Europ. J. Cancer Vol. 11, pp. 175-179. Pergamon Press 1975. Printed in Great Britain
Cytokinetic Considerations of Murine Neuroblastoma (C1300) as a Screening Model for the Childhood Disease* JERRY Z. FINKLESTEIN t and J O H N WEINER~. t Department of Pediatrics, University of California, Los Angeles, and Harbor General Hospital, Torrance, California, and +the University of Southern California, U.S.A. Abstra©t--Kineti,~ studies are presented for the Murine Neuroblastoma C1300 system. Animals receiving subcutaneous inoculations of 106 cells develop a palpable tumor with a mean diameter of 1.3 an in 10 days. The growth characteristics of palpable disease are consistent with a Gompertzian curve distribution; the doubling time of palpable disease is 10 days. Autoradiographic studies demonstrated a D N A synthesis time 0f 4"52 hr and a total generation time of 14-1 hr. These considerations and the sensitivity of the model to cell cycle nonspecific agents suggest that the C1300 system deservesfurther study as a potential model of the human disease.
blastoma will respond [4]. BCNU and cytosine arabinoside have not received an adequate trial in children with this entity. Although vincristine has been used alone with limited success, most clinicians prefer to use it with cyclophosphamide [5]. These results indicate that further study of the C1300 model is warranted, to determine whether it is a valid model for the screening of chemotherapeutic regimens proposed for use in children with metastatic disease. The murine neuroblastoma model has a number of factors which make it unique among animal tumor systems. The tumor cells produce acetylcholine esterase and the enzymes necessary for the synthesis of neurotransmitters in vitro, and under certain culture conditions axon formation takes place [6]. Morphological maturation can be accelerated in vitro and differentiating ceils can be selected with B U D R [7, 8]. Histopathologic and electron microscopic chemical studies of the tumor in vivo reveal the presence of catecholamines [9]. These biological properties suggest that the murine neuroblastoma model has m a n y features of the h u m a n disease. The purpose of this communication is to report results of growth kinetic studies of the C 1300 model and to compare its characteristics
INTRODUCTION
THE C130O mm~ne neuroblastoma system has been proposed as a model for the h u m a n disease [1]. In this model cyclophosphamide, BCNU and cytosine arabinoside prolong significantly the median duration of survival of animals treated with measurable tumor, whereas vincrisfine, actinomycin D, methotrexate, imidazole carboxamide and cyanocoblamin are ineffective in the dosage and regimes used [1]. All the above agents, with the exception of cyanocoblamin, are active in one or more of the classical animal tumor systems; murine L 1210 leukemia, murine P388 leukemia, murine B16 melanoma, murine Lewis lung carcinoma, Rat Walker 256 tumor and murine carcinoma 755 [2, 3]. However, only cyclophosphamide is generally accepted as the primary agent to which a significant number of children with metastatic neuroAccepted 18 October 1974. *Supported in part by Public Health Service Grants CA-14658, CA-14560 and CA-14089 from the National Cancer ][nstitute. Reprint address: Jerry Z. Finklestein, M.D., Department of Pediatric.,;, Harbor General Hospital, 1000 West Carson Street, Torrance, California 90509, U.S.A. 175 D
176
Jerry Z . Finklestein and John Weiner
with the published data of various common animal and human tumors.
