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Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

Naidoo K, Malindisa ST, Otgaar TC, Bernert M, Da Costa Dias B, Ferreira E, Reusch U, Knackmuss S, Little M, Weiss SF, Letsolo BT - PLoS ONE (2015)

Bottom Line: FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT.Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity.These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, Republic of South Africa.

ABSTRACT
Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

No MeSH data available.


Related in: MedlinePlus

Flag® Immunoprecipitation assays confirming an interaction between LRP/LR and hTERT.Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.
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pone.0141618.g003: Flag® Immunoprecipitation assays confirming an interaction between LRP/LR and hTERT.Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.

Mentions: To assess whether the observed co-localization of the two proteins indicated interaction/association with each other, FLAG® co-immunprecipitation/pull down assays were performed (Fig 3). The presence of the LRP::FLAG and hTERT proteins was detected by corresponding antibodies. h-TERT (panel A, bound protein) and LRP::FLAG (panel B, bound protein) both bound to FLAG®M2- beads, in pCIneo-moLRP::FLAG transfected cells, whereas both proteins failed to bind to FLAG®-M2 beads in non-transfected cells (bound protein panel A and B, non-transfected cells). This strongly indicates an interaction/association between hTERT and LRP::FLAG.


Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity.

Naidoo K, Malindisa ST, Otgaar TC, Bernert M, Da Costa Dias B, Ferreira E, Reusch U, Knackmuss S, Little M, Weiss SF, Letsolo BT - PLoS ONE (2015)

Flag® Immunoprecipitation assays confirming an interaction between LRP/LR and hTERT.Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636255&req=5

pone.0141618.g003: Flag® Immunoprecipitation assays confirming an interaction between LRP/LR and hTERT.Pull down assays were used to detect LRP::FLAG as well as any associated proteins bound to the anti-M2 flag beads. A loading control of crude HEK293 lysate was incorporated to ensure the validity of the blots. Panel C indicates the positive and negative controls, where the Bound protein shows the detection of the BAP fusion protein (50 kDa) to the anti-FLAG beads. Panel B indicates that the LRP::FLAG protein was only present in the HEK293 transfected samples, where FLAG was detected on the anti-FLAG beads (Bound protein). Panel A illustrates the detection of a ±140 kDa band (Bound protein) showing a pull down of hTERT for the HEK293 transfected cell line, whereas no signal was detected for the non-transfected HEK293 cell line.
Mentions: To assess whether the observed co-localization of the two proteins indicated interaction/association with each other, FLAG® co-immunprecipitation/pull down assays were performed (Fig 3). The presence of the LRP::FLAG and hTERT proteins was detected by corresponding antibodies. h-TERT (panel A, bound protein) and LRP::FLAG (panel B, bound protein) both bound to FLAG®M2- beads, in pCIneo-moLRP::FLAG transfected cells, whereas both proteins failed to bind to FLAG®-M2 beads in non-transfected cells (bound protein panel A and B, non-transfected cells). This strongly indicates an interaction/association between hTERT and LRP::FLAG.

Bottom Line: FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT.Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity.These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, Republic of South Africa.

ABSTRACT
Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85-90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.

No MeSH data available.


Related in: MedlinePlus