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.

Embryonic inductive tissues that cause histologic differentiation of murine mammary carcinoma in vitro.

A murine mammary tumor was cultured in vitro for 14 days, either in direct combination with various embryonic murine inductive tissues or separated by a Millipore filter from these tissues. From 456 test cultures and 269 control cultures of tumor alone, morphologic, histochemical, and autoradiographic evidence for cytodifferentiation was obtained in the tumor after exposure to inductive tissues directly or through the filter. There appeared to be a gradient in potency of the inductive tissues; embryonic mammary mesenchyme was the most active of the tissues tested. Tumor growth was not different from that of controls, however, when the cultured, inductive tissue-exposed neoplasm was returned to the murine host.

Aging in rodent brain: alteration in astrocyte population.

A method for the preparation of bulk isolated glial cells was modified to provide astrocytes suitable for biochemical analysis. Using this method, the astrocyte-enriched fraction from senescent mouse or rat brain stem could not be recovered from the sucrose interface at which 6 month brain stem astroglia accumulated. An alteration in the buoyant density of the senescent glial cells was demonstrated by using continuous diatrizoate gradients. The involvement of the astrocyte cell population in this age-related shift in buoyant density was confirmed using antiserum specific for glial fibrillary acidic protein.

Alteration of the activity and molecular from 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.

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.

Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells.

We previously reported that short term exposure of cultured rat adrenal medullary endothelial cells (RAMEC) to thrombin enhances the subendothelial deposition of extracellular matrix (ECM) proteins fibronectin, laminin, and collagen types I (C-I) and IV (C-IV) (Papadimitriou et at., 1997). In this work, we extended our previous studies on factors that affect ECM protein deposition to include agents that activate or inhibit some of the most common intracellular signals such as cAMP, protein kinase C (PKC) and calcium. Furthemore, we investigated the possible link between the observed alterations in ECM protein deposition and the secretion of matrix metalloproteinase-2 (MMP-2). Forskolin (adenylyl cyclase activator) caused a dose-dependent increase in the deposition of all four ECM proteins studied. Isoproterenol (beta-adrenergic receptor agonist) and the membrane-permeant cAMP analogue dibutyryl-cAMP, significantly increased the deposited amounts of ECM proteins at low concentrations, and this increase was reversed at higher concentrations of both agents. All these agents had the opposite effect on MMP-2 secretion, increasing it at doses where they decreased ECM protein deposition and vice-versa. However, elevation of cAMP by the phosphodiesterase inhibitor IBMX had no effect neither on the deposited amounts of any of the ECM proteins studied nor on MMP-2 secretion. Activation of PKC by phorbol ester (PMA) resulted in a decrease in ECM protein deposition and an increase in MMP-2 secretion. Finally, chelation of intercellular calcium with BAPTA-AM resulted in an increased ECM deposition and a decrease in MMP-2 secretion, Our results show a complex pattern of regulation of ECM protein deposition by cAMP-mobilizing agents, and also indicate an inverse correlation between ECM protein deposition and secretion of MMP-2. The concerted regulation of both these processes is essential in the formation of new blood vessels and for the integrity of the vascular wall.

Regulation of extracellular matrix remodeling and MMP-2 activation in cultured rat adrenal medullary endothelial cells.

We previously reported that short-term exposure of cultured rat adrenal medullary endothelial cells (RAMEC) to thrombin enhances the subendothelial deposition of extracellular matrix (ECM) proteins fibronectin, laminin, and collagen types I (C-I) and IV (C-IV) (Papadimitriou et al. 1997). In this work, we extended our previous studies on factors that effect ECM protein deposition to include agents that activate or inhibit some of the most common intracellular signals such as cAMP, protein kinase C (PKC), and calcium. Furthermore, we investigated the possible link between the observed alterations in ECM protein deposition and the secretion of matrix metalloproteinase-2 (MMP-2). Forskolin (adenylyl cyclase activator) caused a dose-dependent increase in the deposition of all four ECM proteins studied. Isoproterenol beta-adrenergic receptor agonist) and the membrane permeant cAMP analogue dibutyryl-cAMP significantly increased the deposited amounts of ECM proteins at low concentrations, and this increase was reversed at higher concentrations of both agents. All these agents had the opposite effect on MMP-2 secretion, increasing it at doses where they decreased ECM protein deposition and vice versa. However, elevation of cAMP by the phosphodiesterase inhibitor IBMX had no effect either on the deposited amounts of any of the ECM proteins studied or on MMP-2 secretion. Activation of PKC by phorbol ester (PMA) resulted in a decrease in ECM protein deposition and an increase in MMP-2 secretion. Finally, chelation of intercellular calcium with BAPTA-AM resulted in an increased ECM deposition and a decrease in MMP-2 secretion. Our results show a complex pattern of regulation of ECM protein deposition by cAMP-mobilizing agents and also indicate an inverse correlation between ECM protein deposition and secretion of MMP-2. The concerted regulation of both of these processes is essential in the formation of new blood vessels, and for the integrity of the vascular wall.

