Walfried A. Linden

Analysis of PCP-Data to Determine the Fraction of Cells in the Various Phases of Cell Cycle*

SummaryMathematical models for the analysis of pulse-cytophotometric (PCP) data are described. With computer programs based on these models the fractions of cells in G1-, S- and(G2 + M)-phases are obtained. The methods are applied to PCP measurements of Ehrlich ascites tumor cells, human bone marrow cells and L-929-cells in culture. The results of the L-cell experiment are compared with autoradiographic results; for both methods the duration of the various phases has been calculated. Two different mathematical models for PCP-data evaluation and the autoradiographic method yielded results agreeing within statistical error. The application of the two models on different types of DNA-histograms is discussed: One model is suitable for asynchronous cell populations with a low fraction of S-phase cells, the other can be applied for partially synchronized cells and high S-phase fractions as well.

Die dauer der phasen im zellzyklus von l-929-zellen

SummaryThe mean duration of the different phases of the mitotic cycle in L-929 cells cultivatedin vitro was determined autoradiographically and also by impulsecytophotometry. The mean values obtained with the two methods each involving 5 experiments were within the frame of statistical error. The reproducibility of the ICP measurements was better. The importance in clinical application is discussed.ZusammenfassungDie mittlere Dauer der Phasen des Zellzyklus von L-929-Zellen in Kultur wurde autoradiographisch und impulscytophotometrisch bestimmt. Die Mittelwerte von fünf Untersuchungen wichen biei beden Verfahren nur im Rahmen des statistischen Fehlers voneinander ab. Die Reproduzierbarkeit der ICP-Messungen war besser. Auf Konsequenzen für eine klinische Anwendung wird hingewiesen.

Radiosensitivity and recovery of mouse L cells during the cell cycle

SummaryMouse fibroblasts, subline L-929 F were synchronized by mitotic detachment. The synchronized cell cultures were irradiated with 200 kVp X-rays at different time after mitosis, and age reponse functions and dose effect curves were determined using the colony test. The cell age in the mitotic cycle was obtained from a computer analysis of flow cytometric DNA histograms. Both intrinsic radiosensitivity 1/D0 and extrapolation numbern were found to vary during the cell cycle. TheD0 has a maximum value of 176 ± 1 rad in the middle ofG1 phase and a minimum of 71 ± 1 rad at theS/G2 transition, while the extrapolation number is rather constant from the beginning ofG1 phase (1.9 ± 0.1) to the middle ofS phase (2.3 ± 0.1) and reaches a steep maximum of 9.3 ± 1.1 atS/G2 transition. The values ofn in the various phases of cell cycle are compared with the respective values of the recovery factorγ determined after fractionated irradiation. - Cell survival after a single dose of 616 rad has minima for irradiation atG1/S transition and in earlyG2 phase; the survival in earlyG2 being about 40 times smaller than in earlyG1 phase. Implications for a cell cycle specific therapy are discussed.

Impulsecytophotometric studies on the effects of daunomycin on synchronised L-cells.

Abstract Mechanically synchronized cultures of L-cells were treated in various phases of the cell cycle with daunomycin. After incubation for 4 hr at a concentration of 1 μg/ml the percentage of cells in the various phases was determined using an impulse-cytophotometer † . Applying daunomycin in G1 phase produced an arrest of cells in G1 that had not been observed so far or had been considered negligible. When daunomycin was applied in S phase the progression of cells throughout this phase was not influenced and the end of S phase was not delayed, indicating that DNA synthesis of cells having already started DNA synthesis, is not disturbed by daunomycin at this low concentration. Daunomycin treatment in the late S phase or in the G2 phase resulted in an accumulation of cells in G2 (G2 block).

Radiation-induced synchronization of the Walker carcinoma in vivo

Abstract Exponentially growing Walker tumours of a weight of 1·0 ± 0·3 g were irradiated at a 200 kV p X-ray unit with single and fractionated doses ranging from 50 to 900 rads. The influence of the radiation on the cell cycle of the carcinoma was studied using a pulse-cytophotometer. By this method DNA distribution patterns of the tumour cells are obtained. The mathematical analysis of these DNA distributions using a computer program gives the percentage of cells belonging to the various phases of the cell cycle. The results show a reversible accumulation of cells in the G 2 -phase and a partly synchronous passage of cells from G 2 -phase into mitosis and G 1 -phase. With the highest dose applied, the fraction of cells in G 2 could be increased from 14 to 48% . The degree of synchrony obtained is compared with the values determined after chemical treatment in vivo .

DNA-synthesis in synchronized L-cells after irradiation during the G1-phase of the cell cycle

SummaryMouse fibroblast L-929 cells synchronized by mitotic selection were irradiated during the G1-phase of the cell cycle with a dose of 1000 rad. The rate of DNA synthesis was measured by3H-thymidine incorporation, and the progression of the cells through the cell cycle was determined using a pulse-cytophotometer. Irradiation caused a decrease in the rate of DNA synthesis to half the control value, and an extension of the S-phase to twice its normal duration.

