HS Sultan Research Articles

Inhibition of PIM Kinases Mitigates DNA Repair Responses Following Anthracycline-Induced DNA Damage and Enhances the Anti-Tumor Activity of Doxorubicin Against Lymphoma Cells

The PIM kinases are a family of proteins recently identified as promising therapeutic targets in several cancers, including pancreatic, B-cell malignancies, acute leukemia, and prostate cancer among others. The family of PIM kinases is composed of three different members (PIM-1, -2, and -3) that are short-lived serine/threonine kinases involved in the regulation of a number of cellular pathways that are important for cancer cell growth and survival. The PIM kinases show high homology with each other, and exhibit functional redundancy in vitro and in vivo. Overexpression of PIM kinases promotes tumor growth through activation of several key cell-cycle progression and anti-apoptotic proteins, including BAD, p21, p27KIP, c-Myc, and AKT-1. Recently, overexpression of PIM-2 has been shown to have a protective effect against ultraviolet light induced DNA damage (Zirkin et al. J Biol Chem288:21770-83, 2013). We investigated the protective role of PIM kinases in chemotherapy-induced DNA damage, and whether inhibition of PIM kinases enhances anthracycline-induced DNA damage by inhibiting DNA repair, thus enhancing cell death in lymphoma cells lines.Using immunobloting and RT-PCR, we found similarly low levels of PIM-1 and PIM-3, but a wide range of PIM-2 expression, in a panel of non-Hodgkin lymphoma (NHL) cell lines, including Raji, HS Sultan, Daudi, Farage, Granta519, and Toledo. Treatment of cells with doxorubicin (200-400 nM) resulted in up to a five fold increase gene transcription and expression of PIM-1 and PIM-2, which was maximal at 6 hours, and was associated with an increase in DNA damage as detected using acridine orange flow cytometry assay. We also tested the single agent effect of the pan-PIM kinase inhibitor, CX6258 on the cell lines. CX6258 alone inhibited cell growth in all NHL cell lines with varying degrees of potency with IC50ranging from 0.2 – 12.9 µM. The anti-cancer was associated most with PIM-2 expression, with the most sensitive cell lines, Daudi and Toledo, expressing the most PIM-2. Suppression of PIM-2 expression by shRNA significantly decreased proliferation, indicating that PIM-2 is a significant factor in cell growth. Treatment of NHL cells with CX6258 resulted in increased caspase-3 activation and PARP cleavage, decreased BAD phosphorylation, and apoptosis. Treatment with CX6258 also increased expression of p21, decreased expression of cyclins A1 and B1, and induced G2-M cell cycle arrest. The effect of combinations of CX6258 (5-50 µM) and doxorubicin (50-500 nM) on DNA damage and cell death was tested on HS sultan and Daudi cells. While doxorubicin alone resulted in a two-fold increase in DNA damage, this was significantly increased in the presence of CX6258 (12 fold). The addition of CX6258 inhibited the phosphorylation of the DNA repair proteins H2.AX, ATM, and Chk2 that occurred when the cells were treated with doxorubicin alone. The combination of CX6258 and doxorubicin was synergistic in inducing lymphoma cell death, with combination indexes ranging from 0.32-0.85.Our findings suggest a mechanism for synergy where doxorubicin damages cellular DNA and initiates the DNA damage response, while CX6258 inhibits the upregulated PIM kinases from activating the proteins involved in the response. This synergistic anti-tumor activity is further strengthened by the CX6258 inhibition of cell cycle progression and anti-apoptotic proteins activated by the PIM kinases. Taken together, our results provide pre-clinical rationale for clinical testing of PIM kinase inhibitors in combination with doxorubicin in patients with NHL. It also suggests that CX6258 may similarly enhance the anti-cancer effects of other DNA damaging agents. DisclosuresNo relevant conflicts of interest to declare.

Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell ApoptosisIn VivoPrimarily by Inhibiting VEGF Expression and Angiogenesis

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma line in vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cells in vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

The Internal Ribosome Entry Site-Medaited Expression of VEGF Rescues HS Sultan Tumor Cells From Mammalian Target of Rapamycin (mTOR)-Inhibitors Induced Apoptosis in Vivo

Abstract 1659An important molecular target of mTOR inhibitors in cancer therapy is VEGF expression and neo-angiogenesis. In prior studies, we demonstrated that, although rapalog mTOR inhibitors only induce G1 arrest in B-cell tumor lines, their administration in vivo in xenograft models resulted in tumor cell apoptosis that correlated with inhibition of neo-angiogenesis and VEGF expression within the tumor bed. Other in vitro studies have shown that IRES-dependent translation of myc and D-cyclins can provide a fail-safe mechanism for expression when mTOR inhibitors prevent cap-dependent translation and may act as a resistance mechanism to induction of G1 arrest in vitro. We, thus, tested if VEGF IRES activity could likewise regulate induction of anti-tumor effects and apoptosis in vivo. To test if VEGF IRES activity can regulate anti-tumor responses in vivo, we utilized the HS-Sultan B cell lymphoma line that is PTEN null. Its heightened AKT activity disarms the VEGF IRES, preventing this fail-safe mechanism and sensitizing to mTOR inhibitors. We ectopically expressed a version of the VEGF ORF in these cells fused to the p27 IRES (p27-VEGF), an IRES which is insensitive to AKT and effective in PTEN-null tumors. p27-VEGF transfected tumor cells were used as subcutaneous challenges in immunodeficient mice and results of mTOR inhibitor treatment compared to control mice challenged with tumor cells transfected with the VEGF ORF but without an AKT-resistant IRES. The anti-tumor responses were enumerated by assessing tumor size and tumor apoptosis, neo-angiogenesis and VEGF expression assessed by immunohistochemistry, ELISA, and Western blot analysis of tumor lysate. Ectopic expression of VEGF fused to the p27 IRES specifically enhanced VEGF expression and neo-angiogenesis in tumors of mice treated with the rapalog temsirolimus or active site mTOR inhibitor pp242 and significantly reduced tumor cell apoptosis and anti-tumor responses. The results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and, furthermore, underscore the importance of IRES activity as a resistance mechanism to such targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Pomalidomide: A new IMiD with remarkable activity in both multiple myeloma and myelofibrosis

Pomalidomide: A new IMiD with remarkable activity in both multiple myeloma and myelofibrosis

A Highly Efficient, Clinically Applicable Transfection Method to Redirect the Specificity of Immune Cells and Enhance Their Anti-Tumor Capacity

