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H L Leffert

Publications and source records attributed to H L Leffert.

At least 19 recordsLinked to original sources
PubMed1992

Differential expression of the transfected liver-specific alpha 1-inhibitor III gene in normal hepatocytes and hepatoma cells in culture.

Normal and malignant hepatocytes were transfected during log phase culture with a nested series of DNA plasmids containing 5'-flanking regions of the rat liver-specific acute phase plasma proteinase alpha 1-inhibitor III (alpha 1 I3) gene. Under these conditions, luciferase reporter gene expression in primary adult rat and mouse hepatocytes was 10-fold higher than luciferase expression in hepatoma lines (human HepG2 and Hep3B; rat FAZA). Optimal expression in primary rat hepatocytes required regions stretching 2214 bp 5'-upstream of the transcription start site. Shorter 5'-flanking sequences were optimal for expression in hepatoma cells (-1025 and -186 bp for rat and human lines, respectively) and primary mouse hepatocytes (-225 bp). In contrast, regions from -186 to -225 bp drove luciferase expression in primary rat hepatocytes, but only 20-75% of optimal levels. Qualitative differences were unaccounted for by non-equivalent uptake of plasmid DNA, suggesting that tissue specific gene expression is regulated differently in normal and malignant cells, and with apparent species specificity.

Acute-Phase Proteins↗
PubMed1992

Induction of cyclin mRNA and cyclin-associated histone H1 kinase during liver regeneration.

Cyclins and cyclin-associated cdc kinases are key regulators of oocyte maturation (Maller, J. L. (1990) in The Biology and Medicine of Signal Transduction (Nishizuka, Y., Endo, M., and Tanaka, C., eds) pp. 323-328, Raven Press, New York), yeast cell cycles (Nurse, P. (1990) Nature 344, 503-508), DNA replication in cell-free systems (D'Urso, F., Marraccino, R. L., Marshak, R. R., and Roberts, J. M. (1990) Science 250, 786-791), and amphibian cell proliferative transitions (Hunt, T. (1991) Nature 350, 462-463). The extent to which these regulatory molecules participate in the growth control of differentiated epithelial cells like hepatocytes is unknown. Therefore, we investigated the expression of "G1" (E, C, and D) and "G2/M" (A, B1, and B2) cyclin mRNAs, the relative levels of cyclin A- and B1-associated histone H1-kinase activity, and the appearance of cyclin-associated kinases (p32/p33cdk2 and p33/p34cdc2) in regenerating rat liver and in control tissues from sham hepatectomized rats. To do this, we exploited a battery of human cyclin cDNAs and cyclin antisera that recognize rat molecules. The results suggest an apparent sequence of regeneration-specific changes: 1) elevated and induced expression of cyclins E (2.1 kilobases (kb)) and C (4 kb), and D mRNAs (4 kb), within 12 h, respectively; 2) induction of cyclins A (3.4 and 1.8 kb), B1 (2.5 and 1.8 kb), and B2 (1.9 kb) mRNAs at 24 h; 3) induction of cyclin A- and B1-associated nuclear histone H1 kinase at 24 h; and 4) enhanced levels of PSTAIRE-containing proteins of Mr approximately 32-33 and 33-34 kDa in nuclear extracts from 24-h regenerating liver that co-immunoprecipitate with cyclin A and B1 antisera, respectively. These observations provide an intellectual framework that unifies the biology of hepatocyte mitogenesis, proto-oncogene expression, and the machinery of the cell cycle.

Animals↗
PubMed1991

Induction of sodium pump beta 1-subunit mRNA expression during hepatocellular growth transitions in vitro and in vivo.

