State Of Terminal Differentiation Research Articles

MiR-744-5p promotes T-cell differentiation via inhibiting STK11

T cells utilized in adoptive T cell immunotherapy are typically activated in vitro. Although these cells demonstrate proliferation and anti-tumor activity following activation, they often face difficulties in sustaining long-term survival post-reinfusion. This issue is attributed to the induction of T cells into a terminal differentiation state upon activation, whereas early-stage differentiated T cells exhibit enhanced proliferation potential and survival capabilities. In previous study, we delineated four T cell subsets at varying stages of differentiation: TN, TSCM, TCM, and TEM, and acquired their miRNA expression profiles via high-throughput sequencing. In the current study, we performed a differential analysis of miRNA across these subsets, identifying a distinct miRNA, hsa-miR-744-5p, characterized by progressively increasing expression levels upon T cell activation. This miRNA is not expressed in TSCM but is notably present in TEM. Target genes of miR-744-5p were predicted, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, revealing that these genes predominantly associate with pathways related to the ’Wnt signaling pathway’. We established that miR-744-5p directly targets STK11, influencing its expression. Further, we investigated the implications of miR-744-5p on T cell differentiation and functionality. Overexpression of miR-744-5p in T cells resulted in heightened apoptosis, reduced proliferation, an increased proportion of late-stage differentiated T cells, and elevated secretion of the cytokine TNF-α. Moreover, post-overexpression of miR-744-5p led to a marked decline in the expression of early-stage differentiation-associated genes in T cells (CCR7, CD62L, LEF1, BCL2) and a significant rise in late-stage differentiation-associated genes (KLRG1, PDCD1, GZMB). In conclusion, our findings affirm that miR-744-5p contributes to the progressive differentiation of T cells by downregulating the STK11 gene expression.

Convergent alteration of the mesenchymal stem cell heterogeneity in adipose tissue during aging.

Adipose-derived stem cells (ASCs) from distinct age groups possess different characteristics; however, the age-associated changes in ASCs heterogenicity remain largely unknown. In this study, several publicly available single-cell RNA sequencing (RNA-seq) data cohorts of inguinal adipose tissues, including young (2 weeks), adult (8 weeks), and old (18 months) C57BL/6 mice, were analyzed. Transcriptomic clustering of integrated single-cell RNA-seq data from different age groups revealed the existence of five ASCs subtypes. Interestingly, ASCs showed a loss of heterogeneity with aging, and ASCs subtype 4 (ASC-4) was the dominant subpopulation accounting for more than 98% of aged ASCs converging to the terminal differentiation state. The multidirectional differentiation potentials of different ASCs subtypes were largely distinct while the adipogenic ability of ASC-4 increased with age persistently. Regulon analysis of ASC subtypes further identified Cebpb as the ASC-4-specific transcription factor, which was known as one of the major adipogenic regulators. Analysis of ligand-receptor pairs between ASCs and other cell types in adipose tissue identified age-associated upregulation of inflammatory responses-associated factors including CCL2 and CCL7. Treatment with 100 ng/mL CCL2 in vitro could significantly promote the adipogenesis of ASCs through enhanced phosphorylation of AKT and decreased expression of β-catenin. In addition, supplementation of 100 ng/mL CCL7 could significantly increase the expression of inflammatory genes and ASC-4-specific transcriptional factors in 2-week-old ASCs, potentially acting as a driver of ASCs convergence. Our findings help to delineate the complex biological processes of ASCs aging and shed light on better regenerative and therapeutic applications of ASCs.

The adhesion G protein-coupled receptor GPR56/ADGRG1 in cytotoxic lymphocytes.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor connected to brain development, haematopoiesis, male fertility, and tumorigenesis. Nevertheless, expression of GPR56 is not restricted to developmental processes. Studies over the last years have demonstrated a marked presence of GPR56 in human cytotoxic NK and T cells. Expression of GPR56 in these cells is driven by the transcription factor HOBIT, corresponds with the production of cytolytic mediators and the presence of CX3 CR1 and CD57, indicates a state of terminal differentiation and cellular exhaustion, and disappears upon cellular activation. Functional studies indicate that GPR56 regulates cell migration and effector functions and thereby acts as an inhibitory immune checkpoint. We here discuss the current state of knowledge regarding GPR56 in cytotoxic lymphocytes.

