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G J Dougherty

Publications and source records attributed to G J Dougherty.

At least 19 recordsLinked to original sources
PubMed1997

Molecular mechanisms regulating TNF-alpha production by tumor-associated macrophages.

The molecular mechanisms that regulate the production and/or functional activity of intratumoral tumor necrosis factor-alpha (TNF-alpha) remain poorly defined. To begin to address this issue we have examined the level of TNF-alpha mRNA and protein produced by macrophages present within immunogenic Fsa-R and non-immunogenic Fsa-N tumors grown in syngeneic Lps(d) C3H/HeJ and Lps(n) C3H/HeN mice. The results obtained indicate that macrophages isolated from tumors grown in Lps(d) C3H/HeJ mice express 5-10-fold less TNF-alpha than equivalent cells present in tumors grown in Lps(n) C3H/HeN mice. These data suggest that the mechanisms that operate within the tumor microenvironment to induce the production of TNF-alpha act, at least in part, via the same signal transduction pathway that is defective in Lps(d) C3H/HeJ mice. Interestingly, despite such differences in TNF-alpha production, tumors inoculated into C3H/HeJ and C3H/HeN mice grew at a similar rate and contained an almost identical proportion of macrophages. Moreover, tumor cells purified from tumors grown in C3H/HeJ and C3H/HeN mice produced similar quantities of the TNF-alpha-inducible cytokine GM-CSF. Thus, although differences in the level of TNF-alpha produced within tumors grown in C3H/HeN and C3H/HeJ mice are readily demonstrable, such differences appear to have little direct impact on the outcome of tumor growth.

Animals↗
PubMed1996

The role of host lymphoid populations in the response of mouse EMT6 tumor to photodynamic therapy.

Photodynamic therapy (PDT) treatment of murine EMT6 mammary sarcoma using Photofrin (10 mg/kg) and light (110 J/cm2) cured all these lesions growing in syngeneic BALB/c mice. In contrast, the same treatment produced initial ablation but no long-term cures of EMT6 tumors growing in either scid or nude mice, the immunodeficient strains sharing the same genetic background with BALB/c mice. No difference was detected in either the level of Photofrin accumulated per g of tumor tissue or the extent of tumor cell killing during the first 24 h after PDT of EMT6 tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in scid and nude mice was supported by the results of experiments involving the adoptive T-cell transfer into scid mice or bone marrow transfer between BALB/c and scid mice. The adoptive transfer of splenic virgin T lymphocytes from BALB/c mice into scid mice performed 9 days before PDT of EMT6 tumors growing in the recipients was successful in delaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better improvement and a high cure rate of PDT-treated tumors was obtained with scid mice reconstituted with BALB/c bone marrow. In contrast, a marked drop in tumor cure rate was observed with BALB/c mice reconstituted with scid bone marrow. These results suggest that the activity of host lymphoid populations was essential for preventing the recurrence of EMT6 tumors following the PDT treatment used in this study. The contribution of PDT-induced immune reaction may, therefore, be of critical importance for the cure with at least some tumors.

Adoptive Transfer↗
PubMed1996

Potentiation of photodynamic therapy-elicited antitumor response by localized treatment with granulocyte-macrophage colony-stimulating factor.

Murine squamous cell carcinoma (SCCVII) cells were genetically engineered to produce marine granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF immunotherapy, based on the peritumoral injection of lethally irradiated GM-CSF-producing SCCVII cells, was examined as adjuvant to photodynamic therapy (PDT) treatment of this tumor. The GM-CSF immunotherapy administered three times in 48-h intervals, starting 2 days before the light treatment, substantially improved the curative effect of Photofrin-mediated PDT. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative-mediated PDT. The tumor-localized GM-CSF immunotherapy alone had no obvious effect on the growth of parental SCCVII tumors. This treatment did not significantly alter the differential peripheral WBC count and appeared not to affect tumor leukocyte infiltration. However, GM-CSF treatment did increase the cytotoxic activity of tumor-associated macrophages against SCCVII tumor cells. It appears, therefore, that tumor-localized immune stimulation by GM-CSF amplifies a PDT-induced antitumor immune reaction, which has a potentiating effect on tumor control.

