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Biomedical subjects

R G Hawley

Publications and source records attributed to R G Hawley.

At least 91 records · Page 5Linked to original sources
PubMed1993

Bidirectional modulation of TNF-alpha production by alveolar macrophages in asbestos-induced pulmonary fibrosis.

Bronchoalveolar lavage (BAL) cells were isolated from rats 1, 3, and 6 weeks after a single intratracheal instillation of saline, UICC chrysotile asbestos (5 mg), or silica (5 mg). In asbestos-exposed rats, the pulmonary response was characterized by a significant increase in the number of alveolar macrophages (AMs) and the appearance of fibrotic lesions within 1 week. By contrast, mixed macrophage and neutrophil accumulations were observed in the silica group without evidence of fibrosis. Tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS)-stimulated BAL cells from asbestos-treated rats was significantly lower than controls 1 and 3 weeks after exposure. However, by 6 weeks higher levels of TNF-alpha production were noticeable in this group. Decreases in LPS-induced TNF-alpha production were also observed with BAL cells from silica-treated animals at all time points studied. Lower levels of TNF-alpha were not related to decreased BAL cell viability or the presence of a significant proportion of neutrophils in the silica group. Furthermore, biphasic changes in TNF-alpha production seen in the asbestos group were correlated with concomitant decreases (3 weeks) and increases (6 weeks) in levels of TNF-alpha mRNA in AMs. These data indicate that lower levels of TNF-alpha resulted from inhibition at the gene expression level and provide evidence for bidirectional modulation of TNF-alpha production by AMs during inflammatory reactions.

Animals↗
PubMed1993

Establishment and characterization of first trimester human trophoblast cells with extended lifespan.

We established trophoblast cell cultures with extended lifespans by introducing into first trimester human trophoblasts the gene encoding simian virus 40 large T antigen. The transfected trophoblasts were characterized according to their expression of various morphological and functional markers. Both parental (HTR-8) and transfected (HTR-8/SVneo) lines were morphologically similar and positive for cytokeratin, confirming their epithelial (trophoblastic) identity. Whereas the parental cells senesced after 12-14 passages, the transfectants have been in culture for over 32 passages. Human chorionic gonadotrophin was detected only in the HTR-8/SVneo cells and not in the parental cells. Both lines required at least 5% serum in order to sustain growth in vitro and responded to transforming growth factor-beta (TGF-beta) with reduced [3H]-thymidine incorporation in a dose-dependent manner. Treatment with TGF-beta also resulted in decreased secretion of plasminogen activators (PAs) and reduced PA activity by both lines. Both cell lines secreted mostly 72-kDa type IV collagenase as determined by substrate gel zymography, but the level of secretion of this enzyme was not significantly affected by TGF-beta in either line. Even though both lines exhibited similar in vitro invasive abilities, only the invasiveness of the parental cells was reduced by TGF-beta. Neither parental or transfected cells were capable of growth in soft agar and no sign of tumor formation was evident more than 5 months after subcutaneous inoculation of the transfected cells into nude mice. These results indicate that apart from their ability to sustain prolonged growth in culture, the transfected HTR-8/SVneo cells share a number of phenotypic properties with the parental trophoblast cells. For this reason, these transfected trophoblasts may prove to be an important tool for the study of placental function and/or tumor progression.

Animals↗
PubMed1993

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Establishment of a novel factor-dependent myeloid cell line from primary cultures of mouse bone marrow.

We describe here a novel myelomonocytic cell line (OTT1) obtained from primary cultures of mouse bone marrow cells infected with a retroviral vector carrying the mouse interleukin (IL)-1 alpha gene. OTT1 cells are dependent for their survival and proliferation on IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) or, unexpectedly, IL-5. Despite their IL-5 dependency, OTT1 cells form colonies showing predominantly monocyte maturation when plated in methylcellulose. It is suggested that constitutive expression of the exogenous IL-1 alpha gene may predispose to a monocytic phenotype. OTT1 cells should be a useful experimental model to investigate the molecular mechanisms of IL-5 signal transduction and the possible interrelationships between this signal pathway and those utilized by IL-3 and GM-CSF.

Animals↗
PubMed1991

Expression of retrovirally transduced IL-1 alpha in IL-6-dependent B cells: a murine model of aggressive multiple myeloma.

