Stable expression of Epstein-Barr virus BZLF-1-encoded ZEBRA protein activates p53-dependent transcription in human Jurkat T-lymphoblastoid cells.

Interaction between viral proteins and tumor suppressor p53 is a common mechanism of viral pathogenesis. The Epstein-Barr virus (EBV) BZLF-1 ORF-encoded ZEBRA protein (also denoted EB1, Z, Zta) binds to p53 in vitro and has been associated with the altered transcription of p53-regulated genes in B lymphocytes and epithelial cells. In this work, Jurkat T-lymphoblastoid cells that express ZEBRA were characterized by the use of transiently transfected p53 and p53 reporter genes. Stable expression of ZEBRA was associated with the activation of p53-dependent transcription and increased p53 dependent apoptotic cell death. In Jurkat cell lines, stably expressed ZEBRA protein was apparently localized to the cell cytoplasm, in contrast to the typical nuclear localization of this protein in other cell types. Previous studies have suggested that EBV infection of T lymphocytes may contribute to the malignant transformation of T cells and the increased replication of human immunodeficiency virus. Our observations suggest a mechanism through which ZEBRA protein expressed in human T lymphocytes could alter T-cell proliferation and apoptosis during EBV infection. (Blood. 2000;96:625-634)

Inactivation of NF-kappaB by EBV BZLF-1-encoded ZEBRA protein in human T cells.

We have previously shown that the EBV ZEBRA protein (also denoted EB1, Z, or Zta) encoded by the BZLF open reading frame is expressed in primary human thymocytes and in human T lymphoblastoid cell lines infected by EBV. Expression of EBV-encoded gene products in T lymphocytes could contribute to viral pathogenesis during acute EBV infection as well as in individuals coinfected with EBV and HIV. HPB-ALL and Jurkat T lymphoblastoid cell lines transiently and stably expressing ZEBRA were characterized in this work. Expression of ZEBRA protein in human T lymphoblastoid cells was associated with decreased expression of an NF-kappaB reporter gene, altered expression of the NF-kappaB p50 protein subunit, and decreased DNA binding by components of NF-kappaB. These observations suggest that inactivation of NF-kappaB transcription by ZEBRA in EBV-infected T cells may be a novel mechanism of viral pathogenesis analogous in part to over-expression of the endogenous cytoplasmic inhibitor of NF-kappaB, IkappaBalpha.

Definition and adjustment of Cesarean section rates and assessments of hospital performance.

BACKGROUND: Demand is growing for comparative data such as Cesarean section rates, but little effort has been made to develop either standardized definitions or risk adjustment approaches. OBJECTIVE: To determine to what extent a seemingly straightforward indicator like Cesarean section rate will vary when calculated according to differing definitions used by various performance measurement systems. DESIGN: Retrospective data abstraction of 200 deliveries per hospital. SETTING: Fifteen acute care hospitals including two from outside the USA. MEASUREMENTS: Four widely-used performance measurement systems provided specifications for their Cesarean section indicators. Indicator specifications varied on inclusion criteria (whether the population was defined using Diagnostic Related Groups or ICD-9-CM procedure codes or ICD-9-CM diagnosis codes) and risk-adjustment methods and factors. Rates and rankings were compared across hospitals using different Cesarean section indicator definitions and indicators with and without risk adjustment. RESULTS: Calculated Cesarean section rates changed substantially depending on how the numerator and denominator cases were identified. Relative performance based on Cesarean section rankings is affected less by differing indicator definitions than by whether and how risk adjustment is performed. CONCLUSIONS: Judgments about organizational performance should only be made when the comparisons are based upon identical indicators. Research leading to a uniform indicator definition and standard risk adjustment methodology is needed.

Dissociation of EBV genome replication and host cell proliferation in anti-IgG-stimulated Akata cells.

The Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cell line, Akata, was treated with dimethyl sulfoxide (DMSO) for 96 hr in order to reversibly arrest cell cycle progression in G1 phase. Stimulation of the cells with anti-IgG antibody induced a marked and synchronous replication of EBV DNA within 12 hr, before the cells entered into S-phase after release from DMSO-induced arrest. Furthermore, a reduced efficiency of productive replication was demonstrated if anti-IgG stimulation was delayed after release. The results indicate that entry into S-phase of host cells is not only unnecessary for, but also may have negative consequences for the productive phase of EBV infection. Also, it was shown that addition of acyclovir, an inhibitor of the EBV-encoded DNA polymerase, to anti-IgG-stimulated Akata cells inhibited the productive replication of EBV DNA, but had no effect on the expression of early genes of the virus, including BZLF1, BRLF1, BMRF1, and BHRF1.

