Assessment of the sensitivity of leukaemic cells to cytotoxic drugs by bioluminescence measurement of ATP in cultured cells

1986 ◽  
Vol 71 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Rudolf Kuzmits ◽  
Paul Aiginger ◽  
Matthias M. Müller ◽  
Günter Steurer ◽  
Werner Linkesch
Keyword(s):  
Cultured Cells ◽  
Dose Effect ◽  
Blue Dye ◽  
Response Patterns ◽  
Cytostatic Agents ◽  
Drug Induced ◽  
Atp Assay ◽  
Leukaemic Cells

1. A short-term test in vitro is described, which can be used to detect resistance to cytostatic agents in leukaemic cells. Leukaemic cell suspensions were incubated with cytostatic agents and the resulting intracellular ATP concentrations were measured by a bioluminescence ATP assay. 2. There was a clear dose-effect relationship in acute leukaemia and chronic lymphocytic leukaemia cells for drugs used in the treatment of leukaemias. A good correlation was found between the ATP content of leukaemic cells and cell viability as determined by the trypan blue dye exclusion test. 3. Preliminary individual clinical correlations suggest a correlation between the chemosensitivity in vitro and the response patterns in vivo in leukaemia patients. This simple, fast and sensitive method may have application for determination of drug-induced cytotoxicity in leukaemic cells in vitro.

scholarly journals Modulation of the combinatorial code of odorant receptor response patterns in odorant mixtures

2019 ◽  
Author(s):  
Claire A. de March ◽  
William B. Titlow ◽  
Tomoko Sengoku ◽  
Patrick Breheny ◽  
Hiroaki Matsunami ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Odorant Receptor ◽  
Response Patterns ◽  
Independent Evidence ◽  
Receptor Response ◽  
Pharmacological Interactions ◽  
Combinatorial Codes ◽  
Mixture Suppression

AbstractThe perception of odors relies on combinatorial codes consisting of odorant receptor (OR) response patterns to encode odor identity. The modulation of these patterns by odorant interactions at ORs potentially explains several olfactory phenomena: mixture suppression, unpredictable sensory outcomes, and the perception of odorant mixtures as unique objects. We determined OR response patterns to 4 odorants and 3 binary mixtures in vivo in mice, identifying 30 responsive ORs. These patterns typically had a few strongly responsive ORs and a greater number of weakly responsive ORs. The ORs responsive to an odorant were often unrelated sequences distributed across several OR subfamilies. Mixture responses predicted pharmacological interactions between odorants, which were tested in vitro by heterologous expression of ORs in cultured cells. These tests provided independent evidence confirming odorant agonists for 13 ORs and identified both suppressive and additive effects of mixing odorants. This included 11 instances of antagonism of ORs by an odorant, 1 instance of additive responses to a binary mixture, 1 instance of suppression of a strong agonist by a weak agonist, and the discovery of an inverse agonist for an OR. These findings demonstrate that interactions between odorants at ORs are common.

Ultrastructural Correlations between Clonal Human Tumor Colonies and in Vivo Neoplasms

1982 ◽  
Vol 40 ◽  
pp. 210-211
Author(s):  
M.J. Murphy ◽  
R.R. Price ◽  
J.C. Sloman
Keyword(s):  
Cultured Cells ◽  
Human Tumor ◽  
Cell Type ◽  
Tumor Mass ◽  
Initial Cell ◽  
Cloning Assay ◽  
Human Tumor Cloning ◽  
The Relationship

The in vitro human tumor cloning assay originally described by Salmon and Hamburger has been applied recently to the investigation of differential anti-tumor drug sensitivities over a broad range of human neoplasms. A major problem in the acceptance of this technique has been the question of the relationship between the cultured cells and the original patient tumor, i.e., whether the colonies that develop derive from the neoplasm or from some other cell type within the initial cell population. A study of the ultrastructural morphology of the cultured cells vs. patient tumor has therefore been undertaken to resolve this question. Direct correlation was assured by division of a common tumor mass at surgical resection, one biopsy being fixed for TEM studies, the second being rapidly transported to the laboratory for culture.

