Daudi Burkitt's Lymphoma Cells Research Articles

Recognition by human Vgamma9/Vdelta2 T cells of melanoma cells upon fusion with Daudi cells.

Daudi Burkitt's lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi x MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vgamma9/Vdelta2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi x melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the gamma/delta T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi x melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vgamma9/Vdelta2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level.

Requirement for CD4+T Cells in the γδ T Cell Proliferative Response to Daudi Burkitt's Lymphoma

Daudi Burkitt's lymphoma cells stimulate the proliferation of Vγ2 Vδ2 T cells in human peripheral blood mononuclear cells (PBMC). Transfection studies have established that the γδ T cell receptor is sufficient for recognition of a ligand associated with Daudi cells. Since other studies have demonstrated the need for cooperation between αβ T cells and Vγ2 Vδ2 T cells in the proliferative response to mycobacterial antigens, we used human, unfractionated PBMC and PBMC depleted of specific T cell subsets to determine whether the γδ T cell response to Daudi cells also requires cooperation between αβ and γδ T cells. In the current study, we demonstrate that depletion of CD4+T cells substantially reduces the overall proliferative response of PBMC to Daudi cells and subsequent γδ T cell expansion. IL-2 production by PBMC in response to Daudi cells was also decreased by depletion of CD4+T cells, but not by depletion of CD8+or γδ T cells. In the CD4+T-cell-depleted PBMC, proliferation in response to Daudi cells was restored by the addition of IL-2. These data stress the importance of cooperation between αβ and γδ T cells in the response to some complex antigens.

Alpha Interferon Suppresses the Cyclin D3 and cdc25A Genes, Leading to a Reversible G0-Like Arrest

Alpha interferon is a potent growth inhibitor of Daudi Burkitt's lymphoma cells. We show here that alpha-interferon signaling interacted simultaneously with several components of the basic cell cycle machinery, causing cells to enter into a state that had many features characteristic of the G0 state. Within a few hours after alpha-interferon treatment, cyclin D3 mRNA and protein levels dropped to undetectable levels and, in parallel, the activities of cyclin A- and cyclin E-associated kinases were significantly reduced. The latter resulted from the rapid alpha-interferon-mediated elimination of cdc25A, a phosphatase that is required for antagonism of negative tyrosine phosphorylation of cdk2 in cyclin-cdk complexes. This regulation represents a novel mechanism through which an external inhibitory cytokine interacts with the cell cycle machinery. At later time points after alpha-interferon treatment, the levels of the 55-kDa slowly migrating hyperphosphorylated form of cyclin E and of cyclin A were also reduced. The antiproliferative effects were reversible, and cultures from which alpha interferon was removed reentered S phase after a lag that typically corresponded to approximately two doubling times. During this lag period, the expression of cyclin D3 and cyclin A, as well as of the cdc25A phosphatase, continued to be switched off, in spite of the removal of alpha interferon from the cell surface. In contrast, c-myc, which represents another downstream target gene that is subjected to negative regulation by alpha interferon, was relieved from suppression much earlier, concomitant with the decay in early signaling of the cytokine. The delayed pattern of cyclin reexpression provides evidence that alpha-interferon signaling imposes a G0-like state on this system.

Extracellular Ubiquitin Regulates the Growth of Human Hematopoietic Cells

We investigated the effects of extracellular ubiquitin on the colony formation of human hematopoietic progenitor cells (CFU-GM, CFU-E and BFU-E) and a granulocyte-colony stimulating factor (G-CSF)-dependent human myeloblastic leukemic cell line, OCI/AML/1a. Ubiquitin exhibited inhibitory effects on CFU-GM, CFU-E, BFU-E and OCI/AML/1a colony formation. Extracellular ubiquitin also inhibited the growth of KT3 (T lymphoblast), K562 (erythroleukemia) and Daudi (Burkitt's lymphoma) cells, stimulated the growth of HL60 (promyelocytic leukemia) and Jurkat (T-ALL) cells and showed no effect on the growth of U937 cells (monocytic leukemia). These results indicate that another, previously unrecognized function of extracellular ubiquitin is to regulate hematopoiesis.

