Inl. .I. Radialion
Oncology Biol. Phys.
1977. Vol. 2. pp. I IO!LI 114.
Pergamon Press.
Printed in the U.S.4
l Original Contribution
RADIATION RESPONSE OF CULTURED HUMAN CARCINOEMBRYONIC ANTIGEN-PRODUCING COLON ADENOCARCINOMA CELLSt BENJAMIN
DREWINKO, and MARVIN
Departments
M.D., M.
LI-YING
Ph.D.+
ROMSDAHL,
M.D.,
YANG,
M.S.S
Ph.D.9
of Laboratory Medicine and Surgery, The University of Texas System Cancer, Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030, U.S.A.
The authors investigated the radiation response of cultured colon carcinoma cells in order to provide clues for the usual poor results of radiation therapy in the treatment of large bowel malignancies. The survival curve of asynchronous exponentially growing cells was a shouldered-exponential pattern typical of mammalian cells (0, = 170 rad and 0, = 100 rad). Qualitatively similar curves were obtained for cells irradiated in stationary phase of growth. Exponentially growing cells exhibited a significant capacity to recover from a “priming” dose of radiation with considerable restitution of the shoulder region of dose-dependent survival curves. Stationary phase cultures were unable to repair potentially lethal damage. There were moderate cell cycle stage-dependent fluctuations in survival for exponentially growing cells. The most sensitive elements were cells in early S phase and the most radioresistant were cells in late G,. Our studies indicate that cultured colon carcinoma cells have radiobiological paLrameters similar to those of other mammalian cells and suggest that the clinically observed radioresistance is not a consequence of intrinsic cellular properties or certain cell kinetics factors. Colon carcinoma,
Radiobiology,
Tissue culture, Colony formation.
INTRODUCTION
METHODS
Radiation therapy is considered ineffective as the primary treatment of large bowel malignancies.‘,‘9 The reason for this ineffectiveness is unknown. It has been advanced that a major determinant of radiocurability is the intrinsic cellular radiosensitivity of the different tumor types.4.‘2 The intrinsic radiosensitivity is a function of several cellular parameters, i.e. the nature of the radiosensitive targets, the capacity of the cell to absorb and repair radiodamage, the proliferative status, of the irradiated cell popucan be inlation, etc.” Some of these parameters vestigated in vitro on established cell cultures. A few cell lines derived from large bowel neoplasms9.“.13.2’.25 are currently available to be used for this sort of investigation. This report summarizes our studies on the radiation response of one such established cell line (LoVo cells) derived from a human adenocarcinema of the colon.’ The line has morphological and physiological characteristics corresponding to type 1 of Leibovitz et al.‘s classification of colorectal carcinoma cell lines.16
Cell
AND
MATERIALS
line
Cells used in this investigation were grown in Ham’s F-10 medium supplemented by 20% fetal calf serum, glutamine, vitamins and antibiotics. Under these conditions, freshly passaged cultures exhibit a lag time of 36 hr; exponential growth lasts for about 5 days, with an average doubling time of 37 hr, after which time unfed cultures enter stationary growth phase as defined by no net change in cell counts. The MI,T[ the LI, and cell viability (trypan blue exclusion) defined on monolayer cultures at each growth phase reveal that cells in lag phase (24 hr) have no mitotic cells; an LI of 31%, and cell viability of 92% were recorded for exponentially growing cultures (3 days). Cells in stationary phase showed an MI of O.l%, an LI of 1010,and cell viability of 80%. Stationary phase cells show increments in the LI to 50-60%, 8-16 hr after replating in fresh medium.’ Cells in exponential growth have an average generation time of 30 hr and 90% of the cells can be labeled by continuous incubation with [3H]TdR. Cells in stationary phase of
tThis investigation was supported by Grant CA 16763 from the National Cancer Institute, DHEW, through the National Large Bowel Cancer Project. *Department of Laboratory Medicine.
§Department of Surgery. IAbbreviations used are: MI, mitotic index; LI, labeling index; PE, plating efficiency; TdR, thymidine. 1109
I I IO
Radiation Oncology
growth form acinar structures’ 88 ng/106 cells of carcinoembryonic
0 Biology
and produce antigen.8
0 Physics
about
Radiation source We used a Phillips X-ray machine operating at 250 kVp with added filtration of 0.35 mm Cu (half value layer = 1.12 mm Cu) yielding a dose rate of 90.4rad/min at the level of the cells. Cells were irradiated at room temperature on a platform of tissue-equivalent wax. The exposure rate was measured with a Victoreen r-meter in air. The absorbed dose was calculated using conversion factors listed in National Bureau of Standards Handbook No. 62.*’ Dose-response survival of asynchronous cells 5 x lo6 cells were seeded in 60 mm Petri dishes and incubated at 37°C in a 5% CO2 in air, humidified atmosphere. When exponential growth was achieved, replicate dishes were irradiated and the cells were harvested as described before.’ The cell suspension was counted in an electronic particle counter (Model ZBI, Coulter Electronics, Inc., Hialeah, Florida) and appropriate aliquots were seeded in triplicate Petri dishes containing 5 ml of medium. The cells were incubated for 21 days, the supernatant discarded, the dishes rinsed with 0.9% NaCl solution, and the colonies stained with 2% crystal violet in 95% ethanol. Colonies were scored under a stereomicroscope. Surviving cells were those which gave rise to a colony composed of 50 or more cells. In all experiments, 12 plating efficiency (PE) controls were run in parallel. The survival of the radiated cells was calculated in reference to control cultures as the radio between the PE of treated and control cells. PE was defined as the ratio between visible colonies over the number of plated cells. Control cultures underwent all of the mechanical manipulations undergone by the treated cells, but without receiving X-radiation. The PE of control cultures for the series of experiments detailed herein ranged from 35 to 53%. In another series of experiments, cells were exposed to increasing doses of radiation while in stationary phase of growth (10 days after subculture). These cells were harvested and seeded for colony formation immediately after radiation or were maintained in spent medium at 37°C for 15 and 24 hr before harvesting and replating.” Colony formation was conducted as described above and survival was calculated in reference to stationary phase control cells. The PE of stationary phase control cultures ranged from 16 to 37%. Fractionated treatment 5 x 10’ asynchronously growing LoVo cells were exposed to a first “conditioning” dose of 300rad at the beginning of the experiment (0 hr). The dishes were returned to the incubator and, at hourly inter-
November-December
1977, Volume 2. No.
