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Biomedical subjects

D L Longo

Publications and source records attributed to D L Longo.

At least 37 records · Page 2Linked to original sources
PubMed1999

Abrogation of the hematological and biological activities of the interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein PIXY321 by neutralizing anti-PIXY321 antibodies in cancer patients receiving high-dose carboplatin.

This dose-escalation study was performed to evaluate the hematologic activity, biological effects, immunogenicity, and toxicity of PIXY321 (an interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein) administered after high-dose carboplatin (CBDCA) treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In part A of the study, patients were treated with CBDCA alone during cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 micrograms/m2/d were administered to successive cohorts of 3 to 6 patients in part A. In part B, patient groups received PIXY321 doses of 750, 1,000, and 1,250 micrograms/m2/d. The hematologic effects of PIXY321 were assessed in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was assessed by enzyme-linked immunosorbent assay (ELISA) and neutralization assay. Of the 49 patients enrolled, 31 were fully evaluable for hematologic efficacy. When comparing the first B cycle (cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/microL but was able to speed the time of recovery of platelet counts to 100,000/microL (15 v 20 days; P =.01). Significant improvements in neutrophil nadir and duration of ANC less than 500 were observed in cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/microL by an average of at least 8 days as compared with cycle A-1 (without PIXY321; P </=.004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The immunogenicity of this fusion protein provides a cautionary warning that clinical development of bioengineered human molecules requires thorough testing for immune neutralization.

Adult↗
PubMed1999

Inhibition of human breast carcinoma growth by a soluble recombinant human CD40 ligand.

CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-gamma. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.

Animals↗
PubMed1999

Association of polo-like kinase with alpha-, beta- and gamma-tubulins in a stable complex.

The polo-like kinase (Plk) family has been shown to have an important role in the regulation of the cell-division cycle, especially in organization of the spindle structure, in species from fungi to humans. Recent reports have demonstrated that in mammalian cells Plk is associated with components of the anaphase-promoting complex and a peptidyl-prolyl isomerase, Pin1. To characterize a putative Plk-containing complex, we fractionated mitotic cell lysates on a gel-filtration column. The Plk complex was eluted from the column at molecular sizes ranging from 669 to 2500 kDa in the presence of detergent and high concentrations of salt. Specific associations of Plk with alpha-, beta- and gamma-tubulins in both interphase and mitotic cells were shown by reciprocal immunoprecipitations and immunoblottings and were independent of the microtubule polymerization state, whereas binding assays in vitro indicated that Plk interacts with alpha- and beta-tubulins directly. In addition, mitotic Plk was able to phosphorylate associated tubulins in vitro. Finally, we show that the kinase domain of the Plk molecule is both required and sufficient for its binding to tubulins in vivo. The specific interaction between Plk and tubulins might provide a molecular basis for the physiological functions of Plk in regulating the cell cycle, particularly in establishing the normal bipolar spindle.

Amino Acid Sequence↗
PubMed1999

Deletion of the loop region of Bcl-2 completely blocks paclitaxel-induced apoptosis.

At high concentrations, the tubule poison paclitaxel is able to kill cancer cells that express Bcl-2; it inhibits the antiapoptotic activity of Bcl-2 by inducing its phosphorylation. To localize the site on Bcl-2 regulated by phosphorylation, mutant forms of Bcl-2 were constructed. Mutant forms of Bcl-2 with an alteration in serine at amino acid 70 (S70A) or with deletion of a 60-aa loop region between the alpha1 and alpha2 helices (Deltaloop Bcl-2, which also deletes amino acid 70) were unable to be phosphorylated by paclitaxel treatment of MDA-MB-231 cells into which the genes for the mutant proteins were transfected. The Deltaloop mutant completely inhibited paclitaxel-induced apoptosis. In cells expressing the S70A mutant, paclitaxel induced about one-third the level of apoptosis seen with wild-type Bcl-2. To evaluate the role of mitogen-activated protein kinases (MAPKs) in Bcl-2 phosphorylation, the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 was examined. Paclitaxel-induced apoptosis was associated with phosphorylation of Bcl-2 and activation of ERK and JNK MAPKs. If JNK activation was blocked by transfections with either a stress-activated protein kinase kinase dominant-negative (K-->R) gene (which prevents the activation of a kinase upstream of JNK) or MAPK phosphatase-1 gene (which dephosphorylates and inactivates JNK), Bcl-2 phosphorylation did not occur, and the cells were not killed by paclitaxel. By contrast, neither an ERK inhibitor (PD098059) nor p38 inhibitors (SB203580 and SB202190) had an effect on Bcl-2 phosphorylation. Thus, our data show that the antiapoptotic effects of Bcl-2 can be overcome by phosphorylation of Ser-70; forms of Bcl-2 lacking the loop region are much more effective at preventing apoptosis than wild-type Bcl-2 because they cannot be phosphorylated. JNK, but not ERK or p38 MAPK, appear to be involved in the phosphorylation of Bcl-2 induced by paclitaxel.

