Characterization of the MM.1 human multiple myeloma (MM) cell lines Stephanie Greenstein, Nancy L Krett, Yoshihiro Kurosawa, Chunguang Ma, Dharminder Chauhan, Teru Hideshima, Kenneth C Anderson, Steven T Rosen  Experimental Hematology  Volume 31, Issue 4, Pages 271-282 (April 2003) DOI: 10.1016/S0301-472X(03)00023-7

Figure 1 Morphological features of the MM.1S cultured cells. MM1.S cells were fixed with 10% formaldehyde and prepared by cytospin preparation. Cells were stained with H and E staining (hematoxylin and eosin) for observation of morphological features. The MM.1S cells have typical myeloma cell morphology. They are round with eccentrically located nuclei, and many cells are binucleated or multinucleated. Arrows indicate a binucleated cell and a multinucleated cell. Experimental Hematology 2003 31, 271-282DOI: (10.1016/S0301-472X(03)00023-7)

Figure 2 IL-6-induced signal transduction in MM cells. IL-6 is a growth and survival factor for MM cells. In its capacity as a multifunctional cytokine, IL-6 is responsible for regulating various aspects of growth, differentiation, and proliferation. There are three pathways downstream of the initial receptor-binding event that are likely involved in MM growth. (A) The mitogen-activated protein kinase (MAPK) pathway, which involves the sequential activation of SHC, Grb2, Sos, Ras, Raf, MEK, and MAPK, is involved in MM cell growth and proliferation. (B) The jun-activated kinase (JAK) pathway that activates STAT3 and STAT1 transcription factors regulates cell survival. The predominant form is a STAT 3 homodimer; however, STAT 1 is present to a lesser extent as a homodimer and also as a heterodimer with STAT 3. (C) IL-6 triggers anti-apoptotic/pro-survival signals via activation of the phosphatidyl 3-kinase (PI3K)/Akt kinase pathway. IL-6 induces Akt phosphorylation, which results in the phosphorylation of downstream target molecules such as caspase 9, BAD, GSK-3β, and Forkhead family of pro-apoptotic transcription factors (FKHR). A decrease in FKHR results in a decrease in p27 allowing cell-cycle progression and growth. Akt activation also leads to NF-κB activation, and transcription of pro-survival/anti-apoptotic proteins such as cytokines, IL-6, IAPs, and members of the bcl-2 family. Experimental Hematology 2003 31, 271-282DOI: (10.1016/S0301-472X(03)00023-7)

Figure 3 Dex-induced apoptotic signaling in MM Cells. (A) The cytoplasmic domain of gp130 contains phosphotyrosine motifs for recruitment of a protein-tyrosine phosphatase SHP2. Normally, IL-6 activates SHP2, and promotes MM growth and proliferation. IL-6 also induces binding of SHP2 to RAFTK, resulting in dephosphorylation and inactivation of RAFTK. When MM cells are treated with Dex, RAFTK is phosphorylated and activated. Active RAFTK dissociates from SHP2 and participates in the apoptotic cascade. (B) Dex-induced apoptosis is associated with a significant decrease in MAPK growth activity. (C) IL-6 triggers anti-apoptotic/pro-survival signals via activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt kinase pathway. Akt phosphorylation results in the phosphorylation and inactivation of downstream pro-apoptotic target molecules such as BAD, caspase 9, GSK-3β, and FKHR. Dex blocks the PI3K/Akt kinase pathway, and leads to FKHR activation, upregulation of p21KIP1, and G1 growth arrest. (D) Dex induces also apoptosis by blocking NF-κB-mediated transcription of pro-survival/anti-apoptotic genes. Interactions between Dex-bound GR and NF-κB result in transcriptional interference. Activated GR disrupts essential contacts between NF-κB and factors of the basal transcription machinery, a process known as tethering. Inhibition of NF-κB-mediated transcription results in apoptosis. Experimental Hematology 2003 31, 271-282DOI: (10.1016/S0301-472X(03)00023-7)

Figure 4 Actions of anti-MM agents. In its resting state, NF-κB is sequestered in the cytoplasm by its inhibitor IκBα, which maintains the NF-κB protein in an inactive conformation. The activation cascade begins when IκBα is targeted for phosphorylation and subsequent ubiquitinylation, which identifies the inhibitor for degradation by proteases, and leads to a conformational change and the release of NF-κB. NF-κB then translocates into the nucleus, where it exerts its influence on transcription of pro-inflammatory/survival (IL-6, cytokines) and anti-apoptotic genes (bcl-xL, IAPs). Suppression of NF-κB activity is a common event among apoptosis-inducing agents for MM, including thalidomide/IMiDs, proteasome inhibitors, and Dex. Inhibition of NF-κB by thalidomide/IMiDs decreases expression of these anti-apoptotic mediators and facilitates apoptosis. PS-341 inhibits NF-κB activity and induces apoptosis by preventing the proteasome-mediated degradation of the NF-κB inhibitory protein, IκBα. PS-1145, an IκBα kinase inhibitor, acts by impeding IκBα degradation, which thereby prevents the release of NF-κB and abrogates NF-κB-mediated transcription of pro-survival and anti-apoptotic genes. SN-50 is a cell-permeable specific NF-κB inhibitor, which blocks NF-κB activity by preventing nuclear translocation. Interactions between Dex-bound GR and NF-κB result in transcriptional interference, which blocks NF-κB-mediated trans- cription of pro-survival/anti-apoptotic genes. Experimental Hematology 2003 31, 271-282DOI: (10.1016/S0301-472X(03)00023-7)