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  • 1
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    Strong Antibody Responses Against a Unique Region of CT-Antigen SSX-2 Are Induced After Allogeneic Stem Cell Transplantation and Correlate with Clinical Remission In Patients with Multiple Myeloma
    American Society of Hematology ; 2010
    In:  Blood Vol. 116, No. 21 ( 2010-11-19), p. 1900-1900
    Details
    In: Blood, American Society of Hematology, Vol. 116, No. 21 ( 2010-11-19), p. 1900-1900
    Abstract: Abstract 1900 Cancer-testis (CT) antigens show an expression restricted to malignancies and the human germline among healthy tissues and, therefore, represent attractive targets for cancer vaccines. CT antigen SSX-2 is expressed in about 20% of multiple myeloma patients (MM), however, little is known about the occurrence of spontaneous humoral immune responses against SSX-2 in these patients. Moreover, no information is available regarding the functionality of anti-SSX2 antibody responses and a possible impact on the course of the disease. Screening 1098 peripheral blood samples and 200 bone marrow samples of 194 MM patients, as well as 100 healthy donors serving as controls, we found six patients (3%) to present with SSX-2 specific antibodies. When we mapped the target epitopes using overlapping peptides for the whole SSX-2 sequence, we found the antibodies to be exclusively directed against amino acids 81–90. Remarkably, all antibodies were of the IgG3 subtype. In addition, SSX-2 antibodies were strictly antigen-specific and represented potent activators of the complement system. Correlating the antibody responses with clinical events, we found that the majority (5 out of 6) of seropositive patients had been antibody-negative before allogeneic stem cell transplantation (alloSCT) and had only developed anti-SSX2 antibodies after transplantation. Donor-derived antibody responses increased with depth of remission and, in case of recurrence of the disease, dropped to low or undetectable levels. Our data suggest that alloSCT is able to induce immune responses against myeloma specific SSX-2 antigen which is of low immunogenicity under normal conditions. Such antibody responses are of an effective phenotype and seem to correlate with protection from disease recurrence. To further characterize the biological role of transplantation-induced anti-SSX2 immune responses, we will analyze our patients for the presence of SSX-2-specific T cells which might be induced together with serological responses in the framework of an integrated immune response. Active immunotherapy might contribute to amplifying and shaping anti-SSX2 immune responses in myeloma patients, particularly after alloSCT. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V116.21.1900.1900
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2010
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  • 2
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    Expression of Cancer-Testis Antigens MAGE-C1/CT7 and MAGE-A3 Is Central to the Survival of Myeloma Cells and Their Resistance to Chemotherapy
    American Society of Hematology ; 2008
    In:  Blood Vol. 112, No. 11 ( 2008-11-16), p. 3668-3668
    Details
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 3668-3668
    Abstract: The cancer-testis (CT) class of tumor antigens is a group of proteins, the expression of which is characteristically restricted to cancer and the human germline. Based on their immunogenicity and restricted tissue expression, CT antigens seem ideal targets for immunotherapeutic approaches. This is particularly the case in multiple myeloma (MM) where these tumor antigens are frequently and strongly expressed. Unfortunately, very little is known about the biological function of CT antigens which seems surprising given the strong impact of CT antigen expression on the prognosis of a variety of malignancies including MM. We examined for the first time the impact of the expression of two CT antigens frequently found in MM, MAGE-C1/CT7 and MAGE-A3, on the biology of the disease conducting knock-down experiments using RNA interference (RNAi) technology. As read-out assays, Western Blots as well as a large variety of tests measuring proliferation, cytotoxicity, cell adhesion, and clonogenic growth were performed. Transfecting