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Clinical Cancer Research Vol. 10, 508-520, January 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

CD26 Expression Correlates with a Reduced Sensitivity to 2'-Deoxycoformycin-Induced Growth Inhibition and Apoptosis in T-Cell Leukemia/Lymphomas

Donatella Aldinucci, Dalisa Poletto, Debora Lorenzon, Paola Nanni, Massimo Degan, Karin Olivo, Barbara Rapanà, Antonio Pinto and Valter Gattei

Clinical and Experimental Hematology Research Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, Aviano, Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose and Experimental Design: dCF (2'-deoxycoformycin) is a potent inhibitor of ADA (adenosine deaminase), an enzyme regulating intra- and extracellular concentrations of purine metabolites. ADA exists as cytosolic and extracellular forms, the latter colocalized on the cell surface with CD26. Once the surface expression of CD26 and ADA in a panel of cell lines and primary samples of T-cell leukemia/lymphoma was defined, we correlated this expression with the antiproliferative and apoptotic effect of dCF.

Results: Surface expression of CD26 inversely correlated with the capability of dCF to inhibit cell growth and induce apoptosis both in T-cell lines and primary samples of T-cell malignancies. This conclusion was sustained by a decreased sensitivity to dCF-mediated proapoptotic and/or antiproliferative in vitro effects of: (a) leukemia/lymphoma T-cell lines expressing surface CD26/ADA complex; (b) primary CD26+ T cell malignancies; and (c) normal T cells (CD26+) as compared with tumor T cells (CD26-) in unpurified samples from three cases of T-cell receptor {gamma}{delta}+ T-cell malignancies characterized by a mixture of normal and neoplastic cells. This latter point was confirmed in vivo, in a patient affected by CD26- T-cell receptor {gamma}{delta}+ hepatosplenic {gamma}{delta}+ T-cell lymphomas treated on a compassionate basis with dCF. The inverse correlation between CD26 expression and sensitivity to dCF was also demonstrated in a lymphoblastic lymphoma case in which CD26 was expressed on circulating blasts at relapse but not at diagnosis, as well as in two H9 T-cell clones expressing or not expressing CD26 mRNA and protein.

Conclusions: This study corroborates the notion of CD26 as a marker of poor prognosis for T-cell malignancies and delineates a role for CD26 as a predictor of poor response to dCF.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
dCF (2'-deoxycoformycin; Pentostatin) is a chemotherapeutic agent with activity of ADA (adenosine deaminase) inhibitor that, for the peculiar sensitivity of T cells to ADA inhibitors (1) , displays a striking cytotoxic activity against different types of T-cell malignancies. These include peripheral and cutaneous T-cell lymphomas, T-prolymphocytic leukemia, large granular lymphocyte leukemia, and, recently, HSL (hepatosplenic {gamma}{delta}+ T-cell lymphoma; Refs. 1, 2, 3) .

ADA, an enzyme involved in purine metabolism, catalyzes the hydrolytic deamination of Ado (adenosine) and dAdo (2'-deoxyadenosine) to their corresponding hypoxanthine derivatives, inosine and 2'-deoxy-inosine (4 , 5) . Therefore, drug-mediated inhibition of ADA leads to the accumulation of dAdo and its triphosphate form, dATP, in cell cytoplasms (6 , 7) . These metabolites are able to determine a decrease of DNA synthesis with a subsequent impairment of cell proliferation (6 , 7) . In the case of dATP, this happens through a direct block of the activity of ribonucleotide reductase, the enzyme catalyzing the synthesis of DNA precursor deoxynucleotides (8) . Moreover, increased levels of Ado and dAdo can lead to an unbalanced ratio between S-adenosylmethionine and S-adenosylhomocysteine in favor of this latter molecule, thus impairing the synthesis of methylated nucleosides (6, 7, 8) . Induction of DNA strand breaks, alterations in DNA repair processes, inhibition of RNA transcription, and triggering of apoptotic death have also been shown to occur in different target cells exposed to the drug (9, 10, 11, 12, 13) .

In addition to cell cytosols (5) , ADA has been found in membrane fractions physically associated with the T-cell activation antigen CD26, a Mr 105,000–110,000 cell surface glycoprotein with diverse functional properties (4 , 14) . CD26 corresponds to the surface DPPIV (dypeptidyl peptidase IV; Ref. 14 ), and can act as a binding structure for extracellular matrix components (15) and coreceptor for HIV (16 , 17) . CD26 is primarily expressed by CD3+ medullary thymocytes (18) and by different subsets of resting or activated peripheral T cells (19 , 20) .

It has been demonstrated that CD26, as expressed by neoplastic cells in a fraction of T-cell neoplasms (21 , 22) , is associated with lower survivals and significantly shorter duration of complete remissions, as compared with patients with CD26- diseases receiving similar chemotherapeutic regimens (22 , 23) .

In the present study, by investigating the sensitivity of a panel of T-cell leukemia/lymphoma cell lines and primary samples of T-cell malignancies to dCF, we demonstrated that the expression of CD26 by tumor cells correlated with a reduced dCF-mediated growth inhibition and apoptosis. These data corroborate the notion of CD26 as a marker of poor prognosis for T-cell malignancies and delineate a role for CD26 as a predictor of response to dCF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drugs.
dCF (Nipent; Wyeth Lederle, Aprilia, Italy), dAdo (Sigma Aldrich, Milan, Italy), and the inhibitor of DPPIV enzymatic activity, Propidine (gift of Ingrid De Meester, Wilrijk, Belgium), were dissolved in IMDM (Biochrome KG, Berlin, Germany) and filter sterilized (0.2 µm) immediately before use.

