Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Integrin activation controls metastasis in human breast cancer

Metastasis is the primary cause of death in human breast cancer. Metastasis to bone, lungs, liver, and brain involves dissemination of breast cancer cells via the bloodstream and requires adhesion within the vasculature. Blood cell adhesion within the vasculature depends on integrins, a family of transmembrane adhesion receptors, and is regulated by integrin activation. Here we show that integrin αvβ3 supports breast cancer cell attachment under blood flow conditions in an activation-dependent manner. Integrin αvβ3 was found in two distinct functional states in human breast cancer cells. The activated, but not the nonactivated, state supported tumor cell arrest during blood flow through interaction with platelets. Importantly, activated αvβ3 was expressed by freshly isolated metastatic human breast cancer cells and variants of the MDA-MB 435 human breast cancer cell line, derived from mammary fat pad tumors or distant metastases in severe combined immunodeficient mice. Expression of constitutively activated mutant αvβ3D723R, but not αvβ3WT, in MDA-MB 435 cells strongly promoted metastasis in the mouse model. Thus breast cancer cells can exhibit a platelet-interactive and metastatic phenotype that is controlled by the activation of integrin αvβ3. Consequently, alterations within tumors that lead to the aberrant control of integrin activation are expected to adversely affect the course of human breast cancer.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

Assembly and transport of a premessenger RNP particle

Salivary gland cells in the larvae of the dipteran Chironomus tentans offer unique possibilities to visualize the assembly and nucleocytoplasmic transport of a specific transcription product. Each nucleus harbors four giant polytene chromosomes, whose transcription sites are expanded, or puffed. On chromosome IV, there are two puffs of exceptional size, Balbiani ring (BR) 1 and BR 2. A BR gene is 35–40 kb, contains four short introns, and encodes a 1-MDa salivary polypeptide. The BR transcript is packed with proteins into a ribonucleoprotein (RNP) fibril that is folded into a compact ring-like structure. The completed RNP particle is released into the nucleoplasm and transported to the nuclear pore, where the RNP fibril is gradually unfolded and passes through the pore. On the cytoplasmic side, the exiting extended RNP fibril becomes engaged in protein synthesis and the ensuing polysome is anchored to the endoplasmic reticulum. Several of the BR particle proteins have been characterized, and their fate during the assembly and transport of the BR particle has been elucidated. The proteins studied are all added cotranscriptionally to the pre-mRNA molecule. The various proteins behave differently during RNA transport, and the flow pattern of each protein is related to the particular function of the protein. Because the cotranscriptional assembly of the pre-mRNP particle involves proteins functioning in the nucleus as well as proteins functioning in the cytoplasm, it is concluded that the fate of the mRNA molecule is determined to a considerable extent already at the gene level.

NADH-Monodehydroascorbate Oxidoreductase Is One of the Redox Enzymes in Spinach Leaf Plasma Membranes1

Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.

A tumor-selective somatostatin analog (TT-232) with strong in vitro and in vivo antitumor activity.

We report a series of new in vitro and in vivo data proving the selective antitumor activity of our somatostatin structural derivative, TT-232. In vitro, it inhibited the proliferation of 20 different human tumor cell lines in the range of 50-95% and induced a very strong apoptosis. In vivo TT-232 was effective on transplanted animal tumors (Colon 26, B16 melanoma, and S180 sarcoma) and on human tumor xenografts. Treatment of MDA-MB-231 human breast cancer xenografted in mice with low submaximal doses of TT-232 [0.25 and 0.5 mg/kg of body weight (b.w.)] caused an average 80% decrease in the tumor volume resulting in 30% tumor-free animals surviving for longer than 200 days. Treatment of prostate tumor (PC-3) xenografted animals with 20 mg/kg of b.w. of TT-232 for 3 weeks resulted in 60% decrease in tumor volume and 100% survival even after 60 days, while 80% of nontreated animals perished. We have demonstrated that TT-232 did not bind to the membrane preparation of rat pituitary and cortex and had no antisecretory activity. TT-232 was not toxic at a dose of 120 mg/kg of b.w. in mice. Long-term incubation (24 h) of tumor cells with TT-232 caused significant inhibition of tyrosine kinases in good correlation with the apoptosis-inducing effect. The level of p53 or KU86 did not change following TT-232 treatment, suggesting a p53-independent apoptotic effect. Preincubation of human breast cancer cells (MDA-MB-453) with TT-232 for 2 h decreased the growth factor receptor autophosphorylation. All of these data suggest that TT-232 is a promising and selective antitumor agent.

Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.

Expression of the fructose transporter GLUT5 in human breast cancer.

The primary metabolic characteristic of malignant cells is an increased uptake of glucose and its anaerobic metabolism. We studied the expression and function of the glucose transporters in human breast cancer cell lines and analyzed their expression in normal and neoplastic primary human breast tissue. Hexose uptake assays and immunoblotting experiments revealed that the breast carcinoma cell lines MCF-7 and MDA-468 express the glucose transporters GLUT1 and GLUT2, isoforms expressed in both normal and neoplastic breast tissue. We also found that the breast cancer cell lines transport fructose and express the fructose transporter GLUT5. Immunolocalization studies revealed that GLUT5 is highly expressed in vivo in human breast cancer but is absent in normal human breast tissue. These findings indicate that human breast cancer cells have a specialized capacity to transport fructose, a metabolic substrate believed to be used by few human tissues. Identification of a high-affinity fructose transporter on human breast cancer cells opens opportunities to develop novel strategies for early diagnosis and treatment of breast cancer.