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The activity of the NF-kappaB family of transcription factors is regulated principally by phosphorylation and subsequent degradation of their inhibitory IkappaB subunits. Site-specific serine phosphorylation of IkappaBs by two IkappaB kinases (IKKalpha [also known as CHUK] and IKKbeta) targets them for proteolysis. IKKalpha and -beta have a unique structure, with an amino-terminal serine-threonine kinase catalytic domain and carboxy-proximal helix-loop-helix (HLH) and leucine zipper-like (LZip) amphipathic alpha-helical domains. Here, we describe the properties of two novel cellular isoforms of IKKalpha: IKKalpha-DeltaH and IKKalpha-DeltaLH. IKKalpha-DeltaH and IKKalpha-DeltaLH are differentially spliced isoforms of the IKKalpha mRNA lacking its HLH domain and both its LZip and HLH domains, respectively. IKKalpha is the major RNA species in most murine cells and tissues, except for activated T lymphocytes and the brain, where the alternatively spliced isoforms predominate. Remarkably, IKKalpha-DeltaH and IKKalpha-DeltaLH, like IKKalpha, respond to tumor necrosis factor alpha stimulation to potentiate NF-kappaB activation in HEK293 cells. A mutant, catalytically inactive form of IKKalpha blocked IKKalpha-, IKKalpha-DeltaH-, and IKKalpha-DeltaLH-mediated NF-kappaB activation. Akin to IKKalpha, its carboxy-terminally truncated isoforms associated with the upstream activator NIK (NF-kappaB-inducing kinase). In contrast to IKKalpha, IKKalpha-DeltaLH failed to associate with either itself, IKKalpha, IKKbeta, or NEMO-IKKgamma-IKKAP1, while IKKalpha-DeltaH complexed with IKKbeta and IKKalpha but not with NEMO. Interestingly, each IKKalpha isoform rescued HEK293 cells from the inhibitory effects of a dominant-negative NEMO mutant, while IKKalpha could not. IKKalpha-DeltaCm, a recombinant mutant of IKKalpha structurally akin to IKKalpha-DeltaLH, was equally functional in these assays, but in sharp contrast, IKKbeta-DeltaCm, a structurally analogous mutant of IKKbeta, was inactive. Our results demonstrate that the functional roles of seemingly analogous domains in IKKalpha and IKKbeta need not be equivalent and can also exhibit different contextual dependencies. The existence of cytokine-inducible IKKalpha-DeltaH and IKKalpha-DeltaLH isoforms illustrates potential modes of NF-kappaB activation, which are not subject to the same in vivo regulatory constraints as either IKKalpha or IKKbeta.

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.

Murine bone marrow was either singly or doubly infected with retroviral vectors expressing v-myc (OK10) or v-Ha-ras. The infected bone marrow was cultured in a system that supports the long-term growth of B-lineage lymphoid cells. While the v-myc vector by itself had no apparent effect on lymphoid culture establishment and growth, infection with the v-Ha-ras vector or coinfection with both v-myc and v-Ha-ras vectors led to the appearance of growth-stimulated cell populations. Clonal pre-B-cell lines stably expressing v-Ha-ras alone or both v-myc and v-Ha-ras grew out of these cultures. In comparison with cell lines expressing v-Ha-ras alone, cell lines expressing both v-myc and v-Ha-ras grew to higher densities, had reduced dependence on a feeder layer for growth, and had a marked increase in ability to grow in soft-agar medium. The cell lines expressing both oncogenes were highly tumorigenic in syngeneic animals. These experiments show that the v-myc oncogene in synergy with v-Ha-ras can play a direct role in the in vitro transformation of murine B lymphoid cells.

