Larval Bradysia impatiens (Diptera: Sciaridae) potential for vectoring Pythium root rot pathogens.

A series of laboratory experiments were conducted to investigate the capacity of Bradysia impatiens (Johannsen) larvae to ingest propagules from two strains each of Pythium aphanidermatum (Edson) Fitzp. and P. ultimum Trow and transmit the pathogens to healthy geranium seedlings on a filter-paper substrate in petri dishes. The capacity of fungus gnat larvae to transmit P. aphanidermatum to seedlings rooted in a commercial peat-based potting mix and germination of Pythium oospores and hyphal swellings before and after passage through the guts of larval fungus gnats were also examined. Assays revealed that Pythium spp. transmission by larval fungus gnats varied greatly with the assay substrate and also with the number and nature of ingested propagules. Transmission was highest (65%) in the petri dish assays testing larvae fed P. aphanidermatum K-13, a strain that produced abundant oospores. Transmission of strain K-13 was much lower (<6%) in plug cells with potting mix. Larvae were less efficient at vectoring P. ultimum strain PSN-1, which produced few oospores, and no transmission was observed with two non-oospore-producing strains: P. aphanidermatum Pa58 and P. ultimum P4. Passage of P. aphanidermatum K-13 through larval guts significantly increased oospore germination. However, decreased germination of hyphal swellings was observed following larval gut passage for strains of P. ultimum. These results expand previous studies suggesting that larval fungus gnats may vector Pythium spp.

Fungus gnat feeding and mechanical wounding inhibit Pythium aphanidermatum infection of geranium seedlings.

ABSTRACT A series of laboratory tests were conducted to investigate potential effects of fungus gnat (Bradysia impatiens) feeding damage on susceptibility of geranium seedlings (Pelargonium x hortorum) to infection by the root rot pathogen Pythium aphanidermatum. Effects were compared with those from similar tests in which the seedlings were mechanically wounded by severing the root tip with a scalpel. Assays of geranium seedlings in petri dishes revealed a pronounced negative fungus gnat-Pythium interaction, with exposure to fungus gnat larvae 24 h prior to inoculation with P. aphanidermatum zoospores resulting in up to 47% fewer seedling deaths than would have been expected if the two agents had acted independently. Similar results were observed when seedlings were subjected to mechanical wounding 24 h prior to zoospore inoculation. In contrast, no interaction occurred when seedlings were mechanically wounded immediately prior to inoculation. The degree of plant damage inflicted by the feeding activities of the larval fungus gnats had no significant effect on the combined damage from fungus gnats and Pythium in petri dishes. Ancillary studies showed that Pythium development on V8 agar was not inhibited by the presence of fungus gnat-associated microorganisms, nor were seedlings inoculated with these microbes less susceptible to Pythium infection. The precise mechaism or mechanisms underlying the observed interactions were not elucidated; however, the results strongly suggest that both fungus gnat feeding and mechanical wounding activated systemic defenses that made the seedlings more resistant to Pythium infection.

Antileukemic activity of Flt3 ligand in murine leukemia.

Flt3-ligand (Flt3-L) is an early acting costimulatory cytokine that has been shown to possess antitumor properties in murine solid tumor models. Flt3-L is a trans-membrane protein (tm) but can be proteolytically cleaved to a soluble form, which is also biologically active. In this study, the antitumor effect of both soluble and tmFlt3-L was evaluated in a mouse leukemia model. To mimic the multiorgan involvement characteristic of human leukemia, a factor-dependent cell line FDC.P1 was made leukemogenic by transfection with the human BCR/ABL gene. The resulting cell line, AW, expresses BCR/ABL RNA and protein. It maintains a similar in vitro growth rate as the parent cell line, but unlike the parent cell line, AW cells are factor independent and tumorigenic. Growth of FDC.P1 and AW cells are unaffected by the addition of soluble human Flt3-L to the culture medium. Also, AW growth is unaltered after transduction with a retroviral vector expressing the tm isoform of human Flt3-L (AW/tmFlt3-L). When 10(5) AW cells were i.v. injected into syngeneic DBA/2 mice, fatal leukemia developed in nine of nine (100%) mice within 4-6 weeks with involvement of the blood, bone marrow, spleen, and thymus. Systematic administration of soluble human Flt3-L (500 microg/kg/day) for 10 days protected mice from leukemia, with 11 of 17 mice tumor free at week 8 (64.7%) The tm isoform of Flt3-L also was protective. When 10(4) AW/tmFlt3-L cells were injected i.v. into mice, only 35.7% (5 of 14) developed leukemia versus 100% in control groups. Adoptive transfer of immunity was also demonstrated; T cells obtained from tumor-free animals conferred protection to 87% (seven of eight) naive mice challenged with AW cells. These results demonstrate that both soluble and membrane-bound human Flt3-L has antitumor activity in this leukemia model.

