Heparanase promotes lymphangiogenesis and tumor invasion in pancreatic neuroendocrine tumors.

Heparan sulfate proteoglycans are an important and abundant component of the extracellular matrix, which undergo substantial remodeling throughout tumorigenesis via the enzymatic activity of heparanase. Heparanase has been shown to be upregulated in many human cancers; however, its specific functions in human pancreatic neuroendocrine tumors (PanNETs) and spontaneous mouse models of cancer have not been evaluated. Here, we investigated the role of heparanase in PanNETs using patient samples and the RIP1-Tag2 (RT2) PanNET-transgenic mouse model. High heparanase expression significantly correlated with more advanced tumor stage, higher tumor grade and the presence of distant metastasis in PanNET patients. We genetically manipulated heparanase levels in the RT2 model using heparanase-transgenic mice, which constitutively overexpress heparanase, and heparanase-knockout mice. Heparanase was found to have a critical role in promoting tumor invasion, through both macrophage and cancer cell sources in the tumor microenvironment. In addition, elevated heparanase levels significantly increased peritumoral lymphangiogenesis in vivo and promoted the trans-differentiation of macrophages into lymphatic endothelial cell-like structures in culture. Conversely, we found that heparanase deletion led to increased angiogenesis and pericyte coverage. Together, these data identify important roles for heparanase in regulating several critical aspects of tumorigenesis, demonstrating that heparanase represents a potential therapeutic target for PanNET patients.

Deficiency of the macrophage growth factor CSF-1 disrupts pancreatic neuroendocrine tumor development.

Tumor-associated macrophages have recently emerged as a key regulatory cell type during cancer progression, and have been found to promote tumor malignancy in the majority of studies performed to date. We show in this study that CD68(+) macrophages positively correlate with tumor grade and liver metastasis in human pancreatic neuroendocrine tumors (PNETs). To investigate the potential mechanisms whereby macrophages can promote PNET progression, we crossed the RIP1-Tag2 (RT2) mouse model of pancreatic islet cancer to colony-stimulating factor-1 (CSF-1)-deficient Csf1(op/op) mice, which have reduced numbers of tissue macrophages. Csf1(op/op) RT2 mice had a substantial reduction in cumulative tumor burden, which interestingly resulted from a significant decrease in angiogenic switching and tumor number, rather than an evident effect on tumor growth. In the tumors that did develop in CSF-1-deficient animals, however, there were no significant differences in tumor cell proliferation, apoptosis, angiogenesis or invasion. CSF-1 deficiency decreased macrophage infiltration by approximately 50% during all stages of RT2 tumor progression. Interestingly, several cytokines were upregulated in CSF-1-deficient RT2 tumors, and neutrophil infiltration was increased. These results show that macrophages are important for promoting PNET development and suggest that additional factors contribute to the recruitment and survival of myeloid cells in RT2 tumors in the absence of CSF-1.

(15)O water positron emission tomography in language localization: a study comparing positron emission tomography visual and computerized region of interest analysis with the Wada test.

We compared (15)O water positron emission tomography (PET) auditory and visual confrontational naming activation with an intracarotid amobarbital (Amytal) injection procedure (IAP) for language lateralization in 12 patients with intractable epilepsy. PET scans were evaluated by three raters experienced in functional imaging as well as by a region of interest (ROI) approach. Compared with IAP, raters' positive predictive value for language lateralization ranged from 88 to 91%. ROI analysis had a positive predictive value of 80%. Six patients had surgery; 1 with right-sided IAP language dominance but left-sided PET activation had dysphasia for 6 months after left temporal lobectomy.

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH.

To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2-dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9.5 mg kg-1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.

Evidence that a major portion of cellular potassium is "bound".

In this report we briefly review recent evidence which shows that a substantial proportion of intracellular K+ is "bound" or perturbed from the physicochemical properties expected in dilute aqueous solutions. In addition, we present evidence from electron probe x-ray microanalysis of thin cryosections of cells which indicates that the binding of K+ to anionic groups either carboxyl groups (HCO2) on proteins or to phosphate groups in creatine phosphate (CrP), in adenosine triphosphate, (ATP), in protein and in nucleic acids, are the main determinants of the maintenance of (as differentiated from the generated of) the well known intra- to extracellular K+ concentration difference. The collective evidence suggests that much of cellular K+ is reduced in its mobility and in its chemical activity due to association with negative charge groups (e.g. carboxyl and phosphates). This fact forces abandonment of the misleading assumption that the majority of intracellular K+ and other inorganic ions are as free as would be expected under ideal solution conditions. This realization should have far reaching consequences toward understanding transmembrane movement of water and solutes in cells.

