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Keywords:

  • natural killer cell tumour;
  • l-asparaginase;
  • chronic NK lymphocytosis;
  • apoptosis;
  • asparagine synthetase

Summary

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

We examined the effectiveness of various anti-tumour agents to natural killer (NK)-cell tumour cell lines and samples, which are generally resistant to chemotherapy, using flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Although NK-YS and NK-92 were highly resistant to various anti-tumour agents, l-asparaginase induced apoptosis in these two NK-cell lines. NK-cell leukaemia/lymphoma and acute lymphoblastic leukaemia (ALL) samples were selectively sensitive to l-asparaginase and to doxorubicin (DXR) respectively. Samples of chronic NK lymphocytosis, an NK-cell disorder with an indolent clinical course, were resistant to both drugs. Our study clearly separated two major categories of NK-cell disorders and ALL according to the sensitivity to DXR and l-asparaginase. We examined asparagine synthetase levels by real-time quantitative polymerase chain reaction (RQ-PCR) and immunostaining in these samples. At least in nasal-type NK-cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l-asparaginase. In aggressive NK-cell leukaemia, although asparagine synthetase expression was high at both mRNA and protein levels, l-asparaginase induced considerable apoptosis. Furthermore, samples of each disease entity occupied a distinct area in two-dimensional plotting with asparagine synthetase mRNA level (RQ-PCR) and in vitrol-asparaginase sensitivity (TUNEL assay). We confirmed rather specific anti-tumour activity of l-asparaginase against NK-cell tumours in vitro, which provides an experimental background to the clinical use of l-asparaginase for NK-cell tumours.

Natural killer (NK) cells are characterised by large granular lymphocyte morphology with CD3 CD56+ phenotype, non-major histocompatibilty complex-restricted cytotoxity, and germ-line configuration of T-cell receptor genes (Hercend & Schmidt, 1988; Trinchieri, 1989; Robertson & Ritz, 1990). NK-cell disorders consist of two main categories: chronic NK lymphocytosis and NK-cell leukaemia/lymphoma. Chronic NK lymphocytosis is a rather benign disorder with persistent excess of mature NK cells (CD3 CD16/CD56+) in the peripheral blood (Tefferi et al, 1994). In contrast, NK-cell leukaemia/lymphoma is a group of aggressive disorders, consisting of aggressive NK-cell leukaemia, nasal-type NK-cell lymphoma and blastic NK-cell lymphoma (Jaffe et al, 2001). Aggressive NK-cell leukaemia takes a highly aggressive clinical course accompanied with fever and hepatosplenomegaly (Imamura et al, 1990; Chan et al, 1997; Kwong et al, 1997; Siu et al, 2002; Oshimi, 2003). Nasal-type NK-cell lymphoma occurs mainly in the nasal cavity with extensive necrosis and angioinvasion, and rapidly disseminates to various sites. Blastic NK-cell lymphoma, which may originate from the precursors of plasmacytoid dendritic cells, tends to involve multiple sites, including the skin (Chaperot et al, 2001; Feuillard et al, 2002). Poor outcome of these NK-cell leukaemia/lymphomas has been attributed not only to their rapid progression but also to the refractoriness to standard combination chemotherapy (Kwong et al, 1997). High expression of P-glycoprotein (P-gp), which extrudes various cytotoxic agents, may contribute to their refractoriness to various anti-cancer agents (Yamamoto et al, 1993; Yamaguchi et al, 1995; Egashira et al, 1999). Some reports have recently shown that l-asparaginase, a commonly used agent for childhood acute lymphoblastic leukaemia (ALL), is effective against refractory and relapsed cases of NK-cell lymphoma (Matsumoto et al, 2003; Yong et al, 2003). As the agent depletes asparagine by its hydrolysis, tumour cells that depend on exogenous asparagine are sensitive to this agent (Clarkson et al, 1970; Ertel et al, 1979). Treatment with l-asparaginase induces apoptotic morphology in canine malignant lymphoma cells (Story et al, 1993). Flow cytometric terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL) assay confirmed that l-asparaginase induced G1 arrest and apoptosis in murine leukaemia cells (Ueno et al, 1997). In the present study, we applied the TUNEL assay to two NK-cell lines, NK-YS (Tsuchiyama et al, 1998) and NK-92 (Gong et al, 1994) derived from nasal-type NK-cell lymphoma and aggressive NK-cell leukaemia. Among various anti-tumour drugs, only l-asparaginase induced considerable amount of apoptosis in these NK-cell lines. Furthermore, considerable portion of NK-cell tumour samples was sensitive to in vitro treatment with l-asparaginase.