Thrombopoietin-induced conformational change in p53 lies downstream of the p44/p42 mitogen activated protein kinase cascade in the human growth factor-dependent cell line M07e.

Thrombopoietin is a cytokine with potent megakaryocytopoietic and thrombopoietic activities in vivo. Wild-type p53 is a conformationally flexible, anti-oncogenic transcription factor that plays a principal role in mediating growth factor withdrawal-induced apoptosis in factor-dependent hematopoietic cells. We recently reported that Tpo induces a conformational change in and functional inactivation of p53, coincident with its anti-apoptotic effects, in the human factor-dependent cell line M07e. In an effort to identify potential signaling cascades through which Tpo illicits these effects on p53, we report here that treating M07e cells with MAPK kinase inhibitor PD98059 dramatically suppressed Tpo-induced conformational change in p53 as well as Tpo-enhanced viability in M07e cells in a p53-dependent manner. Furthermore, the expression of constitutively active Raf1 in M07e cells induced conformational change in p53 independent of Tpo stimulation. Inhibition of the JAK/STAT pathway revealed that JAK/STAT signaling plays an insignificant role in conformational modulation of p53 and apoptosis suppression. Inhibition of phosphatidylinositol-3 kinase did not have a significant effect on p53 conformation but did have a weak but significant effect on Tpo-enhanced viability. Cytokine-induced activation of the MAPK pathway and the subsequent functional neutralization of p53, may be an event by which apoptosis is commonly suppressed in hematopoiesis.

Flt3-ligand production by human bone marrow stromal cells.

Bone marrow stromal cells are important sources of cytokines and growth factors which participate in regulation of proliferation and differentiation of hematopoietic stem and progenitor cells. Recently flt3/flk-2-ligand (flt3-L), a new growth factor which uses a membrane tyrosine kinase receptor, was cloned. It is expressed in transmembrane and soluble forms and stimulates/co-stimulates proliferation and colony formation of hematopoietic stem/progenitor cells. It has not been reported whether flt3-L is produced by cells of the hematopoietic bone marrow microenvironment. We demonstrate the expression of flt3-L in bone marrow fibroblasts (BMF) and in stromal cells of adherent layers of long-term bone marrow cultures by RT-PCR, Northern blot, immunocytochemistry and FACS analysis. The latter two methods localized flt3-L intracellularly and on cell membranes. Treatment with interleukin-1 alpha increased the expression of flt3-L in BMF. This demonstrates production and modulation of flt3-L in stromal cells of human bone marrow.

Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells.

Flt3/flk-2 ligand (Flt3-L) co-stimulates and synergizes with cytokines such as granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin in the proliferation of bone marrow and cord blood hematopoietic stem and progenitor cells. To study the biological effects of Flt3-L on the Flt3-L responsive AML5 cell line, the retroviral vector L(Flt3-L)SN was constructed based on the vector LXSN, but containing the human Flt3-L cDNA transcriptionally regulated by the Mo-MLV LTR. High-titer amphotropic producer cells that generated 10(6) cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 micrograms/mL polybrene, and 100 U/mL G-CSF, and expanded 1 week in medium with G-CSF. Transduced cells were selected in medium containing 0.4 mg/mL G418 and then in medium with 1.0 mg/mL G418. Retroviral mediated gene transfer in G418-resistant cells was confirmed after amplification by PCR of neo-specific sequences in genomic DNA. Northern blot analysis demonstrated L(Flt3-L)SN mRNA expression. Soluble Flt3-L was undetectable (< 100 pg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-L was detected on the surface of AML5 cells by FACS analysis. Cells were plated in colony assay with and without 100 ng/mL Flt3-L, 100 U/mL G-CSF, and the combination. Gene transfer or growth factor treatment increased somewhat the clonogenicity of the nontransduced AML5 cells. More strikingly, L(Flt3-L)SN and each growth factor combination greatly increased the size of the resultant colonies such that the size of colonies from AML5/Flt3-L cells without added growth factor approximated that of the AML5 cells stimulated by exogenous soluble Flt3-L. Moreover, MAP kinase activity in L(Flt3-L)SN-transduced cells cultured without soluble Flt3-L was increased to the level induced in control cells by soluble Flt3-L. These results indicate that retroviral mediated gene transfer and autologous expression of the Flt3-L enhances growth and intracellular signaling of AML5 cells, information that should be of value for studying the effects of Flt3-L on immature subsets of primary hematopoietic stem and progenitor cells.

