Transplantable myeloproliferative disease induced in mice by an interleukin 6 retrovirus.

Lethally irradiated mice transplanted with bone marrow cells infected with a novel recombinant retrovirus (murine stem cell virus-interleukin 6 [MSCV-IL-6]) bearing a mouse IL-6 gene developed a fatal myeloproliferative disease within 4 wk of engraftment. The hematologic manifestations of the syndrome included elevated peripheral leukocyte counts (up to 430 x 10(3) cells/mm3) with a predominance of neutrophilic granulocytes, microcytic anemia, and thrombocytosis or thrombocytopenia. The mice showed extensive neutrophil infiltration of the lungs, liver, and occasionally lymph nodes, plus splenomegaly resulting from enhanced splenic myelopoiesis (30-60-fold increase in progenitor numbers). Despite the chronic stimulation of neutrophil excess by IL-6, bone marrow from affected mice was capable of repopulating the hematopoietic tissues (bone marrow and spleen) of lethally irradiated hosts during repeated serial transplantation. In the longest documented case, the progeny of a single MSCV-IL-6-marked cell transferred the myeloproliferative disease to two secondary, four tertiary, and two quaternary recipients (the clone endured for a total of 72 wk). These results, demonstrating considerable proliferative longevity of the IL-6-producing cells, support an in vivo role of IL-6 in the maintenance of hematopoietic precursors. Dysregulated IL-6 production also had significant systemic effects. The mice displayed increased mesangial cell proliferation in the kidney, frequent liver abnormalities, and marked alterations in plasma protein levels. Unlike previous studies where constitutive expression of exogenous IL-6 genes resulted in lymphoproliferative disorders characterized by massive plasmacytosis, minimal plasma cell expansion occurred in the MSCV-IL-6 mice during the observation period. Potential explanations for the differences in disease phenotypes observed in the present and previous studies are different cell types expressing the exogenous IL-6 genes, higher sustained circulating levels of IL-6 achieved using the MSCV-IL-6 retroviral delivery system, and/or the premature death (3-15 wk after transplantation) of the MSCV-IL-6 mice before the onset of plasmacytosis. This animal model should prove useful for further investigation of the function of IL-6 in normal and abnormal hematopoiesis and in inflammatory responses.

Progenitor cell hyperplasia with rare development of myeloid leukemia in interleukin 11 bone marrow chimeras.

Post 5-fluorouracil-treated murine bone marrow cells infected with a recombinant retrovirus (murine stem cell virus-interleukin 11 [MSCV-IL-11]) bearing a human IL-11 gene were transplanted into lethally irradiated syngeneic mice. Analysis of proviral integration sites in DNA prepared from hematopoietic tissues and purified cell populations of long-term reconstituted primary and secondary recipients demonstrated polyclonal engraftment by multipotential stem cells. High levels (100-1,500 U/ml) of IL-11 were detected in the plasma of the MSCV-IL-11 mice. Systemic effects of chronic IL-11 exposure included loss of body fat, thymus atrophy, some alterations in plasma protein levels, frequent inflammation of the eyelids, and often a hyperactive state. A sustained rise in peripheral platelet levels (approximately 1.5-fold) was seen throughout the observation period (4-17 wk). No changes were observed in the total number of circulating leukocytes in the majority of the transplanted animals (including 10 primary and 18 secondary recipients) despite a > 20-fold elevation in myeloid progenitor cell content in the spleen. The exceptions were members of one transplant pedigree which presented with myeloid leukemia during the secondary transplant phase. A clonal origin of the disease was determined, with significant expansion of the MSCV-IL-11-marked clone having occurred in the spleen of the primary host. Culturing of leukemic spleen cells from a quaternary recipient led to the establishment of a permanent cell line (denoted PGMD1). IL-11-producing PGMD1 myeloid leukemic cells are dependent on IL-3 for continuous growth in vitro and they differentiate into granulocytes and macrophages in response to granulocyte/macrophage colony-stimulating factor. The inability of autogenously produced IL-11 to support autonomous growth of PGMD1 cells argues against a mechanism of transformation involving a classical autocrine loop.

