Phorbol ester action is independent of viral and cellular src kinase levels.

Treatment of normal chick embryo fibroblasts with phorbol myristate acetate causes those cells to express many of the phenotypic properties of virally transformed cells and also enhances the expression of transformed properties in Rous sarcoma virus-transformed chick embryo fibroblasts. We report here that phorbol myristate acetate has little or no effect on the level of protein kinases encoded by the viral src or endogenous sarc genes.

Reduced synthesis of pp60src and expression of the transformation-related phenotype in interferon-treated Rous sarcoma virus-transformed rat cells.

Treatment of Rous sarcoma virus-transformed rat cells with rat interferon-alpha (specific activity, 10(6) U/mg of protein) for 24 h caused a 50% reduction in intracellular pp60src-associated protein kinase activity. Staphylococcus aureus V8 protease digestion of pp60src, derived from 32P-labeled monolayer cultures incubated with or without interferon, revealed no differences either in the phosphopeptide pattern or in the phosphoserine-phosphotyrosine ratio. However, [3H]leucine pulse-labeling experiments showed that the synthesis of pp60src was reduced by 42 to 48%, relative to the level of bulk protein synthesis, in the interferon-treated cultures. Rat interferon-alpha also reduced the growth rate of Rous sarcoma virus-transformed rat cells in a dose-dependent manner over a 72-h period. The decrease in growth rate was accompanied by increases in the thickness and number of actin fibers per cell and by a decline in intracellular tyrosine phosphorylation by pp60src. The results suggest that interferon can inhibit the expression of the transformation-related phenotype by selectively reducing the synthesis of the Rous sarcoma virus transforming gene product. However, the interferon effects on the cytoskeletal organization and proliferation of Rous sarcoma virus-transformed cells may be due at least in part to the predominance of interferon-induced phenotypic changes over those caused by pp60src.

Size-variant pp60src proteins of recovered avian sarcoma viruses interact with adhesion plaques as peripheral membrane proteins: effects on cell transformation.

We have shown previously that the membrane association of the src proteins of recovered avian sarcoma viruses (rASVs) 1702 (56 kilodaltons) and 157 (62.5 kilodaltons), whose size variations occur within 8 kilodaltons of the amino terminus, is salt sensitive and that, in isotonic salt, these src proteins fractionate as soluble cytoplasmic proteins. In contrast, wild-type Rous sarcoma virus pp60src behaves as an integral plasma membrane protein in cellular fractionation studies and shows prominent membrane interaction by immunofluorescence microscopy. In this study we have examined the distribution of these size-variant src proteins between free and complexed forms, their subcellular localization by immunofluorescence microscopy, and their ability to effect several transformation-related cell properties. Glycerol gradient sedimentation of extracts from cells infected either with rASV 1702 or rASV 157 showed that soluble src proteins of these viruses were distributed between free and complexed forms as has been demonstrated for wild-type Rous sarcoma virus pp60src. Pulse-chase studies with rASV pp60src showed that, like wild-type Rous sarcoma virus pp60src, it was transiently found in a complexed form. Indirect immunofluorescence showed that size-variant pp60src proteins are localized in adhesion plaques and regions of cell-to-cell contact in rASV 1702- or 157-infected cells. This result is in contrast with the generalized localization of pp60src in plasma membranes of control rASV-infected cells which produce pp60src. Chicken embryo fibroblasts infected by rASVs 1702 and 157 display a partial-transformation phenotype with respect to (i) transformation-related morphology, (ii) cell surface membrane changes, and (iii) retained extracellular fibronectin. It is possible that the induction of a partial-transformation phenotype may be the result of the unique interaction of the src proteins encoded by these viruses with restricted areas of the plasma membrane.

A ribozyme which discriminates in vitro between PML/RAR alpha, the t(15;17)-associated fusion RNA of acute promyelocytic leukemia, and PML and RAR alpha, the transcripts from the nonrearranged alleles.

