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Friend leukemia cells: Relationship between differentiation, clonogenicity and malignancy
Cell Differentiation, 7 (1978) 1--10
1
© Elsevier/North-Holland Scientific Publishers Ltd.
FRIEND LEUKEMIA CELLS: RELATIONSHIP BETWEEN DIFFERENTIATION, CLONOGENICITY AND MALIGNANCY*
Harvey D. PREISLER, Gregory CHRISTOFF, Peter REESE, Paul PAVELIC and Youcef RUSTUM
Departments o f Medicine A and Experimental Therapeutics, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N e w York 14263, U.S.A. Accepted 23 December, 1977
Friend leukemia cells were cultured in vitro in the presence or absence of agents which induce erythroid differentiation. The cultures were harvested and the degree of differentiation determined. Clonogenicity of the cells in vitro and malignancy in vivo were determined as well. There was an inverse exponential relationship between the degree of differentiation and the clonogenicity of the culture. Differentiation was also associated with a modest decline in malignancy. Of interest was the observation that bromodeoxyuridine inhibited the biochemical manifestations of erythroid differentiation, but did not prevent the decline in clonogenicity which accompanied differentiation.
Friend leukemia cells provide a model system for studying the factors which regulate the differentiation of neoplastic cells. This system also provides a means for testing the proposition that an alternate approach to the treatment of neoplasms in man could be the induction of differentiation of malignant cells. In previous publications, we have demonstrated that once the differentiation of Friend cells has begun, the process is irreversible [3] and is associated with a loss of clonogenicity in vitro (Preisler et al., 1975). Other studies from this laboratory have raised the possibility that the decrease in clonogenicity might be related to the toxic effects of DMSO as manifested by a fall in acid-soluble ribonucleotide pools which occurred within 0.5 h of exposure to DMSO (Preisler and Rustum, 1975). The present communication reports studies of the relationship between differentiation, clonogenicity in vitro, and malignancy in vivo in the Friend leukemia system. METHODS
Cells
Friend leukemia cells were grown in suspension cultures as previously * This s t u d y was supported by USPHS Grant No. 5834 from the National Cancer Institute and USPHS Grant No. CA-16056. Abbreviation: PBS, phosphate buffered saline.
described (Preisler and Giladi, 1975). As indicated, cells were cultured in the presence or absence of agents capable of inducing erythroid differentiation in vitro (Preisler and Lyman, 1975; Preisler et al., 1976b). Preliminary studies had demonstrated t h a t the induction of differentiation by the agents employed in the current studies did n o t result in an alteration in acidsoluble ribonucleotide pools (Preisler and Rustum, pers. comm.). Clones IV and 745D were used (Preisler et al., 1976c). Clone IV is induced to differentiate in vitro by DMSO, tetramethylurea, dimethylacetamide and butyric acid. Clone 745D is refractory to DMSO (Preisler et al., 1976c).
Estimate of differentiation and clonogenicity Cultures were harvested after 5 days of growth in the presence or absence of inducers of differentiation and aliquots removed for determination of the total a m o u n t of heme/107 cells (Preisler and Giladi, 1975), the proportion of benzidine positive cells in the culture (Preisler and Giladi, 1975), the clonogenicity of the cells in vitro (Preisler et al., 1976a), and determination of malignant potential.
Assay of malignant potential Syngeneic female DBA2/J mice aged 2--3 months were .inoculated subcutaneously on the flank with either 5 × 10 s or 5 × 104 cells suspended in 0.1 phosphate buffered saline (PBS). The cells were washed with PBS prior to inoculation into the mice. There were 10 mice in each group. They were housed 10 to a cage and provided food and water ad lib. Tumors were measured twice a week with calipers. The mice were autopsied at the time of death and tumors were fixed in formalin, sectioned and stained for pathologic analysis.
