Regulation Of Cell Division Research Articles

Molecular Regulation of Autophagy and Asymmetric Cell Division by Cancer Stem Cell Marker CD133.

CD133, also called prominin-1, is widely known as a cancer stem cell marker, and its high expression correlates with a poor prognosis in many cancers. CD133 was originally discovered as a plasma membranous protein in stem/progenitor cells. It is now known that Src family kinases phosphorylate the C-terminal of CD133. However, when Src kinase activity is low, CD133 is not phosphorylated by Src and is preferentially downregulated into cells through endocytosis. Endosomal CD133 then associates with HDAC6, thereby recruiting it to the centrosome via dynein motors. Thus, CD133 protein is now known to localize to the centrosome as endosomes as well as to the plasma membrane. More recently, a mechanism to explain the involvement of CD133 endosomes in asymmetric cell division was reported. Here, we would like to introduce the relationship between autophagy regulation and asymmetric cell division mediated by CD133 endosomes.

Transcriptome and proteome analyses reveal the potential mechanism of seed dormancy release in Amomum tsaoko during warm stratification

BackgroundIn Amomum tsaoko breeding, the low germination rate is the major limitation for their large-scale reproduction. We found that warm stratification was an effective treatment to break the seed dormancy of A. tsaoko prior to sowing and could be an important component of improving breeding programs. The mechanism of seed dormancy release during warm stratification remains unclear. Therefore, we studied the differences between transcripts and proteomes at 0, 30, 60, and 90 days of warm stratification, to identify some regulatory genes and functional proteins that may cause seed dormancy release in A. tsaoko and reveal their regulatory mechanism.ResultsRNA-seq was performed for the seed dormancy release process, and the number of differentially expressed genes (DEGs) was 3196 in three dormancy release periods. Using TMT-labelling quantitative proteome analysis, a total of 1414 proteins were defined as differentially expressed proteins (DEPs). Functional enrichment analyses revealed that the DEGs and DEPs were mainly involved in signal transduction pathways (MAPK signaling, hormone) and metabolism processes (cell wall, storage and energy reserves), suggesting that these differentially expressed genes and proteins are somehow involved in response to seed dormancy release process, including MAPK, PYR/PYL, PP2C, GID1, GH3, ARF, AUX/IAA, TPS, SPS, and SS. In addition, transcription factors ARF, bHLH, bZIP, MYB, SBP, and WRKY showed differential expression during the warm stratification stage, which may relate to dormancy release. Noteworthy, XTH, EXP, HSP and ASPG proteins may be involved in a complex network to regulate cell division and differentiation, chilling response and the seed germination status in A. tsaoko seed during warm stratification.ConclusionOur transcriptomic and proteomic analysis highlighted specific genes and proteins that warrant further study in fully grasping the precise molecular mechanisms that control the seed dormancy and germination of A. tsaoko. A hypothetical model of the genetic regulatory network provides a theoretical basis for overcoming the physiological dormancy in A. tsaoko in the future.

Cloning of Three Cytokinin Oxidase/Dehydrogenase Genes in Bambusa oldhamii.

Cytokinin oxidase/dehydrogenase (CKX) catalyzes the irreversible breakdown of active cytokinins, which are a class of plant hormones that regulate cell division. According to conserved sequences of CKX genes from monocotyledons, PCR primers were designed to synthesize a probe for screening a bamboo genomic library. Cloned results of three genes encoding cytokinin oxidase were named as follows: BoCKX1, BoCKX2, and BoCKX3. In comparing the exon-intron structures among the above three genes, there are three exons and two introns in BoCKX1 and BoCKX3 genes, whereas BoCKX2 contains four exons and three introns. The amino acid sequence of BoCKX2 protein shares 78% and 79% identity with BoCKX1 and BoCKX3 proteins, respectively. BoCKX1 and BoCKX3 genes are particularly closely related given that the amino acid and nucleotide sequence identities are more than 90%. These three BoCKX proteins carried putative signal peptide sequences typical of secretion pathway, and a GHS-motif was found at N-terminal flavin adenine dinucleotide (FAD) binding domain, suggesting that BoCKX proteins might covalently conjugate with an FAD cofactor through a predicted histidine residue.

The Study on Sea Buckthorn (Genus Hippophae L.) Fruit Reveals Cell Division and Cell Expansion to Promote Morphogenesis.

