Regulation of c-myc transcription by interleukin-2 (IL-2). Identification of a novel IL-2 response element interacting with STAT-4.

Regulation of c-myc expression is known to occur at the level of transcription initiation. However, the participating promoter elements and their cognate binding proteins have not been fully characterized. c-myc transcription can be stimulated by a number of cytokines including interleukin-2 (IL-2). We have identified a novel IL-2-responsive element, located in the 5'-flanking region of the c-myc gene, between nucleotides -1406 and -1387 (relative to the P2 promoter). This element belongs to the family of interferon-gamma activation site-like responsive elements and has the core sequence TTCCAATAA. We confirmed that IL-2-mediated signaling involves activation by phosphorylation of Jak2 tyrosine kinase and subsequently STAT4. The transcription factor STAT4 binds the TTCCAATAA motif within this responsive element and, therefore, is probably involved in enhancing c-myc transcription upon IL-2 stimulation. Our results propose participation of Jak2 and STAT4 in IL-2-induced up-regulation of c-myc.

PTEN transcriptionally modulates c-myc gene expression in human breast carcinoma cells and is involved in cell growth regulation.

A novel tumor suppressor gene, PTEN, has recently been identified at chromosome 10q23, which is inactivated in a number of different tumor types including breast cancer. An investigation of the functional role suggested that PTEN transcriptionally represses both exogenous and endogenous c-myc expressions in human breast carcinoma cells. PTEN, when ectopically expressed in human breast carcinoma cells, exhibited an inhibition of phosphorylation of both activating residues of protein kinase B (PKB)/AKT at Ser-473 and Thr-308 without any significant alteration of AKT expression. Furthermore, introduction of PTEN into human breast carcinoma cells induced apoptotic cell death and inhibited cell growth and tumor formation in nude mice. Taken together, our data suggest that PTEN acts as a transcriptional repressor, inhibits the AKT-mediated cell survival signaling pathway, and negatively regulates human breast carcinoma cell growth. These results further emphasize the potential of PTEN as a gene therapeutic agent.

Autoregulatory circuits in myeloma. Tumor cell cytotoxicity mediated by soluble CD16.

BACKGROUND: Multiple myeloma remains an incurable malignancy due to marked resistance of the tumor to standard doses of chemotherapy. Treatment approaches, using chemotherapeutic dose escalation and hematopoietic stem cell support have resulted in significant augmentation of tumor mass reduction such that complete remissions are effected in approximately 50% of patients. These remissions are however, often not durable. In the setting of minimal residual disease, therefore, adjunctive immunotherapy may be useful. METHODS: Peripheral blood mononuclear cells were studied from 28 untreated patients with multiple myeloma (MM). Mononuclear cell CD16 (FcR gamma III) expression was determined by flow cytometry. The effect of lymphocyte-derived soluble CD16, isolated by affinity chromatography, on MM cell growth and differentiation was assessed. MM cell proliferation, viability, immunoglobulin production and gene expression was studied. RESULTS: Data are presented indicating that cells expressing CD16 are increased in untreated patients with IgG-secreting myeloma. The predominant phenotype of these cells is CD8+ or CD56+. These CD16+ cells can produce a soluble form of the Fc receptor (sFcR, sCD16) that can bind to surface Ig on cultured human IgG-secreting myeloma cells and effect suppression of tumor cell growth and Ig secretion. This effector function is accompanied by concomitant suppression of c-myc as well as IgH and IgL gene transcription. Finally, prolonged exposure to sCD16 causes myeloma tumor cell cytolysis. CONCLUSIONS: sCD16 and possibly other soluble FcR are candidate molecules for adjunctive immunotherapy of myeloma, once complete responses have been effected by intensive cytotoxic therapy, now possible in up to 50% of newly diagnosed patients.

Deletion of the retinoblastoma gene in multiple myeloma.

