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INTRODUCTION |
The cytolethal distending toxins
(CDTs)1 are newly discovered
bacterial protein toxins with the unique ability to induce cell cycle
arrest, thereby inhibiting cell proliferation. CDTs are produced by a
number of bacterial pathogens, including Escherichia coli,
Haemophilus ducreyi, Campylobacter sp.,
Actinobacillus actinomycetemcomitans, Shigella
dysenteriae, and Helicobacter hepaticus. Three linked genes, cdtA, cdtB, and cdtC, encode
three polypeptides, which are responsible for the toxic activity.
Coexpression of all three components is required to confer toxicity
(reviewed in Ref. 1), but the functions of the individual gene products
are still not known. It was reported that purified cdtB from A. actinomycetemcomitans could induce cell cycle arrest in human T
lymphocytes and HeLa cells (2, 3). However, cdtC is absolutely required
for the toxin activity in H. ducreyi, because culture
supernatant fluid from a bacterial strain carrying a mutated cdtC did
not display any cytotoxicity (4).
H. ducreyi is a Gram-negative coccobacillus, which causes
chancroid, a sexually transmitted disease, characterized by genital tissue necrosis and retardation of healing. The pathogenesis of the
disease remains poorly understood. The H. ducreyi CDT
(HdCDT) may represent an important virulence factor, because it is
produced by the majority of H. ducreyi strains from clinical
samples and most patient sera contain HdCDT-neutralizing antibodies,
whereas control sera do not (5, 6). Furthermore, the production of
HdCDT is crucial for cell destruction after adhesion of H. ducreyi to cultured cells, whereas toxin-negative strains adhere but leave the cells intact (7).
Cells exposed to CDTs have been shown to arrest in the G2
phase of the cell cycle, and this is due to accumulation of the tyrosine-phosphorylated form of the cyclin-dependent kinase
(cdc) 2 (reviewed in Ref. 1). A similar response is induced in HeLa cells after exposure to ionizing radiation (IR) (8). In proliferating cells, genotoxic stress activates checkpoint responses, which prevent
progression through the cell cycle until the DNA damage has been
repaired, thus avoiding genetic instability. Checkpoint arrest occurs
at different stages of the cell cycle: the G1/S transition
(G1 checkpoint), the S phase progression, and the
G2/M boundary (G2/M checkpoint) (9, 10). The
closely related protein kinases ATM and ATR are key molecules in
sensing DNA damage. ATM responds mainly to DNA double stranded-breaks
and is inactivated by mutations in ataxia-telangiectasia patients, who
are extremely sensitive to IR and other genotoxic agents. ATM can
activate all the different checkpoints in response to double
stranded-breaks (reviewed in Ref. 11).
G1 arrest induced by DNA damage requires the tumor
suppressor protein p53, which is stabilized via phosphorylation on
serine 20 in an ATM-dependent manner by the chk2 protein
kinase (12-14). Phosphorylated p53 dissociates from the Mdm2 protein
that otherwise will target p53 for degradation via the
ubiquitin-proteasome pathway (15, 16). The G2/M checkpoint
involves the maintenance of cdc2 in an inactive hyperphosphorylated
state. In the unperturbed cell cycle, cdc2 is kept inactive by
phosphorylation on threonine 14 and tyrosine 15 by the wee1 and myt1
kinases. In late G2, the cdc25C phosphatase
dephosphorylates these residues, and thereby activates the
cdc2·cyclin B1 complex (reviewed in Ref. 17). Recent data demonstrate
that the protein kinases chk1 and chk2 are activated via
phosphorylation in vivo in response to DNA damage, and both
kinases are able to phosphorylate and inactivate cdc25C in
vitro (18, 19).
In this study, we demonstrate that HdCDT induces cell growth arrest in
a broad panel of human cell types, including normal keratinocytes and
fibroblasts, which are the possible toxin targets in vivo.
