The cooperation of oncogenes in the transformation of primary rat Schwann cells is a strikingly synergistic process. We have explored the molecular mechanisms involved. Activation of an inducible Raf kinase results in morphologically transformed cells that are arrested in G1 via the induction of p21 ¢ipl and subsequent inhibition of cyclin/cdk activity. In contrast, coexpression of SV40 large T (LT) or a dominant-negative mutant of p53 abolishes p21Cipl induction and alleviates the growth arrest. Moreover in this scenario, Raf activation results in an increase in the specific activity of cyclin/cdk complexes with Raf and LT cooperating to superinduce cyclin A/cdk2 activity and stimulate proliferation in the absence of mitogens. Thus, signaling by Raf and its cooperating partners converges at the regulation of cyclin/cdk complexes, with the cellular responses to Raf modulated by p53.
The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL lOCUS was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (V,) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive v-J joining, such that nearly all v-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo Ig, rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.
Chicken immunoglobulin light chain (IgL) gene rearrangement has been characterized. Rearrangement of the single variable (VL) segment with the single joining (JL) segment within the chicken IgL locus results in the deletion of the DNA between VL and JL from the genome. This deletion is accomplished by a molecular mechanism in which a precise joining of the IgL recombination signal sequences leads to the formation of a circular episomal element. The circular episome is an unstable genetic element that fails to be propagated during B cell development. Evidence was obtained that the formation of the circular episome is accompanied by the addition of a single nonrandom base to both the VL and JL coding segments. The subsequent joining of the VL and JL segments appears to occur at random, as we observed at least 25 unique V-J junction sequences, 11 of which are out-of-frame. A novel recombination mechanism that accounts for the observed features of chicken IgL gene rearrangement is discussed.
We have documented the effect of two nondefective helper viruses, reticuloendotheliosis virus A (REV-A) and chick syncytial virus (CSV) infection on bursal tissue. REV-A infection results in bursal atrophy, destroying both its structural and functional integrity. In contrast, the bursae in CSV-infected chicks, while reduced slightly in size, appear both structurally and functionally normal. REV-A-induced bursal atrophy is not a result of viral replication in the B-lymphocyte as (a) both viruses are capable of inducing, with equal efficiency, the formation of preneoplastic lesions containing proliferating B lymphocytes and (b) it appears that equivalent amounts of viral antigen are expressed in the bursae of chicks infected with either virus. We have examined the phenotype of tumors induced by the replication-defective virus REV-T when replicated by the two different helper viruses, REV-A and CSV. In REV-T(REV-A)-infected chicks, the majority of tumors that develop are negative for IgM expression. In contrast, the majority of tumors induced by REV-T(CSV) infection are IgM+. This finding is confirmed by recovery of IgM- cell lines from REV-T(REV-A)-infected chicks and IgM+ cell lines from REV-T(CSV)-infected chicks. In addition, repopulation studies show that bursal-derived cells that are IgM+ serve as target cells for REV-T(CSV)-induced lymphomas. This study demonstrates, therefore, that REV-T can induce IgM+, B cell lymphomas with high efficiency. We conclude that infections by the helper viruses, REV-A and CSV, differ dramatically in their effects on the composition of the population of cells that serve as targets for REV-T-induced neoplasia.
The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.
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