Nerve growth factor signal transduction in human B lymphocytes is mediated by gp140trk.

Nerve growth factor (NGF) plays an important role in the regulation of the immune system. Recent studies from this laboratory demonstrated the presence of functional NGF receptors on human B lymphocytes; in addition, NGF has been shown to enhance B lymphocyte proliferation. NGF caused both concentration- and time-dependent increases in tyrosine phosphorylation of five proteins of 140, 110, 85, 60 and 42 kDa, which were identified as phospholipase C-gamma 1, phosphatidylinositol-3 kinase and mitogen-activated protein kinase. To elucidate the contribution of the Trk family of tyrosine kinases to the phosphorylation events induced by NGF, we identified gp140trk in human B cells and in human B cell lines. Analysis of specific gp140trk immunoprecipitates indicated that addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of gp140trk and inhibition of this phosphorylation prevented the tyrosine phosphorylation of other proteins. These data identify the central role of gp40trk in NGF signaling of human B lymphocytes.

EBV infection of T cells: potential role in malignant transformation.

The presence of Epstein-Barr virus in different T-cell malignancies is now widely reported. In an effort to ascertain whether T cells are susceptible to EBV infection, we and others have detected the EBV receptor, CD21 on a population of immature thymocytes. We showed that EBV is a cofactor in stimulating proliferation of thymocytes. This proliferation may be a relevant factor in EBV-associated T-cell malignancies as well as EBV causation of acute infectious mononucleosis (AIM). We have further identified a subset of thymocytes that is infectable by EBV in which the genome remains linear in the first weeks after infection. We documented the transcription of the switch protein ZEBRA, an alternatively spliced form, RAZ, and EBNA-1 transcription from the Fp promoter. We hypothesise that EBV may be a cofactor in oncogenesis in T cells through several different pathways.

Expression of novel-transposon-containing mRNAs in human T cells.

The HERV-H family of endogenous retrovirus like elements is the largest such human family known. Using an HERV-H LTR probe, 6 and 4.5 kb transcripts were detected by Northern blot analysis which were induced in normal peripheral T cells after treatment with phytohaemaglutinin (PHA). Expression was not evident 30 min after treatment with phorbol ester, was increased within 3-4 h after treatment, reached a maximum after 8 h and then declined to low levels 24 h after treatment. Expression was inhibited totally by cycloheximide (10 micromolar) and by the immunosuppressant cyclosporin A (1 microgram/ml). Using probes specific for the U3 and U5 regions of the HERV-H LTR, in combination with internal HERV-H probes, evidence was obtained that the 6 and 4.5 kb transcripts are polyadenylated from an HERV-H LTR. A cDNA library was constructed from T cells which had been treated with PHA for 8 h and a 1.7 kb clone was isolated using the HERV-H LTR probe. The insert contained a novel tandem array of an Alu, a LINE-1 element, two endogenous retroviral LTRs and an A-T-rich sequence. The A-T-rich sequence contained multiple copies of AUUUA mRNA regulatory motifs. Because of its high expression level, defined transcription kinetics, novel cassette-like composition and the presence of conserved mRNA stabilization sequences, we hypothesize that the transcript may play a biological role during T cell activation.

Epstein-Barr virus replicative gene transcription during de novo infection of human thymocytes: simultaneous early expression of BZLF-1 and its repressor RAZ.

Epstein-Barr virus (EBV) is known to infect B cells and epithelial cells. We and others have shown that EBV can also infect a subset of thymocytes. Infection of thymocytes was accompanied by the appearance of linear EBV genome within 8 hr of infection. Circularization of the EBV genome was not detected. This is in contrast to the infection in B cells where the genome can circularize within 24 hr of infection. The appearance of the BamHI ZLF-1 gene product, ZEBRA, by RT-PCR, was observed within 8 hr of infection. The appearance of a novel fusion transcript (RAZ), which comprised regions of the BZLF-1 locus and the adjacent BRLF-1 locus, was detected by RT-PCR. ZEBRA protein was also identified in infected thymocytes by immunoprecipitation. In addition, we demonstrated that the EBNA-1 gene in infected thymocytes was transcribed from the Fp promoter, rather than from the Cp/Wp promoter which is used in latently infected B cells. Transcripts encoding gp350/220, the major coat protein of EBV, were identified, but we did not find any evidence of transcription from the LMP-2A or EBER-1 loci in infected thymocytes. These observations suggest that de novo EBV infection of thymocytes differs from infection of B cells. The main difference is that with thymocytes, no evidence could be found that the virus ever circularizes. Rather, EBV remains in a linear configuration from which replicative genes are transcribed.

