Agonistic anti-Fas Antibody Jo2 Research Articles

CD74 interferes with the expression of fas receptor on the surface of lymphoma cells.

BackgroundResistance to Fas-mediated apoptosis limits the efficacy of currently available chemotherapy regimens. We identified CD74, which is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.MethodsCD74 expression was suppressed in human B-lymphoma cell lines, BJAB and Raji, by either transduction with lentivirus particles or transfection with episomal vector, both encoding CD74-specific shRNAs or non-target shRNA. Effect of CD74 expression on Fas signaling was evaluated by comparing survival of mice hydrodynamically transfected with vector encoding full-length CD74 or empty vector. Sensitivity of cells with suppressed CD74 expression to FasL, edelfosine, doxorubicin, and a humanized CD74-specific antibody, milatuzumab, was evaluated by flow cytometry and compared to control cells. Fas signaling in response to FasL stimulation and the expression of Fas signaling components were evaluated by Western blot. Surface expression of Fas was detected by flow cytometry.ResultsWe determined that cells with suppressed CD74 are more sensitive to FasL-induced apoptosis and Fas signaling-dependent chemotherapies, edelfosine and doxorubicin, than control CD74-expressing cells. On the other hand, expression of full-length CD74 in livers protected the mice from a lethal challenge with agonistic anti-Fas antibody Jo2. A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74, suggesting that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. Cells with suppressed CD74 expression showed increased staining of Fas receptor on their surface. Pre-treatment with milatuzumab sensitized BJAB cells to Fas-mediated apoptosis.ConclusionWe anticipate that specific targeting of the CD74 on the cell surface will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis, and thus will increase effectiveness of chemotherapy regimens for hematological malignancies.

CD74 interferes with the expression of fas receptor on the surface of lymphoma cells

BackgroundResistance to Fas-mediated apoptosis limits the efficacy of currently available chemotherapy regimens. We identified CD74, which is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.MethodsCD74 expression was suppressed in human B-lymphoma cell lines, BJAB and Raji, by either transduction with lentivirus particles or transfection with episomal vector, both encoding CD74-specific shRNAs or non-target shRNA. Effect of CD74 expression on Fas signaling was evaluated by comparing survival of mice hydrodynamically transfected with vector encoding full-length CD74 or empty vector. Sensitivity of cells with suppressed CD74 expression to FasL, edelfosine, doxorubicin, and a humanized CD74-specific antibody, milatuzumab, was evaluated by flow cytometry and compared to control cells. Fas signaling in response to FasL stimulation and the expression of Fas signaling components were evaluated by Western blot. Surface expression of Fas was detected by flow cytometry.ResultsWe determined that cells with suppressed CD74 are more sensitive to FasL-induced apoptosis and Fas signaling-dependent chemotherapies, edelfosine and doxorubicin, than control CD74-expressing cells. On the other hand, expression of full-length CD74 in livers protected the mice from a lethal challenge with agonistic anti-Fas antibody Jo2. A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74, suggesting that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. Cells with suppressed CD74 expression showed increased staining of Fas receptor on their surface. Pre-treatment with milatuzumab sensitized BJAB cells to Fas-mediated apoptosis.ConclusionWe anticipate that specific targeting of the CD74 on the cell surface will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis, and thus will increase effectiveness of chemotherapy regimens for hematological malignancies.

CD74 Interferes With The Expression Of Fas Receptor On The Cell Surface

Resistance to Fas-mediated apoptosis manifested by hematopoietic cancers interferes with the efficacy of currently available chemotherapy regimens. We identified CD74, that is known to be overexpressed in hematological malignancies, as one of the factors interfering with Fas-mediated apoptosis.Through stable suppression of CD74 expression in BJAB and Raji cells using shRNA technology, we have determined that cells overexpressing CD74 are more resistant to FasL-induced apoptosis than control non-target shRNA expressing cells (super FasL 10 ng/mL; 36 ± 3.3% vs.18 ± 0.1% and 35 ± 0.1% vs. 20 ± 1.5%; P< 0.01). Transfection of mice with plasmid encoding full-length CD74 protected mice from a lethal challenge with agonistic anti-Fas antibody Jo2. All 5 of vector-transfected mice died within 6 hours from challenge, while 5 out of 6 CD74-transfected mice survived the challenge (P< 0.05). Suppression of CD74 expression also sensitized BJAB cells to Fas-signaling dependent chemotherapies. Doxorubicin (125nM) killed 27 ± 4% of control and 48 ± 5% of CD74 knock-down cells (P< 0.001). Edelfosine (10 uM) induced apoptosis in 36 ± 7% of control and 64 ± 1% of CD74 knock-down cells (P< 0.001).A detailed analysis of Fas signaling in cells lacking CD74 and control cells revealed increased cleavage/activation of pro-caspase-8 and corresponding enhancement of caspase-3 activation in the absence of CD74. This result suggested that CD74 affects the immediate early steps in Fas signaling at the plasma membrane. We next analyzed the levels of Fas receptor on the surface of cells by flow cytometry. Cells with suppressed CD74 expression had 30% higher staining of Fas receptor on their surface; the mean fluorescence intensity increased from 4017 ± 704 to 5122 ± 216 (P< 0.003).We anticipate that specific targeting of the CD74 on the cell surface with milatuzumab will sensitize CD74-expressing cancer cells to Fas-mediated apoptosis and thus will increase effectiveness of chemotherapy regimens for hematological malignancies. Disclosures:No relevant conflicts of interest to declare.

