[CANCER RESEARCH 47, 378-382, January 15, 1987]
Depletion of Sodium Butyrate from the Culture Medium of Friend Erythroleukemia
Cells Undergoing Differentiation1
Charlotte Friend, Maria Zajac-Kaye, J. Gilbert Holland, and Beatriz G-T. Pogo
From the Center for Experimental
New York, New York 10029
Cell Biology and The Molile B. Roth Laboratory, Mount Sinai School of Medicine,
ABSTRACT
Friend erythroleukemia cells can be induced to undergo erythroid
differentiation by a variety of unrelated compounds. The fact that sodium
butyrate causes reversible alterations in growth, morphology, and bio
chemistry in many cell systems prompted us to reexamine its pattern of
induction of differentiation and to compare it to that of dimethyl sulfoxide
(DMSO) and hexamethylbisacetamide (HMBA). By the fourth day of
induction, a peak in hemoglobin accumulation was reached in the cultures
treated with each of these potent inducers. Differences, however, were
noted in cultures in which there had been no change of medium for 7
days. Whereas DMSO or HMBA induced cultures reached a stationary
stage of growth and maintained a high percentage of benzidine positive
cells, butyrate treated cultures resumed active growth and showed a
marked decrease in the percentage of benzidine positive cells. However,
the actual number of terminally differentiated cells remained relatively
constant. The addition of fresh butyrate to 4-day treated cultures pre
vented the decrease in the percentage of benzidine positive cells. Meas
urement of |l4C]butyrate uptake into the cells showed a decrease in the
incorporation of the inducer with time coincident with the decrease in the
percentage of benzidine positive cells and of the butyrate in the medium.
Incorporation of |'11|lliymidine into cells undergoing differentiation for 4
days indicated that butyrate treated cells, but not cells treated with
DMSO or HMBA were capable of active DNA synthesis and growth
after removal of the inducers. These data suggest that butyrate, a natural
fatty acid, is metabolized by the cells and with time its concentration is
reduced to a level below that required to stimulate differentiation. Addi
tional evidence to support this notion are the results obtained with
conditioned medium (CM) from induced cultures. CM-DMSO and CMHMBA retained the capacity to induce differentiation whereas CMhutyrate lost its potency with time.
INTRODUCTION
Murine erythroleukemia
cell lines (FLC)2 originating from
Friend leukemia virus transformed erythroid precursors have
provided a model system for studying differentiation. FLC can
be stimulated to mature along the erythroid pathway when
cultured in the presence of DMSO or a variety of other com
pounds which have a similar capacity to modulate gene expres
sion. These inducers cause morphological and biochemical al
terations that parallel many of those that occur during normal
erythropoiesis, including changes in globin synthesis, heme
synthesis, enzymes involved in nucleotide metabolism, and
membrane associated proteins (for review, see Refs. 1 and 2).
Although the induced FLC acquire the ability to mimic the
functions of RBC and have a limited capacity for self-renewal,
some of the regulatory mechanisms differ from those control
ling normal erythropoiesis. For example, FLC do not respond
to erythropoietin and do not extrude their nuclei at the terminal
stage of differentiation, yet cease to multiply. In addition, the
Received 6/27/86; revised 9/30/86; accepted 10/2/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by National Cancer Institute grants CA
10000, CA 13047, and by the Chemotherapy Foundation.
1The abbreviations used are: FLC, Friend erythroleukemia cells; FL, Friend
leukemia; DMSO, dimethyl sulfoxide; ODC, ornithine decarboxylase; HMBA,
hexamethylbisacetamide; CM, conditioned medium.
City University of New York,
system controlling hemoglobin synthesis is not regulated by
hemin as it is in normal reticulocytes (3). These differences
suggest there may be alternative pathways to hemoglobin syn
thesis in FLC.
