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NEOPLASIA
From the Department of Pediatrics, University
Children's Hospital, Ulm; and the Division of Molecular Oncology,
German Cancer Research Center, Heidelberg, Germany.
In addition to myelosuppression, anticancer drugs cause rapid and
persistent depletion of lymphocytes, possibly by direct apoptosis
induction in mature T and B cells. Induction of apoptosis regulators
was analyzed in peripheral blood lymphocytes from pediatric patients
undergoing first-cycle chemotherapy for solid tumors. In vivo
chemotherapy induced a significant increase in lymphocyte apoptosis ex
vivo. The activation of initiator caspase-8 and effector caspase-3 and
the cleavage of caspase substrates was detected 12 to 48 hours after
the onset of therapy. Caspase inhibition by Z-VAD-fmk did not reduce ex
vivo lymphocyte apoptosis in all patients, indicating the additional
involvement of caspase-independent cell death. No evidence for the
involvement of activation-induced cell death was found in the acute
phase of lymphocyte depletion as analyzed by activation marker
expression and sensitivity for CD95 signaling. Lymphocyte apoptosis in
vivo appeared to be predominantly mediated by the mitochondrial pathway
because a marked decrease of mitochondrial membrane potential
( Anticancer drugs used in chemotherapy for tumors
and leukemias inhibit proliferation and induce cell death in malignant
cells. In addition to the therapeutic effect on malignant cells,
chemotherapy causes severe toxicity in normal tissue, leading to side
effects such as mucositis, hair loss, and myelosuppression. In
addition, chemotherapy induces acute lymphopenia and chronic depletion
of CD4 T cells, leading to increased susceptibility to opportunistic infection.1,2 The molecular mechanisms by which cytotoxic drugs induce depletion of lymphocytes have not been defined and may
involve proliferative arrest in lymphocyte precursor compartments or,
alternatively, direct induction of apoptosis in mature cells.
Regulation of apoptosis or programmed cell death involves different
molecular compartments, such as death receptors,3 Bcl-2 family member proteins,4 mitochondria,5 p53,
and caspases.6 In tissue culture cell lines, cytotoxic
drugs induce the molecular regulators of physiologic
apoptosis7,8 Cytotoxic drug-induced apoptosis in
leukemia9 and carcinoma28 cell lines critically depends on the activation of caspases. Caspases are activated by death
receptor signaling or are a consequence of mitochondrial alterations
including the release of apoptogenic signaling
molecules.10
Cross-linking of the CD95 (APO-1, Fas) death receptor forms a
death-inducing signaling complex containing FADD. This complex recruits
the initiator caspase-8, leading to the activation of caspase-8 and the
cleavage of downstream caspases such as caspase-3 and
substrates.11 Diverse anticancer drugs such as cisplatin, doxorubicin, mitomycin, fluorouracil, and camptothecin have been found
to induce or increase CD95 expression in some tumor cell lines and to
increase the sensitivity for CD95-induced apoptosis. In cytostatic
drug-induced apoptosis in human T-cell leukemia12 and other
tumor cell lines,13-15 doxorubicin and other anticancer drugs have been found to induce CD95 ligand, including CD95
receptor-ligand interaction, a mechanism identified for
activation-induced cell death in T cells.16 Death
receptor-independent cleavage of the proximal caspase-8, however, has
also been found in drug-induced apoptosis.17
Mitochondria-directed apoptotic stimuli induce a variety of
mitochondrial changes, among them the production of oxygen radicals and
the opening of membrane pores, thereby releasing apoptogenic factors
such as cytochrome-c or apoptosis-inducing factor
(AIF). By formation of the apoptosome complex, deregulated
mitochondria initiate the activation of the caspase cascade through the
activation of caspase-9 and caspase-3. Mitochondria-related apoptosis
is regulated by Bcl-2 family members, which are involved in cellular response to a variety of apoptotic stimuli including chemotherapeutic agents.18 Cytotoxic drugs induce Bax
expression,19 and reduction of the mitochondrial membrane
potential The diversity of apoptosis pathways involved in drug-induced
cytotoxicity has so far been investigated in established tumor cell
lines. In this study, we aimed to identify apoptosis pathways activated
in the acute depletion of peripheral blood lymphocytes (PBLs) during in
vivo chemotherapy. We found that in vivo chemotherapy directly induces
apoptosis in mature PBLs involving activation of caspases-3 and -8, reduction of the mitochondrial membrane potential, and induction of
Bax. Lymphocytes were activated and highly sensitive to CD95 after
chemotherapy, but no involvement of CD95-mediated, activation-induced
death could be detected in the acute phase of lymphocyte depletion, nor
was p53 expression increased in resting lymphocytes during in vivo
chemotherapy. In vitro treatment with cytotoxic drugs induced p53
protein and CD95 receptor-ligand interaction in stimulated, but not in
resting, lymphocytes, suggesting differential activation of apoptosis
pathways in mature resting, but not in activated, cycling lymphocytes.
