1Department of Immunology and Microbiology, and the Karmanos Cancer Institute Wayne State University, Detroit, MI 48201

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Key words: genistein, T-cell lymphoblastic leukemia, mitochondria, caspase

Abbreviations: apoptosis activating factor-1, (Apaf-1); B-cell acute lymphoblastic leukemia, (B-ALL); bongkrekic acid, (BA); fetal bovine serum, (FBS); fluorescein isothiocyanate-conjugated annexin V, (annexin V-FITC); permeability transition pore, (PTP); phosphate buffered saline, (PBS); room temperature, (RT); sodium dodecylsulfate, (SDS); T-cell acute lymphoblastic leukemia, (T-ALL)

Epidemiological studies of different populations have indicated that there is a high correlation between a lower incidence of certain types of cancer and a high soy diet (Mills et al, 1989; Lee et al, 1991; Adlercreutz et al, 1995; Wu et al, 1996; Dai et al, 2001; Messina, 2003; Sarkar and Li, 2003; Yamamoto et al, 2003). It was found that Asian women who regularly consumed soy foods had a significantly lower risk of developing breast cancer (Lee et al, 1991; Wu et al, 1996; Dai et al, 2001; Yamamoto et al, 2003). These observations have been supported by additional studies of premenopausal women in different Asian populations, which showed a correlation between a reduced risk of breast cancer and a high intake of soy (Lee et al, 1992; Hirose et al, 1995). In addition, several studies have shown that consumption of soy products reduced the mortality rate from prostate cancer (Mills et al, 1989; Adlercreutz et al, 1993; Hebert et al, 1998; Messina, 2003). Isoflavones have been identified to be the major component of soy that is responsible for lowering the incidence of these cancers (Adlercreutz et al, 1995; Messina, 2003; Yamamoto et al, 2003). More specifically, consumption of genistein, which is the predominant soy isoflavone, strongly correlates with a reduced incidence of cancer in humans and other animals (Adlercreutz et al, 1993; Barnes, 1995; Lamartiniere et al, 1995a,b, 2002; Polkowski and Mazurek, 2000; Mentor-Marcel et al, 2001; Mizunuma et al, 2002; Wang et al, 2002). Because of these observations there has been a strong interest in the use of genistein for cancer prevention.

Besides its potential as a preventive agent for certain cancers, there is considerable interest in the use of genistein for therapeutic purposes as well. This potential use of genistein is based on numerous in vitro and in vivo studies, which have demonstrated that genistein is able to kill malignant cells in contrast with normal cells (Traganos et al, 1992; Constantinou et al, 1998; Davis et al, 1998; Li et al, 1999b; Messina, 1999; Baxa and Yoshimura, 2003). Malignant cell killing by genistein has been detectable for cell lines derived from different types of cancer, including breast (Zava and Duwe, 1997), non-small-cell lung (Lian et al, 1999), head and neck squamous cell (Alhasan et al, 1999), prostate (Geller et al, 1998; Li and Sarkar, 2002), and leukemia (Traganos et al, 1992; Spinozzi et al, 1994; Baxa and Yoshimura, 2003). It has furthermore been demonstrated for many of these cell types that cell killing occurs via apoptosis (Alhasan et al, 1999; Spinozzi et al, 1994; Davis et al, 1998; Li et al, 1999b;). Tumor cell-killing by genistein could be potentially exploited in the treatment of cancers that have become resistant to chemotherapeutic drugs and radiation therapy.

The potential use of genistein as a treatment for cancer was initially recognized by the observation that this reagent is able to specifically inhibit tyrosine kinases (Akiyama et al, 1987), a class of proteins that are frequently involved in the regulation of cellular proliferation (Ullrich and Schlessinger, 1990). Besides this activity, additional studies have demonstrated that genistein can also inhibit DNA topoisomerase II, angiogenesis, metastasis, protein-histidine kinase, and 5-a -reductase (Okura et al, 1988; Huang et al, 1992; Evans et al, 1995; Fotsis et al, 1995; Li et al, 1999a). Other activities of genistein include the downregulation of activated NF-k B and an inhibition of the Akt signaling pathway (Davis et al, 1999; Li and Sarkar, 2002; Baxa and Yoshimura, 2003; Gong et al, 2003). Which of these multiple activities of genistein is responsible for the induction of apoptosis most likely depends on the particular malignant cell type.