myeloma cell line MOLP-8 with RNAi specific for MAGE-C1/CT7 and MAGE-A3 we observed a down-regulation of CT antigen protein expression by 80% and 70%, respectively. Importantly, transfection with MAGE-A3 RNA stealth led to a specific decrease in MAGE-A3 expression while the protein expression of other MAGE genes (MAGE-A4, MAGE-C1/CT7, MAGE-C2/CT10) and non-MAGE CT antigens such as Ropporin-1 was not affected. The same was true for knock-down experiments targeting MAGE-C1/CT7 with the exception of a slight decrease in expression of the highly homologous gene MAGE-C2/CT10. Comparing myeloma cell lines treated with gene-specific versus nonsense RNA stealth, we found that knocking down MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in in about 70% of cells at 72h post transfection as indicated by MTT assays, LDH release assays and Annexin V expression analyzed by flow cytometry. No such effect was observed when transfection was performed with nonsense RNAi (p 〈 0.01). Importantly, CD138-negative myeloma precursors, which also expressed the CT antigens investigated, were also affected by downregulation of CT antigen expression as indicated by an 48% decrease in clonogenic growth (p 〈 0.01). Applying chemotherapy with melphalan to the cell cultures we observed that knock-down of MAGE-C1/CT7 or MAGE-A3 increased the chemosensitivity of MM cell lines by 67% (p 〈 0.01). Importantly, knock-down of MAGE-C1/CT7 or MAGE-A3 specifically affected cell survival and did not alter the proliferative capacity of myeloma cells or cell adhesion. In conclusion, we show here for the first time that CT antigens MAGE-C1/CT7 and MAGE-A3, which are also expressed in the majority of patient myeloma samples, are important for the survival of myeloma cell lines and clonogenic myeloma precursors. In addition, the expression of these CT antigens most likely contributes to promoting resistance to chemotherapy in multiple myeloma. Targeting CT antigens expressed by myeloma cells, either by applying CT antigen-specifric immunotherapy or other targeted therapies, might significantly improve myeloma therapy, i.e. by eliminating chemotherapy-resistant clones.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V112.11.3668.3668
    RVK:
    XA 33000
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    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
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  • 3
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    Multiple Myeloma Patients Treated with Allogeneic Stem Cell Transplantation Show an Increased Frequency of Bone Marrow-Residing CD4+Foxp3+ T Regulatory Cells.
    American Society of Hematology ; 2006
    In:  Blood Vol. 108, No. 11 ( 2006-11-16), p. 5206-5206
    Details
    In: Blood, American Society of Hematology, Vol. 108, No. 11 ( 2006-11-16), p. 5206-5206
    Abstract: BACKGROUND: Immunosuppressive CD4+Foxp3+ T regulatory cells (Treg) play a vital role in immune regulation. Thus, Treg contribute to the prevention of autoimmune disease and graft-versus-host reactions following allogeneic stem cell transplantation (alloSCT) but also to the inhibition of effective anti-tumor T cell responses. It has previously been suggested that the frequency of Treg is increased in the peripheral blood of patients with multiple myeloma (MM). However, little is known about the presence of Treg in the bone marrow and it is unclear whether allogeneic stem cell transplantation might deplete Treg from this immune compartment. METHODS: In the present study, we analyzed percentages of CD4+Foxp3+ Treg as well as Treg expression of CD45RA and CCR7 in the bone marrow (BM) and in the peripheral blood of MM patients who had received alloSCT (N=42), in newly diagnosed MM patients (N=18), and in healthy controls (N=15) using flow cytometry. In addition, we performed inhibition assays in order to test the functional relevance of peripheral and BM-residing Treg. RESULTS: While newly diagnosed MM patients and healthy controls showed no significant difference in the proportions of CD4+Foxp3+ Treg in the bone marrow, percentages of BM-residing CD4+Foxp3+ T regulatory cells were markedly higher (p 〈 0.001 and p 〈 0.01) in patients post alloSCT (3.3±0.3%) than in normal BM (1.0±0.3%) or in BM of untreated MM patients (1.8±0.4%). In both groups of patients (p 〈 0.05) as well as in