Effective immunotherapies rely on triggering specific cytotoxic responses against target cells. Chimeric Antigen Receptors (CAR) composed of antigen recognition domains in the form of scFv portions of monoclonal antibodies and immune cell activation domains provide non-MHC restricted, KIR-independent, immune cellmediated cytotoxicity directed toward target cells expressing specific antigens. It is well demonstrated that CAR transfection into immune cells using viral vectors redirects their specificity and enhances their cytotoxicity. Translation of these findings into clinically feasible treatment strategies is challenged by the labor/costs associated with extended cell processing, and by safety concerns associated with viral vectors. We describe here the use of non-viral methods to deliver a nucleotide sequence encoding CAR into freshly isolated immune cells obtained from peripheral blood leukapheresis collection. Protocols for loading mRNA encoding eGFP (irrelevant control) and a previously described anti-CD19 CAR (Imai et al. Blood 2005) into peripheral blood mononuclear cells (PBMC) and cell subsets (T cells, NK cells, and monocyte-depleted PBMC) were developed and optimized using a clinically validated, automated, closed-system, flow electroporation technology. PBMC were obtained from 5 normal healthy donors and 1 patient with B-cell chronic lymphocytic leukemia (B-CLL). Expression levels of eGFP or anti-CD19-BB-z were analyzed by flow cytometry. Specific cytotoxicity of target cells after 4 hours was measured by flow cytometry using acetoxymethyl-calcein labeled CD19+ target cells which included acute lymphoblastic leukemia (ALL) cell lines, primary B-CLL cells and autologous B cells. Following loading of mRNA encoding eGFP, cell viability was consistently ≥80% for all cell populations. Most cells expressed eGFP: 97±1% (day 1) and 83±2% (day 7). Following loading of mRNA encoding anti-CD19-BB-z, cell viability was 96%±1% for resting T cells (n=2), 93%±10% for PBL (n=2), 94%±8% for PBMC (n=3), 80%±15% for NK cells (n=4). Expression of anti-CD19-BB-z protein receptor was 54%±11% (day 1) in PBMC (n=6). CAR expression decreased over time and was essentially similar to baseline on day 7 post-loading. Cells expressing anti-CD19-BB-z protein (but not those expressing eGFP only), lysed 60%–80% ALL or B-CLL target cells at an effector-to-target ratio (E:T) of 8:1. A shigher E:T ratio of 20:1 was required for 60%–80% lysis of autologous B-cell targets. There was no change in cytolytic activity of CAR mRNA loaded-PBMC during the first 3 days post-loading. Subsequently, cytolytic activity dropped and on day 7 (post-loading) was approximately half of the activity measured on day 3 (post-loading).In vivo testing was performed in a sub-cutaneous tumor grafting model in Beige SCID mice. The mice (N = 5 per group) were transplanted subcutaneously with CD19+ tumor (HS Sultan) cells (1 × 106) co-mixed with PBMC loaded with anti-CD19-BB-z mRNA (doses = 0.74, 2.22, 6.67 and 20 ×106). Controls were tumor cells only or tumor cells co-mixed with unmodified PBMC. Tumors developed and grew in 3/5 animals in each control group and in 2/5 in lowest anti-CD19-BB-z mRNA-loaded dose. No tumor formation was observed in any animal in each of the 3 higher cell doses. More comprehensive animal studies and investigation of engineering additional constructs for chimeric signal receptor with tumor affinities other than anti-CD19 are in progress. These results indicate that CAR can be effectively transfected into immune cells with the methodology that we developed. Because of its simplicity and effectiveness, this methodology is well suited for clinical application and holds promise for improving the specificity and widening the feasibility of cell therapy.

CXCR4 is involved in multiple functions of cancer cells and is a compelling target for monoclonal antibody-based therapeutics

20013 Background: CXCR4 is over-expressed on > 75% of cancers and absent on most non-neoplastic cells. It is a G protein coupled receptor spanning the cell membrane with CXCL-12 as its ligand. It is uniquely involved in 3 vital aspects of cancer: 1) proliferation; 2) motility; and 3) metastasis. Our objective was to assess specific Abs as candidates for clinical development in preclinical models. Methods: Several human cancer cell lines were used to study whether either mAbs specific to the CXCR4 receptor or to CXCL12 could inhibit: 1) proliferation; 2) chemotaxis; and 3) invasion through a reconstituted basement membrane. The ability of anti-CXCR4 Ab to inhibit tumor growth in vivo was also tested. Female Nod/Scid mice were injected IP with 2 × 105 human Namalwa tumor cells with 10 mice per group. Mice were injected with: Group 1) 100μg irrelevant IgG2b isotype control Ab; Group 2) 100μg CX02 anti-CXCR4 Ab; and Group 3) 100 μg CX05 anti-CXCR4 Ab. All mice in each group received an injection of Ab at day 2, 8, 14 and 20. Mice were observed for tumor growth over the following 4–5 months. Results: We originally found that CXCR4 was over expressed in fresh surgical human Glioblastoma, then showing that it was also over expressed in multiple cancer types. Specific anti-CXCR4 monoclonal Abs, CX02 and CX05 as well as anti-CXCL12 Abs demonstrated the ability to block cell proliferation of a variety of human Glioblastoma tumor cells including GB1690, HTB16, and 5GB, but not the proliferation of cells that do not express CXCR4. CX02 and CX05 Abs bind to live tumor cells with high affinity and have an EC50 of 3.2 and 2.7 μg/ml respectively (effective concentration required to provide 50% maximal median fluorescence shift). Both Abs (10 μg/ml) also inhibited by over 90% the motility of HS Sultan cells as demonstrated by the standard chemotaxis assays. In addition, both Abs (10–40 μg/ml) also inhibited the ability of HS Sultan cells to penetrate Matrigel, a reconstituted basement membrane assay used to assess malignant potential of cancer cells. Finally, both Abs were successful in extensively prolonging survival of Nod/Scid mice injected with human Namalwa cells. Conclusions: CXCR4 represents a compelling target for mAb therapy; Phase I clinical trails are pending. [Table: see text]

