Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors.

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

Theory of the acoustic realignment of nematic liquid crystals.

When an ultrasonic wave is applied to a nematic liquid-crystal cell, the molecules change their orientation, leading to a change in the optical intensity transmitted through the cell. Modeling this acousto-optic effect involves three separate theoretical issues: (a) calculating the intensity of sound transmitted through the cell walls into the liquid crystal, (b) determining the consequent realignment of the liquid crystal, and (c) deriving the change in optical transmission through the cell. In this paper, we present a theory that addresses all three of these issues, and thereby reproduces the behavior seen in experiments. The theory shows how the performance depends not only on the liquid-crystal material properties, but also on the geometrical parameters of the system, such as the thickness of the glass walls, thickness of the liquid-crystal layer, angle of the ultrasonic wave, viewing angle, and boundary condition at the glass-liquid crystal interface. The theory predicts that the strong dependence on viewing angle still allows an optical image to be seen for realistic dimensions.

Comparison of the characteristics of the chiral analog of the de Vries type of smectic-A* phase.

In this paper we compare the results of three ferroelectric materials that exhibit unusual smectic-A (SmA) phases. These phases have been assigned to chiral analogs of the de Vries SmA phase. Several experimental techniques have been employed for this investigation. The possible molecular structures of de Vries SmA* phases are discussed.

Acoustic realignment of nematic liquid crystals.

In a nematic liquid-crystal cell, the molecules can be realigned by an ultrasonic wave, leading to a change in the optical transmission through the cell. We present a model for this acousto-optic effect, and show that the magnitude of this effect is controlled by a director-density coupling. We then measure the optical transmission as a function of acoustic intensity for three liquid-crystal materials, and confirm that the data fit the functional form of the theoretical prediction. This fit gives the value of the director-density coupling, which varies greatly from material to material.

Alpha-adrenergic inhibition of proliferation in HepG2 cells stably transfected with the alpha1B-adrenergic receptor through a p42MAPkinase/p21Cip1/WAF1-dependent pathway.

Activation of alpha1B adrenergic receptors (alpha(1B)AR) promotes DNA synthesis in primary cultures of hepatocytes, yet expression of alpha(1B)AR in hepatocytes rapidly declines during proliferative events. HepG2 human hepatoma cells, which do not express alpha(1B)AR, were stably transfected with a rat alpha1B(AR) cDNA (TFG2 cells), in order to study the effects of maintained alpha(1B)AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [3H]thymidine incorporation into DNA. Stimulation of alpha(1B)AR with phenylephrine caused a further large reduction in TFG2 cell growth, whereas no effect on growth was observed in mock transfected cells. Reduced cell growth correlated with increased percentages of cells found in G0/G1 and G2/M phases of the cell cycle. In TFG2 cells, phenylephrine increased p42MAPkinase activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21Cip1/WAF1. Treatment of TFG2 cells with the specific MEKI inhibitor PD98059, or infection with a -/- MEK1 recombinant adenovirus permitted phenylephrine to increase rather than decrease [3H]thymidine incorporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 -/- blunted the ability of phenylephrine to increase p21Cip1/WAF1 expression. In agreement with a role for increased p21Cip1/WAF1 expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21Cip1/WAF1 mRNA blocked the ability of phenylephrine to increase p21Cip1/WAF1 expression and to inhibit DNA synthesis. Antisense p21Cip1/WAF1 permitted phenylephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn off the expression of alpha1B(ARs) in order to prevent the activation of a growth inhibitory pathway. Activation of this inhibitory pathway via alpha1B(AR) appears to be p42MAPkinase and p21Cip1/WAF1 dependent.

Sp1-mediated transcriptional activation from the dominant promoter of the rat alpha1B adrenergic receptor gene in DDT1MF-2 cells.

In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene.

Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.

Hir1p and Hir2p function as transcriptional corepressors to regulate histone gene transcription in the Saccharomyces cerevisiae cell cycle.

The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle. The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the absence of direct DNA binding. When Hir1p and Hir2p were artificially tethered to yeast promoters, each protein repressed transcription, suggesting that they represent a new class of transcriptional corepressors. The two proteins might function as a complex in vivo: Hir2p required both Hir1p and another Hir protein, Hir3p, to repress transcription when it was tethered to an HTA1-lacZ reporter gene, and Hir1p and Hir2p could be coimmunoprecipitated from yeast cell extracts. Tethered Hir1p also directed the periodic transcription of the HTA1 gene and repressed HTA1 transcription in response to two cell cycle regulatory signals. Thus, it represents the first example of a transcriptional corepressor with a direct role in cell cycle-regulated transcription.

Interaction between the C. elegans cell-death regulators CED-9 and CED-4.