MATERIAL
AND METHODS
Tumor induction
Details of the C1300 neuroblastoma tumor have been described previously. Briefly, tumor induction was performed by injecting adult A/J mice (more than eight weeks of age) with 10 6 tumor cells in the interscapular area. Animals were observed for the appearance of palpable disease and the subcutaneous tumors were measured with a vernier calipers at least thrice weekly until the animals expired. T u m o r volume was calculated from the mean diameter taking into account the double thickness of the skin overlying the tumor (2 x 0.5 mm) [10]. Generation time
The generation cycle of the tumor in vivo was calculated by determining the percent labeled mitosis in animals with a palpable tumor. Ninety animals with s.c. tumors of 1 cm in diameter were given single i.p. injections of 2 pCi/gm weight of tritiated thymidine (Sp Act 5 c/mm-Amersham/Searle) ; two mice were sacrificed at various times after injection of the D N A precursor. The tumors were dissected and fixed in Bouin's fixative for two hours, then processed for routine histology. Sections 4 pm thick were prepared and autoradiographs were made b y the dipping method, using Kodak Nuclear Track Emulsion NTB2. After one to three weeks of exposure at 4°C in a plastic fight-tight box, slides were developed in an anhydrous sodium sulfite and potassium bromide developer, with amidol 2,4-diaminophenol dihydrochloride as the developing agent, according to the method of Kopriwa and Leblond [11]. The developed slides were then stained with Harris hematoxylin and 1% alcoholic eosin. Cells with five or more grains were considered labeled. The percent of labeled mitoses (PLM) was determined from the available number of mitoses per slide, which was always over 100. The P L M after the pulse label were ther/plotted against time, and the form of the curve was obtained. The cell cycle time and the components of the cell cycle were determined using the analysis of Mendelsohn and Takashi [12]. RESULTS Animals receiving s.c. inoculations of 10 6
cells develop a palpable tumor with a mean diameter of 1.3 cm in 10 days. The frequency distribution of the measured mean diameters of 119 different mice observed 10 days following injection are given in Table 1. The relationship Table 1. Frequency table f o r the mean diameters o f subcutaneous tumors in 119 mice at 10 days following the injection o f 106 viable neuroblastoma cells. The average diameter was 1.35 cm with a S.D. o f 0.25351 and a range o f 1 to 1.9 cm
Mean dia.
Frequency
Percentage
Cumulative frequency
Cumulative percentage
1.00 l'10 1"20 1.30 1.40 1.50 1.60 1.70 1.80 1'90
15 10 15 17 24 15 8 13 1 I
12.60 8.40 12"60 14-28 20.16 12"60 6.72 10"92 0.84 0"84
15 25 40 57 81 96 104 117 118 119
12.60 21.00 33.61 47.89 68.06 80"67 87.39 98-31 99.15 100"00
between time from injection to maximum tumor size is shown in Fig. 1. The least square line was calculated by the formula: estimated size = 2"14+0.08 (Time--16.66). The measured doubling time is i 0 days.
3.6.~
:.
2.9-. 2.5-
•
• o~j" fi..~o
• O~0/O • ~.JJ~O • •
1.8-
•
OOOO OOO0
1.51.2: I0.0
14.8 24.8 TIME (Days)
36.6
Fig. 1. Maximum tumor size with time in 119 control animals. The scales in the figure are the arithmetic equivalent of the log of the measurements. Each dot reflects one or more observations at the same time and maximum tumor size intervals. The correlation coefficient between time and tumor size is 0.70 (P < 5%). The least square line based on the original arithmetic scales was equivalent with a co~relation of 0-6. The measured doubling time is 10 days.
The percentage of labeled mitosis was determined in tumors from animals sacrificed at various times, from 1 to 40 hr following the injection of the pulse label of tritiated thymidine. Variation of transit time was observed and an asymmetrical curve was obtained (Fig. 2). The
CytokineticConsiderationsof Murine Neuroblastoma (C1300)
177
I00-
,.,o 8 0 -
P-
w 60_.1 _j~j
~
~ 40I..-
i
20-
'2o TIME
Fig. 2.
(Hours)
Cell cycle in vivo of C1300 neuroblastoma. Each point represents the percent labeled mitosis after a developing time of 6, 9 or 21 days.
cell cycle parameters were calculated by the Mendelsohn-Takahashi method [12], using the following estimates: 1. The height of the first peak, H peak = 0.66. 2. The height of the first trough, H trough = 0-12. 3. Time to last peak on the plateau Tn 34 hr. 4. The area to Tn, An = 10.825 hr. Two waves of labeled mitosis were obtained. The peak was reached by 7.5 hr and was equal to 66 %. The cell cycle of the measurable tumor revealed a DNA synthesis time of 4.52 hr and a total generation time of 14.1 hr. The results are compared to other widely used tumor models in Table 2. Also illustrated in the Table is the result of art autoradiographic study of the generation cycle of the embryonic neural tube in a mouse of 10 days' gestation.
