Frederick L. Hall

Epigenetic role of epidermal growth factor expression and signalling in embryonic mouse lung morphogenesis

A major unsolved problem in developmental biology is to determine when and how time- and position-restricted instructions are signaled and received during secondary embryonic inductions such as branching morphogenesis. The mouse embryonic lung rudiment was used to test the hypothesis that endogenous peptide growth factors, specifically epidermal growth factor (EGF), serve as instructive epigenetic signals for morphogenesis. The presence of EGF precursor mRNA transcripts was detected using the reverse-transcriptase-coupled polymerase chain reaction both in E11-E17-day mouse embryo lung tissues in vivo and in E11-day lung cultured for up to 7 days in vitro under chemically defined, serum-free conditions. Immunolocalization identified a position-restricted distribution of EGF in and around the primitive airways both during in vivo lung morphogenesis and in culture. EGF receptors (EGFR) coimmunolocalized with EGF in the primitive airways. Addition of exogenous EGF to lungs in culture resulted in significant concentration-dependent stimulation of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA. Conversely, the addition of tyrphostin (specific EGF receptor kinase antagonist) to lungs in culture resulted in concentration-dependent inhibition of branching morphogenesis, DNA, RNA, and protein content, and in [3H]thymidine incorporation into DNA without apparent cytotoxicity. The inhibition of the EGF signal by tyrphostin was confirmed by immunoprecipitation of tyrosine phosphoproteins. We conclude that early mouse embryo lungs express EGF transcripts and corresponding EGF peptides in a specific position-restricted distribution which coimmunolocalizes with EGFR in the primitive airways, while stimulatory and inhibitory studies indicate a functional role for the transduced EGF signal in the epigenetic regulation of lung branching morphogenesis. We speculate that the peptide growth factor EGF serves a function in secondary embryonic morphogenetic inductions, which may be modulated by interaction with other growth factors.

Spontaneous and Controllable Activation of Suicide Gene Expression Driven by the Stress-Inducible Grp78 Promoter Resulting in Eradication of Sizable Human Tumors

GRP78 is a stress-inducible chaperone protein with antiapoptotic properties that is overexpressed in transformed cells and cells under glucose starvation, acidosis, and hypoxic conditions that persist in poorly vascularized tumors. Previously we demonstrated that the Grp78 promoter is able to eradicate tumors using murine cells in immunocompetent models by driving expression of the HSV-tk suicide gene. Here, through the use of positron emission tomography (PET) imaging, we provide direct evidence of spontaneous in vivo activation of the HSV-tk suicide gene driven by the Grp78 promoter in growing tumors and its activation by photodynamic therapy (PDT) in a controlled manner. In this report, we evaluated whether this promoter can be applied to human cancer therapy. We observed that the Grp78 promoter, in the context of a retroviral vector, was highly activated by stress and PDT in three different types of human breast carcinomas independent of estrogen receptor and p53. Complete regression of sizable human tumors was observed after prodrug ganciclovir treatment of the xenografts in immunodeficient mice. In addition, the Grp78 promoter-driven suicide gene is strongly expressed in a variety of human tumors, including human osteosarcoma. In contrast, the activity of the murine leukemia virus (MuLV) long-terminal repeat (LTR) promoter varied greatly in different human breast carcinoma cell lines, and in some cases, stress resulted in partial suppression of the LTR promoter activity. In transgenic mouse models, the Grp78 promoter-driven transgene is largely quiescent in major adult organs but highly active in cancer cells and cancer-associated macrophages, which can diffuse to tumor necrotic sites devoid of vascular supply and facilitate cell-based therapy. Thus, transcriptional control through the use of the Grp78 promoter offers multiple novel approaches for human cancer gene therapy.

Systemic Administration of a Matrix-Targeted Retroviral Vector Is Efficacious for Cancer Gene Therapy in Mice

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.

Engineering, Expression, and Renaturation of a Collagen-Targeted Human bFGF Fusion Protein

Abstract Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (hbFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6M guanidine-HCI and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [3H]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rhbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [3H]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound healing is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.