I1 and No. 12
vals, 2 dishes received an additional dose of 300rad each and the cells were processed for colony formation. This procedure was repeated for the remaining dishes during the ensuing 7 hr. Controls obtained at 0, 4 and 7 hr were: (a) dishes that were treated in exactly the same manner with the exception of radiation (PE controls); (b) dishes which received a single dose of 300 rad only; and (c) dishes that received the total dose of 600rad at once. After treatment, cells were harvested, counted, seeded, incubated and stained as described above. Survival of irradiated cells was calculated with respect to untreated controls. In order to investigate recovery of the shouldered portion of the dose-response survival curve, a set of dishes received escalating doses of radiation at 0 hr and survival was assessed as usual. The dishes of another set received a total of 500 rad at 0 hr and were returned to the incubator for 4.5 hr. At that time, duplicate dishes were exposed to increasing doses of additional X-radiation and survival assessed as before. Radiation response of synchronized cells 5 x lo5 cells were incubated in 60 mm Petri dishes. When cells achieved exponential growth, they were treated with 7 mM for 24 hr. After removal of the TdR block, synchrony was monitored on replicate cultures by measuring the labeling and mitotic indices and by DNA histography obtained with pulse cytophotometry techniques as described before.* Radioautography revealed a large percentage (>85) of S phase cells (LI) during the first 6 hr; the LI decreased abruptly after 8 hr to values lower (12%) than control asynchronous populations (31%). This plateau of low LI was maintained for the next 10 hr, at which time the LI began increasing to peak values of about 40%, 24 hr following the release of the block. No mitotic figures were noted during the first 4 hr following removal of excess TdR. After 6 hr, the MI steadily increased, peaking at 12 hr (5%) and steadily decreased to 0 or 0.5% after 18 hr. PCP studies showed an accumulation of 85% cells in early S phase. Synchrony was maintained in G2 phase (80% at 11 hr), but rapidly decreased when cells reached G, phase. At 22 hr, less than 75% of the cells were in G, phase. After 24 hr, compartment distribution was similar to that observed for asynchronous cells. Replicate dishes were exposed to increasing doses of X-radiation at 2 hr intervals, for a total period of 26 hr. Control PE was obtained at each time point. Synchrony procedures reduced the PE to 50-70% of control asynchronous cultures. RESULTS The survival growing LoVo dered-exponential
curve of asynchronous exponentially cells was characterized by a shoulcurve typical of the radioresponse
Radioresponse
of cultured
colon
carcinoma
cells 0 B.
of most mammalian cells (Fig. 1A). The curve evidenced a shoulder region with insignificant killing (0, = 170 rad) followed by a steep exponential killing as a function of increasing doses of radiation (0, = 1OOrad). The survival curve of stationary phase cells also evidenced a threshlold exponential pattern (Figs. IB, C and D). However, the 0, (55-112 rad) was significantly lower than that determined for exponentially growing cells. In contrast, the 0, was increased (133-150 rad). In the context of target theory, these findings suggest that while the radiation targets of stationary phase cells are most resistant, the capacity to absorb sublethal damage is severely decreased. Exponentially growing cells treated by fractionated exposure evinced increased survival as the interval between the exposures was lengthened (Fig. 2). After 4 hr, the maximum recovery (64%) had been achieved. Dose-dependent survival experiments were
c
Dq= 55 rod Do;140
rod
I loco
-
1111
et al.
I
300
rad
$
300
rad
$
600
rad
i _---
- -.
+ 300
rad
6
1 t
100%
6
4
recovery
8
Hour
\
500
______
2
B
003’
_--
DREWINKO
,000 Rad
Fig. 1. Dose-response survival of asynchronous exponentially growing and stationiary phase LoVo cells. A depicts the survival curve of exponential cells (48 hr after subculture). B, C and D depict the survival of stationary phase cells (10 days after subculture) harvested immediately after (B), and at 15 (C) and 24 hr (D) after radiation. Points in A represent mean values of 4 independent experiments each with 6 replicates per dose point. B, C and D are the average values of 2 experiments with 6 replicates per dose point. Bars represent standard errors. D,, quasithreshold dose equal to the intercept with the abscissa of the exponential part of survival curve; D,, mean lethal dose equal to the concentration required tlo reduce survival by 63% on exponential part of survival curve.