Animals↗
PubMed1996

Site-specific delivery of cytokines in cancer.

Cytokines can stimulate the immune response by directly activating effector cells and/or by inducing the release of secondary cytostatic or immunomodulatory molecules. Systemic administration of cytokines, however, often causes numerous side effects in the patient and may not provide a constant level at the tumor site, owing to the variable and generally short half-lifes of cytokines. This has led to the development of novel strategies to deliver cytokines specifically to tumor sites in vivo. These include: the use of monoclonal antibodies directed against tumor antigens; cellular vehicles designed to carry genes to tumor sites; genetically engineered tumor cell vaccines; and targetable viral vectors.

Animals↗
PubMed1996

Integrin VLA-6 (alpha 6 beta 1) mediates adhesion of mouse bone marrow-derived mast cells to laminin.

The development of mast cells from bone marrow precursors and their function as the mucosal- or connective-tissue-type mast cell are critically dependent on microenvironmental factors. Extracellular matrix proteins, such as collagen, fibronectin, and laminin, may represent insoluble components of the microenvironment. Recent studies have described multiple isoforms of laminin isolated from different tissues. In the present study, adhesion of mouse bone marrow-derived mast cells (BMMC) and long-term mast cell lines to Engelbreth-Holm-Swarm (EHS) tumor laminin, rat laminin, human merosin, and human placental laminin was compared. The greatest level of adhesion was found with human laminin as the substrate. By use of a newly prepared mouse VLA-alpha 6 integrin-specific mAb (MA6) together with the previously described mAb GoH3, VLA-6 (alpha 6 beta 1) integrin was found to be expressed and utilized by BMMC and long-term mast cell lines. VLA-6 has been described as a major laminin receptor with roles in diverse cell functions including cell growth and differentiation. BMMC have been shown to express a 32/67-kDa laminin receptor. Therefore, in addition to the 32/67-kDa laminin receptor described in early studies, BMMC also express VLA-6 integrin, which may have roles in the regulation of their development.

Animals↗
PubMed1996

Altered patterns of CD44 epitope expression in human chronic and acute myeloid leukemia.

Abnormal expression of different isoforms of CD44 has been found to characterize many types of malignant cells although data for human acute and chronic myeloid leukemia is limited. In this study, we have identified significant, albeit variable, increases in these diseases of the frequency of both light density and CD34+ cells expressing two particular CD44 epitopes, neither of which is commonly found on normal human marrow cells. One of these epitopes is unique to exon v10-containing isoforms of CD44. The other is located in the common region of CD44 and was previously revealed on T cells only after their activation. Interestingly, another T cell activation-associated epitope was found to be expressed on a high proportion of normal marrow cells including the CD34+ subset and this remained the case for most of the primary leukemic samples evaluated. As expected, >90% of cells in all primary normal and leukemic samples expressed high levels of CD44, as shown by their reactivity with an antibody specific for the CD44 hyaluronan-binding site. To begin investigating how expression of the CD44 epitopes seen more commonly on leukemic than on normal CD34+ cells may be modulated, and to identify potentially associated effects on the hyaluronan-binding ability of the CD44 expressed, the effect of phorbol ester treatment on these properties of CD44 were examined. For these studies, a panel of five different human leukemic cell lines that were found to exhibit different patterns of CD44 expression and function in the absence of phorbol ester were used. Both the level and the hyaluronan-binding properties of CD44 could be stimulated in some, but not all, of these leukemic cell lines. Taken together, our findings indicate that CD44 expression is perturbed in a variety of leukemic populations suggesting a possible relationship to some of the pathogenetic features they share.

Biomarkers, Tumor↗
PubMed1995

Differentiation-associated changes in CD44 isoform expression during normal hematopoiesis and their alteration in chronic myeloid leukemia.

