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

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

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes

10.1186/1476-8518-6-2Journal of Immune Based Therapies and Vaccines6

Use of Ultraviolet Light Irradiated Multiple Myeloma Cells as Immunogens to generate Tumor Specific Cytolytic T Lymphocytes

Background: As the eradication of tumor cells in vivo is most efficiently performed by cytolytic Tlymphocytes (CTL), various methods for priming tumor-reactive lymphocytes have been developed. In this study, a method of priming CTLs with ultraviolet (UV)-irradiated tumor cells, which results in termination of tumor cell proliferation, apoptosis, as well as upregulation of heat shock proteins (HSP) expression is described. Methods: Peripheral blood mononuclear cells (PBMC) were primed weekly with UV-irradiated or mitomycin-treated RPMI 8226 multiple myeloma cells. Following three rounds of stimulation over 21 days, the lymphocytes from the mixed culture conditions were analyzed for anti-MM cell reactivity. Results: By day 10 of cultures, PBMCs primed using UV-irradiated tumor cells demonstrated a higher percentage of activated CD8+/CD4- T lymphocytes than non-primed PBMCs or PBMCs primed using mitomycin-treated MM cells. Cytotoxicity assays revealed that primed PBMCs were markedly more effective (p \u3c 0.01) than non-primed PBMCs in killing RPMI 8226 MM cells. Surface expression of glucose regulated protein 94 (Grp94/Gp96) and Grp78 were both found to be induced in UV-treated MM cells. Conclusion: Since, HSP-associated peptides are known to mediate tumor rejection; these data suggest that immune-mediated eradication of MM cells could be elicited via a UV-induced HSP process. The finding that the addition of 17-allylamide-17-demethoxygeldanamycin (17AAG, an inhibitor of HSP 90-peptide interactions) resulted in decreased CTL-induced cytotoxicity supported this hypothesis. Our study, therefore, provides the framework for the development of anti-tumor CTL cellular vaccines for treating MM using UV-irradiated tumor cells as immunogens

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

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

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-4

.5 μg/sample) by trypsin (0.065 μg of Trypsin Gold/reaction up to 20 mins) was then performed using a limited proteolysis protocol from the manufacturer. The reactions were stopped by rapid cooling on ice; and the products of trypsin digestion were resolved by SDS PAGE and immunoblotted S10B1 anti-Ku86 mAb. The effect of protease inhibitors aprotinin (2.0 μg/mL final concentration) plus PMSF (100 μg/mL final concentration) on trypsin digestion was also determined on a larger sample of rhKu86 (13.0 μg/sample). Trypsin digestion (0.065 μg of Trypsin Gold/reaction for 1 min) of rhKu86 was performed as above either without (lane 1) or with (lane 2) protease inhibitors (B).<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells-3

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.<p><b>Copyright information:</b></p><p>Taken from "Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells"</p><p>http://www.cancerci.com/content/8/1/4</p><p>Cancer Cell International 2008;8():4-4.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2386117.</p><p></p

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes-7

cell immunogens for 28 days in 'mixed-culture conditions'. Non-primed, mito-primed, or UV-primed and PBMCs (effector cells) were then co-cultured with viable H-TdR-labeled RPMI 8226 MM cells (target cells) in a re-directed CTL assay. 17-AAG was added at 10 uM to the UV-irradiated RPMI cells for four hours and then washed away before the cells were added to the CTL culture. A standard H-TdR cytotoxicity assays was performed at E:T ratio of 50:1. Experiments were performed triplicate and expressed as the mean ± 2SEM.<p><b>Copyright information:</b></p><p>Taken from "Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes"</p><p>http://www.jibtherapies.com/content/6/1/2</p><p>Journal of Immune Based Therapies and Vaccines 2008;6():2-2.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2383894.</p><p></p

Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes-5

R using actin and grp78 (A) or grp94 (B) primers was performed and data is expressed as calibrator corrected and actin normalized ratios.<p><b>Copyright information:</b></p><p>Taken from "Use of ultraviolet-light irradiated multiple myeloma cells as immunogens to generate tumor-specific cytolytic T lymphocytes"</p><p>http://www.jibtherapies.com/content/6/1/2</p><p>Journal of Immune Based Therapies and Vaccines 2008;6():2-2.</p><p>Published online 28 Apr 2008</p><p>PMCID:PMC2383894.</p><p></p