Comparison of D, JH, and junctional diversity in the fetal, adult, and aged B cell repertoires.

Polymerase chain reaction-amplified cDNA libraries of the IgH genes of fetal, young adult, and aged BALB/c mice were sequenced so that the complimentarity determining region 3 (CDR3) in each could be analyzed. The results show extensive diversity in the CDR3 region in all three libraries examined. A prominent feature of the fetal repertoire is the lack of nucleotide region additions and shorter germline-derived D segments compared with the adult repertoires. Also of interest were distinct differences in D family and JH usage in the three libraries representing different stages of ontogeny. The absence of DFL16.2 in the fetal sequences analyzed was of particular note. Also of note was a substantial underutilization of the largest D family, DSP2, in the aged repertoire. The study provides further evidence that the Ig repertoire is developmentally regulated. In addition, the results indicate that several aspects of the recombination process are different in adult and fetal B lineage cells, suggesting that B cells present early in ontogeny are distinct from those present in the adult.     

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.     

The pattern of joining (JH) gene usage in the human IgH chain is established predominantly at the B precursor cell stage.

Preferential utilization of JH and D genes has been demonstrated in the rearranged IgH chain in human peripheral B cells. We report here that the same hierarchy of JH gene usage is observed in leukemic cells arrested in the B precursor stage of differentiation. Specifically, JH4 and JH6 accounted for 42.9% and 35.7%, respectively, of the JH gene usage in the leukemias compared with an expected frequency of 16.7% assuming unbiased gene usage. Within the D gene families, the DN1 gene appears to be overutilized in both populations, representing about 15% of the total gene usage compared with an expected frequency of 3.2%. Because 21 of the 36 leukemias contained only nonproductive IgH rearrangements, the preferential gene usage could not have arisen from pre-B cells that have undergone clonal selection after a productive rearrangement but before surface Ig expression. Nonproductive rearrangements exhibited the biased gene usage seen for productive rearrangements. These findings suggest that a recombination bias favoring certain segments may be the actual mechanism responsible for the apparent preferential utilization of JH and D genes.     

Rearrangement of immunoglobulin heavy chain genes in human T leukaemic cells shows preferential utilization of the D segment (DQ52) nearest to the J region.

The DNA rearrangements leading to the assembly of genes coding for the immunoglobulin heavy chain (IgH) in B cells and the T cell receptor for antigen in T cells are not completely lineage specific. This probably reflects the use of a common recombinase by IgH and the T cell receptor. This paper describes novel observations on the nature of these cross-lineage rearrangements. A high proportion (though not all) IgH rearrangements in human T leukaemic cells involve the D segment nearest to the J region (DQ52). This same D segment is not involved in B cell IgH rearrangements with one important exception, namely a proportion of B cell leukaemic clones with the most primitive B cell precursor phenotype. These observations have potentially important implications for early lymphoid cell differentiation and in particular support the idea that the 3' D plus J region might lie within a limited window of accessibility of the IgH gene in precursor lymphocytes.     

Regulation of prostaglandin E2 binding to a murine macrophage cell line, P388D1, by insulin.

Preincubation of murine macrophage-like P388D1 cells with physiological amounts of insulin resulted in an increase in prostaglandin E2 binding to these cells, by approximately 2-fold, when compared to untreated cells. Scatchard analysis of the binding of PGE2 to insulin-treated cells indicated that the enhanced binding was due to an increase in receptor number (from 0.30 +/- 0.02 to 0.63 +/- 0.03 fmol/10(6) cells for the high affinity receptor binding sites, and from 2.4 +/- 0.31 to 5.0 +/- 0.41 fmol/10(6) cells for the low affinity receptor binding sites) rather than to an increase in the affinity of the binding sites. The insulin-stimulation of PGE2 binding appeared to be associated with a lowering of the cAMP level in these cells; treatment of cells with insulin lowered the cAMP level by increasing the cAMP phosphodiesterase activity of both the membrane and cytosolic fractions. However, enhanced PGE2 binding to the cells resulted in an increase in cAMP level in the cells. This increase in cAMP level may help to enhance the immunosuppressive action of this prostanoid, as PGE2 is known to suppress many steps in the immune response, including interleukin-1 expression, by raising cAMP levels via activation of receptor-linked adenylate cyclase. Our data suggest that insulin at physiological concentrations may enhance the immunosuppressive action of PGE2.     

