Interferon alpha induces the expression of retinoblastoma gene product in human Burkitt lymphoma Daudi cells: role in growth regulation.

Interferon alpha (IFN-alpha) is a regulatory secretory protein with distinctive biological effects such as antiproliferative actions against many tumor cell lines, including human Burkitt lymphoma Daudi cells. The mechanism underlying growth inhibition by IFN-alpha is not well established. The growth of many mammalian cell types is also regulated by tumor suppressor retinoblastoma (RB) gene product, the RB protein. In the studies presented here, we explored the possible involvement of RB protein in the growth inhibitory action of IFN-alpha in the Daudi cell model system. We observed that IFN-alpha induces a 3- to 10-fold increased expression of RB protein in growth-sensitive Daudi cells but not in the growth-resistant variant of Daudi cells. IFN-alpha-mediated induction of RB protein was an early event that preceded the period of growth inhibition of Daudi cells. IFN-alpha-induced RB protein predominantly exists as the underphosphorylated form. Addition of antibody against IFN-alpha to Daudi cells resulted in the inhibition of constitutive expression of RB protein and stimulation of [3H]thymidine incorporation. These results demonstrate that the induction of RB protein expression in IFN-alpha-treated Daudi cells could constitute an important mechanism of IFN-alpha-mediated growth regulation.     

Assembly and GPIIIa content of cytoskeletal core in platelets agglutinated with bovine von Willebrand factor

The association between occupancy of the von Willebrand factor (vWf) receptor glycoprotein (GP) Ib, agglutination, and the assembly and composition of the cytoskeletal core was studied in 125I-surface-labeled aspirin-treated washed platelets. Binding of ligands to GPIIb-IIIa and platelet aggregation were abolished by addition of EDTA. Platelet agglutination induced by bovine vWf generated a complete cytoskeletal core (Triton-insoluble residue), shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be composed of actin-binding protein (ABP) (260 Kd), 235-Kd protein, myosin heavy chain (200 Kd), alpha-actinin (100 Kd), and actin (43 Kd). In addition, autoradiography of the gels showed a 125I 105-Kd GP, identified by immunoblot as GPIIIa, as well as GPIb, GPIIb, and another band at 87 Kd, probably GPIV. Neither cytoskeletal assembly nor GPIIa incorporation was altered if calpain was inhibited with leupeptin. Platelet suspensions exposed to bovine vWf without stirring (ie, nonagglutinated) or platelets in which agglutination was inhibited with ADP showed smaller cytoskeletons with little ABP, 235 Kd protein, and alpha-actinin. Autoradiographs showed mainly GPIb. Cytochalasin D (CD) and monobromobimane (MB) enhanced agglutination and prevented the inhibitory action of ADP on bovine vWf-induced platelet agglutination. CD markedly inhibited the assembly of the cytoskeletal core as well as GPIIIa retention, whereas MB resulted in a large Triton-insoluble residue which contained GPIIIa. Thus, development of a platelet cytoskeletal core is apparently not required for agglutination, but when a cytoskeletal core is assembled in agglutinated platelets, GPIIIa is retained.     

Interferon-alpha-induced changes in surface antigens in a hairy-cell leukemia (JOK-1), and a Burkitt's lymphoma cell line (Daudi) during in vitro culture.

In further studying the mechanism of action of IFN-alpha in HCL, we cultured the HCL cell line JOK-1 and the IFN-sensitive Burkitt cell line Daudi with and without IFN-alpha and investigated the changes in density of a number of surface antigens by use of mAb and flow cytometry analyses. During culture with IFN-alpha, reproducible changes were induced in both cell lines, which were qualitatively similar but differed quantitatively with small and transient changes in JOK-1. Significant decreases in surface antigen expression were observed for CD 19, 23, 37, and for IgM on both cell lines. Moreover, decreases were seen for CD 10, 22, 45, and MHC class II on Daudi, and for CD 20, 21, 27, and 40 on JOK-1. By contrast, only a few antigens increased in density, including CD 39, A96/G8 and SC9, on both cell lines, CD 22 on JOK-1, and CD 21 on Daudi. The increase in CD 39, A96/G8 and SC9 was probably directly related to the mechanism of action of IFN-alpha, whereas the other changes were most consistent with an unspecific inhibition of protein synthesis, possibly due to an accumulation of cells in G0, even though a differentiating effect cannot be ruled out. Thus, the unique in vivo effect of IFN-alpha in HCL was not paralleled by a specific direct effect on JOK-1 in vitro. Our findings therefore do not support the theory that IFN's mechanism of action in vivo is a direct effect on HC, but suggest that indirect effects are involved.     

