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Genomic instability and cellular stress in organ biopsies and peripheral blood lymphocytes from patients with colorectal cancer and predisposing pathologies.

Lombardi S, Fuoco I, di Fluri G, Costa F, Ricchiuti A, Biondi G, Nardini V, Scarpato R - Oncotarget (2015)

Bottom Line: Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression.We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment.Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.

View Article: PubMed Central - PubMed

Affiliation: Unità di Genetica, Dipartimento di Biologia, University of Pisa, Pisa, Italy.

ABSTRACT
Inflammatory bowel disease (IBD) and polyps, are common colorectal pathologies in western society and are risk factors for development of colorectal cancer (CRC). Genomic instability is a cancer hallmark and is connected to changes in chromosomal structure, often caused by double strand break formation (DSB), and aneuploidy. Cellular stress, may contribute to genomic instability. In colorectal biopsies and peripheral blood lymphocytes of patients with IBD, polyps and CRC, we evaluated 1) genomic instability using the γH2AX assay as marker of DSB and micronuclei in mononuclear lymphocytes kept under cytodieresis inhibition, and 2) cellular stress through expression and cellular localization of glutathione-S-transferase omega 1 (GSTO1). Colon biopsies showed γH2AX increase starting from polyps, while lymphocytes already from IBD. Micronuclei frequency began to rise in lymphocytes of subjects with polyps, suggesting a systemic genomic instability condition. Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression. Lymphocytes did not change GSTO1 expression and localization until CRC formation, where enzyme expression was increased. We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment. Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.

No MeSH data available.


Related in: MedlinePlus

Image analysis for GSTO1 in tissuePanel A. use of ImmunoRatio for quantification of GSTO1 expression. The original image and the corresponding plug-in output, quantified as positive DAB area, are shown at the top and the bottom of the panel, respectively. 100x magnification. Panel B. quantification of GSTO1 localization through fields scoring; example of 1) cytoplasmic field (assigned score 0), 2) field with mixed localization (assigned score 0.5), and 3) field with prevalent nuclear localization (assigned score 1). 400x magnification.
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Figure 7: Image analysis for GSTO1 in tissuePanel A. use of ImmunoRatio for quantification of GSTO1 expression. The original image and the corresponding plug-in output, quantified as positive DAB area, are shown at the top and the bottom of the panel, respectively. 100x magnification. Panel B. quantification of GSTO1 localization through fields scoring; example of 1) cytoplasmic field (assigned score 0), 2) field with mixed localization (assigned score 0.5), and 3) field with prevalent nuclear localization (assigned score 1). 400x magnification.

Mentions: For both γH2AX and GSTO1 evaluation, a quantitative analysis using a computerized image-based method was performed. Images were obtained using a Nikon optical microscope equipped with a reflex digital camera (D60, Nikon). In the case of γH2AX, we proceeded as follows. Two series of 20 pictures of not overlapping fields were obtained from each specimen, starting from opposite extremities of the section (40 pictures per section, 80 pictures per each subject). Pictures were randomly taken, using a 20x objective (magnification of 200x), and non–epithelial biopsy portions were excluded. Images were imported on computer and processed using the ImageJ software (download at http://imagej.nih.gov/ij/). Each RGB-color image was converted into 8-bit gray-scale image. Then an adequate threshold level was set up in order to transform the gray-scale image into a black and white image, where black stains correspond to the darkest gray stains (nuclei) in the preceding image. Stains were segmented using the specific program algorithm in order to separate partially overlapping nuclei. Once we coherently defined size range for stains, nuclei were counted by the program (Figure 6A–D). The number of positive nuclei (showing brown staining) was manually scored for each photo and the results were expressed as percentage of γH2AX positive cells. As for GSTO1 we observed a higher positivity level than for γH2AX, we proceeded using the ImmunoRatio plug-in of the ImageJ software. This plug-in is optimized to evaluate the percentage of positive area on images of sections stained with DAB and haematoxilyn [30]. We obtained 2 series of 10 pictures per section (20 images per section and 40 per subject) using the criteria already described with the exception of decreasing the magnification at 100x (objective 10x). After the proper calibration of the color thresholds required by the plug-in, the output is expressed as GSTO1 positive area (%) (Figure 7A). Localization of the enzyme in biopsies was quantified using 10 pictures obtained at 400x magnification (40x objective) per each section (20 pictures analyzed per each subject) giving them a score of 0 or 1 when enzyme localization was prevalently cytoplasmic or nuclear, respectively; in the case of mixed localization, 0.5 was added for the field (Figure 7B). The sum of scores assigned to each field was divided by the number of fields analyzed (10) and expressed as percentage of GSTO1 nuclear fields.


Genomic instability and cellular stress in organ biopsies and peripheral blood lymphocytes from patients with colorectal cancer and predisposing pathologies.