Programmed cell death (apoptosis) is an evolutionarily conserved process used by multicellular organisms to eliminate cells that are not needed or are potentially detrimental to the organism. Members of the Bcl-2 family of mammalian proteins are intimately involved in the regulation of apoptosis, but, their precise mechanism of action remains unresolved. In Caenorhabditis elegans, the Bcl-2 homologue CED-9 prevents cell death by antagonizing the death-promoting activities of CED-3, a member of the Caspase family of death proteases, and of CED-4, a protein with no known mammalian homologue. Here we show that CED-9 interacts physically with CED-4. Mutations that reduce or eliminate CED-9 activity also disrupt its ability to bind CED-4, suggesting that this interaction is important for CED-9 function. Thus, CED-9 might control C. elegans cell death by binding to and regulating CED-4 activity. We propose that mammalian Bcl-2 family members might control apoptosis in a similar way through interaction and regulation of CED-4 homologues or analogues.

Chiral molecular self-assembly of phospholipid tubules: a circular dichroism study.

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure.

The rat alpha 1B adrenergic receptor gene middle promoter contains multiple binding sites for sequence-specific proteins including a novel ubiquitous transcription factor.

Transcription of the rat alpha 1B adrenergic receptor (alpha 1BAR) gene in the liver is controlled by three promoters that generate three mRNAs. The middle promoter (P2), located between -432 and -813 base pairs upstream from the translation start codon and lacking a TATA box, is responsible for generating the major, 2.7-kilobase mRNA-species expressed in many tissues (Gao, B., and Kunos, G. (1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver nuclear extracts identified three protected regions in P2: footprint I (-432 to -452), footprint II (-490 to -540), and footprint III (-609 to -690). Putative response elements in footprints I and III were not analyzed except the AP2 binding site in footprint III, which could be protected by purified AP2 protein. Footprint II contains four sites corresponding to half of the NF-I consensus sequence, but DNA mobility shift assays indicate that this footprint binds two proteins distinct from NF-I: a ubiquitous CP1-related factor and another novel factor, termed alpha-Adrenergic Receptor Transcription Factor (alpha ARTF), which binds to two separate sites in this region. The alpha ARTF is widely distributed, with the highest amounts found in brain, followed by liver, kidney, lung, and spleen, but no detectable activity in heart. Deletions of alpha ARTF binding sites nearly abolished P2 promoter activity, which suggests that the alpha ARTF is essential for the transcription of the alpha 1BAR gene in most tissues.

The HIR4-1 mutation defines a new class of histone regulatory genes in Saccharomyces cerevisiae.

Both activation and repression have been implicated in the cell cycle-regulated transcription of the histone HTA1-HTB1 locus in Saccharomyces cerevisiae. Transcriptional repressors have been identified through the isolation of recessive mutations in the HIR1, HIR2 and HIR3 genes. These three regulatory genes encode proteins that act at a negative site in the HTA1-HTB1 promoter, and their inactivation results in cell cycle-independent transcription. We report here on the characterization of a fourth HIR mutant. The HIR4-1 mutation is dominant, and the phenotypes that it confers suggest that the mutant gene encodes an altered transcriptional activator. The function of this activator is very specific: it uniquely regulates transcription of the HTA1-HTB1 locus, and it may antagonize repressors that act through the HTA1-HTB1 negative site.

Lipoprotein affinity of human apolipoprotein A-IV during cholesterol esterification.

We have studied the lipoprotein distribution of human apo A-IV during cholesterol esterification by the action of endogenous lecithin-cholesterol acyltransferase. Using immunologic and radiotracer techniques at 4 degrees C, apo A-IV was found in two discrete monomeric and dimeric populations, unassociated with plasma lipoproteins. With incubation at 37 degrees C, apo A-IV initially associated with the high density lipoprotein-3 fraction, but thereafter dissociated from its surface, and reappeared as unbound protein and in association with a complex in the low density lipoprotein size range. Inclusion of LCAT inhibitors in the incubations abolished these changes. We conclude that the changes in lipoprotein distribution of human apo A-IV closely parallel the formation and exchange of plasma cholesteryl esters.

The self-association of human apolipoprotein A-IV. Evidence for an in vivo circulating dimeric form.

We have investigated the self-association properties of human apolipoprotein A-IV using several complementary physical techniques. Sedimentation equilibrium analysis demonstrated that human apolipoprotein A-IV formed oligomeric species in aqueous solution at physiologic pH. Computer analysis established that the best model of self-association is a monomer-dimer-tetramer scheme, with an unusually large monomer-dimer association constant of 2.9 X 10(5) liters/mol. Fluorescence spectroscopy and electrophoretic analysis demonstrated that the rate of monomer-oligomer interconversion is sufficiently slow that a stable population of dimeric protein exists in solution, even at low total protein concentrations, and that the extent of dimerization is minimally influenced by pH. Moreover, these techniques established that the dissociation of oligomeric forms and the unfolding of the monomeric form are discrete and sequential events. In experiments where apolipoprotein A-IV was incubated with human high density lipoproteins, fractionated by gradient gel electrophoresis, and localized by immunoblotting, dimer formation occurred, but very little binding to lipoproteins was observed. Immunoblots of human serum fractionated on acrylamide gradient gels and isopycnic density gradients demonstrated an apolipoprotein A-IV band of size and density consistent with a circulating dimeric form, unassociated with lipid. We conclude that human apolipoprotein A-IV undergoes high affinity self-association in aqueous solutions, and that such self-association likely occurs in vivo. Self-association may thus be important in determining the biologic behavior of human apolipoprotein A-IV by influencing both the kinetics and distribution of its association with plasma lipoproteins.

