Expression of CD26 and Its Associated Dipeptidyl Peptidase IV Enzyme Activity Enhances Sensitivity to Doxorubicin-induced Cell Cycle Arrest at the G₂/M Checkpoint¹

CD26 is a M_r 110,000 surface glycoprotein with an array of diverse functional properties that is expressed on a number of tissues, including epithelial cells and selected leukocyte subsets (1, 2). The extracellular domain of CD26 encodes a membrane-associated ectopeptidase that possesses DPPIV³ activity in its extracellular domain and is able to cleave NH₂ terminal dipeptides from polypeptides with either l-proline or l-alanine at the penultimate position. Although a significant physiological substrate related to the immunological aspects of CD26/DPPIV has not yet been conclusively identified, recent work (3, 4) has shown that CD26 can cleave certain chemokines involved in T-cell and monocyte function. In addition, CD26 is capable of acting as an alternate pathway of T-cell activation under certain experimental conditions, most likely as a result of its physical and functional association with molecules involved in the pathways of T-cell signal transduction, including CD45, mannose 6-phosphate/insulin-like growth factor II receptor and ADA (5, 6, 7, 8, 9, 10, 11, 12, 13). By regulating surface expression of ADA, CD26 potentially plays a key role in immune system function by the catalytic removal of adenosine (14, 15, 16).

Besides its involvement in T-cell physiology, CD26 may also have a role in the development of certain tumors. CD26 expression was detected on differentiated thyroid carcinomas but was absent in almost all of the cases of benign thyroid diseases (17). Aberrant CD26 expression was also seen on hepatocellular carcinomas, with its expression in most cases examined deviating distinctly from its expression on normal hepatocytes (18). B-chronic lymphocytic leukemic cells have high levels of CD26 protein expression and mRNA transcripts (19), whereas certain aggressive T-cell malignancies such as T-cell lymphoblastic lymphomas/acute lymphoblastic leukemias and T-cell CD30+ anaplastic large cell lymphomas have higher CD26 surface expression as compared with more indolent T-cell diseases like mycosis fungoides (20, 21).

Doxorubicin is a widely used anthracycline antibiotic in cancer therapy with broad spectrum antineoplastic activity against solid tumors and hematological malignancies. Previous findings (22, 23) have shown that DNA damage mediated by doxorubicin results in cell cycle arrest at the G₂-M phase by inhibiting p34^cdc2 kinase dephosphorylation and inducing cyclin B1 accumulation. Previous work (16, 20) suggested that CD26 expression is associated with changes in tumor cell line behavior in vitro and that the clinical behavior of selected T-cell tumors may be correlated with differences in CD26 expression, leading to our hypothesis that CD26 expression influences tumor cell biology and potentially its sensitivity to cytotoxic treatments. Because the anthracyclines have well-documented activity in hematological malignancies, being in fact an integral part of chemotherapeutic regimens for these diseases, we investigated the effect of CD26/DPPIV expression on cellular sensitivity to doxorubicin, using as our model CD26-Jurkat transfectants. In this study, we demonstrate that cells expressing CD26/DPPIV displayed enhanced sensitivity to doxorubicin, associated with such disruption of cell cycle-related events as hyperphosphorylation and inhibition of p34^cdc2 kinase activity, cdc25C phosphorylation, and alteration in cyclin B1 expression. Furthermore, the addition of exogenous sDPPIV resulted in increased sensitivity to doxorubicin in tumor cell lines, thus suggesting a potentially useful clinical role for CD26/DPPIV in the treatment of selected human cancers.

