Analysis of the potential role of SET in chromium-induced malignant transformation cells based on quantitative proteomics

Involvement of a novel regulatory cascade consisting of SET-H3K18ac/H3K27ac-53BP1 in Cr(VI)-induced malignant transformation of 16HBE cells

To investigate the effect of poly-ADP-ribosylation in hexavalent chromium Cr(VI) induced cell damage. The study object, poly (ADP-ribose) glycohydrolase (PARG) deficient human bronchial epithelial cells (16HBE cells), was constructed previously by our research group. Normal 16HBE cells and PARG-deficient cells were treated with different doses of Cr (VI) for 24 h to compare the differences to Cr (VI) toxicity, meanwhile set up the solvent control group. On this basis, 5.0 µmol/L of Cr (VI) was selected as the exposure dose, after the exposure treatment, total proteins of both cells were extracted for two dimension fluorescence difference gel electrophoresis (2D-DIGE) separation, statistically significant differential protein spots were screened and identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS), and further validated by Western blot. After Cr (VI) treatment, the survival rate of PARG-deficient cells was higher than normal 16HBE cells. When the doses reached up to 5.0 µmol/L, the survival rate of 16HBE cells and PARG-deficient cells were respectively (59.67 ± 6.43)% and (82.00 ± 6.25)%, the difference between which was significant (t = -4.32, P < 0.05). 18 protein spots were selected and successfully identified after 2D-DIGE comparison of differential proteins between normal 16HBE cells and PARG-deficient cells before and after exposure. The function of those proteins was involved in the maintenance of cell shape, energy metabolism, DNA damage repair and regulation of gene expression. The differential expression of cofilin-1 was successfully validated by Western blot. The expression level of cofilin-1 in the 16HBE cells increased after Cr (VI) exposure with the relative expression quantity of 1.41 ± 0.04 in treated group and 1.00 ± 0.01 in control group, the difference of which was statistically significant (t = -18.00, P < 0.05), while the expression level in PARG-deficient cells had no statistically significant difference (t = -8.61, P > 0.05). Most of the identified differential proteins are closely related to tumorigenesis, suggesting that poly-ADP-ribosylation reaction may resist the cytotoxicity of Cr(VI) by inhibiting Cr (VI) induced tumorigenesis, which provides important reference data to clarify the mechanisms of poly-ADP-ribosylation in Cr (VI) induced cell damage.

Increasing evidence shows that circular RNA (circRNA) plays an important role in the progression of lung cancer. In this study, we found that has_circ_0000043 was highly expressed in 16HBE-T human bronchial epithelial cells that were malignantly transformed by benzo[a]pyrene-trans-7,8-diol-9,10-epoxide via circRNA microarray. We verified that hsa_circ_0000043 was also significantly overexpressed in lung cancer cell lines and tissues. Moreover, hsa_circ_0000043 overexpression was positively correlated with poor clinicopathological parameters, such as tumor-node metastasis stage, distant metastasis, lymph-node metastasis, and overall survival. In vitro assays revealed that hsa_circ_0000043 inhibition suppressed 16HBE-T cell proliferation, migration, and invasion. Furthermore, hsa_circ_0000043 inhibition suppressed tumor growth in a mouse xenograft model. We discovered that hsa_circ_0000043 binds with miR-4492, acting as a miR-4492 sponge. Decreased miR-4492 expression was also associated with poor clinicopathological parameters. Thus, hsa_circ_0000043 was shown to contribute to the proliferation, malignant transformation ability, migration, and invasion of 16HBE-T cells via miR-4492 sponging and BDNF and STAT3 involvement.

: As an important tumor suppressor, the inactivation and mutation of p53 is discovered in more than 50% of cancers. Repression of tumor progression by p53 is mainly through its function as a common transcription factor for regulation of its target genes, involved in cell growth regulation, DNA damage repair and apoptosis process. There are multiple types of post-transcriptional modifications (PTMs) on p53 protein, including phosphorylation, acetylation, mono- and di-methylation, ubiquitylation, sumoylation and so on. These modifications usually do not function alone; they always interplay with other PTMs and collectively regulate p53 function mainly through regulation of p53 stability and transactivity. This paper reviews the function and mechanism of major posttranslational modifications of p53 and the interaction between these posttranslational modifications.

Anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide (anti-BPDE) is the most important metabolite of benzo[a]pyrene which is a ubiquitous environmental pollutant, and may cause human cancer, especially of the lung. Ras genes (H, K, and N) are activated in 40% of human tumors and may contribute to carcinogenesis. Here, we used malignant human bronchial epithelial cells transformed by anti-BPDE (16HBE-T) to help characterize possible molecular mechanisms of carcinogenesis. We compared H-, K-, and N-Ras mRNA and protein expression levels in 16HBE-T cells and untransformed control 16HBE cells (16HBE-N), using reverse transcription-PCR (RT-PCR) and Western blotting. We further used short hairpin RNA to silence N-Ras gene expression in 16HBE-T cells to determine the effects of silencing on the cell cycle, transformation efficiency and tumor growth. We observed overexpression of H-, K-, and N-Ras genes at both mRNA and protein levels in 16HBE-T cells, compared with 16HBE-N cells. Silencing of N-Ras in 16HBE-T cells using stable RNA interference increased the proportion of cells in G(0)/G(1) phase, decreased the proportion in S-phase, decreased transformation efficiency, and inhibited tumor growth. Our findings suggest that overexpression of N-Ras gene plays an important role in malignant transformation of 16HBE cells by anti-BPDE. N-Ras gene may be a useful target for gene therapy.

Skin cancer is the most common form of cancer, with over 5 million cases reported in 2021. UVB exposure derived from sunlight is the major carcinogenic driver of skin cancer and poses a major public health risk. In response to UVB exposure, keratinocytes must activate DNA repair pathways to avoid mutagenesis. In addition to DNA repair pathways, cells initiate well-coordinated DNA damage response pathways to facilitate the repair process, including arresting global transcription. Here we report the use of kethoxal-assisted single-strand DNA-Sequencing (KAS-Seq) technology to profile the transcriptional dynamics that underlie the DNA damage response upon UVB exposure. Mechanistically, KAS-Seq involves the addition of an azide-tagged kethoxal group (N3-kethoxal) to DNA samples, which then reacts with guanines on single-stranded DNA (ssDNA). A “KAS signal” is therefore only obtained when DNA is unwound, allowing the N3-kethoxal reaction with accessible guanines to occur. Accordingly, in response to DNA damage, DNA can form ssDNA bubbles in order to facilitate DNA damage recognition and repair processes. KAS-Seq is therefore a suitable method to profile the transcriptional dynamics that occur in response to UVB-induced DNA damage. Here we show that UVB exposure in normal human keratinocytes (NHEK) cells induces a temporal and dynamic reprogramming in transcription using KAS-Seq. In terms of global transcription, our data show that the greatest loss of KAS signal occurs rapidly between 30mins and 3hrs post-UVB exposure, signifying transcriptional arrest. Interestingly, KAS signal is regained between 6-24hrs post-UVB, which is representative of a transition towards transcriptional recovery and induction of DNA damage processes. Furthermore, a more discrete and targeted analysis shows that the gain and loss of KAS signals primarily occur within the gene body, followed by promoter and intergenic regions. Overall, our data shows that transcriptional control in response to UVB-induced DNA damage is both dynamic and temporal. Further analysis is required to identify which transcriptional phenotype (transcriptional arrest or transcriptional recovery) is the most deleterious to the cell and DNA damage repair processes when disrupted. Additionally, we aim to further elucidate the molecular mechanisms that underlie these global changes in transcription, which will provide expanded insights into the wide-ranging oncogenic mechanisms that underlie UVB exposure. Citation Format: Emma A. Wilkinson, Tong Wu, Gayoung Park, Yan-Hong Cui, Chuan He, Yu-Ying He. Kethoxal-assisted single-stranded DNA sequencing (KAS-seq) reveals dynamic and temporal changes in transcription in response to UVB exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 227.

