Effects of parental light environment on growth and morphological responses of clonal offspring

• Environments experienced by parent ramets of clonal plants can potentially influence fitness of clonal offspring ramets. Such clonal parental effects may result from heritable epigenetic changes, such as DNA methylation, which can be removed by application of DNA de‐methylation agents such as 5‐azacytidine.
• To test whether parental shading effects occur via clonal generation and whether DNA methylation plays a role in such effects, parent plants of the clonal herb Alternanthera philoxeroides were first subjected to two levels of light intensity (high versus low) crossed with two levels of DNA de‐methylation (no or with de‐methylation by application of 5‐azacytidine), and then clonal offspring taken from each of these four types of parent plant were subjected to the same two light levels.
• Parental shading effects transmitted via clonal generation decreased growth and modified morphology of clonal offspring. Offspring responses were also influenced by DNA methylation level of parent plants. For clonal offspring growing under low light, parental shading effects on growth and morphology were always negative, irrespective of the parental de‐methylation treatment. For clonal offspring growing under high light, parental shading effects on offspring growth and morphology were negative when the parents were not treated with 5‐azacytidine, but neutral when they were treated with 5‐azacytidine.
• Overall, parental shading effects on clonal offspring performance of A. philoxeroides were found, and DNA methylation is likely to be involved in such effects. However, parental shading effects contributed little to the tolerance of clonal offspring to shading.

     

The effects of 5‐azacytidine and cadmium on global 5‐methylcytosine content and secondary metabolites in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda

Epigenetic changes are important mechanisms in the regulation of chromatin structure and gene expression. Cytosine methylation is one of the major epigenetic modifications, mediated by DNA methyltransferases, which transfer methyl groups from S‐adenosyl‐L‐methionine (SAM) to the fifth carbon of cytosine. Various external environmental conditions can change the global hypo/hypermethylation pattern of DNA. These alterations may affect the organism's response to stress conditions. In this study, for the first time, we investigated the effects of 5‐azacytidine, a DNA methyltransferase inhibitor, and cadmium, a toxic metal and environmental pollutant, on the growth, biosynthesis of secondary metabolites (phenols, flavonoids, carotenoids), SAM, S‐adenosylhomocysteine, 5′‐methylthioadenosine and global 5‐methylcytosine (5‐mC) in the green microalgae Chlamydomonas reinhardtii and Scenedesmus quadricauda. The studied species showed major differences in 5‐mC content, secondary metabolite content, and antioxidant activity. Cadmium increased GSH (glutathione) content in C. reinhardtii by 60% whereas 5‐azacytidine did not affect GSH. The biosynthesis of GSH in S. quadricauda in response to the stressors was the opposite. Global 5‐mC content of C. reinhardtii was 1%–1.5%, and the content in S. quadricauda was 3.5%. Amount of some investigated methionine cycle metabolites (SAM, S‐adenosyl homocysteine [SAH], methionine) in S. quadricauda distinctly exceeded C. reinhardtii as well. However, chlorophylls a and b, carotenoids, total phenolic content, total flavonoid content and, antioxidant activity were significantly higher in C. reinhardtii than S. quadricauda. Therefore, in further studies it would be advisable to verify whether methylation of cytosine affects the expression of genes encoding certain secondary metabolites.     

DNA methylation as a possible mechanism affecting ability of natural populations to adapt to changing climate

Environmentally induced epigenetic variation has been recently recognized as a possible mechanism allowing plants to rapidly adapt to novel conditions. Despite increasing evidence on the topic, little is known on how epigenetic variation affects responses of natural populations to changing climate. We studied the effects of experimental demethylation (DNA methylation is an important mediator of heritable control of gene expression) on performance of a clonal grass, Festuca rubra, coming from localities with contrasting temperature and moisture regimes. We compared performance of demethylated and control plants from different populations under two contrasting climatic scenarios and explored whether the response to demethylation depended on genetic relatedness of the plants. Demethylation significantly affected plant performance. Its effects interacted with population of origin and partly with conditions of cultivation. The effects of demethylation also varied between distinct genotypes with more closely related genotypes showing more similar response to demethylation. For belowground biomass, demethylated plants showed signs of adaptation to drought that were not apparent in plants that were naturally methylated. The results suggest that DNA methylation may modify the response of this species to moisture. DNA methylation may thus affect the ability of clonal plants to adapt to novel climatic conditions. Whether this variation in DNA methylation may also occur under natural conditions, however, remains to be explored. Despite the significant interactions between population of origin and demethylation, our data do not provide clear evidence that DNA methylation enabled adaptation to different environments. In fact, we obtained stronger evidence of local adaptation in demethylated than in naturally‐methylated plants. As changes in DNA methylation may be quite dynamic, it is thus possible that epigenetic variation can mask plant adaptations to conditions of their origin due to pre‐cultivation of the plants under standardized conditions. This possibility should be considered in future experiments exploring plant adaptations.     

