Jaroslav Janda

Enhanced salt stress tolerance of rice plants expressing a vacuolar H+‐ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel

The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K(+) /Na(+) ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na(+) and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa.

Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice

BackgroundWe report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features.ResultsWe have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype.ConclusionWe anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

Evidence for antisense transcription associated with microRNA target mRNAs in arabidopsis

Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways.

The effect of sulforaphane on histone deacetylase activity in keratinocytes: Differences between in vitro and in vivo analyses

Sulforaphane is a natural product found in broccoli, which is known to exert many different molecular effects in the cell, including inhibition of histone deacetylase (HDAC) enzymes. Here, we examine for the first time the potential for sulforaphane to inhibit HDACs in HaCaT keratinocytes and compare our results with those found using HCT116 colon cancer cells. Significant inhibition of HDAC activity in HCT116 nuclear extracts required prolonged exposure to sulforaphane in the presence of serum. Under the same conditions HaCaT nuclear extracts did not exhibit reduced HDAC activity with sulforaphane treatment. Both cell types displayed down‐regulation of HDAC protein levels by sulforaphane treatment. Despite these reductions in HDAC family member protein levels, acetylation of marker proteins (acetylated Histone H3, H4, and tubulin) was decreased by sulforaphane treatment. Time‐course analysis revealed that HDAC6, HDAC3, and acetylated histone H3 protein levels are significantly inhibited as early as 6 h into sulforaphane treatment. Transcript levels of HDAC6 are also suppressed after 48 h of treatment. These results suggest that HDAC activity noted in nuclear extracts is not always translated as expected to target protein acetylation patterns, despite dramatic inhibition of some HDAC protein levels. In addition, our data suggest that keratinocytes are at least partially resistant to the nuclear HDAC inhibitory effects of sulforaphane, which is exhibited in HCT116 and other cells.

A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes.

One of the primary components of the East Indian sandalwood oil (EISO) is α-santalol, a molecule that has been investigated for its potential use as a chemopreventive agent in skin cancer. Although there is some evidence that α-santalol could be an effective chemopreventive agent, to date, purified EISO has not been extensively investigated even though it is widely used in cultures around the world for its health benefits as well as for its fragrance and as a cosmetic. In the current study, we show for the first time that EISO-treatment of HaCaT keratinocytes results in a blockade of cell cycle progression as well as a concentration-dependent inhibition of UV-induced AP-1 activity, two major cellular effects known to drive skin carcinogenesis. Unlike many chemopreventive agents, these effects were not mediated through an inhibition of signaling upstream of AP-1, as EISO treatment did not inhibit UV-induced Akt or MAPK activity. Low concentrations of EISO were found to induce HaCaT cell death, although not through apoptosis as annexin V and PARP cleavage were not found to increase with EISO treatment. However, plasma membrane integrity was severely compromised in EISO-treated cells, which may have led to cleavage of LC3 and the induction of autophagy. These effects were more pronounced in cells stimulated to proliferate with bovine pituitary extract and EGF prior to receiving EISO. Together, these effects suggest that EISO may exert beneficial effects upon skin, reducing the likelihood of promotion of pre-cancerous cells to actinic keratosis (AK) and skin cancer.

Changes in mitochondrial DNA alter expression of nuclear encoded genes associated with tumorigenesis

We previously reported the presence of a mtDNA mutation hotspot in UV-induced premalignant and malignant skin tumors in hairless mice. We have modeled this change (9821insA) in murine cybrid cells and demonstrated that this alteration in mtDNA associated with mtBALB haplotype can alter the biochemical characteristics of cybrids and subsequently can contribute to significant changes in their behavioral capabilities. This study shows that changes in mtDNA can produce differences in expression levels of specific nuclear-encoded genes, which are capable of triggering the phenotypes such as seen in malignant cells. From a potential list of differentially expressed genes discovered by microarray analysis, we selected MMP-9 and Col1a1 for further studies. Real-time PCR confirmed up-regulation of MMP-9 and down-regulation of Col1a1 in cybrids harboring the mtDNA associated with the skin tumors. These cybrids also showed significantly increased migration and invasion abilities compared to wild type. The non-specific MMP inhibitor, GM6001, was able to inhibit migratory and invasive abilities of the 9821insA cybrids confirming a critical role of MMPs in cellular motility. Nuclear factor-κB (NF-κB) is a key transcription factor for production of MMPs. An inhibitor of NF-κB activation, Bay 11-7082, was able to inhibit the expression of MMP-9 and ultimately decrease migration and invasion of mutant cybrids containing 9821insA. These studies confirm a role of NF-κB in the regulation of MMP-9 expression and through this regulation modulates the migratory and invasive capabilities of cybrids with mutant mtDNA. Enhanced migration and invasion abilities caused by up-regulated MMP-9 may contribute to the tumorigenic phenotypic characteristics of mutant cybrids.

Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.

Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.

Identification of an mtDNA Mutation Hot Spot in UV-Induced Mouse Skin Tumors Producing Altered Cellular Biochemistry

There is increasing awareness of a role of mtDNA alterations in the development of cancer since mtDNA point mutations are found at high frequency in a variety of human tumors. To determine the biological effects of mtDNA mutations in UV-induced skin tumors, hairless mice were irradiated to produce tumors and the tumor mtDNAs were screened for single nucleotide changes using temperature gradient capillary electrophoresis (TGCE) followed by direct sequencing. A mutation hot spot (9821insA) in mt-Tr locus (tRNAArg) was discovered in approximately one third of premalignant and malignant skin tumors. To determine the functional relevance of this particular mutation in vitro, cybrid cell lines containing different mt-Tr (tRNAArg) alleles were generated. The resulting cybrid cell lines contain the same nuclear genotype and differ only in their mtDNA. The biochemical analysis of the cybrids revealed that the mutant haplotype is associated with diminished levels of complex I protein resulting in lower levels of baseline oxygen consumption and lower cellular ATP production. We hypothesize that this specific mtDNA mutation alters cellular biochemistry supporting the development of keratinocyte neoplasia.

Cyclophilin 40 alters UVA-induced apoptosis and mitochondrial ROS generation in keratinocytes.

Abstract The CyP40 protein encoded by PPID gene is a member of the peptidyl-prolyl cis–trans isomerase (PPIase) family. PPIases catalyze the cis–trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. The CyP40 protein has been shown to possess PPIase activity and, similar to other family members, can bind to the immunosuppressant drug cyclosporin A (CsA). In this study, we created keratinocyte cell lines with CyP40 being stably knocked down using viral particles containing shRNA for CyP40 which knocked down the expression level of CyP40 transcripts by 90–99%. The proliferation rates of the cell lines with silenced CyP40 were decreased compared to the control cells. After UVA irradiation, the rate of apoptosis was found to be significantly lower in CyP40 silenced cell lines than it was in control cells. Moreover, mitochondrial membrane potential (MMP) was found to be less dissipated and mitochondrial permeability transition pore (MPTP) less active in cells with knocked down CyP40 than in control cells after UVA irradiation. Also, less mitochondrial superoxide was detected in the cells with silenced CyP40 compared to control cells after UVA exposure. Moreover, silencing of CyP40 partially modulates expression of key genes involved in mitochondrial pore formation including CyPD, ANTs and VDAC family members. The ability of CyP40 to regulate UV induced apoptosis implicates this protein as a potential target for therapy in cancer cells.

Inhibition of Akt enhances the chemopreventive effects of topical rapamycin in mouse skin

The PI3Kinase/Akt/mTOR pathway has important roles in cancer development for multiple tumor types, including UV-induced nonmelanoma skin cancer. Immunosuppressed populations are at increased risk of aggressive cutaneous squamous cell carcinoma (SCC). Individuals who are treated with rapamycin (sirolimus, a classical mTOR inhibitor) have significantly decreased rates of developing new cutaneous SCCs compared with those that receive traditional immunosuppression. However, systemic rapamycin use can lead to significant adverse events. Here, we explored the use of topical rapamycin as a chemopreventive agent in the context of solar-simulated light (SSL)-induced skin carcinogenesis. In SKH-1 mice, topical rapamycin treatment decreased tumor yields when applied after completion of 15 weeks of SSL exposure compared with controls. However, applying rapamycin during SSL exposure for 15 weeks, and continuing for 10 weeks after UV treatment, increased tumor yields. We also examined whether a combinatorial approach might result in more significant tumor suppression by rapamycin. We validated that rapamycin causes increased Akt (S473) phosphorylation in the epidermis after SSL, and show for the first time that this dysregulation can be inhibited in vivo by a selective PDK1/Akt inhibitor, PHT-427. Combining rapamycin with PHT-427 on tumor prone skin additively caused a significant reduction of tumor multiplicity compared with vehicle controls. Our findings indicate that patients taking rapamycin should avoid sun exposure, and that combining topical mTOR inhibitors and Akt inhibitors may be a viable chemoprevention option for individuals at high risk for cutaneous SCC. Cancer Prev Res; 9(3); 215–24. ©2016 AACR.