]DISCUSSION While any number of solid tumors in animals can serve as a useful model for human neoplasia, the lack of correlation between anticancer drug activity in anirr, al screens and in childhood neuroblastoma is striking. The C1300 murine system characteristics which make it attractive to the chemotherapist are: transplantability, uniform lethality', responsiveness to cyclophosphamide [1] and to radiotherapy [13]. Furthermore, the tumor has been found to have catecholamine-like substances on electron microscopic chemical studies [9]. The growth of a tumor depends on the length of the cell cycle, the fraction of proliferating cells and the rate of cell loss. In a uniformly distributed, uniformly cycling population, pulse-labeling with tritiated thymidine results in a "trapezoid" percent labeled mitosis (PLM) curve with 100 % of the cells labeled. However,
if cells are distributed uniformly in cycle, they have transit times which obey Gaussian distributions with variation in the transit times. Methods used to analyze these cell cycle curves have included the "boundary method" including the "half-height method" [12]. Since curves with various transit times demonstrate isocyclic waves, it is difficult to analyze them without arbitrarily extrapolating the tails of the first wave to zero PI, M or inserting an arbitrary boundary between the first and second troughs. However, P L M curves consist not only of variable transit times but also exponential age distributions. The error with the boundary method increases as the height of the first peak lowers; it is for this reason that Mendelsohn and Takahashi proposed the peakheight estimator level based on a synthetic P L M curve; their methodology was utilized in the calculation reported for the C1300 neuroblastoma model. The P L M peak of 66 % is another reflection of a commonly observed finding; namely, damping-out of labeled mitosis. It emphasizes the point that the growth of a tumor is the result of cell production and various types of cell loss. Deviations in the median cell cycle time and the population doubling time of solid tumors may result from a decreasing growth fraction, increasing cell loss, increasing cell cycle time, or any combination thereof [14]. The murine system has a short DNA synthesis (Ts) time, which is similar to that seen in the ten-day murine embryonic neural tube [15]. The 4-0hr Ts in the embryo suggests the intriguing speculation that the murine C1300's kinetic characteristics are consistent with that of undifferentiated neural tissue [16]. The short generation time (Tc) in the embryonic neural tube is in keeping with the observation that during the course of differentiation, the need for specific proteins
Jerry Z. Finklestein and John Weiner
178 Table 2.
Kinetic parameters of the C1300 murine neuroblastoma system and selected animal and human tumors i
Cell mass (days after) implant)
Tumor Murine C1300 L1210 Leukemia LI210 Leukemia Mouse Embryo Neural Tube Lewis Lung Sa 180 BI6 Melanoma Leukemia Man Neuroblastoraa Children
(10 days) ( 0 days) ( 6 days) n/a 340 rag 600 nag 560 nag 1012 Ca
Approximate doubling time (days) 10 0.6 or > n/a 2"9 1"2 1"5 4 or > 4.2
(a) Ts (hr)
4.52 9 8.9 4.0 8.5 7.5 7.0 ca 20
i
(b) Tc (hr)
14.1 12.8 11.8 8.4 18.8 13.0 20"0 ca 40-80 40
w
Ts/Tc
References
0.32 0-7 0.74
[19] [201
0.48 0.45 0.57 0.35 ca .25-.50
ii
[15] [20] [21] [3] [22] [23, 24]
(a) Ts = DNA synthesis time. (b) Tc = Total generation time.
results in a lengthening of the duration of the cell cycle. The cell cycle characteristics of the C1300 system resemble other rodent tumors in that the generation time is between 11 and 20 hr (Table 2). The ratio of Ts/Tc closely parallels those observed in the Lewis lung and B16 melanoma. The doubling time of 10 days was obtained using time of maximum tumor size; this is consistent with a Gompertzian curve distribution. Kinetic data can be of help in the design of chemotherapy. In that respect, the C1300 resembles these rodent tumors since it appears to be preferentially sensitive to cell cycle phase nonspecific agents. This is not surprising since most animal tumor systems are sensitive to relatively high doses of alkylating agents [17]. Nevertheless, the kinetic characteristics and behavior of the C1300 system does provide the advantage of gaining basic knowledge regarding the relationship of kinetic and therapeutic data in a "neural" neoplasm with its short DNA synthesis time. Its relevance to h u m a n disease requires further study.
