Patricia M. Fernandez

Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions.

The 78 kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER−) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER− breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER− tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy.

Tissue factor and angiogenesis in cancer.

Idiopathic thrombosis often precedes the diagnosis of occult cancer by several years. Whether hypercoagulability predisposes for malignancy or the converse holds true is an unresolved paradigm that stems from the known vicious cycle of clot formation and tumor growth. Central to this paradigm is the interplay between tissue factor (TF), the initiator of coagulation, and angiogenesis, the life support of tumors. Both clotting-dependent and -independent mechanisms of TF-induced angiogenesis have been elucidated that may signal through distinct pathways. This review focuses on the latest studies of TF and angiogenesis and highlights recent applications that have led to the development of promising new TF-targeted cancer therapeutics. Finally a cautionary note is given about unexpected complications arising from antiangiogenic therapy that may potentially involve TF.

Pericytic-like angiotropism of glioma and melanoma cells

We have identified in malignant melanoma an angiotumoral complex in which tumor cells occupy a pericytic location along the endothelium of microvessels without evidence of intravasation. We have suggested that this pericytic-like angiotropism could be a marker of an extravascular migration of tumor cells along the abluminal surface of vessels. The extravascular migratory metastasis proposed for melanoma has close analogies with glioma migration. To compare our hypothesis of extravascular migration by melanoma with the migration of glioma cells, we have used the B16 murine melanoma cell line and the GL26 murine glioma cell line in an in vivo murine brain tumor model and in vitro using endothelial cells that have formed capillary-like structures and have been cocultivated with tumor cells. In the brain tumors, a clear progression of glioma and melanoma cells was observed along the abluminal surface of vessels, where they occupied a pericytic location along the periendothelial laminin. In vitro, time-lapse videomicroscopy recorded the migration of tumor cells toward endothelial tubules. After 24 hours, both the melanoma cells and the glioma cells were localized along the external surfaces of the vascular tubules, occupying a pericytic-like location. These similarities between glioma and melanoma support the hypothesis of an extravascular migration of melanoma cells, particularly along the abluminal surface of vessels.

Targeting the Endothelium in Cancer – The Importance of the Interaction of Hemostatic Mechanisms and the Vascular Wall for Tumor Growth and Angiogenesis

angiogenesis include thrombin, protease-activated receptors (PARs) and fibrin. These pathways are schematically represented in figure 1 [4]. While angiogenesis is the central link between blood coagulation and tumorigenesis, coagulation products (via pathways other than those related to angiogenesis) may also promote tumor growth. Clotting-dependent pathways of tumor angiogenesis likely involve activation of the TF receptor via ligand binding, followed by downstream production of thrombin and ensuing clot formation. Clotting-independent pathways appear to involve phosphorylation of the cytoplasmic domain of the TF receptor and subsequent downstream signaling events that occur independent of thrombin production or clot formation, and possibly even independent of ligand activation [5, 7] (fig. 2). The cytoplasmic tail of TF appears to regulate non-clotting-dependent mechanisms, including cytoskeletal reorganization, vascular remodeling, angiogenesis and cellular metastasis [6–9]. Following extracellular binding of the TF receptor, the actin-binding protein 280 (ABP-280) is recruited to the cytoplasmic tail where it participates in the assembly of actin filaments [8]. The carboxyl terminus of ABP-280 associates with the Patients who present with idiopathic venous thromboembolism (VTE) frequently harbor an occult cancer that does not become clinically evident until months or perhaps years later. Although Professor Armand Trousseau first documented this link between coagulation and malignancy in 1865 [1], the mechanisms underlying the association have only recently started to become more apparent. The key mediator of this link appears to be tissue factor (TF), the ubiquitous 47 kd membrane protein receptor for factor VII (and factor VIIa) in the clotting cascade that is overexpressed in tumor cells, tumor-associated macrophages and tumor-associated endothelial cells. In addition to its role in the mediation of VTE in cancer, expression of TF by aberrant (angiogenic) endothelial cells may also be of critical importance both as a novel marker and as a regulator of tumor angiogenesis. Therefore, TF appears to be a useful target for so-called ‘vascular targeting agents’ [2], many of which will be discussed in this first session of the meeting. TF may regulate tumor growth, diapedesis of tumor cells across endothelial barriers and tumor angiogeneis [3–5]. The function of TF in tumor angiogenesis, which is essential for tumor growth and metastasis, is mediated via both clotting-dependent and -independent pathways [3–7]. Other

Enhancing quantification of mammalian cell transfections with chloramphenicol acetyltransferase reporter plasmids

We modified existing techniques to optimize conditions for obtaining quantitative, highly replicable, and sensitive transfections. The processes described may serve as a model for investigators initiating transfection procedures who wish to obtain definitive and quantitative results quickly and efficiently. In our example, we compared specific gene expressions of plasmids with the chloramphenicol acetyltransferase (CAT) reporter. Techniques included measuring CAT activity in transfected mammalian cells, selecting a procedure for extracting plasmids from bacterial cells, evaluating the timing of the transfection, choosing a transfection reagent and the reagent: plasmid DNA ratio, and determining procedures for the extraction of cells.