Search

The loss of skeletal muscle mass during aging is a significant health concern linked to adverse outcomes in older individuals. Understanding the molecular basis of age-related muscle loss is crucial for developing strategies to combat this debilitating condition. Long noncoding RNAs (lncRNAs) are a largely uncharacterized class of biomolecules that have been implicated in cellular homeostasis and dysfunction across a many tissues and cell types. To identify lncRNAs that might contribute to skeletal muscle aging, we screened for lncRNAs whose expression was altered in vastus lateralis muscle from older compared to young adults. We identified FRAIL1 as an aging-induced lncRNA with high abundance in human skeletal muscle. In healthy young and older adults, skeletal muscle FRAIL1 was increased with age in conjunction with lower muscle function. Forced expression of FRAIL1 in mouse tibialis anterior muscle elicits a dose-dependent reduction in skeletal muscle fiber size that is independent of changes in muscle fiber type. Furthermore, this reduction in muscle size is dependent on an intact region of FRAIL1 that is highly conserved across non-human primates. Unbiased transcriptional and proteomic profiling of the effects of FRAIL1 expression in mouse skeletal muscle revealed widespread changes in mRNA and protein abundance that recapitulate age-related changes in pathways and processes that are known to be altered in aging skeletal muscle. Taken together, these findings shed light on the intricate molecular mechanisms underlying skeletal muscle aging and implicate FRAIL1 in age-related skeletal muscle phenotypes., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Age is a major risk factor for abdominal aortic aneurysm (AAA), for which treatment options are limited to surgical intervention for large AAA and watchful waiting for small aneurysms. However, the factors that regulate the expansion of aneurysms are unclear. Development of new therapeutic strategies to prevent or treat small aneurysms awaits a more thorough understanding of the etiology of AAA formation and progression with aging. A variety of structural and functional changes have been reported in aging vasculature, but emerging evidence implicates senescent cells in the formation of AAA through their paracrine effects on vascular wall cell populations. Here we show that aging is associated with transcriptional changes in abdominal aortic tissue consistent with loss of smooth muscle cells, leukocyte adhesion, inflammation, and accumulation of senescent cells in the vascular wall and surrounding perivascular adipose tissue. Furthermore, aged mice demonstrated anatomical and histopathological features of AAA development in response to administration of angiotensin II over 28 days. Importantly, in our study we sought to determine if reducing senescent cells could lessen the severity of AAA in aged mice. We find that pretreatment of aged mice with oral senolytic agents (dasatinib + quercetin) reduced senescent cell abundance in the arterial walls and surrounding tissues and lessened the severity of AAA in response to angiotensin II administration. These data provide important preliminary evidence supporting a role of senescent cells in age-related AAA formation and progression and suggest that strategies to reduce senescent cell burden hold promise to lessen AAA severity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Measurable residual disease (MRD) assessment by marrow-based next-generation flow cytometry (NGF) following autologous stem cell transplantation (ASCT) may lead to false-negative results due to patchy marrow involvement and extramedullary disease in patients with multiple myeloma. We assessed the value of simultaneous MRD evaluation with NGF and serum matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MASS-FIX). Of all 61 complete responders who were NGF-negative for MRD, around day-100 post ASCT, 59% were MASS-FIX-positive. At median follow-up of 26 months, 69% of MASS-FIX(+)/NGF(-) patients were alive and progression-free versus 96% of MASS-FIX(-)/NGF(-) patients, P = 0·02. MASS-FIX, a simple peripheral blood-based assay complements marrow-based NGF to accurately prognosticate patients with myeloma., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Senescent cells accumulate in numerous tissues in several chronic conditions such as aging, obesity, and diabetes. These cells are in a state of irreversible cell-cycle arrest and secrete inflammatory cytokines, chemokines and other immune modulators that have paracrine effects on nearby tissues. Adipose tissue, in particular, harbors senescent cells, which have been linked with numerous chronic conditions and age-related comorbidities. Here we performed a series of in vitro experiments to determine the influence of senescent preadipocytes on key cell types found in vessel walls, including vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages (MQs), and adipose-derived stromal/stem cells (ASCs). Primary human preadipocytes were irradiated to trigger a senescence-like phenotype. VSMCs, ECs, MQs, and ASCs were exposed to conditioned media collected from irradiated preadipocytes or control preadipocytes. Additional experiments were performed where VSMCs, ECs, MQs, and ASCs were co-cultured with irradiated or control preadipocytes. The secretome of irradiated cells induced an inflammatory phenotype, decreased cell viability, disrupted proliferation and migration, and impaired metabolic function of these cell types in vitro. These maladaptive changes in response to senescent cell exposure provide early evidence in support of a hypothesis that senescent preadipocytes trigger phenotypic and functional changes in key cellular components of blood vessels that may contribute to vascular disease., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The genetic abnormalities underlying multiple myeloma (MM) are notoriously complex and intraclonal heterogeneity is a common disease feature. In the current study, we describe the establishment of a monoclonal immunoglobulin A (IgA) kappa (κ) MM cell line designated MC-B11/14. Cytogenetic and fluorescence in situ hybridization analyses of the original and relapse patient samples revealed that the MM clone was nonhyperdiploid and possessed an 11;14 chromosomal translocation. The MC-B11/14 cell line, established from the relapse sample, is tetraploid and houses the t(11;14) abnormality. Given our long-standing interest in Ig function and secretion, we next used CRISPR technology to knock out IgA heavy-chain expression in the MC-B11/14 cells to assess the biological consequences of converting this cell line to one only expressing κ light chains. As expected, secretion of intact IgA was undetectable from MC-B11/14 IgA- cells. Sensitivity to pomalidomide treatment was similar between the MC-B11/14 WT and MC-B11/14 IgA- cells; however, MC-B11/14 IgA- cells were found to be significantly more resistant to bortezomib treatment. This study describes the establishment of a new human MM cell line tool with which to study disease biology and the use of CRISPR technology to create a potentially useful model with which to study MM light-chain escape., (Copyright © 2018 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine 2017;6:1829-1839., (© 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Multiple myeloma (MM) is characterized by the clonal expansion of malignant plasma cells within the bone marrow. There is a growing literature that tumor cells release biologically active microvesicles (MVs) that modify both local and distant microenvironments. In this study, our goals were to determine if MM cells release MVs, and if so, begin to characterize their biologic activity. Herein we present clear evidence that not only do both patient MM cells and human MM cell lines (HMCLs) release MVs, but that these MVs stimulate MM cell growth. Of interest, MM-derived MVs were enriched with the biologically active form of CD147, a transmembrane molecule previously shown by us to be crucial for MM cell proliferation. Using MVs isolated from HMCLs stably transfected with a CD147-GFP fusion construct (CD147GFP), we observed binding and internalization of MV-derived CD147 with HMCLs. Cells with greater CD147GFP internalization proliferated at a higher rate than did cells with less CD147GFP association. Lastly, MVs obtained from CD147 downregulated HMCLs were attenuated in their ability to stimulate HMCL proliferation. In summary, this study demonstrates the significance of MV shedding and MV-mediated intercellular communication on malignant plasma cell proliferation, and identifies the role of MV-enriched CD147 in this process.