Patients
Samples
Analysis of activation markers and T-cell enumeration For each test, 0.5 to 1.0 × 106 cells were incubated with combinations of 4 fluorochrome-conjugated monoclonal antibodies for 25 minutes at 4°C. Antibody combinations used for 4-color fluorescence (fluorescein isothiocyanate [FITC], R-phycoerythrin [PE], PerCP, and allophycocyanine [APC]) were CD4, CD3, CD8, CD45; CD4, HLA-DR, CD8, CD95; CD4, CD25, CD8, CD95; CD4, CD69, CD8, CD95; CD4, CD62L, CD8, CD95; CD45RA, CD95, CD4, CD45RO; CD45RA, CD95, CD8, CD45RO. Antibodies anti-CD4 FITC, -CD3 PE, -CD8 PE, -CD25 PE, -CD69 PE, and -CD45RA FITC were purchased from Coulter/Immunotech, (Krefeld, Germany); all other antibodies were purchased from Becton Dickinson, (Heidelberg, Germany). Samples were washed with HBSS with 2% bovine serum albumin (Serva, Heidelberg, Germany) and 0.2% azide (Merck, Darmstadt, Germany). Samples were fixed with 2% paraformaldehyde and immediately analyzed on a FACSCalibur Cytometer (Becton Dickinson) equipped with a 488-nm argon laser and a 650-nm red diode laser. At least 50 000 events per sample were acquired, stored in list-mode files, and subsequently analyzed with Cell Quest software (Becton Dickinson). Absolute CD4 and CD8 cell numbers were calculated from absolute leukocyte count, percentage of lymphocytes in the differential, and percentage of CD4 and CD8 cells in the lymphocyte gate.Flow cytometric analysis of mitochondrial transmembrane
potential (   m in combination with surface
markers, cells were labeled with anti-CD3, -CD4, -CD8, and -CD95 as
described above, washed with HBSS-FCS, resuspended at a concentration
of 5 × 105/mL, and incubated with
3,3'-dihexyloxacarbocyanine iodide (DiOC6(3), 460 ng/mL;
FL-1) (Molecular Probes, Eugene, OR) for 12 minutes at 37°C in the
dark, followed by immediate flow cytometric analysis. Incubation with
carbonyl cyanide m-chlorophenyl hydrazone mClCCP (Sigma Chemical,
Deisenhofen, Germany) served as control for down-regulated m.
Ex vivo in vitro cell death assays Mononuclear cells from patients undergoing chemotherapy were seeded onto 24-well plates at 1.0 × 106/mL in RPMI 1640 with the standard supplements 10% FCS, 200 mM L-glutamine, and 100 U/mL penicillin-streptomycinand were incubated with 1 µg/mL
apoptosis-inducing anti-CD95 (APO-1 IgG3), 1-10 µg/mL blocking
anti-CD95 (anti-APO Fab2), 1 to 10 µg/mL neutralizing CD95 ligand
antibody Nok2 (BD PharMingen, Heidelberg, Germany), and cytarabine
(kindly provided by Pfizer, Karlsruhe, Germany) as positive control.