Studies of the effect of genistein on malignant cells have been performed predominantly on solid tumors, such as breast and prostate (Kyle et al, 1997; Constantinou et al, 1998; Shao et al, 1998; Li et al, 1999b). Although fewer studies have been conducted on hematopoietic malignancies, it has been shown that genistein induces apoptosis in human T-cell lymphomas as well (Traganos et al, 1992; Spinozzi et al, 1994; Hayon et al, 2003). The mechanism of the induction of apoptosis in T-cells and other cell types is not well understood. To identify some of the early steps in the induction of apoptosis by genistein, we examined murine T-leukemia cell lines and demonstrated that an early step in apoptosis induction is damage to the mitochondrial membrane (Baxa and Yoshimura, 2004). Furthermore, we observed that mitochondrial damage resulted in more downstream apoptotic events, such as activation of caspase-9 and caspase-3, as well as DNA fragmentation. To determine whether this early effect of genistein in murine cells also occurs in human T-leukemia cells, in this study we examined two acute lymphoblastic leukemia (T-ALL) cell lines, Jurkat (Schneider et al, 1977) and CCRF-CEM (Foley et al, 1965). T-ALL accounts for 10 to 15% of newly diagnosed cases of childhood acute lymphoblastic leukemia, but it has a higher relapse rate than in patients with B-cell leukemias (Goldberg et al, 2003). Although with aggressive combinations of cytotoxic chemotherapeutic drugs and radiation, the 5-year survival rate has now reached 75% (Goldberg et al, 2003), there is still a need for more effective and less toxic treatments for these patients and those with refractory disease.

Two well-characterized pathways for apoptosis induction are the extrinsic and intrinsic pathway. The extrinsic pathway involves cell surface signaling, such as in the case of Fas/Fas L and the TNF-a receptor (Ashkenazi and Dixit, 1998), while the intrinsic pathway involves mitochondrial damage (Traganos et al, 1992; Spinozzi et al, 1994; Green and Reed, 1998; Desagher and Martinou, 2000). Activation of the intrinsic pathway is initiated by damage to the mitochondrial membrane, leading to the release of cytochrome c into the cytoplasm (Martinou et al, 2000). Released cytochrome c subsequently binds the apoptosis activating factor-1 (Apaf-1) and ATP, which results in the recruitment and activation of procaspase-9 (Pan et al, 1998). Caspase-9 activation subsequently results in other more downstream events of apoptosis, such as activation of caspase-3, which in turn is responsible for the execution of additional features of apoptosis (Thornberry and Lazebnik, 1998).

A mechanism by which mitochondrial damage can occur involves the sustained opening of the mitochondrial permeability transition pore (PTP) (Kroemer et al, 1998; Bernardi et al, 1999; Desagher and Martinou, 2000). Opening of the PTP leads to mitochondrial depolarization and the influx of solutes, which result in outer membrane damage and the release of cytochrome c (Hirsch et al, 1998). Certain stress factors, such as an increase in intracellular calcium, generation of reactive oxygen species, and changes in cytosolic pH, induce mitochondrial damage via the PTP (Hirsch et al, 1998). In this study, we have made the novel observation that one of the early steps in the induction of apoptosis by genistein in human T-ALL cells involves mitochondrial damage via the permeability transition pore, thus implicating an early involvement of the intrinsic pathway.