Mathematical analysis of correlation between clonogenicity and differentiation For each experimental data set, the natural logarithm of number of colonies/106 cells was regressed on percent benzidine positive cells or heme/ 107 cells by least squares techniques. The models utilized were as follows: Ln (No. of colonies/106) = B0 + B1 (%B +) or
= B0 + B1 (heme/107), where the Bi's are the least squares regression coefficients. The coefficients represent the numeric values which produce 'the best fit' of a straight line function to the data. B0, for example, is an estimate of the number of benzidine positive cells in the absence of an inducing agent.
The hypotheses that there is no simple linear regression of the natural logarithm of the n u m b e r of colonies on either percent benzidine positive cells or heme/107 were tested via an F ratio at the ~ = 0.05 level. RESULTS
The question of the relationship between differentiation and clonogenicity was approached in 2 ways. The first approach involved the culturing of Clone IV cells for 5 days in the presence of one of several inducers of differentiation. The second approach involved the culture of cells in the presence of different concentrations of a single inducer compound. At the end of 5 days of culture, the percent of benzidine positive cells, the heme/107 cells and in the in vitro clonogenicity was measured. Table I gives the results of 2 such experiments. Fig. 1 expresses this data graphically. In both types of experiments, there was an inverse exponential relationship between the a m o u n t of heme in the culture or the percent of benzidine positive cells and the number of colonies formed in vitro. Of interest was the observation that B U d R inhibition of DMSO-induced differentiation failed to result in a reversion of clonogenicity back towards that of the control cultures.
TABLE I R e l a t i o n s h i p b e t w e e n degree o f d i f f e r e n t i a t i o n a n d c l o n o g e n i c i t y o f Clone IV cells %B+ cells
ug h e i n e / 1 0 7 cells
Colonies/103 cells
0,1 56 49 32 26
3 22 24 8.4 3.1
562 11 32 87 150
0 31.5 29 6.5 0 1.5 11
0.53 32.5 14.2 3.9 0.9 1.5 9.8
610 101 272 509 486 137 5.5
Expt. I Control DMSO (0.26 m ) Tetramethylurea (0.0086 m) D i m e t h y l a c e t a m i d e (0.01 m ) B u t y r i c acid (0.005 m )
Expt. H Control DMSO 0.26 m DMSO 0.02 rn DMSO 0.13 m DMSO 0.065 rn B U d R 2 ug/rnl B U d R 2 ug/rnl + DMSO 0.26 m
Cells were s e e d e d at a c o n c e n t r a t i o n o f 1 × 105 cells/ml in t h e p r e s e n c e or a b s e n c e o f an i n d u c e r o f d i f f e r e n t i a t i o n . A f t e r 5 d a y s o f g r o w t h the c u l t u r e s were harvested. F o r E x p . I: F Colonies vs. %B + cells = 1 0 0 . 3 2 " ; F c o l o n i e s vs. ug h e i n e = 1 0 . 9 9 " . F o r E x p . II: F c o l o n i e s vs. %B + cells = 1 1 . 4 2 " ; F c o l o n i e s vs. ug h e m e = 2 4 9 . 7 6 * . These values d o n o t i n c l u d e B U d R c o n t a i n i n g cultures. * F X / I - = 1, V ~ = 3 , ¢ ~ = 0 . 0 5 = 1 0 . 1 3
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The inverse exponential relationship between the number of colonies produced in vitro and the degree of differentiation in the culture when measured either by the percentage of benzidine positive cells in the culture or by the total amount of heme present in the culture was statistically significant in 4 of 4 experiments. This is evidenced by F ratios relating differentiation and clonogenicity given in Table I. Fig. 1 represents these studies graphically. Similar studies were conducted using Clone 7 4 5 D and DMSO as the T A B L E II
Effect of DMSO o n the elonogenicity o f Clone 7 4 5 D cells %B+ Cells
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0 0
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0 0
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Expt. I Control DMSO (0.26 m)
Expt. II Control DMSO ( 0 . 2 6 m )
Expt. III Control DMSO ( 0 . 2 6 m )
inducing agent. Table II illustrates that in 3 of 3 experiments DMSO failed to induce any evidence of differentiation, but clonogenicity was reduced by 23--44%.
