Further Biologic Characteristics of a Human Carcinoembryonic Antigen-Producing Colon Carcinoma Cell Line 1.2 Benjamin Drewinko, 3 Li-Ying Yang, 3 Barthel Barlogie, Mary Anne Malahy,4 and Beppino Giovanella 5.6
Marvin Romsdahl,
3
Marvin Meistrich,
3
Carcinoma of the large bowel is one of the most frequently occurring cancers in this country. The incidence of new cases for 1977 is estimated to be 101.000 patients (1). However, chemotherapy of surgically incurable tumors has remained largely unsatisfactory despite the availability of some potent systemic anticancer agents (2-4). Therefore, new approaches to systemic treatment for unresectable intestinal cancer should be sought. These include the search for new. more effective antitumor agents and the utilization of combinations of drugs and ionizing radiation (4). The magnitude of such a search at the clinical level is hindered by I) the fact that it entails experimentation in human subjects with the consequent difficulties in standardization. reproducibility. and speed. 2) its considerable expense. and 3) the limitations in the number of compounds that can be evaluated. Cell cultures can provide a rapid, efficient. and economic system in initial toxicity screenings and in the elucidation of the mode of action of a drug (5). Although explanation in vitro may disrupt physiologic controls that operate in vivo and the transplanted cells may lose normal identifying characteristics, many cells retain morphologic and functional markers that characterize their cellular origin even after extensive propagation. For GI tract neoplasias. one such marker consists of the capacity of cells to synthesize CEA. Some CEA-producing cell lines have been established in culture (6-11). but most do not appear suitable for studies of drug-induced cell lethality. These cell
MATERIALS AND METHODS
Cell line.-LoVo cells were established in 1972 from a metastatic nodule of a 56-year-old patient with adenocarcinoma of the colon. The cells were normally propagated as monolayer cultures in Ham's F-1O me-
ABBREVIATIONS USED: CEA=carcinoembryonic antigen; CV=coefficient(s) of variation; DT=doubling time; GI=gastrointestinal; GT= generation time; HBSS=Hanks' balanced salt solution; H &: E=he· matoxylin and eosin; LI=labeling indexes; MI=mitotic indexes; PAS= periodic acid-Schiff; PCP=pulse cytophotometry; PE=plating efficiency; PLM=pulse-labeled mitosis; T c=generation time; T G,=time in G,-phase; T G2=time in G 2-phase; T.w=time in M-phase; Ts=time in S-phase; dThd=thymidine.
, Received November 4. 1977; accepted February I. 1978. Supported by Public Health Service grant CAI6763 from the National Large Bowel Cancer Project, National Cancer Institute. 3 The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Ave., Houston, Tex. 77030. 4 National Large Bowel Cancer Project, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute. , SI. Joseph's Hospital. 1919 La Branch, HOllston. Tex. 77002. 6 Stehlin Foundation for Cancer Research, 777 SI. Joseph's Professional Bldg., Houston. Tex. 77002. 2
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lines have been characterized basically in reference to their CEA production. but detailed morphologic and kinetic analyses are rare. Furthermore. a prerequisite for the adequate performance of these investigations is that the cells possess the capacity to form large colonies with a sufficiently high PE (defined as the ratio of the number of colonies counted divided by the number of cells initially plated), because meaningful and relevant information on drug-induced cell killing effects of proliferating populations can be obtained only by the colony-forming techniques (12. 13). We have established and characterized a CEA-producing colon carcinoma cell line (LoVo) which retained many morphologic and functional features expected of malignant cells of GI tract origin (14, 15). Further kinetic properties and colony-formation capacity of the cells have been analyzed, their tumorigenic potential was established by successful implantation into nude mice. and techniques were developed for their synchronization. Thus line LoVo appears to be a unique candidate for an in vitro model of colon carcinoma and is potentially lIseful for a variety of studies concerning cellular pharmacology.
ABSTRACT -Several biologic characteristics of human carclnoembryonic antigen (CEA)-produclng colon adenocarcinoma cells grown In vitro were analyzed. Doubling times of exponentially growing cells ranged from 34 to 38 hours for Initial cell concentrations of 10" through 7X10"/petri dish (4,290-30,030 cells/cm2). The generation time, as analyzed by pulse-labeled mitosis (PLM) techniques and calculated according to the model of Jansson, was 29.3 hours with a coefficient of variation of 22%. Cultures exposed to continuous Incubation with tritiated thymidine had a growth fraction of 90%. Compartment distribution calculated in reference to cell cycle transit times measured by PLM techniques was similar to that defined directly by pulse cytophotometry (PCP). PCP analysis of cells in stationary phase of growth revealed a significant fraction of cells with S-phase DNA content, even though the simultaneously defined labeling index was only 1%. Adequate synchronization In S- and G2-phase was achieved by treatment of the cells with 7 mM thymidine for 24 hours. Centrifugal elutriation and mitotic selection techniques were clearly inferior in terms of reproducibility and cell yield. Colony formation was most efficient for cells plated in fresh medium and incubated for 20 days. Cells inoculated into nude mice produced tumor masses that presented morphologic markers similar to those defined for in vitro cells and were capable of synthesizing CEA.-J Natl Cancer Inst 61: 75-83, 1978.
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76 Drewlnko, Yang, Barlogle, et at
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unlabeled. Data were fitted by means of the multicompartment model of Takahashi as modified by Jansson (16). Continuous labeling of LoVo cells was done with (5H]dThd (2 #LCi/ml; sp act, 6.7 Ci/mmole) dispensed to multiple replicate dishes. Samples were collected every 2 hours for 50 hours and processed for autoradiography. In these experiments, percent labeled mitosis was analyzed by the counting of 50-100 mitotic cells per time point, whereas the LI was defined by the scoring of 500 cells per time point. PCP.-PCP studies were performed on LoVo cells by a modification of the techniques reported for cultured lymphoma cells and freshly obtained human hematopoietic cells (17, 18). We initially experienced some difficulty in obtaining adequate DNA histograms by PCP. These difficulties originated from the approximately 10% of the cell population that was not monodispersed and gave histograms with high CV (>5%). We solved this problem in two ways: a) We briefly exposed cells harvested by method 16 (14) to ultrasound vibrations in a Heat System sonicator (20 sec at 30% full power; Heat Systems-Ultrasonics, Inc., Plainview, N.Y.). This step, however, produced cellular debris identified in the histogram by a small peak to the left of the G, peak. Repeated samples showed that the CV of the various compartments was about 2-3%. b) We used, pepsin for digestion of cell-to-cell junctional complexes, as previously reported by lante et al. (19) for human solid tumors. After decanting the nutrient medium, the dishes were rinsed with HBSS and incubated with 0.5% pepsin for 5 minutes at 22° C. The enzymatic reaction was terminated by the addition of growth medium. Cells were then vigorously pipetted through a Pasteur pipette and subsequently washed twice with 0.9% NaCl. Ice-cold absolute ethanol was then added dropwise to a final concentration of 70%. Fixed cells were stained with mithramycin and ethidium bromide in a concentration of 25 and 12.5 ",g/ml, respectively. The staining solution also contained 3.75 mM MgCh, which is equimolar to the concentration of mithramycin. Routinely, 50,000-100,000 cells were measured in a Phywe ICP II pulse cytophotometer (Phywe AG, Gottingen, Federal Republic of Germany) at a flow rate of 500 cells/second. A modification of Fried's model was employed for histogram evalllation. 7 Synchronization.-Exponentially growing cells were harvested as described before and counted in an electronic particle counter (Coulter Electronics Inc., Model lBI), and aliquots of 5X105 cells were dispensed to petri dishes containing 3 ml medium. After 48 hours in culture, cells were treated with increasing concentrations of dThd (I, 3, 5, 6, 7, 8, and 11 mM) for either 15, 24, or 36 hours. The supernatant was decanted, and the cells were washed twice with fresh medium and reincubated with 3 ml fresh medium. Every 2 hours, cells were pulse labeled for 30 minutes with I #LCi of [3H]Cyd/ml 7 Johnston D, White RA, Barlogie B: Automatic processing and interpretation of DNA distribution: Comparison of several techniques. Presented at Engineering Foundation's 5th Conference on Automated Cytology, Pensacola Beach, Fla., December 12-17, 1976
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dium supplemented by 20% fetal calf serum, vitamms, glutamine, and antibiotics. Under these conditions, the cells were capable of forming acinar structures and signet ring cells (14). The kinetics of CEA production by these cells were previously reported; supernatants of exponentially growing cultures contained about 80 ng of CEAIl06 cells (15). LoVo cells were subcultured serially once a week, and stock cultures were harvested by our previously described method 16 (14). The supernatant was discarded and the cells were rinsed with prewarmed saline solution. The cells were incubated with hyaluronidase (102 IU/ml) for 5 minutes at 37° C and then incubated with trypsin (2.5% in HBSS) for 5 minutes at 37° C. Cells were washed once in medium, resuspended in medium, and split two-fold into new culture vessels. Kinetic studies.-To determine the growth curve of exponentially growing LoVo cells, l-week-old stock cultures were harvested as described above. Aliquots of 105, 5X105 and 7X105 cells were seeded into 6O-mm petri dishes containing 5 ml of nutrient medium (4,290; 21,450; and 30,030 cells/cm 2 , respectively). After cells reached exponential growth, two cell counts in each of two replicates were made every 24 hours for about 160 hours with an electronic particle counter (Coulter Electronics Inc., Hialeah, Fla.; model lBI). Data were analyzed by linear regression techniques and DT was calculated from In2/s10pe. In other experiments, the MI, LI, and cell viability were defined for monolayer cells at various phases of in vitro growth monitored by daily cell counts. Aliquots of 5XI05 cells were seeded into 60-mm petri dishes containing 5 ml of medium. Cells were allowed to progress through all phases (lag, exponential, and stationary) without refeeding. Stationary phase was defined by no net increase in cell number. Cell viability was measured by trypan blue exclusion on replicate aliquots. The LI was determined in duplicate dishes by pulse labeling with 1 ",Ci[3H]dThd/ml (sp act, 3.0 Ci/mmole) for 30' minutes. The cells were harvested, and cytocentrifuge preparations stained with 2% aceto-orcein were processed fQr autoradiography with the use of a 50% solution of Ilford K5 emulsion in distilled water. The LI was scored on 500 cells/slide, and the MI was scored on 1,000 cells/slide. For cell cycle analysis, aliquots of 5X105 cells were seeded in 60-mm petri dishes and allowed to reach exponential growth. All cells were pulse labeled with [3H]dThd (I ",Ci/ml; sp act, 3.0 Ci/mmole) for 30 minutes at 37° C. Cells were washed twice with medium containing 0•. 1 mg/ml of dThd and once with fresh medium and reincubated with fresh medium. Duplicate dishes were harvested every 2 hours and processed for autoradiography. Slides were exposed for 2 weeks and developed in Kodak DI9 (Eastman Kodak, Rochester, N.Y.). Background was estimated from cell-free areas of the slide and from cell-free slides processed simultaneously. A cell was considered labeled if it displayed five or more grains. However, virtllally every labeled mitosis showed greater than IS grains/cell. From 50 to 200 mitotic cells/time point were scored as labeled or