TLX1/HOX11-induced hematopoietic differentiation blockade.

Aberrant expression of the human homeobox-containing proto-oncogene TLX1/HOX11 inhibits hematopoietic differentiation programs in a number of murine model systems. Here, we report the establishment of a murine erythroid progenitor cell line, iEBHX1S-4, developmentally arrested by regulatable TLX1 expression. Extinction of TLX1 expression released the iEBHX1S-4 differentiation block, allowing erythropoietin-dependent acquisition of erythroid markers and hemoglobin synthesis. Coordinated activation of erythroid transcriptional networks integrated by the acetyltransferase co-activator CREB-binding protein (CBP) was suggested by bioinformatic analysis of the upstream regulatory regions of several conditionally induced iEBHX1S-4 gene sets. In accord with this notion, CBP-associated acetylation of GATA-1, an essential regulator of erythroid differentiation, increased concomitantly with TLX1 downregulation. Coimmunoprecipitation experiments and glutathione-S-transferase pull-down assays revealed that TLX1 directly binds to CBP, and confocal laser microscopy demonstrated that the two proteins partially colocalize at intranuclear sites in iEBHX1S-4 cells. Notably, the distribution of CBP in conditionally blocked iEBHX1S-4 cells partially overlapped with chromatin marked by a repressive histone methylation pattern, and downregulation of TLX1 coincided with exit of CBP from these heterochromatic regions. Thus, we propose that TLX1-mediated differentiation arrest may be achieved in part through a mechanism that involves redirection of CBP and/or its sequestration in repressive chromatin domains.

Protein tyrosine phosphatase 2 (SHP-2) moderates signaling by gp130 but is not required for the induction of acute-phase plasma protein genes in hepatic cells.

Signals propagated via the gp130 subunit of the interleukin-6 (IL-6)-type cytokine receptors mediate, among various cellular responses, proliferation of hematopoietic cells and induction of acute-phase plasma protein (APP) genes in hepatic cells. Hematopoietic growth control by gp130 is critically dependent on activation of both STAT3 and protein tyrosine phosphatase 2 (SHP-2). To investigate whether induction of APP genes has a similar requirement for SHP-2, we constructed two chimeric receptors, G-gp130 and G-gp130(Y2F), consisting of the transmembrane and cytoplasmic domains of gp130 harboring either a wild-type or a mutated SHP-2 binding site, respectively, fused to the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor. Rat hepatoma H-35 cells stably expressing the chimeric receptors were generated by retroviral transduction. Both chimeric receptors transmitted a G-CSF-induced signal characteristic of that triggered by IL-6 through the endogenous gp130 receptor; i.e., both activated the appropriate JAK, induced DNA binding activity by STAT1 and STAT3, and up-regulated expression of the target APP genes, those for alpha-fibrinogen and haptoglobin. Notwithstanding these similarities in the patterns of signaling responses elicited, mutation of the SHP-2 interaction site in G-gp130(Y2F) abrogated ligand-activated receptor recruitment of SHP-2 as expected. Moreover, the tyrosine phosphorylation state of the chimeric receptor, the associated JAK activity, and the induced DNA binding activity of STAT1 and STAT3 were maintained at elevated levels and for an extended period of time in G-gp130(Y2F)-expressing cells following G-CSF treatment compared to that in cells displaying the G-gp130 receptor. H-35 cells ectopically expressing G-gp130(Y2F) were also found to display an enhanced sensitivity to G-CSF and a higher level of induction of APP genes. Overexpression of the enzymatically inactive SHP-2 enhanced the signaling by the wild-type but not by the Y2F mutant G-gp130 receptor. These results indicate that gp130 signaling for APP gene induction in hepatic cells differs qualitatively from that controlling the proliferative response in hematopoietic cells in not being strictly dependent on SHP-2. The data further suggest that SHP-2 functions normally to attenuate gp130-mediated signaling in hepatic (and, perhaps, other) cells by moderating JAK action.

