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Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

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PL2L60 promotes pCSC expansion in vitro through up-regulation of Stat3 and Bcl2 gene expressions.The pCSCs (clone 2C4) were transfected with Piwil2 exon 11-specific siRNA or scRNA, mock transfected (Mock), or untransfected (Untr), as previously described [5] and analyzed for PL2L60 expression, gene expression and cell expansion. A, Western blot analysis of PL2L60 in pCSCs treated as described above. B, Quantitation of PL2L60 proteins (AU) detected in A. C, RT-PCR analysis of Piwil2 transcripts in pCSCs using primer pairs specific for E6-14 or E18-21. D, The effects of PL2L60 on pCSC expansion: pCSCs were counted as mean ± SD in triplicate at indicated times after transfection with siRNA. **, p<0.01; as compared to the mock or scRNA transfected groups. E, RT-PCR analysis of transcriptional expression of Stat3, Bcl2 and Bcl-xL genes in pCSCs: H2O indicates that cDNA was omitted. F, Quantitation of Stat3, Bcl-2 and Bcl-xL transcripts (AU) shown in E. For Western blot (A & B) and RT-PCR (C, E & F) analysis, the cells were harvested at 36–48 h after transfection; for cell expansion, the cells were counted at various time points post transfection as indicated. The cells were seeded in triplicate as previously described [5]. AU (Arbitrary Units) was determined by the ratio of a factor tested to β-actin. The data shown are a representative from at least 3 experiments.
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pone-0013406-g005: PL2L60 promotes pCSC expansion in vitro through up-regulation of Stat3 and Bcl2 gene expressions.The pCSCs (clone 2C4) were transfected with Piwil2 exon 11-specific siRNA or scRNA, mock transfected (Mock), or untransfected (Untr), as previously described [5] and analyzed for PL2L60 expression, gene expression and cell expansion. A, Western blot analysis of PL2L60 in pCSCs treated as described above. B, Quantitation of PL2L60 proteins (AU) detected in A. C, RT-PCR analysis of Piwil2 transcripts in pCSCs using primer pairs specific for E6-14 or E18-21. D, The effects of PL2L60 on pCSC expansion: pCSCs were counted as mean ± SD in triplicate at indicated times after transfection with siRNA. **, p<0.01; as compared to the mock or scRNA transfected groups. E, RT-PCR analysis of transcriptional expression of Stat3, Bcl2 and Bcl-xL genes in pCSCs: H2O indicates that cDNA was omitted. F, Quantitation of Stat3, Bcl-2 and Bcl-xL transcripts (AU) shown in E. For Western blot (A & B) and RT-PCR (C, E & F) analysis, the cells were harvested at 36–48 h after transfection; for cell expansion, the cells were counted at various time points post transfection as indicated. The cells were seeded in triplicate as previously described [5]. AU (Arbitrary Units) was determined by the ratio of a factor tested to β-actin. The data shown are a representative from at least 3 experiments.

Mentions: Because PL2L60 was predominantly expressed in the pCSCs (2C4, 3B5C and 3B6C) (Fig. 4), we reasoned that PL2L60 is associated with pCSC expansion in vitro. In order to verify the hypothesis, we knocked down PL2L60 mRNAs in pCSCs (clone 2C4) by transfection with siRNAs targeting exon 11 of Piwil2 transcripts. As a result, PL2L60 expression was reduced at both transcriptional and translational levels in the siRNA-transfected cells, as compared to the controls including the cells transfected with scrambled siRNA (scRNA), mock transfected and un-transfected (Fig. 5A, B & C). Correspondingly, pCSC expansion was also significantly suppressed in the siRNA-transfected pCSCs (Fig. 5D). The reduced pCSC expansion was associated with down-regulation of the transcripts of Stat-3 and Bcl-2 genes but not Bcl-XL gene in the pCSCs (Fig. 5E & F). The Piwil2 exon 11 is located within the putative open reading frame of PL2L60 gene (Fig. 2A). Consistently, the transcriptional products of both E6-14 and E18-21 were also significantly reduced in the pCSCs transfected with siRNA targeting exon 11 (Fig. 5C). The results reveal that the “Piwil2” mRNAs that was previously reported to be stably expressed in pCSC lines [5] de facto represent the mRNAs of PL2L60 gene, which can support pCSC expansion in vitro through promoting Stat-3/Bcl-2 pathway.


Identification of Piwil2-like (PL2L) proteins that promote tumorigenesis.

Ye Y, Yin DT, Chen L, Zhou Q, Shen R, He G, Yan Q, Tong Z, Issekutz AC, Shapiro CL, Barsky SH, Lin H, Li JJ, Gao JX - PLoS ONE (2010)

