Bcl-2, bax and bcl-xL expression in human sensitive and resistant leukemia cell lines.
With the growing understanding of cytostatic drug-induced programmed cell death new drug-resistance mechanisms based on the altered ability of cells to die by apoptosis have been defined. At first, the sensitive and P-glycoprotein (P-gp)-related resistant cell lines were tested to induce apoptosis by a non-P-gp transported drug, such as cytosine arabinoside (ara-C). It was demonstrated that ara-C induces apoptosis in sensitive as well as in P-gp-related resistant cell lines, as expected. Furthermore, the role of bcl-2 and bcl-xL apoptosis inhibitors as well as bax expression (apoptosis inducer) in human sensitive leukemic cell lines (CCRF-CEM and HL-60) as compared to their resistant variants such as CCRF-CEM/ACT400, CCRF-CEM/VCR1000, HL-60/IDA40, HL-60/DNR250 was evaluated. In addition to the P-gp-related resistance, a possible multidrug resistance-associated protein (MRP) and the lung resistance protein (LRP)-related resistance were assessed by flow cytometry using the monoclonal antibodies 4E3.16, MRPr1 and LRP56. Furthermore, the function of P-gp was determined with the rhodamine-123 (R-123) accumulation test. Bcl-2 and bax were analyzed by both flow cytometry and ECL Western blot, bcl-xL by ECL-Western blot alone. Comparison of the two sensitive cell lines demonstrated different bcl-2, bax and bcl-xL patterns. The common characteristic was the increased expression of one of the apoptosis inhibitor proteins, such as bcl-2 or bcl-xL. The sensitive CCRF-CEM showed a high bax level, where a decrease of about 75% in resistant variants was measured. Compared to their sensitive counterpart HL-60, a low bax expression was analyzed, which increased in the resistant variant. The common characteristic of all resistant cell lines was the decreased expression of bax compared to bcl-2 or bcl-xL. In the P-gp-related resistant HL-60/DNR250 only an increase in bcl-xL was seen, whereas in the LRP-expressing as well as P-gp and MRP negative resistant HL-60/IDA40 both apoptotic inhibitor proteins bcl-2 and bcL-xL showed maximum increase, compared to the other resistant cell lines. The P-gp-related resistant cell lines CCRF-CEM/ACT400 and CCRF-CEM/VCR1000 also showed an increased expression of both bcl-2 and bcl-xL. Summarizing these results, it was shown that the examined sensitive human leukemic cell lines and their resistant variants demonstrated a different pattern of markers for preventing and promoting apoptosis. An association between P-gp and possible LRP-expressing leukemic cells as well as apoptosis-preventing markers (bcl-2, bcl-xL) seems to exist. The clinical relevance of the coexpression of various resistance mechanisms remains to be confirmed in large leukemia patient groups.
Nuessler
V
V
Klinikum Grosshadern, Medizinische Klinik und Poliklinik III, Munich, Germany.
Stötzer
O
O
Gullis
E
E
Pelka-Fleischer
R
R
Pogrebniak
A
A
Gieseler
F
F
Wilmanns
W
W
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D017304:Annexin A5 / Q000032:analysis; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D003561:Cytarabine / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005434:Flow Cytometry; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007938:Leukemia / Q000378:metabolism* / Q000473:pathology*; D008856:Microscopy, Fluorescence; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D020394:Vault Ribonucleoprotein Particles; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099187:Major Vault Protein
D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D017304:Annexin A5; D000970:Antineoplastic Agents; D003561:Cytarabine; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D020394:Vault Ribonucleoprotein Particles; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D000099187:Major Vault Protein; C494826:BAX protein, human; C494811:BCL2L1 protein, human