Rhenium-188 as an alternative to Iodine-131 for treatment of breast tumors expressing the sodium/iodide symporter (NIS).

The sodium-iodide symporter (NIS), which transports iodine into the cell, is expressed in thyroid tissue and was recently found to be expressed in approximately 80% of human breast cancers but not in healthy breast tissue. These findings raised the possibility that therapeutics targeting uptake by NIS may be used for breast cancer treatment. To increase the efficacy of such therapy it would be ideal to identify a radioactive therapy with enhanced local emission. The feasibility of using the powerful beta-emitting radiometal (188)Re in the form of (188)Re-perrhenate was therefore compared with 131I for treatment of NIS-expressing mammary tumors. In the current studies, using a xenografted breast cancer model induced by the ErbB2 oncogene in nude mice, (188)Re-perrhenate exhibited NIS-dependent uptake into the mammary tumor. Dosimetry calculations in the mammary tumor demonstrate that (188)Re-perrhenate is able to deliver a dose 4.5 times higher than (131)I suggesting it may provide enhanced therapeutic efficacy.

The role of endothelin in the pathogenesis of Chagas' disease.

Infection with Trypanosoma cruzi causes a generalised vasculitis of several vascular beds. This vasculopathy is manifested by vasospasm, reduced blood flow, focal ischaemia, platelet thrombi, increased platelet aggregation and elevated plasma levels of thromboxane A(2) and endothelin-1. In the myocardium of infected mice, myonecrosis and a vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium are observed. Immunohistochemistry studies employing anti-endothelin-1 antibody revealed increased expression of endothelin-1, most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for prepro endothelin-1, endothelin converting enzyme and endothelin-1 were observed in the infected myocardium. When T. cruzi-infected mice were treated with phosphoramidon, an inhibitor of endothelin converting enzyme, there was a decrease in heart size and severity of pathology. Mitogen-activated protein kinases and the transcription factor activator-protein-1 regulate the expression of endothelin-1. Therefore, we examined the activation of mitogen-activated protein kinases in the myocardium by T. cruzi. Western blot demonstrated an extracellular signal regulated kinase. In addition, the activator-protein-1 DNA binding activity, as determined by electrophoretic mobility shift assay, was increased. Increased expression of cyclins A and cyclin D1 was observed in the myocardium, and immunohistochemistry studies revealed that interstitial cells and vascular and endocardial endothelial cells stained intensely with antibodies to these cyclins. These data demonstrate that T. cruzi infection of the myocardium activates extracellular signal regulated kinase, activator-protein-1, endothelin-1, and cyclins. The activation of these pathways is likely to contribute to the pathogenesis of chagasic heart disease. These experimental observations suggest that the vasculature plays a role in the pathogenesis of chagasic cardiomyopathy. Additionally, the identification of these pathways provides possible targets for therapeutic interventions to ameliorate or prevent the development of cardiomyopathy during T. cruzi infection.

NF-kappaB and cell-cycle regulation: the cyclin connection.

The cyclins are a family of proteins that are centrally involved in cell cycle regulation and which are structurally identified by conserved "cyclin box" regions. They are regulatory subunits of holoenzyme cyclin-dependent kinase (CDK) complexes controlling progression through cell cycle checkpoints by phosphorylating and inactivating target substrates. CDK activity is controlled by cyclin abundance and subcellular location and by the activity of two families of inhibitors, the cyclin-dependent kinase inhibitors (CKI). Many hormones and growth factors influence cell growth through signal transduction pathways that modify the activity of the cyclins. Dysregulated cyclin activity in transformed cells contributes to accelerated cell cycle progression and may arise because of dysregulated activity in pathways that control the abundance of a cyclin or because of loss-of-function mutations in inhibitory proteins.Analysis of transformed cells and cells undergoing mitogen-stimulated growth implicate proteins of the NF-kappaB family in cell cycle regulation, through actions on the CDK/CKI system. The mammalian members of this family are Rel-A (p65), NF-kappaB(1) (p50; p105), NF-kappaB(2) (p52; p100), c-Rel and Rel-B. These proteins are structurally identified by an amino-terminal region of about 300 amino acids, known as the Rel-homology domain. They exist in cytoplasmic complexes with inhibitory proteins of the IkappaB family, and translocate to the nucleus to act as transcription factors when activated. NF-kappaB pathway activation occurs during transformation induced by a number of classical oncogenes, including Bcr/Abl, Ras and Rac, and is necessary for full transforming potential. The avian viral oncogene, v-Rel is an NF-kappaB protein. The best explored link between NF-kappaB activation and cell cycle progression involves cyclin D(1), a cyclin which is expressed relatively early in the cell cycle and which is crucial to commitment to DNA synthesis. This review examines the interactions between NF-kappaB signaling and the CDK/CKI system in cell cycle progression in normal and transformed cells. The growth-promoting actions of NF-kappaB factors are accompanied, in some instances, by inhibition of cellular differentiation and by inhibition of programmed cell death, which involve related response pathways and which contribute to the overall increase in mass of undifferentiated tissue.