Due to its unique flavor and high antioxidant content, the sea buckthorn (genus Hippophae L.) fruit is increasingly favored by consumers. Developing from the perianth tube, the sea buckthorn fruit varies greatly among species in both size and shape. However, the cellular regulation mechanism of sea buckthorn fruit morphogenesis remains unclear. This study presents the growth and development patterns, morphological changes, and cytological observations of the fruits of three Hippophae species (H. rhamnoides ssp. sinensis, H. neurocarpa, and H. goniocarpa). The fruits were monitored every 10-30 days after anthesis (DAA) for six periods in their natural population on the eastern margin of the Qinghai-Tibet Plateau in China. The results showed that the fruits of H. rhamnoides ssp. sinensis and H. goniocarpa grew in a sigmoid mode, while H. neurocarpa grew in an exponential mode under the complex regulation of cell division and cell expansion. In addition, cellular observations showed that the mesocarp cells of H. rhamnoides ssp. sinensis and H. goniocarpa were larger in the area with prolonged cell expansion activity, while H. neurocarpa had a higher cell division rate. Elongation and proliferation of the mesocarp cells were found to be essential factors affecting the formation of fruit morphology. Finally, we established a preliminary cellular scenario for fruit morphogenesis in the three species of sea buckthorn. Fruit development could be divided into a cell division phase and a cell expansion phase with an overlap between 10 and 30 DAA. In particular, the two phases in H. neurocarpa showed an additional overlap between 40 and 80 DAA. The description of the sea buckthorn fruit's transformation and its temporal order may provide a theoretical basis to explore the growth mechanism of fruits and regulate their size through certain cultivation techniques.

Size- and position-dependent cytoplasm viscoelasticity through hydrodynamic interactions with the cell surface

Many studies of cytoplasm rheology have focused on small components in the submicrometer scale. However, the cytoplasm also baths large organelles like nuclei, microtubule asters, or spindles that often take significant portions of cells and move across the cytoplasm to regulate cell division or polarization. Here, we translated passive components of sizes ranging from few up to ~50 percents of the cell diameter, through the vast cytoplasm of live sea urchin eggs, with calibrated magnetic forces. Creep and relaxation responses indicate that for objects larger than the micron size, the cytoplasm behaves as a Jeffreys material, viscoelastic at short timescales, and fluidizing at longer times. However, as component size approached that of cells, cytoplasm viscoelastic resistance increased in a nonmonotonic manner. Flow analysis and simulations suggest that this size-dependent viscoelasticity emerges from hydrodynamic interactions between the moving object and the static cell surface. This effect also yields to position-dependent viscoelasticity with objects initially closer to the cell surface being harder to displace. These findings suggest that the cytoplasm hydrodynamically couples large organelles to the cell surface to restrain their motion, with important implications for cell shape sensing and cellular organization.

A molecular dynamics study of the FGF1/FGF2 heterodimer in human fibroblasts.

A family of mitogenic proteins known as fibroblast growth factors is responsible for regulating cell division and growth throughout the body. These signaling molecules are also crucial for wound healing, repairing, and regenerating damaged tissue quickly in order to prevent chronic injuries. Phylogeny and sequence homology classify them into seven subfamilies 1, 4, 7, 8, 9, 11, and 15. Out of all the FGF members, the FGF1 subfamily, consisting of FGF1 and FGF2, is the most prominent and well-studied. It is believed that FGF1 plays the most significant role in cell proliferation, whereas FGF2 plays a vital role in the angiogenic activity. The half-life of FGF1 and FGF2 is significantly shortened under physiological conditions due to their low thermal and proteolytic stability. Heparin is one of the FGF ligands that induce conformational changes in these molecules, which are needed for the FGFs to interact with the receptors (FGFR1, FGFR2, FGFR3, and FGFR4) and initiate the signaling cascade. Besides the limitations associated with individual FGF1 and FGF2, another disadvantage is that FGF1 binds universally to all FGFR1-4, whereas FGF2 binds only to FGFR2. It may be possible that an FGF1-FGF2 dimer facilitates the binding between FGF2 and FGFR2, allowing FGF1 to bind to any adjacent receptor, thereby activating both FGF1 and FGF2 to promote cell growth and angiogenesis. In this study, I have modeled an FGF1-FGF2 fused protein and used microsecond-level molecular dynamics simulations to study the differential behavior of several engineered protein variants regardless of heparin exposure to achieve high thermal stability and enhanced biological functions. Consequently, I have observed an increase in the stability of each monomer within the dimer protein.