Isoform heterogeneity and lipid affinity of human lymph and plasma apolipoprotein A-IV.

We have compared the physical properties and lipid affinity of apolipoprotein A-IV isolated from lymph chylomicrons and from lipoprotein-depleted plasma. Lymph and plasma apolipoprotein A-IV demonstrated distinctly different charge properties as assessed by anion exchange chromatography and isoelectric focusing. These differences were not attributable to disparities of amino acid or sialic acid content. Lymph apolipoprotein A-IV displayed a significantly higher affinity than plasma apolipoprotein A-IV for particles of a phospholipid-triglyceride emulsion. We conclude that the charge properties of human lymph and plasma apolipoprotein A-IV may determine conformational states which alter its ability to bind to the surface of lipid particles.

Structural properties and lipid binding of human apolipoprotein A-IV.

The in vivo affinity of human apolipoprotein A-IV (apo-A-IV) for plasma lipoproteins is considerably less than that of other apolipoproteins. We have therefore studied its spectroscopic properties and its association with model chylomicrons to investigate its structural characteristics and to define their influence upon its affinity for lipids. Fluorescence emission spectra of apo-A-IV in dilute aqueous solution revealed that its single tryptophan residue resides in a pH-sensitive hydrophobic domain, which is maximally protected from iodide quenching at pH 7.5. Denaturation of apo-A-IV by guanidine hydrochloride caused a multiphasic fluorescence emission red shift, with an unusual enhancement of quantum yield. Circular dichroism spectroscopy of apo-A-IV demonstrated negative ellipticity maxima at 210 and 222 nm, consistent with 54% alpha-helical structure. The alpha-helicity of apo-A-IV as measured by [theta]222 was also pH-sensitive and displayed a distinctive decrease between pH 7.0 and 8.0. Apo-A-IV was exquisitely sensitive to denaturation by guanidine hydrochloride, and its estimated free energy of stabilization in aqueous solution was near zero. Apo-A-IV bound to the surface of Sf greater than 400 particles of a phospholipid-triglyceride emulsion in a noncooperative, concentration-dependent manner. The affinity of apo-A-IV for these model chylomicrons was influenced by changes in pH or addition of guanidine hydrochloride in a manner which correlated well with the structural changes observed under similar conditions. We conclude that human apolipoprotein A-IV possesses several biophysical properties characteristic of the better studied plasma apolipoproteins, yet, apo-A-IV appears to be marginally stable in aqueous solution and its structural characteristics and lipid binding properties are particularly sensitive to environment.

Human apolipoprotein A-IV: displacement from the surface of triglyceride-rich particles by HDL2-associated C-apoproteins.

Human apolipoprotein A-IV rapidly dissociates from the surface of lymph chylomicrons following their entry into circulation by an unknown mechanism. We have therefore investigated the binding of human apoA-IV to triglyceride-rich particles and the interaction of these apoA-IV/lipid complexes with human HDL2. Human apoA-IV was purified from lipoprotein depleted serum (J. Lipid Res. 1983. 24:52-59). Triglyceride-rich particles of well-defined properties were isolated from Intralipid, a commercially available phospholipid-triglyceride emulsion. Various concentrations of radiolabeled human apoA-IV were incubated at 24 degrees C with a fixed quantity of lipid particles; the particles were reisolated by centrifugation, and bound and free apoA-IV were quantitated. In 50 mM Tris, pH 7.4, apoA-IV bound to the triglyceride-rich particles in a non-cooperative manner, with a Kd of 2.0 microM. The calculated maximal binding was 4.96 X 10(-4) mol of apoA-IV bound per mol of phospholipid. The addition of increasing amounts of human HDL2 to the incubations caused the progressive dissociation of apoA-IV from the triglyceride-rich particles. Analysis of the reisolated particles by isoelectric focusing demonstrated the presence of C-apoproteins, suggesting their transfer from HDL2. Addition of purified apoC-III-1 to the incubations at concentrations equivalent to those present in HDL2 caused a similar dissociation of apoA-IV. HDL2 was modified to selectively remove C-apoproteins, without alteration of other physical characteristics. This modified HDL2 was four times less effective in causing apoA-IV dissociation. These results demonstrate that the lipid binding properties of human apoA-IV may be quantitatively examined using triglyceride-rich particles as model chylomicrons. This approach reproduces in vitro the dissociation of apoA-IV that occurs in vivo when mesenteric lymph chylomicrons enter the circulation, and suggests that the primary mechanism for this phenomenon is the transfer of C-apoproteins from high density lipoproteins to the triglyceride-rich particle surface. We hypothesize that this mechanism may play an important role in the modulation of chylomicron apoA-IV content in man.