Rafal Woycicki

Intense THz pulses down-regulate genes associated with skin cancer and psoriasis: a new therapeutic avenue?

Terahertz (THz) radiation lies between the infrared and microwave regions of the electromagnetic spectrum and is non-ionizing. We show that exposure of artificial human skin tissue to intense, picosecond-duration THz pulses affects expression levels of numerous genes associated with non-melanoma skin cancers, psoriasis and atopic dermatitis. Genes affected by intense THz pulses include nearly half of the epidermal differentiation complex (EDC) members. EDC genes, which are mapped to the chromosomal human region 1q21, encode for proteins that partake in epidermal differentiation and are often overexpressed in conditions such as psoriasis and skin cancer. In nearly all the genes differentially expressed by exposure to intense THz pulses, the induced changes in transcription levels are opposite to disease-related changes. The ability of intense THz pulses to cause concerted favorable changes in the expression of multiple genes implicated in inflammatory skin diseases and skin cancers suggests potential therapeutic applications of intense THz pulses.

Immunosenescence is associated with altered gene expression and epigenetic regulation in primary and secondary immune organs.

Deterioration of the immune system (immunosenescence) with age is associated with an increased susceptibility to infection, autoimmune disease and cancer, and reduced responsiveness to vaccination. Immunosenescence entails a reduced supply of naïve T cells from the thymus and increased specialization of peripheral T cell clones. Both thymic involution and peripheral T cell homeostasis are thought to involve cellular senescence. In order to analyze this at the molecular level, we studied gene expression profiles, epigenetic status, and genome stability in the thymus and spleen of 1-, 4-, and 18-month-old Long Evans rats. In the thymus, altered gene expression, DNA and histone H3K9 hypomethylation, increased genome instability, and apoptosis were observed in 18-month-old animals compared to 1- and 4-month-old animals. In the spleen, alterations in gene expression and epigenetic regulation occurred already by the age of 4 months compared to 1 month and persisted in 18-month-old compared to 1-month-old rats. In both organs, these changes were accompanied by the altered composition of resident T cell populations. Our study suggests that both senescence and apoptosis may be involved in altered organ function.

The elucidation of stress memory inheritance in Brassica rapa plants

Plants are able to maintain the memory of stress exposure throughout their ontogenesis and faithfully propagate it into the next generation. Recent evidence argues for the epigenetic nature of this phenomenon. Small RNAs (smRNAs) are one of the vital epigenetic factors because they can both affect gene expression at the place of their generation and maintain non-cell-autonomous gene regulation. Here, we have made an attempt to decipher the contribution of smRNAs to the heat-shock-induced transgenerational inheritance in Brassica rapa plants using sequencing technology. To do this, we have generated comprehensive profiles of a transcriptome and a small RNAome (smRNAome) from somatic and reproductive tissues of stressed plants and their untreated progeny. We have demonstrated that the highest tissue-specific alterations in the transcriptome and smRNAome profile are detected in tissues that were not directly exposed to stress, namely, in the endosperm and pollen. Importantly, we have revealed that the progeny of stressed plants exhibit the highest fluctuations at the smRNAome level but not at the transcriptome level. Additionally, we have uncovered the existence of heat-inducible and transgenerationally transmitted tRNA-derived small RNA fragments in plants. Finally, we suggest that miR168 and braAGO1 are involved in the stress-induced transgenerational inheritance in plants.

Effect of external and internal factors on the expression of reporter genes driven by the N resistance gene promoter.

The role of resistance (R) genes in plant pathogen interaction has been studied extensively due to its economical impact on agriculture. Interaction between tobacco mosaic virus (TMV) and the N protein from tobacco is one of the most widely used models to understand various aspects of pathogen resistance. The transcription activity governed by N gene promoter is one of the least understood elements of the model. In this study, the N gene promoter was cloned and fused with two different reporter genes, one encoding β-glucuronidase (N::GUS) and another, luciferase (N::LUC). Tobacco plants transformed with the N::GUS or N::LUC reporter constructs were screened for homozygosity and stable expression. Histochemical analysis of N::GUS tobacco plants revealed that the expression is organ specific and developmentally regulated. Whereas two week old plants expressed GUS in midveins only, 6-wk-old plants also expressed GUS in leaf lamella. Roots did not show GUS expression at any time during development. Experiments to address effects of external stress were performed using N::LUC tobacco plants. These experiments showed that N gene promoter expression was suppressed when plants were exposed to high but not low temperatures. Expression was also upregulated in response to TMV, but no changes were observed in plants treated with SA.

