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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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Identification and characterization of PL2L proteins of humans and mice.A & B, Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili−/− and mili+/+ mice. B, HeLa cell lysate: Lane 1 & 5: 4 µg/mL antibody without peptide; Lane 2 & 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C, GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili−/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.
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pone-0013406-g003: Identification and characterization of PL2L proteins of humans and mice.A & B, Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili−/− and mili+/+ mice. B, HeLa cell lysate: Lane 1 & 5: 4 µg/mL antibody without peptide; Lane 2 & 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C, GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili−/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.

Mentions: To verify the protein expression of these PL2L genes in humans and mice, we designed a C-terminal Piwil2 peptide with amino acid sequence (15-mers) between PAZ and Piwi domains, which is shared by Piwil2 and putative PL2L proteins as well as homologous between the humans and mice. We immunized two rabbits with the peptide to generate polyclonal antibody (pAb) specific for Piwil2 and PL2L proteins. We obtained a high titer of peptide-specific rabbit pAb from one of the rabbits (RB9926), which was purified by the specific peptide-affinity chromatography and further characterized by Western-blot. As shown in Fig. 3A, the purified pAb can recognize a number of protein bands with estimated molecular weight (MW) of 110, 80, 60, and 50 kDa in both murine testicular cell lysates (Fig. 3A) and human tumor cell lysates (HeLa) (Fig. 3B). These proteins are Piwil2-peptide specific, because 110 and 50 kDa protein bands were not detected in the testicular cell lysates of mili−/− mice [18] and the 80 and 60 kDa protein bands were greatly reduced compared to wild-type (wt) mice (Fig. 3A). Consistently the same protein bands in the human tumor cell lysates were almost completely blocked by Piwil2 peptides, which were used as immunogens (Fig. 3B). The results suggested that the Piwil2-specific polyclonal antibody at least recognize three Piwil2-like proteins (PL2L80, PL2L60, and PL2L50). Among them, PL2L80 could not be predicted from NCBI GeneBank or Genomatix database (http://www.genomatix.de/). Incomplete disruption of PL2L80 and PL2L60 proteins in the mili−/− testis appeared to be associated with the expression of transcripts covering Piwil2 exons from 6 to 23, which were not abrogated at all in mili−/− testis, as well as with the low level expression of Piwil2 exons from 1 to 7 in mili−/− mice, as demonstrated by Piwil2-specific GEM RT-PCR (Fig. 3C), although the underlying mechanism is not yet clear. In the mili−/− mice, Piwil2 was disrupted via homologous recombination with the 5.2-kb BamHI fragment encompassing from exon 2 to exon 5 in the pPNT vector with a neomycin resistance cassette [18]. Thus, the transcripts of PL2L genes or transcripts of Piwil2 truncated at 5′ end, which were controlled by other independent promoters, were not completely suppressed in mili−/− testis (Fig. 2). The results suggest that whole Piwil2 protein, but not its variants such as PL2L80 and PL2L60, are completely disrupted in the mili−/− mice.


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)

Identification and characterization of PL2L proteins of humans and mice.A & B, Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili−/− and mili+/+ mice. B, HeLa cell lysate: Lane 1 & 5: 4 µg/mL antibody without peptide; Lane 2 & 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C, GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili−/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958115&req=5

pone-0013406-g003: Identification and characterization of PL2L proteins of humans and mice.A & B, Western blot analysis of Piwil2 and PL2L proteins expressed in mouse testis (A) and HeLa cells (B), using rabbit polyclonal antibody to Piwil2 peptide. A, Testicular cell lysates of mili−/− and mili+/+ mice. B, HeLa cell lysate: Lane 1 & 5: 4 µg/mL antibody without peptide; Lane 2 & 6: 4 µg/mL antibody with 16 µg/mL Peptide; Lane 3: 2 µg/mL antibody alone; Lane 4: 2 µg/mL antibody with 8 µg/mL Peptide. P: Piwil2 peptide; Ab: antibody to Piwil2 peptide. C, GEM RT-PCR analysis of testicular tissues from wild-type (Lane 1) and mili−/− mice (Lane 2): the primer pairs specific for E1-7, E6-14, E13-21 and E21-23 of Piwil2 were used for GEM RT-PCR analysis, and the primer pair specific for E18-21 was used as a positive control. Lane 3: no cDNA control.
Mentions: To verify the protein expression of these PL2L genes in humans and mice, we designed a C-terminal Piwil2 peptide with amino acid sequence (15-mers) between PAZ and Piwi domains, which is shared by Piwil2 and putative PL2L proteins as well as homologous between the humans and mice. We immunized two rabbits with the peptide to generate polyclonal antibody (pAb) specific for Piwil2 and PL2L proteins. We obtained a high titer of peptide-specific rabbit pAb from one of the rabbits (RB9926), which was purified by the specific peptide-affinity chromatography and further characterized by Western-blot. As shown in Fig. 3A, the purified pAb can recognize a number of protein bands with estimated molecular weight (MW) of 110, 80, 60, and 50 kDa in both murine testicular cell lysates (Fig. 3A) and human tumor cell lysates (HeLa) (Fig. 3B). These proteins are Piwil2-peptide specific, because 110 and 50 kDa protein bands were not detected in the testicular cell lysates of mili−/− mice [18] and the 80 and 60 kDa protein bands were greatly reduced compared to wild-type (wt) mice (Fig. 3A). Consistently the same protein bands in the human tumor cell lysates were almost completely blocked by Piwil2 peptides, which were used as immunogens (Fig. 3B). The results suggested that the Piwil2-specific polyclonal antibody at least recognize three Piwil2-like proteins (PL2L80, PL2L60, and PL2L50). Among them, PL2L80 could not be predicted from NCBI GeneBank or Genomatix database (http://www.genomatix.de/). Incomplete disruption of PL2L80 and PL2L60 proteins in the mili−/− testis appeared to be associated with the expression of transcripts covering Piwil2 exons from 6 to 23, which were not abrogated at all in mili−/− testis, as well as with the low level expression of Piwil2 exons from 1 to 7 in mili−/− mice, as demonstrated by Piwil2-specific GEM RT-PCR (Fig. 3C), although the underlying mechanism is not yet clear. In the mili−/− mice, Piwil2 was disrupted via homologous recombination with the 5.2-kb BamHI fragment encompassing from exon 2 to exon 5 in the pPNT vector with a neomycin resistance cassette [18]. Thus, the transcripts of PL2L genes or transcripts of Piwil2 truncated at 5′ end, which were controlled by other independent promoters, were not completely suppressed in mili−/− testis (Fig. 2). The results suggest that whole Piwil2 protein, but not its variants such as PL2L80 and PL2L60, are completely disrupted in the mili−/− mice.

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