Brian R. Unsworth

Breast Cancer: Induction of Differentiation by Embryonic Tissue

A mouse mammary tumor, adenocarcinoma BW 10232, was maintained in vitro for 14 days, separated from embryonic mammary mesenchyme by a Millipore filter. Tubules developed in the tumor; deoxyibonucleic acid synthisis declined; and a presumptive acid mucopolysaccharide matrix, not evident in the controls, appeared.

Simulated microgravity conditions enhance differentiation of cultured PC12 cells towards the neuroendocrine phenotype.

SummaryWe are studying microenvironmental cues which contribute to neuroendocrine organ assembly and tissue-specific differentiation. As our in vitro model, we cultured rat adrenal medullary PC12 pheochromocytoma cells in a novel cell culture system, the NASA rotating wall vessel (RWV) bioreactors. This “simulated microgravity” environment in RWV bioreactors, characterized by randomizing gravitational vectors and minimizing shear stress, has been shown to favor macroscopic tissue assembly and to induce tissue-specific differentiation. We hypothesized that the unique culture conditions in the RWV bioreactors might enhance the in vitro formation of neuroendocrine organoids. To test our hypothesis, we evaluated the expression of several markers of neuroendocrine differentiation in cultures of PC12 cells maintained for up to 20 d in the slow turning lateral vessel (STLV) type RWV. PC12 cell differentiation was assessed by morphological, immunological, biochemical and molecular techniques. PC12 cells, cultured under “simulated microgravity” conditions, formed macroscopic, tissue-like organoids several millimeters in diameter. Concomitantly, the expression of phenylethanolamine-N-methyl transferase (PNMT), but not of other catecholamine synthesizing enzymes, was enhanced. Increased PNMT expression, as verified on both the gene and protein level, was accompanied by an increase in the specific activity of the enzyme. Furthermore, after 20 d in culture in the STLV, we observed altered patterns of protein tyrosine phosphorylation and prolonged activation of c-fos, a member of the AP-1 nuclear transcription factor complex. We conclude that culture conditions in the RWV appear to selectively activate signal transduction pathways leading to enhanced neuroendocrine differentiation of PC12 cells.

Reactive oxygen species, apoptosis and alte1red NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: AnIn Vitro cellular model for diabetic neuropathy

Diabetic neuropathies, affecting the autonomic, sensory, and motor peripheral nervous system, are among the most frequent complications of diabetes. The symptoms of diabetic polyneuropathies are multi-faceted; the etiology and the underlying mechanisms are as yet unclear. Clinical studies established a significant correlation between the control of the patients’ blood glucose level and the severity of the damage to the peripheral nervous system. Recentin vitro studies suggest that elevated glucose levels induced dysfunction and apoptosis in cultured cells of neuronal origin, possibly through the formation of reactive oxygen species (ROS). Based on these results, we hypothesized that elevated glucose levels impair neuronal survival and function via ROS dependent intracellular signaling pathways. In order to test this hypothesis, we cultured neural crest-derived PC12 pheochromocytoma cells under euglycemic (5 mM) and hyperglycemic (25 mM) conditions. Continuous exposure of undifferentiated PC12 cells for up to 72 h to elevated glucose induced the enhanced generation of ROS, as assessed from the increase in the cell-associated fluorescence of the ROS-sensitive fluorogenic indicator, 2,7-dichlorodihydrofluorescein diacetate. In cells cultured in high glucose, both basal and secreta-gogue-stimulated catecholamine release were enhanced. Furthermore, high glucose, reduced (by ca. 30%) the rate of cell proliferation and enhanced the occurrence of apoptosis, as assessed by DNA fragmentation, TUNEL assay and the activation of an apoptosis-specific protease, caspase CCP32. Elevated glucose levels significantly attenuated nerve growth factor (NGF)-induced neurite extension, as quanti-tated by computer-aided image analysis. Culturing PC12 cells in high glucose resulted in alterations in basal and NGF-stimulated mitogen-activated protein kinase (MAPK) signaling pathways, specifically in a switch from the neuronal survival/differentiation-associated MAPKERK to that of apoptosis/stress-associated MAPKp38and JNK. Based on our results we present a model in which the prolonged, excess formation of ROS represents a common mechanisms for hyperglycemia-induced damage to neuronal cells. We propose that this simplein vitro system might serve as an appropriate model for evaluating some of the effects of elevated glucose on cultured cells of neuronal origin.

