Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription.

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms.

Active lambda and kappa antibody gene rearrangement in Abelson murine leukemia virus-transformed pre-B cell lines.

The two Abelson murine leukemia virus (A-MuLV)-transformed cell lines, BM18-4 and ABC-1, undergo immunoglobulin L-chain gene recombination during passage in tissue culture. BM18-4 cells are capable of kappa gene recombination, whereas ABC-1 cells are capable of both kappa and lambda gene recombination. The expression of H chains is apparently not necessary for continuing L chain gene recombination in either of these cells, although H-chain expression may have been involved in the initiation of L-chain gene recombination. All ABC-1 cells that have lambda gene rearrangements also display recombined kappa alleles, supporting the hypothesis that kappa and lambda gene recombination are initiated in an ordered, developmentally regulated manner in maturing B cells. However, analyses of the ABC-1 line indicate that pre-B cells that have initiated lambda gene recombination do not terminate kappa gene rearrangement. The lambda gene recombinations that occur in the ABC-1 cell line indicate that the germline order of lambda gene segments is: 5' ... V lambda 2 ... J lambda 2C lambda 2-J lambda 4C lambda 4 ... V lambda 1 ... J lambda 3C lambda 3-J lambda 1C lambda 1 ... 3'. In addition, the frequencies of lambda 1, lambda 2, and lambda 3 gene recombinations among ABC-1 cells are quite different than the frequencies of B cells producing lambda 1, lambda 2, and lambda 3 L-chains in the mouse. RS DNA recombinations also occur in the BM18-4 and ABC-1 cell lines, supporting the notion that Ig gene recombinases are involved in RS rearrangement. Recombined RS segments are infrequent among BM 18-4 cells but common among ABC-1 cells, suggesting that RS recombinational events often occur in maturing pre-B cells just before initiation of lambda gene rearrangements. This developmental timing is consistent with the hypothesis that RS recombination may be involved in the initiation of lambda gene assembly.

Heavy chain variable (VH) region diversity generated by VH gene replacement in the progeny of a single precursor cell transformed with a temperature-sensitive mutant of Abelson murine leukemia virus.

Sequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity, and joining (VHDJH) complex generated by VH to VHDJH joining (VH gene replacement) in the progeny derived from a common precursor cell transformed with a temperature-sensitive (ts) Abelson murine leukemia virus (A-MuLV) indicates that endogenous VH gene replacement in vitro generates immunoglobulin gene joints distinct from those generated by the usual VH to DJH joining. Such joints keep the pentamer CAAGA at the 3' end of the donor VH segment and lack a recognizable D segment, as can be seen also in vivo. The results suggest that VH gene replacement participates in generating VH region diversity in vivo, as previously postulated. During the joining process, a unique VH gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries. The basis of these regulatory processes is discussed.

Leukemic challenge unmasks a requirement for PI3Kdelta in NK cell-mediated tumor surveillance.

Specific inhibitors of PI3K isoforms are currently evaluated for their therapeutic potential in leukemia. We found that BCR/ABL(+) human leukemic cells express PI3Kdelta and therefore explored its impact on leukemia development. Using PI3Kdelta-deficient mice, we define a dual role of PI3Kdelta in leukemia. We observed a growth-promoting effect in tumor cells and an essential function in natural killer (NK) cell-mediated tumor surveillance: Abelson-transformed PI3Kdelta-deficient cells induced leukemia in RAG2-deficient mice with an increased latency, indicating that PI3Kdelta accelerated leukemia progression in vivo. However, the absence of PI3Kdelta also affected NK cell-mediated tumor surveillance. PI3Kdelta-deficient NK cells failed to lyse a large variety of target cells because of defective degranulation, as also documented by capacitance recordings. Accordingly, transplanted leukemic cells killed PI3Kdelta-deficient animals more rapidly. As a net effect, no difference in disease latency in vivo was detected if both leukemic cells and NK cells lack PI3Kdelta. Other tumor models confirmed that PI3Kdelta-deficient mice succumbed more rapidly when challenged with T- or B-lymphoid leukemic or B16 melanoma cells. Thus, the action of PI3Kdelta in the NK compartment is as relevant to survival of the mice as the delayed tumor progression. This dual function must be taken into account when using PI3Kdelta inhibitors as antileukemic agents in clinical trials.

Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.

Transformation by Abelson murine leukemia virus (Ab-MLV) is a multistep process in which growth-stimulatory signals from the v-Abl oncoprotein and growth-suppressive signals from the p19(Arf)-p53 tumor suppressor pathway oppose each other and influence the outcome of infection. The process involves a proliferative phase during which highly viable primary transformants expand, followed by a period of marked apoptosis (called "crisis") that is dependent on the presence of p19(Arf) and p53; rare cells that survive this phase emerge as fully transformed and malignant. To understand the way in which v-Abl expression affects p19(Arf) expression, we examined changes in expression of Arf during all stages of Ab-MLV transformation process. As is consistent with the ability of v-Abl to stimulate Myc, a transcription factor known to induce p19(Arf), Myc and Arf are induced soon after infection and p19(Arf) is expressed. At these early time points, the infected cells remain highly viable. The onset of crisis is marked by an increase in p19(Arf) expression and a change in localization of the protein from the nucleoplasm to the nucleolus. These data together suggest that the localization and expression levels of p19(Arf) modulate the effects of the protein during oncogenesis and reveal that the p19(Arf)-mediated response is subject to multiple layers of regulation that influence its function during Ab-MLV-mediated transformation.

Mutations affecting the MA portion of the v-Abl protein reveal a conserved role of Gag in Abelson murine leukemia virus (MLV) and Moloney MLV.

Abelson murine leukemia virus (Ab-MLV) arose from a recombination between gag sequences in Moloney MLV (Mo-MLV) and the c-abl proto-oncogene. The v-Abl oncoprotein encoded by Ab-MLV contains MA, p12, and a portion of CA sequences derived from the gag gene of Mo-MLV. Previous studies indicated that alteration of MA sequences affects the biology of Mo-MLV and Ab-MLV. To understand the role of these sequences in Ab-MLV transformation more fully, alanine substitution mutants that affect Mo-MLV replication were examined in the context of Ab-MLV. Mutations affecting Mo-MLV replication decreased transformation, while alanine mutations in residues dispensable for Mo-MLV replication did not. The altered v-Abl proteins displayed aberrant subcellular localization that correlated to transformation defects. Immunofluorescent analyses suggested that aberrant trafficking of the altered proteins and improper interaction with components of the cytoskeleton were involved in the phenotype. Similar defects in localization were observed when the Gag moiety containing these mutations was expressed in the absence of abl-derived sequences. These results indicate that MA sequences within the Gag moiety of the v-Abl protein contribute to proper localization by playing a dominant role in trafficking of the v-Abl molecule.

Molecular dissection of transcriptional control elements within the long terminal repeat of the retrovirus.

The retroviral long terminal repeat (LTR) contains transcriptional control elements that affect viral gene expression. By deletion mutagenesis of the genome of the cloned Abelson murine leukemia virus, regulatory signals could be mapped to at least three domains within the LTR. A defective 5' LTR that did not sustain transforming gene function was complemented by an intact LTR positioned at the 3' end of the genome. This versatility of the retroviral genome with respect to its transcriptional control elements appears to provide a strong selective advantage for viral gene expression.

Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells.

A substitution mutation has been introduced into the c-abl locus of murine embryonic stem cells by homologous recombination between exogenously added DNA and the endogenous gene, and these cells have been used to generate chimeric mice. It is shown that the c-abl mutation was transmitted to progeny by several male chimeras. This work demonstrates the feasibility of germ-line transmission of a mutation introduced into a nonselectable autosomal gene by homologous recombination.

Chromosomal assignment of the endogenous proto-oncogene C-abl.

