Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.     

Functional diversity within the suppressor phenotype as defined by monoclonal antibody in T-cell prolymphocytic leukemia

A patient with T-cell prolymphocytic leukemia (T-PLL) is described. The malignant T-cells from the patient were predominantly Leu-2-positive, indicating a suppressor phenotype. The cells were then tested to determine their functional capabilities. The patient's Leu-2-positive cells initially suppressed B-cell proliferation, as predicted by their phenotype but later functioned as T helper cells in the pokeweed mitogen system without a change in phenotype. The cells also responded inadequately to alloantigen and mitogen despite addition of exogenous T-cell growth factor (TCGF). Leu-2-positive prolymphocytes from the spleen of the patient were constitutive producers of TCGF. Surface phenotype using monoclonal antibody was inadequate to predict T-cell function of the cells from this patient with T-PLL. In addition, these data suggest there may be functional subpopulations within the OKT8+ phenotype. Constitutive TCGF production by malignant post-thymic T-cells may represent a mechanism by which these cells sustain their own growth.     

Biologic effects of prolonged melphalan treatment of murine long-term bone marrow cultures and interleukin 3-dependent hematopoietic progenitor cell lines

The mechanism of alkylating agent-induced leukemia is unknown. For the determination of whether chronic alkylating agent treatment of hematopoietic stem cells in vitro was detectably leukemogenic, murine long-term bone marrow cultures (LTBMC) and clonal interleukin 3 (IL-3)-dependent multipotential hematopoietic progenitor cell lines [B6SUtA clone (cl) 27 and Ro cl 3-1] derived from LTBMC were chronically pulse treated in vitro with the alkylating agent melphalan [L-phenylalanine mustard (L-PAM)]. Weekly treatment of C3H/HeJ or CD-1 Swiss mouse LTBMC with 3 X 10(-6)M L-PAM significantly decreased cumulative production of nonadherent granulocytes and granulocyte-macrophage progenitor cells responsive to L-cell or WEH1-3 cell colony-stimulating factor compared to the production seen in untreated control cultures; it also significantly reduced the hematopoietic longevity (13 wk compared to greater than 20 wk for untreated control cultures). Weekly, twice weekly, or daily (3 X 10(-6)M) L-PAM treatment of IL-3-dependent cell lines induced gradual L-PAM adaptation in the absence of a detectable change in the maximum binding capacity of 125I-labeled IL-3. No leukemogenic variants of line B6SUtA cl 27 were detectably induced. However, 3 stably expressed marker chromosomes were induced after 12 months of L-PAM treatment of line B6SUtA cl 27. Thus IL-3-dependent hematopoietic progenitor cells slowly adapt to L-PAM when in suspension culture in vitro. Physiologic expression of drug toxicity in LTBMC may prevent this hematopoietic cell gradual adaptation.     

Enhanced induction of growth of B lymphoblasts from fresh human blood by primate type-C retroviruses (gibbon ape leukemia virus and simian sarcoma virus).

Cell lines were established from the fresh peripheral blood of over 50% (11 of 21) of Epstein-Barr virus (EBV) seropositive human donors after exposure to viruses of the gibbon ape leukemia virus (GALV)—simian sarcoma virus/simian sarcoma-associated virus (SiSV/SiSAV) group. The incidence of growth induction upon exposure to baboon endogenous virus (BaEV), feline leukemia virus (FeLV) or to uninfected monolayer cultures was lower than 10%. Leukocytes from the peripheral blood of leukemic patients gave similar results, but exposure of fresh peripheral blood from eight normal EBV seronegative donors to the same viruses did not result in induction of cell growth. The established cell lines were polyclonal B-lympho-blasts as determined by morphological cytochemlcal, immunological and functional properties. The cells expressed EBV nuclear antigen (EBNA test), formed rosettes with complement-activated sheep or bovine erythrocytes (EAC-rosette test), but not with untreated erythrocytes (E-rosette test), and were positive for one or more surface-bound immunoglobullns. They were also karyotypically normal as determined by standard chromosome analysis and by trypsin-Giemsa banding techniques. However, unlike other reported newly-established diploid B-lymphoblast cell lines, cells from approximately 80% of the cultures tested grew as locally invasive tumors when inoculated into 1-to 3-week-old athymic nude mice and formed colonies in semi-solid medium. Lymphoblasts from nude mouse tumors were readily re-established as suspension cultures which retained their lymphoblastic properties and remained karyotypically normal. These studies indicate that under certain conditions, type-C retroviruses of the GALV-SISV/ SISAV group directly or indirectly increase the incidence of transformation of human B lymphocytes.     

