Expression of Cytochrome P450 Genes in CD34+ Hematopoietic Stem and Progenitor Cells
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《干细胞学杂志》
a Biotransformations Group, National Institute of Public Health, Center of Occupational Diseases, robárova, Czech Republic;
b Institute of Pharmacology, Faculty of Medicine, Palacky University at Olomouc and
c Hematooncological Department, Faculty Hospital Olomouc, Olomouc, Czech Republic;
d Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Key Words. CD34+ ? Cytochrome P450 ? Expression ? Progenitor cells ? Real-time polymerase chain reaction
Correspondence: Pavel Souek, Ph.D., Biotransformations Group, National Institute of Public Health, Center of Occupational Diseases, robárova 48, Praha 10, Czech Republic. Telephone: 420-267-082-711; Fax: 420-267-311-236; e-mail: psoucek@szu.cz
ABSTRACT
Cytochromes P450 (P450, EC 1.14.14.1 ) represent the most important group of biotransformation enzymes and play a key role in the metabolism of various endogenous chemicals (e.g., steroids), drugs of medical use, and environmentally related mutagens and carcinogens . This study focused on the presence of four forms of P450 enzymes in CD34+ human hematopoietic stem and early progenitor cells (PBSPCs).
P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis . It is expressed in several human tissues in which cancers typically occur such as prostate, ovary, uterus, and mammary gland .
P450 2E1 is known to be responsible for metabolic activation of many low-molecular-weight compounds, which are either toxic or suspected to act as chemical carcinogens (e.g., chlorinated hydrocarbons and nitrosamines ). P450 2E1 gene (CYP2E1) is expressed at the early stage of human fetus development, which indicates its importance for developing human organism . It is one of the major hepatic P450 enzymes; however, it has also been detected at significant levels in human esophagus, kidney, lung , and brain .
P450 2C9 contributes not only to metabolism of drugs but also to biotransformation of fatty acids, prostanoids, and steroid hormones. It may catalyze potentially toxic bioactivation reactions . P450 2C9 expression was found in the liver, kidney, adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, and epithelial cells in the skin, prostatic ducts, and gastrointestinal tract .
The steroid hydroxylase P450 3A4 is the most abundant P450 enzyme in the human liver, and P450 3A enzymes metabolize more than 50% of prescription drugs. The CYP3A4 is expressed in the liver, gut, colon, prostate, and breast . Structurally similar, P450 3A7 is the major fetal form, which implies the role of P450 3A enzymes in early development .
The ratio of activation/detoxification of chemicals by P450s may modulate individual susceptibility to diseases such as cancer and influence therapeutic response to various drugs. Therefore, it is extremely important to assess expression and activity of these enzymes in biological systems for which use is considered in future human medicine. Stem cells are an excellent example among such systems . The information about expression of genes coding for biotransformation enzymes, especially P450s in population of CD34+ human hematopoietic stem and progenitor cells, is still limited. To date, expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1, and 3A4) was demonstrated in human bone marrow, in various types of macrophages, in cell lines HL-60 and HEL, and in Epstein-Barr virus–transformed B-lymphoblastoid cell lines as well as in mononuclear cells . Presence of P450 2E1 in bone marrow cells was indicated by Western blotting . The present study focused on bridging this gap in our knowledge. We have investigated gene expression and protein levels of four major P450 enzymes in a highly purified population of human CD34+ progenitor and stem cells (devoid of mononuclear leukocytes) obtained from peripheral blood.
MATERIALS AND METHODS
Isolation of CD34+ PBSPCs, Total RNA, and cDNA Quality Assessment
Samples of CD34+ cells were prepared by magnetic bead sorting as described above. The final preparation contained 2.7 x 108 cells with purity over 96% as assessed by flow cytometry with fluorescein isothiocyanate–labeled antibodies. The total RNA was isolated from PBSPCs by Trizol procedure. The yield was 0.82 μg/μl. In total, 108 μg of total RNA was obtained with A260/280 ratio of 1.98 according to spectrophotometry. Agarose electrophoresis showed that RNA was pure and not degraded (no high-molecular-weight bands, distinct bands of 18S and 28S rRNA, no smears under 18S). Total RNA in amount of 0.5 and 1 μg was used for cDNA synthesis. Purity of cDNA was checked by PCR amplification of ubiquitin C fragment. This test proved that synthesized cDNA was present in full length and was not significantly contaminated by genomic DNA, as judged by absence of band of 1,009 bp in negative controls (Fig. 1).
