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Retrotransposition of long interspersed nucleotide element-1 is associated with colitis but not tumors in a murine colitic cancer model.

Otsubo T, Okamura T, Hagiwara T, Ishizaka Y, Dohi T, Kawamura YI - PLoS ONE (2015)

Bottom Line: L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon.L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells.DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Research Center for Hepatitis and Immunology, Research Institute, National Center for Global Health and Medicine, Ichikawa 272-8516, Japan.

ABSTRACT
Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.

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L1-RTP levels in AOM+DSS colitis mouse model.(A) Schematic time schedule of the AOM+DSS colitis mouse model. Arrows indicate the time-points associated with the acute inflammatory and tumor phases for harvesting the colon tissues and/or tumors. (B) L1-RTP levels were analyzed with genomic DNA extracted from whole distal colon tissues and/or tumors. The EGFP copy/genome was calculated by using ß-actin as internal control. Samples labeled “Acute AOM+DSS” were obtained during the acute phase in panel A, and others were collected during the tumor phase. AOM+DSS indicates residual colon tissue after removing visible tumors. Data were shown as mean ± SD. P values were calculated with an unpaired two-tailed t test.
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pone.0116072.g001: L1-RTP levels in AOM+DSS colitis mouse model.(A) Schematic time schedule of the AOM+DSS colitis mouse model. Arrows indicate the time-points associated with the acute inflammatory and tumor phases for harvesting the colon tissues and/or tumors. (B) L1-RTP levels were analyzed with genomic DNA extracted from whole distal colon tissues and/or tumors. The EGFP copy/genome was calculated by using ß-actin as internal control. Samples labeled “Acute AOM+DSS” were obtained during the acute phase in panel A, and others were collected during the tumor phase. AOM+DSS indicates residual colon tissue after removing visible tumors. Data were shown as mean ± SD. P values were calculated with an unpaired two-tailed t test.

Mentions: A line of transgenic mice harboring a human L1-EGFP reporter gene (hL1-EGFP), named Line 4, was developed in our facility [5] and used in all experiments. Mice were kept under specific pathogen-free conditions during the experiments. CAC model protocols are shown in Fig. 1A. Weekly peritoneal injection of azoxymethane (AOM, 10 mg/kg, Sigma-Aldrich Japan) was started at 6 weeks of age and repeated four times. Mice were starved for the 12 h prior to AOM injection to enhance the effect of carcinogenesis with AOM [21]. Mice in the experimental group were given 3.5% (W/V) dextran sulfate sodium (DSS, Sigma-Aldrich Japan) in the drinking water for 5 days at 7 weeks of age, and this treatment was repeated four times on a monthly basis. At 22 weeks of age (tumor phase), colon tissues were obtained. Visible tumors were cut from the mucosa and separately analyzed from the background mucosa. In some experiments, colon tissues were obtained from 8-week-old mice following two injections of AOM and within seven days after stopping DSS treatment (acute phase).


Retrotransposition of long interspersed nucleotide element-1 is associated with colitis but not tumors in a murine colitic cancer model.

Otsubo T, Okamura T, Hagiwara T, Ishizaka Y, Dohi T, Kawamura YI - PLoS ONE (2015)

L1-RTP levels in AOM+DSS colitis mouse model.(A) Schematic time schedule of the AOM+DSS colitis mouse model. Arrows indicate the time-points associated with the acute inflammatory and tumor phases for harvesting the colon tissues and/or tumors. (B) L1-RTP levels were analyzed with genomic DNA extracted from whole distal colon tissues and/or tumors. The EGFP copy/genome was calculated by using ß-actin as internal control. Samples labeled “Acute AOM+DSS” were obtained during the acute phase in panel A, and others were collected during the tumor phase. AOM+DSS indicates residual colon tissue after removing visible tumors. Data were shown as mean ± SD. P values were calculated with an unpaired two-tailed t test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4339839&req=5

pone.0116072.g001: L1-RTP levels in AOM+DSS colitis mouse model.(A) Schematic time schedule of the AOM+DSS colitis mouse model. Arrows indicate the time-points associated with the acute inflammatory and tumor phases for harvesting the colon tissues and/or tumors. (B) L1-RTP levels were analyzed with genomic DNA extracted from whole distal colon tissues and/or tumors. The EGFP copy/genome was calculated by using ß-actin as internal control. Samples labeled “Acute AOM+DSS” were obtained during the acute phase in panel A, and others were collected during the tumor phase. AOM+DSS indicates residual colon tissue after removing visible tumors. Data were shown as mean ± SD. P values were calculated with an unpaired two-tailed t test.
Mentions: A line of transgenic mice harboring a human L1-EGFP reporter gene (hL1-EGFP), named Line 4, was developed in our facility [5] and used in all experiments. Mice were kept under specific pathogen-free conditions during the experiments. CAC model protocols are shown in Fig. 1A. Weekly peritoneal injection of azoxymethane (AOM, 10 mg/kg, Sigma-Aldrich Japan) was started at 6 weeks of age and repeated four times. Mice were starved for the 12 h prior to AOM injection to enhance the effect of carcinogenesis with AOM [21]. Mice in the experimental group were given 3.5% (W/V) dextran sulfate sodium (DSS, Sigma-Aldrich Japan) in the drinking water for 5 days at 7 weeks of age, and this treatment was repeated four times on a monthly basis. At 22 weeks of age (tumor phase), colon tissues were obtained. Visible tumors were cut from the mucosa and separately analyzed from the background mucosa. In some experiments, colon tissues were obtained from 8-week-old mice following two injections of AOM and within seven days after stopping DSS treatment (acute phase).

Bottom Line: L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon.L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells.DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Research Center for Hepatitis and Immunology, Research Institute, National Center for Global Health and Medicine, Ichikawa 272-8516, Japan.

ABSTRACT
Long interspersed element-1 (L1) is a transposable element that can move within the genome, potentially leading to genome diversity and modified gene function. Although L1 activity in somatic cells is normally suppressed through DNA methylation, some L1s are activated in tumors including colorectal carcinoma. However, how L1-retrotransposition (L1-RTP) is involved in gastrointestinal disorders remains to be elucidated. We hypothesized that L1-RTP in somatic cells might contribute to colitis-associated cancer (CAC). To address this, we employed an experimental model of CAC using transgenic L1-reporter mice carrying a human L1-EGFP reporter gene. Mice were subjected to repeated cycles of colitis induced by administration of dextran sodium sulfate (DSS) in drinking water with injection of carcinogen azoxymethane (AOM). L1-RTP levels were measured by a quantitative polymerase chain reaction targeting the newly inserted reporter EGFP in various tissues and cell types, including samples obtained by laser microdissection and cell sorting with flow cytometry. DNA methylation levels of the human L1 promoter were analyzed by bisulfite pyrosequencing. AOM+DSS-treated mice exhibited significantly higher levels of L1-RTP in whole colon tissue during the acute phase of colitis when compared with control naïve mice. L1-RTP levels in whole colon tissue were positively correlated with the histological severity of colitis and degree of neutrophil infiltration into the lamina propria (LP), but not with tumor development in the colon. L1-RTP was enriched in LP mesenchymal cells rather than epithelial cells (ECs), myeloid, or lymphoid cells. DNA methylation levels of the human L1 promoter region showed a negative correlation with L1-RTP levels. L1-RTP was absent from most tumors found in 22-week-old mice. In conclusion, we demonstrated that L1-RTP was induced in the mouse CAC mucosa in accordance with the acute inflammatory response; however, retrotransposition appears not to have direct relevance to colitis-induced cancer initiation.

Show MeSH
Related in: MedlinePlus