The molecular basis of the commitment process of the FLC
is not well understood possibly because so many structurally
and functionally unrelated compounds can trigger the complex
series of events that lead to the initiation of the program for
differentiation. However, the inducers can be divided into 2
classes based on ability to induce differentiation in variant
clones (4) and to stimulate ODC (5). ODC, the rate limiting
enzyme in the synthesis of polyamines, is regulated by many
agents that stimulate cell growth and differentiation. DMSO
and HMBA, inducers belonging to class 1, appear to trigger
differentiation via a mechanism involving polyamine biosyn
thesis enzymes. Sodium butyrate, an inducer belonging to class
2, has little or no effect on ODC and is not inhibited by
dexamethasone
or 12-O-tetradecanoylphorbol-l-acetate
(5)
both of which inhibit class 1 inducers. In addition, it causes
histone hyperacetylation in FLC (6) and at lower concentrations
is not cytostatic and inhibits differentiation by HMBA (7). The
effects of butyrate on FLC and other cell systems have been
reviewed (8).
Whereas exposure to DMSO or HMBA for 12-18 h is
sufficient for the cells to become irreversibly committed to the
erythroid pathway (9), there are a number of studies (10, 11)
which indicate that the cells exposed to butyrate for a similar
period of time are not. If the treated cells were transferred to
medium with or without butyrate, the percentage of benzidine
positive cells in medium without the inducer was lower than
that in the butyrate supplemented medium.
We have reexamined the response of FLC to butyrate in
order to gain a better understanding of the nature of the
regulatory cascade that leads to terminal differentiation. Our
results suggest that butyrate is metabolized by the cells and as
a result the concentration of the compound in the culture
medium is decreased. This decrease may account for the revers
ibility of the changes in growth, morphology, and biochemistry
that butyrate has been reported to induce in a variety of human
and murine cell lines (8, 12-14). We have found that if the
concentration of butyrate is maintained by the addition of the
compound to the cultures after the fourth day, the cells termi
nally differentiated by the seventh day. The possibility that
there may be a component(s) which regulate differentiation and
whose accumulation depends on the optimal concentration of
butyrate is under investigation.
MATERIALS
AND METHODS
Cell Lines and Culture Conditions. The establishment, growth con
ditions, and characterization of FL cell lines have been reviewed (2).
Briefly, the cells of line 5-86, a subclone of line 74SA were maintained
in Glasgow minimum essential medium (Gibco) supplemented with
10% fetal calf serum (Reheis), penicillin (100 units/ml), and strepto
mycin (100 /ig/ml). Cultures were incubated at 37'C in 5% CO2/95%
air. At designated intervals, the number of cells in each culture was
378
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BUTYRATE
REQUIREMENT
FOR STIMULATION
OF FLC INDUCTION
determined by counting in a hemocytometer. Viability was determined
by the trypan blue exclusion assay.
Conditioned Medium. CM from untreated cultures or cultures treated
for 4 days with either DMSO, HMBA, or sodium butyrate was centrifuged and filtered through a Swinnex-HA, 0.45 mm, and then assayed
for inducer activity. CMs were seeded with 1 x IO5 fresh cells/ml and
cells treated with DMSO or HMBA for the same period. These
cells were no longer proliferating and were committed along
the erythroid pathway as the increase in the number of benzidine
positive cells with time indicated.
We also compared the replicative capacity of the virus in the
chronically
infected FL cells undergoing induction of differen
scored for benzidine positivity 4 and 7 days later.
tiation. The reverse transcriptase activity released into the
Reverse Transcripta se Assay. The amount of virus released into the
culture fluid was measured by incorporation of [3H]deoxythymidine
medium of each culture was measured daily for 4 days. As
shown in Fig. 1, virus replication parallels cell growth. Although
monophosphate, using polyriboadenylic acid hybridized to multiple
lengths of deoxythymidylate, 10 residues long as template-primer under
the values varied from experiment to experiment, in general 1.5
the conditions described previously (IS).
HIM sodium butyrate inhibited both cell and virus replication.
