1 Dept. of Molecular Biology Bionetics Research
Foundation 7300 Pearl St. Bethesda, Md. 20014
2 Laboratory of Tumor Cell Biology National Cancer Institute National
Institutes of Health Bethesda, Maryland 20014
Normal function of hemopoietic tissues depends on a continuous
supply of functional mature cells through maturation of progenitor
cells and stem cells. Leukemia is believed to be resulted from a
disturbance in this maturation process. In mouse, the stem cells
are measured by spleen colony technique (CFU-S measurement) and
the progenitor cells by in vitro culture technique (CFU-C measurement).
Sachs and his colleagues reported that human leukemic cells can
be induced to mature in the in vitro culture system in the presence
of colony-stimulating activity (CSA). These findings were partially
confirmed and extented to many leukemic cases. Our attempt to establish
a relationship between the type of leukemia and nature of leukemic
cell maturation in culture was not successful. Using RL V induced
leukemia in mice as a model system, the effect of RL V infection
on the number of CFU-C and CFU-S was studied. Our results showed
that there was an early transitory depression of CFU-S and a long-term
stimulation of CFU-S following RL V infection. These results suggest
that the earliest event following RL V infection is the committment
of CFU-S to CFU-C and therefore CFU-S are target cells of RL v.
However, these conclusions do not exclude the possibility that the
other cell types could also be target cells. The mature granulocytes
derived from leukemic cells show budding virus particles. The budding
virus particles were also found in mature granulocytes and anucleated
erythrocytes obtained from RL V infected mice.
I. Concept of a Stem Cell
The normal function of hemopoiesis is carried out by various kinds
of mature blood cells, such as erythrocytes, granulocytes) megakaryocytes
and lymphoid cells. The life span of these mature cells are relatively
short. Therefore, a continuous supply of these mature cells is essential
for the normal function of the hemopoietic tissue. These mature
cells are derived from a class of early undifferentiated cells through
a sequence of differentiation and proliferation. This class of early
undifferentiated cells are termed "stem cells" (1, 2). It is important
to emphasize that the stem cells are defined according to their
functions. The morphology of this class of cell is not known. A
stem cell should have the following properties. It should be capable
of I) proliferation, 2) differentiation, 3) self-renewal upon proliferation
and 4) response to physiological demands. If a stem cell is capable
of giving rise to more than one type of differentiated cells, it
is termed a multipotent stem cell whereas if it is capable of giving
rise only to one type of differentiated cell it is termed unipotent
stem cell. A multipotent stem cell by definition is a precursor
of a unipotent stem cell (progenitor cell). Their relationship is
diagrammatically illustrated in Fig. 1. This figure depicts the
transition from stem cell to progenitor cell and the transition
from progenitor cell to differentiated cell which are dependent
on the presence of specific factors. The production of these regulatory
factors presumably are under physiological control. Any cause which
disturbs the process of maturation shown in Fig. 1 will naturally
impair the normal function of hemopoietic tissue. Leukemia is believed
to result from a disturbance of this maturation process ( 3-8) .There
are three possible outcomes that could result from this disturbance
( see Fig. 2) .These are: ( A ) blockage of differentiation ( 4-8)
, ( B) aberration of differentiation and (C) reversal of differentiation.
In (A) and (C) , the leukemic cells should represent some cell types
present during the course of normal differentiation and in ( B )
, the leukemic cells should be different from any of the known cell
types. The mnodels in this figure also indicate that the target
cells of leukemogenesis (with respect to their stage of differentiation)
are not well defined. So far there is no conclusive evidence to
support or rule out any of these possibilities or to implicate a
particular stage of cell differentiation as the target cell for
the initiation of leukemogenesis.
Fig. 1: A diagrammatical representation of maturation
of hemopietic stem cells. Abbreviations: S, multipotent stem cells;
P, progenitor cells, unipotent stem cells; D, differentiated cells
and elements, such as granulocytes, erythrocytes, and platelets.
