Point mutations in the c-Myc transactivation domain are common in Burkitt's lymphoma and mouse plasmacytomas.

We have screened the entire coding region of c-myc in a panel of Burkitt's lymphomas (BLs) and mouse plasmacytomas (PCTs). Contrary to the belief that c-myc is wild type in these tumours, we found that 65% of 57 BLs and 30% of 10 PCTs tested exhibit at least one amino acid (aa) substitution. These mutations were apparently homozygous in all BL cell lines tested and two tumour biopsies, implying that the mutations often occur before Myc/Ig translocation in BL. In PCTs, only the mutant c-myc allele was expressed indicating a functional homozygosity, but occurrence of mutations after the translocation. Many of the observed mutations are clustered in regions associated with transcriptional activation and apoptosis, and in BLs, they frequently occur at sites of phosphorylation, suggesting that the mutations have a pathogenetic role.

Binding and suppression of the Myc transcriptional activation domain by p107.

An amino-terminal transactivation domain is required for Myc to function as a transcription factor controlling cell proliferation, differentiation, and apoptosis. A complementary DNA expression library was screened with a Myc fusion protein to identify proteins interacting with this domain, and a clone encoding the Rb-related p107 protein was isolated. The p107 protein was shown to associate with Myc in vivo and to suppress the activity of the Myc transactivation domain. However, mutant forms of Myc from Burkitt lymphoma cells, which contain sequence alterations in the transactivation domain, were resistant to p107-mediated suppression. Thus, disruption of a regulatory interaction between Myc and p107 may be important in tumorigenesis.

Synthesis of kappa light chains by cell lines containing an 8;22 chromosomal translocation derived from a male homosexual with Burkitt's lymphoma.

Three cell lines were derived from a homosexual patient with probable acquired immunodeficiency syndrome and Burkitt's lymphoma. The cell lines produce an unusual strain of Epstein-Barr virus which will both transform cord blood lymphocytes and induce early antigens in Raji cells. Translocations between chromosomes 8 and 22 have occurred in all three lines, but the cells synthesize immunoglobulin M with light chains of the kappa type, in contrast to the usual concordance between a translocation involving chromosome 22 and lambda chain synthesis. Both kappa genes and one lambda gene are rearranged. These findings indicate either that translocation may occur as a separate event from immunoglobulin gene rearrangement or that the proposed hierarchical sequence of immunoglobulin gene rearrangements is not always adhered to. The data also imply that in cells containing a translocation between the long arm of chromosome 8 and a chromosome bearing an immunoglobulin gene, alteration of cellular myc expression may occur regardless of the immunoglobulin gene that is expressed.

B cell differentiation and immunoregulatory T cell function in human cord blood lymphocytes.

The functional maturity of T and B lymphocyte populations from human newborns was evaluated using a reverse hemolytic plaque assay to detect immunoglobulin-secreting cells generated in in vitro cultures stimulated with pokeweed mitogen (PWM), a T cell-dependent polyclonal activator, and the Epstein-Barr virus (EBV), a T cell-independent B cell activator. Cord blood lymphocytes failed to produce immunoglobulin in response to PWM, but did respond with immunoglobulin synthesis to stimulation with EBV. Co-culture experiments demonstrated that cord blood T cells would inhibit immunoglobulin production by adult cells stimulated with PWM, but not with EBV. Cord blood T cells did suppress immunoglobulin production by cord blood B cells when stimulated with a mixture of EBV and PWM, indicating that cord blood, in contrast to adult blood, contains a population of suppressor T cell precursors that are easily activated by PWM. Irradiation of the cord blood T cells with 2,000 rad eliminated the suppressor activity and revealed normal helper function for immunoglobulin (Ig) G, A, and M when these T cells were co-cultured with adult B cells. Cord blood B cells co-cultured with adult T cells or irradiated cord blood T cells did produce immunoglobulin in response to PWM, but the response was significantly lower than that of adult B cells, and only IgM was produced in these cultures. These studies demonstrate that both the T and B cells of the human newborn have significant functional differences compared with the functions of T and B lymphocyte populations in adults.

Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.

Cancer in developing countries: opportunity and challenge.

Epidemiologic observations indicate that environment and lifestyle are the major determinants of the geographical patterns of cancer. The developing countries, which account for 75% of the world's population, have lower incidence rates of cancer compared with the industrialized nations but bear more than half the global cancer burden. Demographic trends resulting from economic progress (decreasing incidence of infectious diseases, population growth, aging, and urbanization), coupled with increased tobacco consumption and dietary changes, indicate that developing countries will bear a continually increasing proportion of the world's cancer burden and its accompanying demand for the provision of costly treatment programs. Yet the developing countries command only 5% of the world's economic resources, and health care programs are already fully extended and frequently inadequate. Thus, cancer control in the developing countries, including preemptive prevention of the anticipated increases in cancers presently more common in the industrialized nations (e.g., lung, breast, and colon), should include much greater emphasis on cancer prevention than is presently the case. But there is another perspective. The developing countries, with their dramatic contrasts in lifestyles and environments and equally diverse patterns of cancer, provide an unparalleled, and often neglected, opportunity for studies directed toward understanding the mechanisms of environmental carcinogenesis. Such an understanding should eventually lead to the development of novel intervention approaches. Unfortunately, cancer research is much more difficult to conduct in the developing countries because of the lack of population-based registries, poor communication and transportation systems, and deficiencies in infrastructure, financial support, and the training of health professionals. These difficulties could be overcome, to the benefit of all, if the extent of collaboration in cancer research between the developing and industrialized nations were to be greatly expanded.

Use of fluoresceinated complement-coated bacteria and sheep erythrocyte-antibody-complement complexes for identification of complement receptors on lymphoid cell lines: differences in binding characteristics between cell lines of normal and malignant origin.

Twenty-four lymphoma-derived cell lines, 11 cord blood lymphocyte lines, and 3 lymphoblastoid cell lines derived from normal adults were examined for complement (C) receptors utilizing fluoresceinated C-coated bacteria (FBC) to determine the optimal conditions for each type of cell line. Incubation of FBC with lymphoma-derived cell lines at 37 and 0.5 degrees C showed that maximal FBC binding at both temperatures was after 120 minutes, and peak reactivity was invariably higher at 37 degrees C. These temperature-dependent differences were similar, both in Epstein-Barr virus nuclear antigen (EBNA)-positive and EBNA-negative lines. EBNA-positive lines, however, expressed higher levels of FBC rosettes than EBNA-negative lines at both temperatures. In contrast, FBC binding to cord blood cell lines after 120-minute incubation was maximal at 0.5 degrees C. Although similar numbers of FBC rosettes were formed after 30 minutes at both 37 and 0.5 degrees C in cord blood cell lines, rosette formation deteriorated after longer periods of incubation at 37 degrees C. The optimal temperature for FBC binding to lymphoblastoid cell lines could not be determined, since bacteria bound spontaneously to these lines at 37 degrees C. Cell lines were also tested simultaneously for sheep erythrocyte-antibody-complement complex (EAC)M and FBC binding at 37 and 0.5 degrees C. FBC reactivity under optimal conditions for each type of cell line correlated well with EACM reactivity at 37 degrees C. The significance of these results is discussed.

Persistence of transforming and nontransforming Epstein-Barr virus in high passages of P3HR-1 cell lines.

At least three laboratories have reported that the P3HR-1 line, which had originally produced transforming Epstein-Barr virus (EBV), now produces only the nontransforming variant. Studies to determine whether these findings were universal or a consequence of specific cell lines or culture conditions were undertaken in P3HR-1 cultures of identical HLA types from five sources. All of the EBV preparations derived from cell lines cultured at 32, 34, and 35 degrees C transformed cord blood lymphocytes, whereas virus propagated at 37 degrees C did not usually transform. Furthermore, indirect immunofluorescence revealed that a monoclonal antibody directed against transforming EBV membrane glycoprotein bound to 10-12% of the P3HR-1 cells that had been continuously propagated at 34 degrees C, but the antibody did not bind to the same cells cultured at 37 degrees C. Although virus expression was completely repressed in transformed cord blood cells, transforming virus could be rescued by superinfection with nontransforming P3HR-1 EBV. Cells transformed with P3HR-1 virus induced poorly differentiated lymphomas in athymic nude mice after seven or eight passages. Whether all P3HR-1 cells have the potential to produce detectable quantities of transforming virus remains to be determined.