Preclinical studies of lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II).

To explore the feasibility of ex vivo lymphocyte gene therapy for mild Hunter syndrome (mucopolysaccharidosis type II), we evaluated retrovirus-mediated gene transfer of the iduronate-2-sulfatase (IDS) coding sequence into peripheral blood lymphocytes from enzyme-deficient individuals (PBLMPS). Moloney murine leukemia virus-derived retroviral vectors were constructed by inserting the IDS cDNA under transcriptional regulation of the long terminal repeat (LTR) (in vector L2SN) or the cytomegalovirus (CMV) early promoter (vector LNC2). High-titer virus-producer cells were generated using amphotropic PA317 packaging cells. After 3 days of in vitro stimulation of T lymphocytes with anti-CD3 antibody and interleukin-2 (IL-2), PBLMPS were transduced once on each of the next 3 days. Seven to 21 days later, cultured PBLMPS were evaluated for gene transfer and IDS specific activity. Heterogeneous populations of L2SN-transduced PBLMPS had high levels of IDS enzyme activity (456 U/mg per hr +/- SD 292) despite a gene transfer efficiency of 5% or less. Owing to overexpression of IDS in that percentage of PBLMPS successfully transduced, IDS activity was increased above the deficiency found in patients with Hunter syndrome (< 20 U/mg per hr) to a level comparable with that of normal individuals (mean activity of uncultured normal leukocytes 807 U/mg per hr; SD 252). Reduced 35SO4-glucosaminoglycan (GAG) accumulation was observed in PBLMPS that had been transduced with L2SN, or when PBLMPS were grown in medium that had been "conditioned" by growth of L2SN-transduced cells. This latter result indicated that metabolic cross-correction occurred by means of intercellular enzyme transfer. These studies of retrovirus-mediated expression and metabolic correction, finding near-normal levels of IDS in cultured PBLMPS and metabolic correction, demonstrate the potential for treatment of mild, nonneuropathic Hunter syndrome by means of ex vivo lymphocyte gene therapy.

Flt3 ligand antitumor activity in a murine breast cancer model: a comparison with granulocyte-macrophage colony-stimulating factor and a potential mechanism of action.

We have shown that Flk2/Flt3 ligand (Flt3L)-transduced tumor vaccine induces transferable T cell protection against a murine breast cancer cell line, but a direct comparison with the potent effector GM-CSF, the activity against preestablished tumors, and the mechanism of antitumor response in this breast cancer model are not known. We compared vaccination with C3L5 cells expressing Flt3L (C3Lt-Flt3L) and GM-CSF (C3L5-GMCSF) by injecting 1 x 10(4) cells subcutaneously into the chest wall and then, after 4 weeks, challenging the contralateral chest of tumor-free mice with parental C3L5 cells. C3L5-Flt3L and C3L5-GMCSF had reduced in vivo growth rates (25% tumor formation each) compared with 100% tumor formation of C3L5 cells expressing only neomycin phosphotransferase (C3L5-G1N). However, when tumor-free animals were challenged with parental C3L5 cells, C3L5-Flt3L vaccination was significantly better at preventing tumor growth (p < 0.05) than C3L5-GMCSF vaccination (33% of C3L5-Flt3L-vaccinated animals developed tumor compared with 77% of C3L5-GMCSF-vaccinated animals). Adoptive transfer of immunity for both vaccines was demonstrated; splenic T cells from tumor-free mice protected naive mice from parental tumor challenge. To simulate minimal disease, parental C3L5 cells at two concentrations (high, 5 x 10(3) cells; or low, 1 x 10(3) cells) were injected into the contralateral chest wall 4 days prior to treatment with C3L5-G1N or C3L5-Flt3L. C3L5-Flt3L treatment decreased contralateral parental tumor formation (high, 67% tumor free; low, 90% tumor free) compared with C3L5-G1N treatment (high and low, 0% tumor free). Immunodepletion of activated natural killer cells with anti-asialo-GM1 blocked C3L5-Flt3L- and C3L5 plus soluble Flt3L-mediated antitumor activity. Thus, Flt3L-transduced tumor cells manifest potent antitumor activity, apparently mediated, at least partially, by natural killer cells.