Functional (mature cell) proteins refer to proteins such as hemoglobin
of erythrocytes, hydrolytic enzymes from granules of granulocytes,
and globulin of antibody-producing cells
II. Measurement of Stem Cells
Currently there are two functional assays available, one for stem
cells, the other for progenitor cells. Both of them are based on
clonal methods. They are summarized in Table 1. In mice, stem cells
can be assayed by the spleen colony technique ( 2) .The assay is
performed by injecting an adequate number of nucleated cells into
a lethally irradiated recipient. After a period of 8-10 days, the
spleen is removed. The macro-nodules which develop on the surface
of the spleen are then counted. These nodules are called spleen
colonies. Each colony was shown to derive from a multipotent stem
cell as judged by a chromosomal analysis using radiation induced
chromosomal aberrations as markers (9) and by studying the cellular
composition of each colony (10,11). Apparently, the development
of a spleen colony represents a response of a stem cell to a physiological
demand after irradiation and is regulated by the microenvironment
(12) which contain cells that produce short-term mediated h umo
ral factors ( 13) . The progenitor cell for granulocytic cells can
be assayed by an in vitro method originally developed by Bradley
and Metacalf (14) and by Pluznik and Sachs (15). A single cell suspension
is prepared in a semi-soft medium ( either 0.3% agar [14, 15] or
0.8% methyl cellulose ) in the presence of colony stimulating
activity (CSA). The CSA is supplied either in the same layer as
the cell suspension ( 16) or in a separate layer of 0.5 % agar containing
conditioned medium or feeder cells ( 14, 15) .The conditioned medium
was generally prepared by growing cell population containing factor
producing cells in culture medium ( either in the presence or in
the absence of
Table 1. Colony Methods for the Assay
of Haemopoietic Stem Cells and Progenitor Cells
Fig. 2: Abbreviations: S, P, and D are the same as those
of Figure 1; L: leukemic cells; solid arrow, direction of normal
differentiation; open arrow, mode of disturbance of normal differentiation.
fetal calf serum) for 4 to 6 days ( 15) .The number of colonies
is generally recorded 8 to 10 days after plating. At this stage,
the colony size may be as large as 104 cells. The colonies are classified
as granulocytic or macrophage colonies depending on the composition
of cells in the colonies. Both of these colony types contain cells
at various stages of maturation. Iferythropoietin is present in
this culture system the second day after plating, some very small
colonies containing erythropoietic cells ( 8 --100 cells ) may develop
( 17, 18) .In mice, colony formation is strictly dependent on the
addition of CSA. CSA for assaying mouse colony forming cells can
be obtained from mouse sera (19), human urine (20), medium from
cultured fibroblasts (21 ), and from embryonic primary culture cells
(22). CSA are glycoproteins which can be purified by use of concanavalin
Asepharose chromatography. The molecular weight estimates of this
activity have indicated heterogeneity ranging from 10,000 to 190,000
(23 ). It is not known whether CSA is a population of heterogenous
glycoproteins or a complex protein containing many subunits. This
in vitro culture assay for progenitor cells has been successfully
applied to human blood cells ( 24-27) .Similar to colonies derived
from mouse cells, the colonies derived from human blood cells (CFU-C)
also contain granulocytic cells at various stages of maturation,
but in general, the colony sizes of human origin are much smaller
( 50-1,000 cells) than those of mouse origin. The specificity of
CSA required for the in vitro colonies of human origin also differs
from that of mouse origin. In most cases, CSA prepared from human
cells can stimulate the growth of colonies of both human and murine
origin, but the CSA prepared from mouse cells stimulates only mouse
colonies. One difficulty in obtaining a quantitative measurement
of human colony forming cells is the lack of an absolute requirement
for exogenous CSA for colony formation. This is due to a contamination
of factor-producing cells in the human blood cell population. A
successful attempt to separate the factor producing cells from the
colony forming cells has recently been reported (28). Obviously,
separation of these two cell populations is essential for a successful
study of the maturation process.
III. Maturation of Human Leukemic Cells in Culture
One of the most important observations made during the course
of studies of human progenitor cells in culture is that apparently
some leukemic blast cells can be induced to mature in culture in
the presence of appropriate CSA to apparently mature normal appearing
granulocytes (25, 29). The evidence that these mature cells are
derived from leukemic cells is based on some chromosomal analyses
(30,31 ). But the argument is not conclusive, because one still
cannot rule out the possibility that the mature cells are derived
from normal cells which contains the same chromosomal aberration.