Immunoelectron Microscopic Localization of the 60-kDa Heat Shock Chaperonin Protein (Hsp60) in Mammalian Cells

The subcellular distribution of the 60-kDa heat shock protein (Hsp60) was examined in a variety of mammalian cells and tissues, including Chinese hamster ovary cells, human fibroblasts, B-SC-1 kidney cells, Daudi Burkitt's lymphoma cells, and rat liver, by immunogold electron microscopy employing six different monoclonal and polyclonal antibodies that are specific for Hsp60. In cryosections or LR Gold sections of different cultured cells, intense labeling of mitochondria was obtained, typically 200–500 gold particles per mitochondrion and accounting for 80–85% of the total gold particles. In addition, however, in all cell types and using all of the antibodies, about 15–20% of the labeling due to Hsp60 was seen at discrete extramitochondrial sites. Such sites included those in close proximity to mitochondrial outer membranes, foci on endoplasmic reticulum, on the cell surface, and in unidentified vesicles. In cryosections of rat liver, specific labeling due to Hsp60 antibodies was also observed within peroxisomes. Labeling of all cellular components by these antibodies could be prevented by preadsorption with purified recombinant mitochondrial Hsp60 indicating that the labeling is specific for Hsp60. Biotin labeling of cell surface proteins results in biotinylation of Hsp60 as analyzed by immunoprecipitation and Western blots, providing further evidence for Hsp60 presence on the plasma membrane. Immunoprecipitation experiments with Hsp60 antibodies show that under normal conditions no detectable precursor Hsp60 protein is present in cells. However, in cells treated with the potassium ionophore nonactin, which blocks mitochondrial import, only the precursor form of Hsp60 accumulates, providing evidence that at least partial mitochondrial import of Hsp60 is necessary for its maturation. These results also provide evidence that no other 60-kDa protein other than mitochondrial Hsp60 is recognized by the antibodies used for electron microscopy. These findings raise interesting questions concerning the possible role of Hsp60 at extramitochondrial sites.

Burkitt lymphoma Daudi cells contain two distinct farnesyltransferases with different divalent cation requirements.

Farnesylation is a lipid posttranslational modification required for the biological function of several signaling proteins including the Ras oncoprotein where this modification is required for malignant transformation. Here we report the identification of two distinct farnesyltransferases (FTases) in Burkitt lymphoma Daudi cells. Separation of Daudi cell cytosolic fractions by ion exchange chromatography resulted in two peaks (FTases I and II) that, on gel filtration, show molecular masses of 90 000 and 250 000 Da, respectively. Immunoblotting experiments showed that FTase I is composed of an alpha/beta-heterodimer of about 50 000 Da each. FTase II contained a beta-subunit that is immunologically indistinguishable from the beta-subunit of FTase I and the previously reported human and rat brain FTase but contained an alpha-subunit that reacted poorly with a rat brain anti-alpha-antibody. As in rat brain FTase, Daudi FTases I and II both required magnesium for enzymatic activity. However, their zinc requirements differed. In the absence of Zn2+, FTase I had little activity (10%) whereas FTase II had 30% of its maximum activity (maximum activity obtained in the presence of Zn2+). Furthermore, whereas both FTases I and II were potently inhibited by KB-Ras C-terminal Cys-Val-Ile-Met tetrapeptide mimics, only FTase I but not FTase II required zinc for peptide binding and inhibition.