Acute promyelocytic leukemia (FAB M3) is distinguished by the presence of the t(15;17) and clinical response to all-trans retinoic acid (RA) treatment. Acute promyelocytic leukemia is associated with a chromosomal translocation which results in the fusion of genes encoding a putative transcription factor (PML) and the retinoic acid receptor alpha (RAR alpha). It is suggested that the PML/RAR alpha fusion protein functions as an inhibitor of myeloid differentiation. The potential use of ribozymes as therapeutic agents has been investigated in the present study. Hammerhead ribozymes, which by hybridizing to both PML and RAR alpha sequences discriminate between the fusion transcript and the normal transcripts from the nonrearranged alleles, were designed and synthesized. Two hammerhead cleavage sites were targeted: site 1, an AUU located 4 nucleotides 3' to the fusion junction; and site 2, a UUC located 26 nucleotides 3' to the junction. Both sites are located in the RAR alpha portion of the fusion transcript. Using a full-length PML/RAR alpha RNA or an RNA corresponding to 788 nucleotides of the PML/RAR alpha mRNA and a full-length RAR alpha RNA or an RNA corresponding to 960 nucleotides of the RAR alpha mRNA as model substrates, the catalytic behavior of several ribozymes was studied. A modified hammerhead directed against site 2 displayed the highest degree of selectivity for PML/RAR alpha. It is hypothesized that ribozyme-mediated inactivation of PML/RAR alpha provides a new approach to study the role of PML/RAR alpha in the deregulated growth and RA response of acute promyelocytic leukemia.

Targeting the PML/RAR alpha translocation product triggers apoptosis in promyelocytic leukemia cells.

The t(15;17) rearrangement found in acute promyelocytic leukemia (APL) yields a fusion transcript, PML/RAR alpha. PML/RAR alpha expression is linked to leukemogenesis and to clinical sensitivity to all-trans retinoic acid (RA). Paradoxically, RA treatment causes transient complete remissions in most t(15;17) APL cases. The precise roles of PML/RAR alpha in triggering leukemia or in causing a maturation block are not yet known. This study explores directly these PML/RAR alpha functions in the growth and differentiation of APL cells using a hammerhead ribozyme to target PML/RAR alpha mRNA in the NB4 APL cell line. When the PML/RAR alpha cleaving but not the non-catalytic control ribozyme is introduced into the NB4 APL cell line, PML/RAR alpha protein expression is reduced. This catalysis signals growth suppression, cytotoxicity, and apoptosis without overcoming the maturation block found in these leukemic cells. These biologic effects depend on the selective pressure used to express the ribozyme from an episomal vector. Introduction of a non-catalytic, control ribozyme into NB4 cells caused no observed phenotype due to anti-sense activities. Expression of the catalytic or non-catalytic ribozymes in control cells lacking PML/RAR alpha mRNA yielded no apparent growth or differentiation effects. Thus, use of a hammerhead ribozyme that targets PML/RAR alpha expression in APL cells reveals the anti-apoptotic function of this translocation product and demonstrates that PML/RAR alpha cleavage is insufficient to overcome the differentiation block observed in these leukemic cells. Taken together, these findings indicate that persistent PML/RAR alpha expression is required to maintain basal leukemic cell growth and point to the therapeutic potential of targeting PML/RAR alpha in APL.

Binding of deoxyribonuclease I to actin: a new way to visualize microfilament bundles in nonmuscle cells.

Intracellular actin-containing fibers can be visualized by indirect immunofluorescence microscopy when they are stained with antibody directed against DNase I. The location of actin-containing fibers in cells appears to be similar to the staining pattern of antibody to actin. Actin fibers were also visualized by direct fluorescent microscopy with rhodamine-conjugated DNase I.

Properties and mechanism of action of viral and cellular tyrosyl protein kinases.