Fig. 2. Survival of LoVo cells after split-dose treatment. Closed triangles represent the survival of cells exposed to 300 rad at the times indicated by the points. Closed circles indicate the survival of cells treated with 600 rad at the time indicated by the points. Open circles represent the survival of cells exposed to 300rad at 0 hr, returned to the incubator, and reexposed to an additional 300 rad at the times indicated by the points. Points are mean values of 2 experiments each with 6 replicate cultures per dose point. Bars represent standard errors.
conducted on cells which had received a “conditioning” dose of 500 rad and had been incubated for 4.5 hr at 37°C before receiving further increasing doses of radiation. The survival of these cells was increased with respect to those cells that received the total dose at once, and the curve showed a return of the shoulder region (Fig. 3). This shoulder was only 30% of the original one, as quantified by the corresponding Q (0, = 220 rad and DL= 65 rad, respectively, for the first and second shoulder). However, the slope of the linear part of the survival curve showed a 30% increase in the 0, value (Do = 95 rad and 0: = 115 rad, respectively, for first and second slopes). LoVo cells showed only moderate cell cycle stage dependent fluctuations in survival following Xirradiation (Fig. 4) with threefold maximum deviation between the lowest (early S and early G,) and the highest survival points (late G,). Dose response survival curves obtained on synchronized cells in S phase (2 hr post release of block), Gt (11 hr), and G, (22 hr) showed a marked decrease in the shoulder region (0, = 30-80 rad), in comparison with that of asynchronous cells (0, = 170 rad). In contrast, the slopes of the linear part of the curve showed an
1112
Radiation
Oncology
‘\ ,
001
0
,
0
OqJ:
65
rad
Do’=
l/5
rod
Biology 0
1200
800
400
Physics
rad
Fig. 3. Shoulder recovery after split-dose treatment of LoVo cells. Points are average values of 2 separate experiments. Open circles represent the survival of cells which received escalating X-ray doses at 0 hr. Closed circles indicate the survival of cells that received a “priming” dose of 500rad at 0 hr and were reincubated for 4.5 hr before receiving additional X-radiation.
20
L--__s
G2
I
Gl
Fig. 4. Age-dependent survival response of LoVo exposed to a single dose of 500rad.
increase of the 0, value values of both I& and 0, late G, phase (Fig. 5’).
(165-230 rad). were observed
1 cells
The largest for cells in
DISCUSSION Even though the intrinsic radiosensitivity of a cell type is recognized as one fundamental factor in the
November-December
1977, Volume
2. No.
II
and No.
12
potential radiocurability of neoplasms, few radiobiological studies have been conducted in tlitro on different human cell types.‘.3.6.‘7,26 This absence of pertinent studies results from the paucity of established human cell lines with suitable properties (i.e. colony formation) for this sort of investigation. We conducted what we believe is the first investigation on the radioresponse of human colon carcinoma cells grown in vitro in the hope of providing clues for the usual failure of radiotherapy in controlling the growth of these types of cells. Doseresponse survival curves obtained on asynchronous exponentially growing LoVo cells yielded parameters well within the range of those determined for other mammalian cells. Furthermore, the cultured colon carcinoma cells have a capacity to absorb sublethal damage (quantified by 0,) and a radiosensitivity of the critical target (quantified by 0,) similar to that of other human cells in vitro. The cells possess a moderate capacity to recover from a “priming” dose of radiation with restitution of the shoulder region of the survival curve. The survival response of synchronized cells showed only moderate (3.5-fold) differences between the most and least sensitive stages of the cycle. Hence, it appears that no intrinsic radiobiological property of colon carcinoma cells justifies the usual poor clinical results obtained by radiotherapy in the treatment of this neoplasm. Another possibility for the different responses noted in vitro and in uivo may reside in the different conditions under which the cells proliferate. While our experiments were conducted on exponentially growing elements under abundant oxygenation and anabolite supply, a great number of cells in uivo grow under anoxic and ischemic conditions which may give rise to a large population of nonproliferating cells.‘8,24 This fact is supported by the low LI values (which quantifies the proportion of cells in DNA synthesis) measured for these tumors in ~ivo.~~ Thus, the radioresistance of colon carcinoma might be a function of population growth kinetics, an explanation suggested also for in vitro-in vivo differences in the radioresponse of human melanoma cells.’ Yet, the survival of LoVo cells in stationary phase of growth was not significantly different from that observed for exponentially growing cells; in fact, it was somewhat lower. This lower survival resulted primarily from a reduction of the capacity to absorb sublethal damage as quantified by a smaller Dq. This effect has been reported for other cell lines and attributed to a decrease in the capacity of stationary phase cells to repair sublethal and potentially lethal damage.15 Our studies would indicate that LoVo cells in stationary phase also have a reduced capacity to repair potentially lethal damage as evidenced by similar survival curves regardless of the interval between irradiation and subsequent harvesting.
Radioresponse of cultured colon carcinoma cells 0 B.
Ol-
0
200
600
1000
200
600
DREWINKO et al.
1000
200
1113
600
1000
rad
Fig. 5. Dose-dependent
survival of synchronized and 11 hr, respectively,
LoVo
cells. G,, S and G, phase cells were selected 22, 2 after release of the TdR block.
