cAMP enhances Cx43 gap junction formation and function and reverses choline deficiency apoptosis.

Previously, it had been shown that acute choline deficiency (CD) induced apoptosis in cultured rat liver epithelial cells, whereas cells that are adapted to survive in low-choline-containing medium acquire resistance to CD apoptosis and undergo malignant transformation. Thus, understanding the mechanisms of action of CD could increase our understanding of the role of choline, an essential nutrient, in the process of malignant transformation. The present experiments were designed to test the hypothesis that CD might function as a pro-apoptotic trigger by altering the localization of connexin 43 gap junction protein and gap junctional intercellular communication (GJIC). Established liver epithelial cells (WB cells; Hep3B cells) were maintained in a defined, serum-free medium control (70 microM choline) or choline deficient medium (CD, 5 microM choline) and the localization of connexin 43 protein (Cx43) was studied by immunocytochemistry and Western blotting. In nontumorigenic WB cells, CD apoptosis was associated with retention of Cx43 in the golgi/ER region of the cytoplasm and decreased GJIC as measured using a preloading fluorescent dye transfer method (calcein AM/DiIC(18)). Cells maintained in CD in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate exhibited restoration of Cx43 at the plasma membrane and increased GJIC and inhibition of apoptosis. These studies show that CD apoptosis in nontumorigenic liver epithelial cells is associated with alterations to Cx43 and GJIC and that an uncoupling of Cx43 localization and GJIC is related to resistance to CD apoptosis in transformed liver epithelial cells.

Maternal choline availability alters the localization of p15Ink4B and p27Kip1 cyclin-dependent kinase inhibitors in the developing fetal rat brain hippocampus.

Previously we have shown that changes in maternal dietary choline are associated with permanent behavioral changes in offspring. Importantly, in adult male rats, feeding a choline-deficient diet increases the localization of cyclin-dependent kinase inhibitors (CDKIs) in the liver, whereas young adult CDKI knockout mice (p15Ink4B or p27Kip1) exhibit behavioral abnormalities. Thus, maternal dietary choline-CDKI interactions could underlie the changes we observe in fetal hippocampal development and cognitive function in offspring. Here, timed-pregnant rats on embryonic day E12 were fed the AIN-76 diet with varying levels of dietary choline for 6 days, and, on E18, fetal brain sections were collected, and the localization of CDKI proteins was studied using immunohistochemistry and an unbiased image analysis method. In choline-supplemented animals compared to controls, the number of cells with nuclear immunoreactivity for p15Ink4b CDKI protein was decreased 2- to 3-fold in neuroepithelial ventricular zones and adjacent subventricular zones corresponding to the fimbria, primordial dentate gyrus and Ammon's horn regions in the fetal hippocampus. In contrast, maternal dietary choline deficiency significantly decreased nuclear p15Ink4b immunoreactivity in the neuroepithelial layer of the dentate gyrus. Unlike p15Ink4b, the CDKI protein p27Kip1 was observed almost exclusively in the cytoplasm, though the protein was distributed throughout the proliferating and postmitotic zones in the E18 fetal hippocampus. Maternal dietary choline supplementation decreased the cytoplasmic staining intensity for p27Kip1 throughout the fetal hippocampus compared to control animals. Choline deficiency increased the staining intensity of p27Kip1 throughout the hippocampus in association with increased expression of MAP-1 and vimentin proteins. These results link maternal dietary choline availability to CDKI protein immunoreactivity and commitment to differentiation during fetal hippocampal development.

Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes.

The mechanism of induction of apoptosis by the novel anti-cancer drug 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigated in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 microM ET-18-OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-18-OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET-18-OCH3 in the presence of the antioxidant alpha-tocopherol. Similar results were achieved when cells were switched acutely to choline-deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosphatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5',6,6'-tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET-18-OCH3 or CD. Taken together, these results suggest that the form of p53-independent apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.

Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

Lactoferrin: a tamoxifen-responsive protein in normal and malignant human endometrial cells in culture.

We investigated the possible role of the estrogen-regulated protein lactoferrin (Lf) in the response of isolated normal human endometrial epithelial cells (NHEC) and established human endometrial carcinoma (EC) cell lines to tamoxifen (TAM). Using confocal laser scanning microscopy and a monospecific antibody, Lf was localized to the cytoplasm of normal and EC cells. Antibody neutralization of secreted Lf inhibited, whereas exogenous Lf (0--100 microg/ml) enhanced, cell proliferation in both classes of cells. Treatment of NHEC with TAM inhibited cell growth via a protein kinase-C-mediated pathway, concomitant with a reduction in the staining intensity for Lf. Importantly, in EC cells, TAM greatly enhanced the staining intensity for Lf, but did not affect cell growth. We propose that stable expression of Lf protein by EC cells may impart a survival advantage to these cells, which may, in part, account for the resistance of these cells to tamoxifen.