Deletion of the retinoblastoma gene (Rb-1) was found in more than 50% (12/23) of patients with multiple myeloma (MM) by fluorescence in situ hybridization (FISH). Myeloma cells were highly purified from bone marrow aspirates by flow cytometry and analyzed using probes specific for the Rb-1 gene and the centromeric region of chromosomes 13 and 21. Routine cytogenetics revealed abnormal chromosome 13 in only 17% (4/23) of these patients. No correlation between Rb-1 deletion and tumor stage, immunoglobulin isotype, anemia, serum beta-2 microglobulin levels, patient age or the extent of prior therapy was found. However, the high incidence of Rb-1 deletion detected by FISH suggests a role of this tumor suppressor gene in the biology of MM. Although allelic loss of the Rb-1 gene is unlikely to be the only genetic change necessary for the development of MM, it may be a relatively early event in MM unrelated to chemotherapeutic intervention. Since the Rb-1 gene suppresses IL-6 production and secretion, Rb-1 deletion may result in deregulation of IL-6 expression and hence expansion of IL-6 dependent myeloma clones.

Immunoregulation of murine and human myeloma.

The studies presented in this review address the issue of immunoregulation of growth and differentiation of myeloma tumor cells by host effector cells (Fig. 1). In the murine myeloma system, substantial, direct evidence is presented that myeloma tumor cell growth and differentiation can be modulated by host idiotype-, antigen-, and isotype-specific immunoregulatory cells. Only isotype-specific immunoregulatory cells, however, represent an endogenous, host-generated response, whereas idiotype- and antigen-specific responses require exogenous manipulation (immunization) of the host. In patients with multiple myeloma, however, little direct evidence is available showing regulation of growth and differentiation of tumor cells. Instead, a substantial body of literature suggests a complex interaction between the host immune system and the tumor that, in principle, may result in regulation of tumor cell growth and/or differentiation, although at best the regulation is clinically ineffective. Changes in the phenotype and functions of T cells, B cells, macrophages, and NK/LAK cells have been demonstrated in patients with multiple myeloma and, at least in vitro, each of these cell populations has been shown to modulate growth/differentiation or survival of human myeloma cells. The future challenge in our studies of the immunobiology of multiple myeloma will be to better understand the mechanisms controlling growth of myeloma tumor cells and the interactions between the tumor cells and the host immune system so that we might develop appropriate strategies to augment antitumor immune responses.

Defective isotype-specific regulation of IgA anti-erythrocyte autoantibody-forming cells in NZB mice.

B cell hyperactivity characterizes many autoimmune diseases. In NZB mice this is manifested by a variety of immunologic aberrations, including increased B cell proliferation and hyper IgM and IgA secretion in vitro. Recent studies have shown that IgA secretion can be suppressed or enhanced in an isotype-specific manner by a soluble factor(s), called IgA-binding factor (IgABF), produced by IgA FcR-bearing T cells. We now show that T cells from young NZB mice, cultured with high concentrations of IgA, produce an IgABF that has aberrant biologic activity when compared to IgABF produced from IgA FcR+ T cells of BALB/c mice. Although BALB/c IgABF normally suppresses proliferation and secretion by IgA-producing B cells, neither proliferation nor IgA secretion from normal murine IgA-B cells is suppressed by NZB IgABF. In fact, IgA secretion is significantly enhanced by NZB IgABF. We also present the first evidence of IgA anti-mouse erythrocyte (anti-MRBC) autoantibody-forming cells present in the spleens of NZB mice. Whereas BALB/c IgABF suppresses the in vitro generation of IgA anti-MRBC autoantibody-forming cells by NZB spleen cells, NZB IgABF enhances this response. Of particular interest is the development of IgA anti-MRBC autoantibody-forming cells in cultures of spleen cells from nonautoimmune BALB/c mice in the presence of NZB IgABF. These studies suggest that isotype-specific T cells factors might play an important role in the development of autoantibody-forming cells.

Changes in gene expression associated with IFN-beta and IL-2-induced augmentation of human natural killer cell function.

