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A flow cytometric study of the rat Yoshida AH-130 ascites hepatoma

A flow cytometric study of the rat Yoshida AH-130 ascites hepatoma
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  Cancer Letters, 72 (1993) 169-173 Elsevier Scientific Publishers Ireland Ltd. 169 A flow cytometric study of the rat Yoshida AH-130 ascites hepatoma Marc Marzabala, Cklia Garcia-Martineza, Jaume Comasb, Francisco J. L6pez-Soriano” and Josep M. Argik” zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ‘Departament de Bioquimica i Fisiologia and hUnitat de Citometria de Flurr, Serveis Cientifico-Thics. Universitut de Burcelonu. Barcelona Spain) (Received 12 January 1993) (Revision received 18 May 1993) (Accepted 1 June 1993) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Summary The implantation of the Yoshida AH- 130 ascites hepatoma to rats results in a marked reduction in body weight in the tumour-bearing hosts. This is associated with an important reduction in both food intake and energetic efficiency in the last period of tumour growth. The growth of the tumour mass has a clear initial exponential phase, in which the maximum cell density is reached, with the majority of cells being in the synthetic phase (S) followed by most of the cells being in the G,,/G, phase, as determined using flow cytometry. Keywords: flow cytometry; Yoshida AH-l 30 ascites hepatoma; tumour; rats Introduction A variety of individual cellular characteristics The Yoshida AH-130 ascites hepatoma is a highly cachectic rat tumour of rapid growth and poorly differentiated cells. The tumour is char- acterized by a relatively short doubling time of one day [l] and is widely used in experimental studies. Correspondence to: Josep M. ArgilCs. Unitat de Bioquimica i Biologia Molecular B. Departament de Bioquimica i Fisiologia. Facultat de Biologia, Universitat de Barcelona, 08071 Barcelona. Spain. can be assessed using flow cytometry. This tech- nique is increasingly used in cancer research to measure cellular DNA content, which in turn can be used for the prognosis, recurrence and survival probability associated with human tumours [2.-41. The aim of the present investigation was to characterize by means of flow cytometric analysis the cell cycle characteristics of the Yoshida AH- 130 tumour cells once they are intraperitoneally in- oculated into a rat and to relate them to the tumour growth and burden in the host. Materials and Methods Animals All animals (Wistar rats) were fed ad libitum on a chow diet consisting (by weight) of 54% carbo- hydrate, 17% protein and 5% fat (the residue was non-digestible material), with free access to drink- ing water, and were maintained at an ambient tem- perature of 22 f 2°C with a 12-h light/l2-h dark cycle (lights on from 08:OO h). Food intake and body weight were measured daily after tumour inoculation. Biochemicals All biochemicals were reagent grade and obtain- ed either from Boehringer Mannheim S.A. (Barcelona) or Sigma Chemical Company (St. Louis, USA).  170 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Tumour impluntation A Yoshida AH-130 ascites hepatoma cell sus- pension (approx, 120 x IO6 cells in 2 ml) was in- jected intraperitoneally, the control rats being injected with 2 ml of 0.9% (w/v) NaCl solution. The Yoshida AH-130 is a rapidly growing tumour with a volume doubling time of 1 day. Total cell content was estimated using Trypan Blue staining. and then decreases progressively until 10 days after inoculation. zyxwvutsrqponmlkjihgfedcbaZYXWV FIQW cytometry Protein and DNA measurements Total protein [S] and DNA [6] content were measured in the tumoral cell mass according to standard procedures. Flow cytometry Flow cytometry analysis was carried out using an Epics Elite flow cytometer (Coulter Electronics Corporation, Hialeah, Florida). Excitation was carried out using a standard 488 nm air-cooled argon-ion laser at 15 mW power. The instrument was set up with the standard configuration. For- ward scatter (FSC), side scatter (SK) and pro- pidium iodide red fluorescence (675 mn bandpass filter) were used. Optical alignment was performed on an optimized signal from 10 nm fluorescent beads (DNA-check, Coulter Electronics Corpora- tion, Hialeah, Florida). Propidium iodide was ap- plied simultaneously with RNase (DNase free) after methanol fixation. Chicken erythrocytes and peripheral rat blood leucocytes were used as a cali- bration standard for DNA diploidy. Time was used as a control of the stability of the instrument in a fluorescence-versus-time dot plot. The cell cycle analysis was carried out using Multicycle Software (Phoenix Flow Systems, San Diego, California). The variation coefficient of the diploid peaks varied from 4.5-6.6, the number of nuclei analyzed being usually 20 000. The results of the Bow DNA analysis of the cell cycle distribution of Yoshida AH-130 cells are shown in Table I and Figs, 2 and 3. It is a clearly an aneuploid tumour with a DNA index of 1.35 of 0.009. The initial inoculum (day 0) show a majority of cells in the GdG, phase, while at day 3 the largest fraction of cells is in the S phase and the minor fraction is in the G?/M phase, Five days after inoculation, when the tumour is still in the exponential growth phase, the majority of cells are again in the GdG, phase. The decrease in the per- centage of cells in the S phase could be accounted zyxwvutsr   8 7 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON 2 4 6 8 days after inoculation Results and Discussion Tumour ~r~u~tiz 2 4 5 8 10 Changes in tumour volume, density, weight and cellularity are depicted in Fig. 1. Following an ini- tial lag phase, the tumour mass and cell number show an exponential growth rate. The density of cells reaches a peak at day 3 following inoculation days after inoculation Fig. I. A); Tumour volume ml x IO) m): i. density cell number x 10x/ml; Cl. dry weight g) and A. cellularity ceil number x 109. B): Cl. Total DNA and protein content of the tumour following inoculation.  