Human T-cell leukemia Jurkat stable transfectants have been described (14, 24, 25). The Jurkat cell lines include: (a) wild-type CD26-transfected Jurkat cell lines (wtCD26); (b) Jurkat cell lines transfected with mutant CD26 containing an alanine at the putative catalytic serine residue at position 630, resulting in a mutant CD26-positive/DPPIV-negative Jurkat transfectant (S630A); (c) Jurkat cell lines transfected with mutant CD26 containing point mutations at ADA-binding site residues 340–343, with amino acids L₃₄₀, V₃₄₁, A₃₄₂, and R₃₄₃ being replaced by amino acids P₃₄₀, S₃₄₁, E₃₄₂, and Q₃₄₃, resulting in a mutant CD26-positive/DPPIV-positive Jurkat transfectant incapable of binding ADA (340-4); (d) vector-only Jurkat transfectant (neo); and (e) nontransfected control Jurkat cells (control). Jurkat transfectants were maintained in culture media, which consisted of RPMI 1640 supplemented with 10% FCS, penicillin (100 units/ml), streptomycin (100 μg/ml), and G418 (0.25 mg/ml; Life Technologies, Inc.). Nontransfected control Jurkat cells were maintained in the same culture media without G418. Jiyoye cells were maintained in the same media but supplemented with 20% FCS, whereas Namalwa cells were maintained in culture supplemented with 7.5% FCS. Anti-p34^cdc2, anti-cdc25C, and anti-cyclin B1 were from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-actin were from Sigma Chemical Co. Tetrazolium salt MTT (Sigma Chemical Co.) was dissolved at a concentration of 5 mg/ml in sterile PBS at room temperature, with the solution being filter-sterilized and stored at 4°C in the dark. Extraction buffer was prepared as follows: 20% w/v of SDS was dissolved at 37°C in a solution of 50% each of N,N-dimethyl formamide (Sigma Chemical Co.) and distilled water; pH was adjusted to 4.7 by the addition of 1 m HCl. sDPPIV was produced by Chinese hamster ovary cells as described previously (12). Doxorubicin was purchased from Calbiochem and was dissolved in sterile PBS.

Cell growth assay was performed as described previously (26). Cells were incubated in microplates in the presence of culture media alone, culture media and sDPPIV (50 μg/ml), culture media and doxorubicin at the indicated concentrations, or culture media, sDPPIV (50 μg/ml), and doxorubicin at the indicated concentrations, for a total volume of 100 μl (50,000 cells/well). After 48 h of incubation at 37°C, 25 μl of MTT was added to the wells at a final concentration of 1 mg/ml. The microplates were then incubated for 2 h at 37°C, followed by the addition of 100 μl of extraction buffer. After overnight incubation at 37°C, A_570/nm measurements at 570 nm were performed, with the SE of the triplicate wells being less than 15%. Cytotoxicity index was calculated as follows:

Cells were incubated in culture media alone or culture media and doxorubicin (0.05 μm) at 37°C for 24 h. Cells were then collected, washed twice with PBS, and resuspended in PBS containing 10 μg/ml propidium iodide, 0.5% Tween 20, and 0.1% RNase at room temperature for 30 min. Samples were then analyzed (FACScan; Becton Dickinson) for DNA content. Cell debris and fixation artifacts were gated out and G₀/G₁, S, and G₂-M populations were quantified using the CellQuest and ModFit LT programs.

After incubation at 37°C in culture media and doxorubicin at the concentrations indicated for 24 h, cells were harvested from wells, washed with PBS, and lysed in lysis buffer, consisting of 1% Brij 97, 5 mm EDTA, 0.02 m HEPES (pH 7.3), 0.15 m NaCl, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm NaF, 10 μg/ml aprotinin, and 0.2 mm sodium orthovanadate. After incubating on ice for 15 min, nuclei were removed by centrifugation and supernatants were collected. Sample buffer (2×) consisting of 20% glycerol, 4.6% SDS, 0.125 m Tris (pH 6.8), and 0.1% bromphenol blue was added to the appropriate aliquots of supernatants. After boiling, protein samples were submitted to SDS-PAGE analysis on an 8% gel under standard conditions using a mini-Protean II system (Bio-Rad). For immunoblotting, the proteins were transferred onto nitrocellulose (Immobilon-P; Millipore). After overnight blocking at 4°C in blocking solution consisting of 0.1% Tween 20 and 5% BSA in Tris-buffered saline, membranes were blotted with the appropriate primary antibodies diluted in blocking solution for 1 h at room temperature. Membranes were then washed with blocking solution, and appropriate secondary antibodies diluted in blocking solution were then applied for 1 h at room temperature. Secondary antibodies were goat antimouse or goat antirabbit horseradish peroxidase conjugates (Dako). Membranes were then washed with blocking solution, and proteins were subsequently detected by chemiluminescence (Amersham Pharmacia Biotech).