Transforming function of the HOX11/TCL3 homeobox gene.

The HOX11/TCL3 homeobox gene was identified at the breakpoint region in pediatric T-cell acute lymphoblastic leukemia harboring 10q24 chromosomal translocations. We previously reported that primary murine bone marrow cells transduced ex vivo with a recombinant HOX11-containing retrovirus, MSCV-HOX11, gave rise to cell lines at high frequency having characteristics of early myeloid cells. Cell lines were also established from the bone marrow and spleen of transplant recipients sacrificed 5 months after engraftment with MSCV-HOX11-transduced bone marrow cells. These latter lines, which exhibited a more differentiated myelomonocytic phenotype, harbored proviruses encoding a smaller HOX11 protein. None of the mice that received HOX11-expressing bone marrow cells or myeloid cell lines developed leukemia during 6-month observation periods. Here, we report that two bone marrow transplant recipients eventually developed T-cell acute lymphoblastic leukemia-like malignancies at 7 and 12 months posttransplant, indicating that progression to a fully malignant state required additional mutations. One tumor synthesized full-length HOX11 whereas the other expressed the smaller version of the protein. The smaller HOX11 protein suffered a carboxyl-terminal truncation. We subsequently constructed MSCV-based retroviral vectors expressing deleted forms of HOX11 and identified an amino-terminal region that was dispensible for generation of myeloid cell lines having a similar phenotype as those induced by full-length HOX11. We thus conclude that regions near the amino and carboxyl termini of HOX11 are not essential for transforming function, nor do they appear to determine the lineage or stage of differentiation of the target cell for transformation.

Hematopoietic transforming potential of activated ras in chimeric mice.

Although activating mutations in ras genes are the most common genetic abnormality in human hematologic malignancies, the role of ras mutations as an initiating event in leukemogenesis remains unclear. To assess the consequences of ectopic expression of an activated ras gene in normal hematopoietic cells in vivo, lethally irradiated mice were reconstituted with bone marrow cells infected with a mutant ras-containing retrovirus [murine stem cell virus (MSCV)-v-H-ras] based on the MSCV retroviral vector which efficiently transduces functional genes into hematopoietic stem/progenitor cells. Despite a marked myeloid leukocytosis detectable in the peripheral blood within 4 weeks of engraftment, none of 22 primary or secondary transplant recipients studied for longer periods of time presented with myeloid neoplasms. Instead, 18 of the MSCV-v-H-ras mice developed pre-T-cell thymic lymphomas and/or pre-B-cell lymphoblastic leukemia/lymphomas between 7 and 12 weeks post-transplantation. The pre-B and pre-T lymphoid tumors that arose in one animal were shown to harbor a common MSCV-v-H-ras provirus, indicating that the target cell for transformation was a bipotential lymphoid precursor. To more precisely examine the effects of activated ras expression on the behavior of hematopoietic progenitors, infected bone marrow cells were assayed in methylcellulose cultures under conditions favorable for growth of multilineage myeloid colonies or were passaged as bulk suspension cultures in the presence of various hematopoietic growth factors, including interleukin (IL)-3, IL-4, IL-6 and IL-7. MSCV-directed expression of v-H-ras selectively promoted the formation of large dense colonies comprised of monocyte-macrophages in methylcellulose cultures. When transferred to liquid cultures, the vast majority of the cells underwent terminal macrophage differentiation. By comparison, tumorigenic B-lymphoid and mixed lymphoid/myeloid cell lines were routinely established from the bulk suspension cultures, with cell lines of predominantly myeloid phenotype emerging only in IL-6-supplemented cultures. These results, considered together with previous findings, suggest that activating ras mutations could be an initiating genetic alteration in human acute lymphoblastic leukemia but are more likely to be a post-initiation change in human acute myeloid leukemia.

