Chloramphenicol Induces Abnormal

Differentiation and Inhibits Apoptosis in

Activated T Cells
Zeng-Rong Yuan and Yufang Shi

DOI: 10.1158/0008-5472.CAN-07-6061 Published June 2008

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 The Aerobic Carbohydrate Metabolism of Leukocytes in Health and Leukemia. I. Glycolysis and
Respiration - March 1, 1953

Abstract
Chloramphenicol is a broad-spectrum antibiotic used for the treatment of many infectious diseases
and has become one of the major seafood contaminants. Hematologic disorders such as aplastic
anemia and leukemia induced by chloramphenicol are a major concern. However, the mechanism
underlying chloramphenicol-induced leukemogenesis is not known. By investigating the effects of
chloramphenicol on the activation of mouse T cells stimulated with anti-CD3 antibody or
staphylococcal enterotoxin B, we found that chloramphenicol induces the differentiation of activated
T cells into lymphoblastic leukemia-like cells, characterized by large cell size, multiploid nuclei, and
expression of CD7, a maker for immature T cells and T-cell lymphocytic leukemia, thus
phenotypically indicating differentiation toward leukemogenesis. High expression of cyclin B1, but
not p53, c-myc, and CDC25A, was detected in chloramphenicol-treated activated T cells, which may
relate to abnormal cell differentiation. Chloramphenicol inhibited the activation-induced cell death of
mouse and human T-cell receptor–activated T cells by down-regulating the expression of Fas ligand.
Our findings show that abnormal cell differentiation and inhibition of apoptosis may contribute to the
development of leukemia associated with clinical applications of chloramphenicol. [Cancer Res
2008;68(12):4875–81]

Introduction
Chloramphenicol is an antibiotic originally isolated from Streptomyces venezuelae in 1947. It has a
bactericidal effect on a broad range of bacteria, as well as Rickettsia, Chlamydia, and Mycoplasma.
Because chloramphenicol is relatively inexpensive to manufacture, highly effective in combating
infections, and able to pass the blood-brain barrier, it is still widely used in developing countries ( 1–
6). However, because of its potential toxicity to the hematopoietic system, chloramphenicol is only
 occasionally used in developed countries as a substitutional therapy for some infections caused
by Haemophilus influenzae, Streptococcus pneumoniae, Salmonella typhi, and Neisseria species. The major
side effects found in patients treated with chloramphenicol include reversible bone marrow
depression, aplastic anemia, and leukemia. Epidemiologic studies indicate that 1:30,000 to 1:45,000
patients receiving chloramphenicol treatment go on to develop leukemia. Recently, chloramphenicol
has become one of the major contaminants of farmed shrimps and fish, with some products
containing excessive chloramphenicol residue, raising new concerns about its toxicity.
Leukemia has been linked to various risk factors, such as heredity status, chronic virus infection,
radiation, chemical contaminants, and medications. Based on epidemiologic studies,
chloramphenicol has been strongly correlated with leukemogenesis ( 7). One research group has
successfully induced leukemia in chloramphenicol-treated toads ( 8). However, the molecular
mechanism through which chloramphenicol induces leukemogenesis is still unclear. Nevertheless,
nitrobenzene is a substituent chemical moiety in chloramphenicol and nitrobenzene is a long known
carcinogen. It is possible that the transforming ability is related to the nitrobenzene group of this
antibiotic. Pharmacologically, chloramphenicol reversibly binds to the 50S ribosomal subunit and
prevents the transfer of amino acids during peptide chain elongation in bacteria. Chloramphenicol
also has effects on protein synthesis in rapidly proliferating but not resting mammalian cells. Thus, it
is possible that leukemia induced by chloramphenicol may directly change the expression of genes
associated with cell cycle and apoptosis.
Much evidence has shown that the development of leukemia is related to gene dysregulation. c-myc
is a transcriptional factor for control of cell proliferation, and high expression of c-myc has been
found in leukemia ( 9, 10). p53 is a tumor suppressor gene, and mutations causing p53 dysfunction
have been detected in patients with different types of leukemia ( 11– 13). Cyclin B1, which plays a
critical role in the regulation of cell cycle progression, was recently identified as an oncogene.
Overexpression and/or unscheduled expression of cyclin B1 are always detected in the cells from
leukemia and other tumors ( 2, 14– 19). Accumulating evidence indicates that CDC25A can be
oncogenic, and its overexpression is frequently shown in a large number of tumors ( 20–23).
Moreover, leukemia is also considered a disorder of dysregulation of apoptosis because alterations
in Fas and Fas ligand (FasL) expression have been found in leukemia ( 24, 25). These data indicate
that leukemia is a multiplex disorder resulting from dysfunction in various genes, often combined
with environmental causes.
To explore the mechanism by which chloramphenicol induces leukemia development, mouse
primary splenocytes were used as a cell model for morphologic and molecular studies in vitro.
Mouse primary splenocytes were activated with anti-CD3 in the presence or absence of
chloramphenicol for a prolonged time. We found that chloramphenicol promoted abnormal T-cell
differentiation and allowed the formation of leukemia-like cells, whereas in cultures without
chloramphenicol all cells eventually died and no abnormal cells were found. These cells survived in
culture for months. They expressed high levels of cyclin B1 and very low level of FasL. In addition,
these abnormal cells also expressed high level of CD7, a hallmark of immature lymphoblastic
leukemia. Therefore, this finding for the first time provides direct evidence that chloramphenicol
induces leukemogenesis in mammalian T cells.