Cells were treated with doxorubicin at the indicated concentrations for 24 h, and cell extracts were prepared in lysis buffer as described above. Total protein (320 μg) was then diluted to 1 mg/ml protein concentration in HB buffer [20 mm HEPES (pH 7.7), 75 mm NaCl, 2.5 mm EDTA (pH 8.0), 0.05% Triton X-100, 20 mm β-glycerophosphate, 0.5 mm sodium orthovanadate, 1 mm DTT, 2 μg/ml leupeptin, and 1 mm phenylmethylsulfonylfluoride] and immunoprecipitated with anti-p34^cdc2 monoclonal antibody (Santa Cruz Biotechnology) and protein A-Sepharose beads for 3–4 h. Immunoprecipitates were washed twice with HB buffer and twice with kinase buffer [25 mm HEPES (pH 7.6), 20 mm MgCl₂, and 20 mm β-glycerophosphate]. Kinase assays were performed with cold ATP (50 μm) and [γ-³²P]ATP (5 μCi) in the presence of 1 μg of histone H1 (Life Technologies, Inc.) for 30 min at room temperature, and the reactions were stopped by boiling in Laemmli buffer. Samples were submitted to SDS-PAGE analysis on 12% gel under standard conditions, and the bands were visualized by autoradiography.

DPPIV enzyme activity was detected by using an Enzyme Overlay Membrane system (Enzyme Systems Products, Dublin, CA) to which the substrate Ala-Pro-7-amino-4-trifluoromethyl coumarin has been coupled, as described previously (27). After incubation at 37°C with media alone or doxorubicin for 24 h, cell lysates were prepared, and sample buffer consisting of 20% glycerol, 4.6% SDS, 0.125 m Tris (pH 6.8), 0.1% bromphenol blue, and 2% 2-mercaptoethanol was added at room temperature. Samples were then submitted to SDS-PAGE analysis on a 8% gel under standard conditions. The Enzyme Overlay Membrane was moistened in 0.5 m Tris-HCl (pH 7.8), placed over the surface of the gel, and incubated at 37°C for 40 min in a humidified atmosphere. The membrane was then removed from the gel and placed atop a long-wavelength UV lamp box to monitor enzymatic reaction, which involves the removal of the dipeptide Ala-Pro from the fluorogenic 7-amino-4-trifluoromethyl coumarin and results in the appearance of fluorescent bands on the membrane.

Immunoprecipitation analysis was performed as described previously (14, 15). Briefly, Jurkat transfectants or control nontransfected Jurkat cells were labeled by lactoperoxidase-catalyzed iodination. The lysates were incubated with 2 μg of anti-CD26 antibody (1F7) before precipitation with goat antimouse coupled to Affi-Gel (Bio-Rad). Immune complexes were then washed extensively, and eluted by boiling for 5 min in 50 μl of SDS sample buffer before analysis by SDS-PAGE.

Using stable Jurkat transfectants, we investigated the effect of CD26 expression on susceptibility to doxorubicin, as determined by MTT assays. Fig. 1 shows that wild-type CD26 transfectants (wtCD26) displayed markedly increased sensitivity to doxorubicin as compared with parental (control) or vector only (neo) Jurkat cells. Intriguingly, CD26 transfectants mutated at the DPPIV catalytic site (S630A) were less sensitive to doxorubicin. On the other hand, Jurkat cells transfected with CD26 mutated at the ADA-binding site while still retaining DPPIV activity (340-4) exhibited greater doxorubicin sensitivity, similar to the wtCD26 transfectants. These data suggested that the presence of CD26, particularly its associated DPPIV enzymatic activity, resulted in enhanced sensitivity to DNA damage mediated by the antineoplastic agent doxorubicin.

Cell cycle analysis by propidium iodide staining demonstrated that the enhancement in doxorubicin sensitivity seen in wtCD26 transfectants was attributable to an increase in the percentage of cells arresting at G₂-M (Fig. 2,A; Table 1). Once again, DPPIV enzymatic activity was required for the increased sensitivity to doxorubicin because the G₂-M block in the S630A CD26 mutant was indistinguishable from control parental Jurkat cells, whereas wtCD26 and 340-4 cells exhibited enhanced G₂-M arrest after doxorubicin treatment. Meanwhile, expression of CD26 and its mutant derivatives in the transfected cells were confirmed by immunoprecipitation analysis (Fig. 2 B) as well as by immunofluorescence studies (data not shown), as has been described previously (14, 24).

Next, we determined the DPPIV enzyme activity on Jurkat transfectants after treatment with doxorubicin. As seen in Fig. 3, wtCD26 transfectants retained DPPIV enzyme activity after incubation with doxorubicin, whereas S630A transfectants as well as control nontransfected Jurkat cells did not exhibit DPPIV enzyme activity. Therefore, these data indicated that the observed differences in doxorubicin sensitivity in these various Jurkat transfectants were associated with differences in DPPIV enzymatic activity.

p34^cdc2 is the key regulator of cell cycle progression through G₂-M (28). Treatment with doxorubicin decreased p34^cdc2 kinase activity, which was associated with cell cycle arrest at the G₂-M checkpoint (22, 23). We thus evaluated p34^cdc2 kinase activity after doxorubicin treatment in Jurkat cells. As seen in Fig. 4 A, inhibition of p34^cdc2 kinase activity occurred at lower concentrations of doxorubicin in wtCD26 Jurkat transfectants as compared with control nontransfectants or S630A transfectants. P34^cdc2 enzyme activity hence correlated with the observed differences in sensitivity of these Jurkat lines to G₂-M arrest after doxorubicin-induced DNA damage.