Jurkat (TIB-152) and CCRF-CEM (CCL-119) cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 supplemented with L-glutamine, 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 10 mM Hepes, and 20 units per ml penicillin-streptomycin at 37° C and 5% CO₂. Cells were cultured at 5 x 10⁵ cells per ml 24 hr prior to the addition of genistein (Toronto Research Chemicals, Toronto, Canada). Cells were washed once with RPMI 1640 and resuspended at 2 x 10⁵ cells per ml at the time of genistein exposure. A genistein stock solution was prepared in DMSO at a concentration of 100 mM. The same volume of DMSO was added to control cells, resulting in a final concentration of DMSO of less than 0.05%. For bongkrekic acid studies, cells were treated with 150 m M bongkrekic acid (EMD Biosciences, Inc., La Jolla, CA) for 1 hr at 37ºC prior to the addition of genistein.

A quantitative analysis of viable, dead, and apoptotic cell populations was conducted by staining cells with propidium iodide (PI) (Sigma-Aldrich, St. Louis, MO) and fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) (PharMingen, BD Biosciences, San Diego, CA). 10⁵ cells were washed twice with cold phosphate buffered saline (PBS) and stained with 0.5 m g per ml PI and 0.6 µg per ml annexin V-FITC in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂). Cells were incubated at room temperature (RT) in the dark for 15 min, after which time 400 m l of binding buffer was added. Cells were analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer (Wayne State University and Karmanos Cancer Institute Flow Cytometry Core Facility) within 1 hr of staining. Data were collected on 2 x 10⁴ cells using the CELLQuest software (Becton Dickinson, BD Biosciences). Gating was established on single color controls.

5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazoly lcarbocyanine iodide (JC-1, Molecular Probes, Inc., Eugene, OR) was used as an indicator dye for mitochondrial depolarization. JC-1 was added to cells in culture to a final concentration of 5 m g per ml for 15 min at RT. Stained cells were pelleted, washed twice in cold PBS, and resuspended in 500 m l PBS. As a positive control, cells were treated with valinomycin (Sigma-Aldrich) at 4 m g per ml final concentration for 4 hr at 37ºC. Cells were analyzed by two-color flow cytometry. Gating was established with untreated and valinomycin treated cells.

2 x 10⁷ cells were collected, washed twice with PBS, and lysed in 200 m l 50 mM Tris-HCl, pH 7.5, containing 0.03% Nonidet P-40 and 1mM dithiothreitol. For positive controls for caspase cleavage, cells were treated with 1 m M staurosporine (Sigma-Aldrich) for 4 hr at 37˚C. Cellular extracts were prepared as described (Pazirandeh et al, 2000). Protein amounts were measured using the bicinchoninic acid protein assay (Pierce, Rockford, IL). 50 m g protein was added to reducing buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% sodium dodecylsulfate (SDS), 0.01% bromophenol blue, 5% b -mercaptoethanol) and boiled for 4 min. Samples were electrophoresed in running buffer of 25 mM Tris, 192 mM glycine, 1% SDS, pH 8.3, through a 12% or 15% SDS-polyacrylamide gel at 120 volts for 1 hr and transferred to polyvinylidene difluoride membrane (BioRad, Hercules, CA) at 350 mAmp for 2 hr at 4˚C. Membranes were probed with a rabbit antibody specific for either caspase-3, caspase-9 (Cell Signaling Technology, Beverly, MA), or b -actin (Sigma-Aldrich) overnight at 4˚C. After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit serum (Pierce) for 1 hr at RT. Protein bands were detected by enhanced chemiluminescence (ECL, Amersham Pharmacia, Piscataway, NJ) and visualized on Biomax MR film (Kodak, Rochester, NY).