STUDIES ON THE POPULATION KINETICS OF THE WALKER CARCINOMA BY AUTORADIOGRAPHY AND PULSE CYTOPHOTOMETRY

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. The transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5‐6 days after transplantation, a PLM curve was performed, yielding estimates of Tc ≅ 18.0 hr, Ts ≅ 6.4 hr, TG2+M≅ 4.1 hr. With the double labelling technique in vitro under 2.2 atm oxygen we obtained: Tc ≅ 18.2hr, Ts ≅ 8.2 hr, TG2+M≅ 2.0hr. From pulse cyto‐photometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: fG1≅ (47.6 ± 1.1)%, fs≅ (34.1 ± 1.0)%, fG2+M≅ (18.3 ± 1.5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0.93 on the seventh and eleventh day. The cell loss factor was φ≅ 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.

Impulscytophotometrische Analyse der Zervixzellen von Frauen aus der Postmenopause

Summary49 suspensions with cells of the cervix uteri exclusively taken from postmenopausal women were analysed with the pulsecytophotometer. 7 of these cases had an histologically verified invasive cancer of the cervix uteri. There were no false negative results, the percentage of false positive measurements was about 30%. This good result may arise from selecting the cell material analized exclusively from postmenopausal patients and the choice of mathematical model for histogram interpretation, which has been constructed analogously to the cycle of mitosis of cells. Comparing the different methods of preparation of the suspensions the standard procedure (RNase, pepsin, ultrasonic) before staining with ethidium bromide seems to be the best one.Zusammenfassung49 Suspensionen mit Zervixzellen von Postmenopause-Patientinnen wurden mit dem Impulscytophotometer untersucht. Darunter waren 7 Krebsfälle. Die Rate falsch negativer Meßergebnisse war null. Der Prozentsatz falsch positiver Resultate betrug ca. 30%. Dieses relativ günstige Ergebnis kann bedingt sein durch die Auswahl des Untersuchungsmaterials und das sich an den Mitosezyklus der Zellen anlehnende mathematische Verfahren der Histogrammauswertung. Unter den verschiedenen Präparationsmethoden liefert das sog. Standardverfahren (RNase, Pepsin, Ultraschall) vor der Ethidiumbromidfärbung bisher noch die besten Resultate.

Impulscytophotometrische DNS-Histogramme normaler und maligner Plattenepithelien der Cervix uteri