Effect of dietary cellulose on cell proliferation and progression of 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

The effects of different levels of dietary cellulose on colonic crypt mitotic activity and colon carcinogenesis were studied in 190 male Sprague-Dawley rats. Rats were divided into groups and fed a basal fiber-free diet supplemented with either 0, 5, or 15% pure cellulose (w/w), for periods of 10 weeks (initiation stage) or 32 weeks (promotional stage). Half of the rats in each group were given weekly s.c. injections of 9.5 mg 1,2-dimethylhydrazine (the base) (DMH) for 8 weeks. Some of the rats were killed at 10 weeks while most were killed 22 weeks later. In some groups the dietary cellulose level was changed to a different level at 10 weeks. Food intake and body weight data showed that the rats within each experiment were isocalorically fed. There was a direct correlation between crypt height and the percentage of cellulose in the diet. Addition of 5 or 15% dietary cellulose during the initiation stage of carcinogenesis resulted in a significant increase in crypt height. Increasing dietary cellulose after the initiation stage (0 to 5% and 5 to 15%) or maintaining a high dietary cellulose level throughout both the initiation and promotional stages (15%) resulted in a significant increase in crypt height. A DMH-induced increase in mitotic activity that was observed during the initiation stage was no longer evident after the 22-week promotional stage. The significant DMH-induced increases in proliferative zone height and crypt height that were initially observed during the initiation stage were also observed after the 22-week promotional stage. These data indicate that the initial DMH-induced increases observed in proliferative zone height and crypt height are irreversible. Addition of 5 or 15% cellulose was found to suppress DMH-enhanced mitotic activity in the crypts of the descending colon during the initiation stage of carcinogenesis. This finding was correlated with a significantly lower incidence of adenocarcinomas in rats maintained on 5 or 15% cellulose throughout both the initiation and promotional stages.

Suppression of a a carcinogen (1,2-dimethylhydrazine dihydrochloride)-induced increase in mitotic activity in the colonic crypts of rats by addition of dietary cellulose.

Serial injections of the colon carcinogen, 1,2-dimethylhydrazine (DMH), have been reported to increase the proliferative activity in the colonic crypts preceding development of tumors. Can addition of purified cellulose to a fiber-free AIN-76 rat diet be used to suppress this increase in proliferative activity? To answer this question rats were divided into two groups, and one group was given eight weekly injections of the DMH base at 9.5 mg/kg of body weight. Throughout this period and for 2 additional wk the rats were isocalorically fed a defined nutritionally complete diet both with and without different dietary levels of cellulose (0, 5, and 15%). The rats were given injections of colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. Analysis of variance was performed on various morphometric parameters obtained from histological sections of midaxial crypts from the descending colon. Our results confirm that DMH induced a significant increase in the mitotic activity as measured by the number of metaphase figures per crypt. The presence of dietary cellulose did cause a significant suppression of the DMH-induced increase in the crypt mitotic activity.

The influence of macromolecular polymerization of spin-lattice relaxation of aqueous solutions.

The docking or polymerization of globular proteins is demonstrated to cause changes in proton NMR spin-lattice (T1) relaxation times. Studies on solutions of lysozyme, bovine serum albumin, actin, and tubulin are used to demonstrate that two mechanisms account for the observed changes in T1. Polymerization displaces the hydration water sheath surrounding globular proteins in solution that causes an increase in T1. Polymerization also slows the average tumbling rate of the proteins, which typically causes a contrary decrease in T1. The crystallization reaction of lysozyme in sodium chloride solution further demonstrates that the "effective" molecular weight can either decrease or increase T1 depending on how much the protein is slowed. The displacement of hydration water increases T1 because it speeds up the mean motional state of water in the solution. Macromolecular docking typically decreases T1 because it slows the mean motional state of the solute molecules. Cross-relaxation between the proteins and bound water provides the mechanism that allows macromolecular motion to influence the relaxation rate of the solvent. Fast chemical exchange between bound, structured, and bulk water accounts for monoexponential spin-lattice relaxation. Thus the spin-lattice relaxation rate of water in protein solutions is a complex reflection of the motional properties of all the molecules present containing proton magnetic dipoles. It is expected, as a result, that the characteristic relaxation times of tissues will reflect the influence of polymerization changes related to cellular activities.