Activation of human thymocytes after infection by EBV.

The discovery of EBV in certain T cell malignancies and the expression of the EBV receptor, CR2/CD21, on a population of immature thymocytes, T lymphoblastoid cell lines, and childhood acute T lymphoblastic leukemia cells suggested that EBV-receptor interactions on T cells may be of importance. We have shown that, within the thymus, a population of large, immature cells expresses CD21. EBV altered the activation responses of immature thymocytes in vitro. Triggering through CD2 is mitogenic for mature, but not immature, T cells. However, during infection by EBV, ligation of CD2 caused thymocytes to proliferate in the absence of exogenous cytokines. This function was a result of the interaction of EBV with its receptor, CD21, but was caused by infection rather than surface signaling, because neither specific mAb nor the P3HR-1 strain of virus mimicked the effect of B95-8. Immature thymocytes were infected by EBV, as determined by the internalization of the viral genome and its transcriptional activity. Consistent with the activity of B95-8, EBNA-2 transcripts were identified within infected thymocyte populations. In addition, components of the viral replicative pathway were expressed during infection of thymocytes. These components included transcription of BZLF-1, an early gene that characterizes EBV-infected B cells after disruption of latency. A second transcript was identified as encoding the recently characterized RAZ, which also is associated with replicative infection. The consequences of EBV infection of T cells at an early stage of differentiation may lead to failure of normal T cell repertoire development, autoimmunity, or malignancy.

Effects of rGM-CSF and rG-CSF on the cisplatin sensitivity of the blast cells of acute myeloblastic leukemia.

Recombinant growth factors have been shown to alter the sensitivity of acute myeloblastic leukemia (AML) blast cells to cytosine arabinoside (ara-C) in culture. The mechanism is controversial and suggestions for it include changes in ara-C metabolism, changes in cell cycle parameters, and changes in the balance between self-renewal and determination in blast stem cells. We addressed this issue by measuring the cisplatin sensitivity of freshly obtained AML blasts in rG-CSF, rGM-CSF, or the two together. For comparison, simultaneous measurements of ara-C sensitivity were made. We found that exposure to different factors in suspension altered the cisplatin sensitivity of AML blasts in the same direction as the change observed in ara-C sensitivity. Similar changes in cisplatin sensitivity were seen when cells were briefly exposed to the drug, washed, and then grown in suspension in the presence of different growth factors. Control experiments showed that the conditions in suspension, not in the clonogenic assay in methylcellulose, were responsible for the changes in cisplatin sensitivity. The capacity of high specific activity to inactivate clonogenicity was tested at several times under growth conditions which altered the sensitivity of cells to cisplatin. Whereas changes in survival after 3HTdR and cisplatin both were seen with time, growth conditions that altered cisplatin sensitivity were not associated with changes in 3HTdR toxicity. The data do not support explanations of the effects of growth conditions on drug toxicity which depend either on drug metabolism or cell cycle effects. Instead, the findings are consistent with a model that postulates an association between drug sensitivity and the balance between self-renewal and differentiation in the blast population.

Autonomous expression of RTVL-H endogenous retroviruslike elements in human cells.

