COMPARATIVE LEUKEMIA RESEARCH 1973, LEUKEMOGENESIS, BIBL. HAEMAT., NO. 40, ED. Y. PTO AND R. M. DUTCHERt, UNIV. OF TOKYO PRESS, TOKYO/KARGER, BASEL, PP. 179-183 (1975)
Attempts to Transform Murine Hemopoietic Cells by Rauscher Leukemia Virus
P. BENTVELZEN, A. M. AARSSEN, J. BRINKHOF, K. NOOTER, C. ZURCHER, and G. J. VAN DEN ENGH Radiobiological Institute TΝO and Institute for Experimental Gerontology ΤΝΟ* Primary Rauscher leukemia virus (RLV)-induced myeloid leukemias can produce many small clones in agar in the absence of a factor needed for the proliferation of normal myeloid cells. It seems that leukemic cells can more efficiently utilize the small amount of colony-stimulating factor (CSF) that is produced by them. At optimal stimulation by exogenous CSF, leukemic cells exhibit a poorer rate of proliferation than normal bone marrow cells. Hemopoietic cells can replicate the virus after infection in vitro. In some experiments, infection of normal bone marrow cells leads to the production of some cells with the same growth pattern as cells from primary leukemias.
* Rijswijk (ZH), The Netherlands.
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
Murine leukemia viruses can replicate well in embryonic fibroblasts but, in contrast
to the sarcoma viruses, rarely transform them. It is obvious that epigenetic factors are highly important for the oncogenic expression of the somatic proviruses of oncornaviruses. We are presently engaged in the study of the influence of cell differentiation on transformation of hemopoietic cells by murine leukemia viruses. At this time, there is no good in vitro system available to assess the oncogenic potential of a leukemia virus preparation. Since murine hemopoietic cells can now be grown in culture and especially the differentiation into the myeloid series can be studied in great detail (1, 2, 8), we thought it worthwhile to attempt in vitro transformation of hemopoietic cells by a leukemia virus. The development of granulocyte/macrophage colonies in agar is dependent upon the presence of a colony-stimulating factor (CSF), which can be obtained from human urine, mouse embryos, or conditioned medium from mouse embryonic fibroblast cul-
180
ΒENTVELΖΕΝ/ΑΑRSSEN/ERIΝΚΗΟF/ΝΟΟΤΕR/ΖURCHER/VAN DEN ENGH
tures (6, 10, 11). It is assumed that this CSF is in vivo a granulopoietic hormone. There are three established murine myeloid leukemia cell lines that can form colonies in the absence of CSF (3, 5, 7). This property of autonomous growth is comparable to the capacity for growth in agar of virus-transformed fibroblasts, widely utilized for the assay of the transforming capacity of a virus (4). It is becoming increasingly clear that transformation in vitro corresponds only with an early event in viral carcinogenesis. It was assumed, therefore, that established tumor lines do not offer a good model for the detection of transformed hemopoietic cells. Rauscher Virus-induced Myeloid Leukemias We decided to study primary myeloid tumors that were induced by an oncornavirus. It was observed that inoculation of irradiated C57BL mice (300 rad, whole body) with Rauscher leukemia virus (RLV) leads to the development of myeloid leukemia within a few months. Several mice were found to have chloromas at autopsy. Our standard RLV preparation does not seem to induce myeloid leukemia in BALB/c mice, but we fortuitously obtained an RLV preparation (RLV-K) that induces a transient erythroblastosis from which only 10% of the animals die and which is followed approximately 6 months later by a great variety of reticuloendothelial malignancies, among them myeloid leukemia with a great range of differentiation arrest and with or without involvement of the peripheral blood. Only in 4 of 34 attempts have we succeeded in developing a transplantable tumor from a granulocytic leukemia. This is not due to strong antigenicity, since in experiments with mice strongly immunosuppressed by antilymphocyte serum, we succeeded in only 2 out of 10 attempts. TABLE
I. CSF Titration on Various Types of RLV-induced Leukemia Number of clones per 105 cells
CSF dilution 8 64 256
Normal
Aleukemic leukemia
True leukemic picture
Transplant. leukemia
Colonies Clusters
Colonies Clusters
Colonies Clusters
Colonies Clusters
157 40 0
n 135 1
64 4 0
n n 11
34 6 0
76 49 5
12 13 12
69 47 37
n : Too numerous to be counted.