Advanced Phase I/II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Gemcitabine-resistant Metastatic Pancreatic Cancer

Rexin-G, a nonreplicative pathology-targeted retroviral vector bearing a cytocidal cyclin G1 construct, was tested in a phase I/II study for gemcitabine-resistant pancreatic cancer. The patients received escalating doses of Rexin-G intravenously from 1 × 1011 colony-forming units (cfu) 2–3× a week (dose 0–1) to 2 × 1011 cfu 3× a week (dose 2) for 4 weeks. Treatment was continued if there was less than or equal to grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR), or vector-neutralizing antibodies were noted. In nine evaluable patients, 3/3 patients had stable disease (SD) at dose 0–1. At dose 2, 1/6 patients had a partial response (PR) and 5/6 patients had SD. Median progression-free survival (PFS) was 3 months at dose 0–1, and >7.65 months at dose 2. Median overall survival (OS) was 4.3 months at dose 0–1, and 9.2 months at dose 2. One-year survival was 0% at dose 0–1 compared to 28.6% at dose 2, suggesting a dose–response relationship between OS and Rexin-G dosage. Taken together, these data indicate that (i) Rexin-G is safe and well tolerated, and (ii) Rexin-G may help control tumor growth, and may possibly prolong survival in gemcitabine-resistant pancreatic cancer, thus, earning US Food and Drug Administrations (FDA) fast-track designation as second-line treatment for pancreatic cancer.

Cyclin D1 antisense RNA destabilizes pRb and retards lung cancer cell growth

To investigate the role of cyclin D1 in the regulation of lung cancer cell growth, we created five stably transfected cell lines carrying a cyclin D1 antisense construct. The transfected cells exhibited a marked decrease in the rate of cell growth, in contrast to the original lines (A549 and NCI-H441). The expression of several cell cycle-regulating proteins, including cyclin A, the cyclin-dependent kinases (cdk) 2 and cdk4, in addition to cyclin D1 itself, was markedly decreased. The expression of one cdk inhibitor, p21WAF1/CIP1, increased in the A549-derived cell lines. A specific target of cyclin D1 activity, the growth-suppressing product of the retinoblastoma gene, pRb, exhibited decreased expression and a decreased level of phosphorylation in the transfected cells. Decreased expression of pRb due to a significant increase in its turnover rate suggested that the stability of the protein may depend on phosphorylation by cyclin D1-dependent cdk activity. In addition to the impact on pRb stability, decreased expression of cyclin D1 induced susceptibility to cell death after withdrawal of exogenous growth factors in the antisense transfected cell lines, a response that was not observed in the original cancer cell lines. We conclude that abrogation of cyclin D1 overexpression in lung cancer cells disrupts several key pathways that are required for uncontrolled cell growth and induces those that lead to cell death after growth factor deprivation. Therefore, we speculate that use of antisense cyclin D1 expression in appropriate gene vectors could be a useful method for retarding lung cancer cell growth in accessible tumors such as those of the lung epithelium.

Incorporation of Tumor Vasculature Targeting Motifs into Moloney Murine Leukemia Virus Env Escort Proteins Enhances Retrovirus Binding and Transduction of Human Endothelial Cells

ABSTRACT Adhesion receptors expressed on the surfaces of tumor-activated endothelial cells provide an advantageous locus for targeting gene therapy vectors to angiogenic tissues and/or tumor vasculature. In this study, we engineered a series of Asn-Gly-Arg (NGR)-containing congeners of the presumptive cell binding motif contained within the ninth type III repeat of fibronectin and displayed these tumor vasculature targeting motifs (TVTMs) within the context of Moloney murine leukemia envelope “escort” proteins. Comparative studies of envelope incorporation into viral particles and evaluation of the cell binding properties of the targeted vectors revealed critical structural features, thus identifying a subset of optimal TVTMs. Utilizing a modified ELISA to evaluate viral binding to target cells, we observed a significant down-regulation of TVTM-virion binding to human endothelial cells following sustained (48-h) exposure to VEGF. Normalized for equivalent titers (106 CFU/ml), as assayed on NIH 3T3 cells, vectors displaying TVTM escort proteins significantly enhanced the transduction efficiency from 12.2 to 37.4% in human KSY-1 endothelial cell cultures (P < 0.001) and from 0.4 to 4.1% in human umbilical vein endothelial cell (HUVEC) cultures (P < 0.001). In summary, these studies utilized an engineering approach to identify a subset of TVTMs that are stably incorporated as envelope “escort” proteins into retroviral vectors and that, by functioning to improve the binding efficiency and transduction of both HUVEC and KSY1 endothelial cells, may have therapeutic potential for targeting gene delivery to the tumor-associated vasculature.