Cell Lines and in Vitro Activation of Normal T Cells.
The human cutaneous lymphoma T-cell lines HUT 78 (Sezary syndrome), H9 (HUT78 subclone; Ref. 24 ), and HUT102 (mycosis fungoides); the CD30+ anaplastic large-cell lymphoma cell line with T-cell phenotype Karpas 299; and the T-LBL (lymphoblastic lymphoma) cell lines KE37, FRO, CEM (common thymocyte phenotype), and Jurkat (post-thymic phenotype) were maintained in IMDM (Biochrome KG, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Biochrome KG), 0.2 mg/ml penicillin/streptomycin, and 0.1% (w/v) L-glutamine (Biochrome KG) at 37°C in a 5% CO2 fully humidified atmosphere. Sources and phenotypic characterization of all of the above cell lines have been reported in detail elsewhere (22 , 25) .

As positive controls for CD26 expression, normal peripheral blood (PB) mononuclear cells obtained by healthy donors were activated in vitro by exposure to 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) and ionomycin (1 µg/ml) as described (22) .

Isolation and Characterization of CD26+ and CD26- H9 T-Cell Clones.
With the aim to isolate subclones of the same T-cell line characterized by different levels of CD26 expression, an approach of limiting dilution cloning was performed. In particular, H9 T cells were plated (100 µl/well) at very low density (3.33 cells/ml) in a U-bottomed 96-well tissue culture plate in IMDM supplemented with 10% FCS. After 2 weeks, cells from positive wells were collected, expanded in vitro, and extensively characterized for CD26 and T-cell antigen expression in flow cytometry and reverse-transcription PCR (see below). These procedures allowed the identification of two H9 T cell clones, named CD26+H9 and CD26-H9, which were used for additional experiments.

Fresh Leukemic Cell Isolation and Purification.
The study included cellular samples obtained from PB or bone marrow samples of 6 patients affected by T-LBL, 2 patients with peripheral T cell lymphoma (PTCL), and 3 with HSL. Diagnoses were based on correlative analysis of the morphological, immunophenotypic, and, when necessary, genotypic characteristics of neoplastic cells, as reported previously (26, 27, 28, 29) . Mononuclear cells were isolated by centrifugation on a Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient, washed twice with HBSS, and used for immunophenotyping and tissue culture studies.

For experiments involving [3H]thymidine incorporation and clonogenic assays, tumor T-cells were additionally purified by removing the normal T-cell fraction, essentially as described previously (2) . In particular, according to the immunophenotyping profile of the neoplastic component, specific strategies of positive or negative selections with monoclonal antibody (mAb) -conjugated immunomagnetic beads (Myltenyi Biotec) were chosen. In the case of T-LBL, neoplastic cells were purified by an indirect positive selection with anti-CD1a mAbs used in conjunction with immunomagnetic bead-conjugated secondary mAbs or by a direct depletion of normal T cells by using immunomagnetic bead-conjugated anti-CD4 and anti-CD8 mAbs. HSL cells were purified exactly as described (2) , whereas in a case of T-cell receptor (TCR) {gamma}{delta}-expressing PTCL, neoplastic cells were purified with anti-TCR{gamma}{delta} mAbs used along with immunomagnetic bead-conjugated secondary mAbs. In all of the cases, the resulting cell preparations contained >95% of tumor T cells, as assessed by flow cytometry.

Flow Cytometry Analysis.
CD26 expression on leukemia/lymphoma T-cell lines, H9 T-cell clones, and primary fresh samples of T-cell neoplasms was evaluated in single or double fluorescence staining by using the FITC-conjugated anti-CD26 mAb L272 (Becton-Dickinson, San Jose, CA) or phycoerythrin (PE) -conjugated anti-CD26 mAb BA5 (Coulter Immunotech). ADA surface expression was evaluated by indirect immunofluorescence using a goat polyclonal antihuman ADA antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) used along with a PE-conjugated donkey antigoat IgG secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA), as described (30) . Other mAbs used in this study were CD1a-PE, CD54-PE, CD4-PE, CD28-PE, CD8-PE (Becton-Dickinson), CD29-PE (Coulter Immunotech) CD30-FITC, CD45R0-PE, and CD71-FITC (DAKO). In the case of TCR{gamma}{delta}-expressing neoplasms, primary samples from patients were stained with anti-CD26 mAbs used in conjunction with the FITC-conjugated anti-TCR{alpha}ß mAb WT31 or the anti-TCR{gamma}{delta} mAb 11F2 (Becton-Dickinson) to distinguish between normal, TCR{alpha}ß+, and neoplastic, TCR{gamma}{delta}+, T-cell components. Nonspecific isotype-matched immunoglobulins (Becton-Dickinson) were used as controls. Viable, antibody-labeled cells were identified according to their forward and right angle scattering, electronically gated, and analyzed on a FACScan flow cytometer (Becton-Dickinson) by the CellQuest software (Becton-Dickinson).

RNA Isolation and Reverse Transcription-PCR.
Total RNA (1 µg) extracted by the guanidinium thiocyanate method (31) was reverse transcribed and amplified with 25 pmol of specific primers for CD26 (sense, 5'-CGGTCCTGGTCTGCCCCTCTA-3', region 1429–1449; antisense, 5'-CGCCACGGCTATTCCACACTT-3', region 1972–1952). The conditions for PCR reactions were 4 min at 94°C, followed by 30 cycles of 30 s at 62°C, 1 min at 72°C, 30 s at 94°C, and a final extension of 10 min at 72°C. Ten µl of amplified cDNAs were run in a 1.5% agarose gels and stained with ethidium bromide. All of the cDNAs were checked for first-strand synthesis, exactly as reported (32) .

