Apoptosis. Fascinating death factor.
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Biomedical subjects
S Cory
Publications and source records attributed to S Cory.
B-lymphoid to granulocytic switch during hematopoiesis in a transgenic mouse strain.
B-lymphoid and myeloid development are markedly perturbed in a unique transgenic mouse strain, max 41. Pro-B, pre-B, and B lymphocytes were severely reduced but granulocytes were greatly elevated. This phenotype could be adoptively transferred with bone marrow cells. It was not alleviated by bcl-2 or myc transgenes that promote lymphocyte survival or proliferation. Bitransgenic myc/max 41 mice developed pre-B cell lymphoma. An accompanying massive granulocytosis unexpectedly proved to be clonally derived from the pre-B lymphoma cells. These observations suggest that B lymphopoiesis in max 41 mice has been diverted to granulocyte production. Since neither cell type expressed the transgene, this novel lymphomyeloid deviation probably reflects insertional alteration of a hematopoietic regulatory gene.
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Enhanced cell survival and tumorigenesis.
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Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow.
Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the "lineage-specific" markers CD45R (B220), Gr-1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with "compromised" marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.
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Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death.
In transgenic mice, self-reactive B lymphocytes are eliminated if they encounter membrane-bound self antigens during their development within the bone marrow. We show here that two separate and sequential events, arrested development and cell death, bring about B cell elimination. Developmental arrest is an early outcome of antigen binding in immature B cells, blocks acquisition of adhesion molecules and receptors important for B cell migration and activation, and is rapidly reversible by removal of antigen. Death of the arrested B cells occurs within 1 to 3 days and can be delayed by expression of a bcl-2 transgene, which results in escape of large numbers of self-reactive B cells from the bone marrow but fails to override the developmental arrest. These findings define a novel pathway for B cell elimination, involving an initial stage vulnerable to breakdown in autoimmune disease.
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Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox-2.4 homeobox gene.
The murine myelomonocytic cell line WEHI-3B exhibits ectopic expression of the genes encoding the homeobox protein, Hox-2.4, and the myeloid growth factor, interleukin-3 (IL-3). We showed previously that concomitant expression of IL-3 and Hox-2.4 in bone marrow cells induced the development of transplantable growth factor-independent tumours resembling the WEHI-3B tumour. We have now investigated the effect of enforced expression of Hox-2.4 alone. Bone marrow cells were infected with Hox-2.4 retrovirus and then either cultured in agar or transplanted into irradiated mice. In vitro, colonies derived from virus-infected cells readily yielded IL-3-dependent, non-tumorigenic cell lines of the myelomonocytic, megakaryocytic and mast cell lineages. Surprisingly, both the establishment and maintenance of these lines required very high concentrations of IL-3 and reduced levels promoted differentiation. Transplanted mice analysed after 3 months appeared normal but their spleen and bone marrow contained abundant provirus-bearing progenitor cells, from which IL-3-dependent long-term cell lines could readily be established in vitro. Four of 18 animals monitored for up to 12 months eventually developed clonal leukaemia, associated in three cases with IL-3 production. Thus ectopic expression of Hox-2.4 enhances self-renewal of immature myeloid progenitors and progression to a fully malignant state is favoured by somatic mutations conferring autocrine production of IL-3.
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E mu-bcl-2 transgene facilitates spontaneous transformation of early pre-B and immunoglobulin-secreting cells but not T cells.
To assess the lymphoid tumorigenic potential of bcl-2, mice of five independent strains expressing a bcl-2 transgene in B and/or T cells were monitored for disease up to 12 months of age. Lymphoma prevalence was minimal in the T lineage but significant, although low (3-15%), in the B lineage. The principal types of tumors were plasmacytomas secreting immunoglobulin and novel lymphomas that expressed markers such as Sca-1, CD4, Thy-1, CD34 and CD45(B220), consistent with an origin very early in B-lymphoid development. Rearrangement of the c-myc gene was common in the plasmacytomas, implying a synergistic role for myc and bcl-2 in their etiology, but was not detected in the lymphomas.