Retrovirally mediated gene transfer of Arg22 and Tyr22 forms of dihydrofolate reductase into the hematopoietic cell line K562: a comparison of methotrexate resistance.

Mutations in the enzyme dihydrofolate reductase (DHFR) can confer resistance to the inhibitory effects of folate analogs such as methotrexate (Mtx) and trimetrexate (Ttx). Retroviral vectors expressing the DHFR-Arg22 mutants and the newly described DHFR-Tyr22 mutant were used to transduce the hematopoietic cell line K562. In vitro selection of vector-containing cells was documented via polymerase chain reaction and Southern analysis. When proliferation of selected vector-containing cells was evaluated over a range of Mtx concentrations (0.01 to 10 micromol/L), both Arg22 and Tyr22 provided protection from Mtx, but Tyr22 proved superior to Arg22 in conferring Mtx resistance at low concentrations. Ttx proved to be 10- to 100-fold more potent than Mtx in inhibiting proliferation of nontransduced K562, but the relative effectiveness of individual mutants in conferring drug resistance was similar to that of Mtx. Decreasing the amount of folate in the culture medium to more physiological concentrations increased the potency of administered Mtx and Ttx. Drug resistance in retrovirally transduced K562 cells is consistent with the enzymatic characteristics of the individual mutants. Our findings suggest that the new Tyr22 form of DHFR may prove better in conferring drug resistance than the previously reported Arg22 mutant.

Commentary: Immunohistochemistry represents a useful tool to study human cell engraftment in SCID mice transplantation models.

Human cord blood cells have been shown to highly engraft the marrows of sublethally irradiated SCID mice. Herein we report our experience with this system and the use of immunohistochemistry to identify human cell engraftment. Immunohistochemistry results correlated well with those of flow cytometry, human progenitor-cell cultures, and molecular analysis of human specific markers. Immunohistochemistry should play a useful role in the in vivo analysis of human stem/progenitor cell engraftment in xenogeneic transplantation models.

Gene therapy strategies for leukemia.

A number of diverse gene therapy strategies are being evaluated in the search for novel therapeutic approaches to leukemia. Antisense oligonucleotides, ribozymes and retroviral vectors are approaches directed at the molecular mechanisms of cancer. Transfer of genes encoding cytokines and human leukocyte antigens (HLAs) could also be used to elicit immunity against tumor cells. Gene marking strategies have been useful in elucidating the biology of disease relapse after autologous bone marrow transplantation. Suicide genes, such as the herpes simplex thymidine kinase gene, have been used to modulate graft-versus-host disease after allogeneic bone marrow transplantation. Although gene delivery remains a major challenge to gene therapy, some modifications have been implemented to overcome this issue. This review will summarize these gene therapy strategies aimed at increasing the survival of patients with leukemia.

"Supercharged Cells" for delivery of recombinant human iduronate-2-sulfatase.

Expression of iduronate-2-sulfatase (IDS) from three different promoters in four retroviral vectors was studied in peripheral blood lymphocytes from patients with Hunter syndrome (PBL(MPS)), i.e., the LTR in vectors L2SN and L2, avian beta-actin promoter in LB2, and the CMV early promoter in LNC2. PBL(MPS) were exposed to packaging cell supernatant resulting in transduction frequencies ranging 10-fold from 5 to 49%. Surprisingly, IDS activities were equally high in all transduced lymphocyte populations: 515 U/mg/h in PBL(MPS)-L2SN, 734 in PBL(MPS)-LB2, 352 in PBL(MPS)-L2, and 389 in PBL(MPS)-LNC2 compared to controls (<10 in PBL(MPS)-LXSN or PBL(MPS)). The half-life of endocytosed IDS in PBL(MPS) was 1.9 days. However, the level of lymphocyte IDS activity from proviral expression was found to be only a fraction of the total, a large portion being derived from reuptake of enzyme from murine packaging cells, i.e., a "second source" of enzyme. Therefore, measurement of transgene lysosomal enzyme soon after exposure of target cells to vector supernatant may yield a gross overestimate of long-term transgene expression by transduced cells. Nevertheless, patient fibroblasts cocultured with transduced PBL(MPS) had reduced (35)SO(4)-GAG accumulation, levels similar to those of normal fibroblasts. These studies revealed a broadly applicable phenomenon: cells can be charged with a lysosomal enzyme to levels much higher than those found in nature. By "supercharging" cells with a lysosomal protein (or other molecule bearing the mannose-6-phosphate ligand), such cells may be exploited as vehicles for systemic delivery of therapeutic or diagnostic agents.

