Transcriptomics and Cell Transformation Assay: an Integrated Approach to Evaluate the Effects of Low Dose Ionizing Radiation

Background and aims: Ionizing radiation (IR) are a well-known carcinogenic agent, acting through genotoxic mechanisms. In the last years, great attention has been paid to the effects of IR at low doses and to the non-monotonic dose-response curve for IR exposures. To improve the knowledge of IR-mediated effects and possibly identify biomarkers for IR effects, we combined the Cell Transformation Assay (CTA) with transcriptomics, to correlate cytotoxicity and transformation endpoints with the modulation of gene profiles after IR exposure. Methods: BALB/c3T3 cells were exposed to ionizing radiation ranging from 0.25Gy and 6Gy. Irradiated cells were seeded for the CTA 20h later. At the same time, RNA was extracted for microarray experiments. The cell clonal survival was significantly increased in 0.25Gy IR exposed cells, while the 3Gy dose strongly inhibited cellular growth. Cell transformation was observed only at the highest dose (3Gy). Results: Cell’s transformation was observed at 1.5, 2 and 3Gy doses. The 0.25Gy dose, which was able to induce an increment of clonal efficiency, did not induce cell transformation. The gene expression profile, which was obtained by comparing cells treated with the highest tested dose of 3Gy with the cells exposed to the lowest, not transforming, dose of 0.25Gy, identified several genes related to mitotic cell cycle and cholesterol biosynthesis. Conclusion: Our study showed that the up-regulation of genes belonging to the Spindle Assembly Checkpoint and mitosis progression could support the transforming ability of the 3Gy BALB/c3T3 exposed cells, probably through the involvement of genomic instability. Gene transcripts involved into cholesterol biosynthesis appear to be critical, as well. All these transcripts may be regarded as potential biomarkers of IR effects.

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The Cell Transformation Assay: Toward a Statistical Classification of Mixed and Intermediate Foci Images

Alternatives to Laboratory Animals ◽

10.1177/026119291103900118 ◽

2011 ◽

Vol 39 (1) ◽

pp. 23-36 ◽

Cited By ~ 3

Author(s):

Claudio Procaccianti ◽

Federico M. Stefanini ◽

Chiara Urani

Keyword(s):

Cell Transformation ◽

Statistical Classification ◽

Transformation Assay

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The testing of chemicals in the Syrian hamster embryo (SHE) cell transformation assay for assessment of carcinogenic potential

Toxicology in Vitro ◽

10.1016/j.tiv.2003.08.001 ◽

2004 ◽

Vol 18 (2) ◽

pp. 213-218 ◽

Cited By ~ 14

Author(s):

G. Engelhardt ◽

K.-R. Schwind ◽

B. Mußler

Keyword(s):

Syrian Hamster ◽

Cell Transformation ◽

Transformation Assay ◽

Carcinogenic Potential

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Consequences of mitotic slippage for antimicrotubule drug therapy

Endocrine Related Cancer ◽

10.1530/erc-17-0147 ◽

2017 ◽

Vol 24 (9) ◽

pp. T97-T106 ◽

Cited By ~ 21

Author(s):

Bing Cheng ◽

Karen Crasta

Keyword(s):

Cell Death ◽

Spindle Assembly Checkpoint ◽

Mitotic Cell ◽

G1 Arrest ◽

Front Line ◽

Cell Fates ◽

Mitotic Slippage ◽

Drug Treatment Outcomes ◽

Antimicrotubule Agents ◽

Sustained Activation

Antimicrotubule agents are commonly utilised as front-line therapies against several malignancies, either by themselves or as combination therapies. Cell-based studies have pinpointed the anti-proliferative basis of action to be a consequence of perturbation of microtubule dynamics leading to sustained activation of the spindle assembly checkpoint, prolonged mitotic arrest and mitotic cell death. However, depending on the biological context and cell type, cells may take an alternative route besides mitotic cell death via a process known as mitotic slippage. Here, mitotically arrested cells ‘slip’ to the next interphase without undergoing proper chromosome segregation and cytokinesis. These post-slippage cells in turn have two main cell fates, either cell death or a G1 arrest ensuing in senescence. In this review, we take a look at the factors determining mitotic cell death vs mitotic slippage, post-slippage cell fates and accompanying features, and their consequences for antimicrotubule drug treatment outcomes.

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Effects of Ionizing Radiation and Long-Term Storage on Hydrated vs. Dried Cell Samples of Extremophilic Microorganisms

Microorganisms ◽

10.3390/microorganisms10010190 ◽

2022 ◽

Vol 10 (1) ◽

pp. 190

Author(s):

Ida Romano ◽

Carlo Camerlingo ◽

Lisa Vaccari ◽

Giovanni Birarda ◽

Annarita Poli ◽

...

Keyword(s):

Ionizing Radiation ◽

Galactic Cosmic Rays ◽

Cellular Growth ◽

Space Environment ◽

Water Medium ◽

Space Condition ◽

Long Term Storage ◽

Extremophilic Microorganisms ◽

Term Storage

A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the “STARLIFE” project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core’s content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage.

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An Optimised Data Analysis for the Balb/c3T3 Cell Transformation Assay and its Application to Metal Compounds

International Journal of Immunopathology and Pharmacology ◽

10.1177/039463200702000403 ◽

2007 ◽

Vol 20 (4) ◽

pp. 673-684 ◽

Cited By ~ 6

Author(s):

J. Ponti ◽

B. Munaro ◽

M. Fischbach ◽

S. Hoffmann ◽

E. Sabbioni

Keyword(s):

Data Analysis ◽

Cell Transformation ◽

T Test ◽

Alternative Methods ◽

Metal Compounds ◽

Exact Test ◽

Fisher's Exact Test ◽

Transformation Assay ◽

Carcinogenic Potential ◽

Fisher’S Exact Test

The Balb/c3T3 cell transformation assay (CTA) is an available in vitro system to detect the carcinogenic potential of chemicals. Currently, the European Centre for the Validation of Alternative Methods (ECVAM) is validating this test, assessing its reliability and relevance. Its endpoint is the formation of type III foci, which is, when using clone A31-1-1, a very rare event that usually does not occur at all for negative controls. The carcinogenic potential of a compound tested is assessed by comparing the number of foci in treated and untreated cells. The objective of the present work is to optimise the data analysis for this endpoint by applying the most commonly used approach by a t-test and the Fisher's exact test as an alternative approach. For this purpose selected metal compounds classified as carcinogenic (NaAsO2, CdCl2 cisPt), as suspected carcinogenic (C6H5)4AsCl, CH3HgCl), or as compounds without evidence of carcinogenic properties in humans ((NH4)2PtCl6, NaVO3) as well as a non-carcinogenic (AgNO3) were analysed. Our evaluation revealed that the t-test approach, which assumes normality of data, is not appropriate. The results demonstrated that the statistical analysis by Fisher's exact test better reflects the data properties and greatly facilitates the interpretation of Balb/c3T3 CTA data regarding carcinogenic potential.

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