G. Kellner

Radiochemical investigation of the utilisation of glucose by tissue cultures

Abstract The aerobic production of carbon dioxide and of lactic acid from radioactive glucose by chicken fibroblasts and by HeLa tumour in vitro has been investigated. The ratio of glucose carbon converted into lactic acid and into carbon dioxide, respectively, has been called q-value. Generally, the q-values of fibroblasts are low in conditions favourable for survival. After explantation or transplantation, the q-values are high, and go down only as the tissue adapts itself to new conditions. However, scarcity of the metabolic fuel glucose also tends to decrease the value of q, and very low q-values are observed when the fibroblasts are kept starving in isotonic salt solution. With HeLa, no conditions have been found where respiration predominates over glycolysis. Though for both HeLa and fibroblasts conditions can be adjusted so that far more glucose is glycolysed than oxidised, respiration seems to play a role in fibroblasts, which is qualitatively different from that in HeLa. In view of the strong dependence of q on experimental conditions, these must be standardised strictly, if different kinds of tissue are to be compared.

Effects of trypsin treatment on tissue in culture

Abstract The influence of controlled damage on the growth and the energy metabolism of tissue in culture was studied. In the preparation of monolayers of chick mesenchyma tissue the time of treatment with trypsin was varied. Thereafter, the cells in the roller tubes were counted at time intervals. At first, fewer cells were found in the tubes where trypsin treatment had been more prolonged, but after 5 days all cell counts were similar. The production of CO 2 and lactic acid from radioactive glucose were also measured in the time interval from the third to the fifth day after treatment with trypsin. Depressed respiration and increased fermentation were found. It is suggested that the damage to respiration is, in part, compensated by an increase in fermentation.

Effects of hydrogen ion concentration on tissue in culture.

Abstract Monolayer cultures of chick mesenchyma tissue were grown in media of various pH values (initially 6.0–8.5) but otherwise identical. The tissue displays a remarkable power to adjust the pH value of the medium in the direction of pH 7.5. The fastest increase in cell counts was obtained when the initial pH value was about 7.5. In some experiments, radioglucose was contained in the medium. The amount of radioactive CO 2 and lactic acid produced during incubation increased with increasing pH value. In tubes containing large numbers of cells, fermentation was more enhanced by increasing alkalinity than was respiration.

Production of carbon dioxide and lactic acid from radioactive glucose by tissue in culture

Abstract Chick embryo mesenchyma tissue in culture (mostly monolayers with known numbers of cells) was incubated in growth-promoting nutrient media with radioactive glucose, the carbon dioxide and the lactic acid isolated, and their radioactivities determined with the gas Geiger counter. Both “synthetic” and “natural” media were used. Some experiments were also done with tissue kept in salt solution. As the number of cells per unit quantity of glucose increases, glycolysis per cell decreases and respiration per cell increases, as far as glucose serves as the substrate. Thus the Crabtree effect in tissue culture is at least partly due to a switch from the utilization of glucose by respiration to its utilization by glycolysis (“one-substrate” theory). The effects of controlled damage by trypsin resemble those of an increase in the supply of glucose. In all experiments with growth-promoting media more glucose is glycolyzed than respired, but more free energy is usually derived from respiration than from glycolysis. The total amount of free energy derived from glucose, per cell, does not change significantly as the supply of glucose per cell is varied. No major differences in the utilization of glucose are observed in the given conditions between monolayers and solid tissue pieces.

Chromatographic separation of pyruvic, oxalacetic and alpha-ketoglutaric acid from tissue cultures

Abstract A new method for the isolation and separation of α-ketoacids participating in the citric acid cycle is described. The dinitrophenylhydrazones (DNPH) formed in the cold aqueous system are extracted with ether, removed from the ether with sodium carbonate, re-extracted with ether after acidification, and subjected to descending chromatography on a paper impregnated with phosphate buffer. The solvent consists of n-butanol pre-treated with an equal volume 3% aqueous ammonia. Pyruvic DNPH gives two spots (RF = 0.61 and 0.75), oxalacetic and α-ketoglutaric DNPH each one spot only (RF = 0.10 and 0.07, respectively). After elution, the hydrazones are determined by colorimetry. Two micrograms of the acids can still be determined, and a check on recovery is possible by isotope dilution. Oxalacetic DNPH is stable at room temperature, even when dissolved in 10% Na2CO3, but is decomposed to pyruvic DNPH by moderate heat. Therefore, in previous investigations where oxalacetic DNPH had been exposed to heat during analysis, major errors must have occurred.