The effect of alpha and gamma-interferon on proliferation and production of IgE and beta 2-microglobulin in the human myeloma cell line U-266 and in an alpha-interferon resistant U-266 subline

An IFN-resistant subline (U-266r alpha) was established from the IFN-alpha-sensitive myeloma cell line U-266 by subculturing U-266 cells with increasing doses of INF-alpha. The U-266r alpha secreted IgE at a higher rate than the U-266 (7.2 X 10(-13) g/c/8 h as compared to 3.3 X 10(-13) g/c/8 h). The 2 cell lines were found to be equally high producers of beta 2m (9.2 and 9.6 X 10(-13) g/c/8 h). The U-266 produced 2.9 times less IgE and 5 times more beta 2m compared to the initial production rates at establishment. INF-alpha and recombinant IFN-alpha 2 (rIFN-alpha 2) inhibited proliferation and concomitantly decreased the rate of IgE and beta 2m secretion in U-266 but not in U-266 IFNr alpha, which in contrast was slightly stimulated by IFN-alpha with respect to growth, IgE and beta 2m secretion. In addition, IFN-alpha at a concentration of 100 U/ml was shown to decrease the IgE and beta 2m production without exerting more than minimal cytotoxicity on U-266 cells. No antiproliferative effect was found for IFN-gamma or recombinant IFN-gamma (rIFN-gamma) on either of the 2 cell lines. IFN-gamma and rIFN-gamma were, however, found to stimulate the production of beta 2m. Our results show that the U-266 and the derived IFN-alpha-resistant subline can be used as models for studying some of the biological effects of IFN-alpha and -gamma in vitro. The clinical implications of these in vitro results, in particular the usefulness of serum determinations of immunoglobulin and beta 2m concentrations for monitoring the tumor cell mass, are discussed.     

Contribution of negative cooperativity to the thyrotropin-receptor interaction in normal human thyroid: kinetic evaluation.

The kinetics of 125I-labeled thyrotropin (125I-TSH) binding to human thyroid receptors are presented. At pH 6.0, binding was maximal (30--35%) and there was one class of binding sites [Kd = 6.8 X 10(-9) M; binding capacity (Ro) = 57 pmol/mg of protein]. At pH 7.4, Scatchard plots of binding were nonlinear, indicating either a single class of negatively cooperative sites (Kd = 3.7 X 10(-9) M; Ro = 26 pmol/mg of protein) or, alternatively, independent high- (Kd = 5.0 X 10(-10) M; Ro = 3 pmol/mg of protein) and low-affinity (Kd = 1.7 X 10(-8) M; Ro = 26 pmol/mg of protein) binding sites. The role of negative cooperativity was evaluated from the rates of association and dissociation at pH 7.4. The kinetically determined binding constants (Kd = 1.7 X 10(-11) M; Ro = 2 pmol/mg of protein) were more similar to those determined for the high-affinity component than to those predicted from the negative cooperativity model. Dissociation of bound TSH was independent of initial site occupancy over a 40-fold range, corresponding to a 100-fold range of free TSH concentration. The dissociation rate of 125I-TSH was enhanced by unlabeled TSH to a similar degree, irrespective of initial binding site occupancy. Because the negative cooperativity model does not accommodate these data, it is concluded that TSH receptors in human thyroid behave kinetically and at equilibrium as a single class of high-affinity sites up to TSH concentrations well above the physiological range.     