Most of the previous studies of the effects of genistein on human T-cell leukemia lines have identified cellular changes that occurred after 24 hr or more of exposure (Traganos et al, 1992; Spinozzi et al, 1994; Markovits et al, 1995). Various studies have shown that after this exposure time, genistein has multiple effects on cells, including cell cycle arrest, inhibition of tyrosine kinases and DNA topoisomerase II, downregulation of NF-k B, and induction of apoptosis (Akiyama et al, 1987; Okura et al, 1988; Traganos et al, 1992; Spinozzi et al, 1994; Davis et al, 1999; Polkowski and Mazurek, 2000; Baxa and Yoshimura, 2003). Because there are few studies of some of the effects that genistein produces in cells at early times after exposure, we undertook this study of human T-ALL cells to identify the early steps involved in cell death signaling. We chose this leukemic cell type to study because an understanding of the mechanism of cell killing by genistein should provide insights into its potential use for the treatment of this type of childhood T-cell leukemia, which normally has a poorer prognosis than childhood leukemia of B-cell origin (DeVita, 2001).

For this study we chose the Jurkat and CCRF-CEM cell lines, which were derived from two different patients with childhood T-cell leukemia (Foley et al, 1965; Schneider et al, 1977). Similar to previous results from other laboratories, we observed that genistein induced cell killing in Jurkat and CCRF-CEM cells at concentrations of 15 m M and greater in a dosage- and time-dependent manner (Figure 1A and B). In addition, flow cytometric analysis of cells stained with Annexin V-FITC and PI confirmed that cell killing occurred via apoptosis (Figure 1C and D). It has been shown that flow cytometric analysis of cells stained with Annexin V and PI is an effective method to distinguish between live (PI^-, annexin V^-), apoptotic (PI^-, annexin V⁺), and dead (PI⁺, annexin V⁺) cells (Koopman et al, 1994). Our detection of apoptosis for Jurkat cells confirmed the results of Spinozzi et al, 1994, who examined Jurkat cells at 48 hr after genistein treatment. Although the percentage of apoptotic cells peaked at 48 hr for both cell lines, our data showed that apoptosis occurred at an earlier time as well.

As an independent assay for apoptosis, we examined caspase-3 activation, which is a hallmark of apoptosis that occurs via both the extrinsic and intrinsic pathways (Thornberry and Lazebnik, 1998). Activation of caspase-3 occurs by cleavage of the 35 kDa procaspase form to 17 kDa and 12 kDa protein products (Nicholson et al, 1995; Sun et al, 1999). Protein extracts were prepared from Jurkat and CCRF-CEM cells treated with 60 m M genistein for 24, 48, and 72 hr. The 35 kDa procaspase form and 17 kDa cleavage product were detectable by immunoblotting with a polyclonal antibody specific for caspase-3 (Cell Signaling Technology, Inc.) (Figure 2). An intermediate 19 kDa cleavage product was also detectable for our cell extracts. For both cell lines, we detected increasing amounts of the 19 kDa and 17 kDa cleavage products over time, which confirmed that genistein was inducing apoptosis in these cells. The largest amount of activated caspase-3 was detectable at 72 hr of genistein treatment for both cell lines (Figure 2A and B, lane 6). As a positive control for caspase-3 activation, we treated Jurkat and CCRF-CEM cells for 4 hr with 1 m M staurosporine, which is known to activate caspase-3 and induce apoptosis (Bijur, 2000) (Figure 2A and B, lane 7).

In a study of the effects of genistein on murine thymic lymphoma cells, we observed that mitochondrial damage occurred as an early step in the induction of apoptosis (Baxa and Yoshimura, 2004). To determine

Figure 1. Genistein kills T-ALL cells via apoptosis. Number of viable Jurkat (A) or CCRF-CEM (B) cells after treatment with different concentrations of genistein for various times. 4 x 10⁵ cells for each cell line were treated with genistein at concentrations ranging from 0 to 60 m M. Cells were collected and stained with trypan blue at days 1 through 4 after genistein exposure. Percentage of apoptotic Jurkat (C) or CCRF-CEM (D) cells treated with 60 m M genistein for various times. Percentage of apoptotic cells was determined by flow cytometric analysis of cells stained with annexin V-FITC and PI. The results shown are the mean values and standard deviations calculated from duplicate samples from two independent experiments.