CD44 is a widely expressed, multifunctional, cell-surface glycoprotein that has been implicated in the regulation of normal hematopoiesis. In addition, expression of particular isoforms of CD44 has been associated with malignant transformation and/or the acquisition of metastatic potential. In this study, we used two recently developed monoclonal anti-CD44 antibodies, one reactive with an epitope shared by many CD44 isoforms and the other with an epitope unique to CD44 isoforms containing amino acids encoded by the alternatively spliced exon v10, to compare the expression of CD44 on primitive hematopoietic cells from the marrow of normal individuals and their neoplastic counterparts present in the peripheral blood of patients with chronic myeloid leukemia (CML). Multiparameter fluorescence-activated cell sorter (FACS) analysis and cell sorting studies showed that CD44 is normally expressed at high to very high levels on both long-term culture-initiating cells (LTC-IC) and granulopoietic colony-forming cells (granulocyte-macrophage colony-forming units [CFU-GM]). In contrast, primitive erythropoietic progenitors (burst-forming units-erythroid [BFU-E]) in normal marrow were more homogeneous in their expression of CD44, and very few (less than 5%) showed the very high levels of CD44 seen on 20% to 25% of LTC-IC and CFU-GM. Antibody staining showed the expression of exon v10-containing CD44 isoforms to be restricted to a small subpopulation (4% to 8%) of morphologically recognizable mature (CD34-) myeloid cells within the light-density fraction of normal marrow cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed the presence of two exon v10-containing mRNA species. In CML, a significantly greater proportion of the circulating neoplastic CFU-GM expressed very high levels of CD44, and these CFU-GM were accompanied by an increased number of light density v10+ cells, including some that coexpressed CD34. Nonmalignant hematopoietic progenitors mobilized by prior chemotherapy and growth factor treatment of patients with Hodgkin's disease or acute myeloid leukemia in remission showed no changes in CD44 expression relative to normal marrow progenitors. These results provide evidence of early differentiation-associated changes in CD44 expression during normal hematopoiesis in vivo that may be deregulated in the neoplastic clone of patients with CML.

Base Sequence↗
PubMed1995

Adhesive interactions between alternatively spliced CD44 isoforms.

Alternative splicing of a series of 10 contiguous exons present within the CD44 gene can generate a large number of differentially expressed CD44 isoforms that contain additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule proximal to the membrane spanning domain. Although distinct functions have been ascribed to certain of these isoforms, the effect of particular inserted domains on the ligand-binding specificity of the CD44 molecule remains unclear. In the present study, we demonstrate that while CD44H, the major CD44 isoform expressed on resting hemopoietic cells, and CD44R1, an alternatively spliced isoform present on transformed epithelial cells and certain activated and/or malignant hemopoietic cell types, can both bind avidly to hyaluronan, only CD44R1 can promote homotypic cellular aggregation when expressed in the CD44-negative murine lymphoma cell line TIL1. Experiments in which TIL1 cells transduced with different CD44 isoforms were tested for their ability to adhere to one another or to COS7 cells transfected with CD44R1, indicated that CD44R1 can recognize and bind a common determinant present on both CD44H and CD44R1. Monoclonal antibody blocking studies suggest further, that the determinant recognized by CD44R1 is located in a region of the CD44 molecule distinct from that involved in hyaluronan binding.

Alternative Splicing↗
PubMed1995

Down-regulation of murine fibrosarcoma transforming growth factor-beta 1 expression by interleukin 7.