PL2L60 promotes pCSC expansion in vitro through up-regulation of Stat3 and Bcl2 gene expressions.The pCSCs (clone 2C4) were transfected with Piwil2 exon 11-specific siRNA or scRNA, mock transfected (Mock), or untransfected (Untr), as previously described [5] and analyzed for PL2L60 expression, gene expression and cell expansion. A, Western blot analysis of PL2L60 in pCSCs treated as described above. B, Quantitation of PL2L60 proteins (AU) detected in A. C, RT-PCR analysis of Piwil2 transcripts in pCSCs using primer pairs specific for E6-14 or E18-21. D, The effects of PL2L60 on pCSC expansion: pCSCs were counted as mean ± SD in triplicate at indicated times after transfection with siRNA. **, p<0.01; as compared to the mock or scRNA transfected groups. E, RT-PCR analysis of transcriptional expression of Stat3, Bcl2 and Bcl-xL genes in pCSCs: H2O indicates that cDNA was omitted. F, Quantitation of Stat3, Bcl-2 and Bcl-xL transcripts (AU) shown in E. For Western blot (A & B) and RT-PCR (C, E & F) analysis, the cells were harvested at 36–48 h after transfection; for cell expansion, the cells were counted at various time points post transfection as indicated. The cells were seeded in triplicate as previously described [5]. AU (Arbitrary Units) was determined by the ratio of a factor tested to β-actin. The data shown are a representative from at least 3 experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958115&req=5

pone-0013406-g005: PL2L60 promotes pCSC expansion in vitro through up-regulation of Stat3 and Bcl2 gene expressions.The pCSCs (clone 2C4) were transfected with Piwil2 exon 11-specific siRNA or scRNA, mock transfected (Mock), or untransfected (Untr), as previously described [5] and analyzed for PL2L60 expression, gene expression and cell expansion. A, Western blot analysis of PL2L60 in pCSCs treated as described above. B, Quantitation of PL2L60 proteins (AU) detected in A. C, RT-PCR analysis of Piwil2 transcripts in pCSCs using primer pairs specific for E6-14 or E18-21. D, The effects of PL2L60 on pCSC expansion: pCSCs were counted as mean ± SD in triplicate at indicated times after transfection with siRNA. **, p<0.01; as compared to the mock or scRNA transfected groups. E, RT-PCR analysis of transcriptional expression of Stat3, Bcl2 and Bcl-xL genes in pCSCs: H2O indicates that cDNA was omitted. F, Quantitation of Stat3, Bcl-2 and Bcl-xL transcripts (AU) shown in E. For Western blot (A & B) and RT-PCR (C, E & F) analysis, the cells were harvested at 36–48 h after transfection; for cell expansion, the cells were counted at various time points post transfection as indicated. The cells were seeded in triplicate as previously described [5]. AU (Arbitrary Units) was determined by the ratio of a factor tested to β-actin. The data shown are a representative from at least 3 experiments.
Mentions: Because PL2L60 was predominantly expressed in the pCSCs (2C4, 3B5C and 3B6C) (Fig. 4), we reasoned that PL2L60 is associated with pCSC expansion in vitro. In order to verify the hypothesis, we knocked down PL2L60 mRNAs in pCSCs (clone 2C4) by transfection with siRNAs targeting exon 11 of Piwil2 transcripts. As a result, PL2L60 expression was reduced at both transcriptional and translational levels in the siRNA-transfected cells, as compared to the controls including the cells transfected with scrambled siRNA (scRNA), mock transfected and un-transfected (Fig. 5A, B & C). Correspondingly, pCSC expansion was also significantly suppressed in the siRNA-transfected pCSCs (Fig. 5D). The reduced pCSC expansion was associated with down-regulation of the transcripts of Stat-3 and Bcl-2 genes but not Bcl-XL gene in the pCSCs (Fig. 5E & F). The Piwil2 exon 11 is located within the putative open reading frame of PL2L60 gene (Fig. 2A). Consistently, the transcriptional products of both E6-14 and E18-21 were also significantly reduced in the pCSCs transfected with siRNA targeting exon 11 (Fig. 5C). The results reveal that the “Piwil2” mRNAs that was previously reported to be stably expressed in pCSC lines [5] de facto represent the mRNAs of PL2L60 gene, which can support pCSC expansion in vitro through promoting Stat-3/Bcl-2 pathway.

Bottom Line: Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers.Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB.These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Ohio State University Medical Center, Columbus, Ohio, United States of America.

ABSTRACT
PIWIL2, a member of PIWI/AGO gene family, is expressed in the germline stem cells (GSCs) of testis for gametogenesis but not in adult somatic and stem cells. It has been implicated to play an important role in tumor development. We have previously reported that precancerous stem cells (pCSCs) constitutively express Piwil2 transcripts to promote their proliferation. Here we show that these transcripts de facto represent Piwil2-like (PL2L) proteins. We have identified several PL2L proteins including PL2L80, PL2L60, PL2L50 and PL2L40, using combined methods of Gene-Exon-Mapping Reverse Transcription Polymerase Chain Reaction (GEM RT-PCR), bioinformatics and a group of novel monoclonal antibodies. Among them, PL2L60 rather than Piwil2 and other PL2L proteins is predominantly expressed in various types of human and mouse tumor cells. It promotes tumor cell survival and proliferation in vitro through up-regulation of Stat3 and Bcl2 gene expressions, the cell cycle entry from G(0/1) into S-phase, and the nuclear expression of NF-κB, which contribute to the tumorigenicity of tumor cells in vivo. Consistently, PL2L proteins rather than Piwil2 are predominantly expressed in the cytoplasm or cytoplasm and nucleus of euchromatin-enriched tumor cells in human primary and metastatic cancers, such as breast and cervical cancers. Moreover, nuclear PL2L proteins are always co-expressed with nuclear NF-κB. These results reveal that PL2L60 can coordinate with NF-κB to promote tumorigenesis and might mediate a common pathway for tumor development without tissue restriction. The identification of PL2L proteins provides a novel insight into the mechanisms of cancer development as well as a novel bridge linking cancer diagnostics and anticancer drug development.

Show MeSH
Related in: MedlinePlus