Materials and methods

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Cell lines

Jurkat, K562, NK-92 and NK-YS cell lines were used. Jurkat and K562 were grown in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 μg/ml streptomycin. NK-92 and NK-YS, kindly provided by Dr Jiang-Hong Gong (University of British Columbia, Vancouver, Canada) and Dr Junjiroh Tsuchiyama (Okayama University Medical School, Japan), respectively, were grown in Iscove's modified Dulbecco's medium supplemented with 10% FCS, 100 U/ml of interleukin-2 (Takeda, Osaka, Japan), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were split to keep the cell density at 2 × 105 to 1 × 106 cells/ml.

Patients and tumour samples

Eighteen patients with NK-cell disorders were entered into the study. Informed consent was obtained in all cases. T-cell receptor genes of all cases were all in germline configuration. Ten patients had chronic NK lymphocytosis, four nasal-type NK-cell lymphoma, two aggressive NK-cell leukaemia and two blastic NK-cell lymphoma. The percentages of CD56 or CD16 positive cells were more than 85% in eight patients, 60–85% in six, 50–60% in three and 30% in one. We also used ALL cells from six patients: two patients with T-ALL and four with B-ALL. Karyotypes of ALL samples were normal except for one which was t(9;22)(q34;q11).

Anti-cancer agents and drug treatment

We used doxorubicin (DXR), daunorubicin (DNR), vincristine (VCR), methotrexate (MTX), cytosine β-d-arabinofuranose (Ara-C) (all reagents were purchased from Sigma, St Louis, MO, USA), and l-asparaginase (Kyowa Hakkou, Tokyo, Japan). Logarithmically growing cells were treated for the indicated times with these drugs.

Cell fixation

Cells treated with the indicated conditions were harvested by centrifugation for 8 min at 4°C at 400 g, washed once with phosphate-buffered saline (PBS), and then fixed in 1% formaldehyde in PBS (pH 7·4) for 15 min on ice. After washing in PBS, cells were resuspended in 70% cold (−20°C) ethanol and immediately transferred to the freezer. The cells were stored at −20°C for 1 day before being subjected to the TUNEL assay.

Flow cytometric TUNEL assay

Cells were washed twice in PBS, resuspended in 50 μl of a cacodylate buffer containing 0·2 mol/l potassium cacodylate, 25 mmol/l Tris-HCl (pH 6·6), 2·5 mmol/l CoCl2, 0·25 mg/ml bovine serum albumin, 5 U of terminal deoxynucleotidyl transferase (TdT) and 0·5 nmol of biotin-dUTP (all reagents were purchased from Boehringer Mannheim, Indianapolis, IN, USA). The cells were incubated in this solution at 37°C for 30 min, rinsed in PBS, resuspended in 100 μl of a solution containing 4 μl concentrated saline-sodium citrate buffer, 2·5 μg/ml fluoresceinated avidin (Boehringer Mannheim), 0·1% Triton X-100, and 5% (w/v) non-fat dry milk, and incubated in this solution for 30 min at room temperature in the dark. After incubation in the solution, the cells were rinsed in PBS containing 0·1% Triton X-100 and resuspended in 1 ml of PBS containing 5 μg/ml of propidium iodide (PI) and 200 μg/ml of RNase A (both from Sigma). This procedure essentially followed that of previous reports (Sugimoto et al, 1998, 2002).

Flow cytometry was performed on a CYTRON ABSOLUTE flow cytometer (Ortho, Raritan, NJ, USA). The PI signal of orange fluorescence and TUNEL signal of green fluorescence of each cell were measured using linear and logarithmic amplifications of the standard optics of the CYTORON ABSOLUTE. Even without anti-tumour drug treatment, NK-cell samples often underwent apoptosis spontaneously in in vitro culture conditions. We thus evaluated the anti-tumour effect on NK-cell tumour and ALL samples using the corrected apoptotic cell percentage, which we defined as [drug-induced apoptosis (%) − spontaneous apoptosis (%)]/[100 − spontaneous apoptosis (%)].