The bone marrow of 63 leukemic mice has been tested in vitro for their ability to produce clones in agar at different concentrations of CSF. Detailed information about this experiment will be published elsewhere, but some cases are presented in Table I. The general picture is that, at levels of CSF optimal for the growth of normal bone marrow, the leukemic material produces fewer colonies than the controls. Often bone marrow
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
CSF-responsiveness of RLV-induced Myeloid Leukemia
ATTEMPTS TO TRANSFORM MURINE HEMOPOIETIC CELLS 181 from leukemic mice produces a very large number of small clusters (less than 50 cells) at high concentrations of CSF. Cluster formation in the Absence of CSF In 34 of the 63 cases we found that at low levels of CSF (or even in the absence of this factor), the leukemic material produces many more small clusters than the controls. This property of growth in the absence of exogenous CSF becomes especially manifest when high cell numbers are plated (Table II). Since bone marrow contains CSFTABLE
II. Influence of Cell Density on Colony Formation in Agar Normal bone marrow
Number of cells plated
105 00
1,
64 256 No CSF
Ñ
106
U
ο Ú
)
Ú
Ñ ~ ο U
14 0 0
19 2 2
16 1 1
58 17 5
40 22 3
Ν
CSF dilution
2X105
.
4 a41 ~
εη. Ν
~
Leukemic bone marrow 105 $_ ~ ο C) n n 8
W
ö U
3η.ι ~ ο Ú
)
®Ν ~ ~ C)
0 2 1
27 13 11
4 10 7
83 87 62
W
a4J
2X105 aN -
108 W 4 Ψ
ζ
ö
ο
)
Ú
14 23 38
n n n
n : Too numerous to be counted.
producing cells, the observed autostimulation might be due to an excessive release of CSF by the leukemic cells, such as the release of the overgrowth factor by Rous sarcoma virus-transformed chicken cells into the medium (9). When feeder layers of irradiated bone marrow cells from either normal or leukemic mice are used, no difference in the production of a stimulating factor can be detected TABLE
III. CSF Production by Feeder Layers of Normal or Leukemic Bone Marrow Number of clones produced by 5 x 105 Normal bone marrow cells
Feeder layer
Leukemic bone marrow cells
Colonies
Clusters
Colonies
Clusters
0 0 2
40 43 20
28 144 128
n n n
None 5 X 105 irradiated normal bone marrow cells 5 X 105 irradiated leukemia bone marrow cells n : Too numerous to be counted.
Virus Replication in Cultures of Hemopoietic Cells We reasoned that the relative capacity for autonomous growth could be utilized in discerning transformed hemopoietic cells from normal cells. In order to achieve virus-
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
(Table III). It seems that the leukemic cells are more efficient in their utilization of the small amounts of CSF released by some bone marrow cells compared to the lesser need for a multiplication-stimulating factor by transformed fibroblasts.
182 BENTVELZEN/AARSSEN/BRINKHOF/NOOTER/ZURCHER/VAN DEN ENGH
induced transformation in vitro we first investigated the capacity of hemopoietic cells to become infected in vitro and to reproduce the virus. Therefore, we employed two different systems. In the first system, a monolayer of mouse embryonic fibroblasts was irradiated (2,000 rad) and then overlayed with 1 % agar. On top of the agar, a liquid suspension of bone marrow cells (105/mí) was deposited to which was added 5 logs of RLV. The virus dose was 5 logs per culture as estimated by the induction of splenomegaly within 3 weeks in BALB/c mice. After 2 and 3 days of culture, the supernatant contained 2-3 logs of virus, but no virus was detectable after 1 day. The rate of virus replication proved to be inferior to that in cultures of secondary mouse embryonic fibroblasts. The other system was a liquid suspension of only bone marrow cells to which an optimal amount of CSF was added. Such cultures yielded virus after 3 days. Omission of CSF inhibited virus replication, although approximately 30% of the cells retained their proliferative capacity as tested by subsequent addition of CSF. We assume that DNA synthesis is necessary for successful infection of hemopoietic cells by RLV. In control experiments for both systems with uninfected cells, we never observed splenomegaly after inoculation of the respective supernatants. Our culture conditions do not seem to activate an endogenous splenomegaly-inducing virus. Cells kept in the first culture system for 2 weeks were replated in agar without CSF for the detection of transformed cells. In three separate experiments we obtained about 150 small clusters from the virus-incubated cultures, whereas in the controls, this number was always less than 10. In 9 other experiments, no significant increase in the number of clusters was observed after exposure to RLV. We are at present varying culture conditions in order to enhance the rate of transformation and to obtain good reproducibility.