The HOX11 homeobox-containing gene of human leukemia immortalizes murine hematopoietic precursors.

The t(10;14) chromosomal translocation of T-cell acute lymphoblastic leukemia joins the T-cell receptor delta gene to a region upstream of a diverged homeobox-containing gene called HOX11. To better understand the pathogenetic role of HOX11 in leukemogenesis, post 5-fluorouracil-treated murine bone marrow cells were infected with a replication-defective retrovirus bearing the gene. Constitutive expression of HOX11 in hematopoietic precursors yielded cell lines at high frequency consisting of immature cells belonging to the myeloid lineage. HOX11-transformed cell lines displayed a strict dependence on IL-3 for their survival and proliferation in culture and were not leukemogenic. The results support the hypothesis that the transforming capacity of HOX11 derives from its ability to alter the expression of genes regulating hematopoietic differentiation and that secondary mutations promoting cell survival or stimulating proliferation are required for progression to malignancy. The findings further suggest that the oncogenic activity of HOX11 might not be restricted to T-cell leukemias in humans.

Regulation of a metallothionein-rasT24 fusion gene by zinc results in graded alterations in cell morphology and growth.

We constructed fusion genes consisting of the mouse metallothionein I (MT) 5' region and the coding region of either the human H-ras gene (c-rasP3) or a mutated allele (c-rasT24); both ras genes lacked the initial (non-coding) exon and the first 30 bp of the non-coding region of the second exon. Transfection of Rat-1 cells produced foci only with pMT-rasT24, and selection in soft agar yielded clones in which MT-rasT24 expression was zinc-regulatable. In response to increasing concentrations of ZnSO4, these lines showed increasingly altered morphology (conversion to fusiform or spheroidal morphology), progressively higher maximal cell density, and an increasingly greater fraction of cells in the S + G2 + M portion of the cell cycle at high density. MT-rasT24 RNA levels in zinc-responsive lines were increased between 4- and 6-fold by the addition of ZnSO4 (final concentration = 100 microM) to the medium. Replating cells in the absence of zinc reversed the biological effects and resulted in reduction in MT-rasT24 RNA levels. Thus, graded alterations in phenotype result from increasing levels of MT-rasT24 gene expression.

Enhanced immunogenicity of B cell lymphoma genetically engineered to express both B7-1 and interleukin-12.

The A20 murine B cell lymphoma was transfected with B7-1 and subsequently these variants and vector control variants were retrovirally infected to express murine interleukin-12 (mIL-12). In vitro data showed that the B7-1 variants enhanced secretion of IL-2 and IL-4 by allogeneic T cells in mixed lymphocyte tumor cultures. While IL-12 variants stimulated IFN-gamma, variants expressing both B7-1 and IL-12 stimulated IFN-gamma, IL-2, and IL-4 secretion. Tumorigenicity experiments showed that whereas B7-1 delayed tumor onset, only the mIL-12 variants with or without B7-1 were completely rejected in syngeneic hosts. In addition, tumor-free mice were protected against subsequent challenge with the parental unmodified cells and had enhanced cytotoxic T lymphocyte (CTL) lysis activity. Results from minimal disease mixing experiments demonstrated that only the A20/B7-1/mIL-12 variant was able to reject A20 unmodified cells inoculated at the same site, whereas prolonged survival was observed when the A20 parental cells were inoculated at different sites. Depletion studies and injections into nu-/nu- mice demonstrated that both CD4+ and CD8+ T cells may mediate immunity. These data suggest that vaccinations with tumor cells genetically modified to express both B7-1 and IL-12 may alter cytokine profiles and generate CTL activity and, thus, the mechanisms of enhanced antitumor immunity may be multifactorial.

Transmissibility of murine stem cell virus-based retroviral vectors carrying both interleukin-12 cDNAs and a third gene: implications for immune gene therapy.