Regulation of low-density lipoprotein receptors and assessment of their functional role in Burkitt's lymphoma cells

The status of low-density lipoprotein receptors (LDLR) in Daudi Burkitt's lymphoma (BL) cells was examined using a flow cytometric assay employing the fluorescent ligand DiI-LDL, and a radioligand-binding assay using [ 125I]LDL. The binding is concentration-and time-dependent; and is specific, as judged by its competitive displacement in the presence of unlabeled LDL, and inhibition by heparin, EGTA, and 4°C incubation. The regulation of the receptor and its functional role were then explored. Our results suggest the following: (a) In sharp contrast to normal peripheral blood lymphocytes, the LDLR levels in BL cells are basally elevated when cultured in fetal bovine serum (FBS) medium. (b) In accord with normal peripheral blood lymphocytes, incubation in lipoprotein-deficient serum (LPDS) medium further up-regulates the level of the receptor in BL cells, and co-incubation with LDL or 25-hydroxycholesterol down-regulates the receptor level. The magnitude of the up-regulation is significantly smaller than in normal peripheral blood lymphocytes. (c) Northern blots using a plasmid-DNA probe for LDLR mRNA point to a similar pattern for message regulation as is observed in direct binding studies. (d) Although the LDLR level is constitutively high in BL cells, availability of LDL, unlike transferrin, is not a growth requirement since incubation of cells in LPDS medium does not prevent proliferation of these cells. (e) In contrast to anti-transferrin receptor antibody which results in apoptosis upon binding, anti-LDLR antibody does not inhibit growth or induce apoptosis. Our results suggest LDLR is expressed at a significantly higher level in BL cells than in normal peripheral blood lymphocytes. Although up-regulation and down-regulation of LDLR are observed, this applies only to a small population of LDLR. The bulk receptor population is significantly resistant to down-regulation. Furthermore, notable differences in the functional role of the LDLR are found relative to the transferrin receptor which is also up-regulated in BL cells.

The effect of a mesogenic and a lentogenic Newcastle disease virus strain on Burkitt lymphoma Daudi cells.

The destructive effect of Newcastle disease virus (NDV) strains on Burkitt lymphoma Daudi cells was investigated. Interaction of an active and UV-inactivated mesogenic strain (Roakin), as well as an active attenuated lentogenic strain (B1), grown in the allantoic sac of embryonated eggs, at high multiplicity, caused inhibition in cellular DNA synthesis and arrest in cell multiplication, eventually killing of the cells. The lentogenic strain cultivated in chicken fibroblasts exhibited only a moderate activity. The mechanism of the cytolytic effect is presumably linked to the increase in cell membrane permeability indicated by the elevation in 51Cr release. Thus it appears that the massive adsorption and/or penetration of viral particles, active or UV-inactivated (or possibly a toxic component that resides in the virion), damages the plasma membrane and may be responsible for the killing of the cells.

Dimethyl Sulfoxide Suppresses Apoptosis in Burkitt's Lymphoma Cells

Previous reports have suggested that dimethyl sulfoxide (DMSO) may be a useful reversible G 1 arresting agent for synchronizing Raji Burkitt's lymphoma cells (K. Takase et al. (1992) Cell Growth Differ. 3, 515-521; M. Sawai et al. (1990) Exp. Cell Res. 187, 4-10). We have therefore critically evaluated several aspects of DMSO's effects using Daudi and Ramos Burkitt's lymphoma (BL) cells. In BL cells starved in the presence or absence of DMSO for 4 to 6 days (approximately four to six doubling times), the following observations were noted: (A) Both Daudi and Ramos cells show increased cell synchrony accompanied by apoptosis when starved in RPMI 1640 supplemented with 10% fetal calf serum (FCS). Inclusion of 1.5% DMSO causes a diminution in apoptosis with minimal effects on synchrony. (B) Lowering the FCS concentration to 5% induces apoptosis. DMSO-mediated protection from apoptosis is observed in Daudi but not in Ramos. (C) When human serum (10%) is used instead of FCS, Daudi cells show no apoptosis and DMSO is without effect on cell cycle distribution. By contrast, Ramos cells show significant apoptosis, which is prevented by the inclusion of DMSO. (D) When starved in a chemically defined medium (AIM-V), both Daudi and Ramos cells show significant apoptosis. DMSO protects Ramos from apoptosis under these conditions. (E) Upon removal of DMSO, both Daudi and Ramos cells reenter the cell cycle but with significant apoptosis. (F) The protective effect of DMSO from apoptosis is observed in a narrow range of concentration between 1 and 2%. At higher concentration, DMSO itself induces apoptosis. These results suggest that DMSO itself prevents apoptosis, an effect which may present as an apparent effect on cell synchrony.

