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Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi's sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P =.01), CD86 (P =. 0003), and CD14 (P =.04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.

Whether Kaposi's sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients.

Monoclonal antibodies (MoAbs) that selectively identify Muc-1 core protein (MoAbs DF3-P, VU-4H5) determinants were used to identify the Muc-1 glycoform present on 7 multiple myeloma (MM) cell lines, 5 MM patient plasma cells, 12 MM patient B cells, as well as 32 non-MM cell lines and normal hematopoietic cells. Flow cytometry studies demonstrated that all MM cell lines, MM patient plasma cells, and MM patient B cells expressed Muc-1 core protein epitopes. Circulating B cells from 4 normal donors also expressed Muc-1 core protein. In contrast, Muc-1 core protein was absent on 28 of 32 non-MM neoplastic cell lines, 17 of which expressed Muc-1. Splenic and tonsillar B cells, CD34(+) stem cells, resting T cells, and bone marrow plasma cells obtained from normal donors both lacked Muc-1 glycoforms. We next studied the effects of estrogen, progesterone, and glucocorticoid receptor agonists and antagonists on Muc-1 expression, because consensus sequences for the response elements of these steroids are present on the Muc-1 gene promoter. These studies showed that dexamethasone (Dex) induced Muc-1 expression on MM cell lines, as determined by both flow cytometry and Western blot analyses. Dex also induced upregulation of Muc-1 on prostate and ovarian cancer cell lines. Time and dose-response studies demonstrated that Dex induced maximal cell surface Muc-1 expression by 24 hours at concentrations of 10(-8) mol/L. Dex induced Muc-1 upregulation could be blocked with a 10-fold excess of the glucocorticoid receptor antagonist RU486, confirming that Dex was acting via the glucocorticoid receptor. No changes in Muc-1 expression were observed on MM cells treated with estrogen and progesterone receptor agonists and antagonists or with RU486. These studies provide the framework for targeting Muc-1 core protein in vaccination and serotherapy trials in MM. In addition, the finding that Muc-1 expression on MM cells can be augmented by Dex at pharmacologically achievable levels suggests their potential utility in enhancing treatments targeting Muc-1 in MM.

Adenoviruses are efficient gene delivery agents for a variety of neoplasms. In the present study, we have investigated the use of adenoviruses for the delivery of the thymidine kinase (tk) gene into multiple myeloma (MM) cells. We first demonstrated that MM cell lines and MM patient cells express both adenovirus receptors as well as the DF3/MUC1 protein, thus providing a rationale for using adenoviruses to selectively deliver genes under the control of the DF3 promoter. By using an adenoviral construct containing beta-galactosidase (beta-gal) gene driven by the DF3 promoter (Ad. DF3-betagal), we demonstrate greater than 80% transduction efficiency in OCI-My5 and RPMI 8226 MM cell lines at a multiplicity of infection of 1 to 100. Importantly, transduction with the tk gene driven by the DF3 promoter (Ad.DF3-tk) followed by treatment with 50 micromol/L ganciclovir (GCV) purged >/=6 log of contaminating OCI-My5 and RPMI 8226 MM cells within bone marrow mononuclear cells. In contrast, normal human hematopoietic progenitor cell number was unaffected under these conditions. Selectivity of DF3/MUC1 promoter was further confirmed, because Ad.DF3-betagal or Ad.DF3-tk did not transduce MUC1-negative HeLa cervical carcinoma cells. In addition, GCV treatment of Ad.DF3-tk-transduced RPMI 8226 MM cells did not induce a significant bystander effect. These findings demonstrate that transduction with Ad vectors using a tumor-selective promoter provides a highly efficient and selective approach for the ex vivo purging of MM cells.