Materials and Methods

Cell culture and stimulators in vitro. Spleens were taken from 6- to 8-week-old BALB/c mice and
pulverized between two autoclaved grass slides in RPMI 1640 (Invitrogen), and a single-cell
suspension was obtained by passing through a 40-μm-diameter nylon mesh (BD Biosciences). The
cells were washed twice in RPMI 1640 and then cultured at 2 × 106 to 5 × 106 per mL density in
complete RPMI 1640, containing 10% fetal bovine serum, 10% L-glutamine, penicillin, and
streptomycin. Antibody against CD3 (145-2c-11) at 2.5 μg/mL or staphylococcal enterotoxin B (SEB;
Sigma-Aldrich) at 5 μg/mL was used to activate T cells in the presence or absence of
 chloramphenicol at 500 μg/mL for 1 week. The cells were then maintained in complete RPMI 1640
with interlekin-2 (IL-2) at 50 to 100 units/mL for cell survival studies. Cell differentiation and
morphologic changes were observed by light microscopy.
Cell staining. For morphologic study, cell smears were prepared by spreading of cultured cells on a
glass slide and allowing them to air dry. The Giemsa reagent (Sigma-Aldrich) was used to stain the
cells according to the manufacturer's protocol. Cellular morphologic characteristics were examined
under light microscopy. For immunofluorescence staining, freshly cultured cells were washed twice
in ice-cold staining buffer (1× PBS with 2% fetal bovine serum and 0.01% sodium azide). Cell
staining was carried out in the same staining buffer with FITC-conjugated anti-CD3 (eBioscience)
and anti-CD7 plus phycoerythrin (PE)-conjugated anti-goat IgG (Santa Cruz Biotechnology) following
the supplier's protocols. After washing twice with cold staining buffer, the cells were mounted with
ProLong antifade reagent (Molecular Probes) and covered with a glass coverslip. Fluorescence was
analyzed under a Nikon fluorescence inverted microscope (Eclipse TE 2000-S, Nikon).
Flow cytometric analysis. Human T-cell lines Jurkat and Jcam (kind gifts of Dr. Gordon Mills, M. D.
Anderson Cancer Center, Houston, TX) were activated by ionomycin at 100 nmol/L each in culture
medium and mouse T-cell lines A1.1 and IE5 in plates coated with anti-CD3 at 2.5 μg/mL. Different
concentrations of chloramphenicol were added with the stimulators as indicated. After incubation at
37°C for overnight, the cells were harvested and washed twice with cold 1× PBS. The cell pellets
were then resuspended in propidium iodide staining buffer (1× PBS with 20 μg/mL propidium iodide,
0.2% saponin, and 50 μg/mL RNase) and incubated for 30 min at room temperature. Flow cytometry
analysis was carried out with a FACScan (Becton Dickinson). Cell cycle distribution is indicated by
DNA content revealed by propidium iodide staining. After gating out cell fragments, the population of
sub-G0 phase on the histogram plot represents apoptotic cells. For cell surface marker testing, the
mouse cell line IE5 was stimulated with and without anti-CD3 in the presence or absence of
chloramphenicol at 500 μg/mL. After incubation at 37°C for 6 h, the cells were harvested and
washed twice with cold staining buffer. The FITC-conjugated anti-Fas antibody and PE-conjugated
anti-FasL antibodies (BD Biosciences) were used for cell surface staining and analyzed by flow
cytometry.
Northern blot. Mouse splenocytes were stimulated with anti-CD3 in the presence or absence of
chloramphenicol at 500 μg/mL for 1 week. Total RNA was extracted using TriPure reagent and its
protocol (Roche). RNA (20 μg) of each sample was separated on a 1% denaturing formaldehyde
agarose gel and then transferred to a nylon membrane (Amersham Biosciences). The membrane
was repeatedly hybridized with [32P]dCTP-labeled probes for cyclin B1, p53, c-myc, and CDC25A.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal quantitative control.
Regulatory T-cell population. BALB/c mouse splenocytes were stimulated with anti-CD3 alone (2.5
μg/mL) or plus chloramphenicol (500 μg/mL) in complete RPMI 1640 for 1 week. The population of
CD4+CD25+ cells was measured by flow cytometric analysis after staining with FITC-conjugated anti-
CD4 and PE-conjugated anti-CD25 (eBioscience). FoxP3 expression in CD4+CD25+ cells was further
analyzed by real-time quantitative PCR using SYBR Green PCR Master Mix (Applied Biosystems).
The primer sequences were the following: TTCATGCATCAGCTCTCCAC (sense) and
CTGGACACCCATTCCAGACT (antisense). GAPDH expression was analyzed simultaneously as an
internal quantitative control.

