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Biomedical subjects

G J Kaspers

Publications and source records attributed to G J Kaspers.

At least 19 recordsLinked to original sources
PubMed2003

Expression of deoxycytidine kinase in leukaemic cells compared with solid tumour cell lines, liver metastases and normal liver.

Deoxycytidine kinase (dCK) is required for the phosphorylation of several deoxyribonucleoside analogues that are widely employed as chemotherapeutic agents. Examples include cytosine arabinoside (Ara-C) and 2-chlorodeoxyadenosine (CdA) in the treatment of acute myeloid leukaemia (AML) and gemcitabine to treat solid tumours. In this study, expression of dCK mRNA was measured by a competitive template reverse transcriptase polymerase chain reaction (CT RT-PCR) in seven cell lines of different histological origin, 16 childhood and adult AML samples, 10 human liver samples and 11 human liver metastases of colorectal cancer origin. The enzyme activity and protein expression levels of dCK in the cell lines were closely related to the mRNA expression levels (r=0.75, P=0.026 and r=0.86, P=0.007). In AML samples, dCK mRNA expression ranged from 1.16 to 35.25 (x10(-3)xdCK/beta-actin). In the cell line panel, the range was 2.97-56.9 (x10(-3)xdCK/beta-actin) of dCK mRNA expression. The enzyme activity in liver metastases was correlated to dCK mRNA expression (r=0.497, P=0.05). In the liver samples, these were not correlated. dCK mRNA expression showed only a 36-fold range in liver while a 150-fold range was observed in the liver metastases. In addition, dCK activity and mean mRNA levels were 2.5-fold higher in the metastases than in the liver samples. Since dCK is associated with the sensitivity to deoxynucleoside analogues and because of the good correlation between the different dCK measurements in malignant cells and tumours, the CT-RT PCR assay will be useful in the selection of patients that can be treated with deoxycytidine analogues.

Adult↗
PubMed2001

A possible role for methotrexate in the treatment of childhood acute myeloid leukaemia, in particular for acute monocytic leukaemia.

Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.

Antimetabolites, Antineoplastic↗
PubMed2001

Post-induction residual leukemia in childhood acute lymphoblastic leukemia quantified by PCR correlates with in vitro prednisolone resistance.

Most prognostic factors in childhood acute lymphoblastic leukemia (ALL) are informative for groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy for the individual patient. The residual leukemia following 4 weeks of induction therapy with prednisolone, vincristine, doxorubicin and i.t. methotrexate and the in vitro resistance to prednisolone, vincristine, and doxorubicin were measured in 30 boys and 12 girls with B (n = 34) or T lineage (n = 8) ALL. The residual leukemia was quantified after 2 (MRD-D15, n = 29) and 4 weeks (MRD-PI, n = 42) of induction therapy with a precise and reproducible clone-specific PCR technique. The median MRD-D15 and MRD-PI were 0.50% (75% range 0.0088.1%) and 0.014% (75% range 0.001-2.0%), respectively, and these levels correlated significantly (n = 29, rs = 0.75, P < 0.001). Both the MRD-D15 and the MRD-PI were related to the age of the patient (MRD-D15: rs= 0.48, P= 0.009; MRD-PI: rs = 0.45, P = 0.003). Patients with T lineage ALL had higher MRD-PI than those with B lineage ALL (median MRD-PI: 0.5% vs 0.01%, P = 0.05). The median LC50 (concentration lethal to 50% of cells) for prednisolone was 2.3 microg/ml (75% range 0.05-668). Both MRD-D15 and MRD-PI correlated significantly with the in vitro resistance to prednisolone (MRD-D15: rs = 0.41, P = 0.03; MRD-PI: rs = 0.39, P = 0.01); but not to in vitro vincristine or doxorubicin resistance. The correlations between MRD and in vitro prednisolone resistance were even more pronounced when B cell precursor and T cell leukemia were analyzed separately (B cell precursor ALL: MRD-PI vs prednisolone LC50: n = 33, rs = 0.47, P = 0.006; T cell ALL: MRD-PI vs prednisolone resistance: n = 8, rs = 0.84, P = 0.009). After a median follow-up of 5.0 years (75% range 3.2-6.9) eight patients have relapsed. All of the 21 patients with a MRD-PI < or =0.5% and a prednisolone LC50 < or =10 microg/ml have remained in remission whereas the 7 year event-free survival for the remaining 20 patients was 0.45 +/- 0.16 (P= 0.002) Prospective studies in childhood ALL are needed to clarify whether combined monitoring of in vitro drug resistance and residual leukemia early during chemotherapy could offer new ways to classify patients and stratify the intensity of therapy.