Immunostaining

A monoclonal antibody 3G6 against asparagine synthetase was prepared by a standard hybridoma cell formation method (Sheng et al, 1992; Hutson et al, 1997). As previously described, specimens of cell lines prepared using Cytospin 3 (Shandon, Pittsburgh, PA, USA) and bone marrow smears were fixed with a methanol and acetone mixture (1:1) for 90 s at 4°C using the EX-IHC automated immunohistochemistry system (Ventana Medical Systems, Tucson, AZ, USA). In lymphoma cases, formalin-fixed, paraffin-embedded tissue specimens were prepared. The slides were inhibited with H2O2, incubated with the primary monoclonal antibody against asparagine synthetase for 16 min at 37°C, and detected by the diaminobenzidine (DAB) Detection Kit (Ventana Medical Systems) (Irino et al, 2004). The intensity of the asparagine synthetase signal was determined by inspection in comparison with the controls.

Real-time quantitative polymerase chain reaction assay of asparagine synthetase mRNA

Total RNA was isolated using RNA STAT-60 (Tel-Test, Inc.). First strand cDNA was synthesised from 1 μg of RNA using Super Script II (Gibco BRL, Carlsbad, CA, USA). Real-time quantitative polymerase chain reaction (RQ-PCR) was performed with the LightCycler FastStart DNA Master SYBR Green I kit, containing the SYBR Green I dye (Roche Molecular Biochemicals, Mannheim, Germany). For each sample, reactions were set up in capillaries with the following reaction mix: 2 μl of DNA template; 0·5 μl each of 10 μmol/l sense and antisense primers; 2·4 μl of 25 mmol/l MgCl2; 2 μl of LightCycler-DNA Master SYBR Green I reaction mix; and 12·6 μl sterile water. The amplification conditions were 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 10 s and extension at 72°C for 6 s. All primers were purchased from TAKARA BIO Inc (Otsu, Japan). Primers for the amplification of asparagine synthetase cDNA were sense, 5′-CGGTAGTTGACCCGCTGTTTG-3′, and antisense, 5′- CACCATCCACTTTGGTCTGGTATTC-3′. The calculated number of transcripts of asparagine synthetase was normalised to the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH).

Results

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

We examined apoptotic cell percentages of Jurkat and two NK-cell lines after treatment with various anti-cancer agents including VCR, MTX, Ara-C, DNR, DXR and l-asparaginase using flow cytometric TUNEL assay (Sugimoto et al, 1998, 2002). At first, we determined the treatment conditions (drug concentration and treatment duration), which caused 30–70% of apoptosis in Jurkat cells. NK-YS and NK-92 cells were extremely resistant to these drugs, and at most 10% of them were apoptotic. On the contrary, 84% of NK-YS and 67% of NK-92 cells underwent apoptosis after 48 h of incubation with 0·01 IU/ml of l-asparaginase, although the same treatment induced apoptosis only in 19% of Jurkat cells. Even at different concentrations and treatment durations, l-asparaginase induced apoptosis rather specifically in those NK-cell lines (Fig 1A). Essentially the same results were obtained in four independent experiments. Among various anti-tumour agents tested here, only l-asparaginase was more effective to the two NK-cell lines than to Jurkat cells. As MTX and Ara-C, whose effects are known to be independent of P-gp status (Pastan & Gottesman, 1994), had little effect on these two NK-cell lines, high expression of P-gp cannot fully explain the refractoriness of NK-cell tumours to various anti-tumour agents. However, l-asparaginase was obviously effective to NK-cell tumour cell lines, regardless of any mechanisms of drug resistance. Figure 1B shows the results of flow cytometric TUNEL assay on NK-YS cells. Treatment with 300 nmol/l of DXR for 24 h caused a small decrease in the G1 peak and small amount of apoptosis in NK-YS cells (b, e). Although treatment with l-asparaginase did not apparently change the cell cycle distribution of NK-YS (Fig 1B-c), most of the non-G1 cells underwent apoptosis (Fig 1B-f). Similar results were obtained in NK-92 cells (data not shown). We next examined the effectiveness of l-asparaginase to NK-cell tumour samples. Even without anti-tumour drug treatment, NK-cell samples often underwent apoptosis spontaneously in in vitro culture conditions. We thus evaluated the anti-tumour effect of l-asparaginase using the corrected apoptotic cell percentage, as indicated in the study design. Figure 2 shows the corrected apoptotic cell percentages of four nasal-type NK-cell lymphoma, two aggressive NK-cell leukaemia, two blastic NK-cell lymphoma, 10 chronic NK lymphocytosis and six ALL samples, treated with 450 nmol/l of DXR or with 10 IU/ml of l-asparaginase for 24 h. The corrected apoptotic cell percentages after DXR treatment were nearly 0% in chronic NK lymphocytosis, <10% in NK-cell tumours and about 40% in ALL. In contrast, l-asparaginase treatment caused corrected apoptotic cell percentages of 20–60% in NK-cell tumour samples except one, and around 10% of apoptosis in chronic NK lymphocytosis and ALL samples. Therefore, our results showed that l-asparaginase was more effective than DXR not only to NK-cell lines but also to patient NK-cell tumour samples. On the contrary, ALL samples were more sensitive to DXR than to l-asparaginase. Chronic NK lymphocytosis was resistant to both agents. The characteristics, diagnosis, treatment regimen(s), survival duration of each patient are shown in Table I. According to the sensitivity to these two agents, ALL, NK-cell tumours, and chronic NK lymphocytosis could be clearly divided into three categories; i.e. DXR-sensitive, l-asparaginase-sensitive and resistant to both agents respectively (Fig 2).