Western Blot Analysis.
Cell lysates from leukemia/lymphoma T-cell lines were prepared as described (33) . Equal amounts of protein (50 µg) were subjected to10% SDS-PAGE. Proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), and residual binding sites were blocked by incubation for 2 h in 0.5% casein dissolved in PBS-0.1% Tween 20. Membranes were then incubated for 2 h with 1.5 µg/ml of goat polyclonal anti-human ADA (Santa Cruz Biotechnology), and specific binding was revealed by horseradish peroxidase-conjugated antigoat IgG (Santa Cruz Biotechnology) by using an enhanced chemiluminescence system (ECL-Plus; Amersham).

Colony Assay and [3H]Thymidine Incorporation.
Clonogenic growth of leukemia/lymphoma T-cell lines, H9 T-cell clones, and primary leukemic T cells was assayed as described previously (2 , 33) . Briefly, 2.0 x 105/ml purified tumor T cells or 5.0 x 103/ml T-cell lines, were suspended in 1 ml of IMDM containing 0.8% methyl-cellulose and 15% FCS in the presence of a combination of dCF (1–100 µM) plus dAdo (10 µM) and seeded in 100 µl aliquots (8 replicates) in 96-well flat-bottomed microplates. After 14 days of incubation, plates were observed under phase-contrast microscopy, and aggregates with >40 cells were scored as colonies.

In a case of TCR{gamma}{delta}-expressing T-LBL, the neoplastic components were cultured in the presence of propidine, an inhibitor of DPPIV enzymatic activity (34) , and the effects of this drug evaluated by a standard [3H]thymidine incorporation assay. Briefly, 1 x 106/ml tumor cells were plated in triplicate into 96-well U-bottomed microplates in IMDM plus 10% FCS, in the presence of different concentrations (10–100 µM) of propidine (34) . After 72 h, cells were pulsed with 1 µCi/well [3H]thymidine (specific activity 25 Ci/mmol; Amersham Life Science, Amersham, United Kingdom) for the final 12 h of culture, harvested onto glass fiber membranes, and counted in a liquid scintillator ß-counter (Tri-Carb 1600TR; Camberra-Packard, Meridien, CT). Results are expressed as mean cpm + SD [3H] thymidine incorporation of triplicate cultures.

Cell Cultures.
Mononuclear cells (1.0 x 106/ml) from PB samples of patients affected by TCR{gamma}{delta}+ T-cell malignancies or 2.0 x 105/ml cells from leukemia/lymphoma T-cell lines and H9 T-cell clones were incubated in medium alone, in the presence of dCF (25 µM), dAdo (25 µM), and a combination of dCF and dAdo. After 72 h of culture, cells were harvested, assessed for viability by the trypan blue-exclusion test, and, in the case of primary tumor samples, double stained with a combination of anti-CD26-PE, anti-TCR{alpha}ß-FITC, and anti-TCR{gamma}{delta}-FITC mAbs.

Measurement of in Vitro Apoptosis.
Two x 105/ml cells from leukemia/lymphoma T-cell lines and H9 T-cell clones, in exponential growth phase, were incubated in medium alone, and in the presence of dCF (25 µM), dAdo (25 µM), and a combination of dCF plus dAdo. After 72 h, DNA fragmentation was determined by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated nick end labeling. For DNA fragmentation, cells were collected, washed twice with PBS, and resuspended in PBS containing 5 µg/ml propidium iodide, 0.1% sodium citrate, 0.2% NP40, 5 mM EDTA, and 0.1% RNase for 30 min at room temperature. Samples were then analyzed by flow cytometer by gating out cell debris and fixation artifacts, and scoring the number of apoptotic cells as the percentage of events falling in an area immediately preceding the G0-G1 peak of DNA content histograms. In parallel experiments, DNA fragments present in apoptotic cells were labeled with FITC-dUTP by using the MEBSTAIN apoptosis kit Direct (Coulter Immunotech) according to the manufacturer’s instructions and subsequently analyzed by flow cytometry.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CD26 and ADA in Leukemia/Lymphoma T-Cell Lines.
Four of 8 leukemia/lymphoma T-cell lines (Karpas 299, H9, HUT78, and HUT102) expressed CD26 surface protein in flow cytometry (Fig. 1ACitation , left panels). The highest levels of CD26 expression were detected in the Karpas299 and H9 cell lines. Surface expression of ADA correlated directly with the presence of CD26 (Fig. 1ACitation , right panels). Consistently, in Karpas299 and H9 cells, dual color immunofluorescence experiments revealed the coexpression of ADA and CD26 on the surface of the same cell populations (Fig. 1B)Citation . When CD26 expression was assessed at mRNA level by reverse transcription-PCR, results were consistent with flow cytometry data, and high amounts of the expected 544-bp amplified fragment were detected in cDNAs from Karpas299, H9, HUT78, and HUT102 T cell lines. A weak specific band was also found in two additional cell lines (KE37 and Jurkat) but not in CEM and FRO cells, which were CD26- in flow cytometry (Fig. 1C)Citation . Conversely, by investigating in whole cell lysates the total pool of ADA protein by Western blotting, the enzyme was found to be expressed, as a single component of Mr ~41,000, in all of the leukemia/lymphoma T-cell lines analyzed (Fig. 1D)Citation .



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  		Fig. 1. Expression of CD26 and adenosine deaminase (ADA) in human leukemia/lymphoma T cell lines. A, flow cytometric detection of surface CD26 and ADA proteins. Cells were stained with phycoerythrin-conjugated anti-CD26 mAb BA5 (left panels, bold lines) and goat polyclonal antihuman ADA antibodies used along with phycoerythrin-conjugated donkey antigoat IgG secondary antibodies (right panels, bold lines). Thin lines indicate background fluorescence, as determined by isotype-matched control immunoglobulins. The X- and Y-axes indicate the logarithm of the relative intensity of red fluorescence and relative cell number, respectively. B, simultaneous detection of surface CD26 and ADA proteins by flow cytometry. Karpas299 and H9 cell lines were double-stained with FITC-conjugated anti-CD26 mAb L272 and goat polyclonal antihuman ADA antibodies used as above. C, detection of CD26 mRNA by reverse transcription-PCR. cDNAs from leukemia/lymphoma T-cell lines were amplified with primer pairs specific for CD26. Peripheral blood T-cells activated with 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) and ionomycin (1 µg/ml) were used as positive control (+). Amplifications carried out in the absence of cDNA were used as negative controls (-). Amplified products were visualized on ethidium bromide-stained agarose gels. D, detection of ADA protein in whole lysates. Cell lysates (50 µg/lane) from T-cell lines were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Blots were then incubated with the goat polyclonal antihuman ADA and shown by chemiluminescence.