Adolescent↗
PubMed2000

Cellular drug resistance profiles in childhood acute myeloid leukemia: differences between FAB types and comparison with acute lymphoblastic leukemia.

Determining in vitro drug resistance may reveal clinically relevant information in childhood leukemia. Using the methyl-thiazol-tetrazolium assay, the resistance of untreated leukemic cells to 21 drugs was compared in 128 children with acute myeloid leukemia (AML) and 536 children with acute lymphoblastic leukemia (ALL). The differences between 3 French-American-British (FAB) types (M1/M2, M4, and M5) were also compared. AML was significantly more resistant than ALL to the following drugs, as noted by the median resistance: glucocorticoids (greater than 85-fold), vincristine (4.4-fold), L-asparaginase (6.9-fold), anthracyclines (1.8- to 3.4-fold), mitoxantrone (2.6-fold), etoposide (4.9-fold), platinum analogues (2.4- to 3.4-fold), ifosfamide (3.5-fold), and thiotepa (3.9-fold). For cytarabine and thiopurines, the median LC50 values (the drug concentration that kills 5% of the cells) were equal. Also, busulfan, amsacrine, teniposide, and vindesine showed no significant differences, but the numbers were smaller, and the median LC50 values were 1.3- to 5.2-fold higher in AML. None of the drugs demonstrated greater cytotoxicity in AML. FAB M5 was significantly more sensitive than FAB M4 to most drugs frequently used in AML, as indicated by the following ratios of median sensitivities: the anthracyclines (2.6- to 3.2-fold), mitoxantrone (12.5-fold), etoposide (8.7-fold), and cytarabine (2.9-fold). For etoposide and cytarabine (5.4- and 3.4-fold, respectively) FAB M5 was also significantly more sensitive than FAB M1/M2. FAB M5 was equally sensitive to L-asparaginase and vincristine as ALL. Only 15% of the AML samples were "intermediately" sensitive to glucocorticoids, mainly in FAB M1/M2. The poorer prognosis of childhood AML is related to resistance to a large number of drugs. Within AML, FAB M5 had a distinct resistance pattern. These resistance profiles may be helpful in the rational design of further treatment protocols. (Blood. 2000;96:2879-2886)

Acute Disease↗
PubMed2000

Asparagine synthetase activity in paediatric acute leukaemias: AML-M5 subtype shows lowest activity.

Lack of sufficient cellular activity of asparagine synthetase (AS) in blast cells compared with normal tissue is thought to be the basis of the antileukaemic effect of L-asparaginase in acute lymphoblastic leukaemia (ALL). Although L-asparaginase is routinely used in ALL, its role and value in the treatment of acute myelogenous leukaemia (AML) is still being discussed. To evaluate the pharmacological basis for L-asparaginase treatment, we established pretreatment monitoring of the intracellular AS activity in blast cells of patients with AML and ALL. There was no general difference in AS activity between ALL and AML samples. Significantly lower AS activity, however, was found in the B-lineage ALL subgroups as well as AML-M5.

Adolescent↗
PubMed1999

Circumvention of methotrexate resistance in childhood leukemia subtypes by rationally designed antifolates.

Cellular methotrexate (MTX) resistance may cause treatment failure in childhood common/preB-acute lymphoblastic leukemia (c/preB-ALL), T-lineage ALL (T-ALL), and acute myeloid leukemia (AML). The ex vivo potency of several antifolates (MTX, trimetrexate [TMQ], GW1843U89, multitargeted antifolate [MTA], Raltitrexed, and ZD9331) was studied via in situ inhibition of thymidylate synthase (TS). After short-term exposure, relapsed c/preB-ALL (rALL, n = 21), T-ALL (n = 22), and AML (n = 22) were 3-fold, 10-fold, and 6-fold less sensitive to MTX (P </=.01) compared with initial c/preB-ALL (n = 43). This difference in resistance was not observed for TMQ. Also for GW1843U89 and MTA, no resistance was observed in rALL and AML compared with c/preB-ALL. T-ALL compared with c/preB-ALL tended to be less resistant to GW1843U89 (3-fold) and MTA (6-fold) than to MTX (10-fold) (P =.06). Raltitrexed was more active against c/preB-ALL compared with the other subtypes. After 21 hours continuous incubation, T-ALL and AML samples were equally sensitive as c/preB-ALL to MTX, but rALL was 3-fold resistant to MTX compared with initial c/preB-ALL (P =.003). The resistance of rALL was circumvented by TMQ (1-fold; P =.03) and GW1843U89 (1.4-fold; P =. 004). Novel antifolates, except MTA, displayed a more potent TS inhibition than MTX during continuous exposure. These results suggest that MTX resistance in AML and T-ALL can be circumvented by continuous exposure, and that novel antifolates should be explored further for MTX-resistant T-ALL, rALL, and AML cells.