1 BRADLEY, T. R. and METCALF, D. The growth of mouse bone marrow cells in vitro. flust. J. Exp. Bíοl. Med. Sci., 44: 287-300 (1966). 2 DICKE, K. A., PLATENBURG, M. G. C., and VAN BEKKUM, D. W. Colony formation in agar: In vitro assay for heemopoietic stem cells. Cell Tiss. Kinet., 4: 463-477 (1971). 3 IcrnKAwA, Y. Differentiation of leukemic myeloblasts. Gann Monograph on Cancer Research, 12: 215-229 (1972). 4 MACPHERSON, I. The characteristics of animal cells transformed in vitro. Adv. Cancer Res., 13: 169-215 (1970). 5 METCALF, D., MOORE, M. A. S., and WARNER, N. L. Colony formation in vitro by myelomonocytic leukemic cells. J. Natl. Cancer Inst., 43: 983-997 (1969). 6 PIKE, Β. L. and RοBτNSON, W. A. Human bone marrow colony growth in agar-gel. J. Cell Comp. Physiol., 76: 77-84 (1970). 7 PLUZNIK, D. H. The effect of a growth-enhancing substance from leukemic cells and normal mouse embryo fibroblasts, on leukemic cells in tissue culture. Israel J. Med. Sci., 5: 306-312 (1969). 8 PLUZNIK, D. H. and SACHS, L. The cloning of normal "mast" cells in tissue culture. J. Cell. Comp. Physiol., 66: 319-324 (1965).
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
REFERENCES
ATTEMPTS TO TRANSFORM MURINE HEMOPOIETIC CELLS 183
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
9 RUBIN, H. Overgrowth-stimulating activity of disrupted chick embryo cells and cells infected with Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S., 67: 1256-1263 (1970). 10 SHERIDAN, J. W. and STANLEY, Ε. R. Tissue sources of bone marrow colony stimulating factor. J. Cell. Comp. Physiol., 78: 451-460 (1971). 11 STANLEY, Ε. R. and METCALF, D. Partial purification and some properties of the factor in normal and leukaemic human urine stimulating mouse bone marrow colony growth in vitro. flust. J. Exp. Bíol. Med. Sci., 47: 467-483 (1969).
Attempts to Transform Murine Hemopoietic Cells by Rauscher Leukemia Virus
P. BENTVELZEN, A. M. AARSSEN, J. BRINKHOF, K. NOOTER, C. ZURCHER, and G. J. VAN DEN ENGH Radiobiological Institute TΝO and Institute for Experimental Gerontology ΤΝΟ* Primary Rauscher leukemia virus (RLV)-induced myeloid leukemias can produce many small clones in agar in the absence of a factor needed for the proliferation of normal myeloid cells. It seems that leukemic cells can more efficiently utilize the small amount of colony-stimulating factor (CSF) that is produced by them. At optimal stimulation by exogenous CSF, leukemic cells exhibit a poorer rate of proliferation than normal bone marrow cells. Hemopoietic cells can replicate the virus after infection in vitro. In some experiments, infection of normal bone marrow cells leads to the production of some cells with the same growth pattern as cells from primary leukemias.
* Rijswijk (ZH), The Netherlands.