Abstract 595: Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC

Abstract Immune-checkpoint blockade (ICB) has proved a major success, especially in highly mutated tumors such as lung cancer. Nevertheless, not all patients respond to ICB. It is possible that regulatory T cells (Tregs) play a role in this lack of response by suppressing tumor-reactive cytotoxic T cells, however the specific mechanisms that lead to this suppression remain elusive. It is therefore necessary to understand the functional programming and suppressive nature of Treg subsets in the tumor microenvironment to define targetable molecules for future biomarker-driven therapeutics. In this study we performed single cell RNA-sequencing on T cells isolated from resected tissue and peripheral blood from 15 neoadjuvant nivolumab (anti-PD1)-treated and 10 untreated non-small cell lung cancer (NSCLC) patients. We identified and analyzed 71,251 CD4+ FoxP3+ Tregs. Refined clustering was performed, and we used pseudotime and differential gene analyses to understand the transcriptional relationship between clusters and patient groups. With our highly refined clustering approach, we identified 8 separate Treg clusters that reflect differing functionalities within the tumor microenvironment. We demonstrate distinct Treg subsets that diverge towards either an activated state, expressing members of the tumor necrosis factor receptor (TNFR) superfamily: OX40, 41BB, GITR, or a resting state. Patients who respond to ICI have a decreased activated Treg score and demonstrate RNA velocity trajectory from activated Tregs towards more in-active and resting populations. Untreated patients conversely show a high activated Treg score with RNA velocity demonstrating activated Tregs are a terminal differentiation state. We hypothesize that ICI treatment pushes Tregs away from an activated phenotype towards more quiescent and that the efficiency of this transition may predict response to ICI. We plan to stimulate receptors associated with non-response using TNFR agonist ligands and hypothesize that their induced signaling will result in transcriptional program changes altering the suppressive ability of Tregs. In addition, we show that Tregs who experience antigen within the tumor microenvironment are more suppressive than bystander Tregs that home to the tumor without antigen stimulation. Together, this study provides an in-depth look at the Treg-derived suppressive mechanisms governing their function in the TME of anti-PD-1-treated vs. untreated tumors. This in-depth analysis of tumor Tregs has identified specific targetable biomarkers which could be used to improve ICB response while mitigating off-target immune adverse events by specifically inhibiting a small subset of Tregs without disturbing systemic immune homeostasis. Citation Format: Arbor G. Dykema, Jiajia Zhang, Boyang Zhang, Taibo Li, Justina X. Caushi, Laurene S. Cheung, Hongkai Ji, Zhicheng Ji, Kellie N. Smith, Drew M. Pardoll. Distinct tumor infiltrating Treg lineages are associated with response to anti-PD1 checkpoint blockade in NSCLC [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 595.

Senescence-Associated Molecules and Tumor-Immune-Interactions as Prognostic Biomarkers in Colorectal Cancer

