Antiimmunoglobulin inhibition of Burkitt's lymphoma cell proliferation and concurrent reduction of c-myc and mu heavy chain gene expression.

We have demonstrated that polyvalent antiimmunoglobulin antibodies directed at appropriate cell surface light (L) or heavy (H) immunoglobulin (Ig) chains will inhibit proliferation and the expression of c-myc and mu-Ig chain mRNA in Burkitt's lymphoma (BL) cell lines bearing 8;14 chromosomal translocations. This effect was not observed in BL cell lines bearing 8;22 translocations or in BL cell lines which did not express surface Ig or in karyotypically normal Epstein-Barr virus-transformed lymphoblastoid cell lines. The antiproliferative effect was reproducible and resulted in cell death in the most sensitive cell lines. The decrease in gene expression preceded the antiproliferative effect. The effect of anti-Ig on gene expression was relatively specific since the level of total (shown by Northern blots) and cytoplasmic (dot blots) mRNA of several other genes (beta-actin, G6PD, kappa-L chain) and the first exon of c-myc (in cell lines in which this exon is expressed separately from the second and third exons) was not changed in these same BL cell lines. Expression of both c-myc and mu was maximally inhibited between 3 and 6 h after the addition of anti-Ig. In the most sensitive BL cell line, concurrent reduction in c-myc and mu mRNA was noted as early as 1 h after anti-Ig and the nadir of expression of these genes occurred at 3 h. These results indicate that the deregulated high constitutive expression of c-myc in some BLs can be down-regulated by anti-Ig resulting in inhibition of proliferation and cell death. In addition these data are consistent with the possibility that in at least some 8;14 bearing BLs the malignant transformation occurs in an immature B-cell undergoing antigen-independent differentiation.

Complement receptor expression by neoplastic and normal human cells.

Complement receptor (CR) expression in cell lines derived from Burkitt's lymphoma (BL), Epstein Barr virus-transformed cord blood lymphocytes (CB), and peripheral lymphocytes from patients with infectious mononucleosis (IM) was examined. Red cell intermediates bearing various densities of C4b, C3b, C3bi, or C3d were tested for rosette formation with the cell lines. In addition, a series of studies was performed under conditions that precluded the cleavage of cellbound C3b by Factor I (C3bINA). These conditions did not alter rosetting by the cells that were tested. RAJI cells rosetted with EAC3bi greater than EAC3d greater than EAC3b, but not with EAC4b. EAC3b/RAJI rosette formation required much greater quantities of C3b bound to red cells than did CB and IM lines, which unlike RAJI, also bound EAC4b. All of the BL lines failed to bind EAC4b even at a C4b density of 50,000 molecules/cell, but several lines did form rosettes with EAC3b, and most formed rosettes with EAC3bi and EAC3d. Fluid phase C3b blocked RAJI/EAC3b rosetting while having little effect on RAJI/EAC3bi rosette activity. Moreover, fluid phase C3b, as well as C4b, blocked RAJI/EAC3b rosettes more effectively than CB/EAC3b rosettes. The results indicate that the RAJI cell line has a receptor for C3b, with characteristics that differ markedly from the C3b receptor of cell lines derived from CB lymphocytes and of lymphoblastoid cell lines derived from patients with IM. This receptor is capable of interacting with soluble but not cellbound C4b. In these studies, rosette formation was examined under various ionic conditions. RAJI/EAC3b rosette formation was severely reduced as ionic strength was increased, whereas RAJI/EAC3bi binding was only moderately decreased at physiologic ionic strength. In striking contrast, EAC3bi binding to monocytes, PMN, and human erythrocytes was markedly reduced as ionic strength increased, but EAC3b binding to these cells was less sensitive to changes in ionic strength. Under conditions of physiologic ionic strength, the C3bi receptor of phagocytic cells may be at a functional disadvantage in the binding of C3bi-coated particles. This may have major physiologic implications.

Induction of complement receptor expression in cell lines derived from human undifferentiated lymphomas. II. Characterization of the induced complement receptors and demonstration of the simultaneous induction of EBV receptor.

We have studied the specificity of complement receptors induced by theophylline in 2 cell lines derived from undifferentiated lymphomas, one of Burkitt's type, and compared it to that of complement receptors in other cell types. Both C3b and C3d receptors were induced. The induced C3b receptor differed from the C3b receptor of mature normal lymphocytes, polymorphonuclear leukocytes and the cells of a nodular lymphoma in 2 respects. Firstly, it bound C3b much less avidly (by a factor of several hundred-fold) and secondly, we were unable to demonstrate C4b binding. EBV receptors were induced at the same time as complement receptors, and permitted the conversion of a greater fraction of cells to EBNA positivity after experimental infection with EBV. The induction of receptors was not associated with a change in the fluidity of the plasma membranes and our data do not favor a different orientation of induced receptors within the membrane as compared to receptors of other cell types--a potential explanation for the different specificities. Our findings are consistent with the possibility that the complement receptors of lymphocyte precursors differ from these of mature lymphocytes.

