A novel member of the Swi6p family of fission yeast chromo domain-containing proteins associates with the centromere in vivo and affects chromosome segregation.

We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates. Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins. Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization. The S. pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome. Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p. Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.

Expression of functional formyl peptide receptors by human astrocytoma cell lines.

Activation of astrocytes is important in the pathogenesis of a variety of diseases in the central nervous system, such as infection and neurodegeneration. We found that the bacterial chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLF) induced potent migration and Ca(2+) mobilization in human astrocytoma cell lines. The effect of fMLF was pertussis toxin-sensitive, suggesting the involvement of seven transmembrane, G protein-coupled receptor(s) for fMLF. Scatchard analyses revealed that astrocytoma cell lines express both high- and low-affinity binding sites for [3H]fMLF. RT-PCR confirmed the expression of transcripts of fMLF receptors, the high-affinity FPR and the low-affinity FPRL1 by these cells. Both fMLF and F peptide, a synthetic peptide domain of HIV-1 envelope protein which specifically activates FPRL1, increased secretion of IL-6 by astrocytoma cells. Our study demonstrates for the first time that FPR and FPRL1 expressed by astrocytoma cell lines are functional, and suggests a molecular basis for the involvement of these receptors in host defense in the brain.

Differential expression and responsiveness of chemokine receptors (CXCR1-3) by human microvascular endothelial cells and umbilical vein endothelial cells.

The basis for the angiogenic effects of CXC chemokines such as interleukin 8 (IL-8) and for angiostatic chemokines such as interferon-inducible protein 10 (IP-10) has been difficult to assess. We recently reported, based on an RNase protection assay, that human umbilical vein endothelial cells (HUVECs) did not express detectable mRNA for the IL-8 receptors CXCR1 and CXCR2. This raised the possibility of heterogeneity of receptor expression by different endothelial cell (ECs) types. Since systemic angiogenesis induced by IL-8 would more likely involve microvessel ECs, we investigated CXC receptor expression on human microvascular dermal endothelial cells (HMECs). By confocal microscopy and immunofluorescence we observed that HMECs consistently expressed high levels of CXCR1 and CXCR4 (mean fluorescence intensity of 261+/-22.1 and 306.2+/-19, respectively) and intermediate levels of CXCR3 and CXCR2 (173.9+/-30. 2 and 156+/-30.9, respectively). In contrast, only a small proportion of HUVEC preparations expressed low levels of CXCR1, -2, and -3 (66+/-19.9; 49+/-15, and 81.4+/-17.9, respectively). However, both HMECs and HUVECs expressed equal levels of CXCR4. As expected, HMECs had more potent chemotactic responses to IL-8 than HUVECs, and this was correlated with the levels of IL-8 receptors on the ECs. Antibodies to CXCR1 and CXCR2 each had inhibitory effects on chemotaxis of HMECs to IL-8, indicating that both IL-8 receptors contributed to the migratory response of these cells toward IL-8. Assessment of the functional capacity of CXCR3 unexpectedly revealed that HMECs migrated in response to relatively higher concentrations (100-500 ng/ml) of each of the 'angiostatic' chemokines IP-10, ITAC, and MIG. Despite this, the 'angiostatic' chemokines inhibited the chemotactic response of HMECs to IL-8. IL-8 and SDF-1alpha but not IP-10 induced calcium mobilization in adherent ECs, suggesting that signaling events associated with calcium mobilization are separable from those required for chemotaxis. Taken together, our data indicated that functional differences among EC types is dependent on the level of the expression of CXC chemokine receptors. Whether this heterogeneity in receptor expression by ECs reflects distinct differentiation pathways remains to be established.

Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels.