However, since the disclosure of maturation of human leukemic cells
in culture, many studies have been carried out to establish a pattern
as to which type of leukemic cells seems to be "inducible" to mature.
The result of these studies are also inconclusive. A summary of
such studies carried out in our laboratory in the past year is shown
in Table 2. The capacity of colony formation varies with various
types of leukemia and also varies within the same type of leukemia.
This variation might be due to either difficulty in assuring reproducibly
standardized in vitro colonies assay
Table 2. Differentiation of Leukemic
Cells in Culture
systems for human blood cells or a lack of precise standards for
classification of leukemia ( classifications presently being based
almost entirely on morphology) .For example, leukemic cells from
some patients with acute myelocytic leukemia (AML ) are not able
to form any colonies while some are able to produce many more colonies
than cells derived from the blood or bone marrow of normal individuals.
In general, the number of colony forming cells from AML blood is
significantly lower than that from normal individuals. Moreover,
colonies derived from AML cells sometimes do not contain mature
granulocytes. In remission, the efficiency of colony formation and
maturation of colony cells returns to normal. The remission is defined
as the return of blood cell count to a normal level and disappearance
of blast type leukemic cells. In acute monomyelocytic leukemia (AMML)
, the leukemia cells behave similarly to normal cells with respect
to colony formation. In chronic myelocytic leukemia, the leukemia
blood cells contain less colony forming cells than that of normal
blood cells, and they contain even less colony forming cells when
blast crisis occurs. At this stage, most cells in colonies are not
mature (however, there are exceptions), patients with acute lymphocytic
leukemia do not form any colonies. This is not surprising if the
in vitra CSA dependent colonies are, in fact, derived from granulocytic
cells. These findings are more or less similar to those reported
from other laboratories, e. g., Robinson (32). We have examined
human leukemic cells not only for their ability to respond to factor
(CSA) but also for their ability to produce CSA. These results are
also shown in Table 2. Leukemic cells are poor producers of CSA.
Since this information in the table is still incomplete and since
one cannot rule out the possibility of inhibitors of CSA released
by leukemic cells, this conclusion is tentative. It would seem that
both colony forming (factor responding) cells (CFC) and factor producing
cells (FPC) are required to achieve maturation in vitra and probably
in viva. At the cellular level the defect in leukemia may be of
two general types: one, a defect in factor production, and the other,
a defect in response to factor stimulation. Leukemias might be classified
in this manner and further, according to the degree of responsiveness
to CSA. Reclassification of leukemia based on detectable physiological
functions may be helpful in obtaining meaningful conclusions from
these in vitro cell maturation systems.
IV. RNA Oncogenic Viruses and Human Leukemogenesis
The apparent inducement of human leukemic cells to develop into
seemingly normal mature cells supports the idea that leukemia is
due to a disturbance in the maturation process. The cause of this
disturbance is not known. However, there are now strong reasons
for suspecting a role for type-C RNA tumor viruses or information
derived from these viruses in the pathogenesis of the disease. Expression
of viral genomes in human leukemic cells either from endogenous
genetic information or from an exogenous infection has been recently
supported from results of extensive studies in searching for the
footprints of viral information in human leukemic cells by Gallo
and colleagues and Spiegelman and his colleagues. The evidence for
the existence of viral information in human cells is as follows:
1) Virus-like reverse transcriptases have been isolated from some
human leukemic cells but not from normal proliferative cells. These
enzymes have the biochemical characteristics like most of known
mammalian viral reverse transcriptases (33-35). For example, this
enzyme activity is sensitive to RNAse. The enzymes are able to transcribe
the heterogenous part of 70S RNA isolated from primate and mammalian
viruses. They prefer ( dT) 12 -18. (rA)n as template-primer over
( dT) 12 -18. ( dA)n and are able to use ( dG ) 12 -18. ( rC)n.
These are the characteristics of viral reverse transcriptase ( 35,
36) ; 2) The leukemic reverse transcriptase is immunologically related
to reverse transcriptase of primate type-C RNA tumor viruses (37,
38). (They were inhibited by antibodies against viral reverse transcriptase
expecially of primate, but also of murine RNA tumor viruses [37,38].