Models of disuse: a comparison of hindlimb suspension and immobilization.

The effects of 1 and 2 wk of hindlimb suspension (HS) on rat skeletal muscle function were determined and the results compared with those obtained previously with hindlimb immobilization (HI). Both models of disuse (HS and HI) primarily affected slow-twitch muscle. Each decreased the isometric twitch duration in the slow-twitch soleus; however, the HS-mediated effect was entirely a result of a shortened contraction time (CT), whereas HI reduced one-half relaxation time (1/2 RT) as well as CT. Soleus muscle mass and peak tetanic tension (Po) declined with disuse. The HS effect on muscle mass and Po was variable, however, for all experiments HS produced atrophy equal to or greater than HI. A major difference existed in the effects of HS and HI on the maximal speed of soleus muscle shortening (Vmax). One and 2 wk of HS produced increases in Vmax to 4.45 +/- 0.34 and 6.83 +/- 0.74 fiber lengths/s, respectively, compared with control velocities of 3.05 +/- 0.08. By contrast over a similar time period, HI had no significant effect on soleus Vmax. The increase in Vmax at 14 days of HS was associated with, and perhaps caused by, the increased expression of a second faster migrating isozyme of myosin. The new native isozyme comigrated with fast myosin, but its light chain subunits contained only LC1s and LC2s. The mechanism responsible for the increase is unknown. One plausible explanation is that the apparent HS-mediated modification in muscle fiber type is dependent on the elimination of loadbearing or isometric contractions, a condition that does not exist during HI.

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 in adrenergic neurons in the brain and in adrenal chromaffin cells. Using reverse transcription-polymerase chain reaction-based techniques, we detected in the prenatal developing rat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicing mechanism known as intron retention. The spliced, intronless message was downregulated postnatally, while the intron-retained mRNA species continued to be constitutively expressed through adulthood. By contrast in the adrenals, at all stages of development examined, only the intronless message was expressed. In line with previous reports on the failure of glucocorticoids to induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants was not affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiated sympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNA variants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells to dexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a 3-fold increase in PNMT enzymatic activity. In contrast, treatment of PC 12 cells with nerve growth factor (NGF) enhanced the expression of both the intron-retained and the intronless mRNA species without changes in the basal enzyme activity. This latter result suggests that the translation of the intronless mRNA species may be regulated by the intron-retained mRNA species, 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 rare alternative splicing mechanism termed intron retention, and that in the adrenal, but not in the brain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquely suited for studying the hormonal control of tissue-specific splicing in the nervous system.

Radioactive puromycin formation as an assay for the proportion of active ribosomes in mammalian cells: correlation with polysome profiles under different physiological conditions.

We have established optimal conditions for the in vitro formation of peptidyl-[3H] puromycin by mammalian ribosomes. The growth conditions of cultured Ehrlich ascites tumor cells were manipulated to produce changes in the polysome profiles. The correlation between polysome content and peptidyl-[3H] puromycin formation was linear and excellent when different cell densities were compared. The percentage of ribosomes actively engaged in protein synthesis, calculated from the number of 3H-peptide bonds formed, was similar in rapidly growing Ehrlich cells (47%) and in young rat gastrocnemius muscle (44%). Starvation resulted in a 50% reduction in the number of puromycin-reactive ribosomes in rat gastrocnemius.

Reactive oxygen species, apoptosis and altered NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: an in 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. Recent in 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 secretagogue-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 quantitated 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 MAPK ERK to that of apoptosis/stress-associated MAPK p38 and JNK. Based on our results we present a model in which the prolonged, excess formation of ROS represents a common mechanism for hyperglycemia-induced damage to neuronal cells. We propose that this simple in vitro system might serve as an appropriate model for evaluating some of the effects of elevated glucose on cultured cells of neuronal origin.

Tissue factor expression is differentially modulated by cyclic mechanical strain in various human endothelial cells.

