The mouse lymphoma assay

In this paper, the current status of the protocol for the Mouse Lymphoma Assay is discussed. A brief history describes the events leading to current protocol recommendations. Areas for further development such as cytotoxicity, 24-h treatments, acceptability criteria and statistical analysis are also considered. Recent guidelines are reviewed, and consensus issues from the Mouse Lymphoma workgroup assembled as part of the International Workshop on Genotoxicity Test Procedures (IWGTP) are included. There are two versions of the assay - soft agar and microwell - and both will be discussed. For assay procedures, the emphasis will be on a typical microwell protocol but an attempt will be made to highlight protocol variations between laboratories and between the microwell and agar versions of the assay.     

An assay for leukoagglutinating lectins using suspension cultured mouse lymphoma cells (BW5147) stained with neutral red

A sensitive and rapid assay for leukoagglutinating lectins has been developed. This assay utilizes neutral red-stained mouse lymphoma cells from the suspension cultured cell line BW5147. The agglutination of the stained cells can be monitored visually in a manner similar to that for conventional assays for erythroagglutinating lectins using erythrocytes. The activity of lekoagglutinating lectins that are not capable of agglutinating erythrocytes can be quantified by this assay. The utility of the assay was demonstrated using leukoagglutinating and erythroagglutinating lectins from the seeds of Phaseolus vulgaris and Maackia amurensis.     

A statistical analysis for the mouse lymphoma cell forward mutation assay

This paper illustrates the usefulness of solvent control trials and presents a statistical analysis for mouse L5178Y lymphoma data. Examination of solvent control trials establishes that the natural logarithm of mutant frequency is approximately normally distributed and that both the mean and variance of mutant frequency vary by trial. There is little evidence of downturns at higher doses in the dose-response curves studied; therefore, a trend test is proposed for the detection of an increasing dose-response curve. A Monte Carlo investigation confirms that the proposed trend test is better able to detect an increasing dose-response than 4 alternate methods of analysis.     

Selective extraction, concentration, and assay of orthophosphate from microliter quantities of cultured mammalian cells

A colorimetric procedure is described for determination of orthophosphate (0.2-2.5 nmol) in sample volumes up to 400 microliters. Orthophosphate is selectively extracted (in the form of phosphomolybdate) into an organic solvent mixture (2-methylpropan-1-ol and petroleum spirit) leaving interfering substances, such as labile organic phosphates, in the aqueous phase. Orthophosphate is then back-extracted into a small volume of aqueous sodium hydroxide. By keeping this volume small, orthophosphate from large dilute samples can be concentrated into small volumes and assayed colorimetrically in microcuvettes using the dye malachite green. The procedure is highly reproducible and insensitive to interfering substances, as shown by comparison with a conventional malachite green assay without the solvent extraction.     

Characterization of mouse lymphoma cells with altered nucleoside transport

A mutant clone (NT-1) of a T-cell lymphoma was selected for its ability to grow in HAT medium (hypoxanthine, aminopterin and thymidine) in the presence of the nucleoside transport inhibitor P-nitrobenzyl-6-mercaptoinosine (NBMI). NT-1 cells contain half the number of NBMI binding sites present on the parental S49 cells and are partially able to transport nucleosides in the presence of the transport inhibitor (NBMI). These observations suggest that the mutant cells are heterozygous for nucleoside transport proteins and contain two types of transport proteins: the first protein can both bind and is inhibited by NBMI similar to the wild type phenotype, and the second is an altered protein. The altered transport protein apparently lost its NBMI binding sites without a parallel loss of nucleoside transport ability suggesting that the nucleoside transported sites are separate from the binding sites of the transport inhibitor.     