BiologIc PropertIes of Cultured Colon Cells 77
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minidase (550 V/ml) at 37° C for 5 minutes followed by trypsin (0.25%) for 10 minutes at 37° C. Cells were then washed and resuspended in medium. In method 17 the cells were rinsed with 0.9% NaCI and then incubated with 2.5% Viokase (a bovine pancreatic extract; GIBCO, Grand Island, N.Y.) for 5 minutes at 37° C and trypsin (2.5%) at 37° C for 5 minutes. Cells were washed and resuspended in medium. Method 19 consisted of discarding the supernatant and adding 2.5% Viokase for 5 minutes at 37° C, decanting, and adding prewarmed trypsin 0.25% in HBSS for 10 minutes at 37° C. In all instances, cells were counted in an electronic particle counter, and the size (e.g., singlets, doublets) of the potential colony-forming units (multiplicity) was assessed by direct examination under light microscopy before being seeded for colony formation. Different cell concentrations (50, 100, 200, 500, and 1,000 cells/dish) were dispensed to 60-mm petri dishes and various subsequent culturing conditions were investigated. The petri dishes contained: .a) 5 ml of fresh medium (unchanged throughout the entire incubation period); b) 8 ml of fresh medium (unchanged throughout the entire incubation period); c) 5 ml of fresh medium' (changed every 4 days); d) 5 ml of fresh medium to which I drop of L-glutamine (200 mM) was added every 4 days; e) a mixture of I: I or 1:2 conditioned and fresh medium (conditioned medium was obtained from 2-week-old stock cultllfes, centrifuged, and filtered); or f) a feeder layer of LoVo cells 2X10 4 cells/dish) previously irradiated with 10 4 rads (cells were irradiated with a Phillips X-ray machine operating at 250 k VP at a dose rate of 90.4 rads/min at the level of the cells). After various incubation intervals (l8, 21, 22, 23, and 27 days), the colonies were stained with 2% crystal violet in 95% ethanol and examined under a stereomicroscope. Transplantation into athymic (nude) mice.-Stock monolayer cultures were harvested, washed, and counted. Aliqllots (I ml containing 10 7 cells) were injected sc into the backs of nude mice bred in the laboratory of Dr. B. Giovanella, at the Stehlin Foundation for Cancer Research (23). The mice were placed in separate cages and examined weekly for visible tumor growth. After 3 weeks, all of the mice developed tumor nodules that were allowed to progress until reaching a diameter of 9-10 cm. Blood was collected by intracardiac puncture, and the serum was assayed for CEA by the method of Chu and Reynoso (24). The animals were killed and tumor nodules were excised and processed by routine histologic techniques. Sections were stained with H & E, PAS, diastase digestion of PAS, mucicarmine, and Alcian blue. RESULTS
Growth KinetiCS DT calculated by linear regression analysis of seq uential cell count data from dishes containing three different cell titers ranged from 34 to 38 hours, with a mean of 36.3 hours. This result was very close to the 37
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(sp act, 15.1 Cilmmole). Cells were harvested and aliquots were processed for autoradiography to determine LI (per 500 cells) and MI (per 1,000 cells) and for PCP. On the basis of these experiments (see "Results"), improved synchronization with a double block with dThd was attempted (20). Exponentially growing cells were treated with 7 mM dThd for 24 hours. The supernatant was decanted, and the cells were washed twice and reincubated with fresh medium at 37° C for II hours, at which time they were reexposed to another treatment of 7 mM dThd for an additional 24 hours. The second dThd block was terminated when the supernatant was decanted, washed with medium, and reincubated with fresh medium. The degree of synchrony was subsequently determined in the same manner as for cells treated with a single dThd block. At the time of termination of all synchronization treatments, aliquots were also processed for colony formation determinations to define the effects of prolonged dThd treatment on cell survival as reflected by changes in PE. To synchronize LoVo cells by the mitotic selection technique (21), aliquots of 107 cells were seeded in 16ounce Owens' bottles and allowed to reach exponential growth. Each bottle was agitated horizontally for about 15 seconds, and the supernatant was collected. Fresh medium was added, and the cells were reincubated at 37° C. This operation was repeated every 30 minutes for the ensuing 4 hours. The collected cell suspensions were counted, stained for scoring the MI, and processed for colony formation. In other studies, we utilized the centrifugal elutriation method described by Meistrich et al. (22). A suspension of 5X10 7 monodispersed LoVo cells were separated according to volume with a JE-6 elutriator rotor in a J-2IB centrifuge (Beckman Instruments, Inc., Palo Alto, Calif.) at 4° C with a rotor speed of 2,240 rpm. The instrument had been sterilized overnight with 70% ethanol. Cells were suspended in Ham's F-1O medium containing 5% fetal calf serum and 5 mM naphthol disulfonic acid. A series of 13 fractions (50 ml each) were collected sequentially at a flow rate of 3.4 mllminute. Cells from each fraction were counted in an electronic particle counter (Coulter Counter Model ZEI; Coulter Electronics Inc.), their volume distribution was analyzed with a Coulter Counter attached to a multichannel particle size discriminator (Channelyzer; Coulter Electronics Inc.), their position in the cell cycle was defined by PCP, and an aliquot was processed for colony formation. Colony formation.- To conduct valid studies on cell killing effects of antitumor drugs, the colony-formation technique is required (12). For this purpose, it is necessary to use harvesting methods that will provide as close to a single cell suspension as possible with the least impairment to cellular reproductive capacity. For line Lo Vo, this was a difficult enterprise because cells adhere to each other by tight desmosomal junctions (14). We had previously investigated 18 harvesting techniques (14) and selected methods 2, 16, 17, and 19 to study colony-formation capacity. Method 16 was described above. Method 2 consisted of rinsing of stock cultures with 0.9% NaCl and incubation with neura-
78 Drewlnko, Yang, Barlogie, et al. 1. -Kinetic parameters of Lo Vo cells in various phases of growth"
TABLE
Phase Lag Exponential Stationary
Duration
Cell viability by trypan blue exclusion, %b
MI, %b
LI, %b
=36 hr =5 days >6 days
80 92 80
0.0 0.8 0.1
43 31 1
" Multiple replicate cultures containing 5x101 cells/dish were allowed to grow for 12 days without refeeding. Cell number was ~onitored w.ith an elec~ronic particle counter. Lag phase was defmed from time of seedmg (day 0) to initiation of exponential increments in cell number. Stationary phase began when cell increm~nts were no longer apparent (plateau). Determined on day 1 for lag phase; on day 5 for exponential phase; and on day 8 for stationary phase.
2.-Computer-estimated cell cycle parameters according to the Jansson model
Cell cycle stage
Transit time, hr
SD, hr
G, S G2+M GT
14.7 10.7 4.8 29.3
4.4 3.8 2.3 6.3
mean value of 90%. The length of G2-phase calculated at the 50% level of labeled mitosis recorded for cells incubated continllollsly with [3H]dThd was about 4.5 hours. Pulse Cytophotometry
DNA histograms obtained on cells treated with pepsin were selected as our choice' method. Repeated samples showed that the CV of the various compartments w~s less than 2~. Text-figure 2 shows a typical DNA hIstogram obtamed for cells in exponential growth (4 days after subculture). The calculated values for each compartment were: G I/O = 60%, S = 31 %, and G2+M = 9%. By use of cell cycle values defined by PLM techniques, the predicted proportions of cells in each com~artI?ent can be calculated by (length of phase transIt tlme+length of cell cycle time)XO.9, where 0.9 represents the proportion of proliferating cells obtained from continuous labeling experiments. The calculated values are: G I = 45.2%, S = 32.7%, and G 2+M = 12.1%. Inasmuch as nonproliferating cells (10%, from continuous labeling experiments) are measured by PCP within the G I compartment, the total population for that compartment is 55.2%. Thus for LoVo cells, values ob~ained from PCP corresponded closely to those predicted In reference to cell cycle times defined by PLM. Compartment distribution for cells in stationary phase of growth (text-fig. 3) revealed a small increase
Q; ~ ~
0
..c;
PLM% 100
Exponential Phase (4 Days)
(,)
'" '" E ~
•
80
Q.
.0
GI/O =60
z
G2 = 9
~
S
~
=31
'"
60
(,)
40 20
o 3 6 9 12 15 '18 21 24 27 30333639424548 Hours I.-Computer-fitted curve for the calculation of cell cycle stage Urnes analyzed by PLM. Points represent mean values of cells from duplicate dishes/time point.
TEXT-FIGURE
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10
20
30 40 50 60 Channel Number
70
80
TEXT-FIGURE 2:-DNA histogram obtained bv PCP analysis on cells 10 IOxponenual phase of growth (4 days after inoculation). GIlD
=
restmg phase + pre-DNA synthesis phase compartment; S = DNA syntheSIS phase compartment; G, = post-DNA synthesis compartment.
