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Basal and luminal subtypes of invasive bladder tumors have significant prognostic and predictive impacts for patients. However, it remains unclear whether tumor subtype commitment occurs in noninvasive urothelial lesions or in carcinoma in situ (CIS) and which gene pathways are important for bladder tumor progression. To understand the timing of this commitment, we used gene expression and protein analysis to create a global overview of 36 separate tissues excised from a whole bladder encompassing urothelium, noninvasive urothelial lesions, CIS, and invasive carcinomas. Additionally investigated were matched CIS, noninvasive urothelial lesions, and muscle-invasive bladder cancers (MIBC) from 22 patients. The final stage of subtype commitment to either a luminal or basal MIBC occurred at the CIS transition. For all tissues combined, hierarchical clustering of subtype gene expression revealed three subtypes: “luminal,” “basal,” and a “luminal p53-/extracellular matrix (ECM)-like” phenotype of ECM-related genes enriched in tumor-associated urothelium, noninvasive urothelial lesions, and CIS, but rarely invasive, carcinomas. A separate cohort of normal urothelium from noncancer patients showed significantly lower expression of ECM-related genes compared with tumor-associated urothelium, noninvasive urothelial lesions, and CIS. A PanCancer Progression Panel of 681 genes unveiled pathways specific for the luminal p53-/ECM-like cluster, for example, ECM remodeling, angiogenesis, epithelial-to-mesenchymal transition, cellular discohesion, cell motility involved in tumor progression, and cell proliferation and oncogenic ERBB2/ERBB3 signaling for invasive carcinomas. In conclusion, this study provides insights into bladder cancer subtype commitment and associated signaling pathways, which could help predict therapy response and enhance our understanding of therapy resistance. Significance: This study demonstrates that CIS is the stage of commitment for determining MIBC tumor subtype, which is relevant for patient prognosis and therapy response.

Mechanical forces play an important role in the organization, growth, maturation, and function of living tissues. At the cellular level, the transmission of forces from outside the cell through cell-matrix and cell-cell contacts are believed to control spreading, motility, maturation as well as intracellular signaling cascades that may change many characteristics in cells. We looked at cell populations of mouse embryonic fibroblasts that are deficient of focal adhesion kinase (FAK) and examined their mechanical profile. We observed that the lack of FAK induces a mesenchymal-epithelial switch including the regulation of adherens junctions via E-cadherin, leading to increased cell-cell-cohesion. Our results show that the absence of FAK influences the macroscopic cell colony spreading in two (2D) and three (3D) dimensions as well as the velocity fields of the tissue, the single cell persistence and correlation length, changing from an independent to a collective mode of migration. Additionally, the single cell size in the sheet decreases significantly.

Congenital genital malformations occurring in the female population are estimated to be 5 per 1000 and associate with infertility, abortion, stillbirth, preterm delivery and other organ abnormalities. Complete aplasia of the uterus, cervix and upper vagina (Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome) has an incidence of 1 per 4000 female live births. The molecular etiology of congenital genital malformations including MRKH is unknown up to date. The homeobox (HOX) genes HOXA10 and HOXA13 are involved in the development of human genitalia. In this investigation, HOXA10 and HOXA13 genes of 20 patients with the MRKH syndrome, 7 non-MRKH patients with genital malformations and 53 control women were sequenced to assess for DNA variations. A total of 14 DNA sequence variations (10 novel and 4 known) within exonic and untranslated regions were detected in HOXA10 and HOXA13 among our cohorts. Four HOXA10 and two HOXA13 DNA sequence variations were found solely in patients with genital malformations. In addition to mutations resulting in synonymous amino acid substitutions, in the HOXA10 gene a missense mutation was identified and predicted by computer analysis as probably damaging to protein function in two non-MRKH patients, one with a bicornate and the other patient with a septated uterus. A novel exonic HOXA10 cytosine deletion was also identified in a non-MRKH patient with a septate uterus and renal malformations resulting in a premature stop codon and loss of the homeodomain helix 3/4. This cytosine deletion and the missense mutation in HOXA10 were analysed by real time PCR and sequencing, respectively, in two additional larger cohorts of 103 patients with MRKH and 109 non-MRKH patients with genital malformations. No other patients were found with the cytosine deletion however one additional patient was identified regarding the missense mutation. Rare DNA sequence variations in the HOXA10 gene could contribute to the misdevelopment of female internal genitalia.