We have demonstrated the usefulness of several angiotensin analogs as substrates for a number of tyrosyl protein kinases of viral or cellular origin. Results of initial rate studies with pp60src at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 microM respectively and Vmax was 1.0 nmol/min/mg. The end-product ADP and the ATP analog, AMP-PNP, were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]-angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. The available evidence allowed us to conclude that while pp60src contained essential histidine and lysine residues in its active site, the kinase reaction does not involve a phosphoryl enzyme intermediate. Phosphorylation of the angiotensin peptides in vitro also has allowed us to demonstrate the presence of at least two tyrosyl protein kinases in the cytoplasm of normal rat liver cells. These kinases appear to be novel in that they are present in normal cells and are not stimulated by growth factors. Also, results of preliminary experiments indicate that these kinases are not immunologically related to the transforming gene products of Rous and Fujinami sarcoma viruses (unpublished observations). The identification of these new kinases represents another application of the angiotensin peptides as substrates for tyrosyl kinases (13). The results obtained do not exclude the possibility that there exist in rat liver other tyrosyl kinases that do not phosphorylate these particular peptide substrates. No attempt has been made to characterize tyrosyl kinases associated with the plasma membrane fraction. Although they represent only a small fraction of the total activity of liver cells, the plasma membrane kinases have a relatively high specific activity. These kinases may be identical with growth factor receptor kinases previously identified in liver cell membranes (5). The most abundant tyrosyl kinase in rat liver cytoplasm has a molecular weight of 75,000 daltons and was found in cytosol and microsomal salt-wash fraction. The observation that the purified 75 Kd enzyme phosphorylates a 75 Kd protein on tyrosine residues suggests that the enzyme may possess autophosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Tyrosyl protein kinases in normal rat liver: identification and partial characterization.

Rat livers were fractionated and subcellular components were assayed for tyrosyl protein kinase activity. About 60% of the kinase activity in the cytoplasm sedimented with the microsomal fraction, whereas 40% remained in the supernatant. Purification of cytosolic and microsomal kinases by ion-exchange and gel filtration chromatography resolved a major species whose molecular mass was 75 kilodaltons (referred to as TPK 75) and a minor one whose molecular mass was greater than 160 kilodaltons. Partially purified TPK 75 phosphorylated a protein of the same molecular mass on tyrosine residues. The activity associated with TPK 75 was not stimulated by growth factors and was sensitive to thiol re-agents.

In vitro phosphorylation of angiotensin analogs by tyrosyl protein kinases.

Peptide analogs of angiotensin were phosphorylated in vitro by the src gene product, pp60src, of Rous sarcoma virus. The Km for the phosphorylation reaction varied from 1 to 5 mM and the Vmax varied from 2 to 10 nmol/min/mg. Tyrosine was the only residue phosphorylated in all analogs that were examined. The peptides were phosphorylated by tyrosyl protein kinases associated with several avian sarcoma viruses and by the epidermal growth factor-receptor kinase of A431 cells. Peptide substrate also was used to investigate the effectiveness of three different phosphatase inhibitors. Assay of tyrosyl kinase activities in whole cell lysates indicated that both p-nitrophenyl phosphate and sodium vanadate were potent inhibitors of phosphotyrosine phosphatases.

Rous-sarcoma-virus-transformed fibroblasts having low levels of plasminogen activator.

Several clones of transformed chick embryo fibroblasts infected with wild-type B77-C or Prague-C strain of Rous sarcoma virus have been isolated from soft agar suspension. These clones were screened for plasminogen activator activity by overlaying monolayer cultures with medium containing agar, casein, and chicken plasminogen. Twenty-three percent of all of the isolated clones showed little caseinolytic activity, 42% had intermediate activity, and 35% had high activity. Although the clones with low plasminogen activator activity had no more than twice the activity shown by uninfected fibroblasts, they did not differ significantly from clones possessing high levels of plasminogen activator in their morphology, 2-deoxyglucose transport, or efficiency of colony formation in soft-agar.