Immunoglobulin delta expression in Burkitt's lymphoma cell lines.

Eighteen Burkitt's lymphoma (BL) cell lines were analyzed for rearrangement and expression of the delta gene. None had rearrangement of the delta gene locus within the 9.0-kb BamHI restriction fragment. Cell lines that expressed the delta gene contained both mu alleles, with at least one productively rearranged. Thirteen of 18 cell lines had detectable transcripts hybridizing with the delta probe. In 10 of the 13 cell lines with delta transcripts, cytoplasmic delta chains were detected, but only two of these expressed delta chains strongly on the surface. All 13 lines made cytoplasmic mu chains, and all except one made cytoplasmic light chains. Surface IgM was detected in all except two of the 13 cell lines. Although BL has generally been considered not to express IgD, except in occasional cases, previous studies have been confined to examination of the cell surface. Many of the cell lines that we examined express delta mRNA transcripts as well as produce cytoplasmic delta chains but no detectable surface IgD. This suggests that delta chains are detectable in the cytoplasm prior to being apparent on the surface. Our findings argue against an origin of BL from germinal center cells since IgD is almost totally lacking in normal B cells present in germinal centers.

Serial cytogenetic studies of nonendemic Burkitt's lymphoma cell lines.

Cytogenetic study of nonendemic Burkitt's lymphoma (BL) cell lines showed a translocation of chromosomes #8 and #14, t(8;14), present in 16 of the 18 lines examined. Serial cytogenetic studies of nine of these lines showed the t(8;14) to be stable and present in all cells examined. Although many other chromosome aberrations were present, they did not demonstrate the stability or the pervasiveness of the t(8;14). The significance of these results and previously reported cytogenetic studies on the etiology of BL was discussed.

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.

Epstein-Barr virus-associated and other antiviral antibodies during intense BCG administration to patients with Burkitt's lymphoma in remission.

Patients with Burkitt's lymphoma in chemotherapy-induced remission received through dermal scarifications one or two doses per week of approximately 3 X 10(8) living BCG organisms (Pasteur Institute vaccine). This treatment was always followed by usually rapid increases by 1--4 log2 steps in the antibody titers to Epstein-Barr virus (EBV)-associated cell membrane antigens. Titer increases of less than 2.5 log2 steps within the first month after the start of BCG treatment correlated with a significantly elevated frequency of extradural relapse as compared to that seen in patients with larger titer rises. During this time, antibodies to EBV-associated viral capsid antigens and early antigens of D and R specificity, as well as antibodies against herpes simplex, varicella, cytomegalovirus, measles, and respiratory syncytial virus antigens, did not show any consistent or impressive changes.

Molecular basis of lymphomagenesis.

Lymphoid neoplasms, like all malignant tumors, arise as a consequence of the accumulation, in a single cell, of a set of genetic lesions that result in altered proliferation or increased clonal life span. The most frequently observed genetic abnormalities among the malignant non-Hodgkin's lymphomas are translocations, which appear to be lineage and, to a large extent, lymphoma specific. Recombinases that normally mediate the process of antigen receptor gene rearrangement appear to have an important (but not exclusive) role in the mediation of these translocations and of other types of gene fusion (e.g., deletion of intervening DNA). Frequently, such fusions result in the increased or inappropriate expression of crucially important proteins, many of which are transcription factors that regulate the expression of other genes. These abnormalities, however, do not appear to be sufficient to induce lymphoma, and it is likely that the additional genetic lesions required differ from one tumor to another. The likelihood of any given clone of cells accumulating a sufficient number of relevant genetic lesions to give rise to a lymphoma is probably a function of its life span. Prolonged survival of a cell clone may be mediated by viral genomes (e.g., Epstein-Barr virus and human T-cell leukemia/lymphoma virus type 1), by the abnormal expression of cellular genes that inhibit apoptosis (e.g., bcl-2), or by the mutation or deletion of cellular genes that are necessary for apoptosis, e.g., p53. The background rate at which genetic lesions occur is amplified by the interaction of inherited and environmental factors, the latter appearing to be the major determinant of incidence rates. However, inherited factors that influence lymphomagenesis, including variability in the ability to repair DNA damage or in the fidelity of antigen receptor recombinases for their signal sequences, may be crucial determinants of which particular individuals in a given environmental setting develop lymphoma.