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
Murine leukemia viruses can replicate well in embryonic fibroblasts but, in contrast
to the sarcoma viruses, rarely transform them. It is obvious that epigenetic factors are highly important for the oncogenic expression of the somatic proviruses of oncornaviruses. We are presently engaged in the study of the influence of cell differentiation on transformation of hemopoietic cells by murine leukemia viruses. At this time, there is no good in vitro system available to assess the oncogenic potential of a leukemia virus preparation. Since murine hemopoietic cells can now be grown in culture and especially the differentiation into the myeloid series can be studied in great detail (1, 2, 8), we thought it worthwhile to attempt in vitro transformation of hemopoietic cells by a leukemia virus. The development of granulocyte/macrophage colonies in agar is dependent upon the presence of a colony-stimulating factor (CSF), which can be obtained from human urine, mouse embryos, or conditioned medium from mouse embryonic fibroblast cul-
180
ΒENTVELΖΕΝ/ΑΑRSSEN/ERIΝΚΗΟF/ΝΟΟΤΕR/ΖURCHER/VAN DEN ENGH
tures (6, 10, 11). It is assumed that this CSF is in vivo a granulopoietic hormone. There are three established murine myeloid leukemia cell lines that can form colonies in the absence of CSF (3, 5, 7). This property of autonomous growth is comparable to the capacity for growth in agar of virus-transformed fibroblasts, widely utilized for the assay of the transforming capacity of a virus (4). It is becoming increasingly clear that transformation in vitro corresponds only with an early event in viral carcinogenesis. It was assumed, therefore, that established tumor lines do not offer a good model for the detection of transformed hemopoietic cells. Rauscher Virus-induced Myeloid Leukemias We decided to study primary myeloid tumors that were induced by an oncornavirus. It was observed that inoculation of irradiated C57BL mice (300 rad, whole body) with Rauscher leukemia virus (RLV) leads to the development of myeloid leukemia within a few months. Several mice were found to have chloromas at autopsy. Our standard RLV preparation does not seem to induce myeloid leukemia in BALB/c mice, but we fortuitously obtained an RLV preparation (RLV-K) that induces a transient erythroblastosis from which only 10% of the animals die and which is followed approximately 6 months later by a great variety of reticuloendothelial malignancies, among them myeloid leukemia with a great range of differentiation arrest and with or without involvement of the peripheral blood. Only in 4 of 34 attempts have we succeeded in developing a transplantable tumor from a granulocytic leukemia. This is not due to strong antigenicity, since in experiments with mice strongly immunosuppressed by antilymphocyte serum, we succeeded in only 2 out of 10 attempts. TABLE
I. CSF Titration on Various Types of RLV-induced Leukemia Number of clones per 105 cells
CSF dilution 8 64 256
Normal
Aleukemic leukemia
True leukemic picture
Transplant. leukemia
Colonies Clusters
Colonies Clusters
Colonies Clusters
Colonies Clusters
157 40 0
n 135 1
64 4 0
n n 11
34 6 0
76 49 5
12 13 12
69 47 37
n : Too numerous to be counted.
The bone marrow of 63 leukemic mice has been tested in vitro for their ability to produce clones in agar at different concentrations of CSF. Detailed information about this experiment will be published elsewhere, but some cases are presented in Table I. The general picture is that, at levels of CSF optimal for the growth of normal bone marrow, the leukemic material produces fewer colonies than the controls. Often bone marrow
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
CSF-responsiveness of RLV-induced Myeloid Leukemia
ATTEMPTS TO TRANSFORM MURINE HEMOPOIETIC CELLS 181 from leukemic mice produces a very large number of small clusters (less than 50 cells) at high concentrations of CSF. Cluster formation in the Absence of CSF In 34 of the 63 cases we found that at low levels of CSF (or even in the absence of this factor), the leukemic material produces many more small clusters than the controls. This property of growth in the absence of exogenous CSF becomes especially manifest when high cell numbers are plated (Table II). Since bone marrow contains CSFTABLE
II. Influence of Cell Density on Colony Formation in Agar Normal bone marrow
Number of cells plated
105 00
1,
64 256 No CSF
Ñ
106
U
ο Ú
)
Ú
Ñ ~ ο U
14 0 0
19 2 2
16 1 1
58 17 5
40 22 3
Ν
CSF dilution
2X105
.
4 a41 ~
εη. Ν
~
Leukemic bone marrow 105 $_ ~ ο C) n n 8
W
ö U
3η.ι ~ ο Ú
)
®Ν ~ ~ C)
0 2 1
27 13 11
4 10 7
83 87 62
W
a4J
2X105 aN -
108 W 4 Ψ
ζ
ö
ο
)
Ú
14 23 38
n n n
n : Too numerous to be counted.
producing cells, the observed autostimulation might be due to an excessive release of CSF by the leukemic cells, such as the release of the overgrowth factor by Rous sarcoma virus-transformed chicken cells into the medium (9). When feeder layers of irradiated bone marrow cells from either normal or leukemic mice are used, no difference in the production of a stimulating factor can be detected TABLE
III. CSF Production by Feeder Layers of Normal or Leukemic Bone Marrow Number of clones produced by 5 x 105 Normal bone marrow cells
Feeder layer
Leukemic bone marrow cells
Colonies
Clusters
Colonies
Clusters
0 0 2
40 43 20
28 144 128
n n n
None 5 X 105 irradiated normal bone marrow cells 5 X 105 irradiated leukemia bone marrow cells n : Too numerous to be counted.