Changes in microfilament organization and surface topogrophy upon transformation of chick embryo fibroblasts with Rous sarcoma virus.

A series of morphological changes occurred when chick embryo fibroblasts infected with the NY68 mutant of Rous sarcoma virus were shifted from nonpermissive temperature (41degrees) to permissive temperature (37 degrees). We observed three distinct stages in cell morphology and surface topography that were correlated with a reduction in the organization and assembly of actin-containing microfilament bundles. Our observations suggest that control of microfilament organization and surface topography are responsive to the presence of a functioning transforming gene (src) product of Rous sarcoma virus.

Kinetics and mechanism of angiotensin phosphorylation by the transforming gene product of Rous sarcoma virus.

We have studied steady state kinetics of phosphorylation of [Val5]angiotensin II by pp60src, the transforming gene product of Rous sarcoma virus. Results of initial rate studies at varying substrate concentrations indicated that the mechanism was sequential; Michaelis constants for ATP and peptide were 7 microM and 0.24 mM, respectively, and Vmax was 1.0 nmol/min/mg. The end product ADP and the ATP analog AMP-PNP were competitive inhibitors at varying ATP concentrations and noncompetitive inhibitors at varying peptide concentrations. A dead-end analog of angiotensin II, [delta Phe4]angiotensin II, was a noncompetitive inhibitor at varying ATP concentrations, but induced substrate inhibition at varying peptide concentrations. The kinetic data allowed us to conclude that the reaction proceeded via an Ordered Bi Bi mechanism with ATP as the first binding substrate. We also presented evidence that, while pp60src contained essential histidine and/or lysine residues in its active site, the mechanism does not involve a phosphoryl enzyme intermediate.

Purification and characterization of the major species of tyrosine protein kinase in rat liver.

The major species of tyrosine protein kinase of rat liver, has been purified to near homogeneity from liver cytosol. When the kinase was incubated with MnCl2 and [gamma-32P]ATP, two phosphoproteins with molecular masses of 72 and 75 kilodaltons were observed. The purified kinase, called p75 kinase, phosphorylates [Val5]angiotensin II, casein, vinculin, and a 34-kilodalton protein isolated from chicken embryo fibroblasts. However, it does not phosphorylate histones or IgG from Rous sarcoma virus tumor-bearing rabbits. The kinase does not contain any of the major antigenic determinants found in retroviral tyrosine protein kinases or in epidermal growth factor-receptor kinase. p75 kinase activity, as well as viral tyrosine protein kinase activity, is stimulated by heparin. Phosphorylation of angiotensin is also stimulated by high ionic strength. In contrast, casein phosphorylation by the kinase appeared to be inhibited by high salt. Kinetic properties of p75 kinase have been determined and have revealed some striking differences from those of most other tyrosine protein kinases. For instance, p75 kinase exhibits rather stringent dependence for its activity on ATP as phosphoryl donor and Mn2+ as divalent cation.

Synthetic peptide fragment of src gene product inhibits the src protein kinase and crossreacts immunologically with avian onc kinases and cellular phosphoproteins.

All the known avian sarcoma viruses have associated protein kinase activities that phosphorylate tyrosine residues of their target proteins. A decapeptide fragment of pp60src of Rous sarcoma virus (RSV), residues 415-424, and an analog of that sequence have been chemically synthesized by solid-phase methods. The two decapeptides were not phosphorylated by pp60src of RSV, P90 of Y73 avian sarcoma virus, or P140 of Fujinami sarcoma virus. However, both peptides were able to inhibit competitively the kinase activities associated with the transforming proteins. Antiserum was raised against one of the peptides and IgG was purified from the serum by affinity chromatography. The antibody was able to precipitate pp60src of RSV as well as P90 of Y73 virus from cells infected with these viruses. The antibody also precipitated a number of high molecular weight phosphoproteins from normal chicken and rat fibroblasts and from several lines of virus-transformed cells.