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[Cancer Research 61, 7333-7338, October 1, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Thioredoxin Expression in Primary T-Cell Acute Lymphoblastic Leukemia and Its Therapeutic Implication1

Li-en Shao, Mitchell B. Diccianni, Tetsuya Tanaka, Ruby Gribi, Alice L. Yu, Jeanette D. Pullen, Bruce M. Camitta and John Yu2

Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 [L. S., T. T., R. G., J. Y.]; Department of Pediatrics, University of California at San Diego Medical Center, San Diego, California 92103 [M. B. D., A. L. Y.]; University of Mississippi Medical Center, Jackson, Mississippi 39216 [J. D. P.]; and Midwest Children’s Cancer Center, Milwaukee, Wisconsin 53226 [B. M. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased expression of intracellular thioredoxin has been implicated in the inhibition of apoptosis and in a decrease in the sensitivity of the malignancies to drug-induced apoptosis. In the present studies, we analyzed expression of thioredoxin in samples from 28 children with T-cell acute lymphoblastic leukemia and analyzed their sensitivity toward inhibition of thioredoxin expression. Thioredoxin was expressed in variable amounts. Higher expression was associated with higher WBC counts. Exogenously added thioredoxin stimulated proliferation of clonogenic cells among the T-cell acute lymphoblastic leukemia samples expressing relatively lower levels of intracellular thioredoxin, whereas there was no effect on the clonogenic cells expressing high levels of thioredoxin. In addition, there was differential sensitivity of the leukemia clonogenic cells toward 1-methylpropyl 2-imidazolyl disulfide, an inhibitor of thioredoxin expression, as compared with normal hematopoietic progenitors. This suggests the possibility of using this approach for treatment. Because overexpression of thioredoxin is associated with resistance to many anticancer drugs, the inhibition of thioredoxin expression may overcome this drug resistance and probably sensitize leukemia cells to other chemotherapeutic agents.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ALL3 is the most common type of cancer in children. The well-known prognostic factors include WBC count, age, gender, and specific cytogenetic changes. The majority of childhood ALL is B-cell lineage. T-ALL comprises approximately 10–15% of all of the cases of ALL. Because T-ALL is a more aggressive disease, patients require more intensive chemotherapy and, should they relapse, their clinical outcome is very dismal. A better understanding of the biology of T-cell leukemia may facilitate the development of a selective therapy that exploits specific biological properties.

Thioredoxin is an approximately Mr 12,000 redox protein that plays an important role in cell viability, activation, and proliferation (1) . Thioredoxin, known as a dithiol hydrogen donor, is the major reducing protein for many targets, including ribonucleotide reductase, essential for DNA synthesis (2) . It is also required for the interaction between transferrin receptor mRNA and the iron response element-binding protein, serving to promote cellular proliferation (3) . Furthermore, thioredoxin regulates the activity of transcription factors including nuclear factor-{kappa}B, TFIIIC, BZLF1, and the glucocorticoid receptor (4, 5, 6, 7) . The binding of Fos/Jun to the AP-1 site is also subject to redox control by redox factor 1, which is reduced by thioredoxin (8 , 9) . Interestingly, thioredoxin is induced by various stress conditions, such as viral infection (10, 11, 12) , and is also secreted from the cells. Furthermore, several reports (13 , 14) have demonstrated that thioredoxin-transfected cells are more resistant to anticancer drugs than are the control cells. On the other hand, antisense-transfected cells become sensitive again to the same agents.

