Pluripotency Stemness and Cancer: More Questions than Answers.

Embryonic stem cells and induced pluripotent stem cells provided us with fascinating new knowledge in recent years. Mechanistic insight into intricate regulatory circuitry governing pluripotency stemness and disclosing parallels between pluripotency stemness and cancer instigated numerous studies focusing on roles of pluripotency transcription factors, including Oct4, Sox2, Klf4, Nanog, Sall4 and Tfcp2L1, in cancer. Although generally well substantiated as tumour-promoting factors, oncogenic roles of pluripotency transcription factors and their clinical impacts are revealing themselves as increasingly complex. In certain tumours, both Oct4 and Sox2 behave as genuine oncogenes, and reporter genes driven by composite regulatory elements jointly recognized by both the factors can identify stem-like cells in a proportion of tumours. On the other hand, cancer stem cells seem to be biologically very heterogeneous both among different tumour types and among and even within individual tumours. Pluripotency transcription factors are certainly implicated in cancer stemness, but do not seem to encompass its entire spectrum. Certain cancer stem cells maintain their stemness by biological mechanisms completely different from pluripotency stemness, sometimes even by engaging signalling pathways that promote differentiation of pluripotent stem cells. Moreover, while these signalling pathways may well be antithetical to stemness in pluripotent stem cells, they may cooperate with pluripotency factors in cancer stem cells - a paradigmatic example is provided by the MAPK-AP-1 pathway. Unexpectedly, forced expression of pluripotency transcription factors in cancer cells frequently results in loss of their tumour-initiating ability, their phenotypic reversion and partial epigenetic normalization. Besides the very different signalling contexts operating in pluripotent and cancer stem cells, respectively, the pronounced dose dependency of reprogramming pluripotency factors may also contribute to the frequent loss of tumorigenicity observed in induced pluripotent cancer cells. Finally, contradictory cell-autonomous and non-cell-autonomous effects of various signalling molecules operate during pluripotency (cancer) reprogramming. The effects of pluripotency transcription factors in cancer are thus best explained within the concept of cancer stem cell heterogeneity.

Urothelial Cancer Stem Cell Heterogeneity.

Urothelial carcinoma is a tumor type featuring pronounced intertumoral heterogeneity and a high mutational and epigenetic load. The two major histopathological urothelial carcinoma types - the non-muscle-invasive and muscle-invasive urothelial carcinoma - markedly differ in terms of their respective typical mutational profiles and also by their probable cells of origin, that is, a urothelial basal cell for muscle-invasive carcinomas and a urothelial intermediate cell for at least a large part of non-muscle-invasive carcinomas. Both non-muscle-invasive and muscle-invasive urothelial carcinomas can be further classified into discrete intrinsic subtypes based on their typical transcriptomic profiles. Urothelial carcinogenesis shows a number of parallels to a urothelial regenerative response. Both of these processes seem to be dominated by specific stem cell populations. In the last years, the nature and location of urothelial stem cell(s) have been subject to many controversies, which now seem to be settled down, favoring the existence of a largely single urothelial stem cell type located among basal cells. Basal cell markers have also been amply used to identify urothelial carcinoma stem cells, especially in muscle-invasive disease, but they proved useful even in some non-muscle-invasive tumors. Analyses on molecular nature of urothelial carcinoma stem cells performed till now point to their great heterogeneity, both during the tumor development and upon intertumoral comparison, sexual dimorphism providing a special example of the latter. Moreover, urothelial cancer stem cells are endowed with intrinsic plasticity, whereby they can modulate their stemness in relation to other tumor-related traits, especially motility and invasiveness. Such transitional modulations suggest underlying epigenetic mechanisms and, even within this context, inter- and intratumoral heterogeneity becomes apparent. Multiple molecular aspects of urothelial cancer stem cell biology markedly influence therapeutic response, implying their knowledge as a prerequisite to improved therapies of this disease. At the same time, the notion of urothelial cancer stem cell heterogeneity implies that this therapeutic benefit would be most probably and most efficiently achieved within the context of individualized antitumor therapy.

Ovarian Cancer Stem Cell Heterogeneity.