Time course of the utilization of radioglucose by tissue in culture.

Abstract The aerobic production of lactic acid and of carbon dioxide from radioglucose by monolayers of chick mesenchyma tissue and of HeLa human carcinoma tissue during periods of incubation between 8 and 72 hr with synthetic nutrient medium has been measured. At the same time the numbers of the cells were counted so that the mean utilization of the glucose by single cells could be calculated. The utilization of glucose per cell and hr for glycolysis decreases, and for respiration increases, with increasing number of cells/roller tube and with increasing time; the relationship of this result to the Crabtree effect is discussed. The amounts of free energy available to the single cell per hr from the utilization of glucose were calculated. In the case of mesenchyma this amount does not change with time, irrespective of the share of respiration in the total supply of free energy. In contrast, the total supply of free energy to the single HeLa cell, after a transient increase, falls off with time, i.e. with increasing scarcity of metabolic fuel. In all cases, the supply of free energy from the glucose is far inferior to that calculated from manometric experiments of previous authors. It is suggested that the difference is covered mainly by the amino acids in the medium. In further experiments, the effects of varying concentrations of cyanide on the two kinds of tissue were studied. To judge both by the decrease in the cell numbers and by the free energy supply per cell from glucose, HeLa proved more resistant than mesenchyma. It is suggested that both the lesser adaptation of HeLa to scarcity of glucose and the higher resistance to cyanide reflect the fact that HeLa relies for its energy supply on aerobic glycolysis rather than on respiration to a higher extent than normal tissue.

Anwesenheit bestimmter Enzymsysteme in Gewebekulturen

Kulturen von embryonalen Huhnchenfibroblasten (Extremitatenmesenchym) in einem aus Embryonalextrakt und Aszitesflussigkeit bestehenden Nahrmedium wurden mit radioaktiven Substraten versetzt, und es wurde gepruft, ob radioaktives Kohlendioxyd erzeugt wird. Dies war bei Verwendung von Glukose, Fruktose, Mannose und Glyzerin als Substrat der FAll, nicht dagegen bei Verwendung von Saccharose. Es wird geschlossen, das das Gewebe zwar uber Enzymsysteme zur „Verwertung” der erstgenannten Stoffe, aber uber kein System zur Verwertung von Rohrzucker verfugt.

Fraktionierung der makromolekularen Bestandteile von HeLa-Krebszellen mittels DEAE-Chromatographie und Gel-Filtration