Molecular diagnosis of mucopolysaccharidosis type II (Hunter syndrome) by automated sequencing and computer-assisted interpretation: toward mutation mapping of the iduronate-2-sulfatase gene.

Virtually all mutations causing Hunter syndrome (mucopolysaccharidosis type II) are expected to be new mutations. Therefore, as a means of molecular diagnosis, we developed a rapid method to sequence the entire iduronate-2-sulfatase (IDS) coding region. PCR amplicons representing the IDS cDNA were sequenced with an automatic instrument, and output was analyzed by computer-assisted interpretation of tracings, using Staden programs on a Sun computer. Mutations were found in 10 of 11 patients studied. Unique missense mutations were identified in five patients: H229Y (685C-->T, severe phenotype); P358R (1073C-->G, severe); R468W (1402C-->T, mild); P469H (1406C-->A, mild); and Y523C (1568A-->G, mild). Non-sense mutations were identified in two patients: R172X (514C-->T, severe) and Q389X (1165C-->T, severe). Two other patients with severe disease had insertions of 1 and 14 bp, in exons 3 and 6, respectively. In another patient with severe disease, the predominant (> 95%) IDS message resulted from aberrant splicing, which skipped exon 3. In this last case, consensus sequences for splice sites in exon 3 were intact, but a 395 C-->G mutation was identified 24 bp upstream from the 3' splice site of exon 3. This mutation created a cryptic 5' splice site with a better consensus sequence for 5' splice sites than the natural 5' splice site of intron 3. A minor population of the IDS message was processed by using this cryptic splice site; however, no correctly spliced message was detected in leukocytes from this patient. The mutational topology of the IDS gene is presented.

Differential in vitro maturation of hematopoietic stem cells from wild-type and immunoglobulin transgenic mice.

During B-cell lymphopoiesis, hematopoietic stem cells commit to the B-cell lineage as monitored by the expression of phenotypic cell surface antigens and the production of immunoglobulin chains. Two cytokines, interleukin-7 (IL-7) and Flt-3 ligand (FL), appear to act in conjunction to drive this development process. Using an in vitro, stroma-free culture system and these cytokines, the commitment of murine Sca+ Lin- bone marrow cells to the B-cell lineage was examined with stem cells from immunoglobulin (Ig) transgenic and wild-type mice. After 12 days of culture in IL-7 and FL, stem cells from wild-type animals had matured to express surface B220, CD19, CD43, BP-1 and heat-stable antigen (HSA). These cells lacked detectable intracellular mu chains while exhibiting partial D-J rearrangement. In contrast, Sca+Lin- cells from Ig transgenic mice that were cultured similarly expressed B220, CD19, IgD, intracellular and surface mu, HSA but not CD43 or BP-1. These results suggest that expression of the Ig transgene during in vitro development overcame a block in B-cell lymphopoiesis and recapitulated in vivo events. Thus, IL-7 and FL treatment allowed uncommitted stem cells to progress to the early pre-B-cell stage while similarly treated Ig transgenic cells progressed completely to the mature B-cell stage.

Comparative effects of retroviral-mediated gene transfer into primary human stromal cells of Flt3-ligand, interleukin 3 and GM-CSF on production of cord blood progenitor cells in long-term culture.