BACKGROUND: Cytokine genes encode proteins that modulate immune system responses. Modification of tumor cells by the introduction of cytokine genes has been used as a strategy to augment host immunity. Interleukin 7 (IL-7) gene transfer enhances the immune response to tumor cells and can result in tumor regression. Transforming growth factor-beta 1 (TGF-beta 1) is a potent immunosuppressive cytokine produced by many tumors. We have previously reported that recombinant IL-7 decreases the expression of TGF-beta 1 by murine macrophages. PURPOSE: This study investigates the inhibition of tumor-derived TGF-beta 1 production as a possible mechanism for the enhanced antitumor immunity that accompanies IL-7 gene transfer. METHODS: A fibrosarcoma cell line (FSA-JmIL-7) genetically modified to produce IL-7 was used to evaluate the effects of IL-7 on tumor production of TGF-beta 1. The control cell line (FSA-Jneo) originated from the same parental fibrosarcoma cell line (FSA) and was produced by transduction with the same retroviral vector without the IL-7 gene. FSA-Jneo and FSA-JmIL-7 tumor cells were evaluated for the expression of TGF-beta 1 messenger RNA (mRNA). To determine if the observed change in TGF-beta 1 mRNA was associated with an alteration in protein secretion, we compared supernatants from tumor cell cultures for TGF-beta 1 production. Specific anti-TGF-beta 1 monoclonal antibody (MAb) was used to confirm the role of TGF-beta 1 in these assays. RESULTS: Compared with FSA parental and FSA-Jneo cells, FSA-JmIL-7 cells expressed TGF-beta 1 mRNA at a lower level. Compared with supernatants from FSA-Jneo cells, FSA-JmIL-7 supernatants contained consistently lower levels of TGF-beta 1 activity (P < .05). In addition, FSA-Jneo supernatants suppressed lymphocyte proliferation to a significantly greater degree than supernatants from FSA-JmIL-7 cells (P < .05). Studies with anti-TGF-beta 1 MAb added to the supernatants confirmed the role of TGF-beta 1 in inhibition of lymphocyte proliferation. CONCLUSION: These findings suggest that IL-7 gene transfer inhibits the production of TGF-beta 1 by tumor cells and thus may enhance the efficacy of the host's antitumor immune response. IMPLICATION: The regulation of endogenous tumor-derived cytokines in response to cytokine gene transfer may contribute to altered immune responses in the tumor microenvironment and thus may be an important additional parameter to assess in gene therapy.

Animals↗
PubMed1995

Molecular mechanisms regulating the hyaluronan binding activity of the adhesion protein CD44.

In the present study, we describe the isolation and characterization of a cDNA clone designated B6F1.3, that appears to 'activate' the hyaluronan-binding capacity of CD44 upon transfection into the murine fibroblastoid cell line MOP8. Sequence analysis indicates that the putative regulatory molecule encoded by this clone is identical to the murine interleukin-2 receptor gamma chain (mIL-2R gamma), a recently described type 1 transmembrane protein that constitutes an integral component of the cell surface receptors that bind a number of cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and perhaps also IL-13. Mutations in this molecule have been shown to be responsible for X-linked severe combined immunodeficiency (XSCID) in humans. With the exception of bone marrow, the mIL-2R gamma chain was found to be expressed at high levels on all hemopoietic cell lines and tissue types examined. Non-hemopoietic tissues are generally negative. FACS analysis and Western blot analysis indicated respectively that B6F1.3 does not mediate its effects by upregulating the expression of CD44 or by altering the alternative splicing of the molecule. Removal of the cytoplasmic tail of the mIL-2R gamma chain, including a Src homology region 2 (SH2) subdomain, abolished its ability to enhance CD44-mediated binding to hyaluronan suggesting the involvement of signal transduction events triggered via the cytoplasmic domain in the 'activation' process. Determining whether activating molecules such as B6F1.3 are co-expressed within tumor cells may help improve the potential value of CD44 as a diagnostic marker of metastatic disease.

Alternative Splicing↗
PubMed1995

Gene therapeutic approach to primary and metastatic brain tumors: I. CD44 variant pre-RNA alternative splicing as a CEPT control element.

Our laboratory and others have shown alternative splicing of up to ten exons at a discrete extracellular site to be primarily responsible for the generation of CD44 variant (CD44v) isoforms. Based on clear differences in the expression of these CD44v isoforms between normal and malignant tissues, we believe that elucidation of the mechanisms underlying the regulation of CD44 alternative splicing may provide a new gene therapeutic targeting approach based on CD44 pre-mRNA processing in vivo. This strategy incorporates utilization of CD44 alternative splicing control elements into a chimeric enzyme/prodrug therapy (CEPT), a novel modification of the virus-directed enzyme/prodrug therapy (VDEPT) approach for the treatment of brain metastases from tumors of systemic origin. As initial steps towards the development of a gene therapeutic approach based on targeting tumor cell expression of specific CD44v alternatively spliced isoforms, we have: (1) developed a novel in vivo assay system that allows the rapid analyses of potentially therapeutic CD44 alternative splicing minigene constructs; and (2) cloned the E. coli cytosine deaminase (CD) gene and fused its enzymatically active domain to alternatively spliced CD44 exons (CD44/CD). Deamination of cytosine by this CD44/CD chimeric fusion protein is demonstrated in E. coli cell lysates to be equal to that of wild type cytosine deaminase.