In an earlier study, we found that hepatitis C virus core protein, HCV-C, participated in the malignant transformation of HCV-C transfected normal human biliary epithelial (hBE) cells by activating telomerase. Here we further investigated the signaling of the malignant transformation. Reverse transcription-polymerase chain reaction (RT-PCR), western blotting and immunoprecipitation were used to analyze the expression of HCV-C, human telomerase reverse transcriptase (hTERT), nuclear factor-kappaB (NF-kappaB) and NF-kappaB inhibitor alpha (IkappaBalpha) genes and the phosphorylation level of IkappaBalpha protein. Electrophoretic mobility shift assays (EMSA) and NF-kappaB-linked luciferase reporter assays were carried out to measure NF-kappaB activity. The expression of HCV-C and hTERT was detected only in HCV-C-transfected hBE (hBE-HCV-C) cells but not in vector-transfected or parental hBE cells. More NF-kappaB protein accumulated in nuclear extracts of hBE-HCV-C cells rather than in those of control cells, though total NF-kappaB protein level showed no difference among these cells. DNA binding activity of NF-kappaB and the NF-kappaB-linked luciferase activity were much higher in HCV-C-transfected hBE cells than those in vector- or non-transfected hBE cells. In addition, the IkappaBalpha phosphorylation level, but not the IkappaBalpha mRNA or protein levels, was increased after HCV-C transfection. Hepatitis C virus core protein activates NF-kappaB pathway in hBE cells by increasing the phosphorylation of IkappaBalpha. The pathway may be responsible for HCV-C-induced malignant transformation of hBE cells.

DNA damage occurs frequently resulting from both exogenous and endogenous factors, which induce a series of downstream responses including autophagy and DNA damage repair. In the past few years, increasing evidence has indicated that the interplay between autophagy and DNA damage repair is essential for maintaining genome stability as well as cellular homeostasis, and have significant effects on cell fate. On one hand, autophagy is induced during the process of DNA damage repair, and can act as an upstream factor of DNA damage repair as well. On the other hand, autophagy plays a rather dual role in regulating DNA damage repair, as mild and repairable DNA damage repair can be restored with facilitation of autophagy, hyperactivation of autophagy can be cytotoxic and have negative impact on DNA damage repair. In this article, we review current understandings about the cross talk between autophagy and DNA damage repair, with particular attention to their significance to genome integrity and effects on cell fate.

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

Objective To screen and identify serum protein biomarkers for the differential diagnosis between ischemic colitis (IC) and ulcerative colitis (UC) by tandem mass tag (TMT) combined with liquid chromatography/tandem mass spectrometry (LC-MS/MS). Methods From January 2018 to January 2019, at the First Affiliated Hospital of School of Medicine of Zhejiang University, patients with UC or IC, and health controls, each 10 cases, were enrolled into UC group, IC group and normal control (NC) group, respectively. Fasting serum samples of all the subjects were collected. After removal of high-abundance protein, followed by proteolysis, peptide labeling and fractionating, the samples were then processed by mass spectrometry. The protein with TMT data of three groups was obtained and protein with TMT value 0 were removed. Heat map of protein was constructed. The differential protein was defined as the protein fold change over 1.5 or less than 0.67. The Reactome database was used to cluster the pathways of differential proteins among groups. Statistical methods included t test, hypergeometry test and corrected by BH multiple test. Results A total of 357 serum proteins were identified by proteomic profiling. There were 27 differential proteins between the IC group and the NC group, including six up-regulated proteins and 21 down-regulated proteins. There were 228 differential proteins between the UC group and the NC group, including 75 up-regulated proteins and 153 down-regulated proteins. There were 49 differential proteins between UC group and IC group, including 22 up-regulated proteins and 27 down-regulated proteins. In the comparison of differential proteins between the NC group, IC group and UC group, only the expression of fibrin 3 was statistically significant (the fold change between UC and NC, between UC and IC, between IC and NC were 0.24, 0.46 and 0.53, respectively; t=-5.089, -7.298 and -3.919, all P<0.01). The results of pathway cluster analysis showed that in the comparison of differential proteins between IC group and NC group, only the platelet degranulation pathway was enriched, and 10 proteins were involved in this pathway (P<0.01). In the comparison of differential proteins between UC group and NC group, there were 58 pathways enriched, of which 38 proteins were involved in the platelet degranulation pathway, 16 proteins were involved in the initial complement trigger pathway, 13 proteins were involved in the complement cascade pathway, and 11 proteins were involved in antibody-mediated complement activation pathway (all P<0.01). In the comparison of differential proteins between UC group and IC group, three different pathways were obtained. Among them, nine proteins were involved in the platelet degranulation pathway, seven proteins were involved in the initial complement trigger pathway, and five proteins were involved in the complement cascade pathway (all P<0.01). Conclusions The difference in serum proteome between IC patients and UC patients was significant, and the differential proteins are mainly involved in platelet activation and complement activation. The candidate proteins identified in this study may be used as biomarkers for the differential diagnosis of UC and IC in the future. Key words: Colitis, ischemic; Colitis, ulcerative; Proteomics; Markers, serum