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hours previously reported for this cell line (14). The MI, LI, and cell viability (trypan blue exclusion) defined on monolayer cultures at each growth phase (table I) revealed that cells in lag phase (24 hr) had no mitotic cells; LI was 43% (range, 40-50), and viability was approximately 80%. An MI of 0.8% (range, 0.3-1.1), an LI of 31% (range, 28-40), and 92% viable cells were recorded for exponentially growing cultures (3 days). Cells in stationary phase (8 days) showed an MI of 0.1 % (range, 0.0-0.23), an LI of 1%, and 80% viable cells. ~he GT and length of each cell cycle stage were ?ehned by the PLM technique on exponentially growmg cells. PLM values were calculated by pooling of raw data fr?m 4-6 slid~s/time point (text-fig. I). Data points were fHted accordmg to the model of Jansson, which calculated cell cycle stage times shown on table 2. The CV of the GT was about 22%, whereas the CV corresponding to each stage transit time varied from 30% in GI-phase to about 50% in Grphase. From the data in table 2, the expected LI calculated as the ratio TslTc was 36.5% and T M calculated from (MIXTcl/ln2 was 0.33 hour. . The LI of cells exposed continuously to [3H]dThd mcreased at the rate of 3.2%/hour. After about 20 hours, a plateau of labeled nuclei was reached. In different experiments, the plateau varied from 88 to 92%, with a
TABLE
Biologic Properties of Cultured Colon Cells 79
Q)
c:: c::
Stationary Phase (" Days)
0
.c
u
~
Q)
Q.
GI/O= 69 =15 G2 =16
Q;
S
.0
E
:::>
z
Q)
u
10
20 30 40 50 Channel Number
60
70
TEXT·FIG!'RE 3.-DNA histogram for cells in stationary phase of growth (II days after inoculation).
in G 1 (69%), a significant increase in G 2 + M (16%), and 15% of the cell population in the S-phase compartment. However, the simultaneously defined LI was 1%. Thus our data reveal that cells in stationary phase of growth do not necessarily accumulate in G1-phase. Apparently, many cells in stationary phase can be delayed in G 2phase (no mitoses were detectable in stationary cultures) whereas other are stopped in the midst of synthesizing DNA [U-cells (25)] or synthesize DNA very slowly. Synch ronization
Synchronization of LoVo cells was attempted by the blocking of cell cycle traverse with excess dThd (1-11 mM) for 15, 24, and 36 hours. The degree of synchrony obtained by this technique was dose- and time-dependent. Incubation for 15 hours achieved only 60-70% cells in S-phase at the end of the synchrony procedure. Incubation for 24 hours resulted in 80-85% cells in DNA synthesis at the end of the block for concentrations of 6, 7, and 8 mM (text-fig. 4). Treatment for 36 hours resulted in 70-80% cells in S-phase. The kinetics of cell cycle traverse following removal of the blocking agent after 24 hours of incubation were followed for the ensuing 28 homs by autoradiography (LI and MI) and PCP. Autoradiography revealed a large percentage of Sphase cells (LI) during the first 6 hours; the LI decreased abruptly after 8 hours to values lower than control asynchronous populations (12%). This plateau of low LI was maintained for the next 10 hours, at which time it increased to peak values of about 40%, 24 hours after the release of the block. No mitotic figures were noted during the first 4 hours after removal of excess dThd. After 6 hours, the MI steadily increased, peaked at 12 hours (5%), and steadily decreased to 0 or 0.5% after 18 hours. PCP studies on cells blocked for 15 hours with dThd revealed poor synchronization with LoVo cells spread throughout S-phase. Sharpening of VOL 61, NO. I. JULY 1978
100
'"
Q)
90%). Yet the cell yield was not greater than 9X105 cells/shake, i.e., less than 10% of the expected yield based on the MI of asynchronous populations. Colony formation by these cells was similar to that of control cells. Centrifugal elutriation failed to yield optimal synchronization results. Cell recovery was good with 90% of the cells loaded into the chamber retrieved in fractions 1-13. Damaged (trypan blue-positive) cells were separated in fractions 1-4. Colony formation of cells retrieved in fractions 5-13 was equal to that of unseparated cells. Highly enriched populations of G1-phase cells (86-95%) were obtained in fraction 5 (text-fig. 5), but the maximum content of "enriched" S-phase cells (fraction 7) was only 37%. G 2+M cells were purified to 56% in fraction 9 (text-fig. 6). However, by microscopic
80 Drewlnko, Yang, Barlogle, et al. TABLE 3.-Colcmy formation of Lo Vo cells as a functicm of different harvesting methods"
PE
Colony-forming units
Harvesting method No. b
Singlet
Doublet
Triplet
2 19 17 16
70 37.5 37.5 83
18 22.5 26.5 12
8 14.3 14.7 5
>4 4 25.7 21.3 0
50 0 10-38 2-12 43-62
100 0 60-71 50-93 56-68
range, %C
200 0 73-85 70-80 47-73
500
1,000
1-2 42-50 40-82 53-69
3-4 31-36 22-64 49-70
" Cells were incubated for 20 days at 37° C with 5 ml of medium without refeeding. b For details of technique, see text. C A function of initial seed titer. 1.0
0.8
0.6 61=86% S =14%
u
u:
0.4
0.2
0.0 +----r'----,----"=r='-~----,
o
20
40 60 Channel Number
80
5.-DNA histogram of cells separated by centrifugal elutriation (fraction 5). Eighty-six percent of cells exhibit G 1 compartment-DNA content.
TEXT·FlGURE
examination, about 20% of these cells were doublets, which suggested that a significant fraction of the population consisted of paired Gt-cells. Furthermore, the volume distributions of cells in the individual fractions were not as sharp as those obtained previously with L-P59 cells (22). The width of the volume distribution profiles (full width at half maximum + modal channel number) ranged between 0.65 and LOO. In addition, cell aggregation was substantiaL A clump of cells remained at the bottom of the chamber and was washed out at the end of the run. This clump contained about 10-20% of the population, most of which were large cells or cell aggregates. To investigate the absence of efficient separation by the elutriation technique, cells were synchronized by the excess dThd method and allowed to progress through the cycle; the volume distribution of the synchronized cells was defined at various points of the cycle. Again, volume distributions were wide with no sharp peaks indicative of correspondence between cell volume and cell cycle stage.
1.0
0.8
c
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0.6
o
u
u:
0.4
0.2
00 - t - - - - , - ' - - - - , - - - , - - - - - - " - , 80 40 60 20 o Channel Number
Colony Formation
For all harvesting methods employed (with the exception of harvesting method 2, which failed under
61=23% S =2/ % 62 +M=56%
6.-DNA histogram of cells separated by centrifugal elutriation (fraction 9).
TEXT·FlGURE
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c:
o
any subsequent culturing conditions), the best results in terms of PE were noted when the plated cells were cultured continuously in fresh medium. No PE differences were noted for cells incubated with either 5 or 8 ml of medium, though incubation with 8 ml of medium yielded larger colonies. Incubation with both conditioned medium and feeder layers resulted in a low PE, with small colonies frequently composed of less than 32 cells. Changing medium or the addition of glutamine every 4 days did not improve the PE and produced significant contamination. Table 3 demonstrates PE of LoVo cells incubated for 20 days in 5 ml of fresh medium as a function of harvesting method. Method 2 produced only a few small colonies at high cell inocula. Both methods 17 and 19 yielded a large PE, but the multiplicity (27) of the originating colony-forming units was tOO great to allow clear-cut conclusions on single-cell killing effects. The multiplicity of cells harvested by method 16 was a negligible 1.22; colonies were compact and composed of greater than 500 cells (fig. 1). Only method 16 gave a linear correlation between PE and seed titer for cell concentrations ranging from 50 to 1,000/dish. No difference in PE occurred when colonies were stained after 18 through 23 days of incubation.
Biologic Properties of Cultured Colon Cells 81 Xenogeneic Transplantation The tumorigenic potential of LoVo cells was established by their transplantation into nude mice. Inoculated cells grew relatively slowly, and visible tumor nodules appeared after 3 weeks. Transplanted cells retained the morphologic features of the original tumor. Histologic examination demonstrated large cuboidal-tocolumnar cells with vesicular nuclei, formation of glandular structures, and the rare presence of signet ring cells (fig. 2). Yet no mucin production was demonstrated by histochemical techniques. Sera from 3 LoVo cell-bearing mice assayed for CEA when the animals were killed revealed 31.3, 40.0, and 87.5 ng CEA/mg, respectively, whereas no CEA was detected in control mICe.