Results
Chloramphenicol promotes abnormal differentiation of leukemic cells. The most suspected
transformations induced by chloramphenicol are acute lymphoblastic leukemia and acute
myelogenous leukemia, both of which are derived from proliferating cells. Because our preliminary
experiments with in vitro culture of mouse splenocytes with chloramphenicol alone did not produce
abnormal cells, no matter what concentration of chloramphenicol was used or how long the cultures
were maintained, we believe that the effect of chloramphenicol is exerted on proliferating cells.
Epidemiologic study of chloramphenicol-induced leukemia has shown that it is always related to a
history of bacterial infection. We thus believe that some bacterial superantigens, such as SEB, may
work synergistically with chloramphenicol by inducing T-cell proliferation. In these proliferating cells,
chloramphenicol may promote abnormal differentiation by altering the expression of some cell cycle–
related and apoptosis-related genes. By stimulating mouse splenocyte with anti-CD3 or SEB in the
presence or absence of chloramphenicol for 1 week, we found that chloramphenicol at 400 to 600
μg/mL resulted in the appearance in culture of many large cells, which were not present in control
cultures activated without chloramphenicol or treated with chloramphenicol alone ( Fig. 1A ). These
abnormal cells were morphologically similar to leukemic cells, characterized by very large cell size
and multiploid nuclei. The same cultured cells were also analyzed for DNA content with propidium
iodide staining. The results show that chloramphenicol not only induces additional mitosis, as
indicated by the high diploid peak, but also results in many multiploid cells, changes that were
identical to those revealed by microscopy ( Fig. 1B). Significantly, when we continued to culture the
chloramphenicol-treated activated T cells supplemented with IL-2 for up to a month, many large cells
continued to survive ( Fig. 2A ), whereas all cells activated in the absence of chloramphenicol died
off ( Fig. 2B). Furthermore, by immunofluorescence staining, we found that all of the surviving cells
express CD3 and only the large cells also express CD7, a marker of immature T leukemic cells ( Fig.
2C and D). This phenotypic change may represent a differentiation process toward leukemogenesis.
Taken together, these results show that chloramphenicol induces abnormal cell differentiation to
leukemia-like cells.

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Figure 1.
Chloramphenicol induces abnormal cell differentiation. Mouse splenocytes were cultured for 1 wk in
different conditions, as indicated. Cell morphology was observed under light microscopy. A, the
presence of chloramphenicol (CAP) in the culture induces many large cells, which were not present
in control cultures. B, top,chloramphenicol-stimulated cells were much larger with multiploid
nuclei; bottom, flow cytometric analysis shows a significantly right shift in chloramphenicol-treated
cells in forward scatter. The phenotype of abnormal large cells is as a leukemia-like cell
morphocytologically.