miR-34a directly targets tRNAiMet precursors and affects cellular proliferation, cell cycle, and apoptosis

Significance This work identifies tRNAiMet precursors as a direct target of tumor suppressor miR-34a, and indicates that targeted suppression of tRNAiMet levels attenuates cell proliferation while inducing cell cycle arrest and apoptosis. The tRNAiMet may act as an oncogene and contribute significantly to tumorigenesis. Our findings provide conceptual and mechanistic insights into biology and cancer epigenetics and might have significant therapeutic implications. It remains unknown whether microRNA (miRNA/miR) can target transfer RNA (tRNA) molecules. Here we provide evidence that miR-34a physically interacts with and functionally targets tRNAiMet precursors in both in vitro pulldown and Argonaute 2 (AGO2) cleavage assays. We find that miR-34a suppresses breast carcinogenesis, at least in part by lowering the levels of tRNAiMet through AGO2-mediated repression, consequently inhibiting the proliferation of breast cancer cells and inducing cell cycle arrest and apoptosis. Moreover, miR-34a expression is negatively correlated with tRNAiMet levels in cancer cell lines. Furthermore, we find that tRNAiMet knockdown also reduces cell proliferation while inducing cell cycle arrest and apoptosis. Conversely, ectopic expression of tRNAiMet promotes cell proliferation, inhibits apoptosis, and accelerates the S/G2 transition. Moreover, the enforced expression of modified tRNAiMet completely restores the phenotypic changes induced by miR-34a. Our results demonstrate that miR-34a directly targets tRNAiMet precursors via AGO2-mediated cleavage, and that tRNAiMet functions as an oncogene, potentially representing a target molecule for therapeutic intervention.

Adverse effects of paternal chemotherapy exposure on the progeny brain: intergenerational chemobrain

Recent advances in cancer treatments have led to significant increases in cure rates. Most cancer patients are treated with various cytotoxic chemotherapy regimens. These treatment modalities are mutagenic and genotoxic and cause a wide array of late-occurring health problems, and even exert a deleterious influence on future offspring. The adverse effects from exposed parents on offspring are referred to as transgenerational effects, and currently little is known about chemotherapy-induced transgenerational effects. Furthermore, transgenerational effects have not been studied in the brains of progeny of exposed parents. In this study, we analyzed the existence and molecular nature of transgenerational effects in the brains of progeny of animals exposed to three common chemotherapy agents: cyclophosphamide (CPP), procarbazine (PCB) and mitomycin C (MMC). For the first time, our results show that paternal exposure to chemotherapy drugs causes transgenerational changes in the brain of unexposed progeny. Although no DNA damage was observed in terms of γH2AX levels, some alterations were found in levels of PCNA, protein involved in DNA repair, replication and profileration. Furthermore, there were changes in proliferation and apoptosis proteins BCL2 and AKT1, the proteins associated with DNA methylation, DNMT1 and MeCP2. Some altered expression trends were noted in proteins involved in myelin biogenesis, MBP and MYT1L. Moreover, global transcriptome profiling revealed changes in over 200 genes in the whole brains of progeny of animals exposed to CPP, and the changes in the levels of FOXP2 and ELK1proteins were confirmed by western blot analysis. These findings suggest that paternal chemotherapy significantly affects offspring brain development and may affect brain functioning. This research provides a key roadmap for future investigations of the novel phenomenon of transgenerational effects of chemotherapy in the brain of progeny of exposed parents.

Effect of intense THz pulses on expression of genes associated with skin cancer and inflammatory skin conditions

The growing experimental evidence suggests that broadband, picosecond-duration THz pulses may influence biological systems and functions. While the mechanisms by which THz pulse-induced biological effects are not yet known, experiments using in vitro cell cultures, tissue models, as well as recent in vivo studies have demonstrated that THz pulses can elicit cellular and molecular changes in exposed cells and tissues in the absence of thermal effects. Recently, we demonstrated that intense, picosecond THz pulses induce phosphorylation of H2AX, indicative of DNA damage, and at the same time activate DNA damage response in human skin tissues. We also find that intense THz pulses have a profound impact on global gene expression in human skin. Many of the affected genes have important functions in epidermal differentiation and have been implicated in skin cancer and inflammatory skin conditions. The observed THzinduced changes in expression of these genes are in many cases opposite to disease-related changes, suggesting possible therapeutic applications of intense THz pulses.