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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

Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines. RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 106 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.
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Figure 4: Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines. RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 106 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.

Mentions: In order to investigate other post-translational modifications that might be responsible for the generation of 69-kDa Ku86v, we looked at the intracellular proteolytic degradation of Ku86. Since our current data (Fig. 1B and 2A) have demonstrated that various in vitro protease inhibition strategies using fresh cell lysates did not reduce the generation of 69-kDa Ku86v, we investigated the effects of various inhibitors in whole MM cells. Within the MM cell, the ubiquitin-proteasome pathway is amongst the most active intracellular protein degradation systems. Surprisingly, incubation of whole MM cells with 2 to 24 ng/mL of bortezomib (Velcade®, Millennium Pharmaceuticals, MA, USA), a proteasome inhibitor, for as much as 48 hrs, had no effect on proteolytic generation of Ku86 or Ku86v (data not shown); suggesting that other proteolytic systems could be active in the MM cell. However, simultaneous inhibition of several classes of proteases; namely serine, cysteine and metallo- proteases using Complete™ tablets for 18 hrs was associated with 3- to 7-fold decreased expression of 69-kDa Ku86v (Fig. 4A lanes 2 and 3), as compared to the mock experiment (Fig. 4A lane 1). The data again suggest that 69-kDa Ku86v is generated constitutively within living MM cells. Similarly, broad-based inhibition of serine proteases via aprotinin plus PMSF (Fig. 4B lane 4), but not inhibition of cysteine proteases via antipain plus leupeptin (Fig. 4B lane 3), for 24 hrs is associated with 3- to 4-fold decreased expression of 69-kDa Ku86v; further suggesting that serine proteases (but not cysteine proteases) could be involved in generation of 69-kDa Ku86v. Importantly, protease inhibition did not affect cell viability (data not shown), or expression of 86-kDa Ku86 or actin. These data are consistent with a report that suggests that nuclear serine proteases are responsible for in vitro generated forms the Ku86 variant [22].


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)

Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines. RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 106 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.
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Related In: Results  -  Collection

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Figure 4: Proteolysis plays an important role in the intracellular generation of the 69-kDa Ku86v variant protein in MM cell lines. RPMI 8226 (A) and SGH-MM5 (data not shown) MM cell lines (5.0 × 106 cells/sample) were first incubated for 18 hrs with either 1× or 2× Complete™ protease inhibitor tablets (lanes 2 and 3); and then washed and checked for viability using trypan blue exclusion assay. Mock experiments in which cell lines were incubated for 18 hrs with media alone (lane 1) served as negative controls. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. The RPMI 8226 MM cell line s (B) was also incubated for 24 hrs with 2× Complete™ protease inhibitor tablets (lane 2), antipain (2.0 μg/mL final concentration) plus leupeptin (2.0 μg/mL final concentration) (lane 3), or aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) (lane 4) and analyzed in the same way as above. Mock experiments in which cell lines were incubated for 24 hrs with media alone (lane 1) again served as negative controls. Membranes were stripped and re-probed using anti-actin mAb (control) to confirm equal protein loading. Cell lysates were resolved by SDS-PAGE and immunoblotted using S10B1 anti-Ku86 mAb. Relative expression of 69-kDa Ku86v-DNA complexes (normalized to weakest band) was determined using image densitometry. All experiments were performed in triplicate.
Mentions: In order to investigate other post-translational modifications that might be responsible for the generation of 69-kDa Ku86v, we looked at the intracellular proteolytic degradation of Ku86. Since our current data (Fig. 1B and 2A) have demonstrated that various in vitro protease inhibition strategies using fresh cell lysates did not reduce the generation of 69-kDa Ku86v, we investigated the effects of various inhibitors in whole MM cells. Within the MM cell, the ubiquitin-proteasome pathway is amongst the most active intracellular protein degradation systems. Surprisingly, incubation of whole MM cells with 2 to 24 ng/mL of bortezomib (Velcade®, Millennium Pharmaceuticals, MA, USA), a proteasome inhibitor, for as much as 48 hrs, had no effect on proteolytic generation of Ku86 or Ku86v (data not shown); suggesting that other proteolytic systems could be active in the MM cell. However, simultaneous inhibition of several classes of proteases; namely serine, cysteine and metallo- proteases using Complete™ tablets for 18 hrs was associated with 3- to 7-fold decreased expression of 69-kDa Ku86v (Fig. 4A lanes 2 and 3), as compared to the mock experiment (Fig. 4A lane 1). The data again suggest that 69-kDa Ku86v is generated constitutively within living MM cells. Similarly, broad-based inhibition of serine proteases via aprotinin plus PMSF (Fig. 4B lane 4), but not inhibition of cysteine proteases via antipain plus leupeptin (Fig. 4B lane 3), for 24 hrs is associated with 3- to 4-fold decreased expression of 69-kDa Ku86v; further suggesting that serine proteases (but not cysteine proteases) could be involved in generation of 69-kDa Ku86v. Importantly, protease inhibition did not affect cell viability (data not shown), or expression of 86-kDa Ku86 or actin. These data are consistent with a report that suggests that nuclear serine proteases are responsible for in vitro generated forms the Ku86 variant [22].

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