Dietary antioxidant depletion: enhancement of tumor apoptosis and inhibition of brain tumor growth in transgenic mice.

Apoptosis, or regulated cell suicide, eliminates unwanted and damaged cells, including precancerous and cancerous cells. Since reactive oxygen species (ROS) act as essential apoptotic mediators, we reasoned that increasing the ROS level might enhance apoptosis and thereby slow down tumor growth. Here, using a defined transgenic brain tumor model with known tumor apoptosis rates, we test the impact of antioxidant-depleted diet, capable of increasing ROS levels, or antioxidant-enriched diets on tumor growth. Dramatically increased apoptosis occurs within tumors, but not in normal tissues of antioxidant-depleted mice. The presence of detectable increased oxidant stress within tumors indicates that the likely mechanism of enhanced tumor apoptosis is via ROS and DNA oxidative impairment. Importantly, due to the ROS-enhanced apoptosis, tumor growth is inhibited in mice fed an antioxidant-depleted diet. In clear contrast, an antioxidant-rich diet had no impact on tumor growth.

Maternal dietary choline availability alters mitosis, apoptosis and the localization of TOAD-64 protein in the developing fetal rat septum.

Maternal changes in dietary choline availability alter brain biochemistry and hippocampal development in the offspring resulting in lifelong behavioral changes in the offspring. In order to better understand the relationship between maternal diet, brain cytoarchitecture and behavior, we investigated the effects of choline availability on cell proliferation, apoptosis and differentiation in the fetal rat brain septum. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the progenitor neuroepithelium adjacent to the septum. In addition, we found an increased number of apoptotic cells in the septum of CD animals compared to controls (3.5+/-0.5 vs. 1.7+/-0.5 apoptotic cells per section; p<0.05). However, CD had no effect on apoptosis in the indusium griseum (IG), a region of cortex dorsal to the septum. Using an unbiased image analysis method and a monoclonal antibody we found a decreased expression of the TOAD-64 kDa protein, a marker of commitment to neuronal differentiation during fetal development, in the dorsal lateral septum of CD animals. CD also decreased the expression of TOAD-64 kDa protein in the IG and cortical plate adjacent to the septum. These results show that dietary choline availability during pregnancy alters the timing of mitosis, apoptosis and the early commitment to neuronal differentiation by progenitor cells in regions of the fetal brain septum, as well as hippocampus, two brain regions known to be associated with learning and memory.

Choline availability alters embryonic development of the hippocampus and septum in the rat.

Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on cell proliferation, migration, and apoptosis in neuronal progenitor cells in the hippocampus and septum were analyzed in fetal brains at different stages of embryonic development. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days, and, on days E18 or E20, fetal brain sections were collected. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the neuroepithelium adjacent to the hippocampus. An increased number of apoptotic cells were found in the region of the dentate gyrus of CD hippocampus compared to controls (5.5+/-0.7 vs. 1.9+/-0.3 apoptotic cells per section; p<0.01). Using a combination of bromodeoxyuridine (BrdU) labeling and an unbiased computer-assisted image analysis method, we found that modulation of dietary choline availability changed the distribution and migration of precursor cells born on E16 in the fimbria, primordial dentate gyrus, and Ammon's horn of the fetal hippocampus. CD also decreased the migration of newly born cells from the neuroepithelium into the lateral septum, thus indicating that the sensitivity of fetal brain to choline availability is not restricted to the hippocampus. We found an increase in the expression of TOAD-64 protein, an early neuronal differentiation marker, in the hippocampus of CD day E18 fetal brains compared to controls. These results show that dietary choline availability alters the timing of the genesis, migration, and commitment to differentiation of progenitor neuronal-type cells in fetal brain hippocampal regions known to be associated with learning and memory processes in adult brain.

Choline deficiency induces apoptosis and decreases the number of eosinophilic preneoplastic foci in the liver of OXYS rats.