Ovarian carcinoma features pronounced clinical, histopathological, and molecular heterogeneity. There is good reason to believe that parts of this heterogeneity can be explained by differences in the respective cell of origin, with a self-renewing fallopian tube secretory cell being likely responsible for initiation of an overwhelming majority of high-grade serous ovarian carcinomas (i.e., type II tumors according to the recent dualistic classification), whereas there are several mutually non-exclusive possibilities for the initiation of type I tumors, including ovarian surface epithelium stem cells, endometrial cells, or even cells of extra-Müllerian origin. Interestingly, both fallopian tube self-renewing secretory cells and ovarian surface epithelium stem cells seem to be characterized by an overlapping array of stemness signaling pathways, especially Wnt/ß-catenin. Apart from this variability in the respective cell of origin, the particular clinical behavior of ovarian carcinoma strongly suggests an underlying stem cell component with a crucial impact. This becomes especially evident in high-grade serous ovarian carcinomas treated with classical chemotherapy, which entails a gradual evolution of chemoresistant disease without any apparent selection of clones carrying obvious chemoresistance-associated mutations. Several cell surface markers (e.g., CD24, CD44, CD117, CD133, and ROR1) as well as functional approaches (ALDEFLUOR™ and side population assays) have been used to identify and characterize putative ovarian carcinoma stem cells. We have recently shown that side population cells exhibit marked heterogeneity on their own, which can hamper their straightforward therapeutic targeting. An alternative strategy for stemness-depleting interventions is to target the stem cell niche, i.e., the specific microanatomical structure that secures stem cell maintenance and survival through provision of a set of stem cell-promoting and differentiation-antagonizing factors. Besides identifying direct or indirect therapeutic targets, profiling of side population cells and other ovarian carcinoma stem cell subpopulations can reveal relevant prognostic markers, as exemplified by our recent discovery of the Vav3.1 transcript variant, which filters out a fraction of prognostically unfavorable ovarian carcinoma cases.

Sarcoma Stem Cell Heterogeneity.

Sarcomas represent an extensive group of divergent malignant diseases, with the only common characteristic of being derived from mesenchymal cells. As such, sarcomas are by definition very heterogeneous, and this heterogeneity does not manifest only upon intertumoral comparison on a bulk tumor level but can be extended to intratumoral level. Whereas part of this intratumoral heterogeneity could be understood in terms of clonal genetic evolution, an essential part includes a hierarchical relationship between sarcoma cells, governed by both genetic and epigenetic influences, signals that sarcoma cells are exposed to, and intrinsic developmental programs derived from sarcoma cells of origin. The notion of this functional hierarchy operating within each tumor implies the existence of sarcoma stem cells, which may originate from mesenchymal stem cells, and indeed, mesenchymal stem cells have been used to establish several crucial experimental sarcoma models and to trace down their respective stem cell populations. Mesenchymal stem cells themselves are heterogeneous, and, moreover, there are alternative possibilities for sarcoma cells of origin, like neural crest-derived stem cells, or mesenchymal committed precursor cells, or - in rhabdomyosarcoma - muscle satellite cells. These various origins result in substantial heterogeneity in possible sarcoma initiation. Genetic and epigenetic changes associated with sarcomagenesis profoundly impact the biology of sarcoma stem cells. For pediatric sarcomas featuring discrete reciprocal translocations and largely stable karyotypes, the translocation-activated oncogenes could be crucial factors that confer stemness, principally by modifying transcriptome and interfering with normal epigenetic regulation; the most extensively studied examples of this process are myxoid/round cell liposarcoma, Ewing sarcoma, and synovial sarcoma. For adult sarcomas, which have typically complex and unstable karyotypes, stemness might be defined more operationally, as a reflection of actual assembly of genetically and epigenetically conditioned stemness factors, with dedifferentiated liposarcoma providing a most thoroughly studied example. Alternatively, stemness can be imposed by tumor microenvironment, as extensively documented in osteosarcoma. In spite of this heterogeneity in both sarcoma initiation and underlying stemness biology, some of the molecular mechanisms of stemness might be remarkably similar in diverse sarcoma types, like abrogation of classical tumor suppressors pRb and p53, activation of Sox-2, or inhibition of canonical Wnt/ß-catenin signaling. Moreover, even some stem cell markers initially characterized for their stem cell enrichment capacity in various carcinomas or leukemias seem to function quite similarly in various sarcomas. Understanding the biology of sarcoma stem cells could significantly improve sarcoma patient clinical care, leading to both better patient stratification and, hopefully, development of more effective therapeutic options.

Truncated isoform Vav3.1 is highly expressed in ovarian cancer stem cells and clinically relevant in predicting prognosis and platinum-response.

