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Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer.

Chen L, Dahlstrom JE, Chandra A, Board P, Rangasamy D - Breast Cancer Res. Treat. (2012)

Bottom Line: The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %.Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression.Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia.

ABSTRACT
Long interspersed nuclear element 1 (L1) belongs to a family of retrotransposons. Expression of the normally repressed L1 retrotransposons has been shown to induce genome instability by creating DNA double-stranded breaks and chromosomal rearrangements through the process of retrotransposition. At present, little is known about the expression of L1-encoded ORF1p and ORF2p which are indispensable for its retrotransposition activity. Given its potentially harmful effects on the genome, we investigated the implications of both ORF1p and ORF2p expression and their subcellular localization in a range of breast cancer cell lines and breast tumor tissues including 15 normal breast tissues, 25 fibroadenomas, 25 ductal carcinomas in situ (DCIS), and 95 invasive cancers. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of ORF1p and ORF2p using univariate and multivariate analysis. High cytoplasmic expression of ORF1p and ORF2p was seen in DCIS tumors, but they were not related with survival outcome. The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %. Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression. This is the first study examining the effects of both ORF1p and ORF2p expression in breast cancer tissues. Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival. The differing patterns of both cytoplasmic and nuclear ORF1p and ORF2p expression indicate that further studies of the biology and function of L1 retrotransposons are required in breast cancer.

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Anti-ORF1p and Anti-ORF2p antibodies specifically recognize L1-encoded proteins. A Structure of a functional human L1 retrotransposon showing the 5′-UTR promoter, ORF1- and ORF2-encoding genes, and the 3′ poly-A tail. EN endonuclease, RTase reverse transcriptase, An poly-A tail. B Recombinant 43-kDa ORF1p and 150-kDa ORF2p were used for the generation of antibodies. C Western blot analysis examining the specificity of the generated anti-ORF1p and anti-ORF2p antibodies. Lanes 1 and 4 were loaded with 50 μg of bacterial extracts isolated from uninduced cells, and lanes 2 and 5 loaded with the induced bacterial extracts of 69 kDa GST-ORF1p (left panel) and 150 kDa 6xHis-ORF2p (right panel) fusion proteins. No staining was seen in the uninduced bacterial extracts and detection of the fusion proteins of GST-ORF1p and 6xHis-ORF2p in the induced bacterial extracts. Lanes 3 and 6 were loaded with 25 ng of 43-kDa ORF1p and 150-kDa ORF2p as positive controls. D The increased expression levels of ORF1p and ORF2p were detected by western blotting of the L1RP-transfected HeLa cells. N.Tera.2D1 cells were used as positive controls. For protein normalization, α-tubulin was used. E Immunofluorescence detection of transiently expressed ORF1p and ORF2p in the cytoplasm of HeLa cells using the anti-ORF1p (panel a) and anti-ORF2p (panel b) antibodies. Nuclei were stained with DAPI (blue) and ORF1p and ORF2p (Texas Red). As negative controls, the immunofluoresence assays were performed on the L1RP-transfected HeLa cells with the antigen-depleted anti-ORF1p (panel c) and anti-ORF2p (panel d) antibodies
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Fig1: Anti-ORF1p and Anti-ORF2p antibodies specifically recognize L1-encoded proteins. A Structure of a functional human L1 retrotransposon showing the 5′-UTR promoter, ORF1- and ORF2-encoding genes, and the 3′ poly-A tail. EN endonuclease, RTase reverse transcriptase, An poly-A tail. B Recombinant 43-kDa ORF1p and 150-kDa ORF2p were used for the generation of antibodies. C Western blot analysis examining the specificity of the generated anti-ORF1p and anti-ORF2p antibodies. Lanes 1 and 4 were loaded with 50 μg of bacterial extracts isolated from uninduced cells, and lanes 2 and 5 loaded with the induced bacterial extracts of 69 kDa GST-ORF1p (left panel) and 150 kDa 6xHis-ORF2p (right panel) fusion proteins. No staining was seen in the uninduced bacterial extracts and detection of the fusion proteins of GST-ORF1p and 6xHis-ORF2p in the induced bacterial extracts. Lanes 3 and 6 were loaded with 25 ng of 43-kDa ORF1p and 150-kDa ORF2p as positive controls. D The increased expression levels of ORF1p and ORF2p were detected by western blotting of the L1RP-transfected HeLa cells. N.Tera.2D1 cells were used as positive controls. For protein normalization, α-tubulin was used. E Immunofluorescence detection of transiently expressed ORF1p and ORF2p in the cytoplasm of HeLa cells using the anti-ORF1p (panel a) and anti-ORF2p (panel b) antibodies. Nuclei were stained with DAPI (blue) and ORF1p and ORF2p (Texas Red). As negative controls, the immunofluoresence assays were performed on the L1RP-transfected HeLa cells with the antigen-depleted anti-ORF1p (panel c) and anti-ORF2p (panel d) antibodies

