Investigations of different types of AgBr-layers for use in electron microscope autoradiography

On account of the low grain size three commercial emulsions GEVAERT NUC 307, ILFORD L4 and KODAK NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers. In order to investigate the sensitivities of the three emulsions monolayer preparations were irradiated in the electron microscope using an energy of 5.7 keV, corresponding to the mean β-energy of Tritium. After exposure the specimens were developed but left unfixed. The sensitivity may then be determined, using the ratio of developed to the total number of grains. For the formation of one latent image the ILFORD L4 emulsion must be hit on the average by 1-1.4 electrons per AgBr-grain; the corresponding figures for GEVAERT NUC 307 and KODAK NTE are 2-3 and 4-5, respectively. The problem of resolution of point and plane sources of radioactivity is discussed

Autoradiography with the electron microscope: properties of photographic emulsions

In this chapter, some current techniques of autoradiography with the electron microscope will be discussed, as well as some properties of the three photographic emulsions most commonly used for this purpose, namely, Gevaert NUC 307, Ilford LA, and Kodak NTE

Characteristics of three nuclear emulsions for autoradiography at the electron microscope

Drei handelsübliche Kernspuremulsionen, Gevaert NUC 307, Ilford L4 und Kodak NTE, wurden wegen ihrer geringen Korngröße auf ihre Eignung zur elektronenmikroskopischen Autoradiographie untersucht. Korngrößenverteilungskurven wurden aufgenommen und ein geeigneter Entwickler ausgesucht. Zur Bestimmung der Empfindlichkeit dieser drei Emulsionen wurden Einkornschichten im Elektronenmikroskop mit Elektronen einer Energie von 5,7 keV, der mittleren beta-Energie des Tritiums, bestrahlt. Anschließend wurden die Emulsionen entwickelt, aber nicht fixiert. Mit dem Anteil der entwickelten AgBr-Körner kann dann über Trefferkurven die Empfindlichkeit der Emulsionen bestimmt werden. Man benötigt zur Bildung eines latenten Bildkeimes für die Ilford L4-Emulsion 1 - 1,4 Elektronen pro AgBr-Korn, für die Gevaert NUC 307-Emulsion 2 - 3 und für die Kodak NTE-Emulsion 4 - 5 Elektronen pro AgBr-Korn. Folgerungen für das Auflösevermögen bei radioaktiven Punkt- und Flächenquellen werden diskutiert. Fortschritte in der Mikroautoradiographie werden von der Entwicklung feinkörniger Emulsionen abhängen, deren Empfindlichkeit bei etwa einem Elektron pro AgBr-Korn liegen sollte.On account of their low grain size three commercial emulsions, Gevaert NUC 307, Ilford L4 and Kodak NTE have been investigated to assess their qualities for electron microscope microautoradiography. Grain size distribution curves were determined and a developer suitable for microautoradiography was selected after having tested different types of developers. In order to investigate the sensitivities of the three emulsions, monolayer preparations were irradiated in the electron microscope, using an energy of 5.7 keV corresponding to the mean β-energy of tritium. After exposure the specimens were developed but left unfixed. The sensitivity may then be determined using the percentage of developed grains. For the formation of one latent image the Ilford L4 emulsion must be hit on the average by 1 - 1.4 electrons per AgBr grain; the corresponding figures for Gevaert NUC 307 and Kodak NTE are 2 - 3 and 4 - 5 respectively. The problem of resolution of point and plane sources of radioactivity is discussed. Future advances in microautoradiography will depend on the development of emulsions with lower grain sizes, but such improvement must not be at the expense of sensitivity

Studies on synchronisation in vivo: temporary inhibition of DNA synthesis in normal and malignant mammalian cell systems with hydroxyurea