This suggests that the human leukemic enzyme shares a common antigenic
determinant with primate and murine viral enzymes. ) ; 3) Human
leukemic cells contain an RNA sequence homologous to DNA products
prepared from an endogenous reaction of mammalian and primate RNA
oncogenic viruses (39,40,41); 4) The size of the RNA template-primer
for the leukemic reverse transcriptase appears to be the same as
viral RNA ( 42, 43) ; 5) The reverse transcriptase and associated
RNA are present in a post-mitochondrial cytoplasmic particle which
retains morphological integrity on repeated high speed centrifugation
and has a density of 1.15 to 1.18 ( 43) , characteristics typical
of type-C RNA tumor viruses. It now appears unequivocal that these
particles are viral-related. This information has been recently
reviewed in detail elsewhere ( 44 ).
V. RNA Oncogenic Viruses and Murine Stem Cells
With the above background in mind, it is evident that studies designed
to determine the affect of animal type-C viruses on normal hemopoiesis
are of immediate interest. The data described below was derived
from our beginning attempts in this direction in mice. One advantage
of an animal system is that one can follow a defined course of leukemic
development, especially the early events following viral infection.
Fig. 3: Experimental design for the effect of RL V infection
on cru-s and cru-c compartments. Eight-week old NIH Swiss mice were
used for this experiment. RL V was injected intravenously through
the tail vein. To prepare cell suspensions, at least five uninfected
and infected mice were used, respectively, Spleen colony technique
was performed as described previously (2). The cru-c was measured
according to the procedures described by Pluznik and Sachs ( 15) ,
except that CSA was prepared from primary culture of BALB/C embryonic
Fig.4: Effect of RLV infection on CFU-S from marrow and
spleens. The spleen colony assay was carried out as described previously
( 2) .The spleens were fixed in Bouin solution. Each point is an
average of 7 spleens. Day O started with injection of RLV.
tion relating to the earliest events may be the most important
to our understanding of leukemogenesis. We induced leukemia in NIH
Swiss mice with Rauscher leukemia virus (RL V) and used it as a
model system to study the effect of leukemia development on the
process of leukocyte maturation. The experimental design of this
experiment is shown in Figure 3. RL V was injected intravenously
into NIH Swiss mice, and various days after injection, marrow and
spleen cells of infected mice were harvested and a single-cell-suspension
was made. Aliquots of the cell suspension were: ( a) injected into
the lethally irradiated mice to measure the content of spleen colonies,
and (b) plated on agar medium to measure the content of progenitor
cells. The result
Fig. 5: Effect of RL V infection on CFU-C from marrow
of infected and uninfected mice. CFU-C was measured as described
in the legend of Figure 3. Each point is an average of two plates.
Normal control are those marrow cells obtained from uninfected mice.
of this experiment for spleen colony formation is shown in Figure
4. There is an initial dip in the number of colony forming units
(CFU-S) per unit cell number injected both from spleen and marrow
cells on the second and third days after injection of RL v. However,
the CFU-S per unit cell number then gradually recovered to a normal
level. A short period of "overshoot" was observed on the fourth
and fifth day after RL V injection. Since the cell number increased
during the course of leukemia development, the total CFU-S, in fact,
increased more than 10 fold by one month following the development
of leukemia. The effect of RL V infection on the progenitor compartment
of marrow is shown in Figure 5. Soon after viral infection, the
number of CFU-C (granulocytic progenitor cells) per 105 nucleated
cells increased about 10 fold. This level was maintained for a few
weeks and then gradually decreased by the fifth week after infection.
We did not determine whether CFU-C eventually decreases to a normal
or a subnormal level since infected animals started to die by the
eighth week after inoculation with RL V. This rise and decline of
CFU-C following leukemic development might provide an explanation
for the variation in measuring CFU-C in human leukemic cell populations.
Fig. 6: Effect of RL V infection on CFU -C from spleens
of uninfected and infected mice. The procedures are the same as
those described in Figure 5.