Background and AimsThe initiation of cellular senescence in response to protumorigenic stimuli counteracts malignant progression in (pre)malignant cells. Besides arresting proliferation, cells entering this terminal differentiation state adopt a characteristic senescence-associated secretory phenotype (SASP) which initiates alterations to their microenvironment and effects immunosurveillance of tumorous lesions. However, some effects mediated by senescent cells contribute to disease progression. Currently, the exploration of senescent cells' impact on the tumor microenvironment and the evaluation of senescence as possible target in colorectal cancer (CRC) therapy demand reliable detection of cellular senescence in vivo. Therefore, specific immunohistochemical biomarkers are required. Our aim is to analyze the clinical implications of senescence detection in colorectal carcinoma and to investigate the interactions of senescent tumor cells and their immune microenvironment in vitro and in vivo.MethodsSenescence was induced in CRC cell lines by low-dose-etoposide treatment and confirmed by Senescence-associated β-galactosidase (SA-β-GAL) staining and fluorescence activated cell sorting (FACS) analysis. Co-cultures of senescent cells and immune cells were established. Multiple cell viability assays, electron microscopy and live cell imaging were conducted. Immunohistochemical (IHC) markers of senescence and immune cell subtypes were studied in a cohort of CRC patients by analyzing a tissue micro array (TMA) and performing digital image analysis. Results were compared to disease-specific survival (DSS) and progression-free survival (PFS).ResultsVarying expression of senescence markers in tumor cells was associated with in- or decreased survival of CRC patients. Proximity analysis of p21-positive senescent tumor cells and cytotoxic T cells revealed a significantly better prognosis for patients in which these cell types have the possibility to directly interact. In vitro, NK-92 cells (mimicking natural killer T cells) or TALL-104 cells (mimicking both cytotoxic T cells and natural killer T cells) led to dose-dependent specific cytotoxicity in >75 % of the senescent CRC cells but <20 % of the proliferating control CRC cells. This immune cell-mediated senolysis seems to be facilitated via direct cell-cell contact inducing apoptosis and granule exocytosis.ConclusionCounteracting tumorigenesis, cellular senescence is of significant relevance in CRC. We show the dual role of senescence bearing both beneficial and malignancy-promoting potential in vivo. Absence as well as exceeding expression of senescence markers are associated with bad prognosis in CRC. The antitumorigenic potential of senescence induction is determined by tumor micromilieu and immune cell-mediated elimination of senescent cells.

Elucidating multi-input processing 3-node gene regulatory network topologies capable of generating striped gene expression patterns.

A central problem in developmental and synthetic biology is understanding the mechanisms by which cells in a tissue or a Petri dish process external cues and transform such information into a coherent response, e.g., a terminal differentiation state. It was long believed that this type of positional information could be entirely attributed to a gradient of concentration of a specific signaling molecule (i.e., a morphogen). However, advances in experimental methodologies and computer modeling have demonstrated the crucial role of the dynamics of a cell’s gene regulatory network (GRN) in decoding the information carried by the morphogen, which is eventually translated into a spatial pattern. This morphogen interpretation mechanism has gained much attention in systems biology as a tractable system to investigate the emergent properties of complex genotype-phenotype maps. In this study, we apply a Markov chain Monte Carlo (MCMC)-like algorithm to probe the design space of three-node GRNs with the ability to generate a band-like expression pattern (target phenotype) in the middle of an arrangement of 30 cells, which resemble a simple (1-D) morphogenetic field in a developing embryo. Unlike most modeling studies published so far, here we explore the space of GRN topologies with nodes having the potential to perceive the same input signal differently. This allows for a lot more flexibility during the search space process, and thus enables us to identify a larger set of potentially interesting and realizable morphogen interpretation mechanisms. Out of 2061 GRNs selected using the search space algorithm, we found 714 classes of network topologies that could correctly interpret the morphogen. Notably, the main network motif that generated the target phenotype in response to the input signal was the type 3 Incoherent Feed-Forward Loop (I3-FFL), which agrees with previous theoretical expectations and experimental observations. Particularly, compared to a previously reported pattern forming GRN topologies, we have uncovered a great variety of novel network designs, some of which might be worth inquiring through synthetic biology methodologies to test for the ability of network design with minimal regulatory complexity to interpret a developmental cue robustly.