Direct Suppression of Fas-Mediated Apoptosis by PMLRARa through Forming An Apoptotic Inhibitory Complex with c-FLIP In Acute Promyelocytic Leukemia

AbstractAbstract 3148Fas plays a critical role in cell proliferation and in the selective killing of autoreactive lymphocytes and abnormal cells, including infected cells. To explain the common expression of Fas and the resistance to the Fas-induced apoptosis observed in some normal and cancer cells, we screened cells for potential regulators of the Fas death receptor. By using mass spectroscopy analysis of Fas-associated proteins, we identified peptides derived from promyelocytic leukemia (PML).PML enhances pro-apoptotic signaling, while its dominant negative form, promyelocytic leukemia–retinoic acid receptor α (PMLRARα) fusion protein, activates pro-survival pathways. Given these opposing functions, we tested whether PMLRARα blocks Fas-mediated apoptosis.Co-immunoprecipitation analysis demonstrated that PMLRARα interacts with Fas in acute promyelocytic leukemia (APL)-derived NB4 cells, U937/PR9 cells and APL primary cells isolated from patients. The PMLRARα-Fas binding was mapped to the PML B-box domain of PMLRARα and death domain of Fas. Flow cytometry analysis of propidium iodide- and Annexin V-stained cells challenged with Fas ligand (FasL) or agonistic anti-Fas antibody CH-11 indicated that PMLRARα blocks Fas-mediated apoptosis at early and late stages. In line with this finding, knockdown of PMLRARα with shRNA sensitized the NB4 cells to Fas-mediated apoptosis. Detailed analysis showed that expression of PMLRARα prevents procaspase-8 from binding to the Fas complex upon stimulation with the agonistic anti-Fas antibody (CH-11) and thus, also prevents cleavage/activation of procaspase-8. Further analysis indicated that PMLRARα recruits caspase-8 inhibitor c-FLIPL/S to Fas to suppress Fas signaling. A significantly higher number of mice transfected with PMLRARα-expressing plasmid than mice transfected with empty vector survived the treatment with the mouse agonistic anti-Fas antibody Jo2 (11 of 12 vs. 0 of 12; P < 0.001). Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with vector-transfected mice.These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. Our results may provide an explanation for the long-known role of PMLRARα and PML in the regulation of Fas signaling, which, as we have shown, can occur by regulation via direct interaction. The newly-discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptossis. By neutralizing the effect of death receptor inhibitors such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

Peptides Compete in Human Herpesvirus 8 K1-Fas Complexes Associated with Hyperplasia and Restore Apoptosis.

Abstract 2965Poster Board II-941The long-term expression of human herpesvirus 8 (HHV-8) K1 produces hyperplasia of lymph nodes, splenomegaly, and lymphomas in mice. The mechanism of how K1 causes hyperplasia and lymphomas. The immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to activate of Akt and nuclear factor kappa B (NF-kB). However, we have recently shown that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survive a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by altered Fas signaling.Examination of mice expressing K1 via a ubiquitous promoter showed that K1 transgenic mice (n=10) had 90% developed lymphoid hyperplasia (at least 3 lymph nodes >3 mm) and 60% developed lymphomas after 18 months, while all (26) control nontransgenic mice remained free of lymph node hyperplasia, splenomegaly, and lymphoma. Some K1 mice developed liver or mesenteric tumors (4 of 10 mice). The spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-K1 transgenic control mice. Hematoxylin and eosin staining of spleen sections showed lymphocyte expansion in the periarteriolar lymphocyte sheath with disruption of normal spleen architecture. Anti-kappa and anti-lambda light chain antibodies revealed the presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but no foci were present in 4 control non-transgenic mice. Moreover, K1 protein was expressed in approximately 10% of splenic cells after staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein confers resistant to Fas-mediated apoptosis, splenic cells of 6-month-old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes showed morphology of apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2-induced apoptosis compared with splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 out of 12 K1 transgenic (30%) and 13 out of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.We mapped the region that K1 uses to bind to Fas to an immunoglobulin (Ig) chain-like domain by expressing deletion mutants of K1. Overexpression of an Ig domain-containing protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. K1 splenocytes incubated with N251 and N253 showed enhanced rates of Fas-mediated apoptosis over control peptide-treated K1, and peptides did not enhance the apoptosis rates of control non-K1 expressing splenocytes.This analysis indicates a key role of K1 in lymphoid hyperplasia and lymphoma and that K1 direct binding to Fas occurs in vivo, which is effectively targeted by competing peptides to restore apoptosis. Disclosures:No relevant conflicts of interest to declare.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi s sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF -B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer s patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 200 mg vs. 94 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with antikappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 1% of splenocytes from K1 mice versus 17 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 0.6% vs. 50 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Human Herpesvirus 8 Protein K1 Interferes with Fas-Mediated Apoptosis Induces Clonal Growth and Lymphoid Hyperplasia