Modeling the mouse lymphoma forward mutational assay: the Gene-Tox program database

An SAR model of the induction of mutations at the tk(+/-) locus of L5178Y mouse lymphoma cells (MLA, for mouse lymphoma assay) was derived based upon a re-evaluation of experimental results reported by a Gene-Tox (GT) working group [A.D. Mitchell, A.E. Auletta, D. Clive, P.E. Kirby, M.M. Moore, B.C. Myhr, The L5178Y/tk(+/-) mouse lymphoma specific gene and chromosomal mutation assay. A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res. 394 (1997) 177-303.]. The predictive performance of the GT MLA SAR model was similar to that of a Salmonella mutagenicity model containing the same number of chemicals. However, the structural determinants (biophores) derived from the GT MLA SAR model include both electrophilic as well as non-electrophilic moieties, suggesting that the induction of mutations in the MLA may occur by both direct interaction with DNA and by non-DNA-related mechanisms. This was confirmed by the observation that the set of biophores associated with MLA overlapped significantly with those associated with phenomena related to loss of heterozygosity, chromosomal rearrangements and aneuploidy. The MLA SAR model derived from the GT data evaluation was significantly more predictive than an SAR model previously derived from MLA data reported by the US National Toxicology Program [B. Henry, S.G. Grant, G. Klopman, H.S. Rosenkranz, Induction of forward mutations at the thymidine kinase locus of mouse lymphoma cells: evidence for electrophilic and non-electrophilic mechanisms, Mutation Res. 397 (1998) 331-335.]. Moreover, the latter model appeared to be more complex than the former, suggesting that the GT induction data was both simpler mechanistically and more homogeneous than that of the NTP.     

A double-reporter splicing assay for determining splicing efficiency in mammalian cells

Changes in alternative splicing patterns can result from both inherited and acquired defects, and they are increasingly recognized as causes of human diseases. Hence, improvements in the understanding of alternative splicing regulation may provide opportunities for restoring productive patterns of splicing. The identification of factors (such as proteins, nucleic acids or small molecules) that modulate the splicing pattern would be facilitated by systems with which many samples can be screened. The absence of reliable systems prompted us to develop an assay system based on dual enzymatic activities. Two distinct signals derived from spliced and unspliced RNA are measured, providing the basis for a robust, rapid and convenient assay for investigating splicing. This protocol describes how to use this system; the time required for lysing the cells and recording enzymatic activity is about 2 hours.     

Immunochemotherapy studies with murine lymphoma cells growing in mouse brain

Summary The present study was undertaken to explore the possible interaction between tumor immunity and antineoplastic agents at the brain level in murine lymphoma models. Host immunity against intracerebral lymphoma graft was directed against tumor-associated histocompatibility antigens, using an appropriate design of genetic distance between host and tumor. It was revealed that primary graft response against lymphoma cells can be demonstrated in the central nervous system. Immunochemotherapy synergism can occur at the mouse brain level, when allogeneic lymphomas are inoculated intracerebrally and the recipient hosts are treated with antineoplastic agents capable of crossing the blood-brain-barrier.Supported by C.N.R. Italia-USA Contract: n. 79.02381.65 Abbreviations used: ADM = Adriamycin BBB = Blood-brain-barrier BCNU = 1,3-bis-(2 chloroethyl)-1-nitrosourea Cy = Cyclophosphamide CNS = Central nervous system D/T = Dead mice over total animals tested DTIC = 5(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide HTHI = Host tumor histoincompatibility IC = Intracerebrally MMHL = Multiple minor histocompatible loci MST = Median survival time in days NS = Not significant NT = Not tested TAHA = Tumor-associated histocompatibility antigens TATA = Tumor-associated transplantation antigens     

Gene-mutation induction by arsenic compounds in the mouse lymphoma assay

Arsenic compounds are generally considered as poor inducers of gene mutations. To investigate the mutagenicity of several arsenic compounds at the thymidine kinase (Tk) gene, a reporter gene for mutation induction, we used the mouse lymphoma assay (MLA). This test is widely applied and detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The selected arsenic compounds were two inorganic (sodium arsenite and arsenic trioxide) and four organic compounds (monomethylarsonic acid, dimethylarsinic acid, tetraphenylarsenium and arsenobetaine). The results show that sodium arsenite, arsenic trioxide, monomethylarsonic acid and dimethylarsinic acid are mutagenic, showing a clear dose-response pattern. On the other hand, tetraphenylarsenium and arsenobetaine are not mutagenic. Inorganic arsenic compounds are the more potent agents producing significant effects in the micromolar range, while the mutagenic organic arsenic compounds induce similar effects but in the millimolar range.     