Binding of thyroid hormones to nuclear extracts of thyroid cells

Binding of T3 and T4 to soluble nuclear extracts of FRTL-5 cells, rabbit thyroid glands, and rat liver was studied. [125I]Iodo-T3 or [125I]iodo-T4 in concentration ranges of 100-fold (10-fold on each side of measured Kd) was incubated with extract at 4 C, pH 8.2, and the quantity of bound hormone was determined by collection on nitrocellulose filters. The results were corrected for nonspecific binding. Steady state (equilibrium) binding was achieved by 36 h. Apparent dissociation constants (Kd) were determined from Scatchard analysis of data pertaining to extent of binding at 36 h as a function of hormone concentration and were also calculated from kinetics of binding as the ratio of rate constants. A single class of saturable, high affinity hormone-binding sites was found. Kd values for T3 and nuclear extracts of FRTL-5 cells, rabbit thyroid gland, and rat liver were, respectively, 3.9 X 10(-11) M, 2.8 X 10(-11) M, and 4.3 X 10(-11) M from Scatchard analysis; when calculated from kinetics of hormone association, the value was 3.6 X 10(-11) M for both FRTL-5 cell and rat hepatic nuclear extract. No analysis of the time course of binding of T3 to rabbit thyroid nuclear extract was made. Kd values for T4 and FRTL-5 cell extract were 6.2 X 10(-10) M from Scatchard analysis and 5.0 X 10(-10)M from kinetic data. Half-times (t1/2) of dissociation of T3 from FRTL-5 cell and rat liver nuclear extract, calculated from association curves, were 7 and 5 h, respectively, while corresponding values determined directly and experimentally were 10.5 and 13 h. For T4 and FRTL-5 cell extract, the t1/2 of dissociation calculated from kinetics of association was 5 h; no direct experimental determination of the value was made. Numbers of T3-binding sites of FRTL-5 cell, rabbit thyroid gland, and rat liver nuclear extracts were, respectively, 71 X 10(-15), 62 X 10(-15), and 208 X 10(-15) mol/mg protein. For T4 and FRTL-5 cell extract, the value was 70 X 10(-15) mol/mg protein. The data indicate that the reaction of T3 and T4 with the various nuclear extracts can be described as reversible and bimolecular. The presence in thyroid cells of thyroid hormone nuclear binding sites suggests that they may be receptors that mediate cellular actions of these hormones within the gland itself.     

Expression and signaling specificity of the IFNAR chain of the type I interferon receptor complex.

The IFNAR chain of the type I interferon (IFN) receptor (IFNIR) undergoes rapid ligand-dependent tyrosine phosphorylation and acts as a species-specific transducer for type I IFN action. Using the vaccinia/T7 expression system to amplify IFNAR expression, we found that human HeLa-S3 cells transiently express high levels of cell surface IFNAR chains (approximately 250,000 chains per cell). Metabolic labeling and immunoblot analysis of transfected HeLa cells show that the IFNAR chain is initially detected as 65-kDa and 98-kDa precursors, and then as the 130-kDa mature protein. Due to variation in N-glycosylation, the apparent molecular mass of the mature IFNAR chain varies from 105 to 135 kDa in different cells. IFNIR structure was characterized in various human cell lines by analyzing 125I-labeled IFN cross-linked complexes recognized by various antibodies against IFNIR subunits and JAK protein-tyrosine kinases. Precipitation of cross-linked material from Daudi cells with anti-IFNAR antibodies showed that IFNAR was present in a 240-kDa complex. Precipitation of cross-linked material from U937 cells with anti-TYK2 sera revealed a 240-kDa complex, which apparently did not contain IFNAR and was not present in IFN-resistant HEC1B cells. The tyrosine phosphorylation and down-regulation of the IFNAR chain were induced by type I IFN in several human cell lines of diverse origins but not in HEC1B cells. However, of type I IFNs, IFN-beta uniquely induced the tyrosine phosphorylation of a 105-kDa protein associated with the IFNAR chain in two lymphoblastoid cell lines (Daudi and U266), demonstrating the specificity of transmembrane signaling for IFN-beta and IFN-alpha through the IFNAR chain.     