After 24 hours, cells were washed and stained with anti-CD3, -CD4, and
-CD8, and apoptosis was analyzed by phosphatidylserine externalization
with annexin V-FITC (Boehringer Mannheim, Mannheim, Germany) and
forward-side scatter changes.
Western blot analysis Mononuclear cells were lysed for 30 minutes at 4°C in phosphate-buffered saline with 0.5% Triton-X 100 (Serva) and 1 mM phenylmethylsulfonyl fluoride (Sigma) followed by high-speed centrifugation. In the analyses directly ex vivo, monocytes were not depleted because the percentages of monocytes in the samples were lower than 10% and the cultivation for adherence technique or manipulation in a separation procedure would have induced apoptosis in some of the cells. Protein concentration was assayed using bicinchoninic acid (Pierce, Rockford, IL). Forty micrograms protein per lane was separated by 12% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was electroblotted onto nitrocellulose (Amersham Pharmacia, Freiburg, Germany). Equal protein loading was controlled by Ponceau red staining of membranes. After blocking for 1 hour in phosphate-buffered saline supplemented with 2% bovine serum albumin (Sigma) and 0.1% Tween 20 (Sigma), immunodetection of CD95 ligand, Bax, caspases-3 and -8, and poly (ADP-ribose) polymerase (PARP) was performed using mouse anti-CD95L monoclonal antibody (1:250; PharMingen G247) mouse anti-caspase-8 monoclonal antibody C1526 (1:5 dilution of hybridoma supernatant), mouse anti-caspase-3 monoclonal antibody (1:1000; Transduction Laboratories, Lexington, KY), rabbit anti-PARP polyclonal antibody (1:10 000; Enzyme Systems Products, Dublin, CA), and mouse anti-p53 monoclonal antibody (Transduction Laboratories). Goat anti-mouse immunoglobulin G (IgG) or goat anti-rabbit IgG (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA) followed by enhanced chemiluminescence (Amersham Pharmacia) was used for detection.In vitro studies of drug-induced apoptosis Mononuclear cells were isolated from buffy coat of healthy blood donors, and monocytes were depleted by culture in plastic flasks for 45 minutes in RPMI 1640 and by harvest of nonadherent cells. One part of the PBLs was directly processed for culture with cytotoxic drugs, whereas the other was stimulated with 5 µg/mL phytohemagglutinin (PHA; Sigma) and 30 U/mL interleukin-2 (IL-2) (Pepro Tech) for 72 hours (day 3 blasts). After stimulation, apoptotic cells were removed by Ficoll-Hypaque density centrifugation, the blasts washed twice, resuspended in complete medium with IL-2, and cultured for 6 and 12 hours at 106 cells/mL with medium, 10 µg/mL cytarabine (kindly provided by Pfizer), and 10 µg/mL etoposide (kindly provided by Bristol Meyer Squibb, Regensburg, Germany).Because PHA blasts were all CD4 CD45RO memory T-cell type, CD4 T memory
cells were identified among PHA blasts and resting PBLs using 4-color
flow cytometry with anti-CD3, -CD4, and -CD45RO, and apoptosis was
measured by annexin V binding. Drug-specific apoptosis was calculated
as follows: [% apoptosis in drug-treated samples For analysis of CD95 sensitivity, PBLs and PHA blasts were cultured
with medium, cytarabine, and etoposide and were subsequently cultured
with and without apoptosis-inducing anti-APO-1 (IgG3) for another 4 hours. Apoptosis in CD4 T memory cells was measured, and CD95-specific
apoptosis was calculated as follows: [% apoptosis with
anti-APO-1 For analysis of CD95 receptor-ligand interaction, D3 blasts were cultured with 10 µg/mL etoposide in medium alone, with 0.1 µg/mL apoptosis-inducing anti-APO-1; 1, 5, and 10 µg/100 µL neutralizing CD95 ligand antibody NOK-2; and 10 µg/mL CD95 receptor-blocking anti-APO-1 Fab for 6 and 12 hours. Apoptosis was measured by flow cytometry in CD4 T memory cells as described above.