Rho family GTPases regulate mammary epithelium cell growth and metastasis through distinguishable pathways.

BACKGROUND: Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed. MATERIALS AND METHODS: We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size. RESULTS: Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the co-localization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU. CONCLUSIONS: These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Epidermal growth factor receptor distribution during chemotactic responses.

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

Trypanosoma cruzi infection (Chagas' disease) of mice causes activation of the mitogen-activated protein kinase cascade and expression of endothelin-1 in the myocardium.

Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore. in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mitogen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice. These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection.

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.

Transforming growth factor-beta1 recruits histone deacetylase 1 to a p130 repressor complex in transgenic mice in vivo.

Transforming growth factor (TGF)-beta1 functions as a tumor suppressor in vivo. Using transgenic mice, we show that hepatic TGF-beta1 overexpression inhibits abundance of the cyclin-dependent kinase activating tyrosine phosphatase cdc25A protein. The reduction in cdc25A protein levels was associated with increased binding of histone deacetylase 1 to p130 in the hepatic extracts. In cultured cells, HDAC1/p130 overexpression inhibited activity of the cdc25A promoter through an E2F site. TGF-beta1 treatment enhanced p130 binding to the cdc25A promoter E2F site assessed in chromatin immunoprecipitation assays. Hepatic proliferation induced by partial hepatectomy was associated with a decrease in the amount of HDAC1 bound to p130, without a significant decrease in p130 abundance, suggesting that HDAC1 binding to p130 may be regulated by proliferative stimuli. The induction of cdc25A abundance induced by partial hepatectomy correlated with the induction of DNA synthesis. These studies suggest that TGF-beta1 may enhance HDAC1 binding to p130 in vivo, thereby inhibiting cdc25A gene expression. TGF-beta1 regulation of HDAC1/pocket protein associations may provide a link between chromatin remodeling proteins and cdk inhibition through induction of cdc25A in vivo.

The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.

The application of a lentiviral vector for gene transfer in fetal human hepatocytes.

BACKGROUND: The applications of traditional retroviral vectors are limited because proviral integrations into the host genome require DNA synthesis. Lentiviruses are considered to be advantageous because of their ability to infect non-dividing cells. METHODS: To demonstrate the potential of lentiviral vectors, we used a human immunodeficiency virus (HIV)-1 virus encoding the green fluorescence protein (GFP) to infect fetal human hepatocytes. GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection. RESULTS: Primary fetal hepatocytes incorporated the GFP reporter with high (30-40%) efficiency. A cell line derived from human fetal liver (HFL) exhibited similar transduction efficiency to the lentiviral vector. To demonstrate the relationship between lentiviral gene transfer and cell proliferation, cells were subjected to gamma-irradiation, which attenuated the replication of primary fetal hepatocytes. However, lentiviral gene transfer was unaffected by this decrease in cell proliferation. GFP expression in transduced cells was preserved during multiple passages in cell culture. When GFP-expressing cells were transplanted into the liver of Balb/C SCID mice via intrasplenic injection, GFP expression was observed throughout the 3 week duration of the study. CONCLUSION: These studies establish that human hepatocytes are amenable to lentiviral gene transfer with sustained transgene expression. Incorporation of lentiviral vectors will be helpful in testing strategies for hepatic gene therapy.

Cell cycle molecules and diseases of the cardiovascular system.

Injury to the cardiovascular system causes an elevated expression of endothelin-1 (ET-1) and activation of several important signaling pathways including the mitogen-activated kinase (MAPK) cascade. The activation of these pathways has been implicated in the pathogenesis of cardiovascular disease caused by hypoxia, infections, and ischemia /reperfusion injury, cardiomyopathy and restenosis after balloon angioplasty. Important downstream targets of the MAPK and ET-1 pathways are the cell cycle regulatory molecules (cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors). Regulation of these molecules contributes to remodeling throughout the cardiovascular system. In addition, cell cycle molecules are important in the regulation of angiogenesis. These new data have led to the development of potential therapeutic modalities targeting these regulatory molecules in order to ameliorate various cardiovascular disease states.

Caveolin-1 inhibits epidermal growth factor-stimulated lamellipod extension and cell migration in metastatic mammary adenocarcinoma cells (MTLn3). Transformation suppressor effects of adenovirus-mediated gene delivery of caveolin-1.

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.

Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.