Combining single-cell RNA sequencing of peripheral blood mononuclear cells and exosomal transcriptome to reveal the cellular and genetic profiles in COPD

BackgroundIt has been a long-held consensus that immune reactions primarily mediate the pathology of chronic obstructive pulmonary disease (COPD), and that exosomes may participate in immune regulation in COPD. However, the relationship between exosomes and peripheral immune status in patients with COPD remains unclear.MethodsIn this study, we sequenced plasma exosomes and performed single-cell RNA sequencing on peripheral blood mononuclear cells (PBMCs) from patients with COPD and healthy controls. Finally, we constructed competing endogenous RNA (ceRNA) and protein–protein interaction (PPI) networks to delineate the interactions between PBMCs and exosomes within COPD.ResultsWe identified 135 mRNAs, 132 lncRNAs, and 359 circRNAs from exosomes that were differentially expressed in six patients with COPD compared with four healthy controls. Functional enrichment analyses revealed that many of these differentially expressed RNAs were involved in immune responses including defending viral infection and cytokine–cytokine receptor interaction. We also identified 18 distinct cell clusters of PBMCs in one patient and one control by using an unsupervised cluster analysis called uniform manifold approximation and projection (UMAP). According to resultant cell identification, it was likely that the proportions of monocytes, dendritic cells, and natural killer cells increased in the COPD patient we tested, meanwhile the proportions of B cells, CD4 + T cells, and naïve CD8 + T cells declined. Notably, CD8 + T effector memory CD45RA + (Temra) cell and CD8 + effector memory T (Tem) cell levels were elevated in patient with COPD, which were marked by their lower capacity to differentiate due to their terminal differentiation state and lower reactive capacity to viral pathogens.ConclusionsWe generated exosomal RNA profiling and single-cell transcriptomic profiling of PBMCs in COPD, described possible connection between impaired immune function and COPD development, and finally determined the possible role of exosomes in mediating local and systemic immune reactions.

Identification of Biomarkers Controlling Cell Fate In Blood Cell Development.

A blood cell lineage consists of several consecutive developmental stages starting from the pluri- or multipotent stem cell to a state of terminal differentiation. Despite their importance for human biology, the regulatory pathways and gene networks that govern these differentiation processes are not yet fully understood. This is in part due to challenges associated with delineating the interactions between transcription factors (TFs) and their corresponding target genes. A possible step forward in this case is provided by the increasing amount of expression data, as a basis for linking differentiation stages and gene activities. Here, we present a novel hierarchical approach to identify characteristic expression peak patterns that global regulators excert along the differentiation path of cell lineages. Based on such simple patterns, we identified cell state-specific marker genes and extracted TFs that likely drive their differentiation. Integration of the mean expression values of stage-specific “key player” genes yielded a distinct peaking pattern for each lineage that was used to identify further genes in the dataset which behave similarly. Incorporating the set of TFs that regulate these genes led to a set of stage-specific regulators that control the biological process of cell fate. As proof of concept, we considered two expression datasets covering key differentiation events in blood cell formation of mice.

The Morphology of Cell Differentiation, Terminal Differentiation and Ageing Seems To Reflect the Same Process: a Short Note

Based on simple microscopic cell morphology in blood and bone marrow smear preparations, it seems to be likely that the cell differentiation and terminal differentiation in human blood cells, and particularly in erythroid or granulocytic lineages, simultaneously reflect ageing of the lineage progenitors and terminal differentiation steps. The terminal differentiation stages of both these lineages actually appear as senescent cells. Abnormal ageing of progenitor cells may represent one of the “dysplastic” phenomena of the premature terminal differentiation state. Such state is characterized by heterochromatin condensation and nucleolar morphology similar to that in fully differentiated terminal cells of granulocytic or erythroid lineages. It should also be mentioned that in some known erythropoietic disorders, less differentiated erythroblasts may lose nuclei similarly as “normal” fully terminally differentiated cells of the erythroid cell lineage. It seems to be clear that cells in both abnormal less differentiated and terminally differentiated stages of erythroid or granulocytic lineages lose the ability to multiply similarly as senescent cells. On the other hand, the background of cell ageing and differentiation is very complicated and requires a different approach than the simple microscopic morphology at the single cell level. However, the morphology and clinical cytology at the single cell level might still contribute with complementary data to more sophisticated complex studies of that topic. In addition, the morphological approach facilitates the study of the main components of single cells in various states, including the differentiation steps or ageing.