Lymphocyte depletion and in vitro apoptosis To estimate the kinetics of lymphocyte depletion in patients undergoing first-cycle chemotherapy (Table 1), the absolute number of lymphocytes was calculated from PBL differential counts. Unexpectedly, we found a rapid decrease of lymphocyte numbers, from mean values of 2700/µL to 700/µL within 72 hours of treatment. Because the predominant depletion of CD4 T cells after chemotherapy has been reported,2 we investigated, whether CD4 and CD8 cells were differentially reduced. As shown in Figure 1B, CD4 and CD8 cells were equally reduced in the acute phase of chemotherapy. In addition, naive resting T cells (CD4+CD45RA+) and memory T cells (CD4+CD45RO+) were equally reduced (data not shown). Thus, chemotherapy rapidly causes a reduction of mature lymphocytes in peripheral blood irrespective of the lymphocyte subtype.
Rapid reduction of mature lymphocytes suggested direct induction of lymphocyte death rather than inhibition of precursor cells. Detection of apoptosis in vivo by morphologic changes, DNA fragmentation, or phosphatidylserine externalization is prevented by the rapid clearance of apoptotic cells from peripheral blood. Therefore, we analyzed ex vivo apoptosis of PBLs after 24-hour cultivation during in vivo chemotherapy. As shown in Figure 1C, CD4 T-cell apoptosis significantly increased to above normal levels during treatment, indicating the induction of pro-apoptotic changes in mature peripheral T lymphocytes by in vivo chemotherapy. Similar results were obtained for CD8 T cells, which exhibited a higher rate of spontaneous apoptosis before therapy (data not shown). Activation of caspases To detect caspase activity and to identify caspases activated during chemotherapy, mononuclear cells of patients undergoing chemotherapy were analyzed by immunoblot directly ex vivo. Given that the samples consisted of more than 90% lymphocytes, depletion procedures were not performed so as not to confound the results by additional in vitro manipulation. As shown in Figure 2A, caspase activity was detected between 12 and 48 hours after the onset of chemotherapy by the p85 cleavage product of PARP. Activation of the effector caspase 3, shown by the appearance of p17 and p10 subunits, was detected from 36 hours after the start of chemotherapy. Caspase-8 activation, shown by the appearance of the active p18 subunit, could be detected from 12 hours (patients 2 and 5) to 48 hours (patient 3). To estimate the contribution of caspase activation to apoptosis induction in lymphocytes, the effect of caspase inhibition on ex vivo apoptosis in CD4 T cells of 7 patients during chemotherapy was studied. We observed a marked heterogeneity in apoptosis reduction by the caspase inhibitor; Figure 2B-D shows 3 representative examples. In 2 patients, we observed a pattern similar to caspase-dependent cell death of cell lines, whereby the increase of apoptosis during chemotherapy was significantly reduced on incubation with Z-VAD-fmk (Figure 2B). In other patients, caspase inhibition reduced apoptosis 24 hours after the start of chemotherapy but not later (Figure 2C). Finally, in other patients, caspase inhibition did not reduce the chemotherapy-induced increase of ex vivo apoptosis at all (Figure 2D). This heterogeneity indicates the additional involvement of caspase-independent pathways, which may depend on either individual variability or different cytotoxic drugs used for treatment.
Analysis of activation and CD95-induced cell death in T cells during in vivo chemotherapy Several investigators have reported an accumulation of activated T cells after chemotherapy with high sensitivity for activation-induced cell death. Early cleavage of caspase-8 may be indicative of CD95 receptor signaling and activation-induced death in T cells. We studied the activation of peripheral blood T cells in 10 patients (A1-A10) during in vivo chemotherapy by flow cytometry. As shown in Figure 3A, the proportion of CD4 T cells expressing CD69 was significantly higher before the second or third cycle of chemotherapy than before the first. There was, however, no increase in CD69 expression during the acute phase of lymphocyte reduction in the first course of chemotherapy (Figure 3B). Analyses were performed for the expression of HLA-DR, CD25, CD62L, and CD95 on CD4/CD45RA , CD4/CD45RA+, and CD8 T cells with
essentially the same results (data not shown). Thus, neither activation
markers nor CD95 receptor expression was directly induced by
chemotherapy in the acute phase of lymphocyte depletion.