Induction of Differentiation. Three compounds known to be potent
In addition, we have examined the properties of the Friend
inducers of the erythroleukemia cells were used: DMSO, Fischer Sci
leukemia virus genome and its expression during differentia
entific Company (1.5% vol/vol); HMBA, kindly provided by Dr. R. tion. The integration patterns and the number of proviral copies
Rubin (4 HIM); and sodium butyrate, Baker Chemical Co. (1.5 IBM).
Each was added directly to the culture at the time of seeding 1-2 x 10* remain unchanged during induction by each of the inducers
(18).
cells/ml and where indicated fresh butyrate was added on day 4. The
Comparison of Ability of Induced Cells to Synthesize DNA.
number of benzidine positive cells in cultures containing inducers was
To ascertain whether the treated cells were terminally differ
determined at the times indicated in the text by the method previously
entiated and DNA synthesis was at a minimal level, we com
described (16, 17).
Labeling of Cells. To assay for DNA replication, the cells were pulse
pared the extent of DNA synthesis in the cells induced for 4
labled with 1 /¿Ci/ml[m€f/ry/-3H]thyinidine(specific activity, 20 /»Ci/ days to those transferred to fresh medium in the presence or
nimol) for l h at 37*C at the intervals indicated in the text. The cultures
absence of inducer. The incorporation [3H]thymidine into the
were then centrifugea and the supernatant fluid aspirated. After washing
the cells with phosphate buffered saline, the pellets were resuspended
in 10% cold trichloroacetic acid and kept on ice for 20-30 min. The
precipitates were collected on Millipore filters and washed several times
with cold 10% trichloroacetic acid, dried, and the radioactivity was
determined by means of a scintillation counter.
To determine butyrate uptake, n-butyric acid, sodium salt, 1-14C,
(specific activity, 39 mCi/mmol; ICN Radiochemical) was added at 1
ftCi/ml together with cold butyrate to give a final concentration of 1.5
IHMsodium butyrate at the time the cultures were seeded. At designated
intervals, 2-ml aliquota were centrifugea, and the cells were washed in
phosphate buffered saline and collected on Millipore filters. Radioac
tivity was determined by scintillation counting.
The radioactivity remaining in the tissue culture medium, after
centrifugaron of the cells, was also determined using Biofluor (New
England Nuclear) as scintillant. Quenching by the tissue culture me
dium components was determined by appropriate controls.
RESULTS
Patteras of Induction of Differentiation. The effect of exposure
to the 3 potent inducers was compared in the first series of
experiments. Because a more prolonged arrest in G t is observed
with DMSO and butyrate than with HMBA, cultures treated
with these compounds were generally initiated with twice the
number of cells (2 x IO5cells/ml) to provide a sufficient amount
of cells for the assays. Butyrate at the concentration used (1.5
HIM)was the most cytostatic of the 3 inducers. Although the
cell counts of the treated cultures were not always reproducible,
the pattern of induction each followed was. All experiments
were performed a minimum of 3 times. The data from repre
sentative experiments are shown. The response of the cells to
each of the inducers was not affected by the difference in the
number of cells used to seed the cultures. In the experiments
shown in Table 1, there was no change of medium during the
7-day observation period. For some unexplained reason HMBA
was more cytotoxic than usual in this experiment. However,
each inducer caused substantial accumulation of hemoglobin by
the fourth day as detected by the benzidine positive reaction.
After longer periods of exposure, however, striking differences
were observed. In cultures treated with butyrate, an acceleration
of growth accompanied by a decrease in the percentage of
benzidine positive cells was observed by the sixth and seventh
day of exposure. This was in contrast to the behavior of the
acid insoluble fraction of cells pulse labeled for l h was mea
sured (Table 2). No significant differences in the incorporation
between the control and treated cells was observed on the fourth
day, when all cultures generally reach a stationary stage. Dif
ferences were apparent, however, when the cells transferred to
fresh medium with or without the inducer were assayed. After
2 further days of growth in the presence of the inducers (day
6), there was a 2- to 3-fold increase in incorporation of thymidine in all cultures. Growth was limited as shown in Table 1
and accordingly the capacity for DNA synthesis. The capacity
for DNA synthesis remained at approximately the same level
in DMSO or HMBA treated cells which had been transferred
into fresh medium without inducer. In contrast, butyrate treated
cells transferred into fresh medium without inducer showed a
5-fold increase in incorporation comparable to that which oc
curred in the control untreated cells transferred to fresh medium
at the same time.