Die Bearbeitung einer Reihe von Problemstellungen der experimentellen und klinischen Krebsforschung setzt die Möglichkeit einer Synchronisation proliferierender Zellsysteme in vivo voraus. Dies gilt z. B. für die Frage, ob bei Säugerzellen als Funktion ihrer Position im Zellcyclus Empfindlichkeitsunterschiede vorhanden sind, und zwar sowohl hinsichtlich der Auslösbarkeit des Prozesses der malignen Transformation durch Cancerogene, als auch in bezug auf die Inaktivierbarkeit maligner Zellen durch cytocide Agentien oder ionisierende Strahlung. In der vorliegenden Arbeit wird über Untersuchungen zur in vivo-Synchronisation verschiedener Gewebe (Embryo; Leber; Milz; transplantabler BICR/M1R-Tumor) der Ratte durch temporäre Blockade der DNA-Synthese mit Hydroxyharnstoff (HU) berichtet.The synchronous passage of proliferating cells through defined phases of the cell cycle is a prerequisite for the study of a number of problems associated with carcinogenesis and cancer therapy. It is particularly required for investigations of the differential sensitivity of mammalian cells in specific phases of the cell cycle to agents capable of initiating the process of malignant transformation, or causing cell death.The present study is concerned with the in vivo synchronisation of different rat tissues (embryo; liver; spleen; transplantable BICR/M1R tumor) by temporary specific inhibition of DNA synthesis with hydroxyurea (HU)

Synchronisation in vivo: temporary inhibition of DNA synthesis in the rat embryo with hydroxyurea

As part of experimental studies on the synchronisation of mammalian cell populations in vivo, the proliferative response of rat embryo cells (18th day of gestation) was investigated following transplacental administration of hydroxyurea (HU). DNA synthesis was strongly inhibited by i.p. injection of a single dose of 0.25 or 0.5 mg HU/g body weight, for periods of 2.5 and 4.5 h, respectively. Reversal of blocks occurred rapidly due to the short half-life (45 min) of the inhibitor in the embryo. The resulting synchronizing effects were analysed in terms of mitotic indices and rate of 3H-TdR incorporation for a period of 18 h after administration of HU

Electrophysiological properties of ethylnitrosourea-induced, neoplastic neurogenic rat cell lines cultured in vitro and in vivo

A comparative analysis was performed on the electrophysiological properties of 11 neoplastic neurogenic cell culture lines and five other cell lines of different origin (HV1C, rat bile duct carcinoma; BICR/M1 RK , rat mammary tumor; HeLa, human cervix carcinoma; 3T3, mouse embryo; REe, rat embryo). Neurogenic lines were derived either from N-ethyl-N-nitrosourea-induced neoplasms of the nervous system or from cultured fetal rat brain cells that had undergone neoplastic transformation in vitro after exposure to Nethyl-N-nitrosourea in vivo. Electrical membrane excitability was lacking in all neurogenic cells analyzed. Their membrane potential and input resistance values were similar to those of the nonneurogenic lines. Intercellular ionic coupling was consistently observed between cells of a fibroblastoid shape or cells bearing multiple cytoplasmic processes (i.e., all neurogenic lines HV1C, BICR/M1RK , and 3T3). Epithelioid cells (i.e., HeLa, REe, an NV1C subpopulation, and a GV1C1 variant) showed no such intercellular communication. In vivo monolayer cultures on glass coverslips were obtained by a modified i.p. diffusion chamber technique. Under these conditions, the cells (with the exception of a glioma-derived cell line) retained the morphological appearance and electrophysiological properties observed in vitro

Molecular and cellular mechanisms in nervous system-specific carcinogenesis by N-ethyl-N-nitrosourea

A single pulse of N-ethyl-N-nitrosourea (ENU), applied to BDIX rats during the perinatal age, specifically results in a high incidence of neuroectodermal neoplasms in the central and peripheral nervous system (NS). The pronounced sensitivity of the developing NS suggests a dependence of the carcinogenic effect on the proliferative and/or differentiative state of the target cells at the time of the ENU pulse. The specificity of ENU for the NS cannot be due to tissue variations in the degree of carcinogen-cell interactions, since the reactive, electrophilic ethyl cation is produced by rapid, nonenzymatic decomposition of ENU indiscriminately in all tissues. Correspondingly, the initial molar fractions of ethylated purine bases are similar in the DNA of "high-risk" (perinatal brain) and "low-risk" tissues (e.g., liver; adult brain). However, while the respective half lives in DNA of N7-ethylguanine and N3-ethyladenine show only minor differences for both types of tissues, the mutagenic ethylation product 06-ethylguanine is removed from brain DNA very much more slowly than from the DNA of other tissues. Together with their high rate of DNA replication during the perinatal age, the incapacity of rat brain cells for enzymatic elimination of 06-alkylguanine from their DNA could account for an increased probability of neoplastic conversion, and hence for the NS specificity of ENU in the rat