The recent PLM data were fitted with the computerized model of Jansson. Values defined for Tc and T G2 were similar to those previously reported for the early passages of line LoVo (14). However, values for Ts and T GI were different in that T s was now shortened by about 7 hours, the time gained by the newly determined T GI. In part, this difference can be attributed to the manual calculation utilized in our previous report, whereas present values were defined more precisely by a computer program utilizing minimization techniques. Examination of chart 3 in our previous publication (14) reveals a "hump" in the descending limb of the first wave of labeled mitosis, just about the 50% level, which increased the value of Ts. The validity of the shorter Ts value computed in the present study is supported by the 8- to lO-hour S-phase transit time observed for synchronized Lo Vo cells. The CV of the G T and the phases of the cycle are among the largest reported for mammalian cells (27) and explains the rapid synchrony decay after release of the dThd block once Lo Vo cells traverse G2phase (28). In contrast to the results obtained for TI cells, a human lymphoma line (29), LoVo cells did not present differences in T G2 measured by the PLM or continuous labeling techniques. Apparently, LoVo cells are not susceptible to the changes in pH and temperature involved in the washing procedures employed in the PLM method, and their G 2 transit is unaffected. It has long been accepted that mammalian cells in stationary phase of growth accumulate in GI-phase and that this phenomen can be used to effectively synchronize the cultures (30). This contention originated from the fact that cells in stationary phase failed to incorporate labeled DNA precursors and, after subculturing, entered DNA phase in a synchronized wave. Our studies on LoVo cells in stationary phase demonstrated that failure to incorporate [3H]dThd cannot be considered evidence of non-S-stage position, because many cells were shown by PCP to have S-phase DNA content. Either such cells are blocked during their S-phase transit or their nucleic acid metabolism is modified in Stich a manner that they cannot utilize exogenous nucleosides. VOL. 61. NO. I. JULY 1978
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DISCUSSION
In contrast to nonhuman cell lines, which possess short GT with low CV and less fastidious culturing requirements (31), the excess dThd technique was, thus far, the only successful synchronization method for LoVo cells in terms of consistency, reliability, and synchronized cell yield. Although the method reduced the PE of LoVo cells and chemical synchronization may be undesirable for drug studies involving certain antimetabolites, the excess dThd technique presently remains the sole procedure that can be used for these sorts of investigations. The reason why centrifugal elutriation was unsuccessful in providing purified populations in all stages of the cycle resides in the nature of the LoVo cells. Different from rodent cell lines, which possess relatively uniform volumes, LoVo cells have a significant fraction of multinucleated cells and present considerable variations in volume and density from cell to cell. These variations are independent of position in the cycle as shown by volume determinations utilizing a multichannel particle size discriminator. Cell cycle traverse-dependent increments in cell volume are an absolute requirement for successful separation by centrifugal elutriation. Inasmuch as LoVo cells do not display this property, the failure of the elutriation method in providing purified populations in all stages is thus explained. Although we have not yet abandoned this technique, because highly purified GI-cells can be reseeded and allowed to progress synchronously through the cycle, one of our aims, that of rapidly obtaining large fractions of cells purified in specific stages of the cycle, has not been met; therefore, we must resort to alternative techniques. The fact that a purified cell yield in mitosis could be obtained by the mitotic selection technique suggests that it might be feasible to synchronize large numbers of LoVo cells by the use of automatic batch cell processor devices similar to that described by Klevecz (32). Colony formation by LoVo cells was most efficient when cells were incubated in fresh medium. In a fashion similar to that reported for human lymphoma cells (33), supplementation with conditioned medium or feeder layers decreased the PE and the size of the colonies. Thus the growth of LoVo cells may possibly be regulated by a molecular mediator present in supernatants of aged cultures or synthesized by the radiated cells in the feeder layer. The tumorigenic potential of LoVo cells was substantiated by successful transplantation to nude mice, a test currently accepted as the most reliable and meaningful indicator of malignant potential (34, 35). Interestingly, LoVo cells maintained the capacity to synthesize and release CEA and to originate glandular structures in vivo and in vitro (14) in spite of more than 100 passages in vitro. These results are similar to those reported for other colon carcinoma cell lines (36, 37). The fact that purely epithelial cell lines can develop organized structures in vivo and in vitro indicates that this capacity does not require the activity of inducer tissues. Although the xenografts of Lo Vo cells revealed glandular structures and signet ring cells, no mucin production was detected by standard histochemical techniques. The
82 Drewinko, Yang, Barlogie, et al.
absence of mucin had also been observed previously for the monolayer cultures of LoVo cells (14). This phenomenon suggests that signet ring cells do not necessarily result from the accumulation of mucinous material. We have characterized all of the necessary biologic parameters of an established human CEA-producing cell line in preparation for detailed studies of the effects of antitumor agents at the cellular level. We believe that LoVo cells will provide a screening system for antitumor drugs useful in the treatment of colon carcinoma superior to systems presently available.
(17) BARLOGIE B. DREWINKO B. BUCHNER T, et al: Pulse cytophoto-
REFERENCES
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(18) (19)
(20)
(21)
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(23)
(24)
(25)
(26) (27)
(28) (29)
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(31)
(32)
10:157-172, 1975
(33) DREWINKO B: Colony-forming ability of human immunoglobulin producing cells. Lab Invest 25:533-535. 1971 (34) GIOVANELLA BC. STEHLIN JS. WILLIAMS LJ: Heterotransplantation of human malignant tumors in "nude" thymus less mice. II. Malignant tumors induced by injections of cell cultures derived from human solid tumors. j Nat! Cancer Inst 52:921-930. 1974 (35) STILES CF. DESMOND W. CHUMAN LM. et al: Relationship of cell growth behavior in vitro to tumorigenicity in athymic nude mice. Cancer Res 36:3300-3305, 1976 (36) CARREL S. SORDAT B. MERENDA C: Establishment of a cell line (Co-1l5) from a human colon carcinoma transplanted into nude mice. Cancer Res 36:3978-3984, 1976 (37) ROSENTHAL KL. TOMPKINS WA. FRANK GL. et al: Variants of a human colon adenocarcinoma cell line which differs in morphology and carcinoembryonic antigen production. Cancer Res 37:4024-4030. 1977
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(2) MOERTEL CG: Large bowel. In Cancer Medicine (Holland JF, Frei E, eds). Philadelphia: Lea Be Febiger, 1973, pp 1597-1627 (3) HORTON J, HACKER B, CUNNINGHAM TJ, et al: The chemotherapy of large bowel cancer. Present status and future prospects. Digest Dis 19:1040-1046, 1974 (4) VAITKEVICIUS VK, BAKER LH, BUROKER TR, et al: Chemotherapy of gastrointestinal adenocarcinoma. In Cancer Chemotherapy. Fundamental Concepts and Recent Advances. Chicago: Year Book Medical Publishers. 1975. pp 263-278 (5) DAWSON M: Cellular Pharmacology. The Effects of Drugs on Living Vertebrate Cells In Vitro. Springfield, Ill.: Thomas. 1972 (6) LAING CA. HEPPNER GH. Kopp LE. et al: Detection of carcinoembryonic antigen in the media of cultures of carcinomatous cells of digestive system origin. J Nat! Cancer Inst 48:19091911, 1972 (7) EGAN ML, TODD CW: Carcinoembryonic antigen: Synthesis by a continuous line of adenocarcinoma cells. J Nat! Cancer Inst 49:887-889. 1972 (8) TOMPKINS WA. WATRACH AM, SCHMALE JD, et al: Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J Nat! Cancer Inst 52:1101-11IO, 1974 (9) MCCoMBS WB, LEIBOVITZ A, McCoy CE, et al: Morphologic and immunologic studies of a human colon tumor cell line (SW-48). Cancer 38:2316-2327, 1976 (10) TOM BH, RUTZKY LP, JAKSTYS MM, et al: Human colonic adenocarcinoma cells. I. Establishment and description of a new line. In Vitro 12:180-191. 1976 (11) LEIBOVITZ A, STINSON jC, MCCoMBS WB, et al: Classification of human colorectal adenocarcinoma cell lines. Cancer Res 36:4562-4569, 1976 (12) ROPER P, DREWINKO B: Comparison of in vitro methods to determine drug-induced cell lethality. Cancer Res 36:2182-2188, 1976 (13) BHUYAN BK, LOUGHMAN BE, FRASER TJ. et al: Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp Cell Res 97:275-280. 1976 (14) DREWINKO B, ROMSDAHL MM. YANG LY. et al: Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line. Cancer Res 36:467-475, 1976 (15) DREWINKO B. YANG LY: Restriction of CEA synthesis to the stationary phase of growth of cultured human colon carcinoma cells. Exp Cell Res 101:414-416. 1976 (16) JANSSON B: Mathematical models in cell cycle kinetics. In Mathematical Models in Biology and Medicine (Bailey NT. Sendov B. Tsanev R. eds), Amst.erdam: North Holland. 1974, pp 21-39
metric analysis of synchronized cells in vitro. Cancer Res 36:1181, 1976 BARLOGIE B, SPITZER G, HART JS. et al: DNA histogram analysis of human hemopoietic cells. Blood 48:245-258. 1976 ZANTE J. SCHUMANN J, BARLOGIE B, et al: New preparation and staining procedures for specific and rapid analysis of DNA distributions. In Second International Symposium on Pulse Cytophotometry (Gohde W, Schumann J and Buchner T, eds). Ghent. Belgium: Europ Press Medikon. 1976, pp 97-106 BooTSMA D. BUDKE L. VOSS 0: Studies on synchronous division of tissue culture cells initiated by excess thymidine. Exp Cell Res 33:301-309. 1964 TERASIMA T, TOLMACH LJ: Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp Cell Res 30:344-362. 1963 MEISTRICH ML. GRDINA DJ, MEYN RE: Application of cell separation methods to the study of cell kinetics and proliferation. In Growth Kinetics and Biochemical Regulation of Normal and Malignant Cells (Drewinko B. Humphrey RM. eds). Baltimore: Williams Be Wilkins. 1977. pp 131-142 GIOVANELLA B, STEHLIN JS: Heterotransplantation of human malignant tumors in "nude" thymusless mice. I. Breeding and maintenance of "nude" mice. J Natl Cancer Inst 51:615-619. 1973 CHU TM. REYNOSO G: Evaluation of a new radioimmunoassay method for carcinoembryonic antigen in plasma with use of zirconyl phosphate gel. Clin Chern 18:918-922. 1972 ERNST p, FAILLE A. KILLMAN SA: Perturbation of cell cycle of human leukemic myeloblasts in vivo by cytosine arabinoside. Scand J Hematol 10:209-218. 1973 ELKIND MM, WHITMORE GF: The Radiobiology of Cultured Mammalian Cells. New York: Gordon and Breach. 1967 DAWSON KB, MADOC-jONES H, FIELD EO: Variations in the generation times of a strain of rat sarcoma cells in culture. Exp Cell Res 38:75-84. 1965 ENGELBEKG J: The decay of synchronization of cell division. Exp Cell Res 36:647-662. 1964 DREWINKO B, BOBO B. ROPER p. et al: Analysis of growth kinetics of a human lymphoma cell line. Cell Tissue Kinet 1l:177191. 1978 STUBBLEFIELD E: Synchronization methods for mammalian cell cultures. Methods Cell Physiol 3:25-43. 1968 MEISTRICH ML, MEYN RE, BARLOGIE B: Synchronization of mouse LP-59 cells by centrifugal elutriation separation. Exp Cell Res 105:169-177. 1977 KLEVECZ RR: Automated cell cycle analysis. Methods Cell Bioi
Biologic Properties of Cultured Colon Cells 83 FIGURE I.-Colony (ormalion by LoVo cells harvesled by mel hod 16 and incubaled in fresh medium (or 21 days. Cryslal viole!. X 12
FIGURE 2.-Morphologic aspecls of a lumor nodule oblained from Ihe inoculalion of cullured LoVo cells imo nude mice. Note giandlilar slrUClUres. H & E. X 200
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1
Marvin Romsdahl,
3
Marvin Meistrich,
3
Carcinoma of the large bowel is one of the most frequently occurring cancers in this country. The incidence of new cases for 1977 is estimated to be 101.000 patients (1). However, chemotherapy of surgically incurable tumors has remained largely unsatisfactory despite the availability of some potent systemic anticancer agents (2-4). Therefore, new approaches to systemic treatment for unresectable intestinal cancer should be sought. These include the search for new. more effective antitumor agents and the utilization of combinations of drugs and ionizing radiation (4). The magnitude of such a search at the clinical level is hindered by I) the fact that it entails experimentation in human subjects with the consequent difficulties in standardization. reproducibility. and speed. 2) its considerable expense. and 3) the limitations in the number of compounds that can be evaluated. Cell cultures can provide a rapid, efficient. and economic system in initial toxicity screenings and in the elucidation of the mode of action of a drug (5). Although explanation in vitro may disrupt physiologic controls that operate in vivo and the transplanted cells may lose normal identifying characteristics, many cells retain morphologic and functional markers that characterize their cellular origin even after extensive propagation. For GI tract neoplasias. one such marker consists of the capacity of cells to synthesize CEA. Some CEA-producing cell lines have been established in culture (6-11). but most do not appear suitable for studies of drug-induced cell lethality. These cell
MATERIALS AND METHODS
Cell line.-LoVo cells were established in 1972 from a metastatic nodule of a 56-year-old patient with adenocarcinoma of the colon. The cells were normally propagated as monolayer cultures in Ham's F-1O me-
ABBREVIATIONS USED: CEA=carcinoembryonic antigen; CV=coefficient(s) of variation; DT=doubling time; GI=gastrointestinal; GT= generation time; HBSS=Hanks' balanced salt solution; H &: E=he· matoxylin and eosin; LI=labeling indexes; MI=mitotic indexes; PAS= periodic acid-Schiff; PCP=pulse cytophotometry; PE=plating efficiency; PLM=pulse-labeled mitosis; T c=generation time; T G,=time in G,-phase; T G2=time in G 2-phase; T.w=time in M-phase; Ts=time in S-phase; dThd=thymidine.
