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Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells.

Gullo CA, Ge F, Cow G, Teoh G - Cancer Cell Int. (2008)

Bottom Line: Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM.Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein.These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Research (DCR), Cancer Immunology Laboratory, Singapore General Hospital (SGH), Outram Road, Singapore 169608, Singapore. charles.gullo@sgh.com.sg

ABSTRACT

Background: Truncated variants of Ku86 protein have previously been detected in 86% to 100% of freshly isolated patient multiple myeloma (MM) cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM.

Results: Although, a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in human lymphocytes, we now show using whole cell immunoblotting that the RPMI-8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as a 69-kDa Ku86v; a C-terminus truncated 69-kDa variant Ku86 protein. In contrast, Ku86v proteins were not detected in the freshly isolated lymphocytes as was previously reported. Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein.

Conclusion: These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing.

No MeSH data available.


Related in: MedlinePlus

The 69-kDa Ku86v in MM cell lines is present in the cytosolic, nuclear and membrane fractions and binds DNA. Having determined that MM cell lines constitutively express 69-kDa Ku86v, we next studied the subcellular location of Ku86v. Moreover, since CD40 triggering induces expression of 86-kDa Ku86 on the cell membrane of MM cells, we also investigated whether CD40 triggering affected the subcellular location Ku86v. Fresh whole cell (W), cytosolic (C), nuclear (N) and membrane (M) cell lysate fractions were obtained from sCD40L-triggered (5.0 μg/mL for 4 hrs) and resting RPMI 8226 MM cells, and subjected to normal SDS-PAGE (A). Full-length and variant forms of Ku86 were detected by western immunoblotting using S10B1 anti-Ku86 mAb. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Relative expression of 69-kDa Ku86v (normalized to weakest band) was determined using image densitometry and expressed in bar chart format. The presence of Ku86/Ku86v protein-DNA complexes were next detected using EMSA (B and C). Cytosolic (C), nuclear (N) or membrane (M) protein extracts (4.0 μg/sample) were first obtained from CESS (negative control), CD40-triggered (5.0 μg/mL sCD40L for 4 hrs) or non-CD40-triggered RPMI 8226 MM cell lines. Non-competitive binding of Ku86/Ku86v to DNA (B) was detected using a specific biotin end-labeled DNA probe (20.0 fmol/sample), native PAGE, and hrp-conjugated streptavidin chemiluminescence imaging. Full-length Ku86-DNA complexes are found at position I, whereas DNA complexes with the truncation variant of Ku86 are found at position II. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. To confirm the specificity of DNA binding, a competitive assay (C), in which a variable amount of cell lysate (up to 5.0 μg/sample) was mixed with a fixed amount (4.0 pmol/sample) of non-biotin-labeled DNA, was also performed in the same fashion. All experiments were repeated with the SGH-MM5 MM cell line (data not shown), showed similar results, and performed in triplicate.
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Figure 2: The 69-kDa Ku86v in MM cell lines is present in the cytosolic, nuclear and membrane fractions and binds DNA. Having determined that MM cell lines constitutively express 69-kDa Ku86v, we next studied the subcellular location of Ku86v. Moreover, since CD40 triggering induces expression of 86-kDa Ku86 on the cell membrane of MM cells, we also investigated whether CD40 triggering affected the subcellular location Ku86v. Fresh whole cell (W), cytosolic (C), nuclear (N) and membrane (M) cell lysate fractions were obtained from sCD40L-triggered (5.0 μg/mL for 4 hrs) and resting RPMI 8226 MM cells, and subjected to normal SDS-PAGE (A). Full-length and variant forms of Ku86 were detected by western immunoblotting using S10B1 anti-Ku86 mAb. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Relative expression of 69-kDa Ku86v (normalized to weakest band) was determined using image densitometry and expressed in bar chart format. The presence of Ku86/Ku86v protein-DNA complexes were next detected using EMSA (B and C). Cytosolic (C), nuclear (N) or membrane (M) protein extracts (4.0 μg/sample) were first obtained from CESS (negative control), CD40-triggered (5.0 μg/mL sCD40L for 4 hrs) or non-CD40-triggered RPMI 8226 MM cell lines. Non-competitive binding of Ku86/Ku86v to DNA (B) was detected using a specific biotin end-labeled DNA probe (20.0 fmol/sample), native PAGE, and hrp-conjugated streptavidin chemiluminescence imaging. Full-length Ku86-DNA complexes are found at position I, whereas DNA complexes with the truncation variant of Ku86 are found at position II. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. To confirm the specificity of DNA binding, a competitive assay (C), in which a variable amount of cell lysate (up to 5.0 μg/sample) was mixed with a fixed amount (4.0 pmol/sample) of non-biotin-labeled DNA, was also performed in the same fashion. All experiments were repeated with the SGH-MM5 MM cell line (data not shown), showed similar results, and performed in triplicate.