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A rapid procedure for screening fibroblast packaging cell lines for secretion of selectable retrovirus.
A simple method is described for rapidly screening fibroblast packaging cell line clones for high-titer secretion of retroviruses carrying a selectable marker. Virus-containing supernatants are used to infect FDC-P1 myeloid cells in 24-well tissue culture plates, selection is applied and wells containing live cells (infected by retrovirus and thus expressing the selectable marker) are detected after 5 to 10 days. The number of live cells in each well is proportional to the retroviral titer of the infecting supernatant. The assay is quick to set up and allows simultaneous screening of many samples.
3T3 Cells↗
bcr-abl-Induced cell lines can switch from mast cell to erythroid or myeloid differentiation in vitro.
The chimeric bcr-abl gene formed by the Philadelphia translocation is thought to initiate chronic myeloid leukemia. Engraftment of mice with bone marrow cells infected with a bcr-abl retrovirus has been shown to elicit multiple hematopoietic disorders, including a clonal but nontransplantable hyperproliferation of erythroid and/or mast cells. Culture of spleen and bone marrow cells from such mice usually yielded mast cell lines, even when erythroid disease dominated the primary animal. The mast cells, which carried the same proviral insert as the primary disease, generally grew slowly and were neither transplantable nor clonogenic in agar until they had been cultured for several months. Unexpectedly, several bcr-abl-induced lines switched in vitro from mast cell to megakaryocytic and/or erythroid character, and one became myeloid. The dramatic phenotypic shifts seem likely to involve changes occurring within progenitor cells maintaining the clone, rather than mutation of mature mast cells. The variant lines exhibited substantial spontaneous differentiation, despite being readily transplantable and therefore fully transformed. The production of hematopoietic growth factors by the mast cell lines and their phenotypic variants may implicate an autocrine loop in their evolution. These novel bcr-abl cell lines should aid in the study of genetic events in the progression from chronic to acute leukemia and facilitate analysis of hematopoietic lineage commitment.
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The role of bcl-2 in lymphoid differentiation and neoplastic transformation.
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Hematologic disease induced in BALB/c mice by a bcr-abl retrovirus is influenced by the infection conditions.
Irradiated mice reconstituted with bone marrow cells infected with a retrovirus carrying the bcr-abl oncogene of human chronic myeloid leukemia are subject to a range of neoplastic hematopoietic diseases, both myeloid and lymphoid. Comparison of DBA/2 and C57BL/6 mice has revealed a marked strain difference in susceptibility to the various tumor types. The present study, performed with BALB/c mice, indicates that the kinetics and nature of the induced disease can be modulated by the infection procedure, as well as the genetic background, and that retroviral regulatory sequences may influence the outcome. A distinctive clonal myeloproliferative disorder, somewhat akin to chronic myeloid leukemia but with prominent erythroid and mast cell components, as well as granulocytic excess, was characterized.
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Oncogene co-operation in leukaemogenesis.
The multistep development of haematopoietic malignancies, like other neoplasms, reflects sequential mutations that either activate proto-oncogenes or disrupt tumour suppressor genes. In a few spontaneous leukaemias or lymphomas, more than one mutation has now been identified, and the experimental analysis of oncogene co-operation is advancing rapidly via retroviral gene delivery and characterization of transgenic mice bearing oncogenes. In transgenic models, tumorigenesis can be accelerated by introducing another oncogene or by using a retrovirus as an insertional mutagen to identify cellular genes that collaborate with the transgene. Leukaemogenesis can be promoted by some ten pairs of oncogenes. The myc nuclear oncoprotein, for example, can collaborate with cytoplasmic oncoproteins such as ras, raf, bcl-2, pim-1 and v-abl, as well as with nuclear products such as bmi-1 or the tumour suppressor p53. The genes in such partnerships seem to provide complementary functions. For example, myc seems to prevent cells from becoming quiescent, whereas bcl-2 blocks programmed cell death; and others, for example ras, may diminish growth factor requirements. The products of genes that collaborate may lie on separate signal transduction pathways, leading to distinct nuclear targets. Key targets are postulated to be regulators of the cell cycle, especially the cyclins and associated kinases that govern progression in the G1 phase.