, Received November 4. 1977; accepted February I. 1978. Supported by Public Health Service grant CAI6763 from the National Large Bowel Cancer Project, National Cancer Institute. 3 The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Ave., Houston, Tex. 77030. 4 National Large Bowel Cancer Project, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute. , SI. Joseph's Hospital. 1919 La Branch, HOllston. Tex. 77002. 6 Stehlin Foundation for Cancer Research, 777 SI. Joseph's Professional Bldg., Houston. Tex. 77002. 2
75
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lines have been characterized basically in reference to their CEA production. but detailed morphologic and kinetic analyses are rare. Furthermore. a prerequisite for the adequate performance of these investigations is that the cells possess the capacity to form large colonies with a sufficiently high PE (defined as the ratio of the number of colonies counted divided by the number of cells initially plated), because meaningful and relevant information on drug-induced cell killing effects of proliferating populations can be obtained only by the colony-forming techniques (12. 13). We have established and characterized a CEA-producing colon carcinoma cell line (LoVo) which retained many morphologic and functional features expected of malignant cells of GI tract origin (14, 15). Further kinetic properties and colony-formation capacity of the cells have been analyzed, their tumorigenic potential was established by successful implantation into nude mice. and techniques were developed for their synchronization. Thus line LoVo appears to be a unique candidate for an in vitro model of colon carcinoma and is potentially lIseful for a variety of studies concerning cellular pharmacology.
ABSTRACT -Several biologic characteristics of human carclnoembryonic antigen (CEA)-produclng colon adenocarcinoma cells grown In vitro were analyzed. Doubling times of exponentially growing cells ranged from 34 to 38 hours for Initial cell concentrations of 10" through 7X10"/petri dish (4,290-30,030 cells/cm2). The generation time, as analyzed by pulse-labeled mitosis (PLM) techniques and calculated according to the model of Jansson, was 29.3 hours with a coefficient of variation of 22%. Cultures exposed to continuous Incubation with tritiated thymidine had a growth fraction of 90%. Compartment distribution calculated in reference to cell cycle transit times measured by PLM techniques was similar to that defined directly by pulse cytophotometry (PCP). PCP analysis of cells in stationary phase of growth revealed a significant fraction of cells with S-phase DNA content, even though the simultaneously defined labeling index was only 1%. Adequate synchronization In S- and G2-phase was achieved by treatment of the cells with 7 mM thymidine for 24 hours. Centrifugal elutriation and mitotic selection techniques were clearly inferior in terms of reproducibility and cell yield. Colony formation was most efficient for cells plated in fresh medium and incubated for 20 days. Cells inoculated into nude mice produced tumor masses that presented morphologic markers similar to those defined for in vitro cells and were capable of synthesizing CEA.-J Natl Cancer Inst 61: 75-83, 1978.
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76 Drewlnko, Yang, Barlogle, et at
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unlabeled. Data were fitted by means of the multicompartment model of Takahashi as modified by Jansson (16). Continuous labeling of LoVo cells was done with (5H]dThd (2 #LCi/ml; sp act, 6.7 Ci/mmole) dispensed to multiple replicate dishes. Samples were collected every 2 hours for 50 hours and processed for autoradiography. In these experiments, percent labeled mitosis was analyzed by the counting of 50-100 mitotic cells per time point, whereas the LI was defined by the scoring of 500 cells per time point. PCP.-PCP studies were performed on LoVo cells by a modification of the techniques reported for cultured lymphoma cells and freshly obtained human hematopoietic cells (17, 18). We initially experienced some difficulty in obtaining adequate DNA histograms by PCP. These difficulties originated from the approximately 10% of the cell population that was not monodispersed and gave histograms with high CV (>5%). We solved this problem in two ways: a) We briefly exposed cells harvested by method 16 (14) to ultrasound vibrations in a Heat System sonicator (20 sec at 30% full power; Heat Systems-Ultrasonics, Inc., Plainview, N.Y.). This step, however, produced cellular debris identified in the histogram by a small peak to the left of the G, peak. Repeated samples showed that the CV of the various compartments was about 2-3%. b) We used, pepsin for digestion of cell-to-cell junctional complexes, as previously reported by lante et al. (19) for human solid tumors. After decanting the nutrient medium, the dishes were rinsed with HBSS and incubated with 0.5% pepsin for 5 minutes at 22° C. The enzymatic reaction was terminated by the addition of growth medium. Cells were then vigorously pipetted through a Pasteur pipette and subsequently washed twice with 0.9% NaCl. Ice-cold absolute ethanol was then added dropwise to a final concentration of 70%. Fixed cells were stained with mithramycin and ethidium bromide in a concentration of 25 and 12.5 ",g/ml, respectively. The staining solution also contained 3.75 mM MgCh, which is equimolar to the concentration of mithramycin. Routinely, 50,000-100,000 cells were measured in a Phywe ICP II pulse cytophotometer (Phywe AG, Gottingen, Federal Republic of Germany) at a flow rate of 500 cells/second. A modification of Fried's model was employed for histogram evalllation. 7 Synchronization.-Exponentially growing cells were harvested as described before and counted in an electronic particle counter (Coulter Electronics Inc., Model lBI), and aliquots of 5X105 cells were dispensed to petri dishes containing 3 ml medium. After 48 hours in culture, cells were treated with increasing concentrations of dThd (I, 3, 5, 6, 7, 8, and 11 mM) for either 15, 24, or 36 hours. The supernatant was decanted, and the cells were washed twice with fresh medium and reincubated with 3 ml fresh medium. Every 2 hours, cells were pulse labeled for 30 minutes with I #LCi of [3H]Cyd/ml 7 Johnston D, White RA, Barlogie B: Automatic processing and interpretation of DNA distribution: Comparison of several techniques. Presented at Engineering Foundation's 5th Conference on Automated Cytology, Pensacola Beach, Fla., December 12-17, 1976
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dium supplemented by 20% fetal calf serum, vitamms, glutamine, and antibiotics. Under these conditions, the cells were capable of forming acinar structures and signet ring cells (14). The kinetics of CEA production by these cells were previously reported; supernatants of exponentially growing cultures contained about 80 ng of CEAIl06 cells (15). LoVo cells were subcultured serially once a week, and stock cultures were harvested by our previously described method 16 (14). The supernatant was discarded and the cells were rinsed with prewarmed saline solution. The cells were incubated with hyaluronidase (102 IU/ml) for 5 minutes at 37° C and then incubated with trypsin (2.5% in HBSS) for 5 minutes at 37° C. Cells were washed once in medium, resuspended in medium, and split two-fold into new culture vessels. Kinetic studies.-To determine the growth curve of exponentially growing LoVo cells, l-week-old stock cultures were harvested as described above. Aliquots of 105, 5X105 and 7X105 cells were seeded into 6O-mm petri dishes containing 5 ml of nutrient medium (4,290; 21,450; and 30,030 cells/cm 2 , respectively). After cells reached exponential growth, two cell counts in each of two replicates were made every 24 hours for about 160 hours with an electronic particle counter (Coulter Electronics Inc., Hialeah, Fla.; model lBI). Data were analyzed by linear regression techniques and DT was calculated from In2/s10pe. In other experiments, the MI, LI, and cell viability were defined for monolayer cells at various phases of in vitro growth monitored by daily cell counts. Aliquots of 5XI05 cells were seeded into 60-mm petri dishes containing 5 ml of medium. Cells were allowed to progress through all phases (lag, exponential, and stationary) without refeeding. Stationary phase was defined by no net increase in cell number. Cell viability was measured by trypan blue exclusion on replicate aliquots. The LI was determined in duplicate dishes by pulse labeling with 1 ",Ci[3H]dThd/ml (sp act, 3.0 Ci/mmole) for 30' minutes. The cells were harvested, and cytocentrifuge preparations stained with 2% aceto-orcein were processed fQr autoradiography with the use of a 50% solution of Ilford K5 emulsion in distilled water. The LI was scored on 500 cells/slide, and the MI was scored on 1,000 cells/slide. For cell cycle analysis, aliquots of 5X105 cells were seeded in 60-mm petri dishes and allowed to reach exponential growth. All cells were pulse labeled with [3H]dThd (I ",Ci/ml; sp act, 3.0 Ci/mmole) for 30 minutes at 37° C. Cells were washed twice with medium containing 0•. 1 mg/ml of dThd and once with fresh medium and reincubated with fresh medium. Duplicate dishes were harvested every 2 hours and processed for autoradiography. Slides were exposed for 2 weeks and developed in Kodak DI9 (Eastman Kodak, Rochester, N.Y.). Background was estimated from cell-free areas of the slide and from cell-free slides processed simultaneously. A cell was considered labeled if it displayed five or more grains. However, virtllally every labeled mitosis showed greater than IS grains/cell. From 50 to 200 mitotic cells/time point were scored as labeled or