The Protein Kinase C beta (PKCβ) Inhibitor Enzastaurin HCl (LY317615) Augments Chemosensitivity of Multiple Myeloma (MM) Cells to Dexamethasone and the Proteasome Inhibitor Bortezomib.

PKCβ isoforms are important in B-cell signaling and proliferation. PKCβ overexpression in diffuse large B-cell lymphoma relates with poor prognosis but the clinical significance of PKCβ in MM is not yet established. Herein, we report antimyeloma activity of the PKCβ inhibitor enzastaurin HCl (LY317615) both as a single agent and in combination with bortezomib and dexamethasone.Methods: Six MM cell lines (NCI-H929, RPMI8226, IM9, U266, ARH-77 and HS Sultan) with varying degree of PKC activity were treated with graded dose of enzastaurin and studied for induction of apoptosis by flow cytometric analysis of annexin and propidium iodide stainings, and inhibition of proliferation by MTT assay.Results: IC50 doses of both effects were low at 1-5 μM in all cell lines and did not correlate with the baseline PKC activity. Interestingly, the combination of enzastaurin at non-toxic concentrations with varying doses of dexamethasone and bortezomib yielded synergistic enhancement of cell death and inhibition of proliferation in all cell lines. Multiparameter immunoblottings of cell lysate after enzastaurin treatment showed that, at these functional doses, PI3/AKT, Erk and JNK pathways were unaffected. Additional studies are ongoing to evaluate the effect of enzastaurin on activities of apoptotic proteins, and verify its effect in primary samples.Conclusions: Our preliminary data indicate that the PKCβ inhibitor enzastaurin demonstrates single-agent antimyeloma activity and can sensitize MM cells to bortezomib and dexamethasone. Enzastaurin is promising for further studies as an approach to increase activity and decrease toxicity of MM therapy. [Display omitted]

Mechanisms of IL-21 Enhancement of Rituximab Efficacy in a Lymphoma Xenograft Model.