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bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship.
Early death is the fate of most developing T lymphocytes. Because bcl-2 can promote cell survival, we tested its impact in mice expressing an E mu-bcl-2 transgene within the T lymphoid compartment. The T cells showed remarkably sustained viability and some spontaneous differentiation in vitro. They also resisted killing by lymphotoxic agents. Although total T cell numbers and the rate of thymic involution were unaltered, the response to immunization was enhanced, consistent with reduced death of activated T cells. No T cells reactive with self-superantigens appeared in the lymph nodes, but an excess was found in the thymus. These observations, together with previous findings on B cells, suggest that modulated bcl-2 expression is a determinant of life and death in normal lymphocytes.
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Transgenic models of tumor development.
Numerous cancer-prone strains of mice have been created by the introduction of candidate tumor-promoting genes into fertilized eggs. Each transgenic strain is predisposed to develop specific types of tumors, but they usually arise stochastically because of the need for spontaneous mutation of genes that collaborate with the introduced oncogene. These mice are providing insights into the effects of individual oncogenes on cellular proliferation, differentiation, and viability, as well as on oncogene cooperativity. Their predisposed state imposes sensitivity to viral and chemical carcinogenesis, and the mice should prove valuable in tests of potential carcinogens, therapies, and preventive measures.
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Enforced BCL2 expression in B-lymphoid cells prolongs antibody responses and elicits autoimmune disease.
The biological functions of the BCL2 gene were investigated in transgenic mice harboring human BCL2 cDNA under the control of an immunoglobulin heavy chain enhancer (E mu). Mice of a representative transgenic strain, E mu-bcl-2-22, had a great excess of B lymphocytes, immunoglobulin-secreting cells, and serum immunoglobulins, attributable to increased longevity of B-lineage cells. Pre-B and plasma cells as well as B cells exhibited prolonged survival in culture. Immunized animals produced an amplified and protracted antibody response. Within the first year of life, most mice spontaneously produced antibodies to nuclear antigens, and 60% developed kidney disease, diagnosed as immune complex glomerulonephritis. Thus E mu-bcl-2-22 mice constitute a transgenic model for a systemic autoimmune disease resembling the human disorder systemic lupus erythematosus.
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Transgenic models for haemopoietic malignancies.
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Murine erythroid cell lines derived with c-myc retroviruses respond to leukemia-inhibitory factor, erythropoietin, and interleukin 3.
The transforming potential of the c-myc gene is shown here, for the first time, to include murine erythroid cells. Continuously growing cell lines were reproducibly generated by infection of day 13 CBA fetal liver cells with novel recombinant c-myc retroviruses. By cytostaining, most cells resembled early erythroblasts, but certain lines also contained significant numbers of hemoglobinized cells. RNA analysis revealed substantial expression of the genes encoding beta-globin and the erythroid-specific transcription factor GF-1. Although apparently immortal, the lines were not initially transplantable. Thus, constitutive myc expression in early erythroid cells can enhance their self-renewal capacity but is insufficient to fully transform them. The cell lines proliferated without the addition of exogenous factors, but their clonogenicity in semisolid medium was enhanced in the presence of erythropoietin, interleukin 3, and/or leukemia-inhibitory factor. In combination with either interleukin 3 or erythropoietin, leukemia-inhibitory factor also facilitated differentiation of certain lines. These results suggest that leukemia-inhibitory factor may have a previously unsuspected role in the regulation of erythropoiesis and could be considered as a possible therapeutic agent for the clinical management of erythroleukemia.
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Novel primitive lymphoid tumours induced in transgenic mice by cooperation between myc and bcl-2.
The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.
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