BiologIc PropertIes of Cultured Colon Cells 77
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minidase (550 V/ml) at 37° C for 5 minutes followed by trypsin (0.25%) for 10 minutes at 37° C. Cells were then washed and resuspended in medium. In method 17 the cells were rinsed with 0.9% NaCI and then incubated with 2.5% Viokase (a bovine pancreatic extract; GIBCO, Grand Island, N.Y.) for 5 minutes at 37° C and trypsin (2.5%) at 37° C for 5 minutes. Cells were washed and resuspended in medium. Method 19 consisted of discarding the supernatant and adding 2.5% Viokase for 5 minutes at 37° C, decanting, and adding prewarmed trypsin 0.25% in HBSS for 10 minutes at 37° C. In all instances, cells were counted in an electronic particle counter, and the size (e.g., singlets, doublets) of the potential colony-forming units (multiplicity) was assessed by direct examination under light microscopy before being seeded for colony formation. Different cell concentrations (50, 100, 200, 500, and 1,000 cells/dish) were dispensed to 60-mm petri dishes and various subsequent culturing conditions were investigated. The petri dishes contained: .a) 5 ml of fresh medium (unchanged throughout the entire incubation period); b) 8 ml of fresh medium (unchanged throughout the entire incubation period); c) 5 ml of fresh medium' (changed every 4 days); d) 5 ml of fresh medium to which I drop of L-glutamine (200 mM) was added every 4 days; e) a mixture of I: I or 1:2 conditioned and fresh medium (conditioned medium was obtained from 2-week-old stock cultllfes, centrifuged, and filtered); or f) a feeder layer of LoVo cells 2X10 4 cells/dish) previously irradiated with 10 4 rads (cells were irradiated with a Phillips X-ray machine operating at 250 k VP at a dose rate of 90.4 rads/min at the level of the cells). After various incubation intervals (l8, 21, 22, 23, and 27 days), the colonies were stained with 2% crystal violet in 95% ethanol and examined under a stereomicroscope. Transplantation into athymic (nude) mice.-Stock monolayer cultures were harvested, washed, and counted. Aliqllots (I ml containing 10 7 cells) were injected sc into the backs of nude mice bred in the laboratory of Dr. B. Giovanella, at the Stehlin Foundation for Cancer Research (23). The mice were placed in separate cages and examined weekly for visible tumor growth. After 3 weeks, all of the mice developed tumor nodules that were allowed to progress until reaching a diameter of 9-10 cm. Blood was collected by intracardiac puncture, and the serum was assayed for CEA by the method of Chu and Reynoso (24). The animals were killed and tumor nodules were excised and processed by routine histologic techniques. Sections were stained with H & E, PAS, diastase digestion of PAS, mucicarmine, and Alcian blue. RESULTS
Growth KinetiCS DT calculated by linear regression analysis of seq uential cell count data from dishes containing three different cell titers ranged from 34 to 38 hours, with a mean of 36.3 hours. This result was very close to the 37
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(sp act, 15.1 Cilmmole). Cells were harvested and aliquots were processed for autoradiography to determine LI (per 500 cells) and MI (per 1,000 cells) and for PCP. On the basis of these experiments (see "Results"), improved synchronization with a double block with dThd was attempted (20). Exponentially growing cells were treated with 7 mM dThd for 24 hours. The supernatant was decanted, and the cells were washed twice and reincubated with fresh medium at 37° C for II hours, at which time they were reexposed to another treatment of 7 mM dThd for an additional 24 hours. The second dThd block was terminated when the supernatant was decanted, washed with medium, and reincubated with fresh medium. The degree of synchrony was subsequently determined in the same manner as for cells treated with a single dThd block. At the time of termination of all synchronization treatments, aliquots were also processed for colony formation determinations to define the effects of prolonged dThd treatment on cell survival as reflected by changes in PE. To synchronize LoVo cells by the mitotic selection technique (21), aliquots of 107 cells were seeded in 16ounce Owens' bottles and allowed to reach exponential growth. Each bottle was agitated horizontally for about 15 seconds, and the supernatant was collected. Fresh medium was added, and the cells were reincubated at 37° C. This operation was repeated every 30 minutes for the ensuing 4 hours. The collected cell suspensions were counted, stained for scoring the MI, and processed for colony formation. In other studies, we utilized the centrifugal elutriation method described by Meistrich et al. (22). A suspension of 5X10 7 monodispersed LoVo cells were separated according to volume with a JE-6 elutriator rotor in a J-2IB centrifuge (Beckman Instruments, Inc., Palo Alto, Calif.) at 4° C with a rotor speed of 2,240 rpm. The instrument had been sterilized overnight with 70% ethanol. Cells were suspended in Ham's F-1O medium containing 5% fetal calf serum and 5 mM naphthol disulfonic acid. A series of 13 fractions (50 ml each) were collected sequentially at a flow rate of 3.4 mllminute. Cells from each fraction were counted in an electronic particle counter (Coulter Counter Model ZEI; Coulter Electronics Inc.), their volume distribution was analyzed with a Coulter Counter attached to a multichannel particle size discriminator (Channelyzer; Coulter Electronics Inc.), their position in the cell cycle was defined by PCP, and an aliquot was processed for colony formation. Colony formation.- To conduct valid studies on cell killing effects of antitumor drugs, the colony-formation technique is required (12). For this purpose, it is necessary to use harvesting methods that will provide as close to a single cell suspension as possible with the least impairment to cellular reproductive capacity. For line Lo Vo, this was a difficult enterprise because cells adhere to each other by tight desmosomal junctions (14). We had previously investigated 18 harvesting techniques (14) and selected methods 2, 16, 17, and 19 to study colony-formation capacity. Method 16 was described above. Method 2 consisted of rinsing of stock cultures with 0.9% NaCl and incubation with neura-
78 Drewlnko, Yang, Barlogie, et al. 1. -Kinetic parameters of Lo Vo cells in various phases of growth"
TABLE
Phase Lag Exponential Stationary
Duration
Cell viability by trypan blue exclusion, %b
MI, %b
LI, %b
=36 hr =5 days >6 days
80 92 80
0.0 0.8 0.1
43 31 1
" Multiple replicate cultures containing 5x101 cells/dish were allowed to grow for 12 days without refeeding. Cell number was ~onitored w.ith an elec~ronic particle counter. Lag phase was defmed from time of seedmg (day 0) to initiation of exponential increments in cell number. Stationary phase began when cell increm~nts were no longer apparent (plateau). Determined on day 1 for lag phase; on day 5 for exponential phase; and on day 8 for stationary phase.
2.-Computer-estimated cell cycle parameters according to the Jansson model
Cell cycle stage
Transit time, hr
SD, hr
G, S G2+M GT
14.7 10.7 4.8 29.3
4.4 3.8 2.3 6.3
mean value of 90%. The length of G2-phase calculated at the 50% level of labeled mitosis recorded for cells incubated continllollsly with [3H]dThd was about 4.5 hours. Pulse Cytophotometry
DNA histograms obtained on cells treated with pepsin were selected as our choice' method. Repeated samples showed that the CV of the various compartments w~s less than 2~. Text-figure 2 shows a typical DNA hIstogram obtamed for cells in exponential growth (4 days after subculture). The calculated values for each compartment were: G I/O = 60%, S = 31 %, and G2+M = 9%. By use of cell cycle values defined by PLM techniques, the predicted proportions of cells in each com~artI?ent can be calculated by (length of phase transIt tlme+length of cell cycle time)XO.9, where 0.9 represents the proportion of proliferating cells obtained from continuous labeling experiments. The calculated values are: G I = 45.2%, S = 32.7%, and G 2+M = 12.1%. Inasmuch as nonproliferating cells (10%, from continuous labeling experiments) are measured by PCP within the G I compartment, the total population for that compartment is 55.2%. Thus for LoVo cells, values ob~ained from PCP corresponded closely to those predicted In reference to cell cycle times defined by PLM. Compartment distribution for cells in stationary phase of growth (text-fig. 3) revealed a small increase
Q; ~ ~
0
..c;
PLM% 100
Exponential Phase (4 Days)
(,)
'" '" E ~
•
80
Q.
.0
GI/O =60
z
G2 = 9
~
S
~
=31
'"
60
(,)
40 20
o 3 6 9 12 15 '18 21 24 27 30333639424548 Hours I.-Computer-fitted curve for the calculation of cell cycle stage Urnes analyzed by PLM. Points represent mean values of cells from duplicate dishes/time point.
TEXT-FIGURE
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10
20
30 40 50 60 Channel Number
70
80
TEXT-FIGURE 2:-DNA histogram obtained bv PCP analysis on cells 10 IOxponenual phase of growth (4 days after inoculation). GIlD
=
restmg phase + pre-DNA synthesis phase compartment; S = DNA syntheSIS phase compartment; G, = post-DNA synthesis compartment.