To investigate whether in vivo chemotherapy sensitizes for the CD95 signal, we analyzed in vivo-treated lymphocytes for CD95 sensitivity. Again, sensitivity to CD95-mediated cell death was high before the second or third cycle of chemotherapy compared with the first cycle (Figure 3C), but it did not significantly increase during lymphocyte depletion of the first cycle. When we analyzed the influence of receptor blockade on the increase of spontaneous apoptosis during chemotherapy by cultivating in vivo-treated lymphocytes with blocking anti-APO (Fab)2 fragments, no reduction of increased spontaneous apoptosis was found (data not shown). In order not to miss death receptor-mediated lymphocyte apoptosis at early time points, we investigated lymphocytes at 3, 6, and 12 hours after the start of chemotherapy in single patients. Moreover, lymphocytes drawn from patients after the start of chemotherapy were incubated for 3, 6, and 18 hours and were studied for CD95 sensitivity. These results were essentially the same as those in the analysis after 24 hours. Taken together, we found no evidence of activation-induced CD95-mediated death in the acute phase of chemotherapy-induced lymphocyte depletion. Analysis of mitochondrial membrane potential in T lymphocytes during chemotherapy In cell lines, cytotoxic drugs induce apoptosis-related mitochondrial alterations including the mitochondrial transmembrane potential (M), which is generated by the proton
gradient over the inner mitochondrial membrane. The lipophilic cationic
dye DiOC6(3) accumulates in the mitochondrial matrix driven
by the M, and reduction in fluorescence intensity
indicates M dissipation. Because a sequence of
M reduction, phosphatidylserine externalization, and
cell death has been found in primary lymphocytes in vitro, we
investigated whether M reduction is induced in PBLs
by in vivo chemotherapy.
In peripheral blood mononuclear cells obtained during chemotherapy,
T-cell subsets were identified by expression of CD3, CD4, and CD8 and
were analyzed for reduced binding of DiOC6(3), as described
in "Patients and methods." Figure 4A
shows 3-color analysis of lymphocytes from patient A10 demonstrating an
increasing frequency of cells with lowered mitochondrial membrane
potential starting within 24 hours of chemotherapy, with a maximum at
48 hours. Using similar analysis in 7 patients (patients A4-A10), a
persistent increase in the percentage of cells with low mitochondrial
membrane potential during 48 hours of chemotherapy was detected in CD4 and in CD8 T lymphocytes (Figure 4B). Thus, in mature PBLs,
pro-apoptotic mitochondrial alterations known to initiate caspase
activation and cell death are induced by in vivo chemotherapy.
Induction of Bax and p53 by in vivo chemotherapy Because mitochondrial changes are regulated by pro-apoptotic Bcl-2 family members, we further investigated whether Bax and its regulator, p53, were induced by chemotherapy. Mononuclear cells obtained during chemotherapy were analyzed for Bax and p53 expression by immunoblot. As shown in Figure 5, Bax expression increased in all patients as early as 12 hours after the start of chemotherapy. Induction was most pronounced in patients B3 and B5, who were treated with the alkylating substances cyclophosphamide and busulfan. Intracellular levels of Bcl-2 measured by flow cytometry remained unchanged during the first 4 days of chemotherapy (data not shown). Interestingly, however, protein levels of p53 remained undetectable during chemotherapy in all patients analyzed. Additional flow cytometric analyses detecting cytoplasmic and nuclear accumulation of p53 confirmed the lack of p53 induction during chemotherapy (data not shown). Thus, in addition to mitochondrial alterations, in vivo chemotherapy induces Bax expression in the absence of detectable increases in p53.