Figure 1. Quality control of CD34+ cDNA using polymerase chain reaction amplification of ubiquitin C fragment. Lane 1, cDNA-negative control from 1 μg of total RNA; lane 2, cDNA-negative control from 0.5 μg of total RNA; lane 3, cDNA from 1 μg of total RNA; lane 4, cDNA from 0.5 μg of total RNA; lane 5, MW, X174DNA/HaeIII digest.
QRTPCR Determination of Cyclophilin A and CYP Expression
Samples of PBSPC cDNA described above were diluted 10 times with Milli-Q ultrapure water and analyzed by QRTPCR. Assay of cyclophilin A expression was used as internal control of gene expression in various clinical samples . In the cells, cyclophilin A expression was very high, suggesting that during handling and processing of samples, no significant RNA degradation occurred (Table 1). Thus, expression levels of CYPs were determined in subsequent experiments. Results revealed very high expression of CYP1B1 (more than 750,000 copies/μg of RNA), which was in stem cells not reported so far (Table 1). CYP2E1 expression was moderate (less than 10,000 copies/μg of RNA), and expression levels of CYP2C9 and CYP3A4 were below the limit of quantification (Table 1). Reaction efficiency was consistent in all assays, and calibration curves showed very good linearity (Table 1). Also, performance of all tests in samples representing 0.5 and 1 μg of total RNA was satisfactory, including zero signals in negative cDNA controls. Control experiment showed no expression of CYP1B1 in breast cancer cell line MDA-MB-435, high expression in resistant breast cancer cell line NCI/ADR-RES, and moderate expression in intestinal Caco-2 cell line (results not shown). The expression of CYP2C9 and 2E1 varied from low to high in these cells (up to 20 times difference, results not shown). CYP3A4 expression in the three cell lines used was negligible.
Table 1. Gene expressions of CYP1B1, 2C9, 2E1, 3A4, and cyclophilin A in CD34+ cells
Results of Immunoblotting Experiments
Western blotting was performed with cells washed and lysed as stated under Materials and Methods to liberate the P450 enzymes from membranes of endoplasmic reticulum. The procedure was sufficiently reliable to get samples yielding positive traces for P450 1B1 (Fig. 2A) in all samples studied as well as for the P450 2E1 (Fig. 2B). Under the same conditions, no expression of P450 2E1 was observed in human keratinocytes derived from HaCaT cell line serving as negative control (results not shown). On the other hand, no bands for P450 3A4/7 were observed. Also, no traces indicating the presence of the P450 2C9 protein were found, which is in line with the preliminary results showing absence of the specific activity of P450 2C9 in the PBSPC microsomes. Hence, the Western blotting confirmed the expression of two P450 forms, P450 1B1 and P450 2E1, in the CD34+ PBSPCs. In parallel, the Northern blotting experiments were performed also, confirming the same results obtained with Western blotting as well as with the QRTPCR (presence of the CYP2E1 and CYP1B1 mRNA and absence of the CYP3A4, CYP3A7, and CYP2C9 mRNA in the sample of the PBPSC total RNA, results not shown).
Figure 2. Immunoblotting of CD34+ cell lysate stained with antibodies against P450 1B1 (A) and P450 2E1 (B). (A): From left to right: lane 1, P450 1B1 standard (positive control); lanes 2–4, human liver microsomes (negative controls); lanes 5, 6, CD34+ cell lysate (two different protein concentrations, corresponding to 3 and 7 μg of protein/lane). (B): From left to right: lane 1, P450 2E1 standard (positive control); lane 2, human liver microsomes (positive control); lanes 3, 4, empty lanes; lanes 5–7, CD34+ lysate (5 μg of protein/lane in triplicate).
DISCUSSION
Our results show expression of two environmentally important P450s but not of other pharmacologically active ones (P450 3A4/7, P450 2C9) in CD34+ hematopoietic stem and progenitor cells. The presence of some P450 forms in the earliest phase of hematopoiesis may also suggest that besides the known protective role of P450 enzymes, both expressed forms may have so far unknown essential roles in development and maturation of hematopoietic cells.