Imaging Ligands Targeting Glypican-3 (GPC3) Receptor Expression in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality and is increasing in incidence worldwide. Early detection of HCC is important since potentially curative therapies exist in the initial stages of HCC; no curative therapies exist for late-stage HCC. However, the initial detection of HCC remains challenging due to the lack of symptoms during the early stage of the disease. Other methods of screening and detecting HCC, including blood serum tests and conventional imaging methods, remain inadequate due to genetic differences between patients and the high background activity of liver tissues. Thus, there is a need for an accurate imaging agent for the diagnosis, staging, and prognosis of HCC. Glypican-3 (GPC3) is an oncofetal receptor responsible for regulating cell division, growth, and survival. GPC3 is a clinically relevant biomarker for imaging and therapeutics, as its expression is HCC tumor-specific and absent from normal and other pathological liver tissues. The development of novel GPC3-targeting imaging agents has encompassed three classes of biomolecules: peptides, antibodies, and aptamers. These biomolecules serve as constructs for diagnostic imaging (demonstrating potential as positron emission tomography [PET], single-photon emission tomography [SPECT], and optical imaging agents) and HCC treatment delivery. At least 20 unique ligands have been identified in the literature as showing specificity for the GPC3 receptor. Although several ligands are currently under clinical investigation as therapies for HCC, clinical translation of GPC3-targeting ligands as imaging agents is lacking. This review highlights the current landscape of ligands targeting GPC3 and describes their promising possibilities as imaging agents for HCC.

GRAS transcription factors regulate cell division planes in moss overriding the default rule

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

The transcription factor AtMYB12 is part of a feedback loop regulating cell division orientation in the root meristem vasculature.

Transcriptional networks are crucial to integrate various internal and external signals into optimal responses during plant growth and development. In Arabidopsis thaliana, primary root vasculature patterning and proliferation are controlled by a network centred around the basic Helix-Loop-Helix transcription factor complex, formed by TARGET OF MONOPTEROS 5 (TMO5) and LONESOME HIGHWAY (LHW), which control cell proliferation and division orientation by modulating the cytokinin response and other downstream factors. Despite recent progress, many aspects of the TMO5/LHW pathway are not fully understood. In particular, the upstream regulators of TMO5/LHW activity remain unknown. Here, using a forward genetics approach to identify new factors of the TMO5/LHW pathway, we discovered a novel function of the MYB-type transcription factor, MYB12. MYB12 physically interacts with TMO5 and dampens the TMO5/LHW-mediated induction of direct target gene expression, as well as the periclinal/radial cell divisions. The expression of MYB12 is activated by the cytokinin response, downstream of TMO5/LHW, resulting in a novel MYB12-mediated negative feedback loop that restricts TMO5/LHW activity, to ensure optimal cell proliferation rates during root vascular development.

Gradient Magnetic Field Accelerates Division of E. coli Nissle 1917.

Cell-cycle progression is regulated by numerous intricate endogenous mechanisms, among which intracellular forces and protein motors are central players. Although it seems unlikely that it is possible to speed up this molecular machinery by applying tiny external forces to the cell, we show that magnetic forcing of magnetosensitive bacteria reduces the duration of the mitotic phase. In such bacteria, the coupling of the cell cycle to the splitting of chains of biogenic magnetic nanoparticles (BMNs) provides a biological realization of such forcing. Using a static gradient magnetic field of a special spatial configuration, in probiotic bacteria E. coli Nissle 1917, we shortened the duration of the mitotic phase and thereby accelerated cell division. Thus, focused magnetic gradient forces exerted on the BMN chains allowed us to intervene in the processes of division and growth of bacteria. The proposed magnetic-based cell division regulation strategy can improve the efficiency of microbial cell factories and medical applications of magnetosensitive bacteria.

A potent ion channel blocker, hydroquinidine, exhibits strong anti-cancer activity on colon, pancreatic, and hepatocellular cancer cells.