Lombardi S, Fuoco I, di Fluri G, Costa F, Ricchiuti A, Biondi G, Nardini V, Scarpato R - Oncotarget (2015)

Image analysis for GSTO1 in tissuePanel A. use of ImmunoRatio for quantification of GSTO1 expression. The original image and the corresponding plug-in output, quantified as positive DAB area, are shown at the top and the bottom of the panel, respectively. 100x magnification. Panel B. quantification of GSTO1 localization through fields scoring; example of 1) cytoplasmic field (assigned score 0), 2) field with mixed localization (assigned score 0.5), and 3) field with prevalent nuclear localization (assigned score 1). 400x magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4558120&req=5

Figure 7: Image analysis for GSTO1 in tissuePanel A. use of ImmunoRatio for quantification of GSTO1 expression. The original image and the corresponding plug-in output, quantified as positive DAB area, are shown at the top and the bottom of the panel, respectively. 100x magnification. Panel B. quantification of GSTO1 localization through fields scoring; example of 1) cytoplasmic field (assigned score 0), 2) field with mixed localization (assigned score 0.5), and 3) field with prevalent nuclear localization (assigned score 1). 400x magnification.
Mentions: For both γH2AX and GSTO1 evaluation, a quantitative analysis using a computerized image-based method was performed. Images were obtained using a Nikon optical microscope equipped with a reflex digital camera (D60, Nikon). In the case of γH2AX, we proceeded as follows. Two series of 20 pictures of not overlapping fields were obtained from each specimen, starting from opposite extremities of the section (40 pictures per section, 80 pictures per each subject). Pictures were randomly taken, using a 20x objective (magnification of 200x), and non–epithelial biopsy portions were excluded. Images were imported on computer and processed using the ImageJ software (download at http://imagej.nih.gov/ij/). Each RGB-color image was converted into 8-bit gray-scale image. Then an adequate threshold level was set up in order to transform the gray-scale image into a black and white image, where black stains correspond to the darkest gray stains (nuclei) in the preceding image. Stains were segmented using the specific program algorithm in order to separate partially overlapping nuclei. Once we coherently defined size range for stains, nuclei were counted by the program (Figure 6A–D). The number of positive nuclei (showing brown staining) was manually scored for each photo and the results were expressed as percentage of γH2AX positive cells. As for GSTO1 we observed a higher positivity level than for γH2AX, we proceeded using the ImmunoRatio plug-in of the ImageJ software. This plug-in is optimized to evaluate the percentage of positive area on images of sections stained with DAB and haematoxilyn [30]. We obtained 2 series of 10 pictures per section (20 images per section and 40 per subject) using the criteria already described with the exception of decreasing the magnification at 100x (objective 10x). After the proper calibration of the color thresholds required by the plug-in, the output is expressed as GSTO1 positive area (%) (Figure 7A). Localization of the enzyme in biopsies was quantified using 10 pictures obtained at 400x magnification (40x objective) per each section (20 pictures analyzed per each subject) giving them a score of 0 or 1 when enzyme localization was prevalently cytoplasmic or nuclear, respectively; in the case of mixed localization, 0.5 was added for the field (Figure 7B). The sum of scores assigned to each field was divided by the number of fields analyzed (10) and expressed as percentage of GSTO1 nuclear fields.

Bottom Line: Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression.We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment.Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.

View Article: PubMed Central - PubMed

Affiliation: Unità di Genetica, Dipartimento di Biologia, University of Pisa, Pisa, Italy.

ABSTRACT
Inflammatory bowel disease (IBD) and polyps, are common colorectal pathologies in western society and are risk factors for development of colorectal cancer (CRC). Genomic instability is a cancer hallmark and is connected to changes in chromosomal structure, often caused by double strand break formation (DSB), and aneuploidy. Cellular stress, may contribute to genomic instability. In colorectal biopsies and peripheral blood lymphocytes of patients with IBD, polyps and CRC, we evaluated 1) genomic instability using the γH2AX assay as marker of DSB and micronuclei in mononuclear lymphocytes kept under cytodieresis inhibition, and 2) cellular stress through expression and cellular localization of glutathione-S-transferase omega 1 (GSTO1). Colon biopsies showed γH2AX increase starting from polyps, while lymphocytes already from IBD. Micronuclei frequency began to rise in lymphocytes of subjects with polyps, suggesting a systemic genomic instability condition. Colorectal tissues lost GSTO1 expression but increased nuclear localization with pathology progression. Lymphocytes did not change GSTO1 expression and localization until CRC formation, where enzyme expression was increased. We propose that the growing genomic instability found in our patients is connected with the alteration of cellular environment. Evaluation of genomic damage and cellular stress in colorectal pathologies may facilitate prevention and management of CRC.

No MeSH data available.


Related in: MedlinePlus