Previous suggestions (Hubert, J. J., Schenk, D. B., Skelly, H., and Leffert, H. L. (1986) Biochemistry 25, 4156-4163) of tissue-specific isoforms or nonexistence of hepatic Na,K-ATPase beta 1-subunits were reevaluated by quantifying beta 1-subunit mRNA levels in quiescent and proliferating liver. RNA was extracted from caudate liver lobes of sham or 67% hepatectomized adult rats and from primary cultures of adult rat hepatocytes that simulate developmental and regenerating growth transitions. Northern blot analysis with a 32P-labeled full-length Na,K-ATPase beta 1-cDNA probe (Mercer, R. W., Schneider, J. W., Savitz, A., Emmanuel, J., Benz, T.J., and Levenson, R. (1986) Mol. Cell. Biol. 6, 3884-3890) revealed four (approximately 2.7, 2.4, 1.7-1.8, and 1.5 kilobases) low abundance mRNA species in quiescent tissue, freshly isolated hepatocytes, and cultured hepatocytes derived from lag or late stationary phase (1-2 days or 11-12 days postplating, respectively). In contrast, proliferating liver from 4 h post-67% hepatectomized rats or cultured hepatocytes in logarithmic growth phase contained levels of beta 1-subunit mRNA which exceeded quiescent levels by 4-35-fold. Membrane Na,K-ATPase activity also increased 2-3-fold during liver regeneration 12-24 h after partial hepatectomy. When proliferation in vitro was augmented by transforming growth factor-alpha, a hepatocyte mitogen, or reinitiated in late stationary phase by a change to fresh culture medium containing rat serum, beta 1-subunit mRNA expression was restimulated 4-20-fold. Parallel measurements of alpha-tubulin mRNA induction showed relatively nonsynchronous or invariant changes during hepatocyte proliferative transitions; similar results were obtained after Northern blots with a sodium pump alpha I-subunit cDNA probe. No detectable hybridization signals were observed when either rat kidney or hepatocyte RNAs from freshly isolated and cultured cells or regenerating tissues were probed for the sodium pump 3.4-kilobase mRNA beta 2-isoform. These observations suggest that enhanced hepatic beta 1-subunit gene expression is linked specifically to growth-associated increases in Na,K-ATPase activity, hepatocyte proliferation, and mitogen activation.

Animals↗
PubMed1991

Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes.

Retroviral vectors were used to transduce recombinant DNA encoding firefly luciferase, Escherichia coli beta-galactosidase or human factor IX into fetal rat hepatocytes in primary culture. Hepatocytes were transduced optimally during a restricted time interval, 2-4 days post-plating. Although efficient and stable expression of reporter gene products was observed in vitro, it was affected differentially by culture conditions (plating density, media constituents) and chemical modulators of hepatocyte growth and differentiation (gelatin, hydrocortisone, isobutylmethylxanthine). Cultured cells, mock-infected or infected with a luciferase-expressing vector, were harvested non-enzymatically and injected subcutaneously into the dorsal neck fascia of neonatal syngeneic rats. Tissue isolated from injection sites one week later contained hepatocyte foci. In animals transplanted with infected cells, the preliminary results suggest that luciferase activity was present at these sites in proportion to the numbers of injected cells. These findings and previous observations made with hepatocytes from neonatal and adult primary cultures, indicate that from day 19 in utero through maturity the transient temporal 'period of susceptibility' to infection in vitro is independent of the developmental state of starting tissue. Transplantability of cultured fetal hepatocytes infected with retroviral vectors and stably expressing reporter gene products suggests that such cells might provide promising models for liver gene therapy.

Animals↗
PubMed1990

DNA-mediated gene transfer into adult rat hepatocytes in primary culture.

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.

Animals↗
PubMed1990

Expression of collagen genes in the liver.

