Quantitative analysis of breast cancer resistance protein and cellular resistance to flavopiridol in acute leukemia patients.
OBJECTIVE
Flavopiridol is a cyclin-dependent kinase inhibitor currently undergoing human clinical trials. As clinical development is pursued, it becomes important to evaluate resistance mechanisms to flavopiridol. To elucidate the contribution of breast cancer resistance protein (BCRP) to cellular resistance to flavopiridol in acute myeloid leukemia, we studied the relationship between cellular resistance to flavopiridol and mRNA expression of BCRP or P-glycoprotein (P-gp, product of MDR1gene) in blast cells from adult patients with acute leukemia.


METHODS
Twenty-one blast cell samples from 20 patients were studied. The expression of BCRP, P-gp, or beta-actin mRNA was determined by real-time reverse transcription-PCR, using fluorescent hybridization probes to evaluate codon 482, a known site of mutations in BCRP mRNA. In vitro cell viability and apoptosis were examined after 24 h exposure to flavopiridol.


RESULTS
BCRP mRNA expression varied over a 200-fold range. In the blast cell samples with BCRP mRNA expression > 10000 copies/pg beta-actin (n = 9), BCRP mRNA correlated proportionally with cell viability in the presence of 250 nM flavopiridol (r = 0.86, P = 0.003) and with apoptosis induced by flavopiridol (r = 0.71, P = 0.031). In contrast, MDR1mRNA expression did not correlate with either flavopiridol cytotoxicity or induction of apoptosis. Melting point analysis of the hybridization probes determined that all 21 patient samples had arginine at codon 482 of BCRP mRNA, the wild-type form.


CONCLUSIONS
These results suggest that unlike P-gp, BCRP may play a role in leukemia cellular resistance to flavopiridol. No mutations at codon 482 were observed in BCRP mRNA in this group of patients.
Nakanishi
Takeo
T
Department of Medicine, Division of Hematology/Oncology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Karp
Judith E
JE
Tan
Ming
M
Doyle
L Austin
LA
Peters
Todd
T
Yang
Weidong
W
Wei
David
D
Ross
Douglas D
DD
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis*; D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival; D003062:Codon; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000627:therapeutic use*; D018922:HL-60 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009363:Neoplasm Proteins / Q000096:biosynthesis*; D010880:Piperidines / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature; D013997:Time Factors
C116614:ABCG2 protein, human; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000199:Actins; D000970:Antineoplastic Agents; D003062:Codon; D018076:DNA, Complementary; D005419:Flavonoids; D009363:Neoplasm Proteins; D010880:Piperidines; D012333:RNA, Messenger; C077990:alvocidib