NK function can be augmented by a variety of agents, including the cytokines IL-2 and IFN. The mechanisms associated with IL-2- and IFN-mediated augmentation of NK function are largely unknown. In order to learn more about the regulation of NK activity, we have studied changes in gene expression that occur upon treatment of a cloned line of NK cells (NK 3.3) with rIL-2 and rIFN-beta. Both IL-2 and IFN-beta induced rapid augmentation of lysis mediated by NK 3.3, which was significant within 1 h, peaked at 6 h of treatment, and declined by 12 h. This enhancement of lytic function was independent of proliferation and associated with a corresponding increase in steady state levels of RNA coding for both the nuclear proto-oncogene c-myb and for the IL-2R. These changes were specific in that RNA levels of another nuclear proto-oncogene, c-myc, were increased by IL-2 but not by IFN-beta, whereas HLA class I RNA levels were relatively unchanged by either IL-2 or IFN-beta treatment. Treatment of NK 3.3 with the combination of IL-2 and IFN enhanced both lysis and c-myb expression in an additive fashion. These findings suggest that c-myb may play a regulatory role in the cytolytic activity of NK cells.

Modulation of Ig gene expression by Ig binding factors. Suppression of alpha-H chain and lambda-2-L chain mRNA accumulation in MOPC-315 by IgA-binding factor.

Previous reports by a number of laboratories have shown that Ig-binding factors may play a role in the regulation of Ig production by B cells. Although numerous studies have addressed the specificity and biologic function of Ig-binding factors at the cellular level, little information is available regarding the mechanism whereby Ig-binding factor modulates Ig production by B cells at the molecular level. Herein we have examined the specificity and molecular mechanism of the suppression of IgA production mediated by IgA-binding factor. Using the IgA-secreting plasmacytoma, MOPC-315, as a target cell, we have demonstrated that: 1) IgA-binding factor binds to IgA, but not to IgG, IgM, or BSA; 2) IgA-binding factor can suppress proliferation as well as IgA production by MOPC-315; 3) soluble IgA, but not IgG or IgM can inhibit the action of IgA-binding factor; 4) suppression of Ig production by IgA-binding factor is maximal within 8 to 12 h after exposure to the factor and is reversible; 5) IgA-binding factor suppresses IgA production by selectively down-regulating synthesis of IgA H and L chain proteins; 6) IgA-binding factor selectively suppresses transcription of alpha-H chain and lambda-2-L chain genes; 7) IgA-binding factor suppresses accumulation of c-myc mRNA. These findings suggest that IgA-binding factor binds selectively to surface IgA on MOPC-315 and suppresses IgA production by down-regulating transcription of H and L chain genes. Suppression of MOPC-315 proliferation by IgA-binding factor may be related to the concomitant down-regulation of the expression of the c-myc gene. c-myc is deregulated in MOPC-315 by virtue of the reciprocal 15:12 chromosomal translocation present in MOPC-315 where the c-myc gene is translocated and rearranged into the alpha-H chain gene complex. Simultaneous suppression of the expression of c-myc and alpha-H chain genes suggests that these two genes may be coordinately modulated, in plasmacytomas, by IgA-binding factor.

Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia.

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non-neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high-performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogens.

Mitogens evoke many alterations in gene expression in eukaryotic cells. Genes which are activated rapidly and transiently, that are evolutionarily conserved and whose induction is shared by diverse cell types when exposed to different growth stimuli are likely to be of critical importance in transducing mitogenic signals and regulating cellular proliferation. c-myc and c-fos are the only known genes fulfilling these criteria. We report on the molecular cloning of a novel early growth response (egr) gene which also satisfies these conditions. In response to serum, its 3.7 kb mRNA is induced dramatically in mouse fibroblasts reaching a peak level at about 30 minutes that is ten times higher than the maximal value attained by c-fos mRNA. This transcript is induced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate and is "superinduced" by serum and cycloheximide together. Importantly, the gene is highly induced by different mitogens in a wide array of cell types: insulin stimulated rat hepatoma cells, adenosine diphosphate treated monkey kidney epithelial cells, and phytohemagglutinin stimulated human peripheral blood lymphocytes. Given the many properties that this gene shares with c-myc and c-fos, it may play a key role in the control of cell growth and perhaps in oncogenesis.

Differential promoter utilization by the c-myc gene in mitogen- and interleukin-2-stimulated human lymphocytes.