Losliche makromolekulare Bestandteile von HeLa-Krebszellen wurden mittels Chromatographie auf DEAE-Cellulose und Gel-Filtration auf Sephadex G-200 aufgetrennt. Die durch Ultrazentrifugierung erhaltenen Mikrosomen und der 105000 g-Uberstand verhielten sich chromatographisch verschieden. Komponenten, die im Bereich der Puffer von 0,01–0,1 M eluiert werden, kommen praktisch nur im Uberstand vor, Bestandteile, die von konzentrierteren Puffern eluiert werden, dagegen auch in den Mikrosomen. Bei der Gel-Filtration vom HeLa-Homogenat werden drei Fraktionen beobachtet. Das Filtrogramm des 105000 g-Uberstandes besteht ebenfalls aus drei Fraktionen, wahrend bei der Filtration der Mikrosomen nur ein symmetrischer Peak gefunden wird.Lösliche makromolekulare Bestandteile von HeLa-Krebszellen wurden mittels Chromatographie auf DEAE-Cellulose und Gel-Filtration auf Sephadex G-200 aufgetrennt. Die durch Ultrazentrifugierung erhaltenen Mikrosomen und der 105000 g-Überstand verhielten sich chromatographisch verschieden. Komponenten, die im Bereich der Puffer von 0,01–0,1 M eluiert werden, kommen praktisch nur im Überstand vor, Bestandteile, die von konzentrierteren Puffern eluiert werden, dagegen auch in den Mikrosomen. Bei der Gel-Filtration vom HeLa-Homogenat werden drei Fraktionen beobachtet. Das Filtrogramm des 105000 g-Überstandes besteht ebenfalls aus drei Fraktionen, während bei der Filtration der Mikrosomen nur ein symmetrischer Peak gefunden wird. Nach kurzer Inkubation von HeLa-Zellen mit Radioeisen verhielt sich die im Homogenat vorkommende makromolekulare radioaktive Substanz elektrophoretisch und chromatographisch wie Ferritin. Bei der Gel-Filtration erscheint in den Filtrogrammen von Mikrosomen und 105000 g-Überstand ein relativ scharfer Aktivitätspeak (Ferritin) im Bereich der Moleküle, mit einem Molekulargewicht>200000. Die Versuche mit 59Fe-markiertem HeLa-Ferritin haben gezeigt, daß Chromatographie auf DEAE-Cellulose und Gel-Filtration auf Sephadex G-200 gut geeignet sind, ein spezifisches Protein von anderen makromolekularen Bestandteilen abzutrennen. Soluble macromolecular components of HeLa cancer cells were fractionated by means of chromatography on DEAE cellulose and on Sephadex G-200. Different chromatographic patterns were obtained for the microsomal fraction and for the 105,000 g supernatant fraction, which were separated by ultracentrifugation. Components eluted in the 0.01–0.1 M buffer range were found primarily in the supernatant; in contrast, the components eluted by the more concentrated buffers were found in the microsomal fraction. On gel filtration of the HeLa cell homogenate three fractions were observed. Gel filtration of the microsomes yielded only one symmetrical peak, whereas the 105,000 g supernatant could be separated into three peaks. After a short incubation of HeLa cells with radioactive iron (59Fe), the macromolecular radioactive substance in the homogenate behaved electrophoretically and chromatographically as ferritin. The distribution of radioactive iron in the filtrograms of microsomes and the 105,000 g supernatant was similar, with a relatively sharp peak of ferritin appearing in the range of molecules with a molecular weight>200,000. The experiments with HeLa cells labelled with 59Fe-ferritin have shown, that chromatography on DEAE cellulose and gel filtration on Sephadex G-200 are well suited for the separation of a specific protein from other macromolecular cell components.

Untersuchung der Proteinbildung durch Gewebekulturen mit Hilfe von Radiokohlenstoff

Der Einbau von Radiokohlenstoff aus Glukose, Glykokoll, Alanin und Tyrosin in die loslichen Proteine von Huhnermesenchym-und (menschlichem) HeLa-Tumorgewebe bei Bebrutung mit Nahrmedium wurde untersucht. Das Medium bestand aus einer Mischung vonGeyscher Salzlosung, Aszitesflussigkeit, Nabelstrangserum und Extrakt von Huhnerembryonen. Die Proteine wurden durch Ultrafiltration unter Druck abgeschieden, durch Hochspannungselektrophorese in Starke-Gel aufgetrennt und nach Verbrennung im Gas-Geiger-Zahlrohr auf Aktivitat gepruft. Auch gewebefreies Medium nimmt Radiokohlenstoff in Eiweis auf, doch handelt es sich dabei, wie Kontrolle durch die Ninhydrinprobe ergab, nicht um echten Einbau von Aminosaureresten in peptidischer Bindung. Hingegen bewirkt das Gewebe echten Einbau. Dieser kann auf die Zahl oder Masse der Zellen bezogen werden und ist bei Mesenchym-und Tumorgewebe von der gleichen Grosenordnung. Den grosten Einbau an Radiokohlenstoff weisen die Albumin- sowie die γ-Globulinfraktion (beide durch das elektrophoretische Verhalten definiert) auf. HeLa nimmt mehr Kohlenstoff aus Glykokoll in γ-Globulin auf als Mesenchym. Da im Gegensatz zu Untersuchungen an Organismen oder Gewebeschnitten mit isoliertem Gewebe gearbeitet wird, besteht keine Moglichkeit der Storung durch Wechselwirkung von Geweben miteinander, insbesondere von Tumorgewebe mit gesundem Gewebe.

Influence of ionizing radiation on protein production by HeLa cells in culture

Bei der Züchtung von HeLa-Krebszellen in tritiumhaltiger Nährlösung während 48 h wurde festgestellt, dass die Mitoserate durch viel kleinere Strahlendosen nachweisbar herabgesetzt wird als die radiochemisch gemessene Rate der Proteinsynthese.