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.

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.

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.

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.

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.

Switching viral latency to viral lysis: a novel therapeutic approach for Epstein-Barr virus-associated neoplasia.

We describe an EBV-driven lytic system (LySED) that can be used to specifically target therapy to EBV- containing tumors. This system takes advantage of the transactivating properties of EBNA-1, a latency protein expressed in all EBV-containing cells, to drive the expression of Zta, a gene sufficient for inducing the EBV lytic cycle. Thus, EBV provides both the target and the executor for mediating tumor-specific cell death, markedly increasing the specificity of the system. Transfection of EBV-positive cell lines with the LySED construct resulted in a switch to lytic cycle and subsequent cell death, even in the presence of an inhibitor of EBV thymidine kinase (acyclovir) without an increase in virion production. In contrast, growth of EBV-negative B-cell lines was not affected.

Theophylline induced differentiation provides direct evidence for the deregulation of c-myc in Burkitt's lymphoma and suggests participation of immunoglobulin enhancer sequences.

Most of the evidence that supports the hypothesis that the c-myc gene is abnormally regulated in Burkitt's lymphoma (BL) is indirect. The putative abnormal expression of c-myc is likely, at least in part, to be a consequence of the usurpation of its regulatory sequences by immunoglobulin enhancer elements, which are invariably juxtaposed to c-myc by the translocations associated with this tumor (C. M. Croce, J. Erikson, A. Ar-Rushdi, D. Aden, and K. Nishikura, Proc. Natl. Acad. Sci. USA, 81: 3170-3174, 1984). We have developed a differentiation induction model system to examine this issue more directly. In a variety of non-BL cell lines, differentiation induction results in the down-regulation of c-myc (G. P. Studzinski, A. K. Bhandal, and Z. S. Brelvi, Proc. Soc. Exp. Biol. Med., 179: 288-295, 1985; Y. Matsui, R. Takahasi, K. Minara, T. Nakagawa, T. Koizumi, Y. Nakao, T. Sugiyama, and T. Fugita, Cancer Res., 49: 1366-1371, 1985; T. Mitchell, E. Sariban, and D. Kufe, Mol. Pharmacol., 30: 398-402, 1986; Z. S. Brelvi, and G. P. Studzinski, J. Cell. Physiol., 128: 171-179, 1986). Since BL is of B-cell origin, differentiation is associated with persistent or increased expression of immunoglobulin genes. Therefore, if c-myc and c-mu are coregulated in BL via immunoglobulin enhancer sequences, persistent or increased expression of the c-myc gene, rather than down-regulation, should occur in differentiated BL cells. Differentiation was induced in four BL cell lines with theophylline (7 x 10(-3) M), and mRNA was examined by Northern blot analysis. In all four BL lines studied (JD38, AG876, KK124, and Daudi), there was persistent or increased expression of both c-mu and c-myc genes (detected with a third exon c-myc probe), in contrast to the decreased expression of the c-myc gene observed in the three Epstein-Barr virus transformed lines studied (A3317, TC84, and CB34). In the BL cell line, JD38, the c-myc gene is truncated (the second and third exons are translocated to chromosome 14 while the first exon remains on chromosome 8). In this line, we demonstrated that theophylline induced differentiation results in down-regulation of the first exon while the level of expression of the translocated second and third exons remains unchanged or increases.(ABSTRACT TRUNCATED AT 400 WORDS)

The pattern of p53 mutations in Burkitt's lymphoma differs from that of solid tumors.

Available evidence suggests that, among hematological malignancies, p53 is most often mutated in Burkitt's lymphoma (BL). However, much of the published data is based on cell lines. We have, therefore, analyzed BL biopsies to determine more accurately the frequency and pattern of p53 mutations in primary tumors and to determine whether there are differences among the various subtypes of BL. Among 27 BL biopsies from South Africa, we have observed mutations in the p53 gene (exons 5 through 8) in 37% of tumors. The higher frequency of mutations in cell lines (70%) suggests that mutation of p53 may be associated with tumor progression. Summarizing available data we conclude that the presence of mutated p53 in BL is independent of the geographic origin of the tumor, the 8;14 chromosomal breakpoint locations and Epstein-Barr virus association. We also find that the mutational spectrum of p53 in BL differs from that observed in nonlymphoid tumors. More than 50% of mutations in BL are clustered in a small stretch of 33 amino acids (codons 213 to 248). Interestingly, codon 213 appears to be as frequently mutated as codon 248. Conversely, codon 273, often mutated in solid tumors, is rarely involved in BL.

Infrequent p53 mutation in mouse tumors with deregulated myc.