V gamma 2V delta 2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer.

The predominant subpopulation of gamma delta T cells in human peripheral blood expresses TCR V region genes V gamma 2 paired with V delta 2. Previous studies have shown that these V gamma 2V delta 2+ T cells proliferate in response to Daudi Burkitt lymphoma cells, synthetic alkyl phosphate molecules including monoethylphosphate (MEP), and an Ag chemically similar to MEP purified from mycobacterial extracts of several species including Mycobacterium tuberculosis. This proliferation is polyclonal and determined by the TCR V gene. However, because these alkyl phosphate molecules are so distinct from conventional peptides and superantigens, we questioned whether these substances induce gamma delta T cell proliferation via TCR-dependent recognition. Here we report that transfection of TCR- Jurkat T cells with cDNA constructs encoding a V gamma 2V delta 2 TCR enabled the transfectants to produce IL-2 in response to Daudi cells, mycobacterial extract, and MEP. The responses were dose dependent and Ag specific. These results demonstrate an essential role for the gamma delta TCR in V gamma 2V delta 2 T cell-mediated recognition of non-peptide Ags by human T cells and suggest a structural similarity or cross-reactivity between cellular and microbial Ags recognized by these gamma delta T cells.

Flow cytometric evaluation of LDL receptors using DiI-LDL uptake and its application to B and T lymphocytic cell lines

Low density lipoprotein receptors (LDL-R) on Daudi Burkitt's lymphoma (BL) cells were assessed using fluorescent DiI (3,3′-dioctadecylindocarbocyanine iodide)-LDL and flow cytometric analysis. Receptor-specific binding of DiI-LDL is followed by internalization of the bound complex and lysosomal hydrolysis of the ligand. Increase in the fluorescence intensity per cell is hence used as a measure of DiI-LDL uptake and, implicity, as an indication of LDL-R presence. Our results show that uptake was observed in > 98% of the Daudi cells, and the level of uptake was significant and clearly distinguishable from autofluorescence, suggesting that: 1. (a) this assay is comparable to the iodinated LDL uptake assay, although the ED 50 values for the ligands are different; 2. (b) this assay is comparable to the flow-cytometric detection of LDL-R using a commercial antibody directed against the receptor itself, and superior to a similar assay based on an antibody directed against membrane-bound LDL; 3. (c) LDL uptake could be monitored along with transferrin uptake, suggesting that multiple endocytic receptor activities can be concurrently studied; 4. (d) DiI-LDL uptake can be examined along with fluorescein-conjugated anti-CD10, -CD19, and -CD71, with little cross-interference, offering the added advantage that endocytic uptake and phenotyping can be simultaneously monitored; 5. (e) the expression of LDL-R is intrinsically elevated in diverse cell lines such as Daudi, Raji, Ramos, Jurkat, and WIL2-NS, but not in normal lymphocytes. Our results therefore indicate that flow cytometric analysis of DiI-LDL uptake has potentially useful applications in the detection and study of endocytic receptor LDL-R in B and T lymphocytic cell lines.

Cell cycle-dependent modulation of alpha-interferon-inducible gene expression and activation of signaling components in Daudi cells.