Receptors for 17beta-estradiol in prolactin-secreting rat pituitary cells

Estrogens stimulate prolactin (PRL) synthesis by GH3 cells, a clonal strain of rat pituitary cells grown in culture. At 4 degrees C the binding of [3H]17 beta-estradiol to monolayer cultures of GH3 cells was specific and of limited capacity, with half-maximal and maximal binding after 1--2 h and 12 h, respectively. Scatchard analysis showed one single class of binding sites with Kd = 3.1 X 10(-10) M and n = 309 X 10(-15) mol 17 beta-estradiol/mg cell protein, calculated to give approx. 25,000 binding sites per cell. At 4 degrees C less than 10% of the specifically bound [3H]17 beta-estradiol was found in the nuclear fraction. When the incubation temperature was raised to 37 degrees C, the amount of radioactivity in the nucleus increased to 25% within 30 min with a corresponding reduction in the cytoplasm. The cytosol fractions from monolayer cultures as well as from tumors of GH3 cells contained specific 17 beta-estradiol binding proteins, having a sedimentation constant close to 8S in a salt-free buffer and 4S in the presence of 0.5 M KCl. scatchard analysis showed one single class of binding sites with Kd = 3.6 X 10(-10) M and n = 258 X 10(-15) mol 17 beta-estradiol/mg cytosol protein (GH3 tumor tissue). Thus, GH3 cells grown in culture and in the intact animal have similar binding characteristics as judged from the data for binding affinity, capacity and specificity. After the in vivo administration of [3H]17 beta-estradiol to GH3 tumor-bearing rats, radioactivity could be extracted (0.5 M KCl) from purified nuclei bound to 4.5S macromolecules. We suggest that the action of 17 beta-estradiol on GH3 cells involves an initial binding of the steroid to specific receptors in the cytoplasm, followed by transport of a fraction of the hormone-receptor complexes to the nucleus involving a temperature-sensitive step.     

Monoclonal human thyroid cell line GEJ expressing human thyrotropin receptors.

By using the hybridoma technology, a monoclonal human thyroid cell line was obtained by fusing fresh normal human thyroid cells with a human lymphoblastoid cell line. The resultant cell line, called GEJ, has been selected for its expression of thyrotropin (TSH) receptors and has morphological and functional characteristics of normal human thyroid cells. In the presence or absence of human TSH, the GEJ cell line accumulates iodide, produces thyroid hormones, expresses thyroid membrane antigens, and binds approximately equal to 600 molecules of TSH per cell. The binding site for TSH has all the characteristics of specific receptor (i.e., temperature and time dependencies, dissociation of bound TSH only by high amounts of TSH, and a glycoprotein nature). Scatchard analysis described a curvilinear graph with two dissociation constants (Kd = 0.12 X 10(-9) M and 1.6 X 10(-9) M) with, respectively, 1.2 X 10(3) and 7.2 X 10(3) binding sites per cell. This human thyroid cell line that expresses TSH receptors could be a useful tool for the study of human thyroid disorders.     

Protein malnutrition in the domestic fowl induces alterations in adrenocortical cell adrenocorticotropin receptors