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hours previously reported for this cell line (14). The MI, LI, and cell viability (trypan blue exclusion) defined on monolayer cultures at each growth phase (table I) revealed that cells in lag phase (24 hr) had no mitotic cells; LI was 43% (range, 40-50), and viability was approximately 80%. An MI of 0.8% (range, 0.3-1.1), an LI of 31% (range, 28-40), and 92% viable cells were recorded for exponentially growing cultures (3 days). Cells in stationary phase (8 days) showed an MI of 0.1 % (range, 0.0-0.23), an LI of 1%, and 80% viable cells. ~he GT and length of each cell cycle stage were ?ehned by the PLM technique on exponentially growmg cells. PLM values were calculated by pooling of raw data fr?m 4-6 slid~s/time point (text-fig. I). Data points were fHted accordmg to the model of Jansson, which calculated cell cycle stage times shown on table 2. The CV of the GT was about 22%, whereas the CV corresponding to each stage transit time varied from 30% in GI-phase to about 50% in Grphase. From the data in table 2, the expected LI calculated as the ratio TslTc was 36.5% and T M calculated from (MIXTcl/ln2 was 0.33 hour. . The LI of cells exposed continuously to [3H]dThd mcreased at the rate of 3.2%/hour. After about 20 hours, a plateau of labeled nuclei was reached. In different experiments, the plateau varied from 88 to 92%, with a
TABLE
Biologic Properties of Cultured Colon Cells 79
Q)
c:: c::
Stationary Phase (" Days)
0
.c
u
~
Q)
Q.
GI/O= 69 =15 G2 =16
Q;
S
.0
E
:::>
z
Q)
u
10
20 30 40 50 Channel Number
60
70
TEXT·FIG!'RE 3.-DNA histogram for cells in stationary phase of growth (II days after inoculation).
in G 1 (69%), a significant increase in G 2 + M (16%), and 15% of the cell population in the S-phase compartment. However, the simultaneously defined LI was 1%. Thus our data reveal that cells in stationary phase of growth do not necessarily accumulate in G1-phase. Apparently, many cells in stationary phase can be delayed in G 2phase (no mitoses were detectable in stationary cultures) whereas other are stopped in the midst of synthesizing DNA [U-cells (25)] or synthesize DNA very slowly. Synch ronization
Synchronization of LoVo cells was attempted by the blocking of cell cycle traverse with excess dThd (1-11 mM) for 15, 24, and 36 hours. The degree of synchrony obtained by this technique was dose- and time-dependent. Incubation for 15 hours achieved only 60-70% cells in S-phase at the end of the synchrony procedure. Incubation for 24 hours resulted in 80-85% cells in DNA synthesis at the end of the block for concentrations of 6, 7, and 8 mM (text-fig. 4). Treatment for 36 hours resulted in 70-80% cells in S-phase. The kinetics of cell cycle traverse following removal of the blocking agent after 24 hours of incubation were followed for the ensuing 28 homs by autoradiography (LI and MI) and PCP. Autoradiography revealed a large percentage of Sphase cells (LI) during the first 6 hours; the LI decreased abruptly after 8 hours to values lower than control asynchronous populations (12%). This plateau of low LI was maintained for the next 10 hours, at which time it increased to peak values of about 40%, 24 hours after the release of the block. No mitotic figures were noted during the first 4 hours after removal of excess dThd. After 6 hours, the MI steadily increased, peaked at 12 hours (5%), and steadily decreased to 0 or 0.5% after 18 hours. PCP studies on cells blocked for 15 hours with dThd revealed poor synchronization with LoVo cells spread throughout S-phase. Sharpening of VOL 61, NO. I. JULY 1978
100
'"
Q)
90%). Yet the cell yield was not greater than 9X105 cells/shake, i.e., less than 10% of the expected yield based on the MI of asynchronous populations. Colony formation by these cells was similar to that of control cells. Centrifugal elutriation failed to yield optimal synchronization results. Cell recovery was good with 90% of the cells loaded into the chamber retrieved in fractions 1-13. Damaged (trypan blue-positive) cells were separated in fractions 1-4. Colony formation of cells retrieved in fractions 5-13 was equal to that of unseparated cells. Highly enriched populations of G1-phase cells (86-95%) were obtained in fraction 5 (text-fig. 5), but the maximum content of "enriched" S-phase cells (fraction 7) was only 37%. G 2+M cells were purified to 56% in fraction 9 (text-fig. 6). However, by microscopic
80 Drewlnko, Yang, Barlogle, et al. TABLE 3.-Colcmy formation of Lo Vo cells as a functicm of different harvesting methods"
PE
Colony-forming units
Harvesting method No. b
Singlet
Doublet
Triplet
2 19 17 16
70 37.5 37.5 83
18 22.5 26.5 12
8 14.3 14.7 5
>4 4 25.7 21.3 0
50 0 10-38 2-12 43-62
100 0 60-71 50-93 56-68
range, %C
200 0 73-85 70-80 47-73
500
1,000
1-2 42-50 40-82 53-69
3-4 31-36 22-64 49-70
" Cells were incubated for 20 days at 37° C with 5 ml of medium without refeeding. b For details of technique, see text. C A function of initial seed titer. 1.0
0.8
0.6 61=86% S =14%
u
u:
0.4
0.2
0.0 +----r'----,----"=r='-~----,
o
20
40 60 Channel Number
80
5.-DNA histogram of cells separated by centrifugal elutriation (fraction 5). Eighty-six percent of cells exhibit G 1 compartment-DNA content.
TEXT·FlGURE
examination, about 20% of these cells were doublets, which suggested that a significant fraction of the population consisted of paired Gt-cells. Furthermore, the volume distributions of cells in the individual fractions were not as sharp as those obtained previously with L-P59 cells (22). The width of the volume distribution profiles (full width at half maximum + modal channel number) ranged between 0.65 and LOO. In addition, cell aggregation was substantiaL A clump of cells remained at the bottom of the chamber and was washed out at the end of the run. This clump contained about 10-20% of the population, most of which were large cells or cell aggregates. To investigate the absence of efficient separation by the elutriation technique, cells were synchronized by the excess dThd method and allowed to progress through the cycle; the volume distribution of the synchronized cells was defined at various points of the cycle. Again, volume distributions were wide with no sharp peaks indicative of correspondence between cell volume and cell cycle stage.
1.0
0.8
c
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0.6
o
u
u:
0.4
0.2
00 - t - - - - , - ' - - - - , - - - , - - - - - - " - , 80 40 60 20 o Channel Number
Colony Formation
For all harvesting methods employed (with the exception of harvesting method 2, which failed under
61=23% S =2/ % 62 +M=56%
6.-DNA histogram of cells separated by centrifugal elutriation (fraction 9).
TEXT·FlGURE
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c:
o
any subsequent culturing conditions), the best results in terms of PE were noted when the plated cells were cultured continuously in fresh medium. No PE differences were noted for cells incubated with either 5 or 8 ml of medium, though incubation with 8 ml of medium yielded larger colonies. Incubation with both conditioned medium and feeder layers resulted in a low PE, with small colonies frequently composed of less than 32 cells. Changing medium or the addition of glutamine every 4 days did not improve the PE and produced significant contamination. Table 3 demonstrates PE of LoVo cells incubated for 20 days in 5 ml of fresh medium as a function of harvesting method. Method 2 produced only a few small colonies at high cell inocula. Both methods 17 and 19 yielded a large PE, but the multiplicity (27) of the originating colony-forming units was tOO great to allow clear-cut conclusions on single-cell killing effects. The multiplicity of cells harvested by method 16 was a negligible 1.22; colonies were compact and composed of greater than 500 cells (fig. 1). Only method 16 gave a linear correlation between PE and seed titer for cell concentrations ranging from 50 to 1,000/dish. No difference in PE occurred when colonies were stained after 18 through 23 days of incubation.
Biologic Properties of Cultured Colon Cells 81 Xenogeneic Transplantation The tumorigenic potential of LoVo cells was established by their transplantation into nude mice. Inoculated cells grew relatively slowly, and visible tumor nodules appeared after 3 weeks. Transplanted cells retained the morphologic features of the original tumor. Histologic examination demonstrated large cuboidal-tocolumnar cells with vesicular nuclei, formation of glandular structures, and the rare presence of signet ring cells (fig. 2). Yet no mucin production was demonstrated by histochemical techniques. Sera from 3 LoVo cell-bearing mice assayed for CEA when the animals were killed revealed 31.3, 40.0, and 87.5 ng CEA/mg, respectively, whereas no CEA was detected in control mICe.