Gene-mutation induction by arsenic compounds in the mouse lymphoma assay

Arsenic compounds are generally considered as poor inducers of gene mutations. To investigate the mutagenicity of several arsenic compounds at the thymidine kinase (Tk) gene, a reporter gene for mutation induction, we used the mouse lymphoma assay (MLA). This test is widely applied and detects a broad spectrum of mutational events, from point mutations to chromosome alterations. The selected arsenic compounds were two inorganic (sodium arsenite and arsenic trioxide) and four organic compounds (monomethylarsonic acid, dimethylarsinic acid, tetraphenylarsenium and arsenobetaine). The results show that sodium arsenite, arsenic trioxide, monomethylarsonic acid and dimethylarsinic acid are mutagenic, showing a clear dose–response pattern. On the other hand, tetraphenylarsenium and arsenobetaine are not mutagenic. Inorganic arsenic compounds are the more potent agents producing significant effects in the micromolar range, while the mutagenic organic arsenic compounds induce similar effects but in the millimolar range.     

A fluorescent assay of proteinases in cultured mammalian cells

We have demonstrated proteinase activity in unfixed cells grown on tissue culture plates with a technique using 5-nitrosalicylaldehyde and peptide derivatives of 4-methoxy-2-naphthylamine. The 4-methoxy-2-naphthylamine liberated by proteinase activity reacts with 5-nitrosalicylaldehyde to form a fluorescent product. The substrates CBZ-alanyl-arginyl-arginyl-4-methoxy-2-naphthylamine and lysyl-alanyl-4-methoxy-2-naphthylamine, were used for the direct visual detection of two arylamidase activities in BALB/c 3T3 and C3H 10T 1/2 cells. With low magnification these enzyme activities can be detected in single clones; with higher magnification the fluorescent product can be seen within the cytoplasm of single cells.     

Fluorine nuclear magnetic resonance-based assay in living mammalian cells

Nuclear magnetic resonance (NMR)-based screening has been recognized as a powerful approach for the identification and characterization of molecules interacting with pharmaceutical targets. Indeed, several NMR methods have been developed and successfully applied to many drug discovery projects. Whereas most of these approaches have targeted isolated biomolecular receptors, very few cases are reported with the screening performed in intact cells and cell extracts. Here we report the first successful application of the fluorine NMR-based assay n-FABS (n-fluorine atoms for biochemical screening) in living mammalian cells expressing the membrane protein fatty acid amide hydrolase (FAAH). This method allows the identification of both weak and potent inhibitors and the measurement of their potency in a physiological environment.     

模型鼠低氧预适应适宜氧气浓度研究

目的:研究低氧预适应训练的适宜氧气浓度。方法:设计了短期和长期两种间歇性低氧暴露模式,研究了一系列不同浓度的低氧环境对模型鼠体重、血氧饱和度、游泳能力等方面的影响,进而探讨低氧预适应效应与氧气浓度之间的内在联系。结果:模型鼠长期暴露于低氧环境中,其体重增长率逐步下降;在15%～8%的低氧浓度区间,模型鼠血氧饱和度随氧气浓度降低呈现平台似缓慢下降趋势;低氧预适应训练后的模型鼠游泳能力显著提高,经在10%低氧环境中进行低氧预适应训练后的昆明小鼠游泳能力提高最为明显。结论:适当浓度的低氧预适应训练可以改善模型鼠低氧耐受能力,显著提高模型鼠运动能力。15%～10%氧气浓度区间可视为低氧预适应有益作用区间。10%氧气浓度为模型鼠低氧预适应训练的较适宜浓度。