The plasmacytoma J558L lacks constitutively active NF-kappa B and is deficient in early response gene activation.

In mature B cells the nuclear factor NF-kappa B which binds within the kappa enhancer is constitutively present in the nucleus. However, the lambda light chain producing myeloma J558L has been found to lack constitutively functional NF-kappa B. Deoxycholate released functional NF-kappa B from cytoplasmic extracts and functional NF-kappa B was present in J558L following cycloheximide but not phorbol ester treatment. J558L was also unable to respond to phorbol ester stimulation with synthesis of mRNA from the early response gene TIS11. J558L differs from S107, another myeloma which was found to be deficient in the synthesis of NF-kappa B but not in the activation of TIS11. Somatic cell hybrids were used to further define the defect in J558L; hybrids were made with the myelomas S107 and S194 and the pre-B cell line 70Z/3. In general, complementation of the defect in J558L was observed; however there was not a direct correlation between the levels of TIS11 mRNA and NF-kappa B expression in the somatic cell hybrids, suggesting that the pathways of activation of these genes, while possibly sharing common elements, are not identical. The defect in J558L was surprising given that it has frequently been used for the expression of transfected light chain genes.     

The role of the kappa enhancer and its binding factor NF-kappa B in the developmental regulation of kappa gene transcription

We report here on a comparison of plasmacytoma cell lines that differ markedly in their ability to express kappa immunoglobulin genes introduced by transfection, but nevertheless express their endogenous kappa genes at comparable levels. The cell line that fails to express exogenous kappa genes is nonpermissive for kappa enhancer function, apparently because it lacks a specific kappa enhancer-binding nuclear factor (NF-kappa B). We show that this same nuclear factor is also lacking in pre-B cells and that treatment of these cells with bacterial lipopolysaccharide induces the appearance of NF-kappa B in nuclear extracts and concomitantly activates the kappa enhancer. These findings indicate that factor NF-kappa B controls kappa enhancer activity, and that this activity is only transiently required during B cell maturation.     

Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation.

NF-kappa B activation is a crucial late step in the induction of immunoglobulin kappa light-chain gene expression in pre-B cells by lipopolysaccharide (LPS). We have analyzed NF-kappa B activation in three independent mutant lines of 70Z/3 pre-B cells which are unresponsive to LPS. All three variant cell lines failed to activate NF-kappa B when induced with LPS or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. However, all three cell lines contained functional NF-kappa B, as revealed by detergent treatment of cytoplasmic extracts. Moreover, cycloheximide induced limited activation of NF-kappa B comparable to that in wild-type 70Z/3 pre-B cells in two of the three variant lines. These results indicate that the mutations blocking kappa gene induction in these variant 70Z/3 pre-B-cell lines affect NF-kappa B activation.     

The SV40 TC-II(kappa B) enhanson binds ubiquitous and cell type specifically inducible nuclear proteins from lymphoid and non-lymphoid cell lines.

We have characterized the complexes resulting from the specific binding in vitro of proteins present in nuclear extracts of several lymphoid and non-lymphoid cell lines to the TC-I and TC-II sequences of the simian virus 40 (SV40) enhancer. No proteins could be detected, binding selectively to the TC-I sequence, but two proteins TC-IIA and TC-IIB were identified interacting specifically with both the TC-II/kappa B enhanson, 5'-GGAAAGTCCCC-3' (important for the activity of the SV40 enhancer in vivo), and with the related H-2Kb enhanson, 5'-TGGGGATTCCCCA-3'. The binding of these two proteins to mutated TC-II enhansons correlates with the effect of these mutations in vivo, suggesting that both proteins may be important for SV40 enhancer activity. The TC-IIA binding activity was present in nuclear extracts of mature lymphoid B cells and was increased in pre-B cell nuclear extracts by lipopolysaccharide (LPS) and cycloheximide treatment. Furthermore, complex formation between the TC-IIA protein and the TC-II enhanson was efficiently competed by the kappa B motif from the kappa chain enhancer, indicating that TC-IIA is the NF-kappa B factor or a closely related protein. However, in contrast to previous reports, a TC-IIA/NF-kappa B-like protein whose properties could not be distinguished from those of the TC-IIA protein present in lymphoid B cells, was found in nuclear extracts of several untreated non-lymphoid cell lines, notably of HeLa cells, but not of undifferentiated F9 embryonal carcinoma (EC) cells [F9(ND)]. The TC-IIA binding activity which was moderately increased in HeLa cell nuclear extracts by 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or cycloheximide treatment could be induced in nuclear extracts of F9(ND) cells by cycloheximide, but not by TPA. Moreover, the TC-IIA binding activity could be induced in cytosolic fractions from F9(ND) cells by treatment with deoxycholate, indicating that these cells contain an inhibitor protein similar to the previously described NF-kappa B inhibitor, I kappa B. The second TC-II enhanson binding protein, TC-IIB, which could be clearly distinguished from the TC-IIA/NF-kappa B-like protein, by a number of differential properties, resembles the previously described KBF1/H2TF1 protein as it binds with a higher affinity to the H-2Kb enhanson than to the TC-II/kappa B enhanson, and its pattern of methylation interference on the H-2Kb and TC-II/kappa B enhansons is identical to that reported for the KBF1/H2TF1 protein.(ABSTRACT TRUNCATED AT 400 WORDS)