GTSF-2: A NEW, VERSATILE CELL CULTURE MEDIUM FOR DIVERSE NORMAL AND TRANSFORMED MAMMALIAN CELLS

SummaryThe aim of this study was to test the versatility of a new basal cell culture medium, GTSF-2. In addition to traditional growth-factors, GTSF-2 contains a blend of three sugars (glucose, galactose, and fructose) at their physiological levels. For these studies, we isolated normal endothelial cells from human, bovine, and rat large blood vessels and microvessels. In addition, GTSF-2 was also tested as a replacement for high-glucose-containing medium for PC12 pheochromocytoma cells and for other, transformed cell lines. The cell growth characteristics were assessed with a novel cell viability and proliferation assay, which is based on the bioreduction of the fluorescent dye, Alamar Blue. After appropriate calibration, the Alamar Blue assay was found to be equivalent to established cell proliferation assays. Alamar Blue offers the advantage that cell proliferation can be measured in the same wells over an extended period of time. For some of the cell types (e.g., endothelial cells isolated from the bovine aorta, the rat adrenal medulla, or the transformed cells), proliferation in unmodified GTSF-2 was equivalent to that in the original culture media. For others cell types (e.g., human umbilical vein endothelial cells and PC12 cells), GTSF-2 proved to be a superior growth medium, when supplemented with simple additives, such as endothelial cell growth supplement (bFGF) or horse serum. Our results suggest that GTSF-2 is a versatile basal medium that will be useful for studying organ-specific differentiation in heterotypic coculture studies.

Time Course and Quantification of Extracellular Matrix Maturation in the Chick Chorioallantoic Membrane and in Cultured Endothelial Cells

Neovascularization, i.e. the formation of new blood vessels either from pre-existing ones or from mesenchymal cells, is associated with the deposition of a subendothelial basement membrane. The sequential expression of extracellular matrix (ECM) proteins, such as fibronectin, laminin, collagen-IV and collagen-I, was quantified both in vivo, in the chick chorioallantoic membrane (CAM) using Western blotting techniques, and in vitro, in cultured rat adrenal medullary endothelial (RAME) cells, using indirect immunofluorescence and enzyme-linked immunoadsorption techniques; the combination of these techniques allowed discrimination between extracellularly and intracellularly located proteins. In the CAM, fibronectin expression increased transiently with the peak at day 7 of development, after which it decreased gradually. By contrast, deposition of laminin and collagen-I increased steadily during development. Quantification at later stages of CAM development showed the predominance of collagen-I, whereas lami...

Tissue-specific alternative mRNA splicing of phenylethanolamine N-methyltransferase (PNMT) during development by intron retention.

The expression of phenylethanolamine N‐methyl transferase (EC 2. 1.1.2.8,PNMT), the final enzyme in the cascade of catecholamine synthesis, is differentially regulated inadrenergic neurons in the brain and in adrenal chromaffin cells. Using reversetranscription‐polymerase chain reaction‐based techniques, we detected in the prenatal developingrat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicingmechanism known as intron retention. The spliced, intronless message was downregulatedpostnatally, while the intron‐retained mRNA species continued to be constitutively expressedthrough adulthood. By contrast in the adrenals, at all stages of development examined, only theintronless message was expressed. In line with previous reports on the failure of glucocorticoidsto induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants wasnot affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiatedsympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNAvariants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells todexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a3‐fold increase in PNMT enzymatic activity. In contrast, treatment of PC12 cells with nervegrowth factor (NGF) enhanced the expression of both the intron‐retained and the intronlessmRNA species without changes in the basal enzyme activity. This latter result suggests that thetranslation of the intronless mRNA species may be regulated by the intron‐retained mRNAspecies, which by itself may yield a truncated, yet enzymatically functional translational product.Our data suggest that the tissue‐specific regulation of PNMT expression is based on a rarealternative splicing mechanism termed intron retention, and that in the adrenal, but not in thebrain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquelysuited for studying the hormonal control of tissue‐specific splicing in the nervous system.