Figure 2. Caspase-3 activation by genistein. Jurkat (A) or CCRF-CEM (B) cells were treated with 60 m M genistein for 24, 48, and 72 hr. 50 m g cell extracts were analyzed by Western blotting with an antibody specific for caspase-3 (Cell Signaling Technology, Inc.). C, control cells; G, genistein-treated cells. STS, staurosporine-treated cells for caspase-3 cleavage control. Arrows indicate the 35 kDa procaspase-3 form, and 19 kDa and 17 kDa cleavage products. b -actin was detected for loading control.

whether this is also an early effect of genistein in T-ALL cells, we used the mitochondrion-specific dye JC-1 (Molecular Probes) to detect mitochondrial membrane damage. JC-1 was chosen for this analysis because it selectively enters normal mitochondria where it forms red fluorescent J-aggregates (Reers et al, 1991; Salvioli et al, 1997). Upon damage to the mitochondrial membrane, which results in a decrease in the transmembrane potential, JC-1 aggregates are disrupted, and monomers that emit a green fluorescence are produced. Thus, a shift of fluorescence in cells from red to green is an indication of depolarization of the mitochondrial membrane.

Jurkat and CCRF-CEM cells were treated with 60 m M genistein for 4 hr before JC-1 staining. Flow cytometric analysis of Jurkat cells showed that a total of 69.5% of Jurkat cells suffered mitochondrial damage as a result of genistein treatment (Figure 3B). Of these cells, 41.3% displayed an increase in green fluorescence, and 28.2% displayed both an increase in green fluorescence and decrease in red fluorescence. These populations represented only 1.4% of control cells (Figure 3A). As a positive control for JC-1 staining, we exposed cells to 4 m g per ml valinomycin, a potassium ionophore, that produces mitochondrial depolarization (Inai et al, 1997). For valinomycin-treated cells, mitochondrial depolarization occurred in a total of 94.4% of cells (Figure 3C).

Figure 3. Genistein causes mitochondrial depolarization in Jurkat cells. 10⁶ cells were treated for 4 hr with either DMSO (A), 60 m M genistein (B), or 4 m g per ml valinomycin (C). JC-1 (5 m g per ml final concentration) was subsequently added to cells for 15 min, after which time two-color flow cytometric analysis was performed. Number in each quadrant indicates the percentage of total cells analyzed. Dot plots are representative data from three independent experiments.

Figure 4. CCRF-CEM cells undergo mitochondrial depolarization by genistein. 10⁶ cells were treated for 4 hr with either DMSO (A), 60 m M genistein (B), or 4 m g per ml valinomycin (C). Cell analysis was performed and displayed as described for Figure 3.

These results thus indicate that mitochondrial depolarization occurred as early as 4 hr after exposure to genistein in Jurkat cells. To determine whether mitochondrial depolarization is an early step that occurs in other T-ALL cells, we performed a similar analysis of CCRF-CEM cells. Flow cytometric analysis of cells treated with 60 m M genistein for 4 hr and subsequently stained with JC-1showed that a total of 66.8% of cells suffered mitochondrial damage (Figure 4B). This was in contrast to a total of only 0.5% of untreated cells (Figure 4A).

We and others have previously observed that genistein is able to induce mitochondrial depolarization in some cell types as a result of association with the membrane PTP (Yoon et al, 2000; Salvi et al, 2002). To examine whether a similar mechanism is involved in the damage to mitochondria by genistein in T-ALL cells, we determined whether bongkrekic acid (BA), a specific inhibitor of the mitochondrial PTP (Zamzami et al, 1996), could reverse the mitochondrial depolarization induced by genistein. As we had seen previously, treatment of Jurkat cells with 60 m M genistein resulted in mitochondrial depolarization in a significant percentage of cells as detectable by JC-1 staining and flow cytometric analysis (Figure 5C). In this representative experiment, genistein produced mitochondrial depolarization in a total of 48.3% of Jurkat cells (Figure 5C), in contrast to control cells, which showed little mitochondrial damage (Figure 5A). On the other hand, Jurkat cells pretreated with 150 m M BA for 1 hr prior to a further 4 hr incubation with genistein (Figure 5D) resembled cells treated with 150 m M BA alone (Figure 5B).