The placenta is a major barrier that prevents potentially infectious agents from causing fetal diseases or related complications during pregnancy. Therefore, we postulated that the placenta might express a broad repertoire of antimicrobial proteins as well as inflammatory chemokines and cytokines to combat invading microorganisms. Here we demonstrate that placental cells indeed express a wide range of AMPs (antimicrobial peptides and proteins) including bactericidal/permeability-increasing protein (BPI), secretory leukocyte protease inhibitor (SLPI), human β-defensin 2 (hBD2), acyloxyacyl hydrolase (AOAH), and cathelicidin (CAP18). In addition, these cells also secrete pro-inflammatory cytokines and chemokines upon stimulation with bacterial ligands. Notably, we show that BPI expression by placental cells could be completely attributed to granulocytes while highly purified placental trophoblasts expressed only a subset of the AMPs like SLPI. Unexpectedly, trophoblast AMPs did not exhibit inducible secretion in response to various TLR ligands and further investigations showed that the unresponsiveness of trophoblasts to lipopolysaccharide (LPS) was due to a lack of TLR4 expression. In summary, we have shown that the expression of different AMPs can be allocated to various cells in the placenta and the repertoire of the AMPs expressed by placental cells is a result of a cooperation of leukocytes as well as cells from embryonic origin.

Objective With an incidence of up to 5% in the general population, genital malformations are a frequent clinical occurrence. The aim of this study was to assess whether a connection could be demonstrated between various degrees of severity of genital malformations and associated abnormalities. Design All patients were classified using the Vagina, Cervix, Uterus, Adnex, and Associated Malformation (VCUAM) classification. Setting University hospital. Patient(s) Two hundred eleven premenopausal patients with female genital malformations. Intervention(s) The patients underwent diagnostic workup for genital malformations using laparoscopy as well as hysteroscopy. Associated malformations were detected by either magnetic resonance imaging (MRI) or ultrasound. Main Outcome Measure(s) Demonstration of a connection between various degrees of severity of genital malformations and associated abnormalities. Result(s) In 72 cases (36%) out of 202 patients with uterine malformations (VCUAM U1–4) we found associated abnormalities. The predominant findings were alterations in the renal system. When vaginal abnormality (VCUAM V1–5) alone was taken into consideration, an associated developmental disturbance in the renal tract was found in 30% of cases (n = 32 from 107). Conclusion(s) A close connection was demonstrated between genital malformations and associated abnormalities. For this reason, the diagnostic workup in patients with malformations should always include the renal system. Depending on the severity of the clinical picture, examinations may need to be extended further.

The University of Chicago high resolution scanning ion microprobe (UC-SIM) was used to image, by Secondary Ion Mass Spectrometry (SIMS), the distribution of Ca 2+ and Mg 2+ in the chromosomes of Indian muntjac (IM) deer mitotic fibroblasts. This is part of a systematic study of the cation composition of mammalian cells and chromosomes throughout the cell cycle, after having shown that Ca 2+ and Mg 2+ appear to be important for chromosome condensation and structure at metaphase. We focus here on a detailed description of the metaphase-anaphase transition at narrow time intervals beyond the G2/M border, made possible by controlled cell synchronization procedures. High-density distributions of chromosome spreads showed progressive stages of mitosis, identified by their morphology, within the same UC-SIM field of view. Subtle differences in cation contents between successive mitotic stages could thus be quantified in identical experimental conditions. Preliminary results indicate maximal chromosomal concentrations of Ca 2+ and Mg 2+ at metaphase, and a progressive decrease of the same with advancing stages of anaphase. Ca 2+ and Mg 2+ distributions were also imaged in the polytene chromosomes of Drosophila melanogaster, whose DNA distribution had been previously studied by BrdU labeling. These cations may play a common role in mitosis from lower eukaryotes to mammals.