Relationship between differentiation and malignancy In 4 individual experiments, groups of 10 mice were inoculated subcutaneously with Clone IV cells which had been cultured in the presence of an inducer of differentiation for 5 days. When 5 X 105 cells were inoculated there was no difference in survival between recipients of cells from control cultures or cells from differentiated cultures. However, mice which were inoculated with 5 X 104 cells from differentiated cultures survived longer than recipients of cells from undifferentiated cultures. Table III gives the survival data of 4 separate experiments along with data on clonogenicity and degree of differentiation of the injected fractions. Fig. 2 gives a survival plot from 2 experiments. While survival differences between controls and recipients of differentiated cultures are readily apparent, there do not appear to be major differences in survival between recipients of the various differentiated cell cultures, regardless of the degree of differentiation or in vitro clonogenicity of the donor culture. The malignancy of the Clone 745D cells described in Table III was also tested. In 2 of 3 studies there was no difference in malignancy between Clone 745D cells cultured in the presence or absence of DMSO. In one experiment recipients of DMSO-treated cells survived 40% longer than recipients of control cultures.
Biological characteristics of tumors For recipients of Clone IV cells, tumors were first detected several days later in recipients of differentiated cells than for recipients of control cells. Once the tumors appeared, their increase in size tended to parallel each other, although there were great individual differences from mouse to mouse. Reliable t u m o r weights and measurements at the time of death were impossible to obtain since surviving mice tended to eat the tumors of their dead cage mates. The tumors of mice which received differentiated cells exhibited minor morphological differences from those found in recipients of control cell cultures. The former tumors (regardless of the inducing agent employed) contained m a n y multinucleated giant cells (Fig. 3) while tumors produced by control cells contained few. We could not determine whether these cells were foreign body giant cells or perhaps megakaryocytes. Tumors produced by 745D cells (whether or n o t previously exposed to DMSO) differed from those produced by Clone IV cells in that the former contained few giant cells and the tumors grew more slowly. The median survival time for recipients of 5 X 104 control 745D cells were 57, 68 and 56 days, respectively. In 4
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Fig. 2. a: Graphic representation of survival data of Experiment II presented in Table III. b: Graphic representation of survival data of Experiment III given in Table III.
s e p a r a t e e x p e r i m e n t s r e c i p i e n t s o f 5 × 104 Clone IV cells h a d m e d i a n survivals o f 40, 46, 48 a n d 33 d a y s , r e s p e c t i v e l y ; h e n c e C l o n e IV cells were m o r e m a l i g n a n t t h a n D cells. Finally, in n o case did we see a n y e v i d e n c e of e r y t h r o i d d i f f e r e n t i a t i o n in t h e t u m o r s .
Fig. 3a. S e c t i o n o f a t u m o r o b t a i n e d f r o m a m o u s e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 5 × 10 a c o n t r o l C l o n e IV cells. N o t e r e t i c u l u m cells. O r i g i n a l m a g . x 6 7 0 . b: S e c t i o n o f a t u m o r o b t a i n e d From t h e m o u s e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 5 x 104 d i f f e r e n t i a t e d C l o n e I V cells. N o t e r e t i c u l u m cells, g i a n t cells. O r i g i n a l m a g . x 6 7 0 .