Activation of p53 and CD95 by cytotoxic drugs in resting and activated peripheral blood lymphocytes in vitro Because p53 and CD95 have been shown to be induced in drug-induced apoptosis in various tumor cell lines but were not induced in PBLs during chemotherapy, we investigated p53 and CD95 involvement in lymphocyte apoptosis in vitro. Considering that previous studies uniformly used actively cycling cells, we hypothesized that involvement of p53 and CD95 in drug-induced apoptosis might be different in resting lymphocytes than it is in cycling lymphocytes.We first compared drug sensitivity in resting versus cycling
lymphocytes on drug treatment in vitro. Lymphocytes were cultured with
cytotoxic drugs, and drug-specific apoptosis was assessed as described
in "Patients and methods." As shown in Figure
6A, stimulated lymphocytes underwent
cytarabine- and etoposide-induced apoptosis within 6 hours, whereas
significant drug-induced apoptosis in resting lymphocytes was not
detectable before 18 hours of treatment. Although p53 protein was
readily induced by cytarabine and etoposide in stimulated lymphocytes,
expression in resting cells remained low (Figure 6B). We further
investigated p53 induction by flow cytometric detection of nuclear and
cytoplasmic p53 expression, which allows the exclusion of dead cells by
electronic gating. By focusing on the live cell population,
quantification of p53 by flow cytometry is more sensitive than Western
blot analysis. With this method, p53 induction was detected in
stimulated lymphocytes treated with cytarabine or etoposide from 6 to
18 hours (Figure 7B-D), whereas there was
no such induction in resting lymphocytes (Figure 7A) as late as 18 hours. The more rapid and pronounced p53 induction in the
etoposide-treated lymphocytes correlated with the higher cytotoxicity
of etoposide compared to cytarabine shown in Figure 6A. Thus, by 2 independent methods we found differential p53 induction in resting and
stimulated lymphocytes.
CD95 ligand protein remained unchanged in resting lymphocytes on
treatment, whereas the expression of CD95L in stimulated lymphocytes
was further induced by cytotoxic drugs (Figure 6B). A similar pattern
was found for the CD95 death receptor (Figure 8A). CD95 receptor expression was induced
by cytarabine and etoposide in stimulated, but not in resting,
lymphocytes. In resting lymphocytes, CD95 receptor expression on
culture with various drugs, including cytarabine and etoposide, was not
altered even after prolonged culture in vitro (data not shown).
We next investigated whether sensitivity for CD95-mediated apoptosis would be induced by cytotoxic drugs in stimulated lymphocytes. Lymphocytes were stimulated for 3 days with PHA and IL-2 (day 3 blasts). Constitutive CD95 sensitivity in these cells was still low, which is in line with previous studies reporting increased CD95 sensitivity in stimulated lymphocytes beyond day 6.27 Day 3 blasts were treated with cytotoxic drugs for 6 and 12 hours and subsequently were incubated with apoptosis-inducing anti-CD95 antibody. Cytarabine and etoposide induced a marked sensitivity for CD95-mediated apoptosis in stimulated lymphocytes, whereas no such sensitivity could be detected in resting cells (Figure 8B). Finally, we investigated whether CD95-receptor/ligand-mediated cell death contributes to cytotoxic drug-induced apoptosis in stimulated lymphocytes. As shown in Figure 8C, etoposide-induced cell death at 6 and 12 hours was reduced by blocking CD95 with anti-APO-Fab or neutralizing CD95L with an antiligand antibody, indicating that receptor-ligand interaction contributes to drug-induced apoptosis in stimulated lymphocytes. Taken together, the data suggest that drug-induced apoptosis involves p53- and CD95-mediated apoptosis pathways in stimulated, but not in resting, lymphocytes.