Integration of Rac-dependent regulation of cyclin D1 transcription through a nuclear factor-kappaB-dependent pathway.

The small GTP-binding protein Rac1, a member of the Ras superfamily, plays a fundamental role in cytoskeleton reorganization, cellular transformation, the induction of DNA synthesis, and superoxide production. Cyclin D1 abundance is rate-limiting in normal G(1) phase progression, and the abundance of cyclin D1 is induced by activating mutations of both Ras and Rac1. Nuclear factor-kappaB (NF-kappaB) proteins consist of cytoplasmic hetero- or homodimeric Rel-related proteins complexed to a member of the IkappaB family of inhibitor proteins. In the current studies, activating mutants of Rac1 (Rac(Leu-61), Rac(Val-12)) induced cyclin D1 expression and the cyclin D1 promoter in NIH 3T3 cells. Induction of cyclin D1 by Rac1 required both an NF-kappaB and an ATF-2 binding site. Inhibiting NF-kappaB by overexpression of an NF-kappaB trans-dominant inhibitor (nonphosphorylatable IkappaBalpha) reduced cyclin D1 promoter activation by the Rac1 mutants, placing NF-kappaB in a pathway of Rac1 activation of cyclin D1. Specific amino acid mutations in the amino-terminal effector domain of Rac(Leu-61) had comparable effects on NF-kappaB transcriptional activity and activation of the cyclin D1 promoter. The NF-kappaB factors Rel A (p65) and NF-kappaB(1) (p50) induced the cyclin D1 promoter, requiring both the NF-kappaB binding site and the ATF-2 site. Stable overexpression of Rac(Leu-61) increased binding of Rel A and NF-kappaB(1) to the cyclin D1 promoter NF-kappaB site. Activation of Rac1 in NIH 3T3 cells induces both NF-kappaB binding and activity and enhances expression of cyclin D1 through an NF-kappaB and ATF-2 site in the proximal promoter, suggesting a critical role for NF-kappaB in cell cycle regulation through cyclin D1 and Rac1.

The role of corticoids, adrenalectomy, phenobarbital and pentobarbital in the promotion of DEN-hepatocarcinogenesis.

This paper shows there is a good correlation between the disturbance of the proliferation control and the plasma Corticosterone circadian pattern on the one hand and the promotion of diethylnitrosamine (DEN) initiated precancerous lesions on the other. In normal rats, phenobarbital (PB) or adrenalectomy, which both decrease plasma corticosterone levels, induce chronic liver growth, enhance the mitotic response to partial Hepatectomy and eliminate the mitotic circadian rhythms. In rats treated by DEN, the same treatments increase the proliferation advantage of PAS positive precancerous cells and promote tumorogenesis. Daily corticosterone injections inhibits liver growth even after hepatectomy both in normal and DEN-treated rats. Under carcinogenic conditions (DEN for 6 weeks), corticoid injections inhibit the selective growth of precancerous cells and the death by cancer occurs later. Pentobarbital (PE), administrated at the same subsedative doses as PB, does not change plasma corticosterone levels nor the mitotic control and does not enhance the carcinogenesis initiated by DEN. The "promoting effect" is closely linked to the disturbance of the biological corticosterone activity that normally synchronizes the liver cell proliferation by temporary inhibition of DNA synthesis for some hours every day (14-16). The role played by corticosterone in the selective growth of precancerous foci (glycogenesis type I) is discussed in the light of the hypothesis (1-2) according to which modification of the carbohydrate metabolism is closely related to the process of hepatocarcinogenesis.

The antiandrogen cyproterone acetate induces synthesis of transforming growth factor beta 1 in the parenchymal cells of the liver accompanied by an enhanced sensitivity to undergo apoptosis and necrosis without inflammation.

Recently, cases of liver damage and liver tumors have been reported after treatment of prostate cancer patients with the antiandrogen cyproterone acetate (CPA). In rat liver, CPA initiates a wave of DNA synthesis that is accompanied by apoptosis. In apoptotic hepatocytes, a latent form of transforming growth factor beta 1 (TGF-beta 1) is detectable by immunohistochemistry. Injection of a single dose of TGF-beta 1 induces apoptosis in the liver of animals pretreated with CPA but has an insignificant effect in untreated animals. In this study, we show by Northern analysis that there is increased expression of TGF-beta 1 in the liver after CPA treatment. Detection of TGF-beta 1 with in situ hybridization showed that TGF-beta 1 was synthesized in the parenchymal cells. Time course and dose-response experiments performed 48 hours after the last application of CPA showed that apoptotic nuclei with chromatin condensed at the nuclear periphery (AN) were already visible 2 hours after injection (0.13%), and apoptotic bodies (ABs) increased 2 to 9 hours after the injection (from 1.28% to 6.67%) after 25 micrograms TGF-beta 1/kg. At 4.5 hours after injection, an induction of apoptosis could be detected with 0.25 microgram TGF-beta 1/kg and after the maximum dose (250 micrograms TGF-beta 1/kg) ANs (0.24%) and ABs (16.74%) were homogeneously distributed throughout the liver lobe. Irrespective of the dose or time after injection of TGF-beta 1, 82% of the ABs were localized within hepatocytes. Liver enzymes were detected in high amounts in the serum (eightfold elevation of glutamate dehydrogenase, fivefold elevation of alanine transaminase [ALT]) 7 hours after the first visible sign of apoptosis. After an additional 20 hours, the liver contained many necrotic figures. These results suggest that the combination of TGF-beta 1 expression coupled with a strikingly enhanced sensitivity to the induction of apoptosis could be responsible both for the liver damage and the development of liver tumors observed after treatment with CPA.