Northern (RNA) blot analysis of RNA from various human cell lines and tissues has demonstrated that elements belonging to the RTVL-H family of human endogenous retroviruslike sequences are expressed in several cell types. The highest levels of RTVL-H-related RNAs were observed in teratocarcinoma cell line NTera2D1, HeLa cells, two bladder carcinoma cell lines, and normal amniotic tissue. Expression was also observed in normal chorion and in some other cell lines. The RTVL-H transcription pattern varied among the different cell types, but several expressed a unit-length 5.6-kilobase transcript. Characterization of cDNA clones corresponding to transcripts present in NTera2D1 cells indicates that the complex transcription pattern observed in these cells is generated by the following: (i) transcription of both full-length and deleted genomic elements, which is initiated within the 5' long terminal repeat (LTR) and, in all but one case, polyadenylated in the 3' LTR; (ii) the splicing of both unit-length transcripts and transcripts from a deleted element; (iii) transcription involving solo LTR sequences; and (iv) transcription which, in one case, reads through the 3' LTR into flanking cellular sequences. Sequence data obtained from 25 cDNA clones revealed that at least 13 RTVL-H elements are expressed in NTera2D1 cells. The positions of several termination codons within the pol region are the same among nine different elements, indicating that an ancestral RTVL-H element bearing these mutations dispersed within the genome. We also found that RTVL-H expression varied among samples of amnion and chorion tissue isolated from different individuals. These findings demonstrate that regulated autonomous expression of RTVL-H sequences occurs in human cells.

Growth factors influence the sensitivity of leukemic stem cells to cytosine arabinoside in culture.

We have proposed that the blasts in acute myeloblastic leukemia (AML) are renewal populations maintained by a small subpopulation of stem cells. The balance between self-renewal and differentiation in blast stem cells may be an important attribute contributing to treatment outcome. Cytosine arabinoside (ara-C) is included in most chemotherapeutic regimens for the treatment of AML. When ara-C survival curves are constructed, the drug appears to be more toxic when an assay is used that detects principally self-renewing divisions, compared with a procedure that depends on terminal divisions. AML blasts usually respond in culture to myelopoietic growth factors; their response often includes a change in self-renewal, differentiation, or both. These features of the model for AML blasts led to the prediction that growth factors would alter ara-C survival curves in a way that depended on the effects of the culture conditions on self-renewal and differentiation. Four AML blast populations were chosen to test this prediction on the basis of our ability to manipulate them by adding or withholding one or more growth factors. Highly significant changes were seen in the ara-C survival curves, depending on the growth factors present in the cultures as was predicted by the observed effects of the factors on renewal and differentiation.

Heterogeneity in acute myeloblastic leukemia.

Morphological-identified blast populations are the hallmark of the malignant clones that dominate hemopoiesis in acute myeloblastic leukemia (AML). Marked heterogenity is characteristic of AML blasts. Patient-to-patient variation is seen in their biological properties but is particularly evident in the response to treatment. Intraclonal variation is generated during clonal expansion, particularly as blast stem cells either undergo self-renewal or enter into a series of terminal divisions. These two alternative activities can be monitored in cell culture using a clonogenic assay and a suspension assay. The balance between renewal and differentiation can be altered by exposing blast populations to various growth factors in culture. Further, certain drugs, particularly ara-C, appear to be more toxic for self-renewing divisions than cell-cycle events generally. We suggest that both drugs and growth factors should be assessed for their effects on self-renewal as part of preclinical testing.

The effects of combinations of the recombinant growth factors GM-CSF, G-CSF, IL-3, and CSF-1 on leukemic blast cells in suspension culture.

The blast cells of acute myeloblastic leukemia may be considered as a renewal population maintained by stem cells that are capable of both self-renewal and differentiation. Blast stem cells grow in culture usually when stimulated by growth factors normally active on myelopoietic cells. Two culture methods permit an evaluation of the balance between self-renewal and differentiation; previous studies have shown that this balance can be affected by recombinant growth factors. These include interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF), active on early cells in normal myelopoiesis, and G-CSF and CSF-1, restricted in normal hemopoiesis to the granulopoietic and macrophage/monocytic lineages, respectively. In this paper we report the results of evaluating the effects on these recombinant growth factors alone or in mixtures of two at optimal concentrations. The results were obtained either using titrations of colony formation in methylcellulose or growth in suspension. Star diagrams, a technique from exploratory data analysis, were used to provide quantitative and graphic displays of the results of the recombinant factors on the balance between blast self-renewal and differentiation. Blasts from 4 acute myeloblastic leukemia patients and one patient with the blast crisis of chronic myeloblastic leukemia were examined in detail. The great patient-to-patient variation usually observed was seen in both plating efficiency in methylcellulose and growth pattern in suspension. In spite of this variation, a common pattern of response to growth factors emerged. When the early acting factors, IL-3 and GM-CSF, were combined, the effect was quantitatively and qualitatively similar to the largest stimulation seen with either of the factors alone. In contrast, late-acting factors, G-CSF and CSF-1, influenced each other's effects when present together and each affected the activities of GM-CSF and IL-3. Notably, CSF-1, which often led to the accumulation of adherent, terminal cells in suspension, usually maintained or increased this differentiation-like activity in combination. G-CSF also favored differentiation in combination, although the effect was usually to increase the number of colonies in methylcellulose, most of which consist of blast cells incapable of further divisions. The results are discussed as they relate to the postulated structure of the blast population and the normal targets of the recombinant growth factors.

