Contribution of known mitogenic signaling pathways to induction of DNA synthesis in quiescent Chinese hamster fibroblasts.

The mitogenic pathways so far identified in mammalian cells fall into three main categories: tyrosine kinase, kinase C, and the cAMP-dependent pathways. In quiescent murine 3T3 fibroblasts, all three signaling pathways synergize with each other to restart DNA synthesis. In order to establish if the same was true in other rodent fibroblast lines we studied the effects of factors, known to modulate the above-mentioned pathways, on DNA synthesis in Chinese hamster embryo fibroblasts (CHEF/18). The factors examined were: (1) EGF and insulin representative of tyrosine kinase-activating growth factors, (2) TPA as specific activator of protein kinase C, (3) cholera toxin, dibutyryl cyclic AMP, and theophylline as compounds increasing cAMP levels. We found that EGF alone is a strong mitogen in CHEF/18 cells, probably because it can modulate by itself all three pathways. Although cAMP acts as a growth enhancer in 3T3 cells, in CHEF/18 where high levels of cAMP were found, increased concentrations of this second messenger produce strong DNA synthesis inhibition and temporal disturbance of ribosomal protein S6 phosphorylation. Possible interpretations of these findings are presented.

Structural requirements of polynucleotides for the activation of (2' - 5')An polymerase and protein kinase.

Two enzymatic pathways are involved in the inhibitory effects of double-stranded (ds)RNA on protein synthesis in cell extracts derived from interferon-treated human fibroblasts or HeLa cells, an oligonucleotide polymerase that synthesizes (2'-5')An from ATP and a protein kinase that phosphorylates the alpha subunit of initiation factor eIF-2 as well as a polypeptide of Mr = 72,000. We have now evaluated the activation of both the (2'-5')An polymerase and protein kinase by a large variety of polynucleotides, triple-stranded and synthetic dsRNAs, homopolymers, alternating copolymers, triple-stranded polymers, purine-purine duplexes and purine-pyrimidine duplexes with modifications at either the pyrimidine or ribose moieties. All these polynucleotides have been the subject of previous interferon induction studies. Some polynucleotides, i.e. (I)n.(C)n and mycophage dsRNA, which have been recognized as excellent interferon inducers, were also potent activators of both (2'-5')An polymerase and protein kinase, whereas non-inducers such as (A)n. (X)n and (A)n. (br5U)n did not activate either the kinase or the polymerase. However, some polymers like (I)n.(br5C)n, (difl)n(C)n and (dIcl)n (C)n, while potent interferon inducers and kinase activators, behaved poorly as activators of the (2'-5')An polymerase. Other polymers, i.e. (dAfl)n (U)n and (A)n.(U)nl (I)n, that do not induce interferon, activated the kinase but not the polymerase. Finally, (I)n (s2c)n, a relatively potent interferon inducer, did not activate either kinase or polymerase. These findings indicate that there is no simple relationship between the interferon-inducing ability of dsRNAs and their stimulating effects on (2'-5')An polymerase and protein kinase activity.

Activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells by 2'-O-methylated poly (inosinic acid) . poly(cytidylic acid), Correlations with interferon-inducing activity.

An oligonucleotide polymerase and a protein kinase which require double-stranded RNA (dsRNA) for activation are induced in HeLa cells by human fibroblast interferon. The polymerase synthesizes a series of oligonucleotides from ATP, whereas the kinase phosphorylates a polypeptide of Mr = 72,000 and the alpha subunit of initiation factor eIF-2. Partially or fully 2'-O-methylated derivatives of poly(inosinic acid) . poly(cytidylic acid) (rIn . rCn) were used to determine the structural requirements of dsRNA in the activation of these two enzymes. While fully methylated polymers failed to activate either enzyme, partially methylated polymers activated the enzymes in specific manners. The activation of the kinase by the rIn . rCn analogues was affected more severely by the level of methylation than was the activation of the polymerase. Moreover, fully methylated analogues blocked the activation of the kinase by rIn . rCn but not the activation of the polymerase. These observations are consistent with a biphasic model for enzyme activation similar to that proposed for interferon induction, which required the recognition of a relatively small region of rIn . rCn as the last step. Differences in the activation of the polymerase and kinase are explicable on the basis of the polymerase requirement for a smaller recognition region of the rIn . rCn duplex than the kinase. Dependence of polymerase activation on the level of methylation shows striking similarities with the interferon inducing activities of these analogues, suggesting a possible relationship between polymerase activation and interferon induction.

Mechanism of pppA(2'p5'A)n2'p5'AOH synthesis in extracts of interferon-treated HeLa cells.