Growth control and gene expression in a new hepatocellular carcinoma cell line, Hep40: inhibitory actions of vitamin K.

The growth characteristics of a newly established cell line, Hep40, derived from a human hepatoma are described. An absolute requirement was found for serum to mediate cell growth. Neither EGF, TGF-alpha, nor HGF altered cell growth in the presence or absence of serum. A partial suppression of cell growth was achieved by several TGF-beta family proteins. Affinity crosslinking gels using 125I-labeled TGF-beta showed a significant decrease in the TGF-beta cell-surface type II receptor in Hep40 cells, compared to the TGF-beta-sensitive Hep3B cell line. However, growth could be completely suppressed by addition of vitamins K to the culture medium in both Hep40 and several other hepatoma cell lines. Growth suppression by vitamins K was accompanied by an increased level of transcripts for c-myc, c-jun, and prothrombin genes, in contrast to the actions of TGF-beta 1 protein, which caused a decrease in the level of c-myc transcripts. These data show that this new human hepatoma cell line has partial resistance to growth inhibition by TGF-beta with a unique TGF-beta receptor defect. However, growth was completely suppressed by vitamins K. The differing gene expression patterns in response to TGF-beta as compared to vitamin K suggest that these two growth inhibitors act through differing pathways.

Importance of cell kinetics rhythmicity for the control of cell proliferation and carcinogenesis in rat liver (review).

The circadian control of cell Proliferation and Differentiation has been studied principally in rat liver. The comparison between the differentiation by hepatic enzymes and the division by the cell cycle under various experimental conditions (postnatal maturation, regeneration after partial hepatectomy, adrenalectomy, corticosterone treatments etc.) leads to the following conclusions: Under physiological conditions, proliferation and differentiation activities present a mutually exclusive relationship with a specific circadian rhythm. For both functions, the circadian variation of corticosterone plays the role of synchronizer, each evening (peak) it induces the synthesis of tissue specific enzymes in G0 cells and simultaneously inhibits the DNA synthesis in cycling cells. The same parameters have been studied during the different stages of hepatocarcinogenesis induced by Diethylnitrosamine (DEN). After initiation alone, (DEN for 2 weeks) circadian control is unchanged and precancerous cells are not able to reach malignancy. Promotion (DEN for 6 weeks) consists of disturbing the circadian synchronization to liberate the selective growth of initiated precancerous cells. This proliferation advantage favours the accumulation of chromosomal aberrations including those implicated in malignant transformation: i.e. activation of oncogenes or inhibition of antioncogenes.

Nuclear lesions during rat hepatocarcinogenesis. II. Measuring the micronuclei during initiation, promotion and progression of rat hepatocarcinogenesis induced with diethylnitrosamine.

We reported in our companion paper the strong correlation between elevated sister-chromatid exchange (SCE) frequencies and the initiation step of rat hepatocarcinogenesis. We have also shown that SCEs return to normal values during the promotion and the progression stages. In the present study, we evaluated the clastogenic activity of diethylnitrosamine (DEN) during initiation, promotion and progression of rat hepatocarcinogenesis. We measured, at various times after DEN administration, the number of micronuclei (MN) produced by the mitotic response to partial hepatectomy. The results established that the DEN treatment induces a great number of preclastogenic lesions. In subcarcinogenic conditions (initiation alone), the number of MN expressed after partial hepatectomy remains high regardless of the time interval between the end of the DEN treatment and the operation. In this condition, the preclastogenic lesions persist for up to 1 year after the DEN administration is discontinued. Conversely, in carcinogenic conditions (initiation + promotion + progression), the number of MN expressed after partial hepatectomy decreases during the promotion and progression stages. These observations indicate that promotion and progression but not initiation are associated with the expression of persistent preclastogenic lesions, resulting in the production of chromosomally abnormal hepatocytes.