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Figure 2.
Chloramphenicol-treated cells express CD3 and CD7 markers and survive longer. Cells were
cultured as described in Materials and Methods. Most cells stimulated in the presence of
chloramphenicol are still alive and appear healthy after 1 mo (A), whereas cells stimulated with anti-
CD3 alone completely died out (B). Among surviving cells in the chloramphenicol cultures, all cells
expressed CD3 (C) and only larger cells expressed CD7, an immature T-cell marker (D), as
determined by immunofluorescence and flow cytometry.

Overexpression of cyclin B causes abnormal cell differentiation. The precise execution of cell cycle
events is tightly controlled by concerted activation and inhibition of the expression of various genes
and the activities of their products ( 10, 26– 28). Dysregulation of critical genes in this process leads
to abnormal cell proliferation and differentiation. To understand the mechanism of chloramphenicol-
mediated abnormal cell differentiation, we analyzed the expression of several cell cycle–related
genes. We found that the expression of cyclin B1 is dramatically increased in cells treated with anti-
CD3 and chloramphenicol, whereas the expression of other genes, such as p53, CDC25A, and c-myc,
was not significantly altered ( Fig. 3 ). Because cyclin B1 regulates the transition in the mitosis phase
of the cell cycle, overexpression of cyclin B1 may result in excessive nuclear division and the
formation of leukemic cells with multiploid nuclei. It has been shown that treatment of cancer cells
with butyrolactone I causes accumulation of cyclin B1 and formation of cells with multiple nuclei
( 29). Even in plants, when a nondegradable cyclin B1 is expressed, it also causes defective G2-M
and increase in cells with multiple nuclei ( 30).

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Figure 3.
Chloramphenicol induces high cyclin B1 expression. The expression of cyclin B1, p53, c-myc, and
CDC25A was analyzed by Northern blotting in splenocytes treated by anti-CD3 antibody in the
presence or absence of chloramphenicol. Chloramphenicol significantly induced overexpression of
cyclin B1, but not p53, c-myc, and CDC25A. GAPDH served as an internal quantitative control.

Chloramphenicol inhibits activation-induced cell death. Activation-induced cell death (AICD) is a key
mechanism for maintaining cellular homeostasis during lymphocyte development, immune
responses, and tumorigenesis. Although mouse splenocytes at rest do not proliferate, they do exhibit
dramatic proliferation potential following stimulation of anti-CD3 or SEB. Previously activated T cells
will undergo apoptosis after reactivation. This apoptosis, however, can be significantly inhibited by
chloramphenicol in our experiment. We found that activation of chloramphenicol-treated cells
displays much less apoptosis than cells treated with anti-CD3 alone ( Fig. 4A ). To further confirm
this finding as a universal biological phenomenon, different concentrations of chloramphenicol were
also applied to inhibit AICD in the human T-cell lines Jurkat and Jcam as well as the mouse T-cell
hybridomas A1.1 and IE5. As shown in Fig. 4B and C, chloramphenicol clearly blocked AICD in all
four cell lines, although these cells were activated by different stimuli. Our data show that
chloramphenicol not only promotes abnormal differentiation of splenocytes into leukemia-like cells
but also promotes the survival of these cells by inhibiting apoptosis. We believe that these two
properties of chloramphenicol could be key to its leukemia-inducing potential.
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Figure 4.
Chloramphenicol inhibits apoptosis in mouse primary cells and several cell lines. Mouse splenocytes
were stimulated by anti-CD3 with graded concentrations of chloramphenicol, as indicated. DNA
content was determined by flow cytometric analysis after staining with propidium iodide. The sub-
G0 population (M1) represents apoptotic cells. A, chloramphenicol significantly inhibited AICD
compared with the cells stimulated with anti-CD3 in the absence of chloramphenicol. Mouse T-cell
lines A1.1 and IE5 were stimulated with anti–CD3-coated plates, and human T-cell lines Jurkat and
Jcam were stimulated by ionomycin (100 nmol/L). Different concentrations of chloramphenicol were
added to the culture, as indicated. Apoptosis was examined by DNA content as in A. B, the inhibition
of apoptosis by chloramphenicol correlated well with its concentration. C, the effect of
chloramphenicol on AICD in IE5 cells is representative.