 
Taken together, 4 of 8 leukemia/lymphoma T-cell lines (Karpas299, H9, HUT78 and HUT102) expressed detectable amounts of surface CD26/ADA complex, whereas the levels of ADA protein in cell lysates were comparable in all of the cell lines.

Effects of dCF on Cell Growth of T-Cell Lines Expressing Different Levels of the CD26/ADA Complex.
Once we defined the constitutive expression of the complex CD26/ADA in T-cell lines, we investigated the effects of dCF on cell proliferation. In agreement with previous results (35) , exposure of T-cell lines to increasing concentrations of dCF, if used alone, did not significantly impair the clonogenic growth of any of the cell lines tested, even at concentrations of dCF as high as 100 µM (data not shown). Therefore, in vitro experiments were always carried out in the presence of the ADA substrate dAdo (35) , used at a concentration of 10 µM (clonogenic growth) or 25 µM (liquid cultures), which did not directly impair cell survival in our cellular systems (data not shown).

As shown in Fig. 2ACitation , exposure of T-cell lines to increasing concentrations of dCF (1–100 µM) in combination with low doses of dAdo resulted in a strong dose-dependent inhibition of clonogenic growth in T-cell lines lacking surface CD26/ADA complex expression. The doses of dCF required to reduce survival to 50% of control (D50) were 0.5 µM for KE-37 and FRO cell lines, 1.0 µM for the Jurkat cell line, and 11 µM for the CEM cell line. Conversely, the Karpas 299, H9, HUT78, and HUT102 cell lines, all expressing surface CD26/ADA complex, were either resistant or less sensitive to the effects of dCF (D50 values >100 µM in all cases; Fig. 2ACitation ). Similar results were obtained when the cells were exposed in liquid culture to a single concentration (25 µM) of dCF in the presence of 25 µM dAdo. As summarized in Fig. 2BCitation , drug treatment of Karpass299, H9, HUT78, and HUT102 cell lines, expressing the CD26/ADA complex, yielded only a slight decrease of viable cells (ranging from 93% to 72% of control). Conversely, exposure of the CD26- T-cell lines (FRO, Jurkat, CEM, and KE-37) to dCF at the same doses reduced significantly the number of viable cells recovered after 72 h of liquid cultures (values ranging from 24% to 2% of control).



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  		Fig. 2. Growth inhibition of human leukemia/lymphoma. T-cell lines by 2'-deoxycoformycin (dCF) plus 2'-deoxyadenosine (dAdo). A, cells were cultured in semisolid medium in the presence of increasing concentrations of dCF (1–100 µM) plus dAdo (10 µM). After 7 days of incubation, aggregates with >=40 cells were scored as colonies. Results represent the mean of eight replicate wells from three different experiments; bars, ± SE. The data are presented as percentage of growth relative to colony formation obtained in the presence of 10 µM dAdo alone. The negative exponential dose-response survival curves are drawn by linear regression and may be described by a single slope parameter (D50 value). B, cells were cultured in medium alone, 25 µM dAdo alone, and in the presence of dCF (25 µM) plus dAdo (25 µM). After 72 h, cells were counted and cell viability assessed by trypan blue exclusion. Data are reported as percentage of control culture carried out in the presence of dAdo alone (25 µM).

 
Effects of dCF on Apoptosis of T-Cell Lines Expressing Different Levels of the CD26/ADA Complex.
To ascertain whether expression of CD26/ADA complex was also able to influence the sensitivity to dCF-induced apoptosis, we evaluated by propidium iodide and terminal deoxynucleotidyl transferase-mediated nick end labeling methods the DNA fragmentation of the various T-cell lines upon exposure to dCF, dAdo, and their combinations. As summarized in Fig. 3ACitation , incubation with dCF plus dAdo of T-cell lines lacking surface CD26/ADA complex was associated with an increase of the percentage of apoptotic nuclei, identified in flow cytometry as a subdiploid peak in DNA content histograms. In particular, values of apoptotic cells detected in the sub-G1 areas ranged from 42% (CEM) up to 68% (Jurkat; Fig. 3ACitation ). Conversely, dCF/dAdo exposure substantially did not change (Karpass299) or only slightly affected (H9, HUT-78, and HUT-102) the levels of sub-G1 cells in T-cell lines expressing surface CD26/ADA complex.



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  		Fig. 3. Induction of apoptosis of human leukemia/lymphoma T-cell lines by 2'-deoxycoformycin (dCF) plus 2'-deoxyadenosine (dAdo). T-cell lines were cultured in medium alone and in the presence of dCF (25 µM), dAdo (25 µM), and a combination of dCF plus dAdo. A, after 72 h cells were stained with propidium iodide and DNA content analyzed by flow cytometry. Cells with less than diploid DNA content were considered as apoptotic cells. Percentages of apoptotic cells are indicated in histograms. B, after 72 h cells were fixed, permeabilized, and DNA fragments revealed by staining with FITC-conjugated dUTP. Thin lines indicate background fluorescence, as determined by incubation without terminal deoxynucleotidyltransferase enzyme. The X- and Y-axes indicate the logarithm of the relative intensity of green fluorescence and relative cell number, respectively. Percentages of dUTP positive cells are indicated in the histogram.