Previous studies (23, 29, 30) have established that hyperphosphorylation of p34^cdc2 at the inhibitory residues Thr14Tyr15 after doxorubicin-induced DNA damage correlated with inhibition of p34^cdc2 kinase activity. As assessed by Western blot analysis (Fig. 4 B), doxorubicin-treated wtCD26 Jurkat transfectants had higher levels of hyperphosphorylated p34^cdc2, particularly at lower doxorubicin doses as compared with nontransfected Jurkat control cells and S630A transfectants, which only exhibited a slight enhancement in the level of phosphorylated p34^cdc2 at the higher doses of doxorubicin. Our data therefore showed that the relative sensitivity of CD26 Jurkat transfectants to doxorubicin-mediated G₂-M arrest correlated with the relative kinase activity and phosphorylation level p34^cdc2, with increasing p34^cdc2 hyperphosphorylation being associated with decreased p34^cdc2 kinase activity and enhanced G₂-M arrest.

The cdc25C protein phosphatase is a key regulator of p34^cdc2 phosphorylation status and kinase activity by dephosphorylating p34^cdc2 Thr14/Tyr15 residues (29, 30, 31). Although hyperphosphorylation of cdc25C on NH₂ terminal serine and threonine residues increases its intrinsic phosphatase activity (32), phosphorylation on residue serine-216 results in 14-3-3 protein binding and negatively regulates cdc25C phosphatase activity by preventing cdc25C from interacting with p34^cdc2 (33). Asynchronously growing Jurkat cells have been shown to express two major electrophoretic forms of cdc25C, reflecting differences in serine-216 phosphorylation status (33). Consistent with previous findings, we also found that lysates from parental Jurkat cells contained these two major electrophoretic forms of cdc25C (Fig. 4 C). Lysates from wild-type CD26 Jurkat transfectants and S630A transfectants also contained the two forms of cdc25C differing in serine-216 phosphorylation. Importantly, wtCD26 transfectants consistently showed a detectable enhancement in cdc25C serine-216 phosphorylation as compared with S630A transfectants and nontransfected control cells when treated with doxorubicin. These results hence indicated that the increase in cdc25C serine-216 phosphorylation was concordant with enhanced p34^cdc2 phosphorylation and concomitant decrease in its kinase activity in doxorubicin-treated wtCD26 Jurkat transfectants as compared with nontransfectants or S630A transfectants.

Next, we investigated the effect of doxorubicin treatment on cyclin B1 expression in the various CD26 Jurkat transfectants, in view of the fact that the p34^cdc2/cyclin B1 complex plays a key role in regulating cell cycle progression at the G₂-M checkpoint. Fig. 4 D showed that the levels of cyclin B1 were higher in wtCD26 Jurkat transfectants as compared with those in nontransfectants or S630A transfectants after treatment with doxorubicin. In some experiments, we did note that the level of cyclin B1 in untreated wtCD26 transfectants were slightly higher than that seen with untreated nontransfectants or S630A transfectants (data not shown). Significantly, in all of the cases tested, the relative increase in cyclin B1 level in cells treated with doxorubicin as compared with untreated cells was greater in wtCD26 transfectants than it was in parental cells or S630A transfectants. Consistent with previous research that showed that doxorubicin treatment led to cyclin B1 accumulation in murine leukemia P388 cells (22), our data thus indicated that doxorubicin treatment in Jurkat cells resulted in alterations in the regulation of cyclin B1 expression, resulting in cyclin B1 accumulation. Importantly, as was the case with p34^cdc2 phosphorylation status and kinase activity as well as cdc25C serine-216 phosphorylation, we found that the relative level of cyclin B1 expression correlated with the relative sensitivity of CD26 Jurkat transfectants to doxorubicin.