Alternative Splicing↗
PubMed1995

Induction of immune cell infiltration into murine SCCVII tumour by photofrin-based photodynamic therapy.

Cellular populations in the squamous cell carcinoma SCCVII, growing in C3H/HeN mice given Photofrin, were examined at various time intervals during the photodynamic light treatment and up to 8 h later. Cell populations present within excised tumours were identified by monoclonal antibodies directed against cell type-specific membrane markers using a combination of the indirect immunoperoxidase and Wright staining or by flow cytometry. Photofrin-based photodynamic therapy (PDT) induced dramatic changes in the level of different cellular populations contained in the treated tumour. The most pronounced was a rapid increase in the content of neutrophils, which increased 200-fold within 5 min after the initiation of light treatment. This was followed immediately by an increase in the levels of mast cells, while another type of myeloid cells, most likely monocytes, invaded the tumour between 0 and 2 h after PDT. The examination of cytolysis of in vitro cultured SCCVII tumour cells mediated by macrophages harvested from the SCCVII tumour revealed a pronounced increase in the tumoricidal activity of tumour-associated macrophages isolated at 2 h post PDT. It seems, therefore, that the PDT-induced acute inflammatory infiltration of myeloid cells into the treated tumour is associated with functional activation of immune cells.

Animals↗
PubMed1995

Radiotherapy for genes that cause cancer.

Many approaches to cancer gene radiotherapy are possible. Even existing strategies, devised with other goals in mind, can cause radiosensitization by altering the intrinsic radiosensitivity of tumour cells or by modifying the tumour microenvironment. Small increases in the levels of radiosensitization could, over a fractionated radiation course, have major effects on the probability of tumour cure. The clinical application of gene radiotherapy will require use of in vivo gene delivery systems that still need validation but, because radiation is effective at decreasing tumour burden, genes will not have to be expressed in all cells in order to achieve a cure.

Apoptosis↗
PubMed1995

Modification of tumor microenvironment by cytokine gene transfer.

The tumor microenvironment is determined by the interactions between host and tumor cells, a process in which cytokines play a major role. We have used retroviral vectors to insert and express cytokine genes in tumor cells so as to induce predictable changes in the host cells that infiltrate tumors. This frequently caused changes in tumor cell phenotype through autocrine/intracrine pathways. We reasoned that cytokine-induced alterations in tumor cell phenotype and/or in infiltrating host cells might alter the in vitro and in vivo cellular response to irradiation. In the present paper we document some of the effects of expression of interleukin-6 (IL-6) and IL-7 genes in tumor cells in this regard. The studies support the hypothesis that cytokines may play a role in determining both intrinsic tumor radioresponsiveness and the tumor microenvironment and in these ways may influence in vivo tumor irradiation responses. Possible cytokine gene-mediated approaches to radiotherapy cancer are discussed.

Animals↗
PubMed1994

Transfer of the tumor necrosis factor alpha gene into hematopoietic progenitor cells as a model for site-specific cytokine delivery after marrow transplantation.

Relapse of leukemia is the major cause of failure after autologous stem cell transplantation due to reinfusion of residual clonogenic cells and the absence of an immune-mediated graft-versus-leukemia effect. To provide an antileukemia effect, immune-activating cytokines have been given to patients after transplantation. Systemic administration of such cytokines early after transplantation is often accompanied by substantial side effects, and it is unknown whether sufficient concentrations reach the sites of residual disease in the marrow. As a method of site-directed immunotherapy provided early after stem cell transplantation, we have tested in a murine model whether (1) marrow can be retrovirally transduced with the tumor necrosis factor alpha (TNF alpha) gene, (2) local production of TNF alpha by marrow cells after transplantation can be achieved, and (3) adverse effects of TNF alpha occurred. Balb/c mice were treated with 5-fluorouracil and bone marrow (BM) was obtained 4 days later. Whole BM was transduced in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor by coculture with the packaging cell line GP+E-86, producing the cDNA for TNF alpha. Irradiated (1,300 cGy) syngeneic recipient mice were given 10(6) transduced BM cells on day 0. Integration of the TNF alpha gene into the host genome was documented by Southern blotting in spleen and BM cells on days 7 and 12 and in BM on day 40 after marrow infusion, but was no longer found on day 90. Messenger RNA for TNF alpha was present on day 12, but could no longer be shown on day 40 or 90. Although no measurable (L929 bioassay) levels of TNF alpha were found in serum of mice who had received TNF alpha transduced marrow, the supernatant of 10(6) unstimulated BM cells obtained 12 days after marrow infusion was found to have 7 pg of TNF alpha compared with 1.8 pg in nontransduced marrow. Mice that had received TNF alpha transduced marrow showed no side effects suggestive of systemic TNF alpha release, and cellularity of the TNF alpha-transduced marrow was not different from control mice that had received unmanipulated marrow or cells transduced with the neomycin resistance gene only. The studies suggest that gene transfer of immune-activating cytokines into hematopoietic cells could be used as a means to achieve their temporary local production early after transplantation by cells located in the BM.