Regulation of proliferation in a murine colony-stimulating factor-dependent myeloid cell line: superinduction of c-fos by the growth inhibitor 8-Br-cyclic adenosine 3':5' monophosphate

We have investigated the effect of 8-Br-cyclic adenosine 3':5' monophosphate (cAMP), a pharmacological activator of cAMP-dependent protein kinase, on the proliferation and the nuclear proto-oncogene induction in a murine granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line. Cells were growth arrested by granulocyte macrophage colony-stimulating factor and serum deprivation and were allowed to proceed in the cell cycle by addition of the lymphokine in the presence or absence of 8-Br-cAMP. 3H-thymidine incorporation assays showed that addition of 8-Br-cAMP inhibited the entry of cells into S phase and the subsequent proliferation. Northern analysis showed that 8-Br-cAMP had opposite effects on c-fos and c-myc mRNA induction. 8-Br-cAMP induced c-fos in the absence of any GM-CSF. In the presence of GM-CSF, c-fos mRNA was superinduced (30-fold induction compared to four- to fivefold by each signal alone). On the contrary, 8-Br-cAMP was not able to induce c-myc in the absence of growth factor and hardly interfered with the induction of c-myc by GM-CSF. Phorbol myristate acetate (PMA), a pharmacological activator of the lipid and CA++-dependent protein kinase C, was shown to induce nuclear proto-oncogene mRNA in the GM-CSF-dependent cell line. We investigated the effect of 8-Br-cAMP on PMA-induced c-fos and c-myc mRNA levels. When both cAMP dependent and lipid-dependent kinase systems were co-stimulated in the absence of GM-CSF, c-fos message was again superinduced (60-fold induction). On the contrary, c-myc message induction by PMA was inhibited by 80% by coactivation of cAMP-dependent protein kinase with 8-Br-cAMP. Our data indicate that an antiproliferative signal induces or even superinduces c-fos message and hardly interferes with c-myc induction, suggesting that the intracellular pathways resulting in c-fos and c-myc induction may be distinct and that two different pathways can lead to c-fos induction, with synergistic effects when both are activated.     

Preferential induction of apoptosis in the rainbow trout macrophage cell line, RTS11, by actinomycin D, cycloheximide and double stranded RNA

The rainbow trout macrophage cell line RTS11 was found to be considerably more sensitive than rainbow trout fibroblast (RTG-2) and Chinook salmon epithelial (CHSE-214) cell lines to killing by macromolecular synthesis inhibitors, actinomycin D (AMD) and cycloheximide (CHX), a synthetic double stranded RNA (dsRNA), polyinosinic:polycytidylic acid (poly IC), and combinations of poly IC with AMD or CHX. Exposures of 24-30 h to AMD or CHX alone killed RTS11, but not CHSE-214 and RTG-2, in basal medium, L-15, with or without fetal bovine serum (FBS) supplementation. A two-week exposure to poly IC killed RTS11 in L-15, whereas RTG-2 and CHSE-214 remained viable. At concentrations that caused very little or no cell death, CHX or AMD pretreatments or co-treatments sensitized RTS11 to poly IC, causing death within 30 h. In all cases death was by apoptosis as judged by two criteria. H33258 staining revealed a fragmented nuclear morphology, and genomic degradation into oligonucleosomal fragments was seen with agarose gel electrophoresis. With AMD- or CHX-induced death, killing seemed caspase-independent as the pan caspase inhibitor, z-VAD-fmk, failed to block killing. By contrast, z-VAD-fmk almost completely abrogated killing by co-treatments of poly IC and low concentrations of AMD or CHX, suggesting caspase dependence. Killing by both types of treatments was blocked by 2 aminopurine (2-AP), which suggests the involvement of dsRNA-dependent protein kinase (PKR). The sensitizing of RTS11 to poly IC killing by AMD or CHX could be explained by a decrease in the level of a short-lived anti-apoptotic protein(s) and/or by the triggering of a ribotoxic stress.     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Response of cell surface glycosyl transferases to dibutyryl adenosine-3',5' cyclic monophosphate in virus-transformed and normal cells.

Galactosyl and sialyl transferases in the plasma membrane of SV40-transformed mouse cells were inhibited by 0.5 mM dibutyryl adenosine-3′,5′ cyclic monophosphate (db-cAMP) while those of normal cells did not respond to this compound. The differential effects of dibutyryl adenosine-3′,5′ cyclic monophosphate on the membrane-bound glycosyl transferases were observed both in isolated plasma membrane and in intact cell membrane. It is suggested that some of the morphological restorations of normal cell characteristics during reverse transformation are partly due to the direct effect of this compound on the cell membrane.     