In the current study, we analyzed different apoptosis regulators in mature PBLs to identify apoptosis pathways activated by in vivo chemotherapy. In our patients receiving the first cycle of chemotherapy for pediatric solid tumors, lymphocytes rapidly declined within 72 hours of the onset of therapy. Considering the longevity of lymphocytes, this rapid decline of lymphocytes in the acute phase of chemotherapy was suggestive of active destruction of mature lymphocytes by apoptosis rather than proliferative arrest in lymphocyte precursors. In fact, we found increased in vitro apoptosis of PBLs during the acute phase of lymphocyte depletion. In addition, pro-apoptotic mitochondrial alterations were induced in these cells, further indicating a direct induction of apoptosis in mature lymphocytes by chemotherapy. In tumor cell lines, cytotoxic drug-induced apoptosis strictly depends on caspase activation.28-30 In peripheral blood mononuclear cells consisting of more than 90% lymphocytes, we found increased cleavage of caspase substrates and activation of caspases-3 and -8 early after the onset of chemotherapy. However, the effect of caspase inhibition on ex vivo apoptosis of lymphocytes during chemotherapy was heterogeneous. In some patients, caspase inhibition reduced the chemotherapy-induced increase of in vitro cell death, in others, no significant effect could be detected. Interestingly, a caspase-independent pathway has recently been found for CD95-mediated apoptosis in primary T cells.31 Thus, caspase-dependent and -independent death pathways may also contribute to lymphocyte apoptosis mediated by cytotoxic drugs. Inefficiency of caspase inhibition seen in some of the patients probably reflects predominant activation of caspase-independent pathways, which could also vary with the cytotoxic drugs administered. Activated T cells with a high propensity to apoptosis have been identified in the chronic phase of lymphocyte depletion after chemotherapy.32,33 Therefore we investigated whether chemotherapy directly induces activation of T cells and activation-induced death by way of the CD95 system. We found activation and increased CD95-specific cell death in T cells after chemotherapy. Activation and CD95 sensitivity, however, were not induced during the acute phase of lymphocyte depletion of the first cycle of chemotherapy. It cannot be excluded that activated T cells eliminated through death receptor signaling are no more detectable in peripheral blood at the time of analysis. In some cases, though, lymphocytes were analyzed as early as 3 hours to 6 hours after the onset of in vivo chemotherapy. Still, the lack of a significant number of CD95-sensitive lymphocytes during the acute phase of chemotherapy and the lack of CD95 sensitivity of in vitro-treated resting lymphocytes suggest that chemotherapy-induced acute lymphocyte depletion is largely independent of activation-induced death through CD95. This implicates that activation and CD95 sensitivity seen in PBLs after chemotherapy are not direct effects of cytotoxic drugs but are consequences of altered turnover and expansion of progenitor cells. In addition, the data indicate that the marked activation of caspase-8 found during acute lymphocyte depletion occurred independently of CD95 receptor signaling. Death receptor-independent cleavage of caspase-8 downstream of mitochondria has been described for betulinic acid-induced apoptosis in neuroblastoma34 and cytotoxic drug induced death in T-cell leukemia cell lines.17,35 It is, therefore, conceivable that chemotherapy-induced activation of caspase-8 in lymphocytes occurs through downstream effector caspases such as caspase-3 or is directly related to mitochondrial alterations. Mitochondrial alterations such as production of radical oxygen
species, disruption of mitochondrial transmembrane potential ( Permeability transition of the inner mitochondrial membrane
indicated by Several studies have suggested a molecular sequence of apoptosis induction in which DNA-damaging agents induce p53 expression that in turn increases the transcription of Bax for the promotion of apoptosis.24,25 No induction of p53 protein could be detected during chemotherapy, suggesting that in PBLs, Bax, mitochondrial alterations, and caspase activation are independent of p53. In addition, we did not find direct evidence in vivo for activation of the CD95 system, which is partially regulated by p53.41 These data are different from those of several reports in cell lines demonstrating a significant contribution of p53 and the CD95 system in drug-induced apoptosis. Interestingly, marked induction of p53 and CD95 sensitivity by cytotoxic drugs, including receptor-ligand interaction, was detectable only in activated lymphocytes on in vitro stimulation. This suggests that the induction of p53 and CD95 by cytotoxic drugs requires an activated or a cycling state, which is present in cell lines and stimulated lymphocytes in vitro but not in resting lymphocytes. Thus, the lack of detectable activation of p53 or CD95 in PBLs during in vivo chemotherapy is probably caused by differential induction of p53 and CD95 in resting and cycling lymphocytes. In proliferating precursor compartments, however, which could not be analyzed in this study, these death pathways may contribute to the depletion of lymphocyte progenitors. In fact, p53-dependent regulation of recovering bone marrow precursors after chemotherapy has been shown in mice,42 and elevated expression of p53 has been found in human hematopoietic progenitors in the regenerative phase after chemotherapy.43 Recent reports have shown that anthracyclines were equally effective in apoptosis induction in resting and stimulated lymphocytes, whereas topoisomerase inhibitors and antimetabolites were more efficient in activated lymphocytes.44 This suggests that the differential sensitivity of resting and activated lymphocytes, and eventually the degree of p53 induction, may also vary with different drugs. Taken together, our data describe distinct apoptosis pathways operative in the rapid depletion of lymphocytes by in vivo chemotherapy and identify differential involvement of p53 and CD95 in resting and cycling lymphocytes.