Vav3 is a key modulator of GTP-hydrolases of the Rho/Rac family, which are crucially involved in cell proliferation. Vav3 is alternatively spliced in full-length Vav3-alpha and N-terminal truncated Vav3.1 lacking its self-regulatory domains. The aim of our study was to estimate the clinical impact of Vav3 and all other Vav family members in ovarian cancer. Purification of a stem-cell like side-population (SP) from ovarian cancer cell lines was performed by flow cytometry/FACS. Differences in gene expression between SP and NSP were assessed by Gene Array analysis and confirmed by RT-PCR and immunoblot. In addition, Vav mRNA expression was determined in 150 epithelial ovarian cancers. Clinicopathological parameters, platinum-sensitivity and survival were analyzed and associated with Vav expression. SP fractions of ovarian cancer cell lines exhibited marked overexpression of Vav3.1 (p < 0.001). Vav1 and Vav2 did not prove to be of clinicopathologic relevance in ovarian cancer. High Vav3.1 expression correlated with higher FIGO stage and residual disease. Furthermore, Vav3.1 overexpression was associated with poor progression-free (HR = 2.820, p = 0.0001) and overall survival (HR = 2.842, p = 0.0001). Subgroup analyses revealed an impact of Vav3.1 on survival in Type-II but not in Type-I cancers. Notably, platinum-refractory cancers showed marked overexpression of Vav3.1 compared to other subsets of platinum-sensitivity (15.848 vs. 6.653, p = 0.0001). In conclusion, Vav3.1 is over-expressed in stem-cell like SP fractions and is clinically relevant in the pathophysiology of ovarian cancer. The N-terminal truncated Vav3.1 may be decisively involved in mechanisms causing genuine multi-drug resistance.

Reconciling evidence from ancient and contemporary genomes: a major source for the European Neolithic within Mediterranean Europe.

Important gaps remain in our understanding of the spread of farming into Europe, due partly to apparent contradictions between studies of contemporary genetic variation and ancient DNA. It seems clear that farming was introduced into central, northern, and eastern Europe from the south by pioneer colonization. It is often argued that these dispersals originated in the Near East, where the potential source genetic pool resembles that of the early European farmers, but clear ancient DNA evidence from Mediterranean Europe is lacking, and there are suggestions that Mediterranean Europe may have resembled the Near East more than the rest of Europe in the Mesolithic. Here, we test this proposal by dating mitogenome founder lineages from the Near East in different regions of Europe. We find that whereas the lineages date mainly to the Neolithic in central Europe and Iberia, they largely date to the Late Glacial period in central/eastern Mediterranean Europe. This supports a scenario in which the genetic pool of Mediterranean Europe was partly a result of Late Glacial expansions from a Near Eastern refuge, and that this formed an important source pool for subsequent Neolithic expansions into the rest of Europe.

[Cancer stem cells as the therapeutic target of tomorrow]. / Tumorstammzellen als therapeutisches Ziel der Tumortherapie von morgen.

The concept of hierarchical organization of tumour cell population, with cancer stem cells positioned at the apex of the cell hierarchy, can explain at least some crucial aspects of biological and clinical behaviour of cancer, like its propensity to relapse as well as the development of therapeutic resistance. The underlying biological properties of cancer stem cells are crucially dependent on various signals, inhibition of which provides an attractive opportunity to attack pharmacologically cancer stem cells. Currently, a lot of such stemness-inhibitors undergo various phases of clinical testing. Interestingly, numerous old drugs that are in routine use in human and veterinary medicine for non-oncological indications appear to be able to specifically target cancer stem cells as well. As cancer stem cells, at least for most tumours, represent usually only a minor tumour cell fraction, it is quite probable that the main focus of the clinical use of the stemness inhibitors would consist in their rational combinations with traditional anticancer treatment modalities. A highly important goal for the future research is to identify reliable and clinically applicable predictive markers that would allow to apply these novel anticancer drugs on the individual basis within the context of personalized medicine.

Mitochondrial DNA signals of late glacial recolonization of Europe from near eastern refugia.

Human populations, along with those of many other species, are thought to have contracted into a number of refuge areas at the height of the last Ice Age. European populations are believed to be, to a large extent, the descendants of the inhabitants of these refugia, and some extant mtDNA lineages can be traced to refugia in Franco-Cantabria (haplogroups H1, H3, V, and U5b1), the Italian Peninsula (U5b3), and the East European Plain (U4 and U5a). Parts of the Near East, such as the Levant, were also continuously inhabited throughout the Last Glacial Maximum, but unlike western and eastern Europe, no archaeological or genetic evidence for Late Glacial expansions into Europe from the Near East has hitherto been discovered. Here we report, on the basis of an enlarged whole-genome mitochondrial database, that a substantial, perhaps predominant, signal from mitochondrial haplogroups J and T, previously thought to have spread primarily from the Near East into Europe with the Neolithic population, may in fact reflect dispersals during the Late Glacial period, ∼19-12 thousand years (ka) ago.