image

Figure 1. Apoptosis of NK-YS, NK-92 and Jurkat cells treated with various anti-tumour agents including l-asparaginase. (A) Apoptotic cell percentages of NK-YS, NK-92 and Jurkat cells treated with various anti-tumour agents were determined by flow cytometric TUNEL assay. Treatment conditions were as follows: 10 nmol/l of vincristine (VCR) for 24 h, 100 nmol/l of methotrexate (MTX) for 48 h 1 μmol/l of cytosine β-d-arabinofuranose (Ara-C) for 48 h, 120 nmol/l of daunorubicin (DNR) for 24 h, 300 nmol/l of doxorubicin (DXR) for 24 h, 0·01 IU/ml of l-asparaginase for 24 and 48 h, and 0·1 IU/ml of l-asparaginase for 24 h. TUNEL assay was performed in triplicate for all cell lines. The arithmetic means and standard deviations were calculated with three values. NK-YS and NK-92 cells were selectively sensitive to l-asparaginase. (B) Cell cycle positions of apoptotic and non-apoptotic NK-YS cells determined by flow cytometric TUNEL assay. Untreated (a, d), treated with 300 nmol/l of doxorubicin for 24 h (b, e) and 0·01 IU/ml of l-asparaginase for 48 h (c, f). Most NK-YS cells at non-G1 cell cycle positions were apoptotic.

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image

Figure 2. Selective effect of l-asparaginase to NK-cell leukaemia/lymphoma. (A) Corrected apoptotic cell percentages of NK-cell leukaemia/lymphoma, chronic NK lymphocytosis and acute lymphoblastic leukaemia (ALL) samples after the treatment with 450 nmol/l of doxorubicin for 24 h. TUNEL assay was performed in triplicate in all samples. (B) Corrected apoptotic cell percentages of the same samples as (A) after 10 IU/ml of l-asparaginase for 24 h treatment. TUNEL assay was performed in triplicate in all samples.

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Table I.  Patient characteristics and clinical data.
Patient no.Age (years)/sexDiagnosisIn vitro response toClinical responseTreatmentSurvival from diagnosisPositive cells (%)
l-AspDXRl-AspCD16CD56
  1. Patient numbers are identical to those in Figs 2–4, and Table II.

  2. Clinical response to l-asparaginase is shown as CR or NR. Complete remission (CR) means residual leukaemic blasts <5% in leukaemia cases, and complete resolution of signs and symptoms and normalisation of all imaging studies in lymphoma cases after l-asparaginase containing regimen.