Hydrogen ion-mediated enhancement of cytotoxicity of bis-chloroethylating drugs in rat mammary carcinoma cells in vitro

Aerobic glycolysis, a metabolic characteristic of malignant cells, can be exploited to increase the concentration of lactic acid selectively in tumor tissues in vivo by systemic administration of glucose (E. Jähde and M. F. Rajewsky, Cancer Res., 42: 1505-1512, 1982). To investigate whether a more acidic microenvironment can enhance the effectiveness of cytocidal drugs, we have analyzed the colony-forming capacity of M1R rat mammary carcinoma cells exposed to bis-chloroethylating agents in culture as a function of extracellular pH (pHe). At pHe 6.2 the cytotoxicity of 4-hydroperoxycyclophosphamide, as measured by inhibition of colony formation, was potentiated by a factor of ∼200 as compared to pHe 7.4. Similar results were obtained with mafosfamide, nitrogen mustard, nornitrogen mustard, melphalan, and chlorambucil; not, however, with ifosfamide. As indicated by experiments using the ionophor nigericin for rapid equilibration of pHe and intracellular pH (pHi; measured with pH-sensitive microelectrodes), modulation of drug action by varying pHe primarily resulted from the concomitant decrease in pHi. The acidic microenvironment enhanced cytotoxicity most effectively during the phase of cellular drug uptake and monofunctional alkylation of DNA. DNA cross-link formation appeared to be less affected by pH, and lowering of pHe during the phase of cross-link removal was only marginally effective

Newly established human retinoblastoma cell lines exhibit an "immortalized" but not an invasive phenotype in vitro

Retinoblastoma (RB), an intraocular childhood tumor occurring in a hereditary (mostly bilateral) or non-hereditary (unilateral) form, is associated with the inactivation of both alleles of a putative tumor suppressor gene (RB-I) located on chromosome 13q14. Both the process of RB development and the biological characteristics of RB cells are as yet poorly understood. We have established 7 new RBL lines (RBL13, RBL14, RBL18 and RBL30, derived from unilateral RB; and RBL7, RBL15 and RBL20, derived from bilateral RB). Southern blot analyses of restriction fragment length polymorphisms in DNA samples from 6 cell lines revealed loss of constitutional heterozygosity at one or several polymorphic locion chromosome 13 in 4 cases. Gross deletions involving the RB-1 locus and amplification of the N-myc gene were not detected in any of the RBL lines. The phenotypic properties of the RBL lines were analyzed in comparison with cells from the original RB tumors, with 4 RB lines established by others (RB383, RB355, RB247C3 and Y79) and with the adenovirus-EIA-transformed human retinoblast line HER-Xhol-CC2. It was found that RB tumors consist of phenotypically heterogeneous cell subpopulations with varying nutrient requirements and differentiation potential in vitro. All cell lines showed the typical characteristics of established (immortalized) cells. In some cases, cells from original RB tumors or cell lines were able to form colonies when cell aggregates of 2-10 cells were suspended in semi-solid agar medium; however, anchorage-independent colonies never developed from single cells. Cell lines RBL13, RBL18, RB247C3, RB355, RB383 and Y79 were tested for invasion into embryonic chick heart fragments in vitro and found to be non-invasive. None of the RBL or RB lines were tumorigenic in nu/nu (T-) mice. Y79 cells (propagated in culture for many years) exhibited properties distinctly different from those of the other cell lines, and thus cannot be considered phenotypically representative of RB cells