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

To investigate the possible mechanism of "regulating qi and relieving depression" acupuncture underlying improvement of chronic unpredictable mild stress (CUMS)-induced depression in rats by using Tandem Mass Tags(TMT) quantitative proteomics technique. Thirty-six male SD rats were randomly divided into control, model and acupuncture groups, with 12 rats in each group. The depression model was induced by CUMS stress for 21 days. After the depression model was successfully established, the rats in the acupuncture group received manual acupuncture stimulation at "Baihui" (GV20) and "Yintang" (GV24+) for 20 min, once daily for 21 days. Open field test, sugar water preference test and forced swimming test (FST) were used to evaluate the behavioral changes. TMT quantitative proteomics was used to obtain differential proteins in the hippocampus tissue and related signaling pathways enrichment was analyzed, followed by verifying differential protein pathways by using Western blot and immunofluorescence methods. Behavior tests showed that on the 21st and 42nd days, the horizontal crossing times, walking distance and percentage of sugar water consumption were significantly decreased (P<0.05), while the immobility time of FST was obviously increased (P<0.05) in the model group relevant to the control group. After acupuncture intervention, the horizontal crossing times, walking distance and percentage of sugar water consumption were significantly increased (P<0.05), and the immobility time was apparently decreased (P<0.05) in the acupuncture group relevant to the model group. The TMT quantitative proteomics of hippocampus tissue displayed that of the 71 differential proteins (model group vs control group), 32 was down-regulated and 39 up-regulated in the model group; and among the above 71 differential proteins, there were 20 differential proteins between acupuncture group and model group, 15 down-regulated and 5 up-regulated in the acupuncture group (vs the model group). The expression of Mapk8ipl was up-regulated in the model group (vs the control group) and down-regulated in the acupuncture group (vs the model group). GO and KEGG enrichment analysis showed that these acupuncture-related differential proteins mainly involve the regulation of blood coagulation system, MAPK signaling pathway, etc. We selected the MAPK/JNK signaling pathway related to depression for verification. Western blot showed that the expression levels of c-JUN and phosphorylated c-JUN terminal kinase (p-JNK) proteins in the hippocampus were up-regulated in the model group relevant to the control group (P<0.05); while the expression levels of c-JUN and p-JNK proteins in the hippocampus were down-regulated in the acupuncture group relevant to the model group (P<0.05). The results of immunofluorescence showed that the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1, CA3 and DG regions was increased in the model group relevant to the control group (P<0.05), while the mean fluorescence intensity of c-JUN and p-JNK in hippocampal CA1, CA3 and DG regions was obviously lower in the acupuncture group than in the model group (P<0.05). Acupuncture for "regulating qi and relieving depression" can significantly improve depression-like behavior in CUMS-induced depression model rats, which involves multiple targets and multiple pathways, including MAPK/JNK signaling.