We investigated the expression of thioredoxin in T-ALL and studied the sensitivity of these samples toward inhibition of thioredoxin expression. The results show that thioredoxin was expressed in variable amounts in T-ALL, with higher expression associated with higher leukemia cell counts. It was also found that the proliferation of T-ALL clonogenic cells from those samples expressing relatively low intracellular thioredoxin was stimulated significantly by the addition of exogenous thioredoxin. In addition, the sensitivity toward an inhibitor of thioredoxin expression varied substantially between the leukemia clonogenic cells and the normal hematopoietic progenitors, suggesting the possibility of using this approach as a potential therapeutic regimen for treatment. Overall, current studies should facilitate a better understanding of the molecular changes in T-ALL and shed light on the potential molecular targets for new treatment and assessment of clinical outcome.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Leukemic Cells.
Peripheral blood or bone marrow samples were obtained from 28 newly diagnosed patients with childhood T-ALL who enrolled in Pediatric Oncology Group protocol #9400. Informed consent was obtained from the patients and/or their parents. The content of lymphoblasts was generally >90%, as determined by Wright stain. The MNC fraction was isolated from these leukemia samples by Ficoll-Paque density gradient centrifugation (Pharmacia, Piscataway, NJ). In some cases, MNCs of T-ALL samples were cryopreserved and stored in liquid nitrogen before use in the studies. Viability on thawing was generally greater than 80%, as determined by trypan blue dye exclusion.

Western Blot Analysis.
The MNC fraction of leukemia samples was lysed on ice for 1 h in lysis solution of 1% NP40, 1 mM EDTA, and 150 mM NaCl in 50 mM Tris buffer (pH 8.0) containing 20 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 5 µg/ml antitrypsin, 1 µg/ml leupeptin, 1 µg/ml pepstain, and 0.5 µg/ml N{alpha}P-Tosyl-L-lysine chloromethyl ketone (Sigma Chemical Co., St. Louis, MO). After centrifugation for 5 min at 4°C and 12,000 x g, the supernatant was harvested and stored at -80°C until further analysis. About 10–20 µg of each protein extract was analyzed by electrophoresis on a 15% polyacrylamide/SDS gel and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). After blocking in Blotto solution (i.e., 5% nonfat milk, 0.2% Tween 20, 0.05% NP40, 150 mM NaCl, and 10 mM Tris-HCl, pH 8.0) for 1 h at room temperature, the membranes were incubated overnight at 4°C with 1:2000 dilution of rabbit antiserum directed against human thioredoxin. Afterward, membranes were washed six times with 10 mM Tris-HCl (pH 8.4) containing 150 mM NaCl and 0.2% Tween 20 and incubated with affinity-purified, horseradish peroxidase-conjugated, goat antirabbit IgG (H + L; 1:1000; Kirkegaard & Perry Lab, Gaithersburg, MD). Finally, detection was performed with Enhanced Chemiluminescent Western blotting detection reagents (Amersham Life Science, Arlington Heights, IL). Semiquantitative measurements were done with Speedlight gel documentation system (Lightools Research, Encinitas, CA) and ImageQuant (Molecular Dynamics, Sunnyvale, CA). To ensure an equal amount of protein was in each sample loaded into the gel, the same membranes were also incubated with 1:1000 diluted mouse monoclonal antibody against ß-actin (Sigma Chemical Co.) and then reacted with peroxidase-labeled goat antimouse IgG (Kirkegaard & Perry Lab).

Leukemia Colony Assay.
Approximately 5 x 105 primary leukemia cells were cultured in 35-mm Petri dishes at 37°C, 5% CO2, in Iscove’s modified Dulbecco’s medium (ICN Biomedicals, Aurora, OH) supplemented with 1.0% methylcellulose, 0.1 µM {alpha}-thioglycerol, 15% horse serum, 15% FCS, 1% BSA, 10 ng/ml PMA, and 100 units/ml recombinant human IL-2 (Immunex, Seattle, WA; also see Ref. 15 ). Two dishes were set up for each individual data point/experiment. T-ALL colonies with 50 or more cells were enumerated after incubation of the dishes for 10–14 days at 37°C. To examine the effect of recombinant human thioredoxin (American Diagnostica, Greenwich, CT), PMA was omitted in the cultures, and 0.5 µg/ml of recombinant thioredoxin was added. On the other hand, for the inhibitor studies, a series of increasing concentrations of the inhibitor compound IV-2 from 0.01 to 30 µM was added in regular leukemia colony cultures, and the concentration of IV-2 resulting in 50% inhibition of colony formation in the cultures (IC50) was calculated.