Cells in stationary phase of growth generally have been accepted as an adequate in vitro model for in uiuo resting populations.‘s Our results for cultured human colon carcinoma cells would suggest that either stationary phase cultures are not an appropriate reflection of noncycling cells in vivo, or that the poor radioresponse of colon carcinoma is independent of both intrinsic cell properties and some of the population growth kinetics properties. Alternatively, in
vivo radioresistance might be a function of inadequate oxygen s~pply*~ while in vitro sensitivity depends on the oxygenation effect.r4 Additional studies must be conducted under anoxic conditions on both multicellular spheroid cultures (another in vitro system purportedly reflecting in uivo resting cells)23 and on other colon carcinoma cells lines16 in order to corroborate these conclusions.
REFERENCES 1. Barendsen, G.W.: Do,se survival curves of human cells in tissue culture irradiated with alpa, beta, 20 kV and 200 kV X-radiation. Nature 193: 1153-l 155, 1962. 2. Barlogie, B., Drewinko, B., Johnston, D.A., Hauss, W.H., Freireich, E.J.: Pulse cytophotometric analysis of synchronized cells in vitro. Cancer Res. 36: 11761181, 1976. 3. Barranco, S.C., Romsdahl, M.M., Humphrey, R.M.: The radiation response of human malignant melanoma cells grown in vitro. Cancer Res. 31: 830-833, 1971. 4. Byfield, J.E.: The role of radiation repair mechanisms in radiation treatment failures. Cancer Chemoth. Rept. 58: 527-538, 1974. 5. Carter, S.K.: Large bowel cancer. The current status of treatment. .Z. Natl Cancer Znstit. 56: 3-10, 1976. 6. Drewinko, B., Humphrey, R.M., Trujillo, J.M.: The radiation response of a long-term culture of human lymphoid cells--I. Asynchronous populations. Znt. Z. Rad. Biol. 21: 361-3713, 1972. 7. Drewinko, B., Romsdahl, M.M., Yang, L.Y., Ahearn, M.J., Trujillo, J.M.: Establishment of a human carcinoembryonic antigen-producing colon adenocarcinema cell line. Cancer Res. 36: 467-475, 1976. 8. Drewinko, B., Yang, L.Y.: Restriction of CEA synthesis to the stationary phase of growth of cultured
human colon carcinoma cells. Exp. Cell Res. 101: 414416, 1976. 9. Egan, M.L., Todd, C.W.: Carcinoembryonic antigen: synthesis by a continuous line of adenocarcinoma cells. J. Nat1 Cancer Znstit. 49: 887-889,
1972.
10. Elkind, M.M., Whitmore, G.F.: The radiobiology of cultured mammalian cells. New York, Gordon & Breach, 1967. 11. Fogh, J., Trempe, G.: New human tumor cell lines. In Human Tumor Cells in vitro, ed. by Fogh, J. New York, Plenum, 1975, pp. 115-154. 12. Friedman, M.: Aspects of radiation biology and radiation pathology observed during the treatment of cancer in man. Br. J. Radiol. 48: 81-96, 1975. 13. Goldenberg, D.M., Pavia, R.M., Hansen, H.J., Vandevoorde, J.P.: Synthesis of carcinoembryonic antigen in uitro. Nature New Biol. 239: 189-190, 1972.
14. Gray, L.H.: Radiobiologic basis of oxygen as a modifying factor in radiation therapy. Am. J. Roentgen. 85: 803-815, 1961. 15. Hahn, G.M., Little, J.B.: Plateau-phase cultures of mammalian cells. An in vitro model for human cancer. Curr. Topics Rad. Res. Quart. 8: 39-83, 1972.
16. Leibovitz, A., Stimson, J.C., McCombs, W.B., McCoy, C.E., Mazur, K.C., Mabry, M.D.: Classification of
1114
17.
18.
19.
20.
21.
Radiation Oncology
0 Biology
0 Physics
human colorectal adenocarcinoma cell lines. Cancer Res. 36: 4562-4569, 1976. Little, J.B.: Repair of sub-lethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature, Lond. 224: 804806, 1969. Mendelsohn, M.L.: Autoradiographic analysis of cell proliferation in spontaneous breast cancer of C3H mouse-III. Growth fraction. J. Nat/ Cancer Znstit. 28: 1015-1029, 1962. Moertel, C.G.: Large bowel. In Cancer Medicine, ed. by Holland, J.F., Frei, E. Philadelphia, Pennsylvania, Lea & Febiger, 1973, pp. 1597-1631. National Bureau of Standards. Report of the International Commission on Radiological Units and Measurements (ICRU). National Bureau of Standards (U.S.), Handbook No. 62, 1957. Rutzky, L.P., Tom, B.H., Tomita, J.T., Kahan, B.D.: Ch_aracterization of a human colonic cell line: Kinetics of CEA synthesis. In Vitro 12: 328, 1976.
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1977, Volume
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22. Steel, G.G., Lamerton, L.F.: Cell population kinetics and chemotherapy. Nat1 Cancer Znstit. Monogr. 30: 29-50, 1969. 23. Sutherland, R.M., McCredie, J.A., Inch, W.R.: Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J. Nat/ Cancer Znstit. 46: 113-120, 1971. 24. Tannock, I.F.: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. Br. J. Cancer 22: 258-273, 1968. W.A.F., Watrach, A.M., Schmale, J.D., 25. Tompkins, Schultz, R.M., Harris, J.A.: Cultural and antigenic properties of newly established cell strain derived from adenocarcinomas of the human colon and rectum, J. Nat1 Cancer Znstit. 52: 1101, 1974. 26. Weichselbaum, R.R., Epstein, J., Little, J.B.: In vitro cellular radiosensitivity of human malignant tumors. Europ. J. Cancer 12: 47-51, 1976.