The combination of immunotherapy with conventional treatments such as radio- and chemotherapy may be necessary to eradicate minimal residual disease. Interleukin 12 (IL-12) is a heterodimeric cytokine composed of two subunits, p40 and p35. Coordinate expression of the IL-12 p40 and p35 genes in several solid tumor models has been found to induce strong and specific antitumor immune responses. In the interest of obtaining high level IL-12 expression in leukemia/lymphoma cells for use as vaccines in cancer immunotherapy, we evaluated three IL-12 retroviral vector designs based on the murine stem cell virus (MSCV) vector which efficiently transduces functional genes into normal hematopoietic cells. MSCVpac-mlL-12 and MIPV-mIL-12 contain an encephalomyocarditis virus internal ribosome entry site for internal translation of bicistronic mRNA transcripts, while MDCVpac-mIL-12 carries an expression cassette in the U3 region of the 3' long terminal repeat. We found that the MSCVpac-mIL-12 vector directed robust expression of both p40 and p35 genes in several murine tumor cell lines of hematopoietic origin, including a T-cell lymphoma, a B-cell lymphoma, and a plasmacytoma/myeloma. In contrast, genomic instability or promoter interference hampered p40 gene expression in cells transduced with the MIPV-mIL-12 and MDCVpac-mIL-12 vectors, respectively. These findings provide the basis for the design of IL-12 retroviral vectors for the treatment of hematologic malignancies in humans.

Retroviral-mediated transfer of genes encoding interleukin-2 and interleukin-12 into fibroblasts increases host antitumor responsiveness.

The transfer of genes encoding cytokines into tumor cells has emerged as a new strategy to increase in vivo host reactivity to a variety of tumors. Because gene transfer into tumor cells cannot be easily applied in the clinical setting, we have developed an experimental model of gene transfer into fibroblasts and examined the capacity of these engineered cells to elicit an antitumor immune response. Interleukin-12 (IL-12) is a heterodimeric cytokine with pleiotropic activities presenting strong antitumor and antimetastatic effects in murine models. A bicistronic retroviral vector was constructed that contained the cDNAs encoding both chains (p40 and p35) of murine IL-12 separated by an internal ribosomal entry site sequence. Syngeneic cutaneous fibroblasts obtained from newborn mice and transduced to secrete either IL-12 or IL-2 were injected subcutaneously with B16F0 or B16F1 melanoma cells. The time of tumor occurrence and overall survival of mice were significantly prolonged when B16F1 cells were coinjected with cytokine-producing fibroblasts compared with B16F1 alone or B16F1 together with unmanipulated fibroblasts. Systemic effects were seen in the mice injected with either IL-2- or IL-12-secreting fibroblasts, with the highest proliferation capability and interferon-gamma production observed in vitro from splenocytes from recipients of IL-2-secreting fibroblasts. Injection of IL-2-secreting fibroblasts or coinjection of IL-2- and IL-12-producing fibroblasts resulted in a significant increase of survival in the B16F0 model; in some cases, complete disease eradication was observed. These results suggest that cutaneous fibroblasts represent a target of choice for gene transfer and would be useful in the treatment of minimal residual disease in humans.

Tricistronic viral vectors co-expressing interleukin-12 (1L-12) and CD80 (B7-1) for the immunotherapy of cancer: preclinical studies in myeloma.