SummaryThe flow-through fluorescence cytophotometer seems to be an successfully prescreening method for cervical cancer (f.i. ICP 11, Phywe/Göttingen/ FRG). Measurements are based upon the abnormally high DNA content of malignant cells. The results are given as histograms.This report contains data of 22 cell suspensions analysed with the impulse cytophotometer (ICP 11). 8 cases were normal (different times of the menstrual cycle, pregnancy, postmenopausal), the other 14 cases were precancerous ones or invasive carcinoma of the uterine cervix. It is ascertained that cell samples of malignant cells of dysplasia, carcinoma in situ or invasive cancer can be determined by means of a DNA-distribution clearly exceeding the normal range. The time for measuring is calculated in minutes, for preparation of cell suspensions in hours (just more than two hours). The percentage of not readible histograms was 36%.The use of the impulse cytophotometer for sucessfully automated prescreening of cervical cellular material should be improved by reducing the cases with not readible histograms and changing the fluorescent stainer. The rate of false negative histograms could be examined more exactly by calculating the percentage of malignant cells of every sample necessary for indicating malignancy undoubtly. This critical cell number is perhaps unequally high in cases of precancerous dysplasia, carcinoma in situ and invasive carcinoma. The application of brief amounts of ultrasonics on the samples is useful but should be given selectively to different cases. The computer evaluation of the histograms will contribute a great deal to concreting the interpretation of these results of DNA-measurement in future.ZusammenfassungEine apparativ ausgeführte gynäkologische Cytodiagnostik erscheint heute mit Durchflußfluorescenzcytophotometern in den Bereich des praktisch Möglichen gerückt zu sein (z.B. mit dem Impulscytophotometer ICP 11 der Firma Phywe/Göttingen). Entscheidendes Kriterium der Auswertung ist der in Tumorzellen abnorm hohe DNS-Gehalt (Desoxyribonukleinsäuregehalt) der Zellkerne. Der gemessene DNS-Gehalt einer Zellprobe wird in Form eines Histogramms ausgeschrieben.Über die Ergebnisse einer impulscytophotometrischen Analyse von 22 verschiedenen Zellsuspensionen von der Cervix uteri wird berichtet. Darunter waren 8 Fälle normaler Plattenepithels (Cyclusphasen, Gravidität, Postmenopause). Die restlichen 14 Fälle stammten von Präkanzerosen und invasiven Carcinomen. Es wird bestätigt, daß Zellsuspensionen mit malignen Plattenepithelien von Dysplasien, Oberflächencarcinomen und invasiven Carcinomen der Cervix uteri in den Histogrammen an dem größeren Anteil von Zellen mit erhöhtem DNS-Gehalt zu erkennen sind. Die Meßzeit ist höchstens minutenlang. Der Arbeitsaufwand für die präparativen Vorarbeiten beträgt vorerst noch etwas mehr als 2 Std. Der Prozentsatz nicht verwertbarer Histogramme lag bei 36%.Mit dem Impulscytophotometer lassen sich wahrscheinlich bessere Ergebnisse erzielen, wenn die Zahl der nicht verwertbaren Histogramme reduziert wird und evtl. ein anderer Fluorescenzfarbstoff eingesetzt wird. Für die Bestimmung der Rate falsch negativer Histogramme ist das Festlegen der kritischen Zellzahl nötig. Das heißt: wie hoch muß der Anteil maligner Zellen in einer Meßprobe sein, um im Histogramm sicher erfaßt zu werden. Diese kritische Zellzahl ist vermutlich für Dysplasien, Oberflächencarcinome und invasive Carcinome ungleich hoch. Die Ultraschallapplikation vor der Messung hat sich bewährt. Ein differenzierter Einsatz dieses präparativen Hilfsmittels ist anscheinend erforderlich. Die Computerauswertung der Histogramme wird künftig die Aussagesicherheit der DNS-Messungen erhöhen.Eine apparativ ausgefuhrte gynakologische Cytodiagnostik erscheint heute mit Durchflusfluorescenzcytophotometern in den Bereich des praktisch Moglichen geruckt zu sein (z.B. mit dem Impulscytophotometer ICP 11 der Firma Phywe/Gottingen). Entscheidendes Kriterium der Auswertung ist der in Tumorzellen abnorm hohe DNS-Gehalt (Desoxyribonukleinsauregehalt) der Zellkerne. Der gemessene DNS-Gehalt einer Zellprobe wird in Form eines Histogramms ausgeschrieben. Uber die Ergebnisse einer impulscytophotometrischen Analyse von 22 verschiedenen Zellsuspensionen von der Cervix uteri wird berichtet. Darunter waren 8 Falle normaler Plattenepithels (Cyclusphasen, Graviditat, Postmenopause). Die restlichen 14 Falle stammten von Prakanzerosen und invasiven Carcinomen. Es wird bestatigt, das Zellsuspensionen mit malignen Plattenepithelien von Dysplasien, Oberflachencarcinomen und invasiven Carcinomen der Cervix uteri in den Histogrammen an dem groseren Anteil von Zellen mit erhohtem DNS-Gehalt zu erkennen sind. Die Meszeit ist hochstens minutenlang. Der Arbeitsaufwand fur die praparativen Vorarbeiten betragt vorerst noch etwas mehr als 2 Std. Der Prozentsatz nicht verwertbarer Histogramme lag bei 36%. Mit dem Impulscytophotometer lassen sich wahrscheinlich bessere Ergebnisse erzielen, wenn die Zahl der nicht verwertbaren Histogramme reduziert wird und evtl. ein anderer Fluorescenzfarbstoff eingesetzt wird. Fur die Bestimmung der Rate falsch negativer Histogramme ist das Festlegen der kritischen Zellzahl notig. Das heist: wie hoch mus der Anteil maligner Zellen in einer Mesprobe sein, um im Histogramm sicher erfast zu werden. Diese kritische Zellzahl ist vermutlich fur Dysplasien, Oberflachencarcinome und invasive Carcinome ungleich hoch. Die Ultraschallapplikation vor der Messung hat sich bewahrt. Ein differenzierter Einsatz dieses praparativen Hilfsmittels ist anscheinend erforderlich. Die Computerauswertung der Histogramme wird kunftig die Aussagesicherheit der DNS-Messungen erhohen.

Clinical Applications of Flow Cytometry

During the last decade appreciable effort has been undertaken to elucidate the impact of cell kinetic concepts for clinical oncology. As flow cytometry (FCM) has become a valuable and reliable research tool in cell cycle kinetics (for a review see (1,18) and as many of the changes in cell transformation from normal to malignant at least principally can be measured in flow systems, this method has been applied both in cancer diagnostics (as well as estimating of prognosis) and cancer treatment monitoring. It has been demonstrated in previous chapters that flow cytometry is a rapid method for the quantitative determination of biologically important substances in single cells in suspension. In clinical applications of FCM the most important quantitative parameters are nucleic acid content, protein content, cell volume and light scattering and cell surface antigens. The aim of this chapter is to present some typical FCM determinations of those parameters in different fields of interest. A critical discussion of these examples should help to get an answer to the question concerning the present and potential benefit of FCM data for the clinician.