Alanine↗
PubMed1999

Synergistic effects of retinoic acid and 8-Cl-cAMP on apoptosis require caspase-3 activation in human ovarian cancer cells.

We investigated the intracellular mechanisms of retinoic acid (9-cis-RA, 13-cis-RA or all-trans-RA) and a cyclic AMP analog 8-Cl-cAMP on growth-inhibition and apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. The cyclic AMP analog, 8-Cl-cAMP, acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlated with the growth inhibition, cell cycle arrest, and apoptosis in both cell types. In addition, combined treatment of cells with RA plus 8-Cl-cAMP resulted in the release of cytochrome c, loss in mitochondrial membrane potential and activation of caspase-3 followed by cleavage of anti-poly(ADP-ribose)polymerase and DNA-dependent protein kinase (catalytic subunit). Interestingly, inhibition of caspase-3 activation blocked RA plus 8-Cl-cAMP induced apoptosis. Furthermore, mutations in a CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cyclic AMP action in ovarian cancer cells by promoting apoptosis. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion in inducing apoptosis via caspase-3 activation, and may have potential for combination biotherapy for the treatment of malignant disease such as ovarian cancer.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

CD40 stimulation promotes human secondary immunoglobulin responses in HuPBL-SCID chimeras.

Antibodies to CD40 have been demonstrated to promote B-cell growth and differentiation in vitro. In order to determine if CD40 stimulation could promote antigen-specific human immunoglobulin (Ig) production in vivo, we examined the effects of anti-human CD40 MoAb in an in vivo system where human peripheral blood lymphocytes (huPBL) were engrafted into mice with severe combined immune deficiency (SCID). The huPBL-SCID mice were then given various doses of diphtheria-tetanus toxoid (DT) vaccine and were examined for the presence of human DT-specific antibodies by ELISA. Surprisingly, treatment with anti-CD40 significantly lowered background DT responses versus untreated chimeras in unimmunized huPBL-SCID mice. However, after immunization, huPBL-SCID mice treated with anti-CD40 MoAb responded to a significantly greater extent in response to the vaccine compared with control huPBL-SCID mice, although total Ig levels were sometimes lower in anti-CD40-treated mice. The predominant Ig isotype induced after immunization was IgG. Thus, CD40 stimulation promotes human secondary IgG responses in huPBL-SCID mice. These data demonstrate that CD40 stimulation is capable of promoting antigen-specific human B-cell responses in vivo.

Animals↗
PubMed1999

Conditioned immune response to interferon-gamma in humans.

We determined whether a classical conditioning paradigm may be used to condition immunologic responses in normal human subjects receiving an optimal immunostimulating dose of recombinant human interferon-gamma (rhIFN-gamma). We conducted a placebo-controlled, double-blind study of 31 normal volunteers in order to determine whether an initially immune-neutral stimulus, oral propylene glycol (PG), could eventually elicit an immune response as a consequence of its being paired with a known immunostimulatory dose and schedule of rhIFN-gamma. Subjects were randomly assigned to one of three groups: (A) rhIFN-gamma injections paired with PG; (B) normal saline injections paired with PG; (C) rhIFN-gamma injections alone. During the 4-week study, subjects received progressively fewer injections so that, by the final week of the study, no injections were given and groups A and B received only PG. The principal outcome measures were serum concentrations of quinolinic acid (QUIN) and neopterin, two nonspecific but sensitive markers of immune activation, and expression of Fc receptors (CD64) on peripheral blood mononuclear cells. RhIFN-gamma injections produced significant and predictable alterations in each of the measured immune parameters. No group B subject made an immune response. Mean serum QUIN levels were significantly higher at the end of week three for subjects in the experimental condition (group A) than for subjects receiving rhIFN-gamma alone (group C) despite receiving identical doses of rhIFN-gamma. Similarly, the predicted decay in mean serum neopterin levels from the end of week 1 to the end of week 2 was seen in group C but not in group A. The exposure of group A to PG blunted the decline of CD64 expression in week four. The data suggest that the pairing of an unconditioned stimulus (rhIFN-gamma) and a conditioned stimulus (PG) permits the conditioned stimulus alone to prolong a cytokine-induced response in normal humans.