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics.

Sixteen lymphoid cell lines were derived from patients with undifferentiated lymphoma of Burkitt's or non-Burkitt's type. They were obtained directly from tumor biopsies, from serous effusions, or from bone marrow. In 10 of the cell lines, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was undetectable; the remaining 6 lines were EBNA-positive (EB-pos). Of the 16 lines, 15 were aneuploid, with detectable chromosome "14q+ markers (11 had +8;14 translocations). These 15 lines, which included the EBNA-negative (EB-neg) lines, were believed to be of tumor cell origin. The remaining line consisted predominantly of diploid cells derived from normal lymphocytes, but some cells of tumor origin were present. Four EB-pos cell lines derived from EB-neg tumors had an aneuploid karyotype consistent with an origin from tumor cells (including no.8;14 translocation in two), which suggested that either tumor cells were infected with EBV in vitro or a tiny fraction of EB-pos tumor cells (or potential tumor cells) present in vivo gave rise to the predominant cell of the line. EB-neg B-cell lines and EB-pos cell lines established from undifferentiated lymphomas differed greatly. EB-neg lines had consistently smaller electronic mean cell volumes and narrow-angle light scatter than did EB-pos lines. This finding correlated with a lower nuclear:cytoplasmic ratio in EB-pos lines. EB-neg lines also had higher saturation cell densities than did EB-pos lines under standard culture conditions. The data indicate either that EBV influences the morphologic and physiologic characteristics of lymphoid cell lines or that EB-neg B-cell lines and EB-pos cell lines are derived ultimately from different lymphocyte subpopulations or that both may apply.

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. II. Surface markers.

Fifteen of 16 lymphoma-derived cell lines and the Faji and P3HR1 cell lines were characterized with regard to certain surface markers, particularly immunoglobulins, complement receptors, Epstein-Barr virus (EBV) receptors, and Fc receptors. Ten lines positive for EBV nuclear antigen (EB-pos) were stained weakly or not at all by antihuman immunoglobulin fluorescein isothiocyanate conjugates, whereas EBV nuclear antigen negative (EB-neg) cell lines stained brightly. EB-pos lines frequently manifested Fc receptors, particularly for 7S antibody, whereas EB-neg lines did not. Receptors for the C3b component of complement and for EBV, which correlated significantly with each other, were expressed to a much lesser extent by EB-neg lines than by EB-pos lines. These findings are pertinent to an understanding of the infrequent association of this virus with American undifferentiated lymphomas of the Burkitt's and non-Burkitt's types.

Cellular interactions in the proliferative response of human T and B lymphocytes to phytomitogens and allogeneic lymphocytes.

In vitro studies were performed to determine the proliferative responsiveness of human peripheral blood thymus-dependent (T) and thymus-independent (B) lymphocytes to phytomitogens and allogeneic lymphocytes. Recombination of T and B cells, with selective inhibition of proliferation of one of the two populations, was used to identify cellular interactions which may contribute to cell proliferation. The distinctive feature of human T lymphocytes to form rosettes with unsensitized sheep erythrocytes was utilized to separate human peripheral blood lymphocytes into highly purified resetting (T) and non-rosetting (B) cells. The proliferative response of these separated lymphocyte subpopulations to various stimulants was assessed from the uptake of tritiated thymidine into DNA. Phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic lymphocytes stimulated separated T cells, whereas no proliferation was observed with the T-cell-depleted B-cell population. This suggests that it is the human T cell which is activated directly by these stimulants. In the presence of T cells (proliferating or nonproliferating), B cells were capable of proliferation following stimulation with phytomitogens, but not in response to histocompatibility antigens. Thus, T-cell-mediated B-cell proliferation contributes to the overall lymphocyte response in phytomitogen-stimulated T + B cell mixtures, but not in human mixed leukocyte cultures. T-cell activation by allogeneic cells required the presence of monocytes; in contrast, the three tested phytomitogens stimulated T cells in the absence of monocytes. This indicates that direct interaction of mitogens with lymphocyte membrane receptors is sufficient to trigger T cells into proliferative response. However, monocytes considerably enhanced the proliferative response of T cells in a dose-dependent fashion; this monocyte-dependent mechanism of T-cell activation was predominant at lower concentrations of phytomitogens, and contributed relatively less at higher mitogen doses. Both, the direct, monocyte-independent, and the indirect, monocyte-dependent T-lymphocyte activation contribute to the total in vitro response of lymphocyte preparations to phytomitogens.