Treatment of HeLa cells with interferon results in the induction of an enzymatic activity designated 2'5'oligo(A) polymerase. The polymerase requires continuous presence of double-stranded RNA (dsRNA) for activity, since degradation of dsRNA abolishes synthesis of the oligomeric series pppA(2'p5'A)n. These oligonucleotides are formed initially at a constant rate with dimer synthesized faster than trimer, and the latter faster than tetramer. After 45 min, accumulation of the dimer declines whereas that of other oligomers still proceeds at a linear rate. These results suggest that an oligomer remains associated with the enzyme for possible consecutive additions of adenylate, since no significant accumulation of dimer precedes synthesis of trimer. The relative amounts of the different oligomers found at the end of a reaction may reflect an increasing probability of release as the oligomers are elongated. The accumulation of dimer, however, decreases when it becomes a substrate for adenylate addition; incorporation of isolated dimer into 2'5'-oligo(A) was directly shown. Other nucleotides with a blocked p5'A terminus, like A5'ppppp5'A and NADH, can serve as adenylate acceptors in the presence of dsRNA. The adenosine triphosphates 2'-dATP and 3'-dATP are not incorporated efficiently into 2'5'-oligo(A) and inhibit its synthesis.

Metabolic stability of 2' 5'oligo (A) and activity of 2' 5'oligo (A)-dependent endonuclease in extracts of interferon-treated and control HeLa cells.

Extracts of interferon-treated HeLa cells adsorbed to poly(I) . poly(C)-agarose have been used to synthesize 2'5'oligo(A). This oligonucleotide has been characterized by enzymatic digestion with alkaline phosphatase, snake venom phosphodiesterase, T2 ribonuclease and chromatography on DEAE, and PEI-cellulose. The oligonucleotide inhibits protein synthesis in vitro and activates an endonuclease present in extracts of control and interferon-treated cells. The metabolic stability of 2'5'oligo(A) has been investigated in these cell extracts. The oligonucleotide undergoes rapid degradation, particularly in the absence of ATP and of an energy regenerating system. Furthermore, the 2'5'oligo(A)-activated endonuclease reverts to an inactive state under these conditions, but can be reactivated upon further addition of 2'5'oligo(A). A possible role for the degradation of 2'5'oligo(A) in the mechanism of interferon action is discussed.

Two novel growth cycle-reflecting proteins associated with Escherichia coli ribosomes.

The isolation and characterization of two low molecular weight, growth cycle-reflecting proteins which are associated with Escherichia coli ribosomes is described. These proteins which show greatly enhanced stoichiometry in ribosomes derived from post-exponential cells, exhibit molecular weights of 16 600, and 11 700 on sodium dodecyl sulfate (SDS) gel electrophoresis. Both proteins are highly acidic, the larger being the most acidic protein associated with the ribosome. Their amino acid compositions are unique and distinguish them from all other ribosomal proteins. Neither of the proteins showed crossreaction with either anti-L7 or anti-S6 sera although their electrophoretic behavior resembles that of L7 and S6. During the purification we have also isolated small amounts of three low molecular weight proteins showing some immunological homology with L7/L12. The isolated proteins were found to be without effect on the in vitro translation of f2-RNA, indicating that these adaptive modifications of the ribosome do not seriously affect its intrinsic activity.

Metabolic stability of the two forms of initiation factor IF-3 in Escherichia coli during the growth cycle.

Possible alteration in the ratio of the long and short forms of initiation factor IF-3 (FEBS Lett. 79, 264-275, 1977) during the growth cycle of Escherichia coli was examined. The ratio was found to remain unchanged between the exponential and stationary growth phases. Contrary to an earlier report (Eur. J. Biochem. 29, 319-325, 1972), the total amount of IF-3 relative to the ribosome content in stationary phase cells was essentially the same as in midlogarithmic phase cells. The activity of IF-3, assayed after its separation from other initiation factors by chromatography, was also the same in extracts from midlogarithmic and stationary phase cells. The data show that in Escherichia coli the ratio of IF-3/ribosome is maintained constant. The ribosomes themselves have been shown to retain virtually full activity in vitro during this transition indicating that growth-cycle-dependent biochemical modifications of the ribosome do not affect its protein synthetic capacity per se.

Studies on the binding of N-bromoacetylpuromycin and N-bromoacetylaminonucleoside to rat liver ribosomes.

[3H]N-Bromoacetylaminonucleoside and [3H]N-bromoacetylpuromycin have been synthesised as possible alkylating agents in order to study their interactions with rat liver ribosomes. Both compounds bind covalently to ribosomes to a considerable extent. The puromycin derivative binds to the extent of approximately 8 mol per ribosome, while the aminonucleoside derivative binds to the extent of approximately 13 mol per ribosome. Ammonium sulphate precipitation of ribosomes or treatment with puromycin, followed by washing of the ribosomes through NH4Cl-containing sucrose density gradients decreases the binding of both derivatives. Partial unfolding or denaturation of ribosomes by heating at 65 degrees C or through the action of various chemical reagents appears to expose more sites for binding. However, at 15 min of heating the binding of the puromycin derivative decreased by approximately 50% while the binding of the aminonucleoside derivative was almost zero. Binding of both labelled derivatives occurred only with the 50S ribosomal subunit. The extent of binding to the smaller 30S subunit was approximately 4% of that of the 50S subunit. Various other experiments are also described dealing with the binding of [3H]N-acetylphenylalanyl-tRNA to the A site of ribosomes following treatment with the N-bromoacetyl derivatives.