Clonogenic Assay for Normal Hematopoietic Progenitor Cells.
Cultures of hematopoietic progenitor cells were performed as described in previous publications (16) . Briefly, 2 x 105 MNCs from normal bone marrow donors were plated in Petri dishes at 37°C in the presence of increasing concentrations of compound IV-2. At day 14, BFU-E was identified as a large aggregate of more than 64 hemoglobinized cells or as clusters of three or more subcolonies consisting of eight or more hemoglobinized cells/subcolony (16) . The CFU-GM was enumerated as a group of more than 50 granulocytic/monocytic translucent cells (16) .

Statistical Analysis of Data.
Comparisons among groups of data were made with Student’s t test or nonparametric ANOVA (17) . Correlation analysis was performed by linear regression and Pearson correlation with GraphPad InStat software (GraphPad Software Inc., San Diego, CA). A P of less than 0.05% was considered statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Thioredoxin Expression in Primary Leukemia from Patients with T-ALL.
The levels of thioredoxin protein in leukemic samples were measured in the Western blots in a semiquantitative manner. Using different amounts of recombinant thioredoxin, the sensitivity of this semiquantitative assay was in the range of 0.5 to 15 pmol (see Ref. 18 ).

In Fig. 1ACitation , typical results are presented according to the levels of thioredoxin expressed in these cells (Lanes 2–6). Expression of thioredoxin protein ranged from less than 1.5-fold to more than 9-fold greater than the expression in normal MNCs (Fig. 1A)Citation . The fold increase of thioredoxin expression was calculated individually relative to normal MNC samples from healthy donors (n = 5) and normalized against the amount of ß-actin in the same analysis.



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  		Fig. 1. Thioredoxin expression in samples obtained from 28 patients with T-ALL. A, representative samples of T-ALL obtained from patients (Lanes 2–6) were analyzed on Western blot as described in "Materials and Methods." Semiquantitative measurement of thioredoxin expression using specific antihuman thioredoxin antiserum was performed and compared with the protein lysate of the MNCs from normal persons (e.g., in Lane 1). Using recombinant thioredoxin in the gel, the sensitivity of this assay was determined to be from 0.5 to 15 pmol (18) . The same membranes were also incubated with anti-ß actin antibodies to ensure equal amounts of protein lysates used in each lane of the gel. In Lanes 2–6 of T-ALL samples, the amounts of thioredoxin expression correspond to <1.5-, 3.0-, 4.5-, 9.0-, and >9.0-fold increase, relative to control. B, protein extracts prepared from 28 leukemia samples obtained from patients and the same MNC fraction of blood from five normal donors were examined on Western blot with anti-thioredoxin and anti-ß actin antibody, as described in Fig. 1ACitation . The levels of expression of thioredoxin were normalized against the amount of ß-actin and then determined as fold increase for each of all of the leukemia samples, separately, as compared with normal MNC samples (for the distribution of the values of fold increase, see Fig. 4ACitation ). The values of fold increase for thioredoxin expression in patient samples could be separated into group A (<=3.0-fold increase; n = 11) and group B (>3.0-fold increase; n = 17). Kruskal-Wallis test revealed that variations of thioredoxin expression in these 28 samples combined are significantly different from normal controls (P < 0.0001). However, there are two subtypes of T-ALL samples: those with relatively low (group A) and those with relatively high (group B) fold increase in thioredoxin expression in the cells. Arrows refer to the values of the mean ± SE of the fold increase of thioredoxin in these two T-ALL subtypes. The nonparametric ANOVA analysis indicated that although those in group A are not statistically different from the controls (P > 0.05), those in group B (marked with ***) are high expressors for intracellular thioredoxin and have very significant P (< 0.001) when compared with the controls, as well as compared with group A.

 
A Kruskal-Wallis test of all of the data in Fig. 1BCitation indicated that the variations of thioredoxin expression in these samples and the normal values in healthy donors are extremely significant (P = <0.0001). The T-ALL samples could be separated into two major subtypes: group A with relatively low expressions of intracellular thioredoxin (<=3.0-fold increase) and group B with relatively high expressions of intracellular thioredoxin (>3-fold increase; Fig. 1BCitation ). The nonparametric ANOVA did not reveal significantly different levels of thioredoxin repression in group A as compared with that in the controls (P < 0.05). On the other hand, the expression of thioredoxin in group B is significantly higher than that in the controls and those in group A (both P < 0.001; Fig. 1BCitation ).