Our previous work suggests that persistent protein malnutrition in immature domestic fowl (Gallus gallus domesticus) alters ACTH-adrenocortical cell interaction, possibly including ACTH receptors. To investigate this possibility, we measured some ACTH receptor parameters in isolated adrenocortical cells from normal and dietary protein-restricted domestic fowl. White Leghorn cockerels (2 weeks old) were fed isocaloric semipurified diets containing either 8% [low (L)] or 20% [normal (N)] soy protein for 4 weeks ad libitum. Cockerels were quickly killed by decapitation and exsanguination, and adrenal glands were removed and prepared for cell isolation. Highly enriched (greater than 80% pure) adrenocortical cells (collagenase isolated, followed by separation on a Percoll continuous density gradient) were evaluated for ACTH receptors using pharmacological and radioligand approaches. In a pharmacological approach, we measured the influence of the complete, competitive antagonist, human (h) ACTH-(7-38) on hACTH-(7-39)-induced corticosterone production by adrenocortical cells from L and N cockerels. Inhibitor constants of hACTH-(7-38), calculated from Schild plots, were 3.16 X 10(-7) and 9.82 X 10(-7) M for L and N cockerel cells, respectively, thus suggesting differences in ACTH receptor function between the two treatment groups. To characterize ACTH receptors directly, we measured the binding of a monoiodinated ACTH analog [125I-Tyr23]hACTH-(1-39) to domestic fowl adrenocortical cells. Binding was linear with cell concentration, highly specific (only ACTH peptides caused significant displacement), rapid (maximal binding by 1 h), reversible (half-time of dissociation, approximately 40 min), and saturable. Curvilinear Scatchard plots were obtained, and vectorial analysis resolved both high and low affinity sites. The concentrations (femtomoles per 50 micrograms DNA) and dissociation constants (Kd) of both classes of sites were different between N and L bird cells. Values of these receptor parameters for N and L cockerel cells were, respectively, as follows: concentrations of low affinity sites, 7.45 and 11.60; concentrations of high affinity sites, 3.16 and 5.50; Kd of low affinity sites, 2.05 X 10(-8) and 2.58 X 10(-9) M; Kd of high affinity sites, 1.01 X 10(-9) and 1.27 X 10(-10) M. Thus, the overall binding capacity of L bird cells was 65% greater than that of N bird cells. In addition, the overall affinity (1/Kd) of sites of L bird cells was 9 times that of sites of N bird cells. These data indicate that persistent protein malnutrition in the domestic fowl increased both the number and affinity of adrenocortical cell ACTH receptors.     

Insulin receptors in fetal rabbit lung type II cells

Insulin binding was studied in type II pneumocytes isolated from fetal rabbit lungs (27 days of gestation) and grown in monolayers in tissue culture. The mean high affinity receptor site number was 11.8 +/- 1.4 X 10(3) (+/-SEM)/cell, with a Kd of 0.45 +/- 0.07 nM (n = 6). Low affinity sites averaged 432 +/- 10.7 X 10(3)/cell, with a Kd of 45.6 +/- 11.8 nM. Incubation of the cells with 5 X 10(-10) M (Bu)2cAMP (DBcAMP) and 10(-3) M methylisobutylxanthine (MIX) for 18 h led to significant increases in the number of high affinity receptor sites and Kd (P = 0.025 and 0.05, respectively). Incubation of the cells with insulin (1 microgram/ml) for 18 h led to a significant diminution in the mean number of high affinity sites to 3.23 +/- 0.68 X 10(3)/cell (P = 0.0025). There was no significant change in the Kd of the high affinity sites. There was also no significant change in the number or affinity of the low affinity sites. When the cells were incubated with insulin in the presence of DBcAMP (5 X 10(-4) M) and MIX (10(-3) M), there was a significant increase in high affinity binding sites to a mean of 8.87 +/- 2.18 X 10(3)/cell (n = 4) compared to the value after incubation in the presence of insulin alone. There was no significant increase in the Kd of the high affinity sites. The following conclusions were drawn from these experiments. 1) Fetal type II pneumocytes possess receptors with high affinity for insulin. 2) The up-regulation of insulin receptor binding induced by high ambient concentrations of insulin in vivo in rabbit fetal lungs and circulating human monocytes does not occur in vitro when isolated pneumocytes are grown in tissue culture. 3) Insulin binding to type II pneumocytes is enhanced by DBcAMP and MIX. 4) Insulin down-regulation of receptor binding is significantly counteracted by DBcAMP and MIX.     

Interleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high-affinity receptors.