Mentions: Since CD40 activation of MM cell lines results in the membrane expression of Ku86 in MM cells [6,12,12], we also investigated the distribution of 69-kDa Ku86v in various subcellular locations, i.e. cytosol, nucleus and cell membrane, relative to CD40 triggering. As can be seen in Fig. 2A, 69-kDa Ku86v is present in all subfractions but is at higher (2.5-fold) levels in the cytosol after CD40 triggering of the RPMI 8226 MM cell line. In order to define a functional role for this observation, we analyzed the DNA-binding characteristics of Ku86 and Ku86v to a known 25-bp Ku86 binding DNA oligonucleotide using EMSAs (Figs. 2B and 2C) [16,22], in CD40-triggered RPMI 8226 MM cells in various subcellular fractions. We demonstrate binding of DNA to both 86-kDa Ku86 (band position I) as well as 69-kDa Ku86v (band position II); confirming the presence of these proteins in the RPMI 8226 MM cell line. In contrast, only full-length Ku86 was found in the negative control CESS cells. Similar results were obtained for the SGH-MM5 MM cell line (data not shown). Interestingly, no other bands are detected suggesting that if other forms of Ku86 exist they do not bind DNA. Moreover, cold competitor DNA was used to confirm specificity of the 25-bp oligonucleotide for Ku86 binding (Fig. 2C). Since the DNA binding domain of Ku86 is located in the N-terminus and is preserved in both full-length Ku86 as well as the 69-kDa Ku86v, our data not only confirms the presence of truncated Ku86v, but also suggests a functional role for Ku86v.


Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells.

Gullo CA, Ge F, Cow G, Teoh G - Cancer Cell Int. (2008)