The cellular kinetics of the murine model with its short DNA synthesis time m a y explain the lack of activity of cell cycle specific agents. Furthermore, a review of the active agents in children with metastatic disease reveals that cyclophosphamide and other cell cycle nonspecific agents should be attempted in humans with this disease. In fact, an increased response rate in children with metastatic disease is found in patients receiving combination therapy with cyclophosphamide and imidazole carboxamide, two cell cycle nonspecific agents, plus vincristine [18]. The sensitivity of the murine neuroblastoma to cytosine arabinoside [1] may be due to its unique biologic characteristics and emphasizes the need to further evaluate the model as a chemotherapy model.
Aclmowlt~lgement--The authors wish to acknowledge the excellent technical assistance of Mss. Rowie Meshnik, Joan Scher and Karen Tittle.
REFERENCES
1. 2. 3. 4. 5.
J . Z . FINKLESTEIN,E. ARIMA,P. E. BYFIELD,J. E. BYFIELDand E. W. FONKALSRUD, Murinc neuroblastoma: A model of human disease. Cancer Chemotherp. Rep. 57, 405 (1973). S.A. SCHEPARTZ,Screening. Cancer Chemother. Rep. Part 3, 2, 3 (1971). D . P . GRISWOLD,JR., Consideration of the subcutaneously implanted B16 melanoma as a screening model for potential antieaneer agents. Cancer Chemother. Rep. Part 2, 1, 315 (1972). W . G . TmJm'~AN and M. H. DONALDSON,Cyelophosphamide (NSC-26271) therapy for children with neuroblastoma. CancerChemother. Rep. 51~ 399 (I 967). A . E . EVANS,R. M. HEYN, W. A. NEWTON,JR., and S. LEIKIN, Vineristine sulfate and cyclophosphamide for children with metastatic neuroblastoma. J. Amer. reed. Ass. 207, 1325 (1969).
Cytokinetic Considerations of Murine Neuroblastoma ( C 1300) 6. 7. 8. 9.
10. 11. 12.
13. 14. 15. 16. 17.
18. 19. 20. 21.
22. 23. 94.
G. AuovsTI-Tocco and G. SATO,Establishment of functional clonal lines of neurons from mouse neuroblastoma. Proc. nat. Acnd. Sci. (Wash.) 64~ 311 (1969). D. SCHUBERT,S. HUMPHREYS,C. BARONIand M. COHN,In vitro differentiation of a mouse neuroblastoma. Proe. nat. Acad. Sei. (Wash.) ~ 316 (1969). N.W. SEEDS,A. G. GILMAN,T. AMANOand M. W. NIRENBERO,Regulation of axon formation by clonal lines of a neural tumor. Proc. nat. Acad. Sci. (Wash.) 66, 160 (1970). J . Z . FINKLESTEIN, K. L. TITTLE, F. HIROSE, H. ITABASHI,A. MITCHELL and J. WEINER, A new approach to the design of effective treatment for neuroblastoma. Pediat. Res. 8~ 400 (1974). E. FRINDEL, E. P. MALAISE, E. ALPEN and M. TUBIANA, Kinetics of cell proliferation of an experimental tumor. Cancer Res. 27~ 1122 (1967). B.M. KOPRrWAand C. P. LEBLOND,Improvements in the coating technique of radioautography. J. Histochem. Cytochem. 