At SIMS XIV, we reported SIMS evidence of specific Mg 2+ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg 2+ -depletion experiments supported a direct role for Mg 2+ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg 2+ -DNA and Mg 2+ -protein interactions. An in-depth study, reported elsewhere, implicated also Ca 2+ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II. We examine here the association of Mg 2+ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca 2+ and Mg 2+ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca 2+ and Mg 2+ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes.

Objective With an incidence of up to 5% in the general population, genital malformations are a frequent clinical occurrence. However, using the existing published classifications of malformations, difficulties arise in classifying genital malformations appropriately. The aim of the present study was to produce a simple, systematic, and reproducible classification system. Design A systematic arrangement of genital and associated malformaltions, using a structure similar to that in the TNM classification of oncological tumors, was developed and validated. Setting Patients with genital malformations in a university hospital. Patient(s) Ninty-nine premenopausal patients with genital malformations. Intervention(s) Patients were diagnosed for genital malformation using laparoscopy or magnetic resonance imaging. Main Outcome Measure(s) A new classification (VCUAM) is presented to evaluate patients with different genital malformations. Result(s) The external and internal female genital organs were divided into the following subgroups in accordance with the anatomy: vagina (V), cervix (C), uterus (U), and adnexa (A). Associated malformations were assigned to a subgroup (M) relative to each specific organ. The classification was validated in a group of 99 patients with genital malformations. Conclusion(s) The VCUAM classification for the first time makes it possible to reflect even complex malformations in a precise and individual fashion, taking associated malformations into account. The classification makes it easier to provide appropriate clinical care for the affected patients.

Our main research interests have focused on cation–DNA and cation–protein interactions in terms of their roles in higher order chromosomal structure. Our previous study directly analyzed the cation composition of cryo-preserved mammalian interphase and mitotic cells, as well as fractionated untreated and cation-depleted chromosomes at a resolution of 50 nm using the University of Chicago high resolution scanning ion microprobe (UC-SIM). Quantitative direct UC-SIMS signals and subsequent imaging of cryo-preserved cells demonstrated that Na+ and K+ were associated with chromatin throughout the cell cycle, whereas in contrast Ca2+ and Mg2+ exhibited a localization change during interphase and mitosis. Interphase Ca2+ and Mg2+ were found mainly throughout the cytoplasm, but at mitosis associated with chromatin. Our findings of chromatin association of Na+ and K+ support a role of these cations in both interphase and mitotic chromatin compaction, whereas Ca2+ and Mg2+ binding points to an essential function in mitotic chromatin compaction. In our present investigation using SIMS images obtained from both human and Indian muntjac metaphase chromosomes, we reveal specific binding of Mg2+ to chromosomal “p” and “q” arm heterochromatic regions correlating with conventional Giemsa (G-) bands. In addition, detailed Mg2+ image density peaks along the chromosomes indicate that Mg2+ peaks correspond to the location of G-bands. Our results support a direct role for Mg2+ in promoting and maintaining the higher order chromatin structure of heterochromatic regions on chromosome arms represented by G-bands, possibly due to both Mg2+–DNA and Mg2+–protein interactions.