Increased use of the glycolytic pathway, even in the presence of oxygen, has recently been recognized as a key characteristic of malignant cells. However, the glycolytic phenotype results in increased lactic acid production and, in order to prevent cellular acidosis, tumor cells must increase proton efflux via upregulation of pH regulators such as proton-pumps, sodium-proton exchangers, and/or monocarboxylate transporters (MCT) (e.g., MCT1, MCT4). Interestingly, expression of MCT1 and MCT4 has been previously shown to be dependent upon expression of the transmembrane glycoprotein CD147. Recently, we demonstrated that primary patient multiple myeloma (MM) cells and human MM cell lines (HMCLs) overexpress CD147. Therefore, the goal of the current study was to specifically determine if MCT1 and MCT4 were also overexpressed in MM cells. RT-PCR analysis demonstrated both primary patient MM cells and HMCLs overexpress MCT1 and MCT4 mRNA. Notably, primary MM cells or HMCLs were found to express variable levels of MCT1 and/or MCT4 at the protein level despite CD147 expression. In those HMCLs positive for MCT1 and/or MCT4 protein expression, MCT1 and/or MCT4 were found to be associated with CD147. Specific siRNA-mediated downregulation of MCT1 but not MCT4 resulted in decreased HMCL proliferation, decreased lactate export, and increased cellular media pH. However, western blot analysis revealed that downregulation of MCT1 also downregulated CD147 and vice versa despite no effect on mRNA levels. Taken together, these data demonstrate the association between MCT1 and CD147 proteins in MM cells and importance of their association for lactate export and proliferation in MM cells.