Mentions: To investigate breast cancers for expression of L1 retrotransposons, we constructed a synthetic L1 gene expressing either an ORF1p or ORF2p. The codon of gene was synonymously optimized for bacterial expression of full-length protein with an apparent molecular mass 43-kDa of ORF1p and 150-kDa of ORF2p (Fig. 1A, B). Antibodies raised against ORF1p and ORF2p were purified and enriched by saturated ammonium sulfate, followed by antigen-specific affinity chromatography. These antibodies recognized ORF1p and ORF2p at the expected band sizes of 43 and 150 kDa, respectively (Fig. 1C, lanes 3 and 6). As a negative control, we used bacterial cell extract because the bacterial genome does not contain L1 sequences. The specificity of antibodies was tested using the bacterially induced expression of the fusion protein tagged with GST-ORF1p and 6xHis-ORF2p. The fusion proteins, with the expected sizes of 69 kDa for GST-ORF1p and 150 kDa for 6xHis-tagged ORF2p, were recognized in the induced bacterial extracts, but not in uninduced extracts (Fig. 1C, lanes 2 and 5).Fig. 1


Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer.

Chen L, Dahlstrom JE, Chandra A, Board P, Rangasamy D - Breast Cancer Res. Treat. (2012)

Anti-ORF1p and Anti-ORF2p antibodies specifically recognize L1-encoded proteins. A Structure of a functional human L1 retrotransposon showing the 5′-UTR promoter, ORF1- and ORF2-encoding genes, and the 3′ poly-A tail. EN endonuclease, RTase reverse transcriptase, An poly-A tail. B Recombinant 43-kDa ORF1p and 150-kDa ORF2p were used for the generation of antibodies. C Western blot analysis examining the specificity of the generated anti-ORF1p and anti-ORF2p antibodies. Lanes 1 and 4 were loaded with 50 μg of bacterial extracts isolated from uninduced cells, and lanes 2 and 5 loaded with the induced bacterial extracts of 69 kDa GST-ORF1p (left panel) and 150 kDa 6xHis-ORF2p (right panel) fusion proteins. No staining was seen in the uninduced bacterial extracts and detection of the fusion proteins of GST-ORF1p and 6xHis-ORF2p in the induced bacterial extracts. Lanes 3 and 6 were loaded with 25 ng of 43-kDa ORF1p and 150-kDa ORF2p as positive controls. D The increased expression levels of ORF1p and ORF2p were detected by western blotting of the L1RP-transfected HeLa cells. N.Tera.2D1 cells were used as positive controls. For protein normalization, α-tubulin was used. E Immunofluorescence detection of transiently expressed ORF1p and ORF2p in the cytoplasm of HeLa cells using the anti-ORF1p (panel a) and anti-ORF2p (panel b) antibodies. Nuclei were stained with DAPI (blue) and ORF1p and ORF2p (Texas Red). As negative controls, the immunofluoresence assays were performed on the L1RP-transfected HeLa cells with the antigen-depleted anti-ORF1p (panel c) and anti-ORF2p (panel d) antibodies
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3473189&req=5