Synergy between interleukin-12 (IL-12) and B7-1 (CD80) for cancer immunotherapy has previously been demonstrated in animal models of breast cancer, lymphoma, and multiple myeloma. With a view to human clinical application, tricistronic retroviral and adenovirus vectors co-expressing IL-12 (IL-12p40 plus IL-12p35) and CD80 were constructed by utilizing two internal ribosome entry site (IRES) sequences to link the three cDNAs. A murine stem cell virus (MSCV)-based retroviral vector (MSCV-hIL12.B7) utilized distinct IRES sequences from the encephalomyocarditis virus (EMCV) and the foot-and-mouth disease virus (FMCV), whereas Ad5-based adenovirus vectors contained transcriptional units with two EMCV IRES sequences under the control of murine (AdMh12.B7) or human (AdHh12.B7) cytomegalovirus promoters. AdMh12.B7 was found to consistently direct higher levels of IL-12 and CD80 expression than AdHh12.B7 following infection of a number of human tumor cell lines. In preclinical studies, the human myeloma cell line U266 was infected with MSCV-hIL12.B7 and a resulting clonal cell line, U/MSCV-h12.B7, was generated with stable expression of CD80 and secreting IL-12 at 1 ng/24 h/10(6) cells. By comparison, following AdMh12.B7 infection, 81% of infected U266 cells (U/AdMh12.B7) expressed CD80 and secreted IL-12 at 25-50 ng/24 h/10(6) cells. Both engineered myeloma cell lines stimulated enhanced allogeneic mixed lymphocyte proliferation and provoked increases in cytotoxic T-lymphocyte responses and gamma-interferon release from normal donor lymphocytes exposed to parental U266 cells. These results suggest potential clinical utility of AdMh12.B7 in immunotherapy strategies for the treatment of multiple myeloma and other cancers.

Association between ICAM-1 expression and metastatic capacity of murine B-cell hybridomas.

We previously reported that a derivative of the interleukin-6 (IL-6)-dependent B9 B-cell hybridoma (B9/LPNU1L) constitutively expressing an interleukin-1 alpha (IL-1 alpha) gene introduced by retrovirus-mediated gene transfer preferentially metastasized to bone marrow following intravenous injection into unirradiated syngeneic BALB/c mice. B9/LPNU1L cells recovered from the femoral marrow of a recipient with hind limb paralysis (denoted B9/BM1) retained their IL-6-dependency yet displayed enhanced metastatic capacity during serial transplantation in vivo. In contrast, autonomously-growing B9 variants spontaneously arising in vitro or IL-6-independent B9 derivatives created by infection with recombinant IL-6 retroviruses rarely gave rise to experimental metastases in syngeneic BALB/c or nude mice. Examination of cell adhesion molecule profiles by immunofluorescence flow cytometry has revealed high levels of CD44, moderate levels of VLA-4 and low levels of LFA-1 on all B9-series cells. By comparison, ICAM-1 expression was significantly elevated on B9/BM1 cells, with independent isolates stably expressing about 4-fold higher levels which were paralleled by corresponding increases in the steady-state levels of ICAM-1 mRNA. L-Selectin was not expressed by any of the cell lines. Despite higher ICAM-1 levels, cell aggregation assays revealed that LFA-1-ICAM-1 adhesive interactions were not involved in the homotypic adhesion of B9/BM1 cells but rather that binding of CD44 to endogenously-synthesized hyaluronan was responsible. Furthermore, B9/BM1 cells expressing high levels of ICAM-1 were found to be less susceptible to cytolysis by natural killer (NK) cells than their weakly metastatic or nonmetastatic counterparts.

Increased frequency of both total and specific monoclonal antibody producing hybridomas using a fusion partner that constitutively expresses recombinant IL-6.

The addition of auxiliary feeder cells or conditioned medium has been shown to augment the yield of mouse hybridomas obtained following the cell-cell fusion of myeloma and B lymphocytes. The addition of one of these factors, interleukin-6 (IL-6) has been found to increase the proportion of hybridomas secreting monoclonal antibodies of desired specificity. As an alternative genetic approach, we have examined the efficacy of a retroviral infectant of Sp2/0 cells that constitutively expresses recombinant murine IL-6 (Sp2/mIL-6) as fusion partner. The results demonstrated that the yields of both viable Ig-secreting hybridomas, and antigen-specific monoclonal antibodies were increased 3-15-fold and 5-9-fold, respectively, with the Sp2/mIL-6 relative to Sp2/0 or Sp2/neo cells as fusion partner. Sp2/mIL-6 cells generated hybridomas with comparable growth rates, stability, and Ig production. The results of staining nascent hybridoma colonies immunohistochemically for Ig production suggest that Sp2/mIL-6 cells as a fusion partner increased the viability and/or stability of nascent hybrid cells that are producing Ig. Thus the Sp2/mIL-6 cells are an improved myeloma parent for the generation of large numbers of antibody-producing hybridomas against specific antigens.

Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins.

Flow cytometric procedures are described to detect a "humanized" version of a new red fluorescent protein (DsRed) from the coral Discosoma sp. in conjunction with various combinations of three Aequorea victoria green fluorescent protein (GFP) variants--EYFP, EGFP, and ECFP. In spite of overlapping emission spectra, the combination of DsRed with EYFP, EGFP, and ECFP generated fluorescence signals that could be electronically compensated in real time using dual-laser excitation at 458 and 568 nm. Resolution of fluorescence signals from DsRed, EYFP, and EGFP was also readily achieved by single-laser excitation at 488 nm. Since many flow cytometers are equipped with an argon-ion laser that can be tuned to 488 nm, the DsRed/EYFP/EGFP combination is expected to have broad utility for facile monitoring of gene transfer and expression in mammalian cells. The dual-laser technique is applicable for use on flow cytometers equipped with tunable multiline argon-ion and krypton-ion lasers, providing the framework for studies requiring simultaneous analysis of four fluorescent gene products within living cells.

Leukocytosis in mice following long-term reconstitution with genetically-modified bone marrow cells constitutively expressing interleukin 1 alpha or interleukin 6.

Leukemic cells of patients with acute myeloid leukemia have recently been shown to spontaneously produce autostimulatory IL-1 and IL-6. In order to investigate the effects of systemic production of these cytokines on normal hematopoietic cells, mice were engrafted with bone marrow cells infected with high-titer retroviral vectors carrying the murine IL-1 alpha or IL-6 genes and the neomycin phosphotransferase gene. Sustained expression of the introduced IL-1 alpha and IL-6 genes was documented by Northern-blot analysis of RNA from G418-resistant mast cells and T cells, derived from bone marrow and spleen, respectively, of successfully reconstituted mice 6-10 months after transplantation. A single mouse engrafted with IL-1 alpha-infected cells which presented with a dramatic neutrophilic granulocytosis (54-fold elevation in circulating neutrophils) was sacrificed for health concerns 2 months post-transplant. Modest changes in peripheral leukocyte counts (at most a 2-fold rise) were observed in all of the other mice, and they remained healthy throughout the observation period. The majority displayed increased hematopoietic activity in bone marrow and spleen, predominantly granulopoiesis, with moderate lymphoid hyperplasia seen in the spleens of mice constitutively expressing IL-1 alpha. These mouse models provide the opportunity to evaluate the potential of persistent IL-1 alpha and IL-6 expression to contribute to leukemogenic transformation.

Identification of c-myc promoter-binding protein and X-box binding protein 1 as interleukin-6 target genes in human multiple myeloma cells.

Interleukin-6 (IL-6) is implicated in the in vivo proliferation of malignant plasma cells in multiple myeloma. To define the molecular basis of the IL-6-induced mitogenic response in myeloma cells, we applied STAR (subtractive transcriptional amplification of mRNA), a new differential expression analysis technology, to isolate mRNAs preferentially expressed in IL-6-treated versus untreated cultures of the factor-responsive myeloma cell line U266. From the resulting collection of STAR clones, sequence information was obtained for a total of 72 distinct transcripts. Of these, 29 were found to correspond to known genes, 22 matched expressed sequence tags in public databases and 21 showed no sequence similarity to any existing entries. Among the known genes uncovered in the screen were those encoding proteins that function in cell division, cell signalling and gene/protein expression. Northern blot analysis documented that two transcription factor genes chosen for further study, c-myc promoter-binding protein (MBP-1) and X-box binding protein 1 (XBP-1), were up-regulated in U266 cells about 3-fold relative to the cell cycle-dependent beta-actin gene 12 h after IL-6 treatment. Both genes were also similarly up-regulated by IL-6 in factor-dependent ANBL-6 myeloma cells. These results indicate that MBP-1 and XBP-1 are IL-6 genes in myeloma cells; as such, they may play a role in IL-6-mediated growth control in multiple myeloma.

