Response of Carcinogen-Altered Mouse Epidermal Cells to Phorbol Ester Tumor Promoters and Calcium

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1mM After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate Calcium causes these foci to stratify, however, in contrast to normal epidermis, DNA- synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells Cell lines derived from these foci may be considered to be putative initiated cells Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines Proliferation was estimated by the [3H]thymidine labeling index, by incorporation of [3H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, rising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. in 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. in line F, [3H]thymidine incorporation in confluent cultures was inhibited by TRA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. If these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage

Experimental performance of liquid hydrogen and liquid fluorine in regeneratively cooled rocket engines

Performance of liquid hydrogen-fluorine propellant combination in regeneratively cooled rocket engin

Transformation of Epidermal Cells in Culture

Studies performed on mouse skin have indicated that chemical carcinogenesis can be subdivided into two distinct stages, initiation and promotion. Initiation results from exposure to a classical mutagenic carcinogen and is irreversible even after a single exposure. The permanently altered initiated cell and its progeny may never form a tumor or in any way be recognizable in the target tissue. Exposure to tumor promoters permits the expression of the neoplastic change in initiated cells, and tumors develop. In contrast to initiators, promoters must be given repeatedly to be effective; individual exposures are reversible. A similar biology is suggested by epidemiologic studies of certain human cancers, particularly lung, breast, colon, and uterine malignancies. Studies in mouse skin cell culture have provided new insights into the changes associated with initiation and promotion. Initiated cells appear to be resistant to signals for terminal differentiation and can proliferate under conditions where normal epidermal cells are obligated to cease proliferation and begin their maturation program. This change is essential for an epithelial tumor cell since it provides the ability to grow away from a basement- membrane attachment site. In cultured epidermal cells, tumor promoters are capable of selectively stimulating the growth of certain cells, including initiated cells, while simultaneously inducing terminal differentiation in other epidermal cells. The net effect of these responses to promoters is the clonal expansion of cells stimulated to proliferate. In this way, promoters are capable of increasing the clone size of initiated cells. These cell culture data provided a biological framework for understanding initiation and promotion in terminally differentiating epithelial tissues

Untersuchungen zur Expression von Uhrengenen in der kortikotrophen AtT-20-Tumorzelllinie der Maus