 
Terminal deoxynucleotidyl transferase-mediated nick end labeling assay, based on the labeling of DNA strand breaks generated during apoptosis, confirmed that treatment with dCF and dAdo was able to induce apoptosis mainly in those T-cell lines that did not express the CD26/ADA complex at the cell surface. As shown in Fig. 3BCitation , upon dCF/dAdo exposure, the FRO, Jurkat, CEM, and KE-37 cell lines expressed levels of FITC-dUTP ranging from 34% to 97% of positive cells, whereas in all of the cell lines expressing surface CD26/ADA complex, the percentage of stained cells did not exceed the value of 15% (HUT102).

Therefore, both the cytostatic/cytotoxic and apoptotic effects of dCF in T-cell lines inversely correlated with the expression of surface CD26/ADA complex.

Surface Expression of CD26, and Effects of dCF and Propidine on the Clonogenic Growth of Primary T-Cell Malignancies.
Primary PB or bone marrow samples from 11 T-cell malignancies, including T-LBL, PTCL, and HSL expressing a {alpha}ß (5 cases), {gamma}{delta} (5 cases), or null (1 case) TCR configuration (Table 1)Citation , were analyzed for CD26 expression. High amounts of surface CD26 protein were found in 2 cases at diagnosis, namely a T-LBL (patient # 2) and a TCR{gamma}{delta}+ PTCL (patient # 8; Table 1Citation ). Moreover, in a case of T-LBL (patient # 6), CD26 expression, absent at diagnosis (1% CD26+ cells), was strongly expressed at relapse (90% CD26+ cells; Table 1Citation ).


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  		Table 1 T-Cell receptor status and CD26 expression and in vitro effects of 2'-deoxycoformycin (dCF) in primary T-cell malignancies

 
In agreement with data obtained on T-cell lines, exposure of CD26- purified primary tumor T cells to dCF (100 µM) plus dAdo (10 µM) resulted in inhibition of clonogenic growth ranging from 78% to 95% of controls (Table 1)Citation . Significantly lower values were observed when the same experiments were performed with purified CD26+ malignant T cells (growth inhibition of 23% or 45% of controls). By taking advantage of a patient (#6 in Table 1Citation ), in which a shift in CD26 expression by circulating T-cell blasts was observed between diagnosis (CD26-) and relapse (CD26+), we tested, by colony assay and [3H]thymidine incorporation, the differential sensitivity of PB tumor cells to dCF and the inhibitor of CD26/DPPIV activity propidine (34) . As summarized in Fig. 4ACitation , treatment of tumor cells collected at diagnosis (CD26-) with dCF yielded a strong dose-dependent inhibition of clonogenic growth (90% of controls at the highest dCF concentration), whereas propidine was completely unable to affect tumor growth by [3H]thymidine incorporation. An opposite behavior was observed by testing the sensitivity of tumor cells obtained at relapse (CD26+). As shown in Fig. 4BCitation , propidine was able to cause a dose-dependent inhibition of [3H]thymidine uptake up to 97% of controls at the highest dose (100 µM). On the other hand, an inhibition of cell growth not exceeding the 35% of controls was obtained by treating CD26+ tumor T cells with dCF (Fig. 4B)Citation .



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  		Fig. 4. Growth inhibition of primary leukemia/lymphoma tumor T cells by 2'-deoxycoformycin (dCF) and propidine. Tumor T cells (2.0 x 105/ml) of patient #6 at diagnosis (CD26-; A) and at relapse (CD26+; B), were cultured in semisolid medium, in culture medium (10 µM), 2'-deoxyadenosine (dAdo) alone, and in the presence of a combination of dCF (1–100 µM) plus dAdo (10 µM; left histograms). After 14 days of incubation, culture dishes were observed under phase contrast microscopy, and aggregates with >40 cells were scored as colonies. Results, expressed as % controls carried out with dAdo alone, represent the mean of eight replicate wells; bars, + SE. Each experimental point obtained by culturing tumor cells with dCF plus dAdo, reached a statistical significance (Student’s t test) when compared with its corresponding control. In addition, (1.0 x 106/ml) tumor cells were cultured in IMDM containing 10% FCS with increasing concentrations (10–100 µM) of propidine (right histograms), and biological responses were detected by [3H]thymidine incorporation. Results are expressed as c.p.m. of triplicate cultures; bars, ± SE. In all of the cases, a statistical significance (Student’s t test) was reached by comparing the experimental points obtained by culturing blast cells in the presence of propidine with their respective controls.

 
In Vitro Effects of dCF on Primary Tumor Cells from TCR{gamma}{delta}+ T-Cell Malignancies and Normal TCR{alpha}ß+ Residual T Cells.
The peculiar sensitivity to dCF of TCR{gamma}{delta}+ HSL has been demonstrated previously (2) . Expression of TCR{gamma}{delta} by malignant T cells allowed the quantitation of tumor cell by discriminating the residual TCR{alpha}ß+ normal T lymphocytes (2) . Following this reasoning, unpurified PB mononuclear cell samples from a case of CD26-/TCR{gamma}{delta}+T-LBL (patient #6), 2 cases of CD26-/TCR{gamma}{delta}+ HSL (patients #9 and 10), and a case of CD26+/TCR{gamma}{delta}+ PTCL (patient #8), all characterized by a mixture of normal and neoplastic cells, were cultured in vitro for 3 days with dCF and dAdo, and then analyzed by flow cytometry with a combination of anti-CD26 and anti-TCR{alpha}ß or anti-TCR{gamma}{delta} mAbs.