Figure 5. Mitochondrial depolarization occurs via the permeability transition pore in Jurkat cells. 10⁶ Jurkat cells were either untreated (A), treated with 150 m M bongkrekic acid (BA) for 5 hr (B), treated with 60 m M genistein for 4 hr with no BA pretreatment (C), or pretreated with 150 m M BA for 1 hr before the addition of 60 m M genistein for an additional 4 hr (D). Cells were subsequently stained with JC-1 and analyzed by two-color flow cytometry as described for Figure 3. Number in each quadrant indicates the percentage of total cells analyzed. Dot plots are representative data from two independent experiments.

Figure 6. Involvement of permeability transition pore in mitochondrial depolarization in CCRF-CEM cells. 10⁶ CCRF-CEM cells were either untreated (A), treated with 150 m M bongkrekic acid (BA) for 5 hr (B), treated with 60 m M genistein for 4 hr with no BA pretreatment (C), or pretreated with 150 m M BA for 1 hr before the addition of 60 m M genistein for an additional 4 hr (D). Cell analysis was performed and displayed as described for Figure 5.

These data indicate that under these conditions BA nearly completely inhibited mitochondrial depolarization induced by genistein, suggesting that mitochondrial damage occurred via deregulation of the PTP. Analysis of CCRF-CEM cells showed that the PTP is similarly involved in mitochondrial depolarization by genistein in these cells (Figure 6). Treatment with genistein alone induced mitochondrial depolarization in 77.5% of CCRF-CEM cells (Figure 6C). In cells pretreated with BA before the addition of genistein, only 4.8% of cells experienced mitochondrial depolarization (Figure 6D).

Our observation that genistein produces mitochondrial damage as an early event in T-ALL cells suggested that apoptosis proceeds via the intrinsic pathway. To further examine this idea, we evaluated caspase-9 activation, which occurs as a result of cytochrome c release from damaged mitochondria (Li et al, 1997). To perform this analysis, we examined protein extracts from Jurkat and CCRF-CEM cells at various times after exposure to 60 m M genistein (Figure 7). Immunoblot analysis of Jurkat cell extracts with an antibody specific for caspase-9 (Cell Signaling Technology, Inc.) revealed an initial caspase-9 cleavage product of 37 kDa after 24 hr of genistein treatment (Figure 7A, lane 2). We detected an increase in this cleavage product with continuous exposure to genistein (lanes 4 and 6). At 48 hr of genistein exposure, we began to see a smaller 35 kDa cleavage protein, which corresponds to the active enzyme (lane 4) (Li et al, 1997; Sun et al, 1999). Both cleavage proteins began to appear in CCRF-CEM cells after 24 hr of genistein treatment (Figure 7B, lane 2). Both products increased with treatment time (lanes 4 and 6). It is not clear why procaspase-9 is processed differently in the two cell lines. As a positive control, cells were treated with 1 m M staurosporine, which induces apoptosis via the intrinsic pathway and produces caspase-9 cleavage (Figure 7A and B, lane 7) (Bijur et al, 2000; Scarlett et al, 2000).

Figure 7. Caspase-9 activation in T-ALL cells treated with genistein. Western blot detection of caspase-9 from Jurkat (A) or CCRF-CEM (B) cells treated with 60 m M genistein for 24, 48, and 72 hr. 50 m g cell extracts were analyzed with an antibody specific for caspase-9 (Cell Signaling Technology, Inc.). C, control cells; G, genistein-treated cells. STS, staurosporin-treated cells for caspase-9 cleavage control. Arrows indicate the 47 kDa procaspase-9 form, and 37 kDa and 35 kDa cleavage products. b -actin was detected for loading control.