Transcription of the c-myc gene is initiated from two principal promoters, P1 and P2. We demonstrate here that a shift in promoter utilization occurred with time in human peripheral blood mononuclear cells (PBMC) that had been stimulated to proliferate. The P1/P2 ratio reached a maximum of approximately 1.3 at 4 h after phytohemagglutinin stimulation and a minimum of 0.31 at 48 h. Actinomycin decay experiments demonstrated that both P1 and P2 transcripts had similar half-lives at early and late times after mitogen stimulation, indicating that the shift in promoter utilization was probably not posttranscriptionally regulated. Addition of interleukin-2 to previously activated PBMC increased c-myc mRNA, but unlike increases after mitogen stimulation, the P1/P2 ratio stayed less than 0.5. Our findings demonstrated that there was a difference between mitogen- and interleukin-2-stimulated increases in c-myc RNA in PBMC.

Proto-oncogene expression in cloned T lymphocytes: mitogens and growth factors induce different patterns of expression.

We have compared the effects of a mitogenic lectin, concanavalin A (Con A), and a growth factor, interleukin-2 (IL-2), on the expression of the c-myc, c-fos, and c-myb proto-oncogenes in cloned T lymphocytes. Accumulation of c-myc mRNA was induced by both ConA and IL-2 in these cells. In contrast, expression of c-fos was stimulated primarily by ConA, and accumulation of c-myb mRNA was induced predominantly by IL-2. Thus, ConA and IL-2 induce expression of overlapping, but non-identical, sets of proto-oncogenes in T lymphocytes. Investigations with several different cloned T cells revealed that: (1) c-myb is not induced in all T cells stimulated to grow, indicating that its expression may not be absolutely required for their proliferation; and (2) expression of c-myc, even in combination with c-fos, can be insufficient for growth, demonstrating functional differences between cellular and viral oncogenes in T cells. These observations provide insight into the roles of the c-myc, c-fos, and c-myb proto-oncogenes in normal T cell responses.

Effect of cyclosporin A and dexamethasone on interleukin 2 receptor gene expression.

Here we demonstrate that CsA and DEX, at concentrations that markedly inhibited PHA-induced proliferation and IL 2 mRNA accumulation, partially diminished the expression of receptors for IL 2 on PBMC. This inhibition of IL 2 receptor expression occurred at a pretranslational level and involved a reduction in both high affinity and low affinity forms of the receptor. Although both CsA and DEX inhibited IL 2 receptor expression by about 50%, only CsA blocked the PHA-mediated induction of IL 2 responsivity in PBMC cultures. These data provide evidence that 1) CsA and DEX suppress the proliferation of T lymphocytes through distinct (though perhaps overlapping) mechanisms, 2) CsA (but not DEX) blocks the PHA-mediated induction of signals necessary for T cells to become capable of proliferating in response to IL 2, and 3) T cells regulate the expression of their genes for IL 2 and IL 2 receptors, at least in part, through independent mechanisms.

Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes.

The proliferation of non-neoplastic T lymphocytes is regulated, in part, by the coordinated expression of genes encoding T-cell growth factor (interleukin 2, IL2), IL2 receptors (IL2R), and transferrin receptors (TFR). In addition to growth factors and their receptors, protooncogenes may regulate lymphocyte proliferation. We used cloned cDNAs homologous to 21 different protooncogenes to screen for their expression at the mRNA level in human peripheral blood mononuclear cells (PBMC) stimulated with the mitogenic lectin phytohemagglutinin (PHA), and we compared the time course of accumulation of mRNAs for these protooncogenes to that of mRNAs for the IL2, IL2R, TFR, and histone H3 genes. mRNAs for c-abl, c-ets, c-yes, and N-ras were present in unstimulated PBMC. After stimulation of PBMC by PHA, we detected marked increases within 10 min in the levels of mRNA for c-fos and c-myc; within 6 hr for IL2 and IL2R mRNAs; within 14 hr for c-myb, p53, N-ras, and TFR mRNAs; and within 24-36 hr for H3 mRNA. Expression of c-abl, c-ets, and c-yes increased gradually following stimulation with PHA. None of the other protooncogenes tested was expressed in PBMC. Addition of the protein synthesis inhibitor cycloheximide, before the addition of PHA to cultures, abolished the PHA-induced accumulation of mRNAs for c-myb, N-ras, and TFR, but not of mRNAs for c-fos, c-myc, IL2, and IL2R. These data indicate that c-fos, c-myc, IL2, and IL2R belong to a group of genes expressed early, whereas c-myb, N-ras, and TFR belong to a group of genes expressed later in PHA-activated PBMC, and that the products of the c-fos and c-myc protooncogenes are not required for expression of IL2 or IL2R genes. Addition of purified IL2 augmented the expression of the later-expressed genes c-myb, p53, N-ras, and TFR in PHA-stimulated cultures of PBMC, as well as of the early genes c-myc and IL2R, but not of c-fos and IL2, thus suggesting that PHA and IL2 stimulate the expression of overlapping, but nonidentical, sets of genes in PBMC.