Helper-independent and replication-defective erythroblastosis-inducing viruses contained within anemia-inducing Friend virus complex (FV-A)

Helper-independent and replication-defective viruses contained within the anemia-inducing Friend virus complex have been analyzed. Helper-independent ecotropic viruses, F-MuLV, have been isolated which produce rapid hepatosplenomegaly and anemia when injected into newborn Swiss mice, but which do not cause this disease syndrome when injected into adult NIH Swiss mice. The histology of the enlarged spleens shows many erythroblasts and some areas of clear erythroid maturation in the later stages of the disease. A replication-defective virus has also been isolated which is an env gene recombinant virus and codes for a 52,000-dalton glycoprotein which has a specific immunological relationship to the gp70 of MCF murine leukemia viruses. This replication-defective virus (SFFV_FV-A) shows many similarities to the previously studied replication-defective virus found in polycythemia-inducing Friend virus stocks (SFFV_FV-P). However, clear differences between SFFV_FV-P and SFFV_FV-A also exist. Pseudotypes of SFFV_FV-A cause rapid proliferation of erythroid precursors when injected into either newborn or adult Swiss mice. This disease is characterized by splenomegaly, friable spleens, normal or moderately low hematocrits, and splenic foci. Pseudotypes of SFFV_FV-P also cause rapid proliferation of erythroid precursors when injected into either newborn or adult Swiss mice. This disease is characterized by splenomegaly, firm spleens and large foci, and normal or elevated hematocrits. The possible relationships between F-MuLV, SFFV_FV-A, and SFFV_FV-P are discussed.     

Human trophoblasts: cellular source of colony-stimulating activity in placental tissue

Colony-stimulating activity (CSA) is a class of protein factors that stimulates the in vitro growth and differentiation of hematopoietic committed stem cells into mature granulocytes and macrophages. In man, production of CSA is mediated by monocytes-macrophages, lymphocyte, and endothelial cells. One of the best studied and most potent sources of CSA is conditioned media derived from cultured human placenta. The cellular source of this placental CSA was studied using cloned term placental cell lines induced by a simian virus 40 (SV-40) Wild-Type (wt) or temperature sensitive (tsA) mutants. Conditioned media from SV-40 wt-transformed placental cells grown at 37 degrees C and tsA-transformed placental cells grown at 33 degrees C (the temperature at which the cells exhibit the transformed phenotype) and at 40 degrees C (temperature at which the cells express a normal differentiated phenotype) contained high levels of CSA. At the restricted temperature (40 degrees C), these cell lines expressed normal trophoblastic characteristics in that they produced human chorionic gonadotropin, pregnancy-specific-beta1-glyco protein, and placental alkaline phosphatase, indicating that the SV-40 transformation has not interfered with normal cellular functions of these trophoblasts. The CSA released by these cell lines resemble those of CM from fresh placental cells, in that the level of activity is high and that formation of both granulocyte/macrophage and eosinophil colonies is stimulated.     

Normal DBA/2 mouse cells synthesize a glycoprotein which interferes with MCF virus infection

The mechanism of resistance to Friend leukemia virus-induced [mink cell focus-inducing, (MCF)-mediated] leukemogenesis in DBA/2 mice was investigated in cell culture systems. DBA/2 fibroblasts were found to be resistant to infection with MCF viruses but not to ecotropic or amphotropic murine leukemia viruses (MuLV's). Since this resistance has been correlated with the presence of an MCF virus-related gp70 constitutively present on the surface of DBA/2 cells, it seemed possible that the mechanism of resistance in this system involved the saturation of MCF-specific cell surface receptors with the gp70 in analogy to viral interference. Two inhibitors of glycoprotein synthesis, 2-deoxy-d-glucose and tunicamycin, which have been shown to reduce retrovirus-induced interference in productively infected cells, significantly decreased the resistance of DBA/2 cells to productive infection with MCF viruses. This decrease in resistance to MCF virus infection could be correlated with a decrease in the expression of MCF-related gp70 at the cell surface. Cells from several other mouse strains showed neither resistance to MCF virus infection nor enhancement of MCF infectivity following drug treatment. These data indicate that DBA/2 cells are resistant to MCF infection in vitro and, by implication, to MCF-mediated leukemogenesis in vivo by a process analogous to viral interference. Ecotropic pseudotypes of MCF virus were able to productively infect untreated DBA/2 cells, indicating that the cell surface interference-like process described here is the only restriction mechanism responsible for resistance to MCF infection in DBA/2 cells. The results are consistent with the idea that Friend leukemia virus actually causes disease via MCF intermediates.