To define the responses of apoptotic regulatory proteins to different chemotherapeutic agents, we investigated the expression of Bcl-2 family gene products, the release of cytochrome c, and the activation of pro-caspase-3 during apoptosis induced by Taxol and Thiotepa, in the MCF-7 breast carcinoma and the HL-60 leukemia cell lines. The earliest event induced by drug exposure was increase in Bad protein levels, followed by Bcl-2 down-regulation, cytochrome c release, and Bcl-xL and Bax up-regulation. Bak accumulation was a late event. Activation of pro-caspase-3 and cleavage of Bcl-2 protein occurred in the HL-60 cells only, and followed the cytochrome c release. The overall responses were qualitatively similar in both cell types, but MCF-7 cells treated with Taxol showed a significant delay in apoptosis, correlating with early up-regulation of Bcl-2 and delayed release of cytochrome c. We conclude that Bad up-regulation is an early indicator of a cellular response that will lead to cell death, but may be modulated by survival mechanisms, which cumulatively govern the ultimate susceptibility to apoptosis.

A mouse fibroblast line cycles between monolayer and spheroid forms, regulates Met and HGF expression, and releases an attachment and growth-promoting substance.

A subline of mesoderm-derived mouse NIH3T3 fibroblasts was selected for its ability to proliferate in serum-free media. This cell line (SFDH) grows as a monolayer at low density and spontaneously forms dense, multicellular spheroids at high density. Spheroid formation can also be induced by the addition of dexamethasone, polybrene, or heparin. Spheroids eventually detach from the substrate, but will reattach and re-form monolayers when transferred to fresh culture vessels and media, repeating the cycle again upon reaching high density. Thin section analysis of spheroids shows morphologically-distinct regions of cells, including an attenuated outer surface and a cuboidal interior with occasional lumen-like areas. Over time in culture, spheroids express increasing levels of met, the Met ligand-SF/HGF and cytokeratin, an epithelial marker, in comparison to monolayers. Both monolayer and spheroid-derived cells are rapidly tumorigenic in nude mice. Media conditioned by SFDH cells contain factors that stimulate growth and attachment of a variety of tumorigenic and non-tumorigenic cell lines, inducing cells to divide in serum-free media for up to 14 days when plated on tissue culture-treated and nontreated plastic surfaces pre-coated with SFDH conditional media. The growth-stimulating activity fractionates as a single peak over a sepharose column in the presence of 6 m urea, and sediments as a high molecular weight complex. Growth-stimulating activity can be neutralized by several antisera specific for hepatocyte growth factor, and the same sera recognize a novel approximately 37 kD protein in active supernatants. The cyclic, continuous nature of alternating monolayer and spheroid forms makes this cell line appropriate for studying changing gene expression patterns in progressive cell-cell/cell-matrix interactions.

Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells.

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.

Characterization of human N8 protein.

We have shown before that the N8 mRNA is expressed at higher levels in lung tumor and lung tumor-derived cell lines than normal lung cells. In this paper, we have characterized the N8 protein, and studied its properties. The N8 gene encodes a major 24 kDa protein and its expression correlates well with the N8 mRNA expression pattern observed in different cell lines. N8 protein is capable of forming a homodimer or multimeter in vitro. It is a phosphorylated cytoplasmic protein and phosphorylation occurs mainly at serine residues. N8 protein is expressed at higher levels in epithelial cells than in mesenchymal cells. N8 protein expression is induced in a fibroblast cell line expressing adenoviral Ela protein, which acquired epithelial-like characteristics. Furthermore, ectopic expression of N8 protein in NIH3T3 cells converts them into a spheroid form. These spheroids also have some of the characteristic features of epithelial cells. Taken together, these results suggest that the N8 protein may be associated with the development or maintenance of epithelial cell phenotype.

In vitro generation of allospecific human CD8+ T cells of Tc1 and Tc2 phenotype.

We have previously shown that allospecific murine CD8+ T cells of the Tc1 and Tc2 phenotype could be generated in vitro, and that such functionally defined T-cell subsets mediated a graft-versus-leukemia (GVL) effect with reduced graft-versus-host disease (GVHD). To evaluate whether analogous Tc1 and Tc2 subsets might be generated in humans, CD8+ T cells were allostimulated in the presence of either interleukin-12 (IL-12) and transforming growth factor-beta (TGF-beta) (Tc1 culture) or IL-4 (Tc2 culture). Tc1-type CD8 cells secreted the type I cytokines IL-2 and interferon gamma (IFN-gamma), whereas Tc2-type cells primarily secreted the type II cytokines IL-4, IL-5, and IL-10. Both cytokine-secreting populations effectively lysed tumor targets when stimulated with anti-T-cell receptor (TCR) antibody; allospecificity of Tc1- and Tc2-mediated cytolytic function was demonstrated using bone marrow-derived stimulator cells as targets. In addition, both Tc1 and Tc2 subsets were capable of mediating cytolysis through the fas pathway. We therefore conclude that allospecific human CD8+ T cells of Tc1 and Tc2 phenotype can be generated in vitro, and that these T-cell populations may be important for the mediation and regulation of allogeneic transplantation responses.