Chloramphenicol blocks FasL expression induced by T-cell receptor activation. AICD in T cells is
mediated by induction of the expression of Fas and FasL and their subsequent interaction. Blocking
this interaction with TR6, Fas fusion protein, or monoclonal antibodies against FasL can completely
prevent AICD ( 31). Because chloramphenicol is believed to affect mitochondrial protein synthesis,
we examined whether the effect of chloramphenicol on AICD is exerted through an effect on
cytochrome c or the Fas pathway. We found that chloramphenicol did not change
cytochrome c levels and did not inhibit apoptosis induced by anti-Fas antibody. Because AICD is
largely dependent on the Fas-FasL pathway, these results indicate that chloramphenicol inhibits
AICD at a step before activation of the Fas receptor. To understand the molecular mechanism by
which chloramphenicol inhibits apoptosis, we examined changes in Fas and FasL expression in
chloramphenicol-treated cells by immunofluorescence cell staining and flow cytometry. We found
that the high expression of FasL stimulated by anti-CD3 antibody is completely inhibited by
chloramphenicol. However, chloramphenicol does not seem to affect Fas expression ( Fig. 5 ).
Therefore, chloramphenicol must inhibit AICD by blocking FasL expression. Our result shows that
the molecular mechanism of chloramphenicol-mediated inhibition of apoptosis is by blocking FasL
expression.
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Figure 5.
Suppression of FasL expression plays a critical role in apoptosis inhibition by chloramphenicol. IE5
cells were stimulated by anti-CD3 with or without chloramphenicol. The expression of Fas and FasL
was detected by immunofluorescence staining and flow cytometry. Anti-CD3 stimulation induced
high FasL expression, but the presence of chloramphenicol significantly inhibited its expression.

Chloramphenicol promotes a more regulatory T-cell population. Regulatory T cells (Treg) are potent
modulators of immune responses ( 32– 35). Various studies indicate that Tregs are
immunosuppressive. Animals with depleted Tregs spontaneously develop various T-cell–mediated
autoimmune diseases ( 36). Transfusion of Tregs inhibits lethal graft-versus-host disease (GVHD) in
bone marrow transplantation ( 33). Because activation of the T-cell receptor (TCR) can induce T-cell
differentiation, including a population of CD4+CD25+ cells, we examined what would occur in the
presence of chloramphenicol. We found that splenocytes stimulated with anti-CD3 in the presence of
chloramphenicol had significantly more CD4+CD25+ cells ( Fig. 6A ). Moreover, expression of the
transcription forkhead box P3 factor (FoxP3) was also significantly higher than in the controls ( Fig.
6B). Thus, chloramphenicol promotes more Treg differentiation, which may act to suppress possible
immune responses against leukemic cells and thus allow the development of leukemia.
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Figure 6.
Chloramphenicol induces the expansion of Tregs. The induction of Tregs from splenocytes of
BALB/c mouse was done as described in Materials and Methods. A, CD4+CD25+ cells were detected
by flow cytometric analysis following two-color staining. B, FoxP3 expression in CD4+CD25+ cells was
evaluated by real-time quantitative PCR. Chloramphenicol was found to favor the outgrowth of the
Treg population.