DISCUSSION
These experiments clearly demonstrate that the induction of differentiation of Friend leukemia cells in vitro is associated with a marked decline in clonogenicity. The decline appears to have 2 components. The major comp o n e n t is largely due to the process of differentiation per se since it occurs in the absence of apparent toxicity (as measured by soluble ribonucleotide pool sizes) and is mathematically related to the degree of differentiation. The presence of a second and minor toxic c o m p o n e n t is suggested by the observation that DMSO, which does n o t induce 745D cells to differentiate, causes a 20--30% decline in clonogenicity of these cells. These studies have also demonstrated that BUdR itself causes a decline in clonogenicity and that while BUdR can inhibit DMSO-induced differentiation (Conkie et al., 1974; Scher et al., 1973), it does not reverse the DMSOinduced decline in clonogenicity. Either only some aspects of DMSO-induced differentiation are inhibited by BUdR, or cells incubated with BUdR are damaged and the decrease in clonogenicity produced by BUdR is additive with that which occurs with differentiation. Additionally, the fact that Clone IV cells undergo DMSO-induced differentiation w i t h o u t significant alteration in acid-soluble ribonucleotide pool size confirms our earlier report t h a t such DMSO-induced alterations are n o t the result of differentiation (Preisler and Rustum, 1975) and also demonstrates that toxic effects of inducers can vary from cell line to cell line. The effects of DMSO on the malignancy of Friend cells demonstrated in these studies were much less than those reported by others (Friend et al., 1971). The number of animals used by the previous investigators were far fewer than those employed in the current studies and the previous investigators reported the results of only a single experiment. However, the cell line used in the present studies is different from that of the previous investigators and may account for some or all of the observed differences. The characteristics of the growth of the tumors produced by Clone IV cells strongly suggest that tumors produced by cells from differentiated cultures resulted from the proliferation of residual undifferentiated cells in the culture. Hence, the longer survival of mice which received differentiated cells resulted from the inoculation of substantially fewer malignant cells. The clonogenicity studies suggest that the number of residual clonogenic cells in differentiated cultures is several logs less than would be suspected by determining the percentage of benzidine positive cells in the culture. This observation is in accord with our previous studies which demonstrated a decline in clonogenicity prior to the detection of benzidine positive cells in the culture (Preisler et al., 1976c). One finding for which we have no explanation was the presence of giant cells in tumors produced by the inoculation of cells from differentiated cultures. We do not know if they are of host origin or t u m o r origin. If the former were true, then t h e y might represent host recognition of antigenic
10 differences b e t w e e n the h o s t and d i f f e r e n t i a t e d t u m o r cells and w o u l d suggest t h a t i m m u n o l o g i c a l factors m a y play a role in the longer survival of recipients o f d i f f e r e n t i a t e d cells. On the o t h e r h a n d , if the giant cells are o f t u m o r origin, t h e n it is possible t h a t either s o m e o f the d i f f e r e n t i a t e d cells replicated in vivo or alternatively t h a t the g r o w t h p o t e n t i a l o f the u n d i f f e r e n t i a t e d cells was altered b y the i n d u c i n g agents. H e n c e the relationship o f possible alterations in i m m u n o g e n i c i t y o c c u r r i n g d u r i n g d i f f e r e n t i a t i o n of F L C in vitro and the r e s u l t a n t decrease in m a l i g n a n t p o t e n t i a l in vivo is a q u e s t i o n y e t to be answered. ACKNOWLEDGEMENTS The authors would like to thank Mr. Shakerum Alamru, Mr. Bradley Bryant and Ms. Edythe Taylor for their technical assistance. REFERENCES Conkie, D., N. Affara, P.R. Harrison, J. Paul and K. Jones: J. Cell. Biol. 63, 414--419 (1974). Friend, C., W. Scher, J.G. Holland and J. Sato: Proc. Natl. Acad. Sci. (USA) 68, 378--382 (1971). Preisler, H.D., S. Bjornsson, M. Mori and G.H. Lyman: Br. J. Cancer 33,634--645 (1976a). Preisler, H.D., G. Christoff and E. Taylor: Blood 47,363--368 (1976b). Preisler, H.D. and M. Giladi: Cell. Physiol. 85,537--546 (1975). Preisler, H.D., J.D. Lutton, M. Giladi, K. Goldstein and E.D. Zanjani: Life Sci. 16, 1241-1252 (1975). Preisler, H.D. and G.H. Lyman: Cell Differentiation 4,179--185 (1975). Preisler, H.D. and Y. Rustum: Life Sci. 17, 1287--1290 (1975). Preisler, H.D., Y. Shiraishi, M. Mori and A.A. Sandberg: Cell Differentiation, 5, 207--216 (1976c). Scher, W., H.D. Preisler and C. Friend: J. Cell. Physiol. 81, 63--70 (1973).