Results shown in Fig. 5, A and BCitation (top panels) demonstrated that dCF exposure dramatically reduced the number of CD26-/TCR{gamma}{delta}+ tumor T cells from patients #6, 9, and 10 by preserving the normal CD26+/TCR{alpha}ß+ residual T-cell component. On the other hand, tumor cells from patient #8 (CD26+/TCR{gamma}{delta}+) were almost completely resistant to the cytotoxic effects of dCF (Fig. 5, A and B)Citation . Of note, the residual normal T-cell fraction, mainly CD26- in this case, appeared to be significantly reduced, if not completely eliminated upon dCF exposure (Fig. 5, A and BCitation , bottom panels).



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  		Fig. 5. In vitro cytotoxic effects of 2'-deoxycoformycin (dCF) on primary tumor cells from T-cell receptor (TCR) {gamma}{delta}+ T-cell malignancies and normal TCR{alpha}ß+ residual T cells. Unpurified peripheral blood mononuclear cell samples from a case of CD26-/TCR{gamma}{delta}+ lymphoblastic lymphoma (patient #6), 2 cases of CD26-/TCR{gamma}{delta}+ HSL (patients #9 and 10), and a case of CD26+/TCR{gamma}{delta}+ PTCL (patient #8), were cultured for 3 days in medium alone, in the presence of dCF (25 µM), 2'-deoxyadenosine (dAdo; 25 µM), and a combination of dCF plus dAdo. Cells were then harvested, counted, double stained with anti-CD26-phycoerythrin and anti TCR{alpha}ß-FITC or TCR{gamma}{delta}-phycoerythrin monoclonal antibodies and then analyzed by flow cytometer by gating cells on the lymphocyte region of the forward and right-angle scatter plot. A, top panels indicate the relative proportion of viable CD26+/TCR{alpha}ß+ and CD26-/TCR{alpha}ß+ cells (residual normal T cells) in control (dAdo) and dCF-treated (dCF + dAdo) cultures; bottom panels indicate the relative proportion of viable CD26+/TCR{gamma}{delta}+ and CD26-/TCR{gamma}{delta}+ cells (tumor T cells) in control (dAdo) and dCF-treated (dCF + dAdo) cultures. B, representative flow cytometry dot plots of peripheral blood mononuclear cell samples after 3 days of culture in the presence of dAdo alone (dAdo) or dCF (dCF + dAdo); top panels refer to patient #9 (CD26-/TCR{gamma}{delta}+ hepatosplenic {gamma}{delta}+ T-cell lymphoma with a CD26+ component in residual TCR{alpha}ß+ T cells); bottom panels refer to patient #8 (CD26+/TCR{gamma}{delta}+ peripheral T cell lymphoma with a prevalent CD26- component in residual TCR{alpha}ß+ T cells); the relative percentages of CD26+/TCR{alpha}ß+ and CD26-/TCR{alpha}ß+ (residual normal T cells), as well as of CD26+/TCR{gamma}{delta}+ and CD26-/TCR{gamma}{delta}+ (tumor T cells) are indicated in the top right quadrant and in the bottom right quadrant of each dot plot, respectively.

 
In Vivo Effects of dCF in a Patient with CD26-/TCR{gamma}{delta}+ HSL.
We have described previously (2) the therapeutic effects of dCF in a case of TCR{gamma}{delta}+ HSL treated with i.v. dCF on a compassionate basis due to the failure of previous lines of therapy and the refusal of additional multiagent chemotherapy (2) . The neoplastic component of this patient (#9 of the present series) demonstrated an exquisite sensitivity to the cytotoxic effects of dCF, and the ratio between normal (TCR{alpha}ß+) and neoplastic (TCR{gamma}{delta}+) cells appeared completely inverted upon three courses of i.v. dCF administrated in bolus at 7-day intervals (2) . The differential sensitivity of the neoplastic and normal components to the effects of dCF might be explained on the basis of the specific expression of CD26 by residual normal T cells. In fact, as shown in Fig. 6Citation (left panels), ~50% of TCR{alpha}ß+ residual T cells expressed CD26 at a high cellular density. This proportion remained substantially unchanged in a PB sample collected after the third course of dCF therapy, but the percentage of normal T cells increased from 20% to 70% (Fig. 6Citation , top panels). Concurrently, the neoplastic component (CD26-/TCR{gamma}{delta}+) was reduced from 68% to 12% (Fig. 6Citation , bottom panels).



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  		Fig. 6. Relative proportion of tumor CD26-/T-cell receptor (TCR) {gamma}{delta}+ T cells and normal CD26+/TCR{alpha}ß+ residual T cells in a case of TCR{gamma}{delta}+ HSL treated in vivo with 2'-deoxycoformycin (dCF). Patient #9 (TCR{gamma}{delta}+ hepatosplenic {gamma}{delta}+ T-cell lymphoma) was treated with i.v. dCF on a compassionate basis due to the failure of previous lines of therapy and the refusal of additional multiagent chemotherapy (2) . Representative flow cytometry dot plots of peripheral blood mononuclear cell samples collected before therapy (left panels) and 1 week after the third course of i.v. dCF (4 mg/m2) are reported. Cells were double stained with anti-CD26-phycoerythrin and anti-TCR{alpha}ß-FITC or TCR{gamma}{delta}-FITC monoclonal antibodies and then analyzed by flow cytometer by gating cells on the lymphocyte region of the forward and right-angle scatter plot. The relative percentages of CD26+/TCR{alpha}ß+ and CD26-/TCR{alpha}ß+ (residual normal T cells) are indicated in the top right quadrant and in the bottom right quadrant, respectively, of dot plots reported in top panels; the relative percentages of CD26-/TCR{gamma}{delta}+ (tumor T cells) are indicated in the bottom right quadrant of dot plots in the bottom panels.