The biology of the malignant plasma cells (PCs) in multiple myeloma (MM) is highly influenced by the bone marrow (BM) microenvironment in which they reside. More specifically, BM stromal cells (SCs) are known to interact with MM cells to promote MM cell survival and proliferation. By contrast, it is unclear if innate immune cells within this same space also actively participate in the pathology of MM. Our study shows for the first time that eosinophils (Eos) can contribute to the biology of MM by enhancing the proliferation of some malignant PCs. We first demonstrate that PCs and Eos can be found in close proximity in the BM. In culture, Eos were found to augment MM cell proliferation that is predominantly mediated through a soluble factor(s). Fractionation of cell-free supernatants and neutralization studies demonstrated that this activity is independent of Eos-derived microparticles and a proliferation-inducing ligand (APRIL), respectively. Using a multicellular in vitro system designed to resemble the native MM niche, SCs and Eos were shown to have non-redundant roles in their support of MM cell growth. Whereas SCs induce MM cell proliferation predominantly through the secretion of IL-6, Eos stimulate growth of these malignant cells via an IL-6-independent mechanism. Taken together, our study demonstrates for the first time a role for Eos in the pathology of MM and suggests that therapeutic strategies targeting these cells may be beneficial.

Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.

DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.

Lymphocyte enhancer binding factor 1 (LEF-1) plays a crucial role in B lineage development and is only expressed in B cell precursors as B cell differentiation into mature B and plasma cells silences its expression. Chronic lymphocytic leukemia (CLL) cells aberrantly express LEF-1 and its expression is required for cellular survival. We hypothesized that modification of the differentiation status of CLL cells would result in loss of LEF-1 expression and eliminate the survival advantage provided by its aberrant expression. In this study, we first established a methodology that induces CLL cells to differentiate into immunoglobulin (Ig) secreting cells (ISC) using the TLR9 agonist, CpG, together with cytokines (CpG/c). CpG/c stimulation resulted in dramatic CLL cell phenotypic and morphologic changes, expression of cytoplasmic Ig, and secretion of light chain restricted Ig. CpG/c stimulation also resulted in decreased CLL cell LEF-1 expression and increased Blimp-1 expression, which is crucial for plasma cell differentiation. Further, Wnt pathway activation and cellular survival were impaired in differentiated CLL cells compared to undifferentiated CLL cells. These data support the notion that CLL can differentiate into ISC and that this triggers decreased leukemic cell survival secondary to the down regulation of LEF-1 and decreased Wnt pathway activation.

Cancer is the consequence of sequential acquisition of mutations within somatic cells. Mutations alter the relative reproductive fitness of cells, enabling the population to evolve in time as a consequence of selection. Cancer therapy itself can select for or against specific subclones. Given the large population of tumor cells, subclones inevitably emerge and their fate will depend on the evolutionary dynamics that define the interactions between such clones. Using a combination of in vitro studies and mathematical modeling, we describe the dynamic behavior of two cell lines isolated from the same patient at different time points of disease progression and show how the two clones relate to one another. We provide evidence that the two clones coexisted at the time of initial presentation. The dominant clone presented with biopsy proven cardiac AL amyloidosis. Initial therapy selected for the second clone that expanded leading to a change in the diagnosis to multiple myeloma. The evolutionary dynamics relating the two cell lines are discussed and a hypothesis is generated in regard to the mechanism of one of the phenotypic characteristics that is shared by these two cell lines.

Despite palliative treatments, tumor-induced bone disease (TIBD) remains highly debilitating for many cancer patients and progression typically results in death within two years. Therefore, more effective therapies with enhanced anti-resorptive and cytotoxic characteristics are needed. We developed bisphosphonate-chemotherapeutic conjugates designed to bind bone and hydrolyze, releasing both compounds, thereby targeting both osteoclasts and tumor cells. This study examined the effects of our lead compound, MBC-11 (the anhydride formed between arabinocytidine (AraC)-5'-phosphate and etidronate), on bone tumor burden, bone volume, femur bone mineral density (BMD), and overall survival using two distinct mouse models of TIBD, the 4T1/luc breast cancer and the KAS-6/1-MIP1alpha multiple myeloma models. In mice orthotopically inoculated with 4T1/luc mouse mammary cells, MBC-11 (0.04 microg/day; s.c.) reduced the incidence of bone metastases to 40% (4/10), compared to 90% (9/10; p=0.057) and 100% (5/5; p=0.04) of PBS- or similarly-dosed, zoledronate-treated mice, respectively. MBC-11 also significantly decreased bone tumor burden compared to PBS- or zoledronate-treated mice (p=0.021, p=0.017, respectively). MBC-11 and zoledronate (0.04 microg/day) significantly increased bone volume by two- and four-fold, respectively, compared to PBS-treated mice (p=0.005, p<0.001, respectively). In mice systemically injected with human multiple myeloma KAS-6/1-MIP1alpha cells, 0.04 and 4.0 microg/day MBC-11 improved femur BMD by 13% and 16%, respectively, compared to PBS (p=0.025, p=0.017, respectively) at 10 weeks post-tumor cell injection and increased mean survival to 95 days compared to 77 days in mice treated with PBS (p=0.047). Similar doses of zoledronate also improved femur BMD (p< or =0.01 vs PBS) and increased mean survival to 86 days, but this was not significantly different than in PBS-treated mice (p=0.53). These results demonstrate that MBC-11 decreases bone tumor burden, maintains bone structure, and may increase overall survival, warranting further investigation as a treatment for TIBD., (2010 Elsevier Inc. All rights reserved.)