Requirement for Butyrate. Could the cellular effects we were
observing be due to the depletion of butyrate from the medium?
The question was raised since fatty acids, such as butyrate are
natural products that can be metabolized by the cells. To
determine whether this was occurring, the cells treated with
each of the inducers for 4 days were adjusted to 2 x 105/ml and
transferred to fresh medium with or without inducer. Assays
were carried out again on the sixth day after the initiation of
the experiments (Table 3). The cells transferred to medium
containing the inducers proceeded along the pathway to termi
nal differentiation. Cultures maintained in butyrate were growth
inhibited. Those maintained in the presence of medium con
taining either DMSO or HMBA were also inhibited but to a
lesser extent. While all the cells from treated cultures trans
ferred to medium without inducers grew well, the pattern of
differentiation was similar to that shown in Table 1, i.e., the
cells that had been treated with DMSO or HMBA remained
committed whereas there was a sharp decrease in the percentage
of benzidine positive cells in the butyrate treated cultures.
To determine whether butyrate was being depleted from the
medium, the incorporation of [14C]butyrate was followed for a
7-day period with no change of medium in cultures seeded with
2 x IO5 cells/ml. Two sets of experiments were performed in
parallel to assay daily the incorporation of the labeled butyrate
and the percentage of benzidine positive cells (Fig. 2). One set
379
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BUTYRATE
REQUIREMENT
FOR STIMULATION
OF FLC INDUCTION
Table 1 Effect of inducen on cell growth and hemoglobin synthesis
Cells synthesizing hemoglobin were determined by benzidine staining as indicated in "Materials and Methods."
day9
Cells 105/ml25
x
TreatmentNone
DMSO(1.5%,v/v)
HMBA (4 HIM)
Sodium butyrate (1.5 HIM)Fourth
dayCCells
benzidine
positive0
28
15.5
12.55
lOVml20.5
x
70
8173Sixth
24.0
19.2
37.0$
1000
800
600
Ã- 400
I 200
hK
O
i 1200
" 1000
eoo
600
400
200
'
24 48 72 %
24 48 72 96
HOURS
Fig. 1. Cell growth and reverse transcriptase activity in control and induced
cultures. Cultures were seeded with 1-2 x 10* cells/ml and the inducers added at
the following concentrations: DMSO, 1.5%; HMBA, 4 mM; sodium butyrate, 1.5
mM. At the times indicated, samples were taken to determine cell growth (•)and
reverse transcriptase activity (A), as described in "Materials and Methods."
Table 2 Thymidine incorporation into cells induced for 4 and 6 days
Cells were adjusted to 2 x I(>' ml on fourth day of induction and resuspended
in fresh medium with and without inducer at the concentrations
"Materials and Methods."
indicated in
[3H]Thymidine incorporated (cpm/103 cells)
dayTreatmentNone
DMSO
HMBASodium
butyrateFourth
Cells xpositive1823
105/ml
85
85
30Seventh
15
390
benzidine
90
91
19
the culture medium (Table 4) showed that the level of [I4C]-
1200
_
day%
benzidine
positive0
Sixth
day2,000
inducer5,700
inducer13,400
3,700
2,500
3,000With
8,500
6,400No
5,700
7,900
15,000
received no supplemental butyrate whereas 1.5 mM cold butyr
ate was added to the second set on the fourth day when uptake
of the labeled inducers had reached a peak. If no butyrate was
added at this point, there was a decrease in the percentage of
benzidine positive cells whereas those cultures which had re
ceived a supplement of cold butyrate maintained a high level of
benzidine positive cells. The incorporation of labeled inducer
decreased with time. When chased by cold butyrate, the label
remained at the same level for 24 h and then decreased possibly
because of degradation. Determination of the radioactivity in
butyrate decreased 50% after 4 days and 90% after 7 days.