 
Effects of dCF on Cell Growth and Apoptosis of H9 T-Cell Clones Expressing Different Levels of CD26.
Taken together, our results suggest a correlation between CD26 expression and resistance to dCF in leukemia/lymphoma T-cell lines and primary tumors. The heterogeneity of these cellular models, by reflecting different intrinsic biological behaviors, may confound conclusions regarding the inverse relationship between CD26 and dCF responsiveness. Therefore, we have isolated and characterized two clones of the H9 T-cell line expressing (CD26+H9) or not (CD26-H9) CD26, and tested their sensitivity to the antiproliferative and proapoptotic effects of dCF (Fig. 7)Citation . The two clones, although displaying the same immunophenotypic profile (CD1a+, CD4+, CD8-, CD28+, CD29+, CD30+, CD45R0+, CD54+, and CD71+; data not shown), expressed significantly different levels of CD26 mRNA (Fig. 7A)Citation and protein (Fig. 7B)Citation by reverse transcription-PCR and flow cytometry, respectively.



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  		Fig. 7. Expression of CD26 and effects of 2'-deoxycoformycin (dCF) plus 2'-deoxyadenosine (dAdo) in CD26+ and CD26- H9 T cell clones. A, detection of CD26 mRNA by reverse transcription-PCR. cDNAs from both CD26+ and CD26-H9 T-cell clones were amplified with primer pairs specific for CD26. Peripheral blood T-cells activated with 12-O-tetradecanoylphorbol-13-acetate (10 ng/ml) and ionomycin (1 µg/ml) were used as positive control (+). Amplifications carried out in the absence of cDNA were used as negative controls (-). Amplified products were visualized on ethidium bromide-stained agarose gels. B, flow cytometric detection of surface CD26 proteins. H9 T-cell clones were stained with FITC-conjugated anti-CD26 monoclonal antibody L272. Thin lines indicate background fluorescence, as determined by isotype-matched control immunoglobulins. The X- and Y-axes indicate the logarithm of the relative intensity of green fluorescence and relative cell number, respectively. C, growth inhibition of H9 T-cell clones by dCF plus dAdo, as determined by colony assay (left panel) and viable cell counts in liquid culture (right panel); left panel, CD26+ and CD26- H9 T cell clones were cultured in semisolid medium in the presence of increasing concentrations of dCF (1–100 µM) plus dAdo (10 µM). After 7 days of incubation, aggregates with >=40 cells were scored as colonies. Results represent the mean of eight replicate wells from three different experiments; bars, ± SE. The data are presented as percentage growth relative to colony formation obtained in the presence of 10 µM dAdo alone; right panel, CD26+ and CD26- H9 T-cell clones (2.0 x 105 cells/ml) were cultured in 24-well tissue culture plates in the presence of increasing concentrations of dCF (1–100 µM) plus dAdo (25 µM). After 72 h viable cells were counted using the trypan blue dye-exclusion assay. Results represent the mean from three different experiments; bars, ± SE. The data are presented as percentage of viable cells obtained in the presence of 25 µM dAdo alone. D, induction of apoptosis of H9 T-cell clones by dCF plus dAdo. CD26+ and CD26- H9 T-cell clones were cultured in medium alone and in the presence of dCF (25 µM), dAdo (25 µM), and a combination of dCF plus dAdo. After 72 h cells were stained with propidium iodide and DNA content analyzed by flow cytometry. Cells with less than diploid DNA content were considered as apoptotic cells. Percentages of apoptotic cells are indicated in histograms.

 
Exposure to increasing concentrations of dCF (1–100 µM) in combination with low doses of dAdo resulted in a strong dose-dependent growth inhibition of CD26-H9 T cells, as determined by clonogenic growth in semisolid media and determination of viable cells in liquid cultures (Fig. 7C)Citation . Conversely, cells from the CD26+H9 T-cell clone turned out to be almost completely resistant to the effects of dCF, even at dCF doses as high as 100 µM (Fig. 7C)Citation . DNA fragmentation analysis by the propidium iodide method confirmed a sensitivity of CD26-H9 T cells to dCF-induced apoptosis. As shown in Fig. 7DCitation , incubation with dCF plus dAdo of CD26-H9 T cells was associated by an increased percentage of apoptotic nuclei (39% of sub-G1 cells), whereas dCF/dAdo exposure only slightly affected the levels of sub-G1 cells (11%) in CD26+H9 T cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In addition to several functions in T-cell physiology (14 , 20 , 36 , 37) , CD26 has been identified as the binding protein for ADA, an enzyme highly active in T cells (5) , that regulates the levels of Ado and dAdo by catalyzing their irreversible hydrolytic deamination (4 , 5 , 20 , 38 , 39) . Recent studies have emphasized the physiological role of surface ADA as a scavenger enzyme to avoid accumulation of catabolites, which may hamper proliferation of T cells (4, 5, 6, 7) . The notion that dCF is a potent inhibitor of ADA with specific antitumor activity against hematological malignancies of T-cell phenotype (1 , 3 , 40, 41, 42) , suggested the investigation of the relationship, in these diseases, among CD26, ADA expression, and sensitivity to the effects of dCF (42) . In the present study, we demonstrated that the expression of CD26 inversely correlated with the capability of dCF to inhibit cell growth and induce apoptosis in a series of T-cell lines and primary samples of T-cell malignancies.