Intoxication of human cells with HdCDT did not exclusively induce
G2 arrest, because human foreskin and embryonic lung (HL) fibroblasts were arrested also in G1, and B cells underwent
apoptosis. HdCDT induced responses similar to IR in epithelial HEp-2
cells, fibroblasts, and lymphoblastoid cell lines (LCLs). Furthermore, ATM-deficient LCLs were much more resistant to the intoxication than
ATM wild type cells. These data show that HdCDT induces cell cycle
arrest by activating checkpoint responses similar to those induced by
IR, and that rapid intoxication requires a functional ATM protein kinase.
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EXPERIMENTAL PROCEDURES |
Cell Lines
Human foreskin fibroblasts were obtained from the American Type
Culture Collection (HS27; ATCC, Manassas, VA), and human embryonic lung
fibroblasts (HL) were purchased from the Department of Microbiology, Sahlgrenska Hospital, Gothenburg, Sweden. Both lines were used between
passages 15 and 25.
The Burkitt's lymphoma (BL) cell line BL41 was established from an
Epstein-Barr virus-negative tumor biopsy (20). The JAC-B1 and SN-B1
LCLs were obtained by in vitro infection of B lymphocytes from healthy donors with the B95.8 strain of Epstein-Barr virus as
previously described (21).
The AT06LA, AT00LA, AT01LA, and AT13LA LCLs established from
ataxia-telangiectasia patients were kindly provided by Dr. R. A. Gatti, Department of Pathology, UCLA, Los Angeles, CA.
Normal keratinocytes, Ad3 and Ad5, were established from human neonatal
foreskin and used between passages 3 and 7.
The other cell lines were as follows: the human larynx carcinoma HEp-2
(ATCC no. CCL-23), the human cervix carcinoma HeLa (ATCC no. CCL-2),
and the human keratinocyte line HaCaT (kindly provided by Dr. N. E. Fusenig, Heidelberg, Germany). All cell lines, except keratinocytes,
were cultivated in Iscove's modified Dulbecco's medium supplemented
with 10% heat-inactivated fetal calf serum, 5 mM
L-glutamine, penicillin (100 units/ml), and streptomycin (100 µg/ml) (complete medium) (Life Technologies, Inc., Gaithersburg, MD). Keratinocytes were cultivated in keratinocyte-SFM medium supplemented with bovine pituitary extract (25 µg/ml) and recombinant epidermal growth factor (0.2 ng/ml) (Life Technologies, Inc.).
Toxin and Treatments
HdCDT was purified by immunoaffinity chromatography using the
neutralizing monoclonal antibody M4D4, as previously described (22);
Western blot analysis with specific antibodies showed that the purified
product contained both the B and C components, whereas A was not
detectable. The protein concentration of the stock solution was 2 mg/ml.
Toxin Treatment--
Cells were incubated for the indicated time
periods in the presence of HdCDT (2 µg/ml) in complete medium.
Irradiation--
Cells were irradiated (20 Gy), washed once in
phosphate-buffered saline (PBS), and incubated for the indicated time
periods in complete medium.
Hydroxyurea and Nocodazole Treatment--
Cells were incubated
in complete medium supplemented with 2.5 mM hydroxyurea
(Calbiochem, La Jolla, CA) or 100 nM nocodazole (Sigma
Chemical Co., St. Louis, MO) for 24 h.
Caffeine Treatment--
Cells were cooled on ice for 15 min
before addition of the toxin (2 µg/ml for 15 min on ice). After the
binding step, cells were washed three times with PBS to remove unbound
toxin and were further incubated for 24 h in complete medium with
or without 4 mM caffeine (Sigma).
Cell Cycle Analysis
Cells were trypsinized, centrifuged, and washed once with PBS.
The cell pellet was resuspended and fixed on ice for 15 min with 1 ml
of cold ethanol (70%). The cells were subsequently centrifuged and
resuspended in 1 ml of propidium iodide (PI) solution (0.05 mg/ml PI;
0.02 mg/ml RNase; 0.3% Nonidet P-40; 1 mg/ml sodium citrate) for
1 h at 4 °C. Flow cytometry analysis was performed on a FACSort
flow cytometer (Becton Dickinson, Mountain View, CA). Data from
104 cells were collected and analyzed using the CellQuest
software (Becton & Dickinson).