Proceso de agotamiento de las células T durante la infección crónica causada por tripanosomátidos intracelulares

La enfermedad de Chagas y la leishmaniasis, causadas por parásitos protozoarios intracelulares del orden Trypanosomatida, son consideradas dos de las enfermedades tropicales desatendidas más importantes. Estas infecciones conllevan un alto desgaste social, provocando el deterioro de la salud de un gran número de pacientes e incluso su muerte. Los linfocitos T son células fundamentales del sistema adaptativo y son los principales elementos inmunitarios para el control de estas infecciones intracelulares. La presente revisión explora los estudios y resultados obtenidos hasta la fecha del proceso de agotamiento celular durante las infecciones causadas por los parásitos Leishmania spp. y Trypanosoma cruzi. Así, se recoge que la persistente estimulación celular en respuesta a antígenos de estos patógenos conduce a un proceso de pérdida de la capacidad funcional antígeno-específica en las poblaciones de células T CD4+ y CD8+. Numerosos estudios muestran la existencia de una correlación directa entre el nivel de la co-expresión de receptores inhibitorios y la gravedad de estas patologías. Paralelamente, se detecta la pérdida de la capacidad funcional específica de antígeno de estas células T, lo que reduce su potencial linfoproliferativo y su capacidad de producir moléculas protectoras contra estas infecciones. Además, durante el curso de estas infecciones se observa un incremento de la frecuencia de células T de memoria efectora con un grado de diferenciación tardía o terminal. Este balanceo fenotípico, a su vez, afecta a la capacidad funcional de las células T aumentando el número de células con características senescentes y apoptóticas. Así, los estudios realizados hasta la fecha demuestran con certeza la existencia de un proceso de agotamiento que afecta a poblaciones clave para el control parasitario. Sin embargo, actualmente se desconoce con precisión el papel que este proceso de agotamiento juega en el agravamiento de estas patologías. Los medicamentos actuales usados para tratar estas enfermedades protozoarias revierten parcialmente este proceso de agotamiento. Así, tras el tratamiento, numerosos pacientes muestra una reducción en la expresión de receptores inhibitorios y co-expresión de los mismos. También se ha observado una mejoría en la capacidad funcional de las distintas poblaciones de células T, que podría estar relacionada con la reversión del proceso disfuncional. Sin embargo, los estudios realizados hasta la fecha en la evaluación de terapias de bloqueo de los receptores inhibitorios no han conducido a resultados prometedores. Algunos autores proponen evaluar terapias de bloqueo simultáneo de varias vías de señalización, con el fin de ampliar el conocimiento sobre esta herramienta como posible inmunoterapia de control de la infección por los mencionados parásitos. Además, se considera necesario continuar investigando sobre cómo se desencadena exactamente este proceso de agotamiento celular y en qué medida influye en la aparición de la sintomatología de los pacientes y ausencia de control de la infección.

Chemotherapy and terminal skeletal muscle differentiation in WT1-mutant Wilms tumors.

Wilms tumors (WT) with WT1 mutations do not respond well to preoperative chemotherapy by volume reduction, suggesting resistance to chemotherapy. The histologic pattern of this tumor subtype indicates an intrinsic mesenchymal differentiation potential. Currently, it is unknown whether cytotoxic treatments can induce a terminal differentiation state as a direct comparison of untreated and chemotherapy‐treated tumor samples has not been reported so far. We conducted gene expression profiling of 11 chemotherapy and seven untreated WT1‐mutant Wilms tumors and analyzed up‐ and down‐regulated genes with bioinformatic methods. Cell culture experiments were performed from primary Wilms tumors and genetic alterations in WT1 and CTNNB1 analyzed. Chemotherapy induced MYF6 165‐fold and several MYL and MYH genes more than 20‐fold and repressed many genes from cell cycle process networks. Viable tumor cells could be cultivated when patients received less than 8 weeks of chemotherapy but not in two cases with longer treatments. In one case, viable cells could be extracted from a lung metastasis occurring after 6 months of intensive chemotherapy and radiation. Comparison of primary tumor and metastasis cells from the same patient revealed up‐regulation of RELN and TBX2,TBX4 and TBX5 genes and down‐regulation of several HOXD genes. Our analyses demonstrate that >8 weeks of chemotherapy can induce terminal myogenic differentiation in WT1‐mutant tumors, but this is not associated with volume reduction. The time needed for all tumor cells to achieve the terminal differentiation state needs to be evaluated. In contrast, prolonged treatments can result in genetic alterations leading to resistance.