These data suggested that butyrate was being metabolized by
the cells, thus reducing the concentration to a level below that
required to stimulate differentiation.
Effect of Conditioned Medium from Induced Cultures. Addi
tional evidence to support the notion that butyrate was being
depleted from the medium was obtained from experiments in
which CM from untreated cultures or cultures treated for 4
days with either DMSO, HMBA, or sodium butyrate was used.
There were insufficient nutrients remaining in CMs from 4-day
control cultures to support cell growth. CMs from the induced
cultures (Table 5), however, permitted cell proliferation, the
most active of which occurred in CM-butyrate. CM-DMSO
and CM-HMBA had not lost induction potency and retained
optimal concentrations of the respective inducer. By day 7,
there were 68 and 84% benzidine positive cells in CM-DMSO
and CM-HMBA, respectively. In marked contrast, there were
only 8% benzidine positive cells in CM-butyrate cultures by day
7 (Table 5).
Similar results were obtained with CM from cultures treated
for 7 days and scored for benzidine positivity 7 days later. Little
growth occurred in cultures initiated with 7 day CMs. The cell
counts in the CM-DMSO and CM-butyrate cultures were 2.3
x 105/ml and 2.0 x 105/ml, respectively, and 4.3 x 105/ml in
control cultures maintained for the same period in CM from
untreated cells. However, cultures in CM-DMSO contained
63% benzidine positive cells in contrast to 16% benzidine
positive cells in the cultures maintained in CM-butyrate.
In order to determine whether the butyrate was being inacti
vated during incubation, medium containing 1.5 mM butyrate
was incubated at 37°Cfor 7 days before the cells were added.
There was no loss of either induction or cytostatic activities of
the preincubated butyrate (data not shown).
Determination of Actual Number of Benzidine Positive Cells.
Taken together, our data were puzzling for they suggested that
differentiation induced in FLC by butyrate was reversible as
were the effects it induced in a number of other systems. This
was a possibility in view of the fact that hemoglobin accumu
lation does not necessarily lead to terminal differentiation (1921). The notion that the induced cells were capable of "dedifferentiation" was eliminated after we plotted the actual number
rather than the percentage of benzidine positive cells in the
cultures against time. These data clearly revealed that the
Table 3 Effect on cell growth and differentiation of removing inducer from cultures
Cells were adjusted to 2 x l ir nil on Imirili day of induction and resuspended in fresh medium with or without inducer.
dayTreatmentNone
xlOVml40
DMSO
HMBA
Sodium butyrateCells
day%
Fourth
20
29
10%
benzidine
positive0
x lOVml,
with inducer4.5
456575Cells
5.0
2.0Sixth
benzidine
positive0
75
87
85Cells
x lOVml,
no inducer20
benzidine
positive0
24
13
16%
70
76
29
380
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BUTYRATE
REQUIREMENT
FOR STIMULATION
OF FLC INDUCTION
30
Control
HMBA
25
20
15
IO
È 5
° O
¿ 30_
NoButyrole
_ DMSO
ì 25
Fig. 2. Time course of ["Clbutyric acid uptake. At the time of seeding, 1 Ci/
ml ( 1.5 IHM)|14C]butyric acid (sodium salt) was added to the cultures and at times
indicated, samples were collected and processed to determine radioactivity and
benzidine positive cells as described in "Materials and Methods." After 4 days
20
15
(arrow) one set of cultures received supplemental 1.5 IHMcold sodium butyrate.
O, sodium butyrate uptake; •.with additional cold butyrate; A, percentage of
benzidine positive cells; A, with additional cold butyrate.