Expression of surface antigens during the cell cycle in different growth phases of American and African Burkitt's lymphoma cell lines.

We have studied the expression of five surface antigens in eight Burkitt's lymphoma cell lines during different phases of the cell cycle and in different growth phases (logarithmic and stationary). Cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and DNA content (propidium iodide), and dual parameter measurements were performed with a flow cytometer. Analysis of cells in specific cell cycle phases during log-phase growth revealed a 1.6-fold increase in surface antigen expression as cells passaged from G1 to G2/M. This is almost identical to the measured increase in cell surface area which occurs during passage of cells through the cell cycle and indicates that under optimal conditions surface antigen density is maintained during cell doubling. We also observed a consistent reduction, by about 50%, in the expression of surface IgM (mu), k-light chain, and B1 on the cell lines during a 5-day culture period. Cell lines that only weakly expressed surface IgM were found to have a more rapid decrease, and in such cell lines IgM was ultimately completely lost from the cell surface. In contrast, the expression of beta 2-microglobulin and HLA-ABC increased in some cell lines, whereas in others a significant decrease of both beta 2-microglobulin and HLA expression was demonstrated as the cells entered stationary growth phase. Decreased cell volume (and therefore surface area) associated with declining growth rate and fewer late S or G2/M cells could account for 20-30% of the observed reduction in surface IgM, k-light chain, and B1 expression, but the major decrement in fluorescence intensity was due to a reduction in the density of these surface antigens. Thus, the ability to maintain surface antigen densities is frequently lost in suboptimal culture conditions.

Comparison of energy metabolism in human normal and neoplastic (Burkitt's lymphoma) lymphoid cells.

In order to detect possible differences in the energy metabolism between normal and neoplastic lymphoid cells, we studied purified normal human lymphocytes (FL) and transformed lymphoblastoid cell lines derived from umbilical cord blood (CL) and compared them to cell lines derived from American Burkitt's lymphoma (BL). The total adenosine triphosphate production rate by these cells was estimated by measuring O2 consumption and lactic acid production rates. O2 consumption (nmol/min/mg protein) was 4.9 +/- 0.3 (S.D.) in CL, 4.4 +/- 0.3 in FL, and 4.9 +/- 0.3 in BL. Lactic acid production (nmol/min/mg protein) was 30.9 +/- 3.0 in CL, 29.9 +/- 3.0 in FL, and 23.4 +/- 4.0 in BL. Using these values of O2 consumption and lactic acid production, the average adenosine triphosphate production rates (nmol/min/mg protein) were calculated to be 60 in CL, 56 in FL, and 53 in BL. We conclude that the BL do not have more aerobic glycolysis than do normal lymphoid cells, suggesting that the lactic acidosis seen in American Burkitt's lymphoma is not due to a preferential glycolytic metabolism of the tumor. More likely, the lactic acidosis is simply due to the large total mass of these neoplastic cells and not due to a modification of their energy metabolism.

Plasma membrane lipid structural order in doxorubicin-sensitive and -resistant P388 cells.