The recent PLM data were fitted with the computerized model of Jansson. Values defined for Tc and T G2 were similar to those previously reported for the early passages of line LoVo (14). However, values for Ts and T GI were different in that T s was now shortened by about 7 hours, the time gained by the newly determined T GI. In part, this difference can be attributed to the manual calculation utilized in our previous report, whereas present values were defined more precisely by a computer program utilizing minimization techniques. Examination of chart 3 in our previous publication (14) reveals a "hump" in the descending limb of the first wave of labeled mitosis, just about the 50% level, which increased the value of Ts. The validity of the shorter Ts value computed in the present study is supported by the 8- to lO-hour S-phase transit time observed for synchronized Lo Vo cells. The CV of the G T and the phases of the cycle are among the largest reported for mammalian cells (27) and explains the rapid synchrony decay after release of the dThd block once Lo Vo cells traverse G2phase (28). In contrast to the results obtained for TI cells, a human lymphoma line (29), LoVo cells did not present differences in T G2 measured by the PLM or continuous labeling techniques. Apparently, LoVo cells are not susceptible to the changes in pH and temperature involved in the washing procedures employed in the PLM method, and their G 2 transit is unaffected. It has long been accepted that mammalian cells in stationary phase of growth accumulate in GI-phase and that this phenomen can be used to effectively synchronize the cultures (30). This contention originated from the fact that cells in stationary phase failed to incorporate labeled DNA precursors and, after subculturing, entered DNA phase in a synchronized wave. Our studies on LoVo cells in stationary phase demonstrated that failure to incorporate [3H]dThd cannot be considered evidence of non-S-stage position, because many cells were shown by PCP to have S-phase DNA content. Either such cells are blocked during their S-phase transit or their nucleic acid metabolism is modified in Stich a manner that they cannot utilize exogenous nucleosides. VOL. 61. NO. I. JULY 1978
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DISCUSSION
In contrast to nonhuman cell lines, which possess short GT with low CV and less fastidious culturing requirements (31), the excess dThd technique was, thus far, the only successful synchronization method for LoVo cells in terms of consistency, reliability, and synchronized cell yield. Although the method reduced the PE of LoVo cells and chemical synchronization may be undesirable for drug studies involving certain antimetabolites, the excess dThd technique presently remains the sole procedure that can be used for these sorts of investigations. The reason why centrifugal elutriation was unsuccessful in providing purified populations in all stages of the cycle resides in the nature of the LoVo cells. Different from rodent cell lines, which possess relatively uniform volumes, LoVo cells have a significant fraction of multinucleated cells and present considerable variations in volume and density from cell to cell. These variations are independent of position in the cycle as shown by volume determinations utilizing a multichannel particle size discriminator. Cell cycle traverse-dependent increments in cell volume are an absolute requirement for successful separation by centrifugal elutriation. Inasmuch as LoVo cells do not display this property, the failure of the elutriation method in providing purified populations in all stages is thus explained. Although we have not yet abandoned this technique, because highly purified GI-cells can be reseeded and allowed to progress synchronously through the cycle, one of our aims, that of rapidly obtaining large fractions of cells purified in specific stages of the cycle, has not been met; therefore, we must resort to alternative techniques. The fact that a purified cell yield in mitosis could be obtained by the mitotic selection technique suggests that it might be feasible to synchronize large numbers of LoVo cells by the use of automatic batch cell processor devices similar to that described by Klevecz (32). Colony formation by LoVo cells was most efficient when cells were incubated in fresh medium. In a fashion similar to that reported for human lymphoma cells (33), supplementation with conditioned medium or feeder layers decreased the PE and the size of the colonies. Thus the growth of LoVo cells may possibly be regulated by a molecular mediator present in supernatants of aged cultures or synthesized by the radiated cells in the feeder layer. The tumorigenic potential of LoVo cells was substantiated by successful transplantation to nude mice, a test currently accepted as the most reliable and meaningful indicator of malignant potential (34, 35). Interestingly, LoVo cells maintained the capacity to synthesize and release CEA and to originate glandular structures in vivo and in vitro (14) in spite of more than 100 passages in vitro. These results are similar to those reported for other colon carcinoma cell lines (36, 37). The fact that purely epithelial cell lines can develop organized structures in vivo and in vitro indicates that this capacity does not require the activity of inducer tissues. Although the xenografts of Lo Vo cells revealed glandular structures and signet ring cells, no mucin production was detected by standard histochemical techniques. The
82 Drewinko, Yang, Barlogie, et al.
absence of mucin had also been observed previously for the monolayer cultures of LoVo cells (14). This phenomenon suggests that signet ring cells do not necessarily result from the accumulation of mucinous material. We have characterized all of the necessary biologic parameters of an established human CEA-producing cell line in preparation for detailed studies of the effects of antitumor agents at the cellular level. We believe that LoVo cells will provide a screening system for antitumor drugs useful in the treatment of colon carcinoma superior to systems presently available.
(17) BARLOGIE B. DREWINKO B. BUCHNER T, et al: Pulse cytophoto-
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(33) DREWINKO B: Colony-forming ability of human immunoglobulin producing cells. Lab Invest 25:533-535. 1971 (34) GIOVANELLA BC. STEHLIN JS. WILLIAMS LJ: Heterotransplantation of human malignant tumors in "nude" thymus less mice. II. Malignant tumors induced by injections of cell cultures derived from human solid tumors. j Nat! Cancer Inst 52:921-930. 1974 (35) STILES CF. DESMOND W. CHUMAN LM. et al: Relationship of cell growth behavior in vitro to tumorigenicity in athymic nude mice. Cancer Res 36:3300-3305, 1976 (36) CARREL S. SORDAT B. MERENDA C: Establishment of a cell line (Co-1l5) from a human colon carcinoma transplanted into nude mice. Cancer Res 36:3978-3984, 1976 (37) ROSENTHAL KL. TOMPKINS WA. FRANK GL. et al: Variants of a human colon adenocarcinoma cell line which differs in morphology and carcinoembryonic antigen production. Cancer Res 37:4024-4030. 1977
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(2) MOERTEL CG: Large bowel. In Cancer Medicine (Holland JF, Frei E, eds). Philadelphia: Lea Be Febiger, 1973, pp 1597-1627 (3) HORTON J, HACKER B, CUNNINGHAM TJ, et al: The chemotherapy of large bowel cancer. Present status and future prospects. Digest Dis 19:1040-1046, 1974 (4) VAITKEVICIUS VK, BAKER LH, BUROKER TR, et al: Chemotherapy of gastrointestinal adenocarcinoma. In Cancer Chemotherapy. Fundamental Concepts and Recent Advances. Chicago: Year Book Medical Publishers. 1975. pp 263-278 (5) DAWSON M: Cellular Pharmacology. The Effects of Drugs on Living Vertebrate Cells In Vitro. Springfield, Ill.: Thomas. 1972 (6) LAING CA. HEPPNER GH. Kopp LE. et al: Detection of carcinoembryonic antigen in the media of cultures of carcinomatous cells of digestive system origin. J Nat! Cancer Inst 48:19091911, 1972 (7) EGAN ML, TODD CW: Carcinoembryonic antigen: Synthesis by a continuous line of adenocarcinoma cells. J Nat! Cancer Inst 49:887-889. 1972 (8) TOMPKINS WA. WATRACH AM, SCHMALE JD, et al: Cultural and antigenic properties of newly established cell strains derived from adenocarcinomas of the human colon and rectum. J Nat! Cancer Inst 52:1101-11IO, 1974 (9) MCCoMBS WB, LEIBOVITZ A, McCoy CE, et al: Morphologic and immunologic studies of a human colon tumor cell line (SW-48). Cancer 38:2316-2327, 1976 (10) TOM BH, RUTZKY LP, JAKSTYS MM, et al: Human colonic adenocarcinoma cells. I. Establishment and description of a new line. In Vitro 12:180-191. 1976 (11) LEIBOVITZ A, STINSON jC, MCCoMBS WB, et al: Classification of human colorectal adenocarcinoma cell lines. Cancer Res 36:4562-4569, 1976 (12) ROPER P, DREWINKO B: Comparison of in vitro methods to determine drug-induced cell lethality. Cancer Res 36:2182-2188, 1976 (13) BHUYAN BK, LOUGHMAN BE, FRASER TJ. et al: Comparison of different methods of determining cell viability after exposure to cytotoxic compounds. Exp Cell Res 97:275-280. 1976 (14) DREWINKO B, ROMSDAHL MM. YANG LY. et al: Establishment of a human carcinoembryonic antigen-producing colon adenocarcinoma cell line. Cancer Res 36:467-475, 1976 (15) DREWINKO B. YANG LY: Restriction of CEA synthesis to the stationary phase of growth of cultured human colon carcinoma cells. Exp Cell Res 101:414-416. 1976 (16) JANSSON B: Mathematical models in cell cycle kinetics. In Mathematical Models in Biology and Medicine (Bailey NT. Sendov B. Tsanev R. eds), Amst.erdam: North Holland. 1974, pp 21-39
metric analysis of synchronized cells in vitro. Cancer Res 36:1181, 1976 BARLOGIE B, SPITZER G, HART JS. et al: DNA histogram analysis of human hemopoietic cells. Blood 48:245-258. 1976 ZANTE J. SCHUMANN J, BARLOGIE B, et al: New preparation and staining procedures for specific and rapid analysis of DNA distributions. In Second International Symposium on Pulse Cytophotometry (Gohde W, Schumann J and Buchner T, eds). Ghent. Belgium: Europ Press Medikon. 1976, pp 97-106 BooTSMA D. BUDKE L. VOSS 0: Studies on synchronous division of tissue culture cells initiated by excess thymidine. Exp Cell Res 33:301-309. 1964 TERASIMA T, TOLMACH LJ: Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells. Exp Cell Res 30:344-362. 1963 MEISTRICH ML. GRDINA DJ, MEYN RE: Application of cell separation methods to the study of cell kinetics and proliferation. In Growth Kinetics and Biochemical Regulation of Normal and Malignant Cells (Drewinko B. Humphrey RM. eds). Baltimore: Williams Be Wilkins. 1977. pp 131-142 GIOVANELLA B, STEHLIN JS: Heterotransplantation of human malignant tumors in "nude" thymusless mice. I. Breeding and maintenance of "nude" mice. J Natl Cancer Inst 51:615-619. 1973 CHU TM. REYNOSO G: Evaluation of a new radioimmunoassay method for carcinoembryonic antigen in plasma with use of zirconyl phosphate gel. Clin Chern 18:918-922. 1972 ERNST p, FAILLE A. KILLMAN SA: Perturbation of cell cycle of human leukemic myeloblasts in vivo by cytosine arabinoside. Scand J Hematol 10:209-218. 1973 ELKIND MM, WHITMORE GF: The Radiobiology of Cultured Mammalian Cells. New York: Gordon and Breach. 1967 DAWSON KB, MADOC-jONES H, FIELD EO: Variations in the generation times of a strain of rat sarcoma cells in culture. Exp Cell Res 38:75-84. 1965 ENGELBEKG J: The decay of synchronization of cell division. Exp Cell Res 36:647-662. 1964 DREWINKO B, BOBO B. ROPER p. et al: Analysis of growth kinetics of a human lymphoma cell line. Cell Tissue Kinet 1l:177191. 1978 STUBBLEFIELD E: Synchronization methods for mammalian cell cultures. Methods Cell Physiol 3:25-43. 1968 MEISTRICH ML, MEYN RE, BARLOGIE B: Synchronization of mouse LP-59 cells by centrifugal elutriation separation. Exp Cell Res 105:169-177. 1977 KLEVECZ RR: Automated cell cycle analysis. Methods Cell Bioi
Biologic Properties of Cultured Colon Cells 83 FIGURE I.-Colony (ormalion by LoVo cells harvesled by mel hod 16 and incubaled in fresh medium (or 21 days. Cryslal viole!. X 12
FIGURE 2.-Morphologic aspecls of a lumor nodule oblained from Ihe inoculalion of cullured LoVo cells imo nude mice. Note giandlilar slrUClUres. H & E. X 200
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1