Adjuvants, Immunologic↗
PubMed1999

Treatment with tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients.

Dendritic cells (DCs) initiate primary and stimulate secondary T-cell responses. We conducted a phase I trial of tumor necrosis factor (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with cancer to increase DCs in peripheral blood or skin based on in vitro data that showed that CD34(+) hematopoietic precursors require these cytokines to mature into functional antigen-presenting DCs. Eleven patients were treated for 7 days with GM-CSF, 125 microg/m(2) twice daily as subcutaneous injections, and TNF-alpha as a continuous infusion at dose levels of 25, 50, or 100 microg/m(2)/day. The maximum tolerated dose of TNF-alpha was 50 microg/m(2)/day with this dose of GM-CSF; dose-limiting toxicities occurred in both patients treated with 100 microg/m(2)/day. One became thrombocytopenic and the other had transient confusion. Epidermal Langerhans' cells were quantitated by S100 staining of skin biopsies and DC precursors in peripheral blood by colony-forming unit dendritic (CFU-dendritic) assays. S100-positive cells in the epidermis doubled after treatment (2.55 S100(+) cells/high-power field before treatment to 6.05 after treatment, p = 0.029). CFU-dendritic in peripheral blood increased after treatment in 3 colorectal cancer patients but not in 3 patients with melanoma. CD11c(+) or CD123(+), HLA-DR(bright), lineage-negative dendritic cell precursors were not increased in peripheral blood mononuclear cells. This trial demonstrates that treatment with TNF-alpha and GM-CSF can increase the number of DCs in the skin and the number of dendritic cell precursors in the blood of some patients with cancer. This approach may increase the efficacy of vaccination to tumor antigens in cancer patients.

Adult↗
PubMed1999

Prolactin exerts hematopoietic growth-promoting effects in vivo and partially counteracts myelosuppression by azidothymidine.

Prolactin (PRL) is a neuroendocrine hormone that influences immune and hematopoietic development. The mechanism of action of this hormone in vivo remains unclear; therefore, we assessed the effects of PRL on hematopoiesis in vivo and in vitro. Normal resting mice were treated with 0, 1, 10, or 100 microg of recombinant human prolactin (rhPRL) for 4 consecutive days and euthanized on the fifth day for analysis of myeloid and erythroid progenitors in the bone marrow and spleen. Both frequencies and absolute numbers of splenic colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-e) were significantly increased in mice receiving rhPRL compared to the controls that had received saline only. Bone marrow cellularities were not significantly affected by any dose of rhPRL, but the absolute numbers and frequencies of bone marrow CFU-GM and BFU-e were augmented by rhPRL. These results suggest that rhPRL can promote hematopoiesis in vivo. Because rhPRL augments myeloid development in vivo, we examined the potential of the hormone to reverse the anemia and myelosuppression induced by azidothymidine (AZT). Mice were given rhPRL injections concurrent with 2.5 mg/mL AZT in drinking water. rhPRL partially restored hematocrits in the animals after 2 weeks of treatment and increased CFU-GM and BFU-e in both spleens and bone marrow. The experiments with AZT and rhPRL support the conclusion that the hormone increases myeloid and erythroid progenitor numbers in vivo, and they suggest that the hormone is clinically useful in reversing myelosuppression induced by AZT or other myeloablative therapies.

Animals↗
PubMed1999

Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

Adult↗
PubMed1999

Biologic therapy for lymphoma.

Biologic approaches to lymphoma treatment are now a practical reality rather than a vaguely perceived hope for the future. Interferon alfa seems to have been established as a life-extending therapy for selected patients with follicular lymphoma. Humanized anti-CD20 monoclonal antibody (rituximab) has an authentic 50% response rate in patients with follicular lymphoma, frequently works when given a second time, and is demonstrating what seems to be synergistic activity when combined with chemotherapy. Radioimmunoconjugates using iodine-131 and yttrium-90 as payload are producing responses in refractory and aggressive lymphomas. Vaccines and adoptive cellular therapies are also showing promising results in early phase clinical testing.

Antibodies, Monoclonal↗
PubMed1999

Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis.