The structures of the rearranged c-myc gene products derived from a rcp T(12;15) have been investigated in the MPC-11 (IgG2b kappa) plasmacytoma. The rcp T(12;15) in MPC-11 is a reciprocal exchange between the c-myc gene on chromosome 15 and the immunoglobulin gamma 2a switch region (S gamma 2a) on chromosome 12. The c-myc gene is broken within a 5'-nontranslated exon, thereby separating the promoter region of the normal c-myc gene from its protein coding sequences. This reciprocal rearrangement results in the loss of 11 base pairs of c-myc sequence and 300 base pairs of S gamma 2a sequence at the point of recombination. Sequences that represent the promoter region of the normal c-myc gene are present in the 5'-myc reciprocal fragment. A comparison of the nucleotide sequences at the recombination site of a number of c-myc rearrangements reveals a common feature that may have mechanistic importance for these translocation events.

Presented is a detailed molecular analysis of the rearranged c-myc oncogene from ABPC45, an unusual plasmacytoma that was originally classified as translocation-negative. Previous data obtained by high-resolution chromosome banding suggested that this tumor was a member of a small group distinguished by the absence of rcpt (12;15) or (6;15) and further characterized by a band deletion near the c-myc locus on chromosome 15. However, genomic Southern blotting and analysis of the cloned oncogene in the present study reveal that (i) chromosome 12 sequences lie 365 base pairs 5' of the rearranged c-myc; (ii) this DNA consists of immunoglobulin alpha switch region and 5' immunoglobulin mu switch region sequences that are rearranged in an aberrant fashion; and (iii) the immunoglobulin heavy-chain gene enhancer element now resides approximately 2.5 kilobase pairs 5' of c-myc. We infer from these and other data that the rearrangement of c-myc in ABPC45 occurred via a multistep switch region-mediated process and that a reciprocal translocation has indeed taken place. Unlike many other plasmacytomas, this event did not interrupt the normal c-myc transcription unit. Rather, disruption of gene regulation is manifested in part by a change in relative usage of the two promoters normally used by the unrearranged gene. In contrast to several of its counterparts in Burkitt lymphomas, DNA sequence analysis of the translocated c-myc gene of ABPC45 reveals that it has not acquired point mutations in the noncoding first exon. These results strongly imply that a cis-acting regulatory element normally located 5' of exon 1 is lost and that heavy-chain constant region or enhancer sequences exert similar cis effects on translocated c-myc loci.

A retroviral vector, RIM, containing murine c-myc under the control of immunoglobulin heavy-chain gene promoter and enhancer elements and v-Ha-ras driven by a Moloney murine leukemia virus long terminal repeat induced IgM-secreting plasmacytomas in 28% of adult and 83% of 3-week-old pristane-conditioned mice with mean latency periods of 60-70 days. In contrast, the same vector only harboring c-myc or v-Ha-ras was virtually ineffective. RIM-induced plasmacytomas expressed retroviral myc and ras genes while their endogenous c-myc alleles were unrearranged and transcriptionally inactive. These plasmacytomas were clonal as each possessed a unique immunoglobulin heavy-chain joining region rearrangement and a single recombinant provirus. Moloney murine leukemia helper virus did not play an obligatory role in tumorigenesis since insertions of Moloney murine leukemia proviruses were found in only 6 of 24 plasmacytomas induced in adult mice. Taken together, these findings support the view that the v-Ha-ras oncogene can cooperate with an activated myc gene in pristane plasmacytomagenesis.

NF-kappaB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IkappaB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), induces nuclear NF-kappaB expression. TNF-alpha signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-alpha receptor tail, leading to the sequential activation of the downstream NF-kappaB-inducing kinase (NIK) and IkappaB-specific kinases (IKKalpha and IKKbeta). When activated, IKKalpha and IKKbeta directly phosphorylate the two N-terminal regulatory serines within IkappaB alpha, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-kappaB complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKalpha. Such a NIK-T559A mutant also dominantly interferes with TNF-alpha induction of NF-kappaB. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKalpha; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-alpha-mediated induction of NF-kappaB and alone is sufficient to disrupt the physical association of NIK and IKKalpha. Together, these findings provide new insights into the molecular basis for TNF-alpha signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.