Previous studies have demonstrated that genistein is able to induce apoptosis in different types of tumor cells (Traganos et al, 1992; Spinozzi et al, 1994; Barnes, 1995; Zava and Duwe, 1997; Geller et al, 1998; Lian et al, 1998; Alhasan et al, 1999; Li et al, 1999b). Various cellular effects of genistein, such as cell cycle arrest, the inhibition of tyrosine kinases, downregulation of NF-k B, inhibition of Akt kinase, and mitochondrial damage (Akiyama et al, 1987; Traganos et al, 1992; Spinozzi et al, 1994; Davis et al, 1999; Polkowski and Mazurek, 2000; Yoon et al, 2000; Salvi et al, 2002; Baxa and Yoshimura, 2003; Gong et al, 2003) may potentially contribute to apoptosis induction. Another commonly observed effect of genistein, i.e., inhibition of topoisomerase II (Okura et al, 1988; Polkowski and Mazurek, 2000; Sarkar and Li, 2003), does not appear to be involved in apoptosis induction (Salti et al, 2000). Many of the cellular effects of genistein have been detected after exposure of cells to genistein for 24 hr or longer. In this study, our goal was to identify some of the earlier events that occur in T-ALL cells after exposure to genistein to better understand the mechanism of action involved in the induction of apoptosis. This information would be useful for the assessment of the potential use of genistein in the treatment of this type of early childhood leukemia.

Our assays for cell killing and apoptosis confirmed observations by others that pharmacological concentrations of genistein are able to induce apoptosis in T-ALL cells in a dosage- and time-dependent manner. More importantly, examination of genistein-treated cells before apoptosis could be detected led to our novel observation that mitochondrial damage was an early step in apoptotic signaling. Furthermore, flow cytometric analysis of cells treated with bongkrekic acid suggested that damage to mitochondria occurred via the mitochondrial membrane pore. Yoon et al, 2000 showed that this also occurred in genistein-treated RPEJ neuronal cells. A study of the effect of genistein on isolated rat liver mitochondria also supports the idea that genistein can target mitochondrial membrane pores to induce damage in cells (Salvi et al, 2002).

The induction of apoptosis via mitochondrial damage has been well-studied and is known to be initiated by the release of cytochrome c from the mitochondrial membrane into the cytoplasm (Martinou et al, 2000). The subsequent binding of cytochrome c to Apaf-1results in the activation of caspase-9, which in turn, is involved in the execution of additional steps in the apoptosis cascade, such as caspase-3 activation (Pan et al, 1998; Thornberry and Lazebnik, 1998). In our analysis of T-ALL cells, caspase-9 activation was detectable at 24 hr after the addition of genistein (Figure 7). Furthermore, our analysis showed that caspase-3 activation occurred after the activation of caspase-9 (Figure 2). This sequence of events suggests that the induction of apoptosis by genistein in T-ALL cells follows the steps commonly observed for the intrinsic pathway involving mitochondrial damage.

Acute lymphoblastic leukemia, which is derived from either a precursor T or B cell, accounts for approximately 75% of childhood leukemias (DeVita, 2001). The prognosis for ALL of precursor T-cell origin is typically worse than for B-cell acute lymphoblastic leukemia (B-ALL) (DeVita, 2001). Even with aggressive chemotherapy, patients with T-ALL are less likely to enter remission and relapse more quickly than those with B-ALL (Goldberg et al, 2003). Our results from this study suggest that genistein may be effective in the treatment of children with T-ALL because of its ability to induce apoptosis via damage to the mitochondrial membrane.

The author would like to thank Ruchi Rastogi for her technical assistance, and the members of the Flow Cytometry Core Facility, supported by Wayne State University and the Karmanos Cancer Institute, for their help. This work was supported in part by the Children's Research Center of Michigan, Detroit, MI.