Preparation of cultured astrocytes for x-ray microanalysis

1989 ◽  
Vol 47 ◽  
pp. 988-989
Author(s):  
N.K.R. Smith ◽  
K.E. Hunter ◽  
P. Mobley ◽  
L.P. Felpel
Keyword(s):  
Cultured Cells ◽  
Elemental Content ◽  
Elemental Distribution ◽  
Sprague Dawley ◽  
X Ray ◽  
Cultured Astrocytes ◽  
Freeze Dried ◽  
Ultimate Objective

Electron probe energy dispersive x-ray microanalysis (XRMA) offers a powerful tool for the determination of intracellular elemental content of biological tissue. However, preparation of the tissue specimen , particularly excitable central nervous system (CNS) tissue , for XRMA is rather difficult, as dissection of a sample from the intact organism frequently results in artefacts in elemental distribution. To circumvent the problems inherent in the in vivo preparation, we turned to an in vitro preparation of astrocytes grown in tissue culture. However, preparations of in vitro samples offer a new and unique set of problems. Generally, cultured cells, growing in monolayer, must be harvested by either mechanical or enzymatic procedures, resulting in variable degrees of damage to the cells and compromised intracel1ular elemental distribution. The ultimate objective is to process and analyze unperturbed cells. With the objective of sparing others from some of the same efforts, we are reporting the considerable difficulties we have encountered in attempting to prepare astrocytes for XRMA.Tissue cultures of astrocytes from newborn C57 mice or Sprague Dawley rats were prepared and cultured by standard techniques, usually in T25 flasks, except as noted differently on Cytodex beads or on gelatin. After different preparative procedures, all samples were frozen on brass pins in liquid propane, stored in liquid nitrogen, cryosectioned (0.1 μm), freeze dried, and microanalyzed as previously reported.

scholarly journals Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3through CYP3A4 induction in vitro and in vivo: Implications for drug-induced osteomalacia

2013 ◽  
Vol 28 (5) ◽  
pp. 1101-1116 ◽  
Author(s):  
Zhican Wang ◽  
Yvonne S Lin ◽  
Leslie J Dickmann ◽  
Emma-Jane Poulton ◽  
David L Eaton ◽  
...  
Keyword(s):  
Drug Induced ◽  
Cyp3a4 Induction

scholarly journals TAMI-06. PRECLINICAL MODELS REVEAL BRAIN-MICROENVIRONMENT SPECIFIC METABOLIC DEPENDENCIES IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii214-ii214
Author(s):  
Jenna Minami ◽  
Nicholas Bayley ◽  
Christopher Tse ◽  
Henan Zhu ◽  
Danielle Morrow ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Cultured Cells ◽  
Tumor Biology ◽  
Metabolic Reprogramming ◽  
Neoplastic Progression ◽  
Extracellular Milieu ◽  
Metabolic Defects ◽  
Metabolic Heterogeneity

Abstract Metabolic reprogramming is a hallmark of cancer, and malignant cells must acquire metabolic adaptations to fuel neoplastic progression. Mutations or changes in metabolic gene expression can impose nutrient dependencies in tumors, and even in the absence of metabolic defects, cancer cells can become auxotrophic for particular nutrients or metabolic byproducts generated by other cells in the tumor microenvironment (TME). Conventional cell lines do not recapitulate the metabolic heterogeneity of glioblastoma (GBM), while primary cultured cells do not account for the influences of the microenvironment and the blood brain barrier on tumor biology. Additionally, these systems are under strong selective pressure divergent from that in vivo, leading to reduced heterogeneity between cultured tumor cells. Here, we describe a biobank of direct-from-patient derived orthotopic xenografts (GliomaPDOX) and gliomaspheres that reveal a subset of gliomas that, while able to form in vivo, cannot survive in vitro. RNA sequencing of tumors that can form both in vivo and in vitro (termed “TME-Indifferent”) compared to that of tumors that can only form in vivo (termed “TME-Dependent”) revealed transcriptional changes associated with altered nutrient availability, emphasizing the unique metabolic programs impacted by the tumor microenvironment. Furthermore, TME-dependent tumors lack metabolic signatures associated with nutrient biosynthesis, thus indicating a potential dependency of these tumors on scavenging specific nutrients from the extracellular milieu. Collectively, these data emphasize the metabolic heterogeneity within GBM, and reveal a subset of gliomas that lack metabolic plasticity, indicating a potential brain-microenvironment specific metabolic dependency that can be targeted for therapy.