10, 269 (1962). M . L . MENDELSOHNand M. TAKAHASHI,A critical evaluation of the Fraction of labeled mitosis method as applied to the analysis of tumor and other cell cycles. In The Cell Cycle and Cancer. (Edited by R. BASERGA) p. 58. Marcel Dekker~ New York (1971). E. ARIMA, J. E. BYFmLD,J. Z. FINKLESTEINand E. W. FONKAT.SRUD,An experimental model for the therapy of mouse neuroblastoma. J. Pedlar. Surg. 8, 757 (1973). R. BASERGA,The relationship of the cell cycle to tumor growth and control of cell division: A review. Cancer Res. 25~ 581 (1965). S . L . KAUFFM.~N,An autoradiographic study of the generation cycle in the ten day mouse embryo neural tube. Exp. Cell Res. 42~ 67 (1966). D. MALAMUD,Differentiation and the cell cycle, In The Cell Cycle and Cancer. (Edited by R. BASERGA)p. 132. Marcel Dekker, New York (1971). H . E . S~PPER, The cell cycle and chemotherapy of cancer, In The Cell Cycle and Cancer. (Edited by R. BASERGA)p. 358. Marcel Dekker, New York (1971). J. Z. FINKLESTEIN, S. LEIKIN, A. EVANS, M. KLEMPERER, I. BERNSTEIN, R. HITTLEand G. D. HAMMOND,Chemotherapy for metastatic neuroblastoma. Proc. Amer. Ass. Cancer Res. 15~ 44 (1974). G . P . WHEELER, B. J. BOWDON, L. J. WILKOFFand E. A. DUTMADaE, The cell cycle of leukemia L1210 cells in vivo and in vitro. Proc. Soc. exp. Biol. 126~ 903 (1967). R.A. YANKEE, V. T. DEVITAand S. PERRY, The cell cycle of leukemia L1210 cells in vivo. Cancer Res. 27~ 2381 (1967). D . P . GRISWOLD,F. M. SCHABEL,W. S. WILCOX, L. SIMPSON-HERmaN and H. E. SKIPPER, Success and failure in the treatment of solid tumors. I. Effects of cyclophosphamide (NSC-26271) on primary and metastatic plasmacytoma in the hamster. Cancer Chemother. Rep. 52~ 345 (1968). S. KILLMANN,Acute leukemia: The kinetics of leukemic blast cells in man. Ser. Haematol. 1~ 38 (1968). H . P . WAGNER and H. K~ER, Cell proliferation in neuroblastoma. Europ. J. Cancer 6~ 369 (1968). W. AHERN~ and P. BUCK, The potential cell population doubling time in neuroblastoma and nephroblastoma. Brit. J. Cancer 25~ 691 (1971).
179
Cytokinetic Considerations of Murine Neuroblastoma (C1300) as a Screening Model for the Childhood Disease* JERRY Z. FINKLESTEIN t and J O H N WEINER~. t Department of Pediatrics, University of California, Los Angeles, and Harbor General Hospital, Torrance, California, and +the University of Southern California, U.S.A. Abstra©t--Kineti,~ studies are presented for the Murine Neuroblastoma C1300 system. Animals receiving subcutaneous inoculations of 106 cells develop a palpable tumor with a mean diameter of 1.3 an in 10 days. The growth characteristics of palpable disease are consistent with a Gompertzian curve distribution; the doubling time of palpable disease is 10 days. Autoradiographic studies demonstrated a D N A synthesis time 0f 4"52 hr and a total generation time of 14-1 hr. These considerations and the sensitivity of the model to cell cycle nonspecific agents suggest that the C1300 system deservesfurther study as a potential model of the human disease.