Oncology Biol. Phys.
1977. Vol. 2. pp. I IO!LI 114.
Pergamon Press.
Printed in the U.S.4
l Original Contribution
RADIATION RESPONSE OF CULTURED HUMAN CARCINOEMBRYONIC ANTIGEN-PRODUCING COLON ADENOCARCINOMA CELLSt BENJAMIN
DREWINKO, and MARVIN
Departments
M.D., M.
LI-YING
Ph.D.+
ROMSDAHL,
M.D.,
YANG,
M.S.S
Ph.D.9
of Laboratory Medicine and Surgery, The University of Texas System Cancer, Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030, U.S.A.
The authors investigated the radiation response of cultured colon carcinoma cells in order to provide clues for the usual poor results of radiation therapy in the treatment of large bowel malignancies. The survival curve of asynchronous exponentially growing cells was a shouldered-exponential pattern typical of mammalian cells (0, = 170 rad and 0, = 100 rad). Qualitatively similar curves were obtained for cells irradiated in stationary phase of growth. Exponentially growing cells exhibited a significant capacity to recover from a “priming” dose of radiation with considerable restitution of the shoulder region of dose-dependent survival curves. Stationary phase cultures were unable to repair potentially lethal damage. There were moderate cell cycle stage-dependent fluctuations in survival for exponentially growing cells. The most sensitive elements were cells in early S phase and the most radioresistant were cells in late G,. Our studies indicate that cultured colon carcinoma cells have radiobiological paLrameters similar to those of other mammalian cells and suggest that the clinically observed radioresistance is not a consequence of intrinsic cellular properties or certain cell kinetics factors. Colon carcinoma,
Radiobiology,
Tissue culture, Colony formation.
INTRODUCTION
METHODS
Radiation therapy is considered ineffective as the primary treatment of large bowel malignancies.‘,‘9 The reason for this ineffectiveness is unknown. It has been advanced that a major determinant of radiocurability is the intrinsic cellular radiosensitivity of the different tumor types.4.‘2 The intrinsic radiosensitivity is a function of several cellular parameters, i.e. the nature of the radiosensitive targets, the capacity of the cell to absorb and repair radiodamage, the proliferative status, of the irradiated cell popucan be inlation, etc.” Some of these parameters vestigated in vitro on established cell cultures. A few cell lines derived from large bowel neoplasms9.“.13.2’.25 are currently available to be used for this sort of investigation. This report summarizes our studies on the radiation response of one such established cell line (LoVo cells) derived from a human adenocarcinema of the colon.’ The line has morphological and physiological characteristics corresponding to type 1 of Leibovitz et al.‘s classification of colorectal carcinoma cell lines.16
Cell
AND
MATERIALS
line
Cells used in this investigation were grown in Ham’s F-10 medium supplemented by 20% fetal calf serum, glutamine, vitamins and antibiotics. Under these conditions, freshly passaged cultures exhibit a lag time of 36 hr; exponential growth lasts for about 5 days, with an average doubling time of 37 hr, after which time unfed cultures enter stationary growth phase as defined by no net change in cell counts. The MI,T[ the LI, and cell viability (trypan blue exclusion) defined on monolayer cultures at each growth phase reveal that cells in lag phase (24 hr) have no mitotic cells; an LI of 31%, and cell viability of 92% were recorded for exponentially growing cultures (3 days). Cells in stationary phase showed an MI of O.l%, an LI of 1010,and cell viability of 80%. Stationary phase cells show increments in the LI to 50-60%, 8-16 hr after replating in fresh medium.’ Cells in exponential growth have an average generation time of 30 hr and 90% of the cells can be labeled by continuous incubation with [3H]TdR. Cells in stationary phase of
tThis investigation was supported by Grant CA 16763 from the National Cancer Institute, DHEW, through the National Large Bowel Cancer Project. *Department of Laboratory Medicine.
§Department of Surgery. IAbbreviations used are: MI, mitotic index; LI, labeling index; PE, plating efficiency; TdR, thymidine. 1109
I I IO
Radiation Oncology
growth form acinar structures’ 88 ng/106 cells of carcinoembryonic
0 Biology
and produce antigen.8
0 Physics
about
Radiation source We used a Phillips X-ray machine operating at 250 kVp with added filtration of 0.35 mm Cu (half value layer = 1.12 mm Cu) yielding a dose rate of 90.4rad/min at the level of the cells. Cells were irradiated at room temperature on a platform of tissue-equivalent wax. The exposure rate was measured with a Victoreen r-meter in air. The absorbed dose was calculated using conversion factors listed in National Bureau of Standards Handbook No. 62.*’ Dose-response survival of asynchronous cells 5 x lo6 cells were seeded in 60 mm Petri dishes and incubated at 37°C in a 5% CO2 in air, humidified atmosphere. When exponential growth was achieved, replicate dishes were irradiated and the cells were harvested as described before.’ The cell suspension was counted in an electronic particle counter (Model ZBI, Coulter Electronics, Inc., Hialeah, Florida) and appropriate aliquots were seeded in triplicate Petri dishes containing 5 ml of medium. The cells were incubated for 21 days, the supernatant discarded, the dishes rinsed with 0.9% NaCl solution, and the colonies stained with 2% crystal violet in 95% ethanol. Colonies were scored under a stereomicroscope. Surviving cells were those which gave rise to a colony composed of 50 or more cells. In all experiments, 12 plating efficiency (PE) controls were run in parallel. The survival of the radiated cells was calculated in reference to control cultures as the radio between the PE of treated and control cells. PE was defined as the ratio between visible colonies over the number of plated cells. Control cultures underwent all of the mechanical manipulations undergone by the treated cells, but without receiving X-radiation. The PE of control cultures for the series of experiments detailed herein ranged from 35 to 53%. In another series of experiments, cells were exposed to increasing doses of radiation while in stationary phase of growth (10 days after subculture). These cells were harvested and seeded for colony formation immediately after radiation or were maintained in spent medium at 37°C for 15 and 24 hr before harvesting and replating.” Colony formation was conducted as described above and survival was calculated in reference to stationary phase control cells. The PE of stationary phase control cultures ranged from 16 to 37%. Fractionated treatment 5 x 10’ asynchronously growing LoVo cells were exposed to a first “conditioning” dose of 300rad at the beginning of the experiment (0 hr). The dishes were returned to the incubator and, at hourly inter-
November-December
1977, Volume 2. No.