The expression of thioredoxin in T-ALL cells was also analyzed and compared with the level of thioredoxin expression in thymocytes from five nonleukemic patients. The conclusion about the enhanced expression of thioredoxin among leukemia cells relative to its expression in thymocytes was similar to those observed in Fig. 1BCitation using MNCs (data not shown).

Effect of Exogenous Thioredoxin on Leukemia Colony Formation.
Thioredoxin could be released from cells and function as an extracellular growth or autocrine factor for normal and tumor cells (10 , 19 , 20) . Thus, it would be of interest to examine whether exogenously added thioredoxin stimulates proliferation of T-ALL cells. In early experiments using [3H]thymidine incorporation assay, no difference in cell proliferation in 3-day cultures was observed between samples of T-ALL leukemia cells with and without the addition of exogenous thioredoxin, suggesting a lack of effect on the bulk of leukemia cells by thioredoxin (data not shown). Next, we considered that an in vitro leukemia colony-forming assay, where clonogenic leukemia cells can be analyzed, may offer a more appropriate system to evaluate the influence of thioredoxin on cell growth. The clonogenic cells of leukemia samples form colonies in response to growth factors such as IL-2 and PMA, which induces expression of IL-2 receptors (21) . Because thioredoxin is known to increase the expression of IL-2 receptor {alpha} chain (22) , we hypothesized that exogenous thioredoxin may be able to stimulate leukemia colony formation in the absence of PMA added to the cultures. As shown in Fig. 2Citation , there was a striking difference in the in vitro leukemia colony-forming assay according to the state of expression of intracellular thioredoxin in the cells. Notably, it was found that an enhancing effect of thioredoxin in these cultures for leukemia colony formation (244.2 ± 7.4%) was observed in the leukemia samples that had very low levels of intracellular thioredoxin expression (mean fold increase 1.8 ± 0.4 as compared with control). In contrast, no enhancement in leukemia colony formation (104.1 ± 3.3%) was observed in samples with higher thioredoxin expression (e.g., 7.3 ± 1.3-fold increase).



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  		Fig. 2. Selective enhancement of colony formation for T- ALL clonogenic cells by exogenous thioredoxin. Leukemia colony assay for 17 T-ALL samples was performed in methylcellulose culture containing approximately 5 x 105 MNCs, 100 units/ml IL-2 (PMA was omitted in the cultures), and in the presence or absence of 0.5 µg/ml recombinant human thioredoxin as described in "Materials and Methods." After 10 to 14 days of culture, T-ALL colonies with 50 or more cells were enumerated. To calculate the increase of colony formation, the numbers of T-ALL colonies in cultures with exogenous thioredoxin were compared with the numbers obtained from their respective controls without the addition of thioredoxin. The expression of intracellular thioredoxin in these primary leukemia samples was also quantitated as described in Fig. 1Citation . T-ALL samples were then separated into two subtypes, as in Fig. 1BCitation : low versus high levels of thioredoxin expression (<= or >3-fold increase, and a total of nine and eight patients, respectively). Specifically, the mean and SE of the fold increase in thioredoxin expression was 1.8 ± 0.4 (low) and 7.3 ± 1.3 (high) in these samples. Arrows refer to the percentage of increase of colony growth (mean ± SE) of the T-ALL clonogenic cells in these two groups after being cultured with exogenous thioredoxin. Kruskal-Wallis test indicated that these two groups had significantly different amount of increase in leukemia colony formation (P < 0.0001) with the addition of exogenous thioredoxin.

 
Effect of Inhibitor for Thioredoxin Expression on ALL Colony Formation.
Because thioredoxin is a critical regulator for the growth of some human cancers, Oblong et al. (23) and Powis et al. (24) examined the biological activity of asymmetrical disulfides in an attempt to determine their potential for redox regulation. A series of alkyl 2-imidazolyl disulfides inhibited thioredoxin-dependent cell proliferation (23) . It was also shown that incubation of thioredoxin with these disulfides results in an irreversible inhibition of the thioredoxin as a substrate for reduction by thioredoxin reductase (24) .