Interleukin 2 (IL-2) receptors on activated T cells exist in high- and low-affinity configurations, both of which share a ligand-binding component known as the Tac protein. Although almost all binding of IL-2 to such cells was inhibited by an antibody to Tac, the predominant component of binding on the natural killer (NK)-like cell line YT was resistant to this reagent. The ligand-binding component on YT cells also differed from Tac in its affinity constant (Kd approximately 8.2 X 10(-10) M vs. Kd approximately equal to 1.1 X 10(-8) M for low-affinity Tac sites) and in its susceptibility to inhibition by certain antibodies to IL-2. When the YT cells were stimulated in a manner that induced expression of the Tac protein, the IL-2 binding sites were converted to a high-affinity configuration (Kd approximately 1.8 X 10(-11) M). Thus, the original binding component on unstimulated YT cells appeared to combine with Tac and IL-2 to produce a high-affinity receptor complex. Use of bifunctional crosslinking agents following ligand binding to unstimulated YT cells yielded covalent IL-2-receptor complexes of 83 and 90 kDa. These complexes were similar in size to those derived from high-affinity receptors on activated T cells and shared a similar fragmentation pattern upon proteolysis. These results demonstrate the existence of a second IL-2 binding component in addition to the Tac protein and suggest that this component combines with Tac and IL-2 to form high-affinity receptor sites.     

Interferons alpha and beta down-regulate the expression of basic fibroblast growth factor in human carcinomas.

We investigated the influence of interferons alpha, beta, and gamma (IFN-alpha, -beta, and -gamma) on the production of basic fibroblast growth factor (bFGF) by human renal carcinoma cells. The human renal carcinoma cell metastatic line SN12PM6 was established in culture from a lung metastasis and SN12PM6-resistant cells were selected in vitro for resistance to the antiproliferative effects of IFN-alpha or IFN-beta. IFN-alpha and IFN-beta, but not IFN-gamma, down-regulated the expression of bFGF at the mRNA and protein levels by a mechanism independent of their antiproliferative effects. Down-regulation of bFGF required a long exposure (> 4 days) of cells to low concentrations (> 10 units/ml) of IFN-alpha or IFN-beta. The withdrawal of IFN-alpha or IFN-beta from the medium permitted SN12PM6-resistant cells to resume production of bFGF. The incubation of human bladder, prostate, colon, and breast carcinoma cells with noncytostatic concentrations of IFN-alpha or IFN-beta also produced down-regulation of bFGF production.     

Cell-surface-expressed T-cell antigen-receptor zeta chain is associated with the cytoskeleton.

The T-cell antigen receptor zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. zeta chain can associate with certain protein tyrosine kinases and retains the capacity to transduce signals independently of the other receptor subunits. Thus, zeta chain could couple cell-surface-expressed T-cell antigen receptors to the intracellular signal-transduction apparatus by its association with various intracellular molecules in addition to tyrosine kinases. In the process of searching for zeta chain-associated molecules we observed that after lysis of resting T cells with Triton X-100, zeta chain is localized in the detergent-insoluble fraction, in addition to its presence in the detergent-soluble fraction. Treatment of T cells with cytochalasin B, an actin-depolymerizing agent, leads to the complete dissociation of zeta chain from the Triton-insoluble fraction, suggesting a linkage between zeta chain and the cytoskeletal matrix. We have also determined that cytoskeletal-associated zeta chain is expressed on the cell surface. Furthermore, a tyrosine-phosphorylated 16-kDa zeta chain was detected only in the Triton-insoluble cytoskeletal fraction of resting T cells. zeta chain also maintains its association with the cytoskeleton when expressed in COS cells, inferring that the cytoskeletal elements involved in this linkage may be ubiquitous. Finally, we have localized a 42-amino acid region in the intracytoplasmic domain of zeta chain, which is crucial for maximal interaction between zeta chain and the cytoskeleton. Anchorage of cell-surface-expressed zeta chain to the cytoskeleton in resting T cells may facilitate recycling of receptor complexes and/or allow the transduction of external stimuli into the cell.     