blastoma will respond [4]. BCNU and cytosine arabinoside have not received an adequate trial in children with this entity. Although vincristine has been used alone with limited success, most clinicians prefer to use it with cyclophosphamide [5]. These results indicate that further study of the C1300 model is warranted, to determine whether it is a valid model for the screening of chemotherapeutic regimens proposed for use in children with metastatic disease. The murine neuroblastoma model has a number of factors which make it unique among animal tumor systems. The tumor cells produce acetylcholine esterase and the enzymes necessary for the synthesis of neurotransmitters in vitro, and under certain culture conditions axon formation takes place [6]. Morphological maturation can be accelerated in vitro and differentiating ceils can be selected with B U D R [7, 8]. Histopathologic and electron microscopic chemical studies of the tumor in vivo reveal the presence of catecholamines [9]. These biological properties suggest that the murine neuroblastoma model has m a n y features of the h u m a n disease. The purpose of this communication is to report results of growth kinetic studies of the C 1300 model and to compare its characteristics
INTRODUCTION
THE C130O mm~ne neuroblastoma system has been proposed as a model for the h u m a n disease [1]. In this model cyclophosphamide, BCNU and cytosine arabinoside prolong significantly the median duration of survival of animals treated with measurable tumor, whereas vincrisfine, actinomycin D, methotrexate, imidazole carboxamide and cyanocoblamin are ineffective in the dosage and regimes used [1]. All the above agents, with the exception of cyanocoblamin, are active in one or more of the classical animal tumor systems; murine L 1210 leukemia, murine P388 leukemia, murine B16 melanoma, murine Lewis lung carcinoma, Rat Walker 256 tumor and murine carcinoma 755 [2, 3]. However, only cyclophosphamide is generally accepted as the primary agent to which a significant number of children with metastatic neuroAccepted 18 October 1974. *Supported in part by Public Health Service Grants CA-14658, CA-14560 and CA-14089 from the National Cancer ][nstitute. Reprint address: Jerry Z. Finklestein, M.D., Department of Pediatric.,;, Harbor General Hospital, 1000 West Carson Street, Torrance, California 90509, U.S.A. 175 D
176
Jerry Z . Finklestein and John Weiner
with the published data of various common animal and human tumors.
MATERIAL
AND METHODS
Tumor induction
Details of the C1300 neuroblastoma tumor have been described previously. Briefly, tumor induction was performed by injecting adult A/J mice (more than eight weeks of age) with 10 6 tumor cells in the interscapular area. Animals were observed for the appearance of palpable disease and the subcutaneous tumors were measured with a vernier calipers at least thrice weekly until the animals expired. T u m o r volume was calculated from the mean diameter taking into account the double thickness of the skin overlying the tumor (2 x 0.5 mm) [10]. Generation time
The generation cycle of the tumor in vivo was calculated by determining the percent labeled mitosis in animals with a palpable tumor. Ninety animals with s.c. tumors of 1 cm in diameter were given single i.p. injections of 2 pCi/gm weight of tritiated thymidine (Sp Act 5 c/mm-Amersham/Searle) ; two mice were sacrificed at various times after injection of the D N A precursor. The tumors were dissected and fixed in Bouin's fixative for two hours, then processed for routine histology. Sections 4 pm thick were prepared and autoradiographs were made b y the dipping method, using Kodak Nuclear Track Emulsion NTB2. After one to three weeks of exposure at 4°C in a plastic fight-tight box, slides were developed in an anhydrous sodium sulfite and potassium bromide developer, with amidol 2,4-diaminophenol dihydrochloride as the developing agent, according to the method of Kopriwa and Leblond [11]. The developed slides were then stained with Harris hematoxylin and 1% alcoholic eosin. Cells with five or more grains were considered labeled. The percent of labeled mitoses (PLM) was determined from the available number of mitoses per slide, which was always over 100. The P L M after the pulse label were ther/plotted against time, and the form of the curve was obtained. The cell cycle time and the components of the cell cycle were determined using the analysis of Mendelsohn and Takashi [12]. RESULTS Animals receiving s.c. inoculations of 10 6
cells develop a palpable tumor with a mean diameter of 1.3 cm in 10 days. The frequency distribution of the measured mean diameters of 119 different mice observed 10 days following injection are given in Table 1. The relationship Table 1. Frequency table f o r the mean diameters o f subcutaneous tumors in 119 mice at 10 days following the injection o f 106 viable neuroblastoma cells. The average diameter was 1.35 cm with a S.D. o f 0.25351 and a range o f 1 to 1.9 cm
Mean dia.