The effects of IV-2 on colony formation of primary T-ALL cells were determined using the in vitro leukemia colony assay as described (15) . Increasing concentrations of compound IV-2 from 0.01 to 30 µM were added to the 35-mm Petri dishes containing methylcellulose and other additives for leukemia colony-forming cultures. After 14 days, colonies were counted, and the IC50 was determined. On the basis of a study of 19 samples from patients, it was found that the leukemia colony formation of primary T-ALL samples is sensitive to inhibition by compound IV-2, with an average IC50 of 3.2 ± 1.3 µM (Fig. 3)Citation . The average IC50s of T-ALL clonogenic cells toward compound IV-2 were very significantly different from the IC50s of normal hematopoietic progenitor cells (e.g., CFU-GM and BFU-E) from healthy donors (9.4 ± 0.5 and 9.6 ± 0.5 µM, respectively, in Fig. 3Citation ; both P < 0.0001 in unpaired t test). Such differential sensitivity between leukemia cells and normal hematopoietic progenitors suggests the possibility of using disulfide compounds for the treatment of human T-cell leukemia.



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  		Fig. 3. Effect of compound IV-2 on colony formation of primary T-ALL cells obtained from 19 patients. A, leukemia colony assay for primary T-ALL obtained from 19 patients was performed with increasing concentrations of IV-2 in the culture mixture containing methylcellulose, IL-2, and PMA, as described in "Materials and Methods." Similarly, MNCs of bone marrow from 19 healthy donors were also analyzed at the same time for normal hematopoietic CFU-GM and BFU-E cells in culture with increasing amounts of IV-2 as described. The IC50 for IV-2 was determined as the concentrations at which there was 50% inhibition of colony formation for T-ALL and for CFU-GM and BFU-E colonies, respectively. Representative dose-response data from four T-ALL samples (•, {blacktriangleup}, {blacktriangledown}, and +) and from one CFU-GM ({circ}) and one BFU-E ({triangleup}) sample are reported as the means of triplicate cultures; bars, SD. B, the IC50s of IV-2 for T-ALL and for normal CFU-GM and BFU-E are shown. Arrows refer to the values of the mean and SE of the IC50. Unpaired t test indicated that the IC50 for T-ALL is significantly different from those for CFU-GM and BFU-E from normal marrow (both P < 0.0001).

 
Relationship between Increase of Thioredoxin Expression and White Cell Count.
Recent studies (25) to develop uniform risk criteria for children with ALL resulted in a consensus based mainly on age of patients and WBC count at diagnosis. As shown in Fig. 4ACitation , the fold increase of thioredoxin expression is statistically correlated with WBC count at diagnosis among these 28 patients. The correlation coefficient, r, in Pearson correlation analysis is 0.46 with 95% confidence interval shown in the figure; and the two-tailed P is 0.01, which is considered to be significantly correlated. When grouped as presented in Fig. 1BCitation , these data also showed the trend of association of higher thioredoxin expression with increasing number of white cells (Fig. 4B)Citation . An unpaired t test indicated that variations in white blood counts among these groups are significant (P = 0.03).



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  		Fig. 4. The relationship between increase of thioredoxin expression in T-ALL cells and number of peripheral blood white cells. A, the expression of thioredoxin among 28 patients presented in Fig. 1BCitation was correlated with WBC counts at the time of diagnosis. Pearson correlation analysis assuming Gaussian distribution revealed correlation coefficient, r, as 0.46 and the two-tailed P is 0.01. , linear regression; ----, 95% confidence intervals. B, the data of thioredoxin expression of the 28 T-ALL samples were grouped as A and B, as in Fig. 1BCitation , and correlated with WBC counts of these patients at diagnosis. Numbers in the figure refer to the means ± SE of WBC counts within each group. Unpaired t test indicated that variations in WBC counts among these groups are statistically significant (P = 0.03).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our studies have shown that thioredoxin expression is increased in leukemic cells from most children with newly diagnosed T-ALL. Human thioredoxin was cloned as an adult T-cell leukemia-derived factor, produced by HTLV-I (26, 27, 28) . This factor was also shown to induce the expression of high-affinity IL-2 receptor {alpha} chain and Tac antigen (22) . Unlike adult T-cell leukemia, which is caused by HTLV-I, childhood T-ALL has no known viral etiology. However, it is interesting to observe that childhood T-ALL cells expressed high levels of thioredoxin. Levels of thioredoxin protein or mRNA were also elevated in primary tumors or cell lines from patients with breast, cervical squamous cell carcinoma, hepatoma, lung, and colon tumors (11 , 12 , 18 , 29) .