In our previous investigation, we showed for the first time high-resolution analytical images of the cation composition of mammalian interphase and mitotic cells as well as of isolated metaphase chromosomes using the University of Chicago scanning ion microprobe (UC-SIM). In order to preserve the ionic integrity of the analyzed cells and prevent the well known occurrence of analytical artifacts due to the high diffusivity of cations in biological samples we used fast cryo-preservation methods (freeze-drying and freeze fracture), without any pre-fixations or washes. We identified the role of the cations in chromosome structure and maintenance using SIMS imaging and immunfluorescence methodologies. Importantly, we determined that the above cations are essential participants in chromosome condensation and maintenance of chromatin higher order structure, through their presumed function in DNA electrostatic neutralization and the direct interaction of Ca2+, in particular, with structural proteins. In addition, both Ca2+ and Mg2+ showed the same cell cycle regulation where during interphase both cations were enriched in the cytosol, particularly in organelles then at mitosis became specifically bound to chromatin. Our present research interest focuses on a more detailed analysis of the distribution of Ca2+ throughout the different cell cycle stages, e.g. G1, G2 and mitosis. We have chosen the stable isotope 44 Ca as a tracer to follow Ca2+ throughout the cell cycle. This nuclide occurs naturally in the ratio 44 Ca / 40 Ca + 44 Ca of 2.06%, so that incorporation at higher concentrations into chromatin or other cellular components should be easily detected by SIMS. Such incorporation can be obtained either by growing cells in a medium where ordinary Ca is replaced entirely by 44 Ca , or by replacing the cell culture medium with the 44 Ca medium for a gated time span (pulsing), after appropriate cell cycle stage synchronization. In this paper, we describe our experimental approach to the 44 Ca labeling of Indian muntjac deer and human fibroblast cells, which involve: (1) the preparation of culture medium where 40 Ca has been replaced by 44 Ca ; (2) the development of appropriate cell culture labeling protocols; (3) the methodology that enabled us to determine accurate isotopic SIMS measurements on chromosomes; (4) the first results where metaphase chromosomes obtained and imaged by SIMS bind up to 60–70% 44 Ca . We are presently developing cell culture synchronization protocols, which will allow us to analyze the binding dynamics of Ca throughout the cell cycle.

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

SummaryWe show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Scaffold-associated regions (SARs) function at the level of modeling or shaping the chromatin of DNA into loop domains. We have mapped 36 SARs in the human type I interferon ( IFN ) gene complex on chromosome 9, band p21-22, to examine the overall structure of this gene complex. A total of 29 strong SARs and 7 weak SARs were mapped to the flanking regions of the different interferon genes. Twenty-two strong SARs mapped to the flanking regions of 13 interferon ( IFNA ) α genes; 2 strong SARs mapped to one interferon ω ( IFNW ) gene; 2 strong SARs mapped to one interferon α pseudogene ( IFNAP ); and 3 strong SARs mapped to two interferon ω pseudogenes ( IFNWP ). One weak SAR mapped to the flanking region of one IFNA gene, whereas 6 weak SARs flanked four IFN pseudogenes ( P11, P12 P20, P23 ). The IFN SAR structure was comparable between the BV173 leukemia cell line and the U373 glioma cell line. Analysis of two glioma deletion breakpoint junctions, where breaks occur within and outside the IFN gene cluster, revealed an association with SARs. IFN SARs showed evidence for cooperativity among the SARs, while DNA sequence analysis revealed a series of clustered A-tracts within strong SARs. These data suggest that the IFN genes may be organized into a series of small (2–10 kb) DNA loop domains, with each loop containing a coding region flanked by SARs. In our model, the SAR enrichment and the clustering of A-tracts observed at the SARs within the IFN gene complex represent a higher level of chromatin organization, which may predispose this region to breakage.

Objective Uterine benign leiomyomas result from proliferation of a single smooth-muscle cell and their growth is affected by steroid hormones via steroid hormone receptors. This investigation analyzed the +331G/A and the V600L single nucleotide polymorphisms in the progesterone receptor, and correlated their incidence with clinical and tumor parameters as well as disease risk. Study Design Peripheral blood DNA was analyzed for the frequency of both progesterone receptor single nucleotide polymorphisms in 270 women with uterine leiomyomas compared with 163 control women without uterine leiomyomas. Results No correlation was found for both single nucleotide polymorphisms or the risk for developing myoma; however, statistical significant associations were found for single nucleotide polymorphism genotypes with specific clinical and tumor characteristics, eg, endometriosis, number of live births, menstrual cycle disorder, and leiomyoma focality. Conclusion Our findings support that specific genotypes in the progesterone receptor may be involved in tumor growth and metastasis but not in tumor initiation.