the healthy controls (p 〈 0.001) percentages of Treg were higher in the peripheral blood than in the bone marrow. While there were no differences regarding the percentages of peripheral Treg between the remaining groups, patients post alloSCT had higher percentages of peripheral Treg than newly diagnosed patients (5.6±0.8 vs. 3.2±0.7%, p 〈 0.05). More than 90% of these donor-derived peripheral and BM-residing Treg expressed a memory T cell phenotype, being negative for CD45RA and CCR7. Importantly, peripheral as well as BM-residing Treg of patients post alloSCT were capable of inhibiting the proliferation of autologous non-Treg CD4+ T cells. CONCLUSION: Our study demonstrates for the first time an increased frequency of immunosuppressive Treg in the bone marrow of MM patients. Remarkably, in our patients these memory-type Treg were all donor-derived and led to an efficient replenishment of Treg in the periphery. These Treg might be necessary for the prevention of graft-versus-host disease in the transplanted MM patients, however, they might also contribute to the failure of an effective graft-versus-myeloma effect in the majority of the patients.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V108.11.5206.5206
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2006
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  • 4
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    Expression of Cancer/Testis Antigens CT10 and SSX in Multiple Myeloma: Promising Targets for Antigen-Specific Immunotherapy.
    American Society of Hematology ; 2005
    In:  Blood Vol. 106, No. 11 ( 2005-11-16), p. 3468-3468
    Details
    In: Blood, American Society of Hematology, Vol. 106, No. 11 ( 2005-11-16), p. 3468-3468
    Abstract: Background: The introduction of autologous and allogeneic stem cell transplantation has resulted in improved responses to treatment in patients with multiple myeloma (MM). However, despite high remission rates, recurrences are frequent due to remaining minimal residual disease. Myeloma-specific immunotherapies would represent a potentially useful treatment option in these patients. Cancer/testis (CT) antigens are highly attractive targets for T cell-mediated immunotherapy of cancer, due to their immunogenicity and restricted tissue expression. We investigated the expression of a larger number of CT antigens, the expression of which is characteristically restricted to cancer and normal testis, in patients with MM. Methods: We analyzed the expression of 9 different CT antigens (SSX-1 to SSX-5, CT10/MAGE-E1, BAGE, MAGE-3, NY-ESO-1) in 10 myeloma cell lines, 54 bone marrow samples from patients with stage II-III myeloma (plasma cell infiltration 10–100 %), and 18 bone marrow samples from healthy donors using rtPCR. Results: Myeloma cell lines showed a remarkably high expression of the majority of CT antigens. All antigens, with the exception of SSX-3, were expressed in 80–100% of cell lines. SSX-3 was not expressed in any cell line. Analyzing bone marrow samples from MM patients, we observed that CT10 showed a very high expression level with 55% of all malignant samples expressing this antigen. The SSX gene family evidenced high expression levels with 40% (SSX-1), 17% (SSX-2), 13% (SSX-4), and 15% (SSX-5) of patient samples expressing the given SSX family member. SSX-3 was not expressed in any samples. In agreement with previous findings we observed a high expression of MAGE-3 (61%), an intermediate expression of BAGE (18%), and a comparably low expression of NY-ESO-1 (7%). With the exception of SSX-4, which was expressed in 18% of healthy bone marrow samples, none of the CT antigens were expressed in non-malignant samples. Conclusions: We show here for the first time that CT10 and members of the SSX gene family are strongly and specifically expressed in multiple myeloma. Importantly, both CT10 and SSX have been shown to elicit spontaneous immune responses in patients with solid tumors. Therefore, CT10 and SSX-1, SSX-2, and SSX-5 might represent valuable targets for antigen-specific immunotherapy of multiple myeloma.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V106.11.3468.3468
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2005
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  • 5