To investigate cell cycle-dependent expression of interferon (IFN)-induced genes and signaling pathways, we examined the modulation of IFN-inducible gene expression and p91 activation, and JAK-1 and Tyk-2 kinases' activation in human Burkitt's lymphoma Daudi cells which were synchronized at different points in the cell cycle progression. We observed that each one of 100-, 67-, and 46-kDa 2'-5'-oligoadenylate synthetase (2-5(A)-synthetase) proteins had a unique expression pattern in different phases of the cell cycle which was different from the expression of 2-5(A)-synthetase proteins in exponentially growing Daudi cells. The cell cycle-dependent changes in the overall levels of expression of 2-5(A)-synthetase proteins correlated well with the induction of expression of 2-5(A)-synthetase mRNA as well as the appearance of IFN stimulatory gene factor-3 and expression and activation of p91 in cells released from the G1/S block in the absence of exogenous IFN-alpha. In addition, cells from the G1/S phase exhibited increased tyrosine phosphorylation of JAK-1 and Tyk-2 kinases and p91 by IFN-alpha as compared with asynchronized cells. Enhanced stimulation of activation of JAK-1, Tyk-2, and p91 by IFN-alpha was progressively reduced from G1/S to G2/M to pass G2/M. These findings demonstrate that the levels of constitutive and inducible expression of IFN-alpha-inducible genes, activation of p91, and tyrosine phosphorylation of JAK-1 and Tyk-2 kinases are differentially influenced by the state of the cell cycle.

Human Peripheral γδ T Cells Are Stimulated by Daudi Burkitt's Lymphoma and Not by Any Other Burkitt's Lymphoma Tested

Daudi Burkitt's lymphoma cells have molecules on their surface which can stimulate proliferation of human γδ T cells, while Raji, another Burkitt's lymphoma, cannot stimulate human γδ T cells. Human peripheral γδ T cells, coexpressing the V γ9/V δ2 chains of the T cell receptor, lyse Daudi cells but not Raji cells. Here, we have screened four other Burkitt's lymphoma cell lines (HH514, DG75, Ramos, and Wilson), as well as cells derived from a fresh Burkitt's lymphoma, to see if any of them can be recognized by human γδ T cells. Leukemia-derived lines MOLT-4, CEM, and K562 have also been included in these studies. Among the Burkitt's lymphomas tested, only Daudi, DG75, and HH514 could be lysed by V γ9/V δ2 + T cell clones derived from the peripheral blood of healthy donors. These T cell clones were also able to lyse the NK sensitive leukemia lines K562 and MOLT-4. When hulk cultures of peripheral blood mononuclear cells from healthy donors were cultured with different Burkitt's lymphoma or leukemia cell lines, only Daudi stimulated the outgrowth of γδ T cells. Similarly, only Daudi cells could stimulate proliferation of γδ T cell clones, and the response was enhanced significantly in the presence of interleukin-2. These data and our prior observations showing the use of the V γ9/V δ2 TCR type by Daudi-reactive human γδ T cells indicate that Daudi cells are not representative of other Burkitt's lymphoma cell lines.

Changes of cell cycle-regulating genes in interferon-treated Daudi cells.

Interferon (IFN) modulates the expression of several genes and some of them are considered to be responsible for the inhibition of cellular growth. However, the alterations of cell cycle-regulating genes produced by IFN still remain unclear. Accordingly, we studied the expression of cell cycle-regulating genes during IFN-induced growth arrest. Cell cycle synchronized and unsynchronized Daudi Burkitt lymphoma cells were treated with IFN. Both the cell cycle distribution and the expression of cell cycle-regulating genes (cdk2, cdc2, cyclins A, B, C, D3, cdc25, and wee 1) were studied by flow cytometry and by Northern blot hybridization or the reverse-transcription polymerase chain reaction, respectively. Treated cells passed through the first G1 phase and gradually accumulated in the following G1 phase. Expression of cyclins A, B, and D3 oscillated along with the cell cycle progression in control cells, and the alterations of cyclin B expression were especially prominent. Both cdc2 and cdk2 also showed changes, but these were not so distinct as observed with cyclin B. Expression of cdc25 and wee1 was little affected by cell cycle progression. In IFN-treated cells, expression of cyclins A and B were down-regulated, while that of cyclin C was not. Cyclin D3 expression was also down-regulated at 48 h, followed by an increase at 72 h. Expression of both cdc2 and cdk2 was down-regulated, especially that of the later. Wee1 expression was down-regulated by IFN but, the expression of cdc25 remained stable. These findings suggest that the modulation of cell cycle-regulating genes, particular by cyclin A and cdk2, plays an important role in IFN-induced cellular growth arrest.

Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.

Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells.

Interferons and interleukin-6 suppress the DNA-binding activity of E2F in growth-sensitive hematopoietic cells.

Transcription factor E2F binds to cellular promoters of certain growth- and cell cycle-controlling genes and forms distinct heteromeric complexes with other nuclear proteins. We show here that alpha and beta interferons (alpha, beta) and interleukin-6 abolished the E2F-containing DNA-binding complexes in Daudi Burkitt lymphoma cells and in M1 myeloblastic cells, which responded to the cytokines by suppression of c-myc transcription. Time kinetics studies showed that the abolishment of E2F complexes coincided with reduction of c-myc expression and that both molecular events preceded the cell cycle block in G0/G1 phase. In contrast, the pattern of E2F complexes remained unchanged in an interferon-treated growth-resistant Daudi cell mutant that displayed relaxed regulation of c-myc. All of the DNA-binding E2F complexes, including those containing the retinoblastoma protein (pRB), cyclin A-p33cdk2, and the free forms of E2F, were reduced by interferons or interleukin-6. Their abolishment was unperturbed by pharmacological treatments that alleviated the cyclin A and pRB responses to interferon. Thus, changes in cyclin A expression and pRB phosphorylation are not primary events that influence the pattern of E2F responses to cytokines. Addition of EDTA to cell extracts of interferon-treated Daudi cells restored the DNA-binding activity of E2F, resulting in the appearance of a single E2F complex that exclusively contained pRB. It is suggested that the regulation of E2F by growth-inhibitory cytokines that induce cell cycle exit takes place at the level of the DNA-binding activity, and by that mean it differs basically from the phase-specific regulation of E2F in cycling cells.

Close correlation between Daudi and mycobacterial antigen recognition by human gamma delta T cells and expression of V9JPC1 gamma/V2DJC delta-encoded T cell receptors.

Recent studies have demonstrated that a large fraction of human gamma delta PBL recognize Ag of prokaryotic and eukaryotic origins, respectively found in hydrosoluble mycobacterial extracts and on the Daudi Burkitt's lymphoma cells. The structural basis of the recognition of these Ag have been presently studied in detail, through analysis of a large panel of thymus- and peripheral blood-derived gamma delta T-cell clones. Our results suggest that Daudi and mycobacteria-reactive gamma delta subsets are strictly overlapping and hence that gamma delta T-cell responses against these two Ag are closely related. Daudi cells and mycobacteria were recognized by V gamma 9+V delta 2+, but not by V gamma 9+V delta 2-, V gamma 9-V delta 2+, or V gamma 9-V delta 2- PBL clones. However, not all V gamma 9+V delta 2+ clones were reactive and, in particular: 1) the proportion of Ag-reactive lymphocytes was much lower among thymus- than PBL-derived clones (respectively 24/36 vs 72/73); 2) none of the V gamma 9+V delta 2+ clones expressing a V9J2C2 gamma chain (n = 4) were reactive to Daudi or mycobacteria, indicating that expression of a disulfide-linked TCR is probably a prerequisite for recognition of these Ag; and 3) among V gamma 9+V delta 2+ clones bearing disulfide-linked TCR, almost all (50/53) clones expressing a V9JPC1 gamma chain were reactive, whereas a large fraction (6/10) of those expressing a V9J1C1 gamma chain were weakly or nonreactive. Together, these observations suggest that germline residues specific to V gamma 9, V delta 2, and J gamma P elements directly contribute to recognition of Daudi and mycobacterial Ag. Furthermore, these findings may provide an explanation for coordinate use of these gene elements by a large fraction of gamma delta PBL, through peripheral selection events mediated by ligands identical or structurally related to the above Ag.