Expression of the CSF-1 gene in the blast cells of acute myeloblastic leukemia: association with reduced growth capacity.

Myelopoietic growth factors are known to influence the growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for the factor CSF-1 and its receptor fms to study DNA and RNA from the blasts of 25 AML patients. The CSF-1 gene was always in the germline configuration. CSF-1 mRNA was found in about half the blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF-1 expression and poor growth in suspension culture. Most blast populations expressed fms; the number of fms expression negative samples was to small to permit the detection of any association between fms expression and growth or any interaction between the effects of the expression of the growth factor and its receptor. We propose that CSF-1 may be an important part of the mechanism determining the balance between self-renewal and determination in AML blast clones.

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture.

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.

Binding of iodinated recombinant human GM-CSF to the blast cells of acute myeloblastic leukemia.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF) is an effective growth factor for the blasts of acute myeloblastic leukemia (AML). Radioiodinated Chinese hamster ovary (CHO)-cell derived GM-CSF was prepared using Bolton-Hunter reagent to label free amino groups on the protein. Normal human neutrophils and the blast cells from AML patients were examined for binding. We found that there were fewer receptors of higher affinity on blast cells compared with neutrophils. After brief culture in suspension, receptor number increased and affinity decreased. Experiments provided evidence that GM-CSF from Escherichia coli had a higher affinity for neutrophils (kd = 20 pM) than the CHO-cell derived protein (kd = 500 pM-1 nM). This difference was reflected in the increased effectiveness of the E. coli protein over the CHO protein to stimulate colony formation in both normal bone marrow cells and AML blasts.

Stem cell renewal and differentiation in acute myeloblastic leukaemia.

The defining properties of stem cells are capacities for self-renewal and, after determination, a limited number of terminal divisions. The blast cells of acute myeloblastic leukaemia (AML) are maintained by stem cells with these two properties. Since renewal and differentiation can be assessed separately in cultures of AML blasts, these cancer cells provide a useful model for examining stem regulation; such studies have practical importance for future developments in the treatment of AML. This paper considers three aspects of blast cell biology. First, evidence is presented that self-renewal and differentiation are regulated by specific genes; further, the DNA encoding these genes has structural features that affect the chemosensitivity of self-renewal. This sensitivity varies from patient-to-patient and is an important attribute contributing to variation in treatment efficacy. Second, the effects of myelopoietic growth factors on blast stem cells are presented and discussed, as these bear on the regulation of the balance between renewal and differentiation. Finally, models of leukaemic haemopoiesis are considered in light of the experimental findings. The suggestion is advanced that leukaemia can be explained better by abnormalities of gene expression than by blocked differentiation.

Structure and expression of genes of GM-CSF and G-CSF in blast cells from patients with acute myeloblastic leukemia.

The hematopoietic growth factors granulocyte/macrophage colony-stimulating factor (GM-CSF) and G-CSF, available as recombinant products, stimulate the growth in culture of blasts from patients with acute myeloblastic leukemia (AML). We used cDNA probes for each gene to study the genomic organization in blast cells of 22 patients and expression in the blast cells of 18 patients. Alteration in the structure of G-CSF (two instances) and GM-CSF (two instances) was found. In two patients in whom it was possible to study DNA from bone marrow obtained at remission, the new bands detected in the leukemic cells were not found. Fifteen of 18 patients showed no RNA expression of either growth factor. Both patients with GM-CSF abnormalities as seen by Southern analysis expressed an abnormally large GM-CSF message but no G-CSF messages. One patient with an abnormal Southern pattern with G-CSF expressed normal-sized G-CSF and GM-CSF messages. The biologic significance of these findings remains to be determined. Nonetheless, the abnormal Southern patterns may prove to be useful clonal markers in the study of AML.