suppress apoptosis upon IL6- withdrawal, we generated a stable populations of T1165 cells expressing Bcl-2, Bcl-xL, and Mcl-1 (Fig. 5 G ). These stable cells were significantly protected from IL6 withdrawal-induced cell death as compared with the empty vector-expressing cells (Fig. 5 H ). These results suggest that K13-induced NF- B protects Exemestane against IL6 withdrawal-in- duced apoptosis by maintaining the expression of antiapo- ptotic members of the Bcl2 family. K13 Protects B9 Plasmacytoma Cells against IL6 Withdraw- al-induced Apoptosis —To demonstrate that the protective effect of K13 against IL6 withdrawal-induced apoptosis is not limited to T1165 cells, we generated stable clones of IL6-depen- dent B9 plasmacytoma expressing K13 or an empty vector using retroviral gene transfer (Fig. 6 A ). Similar to the T1165-K13 cells.

B9 cells expressing K13 were significantly protected against IL6 withdrawal-induced cell death as compared with the empty vector-expressing cells (Fig. 6 B ). Additionally, the protection conferred by K13 was reversed by NF- B inhibitor Bay-11-7082 and was not associated with phosphorylation of STAT1 and STAT3 (Fig. 6, C and D ). Taken collectively with AUGUST2, 2011 • VOLUME 286 • NUMBER 32 JOURNAL OF BIOLOGICAL  Exemestane 107868-30-4 CHEMISTRY 27993 Downloaded from www.jbc.org at NYU School of Medicine Library, on March 7, 2012 6 NF- B Confers IL6 Independence FIGURE 7. T1165-K13 IL6 cells establish peritoneal plasmacytomas with- out pristane preconditioning and lead to disseminated disease involv- ing visceral organs. A and B , BALB/cAnNCr mice were injected intraperito- neally with the indicated cells, and tumor growth was monitored by physical FIGURE 6. K13 protects the B9 murine plasmacytoma cell line against IL6 withdrawal-induced apoptosis via NF- B activation.

A, expression of FLAG-K13 in B9 cells as revealed by Western blotting with a FLAG antibody. B , B9 cells expressing an empty vector or K13 were grown in triplicate in a buy Exemestane 96-well plate in the presence or absence of IL6, and cell viability was mea- sured 48 h later using an MTS assay. The values shown are mean S.D. of two independent experiments performed in triplicate. , p 0.05 versus vector cells. C, B9-vector and B9-K13 cells were treated in triplicate with the indi- cated concentrations ( M ) of Bay-11-7082, and cell viability was measured after 72 h using an MTS assay. B9-K13 cells were grown in the absence of IL6. , p 0.05. D , immunoblot showing lack of phosphorylation of STAT1 and STAT3 in B9-K13 cells when grown in the absence of IL6. the studies using T1165 cells, the above results demonstrate that although K13 protects cells against IL6 withdrawal-in- duced apoptosis via NF- B activation, this effect is not medi- ated through NF- B-induced endogenous IL6 production. Constitutive NF- B Activation Promotes Peritoneal Plasma- cytomas without Pristane Conditioning.

Murine plasmacy- toma cells are not only dependent on IL6 for their in vitro growth but also require it for their growth in vivo (4). Thus, T1165 cells form peritoneal plasmacytoma only if the perito- neal cavity had been preconditioned with pristane, an inflam- matory agent that induces chronic inflammation with copious IL6 production (4, 38). To study the effect of constitutive NF- B activation on the ability of mouse plasmacytoma cells to grow in vivo in the absence of pristane conditioning, we used retroviral-mediated gene transfer to neutrons  express the firefly lucifer- ase (Luc) gene in the T1165 vector and T1165-K13 IL6 cells. examination ( A ) or bioluminescence imagine ( B ) as described under “Materi- als and Methods.” C , plasmacytomas isolated at autopsy from mice injected with the T1165-Luc-K13 IL6 cells. D , splenomegaly in mice injected with T1165-Luc-K13 IL6 cells ( right panel ) as compared with a normal spleen in those injected with the T1165-Luc-vector cells ( left panel ). E , immunoblot analysis showing the presence of FLAG-tagged K13 in the parental T1165- Luc-K13 IL6 cells and in