1
© Elsevier/North-Holland Scientific Publishers Ltd.
FRIEND LEUKEMIA CELLS: RELATIONSHIP BETWEEN DIFFERENTIATION, CLONOGENICITY AND MALIGNANCY*
Harvey D. PREISLER, Gregory CHRISTOFF, Peter REESE, Paul PAVELIC and Youcef RUSTUM
Departments o f Medicine A and Experimental Therapeutics, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, N e w York 14263, U.S.A. Accepted 23 December, 1977
Friend leukemia cells were cultured in vitro in the presence or absence of agents which induce erythroid differentiation. The cultures were harvested and the degree of differentiation determined. Clonogenicity of the cells in vitro and malignancy in vivo were determined as well. There was an inverse exponential relationship between the degree of differentiation and the clonogenicity of the culture. Differentiation was also associated with a modest decline in malignancy. Of interest was the observation that bromodeoxyuridine inhibited the biochemical manifestations of erythroid differentiation, but did not prevent the decline in clonogenicity which accompanied differentiation.
Friend leukemia cells provide a model system for studying the factors which regulate the differentiation of neoplastic cells. This system also provides a means for testing the proposition that an alternate approach to the treatment of neoplasms in man could be the induction of differentiation of malignant cells. In previous publications, we have demonstrated that once the differentiation of Friend cells has begun, the process is irreversible [3] and is associated with a loss of clonogenicity in vitro (Preisler et al., 1975). Other studies from this laboratory have raised the possibility that the decrease in clonogenicity might be related to the toxic effects of DMSO as manifested by a fall in acid-soluble ribonucleotide pools which occurred within 0.5 h of exposure to DMSO (Preisler and Rustum, 1975). The present communication reports studies of the relationship between differentiation, clonogenicity in vitro, and malignancy in vivo in the Friend leukemia system. METHODS
Cells
Friend leukemia cells were grown in suspension cultures as previously * This s t u d y was supported by USPHS Grant No. 5834 from the National Cancer Institute and USPHS Grant No. CA-16056. Abbreviation: PBS, phosphate buffered saline.
described (Preisler and Giladi, 1975). As indicated, cells were cultured in the presence or absence of agents capable of inducing erythroid differentiation in vitro (Preisler and Lyman, 1975; Preisler et al., 1976b). Preliminary studies had demonstrated t h a t the induction of differentiation by the agents employed in the current studies did n o t result in an alteration in acidsoluble ribonucleotide pools (Preisler and Rustum, pers. comm.). Clones IV and 745D were used (Preisler et al., 1976c). Clone IV is induced to differentiate in vitro by DMSO, tetramethylurea, dimethylacetamide and butyric acid. Clone 745D is refractory to DMSO (Preisler et al., 1976c).
Estimate of differentiation and clonogenicity Cultures were harvested after 5 days of growth in the presence or absence of inducers of differentiation and aliquots removed for determination of the total a m o u n t of heme/107 cells (Preisler and Giladi, 1975), the proportion of benzidine positive cells in the culture (Preisler and Giladi, 1975), the clonogenicity of the cells in vitro (Preisler et al., 1976a), and determination of malignant potential.
Assay of malignant potential Syngeneic female DBA2/J mice aged 2--3 months were .inoculated subcutaneously on the flank with either 5 × 10 s or 5 × 104 cells suspended in 0.1 phosphate buffered saline (PBS). The cells were washed with PBS prior to inoculation into the mice. There were 10 mice in each group. They were housed 10 to a cage and provided food and water ad lib. Tumors were measured twice a week with calipers. The mice were autopsied at the time of death and tumors were fixed in formalin, sectioned and stained for pathologic analysis.
Mathematical analysis of correlation between clonogenicity and differentiation For each experimental data set, the natural logarithm of number of colonies/106 cells was regressed on percent benzidine positive cells or heme/ 107 cells by least squares techniques. The models utilized were as follows: Ln (No. of colonies/106) = B0 + B1 (%B +) or
= B0 + B1 (heme/107), where the Bi's are the least squares regression coefficients. The coefficients represent the numeric values which produce 'the best fit' of a straight line function to the data. B0, for example, is an estimate of the number of benzidine positive cells in the absence of an inducing agent.
