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.     

Induction of DNA double-strand breaks by 157Gd neutron capture

The rationale of boron (10B) neutron capture therapy (BNCT) is based on the high thermal neutron capture cross section of 10B and the limited maximum range (about one cell diameter) of the high LET fission products of the boron neutron capture (NC) reaction. The resulting radiochemical damage is confined to the cell containing the BNC reaction. Although other nuclides have higher thermal neutron capture cross sections than 10B, NC by such nuclides results in the emission of highly penetrating gamma rays. However, gadolinium-157 (157Gd) n-gamma reaction is also accompanied by some internal conversion and, by implication, Auger electron emission. Irradiation of Gd3+-DNA complexes with thermal neutrons results in the induction of DNA double-strand (ds) breaks, but the effect is largely abrogated in the presence of EDTA. Thus, by analogy with the effects of decay of Auger electron-emitting isotopes such as 125I, the Gd NC event must take place in the close proximity of DNA in order to induce a DNA ds break. It is proposed that 157Gd-DNA ligands therefore have potential in NCT. The thermal neutron capture cross section of 157Gd, a nonradioactive isotope, is more than 50 times that of 10B.     

Quantitative survey of sieve tube distribution in foliar terminal veins of ten dicot species

Quantitative data on sieve tubes in foliar terminal veins (vein endings) were added to the meager published information from only five dicot species. Correlations with other minor vein configurations were also explored. Leaf samples from ten species of dicots (Oxalis nelsonii, O. pes-capri, O. rubra, O. stricta [Oxalidaceae], Caesalpinia pulcherrima. Glycine max, Trifolium repens [Leguminosae], Ampelamus albidus [Asclepidaceae], Eupatorium rugosum [Asteraceae], and Polygonum convolvulus [Polygonaceae]) were selected for two quantitative procedures: 1) a survey of the arrangement of terminal veins and distribution of sieve tubes in terminal veins in 100 areoles per species using stained leaf clearings; and 2) a search for correlations of sieve tube distribution with number and branching patterns of terminal veins, and with sizes of areoles using image analysis. Two Oxalis species (O. pes-capri and O. stricta) had the smallest areoles and virtually no sieve tubes in any terminal vein. Polygonum convolvulus, at the other extreme, had sieve tubes extending to the tips of most terminal veins. The other species had various intermediate sieve tube configurations. The data indicate that species with few or no sieve tubes associated with their terminal veins, regardless of the number of terminal veins per areole, have smaller areoles. These results may have implications regarding the entry of leaf photosynthates into the vascular system.     

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.     

Morphological and temporal variation in early embryogenesis contributes to species divergence in Malawi cichlid fishes

The cichlid fishes comprise the largest extant vertebrate family and are the quintessential example of rapid “explosive” adaptive radiations and phenotypic diversification. Despite low genetic divergence, East African cichlids harbor a spectacular intra- and interspecific morphological diversity, including the hyper-variable, neural crest (NC)-derived traits such as coloration and craniofacial skeleton. Although the genetic and developmental basis of these phenotypes has been investigated, understanding of when, and specifically how early, in ontogeny species-specific differences emerge, remains limited. Since adult traits often originate during embryonic development, the processes of embryogenesis could serve as a potential source of species-specific variation. Consequently, we designed a staging system by which we compare the features of embryogenesis between three Malawi cichlid species—Astatotilapia calliptera, Tropheops sp. ‘mauve’ and Rhamphochromis sp. “chilingali”—representing a wide spectrum of variation in pigmentation and craniofacial morphologies. Our results showed fundamental differences in multiple aspects of embryogenesis that could underlie interspecific divergence in adult adaptive traits. First, we identified variation in the somite number and signatures of temporal variation, or heterochrony, in the rates of somite formation. The heterochrony was also evident within and between species throughout ontogeny, up to the juvenile stages. Finally, the identified interspecific differences in the development of pigmentation and craniofacial cartilages, present at the earliest stages of their overt formation, provide compelling evidence that the species-specific trajectories begin divergence during early embryogenesis, potentially during somitogenesis and NC development. Altogether, our results expand our understanding of fundamental cichlid biology and provide new insights into the developmental origins of vertebrate morphological diversity.     

Identification of mRNAs differentially expressed in quiescence or in late G1 phase of the cell cycle in human breast cancer cells by using the differential display method.

BACKGROUND: The decision for a cell to enter the DNA synthesis (S) phase of the cell cycle or to arrest in quiescence is likely to be determined by genes expressed in the late G1 phase, at the restriction point. Loss of restriction point control is associated with malignant cellular transformation and cancer. For this reason, identifying genes that are differentially expressed in late G1 phase versus quiescence is important for understanding the molecular basis of normal and malignant growth. MATERIALS AND METHODS: The differential display (DD) method detects mRNA species that are different between sets of mammalian cells, allowing their recovery and cloning of the corresponding cDNAs. Using this technique, we compared mRNAs from synchronized human breast cancer cells (21 PT) in quiescence and in late G1. RESULTS: Six mRNAs differentially expressed in late G1 or in quiescence were identified. One mRNA expressed 10 hr after serum induction showed 99% homology to a peptide transporter involved in antigen presentation of the class I major histocompatibility complex (TAP-1) mRNA. Another mRNA expressed specifically in quiescence and down-regulated 2 hr following serum induction showed 98% homology to human NADP+ -dependent cytoplasmic malic enzyme (EC1.1.1.40) mRNA, which is an important enzyme in fatty acid synthesis and lipogenesis. Three others showed high homology to different mRNAs in the GeneBank, corresponding to genes having unknown functions. Finally, one mRNA revealed no significant homology to known genes in the GeneBank. CONCLUSIONS: We conclude that DD is an efficient and powerful method for the identification of growth-related genes which may have a role in cancer development.