Virus Replication in Cultures of Hemopoietic Cells We reasoned that the relative capacity for autonomous growth could be utilized in discerning transformed hemopoietic cells from normal cells. In order to achieve virus-
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
(Table III). It seems that the leukemic cells are more efficient in their utilization of the small amounts of CSF released by some bone marrow cells compared to the lesser need for a multiplication-stimulating factor by transformed fibroblasts.
182 BENTVELZEN/AARSSEN/BRINKHOF/NOOTER/ZURCHER/VAN DEN ENGH
induced transformation in vitro we first investigated the capacity of hemopoietic cells to become infected in vitro and to reproduce the virus. Therefore, we employed two different systems. In the first system, a monolayer of mouse embryonic fibroblasts was irradiated (2,000 rad) and then overlayed with 1 % agar. On top of the agar, a liquid suspension of bone marrow cells (105/mí) was deposited to which was added 5 logs of RLV. The virus dose was 5 logs per culture as estimated by the induction of splenomegaly within 3 weeks in BALB/c mice. After 2 and 3 days of culture, the supernatant contained 2-3 logs of virus, but no virus was detectable after 1 day. The rate of virus replication proved to be inferior to that in cultures of secondary mouse embryonic fibroblasts. The other system was a liquid suspension of only bone marrow cells to which an optimal amount of CSF was added. Such cultures yielded virus after 3 days. Omission of CSF inhibited virus replication, although approximately 30% of the cells retained their proliferative capacity as tested by subsequent addition of CSF. We assume that DNA synthesis is necessary for successful infection of hemopoietic cells by RLV. In control experiments for both systems with uninfected cells, we never observed splenomegaly after inoculation of the respective supernatants. Our culture conditions do not seem to activate an endogenous splenomegaly-inducing virus. Cells kept in the first culture system for 2 weeks were replated in agar without CSF for the detection of transformed cells. In three separate experiments we obtained about 150 small clusters from the virus-incubated cultures, whereas in the controls, this number was always less than 10. In 9 other experiments, no significant increase in the number of clusters was observed after exposure to RLV. We are at present varying culture conditions in order to enhance the rate of transformation and to obtain good reproducibility.
1 BRADLEY, T. R. and METCALF, D. The growth of mouse bone marrow cells in vitro. flust. J. Exp. Bíοl. Med. Sci., 44: 287-300 (1966). 2 DICKE, K. A., PLATENBURG, M. G. C., and VAN BEKKUM, D. W. Colony formation in agar: In vitro assay for heemopoietic stem cells. Cell Tiss. Kinet., 4: 463-477 (1971). 3 IcrnKAwA, Y. Differentiation of leukemic myeloblasts. Gann Monograph on Cancer Research, 12: 215-229 (1972). 4 MACPHERSON, I. The characteristics of animal cells transformed in vitro. Adv. Cancer Res., 13: 169-215 (1970). 5 METCALF, D., MOORE, M. A. S., and WARNER, N. L. Colony formation in vitro by myelomonocytic leukemic cells. J. Natl. Cancer Inst., 43: 983-997 (1969). 6 PIKE, Β. L. and RοBτNSON, W. A. Human bone marrow colony growth in agar-gel. J. Cell Comp. Physiol., 76: 77-84 (1970). 7 PLUZNIK, D. H. The effect of a growth-enhancing substance from leukemic cells and normal mouse embryo fibroblasts, on leukemic cells in tissue culture. Israel J. Med. Sci., 5: 306-312 (1969). 8 PLUZNIK, D. H. and SACHS, L. The cloning of normal "mast" cells in tissue culture. J. Cell. Comp. Physiol., 66: 319-324 (1965).
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
REFERENCES
ATTEMPTS TO TRANSFORM MURINE HEMOPOIETIC CELLS 183
Downloaded by: Université de Paris 193.51.85.197 - 1/16/2020 12:09:15 AM
9 RUBIN, H. Overgrowth-stimulating activity of disrupted chick embryo cells and cells infected with Rous sarcoma virus. Proc. Natl. Acad. Sci. U.S., 67: 1256-1263 (1970). 10 SHERIDAN, J. W. and STANLEY, Ε. R. Tissue sources of bone marrow colony stimulating factor. J. Cell. Comp. Physiol., 78: 451-460 (1971). 11 STANLEY, Ε. R. and METCALF, D. Partial purification and some properties of the factor in normal and leukaemic human urine stimulating mouse bone marrow colony growth in vitro. flust. J. Exp. Bíol. Med. Sci., 47: 467-483 (1969).