The hypotheses that there is no simple linear regression of the natural logarithm of the n u m b e r of colonies on either percent benzidine positive cells or heme/107 were tested via an F ratio at the ~ = 0.05 level. RESULTS
The question of the relationship between differentiation and clonogenicity was approached in 2 ways. The first approach involved the culturing of Clone IV cells for 5 days in the presence of one of several inducers of differentiation. The second approach involved the culture of cells in the presence of different concentrations of a single inducer compound. At the end of 5 days of culture, the percent of benzidine positive cells, the heme/107 cells and in the in vitro clonogenicity was measured. Table I gives the results of 2 such experiments. Fig. 1 expresses this data graphically. In both types of experiments, there was an inverse exponential relationship between the a m o u n t of heme in the culture or the percent of benzidine positive cells and the number of colonies formed in vitro. Of interest was the observation that B U d R inhibition of DMSO-induced differentiation failed to result in a reversion of clonogenicity back towards that of the control cultures.
TABLE I R e l a t i o n s h i p b e t w e e n degree o f d i f f e r e n t i a t i o n a n d c l o n o g e n i c i t y o f Clone IV cells %B+ cells
ug h e i n e / 1 0 7 cells
Colonies/103 cells
0,1 56 49 32 26
3 22 24 8.4 3.1
562 11 32 87 150
0 31.5 29 6.5 0 1.5 11
0.53 32.5 14.2 3.9 0.9 1.5 9.8
610 101 272 509 486 137 5.5
Expt. I Control DMSO (0.26 m ) Tetramethylurea (0.0086 m) D i m e t h y l a c e t a m i d e (0.01 m ) B u t y r i c acid (0.005 m )
Expt. H Control DMSO 0.26 m DMSO 0.02 rn DMSO 0.13 m DMSO 0.065 rn B U d R 2 ug/rnl B U d R 2 ug/rnl + DMSO 0.26 m
Cells were s e e d e d at a c o n c e n t r a t i o n o f 1 × 105 cells/ml in t h e p r e s e n c e or a b s e n c e o f an i n d u c e r o f d i f f e r e n t i a t i o n . A f t e r 5 d a y s o f g r o w t h the c u l t u r e s were harvested. F o r E x p . I: F Colonies vs. %B + cells = 1 0 0 . 3 2 " ; F c o l o n i e s vs. ug h e i n e = 1 0 . 9 9 " . F o r E x p . II: F c o l o n i e s vs. %B + cells = 1 1 . 4 2 " ; F c o l o n i e s vs. ug h e m e = 2 4 9 . 7 6 * . These values d o n o t i n c l u d e B U d R c o n t a i n i n g cultures. * F X / I - = 1, V ~ = 3 , ¢ ~ = 0 . 0 5 = 1 0 . 1 3
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The inverse exponential relationship between the number of colonies produced in vitro and the degree of differentiation in the culture when measured either by the percentage of benzidine positive cells in the culture or by the total amount of heme present in the culture was statistically significant in 4 of 4 experiments. This is evidenced by F ratios relating differentiation and clonogenicity given in Table I. Fig. 1 represents these studies graphically. Similar studies were conducted using Clone 7 4 5 D and DMSO as the T A B L E II
Effect of DMSO o n the elonogenicity o f Clone 7 4 5 D cells %B+ Cells
Colonies]103 Cells
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0 0
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Expt. I Control DMSO (0.26 m)
Expt. II Control DMSO ( 0 . 2 6 m )
Expt. III Control DMSO ( 0 . 2 6 m )
inducing agent. Table II illustrates that in 3 of 3 experiments DMSO failed to induce any evidence of differentiation, but clonogenicity was reduced by 23--44%.