Characterization of novel markers of senescence and their prognostic potential in cancer.

Cellular senescence is a terminal differentiation state that has been proposed to have a role in both tumour suppression and ageing. This view is supported by the fact that accumulation of senescent cells can be observed in response to oncogenic stress as well as a result of normal organismal ageing. Thus, identifying senescent cells in in vivo and in vitro has an important diagnostic and therapeutic potential. The molecular pathways involved in triggering and/or maintaining the senescent phenotype are not fully understood. As a consequence, the markers currently utilized to detect senescent cells are limited and lack specificity. In order to address this issue, we screened for plasma membrane-associated proteins that are preferentially expressed in senescent cells. We identified 107 proteins that could be potential markers of senescence and validated 10 of them (DEP1, NTAL, EBP50, STX4, VAMP3, ARMX3, B2MG, LANCL1, VPS26A and PLD3). We demonstrated that a combination of these proteins can be used to specifically recognize senescent cells in culture and in tissue samples and we developed a straightforward fluorescence-activated cell sorting-based detection approach using two of them (DEP1 and B2MG). Of note, we found that expression of several of these markers correlated with increased survival in different tumours, especially in breast cancer. Thus, our results could facilitate the study of senescence, define potential new effectors and modulators of this cellular mechanism and provide potential diagnostic and prognostic tools to be used clinically.

Reprogramming somatic cells to a kidney fate.

Recent years have challenged the view that adult somatic cells reach a state of terminal differentiation. Although the ultimate example of this, somatic cell nuclear transfer, has not proven feasible in human beings, dedifferentiation of mature cell types to a more primitive state, direct reprogramming from one mature state to another, and the reprogramming of any adult cell type to a pluripotent state via enforced expression of key transcription factors now all have been shown. The implications of these findings for kidney disease include the re-creation of key renal cell types from more readily available and expandable somatic cell sources. The feasibility of such an approach recently was shown with the dedifferentiation of proximal tubule cells to nephrogenic mesenchyme. In this review, we examine the technical and clinical challenges that remain to such an approach and how new reprogramming approaches also may be useful for kidney disease.

Appraisal of progenitor markers in the context of molecular classification of breast cancers

Clinical management of breast cancer relies on case stratification, which increasingly employs molecular markers. The motivation behind delineating breast epithelial differentiation is to better target cancer cases through innate sensitivities bequeathed to the cancer from its normal progenitor state. A combination of histopathological and molecular classification of breast cancer cases suggests a role for progenitors in particular breast cancer cases. Although a remarkable fraction of the real tissue repertoire is maintained within a population of independent cell line cultures, some steps that are closer to the terminal differentiation state and that form a majority of primary human breast tissues are missing in the cell line cultures. This raises concerns about current breast cancer models.