  3. No response (NR) indicates more than 25% residual leukaemic blasts in leukaemia cases, and no reduction in measurable tumours in lymphoma cases after l-asparaginase containing regimen.

160/MNasal-type NK-cell lymphomaRRNRI(V)AM + l-asp4 months1·294·6
266/FNasal-type NK-cell lymphomaSRNDCHOP2 months1094
362/MNasal-type NK-cell lymphomaSRCRI(V)AM + l-asp>20 months2·498·3
452/FNasal-type NK-cell lymphomaSRCRI(V)AM + l-asp>8 months7097·9
552/MAggressive NK-cell leukaemiaSRNDPACC, high dose MTX6 months4·129·3
622/MAggressive NK-cell leukaemiaSRCRHyper CVAD, l-asp3 monthsND95·2
755/MBlastic NK-cell lymphomaSRNDCHOP, MTX17 monthsND96·8
842/MBlastic NK-cell lymphomaSRNDIVAM, allo-BMT5 yearsND56·1
950/MChronic NK lymphocytosisRRNDNo treatment>30 months4752
1055/FChronic NK lymphocytosisRRNDNo treatment>10 years5979
1118/FChronic NK lymphocytosisRRNDNo treatment>5 yearsND64
1241/FChronic NK lymphocytosisRRNDNo treatment>18 months5·760·6
1346/MChronic NK lymphocytosisRRNDNo treatment>5 years66·18·4
1458/FChronic NK lymphocytosisRRNDNo treatment>10 years5276
1562/MChronic NK lymphocytosisRRNDNo treatment>5 yearsND85
1662/FChronic NK lymphocytosisRRNDNo treatment>10 years9495
1734/MChronic NK lymphocytosisRRNDNo treatment>10 years3351
1866/MChronic NK lymphocytosisRRNDNo treatment>10 years786
1930/FT-ALLRSNRL17 M6 months2·64·9
2075/MT-ALLRSNDL17 M2 months23·3
2156/FB-ALLRSNRL17 M4 months0·30·3
2230/FB-ALLRSNDL17 M>1 months0·80·7
2320/MB-ALLRSCRL17 M, allo-PBSCT>24 monthsNDND
2425/MB-ALLRSCRL17 M>6 months1·24·4

As expression of asparagine synthetase is one known mechanism for resistance to l-asparaginase (Aslanian et al, 2001), we examined asparagine synthetase expression in the samples described above using RQ-PCR and immunostaining. Table II shows the normalised asparagine synthetase mRNA expression value (AS/GAPDH) of each sample. In nasal-type NK-cell lymphoma, asparagine synthetase mRNA levels of two samples were comparatively high (patients 1 and 2), and those of another two samples were low (patients 3 and 4). In this disease entity, asparagine synthetase mRNA levels inversely correlated with in vitro sensitivity to l-asparaginase. Although the sample number was relatively small, asparagine synthetase mRNA levels were high (>0·05) in two aggressive NK-cell leukaemia and two blastic NK-cell lymphoma cases. Expression of asparagine synthetase mRNA was high in almost all samples of chronic NK lymphocytosis. Then, we created a two-dimensional plot of asparagine synthetase mRNA expression and in vitrol-asparaginase sensitivity. Samples belonging to each disease entity occupied a distinct area in this scheme (Fig 3). In spite of comparatively low asparagine synthetase mRNA expression, ALL cells lacked sensitivity to l-asparaginase in vitro. All nine cases of chronic NK lymphocytosis that were resistant to l-asparaginase, had high expression levels of asparagine synthetase mRNA. On the contrary, although l-asparaginase induced considerable apoptosis in two aggressive NK-cell leukaemia and two blastic NK-cell lymphoma samples, asparagine synthetase mRNA expression of these samples was high. As for nasal-type NK-cell lymphoma, there was good correlation between the results of in vitro TUNEL assay and asparagine synthetase mRNA expression (Table II, Fig 3).

Table II.  Asparagine synthetase expression and in vitro sensitivity to l-asparaginase.
Patient no.DiagnosisAS mRNA (AS/GAPDH)AS immunostainingApoptotic cell percentage after l-asp treatment (%)
  1. Asparagine synthetase mRNA expression level (AS/GAPDH), asparagine synthetase immunostaining, corrected apoptotic cell percentage of samples and apoptotic cell percentage of cell lines and apoptotic cell percentage of cell lines treated with 10 IU/ml of l-asparaginase for 24 h are shown.

