A comparison of the CHO/HGPRT+ and the L5178Y/TK+/? mutation assays using suspension treatment and soft agar cloning: Results for 10 chemicals

1993 ◽  
Vol 9 (3) ◽  
pp. 243-257 ◽  
Author(s):  
T. J. Oberly ◽  
K. C. Michaelis ◽  
M. A. Rexroat ◽  
B. J. Bewsey ◽  
M. L. Garriott
Keyword(s):  
Soft Agar ◽  
Soft Agar Cloning

scholarly journals Transformation of NIH 3T3 fibroblasts by an activated form of p59hck.

1989 ◽  
Vol 9 (6) ◽  
pp. 2724-2727 ◽  
Author(s):  
S F Ziegler ◽  
S D Levin ◽  
R M Perlmutter
Keyword(s):  
Tyrosine Kinases ◽  
Soft Agar ◽  
Protein Tyrosine Kinases ◽  
3T3 Fibroblasts ◽  
Family Protein ◽  
Cloning Assay ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Soft Agar Cloning ◽  
Phenylalanine Residue

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

scholarly journals L-myc cooperates with ras to transform primary rat embryo fibroblasts.

1988 ◽  
Vol 8 (6) ◽  
pp. 2668-2673 ◽  
Author(s):  
M J Birrer ◽  
S Segal ◽  
J S DeGreve ◽  
F Kaye ◽  
E A Sausville ◽  
...  
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Rat Embryo ◽  
Ras Gene ◽  
Reading Frame ◽  
Continuous Cell Lines ◽  
Embryo Fibroblasts ◽  
Soft Agar Cloning

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

An evaluation of the cho/hgprt mutation assay involving suspension cultures and soft agar cloning: Results for 33 chemicals

1990 ◽  
Vol 16 (4) ◽  
pp. 260-271 ◽  
Author(s):  
T. J. Oberly ◽  
M. A. Rexroat ◽  
B. J. Bewsey ◽  
K. K. Richardson ◽  
K. C. Michaelis ◽  
...  
Keyword(s):  
Soft Agar ◽  
Suspension Cultures ◽  
Mutation Assay ◽  
Soft Agar Cloning

Thymidylate synthase gene expression in solid tumors predicts for response to pemetrexed in vitro

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13058-13058 ◽  
Author(s):  
U. Eismann ◽  
O. Oberschmidt ◽  
M. Ehnert ◽  
J. Fleeth ◽  
F. Lüdtke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Statistical Tests ◽  
Human Tumor ◽  
Soft Agar ◽  
Expression Data ◽  
Glycinamide Ribonucleotide Formyltransferase ◽  
Cloning Assay ◽  
Thymidilate Synthase ◽  
Soft Agar Cloning

13058 Background: Pemetrexed (P) is a novel antifolate which targets thymidilate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). The aim of the present study was to identify gene expression thresholds for these enzymes in human tumor specimens in order to separate P-sensitive from P-resistant patients. Methods: Soft-agar cloning assays were performed on freshly biopsied tumor cells exposed one hour to clinically achievable concentrations of P. In parallel, RNA was isolated, transcribed to cDNA and subsequently used for multiplex real-time PCR. Gene expression data were normalized against beta-actin transcripts followed by correlation against cloning assay results. Iterative calculations (fourfold analysis) were done for each enzyme separately to find the best cutoff for prediction of sensitivity to P. Results: Sensitive and resistant tumor samples were statistically significant different in gene expression of TS, DHFR, and GARFT (p < 0.003). 81% of all tumors with a TS copy number < 144 (related to 104 copies β-actin) were sensitive to P in vitro. (specificity = 0.69; chi2 = 14.14). Statistical tests demonstrated that gene expression of TS, DHFR, and GARFT are dependent variables and that TS transcription is the leading variable. The combination of TS, DHFR, and GARFT expression data was not superior to TS alone. Conclusions: TS expression is the most meaningful predictor for sensitivity (≤ 144 copies) or resistance (> 144 copies) to Pemetrexed in fresh tumor tissue. This observation forms a rationale for clinical trials using TS expression as predictor for clinical response. No significant financial relationships to disclose.

scholarly journals Immortalized mesenchymal stem cells: a safe cell source for cellular or cell membrane-based treatment of glioma

2021 ◽  
Author(s):  
Yuxuan Zhang ◽  
Jie Liu ◽  
Yunzhao Mo ◽  
Zetao Chen ◽  
Taoliang Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cell Membranes ◽  
Glioma Cells ◽  
Soft Agar ◽  
Proliferative Capacity ◽  
Soft Agar Cloning ◽  
The Brain