Imidazolidinyl urea activates mast cells via MRGPRX2 to induce non-histaminergic allergy

2021 ◽  
Author(s):  
Jiapan Gao ◽  
Delu Che ◽  
Xueshan Du ◽  
Yi Zheng ◽  
Huiling Jing ◽  
...  
Keyword(s):  
Contact Dermatitis ◽  
Mast Cells ◽  
Pharmaceutical Products ◽  
Drug Induced ◽  
Inflammatory Infiltration ◽  
Local Inflammatory Response ◽  
Allergic Contact ◽  
G Protein Coupled

Abstract Imidazolidinyl urea (IU) is used as an antimicrobial preservative in cosmetic and pharmaceutical products. IU induces allergic contact dermatitis, however, the mechanism has not yet been elucidated. Mas-related G protein-coupled receptor-X2 (MRGPRX2) triggers drug-induced pseudo-allergic reactions. The aims of this study were to determine whether IU activated mast cells through MRGPRX2 to further trigger contact dermatitis. Wild-type (WT) and KitW-sh/HNihrJaeBsmJNju (MUT) mice were treated with IU to observe its effects on local inflammation and mast cells degranulation in vivo. Laboratory of allergic disease 2 cells were used to detect calcium mobilization and release of inflammatory mediators in vitro. WT mice showed a severe local inflammatory response and contact dermatitis, whereas only slight inflammatory infiltration was observed in MUT mice. Thus, MRGPRX2 mediated the IU-induced activation of mast cells. However, histamine, a typical allergen, was not involved in this process. Tryptase expressed by mast cells was the major non-histaminergic inflammatory mediator of contact dermatitis. IU induced anaphylactic reaction via MRGPRX2 and further triggering non-histaminergic contact dermatitis, which explained why antihistamines are clinically ineffective against some chronic dermatitis.

scholarly journals Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shojiro Katoh ◽  
Atsuki Fujimaru ◽  
Masaru Iwasaki ◽  
Hiroshi Yoshioka ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Replicative Senescence ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Optimal Method ◽  
Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

scholarly journals Targeting a Lipid Desaturation Enzyme, SCD1, Selectively Eliminates Colon Cancer Stem Cells through the Suppression of Wnt and NOTCH Signaling

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 106
Author(s):  
Yeongji Yu ◽  
Hyejin Kim ◽  
SeokGyeong Choi ◽  
JinSuh Yu ◽  
Joo Yeon Lee ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Notch Signaling ◽  
Cultured Cells ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Dependent Manner ◽  
Lipid Desaturation ◽  
Novel Target

The elimination of the cancer stem cell (CSC) population may be required to achieve better outcomes of cancer therapy. We evaluated stearoyl-CoA desaturase 1 (SCD1) as a novel target for CSC-selective elimination in colon cancer. CSCs expressed more SCD1 than bulk cultured cells (BCCs), and blocking SCD1 expression or function revealed an essential role for SCD1 in the survival of CSCs, but not BCCs. The CSC potential selectively decreased after treatment with the SCD1 inhibitor in vitro and in vivo. The CSC-selective suppression was mediated through the induction of apoptosis. The mechanism leading to selective CSC death was investigated by performing a quantitative RT-PCR analysis of 14 CSC-specific signaling and marker genes after 24 and 48 h of treatment with two concentrations of an inhibitor. The decrease in the expression of Notch1 and AXIN2 preceded changes in the expression of all other genes, at 24 h of treatment in a dose-dependent manner, followed by the downregulation of most Wnt- and NOTCH-signaling genes. Collectively, we showed that not only Wnt but also NOTCH signaling is a primary target of suppression by SCD1 inhibition in CSCs, suggesting the possibility of targeting SCD1 against colon cancer in clinical settings.

scholarly journals In Vivo and In Vitro Toxicodynamic Analyses of New Quinolone-and Nonsteroidal Anti-Inflammatory Drug-Induced Effects on the Central Nervous System