Interleukin 21 (IL-21) is an IL-2 family cytokine produced by activated CD4+ T cells. Potent effects of IL-21 have been observed on the growth, survival, and functional activation of T cells, B cells, and natural killer (NK) cells. A Phase I clinical trial of IL-21 in metastatic melanoma and renal cell carcinoma is currently in progress. We recently reported that IL-21 significantly enhanced rituximab mediated clearance of CD20+ lymphoma cell lines both in vitro and in vivo, and that these effects were potentially mediated through IL-21 enhancement of NK cell capacity to effect antibody dependent cellular cytotoxicity (ADCC). Specifically, NK cells treated with IL-21 showed increased cytotoxicity, granzyme B and IFNg production. Current studies aim to further evaluate the mechanisms by which IL-21 enhances ADCC. A number of observations suggest a multi-factorial basis for IL-21 synergy with rituximab. In a xenograft tumor model, SCID mice were injected IV with HS Sultan cells on day 0. Treatment with recombinant murine IL-21 (mIL-21; starting day 1) combined with rituximab (starting day 3) resulted in significantly increased survival (70% vs. 20% on day 100), compared to rituximab alone. In separate studies, the spleens of mice treated with mIL-21 showed increased numbers of activated macrophages and granulocytes. As macrophages and granulocytes can participate in ADCC, IL-21 synergy with rituximab in vivo may be partly dependent on its activation of these cell types. We have also evaluated whether direct effects of IL-21 on lymphoma cells contribute to enhancement of rituximab efficacy. The xenogeneic B lymphoma models in which IL-21 plus rituximab exhibited enhanced survival are highly aggressive and these models were not shown to respond to treatment with mIL-21 alone. In vitro studies were performed to determine if IL-21 could potentiate the growth inhibitory and pro-apoptotic effects of rituximab. In the absence of effector cells synergistic interaction was not observed. In addition, we tested the ability of IL-21 to enhance cytotoxicity when combined with antibodies targeting non-hematopoietic tumor cells (e.g. trastuzumab). Human NK cells treated with IL-21 displayed significantly increased cytotoxicity in ADCC assays using trastuzumab to target breast cancer cells expressing varying levels of HER-2 antigen. In summary, the current evidence suggests that IL-21 can enhance antibody-mediated tumor cell lysis through activation of multiple effectors of ADCC. Thus IL-21 may prove to be broadly applicable to monoclonal antibody therapy of cancer.

IL-21 enhances rituximab-mediated killing of B-lymphoma cell lines in vitro and in vivo

2581 Background: IL-21 is an IL-2 family cytokine expressed by activated CD4+ T cells. It has potent immunomodulatory activities on a number of cell types and has shown significant anti-tumor activity in preclinical animal studies. Phase I studies in metastatic melanoma and renal cell carcinoma are intended to commence in the first half of 2004. For NK cells, IL-21 promotes activated characteristics, including enhanced cytolytic activity, IFN-γ production, and expression of cell surface CD16. To examine the possible consequences of IL-21 stimulation on antibody mediated NK effector functions, we carried out in vitro and in vivo studies using rituximab/CD20+ lymphoma as a model system. Methods: NK cells were prepared by negative selection from peripheral blood mononuclear cells of healthy donors, cultured for 3 days ± IL-21, and cytolytic activity was measured against human B-lymphoma cell targets in the presence or absence of rituximab. Additionally, IFN-γ production by NK cells cultured with IL-21 and plate bound IgG was assayed by ELISA. To assess the combined effects of IL-21 and rituximab in vivo, SCID mice (n=10) were injected IV with 106 HS Sultan cells and treated with vehicle, IL-21 (5 mg/kg QD, days 1–5), rituximab (1 mg/kg Q 4th day x 5, starting day 3) or a combination of IL-21 and rituximab. Results: IL-21 enhanced IFN-γ production by NK cells costimulated with plate bound IgG in a dose dependent manner. Compared to control NK cultures, pre-exposure to IL-21 significantly increased lytic activity of NK cells on human B-lymphoma cell targets in the presence of rituximab. Similarly, treatment of HS Sultan bearing mice with a combination of IL-21 and rituximab resulted in significantly increased survival compared to treatment with rituximab alone (70% versus 10% at day 90; p<.004, logrank). Conclusions: In both in vitro and in vivo studies, IL-21 synergistically enhanced rituximab mediated cytotoxicity. These data suggest that antibody mediated NK cell effector functions are augmented by IL-21, and that IL-21 may be clinically useful in the context of antibody based cancer therapy. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration ZymoGenetics ZymoGenetics

Tumour cell/dendritic cell fusions as a vaccination strategy for multiple myeloma.

Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy. The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy. We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays. First, the HS Sultan and SK0-007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs. Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs. The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy. These FCs induced MM-specific cytotoxicity. FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells. More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis. These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway.

Immunomodulatory analogs of thalidomide inhibit growth of Hs Sultan cells and angiogenesis in vivo.