Choline deficiency (CD) was previously shown to trigger apoptosis in rat hepatocytes in culture and in vivo. In the present study we investigated the effects of short-term withdrawal of choline from the diet on the expression of putative preneoplastic foci in OXYS rats, an inbred strain with an inherited overproduction of free radicals. Animals were fed a defined, choline-sufficient (CS, control) or choline-deficient (CD) diet for 6 weeks. Eosinophilic, glutathione S-transferase (pi class) (+) preneoplastic foci were found in histologic sections of control OXYS rat liver. CD caused a 60% decrease in the number of eosinophilic foci per liver section (27.0+/-6.1 vs. 10.6+/-4.6 foci/section) compared to CS controls. Apoptotic bodies were detected in 0.18+/-0.03% of hepatocytes in CD livers compared to 0.05+/-0.009% of hepatocytes in controls. Cells which exhibited an apoptotic morphology in hematoxylin and eosin-stained sections were TUNEL-positive, confirming the induction of apoptosis. Also in CD animals compared to controls, there was an increased expression of p27Kip1 protein, and a reduction in PCNA nuclear labeling and the number of mitotic figures, consistent with an inhibition of cell proliferation in the livers of CD animals. This study shows that the liver of OXYS rats with an inherited overgeneration of free radicals retains sensitivity to CD, and that this p53-independent trigger of apoptosis can decrease the number of eosinophilic foci in the livers of these animals.

Choline availability modulates the expression of TGFbeta1 and cytoskeletal proteins in the hippocampus of developing rat brain.

Choline availability influences long-term memory in concert with changes in the spatial organization and morphology of septal neurons, however little is known concerning the effects of choline on the hippocampus, a region of the brain also important for memory performance. Pregnant rats on gestational day 12 were fed a choline control (CT), choline supplemented (CS), or choline deficient (CD) diet for 6 days and fetal brain slices were prepared on embryonic day 18 (E18). The hippocampus in these brain slices was studied for the immunohistochemical localization of the growth-related proteins transforming growth factor beta type 1 (TGFbeta1) and GAP43, the cytoskeletal proteins vimentin and microtubule associated protein type 1 (MAP1), and the neuronal cell marker neuron specific enolase (NSE). In control hippocampus, there was weak expression of TGFbeta1 and vimentin proteins, but moderately intense expression of MAP1 protein. These proteins were not homogeneously distributed, but were preferentially localized to cells with large cell bodies located in the central (approximately CA1-CA3) region of the hippocampus, and to the filamentous processes of small cells in the fimbria region. Feeding a choline-supplemented diet decreased, whereas a choline-deficient diet increased the intensity of immunohistochemical labeling for these proteins in E18 hippocampus. GAP43 and NSE were localized to peripheral nervous tissue but not hippocampus, indicating that the maturation of axons and neurite outgrowth in embryonic hippocampus were unaffected by the availability of choline in the diet. These data suggest that the availability of choline affects the differentiation of specific regions of developing hippocampus.

Apoptosis is induced by choline deficiency in fetal brain and in PC12 cells.

Treatment of rats with choline during critical periods in brain development results in long-lasting enhancement of spatial memory in their offspring. Apoptosis is a normal process during brain development, and, in some tissues, is modulated by the availability of the nutrient choline. In these studies, we examined whether availability of choline influences apoptosis in fetal brain and in the PC12 cell line derived from a rat pheochromocytoma. Timed-bred Sprague Dawley rats were fed a choline-deficient (CD), choline-control, or choline-supplemented (CS) diet for 6 days and, on embryonic day 18, fetal brain slices were prepared and apoptosis was assessed using terminal dUTP nucleotide end labeling (TUNEL) to detect DNA strand breaks and by counting of apoptotic bodies. TUNEL-positive cells were detected in 15.9% (P < 0.01), 8.7% and 7.2% of hippocampal cells from fetuses of dams fed the CD, control or CS diets, respectively. A similar inverse relationship between dietary intake of choline and TUNEL positive cells was detected in an area of cerebral cortex from these fetal brain slices. Counts of apoptotic bodies in fetal brain slices correlated inversely with choline intake of the mothers (6.2% (P < 0.01), 2.5% and 1.9% of hippocampal cells had apoptotic bodies in fetuses of dams fed the CD, control and CS diets, respectively). PC12 cells were grown in DMEM/F12 media supplemented with 70 microM choline or with 0 microM choline. The number of apoptotic bodies in PC12 cells increased when cells were grown in 0 microM choline medium (1.5%; P < 0.05) compared to 70 microM choline medium (0.55%). In PC12 cells, TUNEL labeling (DNA strand breaks) increased in choline deficient (13.5%, P < 0.05) compared to sufficient medium (5.0%). In addition, cleavage of genomic DNA-into 200 bp internucleosomal fragments was detected in choline-deficient cells. These results show that choline deficiency induces-apoptotic cell death in neuronal-type cells and in whole brain. We suggest that variations in choline availability to brain modulate apoptosis rates during development.

Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes.

The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency.