A centromere DNA-binding protein from fission yeast affects chromosome segregation and has homology to human CENP-B.

Genetic and biochemical strategies have been used to identify Schizosaccharomyces pombe proteins with roles in centromere function. One protein, identified by both approaches, shows significant homology to the human centromere DNA-binding protein, CENP-B, and is identical to Abp1p (autonomously replicating sequence-binding protein 1) (Murakami, Y., J.A. Huberman, and J. Hurwitz. 1996. Proc. Natl. Acad. Sci. USA. 93:502-507). Abp1p binds in vitro specifically to at least three sites in centromeric central core DNA of S. pombe chromosome II (cc2). Overexpression of abp1 affects mitotic chromosome stability in S. pombe. Although inactivation of the abp1 gene is not lethal, the abp1 null strain displays marked mitotic chromosome instability and a pronounced meiotic defect. The identification of a CENP-B-related centromere DNA-binding protein in S. pombe strongly supports the hypothesis that fission yeast centromeres are structurally and functionally related to the centromeres of higher eukaryotes.

Early suppressive effects of chemotherapy on recovery of bone marrow megakaryocyte precursors: possible relationship to platelet recovery.

This study utilized a recently developed culture and quantitation system to detect megakaryocyte precursors in CD34+ bone marrow cells from normal donors and breast cancer patients treated with 5-fluorouracil, leucovorin, adriamycin and cyclophosphamide (FLAC). Bone marrow was obtained from patients before and then after their first cycle of FLAC once blood cell counts had recovered. CD34+ cells were isolated and placed in liquid culture with growth factors to stimulate proliferation and lineage commitment. Absorbance values from an enzyme-linked immunosorbent assay were used to quantitate expression of platelet glycoprotein GPIIb/IIIa. There was an increase in absorbance with increasing numbers of cells seeded per culture that was associated with an increase in the number of megakaryocyte lineage cells produced. After 10 days in liquid culture, absorbance values for expression of GPIIb/IIIa from 2,000 normal donor and pre-chemotherapy CD34+ marrow cells were > or = 1.0. Absorbance values from cultures of post-chemotherapy CD34+ cells from four patients were similar to values from pre-chemotherapy CD34+ cells. In contrast, absorbance values from cultures of post-chemotherapy CD34+ cells from two other patients were low (absorbance < 0.5). Low absorbance values for GPIIb/IIIa expression indicate that megakaryocyte production from those CD34+ cells was reduced. Both of those patients developed prolonged thrombocytopenia and platelet nadirs of less than 20,000/microl during FLAC chemotherapy. In contrast, only one out of four patients whose cultures of post-chemotherapy CD34+ cells had absorbance values > or = 1.0 developed platelet nadirs less than 20,000/microl. These results suggest that low platelet nadirs and delayed platelet recovery may be associated with suppressive effects of chemotherapy on recovery of megakaryocyte precursors.

Signal transduction in c-met mediated motogenesis.

We have begun characterizing the signal transduction pathways used by the c-met receptor in cells in which ligand (HGF-SF) stimulates motogenesis in the absence of mitogenesis. Primary targets (within 10-15 minutes) were identified as PI-3 kinase, GAP, PLC gamma, src, and MAP kinase, substrates which are also activated upon growth factor activation of mitogenic receptor systems. Following HGF-SF treatment, the 85 kD subunit of PI-3 kinase is phosphorylated on tyrosine and PI-3 kinase activity rapidly associates with the c-met receptor. A number of these substrates are implicated in cytoskeletal rearrangements and may be important in the motogenic response to the factor. We have also identified a number of colon carcinoma lines which express unamplified levels of constitutively tyrosine phosphorylated c-met protein. In these and other (gastric) cell lines which express amplified levels of activated receptor protein, we have determined that receptor activation is not due to the autocrine production of ligand.