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    Analysis of Spontaneous Vs. Vaccine-Induced Antibody Responses Against Cancer-Testis Antigen MAGE-A3 in Cancer Patients
    American Society of Hematology ; 2011
    In:  Blood Vol. 118, No. 21 ( 2011-11-18), p. 5087-5087
    Details
    In: Blood, American Society of Hematology, Vol. 118, No. 21 ( 2011-11-18), p. 5087-5087
    Abstract: Abstract 5087 Background: Cancer-testis antigens (CTA) are attractive targets for cancer immunotherapy based on their tumor-restricted expression and immunogenicity. A number of CTA, including Melanoma-associated antigen 3 (MAGE-A3), are already under clinical investigation and CTA have been shown to induce strong T cell and humoral immunity in cancer patients receiving active immunotherapy. However, little is known about the fine specificity and the function of vaccine-induced humoral immune responses and it is unclear how they relate to spontaneous CTA-specific immune responses occurring in a minority of patients. Methods: We have performed a longitudinal analysis of spontaneously occurring antibody responses against the CT antigen MAGE-A3 in sera (N=1537), which were collected from patients with multiple myeloma (N=355) over a period of 6 years. Antibody titers were determined by ELISA technique and a B cell ELISPOT assay was applied to estimate the number of MAGEA3-specific memory B cells in peripheral blood of the patients. Fine specificity of the antibody responses was examined using overlapping 20mer peptides spanning the whole sequence of MAGE-A3. The given IgG subtype was determined, and the quality of MAGE-A3-specific antibodies was analyzed using western blot as well as affinity assays. Results were compared to those obtained with MAGE-A3-specific antibody responses induced by vaccination with full-length MAGE-A3 protein and adjuvants AS02B or AS15 in patients with non-small cell lung cancer (NSCLC; N=15). Results: Out of 355 myeloma patients 4 (1.1%) evidenced spontaneous antibody responses against MAGE-A3 at least at one point during the course of their disease. Spontaneously occurring anti-MAGE-A3 humoral responses were usually of low titer. In contrast, all of the vaccinated patients showed high-titered and persisting antibody responses which usually appeared around week 6 after the first application of the vaccine. Accordingly, we found high frequencies of vaccine-induced MAGE-A3-specific memory B cells in the peripheral blood of NSCLC patients while they remained undetectable in most myeloma patients. Vaccine-induced antibody responses underwent affinity maturation reaching affinity levels of spontaneous immune responses after repeated cycles of treatment. MAGE-A3-specific antibodies consisted of IgG1 and IgG3 〉 IgG2 〉 IgG4 subtypes in vaccinated patients whereas spontaneously occurring antibodies were mainly of the IgG2 subtype. Spontaneous as well as vaccine-induced IgG antibodies both recognized the natural full-length protein. Analysis of the fine specificity of the antibody responses revealed that vaccine-induced antibodies recognized a much larger number of MAGE-A3 epitopes than spontaneously occurring antibodies. However, both, spontaneous as well as vaccine-induced responses, most frequently and strongly recognized a specific region within the MAGE-A3 protein corresponding to amino acids 51–70. Conclusions This study demonstrates for the first time important qualitative differences between spontaneously occurring and vaccine-induced antibody responses against the MAGE-A3 antigen in cancer patients. While the potential of both types of antibody responses to promote antigen uptake and induction of T cell responses by antigen-presenting cells might differ, they both recognized the same restricted region within the MAGE-A3 protein. The latter finding might be of importance for the design of future immunotherapies targeting MAGE-A3. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V118.21.5087.5087
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2011
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  • 6
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    A Characteristic Cytokine/Chemokine Milieu Is Present in the Bone Marrow Environment of Multiple Myeloma Patients Before and After Allogeneic Stem Cell Transplantation.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 2795-2795
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 2795-2795
