Exposure to caffeine and suppression of DNA replication combine to stabilize the proteins and RNA required for premature mitotic events

Caffeine had been shown to induce mitotic events in Syrian hamster fibroblast (BHK) cells that were arrested during DNA replication (Schlegel and Pardee, Science 232:1264-1266, 1986). Inhibition of protein synthesis blocked these caffeine-induced events, while inhibition of RNA synthesis showed little effect. We now report that the protein(s) that are required for inducing mitosis in these cells were synthesized shortly after caffeine addition, the activity was very labile in the absence of caffeine, and the activity was lost through an ATP-dependent mechanism. Caffeine dramatically increased the stability of these putative proteins while having no effect on overall protein degradation. Experiments with an inhibitor of RNA synthesis indicated that mitosis-related RNA had accumulated during the suppression of DNA replication, and this RNA was unstable when replication was allowed to resume. These results suggest that the stability of RNA needed for mitosis is regulated by the DNA replicative state of the cell and that caffeine selectively stabilizes the protein product(s) of this RNA. Conditions can therefore be selected that permit mitotic factors to accumulate in cells at inappropriate times in the cell cycle. Two-dimensional gel electrophoresis has demonstrated several protein changes resulting from caffeine treatment; their relevance to mitosis-inducing activity remains to be determined.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

The role of increased proteolysis in the atrophy and arrest of proliferation in serum-deprived fibroblasts

When cultured fibroblasts are deprived of serum, the degradation of long-lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum-deficient medium (0.5% serum), the rate of degradation of long-lived proteins increased about twofold within 30 min. This increase was reduced by 50-70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24-48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two- to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24-48 hours had about twice the rate of protein synthesis and two- to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475-treated cells were also about 30% larger than the untreated cells. Thus, the protease-inhibitor prevented much of the atrophy induced by serum deprivation. The serum-deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum-deprived cells. Other agents which inhibited protein breakdown in serum-deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475-treated cells began DNA synthesis 1-2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.     

Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

A general method for permeabilizing monolayer and suspension cultured animal cells

A wide variety of animal cells have been successfully permeabilized to non-penetrating molecules, using lysolecithin. The sizes of molecules that can enter the cells can be controlled by varying the concentration of lysolecithin. The cells become permeable to small molecules and maintain viability following treatment with low lysolecithin concentrations. At higher concentrations the cells become permeable to proteins but do not retain viability. Lysolecithin permeabilization should permit many studies of the effects of non-penetrating compounds on cellular processes.     

Systematic Dissection of the Metabolic-Apoptotic Interface in AML Reveals Heme Biosynthesis to Be a Regulator of Drug Sensitivity

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A protocol for differential display of mRNA expression using either fluorescent or radioactive labeling

Since its invention in the early 1990s, differential display (DD) has become one of the most commonly used techniques for identifying differentially expressed genes at the mRNA level. Unlike other genomic approaches, such as DNA microarrays, DD systematically detects changes in mRNA profiles among multiple samples being compared without the need of any prior knowledge of genomic information of the living organism being studied. Here, we present an optimized DD protocol with a fluorescent digital readout as well as traditional radioactive labeling. The resulting streamlined fluorescent DD process offers an unprecedented accuracy, sensitivity and throughput in comprehensive and quantitative analysis of eukaryotic gene expression. Results usually can be obtained within days using a limited number of primer combinations, but a comprehensive DD screen may take weeks or months to accomplish, depending on gene coverage required and the number of differentially expressed genes present within a biological system being compared.     

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

Cell Division of Escherichia coli: Control by Membrane Organization

Cells of certain strains of Escherichia coli, after transfer from 37 to 45 C and incubation for 16 min, were observed to swell and subsequently divide synchronously. This swelling and the resulting stretching of the membrane are proposed to be the basis for the synchronous division. Four lines of evidence support this hypothesis. First, osmotic protection by the addition of either sodium chloride or sucrose at the time of heat shock prevents both swelling and synchrony. Second, a mutant neither swelled nor divided synchronously after heat shock. Third, cells grown for several generations with 10% sucrose in the medium swelled and divided synchronously upon transfer to medium without sucrose. Fourth, the mutant not synchronized by heat shock also swelled and underwent synchronous division after the osmotic shift. A tentative model is suggested for the normal control of division, based on membrane configuration at the septation site.