The effects of different cytokines on growth of human cord blood CD34 cells was studied by performing long-term culture (LTC) with primary human stromal cells transduced with genes for either Flt3-ligand (L) (human transmembrane, murine soluble or murine membrane-bound forms), human interleukin 3 (IL-3) or human GM-CSF. Molecular analysis of genomic DNA from transduced stromal cells using neo-specific polymerase chain reaction demonstrated gene transfer of G418-selected stromal cell populations. Enzyme-linked immunosorbent assay and biological assays of conditioned media from transduced stromal cells indicated expression and release of soluble cytokines. Numbers of both immature and more mature progenitors (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM, BFU-E, CFU-GM) were increased threefold compared to control in the Flt3-L (transmembrane) LTC throughout five weeks of culture. IL-3 and GM-CSF feeders increased progenitor cell output also, but these effects were significantly lower than Flt3-L feeders. The two Flt3-L isoform engineered feeders, Ex6 (soluble isoform) and 5H (membrane-bound isoform), showed a decreased effect compared to the transmembrane Flt3-L feeders and, in particular, Ex6 feeders were similar to control feeders and 5H feeders were comparable to Flt3-L feeders only in the first two weeks of LTC. These results were apparent also by limiting dilution assays that showed a higher frequency of pre-CFU in the transmembrane Flt3-L feeders compared to control and the other cytokine feeders. Exogenous addition of soluble growth factors to suspension cultures without feeder layers, while superior to stromal feeders for short-term expansion of early progenitors, were inferior to the long-term maintenance/output on stromal feeders. Pre-CFU analysis supported these data. These results may be of some significance to understanding the actions of Flt3-L on blood cell production.

H-Ras is involved in the inside-out signaling pathway of interleukin-3-induced integrin activation.

The proto-oncogene product, p21(ras), has been implicated in the cellular mechanism of adhesion, although its precise role has been controversial. Numerous cytokines and growth-factors activate Ras, which is an important component of their growth-promoting signaling pathways. On the other hand, the role of Ras in cytokine-induced adhesion has not been elucidated. We therefore investigated the function of H-Ras in the inside-out signaling pathway of interleukin-3 (IL-3)-induced integrin activation in the murine Baf3 cell line after transfection of cells with either constitutively active, dominant-negative, or wild-type H-Ras cDNAs. Adhesion of Baf3 cells to fibronectin was induced by IL-3 in a dose-dependent manner via very late antigen-4 (VLA-4; alpha4beta1 integrins) and VLA-5 (alpha5beta1 integrins) activation. On the other hand, IL-4 did not induce the adhesion of Baf3 cells to fibronectin, although IL-4 did stimulate the cell proliferation of Baf3 cells. Constitutively active H-Ras-transfected Baf3 cells adhered to fibronectin without IL-3 stimulation through VLA-4 and VLA-5, whereas dominant-negative H-Ras-transfected Baf3 cells showed significantly less adhesion induced by IL-3 compared with wild-type and constitutively active H-Ras-transfected Baf3 cells. Anti-beta1 integrin antibody (clone; 9EG7), which is known to change integrin conformation and activate integrins, induced the adhesion of dominant-negative H-Ras-transfected Baf3 cells as much as the other types of H-Ras-transfected Baf3 cells. 8-Br-cAMP, Dibutyryl-cAMP, Ras-Raf-1 pathway inhibitors, and PD98059, a MAPK kinase inhibitor, suppressed proliferation and phosphorylation of MAPK detected by Western blotting with anti-phospho-MAPK antibody, but not adhesion of any type of H-Ras-transfected Baf3 cells, whereas U-73122, a phospholipase C (PLC) inhibitor, suppressed adhesion of these cells completely. These data indicate that H-Ras and PLC, but not Raf-1, MAPK kinase, or the MAPK pathway, are involved in the inside-out signaling pathway of IL-3-induced VLA-4 and VLA-5 activation in Baf3 cells.

The interphase microtubule damage checkpoint defines an S-phase commitment point and does not require p21(waf-1).

Cell cycle checkpoints ensure orderly progression of events during cell division. A microtubule damage (MTD)-induced checkpoint has been described in G(1) phase of the cell cycle (G(1)MTC) for which little is known. The present study shows that the G(1)MTC is intact in activated T lymphocytes from mice with the p21(waf-1) gene deleted. However, p21(waf-1) gene deletion does affect the ratio of cells that arrest at the G(1)MTC and the spindle checkpoint after MTD. The G(1)MTC arrests T lymphocytes in G(1) prior to cdc2 up-regulation and prior to G(1) arrest by p21(waf-1). Once cells have progressed past the G(1)MTC, they are committed to chromosome replication and metaphase progression, even with extreme MTD. The G(1)MTC is also present in a human myeloid cell line deficient in p21(waf-1) gene expression. The p21-independent G(1)MTC may be important in cellular responses to MTD such as those induced by drugs used to treat cancer.