Relationship between differentiation and malignancy In 4 individual experiments, groups of 10 mice were inoculated subcutaneously with Clone IV cells which had been cultured in the presence of an inducer of differentiation for 5 days. When 5 X 105 cells were inoculated there was no difference in survival between recipients of cells from control cultures or cells from differentiated cultures. However, mice which were inoculated with 5 X 104 cells from differentiated cultures survived longer than recipients of cells from undifferentiated cultures. Table III gives the survival data of 4 separate experiments along with data on clonogenicity and degree of differentiation of the injected fractions. Fig. 2 gives a survival plot from 2 experiments. While survival differences between controls and recipients of differentiated cultures are readily apparent, there do not appear to be major differences in survival between recipients of the various differentiated cell cultures, regardless of the degree of differentiation or in vitro clonogenicity of the donor culture. The malignancy of the Clone 745D cells described in Table III was also tested. In 2 of 3 studies there was no difference in malignancy between Clone 745D cells cultured in the presence or absence of DMSO. In one experiment recipients of DMSO-treated cells survived 40% longer than recipients of control cultures.
Biological characteristics of tumors For recipients of Clone IV cells, tumors were first detected several days later in recipients of differentiated cells than for recipients of control cells. Once the tumors appeared, their increase in size tended to parallel each other, although there were great individual differences from mouse to mouse. Reliable t u m o r weights and measurements at the time of death were impossible to obtain since surviving mice tended to eat the tumors of their dead cage mates. The tumors of mice which received differentiated cells exhibited minor morphological differences from those found in recipients of control cell cultures. The former tumors (regardless of the inducing agent employed) contained m a n y multinucleated giant cells (Fig. 3) while tumors produced by control cells contained few. We could not determine whether these cells were foreign body giant cells or perhaps megakaryocytes. Tumors produced by 745D cells (whether or n o t previously exposed to DMSO) differed from those produced by Clone IV cells in that the former contained few giant cells and the tumors grew more slowly. The median survival time for recipients of 5 X 104 control 745D cells were 57, 68 and 56 days, respectively. In 4
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SO
I ~
i .
60
-
l
i 70
.
80
-
SURVIVAL IN DRYS
Fig. 2. a: Graphic representation of survival data of Experiment II presented in Table III. b: Graphic representation of survival data of Experiment III given in Table III.
s e p a r a t e e x p e r i m e n t s r e c i p i e n t s o f 5 × 104 Clone IV cells h a d m e d i a n survivals o f 40, 46, 48 a n d 33 d a y s , r e s p e c t i v e l y ; h e n c e C l o n e IV cells were m o r e m a l i g n a n t t h a n D cells. Finally, in n o case did we see a n y e v i d e n c e of e r y t h r o i d d i f f e r e n t i a t i o n in t h e t u m o r s .
Fig. 3a. S e c t i o n o f a t u m o r o b t a i n e d f r o m a m o u s e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 5 × 10 a c o n t r o l C l o n e IV cells. N o t e r e t i c u l u m cells. O r i g i n a l m a g . x 6 7 0 . b: S e c t i o n o f a t u m o r o b t a i n e d From t h e m o u s e i n o c u l a t e d s u b c u t a n e o u s l y w i t h 5 x 104 d i f f e r e n t i a t e d C l o n e I V cells. N o t e r e t i c u l u m cells, g i a n t cells. O r i g i n a l m a g . x 6 7 0 .