The nucleolus is the most prominent structure in a cell nucleus. It is the site of ribosomal RNA (rRNA) transcription, pre-rRNA processing and ribosome subunit assembly (reviewed by Olson et al., 2002). The nucleolus is a dynamic structure that assembles around the clusters of rRNA gene repeats during late telophase, persists throughout interphase and then disassembles as cells enter mitosis. Owing to the difference in density between the nucleolus and the surrounding nucleoplasm, it is readily visible in either live or fixed cells viewed by phase contrast or differential interference contrast (DIC) optics (upper-left panel). Thanks to the advent of fluorescent protein (FP) technology, nucleoli can also be detected by fluorescence microscopy in cell lines expressing FP-tagged nucleolar proteins. An example is shown in the inset of the upper-left panel, in which PP1γ, a protein phosphatase that accumulates in the nucleolus, is tagged with YFP and stably expressed in HeLa cells (Trinkle-Mulcahy et al., 2003).FIG1Like all other intranuclear structures, the nucleolus is not membrane enclosed, but the combination of its unique density and robust structure makes it one of the most convenient subcellular structures to purify. Thus, when mammalian nuclei are physically disrupted (e.g. by sonication) in a solution of low salt concentration, nucleoli remain intact even under conditions that disintegrate most other subnuclear structures. Nucleoli can therefore be isolated in essentially pure form by centrifuging sonicated nuclei through a density cushion (middle-left panel). The isolated nucleoli are intact, similar in size and morphology to the nucleoli in live cells and even retain transcriptional activity to some extent (Cheutin et al., 2002).Thanks to the ability to isolate large amounts of purified nucleoli, recent studies have analysed the nucleolar protein composition in great detail using high-throughput mass-spectrometry (MS)-based proteomic techniques (lower-left panel). For example, initial proteomic studies of HeLa nucleoli reported the identification of over 400 proteins (Andersen et al., 2002; Scherl et al., 2002). Ongoing improvements in protein separation methods prior to MS, and in the sensitivity of MS, continue to expand the number of nucleolar proteins identified. An online database describing close to 700 human proteins detected in purified nucleoli is now available (www.lamondlab.com/Nopdb) (Andersen et al., 2005). A proteome of the Arabidopsis thaliana nucleolus has also been identified recently (http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home) (Pendle et al., 2005). One striking discovery from these proteomic studies is that up to 30% of the nucleolar proteins are encoded by previously uncharacterised genes (Andersen et al., 2002; Andersen et al., 2005). This suggests that despite the extensive previous research on the nucleolus extending over almost two centuries, there is still much to be learned about its structure and function. Moreover, with the new availability of human and plant nucleolar proteomes, bioinformatic studies have already started to reveal new insights concerning common motifs found in nucleolar proteins (Leung et al., 2003) and the evolution of this nuclear organelle (Staub et al., 2004).Examination of the proteome provides a glimpse into the functional complexity of the nucleolus. Its central role in ribosome subunit biogenesis is confirmed by the presence of many proteins (more than one third of the nucleolar proteome) involved in different steps in rRNA transcription, rRNA processing and modification, as well as the large and small ribosome subunit proteins themselves (Andersen et al., 2005). However, there are also many proteins that have no obvious relationship with these `classical' nucleolar processes. For example, many proteins related to cell cycle regulation (about 3.5% of the identified proteome), DNA damage repair (about 1%) and pre-mRNA processing (about 5%) are detected in isolated nucleoli. This is consistent with the idea that the nucleolus performs additional roles beyond generating ribosomal subunits (reviewed by Olson et al., 2002; Pederson, 1998). More recent examples of cellular activities linked to the nucleolus include RNA editing (Sansam et al., 2003), DNA damage repair (van den Boom et al., 2004), telomere metabolism (Kieffer-Kwon et al., 2004; Zhang et al., 2004), tRNA processing (Paushkin et al., 2004) and regulation of protein stability (Mekhail et al., 2004; Rodway et al., 2004).The internal structure of the nucleolus has been studied in detail by both transmission and scanning electron microscopy. For example, field-emission scanning electron microscopy (FESEM) analysis of isolated HeLa nucleoli provides a high-resolution (∼1 nm) view of the 3D contour of the nucleolar surface and the interface between the nucleolus and the nucleoplasm (main central image). The internal structure of the nucleolus is revealed by transmission electron microscopy (TEM) of thin sections cut through nucleoli. Based on the morphology revealed by such TEM images, three subcompartments have been identified within the interior of the nucleolus. These include fibrillar centres (FCs), which are surrounded by dense fibrillar components (DFCs), and the FC-DFC complexes are embedded in the granular component (GC). Immuno-EM experiments show that many nucleolar proteins accumulate in one or two of these subcompartments, suggesting they each have distinct protein compositions and functions (Schwarzacher and Mosgoeller, 2000). For example, the RNA polymerase I subunit RPA39 is predominantly localised in the FC, whereas fibrillarin, a protein that is involved in ribose 2′-O-methylation of rRNA, accumulates in the DFC, and nucleolar phosphoprotein B23 is found in the GC. Recently, cell lines have been established that simultaneously express these three proteins tagged with different fluorescent proteins. This allows the assembly and disassembly of each subnucleolar compartment to be analysed in live cells during mitosis by high-resolution light microscopy (Leung et al., 2004). Recent data have also revealed that some factors in nucleoli are localised in subnucleolar regions that do not precisely correlate with any of the three well-known subcompartments (Politz et al., 2002), which suggests that the nucleolus is more complex than a sum of FCs, DFCs and the GC.The nucleolus is a structure in which the interactions and translocation of a large number of proteins and RNAs are conducted and coordinated (reviewed by Fromont-Racine et al., 2003). During ribosome subunit biogenesis, pre-rRNA transcripts are transcribed by RNA polymerase I from the repeated clusters of rDNA genes. This process is vividly demonstrated when eukaryotic cells are swollen and spread in an alkaline, hypotonic solution in the presence of detergent (e.g. Chooi and Leiby, 1981; Mougey et al., 1993; Osheim et al., 2004). Under these conditions (Miller spreads), the rDNA repeats are fully extended and, along this central DNA axis, nascent growing rRNA transcripts can be seen emerging from each rDNA unit. As transcription proceeds from the initiation point, the rRNA transcripts become increasingly longer, resulting in a `Christmas tree' model (reviewed by Trendelenburg et al., 1996). It is not clear how this complex structure is assembled in the nucleolus. The initiation of transcription probably occurs either within the FCs or at the FC-DFC boundary. The resulting pre-rRNA transcripts then emerge into the DFC region, where they are cleaved and modified by the small nucleolar RNPs (snoRNPs) and other processing enzymes. The rRNAs also begin the pathway of assembly with ribosomal proteins in the DFC and continue this as they pass through the GC and are exported to the cytoplasm. The sequential movement of rRNA through the FC, DFC and GC subcompartments can be demonstrated when cells are labelled with a short pulse of halogenated nucleotide, which reveals a wave of nascent rRNA spreading from the FC-DFC complexes to the GC regions (this is shown in three stages in the panel labelled `Nucleolar transcription').Despite major advances in recent years, many important questions remain to be answered about the nucleolus. For example, it is still unclear what components provide the structural integrity of the nucleolus. It is also not known how nucleolar assembly and disassembly is regulated during mitosis, and much remains to be learned concerning the full range of biological processes that either occur within, or else involve, the nucleolus. Similarly, a clearer picture is needed at the molecular level between subnucleolar structure and specific functions. Although a detailed description of the protein content of the nucleolus is now emerging, we still do not know what functions many of these proteins perform, and less is known about the full spectrum of RNAs and DNA sequences that associate with nucleoli. The nucleolus is therefore likely to remain a source of interesting new discoveries and undoubtedly some more surprises for the foreseeable future.We thank Anthony K. L. Leung and Carol E. Lyon for providing some of the images, and other members of the Lamond lab for help and discussions.