Human peripheral gamma delta T cells recognize hsp60 molecules on Daudi Burkitt's lymphoma cells.

Studies with the use of polyclonal rabbit antiserum reactive with 60-kDa heat shock proteins (hsp60) suggested that hsp60-related molecules could be found on the surface of Daudi cells and were involved in their recognition by certain human gamma delta T cells. The present study confirms this finding by using a mAb specifically recognizing hsp60. This mAb can block outgrowth of human gamma delta T cells in response to stimulation with Daudi and in response to an extract of the mycobacteria H37Ra. This anti-hsp60 mAb stains the surface of Daudi cells, but does not stain either Raji or EBV-transformed B cells, cells which do not stimulate gamma delta T cell outgrowth. Anti-hsp60 mAb could immunoprecipitate a 60-kDa molecule from the H37Ra extract but was unable to precipitate this 60-kDa molecule if the mAb was first absorbed on Daudi cells. This mAb also precipitated a 60-kDa molecule from the surface of Daudi cells which shows an electrophoretic mobility pattern consistent with hsp60. These experiments demonstrate that human gamma delta T cells recognize hsp60-related epitopes on the surface of Daudi cells and within mycobacterial extracts.

Expression of genes for the Epstein-Barr virus small RNAs EBER-1 and EBER-2 in Daudi Burkitt's lymphoma cells: effects of interferon treatment.

The relative levels, rates of synthesis and stabilities of the abundant Epstein-Barr virus (EBV)-encoded small RNAs, EBER-1 and EBER-2, were examined in Daudi Burkitt's lymphoma cells. Although both RNAs are transcribed at approximately equal rates, the steady-state level of EBER-1 is at least 10-fold greater than that of EBER-2. This is shown to be due to a much faster rate of turnover of EBER-2. In the presence of actinomycin D, the half-lives of EBER-1 and EBER-2 are 8 to 9 h and 0.75 h, respectively. Following treatment of the cells with human interferon (IFN) alpha the transcription of both RNAs is strongly inhibited. However, the level of EBER-1 increases up to twofold, indicating a further stabilization of this RNA. In IFN-treated cells, EBER-2 accumulates in the form of truncated products. Nuclease protection experiments indicated that this is due to a post-transcriptional modification of the 3' end of the molecule. These data show that the effects of IFN treatment on the expression of these two viral gene products are very complex in cells latently infected with EBV.

Interferon alpha induces the expression of retinoblastoma gene product in human Burkitt lymphoma Daudi cells: role in growth regulation.

Interferon alpha (IFN-alpha) is a regulatory secretory protein with distinctive biological effects such as antiproliferative actions against many tumor cell lines, including human Burkitt lymphoma Daudi cells. The mechanism underlying growth inhibition by IFN-alpha is not well established. The growth of many mammalian cell types is also regulated by tumor suppressor retinoblastoma (RB) gene product, the RB protein. In the studies presented here, we explored the possible involvement of RB protein in the growth inhibitory action of IFN-alpha in the Daudi cell model system. We observed that IFN-alpha induces a 3- to 10-fold increased expression of RB protein in growth-sensitive Daudi cells but not in the growth-resistant variant of Daudi cells. IFN-alpha-mediated induction of RB protein was an early event that preceded the period of growth inhibition of Daudi cells. IFN-alpha-induced RB protein predominantly exists as the underphosphorylated form. Addition of antibody against IFN-alpha to Daudi cells resulted in the inhibition of constitutive expression of RB protein and stimulation of [3H]thymidine incorporation. These results demonstrate that the induction of RB protein expression in IFN-alpha-treated Daudi cells could constitute an important mechanism of IFN-alpha-mediated growth regulation.