Because our data indicated that the presence of DPPIV enzymatic activity was critical to doxorubicin sensitivity, we next examined the effect of exogenous sDPPIV on doxorubicin-treated CD26 Jurkat transfectants. For these experiments, low doses of doxorubicin were used to optimally detect the potential effect of exogenous sDPPIV on doxorubicin sensitivity. Although sDPPIV by itself did not affect MTT uptake in cells incubated in medium alone, we found that the presence of exogenous sDPPIV resulted in significantly enhanced sensitivity to doxorubicin (Fig. 5). These data further supported our conclusion that DPPIV enzymatic activity plays a key role in the relative sensitivity of Jurkat cells to doxorubicin.

In view of the fact that the presence of DPPIV activity led to enhanced sensitivity of Jurkat cells to doxorubicin, we examined the effect of sDPPIV on doxorubicin-mediated growth inhibition of other lymphoid cell lines. As seen in Fig. 6, the addition of sDPPIV also enhanced the growth inhibitory effect of doxorubicin in the B-cell lines Namalwa and Jiyoye. Our data thus demonstrated that DPPIV-mediated enhancement in sensitivity to doxorubicin was not restricted to Jurkat cell lines alone but was applicable to other lymphoid cell lines.

In this study, we present data showing that the presence of DPPIV enzymatic activity of CD26 enhances cellular sensitivity to doxorubicin-induced DNA damage. Using stable Jurkat transfectants, we demonstrated that wild-type CD26 transfectants, as well as Jurkat transfectants expressing a CD26 molecule mutated at the ADA-binding residues yet still retaining DPPIV enzymatic activity, exhibited greater sensitivity to doxorubicin than parental Jurkat cells, vector-only transfectants, or CD26-positive/DPPIV-negative Jurkat transfectants. Although previously published work (20) suggested that the presence of CD26 on tumor cells from patients with selected T-cell neoplasms is correlated with poor prognosis, our present findings indicate that expression of CD26, particularly its DPPIV enzyme activity, may influence tumor cell biology by rendering the Jurkat transfectants more sensitive to doxorubicin. Some potential explanations for this discrepancy include the fact that the clinical evidence was based on a relatively small cohort of patients treated with potentially heterogeneous regimens as well as the fact that, whereas CD26 expression was evaluated on patients’ tumor cells, the status of its associated DPPIV enzyme activity in these samples was unclear. Alternatively, CD26/DPPIV expression may allow for a growth advantage to malignant cells while increasing their susceptibility to selected chemotherapeutic agents targeting rapidly growing cells. Our findings that doxorubicin-mediated growth inhibition in these Jurkat cells was attributable to enhanced G₂-M arrest are consistent with previous reports showing that doxorubicin-induced DNA damage resulted in cell cycle arrest at the G₂-M checkpoint (22, 23). Investigating the mechanisms of enhanced doxorubicin sensitivity associated with DPPIV enzyme activity, we established that the presence of DPPIV resulted in increased doxorubicin-mediated p34^cdc2 hyperphosphorylation and inhibition of its kinase activity. Furthermore, consistent with data published previously (22) showing that doxorubicin-mediated G₂-M arrest was associated with enhanced cyclin B1 protein levels, our work showed that the presence of DPPIV enzymatic activity was correlated with higher levels of cyclin B1 after doxorubicin treatment. Our data involving the Jurkat transfectants therefore suggested that DPPIV enzymatic activity lowered the threshold of cellular sensitivity to doxorubicin by affecting molecular events associated with doxorubicin-mediated G₂-M arrest.

Published reports (3, 4, 34, 35, 36, 37) have also suggested that DPPIV enzymatic activity of CD26 plays a key role in immunoregulation. DPPIV inhibitors altered T-cell proliferation, T-helper function to alloantigen-induced humoral response, cytotoxic activity, and lymphokine production; however, nonspecific side effects may be potential confounding factors (34, 35, 36). Meanwhile, approaches using soluble CD26/DPPIV or CD26 transfectants showed that the presence of DPPIV enzymatic activity resulted in an enhancement in stimuli-induced T-cell proliferation (37). In addition to its direct involvement in T-cell activation, CD26/DPPIV regulates the immune system by cleaving selected chemokines at the NH₂ terminus to modify their biological functions, because DPPIV-processed factors exhibited lower chemotactic potency, impaired signaling effects, and altered receptor specificity (3, 4).

Our findings demonstrated that CD26/DPPIV enhances cellular sensitivity to doxorubicin. In view of its ability to cleave selected biological factors as a serine protease, it is possible that CD26/DPPIV exerts its effect by cleavage of certain as yet unidentified peptide growth factors that may in turn affect cellular response to doxorubicin. The presence of CD26/DPPIV-cleaved proteins may fundamentally alter the cell by affecting selected intracellular pathways, hence rendering it more susceptible to doxorubicin treatment. These factors may be derived from serum. Alternatively, they may arise from the cell itself. Future experiments will be performed to examine this question.