In den unterschiedlichsten Lebewesen, wie Cyanobakterien, Pilzen, Pflanzen und Tieren können tägliche Rhythmen biologischer Aktivität beobachtet werden, die von einem endogenen zirkadianen Oszillator gesteuert werden. Dieser zirkadiane Oszillator residiert bei Säugern im Nucleus suprachiasmaticus (SCN) des Hypothalamus, unterhält auch unter konstanten Bedingungen einen Rhythmus mit einer Periodenlänge von etwa 24 h und wird unter natürlichen Bedingungen an den täglichen Wechsel der Beleuchtungsverhältnisse über neuronale Signale, die von den Augen kommen, angepasst. Für die Generation dieser endogenen Oszillationen konnte die rhythmische Expression von so genannten Uhrengenen verantwortlich gemacht werden. Nach der heute gültigen Vorstellung bilden diese zusammen mit ihren Proteinprodukten interagierende transkriptionelle-translationale Rückkopplungsschleifen, die für einen vollständigen Durchlauf, bis ein neuer Zyklus beginnt, etwa 24 h brauchen. Dabei aktivieren zwei Transkriptionsfaktoren der bHLH-PAS-Familie, CLOCK und BMAL1, zu Beginn eines zirkadianen Zyklus als Heterodimer über eine hochspezifisches E-Box-Promotorelement die Transkription der Uhrengene Per1-3, Cry1-2 und Rev-Erbα. Im Zytosol bilden die Uhrengenprodukte der CRYs und PERs zusammen mit der Caseinkinase Iε (CKIε) einen heterotrimeren Komplex, der im Kern wiederum die CLOCK-BMAL1-abhängige Transkription blockiert. Überraschend ist, dass nicht nur die Neurone des Schrittmachers im SCN diese Rhythmen endogen produzieren können, sondern auch eine Vielzahl peripherer Zellen, selbst, wenn sie über Jahre in Kultur gehalten wurden. Man nimmt an, dass der Rhythmus peripherer Zellen in vivo sowohl über neuronalen Verbindungen als auch über bisher noch nicht identifizierte humorale Faktoren synchronisiert wird. Es ist bis heute weder geklärt, worin die molekularen Unterschiede peripherer Oszillatoren im Vergleich zum SCN bestehen, noch, wie der Synchronisationsprozess dieser Zellen zu Stande kommt. Auf Grund methodischer Schwierigkeiten bei der Untersuchung des SCN wurde zuletzt vermehrt gefordert, sich diesen Fragen zunächst an Hand eines Modellsystems, wie einer Zellkultur aus immortalisierten Zellen zu nähern. In der vorliegenden Arbeit wurde deshalb untersucht, ob sich die kortikotrophe hypophysäre AtT-20 Tumorzelllinie der Maus prinzipiell für die Erforschung zirkadianer Rhythmen und deren Synchronisation eignet, d.h. ob sie selbst über eine stimulierbare rhythmische Uhrengen-Expression verfügen. Weiterhin sollte eine geeignete Methode gefunden werden, um zirkadiane Rhythmen auf mRNA-Ebene darzustellen. In einem ersten Schritt wurde über RT-PCR Technik erstmals nachgewiesen, dass die essen-tiellen Uhrengene Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock und CK1ε endogen in AtT-20 Zellen exprimiert werden. Für jedes dieser Gene wurde nun eine Variante der quantitativen Real-Time-PCR (RTQ-PCR), die ΔΔCT-Methode, validiert, die bei hohem Probendurchsatz zuverlässig Expressionsunterschiede wiedergeben kann. Durch Stimulation mit Forskolin, ei-nem Aktivator der Adenylatzyklase, konnte in dieser Arbeit dokumentiert werden, dass kulti-vierte AtT-20 Zellen in der Lage sind, eine rhythmische Expression von Uhrengenen mit einer Periodenlänge von etwa 24 h für mindestens drei Tage zu zeigen. Von allen hier untersuchten Uhrengenen wiesen alle diejenigen eine oszillierende Schwankung des mRNA-Gehaltes auf, die auch im SCN rhythmisch exprimiert werden, namentlich Per1-3, Cry1-2, Bmal1. Im SCN kon-stitutiv exprimierten Uhrengene (Clock, Ck1ε) fluktuieren auch nicht in AtT-20 Zellen. Dar-über hinaus antworteten Zellen auf das hier angewandte Stimulationsprotokoll mit einer initia-len Hochregulierung der Transkription für das Uhrengen Per1, das im SCN eine prominente Rolle bei der Anpassung des endogenen Rhythmus an die exogenen Beleuchtungsverhältnisse spielt und dort als Antwort auf synchronisierende Lichtpulse in ähnlicher Weise induziert wer-den kann. Zeitlich korreliert die Zunahme von Per1-Transkripten – ebenfalls der Situation im SCN entsprechend – mit einer Aktivierung des Transkriptionsfaktors CREB und der Induktion seines molekularen Gegenspielers Icer. Die zeitlich umschriebene Hochregulation der Transkriptionsrate des Repressors Icer während der ersten Stunden nach Applikation des syn-chronisierenden Reizes spricht dafür, dass dieser womöglich in AtT-20, wie auch bereits für Elemente des zirkadianen Systems beschrieben, eine wichtige Rolle bei der Verarbeitung von Synchronisationsreizes im molekularen Uhrwerk spielt. Die genaue Analyse der hier erhobenen Expressions-Rhythmen von Uhrengenen und deren zeitliches Verhältnis zueinander deuteten darauf hin, dass in AtT-20 Zellen ein funktionsfähiges zirkadianes Uhrwerk existiert, das dem des SCN in weiten Teilen gleicht. Die Möglichkeiten der Stimulation und Manipulation (z.B. durch Transfektion) erheben AtT-20 Zellen zu einem Modellsystem für die Erforschung der molekularen Abläufe in der zirkadianen Rhythmusgeneration und –synchronisation. Erkenntnisse aus dieser Forschung können in den unterschiedlichsten klinischen Disziplinen wichtige Anwendungsmöglichkeiten finden.Daily rhythms of biological activity are driven by an endogenous