Deborah L. Cundiff

Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.

Alternatively spliced human tissue factor promotes tumor growth and angiogenesis in a pancreatic cancer tumor model

INTRODUCTION Tissue Factor (TF) expression is observed in many types of cancer, associated with more aggressive disease, and thrombosis. Alternatively-spliced human tissue factor (asHTF) has recently been identified in which exon 5 is deleted. asHTF is soluble due to the substitution of the transmembrane and cytoplasmic domains of exon 6 with a unique COOH-terminal domain. MATERIALS AND METHODS We examine the expression and function of asHTF and full-length Tissue Factor ((FL)TF) in six human pancreatic cancer cells. Further, we transfected asHTF, (FL)TF, and control expression vectors into a non-expressing, human pancreatic cancer line (MiaPaCa-2). We studied the procoagulant activity of asHTF and (FL)TF and the effect on tumor growth in mice. RESULTS asHTF is expressed in 5 of 6 human pancreatic cancer cell lines, but not in normal human fibroblasts, nor the MiaPaCa-2 line. (FL)TF conferred procoagulant activity, but asHTF did not. Transfected cells were injected subcutaneously in athymic mice. Interestingly, compared with control transfection, (FL)TF expression was associated with reduced tumor growth (mean 7 mg vs 85 mg), while asHTF-expression was associated with enhanced tumor growth (mean 389 mg vs. 85 mg). asHTF expression resulted in increased mitotic index and microvascular density. CONCLUSIONS These data suggests that asHTF expression promotes tumor growth, and is associated with increased tumor cell proliferation and angiogenesis in vivo. Our results raise a new perspective on the understanding of the relationship between TF expression and cancer growth, by showing a dissociation of the procoagulant activity of (FL)TF and the cancer-promoting activity of asHTF.

Cell Surface-Dependent Generation of Angiostatin4.5

Angiostatin4.5 (AS4.5) is a naturally occurring human angiostatin isoform, consisting of plasminogen kringles 1–4 plus 85% of kringle 5 (amino acids Lys78 to Arg529). Prior studies indicate that plasminogen is converted to AS4.5 in a two-step reaction. First, plasminogen is activated to plasmin. Then plasmin undergoes autoproteolysis within the inner loop of kringle 5, which can be induced by a free sulfhydryl donor or an alkaline pH. We now demonstrate that plasminogen can be converted to AS4.5 in a cell membrane-dependent reaction. Actin was shown previously to be a surface receptor for plasmin(ogen). We now show that β-actin is present on the extracellular membranes of cancer cells (PC-3, HT1080, and MDA-MB231), and β-actin can mediate plasmin binding to the cell surface and autoproteolysis to AS4.5. In the presence of β-actin, no small molecule-free sulfhydryl donor is needed for generation of AS4.5. Antibodies to actin reduced membrane-dependent generation of AS4.5 by 70%. In a cell-free system, addition of actin to in vitro-generated plasmin resulted in stoichiometric conversion to AS4.5. Annexin II and α-enolase have been reported to be plasminogen receptors, but we did not demonstrate a role for these proteins in conversion of plasminogen to AS4.5. Our data indicate that membrane-associated β-actin, documented previously as a plasminogen receptor, is a key cell membrane receptor capable of mediating conversion of plasmin to AS4.5. This conversion may serve an important role in regulating tumor angiogenesis, invasion, and metastasis, and surface β-actin may also serve as a prognostic marker to predict tumor behavior.

In vivo generation of angiostatin isoforms by administration of a plasminogen activator and a free sulfhydryl donor: a phase I study of an angiostatic cocktail of tissue plasminogen activator and mesna.

Purpose: Angiostatin4.5 (AS4.5), the endogenous human angiostatin, is derived from plasminogen in a two-step process. A plasminogen activator converts plasminogen to plasmin, then plasmin undergoes autoproteolysis to AS4.5. A free sulfhydryl donor can mediate plasmin autoproteolysis. To translate this process to human cancer therapy, we conducted a phase I trial of administration of a tissue plasminogen activator (tPA) with a free sulfhydryl donor (mesna). Patients and Methods: Fifteen patients with advanced solid tumors were treated. The dose of tPA was escalated (cohorts; 1, 2, 3, 5, and 7.5 mg/h for 6 hours). Mesna was administered as a 240 mg/m2 bolus followed by an infusion of 50 mg/h, concurrent with tPA. Both tPA and mesna were administered 3 consecutive days every 14 days. Results: No dose-limiting toxicity was observed. Two AS4.5 isoforms were generated, Lys-AS4.5 and Glu-AS4.5. Mean baseline Lys-AS4.5 level was 20.4 nmol/L (SE, 2.9). In the 5 mg/h tPA cohort, Lys-AS4.5 levels increased by an average of 143% or 24 nmol/L (SE, 4.9) above baseline. Glu-AS4.5 (Mr ∼ 62,000) was also generated (additional 77 amino acids at amino terminus compared with Lys-AS4.5). Glu-AS4.5 level at baseline was undetectable in four of five patients in the 5 mg/h tPA cohort, but at end of infusion, was ∼67 nmol/L (SE, 20). Two patients in the 5 mg/h tPA cohort experienced decreases in tumor markers with treatment, although no clinical objective responses were observed. Conclusion: This study shows that in vivo generation of AS4.5 is safe in humans and may provide a practical approach to achieve antiangiogenic therapy.

Differential binding of plasminogen, plasmin, and angiostatin4.5 to cell surface β-actin : Implications for cancer-mediated angiogenesis

Angiostatin4.5 (AS4.5) is the product of plasmin autoproteolysis and consists of kringles 1 to 4 and approximately 85% of kringle 5. In culture, cancer cell surface globular beta-actin mediates plasmin autoproteolysis to AS4.5. We now show that plasminogen binds to prostate cancer cells and that the binding colocalizes with surface beta-actin, but AS4.5 does not bind to the cell surface. Plasminogen and plasmin bind to immobilized beta-actin similarly, with a Kd of approximately 140 nmol/L. The binding is inhibited by epsilon-aminocaproic acid (epsilonACA), indicating the requirement for a lysine-kringle domain interaction. Using a series of peptides derived from beta-actin in competitive binding studies, we show that the domain necessary for plasminogen binding is within amino acids 55 to 69 (GDEAQSKRGILTLKY). Substitution of Lys61 or Lys68 with arginine results in the loss of the ability of the peptide to block plasminogen binding, indicating that Lys61 and Lys68 are essential for plasminogen binding. Other actin peptides, including peptides with lysine, did not inhibit the plasminogen-actin interaction. AS4.5 did not bind actin at concentrations up to 40 micromol/L. Plasminogen, plasmin, and AS4.5 all contain kringles 1 to 4; however, kringle 5 is truncated in AS4.5. Isolated kringle 5 binds to actin, suggesting intact kringle 5 is necessary for plasminogen and plasmin to bind to cell surface beta-actin, and the truncated kringle 5 in AS4.5 results in its release from beta-actin. These data may explain the mechanism by which AS4.5 is formed locally on cancer cell surfaces and yet acts on distant sites.

139 The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.