Establishment and characterization of a bladder cancer cell line with enhanced doxorubicin resistance by mevalonate pathway activation.

Resistance to chemotherapy is a major problem in the treatment of urothelial bladder cancer. Several mechanisms have been identified in resistance to doxorubicin by analysis of resistant urothelial carcinoma (UC) cell lines, prominently activation of drug efflux pumps and diminished apoptosis. We have derived a new doxorubicin-resistant cell line from BFTC-905 UC cells, designated BFTC-905-DOXO-II. A doxorubicin-responsive green fluorescent protein (GFP) reporter assay indicated that resistance in BFTC-905-DOXO-II was not due to increased drug efflux pump activity, whereas caspase-3/7 activation was indeed diminished. Gene expression microarray analysis revealed changes in proapoptotic and antiapoptotic genes, but additionally induction of the mevalonate (cholesterol) biosynthetic pathway. Treatment with simvastatin restored sensitivity of BFTC-905-DOXO-II to doxorubicin to that of the parental cell line. Induction of the mevalonate pathway has been reported as a mechanism of chemoresistance in other cancers; this is the first observation in bladder cancer. Combinations of statins with doxorubicin-containing chemotherapy regimens may provide a therapeutic advantage in such cases.

Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains. We therefore generated a transgenic mouse expressing a highly sensitive and specific fluorescent reporter of caspase activity, with peak signal localized to the nucleus. As a proof of concept, we first obtained evidence that NACA influences neurophysiology in an amygdalar circuit. Then focusing on the amygdala, we were able to quantify a sex-specific persistent elevation in caspase activity in females after restraint stress. This simple in vivo caspase activity reporter will facilitate systems-level studies of apoptotic and nonapoptotic phenomena in behavioral and pathologic models.

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.

Fibroblasts in urothelial bladder cancer define stroma phenotypes that are associated with clinical outcome.

Little attention was given to the interaction between tumor and stromal cells in urothelial bladder carcinoma (UBC). While recent studies point towards the existence of different fibroblast subsets, no comprehensive analyses linking different fibroblast markers to UBC patient survival have been performed so far. Through immunohistochemical analysis of five selected fibroblast markers, namely alpha smooth muscle actin (ASMA), CD90/Thy-1, fibroblast activation protein (FAP), platelet derived growth factor receptor-alpha and -beta (PDGFRa,-b), this study investigates their association with survival and histopathological characteristics in a cohort of 344 UBC patients, involving both, muscle-invasive and non-muscle-invasive cases. The data indicates that combinations of stromal markers are more suited to identify prognostic patient subgroups than single marker analysis. Refined stroma-marker-based patient stratification was achieved through cluster analysis and identified a FAP-dominant patient cluster as independent marker for shorter 5-year-survival (HR(95% CI)2.25(1.08-4.67), p = 0.030). Analyses of interactions between fibroblast and CD8a-status identified a potential minority of cases with CD90-defined stroma and high CD8a infiltration showing a good prognosis of more than 80% 5-year-survival. Presented analyses point towards the existence of different stroma-cell subgroups with distinct tumor-modulatory properties and motivate further studies aiming to better understand the molecular tumor-stroma crosstalk in UBC.

Urothelial Carcinoma Stem Cells: Current Concepts, Controversies, and Methods.

Cancer stem cells are defined as a self-renewing and self-protecting subpopulation of cancer cells able to differentiate into morphologically and functionally diverse cancer cells with a limited lifespan. To purify cancer stem cells, two basic approaches can be applied, the marker-based approach employing various more of less-specific cell surface marker molecules and a marker-free approach largely based on various self-protection mechanisms. Within the context of urothelial carcinoma, both methods could find use. The cell surface markers have been mainly derived from the urothelial basal cell, a probable cell of origin of muscle-invasive urothelial carcinoma, with CD14, CD44, CD90, and 67LR representing successful examples of this strategy. The marker-free approaches involve side population sorting, for which a detailed protocol is provided, as well as the Aldefluor assay, which rely on a specific overexpression of efflux pumps or the detoxification enzyme aldehyde dehydrogenase, respectively, in stem cells. These assays have been applied to both non-muscle-invasive and muscle-invasive bladder cancer samples and cell lines. Urothelial carcinoma stem cells feature a pronounced heterogeneity as to their molecular stemness mechanisms. Several aspects of urothelial cancer stem cell biology could enter translational development rather soon, e.g., a specific CD44+-derived gene expression signature able to identify non-muscle-invasive bladder cancer patients with a high risk of progression, or deciphering a mechanism responsible for repopulating activity of urothelial carcinoma stem cells within the context of therapeutic resistance.