Antimetabolites, Antineoplastic↗
PubMed1999

Role of folylpolyglutamate synthetase and folylpolyglutamate hydrolase in methotrexate accumulation and polyglutamylation in childhood leukemia.

Inefficient polyglutamylation is a mechanism of resistance to methotrexate (MTX) in childhood T-lineage acute lymphoblastic leukemia (T-ALL) and in acute myeloid leukemia (AML) in comparison with childhood c/preB-ALL. We analyzed the profile of MTX polyglutamylation in childhood c/preB-ALL, T-ALL, and AML (n = 45, 15, and 14, respectively), the activity of the MTX-polyglutamate synthesizing enzyme folylpolyglutamate synthetase (FPGS) (n = 39, 11, and 19, respectively) and of the MTX-polyglutamate breakdown enzyme folylpolyglutamate hydrolase (FPGH) (n = 98, 25, and 34, respectively). MTX-Glu4-6 accumulation after 24 hours exposure to 1 micromol/L [3H]-MTX in vitro was lower in T-ALL (threefold) and AML (fourfold) compared with c/preB-ALL (P </=.001). The FPGS activity was twofold lower in T-ALL and AML than in c/preB-ALL samples (P <. 01). FPGH activity was not different between c/preB-ALL and T-ALL, but threefold higher in AML (P <.001). FPGS, FPGH, and the ratio FPGS/FPGH were correlated with MTX-Glu4-6 accumulation (r =.49, r = -.34 and r =.61, respectively). Multivariate analysis showed that FPGS, but not FPGH, was an independent contributor for MTX-Glu1-6 accumulation, but not for MTX-Glu4-6 accumulation. In conclusion, low FPGS activity is associated with low accumulation of MTX-Glu4-6 in T-ALL and AML. For the group of AML as compared with the group of ALL, a high FPGH activity can play an additional role.

Antimetabolites, Antineoplastic↗
PubMed1999

Differential methotrexate resistance in childhood T- versus common/preB-acute lymphoblastic leukemia can be measured by an in situ thymidylate synthase inhibition assay, but not by the MTT assay.

Methotrexate (MTX) is not cytotoxic to patient-derived acute lymphoblastic leukemia (ALL) cells in total-cell-kill assays, such as the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, putatively due to the rescue effects of hypoxanthine and thymidine released from dying cells. This was mimicked by a diminished methotrexate (MTX) cytotoxicity for the cell lines HL60 and U937 in the presence of hypoxanthine, thymidine, or lysed ALL cells. However, enzymatic depletion or inhibition of nucleoside membrane transport did not result in MTX dose-dependent cytotoxicity in patient samples. Alternatively, a thymidylate synthase inhibition assay (TSIA), based on inhibition of the TS-catalyzed conversion of 3H-dUMP to dTMP and 3H2O, correlated with the MTT assay for antifolate sensitivity in four human leukemia cell lines with different modes of MTX resistance. For 86 ALL patient samples, TSI50 values after 21 hours exposure to MTX were not different between T- and c/preB-ALL (P =.46). After 3 hours incubation with MTX followed by an 18-hour drug-free period, T-ALL samples were 3.4-fold more resistant to MTX compared with c/preB-ALL samples (P =.001) reflecting the clinical differences in MTX sensitivity. TSI50 values correlated with MTX accumulation (r = -.58, P <.001). In conclusion, the TSIA, but not the MTT assay, can measure dose-response curves for MTX in patient-derived ALL cells and showed relative MTX resistance in T-ALL compared with c/preB-ALL.

Adolescent↗
PubMed1999

BCL-2 expression in childhood leukemia versus spontaneous apoptosis, drug induced apoptosis, and in vitro drug resistance.