    Abstract: Abstract 2795 Poster Board II-771 Background: The interaction of Multiple Myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing, survival, and proliferation of the malignant plasma cells. In this study, we aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT) and might be involved in the pathogenesis of MM and/or Graft-versus-Host Disease (GvHD). Design and Methods: Applying Multiplex Bead-based Luminex technology and Quantikine ELISAs, we determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines as well as in the plasma derived from BM and peripheral blood (PB) samples of 10 newly diagnosed MM patients, 20 MM patients post alloSCT, and 20 healthy donors. Results: Besides factors known to be secreted by myeloma cell lines (IL-1RA, IL-8, MCP-1, MIP-1α, MIP1β, MIP-3α) we observed secretion of other cytokines/chemokines such as EGF, HGF, IL2R, IL-12p40/p70, IL-22, IP-10, MIG, and RANTES. In the BM plasma samples of MM patients, we detected elevated levels of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and MIG (p 〈 0.01 or p 〈 0.05). Most of these factors were significantly increased in the BM compared to PB. In addition to increased levels of the factors mentioned above, the BM plasma of MM patients post alloSCT showed selectively elevated concentrations of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin (p 〈 0.01 or p 〈 0.05). Eotaxin levels were particularly high in patients with chronic GvHD. Correlating the BM cytokine/chemokine levels with the clinical characteristics of MM patients, we observed that BM levels of IL-16 (r=0.829, p=0.003) and HGF (r=0.805, p=0.005) correlated positively with myeloma cell infiltration within the BM. In addition, IP-10 (r=0.770, p=0.009), HGF (r=0.645, p=0.044), and IL-6 (r=0.664, p=0.036) correlated significantly with the initial stage of disease. Conclusions: Our study demonstrates that myeloma cells themselves generate a significantly higher number of cytokines/chemokines than previously described. We show that characteristic cytokine/chemokine patterns exact in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin might not only be developed into diagnostic instruments and/or predictive biomarkers, but might also represent potential targets for future myeloma- or GvHD-specific therapies. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.2795.2795
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
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  • 7
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    Longitudinal Analysis of Cancer-Testis Antigen Expression in Multiple Myeloma.
    American Society of Hematology ; 2007
    In:  Blood Vol. 110, No. 11 ( 2007-11-16), p. 4826-4826
    Details
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 4826-4826
    Abstract: BACKGROUND: Based on their immunogenicity and restricted tissue expression, cancer-testis (CT) antigens seem ideal targets for active immunotherapies. We have recently reported a frequent expression of CT antigens in multiple myeloma (Blood2007;109:1103–12). However, CT antigen expression has not been examined over time in patients with multiple myeloma (MM) or other malignancies. This seems surprising, since data on the persistence of CT antigen expression are needed in order to evaluate their usefulness as diagnostic markers and targets of immunotherapeutic approaches, especially in the case of minimal residual disease (MRD). METHODS: We analyzed 336 bone marrow (BM) samples obtained from 130 myeloma patients for expression of CT antigens. Samples of 41 healthy BM donors were used as controls. Expression of MAGEC1/CT7, MAGEC2/CT10, MAGEA3, and SSX2 was examined using qualitative RT-PCR. Real-time PCR was applied to quantify MAGEC1/CT7 expression over time. RESULTS: In MM patients with significant tumor load ( 〉 = 10% BM plasma cells), MAGEC1/CT7 was expressed in 69%, MAGEA3 in 55%, MAGEC2/CT10 in 44%, and SSX2 in 14% of samples. CT antigens were not expressed in healthy BM. Since expression of the remaining CT antigens was rarely observed without expression of MAGEC1/CT7, this CT antigen seemed to fulfill a ‘gatekeeper’ function. Expression of CT antigens correlated positively with clinical stage and was increased in recurrent disease compared to newly diagnosed MM. Noticeably, 76% of samples from