The p34^cdc2/cyclin B1 complex is the major regulator of cell cycle progression at the G₂-M checkpoint. In particular, activation of p34^cdc2 kinase activity is required for progression from G₂ to M. Phosphorylation of the inhibitory residues Thr14/Tyr15 of p34^cdc2 leads to decreased kinase activity and subsequent arrest at the G₂-M checkpoint (29). Previous reports (23, 30) have shown that DNA damage from treatment with doxorubicin led to a hyperphosphorylation state of p34^cdc2 at residues Thr14/Tyr15, resulting in subsequent inhibition of p34^cdc2 kinase activity. Our data showed that the presence of CD26/DPPIV enhanced sensitivity to doxorubicin-mediated arrest because lower concentrations of doxorubicin were needed to induce p34^cdc2 hyperphosphorylation, inhibition of p34^cdc2 kinase activity, and subsequent arrest in wtCD26 Jurkat transfectants as compared with nontransfectants or S630A transfectants. Our studies also demonstrated doxorubicin treatment induced cyclin B1 accumulation, similar to work published previously (22). Furthermore, as seen in the case with p34^cdc2 phosphorylation status and kinase activity, lower concentrations of doxorubicin were needed to mediate cyclin B1 accumulation in cells expressing CD26/DPPIV.

Lysates of untreated parental Jurkat cells as well as Jurkat transfectants contained the two major electrophoretic forms of cdc25C, findings that are consistent with previous work (33) showing the existence of these two forms of cdc25C differing in serine-216 phosphorylation in asynchronously growing Jurkat cells. Meanwhile, serine-216 phosphorylation of cdc25C was consistently enhanced in doxorubicin-treated wild-type CD26 Jurkat transfectants as compared with S630A transfectants and nontransfected cells. Because phosphorylation of serine-216 leads to 14-3-3 protein binding and negatively regulates cdc25C phosphatase activity by preventing its functional interaction with p34^cdc2 (33), it is likely that the enhanced p34^cdc2 phosphorylation and decreased kinase activity seen in doxorubicin-treated wtCD26 transfectants were attributable at least partially to the effect of increased cdc25C serine-216 phosphorylation. The importance of 14-3-3 binding to cdc25C function is also seen from studies showing that cdc25C mutations that disrupted 14-3-3 binding interfered with cdc25C intracellular localization and its effect on cdc2-cyclin B1 complex (38). Although the two major electrophoretic forms reflecting differences in cdc25C serine-216 phosphorylation were detected, the slower migrating cdc25C species hyperphosphorylated at NH₂ terminal serine and threonine residues and associated with increased cdc25C phosphatase activity (32, 33) was not observed in the cell lysates under our experimental conditions (data not shown).

The observed enhancement of doxorubicin-mediated growth inhibition of Jurkat cells by exogenously added sDPPIV further supported our conclusion that DPPIV enzymatic activity potentiates doxorubicin effect on cell growth. Furthermore, we extended our initial findings with Jurkat cells by showing that sDPPIV enhanced doxorubicin sensitivity in the B-cell lines Namalwa and Jiyoye. Several lines of evidence suggested that the effect of sDPPIV on doxorubicin-induced growth inhibition was not attributable merely to nonspecific toxicity. The addition of sDPPIV by itself, at the indicated doses, did not result in decreased MTT uptake when cells were incubated in media alone. Rather, sDPPIV enhanced the inhibitory effect of cells incubated in media containing doxorubicin. Furthermore, the addition of sDPPIV to doxorubicin-containing media did not universally result in enhanced growth inhibition of all of the cell lines tested. For several cell lines tested, sDPPIV did not have any significant effect on doxorubicin-mediated growth inhibition (data not shown). Meanwhile, our findings that certain lymphoid cell lines of B- and T-cell lineage exhibited enhanced doxorubicin sensitivity after concurrent treatment with sDPPIV may have potential clinical significance, because they suggested that DPPIV may have a role in the treatment of selected hematological malignancies. Along with our recent observation that binding of anti-CD26 antibody leads to in vitro and in vivo growth inhibition of CD26+ cell lines (39), the findings in this study further demonstrate that future treatment strategies targeting CD26/DPPIV may be effective for selected neoplasms.