Impairment in coupled beta-adrenergic receptor and adenylate cyclase system during bleomycin-induced lung fibrosis in hamsters

The purpose of this study was to evaluate the coupled beta-adrenergic receptor (BAR) and adenylate cyclase (AC) system of the lung during the course of the bleomycin-(Bleo) induced pulmonary fibrosis in hamsters. The BAR population, dissociation constants (Kd), AC activity, and its sensitivity to various stimulators were studied at 2, 4, 7, 14, and 21 days after intratracheal administration of either 1 unit of Bleo or an equivalent volume of saline. The BAR population in the lungs of Bleo-treated animals did not differ from control at the early times, but it was significantly reduced to 5.9 X 10(3) fmol and 3.6 X 10(3) fmol from the control values of 1.1 X 10(4) fmol and 1.5 X 10(4) fmol per lung at 14 and 21 days after treatment, respectively. The Kd values for control hamster lung ranged from 2.5 X 10(-11) M to 3.7 X 10(-11) M, and for Bleo-treated hamster lung, from 2.7 X 10(-11) M to 4.8 X 10(-11) M. The Kd at the earliest time, 2 days after treatment, did not differ significantly from the Kd values at the subsequent times in control, while for Bleo-treated hamster lung, the Kd values at 7, 14, and 21 days were significantly higher than the Kd at 2 days after treatment. The Kd values for Bleo-treated hamster lung were also significantly higher than control at 14 and 21 days. The AC activity of the lung in Bleo-treated hamster was significantly reduced to 67%, 40%, 38%, and 50% of their respective controls in response to H2O (basal), GTP (10(-4) M), GTP + isoproterenol (10(-4) M each), and NaF (10 mM) at 21 days after treatment. The extent of AC stimulation in Bleo-treated hamster lung in response to various stimulators was generally less than that of saline control. Reductions in the BAR population and increased Kd values in Bleo-treated hamster lung were attributed to its fibrogenic ability and not to nutritional deficiency, which may partly be accountable for decreased AC activity of the lung in these animals. However, there were qualitative differences in the lung AC activity between Bleo-treated and nutritionally deprived hamsters, since the enzyme from the latter group was generally more responsive to stimulators than the enzyme from the former group. It was concluded from the findings of this study that an impairment in the coupled BAR and AC system of the lung may be partly responsible for the fibrogenic ability of bleomycin.     

The accuracy of protein biosynthesis is limited by its speed: high fidelity selection by ribosomes of aminoacyl-tRNA ternary complexes containing GTP[gamma S]

Guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] ) forms a stable ternary complex with polypeptide chain elongation factor Tu (EF-Tu) and aminoacyl-tRNA, and this complex binds rapidly and tightly to a properly programmed ribosome. However, the rate constant for the subsequent hydrolysis of the beta-gamma pyrophosphate bond (3.9 X 10(-3) s-1 at 5 degrees C) is less than 1/2,500th of that for the analogous reaction of GTP. We have taken advantage of this low rate to determine the rate constant for dissociation of the complex of poly(U)-programed ribosomes, EF-Tu, Phe-tRNAPhe, and GTP[gamma S] (2.7 X 10(-3) s-1) and the second-order rate constant for formation of this complex (3.3 X 10(6) M-1 s-1). Therefore, the Kd of the complex may be calculated to be 8.2 X 10(-10) M. An analogous near-cognate complex with Leu-tRNA2Leu in place of Phe-tRNAPhe has been determined by equilibrium methods to have a Kd greater than 1.7 X 10(-6) M. These results indicate that under equilibrium conditions the ribosome can distinguish cognate and near-cognate ternary complexes with great accuracy. Therefore, its failure to show this high specificity with the physiological ternary complexes containing GTP is due to the speed of GTP hydrolysis being similar to the speed of dissociation of the near-cognate complex. The low specificity of the physiological reaction is corrected by subsequent proofreading. The results reported here suggest that proofreading is necessary not simply for high accuracy but for the combination of speed and accuracy required in protein biosynthesis.     

Suppression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activity.