Fig1: Anti-ORF1p and Anti-ORF2p antibodies specifically recognize L1-encoded proteins. A Structure of a functional human L1 retrotransposon showing the 5′-UTR promoter, ORF1- and ORF2-encoding genes, and the 3′ poly-A tail. EN endonuclease, RTase reverse transcriptase, An poly-A tail. B Recombinant 43-kDa ORF1p and 150-kDa ORF2p were used for the generation of antibodies. C Western blot analysis examining the specificity of the generated anti-ORF1p and anti-ORF2p antibodies. Lanes 1 and 4 were loaded with 50 μg of bacterial extracts isolated from uninduced cells, and lanes 2 and 5 loaded with the induced bacterial extracts of 69 kDa GST-ORF1p (left panel) and 150 kDa 6xHis-ORF2p (right panel) fusion proteins. No staining was seen in the uninduced bacterial extracts and detection of the fusion proteins of GST-ORF1p and 6xHis-ORF2p in the induced bacterial extracts. Lanes 3 and 6 were loaded with 25 ng of 43-kDa ORF1p and 150-kDa ORF2p as positive controls. D The increased expression levels of ORF1p and ORF2p were detected by western blotting of the L1RP-transfected HeLa cells. N.Tera.2D1 cells were used as positive controls. For protein normalization, α-tubulin was used. E Immunofluorescence detection of transiently expressed ORF1p and ORF2p in the cytoplasm of HeLa cells using the anti-ORF1p (panel a) and anti-ORF2p (panel b) antibodies. Nuclei were stained with DAPI (blue) and ORF1p and ORF2p (Texas Red). As negative controls, the immunofluoresence assays were performed on the L1RP-transfected HeLa cells with the antigen-depleted anti-ORF1p (panel c) and anti-ORF2p (panel d) antibodies
Mentions: To investigate breast cancers for expression of L1 retrotransposons, we constructed a synthetic L1 gene expressing either an ORF1p or ORF2p. The codon of gene was synonymously optimized for bacterial expression of full-length protein with an apparent molecular mass 43-kDa of ORF1p and 150-kDa of ORF2p (Fig. 1A, B). Antibodies raised against ORF1p and ORF2p were purified and enriched by saturated ammonium sulfate, followed by antigen-specific affinity chromatography. These antibodies recognized ORF1p and ORF2p at the expected band sizes of 43 and 150 kDa, respectively (Fig. 1C, lanes 3 and 6). As a negative control, we used bacterial cell extract because the bacterial genome does not contain L1 sequences. The specificity of antibodies was tested using the bacterially induced expression of the fusion protein tagged with GST-ORF1p and 6xHis-ORF2p. The fusion proteins, with the expected sizes of 69 kDa for GST-ORF1p and 150 kDa for 6xHis-tagged ORF2p, were recognized in the induced bacterial extracts, but not in uninduced extracts (Fig. 1C, lanes 2 and 5).Fig. 1

Bottom Line: The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %.Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression.Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival.

View Article: PubMed Central - PubMed

Affiliation: John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia.

ABSTRACT
Long interspersed nuclear element 1 (L1) belongs to a family of retrotransposons. Expression of the normally repressed L1 retrotransposons has been shown to induce genome instability by creating DNA double-stranded breaks and chromosomal rearrangements through the process of retrotransposition. At present, little is known about the expression of L1-encoded ORF1p and ORF2p which are indispensable for its retrotransposition activity. Given its potentially harmful effects on the genome, we investigated the implications of both ORF1p and ORF2p expression and their subcellular localization in a range of breast cancer cell lines and breast tumor tissues including 15 normal breast tissues, 25 fibroadenomas, 25 ductal carcinomas in situ (DCIS), and 95 invasive cancers. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of ORF1p and ORF2p using univariate and multivariate analysis. High cytoplasmic expression of ORF1p and ORF2p was seen in DCIS tumors, but they were not related with survival outcome. The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %. Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression. This is the first study examining the effects of both ORF1p and ORF2p expression in breast cancer tissues. Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival. The differing patterns of both cytoplasmic and nuclear ORF1p and ORF2p expression indicate that further studies of the biology and function of L1 retrotransposons are required in breast cancer.

Show MeSH
Related in: MedlinePlus