DISCUSSION
These experiments clearly demonstrate that the induction of differentiation of Friend leukemia cells in vitro is associated with a marked decline in clonogenicity. The decline appears to have 2 components. The major comp o n e n t is largely due to the process of differentiation per se since it occurs in the absence of apparent toxicity (as measured by soluble ribonucleotide pool sizes) and is mathematically related to the degree of differentiation. The presence of a second and minor toxic c o m p o n e n t is suggested by the observation that DMSO, which does n o t induce 745D cells to differentiate, causes a 20--30% decline in clonogenicity of these cells. These studies have also demonstrated that BUdR itself causes a decline in clonogenicity and that while BUdR can inhibit DMSO-induced differentiation (Conkie et al., 1974; Scher et al., 1973), it does not reverse the DMSOinduced decline in clonogenicity. Either only some aspects of DMSO-induced differentiation are inhibited by BUdR, or cells incubated with BUdR are damaged and the decrease in clonogenicity produced by BUdR is additive with that which occurs with differentiation. Additionally, the fact that Clone IV cells undergo DMSO-induced differentiation w i t h o u t significant alteration in acid-soluble ribonucleotide pool size confirms our earlier report t h a t such DMSO-induced alterations are n o t the result of differentiation (Preisler and Rustum, 1975) and also demonstrates that toxic effects of inducers can vary from cell line to cell line. The effects of DMSO on the malignancy of Friend cells demonstrated in these studies were much less than those reported by others (Friend et al., 1971). The number of animals used by the previous investigators were far fewer than those employed in the current studies and the previous investigators reported the results of only a single experiment. However, the cell line used in the present studies is different from that of the previous investigators and may account for some or all of the observed differences. The characteristics of the growth of the tumors produced by Clone IV cells strongly suggest that tumors produced by cells from differentiated cultures resulted from the proliferation of residual undifferentiated cells in the culture. Hence, the longer survival of mice which received differentiated cells resulted from the inoculation of substantially fewer malignant cells. The clonogenicity studies suggest that the number of residual clonogenic cells in differentiated cultures is several logs less than would be suspected by determining the percentage of benzidine positive cells in the culture. This observation is in accord with our previous studies which demonstrated a decline in clonogenicity prior to the detection of benzidine positive cells in the culture (Preisler et al., 1976c). One finding for which we have no explanation was the presence of giant cells in tumors produced by the inoculation of cells from differentiated cultures. We do not know if they are of host origin or t u m o r origin. If the former were true, then t h e y might represent host recognition of antigenic
10 differences b e t w e e n the h o s t and d i f f e r e n t i a t e d t u m o r cells and w o u l d suggest t h a t i m m u n o l o g i c a l factors m a y play a role in the longer survival of recipients o f d i f f e r e n t i a t e d cells. On the o t h e r h a n d , if the giant cells are o f t u m o r origin, t h e n it is possible t h a t either s o m e o f the d i f f e r e n t i a t e d cells replicated in vivo or alternatively t h a t the g r o w t h p o t e n t i a l o f the u n d i f f e r e n t i a t e d cells was altered b y the i n d u c i n g agents. H e n c e the relationship o f possible alterations in i m m u n o g e n i c i t y o c c u r r i n g d u r i n g d i f f e r e n t i a t i o n of F L C in vitro and the r e s u l t a n t decrease in m a l i g n a n t p o t e n t i a l in vivo is a q u e s t i o n y e t to be answered. ACKNOWLEDGEMENTS The authors would like to thank Mr. Shakerum Alamru, Mr. Bradley Bryant and Ms. Edythe Taylor for their technical assistance. REFERENCES Conkie, D., N. Affara, P.R. Harrison, J. Paul and K. Jones: J. Cell. Biol. 63, 414--419 (1974). Friend, C., W. Scher, J.G. Holland and J. Sato: Proc. Natl. Acad. Sci. (USA) 68, 378--382 (1971). Preisler, H.D., S. Bjornsson, M. Mori and G.H. Lyman: Br. J. Cancer 33,634--645 (1976a). Preisler, H.D., G. Christoff and E. Taylor: Blood 47,363--368 (1976b). Preisler, H.D. and M. Giladi: Cell. Physiol. 85,537--546 (1975). Preisler, H.D., J.D. Lutton, M. Giladi, K. Goldstein and E.D. Zanjani: Life Sci. 16, 1241-1252 (1975). Preisler, H.D. and G.H. Lyman: Cell Differentiation 4,179--185 (1975). Preisler, H.D. and Y. Rustum: Life Sci. 17, 1287--1290 (1975). Preisler, H.D., Y. Shiraishi, M. Mori and A.A. Sandberg: Cell Differentiation, 5, 207--216 (1976c). Scher, W., H.D. Preisler and C. Friend: J. Cell. Physiol. 81, 63--70 (1973).