The proteins Bcl-2 and Bcl-X(L) prevent apoptosis, but their mechanism of action is unclear. We examined the role of Bcl-2 and Bcl-X(L) in the regulation of cytosolic Ca(2+), nitric oxide production (NO), c-Jun NH(2)-terminal kinase (JNK) activation, and apoptosis in Jurkat T cells. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum-associated Ca(2+) ATPase, was used to disrupt Ca(2+) homeostasis. TG acutely elevated intracellular free Ca(2+) and mitochondrial Ca(2+) levels and induced NO production and apoptosis in Jurkat cells transfected with vector (JT/Neo). Buffering of this Ca(2+) response with 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) or inhibiting NO synthase activity with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME) blocked TG-induced NO production and apoptosis in JT/Neo cells. By contrast, while TG produced comparable early changes in the Ca(2+) level (i.e., within 3 h) in Jurkat cells overexpressing Bcl-2 and Bcl-X(L) (JT/Bcl-2 or JT/Bcl-X(L)), NO production, late (36-h) Ca(2+) accumulation, and apoptosis were dramatically reduced compared to those in JT/Neo cells. Exposure of JT/Bcl-2 and JT/Bcl-X(L) cells to the NO donor, S-nitroso-N-acetylpenacillamine (SNAP) resulted in apoptosis comparable to that seen in JT/Neo cells. TG also activated the JNK pathway, which was blocked by L-NAME. Transient expression of a dominant negative mutant SEK1 (Lys-->Arg), an upstream kinase of JNK, prevented both TG-induced JNK activation and apoptosis. A dominant negative c-Jun mutant also reduced TG-induced apoptosis. Overexpression of Bcl-2 or Bcl-X(L) inhibited TG-induced loss in mitochondrial membrane potential, release of cytochrome c, and activation of caspase-3 and JNK. Inhibition of caspase-3 activation blocked TG-induced JNK activation, suggesting that JNK activation occurred downstream of caspase-3. Thus, TG-induced Ca(2+) release leads to NO generation followed by mitochondrial changes including cytochrome c release and caspase-3 activation. Caspase-3 activation leads to activation of the JNK pathway and apoptosis. In summary, Ca(2+)-dependent activation of NO production mediates apoptosis after TG exposure in JT/Neo cells. JT/Bcl-2 and JT/Bcl-X(L) cells are susceptible to NO-mediated apoptosis, but Bcl-2 and Bcl-X(L) protect the cells against TG-induced apoptosis by negatively regulating Ca(2+)-sensitive NO synthase activity or expression.

Apoptosis↗
PubMed1999

T-small lymphocyte disorders.

Less than 2% of all lymphoproliferative diseases are indolent or small T-cell disorders, and include T-cell chronic lymphocytic leukemia (CLL)/prolymphocytic leukemia (PLL), large granular lymphocyte (LGL) leukemia, and mycosis fungoides (MF). T-PLL has an aggressive clinical course characterized by high lymphocyte counts, marked hepatosplenomegaly, anemia, thrombocytopenia, and median survival times less than 1 year. The majority of cases are associated with abnormalities of chromosome 14. T-CLL probably represents a small cell variant of T-PLL with a similar aggressive course and similar cytogenetics. T-LGL leukemia is a clonal disorder of CD3+, cytotoxic T lymphocytes. Common clinical features include neutropenia, anemia, splenomegaly, and recurrent bacterial infections. The prognosis is dictated by the severity of the neutropenia, with 10-year actuarial survival rates greater than 80%, and most deaths related to sepsis. A small subset of LGL leukemias have a natural killer (NK) phenotype, are refractory to treatment, and result in multiorgan failure and death in a few months. Mycosis fungoides (MF), the most common of the small T-cell disorders, is a cutaneous T-cell lymphoma with a chronic course, often extending over decades, with most patients eventually succumbing to infection. The small T-lymphocyte disorders represent a rare, diverse group of diseases, which generally have an indolent course, but are not curable.

Chromosome Aberrations↗
PubMed1999

Synergistic effects of retinoic acid and 8-chloro-adenosine 3',5'-cyclic monophosphate on the regulation of retinoic acid receptor beta and apoptosis: involvement of mitochondria.

In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective, even at doses that are toxic to the host. In late stages of breast cancer, patients do not respond to RA because the expression of RA receptor beta (RARbeta) is lost. In the present study, the intracellular mechanism(s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) analogue, 8-chloro-adenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined. Our data demonstrated that hormone-dependent MCF-7 cells, but not hormone-independent MDA-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis. Introduction of the RARbeta gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with all-trans-RA in inducing and activating RARbeta gene expression that correlates with the reduction in mitochondrial membrane potential, redistribution of cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase and DNA-dependent protein kinase (catalytic subunit), and induction of apoptosis. Mutations in the cAMP response element-related motif within the RARbeta promoter resulted in loss of synergy in RARbeta transcription. In addition, inhibition of RARbeta expression by an antisense construct also blocked the antitumor effects of RA + 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RARbeta expression may contribute to the tumorigenicity of human mammary epithelial cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion and may have potential for combination biotherapy for the treatment of malignant diseases.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Degradation of proto-oncoprotein c-Rel by the ubiquitin-proteasome pathway.