Despite recent advances in drug discovery, cancer is still one of the most lethal health problems worldwide. In most cases, standard therapy methods and multi-modal treatments fail, and new therapeutic approaches are required. Ion channels are essential in multiple cellular processes regulating cell division, differentiation, and death. Recent studies on ion-channel modulators emphasize their potential to suppress tumor growth. In that regard, we reasoned that an underinvestigated potassium channel modulator, Hydroquinidine (HQ), may exhibit an anti-carcinogenic activity. HQ's potential as an anti-neoplastic compound was examined using colony formation assay, wound healing assay, soft agar assay, and Annexin-V assay in the colon, pancreatic, and hepatocellular carcinomas. Our findings unveiled a remarkable anti-cancer activity of HQ by decreasing colony-forming ability, migration capacity, tumorigenicity, and proliferation and stimulating cellular death. HQ significantly reduced the formed colonies and tumorigenicity for all cells. It displayed a significant anti-migrative effect on hepatocellular carcinoma cells and promoted apoptosis in pancreatic and liver cancer cells. The altered gene expression profile upon HQ treatment was in accordance with observed cellular effects. Cells incubated with HQ downregulated the genes acting in cell division and survival, whereas the expression level of genes functioning in cell cycle arrest and apoptosis was elevated. Our data indicate HQ's competency to limit cancer growth and suggest its utilization as a novel potent anti-carcinogenic agent. Future studies are necessary to provide new insights into the HQ action mechanism and to evaluate its capacity in in-vivo.

Downregulation of CDC25C in NPCs Disturbed Cortical Neurogenesis.

Cell division regulators play a vital role in neural progenitor cell (NPC) proliferation and differentiation. Cell division cycle 25C (CDC25C) is a member of the CDC25 family of phosphatases which positively regulate cell division by activating cyclin-dependent protein kinases (CDKs). However, mice with the Cdc25c gene knocked out were shown to be viable and lacked the apparent phenotype due to genetic compensation by Cdc25a and/or Cdc25b. Here, we investigate the function of Cdc25c in developing rat brains by knocking down Cdc25c in NPCs using in utero electroporation. Our results indicate that Cdc25c plays an essential role in maintaining the proliferative state of NPCs during cortical development. The knockdown of Cdc25c causes early cell cycle exit and the premature differentiation of NPCs. Our study uncovers a novel role of CDC25C in NPC division and cell fate determination. In addition, our study presents a functional approach to studying the role of genes, which elicit genetic compensation with knockout, in cortical neurogenesis by knocking down in vivo.

Profilin 1 deficiency drives mitotic defects and reduces genome stability

Profilin 1—encoded by PFN1—is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability.

Characterization of Transcriptome Dynamics during Early Fruit Development in Olive (Olea europaea L.).

In the olive (Olea europaea L.), an economically leading oil crop worldwide, fruit size and yield are determined by the early stages of fruit development. However, few detailed analyses of this stage of fruit development are available. This study offers an extensive characterization of the various processes involved in early olive fruit growth (cell division, cell cycle regulation, and cell expansion). For this, cytological, hormonal, and transcriptional changes characterizing the phases of early fruit development were analyzed in olive fruit of the cv. 'Picual'. First, the surface area and mitotic activity (by flow cytometry) of fruit cells were investigated during early olive fruit development, from 0 to 42 days post-anthesis (DPA). The results demonstrate that the cell division phase extends up to 21 DPA, during which the maximal proportion of 4C cells in olive fruits was reached at 14 DPA, indicating that intensive cell division was activated in olive fruits at that time. Subsequently, fruit cell expansion lasted as long as 3 weeks more before endocarp lignification. Finally, the molecular mechanisms controlling the early fruit development were investigated by analyzing the transcriptome of olive flowers at anthesis (fruit set) as well as olive fruits at 14 DPA (cell division phase) and at 28 DPA (cell expansion phase). Sequential induction of the cell cycle regulating genes is associated with the upregulation of genes involved in cell wall remodeling and ion fluxes, and with a shift in plant hormone metabolism and signaling genes during early olive fruit development. This occurs together with transcriptional activity of subtilisin-like protease proteins together with transcription factors potentially involved in early fruit growth signaling. This gene expression profile, together with hormonal regulators, offers new insights for understanding the processes that regulate cell division and expansion, and ultimately fruit yield and olive size.

Multiple Roles of PLK1 in Mitosis and Meiosis.