TALE homeoproteins as HOX11-interacting partners in T-cell leukemia.

The mammalian PBX and Meis proteins belong to the TALE (three-amino acid-loop-extension) superfamily of homeodomain-containing transcription factors. Members of both the PBX and Meis groups have been implicated in tumorigenesis and are known to cooperatively bind DNA with Class I (clustered) HOX homeoproteins. Here we show that PBX and Meis homeoproteins cooperatively bind the PBX-responsive sequence in vitro with the oncoprotein encoded by the non-clustered homeobox gene HOX11 activated by the t(10;14)(q24;q11) chromosomal translocation in T-cell acute lymphoblastic leukemia (T-ALL). An FPWME motif N-terminal to the homeodomain is required for interaction with PBX proteins, which appears to confer DNA-binding specificity to HOX11. PBX proteins are highly expressed in HOX11 immortalized/transformed hematopoietic cells; in particular, the 10q24 translocation-carrying T-ALL Sil and K3P lines were found to selectively express PBX2. Ectopic retroviral-directed overexpression of PBX2 in concert with HOX11 in NIH3T3 cells resulted in decreased contact inhibition of growth as evidenced by focus formation in confluent cell monolayers. The accumulated data are thus consistent with a role of TALE homeoproteins in HOX11-mediated leukemogenesis.

Improved therapeutic outcome following combination immunogene vaccination therapy in murine myeloma.

Increasing evidence suggests a role for immunologic vaccination and therapy in the management of minimal residual myeloma. We have previously demonstrated a synergistic effect of combining the Th1 stimulating cytokine IL-12 with the co-stimulatory molecule CD80 in murine myeloma vaccination therapy. We reasoned that the efficacy of such treatment might be further improved by incorporating additional gene products which enhance the function of antigen presenting cells. Studies were therefore conducted with murine myeloma BM1 cells expressing Flt3L (membrane bound or soluble forms) or GM-CSF and the IL-12 x CD80 combination. Single agent and combined therapeutic approaches were explored. All gene-modified BM1 cells, except BM1/IL-12 x CD80, developed tumors when subcutaneously injected into BALB/c mice. As prophylactic tumor vaccines, the combined use of gene-modified BM1/sFlt3L+GM-CSF+IL-12 x CD80 was most effective, providing 100% protection against subsequent parental BM1 tumor challenge. By comparison, only partial protection was observed with any single gene-engineered tumor vaccine. Notably, IL-12 x CD80 coexpressing BM1 cell vaccines were the most effective therapeutic vaccine in a minimal disease model. Such protective vaccination was achieved by stimulation of lymphocyte proliferation and enhancement of cytotoxic lymphocyte activity.

Targeting the cancer cell cycle by cold atmospheric plasma.

Cold atmospheric plasma (CAP), a technology based on quasi-neutral ionized gas at low temperatures, is currently being evaluated as a new highly selective alternative addition to existing cancer therapies. Here, we present a first attempt to identify the mechanism of CAP action. CAP induced a robust ~2-fold G2/M increase in two different types of cancer cells with different degrees of tumorigenicity. We hypothesize that the increased sensitivity of cancer cells to CAP treatment is caused by differences in the distribution of cancer cells and normal cells within the cell cycle. The expression of γH2A.X (pSer139), an oxidative stress reporter indicating S-phase damage, is enhanced specifically within CAP treated cells in the S phase of the cell cycle. Together with a significant decrease in EdU-incorporation after CAP, these data suggest that tumorigenic cancer cells are more susceptible to CAP treatment.