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Evaluation of LMP1 of Epstein-Barr virus as a therapeutic target by its inhibition.

Hannigan A, Wilson JB - Mol. Cancer (2010)

Bottom Line: These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease.Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1.This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G116NU, UK.

ABSTRACT

Background: The latent membrane protein-1 (LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-kappaB. In so doing it leads to deregulated cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease. LMP1 is expressed in several EBV-associated malignancies, notably in Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). However, the viral protein is only detected in approximately 30%-50% of NPC samples, as such its role in carcinogenesis and tumour maintenance can be questioned and thus its relevance as a therapeutic target.

Results: In order to explore if LMP1 has a continuous function in established tumours, its activity was inhibited through expression of a dominant negative LMP1 mutant in tumour cell lines derived from transgenic mice. LMP1 is the tumour predisposing oncogene in two different series of transgenic mice which separately give rise to either B-cell lymphomas or carcinomas. Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1. Inhibition of LMP1 activity in the transgenic B-cell lines was incompatible with growth and survival of the cells and no clones expressing the dominant negative LMP1 mutant could be established.

Conclusions: LMP1 continues to provide a tumour cell growth function in cell lines established from LMP1 transgenic mouse tumours, of both B-cell and epithelial cell origin. LMP1 can perform this function, even when expressed at such low levels as to be undetectable, whereby evidence of its expression can only be inferred by its inhibition being detrimental to the growth of the cell. This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected. This reinforces the basis for pursuing LMP1 as a therapeutic target in EBV associated LMP1-expressing malignancies.

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GFPdnLMP1 expression is lost from EμLMP1 transgenic B-cell lymphoma cultures. Transgenic EμLMP1 cell lines 39.415 and 3959.48, along with an EBV negative Akata cell line sub clone (AK31), transfected with pGFP or pGFPdnLMP1 were assayed for transfectant expression. (A) Protein extract from 5 × 105 39.415 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected after completion of selection at 3 weeks post transfection (post-tx) and then at weekly intervals (maintaining G418 selective pressure). (B) Bright field (left panel) and green fluorescence (right panel) visualized in pGFPdnLMP1 (top panel) or pGFP (bottom panel) transfected 39.415 cells at 3 weeks post transfection. (C) 40 μg of protein extract from 3959.48 and control AK31 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected at 2, 5 and 21 days post transfection (post-tx) for 3959.48 cells and at 12 weeks post-tx for AK31 cells (all under G418 selection). (D) At four weeks post pGFP or pGFPdnLMP1 transfection 3959.48 cells stained with propidium iodide (viable cells exclude staining, apoptotic cells stain) were analysed by flow cytometry, gating on GFP positive fluorescent cells only. Histograms show y axis (cell counts) and x axis (FL2-H, PI staining). The percentage of PI positive cells (of the GFP positive population) is indicated.
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Figure 5: GFPdnLMP1 expression is lost from EμLMP1 transgenic B-cell lymphoma cultures. Transgenic EμLMP1 cell lines 39.415 and 3959.48, along with an EBV negative Akata cell line sub clone (AK31), transfected with pGFP or pGFPdnLMP1 were assayed for transfectant expression. (A) Protein extract from 5 × 105 39.415 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected after completion of selection at 3 weeks post transfection (post-tx) and then at weekly intervals (maintaining G418 selective pressure). (B) Bright field (left panel) and green fluorescence (right panel) visualized in pGFPdnLMP1 (top panel) or pGFP (bottom panel) transfected 39.415 cells at 3 weeks post transfection. (C) 40 μg of protein extract from 3959.48 and control AK31 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected at 2, 5 and 21 days post transfection (post-tx) for 3959.48 cells and at 12 weeks post-tx for AK31 cells (all under G418 selection). (D) At four weeks post pGFP or pGFPdnLMP1 transfection 3959.48 cells stained with propidium iodide (viable cells exclude staining, apoptotic cells stain) were analysed by flow cytometry, gating on GFP positive fluorescent cells only. Histograms show y axis (cell counts) and x axis (FL2-H, PI staining). The percentage of PI positive cells (of the GFP positive population) is indicated.