circadian oscillator and can be observed in living organisms as diverse as cyanobacteria, fungi, plants and animals. In mammals, this circadian oscillator resides in the suprachiasmatic nucleus of the hypothalamus and produces robust rhythms of a period length of about 24 hours, even under constant conditions. Under natural conditions, neuronal input from the eyes entrains its activity to the environmental light-dark cycle. Endogenous oscillations are generated by rhythmic expression of so called clock genes and their protein products that form interacting transcriptional-translational feedback loops, covering a cycling period close to 24 hours. The circadian cycle starts when two transcription factors of the bHLH-PAS family, CLOCK and BMAL1, enhance transcription by binding as heterodimers to highly selective E-Box elements of Per1-3, Cry1-2 and Rev-Erbα. In turn, a heterotrimeric complex of the clock gene products of CRYs, PERs and CK1ε translocates into the nucleus and blocks CLOCK-BMAL1 driven transcription. Surprisingly, not only SCN pacemaker neurons are capable to exhibit endogenous oscillations but also a multitude of different peripheral cells, even if they have been cultured for years. One can assume that in vivo the SCN synchronizes rhythms of peripheral cells by means of both neuronal connections and yet unknown humoral factors. To date, it is still not elucidated in which way the molecular setup of pacemaker neurons of the SCN differs from that of peripheral oscillators, nor it is clear, how synchronization of these cells is performed. As the use of the SCN harbours certain methodical difficulties, it has been proposed to clarify these questions for the present by means of culturing immortalized cells. The present work therefore aimed to determine whether the hypophyseal corticotroph AtT-20 cell line of the mouse can serve as a model to investigate generation and synchronization proc-esses of circadian rhythms, i.e. whether they exhibit rhythmic circadian expression of clock genes upon stimulation. Further, a suitable method should be found, to analyse circadian mRNA rhythms. Here it has been shown for the first time by means of the RT-PCR technique that AtT-20 cells express endogenously the essential clock genes Per1, Per2, Per3, Cry1, Cry2, Bmal1, Clock and CK1ε. For each of these genes, a variant of quantitative Real-Time PCR, the ΔΔCT method, has been validated, which offers both high-throughput processing of many samples and a reliable display of differences in expression. This work shows that cultured AtT-20 cells are able to exhibit an almost 24 hour rhythm in clock gene expression for at least 3 days upon stimulation with forskolin, an activator of the adenylate cyclase. From all genes investigated, all those (Per1-3, Cry1-2, Bmal1) showed oscillating changes of mRNA levels that are also known to be rhythmic in the SCN. On the other hand, clock genes that are constitutively expressed in the SCN did also not oscillate in AtT-20 cells (Clock, CK1ε). Further, AtT-20 cells showed an initial up regulation of mRNA of the clock gene Per1 upon stimulation, similar to SCN neurons when synchronized by photic input signals. Similarly, the increase of Per1 transcripts correlates with the activation of the transcription factor CREB and the induction of its molecular opponent ICER. In AtT-20 cells the up regulation of Icer expression is limited to the first hours after stimulation, indicating that this transcriptional repressor plays an important role in the processing of synchronizing stimuli to the molecular clock mechanism, as it has been described in other elements of the circadian system. The analysis of the here presented rhythms of clock gene expression and their phase relation demonstrates that a functional circadian oscillator exists in AtT-20 cells that is very similar in structure and function to that of the SCN. The possibilities to stimulate and manipulate AtT-20 cells mark them to a model system for the exploration of molecular processes, involved in gen-eration and synchronization of circadian rhythms. Findings of this research may furthermore be applied in various clinical disciplines

Immunocompromise in Gnotobiotic Pigs Induced by Verotoxin-Producing \u3ci\u3eEscherichia coli\u3c/i\u3e (O111:NM)

A verotoxin-producing Escherichia coli serotype O111:NM strain (strain 10049; verotoxin 1 positive) persistently infected experimentally inoculated gnotobiotic pigs, causing attaching-effacing intestinal lesions and chronic diarrhea. Experiments were performed to determine whether persistent infection might be associated with immunocompromise of the host by this organism. Pigs inoculated with this strain had a significant reduction in peripheral blood lymphocytes and lower antibody titers to sheep erythrocytes compared with control pigs. Compared with pigs given a verotoxin-negative pathogenic strain of the same serotype (O111:NM, strain 2430), pigs inoculated with the verotoxin-positive strain had lower peripheral lymphocyte counts and proliferative responses to concanavalin A, phytohemagglutinin, and pokeweed mitogens. The results of this study suggest that strain 10049 has an immunocompromising effect on gnotobiotic pigs