cells isolated from the abdominal plasmacytoma and spleen of mice injected with the T1165-Luc-K13 IL6 cells. imaging confirmed intra-abdomen growth of tumors in all the T1165-Luc-K13 IL6 -injected mice, whereas no tumor develop- ment was detected in the mice injected with T1165-Luc-vector cells (Fig. 7 B ). Autopsy not only confirmed the Fluorouracil presence of plas- macytomas but also showed the enlargement of spleen and liver in the T1165-Luc-K13 IL6 -injected mice (Fig. 7, C and D , and data not shown).

Finally, T1165-Luc-K13 IL6 cells were easily cultured from the spleen of these mice (Fig. 7 E ). Collectively, the above studies demonstrate that K13-induced constitutive NF- B activity not only allows the T1165 cells to establish peri- toneal plasmacytomas without pristane preconditioning but also promote the development of disseminated disease with visceral involvement. DISCUSSION A key role of IL6 in myeloma pathogenesis is supported by the observations that STAT3 is constitutively active in primary myeloma cells and that inhibition of the IL6R/STAT3 pathway induces apoptosis in certain human myeloma cell lines in vitro We injected 0 7 T1165-Luc-vector and T1165-Luc- (39). Furthermore, although intraperitoneal injections of K13 IL6 cells intraperitoneally into the syngeneic .

Balb/cAnNcr pristane can induce plasmacytomas in the wild-type BALB/c mice ( n 9) and followed the development of plasmacytomas mice (4), it fails to do so in IL6-null mice (40). Similarly, estab- by weekly physical examination and bioluminescence imaging over the ensuing–3 months. The mice injected with the T1165-Luc-vector cells demonstrated no physical abnormali- ties. However, those injected with T1165-Luc-K13 IL6 cells developed enlarged abdomens (Fig. 7 A ). Bioluminescence 27994 JOURNAL OF BIOLOGICAL  Fluorouracil 51-21-8 CHEMISTRY lished IL6-dependent plasmacytoma cell lines, such as T1165 and B9, fail to form plasmacytomas when injected into IL6- deficient mice (38). Collectively, these studies highlighted the potential significance of the IL6 pathway in myeloma pathogen- esis and made it a prime target for therapeutic intervention.

VOLUME 286 • NUMBER 32 • AUGUST2, 2011 Downloaded from www.jbc.org at NYU School of Medicine Library, on March 7, 2012 7 NF- B Confers IL6 Independence However, the results of early clinical trials with IL6-blocking antibodies were disappointing (41) and have led to the sugges- tion that there are IL6-independent signaling pathways that play equally important role in the survival and proliferation of myeloma cells (2). Genetic and epigenetic abnormalities in several genes involved in the NF- B pathway, including TRAF3 , NIK , TRAF2 , CYLD , BIRC2/BIRC3 , CD40 , NFKB1 , NFKB2 , LTBR , and members, thereby protecting them from IL6 withdrawal-in- duced apoptosis. However, in addition to buy Fluorouracil antiapoptotic Bcl2 family members, the K13-induced NF- B pathway is known to induce the expression of a number of other antiapoptotic and growth-promoting genes, such as BIRC3 , IL8 , CCL5 , and GM- CSF (46).

Thus, it is conceivable that additional genes induced by the NF- B pathway might contribute to the protective effect of K13 against IL6 withdrawal-induced apoptosis. TAC1 , are seen in 20% of patients with multiple myeloma and IL6 is one of the known NF- B target genes, and K13 is are associated with constitutive activation of the NF- B path- way (16,7). Inactivation of TRAF3 (TNF receptor-associated factor 3) and overexpression of  helium  NF- B-inducing kinase (NIK) are the two most common abnormalities associated with con- stitutive NF- B activation in myeloma samples and cell lines and have been shown to promote their survival (16,7). How- ever, because TRAF3 and NIK also affect the MAPK signaling pathway (42– 44), it was not clear whether deregulation of the NF- B pathways is solely responsible for the myeloma-promot- ing effects of the mutations observed in the above studies. In this study, we have used the viral proteins K13 and Tax as molecular tools to demonstra