Evaluation of Vav3.1 as prognostic marker in endometrial cancer.

PURPOSE: Vav3 is a guanine nucleotide exchange factor that regulates the activity of Rho/Rac family GTPases. In a study on ovarian cancer, we recently demonstrated pronounced prognostic and predictive value of Vav3.1, a specific truncation variant of the parental Vav3 gene. Here, we sought to investigate the role of Vav3.1 in the most prevalent gynecological tumor entity, endometrial cancer. METHODS: Vav3.1 transcript levels were determined in a large cohort of endometrial cancer patients using variant-specific PCR (n = 239), and non-malignant endometrial tissue served as control (n = 26). Expression levels of Vav3.1 were stratified according to established clinicopathological characteristics and correlated to long-term patient survival (average follow-up of > 7.5 years). Type 1 and type 2 cancers were separately investigated. RESULTS: While Vav3.1 was markedly overexpressed in endometrial cancer tissue, we could not detect associations with clinical parameters related to prognosis, such as FIGO stage and tumor grade. Kaplan-Meier estimators of different measures of survival failed to show prognostic significance of Vav3.1 in endometrial cancer. Lack of prognostic value was observed for both type 1 and type 2 cancers. CONCLUSIONS: Our study shows that Vav3.1 is not suited as a marker of cancer progression and/or treatment response in endometrial cancer. Feasibility and potential benefit of targeting Vav3.1 in endometrial cancer needs to be evaluated in future studies, proceeding from its clear, roughly ten-fold, induction in the malignant endometrium.

Protective Effect of Aspirin Against Oligomeric Aß42 Induced Mitochondrial Alterations and Neurotoxicity in Differentiated EC P19 Neuronal Cells.

BACKGROUND: Amyloid-beta (Aß) induced mitochondrial dysfunction is one of the major causes of neuronal toxicity in Alzheimer's disease. A number of recent reports suggest involvement of mitochondrial alterations through intracellular accumulation of oligomeric Aß. These mitochondrial alterations include increased Reactive Oxygen Species (ROS), mt-DNA depletion, decreased oxidative phosphorylation and ATP production, membrane depolarization, reduced number of mitochondria etc. All these defects cumulatively caused neural toxicity and alterations in cellular energy homeostasis. On the other hand, anti-inflammatory drug aspirin is reported to promote both mitochondrial biogenesis and improvement in cellular energy status. METHODS: Taking altogether the mentioned clues, we evaluated protective effect of aspirin, if any on oligomeric Aß42 induced toxicity and mitochondrial alterations in differentiated neuronal cells. RESULTS: A significant reduction in neuronal viability and increased apoptosis was observed in Aß42 treated cells, as evident by MTT assay, apoptosis ELISA and immunofluorescence from ß-III tubulin antibody staining of neuronal cells. A concomitant decrease was also observed in the intensity of mitotracker red FM staining and mt-DNA to nDNA ratio, suggesting mitochondrial membrane depolarization and/or reduced number of mitochondria along with depletion in mt-DNA. However, simultaneous treatment of 5 µM aspirin to oligomeric Aß42 treated cells protected them from mitochondrial dysfunction and neurotoxicity. CONCLUSION: We suggest mitochondrial biogenesis, changes in mitochondrial membrane potential and / or inhibition of Aß42 aggregation by aspirin as possible underlying mechanism(s).

Evaluating the forensic informativeness of mtDNA haplogroup H sub-typing on a Eurasian scale.

The impact of phylogeographic information on mtDNA forensics has been limited to the quality control of published sequences and databases. In this work we use the information already available on Eurasian mtDNA phylogeography to guide the choice of coding-region SNPs for haplogroup H. This sub-typing is particularly important in forensics since, even when sequencing both HVRI and HVRII, the discriminating power is low in some Eurasian populations. We show that a small set (eight) of coding-region SNPs resolves a substantial proportion of the identical haplotypes, as defined by control-region variation alone. Moreover, this SNP set, while substantially increasing the discriminating efficiency in most Eurasian populations by roughly equal amounts, discloses population-specific profiles.