Phase I/II and Phase II Studies of Targeted Gene Delivery In Vivo: Intravenous Rexin-G for Chemotherapy-resistant Sarcoma and Osteosarcoma

Rexin-G, a pathotropic nanoparticle bearing a cytocidal cyclin G1 construct was tested in a phase I/II study for chemotherapy-resistant sarcomas and a phase II study for chemotherapy-resistant osteosarcoma. Twenty sarcoma patients and 22 osteosarcoma patients received escalating doses of Rexin-G intravenously from 8 × 1011 to 24 × 1011 colony forming units (cfu)/cycle. Treatment was continued if there was ≤ grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR) or vector-neutralizing antibodies were noted. In the phase I/II study, 3/6 patients had stable disease (SD) at the lowest dose; median progression-free survival (PFS) was 1.2 months, and overall survival (OS), 3.3 months. At higher doses, 10/14 patients had SD; median PFS was 3.7 months and median OS, 7.8 months. In this phase I/II study, a dose–response relationship with Rexin-G dosage was observed for progression-free and OS times (P = 0.02 and 0.005, respectively). In the phase II study, 10/17 evaluable patients had SD, median PFS was ≥3 months and median OS, 6.9 months. These studies suggest that Rexin-G is safe, may help control tumor growth, and may possibly improve survival in chemotherapy-resistant sarcoma and osteosarcoma.

Phenotypic Differentiation of TGF-β1-Responsive Pluripotent Premesenchymal Prehematopoietic Progenitor (P4 Stem) Cells from Murine Bone Marrow

On the horizon of modern molecular medicine is the requisite technology to capture multipotent human stem cells that are capable of self-renewal and to direct these stem cells along defined lineages for therapeutic purposes. In this article, we describe the hematopoietic and mesenchymal differentiation potential of a unique population of transforming growth factor-beta1 (TGF-beta1)-responsive stem cells derived from murine bone marrow. Stringent selection of the stem cells was accomplished under low serum conditions by virtue of an inherent survival response to a TGF-beta1-vWF fusion protein that was bound to collagen matrices. The TGF-beta1-responsive stem cells initially exhibited a non-adherent and uniformly blastoid morphology, underwent expansion into colonies upon serum reconstitution, and were capable of overt cytodifferentiation along fibrogenic, osteogenic, chondrogenic, or adipogenic lineages upon growth factor stimulation. Remarkably, these stem cells also underwent rapid expansion in the presence of either hematopoietic stem cell factor (SCF) or interleukin3 (IL-3), and differentiated into myeloid and lymphoid phenotypes upon exposure to the latter. Taken together, these results support the hypothesis that pluripotent premesenchymal prehematopoietic progenitor cells, designated P4 stem cells, are present postnatally in murine bone marrow and, thus, may be summarily isolated for various cell-based experimental therapies.

Engineering, Expression and Renaturation of Targeted TGF-Beta Fusion Proteins

This study reports the expression, purification, and renaturation of biologically active Transforming Growth Factor-beta 1 (TGF-beta 1) fusion proteins from Escherichia coli (E. coli). A prokaryotic expression vector was engineered to produce tripartite fusion proteins consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen binding domain, and (iii) a cDNA sequence encoding the active fragment of human TGF-beta 1. The expressed fusion proteins TGF-B1-F1 and TGF-B1-F2, located in inclusion bodies, were solubilized with 8 M urea and renatured using a glutathione redox-coupled system and protracted dialysis under several experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion proteins on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factor was demonstrated by its ability to inhibit mink lung (Mv1Lu) cell proliferation and/or to stimulate proliferation of NIH-3T3 mouse fibroblasts, where purified human platelet TGF-beta 1 served as a positive control. Purified TGF-B1-F1 and TGF-B1-F2 (collagen-binding) constructs exhibited anti-proliferative activities comparable to purified platelet TGF-beta 1, but at lower specific activities. Binding of the renatured TGF-B1-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of 3H-collagen to the TGF-B1-F2 protein immobilized on a Ni-NTA column. The TGF-B1-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active TGF-beta 1 fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, these results support the concept that auxiliary domains may be used to modulate and/or target TGF-beta 1 for specific applications.