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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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LMP1 expression in transgenic mouse derived cell lines. (A) LMP1 was immunoprecipitated from 300 μg of urea buffer-extracted proteins from seven PyLMP1 transgene-positive line 53 carcinoma cell lines and one transgene-negative cell line (as denoted 53.217neg) and 100 μg of protein from control cell lines: Raji (EBV +ve, containing 50-60 copies of EBV) and Ramos (EBV -ve), using the S12 antiserum. Immunoprecipitated proteins were separated by 10%SDS-PAGE and blotted alongside two total lysate (boiling mix extracted) controls, BL2B95-8 (EBV +ve) and BJAB (EBV -ve) (1 × 105 cells each). The blot was probed with anti-LMP1 antibody 1G6 followed by goat α-rat IgG HRP. The bands corresponding to LMP1 and the immunoglobulin heavy (H) and light (L) chains are indicated on the left. (B) Protein was extracted from B-cell lines 39.415 and 3959.48 with controls Raji and BJAB (the number of cells indicated below each track) using boiling mix and samples western blotted for LMP1 using IG6. The transgenic LMP1 (tgLMP1) is slightly smaller than Raji LMP1 (rLMP1) [3].
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Figure 1: LMP1 expression in transgenic mouse derived cell lines. (A) LMP1 was immunoprecipitated from 300 μg of urea buffer-extracted proteins from seven PyLMP1 transgene-positive line 53 carcinoma cell lines and one transgene-negative cell line (as denoted 53.217neg) and 100 μg of protein from control cell lines: Raji (EBV +ve, containing 50-60 copies of EBV) and Ramos (EBV -ve), using the S12 antiserum. Immunoprecipitated proteins were separated by 10%SDS-PAGE and blotted alongside two total lysate (boiling mix extracted) controls, BL2B95-8 (EBV +ve) and BJAB (EBV -ve) (1 × 105 cells each). The blot was probed with anti-LMP1 antibody 1G6 followed by goat α-rat IgG HRP. The bands corresponding to LMP1 and the immunoglobulin heavy (H) and light (L) chains are indicated on the left. (B) Protein was extracted from B-cell lines 39.415 and 3959.48 with controls Raji and BJAB (the number of cells indicated below each track) using boiling mix and samples western blotted for LMP1 using IG6. The transgenic LMP1 (tgLMP1) is slightly smaller than Raji LMP1 (rLMP1) [3].

Mentions: Carcinomas were induced in transgene positive and negative sibling controls (NSC) in the transgenic PyLMP1 line 53 (PyLMP1.53) [3], by topical treatment with chemical carcinogens [13]. These tumours could be readily established in culture; some retained a cuboidal, squamous morphology while others developed a spindle morphology with more transformed growth characteristics (additional files 1&2, supplementary table S1). LMP1 was difficult to extract from these epithelial cells (as with transgenic skin tissue [5]), suggesting an association with the cytoskeleton and necessitating the use of a urea extraction protocol. LMP1 expression was detected by immunoprecipitation and western blotting in several, but not all of the transgene positive carcinoma cell lines developed (figure 1A). Nevertheless, the cell lines in which expression could not be detected maintained the transgene (not shown). There was no apparent correlation between the carcinoma grade, cell line phenotype and LMP1 expression. For example, cell line 53.278a, derived from an aggressive spindle cell carcinoma and showing rapid spindle cell growth in culture (see supplementary information table 1) showed LMP1 expression as did the more cuboidal cell line 234a (with highest LMP1 expression) derived from a grade 3 carcinoma. However, with cuboidal cell line 53.226b (grade 1 carcinoma) and spindle cell line 53.191 (grade 3 carcinoma), little or no LMP1 expression could be detected.


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

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

LMP1 expression in transgenic mouse derived cell lines. (A) LMP1 was immunoprecipitated from 300 μg of urea buffer-extracted proteins from seven PyLMP1 transgene-positive line 53 carcinoma cell lines and one transgene-negative cell line (as denoted 53.217neg) and 100 μg of protein from control cell lines: Raji (EBV +ve, containing 50-60 copies of EBV) and Ramos (EBV -ve), using the S12 antiserum. Immunoprecipitated proteins were separated by 10%SDS-PAGE and blotted alongside two total lysate (boiling mix extracted) controls, BL2B95-8 (EBV +ve) and BJAB (EBV -ve) (1 × 105 cells each). The blot was probed with anti-LMP1 antibody 1G6 followed by goat α-rat IgG HRP. The bands corresponding to LMP1 and the immunoglobulin heavy (H) and light (L) chains are indicated on the left. (B) Protein was extracted from B-cell lines 39.415 and 3959.48 with controls Raji and BJAB (the number of cells indicated below each track) using boiling mix and samples western blotted for LMP1 using IG6. The transgenic LMP1 (tgLMP1) is slightly smaller than Raji LMP1 (rLMP1) [3].
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: LMP1 expression in transgenic mouse derived cell lines. (A) LMP1 was immunoprecipitated from 300 μg of urea buffer-extracted proteins from seven PyLMP1 transgene-positive line 53 carcinoma cell lines and one transgene-negative cell line (as denoted 53.217neg) and 100 μg of protein from control cell lines: Raji (EBV +ve, containing 50-60 copies of EBV) and Ramos (EBV -ve), using the S12 antiserum. Immunoprecipitated proteins were separated by 10%SDS-PAGE and blotted alongside two total lysate (boiling mix extracted) controls, BL2B95-8 (EBV +ve) and BJAB (EBV -ve) (1 × 105 cells each). The blot was probed with anti-LMP1 antibody 1G6 followed by goat α-rat IgG HRP. The bands corresponding to LMP1 and the immunoglobulin heavy (H) and light (L) chains are indicated on the left. (B) Protein was extracted from B-cell lines 39.415 and 3959.48 with controls Raji and BJAB (the number of cells indicated below each track) using boiling mix and samples western blotted for LMP1 using IG6. The transgenic LMP1 (tgLMP1) is slightly smaller than Raji LMP1 (rLMP1) [3].
Mentions: Carcinomas were induced in transgene positive and negative sibling controls (NSC) in the transgenic PyLMP1 line 53 (PyLMP1.53) [3], by topical treatment with chemical carcinogens [13]. These tumours could be readily established in culture; some retained a cuboidal, squamous morphology while others developed a spindle morphology with more transformed growth characteristics (additional files 1&2, supplementary table S1). LMP1 was difficult to extract from these epithelial cells (as with transgenic skin tissue [5]), suggesting an association with the cytoskeleton and necessitating the use of a urea extraction protocol. LMP1 expression was detected by immunoprecipitation and western blotting in several, but not all of the transgene positive carcinoma cell lines developed (figure 1A). Nevertheless, the cell lines in which expression could not be detected maintained the transgene (not shown). There was no apparent correlation between the carcinoma grade, cell line phenotype and LMP1 expression. For example, cell line 53.278a, derived from an aggressive spindle cell carcinoma and showing rapid spindle cell growth in culture (see supplementary information table 1) showed LMP1 expression as did the more cuboidal cell line 234a (with highest LMP1 expression) derived from a grade 3 carcinoma. However, with cuboidal cell line 53.226b (grade 1 carcinoma) and spindle cell line 53.191 (grade 3 carcinoma), little or no LMP1 expression could be detected.

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