The c-rel proto-oncogene product, c-Rel, belongs to the Rel/NF-kappaB transcription factor family, which regulates a large variety of cellular functions. The activation of NF-kappaB involves the degradation of the inhibitor, IkappaB, through the ubiquitin-proteasome (Ub-Pr)-mediated pathway. Here we report that the turnover of c-Rel is also regulated by the Ub-Pr pathway, thus adding another level of complexity to the regulation of NF-kappaB. High molecular weight ubiquitinated c-Rel conjugates are detected in cells and accumulate in cells treated with proteasome inhibitors. In a cell-free in vitro degradation assay, c-Rel is degraded specifically through the Ub-Pr pathway. N-terminally truncated c-Rel is readily degraded, implying the dispensability of N-terminal sequence; in contrast, a series of deletion mutants missing C-terminal sequences display a reduced susceptibility to the degradation. Interestingly, the sequence between residues 118 and 171 of c-Rel, i.e. the region immediately following the c-Rel/v-Rel homology domain, appears to play an important role in mediating ubiquitin conjugation and the subsequent degradation. Together with our previous study showing an elevated tumorigenic potential for C-terminally truncated mutants, our data suggest that the C-terminal domain of c-Rel plays an important role in mediating c-Rel degradation and growth control.

3T3 Cells↗
PubMed1998

Differential effects of the absence of interferon-gamma and IL-4 in acute graft-versus-host disease after allogeneic bone marrow transplantation in mice.

Graft-versus-host disease (GVHD), in which immunocompetent donor cells attack the host, remains a major cause of morbidity after allogeneic bone marrow transplantation (BMT). To understand the role of cytokines in the pathobiology of GVHD, we used cytokine knockout (KO) mice as a source of donor T cells. Two different MHC-disparate strain combinations were examined: BALB/c (H2(d)) donors into lethally irradiated C57BL/6 (H2(b)) recipients or C57BL/6 (H2(b)) donors into B10.BR (H2(k)) recipients. Donor cells were from mice in which either the interferon-gamma (IFN-gamma) or the IL-4 gene was selectively disrupted to understand the role of these cytokines in acute GVHD. In both strain combinations the same pattern was noted with regard to GVHD onset and morbidity. All mice exhibited the classic signs of acute GVHD: weight loss with skin, gut, and liver pathology resulting in morbidity and mortality. Surprisingly, donor cells obtained from mice lacking IFN-gamma gave rise to accelerated morbidity from GVHD when compared with cells from wild-type control donors. Similar results were obtained using normal donors when neutralizing antibodies to IFN-gamma were administered immediately after the BMT. These results suggest that IFN-gamma plays a role in protection from acute GVHD. In marked contrast, cells obtained from IL-4 KO mice resulted in protection from GVHD compared with control donors. Splenocytes from IFN KO mice stimulated with a mitogen proliferated to a significantly greater extent and produced more IL-2 compared with splenocytes obtained from IL-4 KO or control mice. Additionally, there was increased IL-2 production in the spleens of mice undergoing GVHD using IFN-gamma KO donors. These results therefore indicate, with regard to the TH1/ TH2 cytokine paradigm, the absence of a TH1-type cytokine can be deleterious in acute GVHD, whereas absence of a TH2 cytokine can be protective.

Animals↗
PubMed1998

Expression and phosphorylation of fibroblast-growth-factor-inducible kinase (Fnk) during cell-cycle progression.

Fnk is a member of the polo family of cell-cycle-regulated serine/threonine kinases. We report here that it is present in serum-starved quiescent cells and that mitogenic stimulation of quiescent cells with calf serum results in the modification of a significant fraction of the Fnk pool. This modification results in a slower migrating form when analysed by SDS/PAGE. The modification is transient and by 9 h after stimulation all of the Fnk is again present as the faster migrating form. We also show that the Fnk protein increases in abundance as cells progress from G1 to mitosis and is post-translationally modified as cells enter and exit mitosis. The Fnk modification is again manifested as a slower migrating species by SDS/PAGE and is due to phosphorylation of the protein. The mitotic-specific phosphorylation of Fnk correlates with an increase in its kinase activity, and this activity is dramatically reduced by phosphatase treatment of mitotic Fnk immunoprecipitates. During the later stages of mitosis, Fnk is dephosphorylated such that, by the time the cells enter G1, it is all present as the dephosphorylated form. These results suggest that Fnk has two functions, one during the entry of cells into the cell cycle and a second during mitosis of cycling cells.

3T3 Cells↗