Staurosporine Induces Neurite Outgrowth in Neuronal Hybrids (PC12EN) Lacking NGF Receptors

A novel neuronal model (PC12EN cells), obtained by somatic hybridization of rat adrenal medullary pheochromocytoma (PC12) and bovine adrenal medullary endothelial (BAME) cells, was developed. PC12EN cells maintained numerous neuronal characteristics: they expressed neuronal glycolipid conjugates, synthesized and secreted catecholamines, and responded to differentiative agents with neurite outgrowth. PC12EN lacked receptors for EGF and both the p75 and trk NGF receptors, while FGF receptor expression was maintained. Staurosporine (5–50 nM), but not other members of the K252a family of protein kinase inhibitors, rapidly induced neurite outgrowth in PC12EN, as also found in the parental PC12 cells, but not in BAME cells. Similarly, both acidic and basic FGF (1–100 ng/ml) were neurotropic in PC12EN. In contrast to the mechanism by which FGF promoted neurite outgrowth in PC12EN, the neurotropic effect of staurosporine did not involve activation of established signalling pathways, such as tyrosine phosphorylation of erk (ras pathway) or SNT (a specific target of neuronal differentiation). In addition, staurosporine induced the tyrosine phosphorylation of the focal adhesion kinase p125???. However, since the latter effect was also observed with other protein kinase inhibitors of the K252a family, which induced PC12EN cells flattening but no neurite extension, we propose that FAK tyrosine phosphorylation may be related to ubiquitous changes in cell shape. We anticipate that PC12EN neuronal hybrids will become useful models in neuroscience research for evaluating unique cellular signalling mechanisms of novel neurotropic compounds.

Neurotransmitter enzymes in telencephalon, brain stem and cerebellum during the entire life span of the mouse

Neurochemical analysis of neuronal function was undertaken by measuring the activities of cholinacetyltransferase (CAT), acetylcholinesterase (AChE), and glutamic acid decarboxylase (GAD), in the telencephalon, brain stem and cerebellum of the mouse. Cholinergic activity was first expressed in the 10-day embryonic brain stem, which showed a relatively high CAT activity at birth. Postnatal brain stem development was characterized by a rapid and parallel increase in CAT and AChE. Although AChE peaked at 1 month, CAT activity was no achieved until 1 year. Acetylcholine synthesis was initiated in the 12-day embryonic telencephalon and a steady age-related increase in CAT was maintained until birth. A lag in both CAT and AChE activities was recorded during the first week of postnatal telencephalon development. Cerebellar CAT was low at birth, and increased irregularly to reach a maximum by 1 month. In contrast, postnatal cerebellar AChE activity increased steadily over the same time period. The GABA-ergic neuronal system matured rapidly in each brain region, and was unaffected by aging. Although the brain stem precociously expressed cholinergic activity, it wa the region most susceptible to deterioration during aging. Telencephalon CAT activity was unaffected by aging and in the cerebellum, a significantly reduced level of CAT was only found in truly senescent animals. The decreased cholinergic function during senescence was not due to either increased proteolysis or to alteration in the molecular form of the cholinergic enzymes.

Alteration of the activity and molecular form of thymidine kinase during development and aging in the mouse cerebellum

Protein, DNA and thymidine kinase levels were assayed during development and aging in the mouse cerebellum. A roughly parallel increase in protein and DNA content occurred from birth, reaching a plateau at 18 days; these adult levels increased by 30% in the 23 month-old cerebellum. Thymidine kinase activity reached a maximum at 6 postnatal days, then decreased steadily to reach, at 18 days, the low level that was maintained in the adult. The thymidine kinase synthesized in the aged cerebellum differed from that in the neonate by having (i) an increased specific activity, (ii) a faster migrating species upon electrophoresis, (iii) an inhibition by dCTP, and (iv) a lower affinity for the substrate thymidine (higher Km). Mathematical calculations indicated the appearance of a larger number of smaller sized cells in the aged cerebellum, when compared with the young adult. Histological analysis established that the newly synthesized cells were localized in the molecular layer of the old cerebellum. It appears that senescence in the mouse cerebellum may be associated with an increased synthesis of glial cells.

Teratogenic evaluation of terpenoid derivatives.

Abstract Eight terpenoid derivatives with juvenile hormone activity were tested for teratogenicity. Three of the derivatives were teratogenic when administered to mice as a single intraperitoneal injection of 1 mg/gm on either day 9 or day 10 of pregnancy. However, intubation at 5 mg/gm of the two most teratogenic derivatives resulted in one of the derivatives marginally increasing the incidence of abnormal embryos above peanut oil-intubated controls; the other intubated derivative was without adverse affect. Of the remaining five compounds, two with high juvenile hormone activity were without teratogenic effect when injected at 1 mg/gm. Since teratogenic effects were observed only at a dose above likely exposure levels and by a route of administration not possible in the environment, support is provided for the potential use of terpenoid derivatives in the control of insect pests.