Cells are equipped with a diverse network of signaling and regulatory proteins that function as cell cycle regulators and checkpoint proteins to ensure the proper progression of cell division. A key regulator of cell division is polo-like kinase 1 (PLK1), a member of the serine/threonine kinase family that plays an important role in regulating the mitotic and meiotic cell cycle. The phosphorylation of specific substrates mediated by PLK1 controls nuclear envelope breakdown (NEBD), centrosome maturation, proper spindle assembly, chromosome segregation, and cytokinesis. In mammalian oogenesis, PLK1 is essential for resuming meiosis before ovulation and for establishing the meiotic spindle. Among other potential roles, PLK1 regulates the localized translation of spindle-enriched mRNAs by phosphorylating and thereby inhibiting the translational repressor 4E-BP1, a downstream target of the mTOR (mammalian target of rapamycin) pathway. In this review, we summarize the functions of PLK1 in mitosis, meiosis, and cytokinesis and focus on the role of PLK1 in regulating mRNA translation. However, knowledge of the role of PLK1 in the regulation of meiosis remains limited.

Ekspresija survivina u oralnom skvamocelularnom karcinomu

Introduction: Survivin functions as an apoptosis inhibitor and a regulator of cell division. This study aimed to determine the correlation between survivin expression and clinicopathologic parameters of oral squamous cell carcinoma (OSCC) and determine its potential role in the progression/prognosis of this type of tumor. Materials and methods: Immunohistochemical analysis of survivin expression was performed on 45 surgically obtained paraffin-embedded tissue samples of OSCCs. Data on patients' gender, age, tumor grade, site and stage, disease recurrence, metastasis occurrence , and disease-free interval (DFI) were correlated to survivin expression. Results: Survivin immunoreactivity was observed in 77.8% of samples. No significant correlation between survivin expression and age (p = 0.087), gender (p = 0.334), tumor site (p = 0.175), presence of lymph node metastases (p = 0.201), or disease recurrence (p = 0.451) was found. Survivin expression was observed in well and moderately differentiated tumors and in all clinical stages (p = 0.139). Patients with low survivin expression had better survival rates than the group with medium and high survivin expression, i.e., there was a tendency of a shorter DFI in patients with higher expression of survivin (p = 0.065). Conclusion: There is a tendency for a shorter disease-free period in patients with higher survivin expression. These data suggest that survivin expression in OSCC may act as an additional prognostic parameter that indicates an increased proliferative tumor potential. To further validate survivin as a prognostic marker in OSCC, a study with a larger sample size along with clinical follow-up data is needed.

A YAK1-type protein kinase, triacylglycerol accumulation regulator 1, in the green alga Chlamydomonas reinhardtii is a potential regulator of cell division and differentiation into gametes during photoautotrophic nitrogen deficiency.

Yet another kinase (YAK) 1 is a conserved eukaryotic protein kinase coordinating growth and development. We previously isolated a mutant of Chlamydomonas reinhardtii defective in the YAK1 ortholog triacylglycerol (TAG) accumulation regulator 1 (TAR1). The mutant tar1-1 displayed higher levels of chlorophyll, starch, TAG, and biomass than the parental strain C9 (renamed as C9-3) in photoautotrophic nitrogen (N)-deficient conditions. However, we found that the parental C9-3 showed faster chlorosis upon N-deficiency than the original C9 (C9-1) freshly recovered from cryopreservation, suggesting that C9-3 had acquired particular characteristics during long-term subculturing. To exclude phenotypes dependent on a particular parental strain, we newly created tar1 mutants from two wild-types, C9-1 and CC 125. Like tar1-1, the new tar1 mutants showed higher levels of chlorophyll and TAG/starch than the parental strain. Upon removal of N, Chlamydomonas cells divide once before ceasing further division. Previously, the single division after N-removal was arrested in tar1-1 in photomixotrophic conditions, but this phenotype was not observed in photoautotrophic conditions because of the particular characteristics of the parental C9-3. However, using C9- 1 and CC-125 as parental strains, we showed that cell division after N-removal was impaired in new tar1 mutants in photoautotrophic conditions. Consistent with the view that the division under N-deficiency is necessary for gametic differentiation, new tar1 mutants showed lower mating efficiency than the parental strains. Taken together, TAR1 was suggested to promote differentiation into gametes through the regulation of cell division in response to N-deficiency.

Cyclin-dependent kinases in cancer: Role, regulation, and therapeutic targeting.