Animals↗
PubMed1994

Ligand binding specificity of alternatively spliced CD44 isoforms. Recognition and binding of hyaluronan by CD44R1.

CD44 species of widely differing molecular mass have been identified on various normal and/or transformed cells. Recent studies have demonstrated that much of this heterogeneity is produced as a result of the alternative splicing of a series of 10 exons present within the CD44 gene generating a large number of CD44 isoforms containing additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule. At present, the effect of such insertions on the ligand binding specificity of CD44 remains unclear. CD44H, the major CD44 isoform expressed by most resting cell types, has been shown to function as a receptor for the glycosaminoglycan hyaluronan. In contrast, CD44E, the major isoform expressed by the colon carcinoma cell line HT29, which contains a 132-amino acid insert, is unable to recognize and bind this ligand. In the present study we demonstrate that CD44R1, an isoform isolated from the myelomonocytic cell line KG1a, that differs from CD44E by just 3 amino acid substitutions, is fully capable of mediating the attachment of transfected COS7 cells to hyaluronan-coated plastic. In order to confirm that such binding was directly mediated by the introduced CD44 species, chimeric proteins containing the entire extracellular domain of CD44H or CD44R1 fused in-frame to human bone/liver/kidney alkaline phosphatase were prepared and tested for their ability to bind hyaluronan-coated plastic. Both fusion proteins bound equally well to hyaluronan and in each case their attachment could be readily inhibited by monoclonal antibodies directed against the hyaluronan-binding domain of CD44. These data indicate that the 132-amino acid insert present within the extracellular domain of CD44R1 does not interfere with the hyaluronan binding function of the molecule. Since CD44E contains an identically sized insert but is unable to bind hyaluronan, it is likely that mutation of one or more of the 3 amino acid residues that differ between CD44E and CD44R1 is responsible for the altered functional activity of this particular molecule.

Alternative Splicing↗
PubMed1994

Inhibitory effect of locally produced and exogenous interleukin-6 on tumor growth in vivo.

In order to define the potential antitumor activity of the multifunctional cytokine interleukin-6 (IL-6), retrovirus-mediated gene transfer was used to introduce and express a cDNA encoding human IL-6 in the murine fibrosarcoma cell line Fsa-R. Although these genetically modified tumor cells appeared morphologically and phenotypically identical to control Fsa-R cells and had a similar plating efficiency in vitro, they were found to exhibit greatly reduced tumorigenicity in vivo following intravenous injection into syngeneic recipients. Exogenous IL-6 was shown to produce a similar inhibition of tumor growth in the lung if administered intraperitoneally. In contrast, tumor growth in subcutaneous sites was inhibited only if the tumor cells were engineered to express IL-6 locally, or if IL-6 was administered intratumorally. Intraperitoneal injection of IL-6 had no inhibitory effect. Tumors that did grow from IL-6-producing tumor cell inocula in subcutaneous sites were found to contain large numbers of macrophages. These results demonstrate that the antitumor activity of systemically administered IL-6 varies depending on the site of tumor growth and suggest an important role for IL-6 in the recruitment, proliferation and/or survival of tumor-associated macrophages.

Animals↗