An oncogenic chromosome 8-9 gene fusion isolated following transfection of human ovarian carcinoma cell line DNA.

Transfection of NIH3T3 cells with genomic DNA from the human ovarian adenocarcinoma tumor cell line OVCAR-3 identified ovc, a rearranged human DNA sequence which was generated during transfection and which induced both morphological transformation and tumorigenesis. A human alu repeat positive 10.5 kb EcoRI fragment present in all transformants was cloned, and two alu-free fragments of 1.8 kb and 2.2 kb were subcloned. The cloned 10.5 kb fragment is not biologically active in DNA transfection assays. Probe from the 2.2 kb fragment hybridizes to poly A + RNA from the transformants and several human tumor cell lines, including OVCAR-3. The 2.2 kb fragment maps to a site on human chromosome 9 (9p24) not known to contain oncogenic sequences, and identifies a two allele polymorphic restriction site. The 1.8 kb fragment maps to human chromosome 8. The ovc transforming sequences fail to hybridize to probes to any of 14 known oncogenes, indicating that they may represent a previously unknown human transforming gene.

An NMR blood test for cancer: a critical assessment.

An evaluation of the reproducibility and accuracy of the NMR human blood test for cancer described by Fossel, E. T., Carr, J. M. and McDonagh, J., (New England Journal of Medicine 315, 1369-1376) in 1986 has been conducted jointly at the National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD (NCI-FCRF) and the National Research Council, Ottawa, Canada (NRC). The influences on the test of the following were studied: (a) subject fasting; (b) sample collection, storage and handling; (c) use of plasma or serum; (d) variations of test results from the same individual with time; (e) NMR observation parameters including field strength and temperature; and (f) variations in obtaining the Fossel Index (FI) (a number defined by Fossel and co-workers as the average of the widths at half height of the regions in the NMR spectrum of human plasma at 1.3 and 0.88 ppm) by different people from the same plotted spectrum. This test was found to be reproducible but not accurate for screening a general asymptomatic population. The accuracy is defined in terms of the sensitivity, specificity, and predictive values of the test. The accuracy of the test results from our laboratories is compared with the accuracies from other laboratories including Fossel's. The correlation of the Fossel Index with total triglyceride content in the serum has been confirmed by analysing blood components using the following technologies: KBr density gradient centrifugation, high resolution agarose gel electrophoresis, high performance gel permeation chromatography, and chemical analysis.

Compliance with anticonvulsant therapy by epileptic youth. Relationships to psychosocial aspects of adolescent development.

Independence in daily life, family harmony as perceived by youths and their parents, and self-esteem were investigated in relation to anticonvulsant medication compliance in 25 epileptics aged 9-17 years. Medication compliance was assessed by monthly home saliva sampling for phenobarbital concentrations. Psychosocial issues were assessed by standardized instruments. Each psychosocial issue was highly correlated with compliance. Partial correlation analysis reveals that these findings are not explained by the subject's demographic or clinical characteristics. Medication noncompliance appears to be associated with a restriction of independence in daily life, lack of harmony in family relations, and low self-esteem in teenage epileptics. Clinicians should observe for these conditions and initiate patient and family counseling in order to maximize medication compliance and seizure control.

Neurogenic respiratory failure: a 5-year experience using implantable phrenic nerve stimulators.

During the past 5 years, 20 phrenic nerve stimulators have been implanted in 11 patients who were ventilator dependent because of neurogenic respiratory failure. Ten patients had traumatic spinal cord lesions; the remaining patient suffered from a progressive demyelinating disease. There was no operative mortality. Complications included 1 stimulator malfunction and 1 pneumothorax. In spite of adjacent tracheostomies, there were no infections or wound complications. Of the 20 stimulators implanted, 13 initially produced good diaphragmatic function, 2 had fair function, and 5 had little or not function. Three patients became completely independent of their ventilators; 6 became partially independent, thus simplifying nursing care. There were no late complications. As of December, 1979, 7 patients had benefited or were continuing to benefit from phrenic nerve stimulation.