Mentions: Inhibition of LMP1 activity in the tumour derived B-cell lymphoma cells lines 39.415 and 3959.48 was similarly assessed by transfection of the GFPdnLMP1 or GFP expression vectors. The antibiotic selection process was complete by 3 weeks post transfection at which point the cell lines were assayed for GFPdnLMP1 and GFP expression. Cells were harvested at weekly intervals for four weeks maintaining drug selection. With 39.415 cells, GFP expression could be detected in the control pGFP transfectants consistently for the four week period (figure 5A). However while clear GFPdnLMP1 expression was detected at 3 and 4 weeks post transfection, it disappeared from the transfected culture by 5 weeks post transfection. Similarly, clear green fluorescence could be seen in the pGFP transfectants (3 weeks post transfection) but only weak fluorescence in the pGFPdnLMP1 39.415 transfectants (figure 5B). In contrast, green fluorescence in both pGFP and pGFPdnLMP1 transfectants of the control EBV negative cell line AK31 was clearly visible (additional files 1&2, supplementary figure S5) and could consistently be detected by western to at least 12 weeks after transfection (figure 5C). With the 3959.48 cell line, similarly consistent GFP expression was seen in the controls, but GFPdnLMP1 expression could barely be detected in the transfected cultures at 3 weeks post transfection and was not detected by 4 weeks (not shown). Therefore earlier time points post transfection were examined. At two days post transfection of 3959.48 cells strong expression of GFPdnLMP1 was detected which was considerably reduced by 5 days post transfection and again only low level expression was detected by 3 weeks post transfection (despite plasmid selection), while control GFP expression in this cell line was constant (figure 5C).


Evaluation of LMP1 of Epstein-Barr virus as a therapeutic target by its inhibition.

Hannigan A, Wilson JB - Mol. Cancer (2010)

GFPdnLMP1 expression is lost from EμLMP1 transgenic B-cell lymphoma cultures. Transgenic EμLMP1 cell lines 39.415 and 3959.48, along with an EBV negative Akata cell line sub clone (AK31), transfected with pGFP or pGFPdnLMP1 were assayed for transfectant expression. (A) Protein extract from 5 × 105 39.415 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected after completion of selection at 3 weeks post transfection (post-tx) and then at weekly intervals (maintaining G418 selective pressure). (B) Bright field (left panel) and green fluorescence (right panel) visualized in pGFPdnLMP1 (top panel) or pGFP (bottom panel) transfected 39.415 cells at 3 weeks post transfection. (C) 40 μg of protein extract from 3959.48 and control AK31 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected at 2, 5 and 21 days post transfection (post-tx) for 3959.48 cells and at 12 weeks post-tx for AK31 cells (all under G418 selection). (D) At four weeks post pGFP or pGFPdnLMP1 transfection 3959.48 cells stained with propidium iodide (viable cells exclude staining, apoptotic cells stain) were analysed by flow cytometry, gating on GFP positive fluorescent cells only. Histograms show y axis (cell counts) and x axis (FL2-H, PI staining). The percentage of PI positive cells (of the GFP positive population) is indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2913984&req=5