We have studied the structural order of the lipid phase of plasma membranes from P388 murine leukemia cells and from a Doxorubicin-resistant subline, P388/ADR, using electron spin resonance spectroscopy and fluorescence depolarization measurements. Measurements of the order parameter, S, following incubation of cells from both lines with the N-oxyl-4'-4'-dimethyloxazolidine derivative of 5-ketostearic acid show higher values for the resistant cells at all temperatures where S was measured (4-37 degrees). Fluorescence depolarization measurements following incubation of the cells, or cell fractions, with 1,6-diphenylhexatriene indicate more restricted motion of the probe in resistant cells. These measurements also show increased amounts of cytoplasmic lipid in the resistant cells. The higher degree of structural order in the lipid phase of the plasma membranes of P388/ADR cells and their larger intracellular lipid content may account for the decreased rate of intracellular accumulation of anthracycline drugs (and other compounds) seen in these cells and, in part, for their relative resistance to the cytotoxic effects of these drugs.

Characterization of the Epstein-Barr virus isolated from a cell line derived from a patient with American Burkitt's lymphoma.

Epstein-Barr viral (EBV) DNA is nearly always detectable in African Burkitt's lymphoma (BL) but is infrequently found in the histologically indistinguishable American BL. We have derived a tumor cell line from a patient with American BL which produces EBV, and we have compared this virus isolate [JLP(c)] with African BL EBV. The American JLP(c) virus immortalizes human umbilical cord lymphocytes in vitro, and its DNA is indistinguishable from African BL EBV DNA by nucleic acid hybridization and preliminary restriction endonuclease cleavage analysis.

Role of cytoplasmic lipids in altering diphenylhexatriene fluorescence polarization in malignant cells.

Prior studies of fluorescence anisotropy (polarization) with diphenylhexatriene in normal and malignant cell populations have shown differences which have been attributed to an altered membrane lipid composition in cancer. We studied fresh tumor cells from patients with diverse lymphoid neoplasms and found a discrete range of whole-cell fluorescence polarization values (P values) for each type of neoplasm. Following cell fractionation, however, the P values of isolated plasma membranes from malignant cells did not differ significantly from the values obtained with normal donor lymphocytes. Therefore, the altered whole-cell fluorescence polarization measurements in malignant cells are not likely to be due to gross lipid changes in the plasma membrane. Histochemical staining and cell fractionation revealed the presence of cytoplasmic lipid accumulations, and these had extremely low P values, which could account for the low P values of malignant cells. Complementary studies of lymphoid cell lines showed whole-cell fluorescence polarization measurements to be extremely sensitive to exogenous lipid supplements, but membrane values remained stable. We conclude that alterations in membrane lipid fluidity, as measured by the diphenylhexatriene probe, are not consistently found in lymphoid neoplasms and hence cannot presently be invoked to account for the malignant behavior of these cells. However, intracellular neutral lipid accumulation appears to be a common feature of the lymphoid neoplasm. The lipid alterations described could be characteristic of cell immaturity or proliferation rather than malignancy; nevertheless, they may convey unappreciated biological consequences.

Drug-induced changes in DNA fluorescence intensity detected by flow microfluorometry and their implications for analysis of DNA content distributions.

Chicken erythrocytes, which contain less DNA than mammalian diploid cells, were used as an internal standard to control instrumental and staining variables during flow microfluorometric analysis. With the DNA stain, mithramycin, and with an EPICS II flow microfluorometer, ratios between the modal G1 fluorescence of experimental cells and that of chicken erythrocytes were determined. The results indicate that unperturbed cell populations of L1210 and HeLa cells in vitro and L1210 ascites cells in vivo have relatively stable fluorescence ratios, although there is a significant difference between the ratios of one L1210 cell line in vitro and another in vivo. In contrast, L1210 ascites treated in vivo with different schedules of cyclophosphamide and Adriamycin showed wide fluctuations in the fluorescence intensity ratios for 96 hr after treatment. Also, differences in the fluorescence ratios were observed between less advanced and more advanced L1210 ascites after treatment with the same schedule. These effects indicate an alteration in DNA staining with mithramycin, brought about by drug treatment that could seriously affect the interpretation of DNA histogram data. Nevertheless, changes in mithramycin staining may prove to be a very important probe to detect persistent drug effects.