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B12. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanol-amine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes.

Tamoxifen alters the localization of F-actin and alpha 5/beta 1-integrin fibronectin receptors in human endometrial stromal cells and carcinoma cells.

We have investigated F-actin and the integrin fibronectin receptor as possible targets of tamoxifen (TAM) signaling in a cell-based model of the endometrium. Normal human endometrial stromal cells and RL95-2 human endometrial adenocarcinoma cells were treated for 1 h with TAM, a known antagonist of protein kinase C (PKC), or with staurosporine or HA1004, two broad-spectrum protein kinase antagonists capable of inhibiting PKC and PKA, respectively. We utilized fluorescein-phalloidin and confocal microscopy to visualize the cellular distribution of F-actin. Normal stromal cells and RL95-2 cells differed in the arrangement of F-actin in control cells and in their response to TAM. In control stromal cells, actin stress fibers were well organized throughout the cell, but in RL95-2 cells, they were disorganized and present mainly at the cell periphery. F-actin in RL95-2 cells treated with TAM (0.1 and 1.0 microM) or with staurosporine (0.7 and 7.0 nM) exhibited a reorganization into stress fibers consistent with a more stationary phenotype. In contrast, TAM- or staurosporine-treated normal stromal cells exhibited an increase in the amount of organized F-actin. Interestingly, in normal stromal cells treated with staurosporine but not TAM or HA1004, these F-actin fibers appeared to terminate in dense plaques proximal to the plasma membrane. The alpha 5/beta 1 integrin fibronectin receptor mediates between the extracellular matrix and the actin cytoskeleton. TAM induced clustering of the fibronectin receptor at the plasma membrane in normal stromal cells, but not in carcinoma cells. This study supports the importance of plasma membrane-cytoskeletal protein interactions in the response of normal and carcinoma cells to TAM.

Diet, apoptosis, and carcinogenesis.

It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.

Choline deficiency causes increased localization of transforming growth factor-beta1 signaling proteins and apoptosis in the rat liver.

Dietary restriction is known to decrease cell proliferation and increase apoptosis in the liver; however, the role of withdrawal of single dietary factors on cells of the liver is less well understood. In this study, we investigated the effects of short-term choline deficiency (CD; also for choline deficient) on cell survival, proliferation and the expression of proteins related to the transforming growth factor-beta1 (TGF-beta1) growth-inhibitory signaling pathway in the liver. In animals fed a CD diet for 6 weeks, classical apoptotic bodies were detected in 0.28 +/- 0.04% of hepatocytes in CD livers compared to 0.096 +/- 0.006% of hepatocytes in control rats fed a choline-sufficient (CS) diet. These classical apoptotic cells exhibited DNA fragmentation when probed with an in situ end-labeling immunohistochemical method; TUNEL-positive nuclei were also seen in hepatocytes in CD livers which had accumulated large amounts of lipid, consistent with the known DNA-damaging effects of CD. In CS control livers, TGF-beta1 protein was found only in bile duct epithelium and nonparenchymal-type cells, and not in hepatocytes. However, the majority of hepatocytes in the CD liver expressed high levels of TGF-beta1 protein, as well as TGF-beta1 receptor types I and II. Nuclear localization of p27Kip1 protein, which may link TGF-beta1 expression to apoptosis, showed a 10-fold increase in CD hepatocytes (4.1 +/- 1.1 vs. 0.35 +/- 0.04% of cells) compared to controls. In addition, there was a 5-fold increase (0.54 +/- 0.031 vs. 0.011 +/- 0.007% of cells) in the mitotic index in CD-compared control livers. We conclude that feeding a CD diet for 6 weeks induces apoptosis in hepatocytes in the whole rat liver and that this form of cell death appears to be mediated, in part, by TGF-beta1 and related proteins.

Choline deficiency induces apoptosis in SV40-immortalized CWSV-1 rat hepatocytes in culture.

Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 Micron choline (67-73% of cells) than when grown in 70 Micron choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 Micron and 0 Micron choline for 72h. Cells treated with 0 or 5 Micron choline for 72h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24h, cells grown in 0 or 5 Micron choline synthesize DNA more rapidly than those grown in 70 Micron choline. By 72h, the cells grown in 0 or 5 Micron choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T-antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV-1 hepatocytes in culture by inducing apoptosis via what may be a p53-independent process, and that this process begins in viable cells before they detach from the culture dish.

Human endometrial carcinoma cells release factors which inhibit the growth of normal epithelial cells in culture.