1999 ◽  
Vol 43 (5) ◽  
pp. 1091-1097 ◽  
Author(s):  
Hideki Kita ◽  
Hirotami Matsuo ◽  
Hitomi Takanaga ◽  
Junichi Kawakami ◽  
Koujirou Yamamoto ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Threshold Concentration ◽  
Current Response ◽  
Drug Induced ◽  
Inhibition Ratio ◽  
New Quinolones ◽  
The Central Nervous System ◽  
Anti Inflammatory

ABSTRACT We investigated the correlation between an in vivo isobologram based on the concentrations of new quinolones (NQs) in brain tissue and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for the occurrence of convulsions in mice and an in vitro isobologram based on the concentrations of both drugs for changes in the γ-aminobutyric acid (GABA)-induced current response in Xenopus oocytes injected with mRNA from mouse brains in the presence of NQs and/or NSAIDs. After the administration of enoxacin (ENX) in the presence or absence of felbinac (FLB), ketoprofen (KTP), or flurbiprofen (FRP), a synergistic effect was observed in the isobologram based on the threshold concentration in brain tissue between mice with convulsions and those without convulsions. The three NSAIDs did not affect the pharmacokinetic behavior of ENX in the brain. However, the ENX-induced inhibition of the GABA response in the GABAA receptor expressed in Xenopus oocytes was enhanced in the presence of the three NSAIDs. The inhibition ratio profiles of the GABA responses for both drugs were analyzed with a newly developed toxicodynamic model. The inhibitory profiles for ENX in the presence of NSAIDs followed the order KTP (1.2 μM) > FRP (0.3 μM) > FLB (0.2 μM). These were 50- to 280-fold smaller than those observed in the absence of NSAIDs. The inhibition ratio (0.01 to 0.02) of the GABAA receptor in the presence of both drugs was well-fitted to the isobologram based on threshold concentrations of both drugs in brain tissue between mice with convulsions and those without convulsions, despite the presence of NSAIDs. In mice with convulsions, the inhibitory profiles of the threshold concentrations of both drugs in brain tissue of mice with convulsions and those without convulsions can be predicted quantitatively by using in vitro GABA response data and toxicodynamic model.

scholarly journals Synaptopodin: An Actin-associated Protein in Telencephalic Dendrites and Renal Podocytes

1997 ◽  
Vol 139 (1) ◽  
pp. 193-204 ◽  
Author(s):  
Peter Mundel ◽  
Hans W. Heid ◽  
Thomas M. Mundel ◽  
Meike Krüger ◽  
Jochen Reiser ◽  
...  
Keyword(s):  
Dendritic Spines ◽  
Cultured Cells ◽  
Rat Kidney ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Globular Domain ◽  
Postnatal Maturation ◽  
Human And Mouse

Synaptopodin is an actin-associated protein of differentiated podocytes that also occurs as part of the actin cytoskeleton of postsynaptic densities (PSD) and associated dendritic spines in a subpopulation of exclusively telencephalic synapses. Amino acid sequences determined in purified rat kidney and forebrain synaptopodin and derived from human and mouse brain cDNA clones show no significant homology to any known protein. In particular, synaptopodin does not contain functional domains found in receptor-clustering PSD proteins. The open reading frame of synaptopodin encodes a polypeptide with a calculated Mr of 73.7 kD (human)/74.0 kD (mouse) and an isoelectric point of 9.38 (human)/9.27 (mouse). Synaptopodin contains a high amount of proline (∼20%) equally distributed along the protein, thus virtually excluding the formation of any globular domain. Sequence comparison between human and mouse synaptopodin revealed 84% identity at the protein level. In both brain and kidney, in vivo and in vitro, synaptopodin gene expression is differentiation dependent. During postnatal maturation of rat brain, synaptopodin is first detected by Western blot analysis at day 15 and reaches maximum expression in the adult animal. The exclusive synaptopodin synthesis in the telencephalon has been confirmed by in situ hybridization, where synaptopodin mRNA is only found in perikarya of the olfactory bulb, cerebral cortex, striatum, and hippocampus, i.e., the expression is restricted to areas of high synaptic plasticity. From these results and experiments with cultured cells we conclude that synaptopodin represents a novel kind of proline-rich, actin-associated protein that may play a role in modulating actin-based shape and motility of dendritic spines and podocyte foot processes.

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