Transcription factor NF-IL6 (C/EBPbeta) activates the expression of the mouse MHC class I H2-Kb gene in response to TNF-alpha via the intragenic downstream regulatory element.

The 5'-enhancer-deleted genomic construct of the H2-K(b) gene, stably integrated into the genome of L(tk-) fibroblasts, retains full competence to be induced by tumor necrosis factor-alpha (TNF-alpha) treatment. The only defined regulatory region in this construct is the intragenic downstream regulatory element (H2DRE). Computational inspection uncovered two potential NF-IL6 (C/EBPbeta) binding motifs within the H2DRE. Chloramphenicol acetyltransferase (CAT) reporter gene assay revealed that NF-IL6 is able to elevate transcription from H2DRE. Moreover, transient transfection of an NF-IL6 expression vector increased both constitutive and TNF-alpha-induced mRNA levels of endogenous H2 class I genes, and transfection of an NF-IL6 dominant negative construct decreased the expression of endogenous H2 class I genes in a dose-dependent manner. Using the electrophoretic mobility shift assay (EMSA) and antibody supershift assay, we were able to qualify the two computationally identified NF-IL6 binding motifs as one high-affinity and one low-affinity binding site. We conclude that the H2-K(b) gene belongs to target genes of the NF-IL6 (C/EBPbeta) in the course of the cellular response to TNF-alpha, and we discuss some consequences of this conclusion in a general framework of inducible expression of the H2-K(b) gene.

The inverted CCAAT motif is an indispensable element of the enhancer B of the mouse major histocompatibility I H2-Kb gene.

We have identified a strong binding of nuclear proteins derived from Ltk(-) fibroblasts to the enhancer B of the mouse MHC class I H2-K(b) gene. The inverted CCAAT motif and its adjacent upstream sequences have been revealed as protein-binding sites by electrophoretic mobility-shift, methylation interference, and DNase I footprint assays. Specific mutations in the inverted CCAAT motif as well as in the 5'-flanking cytosine pentanucleotide abrogated the formation of the major DNA-protein complex. Transcription of the chloramphenicol acetyltransferase (CAT) reporter gene driven by the H2-K(b) promoter in the Ltk(-) cell line was reduced substantially when a two-nucleotide mutation was introduced into the CCAAT element (CCAATCgcAT). The indicated two-nucleotide mutation decreased transcription initiated from both the homologous and a heterologous promoter. Furthermore, cotransfected MHC class II transactivator (CIITA) elevated the transcription of the reporter gene under the control of the H2-K(b) upstream sequences in the NIH 3T3 cell line. The intact enhancer B involving both the inverted CCAAT motif and the site alpha was found to play an indispensable role in the CIITA-mediated gene transactivation. The band-shift assay with the enhancer B probe revealed forming of a protein complex in a cooperative manner, which was again prevented by mutations in either element. Our results suggest an essential role of the inverted CCAAT element in the constitutive as well as inducible transcription of the mouse MHC class I genes.

The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.

Cancer stem cells (CSC) are believed to be involved in tumor evasion of classical antitumor therapies and have thus become an attractive target for further improvement of anticancer strategies. However, the existence and identity of CSC are still a matter of controversy. In a systematic screen of 13 ovarian cancer cell lines we show that cells with stem cell properties are reliably detectable as a minor population, characterized by ABC transporter expression resulting in the side population (SP) phenotype. In different cell lines, either ABCG2 or ABCB1 was found to be responsible for this effect. Purified SP cells featured virtually all characteristics of bona fide CSC, including clonogenicity, asymmetric division and high tumorigenicity in vivo. Using in-depth phenotyping by multicolor flow cytometry, we found that among the investigated ovarian cancer cell lines the SP compartment exhibits tremendous heterogeneity and is composed of multiple phenotypically distinct subpopulations. Thus, our study confirms previous results showing that CSC are contained within the SP. However, the exact identity of the CSC is still disguised by the high complexity of the CSC-containing compartment. Further functional studies are needed to determine whether a single cellular subset can unambiguously be defined as CSC or whether multiple stem cell-like cells with different properties coexist. Moreover, the observed heterogeneity may reflect a high level of plasticity and likely influences tumor progression, escape from immune-surveillance and development of resistance to anticancer therapies and should therefore be considered in the development of new treatment strategies.