Autocrine and paracrine interactions between cells are important homeostatic mediators in normal tissues. Alterations to growth factor signalling pathways are likely to play a role in multistep carcinogenesis. In this study normal human endometrial epithelial cells (NHEC) after 3 days in culture were treated with serum-free medium conditioned for 24 h by log phase or confluent cultures of established RL95-2, HEC1A, or AN3CA endometrial carcinoma (EC) cell lines. By day 4, NHEC treated with either log phase or confluent conditioned medium (CM) showed a significant decrease (approximately 50-90% of control) in [3H]thymidine ([3H]TdR) incorporation. DNA synthesis was inhibited more by confluent than by log phase CM. By day 7, NHEC treated with CM exhibited fewer colonies per culture, fewer cells per colony, and an increased percentage of single cells. Several growth-regulatory gene products found in the nucleus or at the cell membrane have been shown to be expressed differently in normal and transformed cells. We selected the p53 and c-Ha-ras p21 proteins to further investigate the mechanism of alteration of proliferation in cells treated with carcinoma CM. Thus, by day 7, the percentage of NHEC with nuclear localization of wild type p53 (wt p53) was elevated by treatment with CM. In contrast, CM-treated EC cells continued to proliferate, and showed a decrease in the percentage of cells expressing nuclear wt p53 and an increase in the cytoplasmic expression of c-Ha-ras p21. Our studies show that EC cell lines release factors which inhibit the proliferation of NHEC, thus favoring the proliferation of EC cells.

Choline and hepatocarcinogenesis in the rat.

Rats fed a choline deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. This is the only nutritional deficiency that, in itself, causes cancer. We suggested that carcinogenesis is triggered, in part, because of abnormalities in cell signals which regulate cell proliferation and cell death. Because choline deficient rats develop fatty liver (choline is needed for hepatic secretion of certain lipoproteins), we examined whether an important lipid second messenger involved in proliferative signaling, 1,2-sn-diacylglycerol, accumulated in liver and resulted in the prolonged activation of protein kinase C. We observed that 1,2-sn-diacylglycerol accumulated in the plasma membrane from the non-tumor portion of livers of rats fed a choline deficient diet, and that unsaturated free fatty acids, another activator of protein kinase C, also accumulated in deficient livers. Protein kinase C in the hepatic plasma membrane and nucleus of choline deficient rats was elevated for months; this is the only model system which exhibits such prolonged activation of protein kinase C. Premalignant, abnormal hepatic foci were detected only in the deficient rats, and 15% of deficient rats (none of the controls) had hepatocellular carcinoma at 1 year on the diet. In rats, an early event in choline deficiency is an increase in the rate of cell death. In liver from choline deficient rats, we observed an increase in the numbers of liver cells with fragmented DNA (characteristic of programmed cell death; apoptosis). We used a cell culture model (immortalized rat hepatocytes) to study the effects of choline deficiency on apoptosis. Liver cells grown in a choline deficient medium became depleted of choline, accumulated triacylglycerol and 1,2-sn-diacylglycerol, and had increased DNA fragmentation and other morphologic and biochemical changes associated with apoptosis. This model has great potential as a tool for studying the underlying link between choline deficiency and the regulation of the balance between cell proliferation and cell death. We suggest that choline deficiency altered the cell proliferation signals mediated by protein kinase C within liver, and altered cell apoptosis. These changes in cell signaling may be the triggering events which result in hepatic carcinogenesis.

Transforming growth factor-beta 1 mediates communication between human endometrial carcinoma cells and stromal cells.

The role of transforming growth factor-beta 1 (TGF-beta 1) in communication between human endometrial carcinoma (EC) cells and normal endometrial stromal cells (NSC) was investigated using a cell culture model. Serum-free conditioned medium (CMe) from EC cells (RL95-2, HEC1A) inhibited the proliferation (cells per colony < 50% of control; mitotic index 25-50% of control) of NSC. In contrast, NSC-conditioned medium (CMn) stimulated the proliferation of EC cells, but inhibited the growth of NSC. The proliferation of EC cells was stimulated by the range of dilutions of CMe which inhibited the proliferation of NSC. Using confocal microscopy and a monoclonal antibody, TGF-beta 1, a known product of differentiation in the female reproductive tract, was localized to the cytoplasm of NSC and EC cells. Using a protein slot-blot chemiluminescence method, secreted TGF-beta 1 was detected in serum-free medium conditioned by the growth of NSC and EC cells. TGF-beta 1 antibody-neutralized CMe or CMn stimulated the proliferation of both NSC and EC cells. This study suggests that endometrial carcinoma-stromal cell interactions involve autocrine-paracrine signaling pathways, and that TGF-beta 1 protein is one mediator of such interactions.