The chromosomal locations of the structural genes coding for the constant portions of mouse heavy (H) and light chain immunoglobulins were studied by molecular hybridization techniques. Complementary DNA probes containing the constant-region sequences of kappa and lambdaI light chain and alpha, gamma2b, and mu heavy chain mRNAs were annealed to a large excess of DNA from a series of eight mouse-human hybrid cell lines that are deficient for various mouse chromosomes. The lines were scored as positive when a high proportion of a probe annealed and negative when an insignificant proportion annealed. Some lines were clearly negative for H and lambda and clearly positive for kappa. Others were positive or intermediate for lambda, positive for kappa and negative for H. Still others, including a line that was selected for the absence of the mouse X chromosome, were positive for all immunoglobulin species. These results demonstrate that the Clambda, Ckappa, and CH genes are located on different autosomes in the mouse. In contrast, the three heavy-chain families exhibited consistently uniform hybridization results, suggesting that the genes for Calpha, Cgamma, and Cmu are located on the same chromosome. A comparison of karyotypic data with hybridization data has limited the possible locations of the Ig genes to only a few chromosomes.

Immunoglobulin heavy chain class switching has been observed in vitro. In the IgG2b-producing MPC-11 mouse myeloma cell line, IgG2a-producing cells arise at a high frequency. In some cases, switch variants producing normal-sized (Mr 55,000) gamma 2a heavy chains have arisen spontaneously from a mutagen-induced "intermediate" (ICR 9.7.1) that produces an unusually large (Mr 75,000) heavy chain. Other switch variants have been isolated directly from the parent cell line. The expressed and unexpressed gamma 2b genes of MPC-11 can be distinguished in restriction endonuclease digests of total genomic DNA so that DNA rearrangements detected in MPC-11 variants can be directly associated with one or the other of these two genes. We describe here DNA rearrangements occurring on the expressed heavy chain chromosome of several MPC-11 gamma 2a switch variants and on the expressed chromosome of the ICR 9.7.1 intermediate. Our data indicate that all of these variants express the parental heavy chain variable region (VH) gene, supporting previous protein studies. We provide mapping data for the expressed gene of both ICR 9.7.1 and one of the IgG2a-producing variant cell lines (ICR 9.9.2.1) derived from it and discuss the advantages of an in vitro switching system for examining the dynamics of the immunoglobulin heavy chain class switch.

Chemically induced differentiation of mouse erythroleukemia (MEL) cells leads to complex changes in c-myc mRNA levels. Within 1-2 hr after the addition of the inducer hexamethylene bisacetamide (HMBA), c-myc mRNA levels decrease 10- to 20-fold and remain low until 12-24 hr, at which time the mRNA reaccumulates to its original level. Thereafter as the cells undergo terminal differentiation, c-myc mRNA again declines to a low level. We have investigated the regulation of these changes by measuring c-myc gene transcription and mRNA turnover. We find that the early rapid decline in c-myc mRNA is due to an increase in the block to elongation of transcription within the c-myc first exon. Effective c-myc transcription is then restored after 2 hr of HMBA treatment to the level present in uninduced cells and is maintained throughout the remainder of the differentiation program. These results demonstrate that, except for the rapid decline in c-myc mRNA immediately following inducer treatment, all subsequent regulation of message levels occurs through post-transcriptional mechanisms. Studies of c-myc mRNA turnover suggest that some post-transcriptional regulation is nuclear.