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge, no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach, we established the genetic relationship between the cell lines and the primary patient cells, and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly, we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity, the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover, these cell lines will provide an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology.

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27(neg) B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.

B cell-activating factor belonging to the TNF family (BAFF) plays a critical role in B cell maturation, yet its precise role in B cell differentiation into Ig-secreting cells (ISCs) remains unclear. In this study, we find that upon isolation human naive and memory B (MB) cells have prebound BAFF on their surface, whereas germinal center (GC) B cells lack detectable levels of prebound BAFF. We attribute their lack of prebound BAFF to cell activation, because we demonstrate that stimulation of naive and MB cells results in the loss of prebound BAFF. Furthermore, the absence of prebound BAFF on GC B cells is not related to a lack of BAFF-binding receptors or an inability to bind exogenous BAFF. Instead, our data suggest that accessibility to soluble BAFF is limited within GCs, perhaps to prevent skewing of the conventional B cell differentiation program. In support of this concept, whereas BAFF significantly enhances ISC differentiation in response to T cell-dependent activation, we report for the first time the ability of BAFF to considerably attenuate ISC differentiation of MB cells in response to CpG stimulation, a form of T cell-independent activation. Our data suggest that BAFF may be providing regulatory signals during specific T cell-independent events, which protect the balance between MB cells and ISCs outside GCs. Taken together, these data define a complex role for BAFF in humoral immune responses and show for the first time that BAFF can also play an inhibitory role in B cell differentiation.

B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) ligand superfamily. Although BLyS costimulates adaptive immune cells, the ability of BLyS to stimulate innate immune cells has not been described. Here, we show that BLyS strongly induces human monocyte survival, and activation as measured by proinflammatory cytokine secretion and up-regulation of costimulatory molecule expression. In addition, monocytes cultured with BLyS differentiated into macrophage-like cells. Regarding BLyS receptor(s) expression, freshly isolated monocytes bound low levels of exogenous BLyS and expressed primarily intracellular TACI, and cell surface TACI levels increased following monocyte activation. Of interest, bone marrow monocytes from some multiple myeloma patients expressed significant levels of cell surface TACI at isolation. Our findings indicate that BLyS plays a role in activating innate immune cells. Moreover, this study may explain more clearly why high BLyS production is often correlated with certain inflammatory autoimmune diseases and B-lymphocyte malignancies.

Survivin is a member of the inhibitor of apoptosis protein (IAP) family and functions both as an apoptosis inhibitor and a regulator of cell division. Survivin overexpression is common in many human tumors and correlates with survival in large cell non-Hodgkin's lymphoma. To evaluate this molecule as a potential therapeutic target in large-cell lymphoma, we evaluated the effect of survivin inhibition both in vitro and in vivo. Using an antisense oligonucleotide (ASO) approach, cell growth was significantly inhibited in the DoHH2, RL and HT lymphoma cell lines. In a lymphoma xenograft model, the development of tumors as well as the growth of established tumors was inhibited in the survivin ASO-treated mice compared to controls. To assess the efficacy of the survivin ASO in combination with other biological agents, we combined the survivin ASO with an anti-CD20 monoclonal antibody, rituximab. The effect of survivin ASO and rituximab in combination was additive in vitro. In vivo, however, suppression of tumor growth with the combination was not significantly superior to controls. We conclude that inhibition of survivin expression is an attractive therapeutic strategy in aggressive non-Hodgkin's lymphomas, and that combining survivin ASO with rituximab may enhance the efficacy of this approach.