Frequency
Percentage
Cumulative frequency
Cumulative percentage
1.00 l'10 1"20 1.30 1.40 1.50 1.60 1.70 1.80 1'90
15 10 15 17 24 15 8 13 1 I
12.60 8.40 12"60 14-28 20.16 12"60 6.72 10"92 0.84 0"84
15 25 40 57 81 96 104 117 118 119
12.60 21.00 33.61 47.89 68.06 80"67 87.39 98-31 99.15 100"00
between time from injection to maximum tumor size is shown in Fig. 1. The least square line was calculated by the formula: estimated size = 2"14+0.08 (Time--16.66). The measured doubling time is i 0 days.
3.6.~
:.
2.9-. 2.5-
•
• o~j" fi..~o
• O~0/O • ~.JJ~O • •
1.8-
•
OOOO OOO0
1.51.2: I0.0
14.8 24.8 TIME (Days)
36.6
Fig. 1. Maximum tumor size with time in 119 control animals. The scales in the figure are the arithmetic equivalent of the log of the measurements. Each dot reflects one or more observations at the same time and maximum tumor size intervals. The correlation coefficient between time and tumor size is 0.70 (P < 5%). The least square line based on the original arithmetic scales was equivalent with a co~relation of 0-6. The measured doubling time is 10 days.
The percentage of labeled mitosis was determined in tumors from animals sacrificed at various times, from 1 to 40 hr following the injection of the pulse label of tritiated thymidine. Variation of transit time was observed and an asymmetrical curve was obtained (Fig. 2). The
CytokineticConsiderationsof Murine Neuroblastoma (C1300)
177
I00-
,.,o 8 0 -
P-
w 60_.1 _j~j
~
~ 40I..-
i
20-
'2o TIME
Fig. 2.
(Hours)
Cell cycle in vivo of C1300 neuroblastoma. Each point represents the percent labeled mitosis after a developing time of 6, 9 or 21 days.
cell cycle parameters were calculated by the Mendelsohn-Takahashi method [12], using the following estimates: 1. The height of the first peak, H peak = 0.66. 2. The height of the first trough, H trough = 0-12. 3. Time to last peak on the plateau Tn 34 hr. 4. The area to Tn, An = 10.825 hr. Two waves of labeled mitosis were obtained. The peak was reached by 7.5 hr and was equal to 66 %. The cell cycle of the measurable tumor revealed a DNA synthesis time of 4.52 hr and a total generation time of 14.1 hr. The results are compared to other widely used tumor models in Table 2. Also illustrated in the Table is the result of art autoradiographic study of the generation cycle of the embryonic neural tube in a mouse of 10 days' gestation.
]DISCUSSION While any number of solid tumors in animals can serve as a useful model for human neoplasia, the lack of correlation between anticancer drug activity in anirr, al screens and in childhood neuroblastoma is striking. The C1300 murine system characteristics which make it attractive to the chemotherapist are: transplantability, uniform lethality', responsiveness to cyclophosphamide [1] and to radiotherapy [13]. Furthermore, the tumor has been found to have catecholamine-like substances on electron microscopic chemical studies [9]. The growth of a tumor depends on the length of the cell cycle, the fraction of proliferating cells and the rate of cell loss. In a uniformly distributed, uniformly cycling population, pulse-labeling with tritiated thymidine results in a "trapezoid" percent labeled mitosis (PLM) curve with 100 % of the cells labeled. However,
if cells are distributed uniformly in cycle, they have transit times which obey Gaussian distributions with variation in the transit times. Methods used to analyze these cell cycle curves have included the "boundary method" including the "half-height method" [12]. Since curves with various transit times demonstrate isocyclic waves, it is difficult to analyze them without arbitrarily extrapolating the tails of the first wave to zero PI, M or inserting an arbitrary boundary between the first and second troughs. However, P L M curves consist not only of variable transit times but also exponential age distributions. The error with the boundary method increases as the height of the first peak lowers; it is for this reason that Mendelsohn and Takahashi proposed the peakheight estimator level based on a synthetic P L M curve; their methodology was utilized in the calculation reported for the C1300 neuroblastoma model. The P L M peak of 66 % is another reflection of a commonly observed finding; namely, damping-out of labeled mitosis. It emphasizes the point that the growth of a tumor is the result of cell production and various types of cell loss. Deviations in the median cell cycle time and the population doubling time of solid tumors may result from a decreasing growth fraction, increasing cell loss, increasing cell cycle time, or any combination thereof [14]. The murine system has a short DNA synthesis (Ts) time, which is similar to that seen in the ten-day murine embryonic neural tube [15]. The 4-0hr Ts in the embryo suggests the intriguing speculation that the murine C1300's kinetic characteristics are consistent with that of undifferentiated neural tissue [16]. The short generation time (Tc) in the embryonic neural tube is in keeping with the observation that during the course of differentiation, the need for specific proteins