Abstract 1094: Transcriptional activation by pRB/p107 and coordination with SWI/SNF

Abstract The retinoblastoma susceptibility gene, Rb1, is closely linked with osteosarcoma as well as retinoblastoma. The basis of such ties between individual susceptibility genes and tumors of specific tissue origin is a central question in cancer biology. Functional analysis of pRB and the closely related family members, p107 and p130, has tended to focus on repression of proliferation, but genetic evidence from our lab and others indicates an active requirement for pRB in osteoblast differentiation that might relate more directly with osteosarcoma susceptibility. Our previous studies linked pRB with activation of the early osteoblast marker, alkaline phosphatase (the product of the Akp2 gene). Here we have investigated the functional relationships between pRB and the SWI/SNF complex in Akp2 activation. Chromatin immunoprecipitation (ChIP) assays reveal the Akp2 promoter is targeted directly by all three pRB family members. Only p130 occupies the promoter prior to induction, while pRB and p107 target Akp2 directly during activation, and concurrent with association of the global co-activator p300. Akp2 is also a direct target of SWI/SNF chromatin remodeling complexes. These ATPase-powered complexes help orchestrate the programmatic shifts in gene expression necessary for progression from a self-renewing precursor state to a state of terminal differentiation. In osteoblasts, SWI/SNF complexes that repress osteogenic gene expression in order to maintain the precursor state are characterized by inclusion of the BRM ATPase. The alternative ATPase, BRG1, characterizes complexes that activate osteogenic genes during terminal differentiation. Thus, the onset of osteoblast differentiation is marked by a switch from BRM to BRG1 complexes on the Akp2 promoter. Our analysis shows that the BRM to BRG1 switch is accomplished in a precise two-step mechanism, with dissociation of BRM-containing SWI/SNF dependent on p300, and association of BRG1-containing SWI/SNF dependent on pRB/p107. Binding of RNA polymerase-II is dependent in turn on BRG1. Thus, in contrast to their widely studied role in cell cycle repression, pRB/p107 play a directly activating role in osteogenic gene expression, specifically in recruitment of the activating SWI/SNF complex. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1094.

Up-regulating Sphingosine 1-Phosphate Receptor-2 Signaling Impairs Chemotactic, Wound-healing, and Morphogenetic Responses in Senescent Endothelial Cells

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P(1-3) receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P(1) and S1P(2) was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P(2) receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P(2) to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P(2)-mediated activation of the lipid phosphatase PTEN.

GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.

Molecular Basis of Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is a serious, life-threatening genetic disease in which extensive epithelial-lined cysts develop in the kidneys and, to a lesser extent, in other organs such as liver, pancreas, and ovaries. In a majority of cases (80-85%), the gene involved is PKD1, which is located on chromosome 16 (16q13.3) and encodes polycystin-1, a large receptor-like integral membrane protein that contains several extracellular motifs indicative of cell-cell and cell-matrix interaction. In the remaining (10-15%) cases, the disease is milder and is caused by mutational changes in another gene (PKD2), which is located at chromosome 4 (4q21-23) and encodes polcystin-2, a transmembrane protein, which acts as a nonspecific calcium-permeable channel. Both polycystins function together in a nonredundant fashion, through a common pathway, and produce cellular responses that regulate proliferation, migration, differentiation, and kidney morphogenesis. Through combined function of polycystins, normal tubular cells are maintained in a state of terminal differentiation, and their proliferation is strictly controlled. Loss of function of either protein due to gene mutations results in the tubular cells reverting to a less differentiated state, which is more prone to proliferation. Patients with ADPKD carry a germ-line mutation in PKD1 or PKD2. A second somatic mutation in some of the tubular cells results in loss of both normal alleles, leading to loss of polycystin function. The affected cells lose the normal terminally differentiated state, revert to less differentiated phenotype, and undergo proliferation, which leads to cyst formation. As the cysts enlarge over many decades, the normal renal parenchyma is progressively destroyed, leading to renal failure. Recently, the crucial role of primary cilia in modulating proliferation, migration, and differentiation of tubular epithelium has been recognized. Most of the tubular cells have one or two primary cilia projecting from the apical surface into the luminal space. The cilia act as mechanoreceptors as they bend with the urinary flow within the tubules. Both polycystins are strategically located within the cilia and act as important mediators of ciliary mechanosensation. Loss of this important function due to mutational changes in PKD1 or PKD2 leads to loss of normal control over cellular proliferation, resulting in cyst formation. Several other ciliary proteins have recently been found to contribute directly to a wide spectrum of human kidney diseases with cystic phenotype, thus underscoring the pivotal role the primary cilia play in maintaining the normal structure and function of the tubular cells and probably other cells in the body.