I1 and No. 12
vals, 2 dishes received an additional dose of 300rad each and the cells were processed for colony formation. This procedure was repeated for the remaining dishes during the ensuing 7 hr. Controls obtained at 0, 4 and 7 hr were: (a) dishes that were treated in exactly the same manner with the exception of radiation (PE controls); (b) dishes which received a single dose of 300 rad only; and (c) dishes that received the total dose of 600rad at once. After treatment, cells were harvested, counted, seeded, incubated and stained as described above. Survival of irradiated cells was calculated with respect to untreated controls. In order to investigate recovery of the shouldered portion of the dose-response survival curve, a set of dishes received escalating doses of radiation at 0 hr and survival was assessed as usual. The dishes of another set received a total of 500 rad at 0 hr and were returned to the incubator for 4.5 hr. At that time, duplicate dishes were exposed to increasing doses of additional X-radiation and survival assessed as before. Radiation response of synchronized cells 5 x lo5 cells were incubated in 60 mm Petri dishes. When cells achieved exponential growth, they were treated with 7 mM for 24 hr. After removal of the TdR block, synchrony was monitored on replicate cultures by measuring the labeling and mitotic indices and by DNA histography obtained with pulse cytophotometry techniques as described before.* Radioautography revealed a large percentage (>85) of S phase cells (LI) during the first 6 hr; the LI decreased abruptly after 8 hr to values lower (12%) than control asynchronous populations (31%). This plateau of low LI was maintained for the next 10 hr, at which time the LI began increasing to peak values of about 40%, 24 hr following the release of the block. No mitotic figures were noted during the first 4 hr following removal of excess TdR. After 6 hr, the MI steadily increased, peaking at 12 hr (5%) and steadily decreased to 0 or 0.5% after 18 hr. PCP studies showed an accumulation of 85% cells in early S phase. Synchrony was maintained in G2 phase (80% at 11 hr), but rapidly decreased when cells reached G, phase. At 22 hr, less than 75% of the cells were in G, phase. After 24 hr, compartment distribution was similar to that observed for asynchronous cells. Replicate dishes were exposed to increasing doses of X-radiation at 2 hr intervals, for a total period of 26 hr. Control PE was obtained at each time point. Synchrony procedures reduced the PE to 50-70% of control asynchronous cultures. RESULTS The survival growing LoVo dered-exponential
curve of asynchronous exponentially cells was characterized by a shoulcurve typical of the radioresponse
Radioresponse
of cultured
colon
carcinoma
cells 0 B.
of most mammalian cells (Fig. 1A). The curve evidenced a shoulder region with insignificant killing (0, = 170 rad) followed by a steep exponential killing as a function of increasing doses of radiation (0, = 1OOrad). The survival curve of stationary phase cells also evidenced a threshlold exponential pattern (Figs. IB, C and D). However, the 0, (55-112 rad) was significantly lower than that determined for exponentially growing cells. In contrast, the 0, was increased (133-150 rad). In the context of target theory, these findings suggest that while the radiation targets of stationary phase cells are most resistant, the capacity to absorb sublethal damage is severely decreased. Exponentially growing cells treated by fractionated exposure evinced increased survival as the interval between the exposures was lengthened (Fig. 2). After 4 hr, the maximum recovery (64%) had been achieved. Dose-dependent survival experiments were
c
Dq= 55 rod Do;140
rod
I loco
-
1111
et al.
I
300
rad
$
300
rad
$
600
rad
i _---
- -.
+ 300
rad
6
1 t
100%
6
4
recovery
8
Hour
\
500
______
2
B
003’
_--
DREWINKO
,000 Rad
Fig. 1. Dose-response survival of asynchronous exponentially growing and stationiary phase LoVo cells. A depicts the survival curve of exponential cells (48 hr after subculture). B, C and D depict the survival of stationary phase cells (10 days after subculture) harvested immediately after (B), and at 15 (C) and 24 hr (D) after radiation. Points in A represent mean values of 4 independent experiments each with 6 replicates per dose point. B, C and D are the average values of 2 experiments with 6 replicates per dose point. Bars represent standard errors. D,, quasithreshold dose equal to the intercept with the abscissa of the exponential part of survival curve; D,, mean lethal dose equal to the concentration required tlo reduce survival by 63% on exponential part of survival curve.