  2. Apoptotic cell percentage was divided into two groups: <15% and >25%.

 1Nasal-type NK-cell lymphoma0·043Positive<15
 2Nasal-type NK-cell lymphoma0·075Negative<15
 3Nasal-type NK-cell lymphoma0·016Negative>25
 4Nasal-type NK-cell lymphoma0·003Negative>25
 5Aggressive NK-cell leukaemia0·054Positive>25
 6Aggressive NK-cell leukaemia0·064Positive>25
 7Blastic NK-cell lymphoma0·07Negative>25
 8Blastic NK-cell lymphoma0·131Negative>25
 9Chronic NK lymphocytosis0·075Negative<15
10Chronic NK lymphocytosis0·055Negative<15
11Chronic NK lymphocytosisNDND<15
12Chronic NK lymphocytosis0·041Negative<15
13Chronic NK lymphocytosis0·086Negative<15
14Chronic NK lymphocytosis0·072Negative<15
15Chronic NK lymphocytosis0·074Negative<15
16Chronic NK lymphocytosis0·072Negative<15
17Chronic NK lymphocytosis0·028Negative<15
18Chronic NK lymphocytosis0·054Negative<15
19Acute lymphoblastic leukaemia0·031ND<15
20Acute lymphoblastic leukaemia0·062Negative<15
21Acute lymphoblastic leukaemia0·009Negative<15
22Acute lymphoblastic leukaemia0·008Positive<15
23Acute lymphoblastic leukaemia0·013Positive<15
24Acute lymphoblastic leukaemia0·029Negative<15
Cell line
 K562Erythroblastic transformation from CML0·968Positive<15
 JurkatAcute lymphoblastic leukaemia0·01Negative<15
 NK-92Aggressive NK-cell leukaemia0·003Negative>25
 NK-YSNasal-type NK-cell lymphoma0·0002Negative>25
image

Figure 3. Asparagine synthetase mRNA expression versusin vitrol-asparaginase sensitivity in patient samples. Correlation between asparagine synthetase mRNA level and in vitro apoptotic cell percentage after treatment with 10 IU/ml of l-asparaginase for 24 h in NK-cell tumour, chronic NK lymphocytosis and ALL samples.

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Figure 4 shows representative results of asparagine synthetase staining in NK-cell tumour cell lines and patient samples. Compared with the positive control (K562 cells; Fig 4A), NK-92, NK-YS (Fig 4B) and Jurkat cells were all negative for asparagine synthetase staining. As summarised in Table II, asparagine synthetase was detectable in one of four cases for nasal NK-cell lymphoma, two of two cases for aggressive NK-cell leukaemia and two of four cases of ALL. Although asparagine synthetase mRNA levels were relatively high in cases positive for asparagine synthetase immunostaining, high levels of asparagine synthetase mRNA expression did not always warrant the positive staining.

image

Figure 4. Immunohistochemical detection of asparagine synthetase protein in cell lines and NK-cell tumours. (A) Positive control (K562 cells), (B) negative control (NK-92 cells), (C) nasal-type NK-cell lymphoma (patient 1), (D) aggressive NK-cell leukaemia (patient 5), (E) nasal-type NK-cell lymphoma (patient 2), (F) blastic NK-cell lymphoma (patient 7). Original magnifications are ×400 for A and B, and ×200 for C–F.

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Discussion

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

As NK-cell leukaemia/lymphoma pursues an aggressive course with poor response and short survival with standard chemotherapy containing anthracycline, optimal treatment modalities need to be determined (Kwong et al, 1997). The present study clearly showed the effectiveness of l-asparaginase to NK-cell leukaemia/lymphoma in vitro using the TUNEL assay. Even at different concentrations and treatment durations, l-asparaginase induced apoptosis selectively in NK-cell lines. As previously reported for murine leukaemia cells, l-asparaginase induced G1 arrest and apoptosis rather specifically in the non-G1 population of NK cells (Ueno et al, 1997). There exist some immanent methodical limitations in in vitrol-asparaginase sensitivity assay. Ammonia released by hydrolysation of asparagine and glutamine, which is rapidly eliminated in vivo, might contribute to the cytotoxicity of in vitrol-asparaginase treatment (Wagner et al, 1999). In our experiments, however, culture medium containing phenolphthalein incubated with l-asparaginase was almost the same colour or even less red than the non-treated, rapidly growing control. This observation may exclude the high concentration of ammonia in the l-asparaginase-containing medium. Furthermore, l-asparaginase is known to induce not only asparagine but also glutamine deprivation, the latter may cause cell shrinkage-dependent CD95 clustering, which leads to the activation of the receptor-mediated apoptotic pathway (Fumarola et al, 2001).