Retroviral-mediated gene transfer was employed to introduce an IL-1 alpha cDNA into an IL-6-dependent murine B-cell line. Bone marrow metastases and bone lesions were frequently observed following intravenous injection of these B cells into syngeneic mice. Because the retroviral vector also contained the neomycin phosphotransferase gene, metastatic cells could be easily recovered from bone marrow by addition of G418 to the culture medium. Interestingly, the metastatic B cells were found to retain their IL-6 dependency through several transplant generations. By comparison, intravenous injection of autonomously-growing B-cell lines generated in vitro by retroviral introduction of an IL-6 cDNA rarely resulted in bone marrow metastases. These results demonstrate that abrogation of growth factor dependency is neither necessary nor sufficient for the in vivo growth and dissemination of tumor cells in this experimental system. It is proposed that the increased metastasis of the IL-1 alpha-producing B-cells to bone marrow is due to alterations in cell adhesion molecules. The B-cell bone marrow metastasis model described here may be useful for studies of bone marrow homing and for evaluation of therapeutic regimens for multiple myeloma.

Animals↗
PubMed1990

Long- and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers.

The proliferative longevity of totipotent hematopoietic stem cells (THSC) is a limiting factor in normal hematopoiesis and in therapy by cell- or gene-replacement, but has not yet been ascertained. We have followed the long-term fate of individual clones of mouse THSC from fetal liver or adult bone marrow, after labeling in culture, followed by engraftment and serial transplantation in unirradiated W/Wv-C57BL/6 hosts. The ancestor cell of each clone and its mitotic progeny were uniquely identifiable retrospectively by the DNA integration pattern experimentally produced by replication-incompetent recombinant murine retroviruses. These viruses provided physiologically neutral markers. The marked clones proved to be derived from THSC, based on their contributions to a wide array of myeloid and lymphoid blood lineages in the hosts. The label also identified the target cells as the population displaying clonal succession. The various labeled stem cell clones proliferated for substantially different periods of time. The longest observed clone endured, after the original cell was marked, for at least 2 1/2 years--the equivalent of a mouse's lifetime. However, the results suggest that THSC clones are not all long-lived and that even the longest-lived ones may not be potentially immortal. Thus, the unpredictable lifespan of any given THSC clone indicates the desirability of introducing multiple clones in therapeutic transplants.

Animals↗
PubMed1990

Suppression of programmed death and G1 arrest in B-cell hybridomas by interleukin-6 is not accompanied by altered expression of immediate early response genes.

The murine B-cell hybridoma B9 requires interleukin-6 (IL-6) for its survival and proliferation in vitro. We show here that withdrawal of IL-6 from B9 cultures results in programmed death, concomitant with arrest of the cells in the G1 phase of the cell cycle. Unlike several other systems that undergo programmed cell death, no induction of transcripts corresponding to the testosterone-repressed message-2 or transglutaminase genes is observed during this process. Upon readdition of IL-6 to G1-arrested B9 cells, viability is maintained and entry into S phase occurs after a lag period of 10 to 12 hr. Northern blot analysis showed that the immediate-early mRNAs normally induced shortly after growth factor stimulation in quiescent fibroblasts (c-fos, c-jun, Egr-1, c-myc, JE, and KC), and other growth-related genes (2F1, c-Ha-ras, and p53), are either not induced or remain unchanged during G1 to S phase progression. A correlation was found, however, between the temporal pattern of expression of several G1/S phase genes (dihydrofolate reductase, thymidine kinase, transferrin receptor, and histone H3) and DNA synthesis. These results demonstrate that IL-6-induced viability and growth of hybridoma (and, presumably, plasmacytoma) cells is mediated via novel signal transduction pathways.

Animals↗
PubMed1990

Effect of different promoters on expression of genes introduced into hematopoietic and marrow stromal cells by electroporation.

Electroporation is a simple and relatively efficient means of introducing genes into hematopoietic cells. However, achieving and maintaining high levels of gene expression in transfected hematopoietic progenitor cells remains problematic. In order to address this problem we examined the effect of different viral and cellular promoters on the transient expression of reporter genes transferred into K562, KG1a, and human marrow stromal cells. We found that although the Rous sarcoma virus long terminal repeat was most active in K562 cells in a transient expression assay, the murine cytomegalovirus immediate early promoter was strikingly more active than the other promoters in KG1a cells as well as in the marrow stromal cell population. Long-term stable gene expression was also demonstrated in stromal cells. We infer that the murine cytomegalovirus immediate early promoter may be highly active in human hematopoietic progenitor cells and that human marrow stromal cells may be an attractive vehicle for gene delivery.

Bone Marrow↗
PubMed1989

Comparative analysis of retroviral vector expression in mouse embryonal carcinoma cells.