Telomerase activity is repressed during differentiation along the hepatocytic and biliary epithelial lineages: verification on immortal cell lines from the same origin

Recent investigations indicate that telomerase activity regulates the life span of cells by compensating for telomere shortening during DNA replication. In addition, as differentiation progresses, telomerase activity is reduced in several different cell lineages. These findings lend support to the theory that more immature cells have greater remaining proliferative capacity and longer life span. However, it has not been directly demonstrated that the differentiation along a hepatocytic or a bile ductal lineage is accompanied by reduction of telomerase activity. In this study, we present direct evidence that telomerase activity is reduced during hepatocytic and biliary epithelial differentiation by using our unique cell lines including a stem-like cell line, ETK-1. When hepatocytic differentiation was induced in ETK-1 by 5-azacytidine, telomerase activity decreased significantly. Similarly, when we compared the telomerase activity on SSP-25 and RBE cell lines from the same origin but representing different maturation stages of cholangiocarcinoma, more mature cells were found to possess significantly lower activity. These results indicate that the generally accepted relationship between telomerase activity and differentiation stage also applies in the hepatocytic and biliary epithelial lineages.     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.     

Promotion of human adipose‐derived stem cell proliferation mediated by exogenous nucleosides

Adult stem cells are becoming the best option for regenerative medicine because they have low tumourigenic potential and permit autologous transplantation, even without in vitro culture. Our objectives were to evaluate the effects of exogenous nucleosides on the proliferation of hASCs (human adipose‐derived stem cells), with or without co‐treatment with 5‐aza (5‐azacytidine), and to analyse the expression of lamin A/C during cardiomyocyte differentiation of these cells. We isolated hASCs from human lipoaspirates that were positive for mesenchymal stem cell markers. We found that 5‐aza induces a dose‐dependent inhibition of hASC proliferation [IC50 (inhibitory concentration 50): 5.37 μM], whereas exogenous nucleosides significantly promote the proliferation of hASCs and partially revert the antiproliferative effect of the drug. Multipotentiality of isolated hASCs was confirmed by adipogenic, osteogenic and cardiomyogenic induction. 5‐Aza‐induced cells expressed cardiac troponins I and T and myosin light chain 2, myocardial markers that were directly correlated with lamin A/C expression. Our results support the importance of the nucleoside supplementation of media to improve conditions for the expansion and maintenance of hASCs in culture. In addition, the quantification of lamin A/C expression appears to be a good marker for the characterization of cardiomyocyte differentiation of stem cells that has rarely been used.     

A comparison of the effects of various stimulatory agents on t-PA secretion by normal and malignant cell lines

Succinyl con A and acetyl con A both stimulated epithelial cells to produce similar yields of tissue plasminogen activator (t-PA) to those previously obtained with native con A. However, unlike con A, the derivatized lectins did not adversely affect cell morphology and viability, and cells treated with succinyl con A could secrete t-PA for a prolonged period. Con A and the two derivatives produced similar morphological effects in Bowes melanoma cells, but t-PA production was not increased. Elevated cyclic nucleotide concentrations did not affect t-PA production from epithelial cells, but calcium ionophore treatment generated t-PA yields similar to those obtained with lectins. Azacytidine, which enhanced t-PA production from epithelial cells, did not increase yields from Bowes melanoma cells, and also sodium butyrate, reported to increase t-PA yields from human endothelial cells, had no effect on either cell line.