signal processing
- 2014
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Ingår i: Apoptosis (London). - : Springer Science and Business Media LLC. - 1360-8185 .- 1573-675X. ; 19:9, s. 1411-1418
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Tidskriftsartikel (refereegranskat)abstract
- Label free time-lapse microscopy has opened a new avenue to the study of time evolving events in living cells. When combined with automated image analysis it provides a powerful tool that enables automated large-scale spatiotemporal quantification at the cell population level. Very few attempts, however, have been reported regarding the design of image analysis algorithms dedicated to the detection of apoptotic cells in such time-lapse microscopy images. In particular, none of the reported attempts is based on sufficiently fast signal processing algorithms to enable large-scale detection of apoptosis within hours/days without access to high-end computers. Here we show that it is indeed possible to successfully detect chemically induced apoptosis by applying a two-dimensional linear matched filter tailored to the detection of objects with the typical features of an apoptotic cell in phase-contrast images. First a set of recorded computational detections of apoptosis was validated by comparison with apoptosis specific caspase activity readouts obtained via a fluorescence based assay. Then a large screen encompassing 2,866 drug like compounds was performed using the human colorectal carcinoma cell line HCT116. In addition to many well known inducers (positive controls) the screening resulted in the detection of two compounds here reported for the first time to induce apoptosis.
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| 6. |
- Aftab, Obaid, 1984-, et al.
(författare)
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Label free quantification of time evolving morphologies using time-lapse video microscopy enables identity control of cell lines and discovery of chemically induced differential activity in iso-genic cell line pairs
- 2015
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Ingår i: Chemometrics and Intelligent Laboratory Systems. - 0169-7439 .- 1873-3239. ; 141, s. 24-32
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Tidskriftsartikel (refereegranskat)abstract
- Label free time-lapse video microscopy based monitoring of time evolving cell population morphology has potential to offer a simple and cost effective method for identity control of cell lines. Such morphology monitoring also has potential to offer discovery of chemically induced differential changes between pairs of cell lines of interest, for example where one in a pair of cell lines is normal/sensitive and the other malignant/resistant. A new simple algorithm, pixel histogram hierarchy comparison (PHHC), for comparison of time evolving morphologies (TEM) in phase contrast time-lapse microscopy movies was applied to a set of 10 different cell lines and three different iso-genic colon cancer cell line pairs, each pair being genetically identical except for a single mutation. PHHC quantifies differences in morphology by comparing pixel histogram intensities at six different resolutions. Unsupervised clustering and machine learning based classification methods were found to accurately identify cell lines, including their respective iso-genic variants, through time-evolving morphology. Using this experimental setting, drugs with differential activity in iso-genic cell line pairs were likewise identified. Thus, this is a cost effective and expedient alternative to conventional molecular profiling techniques and might be useful as part of the quality control in research incorporating cell line models, e.g. in any cell/tumor biology or toxicology project involving drug/agent differential activity in pairs of cell line models.
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| 7. |
- Andersson, Claes, et al.
(författare)
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Assessment in vitro of interactions between anti-cancer drugs and noncancer drugs commonly used by cancer patients
- 2023
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Ingår i: Anti-Cancer Drugs. - : Lippincott Williams & Wilkins. - 0959-4973 .- 1473-5741. ; 34:1, s. 92-102
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Tidskriftsartikel (refereegranskat)abstract
- Cancer patients often suffer from cancer symptoms, treatment complications and concomitant diseases and are, therefore, often treated with several drugs in addition to anticancer drugs. Whether such drugs, here denoted as 'concomitant drugs', have anticancer effects or interact at the tumor cell level with the anticancer drugs is not very well known. The cytotoxic effects of nine concomitant drugs and their interactions with five anti-cancer drugs commonly used for the treatment of colorectal cancer were screened over broad ranges of drug concentrations in vitro in the human colon cancer cell line HCT116wt. Seven additional tyrosine kinase inhibitors were included to further evaluate key findings as were primary cultures of tumor cells from patients with colorectal cancer. Cytotoxic effects were evaluated using the fluorometric microculture cytotoxicity assay (FMCA) and interaction analysis was based on Bliss independent interaction analysis. Simvastatin and loperamide, included here as an opioid agonists, were found to have cytotoxic effects on their own at reasonably low concentrations whereas betamethasone, enalapril, ibuprofen, metformin, metoclopramide, metoprolol and paracetamol were inactive also at very high concentrations. Drug interactions ranged from antagonistic to synergistic over the concentrations tested with a more homogenous pattern of synergy between simvastatin and protein kinase inhibitors in HCT116wt cells. Commonly used concomitant drugs are mostly neither expected to have anticancer effects nor to interact significantly with anticancer drugs frequently used for the treatment of colorectal cancer.
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| 8. |
- Andersson, Claes, et al.
(författare)
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Mebendazole is unique among tubulin-active drugs in activating the MEK-ERK pathway
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases.
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| 9. |
- Andersson, Claes R., et al.
(författare)
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In vitro drug sensitivity-gene expression correlations involve a tissue of origin dependency
- 2007
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Ingår i: Journal of chemical information and modeling. - : American Chemical Society (ACS). - 1549-9596 .- 1549-960X. ; 47:1, s. 239-248
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Tidskriftsartikel (refereegranskat)abstract
- A major concern of chemogenomics is to associate drug activity with biological variables. Several reports have clustered cell line drug activity profiles as well as drug activity-gene expression correlation profiles and noted that the resulting groupings differ but still reflect mechanism of action. The present paper shows that these discrepancies can be viewed as a weighting of drug-drug distances, the weights depending on which cell lines the two drugs differ in.
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| 10. |
- Babina, Arianne M., et al.
(författare)
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Rescue of Escherichia coli auxotrophy by de novo small proteins
- 2023
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Ingår i: eLIFE. - : eLife Sciences Publications Ltd. - 2050-084X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here, we show that by upregulating hisB expression, de novo small proteins (<= 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5 ' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.
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