ACKNOWLEDGMENTS
Anzenbacher P, Anzenbacherová E. Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001;58:737–747.
Guengerich FP, Chun YJ, Kim D et al. Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies. Mutat Res 2003;523-524:173–182.
Shimada T, Hayes CL, Yamazaki H et al. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 1996;56:2979–2984.
Guengerich FP, Kim DH, Iwasaki M. Role of human cytochrome P450 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 1991;4:168–179.
Johnsrud EK, Koukouritaki SB, Divakaran K et al. Human hepatic CYP2E expression during development. J Pharm Exp Ther 2003;307:402–407.
Lieber CS. Cytochrome P450 2E1: its physiological and pathological role. Physiol Rev 1997;77:517–544.
Upadhya SC, Tirumalai PS, Boyd MR et al. Cytochrome P4502E (CYP2E) in brain: constitutive expression, induction by ethanol and localization by fluorescence in situ hybridization. Arch Biochem Biophys 2000;373:23–34.
Kirchheiner J, Brockmoller J. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther 2005;77:1–16.
Enayetallah AE, French RA, Thibodeau MS et al. Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 2004;52:447–454.
Keshava C, McCanlies EC, Weston A. CYP3A4 polymorphisms-potential risk factors for breast and prostate cancer: a HuGE review. Am J Epidemiol 2004;160:825–841.
Kitada M, Kato T, Ohmori S et al. Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers. Biochim Biophys Acta 1992;1117:301–305.
Vollweiler JL, Zielske SP, Reese JS et al. Hematopoietic stem cell gene therapy: progress toward therapeutic targets. Bone Marrow Transplant 2003;32:1–7.
Hodges VM, Molloy GY, Wickramasinghe SN. Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow- derived macrophages and human haemopoietic cell lines. Br J Haematol 2000;108:151–156.
Nguyen LT, Ramanathan M, Weinstock-Guttman B et al. Detection of cytochrome P450 and other drug-metabolizing enzyme mRNAs in peripheral blood mononuclear cells using DNA arrays. Drug Metab Dispos 2000;28:987–993.
Bernauer U, Vieth B, Ellrich R et al. CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals. Arch Toxicol 2000;73:618–624.
Haufroid V, Toubeau F, Clippe A et al. Real-time quantification of cytochrome P4502E1 mRNA in human peripheral blood lymphocytes by reverse transcription-PCR: method and practical application. Clin Chem 2001;47:1126–1129.
Carlsson E, Fredriksson J, Groop L et al. Variation in the calpain-10 gene is associated with elevated triglyceride levels and reduced adipose tissue messenger ribonucleic acid expression in obese Swedish subjects. J Clin Endocrinol Metab 2004;89:3601–3605.
Schmid H, Cohen CD, Henger A et al. Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int 2003;64:356–360.
Kousalová L, Anzenbacherová E, Baranová J et al. Presence of cytochrome P450 enzymes in human CD34+ hematopoietic progenitor cells. Gen Physiol Biophys 2004;23:251–257.
Guillén MI, Donato MT, Jover R et al. Oncostatin M down-regulates basal and induced cytochromes P450 in human hepatocytes. J Pharmacol Exper Ther 1998;285:127–134.
Hukkanen J, Hakkola J, Antilla S et al. Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in humanbronchoalveolar macrophages and peripheral blood lymphocytes. Mol Carcinog 1997;20:224–230.
Nagai F, Hiyoshi Y, Sugimachi K et al. Cytochrome P450 (CYP) expression in human myeloblastic and lymphoid cell lines. Biol Pharm Bull 2002;25:383–385.(Pavel Soueka, Pavel Anzen)
b Institute of Pharmacology, Faculty of Medicine, Palacky University at Olomouc and
c Hematooncological Department, Faculty Hospital Olomouc, Olomouc, Czech Republic;
d Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Key Words. CD34+ ? Cytochrome P450 ? Expression ? Progenitor cells ? Real-time polymerase chain reaction
Correspondence: Pavel Souek, Ph.D., Biotransformations Group, National Institute of Public Health, Center of Occupational Diseases, robárova 48, Praha 10, Czech Republic. Telephone: 420-267-082-711; Fax: 420-267-311-236; e-mail: psoucek@szu.cz
ABSTRACT
Cytochromes P450 (P450, EC 1.14.14.1 ) represent the most important group of biotransformation enzymes and play a key role in the metabolism of various endogenous chemicals (e.g., steroids), drugs of medical use, and environmentally related mutagens and carcinogens . This study focused on the presence of four forms of P450 enzymes in CD34+ human hematopoietic stem and early progenitor cells (PBSPCs).