Regulated cell division is one of the fundamental phenomena which is the basis of all life on earth. Even a single base pair mutation in DNA leads to the production of the dysregulated protein that can have catastrophic consequences. Cell division is tightly controlled and orchestrated by proteins called cyclins and cyclin-dependent kinase (CDKs), which serve as licensing factors during different phases of cell division. Dysregulated cell division is one of the most important hallmarks of cancer and is commonly associated with a mutation in cyclins and CDKs along with tumor suppressor proteins. Therefore, targeting the component of the cell cycle which leads to these characteristics would be an effective strategy for treating cancers. Specifically, Cyclin-dependent kinases (CDKs) involved in cell cycle regulation have been identified to be overexpressed in many cancers. Many studies indicate that oncogenesis occurs in cancerous cells by the overactivity of different CDKs, which impact cell cycle progression and checkpoint dysregulation which is responsible for development of tumor. The development of CDK inhibitors has emerged as a promising and novel approach for cancer treatment in both solid and hematological malignancies. Some of the novel CDK inhibitors have shown remarkable results in clinical trials, such as-Ribociclib®, Palbociclib® and Abemaciclib®, which are CDK4/6 inhibitors and have received FDA approval for the treatment of breast cancer. In this chapter, we discuss the molecular mechanism through which cyclins and CDKs regulate cell cycle progression and the emergence of cyclins and CDKs as rational targets in cancer. We also discuss recent advances in developing CDK inhibitors, which have emerged as a novel class of inhibitors, and their associated toxicities in recent years.

Photoacoustic imaging for investigating tumor hypoxia: a strategic assessment.

Hypoxia causes the expression of signaling molecules which regulate cell division, lead to angiogenesis, and further, in the tumor microenvironment, promote resistance to chemotherapy and radiotherapy, and induce metastasis. Photoacoustic imaging (PAI) takes advantage of unique absorption characteristics of chromophores in tissues and provides the opportunity to construct images with a high degree of spatial and temporal resolution. In this review, we discuss the physiologic characteristics of tumor hypoxia, and current applications of PAI using endogenous (label free imaging) and exogenous (organic and inorganic) contrast agents. Features of various methods in terms of their efficacy for determining physiologic and proteomic phenomena are analyzed. This review demonstrates that PAI has the potential to understand tumor growth and metastasis development through measurement of regulatory molecule concentrations, oxygen gradients, and vascular distribution.

PS-BPB04-6: PROOF OF BIOLOGICAL ACTIVITY AND EXPLORATION OF EARLY SIGNALING EVENTS OF ANGIOTENSIN-(1–5)

Introduction: Recombinant human ACE2 increases the circulating levels of angiotensin-(1–5) [Ang-(1–5)], a peptide thus far regarded as biologically inactive. Since ACE2 is a central component of the protective RAS, we hypothesized that Ang-(1–5) is a new biologically active peptide within this hormonal system. Objective: To investigate biological activity and signaling mechanisms of Ang-(1–5). Methods: In order to show a biological effect and to test whether Ang-(1–5) signals through the AT2-receptor (AT2R), nitric oxide (NO) release was measured by DAF-FM fluorescence in AT2R transfected (AT2R-CHO) or non-transfected (NT-CHO) CHO cells, treated with Ang-(1–5) or C21 (AT2R agonist, positive control) (0.1 nM to 10 μM) for 15 minutes. Vehicle (cell media) treated cells served as negative control. To investigate Ang-(1–5) signaling patterns, human aortic endothelial cells (HAEC) were treated with vehicle or Ang-(1–5) (1 μM) for 1, 3, 5 or 20 minutes. Proteins were harvested, digested and labeled with TMTpro-16plex. Phosphopeptide enrichment was carried out by TiO2. Samples were subjected to LC-MS/MS analysis and the resulting mass spectra were searched against the human SwissProt database. Results: Ang-(1–5) induced a concentration-dependent increase in NO production in AT2-CHO cells (Emax: 65.60 ± 14.02%), thus proving its biological activity. Ang-(1–5) had 69% higher efficacy than the established AT2R agonist C21 (Emax: 38.76 ± 10.24%). Ang-(1–5) seems to signal through the AT2R, because effects on NO release were absent in NT-CHO cells. Treatment of HAEC with Ang-(1–5) significantly modified the phosphorylation status of 831 proteins at 1799 residues. The majority of residues (1079) were dephosphorylated while 729 residues were phosphorylated. Changes in protein phosphorylation in response to Ang-(1–5) occurred at all investigated time points, most of them after 20 minutes. Functional bioinformatic analysis revealed a cluster of proteins involved in cell cycle and cell division regulation. Conclusion: This study provides evidence that Ang-(1–5) is an endogenous AT2R agonist with high efficacy towards the AT2R. The early signaling phosphorylation pattern resembles those of other protective RAS agonists, such as C21 and Ang-(1–7).