Western Blot
Cells were lysed in 300 µl of SDS electrophoresis sample
buffer (23), and samples were boiled for 10 min. The amount of protein
in the cell lysates was determined by the Bio-Rad protein assay
(Bio-Rad, Hercules, CA). Twenty micrograms of total cell lysate was
fractionated by SDS-polyacrylamide gel (23), transferred to
polyvinylidene difluoride membranes (Millipore, Bedford, MA) and probed
with the respective antibodies. To detect the electrophoretic mobility
shift of the chk2 kinase, 10-20% linear gradient SDS-polyacrylamide gels were used (Bio-Rad). Blots were developed with enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech, Sweden), using the
appropriate horseradish peroxidase-labeled secondary antibody,
according to the instructions of the manufacturer. The following
antibodies were used: anti-phosphotyrosine (Upstate Biotechnology, Lake
Placid, NY); anti-cdc2, anti-Cip1/p21, and anti-Kip1/p27 (Transduction Laboratories, Lexington, KY); anti-p53 (Ab-6, Calbiochem, La Jolla, CA); anti-phosphoserine-15 p53 rabbit serum (kind gift of Dr. Yoichi
Taya, University of Japan); anti-chk2 (H-300), anti-cdc25C (C-20), and
anti-wee1 (C-20) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-chk1
rabbit serum (kindly provided by Dr. Stephen J. Elledge, Verna and
Marrs McLean Department of Biochemistry and Molecular Biology and
Department of Molecular and Human Genetics, Howard Hughes Medical
Institute, Baylor College of Medicine, Houston, TX).
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RESULTS |
The effect of HdCDT is cell type-specific--
We tested the
activity of HdCDT in a broad panel of human cell lines, including two
epithelial cell lines, three keratinocyte lines, two
normal fibroblast lines, and three lines
of B cell origin. As shown in Table I and Fig.
1A, HdCDT induced cell death or cell cycle arrest in all the lines, judged by propidium iodide (PI)
staining and flow cytometry analysis 24 h after toxin treatment. Most of the cell lines tested were arrested exclusively in
G2, as detected by accumulation of cells with a 4n DNA
content. The HdCDT-induced G2 arrest was not associated
with the absence of a functional p53 protein, because cells expressing
either wild type or mutated p53 were arrested in G2 (Table
I). A different pattern of cell cycle arrest was observed in two normal
fibroblast lines derived from foreskin and lung. Intoxicated
fibroblasts partially accumulated in G2 24 h after
toxin treatment, however, the block in G2 was not complete,
and, even 48 h post-intoxication, cells were arrested also in S
and G1 (Fig. 1A). B cell lines showed a 50%
decrease of the G1 peak, which was associated with a 2- to
4-fold increase of the apoptotic sub-G1 population 24 h after intoxication. A slight increase in the G2
population could be detected only in some experiments (Figs.
1A and 4A; Table
II).

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Fig. 1.
Effect of HdCDT on human cell lines.
A, effect of HdCDT on human lung embryonic fibroblasts
(HL), HEp-2 cells, and B cell lines. Cells were treated with
HdCDT, and cell cycle distribution was assessed by DNA staining with PI
and flow cytometry analysis at the indicated time points. The
G1 peak was arbitrarily set on the mean fluorescence
intensity value of 50. B, lack of hyperphosphorylated cdc2
in HL fibroblasts. Cells were treated with HdCDT for 24 h, and
Western blot analysis was performed from total cell lysate as described
under "Experimental Procedures." Polyvinylidene difluoride
membranes were probed with anti cdc2 and anti-phosphotyrosine-specific
monoclonal antibodies. Cdc2* indicates the
hyperphosphorylated form. One out of three experiments is shown.
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Table II
Effect of HdCDT on ATM wild type and ATM-deficient LCLs
Data are presented as the ratio between the HdCDT-treated cells and the
untreated control.