Abelson murine leukemia virus (A-MuLV) is a replication-defective retrovirus that transforms lymphocytes of the B-cell lineage. This virus is a recombinant between the parental Moloney murine leukemia virus and a cellular gene termed C-abl. By analysis of a series of mouse x Chinese hamster hybrid celllines containing various mouse chromosomes, we have mapped the C-abl gene to mouse chromosome 2.

The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene.

Chronic myelogenous leukemia (CML) is a human disease associated with a consistent chromosomal translocation that results in sequences from the c-abl locus on chromosome 9 being fused to sequences in a breakpoint cluster region (bcr) on chromosome 22. CML cells have two novel products: an 8.5-kilobase RNA transcript containing both abl and bcr and a 210-kilodalton phosphoprotein (P210) recognized by v-abl-specific antisera. To test whether the P210 is the product of the novel 8.5-kilobase bcr/abl fusion transcript, antibodies were prepared against c-abl and bcr determinants. By using these reagents and v-abl-specific antisera, it was demonstrated that the P210 in CML cells is indeed the protein product of the 8.5-kilobase transcript. By analogy to the gag/abl fusion protein of Abelson murine leukemia virus, the replacement of amino terminal c-abl sequences by bcr sequences in P210 may create a transforming protein involved in CML. A 190-kilodalton phosphoprotein that is a candidate for the normal bcr protein was identified in both HeLa and K562 cells.

DNA rearrangement and altered RNA expression of the c-myb oncogene in mouse plasmacytoid lymphosarcomas.

Three types of tumors termed plasmacytomas (ABPC's), lymphosarcomas (ABLS's), and plasmacytoid lymphosarcomas (ABPL's) arise in BALB/c mice treated with pristane and Abelson murine leukemia virus (A-MuLV). While most ABPC's and BLS's contain integrated A-MuLV proviral genome and synthesize the v-abl RNA, most ABPL's do not. The ABPL tumors were examined for the expression of other oncogenes that may be associated with their transformed state, in the absence of transforming virus. These tumors expressed abundant c-myb RNA of unusually large size and showed DNA rearrangements of the c-myb locus.

The role of beta 2-microglobulin in peptide binding by class I molecules.

Efficient transport of class I major histocompatibility complex molecules to the cell surface requires association of the class I heavy chain with endogenous peptide and the class I light chain, beta 2-microglobulin (beta 2M). A mutant cell line deficient in beta 2M transports low amounts of nonpeptide-associated heavy chains to the cell surface that can associate with exogenously provided beta 2M and synthetic peptide antigens. Normal beta 2M-sufficient cells grown in serum-free media devoid of beta 2M also require an exogenous source of beta 2M to efficiently bind synthetic peptide. Thus, class I molecules on normal cells do not spontaneously bind or exchange peptides.

TYK2 is a key regulator of the surveillance of B lymphoid tumors.

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelson-induced B lymphoid leukemia/lymphoma as well as TEL-JAK2-induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus-transformed (A-MuLV-transformed) TYK2(-/-) cells were unaltered, but the high susceptibility of TYK2(-/-) mice resulted from an impaired tumor surveillance, and accordingly, TYK2(-/-) A-MuLV-induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2(-/-) NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV-induced leukemia/lymphoma faster than RAG2(-/-)TYK2(+/-) mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance.

Novel B-cell precursors blocked at the stage of DJH recombination.

Abelson murine leukemia virus-transformed cells have provided the principal model for study of the early events in immunoglobulin gene rearrangements. In this communication, we describe a new type of Abelson virus-transformed pre-B-cell line that is arrested at the DJH stage of the recombination process. These cells differ from other pre-B transformants with respect to two properties associated with the immunoglobulin rearrangement process. First, in contrast to cell lines undergoing VH-to-DJH joining in vitro, none of these cell lines contained detectable levels of RNAs transcribed from their unrearranged VH genes. Second, only some of the cell lines recombined exogenous heptamer-nonamer sequences, indicating that many of them have lost at least a portion of the enzymatic machinery that mediates recombination. The correlation between the absence of unrearranged VH RNAs and the inability to rearrange endogenous immunoglobulin gene segments suggests that VH gene transcription is required both to maintain an active recombination system and for the final step in variable-region formation.

Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.

Hyperexpression of interleukin-7 is not necessary or sufficient for transformation of a pre-B lymphoid cell line.

Interleukin-7 (IL-7) is a potent stimulator of pre-B-lymphocyte proliferation. Pre-B cells transformed by a variety of oncogenes including those of the ABL protein tyrosine kinase family were screened for endogenous IL-7 mRNA expression by polymerase chain reaction and a sensitive bioassay for secreted IL-7. Some v-abl but none of the BCR/ABL, v-src, v-fms, v-myc, v-ras, or v-raf transformants analyzed contained elevated IL-7 transcripts. None of the cell lines secreted detectable bioactivity. We overexpressed IL-7 via a retroviral vector in an IL-7-dependent pre-B cell line to assess the potential for autocrine growth stimulation and malignant transformation. We achieved dramatic deregulation of IL-7 translational suppression by removing portions of the 5' flanking region. Levels of IL-7 expression much greater than those needed to establish factor-independent growth did not induce colony formation in agar by IL-7-expressing pre-B cell lines, and the majority of these lines were nontumorigenic in syngeneic mice. The same pre-B cell line transformed by v-abl displayed a highly malignant phenotype while containing dramatically lower IL-7 transcript levels. We conclude that endogenous IL-7 expression is not a necessary event in transformation of pre-B cells, nor is it sufficient to explain the malignant phenotype in v-abl-transformed cells. Up regulation of endogenous IL-7 expression in some transformed pre-B cells may be one of several synergistic events which can lead to malignant conversion.

The coupling between enhancer activity and hypomethylation of kappa immunoglobulin genes is developmentally regulated.

Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

Structure and expression of germ line immunoglobulin gamma 2b transcripts.

We have isolated a cDNA copy of a truncated C gamma 2b transcript produced by Abelson murine leukemia virus transformants that spontaneously switch from mu to gamma 2b. The initiation site of this transcript was 2 kilobases 5' to the gamma 2b switch recombination region, demonstrating its germ line origin. Nucleotide sequence analyses suggest that this transcript does not encode a protein. Expression of germ line gamma 2b transcripts in Abelson murine leukemia virus transformants and in normal spleen cells correlated with endogenous gamma 2b class switch activity.

Abelson virus potentiates long-term growth of mature B lymphocytes.

Abelson murine leukemia virus (A-MuLV) infection of mouse bone marrow cells usually leads to transformation of pre-B cells. However, when the environment is modified by the continuous presence of lipopolysaccharide (LPS), two novel types of membrane immunoglobulin (mIg)-positive B cell lines are generated. Because the cells which give rise to these cell lines copurify with mIg-positive bone marrow cells, the cell lines arise as a result of A-MuLV interaction with a new type of in vitro target cell. The cell lines generated fall into two groups which differ in several phenotypic characteristics. Group 1 cells are more differentiated than the typical pre-B cell transformant in that they synthesize mIgM and appear to resemble virgin B cells. The group 1 cells do not secrete immunoglobulin and are independent of LPS for growth. In addition, these cell lines synthesize the Abelson P160 protein, contain integrated abl proviral DNA, and are highly tumorigenic in syngeneic animals. The group 2 cell lines differ markedly from both the group 1 cells and from typical, pre-B cell A-MuLV transformants. These cells are mIgG positive and secrete large amounts of immunoglobulin into the culture medium. The cell lines are comprised of both adherent and nonadherent cells and do not synthesize P160 or contain integrated v-abl sequences. The group 2 cells are nontumorigenic in syngeneic animals and require LPS for growth and viability. Both types of cells have remained in culture for over 2 years with no changes in their phenotypic characteristics. This A-MuLV infection system and the novel mIg-positive cell lines may serve as useful models for studying biochemical and molecular properties of mature B cells.

Immunologically distinct p53 molecules generated by alternative splicing.

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.