The antileukemic activity of cytotoxic drugs is increasingly thought to be the result of induction of apoptosis. Several proto-oncogenes have been related to the regulation of this process. In this study we evaluated the relation between bcl-2 expression, spontaneous and dexamethasone (DXM) induced apoptosis, and in vitro resistance to DXM, prednisolone (PRD) and cytarabine (ARA) determined using the total cell kill colorimetric methyl-thiazol-tetrazolium salt (MTT) assay, in childhood acute lymphoblastic leukemia (ALL). Drug resistance was expressed as the LC50 value, the drug concentration lethal to 50% of the cells. Fourty-six samples taken at initial diagnosis (iALL) and 31 samples taken at relapse (rALL) were incubated in culture medium, with and without DXM. Bcl-2 expression and apoptosis were measured flowcytometrically, the latter using DNA histogram analysis. Bcl-2 expression was 1.4 fold higher in rALL than in iALL (p = 0.008). Both spontaneous and DXM induced apoptosis increased significantly from 0 to 48 hours (in up to 71%, 81% of the cells respectively). Bcl-2 expression was inversely correlated with the extent of spontaneous apoptosis after 24 hours in iALL (r = -0.40, p = 0.05). Relapsed samples, but not samples obtained at presentation, expressing high levels of bcl-2 displayed increased resistance to drug induced apoptosis (r = -0.63, p = 0.02). In iALL high bcl-2 expression appeared to be related to low LC50 values of ARA. No correlations were found for DXM or PRD. In conclusion, DXM excerts its cytotoxic effect at least partly by means of induction of apoptosis. Bcl-2 inhibits drug induced apoptosis in rALL. However in iALL bcl-2 expression is not associated with increased in vitro drug resistance, nor with increased resistance to drug induced apoptosis.

Apoptosis↗
PubMed1999

Lack of cross-resistance between prednisolone and methotrexate in childhood acute lymphoblastic leukemia? A preliminary analysis.

We studied the cross-resistance between prednisolone (PRD) and methotrexate (MTX) in children with newly diagnosed acute lymphoblastic leukemia (ALL). This was done because of a previous observation that such patients could show a good clinical response to systemic PRD monotherapy plus intrathecal MTX, despite in vitro PRD resistant ALL cells (as determined with the MTT assay). This suggests an antileukemic effect of MTX, and thus the lack of cross-resistance between PRD and MTX. A systemic antileukemic effect of intrathecally administered MTX has been reported in the literature. Clinical good responders with PRD resistant ALL cells (n = 15) did not show unfavorable MTX-polyglutamylation nor unfavorable low inhibition of thymidylate synthase by MTX, as compared to a heterogeneous group of newly diagnosed ALL children (n = 47). In addition, we did not find a significant correlation between these two parameters and in vitro PRD resistance within the clinical good responders with PRD resistant ALL cells. In conclusion, we did not find a significant cross-resistance between PRD and MTX in vitro. It therefore may be that the good clinical response to systemic PRD plus intrathecal MTX in patients with in vitro PRD resistant ALL cells was caused by a systemic antileukemic activity of the intrathecally administered MTX.

Antimetabolites, Antineoplastic↗
PubMed1999

Effects of interleukin 3, interleukin 7, and B-cell growth factor on proliferation and drug resistance in vitro in childhood acute lymphoblastic leukemia.

Growth factors have been reported to enhance the cytotoxicity of anticancer agents. In our study we investigated the capacities of interleukin 3 (IL-3), interleukin 7 (IL-7), low-molecular-weight B-cell growth factor (lmw-BCGF), and IL-3 + 7 to induce proliferation and to modulate the drug resistance of childhood acute lymphoblastic leukemia (ALL) cells. Proliferation was assessed with the methyl-thiazole-tetrazolium (MTT) assay and other parameters. Cellular resistance to cytarabine, thioguanine, and prednisolone was measured using the MTT assay. In 19 samples containing >90% leukemic cells the proliferative response and the modulation of drug resistance was markedly heterogeneous between patient samples and between growth factors. All growth factors were able to stimulate proliferation significantly after 5 days of culture. lmw-BCGF was the most potent growth factor in this respect. Cytotoxicity of cytarabine and thioguanine was significantly increased by IL-7, that of thioguanine by IL-3 as well. IL-7 enhanced the cytotoxicity of thioguanine significantly more than IL-3 and lmw-BCGF and that of cytarabine more than IL-3. Cytotoxicity of prednisolone was not significantly influenced by any growth factor. In individual cases, growth factors reduced the cytotoxicity of the drugs. IL-3 + 7 did not add activity to the most potent single growth factor in both proliferation and drug resistance measurements. This study shows that IL-3, IL-7, and lmw-BCGF generally induce and occasionally inhibit proliferation of ALL cells. Furthermore, they may either increase or decrease cytotoxicity of anticancer drugs. This heterogeneous response to growth factors concerning induction of proliferation and modulation of drug resistance should be taken into account in their clinical use.