Multiple myeloma (MM) is a progressive disease that is thought to result from multiple genetic insults to the precursor plasma cell that ultimately affords the tumor cell with proliferative potential despite its differentiated phenotype and resistance to undergoing apoptosis. Altered expression of antiapoptotic factors as well as growth factors have been described in a significant number of patients. However, the key regulatory elements that control myeloma development and progression remain largely undefined. Because of the knowledge that B-lymphocyte stimulator (BLyS), a tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis, promotes the survival of malignant B cells, we began a coordinated study of BLyS and its receptors in MM. All MM cells studied expressed one or more of 3 known receptors (B-cell maturation antigen [BCMA], transmembrane activator and CAML interactor [TACI], and B-cell activating factor receptor [BAFF-R]) for BLyS; however, the pattern of expression was variable. Additionally, we provide evidence that BLyS can modulate the proliferative capacity and survival of MM cells. Finally, we provide evidence that BLyS is expressed by MM cells and is present in the bone marrow of patients with MM. Expression of BCMA, TACI, and BAFF-R by MM taken together with the ability of BLyS to support MM cell growth and survival has exciting implications because they may be potential therapeutic targets.

We have previously demonstrated that the responsiveness of multiple myeloma (MM) cells to interferon-alpha (IFN-alpha) stimulation is variable, with an atypical growth response displayed by some cells. Here we report the ability of IFN-alpha to induce tyrosine phosphorylation of a 180 kDa band in the KAS-6/1 MM cell line, which is growth responsive to IFN-alpha. Further characterization demonstrated that this band corresponds to ErbB3. To our knowledge, this is the first report of ErbB3 expression in a cell type of the hematopoietic lineage. Although ErbB receptors have been shown to crosscommunicate with various other receptors, our results show for the first time that the IFN-alpha receptor can crosscommunicate with ErbB3. To address the significance of these observations, we transfected ErbB3-negative DP-6 MM cells with ErbB3 and used siRNA to silence ErbB3 in the KAS-6/1 cell line. Although IFN-alpha transactivated ErbB3 in the DP-6 transfectants, it did not confer growth responsiveness to IFN-alpha. Interestingly, silencing ErbB3 expression in the KAS-6/1 cells decreased the overall growth response to IFN-alpha and to interleukin-6. These results suggest that ErbB3 expression alone does not uniquely confer IFN-alpha growth responsiveness, but instead may amplify proliferation rates in MM cells that have acquired atypical expression of this receptor.

Insulin-like growth factors (IGF-I, IGF-II) have long been recognized as important mitogens in many types of malignancies. Because the role of IGFs in growth control of myeloma cells has not been extensively examined, we have used a panel of IL-6-responsive myeloma cell lines to address this issue. Initial studies demonstrated that IGF-I and IGF-II significantly enhanced DNA synthesis by each of the four cell lines, even when assayed in the absence of IL-6. The specificity of the IGF response was confirmed using an IGF-I receptor Ab, and additional studies demonstrated that IGF responsiveness did not result from induction of autocrine IL-6 expression. When IL-6 responsiveness was assayed, three of four cell lines synthesized DNA in response to IL-6 alone; however, the magnitude of responsiveness was greatly enhanced by addition of IGFs. Similar results were obtained when proliferation and cell cycle progression were analyzed. By contrast, the KP-6 cell line was responsive to IL-6 only when IGF was present. Finally, we analyzed the effects of IGF-I on normal B lymphocytes. IGF, however, did not stimulate B cell DNA synthesis, suggesting that IGF responsiveness may represent a key difference between normal and malignant B cells. In summary, these studies suggest that IGFs may play an important role in multiple myeloma by virtue of their ability to directly stimulate tumor cell growth as well as modulate the magnitude of IL-6-driven growth.

Although IFN-alpha is commonly used as maintenance treatment for multiple myeloma patients, its effectiveness is varied. In this study, we have used a panel of IL-6 responsive myeloma cell lines that vary remarkably in responsiveness to IFN-alpha. Three cell lines were growth arrested by IFN-alpha; however, IFN-alpha significantly stimulated growth of the fourth cell line, KAS-6/1. Our studies have focused on elucidating the mechanism of differential IFN-alpha responsiveness. First, we have shown that IFN-alpha-stimulated growth of the KAS-6/1 cells did not result from induction of autocrine IL-6 expression. Second, analysis of Stats 1, 2, and 3 and IFN regulatory factor-1 (IRF-1) and IRF-2 activation failed to reveal differences between the IFN-alpha growth-arrested or growth-stimulated cells. Third, although IFN-alpha treatment of the IFN-alpha growth-inhibited cell lines reduced IL-6 receptor (IL-6R) expression, IFN-alpha also reduced KAS-6/1 IL-6R expression. Finally, although IFN-alpha treatment reduced IL-6R numbers on each cell line, analysis of Stat protein activation revealed that the receptors were still functional. We conclude that myeloma cell responsiveness to IFN-alpha is heterogeneous and that mechanisms of IFN-alpha-mediated growth inhibition other than IL-6R downregulation must exist in myeloma. Identification of these mechanisms may allow development of agents that are more universally effective than IFN-alpha.