Thioredoxin, as part of a general protein disulfide-reducing system, plays an important role in maintaining the redox environment in the cell (30 , 31) . It is induced by oxidative stresses, serving as an endogenous antioxidant and providing cytoprotective activity. Thioredoxin is a multifunctional protein. It serves as a dithiol hydrogen donor for many target proteins, including ribonucleotide reductase, which is important for cell proliferation. Thioredoxin increases DNA-binding of redox-sensitive transcription factors such as nuclear factor-{kappa}B and thereby regulates gene transcription (4, 5, 6, 7) . In addition, thioredoxin stimulates the redox factor 1 activity (8 , 9) , which acts as a transcription activator for p53 (32) . Moreover, cotransfection with thioredoxin and p53 enhances induction of p21 protein (33) . It is thus far, however, unclear whether endogenous thioredoxin indeed mediates these cell-cycle regulatory events in the cells. It was speculated that the increased expression of thioredoxin in tumor cells might reflect a compensatory mechanism for p53 dysfunction, as is common in most tumors (33) . However, we have shown previously (34) that p53 mutation is rare in T-ALL at diagnosis.

On the other hand, many studies (35) have reported that overexpression of thioredoxin in cells suppresses apoptosis. Gallegos et al. (36) have shown that breast cancer cells stably transfected with thioredoxin show increased anchorage-independent growth, whereas a redox-inactive mutant of thioredoxin acts in a dominant-negative manner to inhibit proliferation and xenograft tumor formation in mice. It was also found that thioredoxin expression is decreased during dexamethasone-induced apoptosis of thymoma cells (35) . Furthermore, the increased levels of thioredoxin were shown to be associated with resistance to cisplatin, mitomycin C, doxorubicin, and etoposide (13 , 14) . Together with the findings of high levels of thioredoxin expression in tumors, these studies suggest that increased thioredoxin expression may lead to an increased tumor growth through inhibition of spontaneous apoptosis and a decrease in the sensitivity of cancer cells to drug-induced apoptosis. Intriguingly, a recent study (37) showed that caspase-3 activity in cell lysate was suppressed by a thiol-oxidant, diamide, but restored by thio-reducing agents including thioredoxin. This observation seems to be at odds with the fact that thioredoxin protects cells against apoptosis and the increase in caspase activity is responsible for the execution of apoptosis. To solve this dilemma, Baker et al. (38) suggested that thioredoxin might reduce thiol groups on other proteins in the cells that negatively regulate caspase activity indirectly. They showed that the in vitro activation of caspase-3 is a general feature of many reduced proteins and not a specific action of thioredoxin (38) .

Age and WBC are important prognostic factors in children with ALL (25) . Recent studies (39) , however, indicate that these prognostic factors are significantly less important in T-ALL than in B-precursor ALL. Knowledge of the molecular abnormalities in leukemic cells may provide better keys to understanding the treatment successes and failures in childhood leukemia. Because increased expression of thioredoxin in transfected cells is related to resistance to chemotherapy, it is possible that overexpression of thioredoxin may be associated with poor clinical outcome. However, whether thioredoxin is an independent prognostic factor awaits further study in a larger group of T-ALL patients for a clinical correlative analysis.