patients who had not responded to therapy, 28% of samples from patients in partial remission, and only 8% of patients in complete remission expressed at least one CT antigen. Samples of patients who had received chemotherapy alone more frequently expressed CT antigens than samples of patients post autologous stem cell transplantation. The lowest frequency of CT antigen expression was observed in patients post allogeneic stem cell transplantation. Remarkably, in case a patient with significant tumor load had expressed a CT antigen once, 97% (MAGEC1/CT7), 88% (SSX2), 81% (MAGEA3), 67% (MAGEC2/CT10) of the subsequent BM samples of the same patient were positive for the respective antigen. When we analyzed 22 MM patients with at least three consecutive BM samples (median follow-up 21 months [range 4–35 months]) longitudinally and quantitatively for CT antigen expression, we observed a correlation between the BM expression of CT7/MAGEC1 mRNA and the clinical course of the disease as indicated by BM plasma cell infiltration (r=0.51, p 〈 0.01) and, even more significantly, serum paraprotein levels (r=0.73, p 〈 0.01). CONCLUSIONS: Performing the first longitudinal analysis of CT antigen expression in a human cancer, we demonstrate that in myeloma patients expression of MAGEC1/CT7, SSX2, MAGEA3, and MAGEC2/CT10 persists over time and represents an independent tumor marker. These findings suggest that downregulation of CT antigen expression is not a common tumor escape mechanism in myeloma and that CT antigens might, therefore, serve as diagnostic markers and targets for active immunotherapy, i.e. in the clinical setting of MRD.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V110.11.4826.4826
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
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  • 8
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    Cancer-Testis Antigen MAGE-C2/CT10 Promotes Proliferation and Enhances Resistance to p53 Mediated Apoptosis in Multiple Myeloma
    American Society of Hematology ; 2012
    In:  Blood Vol. 120, No. 21 ( 2012-11-16), p. 1808-1808
    Details
    In: Blood, American Society of Hematology, Vol. 120, No. 21 ( 2012-11-16), p. 1808-1808
    Abstract: Abstract 1808 Background: Cancer-testis antigens belonging to the MAGE class I family of genes are commonly expressed in Multiple Myeloma (MM). Expression of MAGE class I genes is associated with an aggressive clinical course of MM and resistance to chemotherapy, suggesting that MAGE genes may confer a survival advantage on myeloma cells. MAGE-C2/CT10 is member of the MAGE class I family of genes thought to be a good candidate for cancer immunotherapy given its very frequent expression in primary myeloma. In normal cells, MAGE-C2/CT10 seems to suppress p53 expression by promoting its polyubiquitination and degradation. However, the function of MAGE-C2/CT10 in malignancies is completely unknown. We, therefore, investigated for the first time the role of MAGE-C2/CT10 in tumor cells derived from patients with MM. Material and Methods: MAGE-C2/CT10 expression was analysed by real-time PCR and western blot in myeloma cell lines (N=8) and in PBMC from healthy donors (N=8). For the functional evaluation of MAGE-C2/CT10 we decided to use myeloma cell line U-266 which constitutively expresses MAGE-C2/CT10 and a missense mutant p53 (A161T) that has partially lost its transcriptional activity. The biological role of MAGE-C2/CT10 was investigated by stably silencing its expression using lentiviral short hairpin RNA (shRNA). The effects of silencing MAGE-C2/CT10 expression on myeloma cell biology were examined by determining the number of viable or apoptotic cells using a colorimetric MTT assay and annexin-V/7AAD staining followed by flow cytometry, In addition, we measured myeloma cell proliferation and the anchorage-independent growth using a BrdU incorporation assay and a colony formation assay, respectively. Finally, we investigated cell cycle phase distribution by flow cytometry and we analyzed the expression of key molecules involved in cell cycle progression and apoptosis using a real-time PCR array as well as western blot. Results: We found MAGE-C2/CT10 to be constitutively expressed in all myeloma cells lines but not in PBMC from healthy donors. Lentivirus-mediated silencing of MAGE-C2/CT10 inhibited