Discussion
It has been known for more than 15 years that there is a link between the use of chloramphenicol
and the development of leukemia ( 37, 38). However, the molecular mechanism for leukemogenesis
induced by chloramphenicol is largely unknown. Because chloramphenicol is still popularly used as
an effective treatment for many bacterial infections in developing countries, or as a substitutional
therapy for drug-resistant bacteria in developed countries, a clear understanding of the mechanism
of leukemogenesis induced by chloramphenicol would be of benefit to the development of new drugs
and to human health worldwide. In addition, the current increase in the use of chloramphenicol in
seafood farming and its associated concerns to human health also demand a better understanding
of the toxicology of this antibiotic. In this report, we present evidence that chloramphenicol induces
leukemia-like cells in activated T cells by promoting abnormal cell differentiation and inhibiting
activation-induced apoptosis.
Leukemia is a type of blood cancer characterized by a large number of abnormal or immature cells
in the bloodstream. Unlike most blood cells, the malignant cells tend to multiply and live for a longer
time, thus leading to accumulation within the body. However, the mechanisms through which most
leukemias develop are still not known. It is well known that cancer cells frequently arise in the body
but they normally die by suicide through apoptosis or by fratricide through immune recognition
before they accumulate in significant numbers. Therefore, leukemia development is closely related
to two key events: unrestrained proliferation and enhanced survival. By using mouse splenocytes as
an experimental model in vitro, we showed that chloramphenicol combined with mitogenic stimuli
significantly induces the formation of leukemia-like cells, characterized by large cell size, multiploid
nuclei, and especially the expression of CD7, a marker for immature T cells and T-cell lymphoblastic
leukemia.
The cell phenotypic changes are associated with aberrant expression of cyclin B1, which helps
control cell mitosis. It has been shown in other cell systems that overexpression of cyclin B1
promotes the transformation of cells with a multiploid nuclei ( 39, 40), a mechanism very likely also
operative in inducing the multiploid cellular phenotype in our experimental system. Another cellular
modification required for leukemogenesis is that the malignant cells must escape apoptosis and
avoid attack by the immune system. We show that chloramphenicol is also a potent inhibitor of
activation-induced apoptosis, which is efficient in primary cells as well as cell lines of human and
mouse. This property of chloramphenicol has not been revealed previously and we believe that it
plays a critical role in chloramphenicol-induced leukemogenesis by allowing proliferating cells to
continuously survive. Furthermore, we showed that the molecular mechanism of chloramphenicol
inhibition of apoptosis is through blocking of FasL expression. Therefore, it is likely that the cellular
changes associated with chloramphenicol make leukemic-like cells more likely to survive long-term
and develop into clinical leukemia.
Chloramphenicol had been a popular antibiotic before realizing that it could cause aplastic anemia. It
is now also believed to be a carcinogen based on limited evidence of carcinogenicity from clinical
studies in humans. Three case reports have shown the development of leukemia after
chloramphenicol therapy. In a case-control study in China, Shu and colleagues ( 37, 38) found
increased risks of childhood leukemia, which correlated with the number of days chloramphenicol
was administered. Two case-control studies revealed high, but nonsignificant, increases in the risk of
aplastic anemia associated with the use of chloramphenicol ( 41, 42). However, other studies found
no association between the use of chloramphenicol and the development of adult leukemia (43, 44).
Interestingly, chloramphenicol also induced leukemia in toads ( 8). When mice were treated with
busulfan and chloramphenicol, they develop transplantable T-cell leukemia, providing clear evidence
that chloramphenicol can indeed induce leukemia in T cells ( 45). Therefore, the evidence of a link
between aplastic anemia and leukemia, and the increased risk of leukemia found in some case-
control studies supports the conclusion that chloramphenicol exposure is associated with an
increased malignancy risk in humans.
Immune surveillance also has an important role in killing malignant cells. Recent studies have shown
that T-cell leukemia, such as that induced by human T-cell lymphotrophic virus, possesses a Treg
phenotype. Tregs are potent modulators of immune responses ( 32– 35). Various studies indicate
that Tregs are hyporesponsive and suppressive. Animals with depleted Tregs spontaneously
develop variable T-cell–mediated autoimmune diseases ( 36). Transfusion of Tregs inhibits lethal
GVHD in bone marrow transplantation ( 33). Because activation of the TCR can induce cell
differentiation, including populations of CD4+CD25+ cell, we examined what would occur in the
presence of chloramphenicol. We found that splenocytes stimulated with anti-CD3 in the presence of
chloramphenicol had significantly more CD4+CD25+ cells. Moreover, expression of the transcription
forkhead box P3 factor FoxP3 was also significantly higher than in the controls. Thus,
chloramphenicol promotes more Treg differentiation, which may act to suppress possible immune
responses against leukemic cells.
In conclusion, we have shown a molecular mechanism of chloramphenicol-induced leukemia,
resulting from overexpression of cyclin B1 during cell differentiation and down-regulation of FasL
expression, thus preventing apoptosis. Disruption of normal immune responses by developing a
Treg phenotype may also promote leukemogenesis by down-regulating the immune surveillance
mechanisms.
 Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.

Acknowledgments
Grant support: NIH research grants CA76492 and AI43384.
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.
We thank Arthur Roberts for his critical review of the manuscript; Guangwen Ren for technical
support; and Drs. Sidney Pestka, Arnold Rabson, and Jerome Langer for discussions.

Footnotes
 Note: Current address for Z-R. Yuan: Department of Cell Biology and Molecular Medicine, New
Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 South Orange
Avenue, Newark, NJ 07103.
 Received November 2, 2007.
 Revision received March 25, 2008.
 Accepted March 27, 2008.
 ©2008 American Association for Cancer Research.

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June 2008
Volume 68, Issue 12
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