Fig. 2. Survival of LoVo cells after split-dose treatment. Closed triangles represent the survival of cells exposed to 300 rad at the times indicated by the points. Closed circles indicate the survival of cells treated with 600 rad at the time indicated by the points. Open circles represent the survival of cells exposed to 300rad at 0 hr, returned to the incubator, and reexposed to an additional 300 rad at the times indicated by the points. Points are mean values of 2 experiments each with 6 replicate cultures per dose point. Bars represent standard errors.
conducted on cells which had received a “conditioning” dose of 500 rad and had been incubated for 4.5 hr at 37°C before receiving further increasing doses of radiation. The survival of these cells was increased with respect to those cells that received the total dose at once, and the curve showed a return of the shoulder region (Fig. 3). This shoulder was only 30% of the original one, as quantified by the corresponding Q (0, = 220 rad and DL= 65 rad, respectively, for the first and second shoulder). However, the slope of the linear part of the survival curve showed a 30% increase in the 0, value (Do = 95 rad and 0: = 115 rad, respectively, for first and second slopes). LoVo cells showed only moderate cell cycle stage dependent fluctuations in survival following Xirradiation (Fig. 4) with threefold maximum deviation between the lowest (early S and early G,) and the highest survival points (late G,). Dose response survival curves obtained on synchronized cells in S phase (2 hr post release of block), Gt (11 hr), and G, (22 hr) showed a marked decrease in the shoulder region (0, = 30-80 rad), in comparison with that of asynchronous cells (0, = 170 rad). In contrast, the slopes of the linear part of the curve showed an
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0
,
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rad
Fig. 3. Shoulder recovery after split-dose treatment of LoVo cells. Points are average values of 2 separate experiments. Open circles represent the survival of cells which received escalating X-ray doses at 0 hr. Closed circles indicate the survival of cells that received a “priming” dose of 500rad at 0 hr and were reincubated for 4.5 hr before receiving additional X-radiation.
20
L--__s
G2
I
Gl
Fig. 4. Age-dependent survival response of LoVo exposed to a single dose of 500rad.
increase of the 0, value values of both I& and 0, late G, phase (Fig. 5’).
(165-230 rad). were observed
1 cells
The largest for cells in
DISCUSSION Even though the intrinsic radiosensitivity of a cell type is recognized as one fundamental factor in the
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potential radiocurability of neoplasms, few radiobiological studies have been conducted in tlitro on different human cell types.‘.3.6.‘7,26 This absence of pertinent studies results from the paucity of established human cell lines with suitable properties (i.e. colony formation) for this sort of investigation. We conducted what we believe is the first investigation on the radioresponse of human colon carcinoma cells grown in vitro in the hope of providing clues for the usual failure of radiotherapy in controlling the growth of these types of cells. Doseresponse survival curves obtained on asynchronous exponentially growing LoVo cells yielded parameters well within the range of those determined for other mammalian cells. Furthermore, the cultured colon carcinoma cells have a capacity to absorb sublethal damage (quantified by 0,) and a radiosensitivity of the critical target (quantified by 0,) similar to that of other human cells in vitro. The cells possess a moderate capacity to recover from a “priming” dose of radiation with restitution of the shoulder region of the survival curve. The survival response of synchronized cells showed only moderate (3.5-fold) differences between the most and least sensitive stages of the cycle. Hence, it appears that no intrinsic radiobiological property of colon carcinoma cells justifies the usual poor clinical results obtained by radiotherapy in the treatment of this neoplasm. Another possibility for the different responses noted in vitro and in uivo may reside in the different conditions under which the cells proliferate. While our experiments were conducted on exponentially growing elements under abundant oxygenation and anabolite supply, a great number of cells in uivo grow under anoxic and ischemic conditions which may give rise to a large population of nonproliferating cells.‘8,24 This fact is supported by the low LI values (which quantifies the proportion of cells in DNA synthesis) measured for these tumors in ~ivo.~~ Thus, the radioresistance of colon carcinoma might be a function of population growth kinetics, an explanation suggested also for in vitro-in vivo differences in the radioresponse of human melanoma cells.’ Yet, the survival of LoVo cells in stationary phase of growth was not significantly different from that observed for exponentially growing cells; in fact, it was somewhat lower. This lower survival resulted primarily from a reduction of the capacity to absorb sublethal damage as quantified by a smaller Dq. This effect has been reported for other cell lines and attributed to a decrease in the capacity of stationary phase cells to repair sublethal and potentially lethal damage.15 Our studies would indicate that LoVo cells in stationary phase also have a reduced capacity to repair potentially lethal damage as evidenced by similar survival curves regardless of the interval between irradiation and subsequent harvesting.
Radioresponse of cultured colon carcinoma cells 0 B.
Ol-
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Fig. 5. Dose-dependent
survival of synchronized and 11 hr, respectively,
LoVo
cells. G,, S and G, phase cells were selected 22, 2 after release of the TdR block.
Cells in stationary phase of growth generally have been accepted as an adequate in vitro model for in uiuo resting populations.‘s Our results for cultured human colon carcinoma cells would suggest that either stationary phase cultures are not an appropriate reflection of noncycling cells in vivo, or that the poor radioresponse of colon carcinoma is independent of both intrinsic cell properties and some of the population growth kinetics properties. Alternatively, in
vivo radioresistance might be a function of inadequate oxygen s~pply*~ while in vitro sensitivity depends on the oxygenation effect.r4 Additional studies must be conducted under anoxic conditions on both multicellular spheroid cultures (another in vitro system purportedly reflecting in uivo resting cells)23 and on other colon carcinoma cells lines16 in order to corroborate these conclusions.