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It has been previously shown that G2 arrest induced by
HdCDT was associated with an accumulation of the hyperphosphorylated form of the cyclin-dependent kinase cdc2 (24).
Consequently, we assessed whether the different pattern of cell cycle
arrest observed in the fibroblasts was associated with a lack of
accumulation of hyperphosphorylated cdc2. As shown in Table I and Fig.
1B, accumulation of tyrosine-phosphorylated cdc2 was
detected by Western blot, using anti-cdc2 and
anti-phosphotyrosine-specific monoclonal antibodies, only in HEp-2,
HeLa, and HaCaT cells 24 h post-intoxication. At this stage, cdc2
was strongly down-regulated in normal keratinocytes and fibroblasts,
and no hyperphosphorylation was observed. However, we detected
accumulation of hyperphosphorylated cdc2 at early time points after
intoxication in a representative line of each cell type (Fig.
2, B and C, and
data not shown).

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Fig. 2.
HdCDT and IR induce similar checkpoint
responses. A, cell cycle distribution. HL fibroblasts
and HEp-2 cells were treated with HdCDT (2 µg/ml), hydroxyurea (2.5 mM), or nocodazole (100 mM) for 24 h, or
irradiated (20 Gy) and incubated for 24 h in complete medium. DNA
was stained with PI, and cell cycle distribution was analyzed by flow
cytometry as described in Fig. 1A. In B and
C, analysis of cell cycle regulators. HL fibroblasts and
HEp-2 cells were either treated with HdCDT or exposed to IR as
described in A. At the indicated time points total cell
lysates were prepared and the levels of known cell cycle regulators
were assessed by Western blot analysis. chk2* indicates the
phosphorylated form. One out of two to three experiments performed for
each cell line is shown.
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HdCDT-induced Cell Cycle Arrest Resembles the Response Induced by
Ionizing Radiation--
To test whether HdCDT induced a response
similar to other agents known to cause cell cycle arrest, we compared
the effect of HdCDT and IR in HEp-2 cells and HL fibroblasts, which
showed different patterns of cell cycle arrest. The cell cycle
distribution of irradiated cells was similar to that observed upon
intoxication in both cell types (Fig. 2A). The block in
G1/S observed in fibroblasts is not an artifact due to the
slow proliferation rate of these cells, because we did not detect a
complete accumulation in G2 even 48 h
post-intoxication (Fig. 1A) and treatment of cells with nocodazole, known to synchronize cells in mitosis, induced accumulation of the majority of fibroblasts in G2/M already 24 h
after treatment (Fig. 2A). It is noteworthy that both cell
types treated with 2.5 mM hydroxyurea, which blocks DNA
synthesis by reducing the intracellular levels of deoxynucleoside
triphosphates, were arrested in early S phase. Thus, HdCDT and IR
induced a similar response, which differed from that caused by
hydroxyurea. This issue was further analyzed in time kinetic
experiments, where we tested the expression of known regulators of the
cell cycle in toxin- and radiation-treated cells.
In HL fibroblasts, accumulation of phosphorylated cdc2 was detected
only at early time points (4 and 6 h) after intoxication, and an
identical response was observed in irradiated cells (Fig. 2B). Increased expression of the tumor suppressor protein
p53 was observed in both intoxicated and irradiated fibroblasts 4 h post-treatment. This was associated with a strong up-regulation of
the cyclin-dependent kinase inhibitor p21, and to a lesser extent p27, and their levels increased over time. IR-induced activation of p53 is mediated by the ATM kinase, which phosphorylates p53 on
serine 15. Using a specific anti-phosphoserine-15 p53 rabbit serum, we
showed that this event occurs with a similar kinetic both in
intoxicated and irradiated cells 4 h after treatment (Fig. 2C).