Table 4 Determination of ("CJbutyrate
IO
5
in culture medium
1234567
1234567
DAYS
Fig. 3. Kinetics of cell growth and benzidine positive (li'} cells during differ
entiation. Cells (1-2 x lOVml) were seeded and inducers added as in Fig. 1. At
times indicated, the total number of cells and the number of benzidine positive
Days in
cells were assayed. The number of benzidine negative ( /( ) cells was determined
culture01234567CPM281,000262,000245,000219,000150,00066,00040,00031,000%100.093.087.078.053.523.514.011.0
by subtracting the benzidine positive cells from the total number of cells.
At the times indicated, O.I-ml aliquots were taken from the culture medium
after the cells were centrifuged, and the radioactivity was measured by scintillation
counting.
Table 5 Effect of conditioned medium from induced cultures on cell growth and
differentiation
Cells were seeded into medium from control or induced 4-day cultures and
scored for benzidine positivity 4 and 7 days after initiation of the cultures.
Four days
Medium
Cells x lOVml
Seven days
% benzidine
% benzidine
positive
Cells x lOVml
positive
ControlDMSOHMBASodium
butyrate5.019.710.523.504575206.024.517.230.7068848
number of benzidine positive cells remained relatively constant
from the fourth to seventh day of treatment with each of the
inducers (Fig. 3), i.e., those cells that had differentiated had
reached a point of no return. During this period, there was an
acceleration of growth in butyrate treated cultures of the cells
not yet committed that was correlated with the decrease in
potency of the inducer as demonstrated by the reduction in the
cytostatic and inducer activities. In contrast, growth in the
untreated cultures and in the cultures treated with DMSO
remained stationary and in the treated cultures, virtually all the
cells were benzidine positive.
DISCUSSION
We have reinvestigated the kinetics of growth and hemoglo
bin accummulation in FL cells induced to differentiate with 3
well known potent inducers. The study was undertaken to
address a number of questions concerning the mechanism of
induction by butyrate since it induces reversible alterations in
growth and morphology in many cell systems (8, 12-14)
whereas the changes induced by DMSO and HMBA to our
knowledge are apparently irreversible.
It is known that histones H3 and H4 from the erythroleukemia
cells as well as from HeLa cells become hyperacetylated when
incubated in the presence of sodium butyrate (6). This hyperacetylation is reversible 24 h after removal of butyrate from the
medium. Using molecular hybridization techniques with RNA
from the erythroleukemia cells treated with butyrate or control
cells and nonrepetitive mouse DNA, about 38% new transcripts
were found (22). Many of the new species of protein were not
detected in control or DMSO treated cells. This effect was also
reversible. The butyrate treated cells soon after transfer to
normal medium showed the uninduced pattern of RNA and
protein synthesis.
Our results provide further evidence that the mechanism of
action of sodium butyrate differs from that of the other induc
ers. As compared to DMSO and HMBA, butyrate, at the
concentration used in this study, is cytostatic for the first 3
days of culture. This is coincidental with the peak of butyrate
uptake and hemoglobin accummulation by the cells (Fig. 2).
After the fourth day of treatment, as the butyrate concentration
is diminishing in the media (Table 4), the cells generally resume
active growth and a decrease in the relative number of benzidine
positive cells occurs. However, this cannot be attributed to
reversibility. If the data are calculated on the basis of the actual
number rather than on the percentage, the benzidine positive
cells in butyrate treated cultures remain at approximately the
same level through day 7, indicating that they are terminally
differentiated (Fig. 3).
Studies on the incorporation of [3H]thymidine demonstrated
that after 4 days of exposure, butyrate treated cells but not cells
treated with DMSO or HMBA were capable of active DNA
synthesis and division following the removal of the inducer.
Further, measurement of the uptake of [14C]butyrate by the cells
and in the culture medium showed a decrease in the incorpo
ration of the inducer with time coincident with the decrease in
the percentage of benzidine positive cells. These data support
the notion that butyrate is readily metabolized, thereby reducing
the concentration required to maintain differentiation.