Cell Count↗
PubMed1999

Bcl-2 family members in childhood acute lymphoblastic leukemia: relationships with features at presentation, in vitro and in vivo drug response and long-term clinical outcome.

We have found that, in addition to Bcl-2 and Bax, the expression levels of apoptosis inducers (Bad, Bak) and inhibitors (Bcl-xL, Mcl-1) were highly variable in blasts from 78 children with newly diagnosed acute lymphoblastic leukemia (ALL). The patients were enrolled in the national study ALL-7 of the Dutch Childhood Leukemia Study Group. In contrast to Bcl-2 that inversely correlated with %S-phase cells and WBC, and was lower in T than in B-lineage ALL, the Bcl-2 family members were not found to be associated with features at presentation. These expression levels were also compared with drug resistance in in vitro MTT (methyl-thiazol-tetrazolium) assays for prednisolone, vincristine and asparaginase in 46 children. Protein expression levels of the Bcl-2 family were not found to correlate with in vitroresistance to the individual drugs or the combined drug resistance profile. In addition, neither peripheral blast reduction after 1 week of prednisone monotherapy nor long-term disease-free interval or survival showed a correlation with protein expression. Our results indicate that the anti-proliferative function of Bcl-2 dominates its anti-apoptotic function in ALL, but neither Bcl-2 nor the Bcl-2 family members gained prognostic information in the risk-adapted protocol ALL-7.

Adolescent↗
PubMed1999

Different expression of glutathione S-transferase alpha, mu and pi in childhood acute lymphoblastic and myeloid leukaemia.

Expression of three major classes of glutathione S-transferases (GSTs), i.e. alpha, mu and pi class, P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) were studied in childhood acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and normal peripheral blood lymphocytes by flow cytometry. In vitro cytotoxicity of 4-hydroxy-ifosfamide (IFOS), daunorubicin (DNR) and prednisolone (PRED) was assessed by the MTT assay. Expression of alpha, mu and pi class GST did not significantly differ between leukaemic cells from 100 initial and 14 unrelated relapse ALL patients (GSTalpha P=026; GSTmu P=O009; GSTpi P=0.13). The expression of GSTalpha (1.4-fold, P=0.0004), GSTpi (13-fold, P = 0001) and to a lesser extent also GSTmu (1.1-fold, P=0.03) was higher in ALL compared with normal peripheral blood lymphocytes. Expression of GSTmu and GST7pi was significantly higher in 18 AML compared with 100 ALL patients at initial diagnosis (respectively 1.3-fold, P=0.0005 and 2-fold, P<0.0001). In contrast, GSTalpha was median 2-fold lower expressed in the AML samples (P< 0.0001). Expression levels of alpha, mu and pi class GSTs were not related to the degree of resistance to IFOS, DNR and PRED nor to immunophenotype, white blood cell count or age at presentation of childhood ALL. One exception was a remarkably low expression of GSTalpha in IFOS-sensitive samples compared with a heterogenous expression in IFOS-resistant samples (P= 0.02). Expression of GSTpi, but not of GSTalpha or GSTmu, weakly correlated with the expression of MRP (Rs 0.36, P = 0.002, n = 74) but not with P-gp. However, a high expression of both GSTpi and MRP was not associated with in vitro resistance to IFOS, DNR or PRED. The present data suggest that expression of GSTs is not linked to the degree of resistance to IFOS, DNR and PRED or clinical risk factors in childhood ALL. Whether the high expression of GSTmu and GSTpi in AML cells contributes to the relative resistance to IFOS, DNR and PRED compared with ALL samples (P < or = 0.0001) warrants further study.

Acute Disease↗
PubMed1999

Cellular drug resistance in childhood acute myeloid leukemia. A mini-review with emphasis on cell culture assays.

Cellular drug resistance is an important limiting factor in the success of chemotherapy in childhood acute myeloid leukemia (AML). We summarize the results of the studies published sofar that have focussed on drug resistance in childhood AML, using cell culture assays. We also briefly report our own results of an ongoing study. Finally, potential applications of cellular drug resistance testing are discussed. It appears that cellular drug resistance differs between AML and acute lymphoblastic leukemia and between subgroups of AML patients, that AML cells of relapsed patients are more resistance to cytarabine than those of untreated patients, and that in vitro resistance to cytarabine and daunorubicin is related to a worse prognosis. However, more and larger studies are required to determine the exact role of cellular drug resistance testing in the treatment of childhood AML.

Antineoplastic Agents↗