A different pattern was detected in HEp-2 cells, consistent with
the fact that the two cell types behaved differently when exposed to
HdCDT or IR (Fig. 2A). Phosphorylation of cdc2 was observed
4 h after treatment and was maintained up to 24 h, confirming previous data (Table I, Fig. 2C, and Ref. 24). Increased
expression of p53 and p21 was detected much later than the accumulation
of hyperphosphorylated cdc2. The strongest accumulation of p53 and p21
was observed in HdCDT-treated cells 24 h post-intoxication (Fig. 2C). IR treatment is known to induce phosphorylation
of the chk2 kinase in an ATM-dependent manner, detected as
a shift of the chk2-specific band in Western blot analysis (19). The chk2 protein was shifted both in intoxicated and irradiated HEp-2 cells
4 h after treatment (Fig. 2C). We did not detect any
relevant change either in the levels of expression or in
electrophoretic mobility of other regulatory molecules of the cell
cycle, such as chk1, cdc25C, and wee1 (data not shown).
Caffeine Partially Overrides the Cell Cycle Arrest Induced by
HdCDT--
Caffeine is known to override the G2/M arrest
induced by DNA damage in mammalian cells by indirectly releasing the
inhibitory cdc2 phosphorylation (25-27). Because we have seen a strong
correlation between the responses evoked by HdCDT and IR, we assessed
whether caffeine could prevent the HdCDT-induced G2 arrest
in HEp-2 cells. Cells were treated with HdCDT for 15 min on ice. After
this binding step, cells were washed three times, to remove unbound
toxin, and incubated in complete medium supplemented with 4 mM caffeine. The level of cdc2 phosphorylation and cell
cycle progression was monitored 24 h post-intoxication by Western
blot and flow cytometry analysis, respectively. The caffeine treatment
completely prevented accumulation of hyperphosphorylated cdc2 (Fig.
3A), and this was associated
with partial release of the HdCDT-induced G2 arrest, because 35% of the cells were found in G1 upon caffeine
treatment compared with 8% in the untreated cells (Fig.
3B).

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Fig. 3.
Caffeine partially overrides HdCDT-induced
cell cycle arrest. HEp-2 cells were exposed to HdCDT (2 µg/ml)
for 15 min on ice and, after extensive washing to remove unbound toxin,
incubated in the presence of 4 mM caffeine for 24 h.
Levels of cdc2 phosphorylation (A) and cell cycle
distribution (B) were assessed as described in Fig.
1B and 1A, respectively. One out of two
experiments is shown.
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HdCDT-induced Cell Cycle Arrest Is Delayed in ATM-deficient
Cells--
The ATM protein is a key activator of cell cycle
checkpoints in response to DNA-damaging agents (reviewed in Ref. 11).
Furthermore, recent data have shown that caffeine exerts its activity
by directly inhibiting the ATM kinase (26, 27). Therefore, we tested
the response to HdCDT in two ATM wild type LCLs and four ATM-deficient cell lines. Cells were exposed to HdCDT for 24 h and subsequently stained with PI, followed by flow cytometry analysis. As shown in Fig.
4 and in Table II, a 2- to 4-fold
increase of the sub-G1 population was detected in the ATM
wild type LCLs. This was associated with a strong decrease of the
G1 peak (50% reduction), although no major changes were
observed in the S-G2-M population. The ATM-deficient cells
were much more resistant to the toxin effect, because we could observe
only minor changes in both the sub-G1 and the
G1 populations 24 after treatment. It has been previously
shown that p53 levels increase with a much faster kinetic in ATM wild
type LCLs than in ATM-deficient cells (12). A similar pattern was observed when the control SN-B1 and the ATM-deficient AT13LA lines were
exposed to HdCDT (Fig. 5, A
and B). A 20-fold increased p53 expression was observed in
SN-B1 LCL compared with the 2-fold increase in the AT13LA line 5 h
after treatment, and this trend was maintained throughout our
experiment.

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Fig. 4.
HdCDT intoxication is delayed in
ATM-deficient cells. Two ATM wild type LCLs (A) and
four ATM-deficient LCLs (B) were incubated with HdCDT for
24 h, as described under "Experimental Procedures." DNA was
stained with PI, and cell cycle distribution was analyzed by flow
cytometry as described in Fig. 1A.