We have previously shown that thalidomide and its potent immunomodulatory derivatives (IMiDs) inhibit the in vitro growth of multiple myeloma (MM) cell lines and patient MM cells that are resistant to conventional therapy. In this study, we further characterize the effect of these drugs on growth of B cell malignancies and angiogenesis. We established a beige-nude-xid (BNX) mouse model to allow for simultaneous in vivo measurement of both anti-tumor and anti-angiogenic effects of thalidomide and its analogs. Daily treatment (50 mg/kg/d) with thalidomide or IMiDs was nontoxic. The IMiDs were significantly more potent than thalidomide in vivo in suppressing tumor growth, evidenced by decreased tumor volume and prolonged survival, as well as mediating anti-angiogenic effects, as determined by decreased microvessel density. Our results therefore show that the IMiDs have more potent direct anti-tumor and anti-angiogenic effects than thalidomide in vivo, providing the framework for clinical protocols evaluating these agents in MM and other B cell neoplasms.

Potent Induction of Apoptosis by β-Lapachone in Human Multiple Myeloma Cell Lines and Patient Cells

Human multiple myeloma (MM) remains an incurable hematological malignancy. We have reported that beta-lapachone, a pure compound derived from a plant, can induce cell death in a variety of human carcinoma cells, including ovary, colon, lung, prostate, pancreas, and breast, suggesting a wide spectrum of anticancer activity. We first studied antisurvival effects of beta-lapachone in human MM cells by colony formation assay. To determine whether the differential inhibition of colony formation occurs through antiproliferative activity, we performed MTT assays. The cytotoxicity of beta-lapachone on human peripheral blood mononuclear cells was also measured by MTT assay. To determine whether the cell death induced by beta-lapachone occurs through necrosis or apoptosis, we used the propidium iodide staining procedure to determine the sub-GI fraction, Annexin-V staining for externalization of phosphatidylserine, and fragmentation of cellular genomic DNA subjected to gel electrophoresis. To investigate the mechanism of anti-MM activity, we examined Bcl-2 expression, cytochrome C release, and poly (ADP ribose) polymerase cleavage by Western blot assay. We found that beta-lapachone (less than 4 microM) inhibits cell survival and proliferation by triggering cell death with characteristics of apoptosis in ARH-77, HS Sultan, and MM.1S cell lines, in freshly derived patient MM cells (MM.As), MM cell lines resistant to dexamethasone (MM.1R), doxorubicin (DOX.40), mitoxantrone (MR.20), and mephalan (LR5). Importantly, after treatment with beta-lapachone, we observed no apoptosis in peripheral blood mononuclear cells in either quiescent or proliferative states, freshly isolated from healthy donors. In beta-lapachone treated ARH-77, cytochrome C was released from mitochondria to cytosol, and poly (ADP ribose) polymerase was cleaved, signature events of apoptosis. Finally, the apoptosis induced by beta-lapachone in MM cells was not blocked by either interleukin-6 or Bcl-2, which confer multidrug resistance in MM. Our results suggest potential therapeutic application of beta-lapachone against MM, particularly to overcome drug resistance in relapsed patients.

Regulation of germline promoters by the two human Ig heavy chain 3' alpha enhancers.

The human IgH 3' enhancers, located downstream of each of the two Calpha genes, modulate germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The regulation of GL alpha1 and alpha2 promoters by different human 3' enhancer fragments was investigated in cell lines representing various developmental stages. Both alpha1HS1,2 and alpha2HS1,2 fragments show equally strong enhancer activity on the GL alpha1 and alpha2 promoters in both orientations when transiently transfected into a number of mature B cell line (DG75, CL-01, and HS Sultan). However, there is no activity in a human pre-B cell line (NALM-6) nor a human T cell line (Jurkat). HS3 shows no enhancer activity by itself in any of the cell lines, whereas a modest effect is noted using HS4 in the three mature B cell lines. However, the combination of the alpha2HS3-HS1,2-HS4 fragments, which together form a potential locus control region, displays a markedly stronger enhancer activity than the individual fragments with a differential effect on the alpha1 and alpha2 promoters as compared with the gamma3 promoter. Our results suggest that the human GL alpha promoter may be regulated by two independent pathways. One pathway is induced by TGF-beta1 which directs IgA isotype switch through activation of the GL alpha promoter and no TGF-beta1-responsive elements are present in the different 3' enhancer fragments. The other route is through the human 3' enhancer regions that cis-up-regulate the GL alpha promoter activity in mature B cells.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30 degrees C) but not at restrictive (37 degrees C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