Retardation of embryogenesis by extremely low frequency 60 Hz electromagnetic fields.

Fertilized Medaka fish eggs were used to determine if electromagnetic fields, designed to simulate those beneath a high voltage power line, have biological effects on vertebrate embryo development. The newly fertilized eggs were exposed to a 60 Hz electrical field of 300 mA/m2 current density, a 60 Hz magnetic field of 1.0 gauss RMS, or to the combined electric plus magnetic fields for 48 hours. No gross abnormalities were observed in any of the embryos as they developed, but significant development delays were seen in those embryos exposed to either the magnetic or to the combined electromagnetic fields; delays were not seen in the embryos exposed to the electrical field. Thus, a 60 Hz magnetic field like that encountered in a man made powerline environment was shown to retard development of fish embryos.

Changes in water proton relaxation times and in nuclear to cytoplasmic element gradients during meiotic maturation of Xenopus oocytes.

Fully grown oocytes 1.2 mm in diameter were removed from Xenopus laevis ovaries and were exposed to progesterone (2.5 micrograms/ml in Ringer's solution) to induce completion of the first maturation division or germinal vesicle breakdown (GVBD). This process required 5.5 +/- 0.5 hr. Neither oocyte volume nor water content was observed to change throughout maturation. At selected times, the oocytes were quick frozen in liquid propane and cryosectioned. The sections were freeze-dried, and analyzed for K, Na, Cl, P, S, and Mg in millimolar per kilogram dry weight content in the nucleus and the yolk-free cytoplasm using electron probe X-ray microanalysis. Unstimulated oocytes showed significant nuclear to yolk-free cytoplasmic content gradients (N/C ratio) for the following elements: K (1.84), P (0.65), and S (1.56), but significant N/C content gradients were not found for Na and Mg. By 10 min after progesterone stimulation, a significant change in the N/C ratio of the following elements had occurred due to a rapid increase in nuclear content: K (2.29), Cl (2.11). A significant N/C ratio for Mg (1.35) had developed by 10 min after progesterone stimulation and a significant N/C ratio for Na (2.07) had developed by 45 min. In addition the following elements showed significant content increases in both the nucleus and the yolk-free cytoplasm from the time prior to progesterone stimulation to the time just prior to GVBD at 240 min: K, Na, Cl, P, S, and Mg. Nuclear magnetic resonance measurements of the spin-lattice relaxation time (T1) of water proton in oocytes showed a significant increase in the T1 time after progesterone exposure. The changes in N/C ratios of specific elements and in the physical parameter of water proton relaxation time suggest that progesterone is responsible for inducing changes in the physicochemical interactions between various macromolecules, specific elements, and water.

Effect of cancer cachexia and amiloride treatment on the intracellular sodium content in tissue cells.

This study was designed to investigate the effects of a growing H6 hepatoma on the intracellular element content in three distinctly different tissue cell populations of the mouse host (hepatocytes, fibroblasts, and crystal enterocytes). X-ray microanalysis measurements of the intranuclear concentrations of several elements (sodium, magnesium, phosphorus, sulfur, chlorine, and potassium) were made. Briefly, the tumor presence significantly increased intranuclear sodium concentration but not the concentration of magnesium, phosphorus, sulfur, chlorine, or potassium in three tissue cell types of mice that were anorectic and cachectic. A second aim of the study was to see if injections of the diuretic amiloride, a drug reported to block passive influx of sodium into mammalian cells, would counteract the effect of the tumor presence and lower the intranuclear concentration of sodium towards that of a non-tumor-bearing host. Amiloride did significantly lower the intranuclear level of sodium in the host tissues to that of non-tumor-bearing mice. The amiloride-caused decrease on intracellular sodium was correlated to a decreased cell proliferation activity in the tumor cells and duodenal enterocytes. A possible relationship between the intracellular concentration of sodium in tissue cells and cancer cachexia is discussed.