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Fig. 5.
Different kinetic of p53 stabilization.
A, the ATM wild type SN-B1 and the ATM-deficient AT13LA LCLs
were treated with HdCDT as described under "Experimental
Procedures." At the indicated time points total cell lysates were
prepared and expression of p53 was assessed by Western blot analysis.
B, densitometric analysis was performed using the ImageQuaNT
software (Molecular Dynamics). Data are presented as the ratio between
the optical density of the specific band in HdCDT-treated cells and the
optical density of the corresponding band in the untreated
control.
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DISCUSSION |
The aim of this study was to clarify the mechanism(s) by which the
cytolethal distending toxin produced by H. ducreyi induces cell cycle arrest and cell death in human cells. The strategy was to
investigate the early events occurring in intoxicated cells and compare
the effects caused by HdCDT with those induced by ionizing radiation.
This is the first report where CDT-intoxicated cells have been
analyzed at short time points after toxin exposure, whereas
all previous studies have been performed mainly at 24 or
72 h post-intoxication (2, 24, 28, 29). Furthermore, we
have tested a broad panel of human cell types, including
normal keratinocytes and fibroblasts, which are conceivable
toxin targets in vivo.
Our data demonstrate that HdCDT activates a cell cycle
checkpoint, which resembles that induced by IR, and highlight a new mechanism of action for bacterial protein toxins. This conclusion is
based on three lines of evidence: i) different cell types responded differently to HdCDT (Table I; Figs. 1A and 2A),
suggesting that HdCDT does not specifically target the G2
phase of the cell cycle, as previously suggested (1); ii) similar
profiles of cell cycle arrest and time kinetic responses were induced
by HdCDT and IR (Fig. 2, A-C); iii)
ATM-deficient cells were much more resistant than ATM wild type cells
to HdCDT (Fig. 4).
Activation of Checkpoint Responses--
We have demonstrated that
the response to HdCDT was cell-specific and not exclusively associated
with the induction of cell cycle arrest in G2. This
observation suggested that HdCDT might target an upstream event that
could subsequently activate several downstream pathways leading to cell
cycle arrest or cell death. This has prompted us to investigate whether
the response of intoxicated cells was similar to any other agent
causing activation of checkpoint responses. In normal fibroblasts, both
HdCDT and IR induced a rapid stabilization of the tumor suppressor gene
p53, which was phosphorylated on serine 15, and an increased
expression of the cyclin-dependent kinase inhibitor p21
(Fig. 2C), suggesting that the checkpoint response in normal
lung fibroblasts is p53-dependent. Functional p53 and p21
proteins are also required to sustain G2 arrest caused by
DNA damage (30). However, this is unlikely to occur in HEp-2 cells,
because p53 and p21 up-regulation was detected much later than the
accumulation of hyperphosphorylated cdc2 (Fig. 2C).
Furthermore, cell lines that carried a nonfunctional p53, such as HeLa
and HaCaT, still arrested in G2 and did not up-regulate
p21, suggesting the involvement of a p53-independent pathway (Table I
and data not shown). In intoxicated HEp-2 cells, we could demonstrate
phosphorylation of the chk2 kinase, which plays a major role in the
induction of G2 checkpoint responses upon exposure to
IR (19). Activation of chk2 can lead to cdc25C inactivation and
accumulation of hyperphosphorylated cdc2. Indeed, inactivation of
cdc25C was previously demonstrated in HeLa cells treated with the
Escherichia coli CDT (29), and overexpression of cdc25B and
cdc25C could prevent cell intoxication (31).