Many of the hemostatic properties of endothelium are modulated by chemical and mechanical stimuli. The nature of such endothelial cell (EC) responses often depends upon the anatomical origin of the cells within the vascular tree. In the present study, we used a chromogenic assay to investigate the effect of cyclic strain or tumor necrosis factor alpha (TNF alpha), or both, on tissue factor (TF) activity in human EC derived from umbilical veins (HUVEC), aortae (HAEC), and dermal microvessels (HMVEC). Basal TF activities were low in all three cell types. Incubation for 5 h with (10 ng/ml) TNF alpha resulted in quantitatively diverse elevation of TF activity in all three EC types. Exposure to cyclic strain for 5 h induced significant elevation of TF activity only in HMVEC and HAEC. Concomitant application of cyclic strain and TNF alpha resulted in synergistic elevation of TF expression only in HMVEC. Pharmacologic elevation of cyclic AMP (cAMP) levels and inhibition of protein kinase C (PKC) levels inhibited TNF alpha-induced TF expression in all EC types. However, none of these treatments affected the stimulatory action of cyclic strain in HMVEC. Thus, we have shown that TNF alpha differentially increases TF activity in human EC of various origins, that cyclic strain variably modulates TF activity in human EC, and that both PKC and cAMP mediate TNF alpha-induced TF activity, whereas cyclic strain acts independently of these pathways. These results show differential modulation of the procoagulant potential of diverse human endothelial cells in vitro by hemodynamic stimuli.

GTSF-2: a new, versatile cell culture medium for diverse normal and transformed mammalian cells.

The 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.

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

We 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.

Rhodanese activity during the embryonic development of mouse liver and kidney.

Changes in specific activity and total activity levels of rhodanese in mouse kidney and liver were studied during development of the embryo, neonate and adult. The enzyme profiles during embryonic development were different for kidney and liver. The kidney showed a lower but constant activity from day 11 to 15, then an increase to reach the fully differentiated level at birth. The liver specific activity increased linearly from day 11 to reach a maximum 1 day before birth. The level of rhodanese may be correlated with the onset of organogenesis. The kidney results are discussed in terms of a protodifferentiated level of activity followed by a biphasic increase in specific protein synthesis.

Changes in ribosome-associated proteins during sea urchin development.

Ribosomes isolated from unfertilised eggs of the sea urchin, Strongylocentrotus purpuratus, have a higher protein: RNA ratio than ribosomes extracted from blastula stage ribosomes. Approximately 64 additional protein equivalents are found per ribosome. Most of the proteins are of high molecular weight and are tightly bound, being resistant to high-salt and EDTA treatment. The majority of the proteins appear to be basic in nature and remain associated with the 40S subunit on dissociation of the ribosomes. The possible physiological significance of the additional proteins is discussed in terms of the activation of protein synthesis following fertilisation. Sea urchin ribosomes, isolated from various stages of development, showed differential protein-labelling patterns. The high molecular-weight proteins had preferentially higher specific activities and one ribosomal protein was particularly highly labelled, reaching a maximum at the gastrula stage of development. The functional role of this highly labelled protein during development is discussed.

Lysosomal enzyme release associated with the invasion of rat liver by Novikoff hepatoma.

The lysosomes of both Novikoff heptoma and liver from Novikoff heptoma-bearing rats were found to be relatively intact structurally, lower in acid phosphatase activity, greatly depleted in number but with nearly normal membrane integrity when compared with normal liver.

Adenylyl cyclase isoforms are differentially expressed in primary cultures of endothelial cells and whole tissue homogenates from various rat tissues.

The expression of five adenylyl cyclase isoforms (types II-VI) was studied with reverse transcription-polymerase chain reaction in whole tissue homogenates and in primary cultures of endothelial cells isolated from rat aorta, vena cava, heart, lung, adipose fat, brain, and adrenal medulla. It was found that: i) all endothelial cell types express all adenylyl cyclase isoforms studied, albeit at different levels depending on the tissue origin of the cells, and ii) the adenylyl cyclase isoform profile of isolated endothelial cells differs from that of homogenates of their parent tissues. Our data show a unique endothelial cell type specificity of AC isoform expression, which varies from that of the whole organ. These results support the idea that one of the factors mediating differential regulation of the cAMP cascade in EC in various locations within the vascular tree might be quantitative differences in the levels of AC isoforms expressed in each EC type.

Microgravity decreases tyrosine hydroxylase expression in rat adrenals.

During spaceflight, alterations in blood and urinary catecholamine (CA) levels have been observed, yet the cellular/molecular mechanisms leading to these changes are not known. We used molecular, immunological, and biochemical approaches to analyze in situ the expression of catecholamine enzymes in adrenal medullary chromaffin cells of rats flown for 6 days on board Space Shuttle mission STS-54. Exposure to microgravity (10(-6) g) resulted in a 35% inhibition of both the expression and the specific activity of tyrosine hydroxylase (TH), the rate-limiting step in the cascade of CA synthesis. By contrast, the expression, specific activity, and immunoreactivity of other catecholamine-synthesizing enzymes, e.g., phenylethanolamine-N-methyl-transferase (PNMT), were not altered. The total tissue CA contents were reduced, concomitant with a decrease in the epinephrine:norepinephrine ratio. These results are in line with reports of other gravity-sensitive cellular effects and suggest that the inhibition of TH expression might be due to a direct effect of microgravity on PKC-dependent signal transduction pathways in chromaffin cells.