In this study, we investigated the role of interleukin-1 beta (IL-1 beta) in the malignant evolution of chronic myelogenous leukemia (CML) and the functional activity of IL-1 inhibitors. Bone marrow (BM) and peripheral blood (PB) low-density cells from 38 CML patients were studied in the colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte colony culture assay. Samples from patients with early stage, interferon-alpha (IFN)-sensitive disease formed hematopoietic colonies in the presence of fetal calf serum (FCS), erythropoietin (Epo), and one of the following: granulocyte-macrophage colony-stimulating factor (10 ng/mL), IL-3 (15 ng/mL), both, or phytohemagglutinin-conditioned medium. The addition of IL-1 beta augmented IFN-sensitive CML colony growth in a dose-dependent manner at concentrations of 10 to 100 U/mL. In sharp contrast, addition of the above growth factors did not augment the colony growth-promoting effect of FCS and Epo in samples from IFN-resistant patients; further, adherent cell fractionation or T-lymphocyte depletion attenuated the "autonomous" colony growth. Lysates of 2.5 x 10(7) low-density cells from each of six IFN-resistant and six IFN-sensitive CML patients and three normal volunteers were tested for intrinsic IL-1 beta content in an enzyme-linked immunosorbent assay and yielded a mean of 610 pg, 54.6 pg, and 49.4 pg of IL-1 beta, respectively (P less than .045). Interestingly, both soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) at concentrations of 5 to 100 ng/mL (sIL-1R) and 10 to 500 ng/mL (IL-1RA) inhibited CML colony growth in a dose-dependent fashion, with maximal inhibition of 64% and 65%, respectively. A similar effect was noted with the use of anti-IL-1 beta neutralizing antibodies. These data implicate IL-1 beta in CML disease progression and suggest that the inhibitory effects of molecules such as sIL-1R and IL-1RA could conceivably be the basis of a novel therapeutic strategy against this disorder.     

Androgen and estrogen receptors in hepatocellular carcinoma and in the surrounding noncancerous liver tissue

Both androgen and estrogen receptors were studied in human hepatocellular carcinoma and noncancerous liver tissue surrounding it. Androgen receptor was detected in the cytosol and/or nucleosol of 4 of 8 cancerous tissues and 1 of 6 noncancerous tissues. The levels of androgen receptor in hepatocellular carcinomas ranged from 3.4 to 37.6 fmoles per mg protein with dissociation constants (Kd) of 0.226 - 51.3 X 10(-9) M. That in the surrounding noncancerous tissue was 2.1 fmoles per mg protein with Kd of 0.941 X 10(-9) M. Estrogen receptor was detected in the cytosol of 1 of 7 cancerous tissues, while it was detected in the cytosol and/or nucleosol of 3 of 7 noncancerous tissues. The level of estrogen receptor in hepatocellular carcinoma was 4.9 fmoles per mg protein with Kd of 1.20 X 10(-9) M, and those in the surrounding noncancerous tissues ranged from 2.6 to 1,073 fmoles per mg protein with Kd of 0.223 - 3.15 X 10(-9) M. The results suggest that the expression of androgen receptor may be augmented in association with malignant transformation of hepatocytes while the expression of estrogen receptor may be rather suppressed and that some of hepatocellular carcinomas may be androgen-dependent.     

Interaction of vasoactive intestinal peptide with human blood mononuclear cells

The binding of vasoactive intestinal peptide (VIP) and the stimulation of adenylate cyclase were studied in mononuclear cells from human peripheral blood. The binding depended on time, temperature and pH, and was reversible, saturable and specific. Binding studies suggested the presence of 2 classes of binding site: a class with high affinity (Kd = 2.4 X 10(-10)M) and low capacity (8 fmoles/10(6) cells), and a class with low affinity (Kd = 8.0 X 10(-8)M) and high capacity (800 fmoles/10(6) cells) at 15 degrees C. Secretin displaced [125I]VIP from the cells with a 400-fold lower affinity than VIP, but glucagon, somatostatin and insulin did not show any effect. VIP was a potent and efficient stimulator of cyclic AMP production. The stimulation was observed at a concentration as low as 3 X 10(-11)M and depended on time, temperature and pH. Maximal cyclic AMP production (4-fold above basal levels) was observed with 10(-9) M at 15 degrees. Half-maximal response was obtained at 10(-10)M VIP. Secretin was an agonist of VIP but exhibited a 7000 times lower potency. Peripheral blood mononuclear cells constitute an easily accessible and suitable system for the study of VIP action in different physiological and pathophysiological conditions.