Background: Expression of Human herpesvirus 8 (HHV-8) K1 causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11 ± 0.6% vs. 50 ± 6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis. We mapped the region that K1 uses to bind to Fas as an immunoglobulin (Ig) chainlike domain by expressing deletion mutants of K1. Overexpression of an Ig domaincontaining protein CD79b competed with K1-Fas binding in a dose-dependent manner. Two 20-amino acid peptides (N251, N253) representing the Ig domain of K1 competed with K1-Fas binding in immunoprecipitation/immunoblotting analysis. The N251 and N253 peptides (100 mM) enhanced anti-Fas antibody (CH-11, 50 ng/mL)-induced apoptosis of BJAB lymphoma cells that expressed K1 but not that of vector-transfected BJAB cells. Ig-deleted K1 (K1dIg)-transfected mice were not protected (0/6), and K1- transfected mice were protected (7/10, P < 0.01) against the lethal effects of agonistic anti-Fas (Jo2) antibody. K1dIg expressed in mice did not form complexes with Fas, suggesting that the Ig domain is essential for K1-Fas binding and suppression of apoptosis.Conclusion: These results confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation. K1 blocks Fas-mediated apoptosis and competing peptides can reinstate apoptosis.

Human Herpesvirus 8 Protein K1 Interference with Fas-Mediated Apoptosis Induces Clonal Growth Which Leads to Lymphoid Hyperplasia.

Background: Human herpesvirus 8 (HHV-8) infection is causally associated with the development of primary effusion lymphoma and Kaposi's sarcoma. A transmembrane protein of HHV-8, K1, is readily expressed in those tumors and the expression of K1 alone causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NFκ-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling.Results: Gross examination of thoracic and abdominal cavities of transgenic mice with K1 expression driven by a ubiquitous promoter were sacrificed at 18 months of age revealed enlarged cervical, mediastinal, renal, mesenteric lymph nodes and spleens. Peyer's patches were also enlarged and readily visible on the outer surface of the ileum. Of 10 K1 mice, 90% developed lymphoid hyperplasia (>3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P < 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4±1% of splenocytes from K1 mice versus 17±2% of control splenocytes were undergoing apoptosis (P<0.01). At 24 hours of treatment, the difference was even more significant (11±0.6% vs. 50±6%, P<0.005). Splenocytes of K1 mice were indeed more resistant to Jo2 - induced apoptosis than splenocytes from age-matched control mice. Of mice inoculated with a lethal dose of Jo2 antibody, 3 of 12 K1 transgenic (30%) and 13 of 22 control mice (60%) died (P<0.05), further confirming the protective effect of K1 against Fas-mediated apoptosis.Conclusion: These results over all confirm that K1 is associated with lymphoid hyperplasia and lymphoma and provide plausible explanation of this phenomenon. Interference of K1 with Fas-mediated apoptosis disrupts the normal life cycle of lymphocytes.

Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis

The etiology of progression from steatosis to steatohepatitis (SH) remains unknown. Using nutritional and genetic models of hepatic steatosis, we show that free cholesterol (FC) loading, but not free fatty acids or triglycerides, sensitizes to TNF- and Fas-induced SH. FC distribution in endoplasmic reticulum (ER) and plasma membrane did not cause ER stress or alter TNF signaling. Rather, mitochondrial FC loading accounted for the hepatocellular sensitivity to TNF due to mitochondrial glutathione (mGSH) depletion. Selective mGSH depletion in primary hepatocytes recapitulated the susceptibility to TNF and Fas seen in FC-loaded hepatocytes; its repletion rescued FC-loaded livers from TNF-mediated SH. Moreover, hepatocytes from mice lacking NPC1, a late endosomal cholesterol trafficking protein, or from obese ob/ob mice, exhibited mitochondrial FC accumulation, mGSH depletion, and susceptibility to TNF. Thus, we propose a critical role for mitochondrial FC loading in precipitating SH, by sensitizing hepatocytes to TNF and Fas through mGSH depletion.