Recombinant interleukin 2 regulates levels of c-myc mRNA in a cloned murine T lymphocyte.

The cellular oncogene c-myc has been implicated in the regulation of growth of normal and neoplastic cells. Recently, it was suggested that c-myc gene expression may control the G0----G1-phase transition in normal lymphocytes that were stimulated to enter the cell cycle by the lectin concanavalin A (ConA). Here we describe the effects of purified recombinant interleukin 2 (rIL2) and of ConA on levels of c-myc mRNA in the noncytolytic murine T-cell clone L2. In contrast to resting (G0) primary cultures of lymphocytes, quiescent L2 cells have a higher RNA content than resting splenocytes and express receptors for interleukin 2 (IL2). Resting L2 cells are therefore best regarded as early G1-phase cells. Purified rIL2 was found to stimulate the rapid accumulation of c-myc mRNA in L2 cells. Levels of c-myc mRNA became maximal within 1 h and declined gradually thereafter. In contrast, ConA induced slower accumulation of c-myc mRNA in L2 cells, with increased levels of c-myc mRNA becoming detectable 4 to 8 h after stimulation. Experiments with the protein synthesis inhibitor cycloheximide demonstrated that the increase in levels of c-myc mRNA that were induced by ConA was a direct effect of this lectin and not secondary to IL2 production. Cyclosporin A, an immunosuppressive agent, markedly reduced the accumulation of c-myc mRNA that was induced by ConA but only slightly diminished the accumulation of c-myc mRNA that was induced by rIL2. Taken together, these data provide evidence that (i) c-myc gene expression can be regulated by at least two distinct pathways in T lymphocytes, only one of which is sensitive to cyclosporine A, and (ii) the accumulation of c-myc mRNA can be induced in T cells by IL2 during the G1 phase of the cell cycle.

Monoclonal antibody OKT11A inhibits and recombinant interleukin 2 (IL 2) augments expression of IL 2 receptors at a pretranslational level.

The expression of receptors for interleukin 2 (IL 2) represents a critical event regulating the growth of normal T lymphocytes. We investigated the effects of the inhibitory monoclonal antibody OKT11A (anti-sheep erythrocyte receptor) and of purified recombinant IL 2 (rIL 2) on the expression of IL 2 receptors by activated T cells at both the protein and the mRNA levels. Adding OKT11A antibody (0.5 microgram/ml) to phytohemagglutinin (PHA)-stimulated cultures of human peripheral blood mononuclear cells (PBMC) markedly suppressed cellular proliferation (assessed by [3H]thymidine incorporation) and IL 2 receptor expression (determined by immunofluorescence assay by using the anti-IL 2-receptor antibody, anti-Tac). Northern blot analysis performed with the use of a cDNA probe specific for the human IL 2 receptor gene demonstrated that OKT11A antibody also decreased the accumulation of IL 2 receptor mRNA induced by PHA in PBMC. Purified rIL 2 (10 U/ml) alone had little effect on the expression of IL 2 receptors in unstimulated PBMC cultures. In combination with PHA or with PHA plus OKT11A, however, rIL 2 augmented both the expression of IL 2 receptor protein on PBMC and the accumulation of IL 2 receptor mRNA in PBMC. Adding anti-Tac antibody to PBMC cultures to block the interaction of IL 2 with its receptor diminished the accumulation of IL 2 receptor mRNA induced by PHA. Taken together, these data demonstrate that OKT11A antibody inhibits and IL 2 augments expression of IL 2 receptors on PHA-stimulated T cells, at least in part, at a pretranslational level.