In this study, TMT (tandem mass tag)-labeled quantitative protein technology combined with LC–MS/MS (liquid chromatography-mass spectrometry/mass spectrometry) was used to isolate and identify the proteins of the hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis grandis) and the bamboo inoculated with the pathogenic fungi Arthrinium phaeospermum. A total of 3320 unique peptide fragments were identified after inoculation with either A. phaeospermum or sterile water, and 1791 proteins were quantified. A total of 102 differentially expressed proteins were obtained, of which 66 differential proteins were upregulated and 36 downregulated in the treatment group. Annotation and enrichment analysis of these peptides and proteins using the GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases with bioinformatics software showed that the differentially expressed protein functional annotation items were mainly concentrated on biological processes and cell components. The LC–PRM/MS (liquid chromatography-parallel reaction monitoring/mass spectrometry) quantitative analysis technique was used to quantitatively analyze 11 differential candidate proteins obtained by TMT combined with LC–MS/MS. The up–down trend of 10 differential proteins in the PRM results was consistent with that of the TMT quantitative analysis. The coincidence rate of the two results was 91%, which confirmed the reliability of the proteomic results. Therefore, the differentially expressed proteins and signaling pathways discovered here may be the further concern for the bamboo-pathogen interaction studies.

High levels of Basic Transcription Factor 3 (BTF3) have been associated with prostate cancer. However, the mechanisms underlying the role of BTF3 as an oncogenic transcription factor in prostate tumorigenesis have not been explored. Herein, we report that BTF3 confers oncogenic activity in prostate cancer cells. Mechanistically, while both BTF3 splicing isoforms (BTF3a and BTF3b) promote cell growth, BTF3b, but not BTF3a, regulates the transcriptional expression of the genes encoding the subunits of Replication Factor C (RFC) family that is involved in DNA replication and damage repair processes. BTF3 knockdown results in decreased expression of RFC genes, and consequently attenuated DNA replication, deficient DNA damage repair, and increased G2/M arrest. Furthermore, knockdown of the RFC3 subunit diminishes the growth advantage and DNA damage repair capability conferred by ectopic overexpression of BTF3b. Importantly, we show that enforced BTF3 overexpression in prostate cancer cells induces substantial accumulation of cisplatin-DNA adducts and render the cells more sensitive to cisplatin treatment both in vitro and in vivo. These findings provide novel insights into the role of BTF3 as an oncogenic transcription factor in prostate cancer and suggest that BTF3 expression levels may serve as a potential biomarker to predict cisplatin treatment response.