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumour tropism, anti-tumour, and immunoregulatory properties. The limited passage and self-differentiation abilities of MSCs in vitro hinder preclinical studies of mesenchymal stem cells, and makes the MSC-based treatment of tumours lack a stable, uniform, and homogeneous source of cells. In this study, we focused on the safety of immortalised mesenchymal stem cells (im-MSCs) and, for the first time, studied the feasibility of im-MSCs as candidates for the treatment of glioma.METHODS: The im-MSCs were constructed by the lentiviral transfection of genes, and the proliferative capacity of immortalised MSCs and the proliferative phenotype of MSCs and MSCs co-cultured with glioma cells (U87) were measured using CCK-8 and EdU assays. After long-term culture, karyotype analysis of im-MSCs was conducted. In addition, the tumourigenicity of engineered MSCs was evaluated using soft agar cloning assays. Moreover, the engineered cells were injected into the brain of female BALB/c nude mice. Finally, the cell membranes of im-MSCs were labelled with DIO or DIR to detect their ability to be taken by glioma cells and target in situ gliomas using an IVIS system.RESULTS: Im-MSCs expressed CD73, CD90, CD105, CD29, and CD44 but did not express CD45, CD34, CD14, CD11b, or CD31. Engineered MSCs maintained the ability to differentiate into mesenchymal lineages in vitro. Im-MSCs showed stronger proliferative capacity than unengineered MSCs without colony formation in soft agar, no tumourigenicity in the brain, and normal chromosomes. MSCs or im-MSCs co-cultured with U87 cells showed enhanced proliferation ability, but did not show malignant characteristics in soft agar cloning experiments. Immortalised cells continued to express homing molecules. The cell membranes of im-MSCs were taken up by glioma cells and targeted in situ gliomas in vivo.CONCLUSIONS: Im-MSCs are promising candidates for cellular anti-glioma therapy. Im-MSCs provide a safe, adequate, quality-controlled, and continuous source of cells or cell membranes for the treatment of glioma.

L-myc cooperates with ras to transform primary rat embryo fibroblasts

1988 ◽  
Vol 8 (6) ◽  
pp. 2668-2673
Author(s):  
M J Birrer ◽  
S Segal ◽  
J S DeGreve ◽  
F Kaye ◽  
E A Sausville ◽  
...  
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
Rat Embryo ◽  
Ras Gene ◽  
Reading Frame ◽  
Continuous Cell Lines ◽  
Embryo Fibroblasts ◽  
Soft Agar Cloning

Recent molecular analysis has revealed that L-myc has several domains of extremely conserved amino acid sequence homology with c-myc and N-myc, suggesting similarity of function. We tested the biologic activity of L-myc by using an expression vector containing a cDNA clone coding for the major open reading frame in the 3.9-kilobase mRNA of L-myc under the control of a strong promoter (Moloney long terminal repeat) and found that L-myc complemented an activated ras gene in transforming primary rat embryo fibroblasts. However, the efficiency of transformation was 1 to 10% of that seen with the c-myc and simian virus 40 (SV40) controls. The L-myc/ras transformants initially grew more slowly than c-myc or SV40 transformants, but once established as continuous cell lines, they were indistinguishable from cell lines derived from c-myc/ras or SV40/ras transfectants as determined by morphology, soft-agar cloning, and tumorigenicity in nude mice.

Transformation of NIH 3T3 fibroblasts by an activated form of p59hck

1989 ◽  
Vol 9 (6) ◽  
pp. 2724-2727
Author(s):  
S F Ziegler ◽  
S D Levin ◽  
R M Perlmutter
Keyword(s):  
Tyrosine Kinases ◽  
Soft Agar ◽  
Protein Tyrosine Kinases ◽  
3T3 Fibroblasts ◽  
Family Protein ◽  
Cloning Assay ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Soft Agar Cloning ◽  
Phenylalanine Residue

Phosphorylation of a tyrosine residue near the carboxy terminus of src-family protein tyrosine kinases is believed to regulate the biological activity of these gene products. Conversion of this tyrosine in p59hck (Tyr-501) to a phenylalanine residue by using oligonucleotide-directed mutagenesis yielded a product (p59hckF501) with very potent transforming activity. Quantitative analysis by a soft-agar cloning assay revealed that p59hckF501 was more than 100-fold more effective than a closely related transforming element, p56lckF505, in colony formation. Cells bearing p59hckF501 had increased levels of protein phosphotyrosine. The ability of p59hckF501 to transform NIH 3T3 cells was abolished by a second mutation believed to destroy the ATP-binding domain.

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