A series of replication-defective retroviral vectors were assessed for their ability to efficiently transfer functional genes into undifferentiated cells. In these vectors (designated handicapped because of a deletion of enhancer and promoter sequences in the viral long terminal repeat) transcription of inserted genes is under the control of internal promoters. Although a composite promoter composed of a mutant polyoma virus enhancer (PyF441) coupled to the herpes simplex virus thymidine kinase promoter was anticipated to function efficiently, it was found to be significantly inferior to the mouse X-chromosome phosphoglycerate kinase (pgk-1) promoter, in its ability to express the selectable neomycin phosphotransferase gene in mouse embryonal carcinoma cells. The pHMB vector, which contains the pgk-1 promoter, was shown to confer the drug-resistant phenotype at high frequencies to F9 and P19 cells. This vector might prove to be of general utility for efficient gene expression in other developmental contexts.

Animals↗
PubMed1987

Handicapped retroviral vectors efficiently transduce foreign genes into hematopoietic stem cells.

Retroviral vectors, designated handicapped, are described. These are genetically defective viruses that allow transfer of nonselectable genes under the transcriptional control of internal promoters. The basic handicapped vector (pHHAM) is derived from Harvey, Abelson, and Moloney murine retroviruses. It contains a 327-base-pair deletion in the 3' long terminal repeat that spans enhancer and promoter sequences. The deletion is successfully transferred to the 5' long terminal repeat after reverse transcription of viral RNA, yielding a provirus incapable of synthesizing viral transcripts. HHAM viruses containing the mouse c-myc gene under the control of immunoglobulin kappa chain gene regulatory elements, along with a selectable gene (neo) driven by a weak promoter (tk), were stably transmitted to cultured mouse B cells. The donor c-myc gene was transcribed from the kappa promoter in these cells. Helper-free virus-producing cell lines were generated at titers favorable for the efficient introduction of HHAM viruses, even without selection, into hematopoietic stem cells from mouse bone marrow. When returned to unirradiated congenic recipient mice, the cells were capable of long-term reconstitution of the myeloid and lymphoid lineages of W/Wv mutants and the lymphoid system of scid mutants.

Animals↗
PubMed1986

Immunoglobulin synthesis in non-B cell lines.

A recombinant vector carrying rearranged immunoglobulin heavy (mu) and light (kappa) chain genes was introduced into several cell lines of non-B lineage including T cell (EL4) and two fibroblast lines (CV-1 and L cells). Our results indicated that intracytoplasmic mu-chains, both secretory and membrane types, were expressed in the recipient cell lines. Small amounts of kappa-chain transcripts, corresponding in size to authentic kappa-chain mRNA, were detected in EL4 and L cell transformants, whereas CV-1 transformants did not express the authentic kappa-chain RNA. EL4 and L cell transformants produced kappa-chain. This system will be quite useful for the analysis of regulatory mechanisms involved in immunoglobulin synthesis in non-B cells.

Animals↗
PubMed1984

Transposition of intracisternal A-particle genes in mouse hybridomas.

A nonfunctional immunoglobulin kappa-chain gene previously shown to contain intracisternal A-particle (IAP) sequences within one of its introns was further characterized by DNA and RNA blot analysis. The results of these experiments indicate that the joining of the IAP sequences to the kappa-chain gene was a consequence of an insertion event which presumably involved a complete IAP gene. It is postulated that the insertion of the IAP gene has altered the secondary or tertiary structure of the kappa-chain pre-mRNA, resulting in the utilization of a "cryptic" 5' splice site. The detection of numerous IAPs in isogenous wild-type hybridoma cells by electron microscopy suggests that transposition of IAP genes might proceed via the reverse transcription of IAP-associated RNA. These findings raise the possibility that transposition of IAP genes might also occur in other IAP-positive mouse cells. In particular, insertional mutagenesis by IAP genes in IAP-producing tumor cells could play a role in tumor cell heterogeneity or tumor progression. We have also investigated the possibility that IAP-related sequences might be present in the human genome. Using nonstringent hybridization conditions, multiple discrete restriction fragments could be detected in human DNA with several mouse IAP-specific probes.

Animals↗
PubMed1984

Transposition of two different intracisternal A particle elements into an immunoglobulin kappa-chain gene.

Each of two severely defective mouse kappa-chain genes has acquired a different intracisternal A particle (IAP) element within one of its introns. One IAP element generated 6-base-pair direct repeats upon insertion. In contrast, the other IAP element was not flanked by direct repeats and was missing a single nucleotide from its 3' terminus. Sequence analysis of the latter IAP element demonstrated that its long terminal repeats were not identical. Nevertheless, the long terminal repeats were organized like proviral long terminal repeats, and this IAP element did contain two regions that were analogous to retroviral priming sites for RNA-directed DNA synthesis. The region that corresponded to a retroviral tRNA primer binding site was complementary to the 3' ends of all mammalian phenylalanine tRNAs. These findings are discussed in the context of the presumed mode of transposition of IAP elements involving the reverse transcription of IAP RNA.

Animals↗