Jerry Z. Finklestein and John Weiner
178 Table 2.
Kinetic parameters of the C1300 murine neuroblastoma system and selected animal and human tumors i
Cell mass (days after) implant)
Tumor Murine C1300 L1210 Leukemia LI210 Leukemia Mouse Embryo Neural Tube Lewis Lung Sa 180 BI6 Melanoma Leukemia Man Neuroblastoraa Children
(10 days) ( 0 days) ( 6 days) n/a 340 rag 600 nag 560 nag 1012 Ca
Approximate doubling time (days) 10 0.6 or > n/a 2"9 1"2 1"5 4 or > 4.2
(a) Ts (hr)
4.52 9 8.9 4.0 8.5 7.5 7.0 ca 20
i
(b) Tc (hr)
14.1 12.8 11.8 8.4 18.8 13.0 20"0 ca 40-80 40
w
Ts/Tc
References
0.32 0-7 0.74
[19] [201
0.48 0.45 0.57 0.35 ca .25-.50
ii
[15] [20] [21] [3] [22] [23, 24]
(a) Ts = DNA synthesis time. (b) Tc = Total generation time.
results in a lengthening of the duration of the cell cycle. The cell cycle characteristics of the C1300 system resemble other rodent tumors in that the generation time is between 11 and 20 hr (Table 2). The ratio of Ts/Tc closely parallels those observed in the Lewis lung and B16 melanoma. The doubling time of 10 days was obtained using time of maximum tumor size; this is consistent with a Gompertzian curve distribution. Kinetic data can be of help in the design of chemotherapy. In that respect, the C1300 resembles these rodent tumors since it appears to be preferentially sensitive to cell cycle phase nonspecific agents. This is not surprising since most animal tumor systems are sensitive to relatively high doses of alkylating agents [17]. Nevertheless, the kinetic characteristics and behavior of the C1300 system does provide the advantage of gaining basic knowledge regarding the relationship of kinetic and therapeutic data in a "neural" neoplasm with its short DNA synthesis time. Its relevance to h u m a n disease requires further study.
The cellular kinetics of the murine model with its short DNA synthesis time m a y explain the lack of activity of cell cycle specific agents. Furthermore, a review of the active agents in children with metastatic disease reveals that cyclophosphamide and other cell cycle nonspecific agents should be attempted in humans with this disease. In fact, an increased response rate in children with metastatic disease is found in patients receiving combination therapy with cyclophosphamide and imidazole carboxamide, two cell cycle nonspecific agents, plus vincristine [18]. The sensitivity of the murine neuroblastoma to cytosine arabinoside [1] may be due to its unique biologic characteristics and emphasizes the need to further evaluate the model as a chemotherapy model.
Aclmowlt~lgement--The authors wish to acknowledge the excellent technical assistance of Mss. Rowie Meshnik, Joan Scher and Karen Tittle.
REFERENCES
1. 2. 3. 4. 5.
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