Thioredoxin is secreted by cells through a unique "leaderless" pathway at a high rate (40 , 41) . Exogenously added thioredoxin was also reported to stimulate cell proliferation by enhancing the sensitivity of the cells to cytokines (42) . Thioredoxin induces expression of high affinity IL-2 receptor (22) and has comitogenicity with IL-1, IL-2, and IL-4 (19 , 27 , 43) . In the present study, we found that the addition of exogenous thioredoxin stimulates the proliferation of ALL clonogenic cells in the absence of PMA, which has generally been considered a required component of the culture media, possibly because of its ability to induce the expression of IL-2 receptor in the leukemia cells.

The enhancing effect of exogenous thioredoxin was observed in the group of T-ALL samples that had a relatively low level of thioredoxin expression but not in the cells expressing high levels of intracellular thioredoxin. Conceivably, thioredoxin is secreted from high expressers of thioredoxin in the cells and stimulates the clonogenic progenitors. This agrees with the observation that thioredoxin stimulates cell proliferation of lymphocytes (including HTLV-I and EBV-transformed cells) and other nonlymphoid cells (11 , 19 , 41) . On the other hand, both normal liver cells and the hepatoma cell line, HepG2, synthesize thioredoxin; however, only the former secretes abundant thioredoxin extracellularly and recombinant thioredoxin was reported to inhibit the proliferation of HepG2 cells (41) . These results indicate that different cell types respond differently to variations in the intracellular redox potential.

We have also shown that except for clonogenic cells, the bulk of leukemia blasts are not activated by thioredoxin to undergo cellular proliferation and DNA synthesis, as measured by a thymidine incorporation assay. It is believed that these colony-forming cells from ALL, though comprising only 0.05 to 1.5% of the bulk blood blasts, represent the in vitro counterparts of the in vivo ALL blast progenitors (44) . High numbers of residual clonogenic ALL blasts in remission bone marrow of T-ALL patients usually reflects poor prognosis (45) . Therefore, the observed leukemia-enhancing activity by thioredoxin for these clonogenic leukemia cells may have clinical relevance.

Leukemia colony formations of primary T-ALL are sensitive to inhibition by compound IV-2, with an IC50 of 3.2 µM, substantially lower than the IC50s for normal hematopoietic progenitors such as CFU-GM and BFU-E. Such differential sensitivity suggests the possibility of using disulfide compounds for treatment of human leukemia. Because overexpression of thioredoxin has been found to be associated with resistance to many anticancer drugs, the inhibition of thioredoxin expression may overcome this drug resistance and may further sensitize leukemia cells to chemotherapeutic agents synergistically.

Characterization of the molecular changes in T-ALL will facilitate the development of a selective therapy that exploits specific biological properties and thereby improves the outlook for this disease. Our findings that thioredoxin was expressed in variable amounts in T-ALL with higher expression associated with higher WBC counts and that there was differential sensitivity toward the disulfide compound IV-2 between primary T-ALL and normal hematopoietic progenitors should facilitate a better understanding of the clinical significance of thioredoxin in this childhood malignancy and shed light on the potential targets for new treatment and assessment of clinical outcome.


   ACKNOWLEDGMENTS
 
We thank Dr. Garth Powis at University of Arizona (Tucson, AZ) for providing antibodies against human thioredoxin and compound IV-2.


   FOOTNOTES
 
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.

1 Supported by NIH Grant CA79951 (to J. Y.), LSA6125 (to A. L. Y.), and the Cindy Matters Fund. Back

2 To whom requests for reprints should be addressed, at Department of Molecular and Experimental Medicine, MEM265, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Phone: (858) 784-7924; Fax: (858) 784-7977; E-mail: johnyu{at}scripps.edu Back

3 The abbreviations used are: ALL, acute lymphoblastic leukemia; BFU-E, erythroid burst-forming unit; CFU-GM, granulocyte/monocyte colony-forming unit; MNC, mononuclear cell; PMA, phorbol 12-myristate 13-acetate; redox, reduction/oxidation; T-ALL, T-cell ALL; IV-2, 1-methylpropyl 2-imidazolyl disulfide; IL, interleukin; HTLV-I, human T-cell leukemia virus type-I. Back

Received 4/17/01. Accepted 7/26/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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