Chromosomally integrated retroviral switch (S) substrates have been developed to reveal switch recombinase-like activities (SRLA) in pre-B and mature B cell lines. Switch substrate retrovectors (SSR) contain a long-terminal repeat-driven neomycin (Neo) gene for proviral chromosomal maintenance (pre- and post-S recombination) and a CMV promoter-driven, chimeric hygromycin-thymidine kinase (Hytk) gene (flanked by S mu and S gamma 2b recombination targets) to select for (ganciclovir) and against (hygromycin B) S region recombination. The retro-substrates' strong, constitutive promoters ensure that variations in cellular switch recombinase activities are independent of S region accessibility control. By initially selecting for proviral integrants in hygromycin followed by shifting into neomycin + ganciclovir to select for S sequence-mediated deletions, switch recombinations can be specifically forestalled in B cell lines whilst most switch-incompetent cells do not survive secondary selection. A qualitative, direct PCR assay reveals that SSR recombinations are stochastic in B cell lines generating a product array akin to natural GH class switching. A semi-quantitative DC-PCR assay detects a significant recombinase activity only in a restricted set of late stage pre-B and mature B cell lines. BCL1B1 mature B cells have the highest level of recombinase activity with 25% or more of proviral integrants accumulating S mu/S gamma 2b substrate recombinations within 10-14 cell generations. The SSR recombinase assay can be performed in a transient fashion wherein extensive, B cell-specific recombination can be visualized within only a few cell divisions post proviral integration. We propose that switch recombinase activity becomes activated during B cell ontogeny independent of or prior to the acquisition of CH locus accessibility and that endogenous S segment targeting to pre-existing recombinase requires a level of accessibility beyond transcriptional activation.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

The J558 plasma cell tumor contains two forms of a translocated c-myc gene which are distinguished by virtue of their 3' flanking sequences. The J558 alpha 4 and alpha 25 myc genes are broken by a 12;15 translocation which links c-myc exon 1 to C alpha switch sequences. Comparative restriction mapping and DNA sequence analyses demonstrated that an intracisternal A-particle (IAP) element inserted approximately 2 kilobases 3' of an alpha 4-type myc gene to generate the alpha 25 gene copy. The steady-state level of truncated myc RNAs in J558 was comparable to that in another plasma cell tumor line (MPC-11) which harbors a translocated c-myc locus without an IAP element. The significance of these observations for the putative role of IAP elements in the genesis or progression or both of plasma cell tumors is discussed.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.

Considerable evidence has accumulated suggesting that the antigen receptor(s) on T cells is coded for by genes for the variable (V) region of the immunoglobulin heavy (H) chains. In B cells, a complete gene for the immunoglobulin VH region is formed by somatic recombination of VH and joining region heavy chain (JH) gene segments [through an intermediate diversity(D) region gene segment]. In an attempt to determine whether a complete immunoglobulin VH region is expressed on T cells that bear an antigen receptor, we analyzed the restriction map of the JH-C mu locus in genomic DNA from two cloned murine cytotoxic T-lymphocyte (CTL) lines specific for the x-ray-induced leukemia RL male 1. We found no rearrangement of the JH C mu locus in the CTL lines, indicating that the T-cell antigen receptor(s) in these CTLs is not coded for by a complete immunoglobulin VH gene formed by joining of VH, (DH), and JH genes. In addition, we determined that C mu genes on both chromosomes were present and that there was no rearrangement of the C alpha, C kappa, or lambda chain genes in these CTL cells.

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.

Activation of the transcription factor NF-kappaB is regulated by the phosphorylation and subsequent degradation of its inhibitory subunit, IkappaB. A large multiprotein complex, the IkappaB kinase (IKK), catalyzes the phosphorylation of IkappaB. The two kinase components of the IKK complex, IKKalpha and IKKbeta, were overexpressed in insect cells and purified to homogeneity. Both purified IKKalpha and IKKbeta specifically catalyzed the phosphorylation of the regulatory serine residues of Ikappa Balpha. Hence, IKKalpha and IKKbeta were functional catalytic subunits of the IKK complex. Purified IKKalpha and IKKbeta also preferentially phosphorylated serine as opposed to threonine residues of Ikappa Balpha, consistent with the substrate preference of the IKK complex. Kinetic analysis of purified IKKalpha and IKKbeta revealed that the kinase activity of IKKbeta on Ikappa Balpha is 50-60-fold higher than that of IKKalpha. The primary difference between the two activities is the Km for Ikappa Balpha. The kinetics of both IKKalpha and IKKbeta followed a sequential Bi Bi mechanism. No synergistic effects on Ikappa Balpha phosphorylation were detected between IKKalpha and IKKbeta. Thus, in vitro, IKKalpha and IKKbeta are two independent kinases of Ikappa Balpha.