The number of CFU-C may be a function of the stage of leukemic
development. Similar results were obtained when spleen cells were
used to study the effect of CFU-C upon viral infection. The result
of this experiment is shown in Figure 6. This graph was plotted
as CFU-C per unit cell number as a function of time. Since spleen
cell number increased at least 20 fold during the development of
leukemia, the total CFU-C increased at least 120 fold. From a practical
point of view, we wonder whether this increase of CFU-C in early
infection might be useful as a diagnostic tool for pre-leukemia
or an early stage of leukemia. The effect on the number of CFU-C
and CFU-S by RL V were not simply due to an antigenic stimulation
since heat-inactivated viruses did not cause such an effect. Some
agents such as Freund's complete adjuvant (45), pertussis vaccine
(46), phenylhydrazine ( 47 ) , and endotoxin ( 48) are able to enhance
the number of CFU-C and CFU-S in vivo. However, these effects are
transitory and, therefore, are different from the long-term effect
of RL V. Our observation on the initial decrease of CFU-S by RL
V infection is in agreement with that reported by Seidel ( 49) as
is our finding on the late effect of CFU-S by RL V infection ( 49
) and this late effect is also similar to that reported by Okunewick,
et at. (50). Many interpretations can account for the initial decrease
of CFU-S accompanying the increase of CFU-C. One interpretation
is that the CFU-S is a target cell of RL V. Upon infection, the
stem cells are affected such that their capabilities for spleen
colony formation are lost. Now, these infected stem cells are still
capable of forming colonies in culture at evidenced by an increase
of CFU-C. Perhaps this occurs by a commitment of the stem cell to
granulocytic progenitors. However, our results did not rule out
the possibilities that the other cell compartment could also be
a target for RL V infection.
VI. Properties of Granulocytic Colonies Derived from Murine Leukemic
1) The colonies contain apparent mature granulocytes and colony
formation is absolutely dependent on addition of exogenous CSA.
We found type-C RNA tumor viruses bud from the membrane of the mature
granulocytes. In addition, on examining the other cell types from
infected mice, we found that besides early immature cells, both
mature granulocytes and anucleated erythrocytes show budding type
virus particles. The phenomenon of the budding of virus particles
from mature erythrocytes is interesting and puzzling.
2) With the H3 -thymidine suicide techniq ue ( 51, 52) , we found
that progenitor cells from uninfected and leukemic mice have the
same fraction of cells in cycle, 50% of CFU-C. Thus, the leukemic
cell population apparently does not contain a larger fraction of
cells in cell cycle. This is in agreement with the notion that leukemia
results from a block in leukocyte maturation. This observation could
be important not only from a physiological point of view, but also
from a chemotherapeutical point of view.
3) Similar to normal granulocytic colonies, the granulocytic colonies
which originated from leukemic populations can only be transferred
in culture 2 to 3 times. Our attempts to establish a clonal line
of colony forming cells has not been successful.
4) An injection of mature colony cells originating from a leukemic
cell population induced leukemia in the recipient mice. However,
it is not known whether this transplantability of leukemia is due
to an infection by viruses associated with the cells or due to the
leukemic nature of the transplanted cells. It is worthwhile to emphasize
that we noted that several transplantable murine tumor cells all
contain type-C virus particles. These cells are L 1210 cells ( 53
) , MI cells ( 54) , and leukemic cells from Rauscher leukemia virus
infected mouse ( data not shown). These viruses may play an important
role in the transplant ability of these cells.
Leukemia appears to result from a disturbance of the normal differentiation
of hemopoietic stem cells. This disturbance might be a blockage,
aberration or reversal of differentiation. In some cases, the blockage
process of some cells can apparently be removed in culture as evidenced
by apparent induction of leukemic cells to produce seemingly normal
mature granulocytes in culture in the presence of CSA. This dependence
of CSA on the maturation of leukocytes may cast some light on the
process of leukemogenesis. The phenomenon of CSA stimulation may
provide avenues for studying the process of leukocyte maturation
at the molecular level and a potential new approach for the therapy
of human leukemia and other blood illnesses. In this respect, however,
it is surprising and puzzling that so far there are no reports evaluating
in vivo results of treatment of leukemic mice with CSA.
Acknoweledgements This work was in part supported by a contract
from cancer therapy area, National Cancer Institute. The authors
wish to thank the technical assistance of Craig Snyder and Ellen
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