Increased expression of phosphoserine-15 p53 in human fibroblasts and
phosphorylation of the chk2 kinase in HEp-2 cells strongly suggested
that the response to HdCDT was mediated by the ATM kinase. Involvement
of ATM for rapid cell intoxication was demonstrated by the delay in
intoxication (Fig. 4) and in p53 stabilization (Fig. 5) observed in
ATM-deficient cells compared with wild type LCLs. This notion was
further supported by the ability of caffeine to alleviate the
G2 block imposed by HdCDT in HEp-2 and HeLa cells (Fig. 3,
A and B, and Ref. 29). Recent studies have
demonstrated that caffeine abolishes the G2 block in
mammalian cells upon DNA damage by inhibiting the activation of the ATM
kinase and consequently blocking the downstream activation of the chk2
protein (26, 27). The delayed intoxication observed in ATM-deficient
cells suggests that this protein is required for an early toxin
response, but checkpoint responses can still be activated in the
absence of functional ATM, probably by homologous molecules.
These data indicate that the response to HdCDT is similar to that
induced by IR and therefore suggest that the toxin acts either at the
level of a central regulator(s) of the cell cycle control machinery or
at the level of DNA, activating a checkpoint response in the target
cells. Sert and colleagues (29) did not detect DNA strand breaks in
cells intoxicated with a CDT derived from E. coli, using the
single cell gel electrophoresis assay ("comet assay"). However, two
recent reports have demonstrated that cdtB from E. coli and
Campylobacter jejuni has structural and functional
homology to the mammalian type I DNase. DNase activity was associated
with the E. coli cdtB, as detected by in vitro digestion of the coiled pGEM-7zf+ plasmid (32), whereas marked chromatin disruption was observed in HeLa cells following transfection or microinjection of low amounts of cdtB from C. jejuni
(33). In both cases, point mutations in conserved residues required for
catalysis or magnesium binding abolished the DNase activity and the
ability of the toxin to induce cell cycle arrest. It is therefore
likely that CDTs induce subtle DNA damage leading to the activation of
cell cycle checkpoint responses, in agreement with the data presented
in our study.
H. ducreyi Pathogenicity--
CDTs derived from A. actinomycetemcomitans and H. ducreyi inhibit T cell
proliferation and induce apoptotic death in the Jurkat T cell line,
respectively (2, 34). We have extended these findings demonstrating
that also B cell lines are sensitive to HdCDT. Interestingly, among all
the cell lines we have tested, the two LCLs and the two Burkitt's
lymphoma-derived cell lines BL41 and Rael were very prone to apoptosis
upon HdCDT treatment judged by accumulation of a sub-G1
population of PI stained cells (Figs. 1A and 4A, and data
not shown).
These data, together with the observation that normal keratinocytes and
fibroblasts are sensitive to HdCDT, suggest that the toxin contributes
to the pathogenesis and immunodeficiency of chancroid by: 1) damaging
the epidermal and dermal layers of the genital mucosa and causing
retardation in healing; and 2) decreasing and delaying the host immune
response by induction of apoptosis in B and T lymphocytes, allowing
bacterial replication and enhancing tissue damage.
CDTs as Biological Tool--
Because CDTs interfere with the cell
cycle control machinery, they can offer a possible new tool to
intervene in all cases of cell cycle deregulation, tumors being one of
the major issues. It is noteworthy that the use of cytotoxic prodrugs
has been considered in the field of cancer gene therapy. Attention has
been focused on the herpes simplex virus-thymidine kinase
(HSV-tk) gene, which induces single-strand breaks in DNA
synthesized in the presence of the nucleoside analogue ganciclovir (35,
36). The murine melanoma cell line B16F10 transfected with the
HSV-tk gene undergoes irreversible G2/M arrest
and cytoskeleton reorganization when treated with ganciclovir (37).
HdCDT produces very similar effects in toxin-sensitive tumor cell lines
(24) and could potentially be used as anti-cancer therapy, if a
selective delivery to tumor cells is provided.
It is also well established that bacterial toxins have been extremely
valuable in dissecting several important aspects of cell biology, such
as the role of small GTPases in the control of cytoskeleton
rearrangement (reviewed in Ref. 38). The cross-talk between molecules
regulating the cytoskeleton and those involved in control of cell cycle
progression/arrest is complex and still poorly understood (reviewed in
Refs. 39, 40), and elucidation of the HdCDT mode of action can be
useful to further study this issue.