171 Table 1. Flow DNA analysis of the cell cycle distribution of Yoshida AH-130 cells Day of growth G,jG, phase S phase zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED 0 n 3 % n 5 % n 7 % n 10 % n 70.1 1.35 84.1 f 1.62 38.3 0.57 594 f 42 63.2 f 1.9 2292 + 266 65.1 f 1.37 3299 f 203 73.4 zt 0.63 4414 f 218 11.8 f 2.75 14.4 f 3.30 49.8 + 0.87 770 f 50 30.4 zt 2.30 1089 f 81 17.6 f 1.99 882 zt 91 5.38 + 0.72 322 f 48 G M phase 18.1 f 1.40 21.7 f 1.68 11.9 0.68 189 f 24 6.38 f 0.65 228 f 23 17.4 f 1.77 884 f 1 I1 20.9 f 1.10 1251 zt 97 Data is expressed as percentage (%) and total number (n) of cells x 106. The results presented for group 0 correspond to the srcinal inoculum (120 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA   IO6 cells in stationary phase of growth). The results are expressed as mean values f S.E.M. DNA Content i- erythrocytes for by a longer doubling time, which in turn might be accounted for by an increase in the length of time the cells are in the GdGi phase. At day 7 the fraction of cells present in the synthetic phase drops sharply, at the same time that the fraction associated with the G2/M phase increases. By day 10 the majority of cells are in the GdGi phase, while those in the S phase constitute only a minori- ty (Table I). The combined representation of all cells either in the S or G2/M phases shows a percentage peak at day 3, when the tumour is growing exponential- ly (Fig. 3). The total number of cells either in the S or G2/M phases reaches a maximum at day 7. The ascites fluid contained, in addition to tumour cells, leucocytes and erythrocytes. In this way the percentage of these cells declined progressively with tumour burden. In the case of erythrocytes, 3 days after inoculation these cells represented 33% of the total but only 15% at day 10. Leucocytes constituted 6% of the total cell count at day 3 and only 3% at day 10. Fig. 2. (A): A histogram from flow cytometric measurements showing the DNA content (arbitrary units) distribution of the Yoshida AH-130 cells. The left peak correspond to chicken erythrocytes (fluorescence standard). (B); A biparametric display of the forward scatter (FSC) parameter (related to cell size) versus red fluorescence due to propidium iodide. Differ- ences in DNA content (arbitrary units) can be related to differ- ences in scattering. The different nuclei populations are separated in rectangles.  0 2 4 6 8 10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCB days after Inoculation 100 75 bp 50 0 E E 25 0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Fig 3 Number and percentage of cells in S plus G,IM Fig. 5. Food intake (A) and energetic effictency (B) ~ body phases. Triangles (a) refer to percentage while black circles (0) weight variation related to the amount of food eaten ~ of rats represent total number of cells ( x 109). bearing the Yoshida AH 130 ascites hepatoma. zyxwvutsrqponmlkjihg Host zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA hanges In spite of the fact that the animals’ total weight increased from day 0 to day 7, the weight exclud- ing the tumour mass dropped sharply after inoc- ulation (Fig. 4). The food intake of the tumour- 0 12 3 4 5 6 7 8 910 days after moculatlon 7-10 days Fig. 4. Body weight with (r) and without ([7) the ascitic tumour of rats bearing the Yoshida AH-130 ascites hepatoma. g 100 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM Lj 0 2 4 6 8 10 days after lnoculatlon bearing animals was progressively lowered, with tumour burden being reduced by 18% at day 4, 65% at day 7 and 99% at day 10 (Fig. 4). The energetic efficiency of the animals ~ calculated as the ratio between the amount of food eaten and the net weight increase of the animals without the tumour ~ decreased progressively with the tumour burden (Fig. 5). Concluding l?emarks This report constitutes the first study characterizing the different phases of the cellular cycle in Yoshida AH- 130 tumoral cells. Flow cyto- metry allows a rapid analysis of cell growth status, which can be easily related to the growth of the tumoral mass. In this study the different stages of cellular growth have also been related to the cachexia induced in the host by the tumour burden. Acknowledgements This work was supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (F.I.S.) (90/663) of the Spanish Health Ministry and from the DIGYCT (PB90- 0497) of the Spanish Ministry of Education and Science. C.G. is the recipient of a predoctoral scholarship from the F.I.S. We are very thankful to the Serveis Cientifico-Tecnics of the University  173 of Barcelona for the flow cytometric analysis facilities. References 1 Tessitore, L., Bonelli, G. and Baccino, F.M. (1987) Regu- lation of protein turnover versus growth state: ascites hepatoma. Arch. Biochem. Biophys., 25, 372-384. 2 Thornthwaite, J.T., Sugarbaker, E.V. and Temple, W.J. 1980) reparation of tissues for DNA flow cytometric an- alysis. Cytometry, 1. 229-237. 3 McDivitt. R.W.. Stone, RR. and Meyer. J.S. (1984) A method for dissociation of human breast cancer cells that produces flow cytometric kinetic information similar to that obtained by thymidine labelling. Cancer Res., 44, 2628-2633. 4 Chin, T.L., Hubert, R.P. and Nava E. Flow cytometric an- alysis of DNA content in human bladder tumors and ir- rigation fluids. Cancer, 56. 1677- 168 I. 5 Bradford, M.N. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utiliz- ing the principle of protein-dye binding. Anal. Biochem., 72, 248-254. 6 Burton, K. (1956) Study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estima- tion of deoxyribonucleic acid. Biochem. J., 62, 315-323.
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