Figure 5: GFPdnLMP1 expression is lost from EμLMP1 transgenic B-cell lymphoma cultures. Transgenic EμLMP1 cell lines 39.415 and 3959.48, along with an EBV negative Akata cell line sub clone (AK31), transfected with pGFP or pGFPdnLMP1 were assayed for transfectant expression. (A) Protein extract from 5 × 105 39.415 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected after completion of selection at 3 weeks post transfection (post-tx) and then at weekly intervals (maintaining G418 selective pressure). (B) Bright field (left panel) and green fluorescence (right panel) visualized in pGFPdnLMP1 (top panel) or pGFP (bottom panel) transfected 39.415 cells at 3 weeks post transfection. (C) 40 μg of protein extract from 3959.48 and control AK31 transfected cells (and non-transfected control: nt) were examined by western blotting sequentially using anti-LMP1 (top panel), α-GFP (middle) and α-beta-tubulin (bottom), as indicated. Cell aliquots were collected at 2, 5 and 21 days post transfection (post-tx) for 3959.48 cells and at 12 weeks post-tx for AK31 cells (all under G418 selection). (D) At four weeks post pGFP or pGFPdnLMP1 transfection 3959.48 cells stained with propidium iodide (viable cells exclude staining, apoptotic cells stain) were analysed by flow cytometry, gating on GFP positive fluorescent cells only. Histograms show y axis (cell counts) and x axis (FL2-H, PI staining). The percentage of PI positive cells (of the GFP positive population) is indicated.
Mentions: Inhibition of LMP1 activity in the tumour derived B-cell lymphoma cells lines 39.415 and 3959.48 was similarly assessed by transfection of the GFPdnLMP1 or GFP expression vectors. The antibiotic selection process was complete by 3 weeks post transfection at which point the cell lines were assayed for GFPdnLMP1 and GFP expression. Cells were harvested at weekly intervals for four weeks maintaining drug selection. With 39.415 cells, GFP expression could be detected in the control pGFP transfectants consistently for the four week period (figure 5A). However while clear GFPdnLMP1 expression was detected at 3 and 4 weeks post transfection, it disappeared from the transfected culture by 5 weeks post transfection. Similarly, clear green fluorescence could be seen in the pGFP transfectants (3 weeks post transfection) but only weak fluorescence in the pGFPdnLMP1 39.415 transfectants (figure 5B). In contrast, green fluorescence in both pGFP and pGFPdnLMP1 transfectants of the control EBV negative cell line AK31 was clearly visible (additional files 1&2, supplementary figure S5) and could consistently be detected by western to at least 12 weeks after transfection (figure 5C). With the 3959.48 cell line, similarly consistent GFP expression was seen in the controls, but GFPdnLMP1 expression could barely be detected in the transfected cultures at 3 weeks post transfection and was not detected by 4 weeks (not shown). Therefore earlier time points post transfection were examined. At two days post transfection of 3959.48 cells strong expression of GFPdnLMP1 was detected which was considerably reduced by 5 days post transfection and again only low level expression was detected by 3 weeks post transfection (despite plasmid selection), while control GFP expression in this cell line was constant (figure 5C).

Bottom Line: These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease.Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1.This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Cellular Biology, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G116NU, UK.

ABSTRACT

Background: The latent membrane protein-1 (LMP1) encoded by Epstein-Barr virus (EBV) is an oncoprotein which acts by constitutive activation of various signalling pathways, including NF-kappaB. In so doing it leads to deregulated cell growth intrinsic to the cancer cell as well as having extrinsic affects upon the tumour microenvironment. These properties and that it is a foreign antigen, lead to the proposition that LMP1 may be a good therapeutic target in the treatment of EBV associated disease. LMP1 is expressed in several EBV-associated malignancies, notably in Hodgkin's lymphoma and nasopharyngeal carcinoma (NPC). However, the viral protein is only detected in approximately 30%-50% of NPC samples, as such its role in carcinogenesis and tumour maintenance can be questioned and thus its relevance as a therapeutic target.

Results: In order to explore if LMP1 has a continuous function in established tumours, its activity was inhibited through expression of a dominant negative LMP1 mutant in tumour cell lines derived from transgenic mice. LMP1 is the tumour predisposing oncogene in two different series of transgenic mice which separately give rise to either B-cell lymphomas or carcinomas. Inhibition of LMP1 activity in the carcinoma cell lines lead to a reduction in clonagenicity and clone viability in all of the cell lines tested, even those with low or below detection levels of LMP1. Inhibition of LMP1 activity in the transgenic B-cell lines was incompatible with growth and survival of the cells and no clones expressing the dominant negative LMP1 mutant could be established.

Conclusions: LMP1 continues to provide a tumour cell growth function in cell lines established from LMP1 transgenic mouse tumours, of both B-cell and epithelial cell origin. LMP1 can perform this function, even when expressed at such low levels as to be undetectable, whereby evidence of its expression can only be inferred by its inhibition being detrimental to the growth of the cell. This raises the possibility that LMP1 still performs a pro-oncogenic function in the 50% to 70% of NPC tumours wherein LMP1 protein expression cannot be detected. This reinforces the basis for pursuing LMP1 as a therapeutic target in EBV associated LMP1-expressing malignancies.

Show MeSH
Related in: MedlinePlus