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

Abstract 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.

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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

Blood ◽

10.1182/blood.v93.3.1067.403k01_1067_1074 ◽

1999 ◽

Vol 93 (3) ◽

pp. 1067-1074 ◽

Cited By ~ 3

Author(s):

Marianne G. Rots ◽

Rob Pieters ◽

Gert-Jan L. Kaspers ◽

Christina H. van Zantwijk ◽

Paul Noordhuis ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Mtt Assay ◽

Thymidylate Synthase ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Human Leukemia ◽

Inhibition Assay ◽

Response Curves ◽

Methotrexate Resistance

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.

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The NK-1 receptor is expressed in human leukemia and is involved in the antitumor action of aprepitant and other NK-1 receptor antagonists on acute lymphoblastic leukemia cell lines

Investigational New Drugs ◽

10.1007/s10637-010-9594-0 ◽

2010 ◽

Vol 30 (2) ◽

pp. 529-540 ◽

Cited By ~ 26

Author(s):

Miguel Muñoz ◽

Ana González-Ortega ◽

Rafael Coveñas

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Antitumor Action ◽

Human Leukemia ◽

Receptor Antagonists ◽

Leukemia Cell Lines

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Homozygous deletion of the retinoblastoma gene in an acute lymphoblastic leukemia (T) cell line

Blood ◽

10.1182/blood.v75.3.730.bloodjournal753730 ◽

1990 ◽

Vol 75 (3) ◽

pp. 730-735

Author(s):

J Cheng ◽

P Scully ◽

JY Shew ◽

WH Lee ◽

V Vila ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Homozygous Deletion ◽

Human Leukemia ◽

Rb Protein ◽

Leukemia Cell Lines ◽

The Status ◽

Rb Gene

Human leukemia cell lines were examined for the status of the retinoblastoma (RB) protein by immunoblotting analysis using antibodies raised against the TrpE-RB fusion protein. One of 16 cell lines examined, the T-cell acute lymphoblastic leukemia (ALL) line HSB-2, lacked the 110-Kd RB protein. Southern blot analysis of genomic DNA extracted from HSB-2 cells showed a large homozygous deletion of the RB gene, stretching from exon 18 beyond exon 27. Northern blot analysis showed multiple, abnormal RB transcripts in HSB-2. A truncated protein (72 Kd) was detected with 35S-methionine labeling but not with 32P- orthophosphate labeling of the HSB-2 cells. The genomic deletion of greater than 85 kb DNA at the RB locus (13q14) was not detectable in the karyotype of the HSB-2 cells. Among the 16 human leukemia cell lines examined for the status of the RB gene, only one, the HSB-2 line, showed an abnormal RB protein. Further study of primary leukemia and lymphoma samples appears to be warranted.

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Homozygous deletion of the retinoblastoma gene in an acute lymphoblastic leukemia (T) cell line

Blood ◽

10.1182/blood.v75.3.730.730 ◽

1990 ◽

Vol 75 (3) ◽

pp. 730-735 ◽

Cited By ~ 43

Author(s):

J Cheng ◽

P Scully ◽

JY Shew ◽

WH Lee ◽

V Vila ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Homozygous Deletion ◽

Human Leukemia ◽

Rb Protein ◽

Leukemia Cell Lines ◽

The Status ◽

Rb Gene

Abstract Human leukemia cell lines were examined for the status of the retinoblastoma (RB) protein by immunoblotting analysis using antibodies raised against the TrpE-RB fusion protein. One of 16 cell lines examined, the T-cell acute lymphoblastic leukemia (ALL) line HSB-2, lacked the 110-Kd RB protein. Southern blot analysis of genomic DNA extracted from HSB-2 cells showed a large homozygous deletion of the RB gene, stretching from exon 18 beyond exon 27. Northern blot analysis showed multiple, abnormal RB transcripts in HSB-2. A truncated protein (72 Kd) was detected with 35S-methionine labeling but not with 32P- orthophosphate labeling of the HSB-2 cells. The genomic deletion of greater than 85 kb DNA at the RB locus (13q14) was not detectable in the karyotype of the HSB-2 cells. Among the 16 human leukemia cell lines examined for the status of the RB gene, only one, the HSB-2 line, showed an abnormal RB protein. Further study of primary leukemia and lymphoma samples appears to be warranted.

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Baicalin-Induced Apoptosis is Mediated by Bcl-2-Dependent, but not p53-Dependent, Pathway in Human Leukemia Cell Lines

The American Journal of Chinese Medicine ◽

10.1142/s0192415x06003801 ◽

2006 ◽

Vol 34 (02) ◽

pp. 245-261 ◽

Cited By ~ 34

Author(s):

Den-En Shieh ◽

Hua-Yew Cheng ◽

Ming-Hong Yen ◽

Lien-Chai Chiang ◽

Chun-Ching Lin

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Cytotoxic Effect ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Chemotherapeutic Agents ◽

Human Leukemia ◽

Potential Candidate ◽

Scutellaria Baicalensis Georgi

Acute lymphoblastic leukemia (ALL), especially T-acute lymphoblastic leukemia (T-ALL), is a common childhood malignant neoplastic disorder. Chemotherapy agents, particularly those that can induce apoptosis, are the major intervening strategy in the treatment of ALL. In this study, we investigated in T-ALL cell line, CCRF-CEM, the in vitro cytotoxic effect and the mechanism of action of baicalin, a compound extracted from Scutellaria baicalensis Georgi and S. rivularis Benth (Labiateae). Results demonstrated that baicalin displayed a remarkable cytotoxic effect in CCRF-CEM, with an IC50value of 10.6 μg/ml. It triggered apoptotic effect by fragmentizing cellular DNA and arrested the cell cycle at G0/ G1phase. Baicalin (37.5 μg/ml)had not effected the expression of p53 and Fas protein. It was shown to decline the expression of Bcl-2 (22.0 pg/ml), which consequently caused the loss (52.7%)of transmembrane potential (ΔΨm) in the mitochondria after 72 hours of treatment. Baicalin (37.5 μg/ml) also elevated the amount of cytosolic cytochrome c (19.2 μg/ml), which finally triggered the activation of caspase-3 (50.1 pmol/min). In conclusion, baicalin was found to induce apoptosis in T-ALL cell lines through multiple pathways. This finding encourages further investigation of baicalin in its role as a potential candidate for chemotherapeutic agents in T-ALL.

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