An invariant genetic lesion in mouse plasmacytomas is deregulated expression of c-myc as a consequence of chromosomal translocation. However, retroviral and transgenic studies suggest that additional genetic lesions may contribute to the genesis of plasmacytomas. The p53 tumor suppressor gene is a likely contributor to this genetic lesion, since there is a high incidence of p53 mutation in Burkitt's lymphomas and B-ALL (L3), both of which contain translocations involving c-myc analogous to those in plasmacytomas. In addition, p53 has been shown to be a transcriptional modulator of c-myc expression. In a survey of 27 mouse plasmacytomas by single-strand conformation polymorphism, we identified a single mutation (3.7% incidence), suggesting that p53 lesions are not frequent contributors to plasmacytomagenesis. A similar study of macrophage-monocyte tumors generated by a c-myc-containing retrovirus also indicates a lack of p53 involvement in deregulated c-myc expression. These results suggest that the specific maturation stage of transformed B-lymphocytes, independent of c-myc deregulation, may be the critical factor which determines the involvement of mutant p53.

Examination of Epstein-Barr virus and C-type proviral sequences in American and African lymphomas and derivative cell lines.

While Epstein-Barr viral (EBV) DNA is nearly always detectable in African Burkitt's lymphoma (BL), the relatively low frequency of BL in EBV-positive African children and the infrequent finding of EBV in American BL suggest that other cofactors may contribute to the malignant transformation. Among these possible cofactors are type C oncornaviruses. To evaluate this possibility, we screened the cellular DNA from 16 lymphomas (2 African, 14 American) and the DNA from 20 lymphoma-derived cell lines (4 African, 16 American) with a radiolabeled viral DNA probe from EBV and two oncornaviral probes (murine amphotropic 1504A virus and simian sarcoma virus). The radiolabeled EBV DNA probe hybridized with 18 of 36 tumor or cell line DNA's. Only 2 of 11 American BL tumors contained detectable EBV sequences. However, the cell lines derived from three EBV-negative tumors converted in vitro to EBV positivity, suggesting that some of the tumor cells could be infected with EBV. In contrast, none of the tumors or the cell lines derived therefrom hybridized with either the 1504A or the simian sarcoma virus probes, decreasing the likelihood that type C viruses are cofactors with EBV.

Active transport of methotrexate from cerebrospinal fluid in humans.

The cerebrospinal fluid (CSF) efflux kinetics of methotrexate (MTX) were studied in three patients with indwelling Ommaya reservoirs. A small dose of MTX was injected intraventricularly several hr after the start of a high-dose continuous i.v. infusion of MTX. In all patients, the CSF antifolate concentration returned to the preinjection level before the end of the i.v. infusion. This result indicated that the efflux of MTX from CSF in humans is independent of plasma drug concentrations. Efflux kinetics were further characterized in one patient. Serially obtained CSF samples after intraventricular injections demonstrated a biphasic disappearance curve with alpha- and beta-phase half-disappearance times of 1.7 and 6.6 hr, respectively. Prolongation of the beta-phase half-time was associated with oral acetazolamide medication and with increased intracranial pressure, indicating that inhibition of CSF production slows MTX clearance. CSF MTX concentration, however, declined more rapidly than that of simultaneously administered diethylenetriaminepentaacetic acid, an extracellular marker substance excreted by bulk flow, indicating that bulk flow excretion alone is insufficient to account for MTX efflux from human CSF. Evidence that there is an active transport component was provided by probenecid pretreatment which also prolonged the CSF MTX half-life. These findings suggest that both passive and active mechanisms govern MTX efflux from the CSF in humans and that they can be inhibited by acetazolamide and probenecid, respectively.

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.

Role of the p53 tumor suppressor gene in the tumorigenicity of Burkitt's lymphoma cells.

Burkitt's lymphoma (BL) cell lines carry a translocated c-myc gene and, in 60-80% of cases, exhibit mutations in the p53 tumor suppressor gene. We examined the potential role of the p53 gene in BL tumorigenicity using an in vitro assay that measures p53-dependent cell cycle arrest in the G1 phase of the cell cycle and an in vivo athymic murine model that detects differences in the tumorigenicity of BL cell lines. A highly significant inverse correlation was found between the ability of BL cells to arrest in G1 after irradiation and their tumorigenicity in athymic mice, consistent with the notion that loss of p53 function is associated with increased tumorigenicity. Inactivation of wild-type (wt) p53 function by expression of the human papillomavirus E6 protein in the AG876V BL cell line, which carries both wt and mutant p53 proteins, rendered the cell line significantly more tumorigenic in athymic mice. Transfection of the wt p53 gene into the p53 mutant and highly tumorigenic BL-41 cell line caused it to acquire wt p53 function and rendered it less tumorigenic in mice. In addition to confirming a role for the loss of p53 function in tumor progression, the data demonstrate that wt p53 protein can reduce BL tumorigenicity in vivo.