Hepatic production of type I collagen is markedly increased in liver cirrhosis. Previous studies using primary liver cell cultures have demonstrated that hepatocytes, lipocytes and endothelial cells are all capable of producing collagen. In this study in situ hybridization and hepatic cell sorting have been used to identify which cells are expressing the type I collagen gene, alpha 1(I), in normal rat liver. Northern blotting of mRNAs from purified hepatic cell populations demonstrated that both hepatocytes and several types of non-parenchymal cells express the collagen alpha 1(I) gene. Calculations based on cell numbers, yields of mRNA, and cellular mRNA concentration demonstrated that the majority of collagen alpha 1(I) mRNA originates from the hepatocytes in the normal liver. Localization of a collagen alpha 1(I) mRNA by in situ hybridization confirmed that both hepatocytes and non-parenchymal cells express this gene. Furthermore, collagen alpha 1(I) gene expression in hepatocytes was obtained by transfecting a reporter gene driven by the collagen alpha (I) 5' regulatory segment in primary liver cell cultures. Future experiments will further characterize the regulation of collagen alpha 1(I) gene expression in the liver.

Animals↗
PubMed1989

Vasopressin inhibits type-I collagen and albumin gene expression in primary cultures of adult rat hepatocytes.

The mechanisms that regulate collagen gene expression in hepatic cells are poorly understood. Accelerated Ca2+ fluxes are associated with inhibiting collagen synthesis selectively in human fibroblasts (Flaherty, M., and Chojkier, M. (1986) J. Biol. Chem. 261, 12060-12065). In suspension cultures of isolated hepatocytes, the Ca2+ agonist vasopressin increases cytosolic levels of free Ca2+ (Thomas, A.P., Marks, J.S., Coll, K.E., and Williamson, J. R. (1983) J. Biol. Chem. 258, 5716-5725). However, whether vasopressin's interactions with plasma membrane V1 receptors attenuate hepatic collagen production is unknown. We investigated this problem by studying vasopressin's effects on collagen synthesis and Ca2+ efflux in long-term primary cultures of differentiated and proliferation-competent adult rat hepatocytes. Twelve-day-old quiescent cultures were exposed to test substances and labeled with [5-3H]proline. Determinations of radioactivity in collagenase-sensitive and collagenase-resistant proteins were used to calculate the relative levels of collagen production. Synthetic [8-arg]vasopressin stimulated 45Ca2+ efflux within 1 min and inhibited hepatocyte collagen production within 3 h by 50%; overall rates of protein synthesis were not affected significantly. In cultures labeled with [35S]methionine, vasopressin also decreased the levels of newly synthesized and secreted albumin, but not fibrinogen, detected in specific immunoprecipitates analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Northern blot analyses using specific [32P]cDNA probes revealed 70% decreases in hybridizable levels of collagen alpha 1(I) mRNA in hepatocyte cultures treated with either vasopressin or Ca2+ ionophore A23187; hybridizable levels of albumin mRNA also fell approximately 50% following vasopressin treatment. Vasopressin did not affect collagen production in quiescent cultures of mouse Swiss 3T3, human myofibroblast or rat smooth muscle cells; and hepatocyte collagen production was unaffected by treatment with glucagon or dibutyryl cAMP. Thus, accelerated Ca2+ fluxes induced by vasopressin are associated with decreased production of hepatocyte collagen and albumin in primary cultures that simulate quiescent adult rat liver.

Aging↗
PubMed1989

Transforming growth factor-alpha stimulates proto-oncogene c-jun expression and a mitogenic program in primary cultures of adult rat hepatocytes.

Human transforming growth factor-alpha (TGF-alpha, MW 5547) initiates a mitogenic program in "quiescent" 11-to 13-day-old primary cultures of adult rat hepatocytes. Using validated growth reinitiation assays and chemically defined conditions (Koch and Leffert, 1979a) that simulate proto-oncogene expression in regenerating liver (Kruijer et al., 1986), we find that 5.4 nM TGF-alpha stimulates: (i) increases in rates of amiloride-sensitive 22Na+ uptake; (ii) a transient induction in steady-state mRNA levels of proto-oncogene c-jun; (iii) specific increases in hepatocyte nuclear [3H]dT labeling indices, augmented synergistically by insulin and glucagon; and (iv) increases in rates of S-phase entry. Comparative studies indicate that TGF-alpha is a more effective hepatocyte growth promoter than mouse epidermal growth factor. These observations, and published reports linking normal and cancerous liver as biosynthetic sources of TGF-alpha, suggest an autocrine or paracrine role for TGF-alpha in the control of hepatic growth, regeneration, and gene expression.