This conclusion was sustained by a low sensitivity to dCF-mediated proapoptotic and/or antiproliferative in vitro effects of: (a) leukemia/lymphoma T-cell lines expressing surface CD26/ADA complexes; (b) primary CD26+ T-cell malignancies; and (c) normal T cells (CD26+) as compared with tumor T cells (CD26-) in unpurified samples from TCR{gamma}{delta}+ T-cell malignancies characterized by a mixture of normal and neoplastic cells. This latter point was also confirmed in vivo, in a patient affected by CD26- TCR{gamma}{delta}+ HSL treated on a compassionate basis with dCF (2) . Therefore, CD26 could be proposed as a predictor of resistance to dCF in T-cell tumors. However, the relative heterogeneity of the cellular models investigated suggested a caution in drawing this conclusion, because it cannot be excluded that a different intrinsic biological behavior of the various T-cell tumors may influence dCF responsiveness independently to CD26 expression. On the other hand, the close correlation between CD26 expression and dCF refractoriness was also demonstrated in a T-LBL case in which CD26 was expressed on circulating blasts at relapse but not at diagnosis, as well as in two H9 T-cell clones expressing or not expressing CD26 mRNA and protein. Additional experiments using specific CD26 transfectants and/or CD26 knock-out of original CD26+ tumor cells could be of help to definitely prove the association between CD26 and decreased sensitivity to dCF in T-cell neoplasms.

The biochemical mechanism(s) that may explain this rather unexpected inverse correlation between CD26 expression and dCF activity remain(s) to be elucidated. Nevertheless, some considerations can be done. As shown in the present and other studies, in vitro exposure to dCF alone is usually not sufficient to impair the growth or induce apoptosis of a given cell population (35) . Therefore, experiments aimed to test the inhibitory effect of dCF usually require the presence of the ADA substrates dAdo and/or Ado (35) used in our as well as in other experiments (2 , 35) , at fixed, nontoxic concentrations. This is in keeping with the main function of ADA to act as a detoxifying agent (4 , 5) . In other terms, the antimetabolic effect of dCF is indirect, being mainly due to the accumulation of catabolic products (dAdo and/or Ado), which, by blocking several metabolic pathways, eventually hamper the duplication of DNA (9, 10, 11, 12, 13) . According to this line of reasoning, a possible explanation, at least of the in vitro results obtained in the present study, could be the rapid inactivation of exogenously added dAdo by membrane-anchored ADA occurring in CD26+ but not in CD26- cell samples. These results are in agreement with the demonstration that the presence of membrane-anchored ADA determines resistance to toxic effects of dAdo in CD26-transfected T cells (4) .

Although such a mechanism may at least in part contribute to the explanation of our findings, other data provided in the present study argue against this as the unique explanation of the relative resistance of CD26+ T cells to dCF: (a) the demonstration of a sensitivity of CD26- cells to the toxic effects of dCF even in the presence of CD26+ cells; this was observed in experiments carried out with primary samples of T-cell malignancies characterized by a mixture of CD26+ and CD26- cell populations; and (b) the killing of CD26- HSL cells and the maintenance of the residual normal CD26+ T-cell population, in a patient in which a compassionate therapy with i.v. dCF was performed (2) . A putative explanation of these findings could be the unbalanced distribution of dCF between cell surface and cytosol (5) . In particular, at least in those cell systems characterized by the expression of high amounts of surface CD26/ADA complex, dCF could be retained at the cell surface bound to extracellular ADA, whereas cytosolic ADA might be relatively free to exert its detoxifying activity on exogenous catabolites (4 , 5) . The demonstration that the total level of ADA protein, as determined by Western blotting, was roughly similar in the various cell lines, without any correlation with the antiproliferative or proapoptotic effects of dCF is consistent with this hypothesis. Uptake experiments with radiolabeled dCF might specifically dissect this issue (13) .

Moreover, it cannot be excluded that binding of ADA to the extracellular domains of CD26 may result in some conformational changes of ADA itself that may render the enzyme more resistant to dCF-mediated inhibition. Alternatively, it could be the binding of dCF to extracellular ADA that may determine some yet unidentified changes in the conformation of the CD26/ADA complex and/or the disruption of the interaction between ADA and some putative Ado receptors expressed by lymphoid cells (5 , 35 , 43) . These events may result in the transmission of intracellular signals eventually leading to an increased synthesis of ADA itself. Specific studies aimed to investigate the modulation of ADA expression on dCF exposure are currently ongoing to explore such hypotheses.

Literature data from our and other groups indicates that CD26 is detectable in neoplastic cells of a fraction of T-cell neoplasms (21 , 22 , 42 , 44) . In particular, CD26 expression has been described to be restricted to those pathological entities characterized by an aggressive clinical behavior, such T-LBL and CD30+ anaplastic large-cell lymphoma (22) . Interestingly, within this group of diseases, expression of CD26 has been demonstrated to be statistically associated with a worse outcome in terms of survival, as well as with a significantly shorter duration of complete remission, as compared with patients with CD26- diseases receiving similar chemotherapeutic regimens (22) . In the present study, by demonstrating that the reduced response to dCF in T-cell malignancies correlates with the expression of surface CD26, we corroborate the notion of CD26 as a marker of poor prognosis for T-cell malignancies and delineate a role for CD26 as a predictor of poor response to dCF. Therapeutic strategies specifically targeting CD26 molecules may be more effective for those T-cell neoplasms expressing the surface CD26/ADA complex. In this sense, the findings reported here of a specific sensitivity of CD26+ primary T-LBL cells for the DPPIV-inhibitor propidine can be added to other observations indicating the capability of anti-CD26 antibodies to inhibit growth of CD26+ cell lines (45) and the enhanced sensitivity to doxorubicin of CD26-expressing transfectants (46) .


   FOOTNOTES
 
Grant Support: Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy and the Ministero della Sanità, Ricerca Finalizzata Istituto di Ricovero e Cura a Carattere Scientifico, Rome, Italy.

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.

Requests for reprints: Donatella Aldinucci, Clinical and Experimental Hematology Research Unit, Centro di Riferimento Oncologico, Istituto di Ricovero e Cura a Carattere Scientifico, via Pedemontana Occidentale 12, Aviano I-33081, Italy. Phone: 434-659413; Fax: 434-659409; E-mail: daldinucci{at}cro.it

Received 5/16/02; revised 9/17/03; accepted 9/18/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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