Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Cellular kinetics,cell cycles and cell killing

Summary Techniques available for the calculation of the time variation of the number of viable mammaliar cells in a cell population are reviewed. Events in the course of the cell's growth may include one or more exposures to ionizing radiations or other cytotoxic agents. The dependence of cell killing upon the cell's position in the cell cycle is emphasized, and a unified model for calculation of cell kinetics and cell survival is discussed. For a cell population not limited in growth by contact inhibition or by nutritional factors, experimental data agree with predictions of the model.The possibility of utilizing the model to arrive at optimum treatment schedules for the management of some malignant diseases is discussed. The conclusion drawn is that the state of knowledge with respect to cellular events in solid tumors is such as to leave most such applications in the realm of speculation.This work was supported by the National Institutes of Health, United States Public Health Service, under Grants CA 5008 and CA 4542.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

Unbalanced cell growth and increased protein synthesis induced by chemotherapeutic agents

Unbalanced cell growth as manifested by an increase in cellular volume and in cellular dry mass following exposure to a variety of chemotherapeutic agents has been shown for neoplastic cells in vitro and human leukemic cells in vivo. The purpose of the present investigation was to test the hypothesis that unbalanced cell growth results from a disassociation of cell growth and cell division due to the blocking effect of chemotherapeutic agents. Monolayer cultures of CHO fibroblasts were studied in terms of their response to two chemotherapeutic agents that differ significantly in their mode of action, adriamycin and chlorambucil. Following exposure to these drugs, cell volume increased at a rate of from 1% to 4% per h; the total cell protein increased at a rate of from 4% to 7% per h. These changes were observed in both log and stationary phase cultures. Thus exposure to adriamycin and chlorambucil was followed by a more rapid rate of protein synthesis relative to the rate of degradation, resulting in larger cells with more protein whether or not the cells were actively in the division cycle. This is inconsistent with the hypothesis that unbalanced growth results simply from a disassociation of the cell division cycle from cell growth. These observations suggest that a final common pathway in the mode of action of chemotherapeutic agents may be the induction of unscheduled protein synthesis resulting in unbalanced cell growth.     

Mode of inhibition of DNA synthesis induced by adenosine diphosphoribosylation of nuclear protein

DNA obtained after complete removal of the proteins from NAD-treated rat liver chromatin possessed template capacity equivalent to that obtained from untreated chromatin. The stepwise extraction of proteins from chromatin affected a gradual removal of labeled ADPR and a parallel decrease of the inhibition. The results of the present study suggest that the inhibition of the template capacity for DNA synthesis following preincubation of chromatin with NAD is dependent upon ADP-ribosylation of the associated proteins. The template capacity of chromatin and nucleoprotein complex (Weiss preparation) for DNA synthesis was inhibited, whereas the capacity for RNA synthesis was apparently not affected.     

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus

Inhibition of host protein synthesis after poliovirus infection has been suggested to be a consequence of the proteolytic degradation of a p220 polypeptide necessary to translate capped mRNAs. However, the synthesis of several adenovirus late proteins on capped mRNAs was resistant to poliovirus inhibition. Thus, the hexon protein was still made 8 h after poliovirus superinfection. The synthesis of other adenovirus proteins such as the fiber was much more sensitive to poliovirus-induced inhibition than the hexon, either in the absence or in the presence of guanidine. Detailed densitometric analyses clearly showed the differential behavior of several adenovirus late mRNAs to poliovirus shut-off of translation. This is striking in view of the fact that a common leader sequence in the 5' termini is present in the adenovirus late mRNAs. The use of 3-methyl quercetin, an inhibitor of poliovirus RNA synthesis (Castrillo, J. L., Vanden Berghe, D., and Carrasco, L. (1986) Virology 152, 219-227), showed that translation of several capped adenovirus mRNAs took place in poliovirus-infected cells after the synthesis of host proteins had ceased. The poliovirus mRNA and the adenovirus mRNA coding for the hexon protein are very efficient mRNAs and have a leader sequence of more than 740 and 250 nucleotides, respectively, with very rich secondary structures making it difficult to predict how the scanning model will operate on these two mRNAs.     

Chloral hydrate mediated inhibition of cell division and of protein synthesis

Summary Chloral hydrate (along with other anaesthetics and hypnotics) is an inhibitor of cell division. We have shown that chloral hydrate is also an inhibitor of protein synthesis. This inhibition is unlikely to be a result either of the disruption of cell division or of interference with the function of microtubules. The ability of chloral hydrate to inhibit cell division may result from its ability to inhibit protein synthesis.This research was supported by USPHS grant No GMO6965.     

Early changes of protein synthesis in myocardium and coronary arteries induced by NO synthase inhibition.

The question was addressed whether short-term (4 hour) NO deficiency, inducing an increase in blood pressure in anaesthetized dogs, does influence proteosynthesis in the myocardium and coronary arteries. A potentially positive answer was to be followed by the study of the supporting role of ornithine decarboxylase for the polyamines pathway. N(G)-nitro-L-arginine-methyl ester (L-NAME) (50 mg/kg per hour) was administered i.v. to inhibit NO synthase. After the first L-NAME dose diastolic blood pressure increased from 131.8+/-2.0 to 149.4+/-3.9 mm Hg (p<0.001) and was maintained at about this level till the end of the experiment. Systolic blood pressure only increased after the first dose (from 150.8+/-1.1 to 175.0+/-5.8 mm Hg, p<0.01), returning thereafter to the control level. Similarly, the heart rate declined only after the first dose (from 190.4+/-5.3 to 147.6+/-4.5 beats/min, p<0.01). Total RNA concentrations increased in the left cardiac ventricle (LV), the left anterior descending coronary artery (LADCA) and left circumflex coronary artery (LCCA) by 15.9+/-0.7, 29.7+/-1.3 and 17.6+/-1.0%, p<0.05, respectively. The same applied to [14C]leucine incorporation (by 86.5+/-5.0, 33.5+/-2.6, 29.3+/-4.1%, p<0.05, respectively). The above parameters indicated an increase of proteosynthesis in the LV myocardium and both coronary arteries LADCA and LCCA after short-term NO deficiency. Surprisingly, the ornithine decarboxylase activity in the LV myocardium decreased significantly by 40.2+/-1.6% (p<0.01) but the changes were not significant in the coronary arteries. This unexpected finding makes the role of polyamines in increasing proteosynthesis during a pressure overload due to NO deficiency questionable.     

Epinephrine and glucagon counteract inhibition of protein synthesis induced by D-galactosamine in isolated mouse hepatocytes.

10 mM D-galactosamine enhibited protein synthesis (1 h incubation time) by 67% in isolated mouse liver cells. Counteracting uridylate deficiency induced by D-galactosamine by preventive administration of 20 mM uridine did not decrease the extent of protein synthesis inhibition. 20 mM D-galactose reverted the inhibition of protein synthesis by D-galactosamine. 10(-5) M epinephrine and 10(-7) M glucagon decreased the incorporation of D-galactosamine into glycogen to 38% and 26% of the control value, respectively, after a 35 min incubation and reduced the inhibition of protein synthesis by D-galactosamine effectively. Experimental evidence supports the view that aminoglycogen formed after D-galactosamine treatment is responsible for the inhibition of protein synthesis.     

Restoration of light induced photosystem II inhibition without de novo protein synthesis

Illumination of isolated spinach thylakoid membranes under anaerobic conditions gave rise to severe inhibition of photosystem II electron transport but did not result in D1-protein degradation. When these photoinhibited thylakoids were incubated in total darkness the photosystem II activity could be fully restored in vitro in a process that required 1-2 h for completion.