significantly the proliferation and the anchorage-independent growth of myeloma cells. Cell cycle analysis demonstrated that the anti-proliferative effect of MAGE-C2/CT10 silencing in U-266 was due to a 70% decrease of cells in the S phase, a cell cycle arrest at both G0/G1 and G2/M transitions and an increase in the subG0/G1 population due to an activation of apoptotic cell death. The serine-threonine checkpoint effector kinase 2 (CHK2) and its substrate, the tumor suppressor protein p53, are essential for cell cycle control, DNA repair and apoptosis. We found that the loss of MAGE-C2/CT10 expression was associated with the activation of CHK2 through phosphorylation at Thr68 as well as the activation of p53 by phosposphorylation at Ser20. Furthermore, we observed a three-fold increase in the endogenous level of p53 protein which correlated with an up-regulation of two transcriptional targets of p53, the cyclin-dependant kinase inhibitor p21WAF1 and the growth arrest and DNA-damage-inducible alpha protein (GADD45A), known to be essential for p53-induced G1 and G2 arrest, respectively. Finally, using the Human Apoptosis Profiler PCR array that contains a number of p53 target genes, we found that apoptosis induced by MAGE-C2/CT10 knockdown was due to a more than two-fold increase in the transcription of pro-apoptotic genes like BAX (Bcl2-associated × protein), BAD (BCL2-associated agonist of cell death), Cytochrome c, APAF1 (Apoptosis activating factors) as well as several caspases, which are the down-stream mediators of p53-dependant apoptosis in response to DNA damage. Conclusions: Our collected findings support an anti-apoptotic function of MAGE-C2/CT10 in MM, likely through the regulation of key molecules involved in cell cycle progression and p53-mediated apoptosis. The central role of MAGE-C2/CT10 in the biology of myeloma strongly suggest that this cancer-testis antigen represents a promising target for myeloma-specific immunotherapies or other targeted modes of therapy for MM. Disclosures: Kröger: Fresenius Biotech: Honoraria, Research Funding.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V120.21.1808.1808
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2012
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  • 9
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    Cancer-testis antigens are commonly expressed in multiple myeloma and induce systemic immunity following allogeneic stem cell transplantation
    American Society of Hematology ; 2007
    In:  Blood Vol. 109, No. 3 ( 2007-02-01), p. 1103-1112
    Details
    In: Blood, American Society of Hematology, Vol. 109, No. 3 ( 2007-02-01), p. 1103-1112
    Abstract: Immunotherapies using cancer-testis (CT) antigens as targets represent a potentially useful treatment in patients with multiple myeloma (MM) who commonly show recurrent disease following chemotherapy. We analyzed the expression of 11 CT antigens in bone marrow samples from patients with MM (n = 55) and healthy donors (n = 32) using reverse transcriptase–polymerase chain reaction (RT-PCR). CT antigens were frequently expressed in MM with 56% (MAGEC2), 55% (MAGEA3), 35% (SSX1), 20% (SSX4, SSX5), 16% (SSX2), 15% (BAGE), 7% (NY-ESO-1), and 6% (ADAM2, LIPI) expressing the given antigen. Importantly, CT antigens were not expressed in healthy bone marrow. Analyzing patients with MM (n = 66) for antibody responses against MAGEA3, SSX2, and NY-ESO-1, we found strong antibody responses against CT antigens preferentially in patients who had received allogeneic stem cell transplantation (alloSCT). Antibody responses against NY-ESO-1 correlated with NY-ESO-1–specific CD4+ and CD8+ T-cell responses against peptide NY-ESO-151-62 and CD4+ responses against NY-ESO-1121-140 in 1 of these patients. These allogeneic immune responses were not detectable in pretransplantation samples and in the patients' stem cell donors, indicating that CT antigens might indeed represent natural targets for graft-versus-myeloma effects. Immune responses induced by alloSCT could be boosted by active CT antigen–specific immunotherapy, which might help to achieve long-lasting remissions in patients with MM.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood-2006-04-014480
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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