2′,2′‐Difluorodeoxycytidine (Gemcitabine) Induces Apoptosis in Myeloma Cell Lines Resistant to Steroids and 2‐Chlorodeoxyadenosine (2‐CdA)

The paucity of effective cytotoxic agents for the treatment of steroid resistant multiple myeloma explains the ongoing search for alternative substances for chemotherapy of this disease. In the present study, the purine antagonist 2-chlorodeoxyadenosine (2-CdA, cladribine) and the pyrimidine antagonist 2',2'-difluorodeoxycytidine (gemcitabine) were tested on four myeloma cell lines (i.e., U 266, OPM 2, RPMI 8226, IM 9), one plasma cell leukemia cell line (HS Sultan) and a myeloid control cell line (HL 60), all of which are resistant to 10-6 M dexamethasone. Gemcitabine has been found to be promising in the chemotherapy of other tumors with low proliferative activity, but its effectiveness against myeloma cells has not been analyzed so far. In our tests, gemcitabine induced a significant degree of apoptosis in all cell lines investigated. After incubation for 48 h with 10 microM gemcitabine, the median numbers of apoptotic cells were in the range of 45% in the OPM 2 and 79% in the U 266 cell line. All of the investigated cell lines were responsive to concentrations of 10 microM gemcitabine even after an exposure of only 30 min, three of them (U 266, HS Sultan, IM 9) also responded to a concentration of 10 nM. Higher concentrations and longer exposure times were necessary to suppress the growth of normal hematopoietic bone marrow progenitor cells. In contrast to gemcitabine, standard concentrations of 2-CdA (i.e., 30 and 300 nM) failed to induce a significant degree of apoptosis in the cell lines investigated but inhibited the growth of myeloid progenitor cells. The results suggest that gemcitabine induces apoptosis in myeloma and plasma cell leukemia lines resistant to steroids and 2-CdA. The fact that tumor cell apoptosis was achieved at concentrations clinically achievable and tolerable, which at the same time do not inhibit the growth of normal CFU-GM progenitor cells, favors the initiation of phase I trials with this drug for the treatment of multiple myeloma.

Human Umbilical Vein Endothelial Cells Secrete Transcobalamin II

Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.

Human umbilical vein endothelial cells secrete transcobalamin II

Transcobalamin II (TC II) is essential for cellular uptake of cobalamin. However, the origin of this transport protein is controversial and many organ sources have been suggested. We studied human umbilical vein endothelial cells cultured in vitro. The cells contained TC II (2.3 pmol/10(8) cells) and released progressively increasing amounts of the protein into the surrounding medium during the 3-day incubation period. This release exceeded the starting intracellular content of TC II. In contrast, endothelial cells did not contain or elaborate R binder, the other major circulating binding protein for cobalamin, Cycloheximide inhibited the elaboration of TC II, suggesting that the endothelial cells synthesize the protein. Thrombin, which stimulates tissue plasminogen activator release, did not enhance TC II release, and neither did endotoxin or mellitin. However, thrombin did appear to partially protect TC II release from inhibition by cycloheximide. Among other cells studied, human fibroblasts also released TC II into the incubation medium, while K562 human leukemia cells, ARH-77 and HS Sultan human plasma cell lines, and Raji strain lymphoblasts did not. The data suggest that endothelial cells are an important source of the metabolically crucial TC II.