Effect of cyclophosphamide on gene expression of cytochromes p450 and beta-actin in the HL-60 cell line.
Many studies have demonstrated that cyclophosphamide (CPA) can affect hepatic cytochrome p450 (CYP) isoenzyme activity in animals. We have investigated the effect of CPA on gene expression of various CYP enzymes as well as beta-actin in the human acute promyelocytic leukemia cell line (HL-60S) and its multidrug-resistant (MDR) phenotype HL-60R. Cells were incubated at different concentrations of CPA ranging between 50 micro g/ml and 5 mg/ml. In determination of cytotoxicity and resistance factor (RF: IC(50) HL-60R/IC(50) HL-60S), concentrations of 100 and 500 micro g/ml CPA were selected to treat HL-60S and HL-60R up to 72 h. CYP gene expression in the cells prior to and after treatment with CPA was determined using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR. Unexposed cell lines did not contain measurable levels of mRNA for CYP2B6, CYP3A4, CYP2C9 and CYP2C19 and no induction was observed after exposure. However, CYP1B1-specific mRNA, which is predominantly expressed in HL-60 cell line, was suppressed after exposure to CPA in a concentration-dependent manner. Beta-actin gene expression was also decreased. The HL-60 RF to CPA was calculated to 0.71, indicating that the multidrug-resistant (MDR) phenotype is not involved in the mechanism of resistance to CPA. No CYPs were induced by CPA in vitro, which probably indicates that the CYP inducibility in blood cells is poor. Our study suggests that suppression of beta-actin gene expression contributes or is involved in the CPA cytotoxicity.
Xie
Han-Jing
HJ
Division of Clinical Pharmacology, Department of Medical Laboratory Sciences and Technology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
Lundgren
Stefan
S
Broberg
Ulrika
U
Finnström
Niklas
N
Rane
Anders
A
Hassan
Moustapha
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects; D003520:Cyclophosphamide / Q000494:pharmacology*; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D010641:Phenotype; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000199:Actins; D018906:Antineoplastic Agents, Alkylating; D018076:DNA, Complementary; D012333:RNA, Messenger; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1