We next examined the effectiveness of l-asparaginase and DXR to NK-cell tumour samples. ALL, NK-cell leukaemia/lymphomas and chronic NK lymphocytosis correlated well with three categories of drug sensitivity; i.e. DXR-sensitive, l-asparaginase-sensitive and resistant to both agents respectively. Therefore, although l-asparaginase is commonly used for the treatment of adult ALL, NK-cell tumour samples were much more sensitive to this agent than ALL samples. Our results strongly indicate that l-asparaginase should be included in the treatment regimen for NK-cell leukaemia/lymphoma. The vast majority of the patients with chronic NK lymphocytosis have long-term survival without any treatment (Tefferi et al, 1994). Furthermore, our results showed poor response of chronic NK lymphocytosis to two representative anti-tumour agents of different mechanism of action. We should avoid anti-tumour treatment to patients with chronic NK lymphocytosis. High levels of asparagine synthetase mRNA expression in chronic NK lymphocytosis may cause the apparent l-asparaginase resistance.

Two-dimensional plotting with asparagine synthetase mRNA level and l-asparaginase sensitivity showed that each disease entity occupied a distinct region in the scheme (Fig 3). Samples of nasal-type NK-cell lymphoma were distributed in a rather elongated ellipse, which shows the inverse correlation between asparagine synthetase mRNA levels and in vitrol-asparaginase sensitivity in this disease entity. Furthermore, although the sample number was only four, these two parameters and asparagine synthetase immunostaining were interrelated with the clinical response to l-asparaginase. Indeed, patient 1, whose tumour cells were extremely resistant to l-asparaginase in vitro and showed high levels of asparagine synthetase mRNA and protein expression, responded very poorly to l-asparaginase, and the overall survival was only 4 months (Table I). ALL cells were resistant to in vitrol-asparaginase treatment in spite of relatively low asparagine synthetase mRNA levels. In aggressive NK-cell leukaemia and blastic NK-cell lymphoma, although asparagine synthetase mRNA levels were high, l-asparaginase induced considerable apoptosis in vitro. The two cases of aggressive NK-cell leukaemia were positive for asparagine synthetase immunostaining, which confirmed that asparagine synthetase expression is really high in these cases. We could explain the sensitivity of these samples to l-asparaginase by glutamine-deprived cell shrinkage-dependent apoptosis, which is independent of asparagine synthetase expression (Fumarola et al, 2001). Recent studies on paediatric ALL showed that asparagine synthetase mRNA expression is linked with l-asparaginase resistance only in TEL-AML1-negative but not -positive ALL (Remarkers-van Woerden et al, 2000; Stams et al, 2003, 2005). Therefore asparagine synthetase level alone should not determine sensitivity to l-asparaginase at least in some subtypes of haematological malignancies.

In this report, we confirmed a rather specific anti-tumour activity of l-asparaginase against NK-cell tumours in vitro. This result provides an experimental background to the clinical use of l-asparaginase for NK-cell tumours. Furthermore, during this study concerning l-asparaginase sensitivity, we made the following observations. (i) At least in nasal-type NK-cell lymphoma, there was a good correlation among asparagine synthetase expression, in vitro sensitivity and clinical response to l-asparaginase. (ii) In aggressive NK-cell leukaemia, although asparagine synthetase expression was high, l-asparaginase induced considerable apoptosis in vitro. (iii) Each disease entity occupied a distinct area in the two-dimensional plot with RQ-PCR of asparagine synthetase and in vitro TUNEL assay. We expect to perform more detailed studies with larger number of samples to confirm these findings in the future.

References

  1. Top of page
  2. Summary
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
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