The expressed immunoglobulin heavy chain genes of five gamma 2b-gamma 2a hybrid chain-producing variants of the mouse myeloma MPC-11 (gamma 2b, kappa) have been characterized by genomic Southern blot analysis. Results show that a hybrid gamma 2b-gamma 2a gene was formed in each variant by recombination between the expressed gamma 2b gene of MPC-11 and a gamma 2a gene. The recombination sites are within regions of marked homology between gamma 2b and gamma 2a genes: at least three and probably four variants show gamma 2b-gamma 2a recombination within the heavy chain constant region 2 (CH2) domain, while the fifth has its recombination site between the penultimate nucleotide of CH1 and the eighth nucleotide of the hinge. An unexpected finding is that the hybrid heavy chain-producing variants fall into two subgroups based on their use of different gamma 2a gene forms in hybrid gene formation. This result leads to the speculation that either a tandem gamma 2a gene duplication was present in MPC-11 prior to variant generation or mitotic recombination between chromosomes occurred in the generation of one variant subgroup. The similarity of hybrid gene formation in MPC-11 variants to that apparently responsible for concerted evolution within multigene families and hybrid protein expression in various individuals is noted, and the possible relationship between hybrid gene formation and the heavy chain class switch is discussed.

A DNA sequence that generates aberrantly rearranged immunoglobulin heavy chain constant region genes in murine plasmacytomas is shown to participate in a chromosome translocation. We have previously termed this DNA sequence NIARD for non-immunoglobulin-associated rearranging DNA. NIARD rearrangements were found frequently in murine plasmacytomas but were not detected in normal lymphocytes. These rearrangements occasionally involve the switch region of the C alpha gene. In this study, DNA samples obtained from mouse-Chinese hamster somatic cell hybrid lines were digested with various restriction endonucleases and analyzed by the Southern transfer technique with a NIARD hybridization probe. These experiments show that NIARD resides on chromosome 15 in the mouse germ line. Since NIARD is found adjacent to the C alpha gene (located on chromosome 12) in some plasmacytomas, it is apparent that a translocation involving these two chromosomes has occurred. We have proposed a rcpT(12;15) model to explain our data. The implications of NIARD rearrangements for malignant transformation are discussed.

We have characterized a class of DNA rearrangements in plasmacytomas. These recombination events involve a DNA sequence whose origin is outside of the locus of the heavy chain constant region genes, CH. Therefore, we choose to refer to this sequence as non-immunoglobulin-associated rearranging DNA (NIARD). We have isolated two abortively rearranged C alpha genes, generated by NIARD events from the alpha-producing J558 myeloma. Restriction endonuclease maps of these sequences reveal two possible recombination sites in NIARD that are separated by approximately 6.5 kilobase pairs of DNA (defined as 5' and 3' sites). A NIARD rearrangement occurs in 15 out of 20 plasmacytomas tested, including gamma 3-, gamma 1-, gamma 2b-, gamma 2a-, and alpha-producers, but this event usually does not involve a CH switch (S) region. In fact, only S alpha appears to accept NIARD. However, NIARD did not undergo a rearrangement in eight IgA-producing hybridomas tested. One germ-line copy of NIARD (a 22-kilobase pair EcoRI fragment) is retained in all plasmacytomas. NIARD does not appear to possess repetitive DNA sequences homologous to S mu or S alpha. We discuss the possible role and implications of NIARD-like sequence rearrangements in allelic exclusion and chromosomal translocation events in plasmacytomas.