REFERENCES 1. Barendsen, G.W.: Do,se survival curves of human cells in tissue culture irradiated with alpa, beta, 20 kV and 200 kV X-radiation. Nature 193: 1153-l 155, 1962. 2. Barlogie, B., Drewinko, B., Johnston, D.A., Hauss, W.H., Freireich, E.J.: Pulse cytophotometric analysis of synchronized cells in vitro. Cancer Res. 36: 11761181, 1976. 3. Barranco, S.C., Romsdahl, M.M., Humphrey, R.M.: The radiation response of human malignant melanoma cells grown in vitro. Cancer Res. 31: 830-833, 1971. 4. Byfield, J.E.: The role of radiation repair mechanisms in radiation treatment failures. Cancer Chemoth. Rept. 58: 527-538, 1974. 5. Carter, S.K.: Large bowel cancer. The current status of treatment. .Z. Natl Cancer Znstit. 56: 3-10, 1976. 6. Drewinko, B., Humphrey, R.M., Trujillo, J.M.: The radiation response of a long-term culture of human lymphoid cells--I. Asynchronous populations. Znt. Z. Rad. Biol. 21: 361-3713, 1972. 7. Drewinko, B., Romsdahl, M.M., Yang, L.Y., Ahearn, M.J., Trujillo, J.M.: Establishment of a human carcinoembryonic antigen-producing colon adenocarcinema cell line. Cancer Res. 36: 467-475, 1976. 8. Drewinko, B., Yang, L.Y.: Restriction of CEA synthesis to the stationary phase of growth of cultured
human colon carcinoma cells. Exp. Cell Res. 101: 414416, 1976. 9. Egan, M.L., Todd, C.W.: Carcinoembryonic antigen: synthesis by a continuous line of adenocarcinoma cells. J. Nat1 Cancer Znstit. 49: 887-889,
1972.
10. Elkind, M.M., Whitmore, G.F.: The radiobiology of cultured mammalian cells. New York, Gordon & Breach, 1967. 11. Fogh, J., Trempe, G.: New human tumor cell lines. In Human Tumor Cells in vitro, ed. by Fogh, J. New York, Plenum, 1975, pp. 115-154. 12. Friedman, M.: Aspects of radiation biology and radiation pathology observed during the treatment of cancer in man. Br. J. Radiol. 48: 81-96, 1975. 13. Goldenberg, D.M., Pavia, R.M., Hansen, H.J., Vandevoorde, J.P.: Synthesis of carcinoembryonic antigen in uitro. Nature New Biol. 239: 189-190, 1972.
14. Gray, L.H.: Radiobiologic basis of oxygen as a modifying factor in radiation therapy. Am. J. Roentgen. 85: 803-815, 1961. 15. Hahn, G.M., Little, J.B.: Plateau-phase cultures of mammalian cells. An in vitro model for human cancer. Curr. Topics Rad. Res. Quart. 8: 39-83, 1972.
16. Leibovitz, A., Stimson, J.C., McCombs, W.B., McCoy, C.E., Mazur, K.C., Mabry, M.D.: Classification of
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21.
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human colorectal adenocarcinoma cell lines. Cancer Res. 36: 4562-4569, 1976. Little, J.B.: Repair of sub-lethal and potentially lethal radiation damage in plateau phase cultures of human cells. Nature, Lond. 224: 804806, 1969. Mendelsohn, M.L.: Autoradiographic analysis of cell proliferation in spontaneous breast cancer of C3H mouse-III. Growth fraction. J. Nat/ Cancer Znstit. 28: 1015-1029, 1962. Moertel, C.G.: Large bowel. In Cancer Medicine, ed. by Holland, J.F., Frei, E. Philadelphia, Pennsylvania, Lea & Febiger, 1973, pp. 1597-1631. National Bureau of Standards. Report of the International Commission on Radiological Units and Measurements (ICRU). National Bureau of Standards (U.S.), Handbook No. 62, 1957. Rutzky, L.P., Tom, B.H., Tomita, J.T., Kahan, B.D.: Ch_aracterization of a human colonic cell line: Kinetics of CEA synthesis. In Vitro 12: 328, 1976.
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22. Steel, G.G., Lamerton, L.F.: Cell population kinetics and chemotherapy. Nat1 Cancer Znstit. Monogr. 30: 29-50, 1969. 23. Sutherland, R.M., McCredie, J.A., Inch, W.R.: Growth of multicell spheroids in tissue culture as a model of nodular carcinomas. J. Nat/ Cancer Znstit. 46: 113-120, 1971. 24. Tannock, I.F.: The relation between cell proliferation and the vascular system in a transplanted mouse mammary tumor. Br. J. Cancer 22: 258-273, 1968. W.A.F., Watrach, A.M., Schmale, J.D., 25. Tompkins, Schultz, R.M., Harris, J.A.: Cultural and antigenic properties of newly established cell strain derived from adenocarcinomas of the human colon and rectum, J. Nat1 Cancer Znstit. 52: 1101, 1974. 26. Weichselbaum, R.R., Epstein, J., Little, J.B.: In vitro cellular radiosensitivity of human malignant tumors. Europ. J. Cancer 12: 47-51, 1976.