Previous studies have suggested that multiple myeloma (MM) cells express estrogen receptors (ER). In the present study, we characterized the effects of estrogen agonists and antagonists (anti-estrogens [AE]) on growth of MM cell lines and MM patient cells. In addition to antagonizing estrogen binding to ER, AE can trigger apoptosis. Hence, we also determined whether estrogens or AE altered MM cell survival. Immunoblotting showed that ER-alpha is expressed in 4 of 5 MM cell lines (ARH-77, RPMI 8226, S6B45, and U266, but not OCI-My-5 cells), as well as in freshly isolated MM cells from 3 of 3 patients. 17beta-estradiol (E2) did not significantly alter proliferation of MM cell lines or MM patient cells. In contrast, two structurally distinct AE, tamoxifen (TAM) and ICI 182,780 (ICI), significantly inhibited the proliferation of all 5 MM cell lines and MM cells from 2 of 2 patients (IC50, 2 to 4 micromol/L). Proliferation of these cell lines was also inhibited by the hydroxylated TAM derivative, 4-hydroxytamoxifen (4HTAM), although this derivative was less potent than TAM (IC50, 3 to 25 micromol/L). In contrast, the dehalogenated TAM derivative toremifene (TOR) did not inhibit MM cell proliferation. We next examined the effects of these agents on MM cell survival. TAM, ICI, and, to a lesser extent, 4HTAM and TOR triggered apoptosis in both ER-alpha-positive as well as ER-alpha-negative MM cell lines and patient MM cells, evidenced both by fluorescence-activated cell sorting (FACS) analysis using propidium iodide staining and the TUNEL assay. TAM-induced growth inhibition and apoptosis of ER-alpha-positive S6B45 MM cells was not blocked by coculture with excess E2. TAM-induced apoptosis of S6B45 MM cells was also unaffected by addition of exogenous interleukin-6. Importantly, both the inhibition of MM cell proliferation and the induction of MM cell apoptosis were achieved at concentrations of TAM (0.5 and 5.0 micromol/L) that did not significantly alter in vitro growth of normal hematopoietic progenitor cells. Similar plasma levels of TAM have been achieved using high-dose oral TAM therapy, with an acceptable toxicity profile. These studies therefore provide the rationale for trials to define the utility of AE therapy in MM., (Copyright 1998 by The American Society of Hematology.)

Interleukin-6 (IL-6) is a growth factor for multiple myeloma (MM) cells and can inhibit MM cell apoptosis. Our recent studies show that IL-6 facilitates MM cell growth via phosphorylation of retinoblastoma protein (pRB); however, the effects of IL-6 on those cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDIs) that are known to regulate phosphorylation of pRB have not been defined in MM cells. In the present report, we cultured MM cell lines and patient cells with IL-6 and/or dexamethasone (Dex) and characterized changes in cell cycle; expression and association of cyclins, CDKs, and CDIs; and phosphorylation of pRB. Dex induced G1 growth arrest in MM cells, whereas IL-6 facilitated G1 to S phase transition; moreover, the effect of Dex was blocked by IL-6. p21WAF1 (p21) protein was constitutively expressed in the majority of MM cells independent of the status of p53. Its expression was upregulated by Dex and downregulated by IL-6; again, IL-6 inhibited the increase in p21 triggered by Dex. These alterations in p21 expression in MM cells were associated with changes in p21 binding to CDK2, CDK4, and CDK6; CDK2, CDK4, and CDK6 kinase activities; and phosphorylation of pRB. In contrast, expression of G1 cell cycle regulatory proteins, including p27KIP1, cyclin D2, and cyclin E, was not altered in MM cells cultured with Dex and/or IL-6. Finally, interferon-gamma (IFN-gamma) also induced G1 growth arrest and upregulated p21 protein expression; as with Dex, affects of IFN-gamma were inhibited by IL-6. Our results therefore show that changes in cell cycle distribution in MM cells triggered by Dex, IL-6, and IFN-gamma correlate with changes in p21 protein expression and implicate p21 in the coupling of Dex-, IL-6-, and IFN-gamma-related signals to G1 cell cycle regulation in MM cells.

Fas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.