Animals↗
PubMed1988

Biochemical localization of hepatic surface-membrane Na+,K+-ATPase activity depends on membrane lipid fluidity.

Membrane proteins of transporting epithelia are often distributed between apical and basolateral surfaces to produce a functionally polarized cell. The distribution of Na+,K+-ATPase [ATP phosphohydrolase (Na+/K+-transporting), EC 3.6.1.37] between apical and basolateral membranes of hepatocytes has been controversial. Because Na+,K+-ATPase activity is fluidity dependent and the physiochemical properties of the apical membrane reduces its fluidity, we investigated whether altering membrane fluidity might uncover cryptic Na+,K+-ATPase in bile canalicular (apical) surface fractions free of detectable Na+,K+-ATPase and glucagon-stimulated adenylate cyclase activities. Apical fractions exhibited higher diphenylhexatriene-fluorescence polarization values when compared with sinusoidal (basolateral) membrane fractions. When 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) was added to each fraction, Na+,K+-ATPase, but not glucagon-stimulated adenylate cyclase activity, was activated in the apical fraction. In contrast, further activation of both enzymes was not seen in sinusoidal fractions. The A2C-induced increase in apical Na+,K+-ATPase approached 75% of the sinusoidal level. Parallel increases in apical Na+,K+-ATPase were produced by benzyl alcohol and Triton WR-1339. All three fluidizing agents decreased the order component of membrane fluidity. Na+,K+-ATPase activity in each subfraction was identically inhibited by the monoclonal antibody 9-A5, a specific inhibitor of this enzyme. These findings suggest that hepatic Na+,K+-ATPase is distributed in both surface membranes but functions more efficiently and, perhaps, specifically in the sinusoidal membranes because of their higher bulk lipid fluidity.

Animals↗
PubMed1986

Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping.

The catalytic alpha-subunit of rat hepatic (Na+, K+)-ATPase (EC 3.6.1.3) has been isolated by immunoaffinity chromatography from microsomes solubilized in n-dodecyl octaethylene glycol monoether. The procedure employs an anticatalytic mouse monoclonal antibody ("9-A5") covalently linked to Sepharose 4B that specifically blocks phosphorylation of the sodium pump's alpha-subunit from [gamma-32P]ATP [Schenk, D. B., Hubert, J.J., & Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951]. The hepatic subunit is virtually identical with purified rat, dog, and human renal alpha-subunits as judged by its apparent molecular weight after polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Mr 92K) and its two-dimensional tryptic and chymotryptic peptide maps on cellulose-coated thin-layer plates. In contrast, the structures of authentic renal beta-subunits from the three species differ significantly from each other as judged by their peptide maps; no detectable homologies are seen between their chymotryptic maps and those of putative hepatic "beta"-subunits (Mr 50K and 55K) eluted from 9-A5-Sepharose. Additional studies of ouabain-sensitive 86Rb+ uptake in primary cultures of adult rat hepatocytes reveal inhibition curves with single inflection points (ID50 = 0.1 mM ouabain) in the absence or presence of pump-stimulating peptides like insulin, glucagon, and epidermal growth factor. These findings indicate that rat hepatocytes express only one of two known structurally conserved forms of catalytic subunit (the renallike alpha form) and, if at all, structurally divergent forms of the sodium pump's beta-subunit. In addition, immunoaffinity chromatography with 9-A5-Sepharose facilitates the isolation of (Na+, K+)-ATPases from nonrenal tissues with low levels of sodium pumps.

Animals↗
PubMed1986

Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes.

Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions.

Animals↗
PubMed1979

Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation.

Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a "burst" of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid "A" transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]

Amiloride↗
PubMed1979

Liver cells.

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Animals↗