The 69-kDa Ku86v in MM cell lines is present in the cytosolic, nuclear and membrane fractions and binds DNA. Having determined that MM cell lines constitutively express 69-kDa Ku86v, we next studied the subcellular location of Ku86v. Moreover, since CD40 triggering induces expression of 86-kDa Ku86 on the cell membrane of MM cells, we also investigated whether CD40 triggering affected the subcellular location Ku86v. Fresh whole cell (W), cytosolic (C), nuclear (N) and membrane (M) cell lysate fractions were obtained from sCD40L-triggered (5.0 μg/mL for 4 hrs) and resting RPMI 8226 MM cells, and subjected to normal SDS-PAGE (A). Full-length and variant forms of Ku86 were detected by western immunoblotting using S10B1 anti-Ku86 mAb. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Relative expression of 69-kDa Ku86v (normalized to weakest band) was determined using image densitometry and expressed in bar chart format. The presence of Ku86/Ku86v protein-DNA complexes were next detected using EMSA (B and C). Cytosolic (C), nuclear (N) or membrane (M) protein extracts (4.0 μg/sample) were first obtained from CESS (negative control), CD40-triggered (5.0 μg/mL sCD40L for 4 hrs) or non-CD40-triggered RPMI 8226 MM cell lines. Non-competitive binding of Ku86/Ku86v to DNA (B) was detected using a specific biotin end-labeled DNA probe (20.0 fmol/sample), native PAGE, and hrp-conjugated streptavidin chemiluminescence imaging. Full-length Ku86-DNA complexes are found at position I, whereas DNA complexes with the truncation variant of Ku86 are found at position II. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. To confirm the specificity of DNA binding, a competitive assay (C), in which a variable amount of cell lysate (up to 5.0 μg/sample) was mixed with a fixed amount (4.0 pmol/sample) of non-biotin-labeled DNA, was also performed in the same fashion. All experiments were repeated with the SGH-MM5 MM cell line (data not shown), showed similar results, and performed in triplicate.
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Figure 2: The 69-kDa Ku86v in MM cell lines is present in the cytosolic, nuclear and membrane fractions and binds DNA. Having determined that MM cell lines constitutively express 69-kDa Ku86v, we next studied the subcellular location of Ku86v. Moreover, since CD40 triggering induces expression of 86-kDa Ku86 on the cell membrane of MM cells, we also investigated whether CD40 triggering affected the subcellular location Ku86v. Fresh whole cell (W), cytosolic (C), nuclear (N) and membrane (M) cell lysate fractions were obtained from sCD40L-triggered (5.0 μg/mL for 4 hrs) and resting RPMI 8226 MM cells, and subjected to normal SDS-PAGE (A). Full-length and variant forms of Ku86 were detected by western immunoblotting using S10B1 anti-Ku86 mAb. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Relative expression of 69-kDa Ku86v (normalized to weakest band) was determined using image densitometry and expressed in bar chart format. The presence of Ku86/Ku86v protein-DNA complexes were next detected using EMSA (B and C). Cytosolic (C), nuclear (N) or membrane (M) protein extracts (4.0 μg/sample) were first obtained from CESS (negative control), CD40-triggered (5.0 μg/mL sCD40L for 4 hrs) or non-CD40-triggered RPMI 8226 MM cell lines. Non-competitive binding of Ku86/Ku86v to DNA (B) was detected using a specific biotin end-labeled DNA probe (20.0 fmol/sample), native PAGE, and hrp-conjugated streptavidin chemiluminescence imaging. Full-length Ku86-DNA complexes are found at position I, whereas DNA complexes with the truncation variant of Ku86 are found at position II. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. To confirm the specificity of DNA binding, a competitive assay (C), in which a variable amount of cell lysate (up to 5.0 μg/sample) was mixed with a fixed amount (4.0 pmol/sample) of non-biotin-labeled DNA, was also performed in the same fashion. All experiments were repeated with the SGH-MM5 MM cell line (data not shown), showed similar results, and performed in triplicate.
Mentions: Since CD40 activation of MM cell lines results in the membrane expression of Ku86 in MM cells [6,12,12], we also investigated the distribution of 69-kDa Ku86v in various subcellular locations, i.e. cytosol, nucleus and cell membrane, relative to CD40 triggering. As can be seen in Fig. 2A, 69-kDa Ku86v is present in all subfractions but is at higher (2.5-fold) levels in the cytosol after CD40 triggering of the RPMI 8226 MM cell line. In order to define a functional role for this observation, we analyzed the DNA-binding characteristics of Ku86 and Ku86v to a known 25-bp Ku86 binding DNA oligonucleotide using EMSAs (Figs. 2B and 2C) [16,22], in CD40-triggered RPMI 8226 MM cells in various subcellular fractions. We demonstrate binding of DNA to both 86-kDa Ku86 (band position I) as well as 69-kDa Ku86v (band position II); confirming the presence of these proteins in the RPMI 8226 MM cell line. In contrast, only full-length Ku86 was found in the negative control CESS cells. Similar results were obtained for the SGH-MM5 MM cell line (data not shown). Interestingly, no other bands are detected suggesting that if other forms of Ku86 exist they do not bind DNA. Moreover, cold competitor DNA was used to confirm specificity of the 25-bp oligonucleotide for Ku86 binding (Fig. 2C). Since the DNA binding domain of Ku86 is located in the N-terminus and is preserved in both full-length Ku86 as well as the 69-kDa Ku86v, our data not only confirms the presence of truncated Ku86v, but also suggests a functional role for Ku86v.

Bottom Line: Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM.Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein.These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical Research (DCR), Cancer Immunology Laboratory, Singapore General Hospital (SGH), Outram Road, Singapore 169608, Singapore. charles.gullo@sgh.com.sg

ABSTRACT

Background: Truncated variants of Ku86 protein have previously been detected in 86% to 100% of freshly isolated patient multiple myeloma (MM) cells. Since, the Ku70/Ku86 heterodimer functions as the regulatory subunit of the DNA repair enzyme, DNA-dependent protein kinase, we have been interested in the altered expression and function of Ku86 variant (Ku86v) proteins in genome maintenance of MM.

Results: Although, a number of studies have suggested that truncated forms of Ku proteins could be artificially generated by proteolytic degradation in vitro in human lymphocytes, we now show using whole cell immunoblotting that the RPMI-8226 and SGH-MM5 human MM cell lines consistently express full-length Ku86 as well as a 69-kDa Ku86v; a C-terminus truncated 69-kDa variant Ku86 protein. In contrast, Ku86v proteins were not detected in the freshly isolated lymphocytes as was previously reported. Data also indicates that the Ku86v was not generated as a result of carbohydrate modification but that serine proteases may act on the full-length form of the protein.

Conclusion: These data confirm that MM cells contain bona fide Ku86v proteins that were generated intracellularly by a post-transcriptional mechanism, which required proteolytic processing.

No MeSH data available.


Related in: MedlinePlus