Although we have pointed out some of the effects of butyrate
that differ from those of the other inducers, there are other
parameters that they affect in common. For instance, during
induction by each of the inducers, the number of proviral copies
and the integration patterns remain unchanged (18). A study of
the DNA-binding proteins from nuclei of control and treated
381
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BUTYRATE
REQUIREMENT
FOR STIMULATION
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Friend erythroleukemia cells. J. Biol. Chem., 254:4283-4290, 1979.
23. Terada, M., Nudel, U., Fibach, E., Rifkind, R. A., and Onaki, P. A. Changes
in DNA associated with induction of erythroid differentiation by dimethyl
sulfoxide in murine erythroleukemia cells. Cancer Res., 38:835-840, 1978.
24. Novogrodsky, A., Dvir, A., Ravid, A., Shkolink, T., Stenzel, K. H., Rubin,
L. R., and Zaizov, R. Effect of polar organic compounds on leukaemic cells:
butyrate induced partial remission of acute myeloid leukaemia in a child.
Cancer (Phila.), 51:9-14, 1983.
cells revealed that 2 extra DNA-binding proteins of M, 25,000
and 38,000 are present in the induced cells along with a decrease
in the DNA-binding capacity of the histones. However, no
major differences have been observed among the 3 inducers we
have used indicating that sodium butyrate has no specific effects
at this level.3 These results are in agreement with the findings
that all the potent inducers cause breakages in the DNA of the
induced cells (23, 17).
In conclusion, the phenomenon we are observing cannot be
attributed to a reversibility of the butyrate effects since the
actual number of cells accumulating hemoglobin in the treated
cultures remain committed. Instead our results suggest that
butyrate is unique among the inducers in that it is a natural
fatty acid that is rapidly metabolized. It remains to be deter
mined whether our observations can be attributed to a process
of degradation which might release a regulatory factor required
to maintain differentiation. This factor may be unstable and
active only in the presence of the inducer. Perhaps direct
measurement of butyrate by Chromatographie methods may
yield further information. Of particular interest in this respect
is the study of Novogrodsky et al. (24) who followed the serum
level in dogs given an infusion of 3% butyrate (29 mg/kg/24
li). After the plasma was infused, the half-life was found to be
extremely rapid, less than 5 min. Nevertheless, butyrate treat
ment of a child with acute myelogenous leukemia induced a
partial remission.
The molecular basis for the differences which distinguish the
effects of the inducers with ability to modulate gene expression
remains unclear. As mentioned above the regulatory mecha
nisms in FL cells differ from those controlling erythropoiesis
in normal RBC (2, 3). This suggests that alternative pathways
to differentiation and cessation of growth may be available to
the erythroleukemia cells as well as to other types of neoplastic
cells. These alternative pathways are being brought to light by
the various compounds which have the ability to repress the
malignant phenotype.
REFERENCES
1. Marks, P., and Rifkind, R. A. Erythroleukemia differentiation. Annu. Rev.
Biochem., ¿7:419-426, 1978.
2. Friend, C. Murine virus induced erythroleukemia cell lines. In: G. Sato (ed.),
Functionally Differentiated Cell Lines, pp. 235-249. New York: Alan R.
l.iss.
llK'..
OF FLC INDUCTION
1981.
3. Stringer, E., and Friend, C. Hemin-independent control of globin synthesis
in Friend erythroleukemia cells induced to differentiate. Proc. Nati. Acuii.
Sci. USA, 79:1839-1843, 1982.
3 Unpublished data.
382
Downloaded from cancerres.aacrjournals.org on April 13, 2017. © 1987 American Association for Cancer Research.
Depletion of Sodium Butyrate from the Culture Medium of
Friend Erythroleukemia Cells Undergoing Differentiation
Charlotte Friend, Maria Zajac-Kaye, J. Gilbert Holland, et al.
Cancer Res 1987;47:378-382.
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