Transcriptional and ERK1/2-dependent synergistic upregulation of p21(cip1/waf1) associated with steel factor synergy in MO7e.

Steel factor (SLF) plus GM-CSF induces proliferative synergy in myeloid progenitors and factor-dependent cell line MO7e. We previously reported that the protein level of cyclin-dependent kinase inhibitor p21(cip1/waf1) (p21) increased synergistically when MO7e cells were stimulated with SLF plus GM-CSF and that p21 induction was required for SLF synergistic responses. Here we show that this p21 induction is regulated at the transcriptional level. Based on use of a multiprobe RNase protection assay, the synergistic increase of p21 mRNA was unique among many cell cycle regulators. While STAT5A and 5B were activated after stimulation with GM-CSF alone or SLF plus GM-CSF, there was no difference in activation between the groups. p44/42 MAP kinase (ERK1/2) was synergistically activated by SLF plus GM-CSF, but SAPK/JNK and p38 MAP kinase were not. Synergistic induction of p21 was significantly decreased with a MEK1 inhibitor, suggesting that the ERK1/2 pathway is involved in the synergistic increase of p21 after GM-CSF plus SLF stimulation.

Thrombopoietin upregulates the promoter conformation of p53 in a proliferation-independent manner coincident with a decreased expression of Bax: potential mechanisms for survival enhancing effects.

Thrombopoietin (Tpo) has proliferative and maturational effects on immature and more committed cells, respectively. We previously reported a role for Tpo as a survival factor in the factor-dependent human cell line M07e by demonstrating that Tpo suppresses apoptosis in the absence of induced proliferation. Wild-type p53 is a tumor suppressor gene that can play a vital role in mediating growth factor withdrawal-induced apoptosis in factor-dependent hematopoietic cells. Wild-type p53 can switch from a suppressor conformation, with an antiproliferative, pro-apoptotic phenotype, to a promoter conformation that has a diminished ability to mediate cell cycle arrest and apoptosis. In an effort to elucidate the mechanisms through which Tpo suppresses apoptosis, we investigated the effects of Tpo treatment on p53-mediated apoptosis in M07e cells. Tpo upregulated the expression of the promoter conformation of p53 in M07e cells coincident with a downregulation of Bax and Mdm2 protein levels. Protein levels of Bcl-2 and Bcl-xL did not significantly vary as a function of growth-factor stimulation. Conversely, the levels of suppressor conformation p53 were maximal when M07e was in a growth arrested state and decreased during factor stimulation. Furthermore, Tpo treatment induced an extranuclear buildup and greatly weakened the DNA binding capacity of p53. p53-specific antisense oligonucleotide treatment recapitulated the effects of Tpo treatment on the levels of Bax, Mdm-2, and Bcl-2. These results suggest that Tpo is suppressing growth factor withdrawal induced-apoptosis, at least in part, by downregulating the expression of pro-apoptotic Bax protein levels, through modulating the conformation of p53, which results in a functional inactivation of its pro-apoptotic abilities.

Metabolic correction and cross-correction of mucopolysaccharidosis type II (Hunter syndrome) by retroviral-mediated gene transfer and expression of human iduronate-2-sulfatase.

To explore the possibility of using gene transfer to provide iduronate-2-sulfatase (IDS; EC 3.1.6.13) enzyme activity for treatment of Hunter syndrome, an amphotropic retroviral vector, L2SN, containing the human IDS coding sequence was constructed and studied for gene expression in vitro. Lymphoblastoid cell lines (LCLs) from patients with Hunter syndrome were transduced with L2SN and expressed high levels of IDS enzyme activity, 10- to 70-fold higher than normal human peripheral blood leukocytes or LCLs. Such L2SN-transduced LCLs failed to show accumulation of 35SO4 into glycosaminoglycan (35SO4-GAG), indicating that recombinant IDS enzyme participated in GAG metabolism. Coculture of L2SN-transduced LCLs with fibroblasts from patients with Hunter syndrome reduced the accumulation of 35SO4-GAG. These results demonstrated retroviral-mediated IDS gene transfer into lymphoid cells and the ability of such cells to provide recombinant enzyme for intercellular metabolic cross-correction.