Expression of cyclin A,B1 and D1 after induction of cell cycle arrest in the Jurkat cell line exposed to doxorubicin

Jurkat human lymphoblastoid cells were incubated in increasing concentrations of doxorubicin (0.05, 0.1 and 0.15 μM) to induce cell death, and their expression of cyclin A, B1 and D1 was evaluated by flow cytometry (cell cycle progression, Annexin V assay, percentages and levels of each of the cyclins), transmission electron microscopy (ultrastructure) and confocal fluorescence microscopy (expression and intracellular localization of cyclins). After low‐dose doxorubicin treatment, Jurkat cells responded mainly by G2/M arrest, which was related to increased cyclin B1, A and D1 levels, a low level of apoptosis and/or mitotic catastrophe. The influence of doxorubicin on levels and/or localization of selected cyclins was confirmed, which may in turn contribute to the G2/M arrest induced by the drug.     

The modulation of transcobalamin II (TC-II) production by cyclic adenosine 3',5'-monophosphate in the murine macrophage cell line J774: relationship to growth behavior

We undertook a study to define the role of cyclic AMP [cAMP] in modulating the secretion of transcobalamin II (TC-II) in the mouse macrophage like cell line J774. J774 was observed to secrete large amounts of TC-II, particularly in the presence of 8-bromo cAMP or cholera toxin or when grown in medium supplemented with low concentrations of horse serum (1% or 5%) or in serum-free medium. Variant cell lines derived from J774 and deficient either in adenylate cyclase (ac -) or cAMP-dependent protein kinase (pk -) activity showed very low and intermediate levels of basal secretory activity of TC-II, respectively, compared to J774. Maximum secretory activity of TC-II was observed in J774 under conditions in which growth was poorest (in the presence of 8-bromo-cAMP or 1% or 5% horse serum-supplemented medium or in serum-free medium). Cells grown in serum-free medium were found to have elevated basal adenylate cyclase activity and cAMP levels compared to those grown in medium supplemented with 20% horse serum. The data from this study demonstrate a negative correlation between growth activity and TC-II secretion in the J774 cell line. The stimulatory effect of exogenous cAMP on TC-II secretion by J774, the reduced secretory activity of the variant lines ac- and pk- and the observed increase in cell cAMP levels under conditions of serum starvation in which TC-II secretion is considerably enhanced, suggest that cell cAMP is an important modulator of TC-II secretion and growth behavior in the J774 cell line.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.     

Deletion mapping of Ig VH gene segments expressed in human CD5 B cell lines. JH proximity is not the sole determinant of the restricted fetal VH gene repertoire.

VH gene segments expressed in a panel of monoclonal human CD5 B cell lines have been positioned on the IgH locus by deletion mapping. The analysis yielded a relative order of VH fragments of the VH2, VH4, VH5, and VH6 gene families that was consistent with, and provided a further refinement of existing maps of the human IgH locus. We demonstrate that four of six VH gene segments expressed in the CD5 B cell lines map > 500 kb from the cluster of JH segments. Two of the gene segments, positioned at approximately 850 kb (58p2) and approximately 500 kb (1-9III) from the JH segments, respectively, belong to the previously identified small cohort of second trimester fetal VH gene segments. The data show that JH proximity is not the sole determinant of restricted VH gene utilization in early human ontogeny.     

The genetic organization of integrated hepatitis B virus DNA in the human hepatoma cell line PLC/PRF/5.

Hepatitis B virus (HBV) DNA is often found integrated in the genome of infected human liver cells and is supposed to be related to the development of primary liver carcinoma (PLC). Four clones of HBV DNA-containing sequences derived from DNA of the human PLC-derived cell line PLC/PRF/5 are discussed. The viral sequences show no intricate rearrangements excepting for a duplication and an inversion in one case, and a deletion in another. In all cases integration of the viral DNA was seen to be in a region which is single-stranded in the unintegrated HBV DNA. Sequence homologies between human and viral DNA flanking the integration sites have been detected. That may have a functional role in integration. Nucleotide sequence analyses of regions encompassing the viral-human junctions reveal open reading frames which consist of viral and/or human information. The possible expression of chimeric or cellular proteins may play a role in tumour development, and offers directions for further investigations.