We thank Tanja Dravits for excellent technical assistance.
Supported by grants from the Deutsche Forschungsgemeinschaft and Bundesministerium für Bildung und Forschung.
Submitted November 6, 2000; accepted July 9, 2001.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Klaus-Michael Debatin, Department of Pediatrics, University Children's Hospital, Prittwitzstr 43, 89070 Ulm Germany; e-mail: klaus-michael.debatin{at}medizin.uni-ulm.de.
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  H. E. Kendall, P. M. Vacek, J. L. Rivers, S. C. Rice, T. L. Messier, and B. A. Finette Analysis of Genetic Alterations and Clonal Proliferation in Children Treated for Acute Lymphocytic Leukemia. Cancer Res., September 1, 2006; 66(17): 8455 - 8461. [Abstract] [Full Text] [PDF]
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S. C. Rice, P. Vacek, A. H. Homans, T. Messier, J. Rivers, H. Kendall, and B. A. Finette Genotoxicity of Therapeutic Intervention in Children with Acute Lymphocytic Leukemia Cancer Res., July 1, 2004; 64(13): 4464 - 4471. [Abstract] [Full Text] [PDF]
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D. J. Waxman and P. S. Schwartz Harnessing Apoptosis for Improved Anticancer Gene Therapy Cancer Res., December 15, 2003; 63(24): 8563 - 8572. [Abstract] [Full Text] [PDF]
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X. W. Meng, J. Chandra, D. Loegering, K. Van Becelaere, T. J. Kottke, S. D. Gore, J. E. Karp, J. Sebolt-Leopold, and S. H. Kaufmann Central Role of Fas-associated Death Domain Protein in Apoptosis Induction by the Mitogen-activated Protein Kinase Kinase Inhibitor CI-1040 (PD184352) in Acute Lymphocytic Leukemia Cells in Vitro J. Biol. Chem., November 21, 2003; 278(47): 47326 - 47339. [Abstract] [Full Text] [PDF]
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K. Elflein, M. Rodriguez-Palmero, T. Kerkau, and T. Hunig Rapid recovery from T lymphopenia by CD28 superagonist therapy Blood, September 1, 2003; 102(5): 1764 - 1770. [Abstract] [Full Text] [PDF]
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A. Zeuner, F. Pedini, M. Signore, U. Testa, E. Pelosi, C. Peschle, and R. De Maria Stem cell factor protects erythroid precursor cells from chemotherapeutic agents via up-regulation of BCL-2 family proteins Blood, July 1, 2003; 102(1): 87 - 93. [Abstract] [Full Text] [PDF]
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J. Bladon and P. Taylor The common pathways, but different outcomes, of apoptosis induced by extracorporeal photopheresis and in vivo chemotherapy may reinforce the important immunomodulatory effect of monocytes Blood, April 15, 2002; 99(8): 3071 - 3072. [Full Text] [PDF]
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Abstract
Introduction
% apoptosis in
untreated samples/100 