Transcriptional Activation of Prion Protein Gene in Growth-Arrested and Differentiated Mouse Erythroleukemia and Human Neoplastic Cells

The prion protein (PrP) is a GPI-anchored sialoglycoprotein that has attracted worldwide attention over the years due to its involvement in the pathogenesis of transmissible spongiform encephalopathies in sheep (scrapie), cattle (BSE), and humans (CJD). To understand the precise role of the Prn-p gene in cell growth and differentiation we investigated the expression pattern of the Prn-p gene in proliferating cells and in cells arrested in growth either by confluency or by induction of terminal differentiation. Viral-transformed mouse spleen hematopoietic cells named murine erythroleukemia (MEL) and other types of inducible cells (human neuroectodermal RD/TE-671, myoid RD cells) were employed. Cells grown exponentially, at confluency, or irreversibly arrested in growth at terminal differentiation state were analyzed by fluorescence cell sorting and Northern blot hybridization to estimate the steady-state level of PrP mRNA at different phases of the cell cycle. MEL cells that failed to differentiate from treatment with N6-methyladenosine (N6mAdo), an inhibitor of differentiation, were also analyzed for PrP mRNA level. Our results indicate the following: (a) growth arrest of cells at G1 phase by confluency or by induction of terminal differentiation led to increased accumulation of PrP mRNA transcripts, an event observed also in differentiated MEL, RD/TE-671, and RD cells independent of the inducer used; (b) treatment of MEL cells with N6mAdo prevented early activation of the Prn-p gene in cells treated with the inducer; and (c) cell-free nuclear run-off studies showed enhanced expression of the Prn-p gene due to transcriptional activation. These findings indicate, for the first time, that the Prn-p gene, which is thought to be a housekeeping gene, is transcriptionally activated in G1 phase in confluent and terminally differentiated cells. This information may be valuable in understanding the overaccumulation of PrP in some differentiated tissues and may let us repress Prn-p gene activation by novel agents.

Diversity of mouse lipoxygenases: identification of a subfamily of epidermal isozymes exhibiting a differentiation-dependent mRNA expression pattern.

By using reverse transcription-polymerase chain reaction technology (RT-PCR) and Northern blot analysis, the tissue-specific mRNA expression patterns of seven mouse lipoxygenases (LOX)--including 5S-, 8S-, three isoforms of 12S-, 12R-LOX, and a LOX of an as-of-yet unknown specificity, epidermis-type LOX-3 (e-LOX-3)--were investigated in NMRI mice. Among the various tissues tested epidermis and forestomach were found to express the broadest spectrum of LOX. With the exception of 5S- and platelet-type 12S-LOX (p12S-LOX) the remaining LOX showed a preference to exclusive expression in stratifying epithelia of the mouse, in particular the integumental epidermis. The expression of the individual LOX in mouse epidermis was found to depend on the state of terminal differentiation of the keratinocytes. mRNA of epidermis-type 12S-LOX (e12S-LOX) was detected in all layers of neonatal and adult NMRI mouse skin, whereas expression of p12S-LOX, 12R-LOX, and e-LOX-3 was restricted to suprabasal epidermal layers of neonatal and adult mice. 8S-LOX mRNA showed a body-site-dependent expression in that it was detected in stratifying epithelia of footsole and forestomach but not in back skin epidermis. In the latter, 8S-LOX mRNA was strongly induced upon treatment with phorbol esters. With the exception of e12S-LOX and p12S-LOX, the isozymes that are preferentially expressed in stratifying epithelia are structurally related and may be grouped together into a distinct subgroup of epidermis-type LOX.