P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis . It is expressed in several human tissues in which cancers typically occur such as prostate, ovary, uterus, and mammary gland .
P450 2E1 is known to be responsible for metabolic activation of many low-molecular-weight compounds, which are either toxic or suspected to act as chemical carcinogens (e.g., chlorinated hydrocarbons and nitrosamines ). P450 2E1 gene (CYP2E1) is expressed at the early stage of human fetus development, which indicates its importance for developing human organism . It is one of the major hepatic P450 enzymes; however, it has also been detected at significant levels in human esophagus, kidney, lung , and brain .
P450 2C9 contributes not only to metabolism of drugs but also to biotransformation of fatty acids, prostanoids, and steroid hormones. It may catalyze potentially toxic bioactivation reactions . P450 2C9 expression was found in the liver, kidney, adrenals, pancreatic islets, pituitary gland, lymphoid tissues, muscles, and epithelial cells in the skin, prostatic ducts, and gastrointestinal tract .
The steroid hydroxylase P450 3A4 is the most abundant P450 enzyme in the human liver, and P450 3A enzymes metabolize more than 50% of prescription drugs. The CYP3A4 is expressed in the liver, gut, colon, prostate, and breast . Structurally similar, P450 3A7 is the major fetal form, which implies the role of P450 3A enzymes in early development .
The ratio of activation/detoxification of chemicals by P450s may modulate individual susceptibility to diseases such as cancer and influence therapeutic response to various drugs. Therefore, it is extremely important to assess expression and activity of these enzymes in biological systems for which use is considered in future human medicine. Stem cells are an excellent example among such systems . The information about expression of genes coding for biotransformation enzymes, especially P450s in population of CD34+ human hematopoietic stem and progenitor cells, is still limited. To date, expression of mRNA for five cytochrome P450s (CYP1A1, 2A6/7, 2D6, 2E1, and 3A4) was demonstrated in human bone marrow, in various types of macrophages, in cell lines HL-60 and HEL, and in Epstein-Barr virus–transformed B-lymphoblastoid cell lines as well as in mononuclear cells . Presence of P450 2E1 in bone marrow cells was indicated by Western blotting . The present study focused on bridging this gap in our knowledge. We have investigated gene expression and protein levels of four major P450 enzymes in a highly purified population of human CD34+ progenitor and stem cells (devoid of mononuclear leukocytes) obtained from peripheral blood.
MATERIALS AND METHODS
Isolation of CD34+ PBSPCs, Total RNA, and cDNA Quality Assessment
Samples of CD34+ cells were prepared by magnetic bead sorting as described above. The final preparation contained 2.7 x 108 cells with purity over 96% as assessed by flow cytometry with fluorescein isothiocyanate–labeled antibodies. The total RNA was isolated from PBSPCs by Trizol procedure. The yield was 0.82 μg/μl. In total, 108 μg of total RNA was obtained with A260/280 ratio of 1.98 according to spectrophotometry. Agarose electrophoresis showed that RNA was pure and not degraded (no high-molecular-weight bands, distinct bands of 18S and 28S rRNA, no smears under 18S). Total RNA in amount of 0.5 and 1 μg was used for cDNA synthesis. Purity of cDNA was checked by PCR amplification of ubiquitin C fragment. This test proved that synthesized cDNA was present in full length and was not significantly contaminated by genomic DNA, as judged by absence of band of 1,009 bp in negative controls (Fig. 1).
Figure 1. Quality control of CD34+ cDNA using polymerase chain reaction amplification of ubiquitin C fragment. Lane 1, cDNA-negative control from 1 μg of total RNA; lane 2, cDNA-negative control from 0.5 μg of total RNA; lane 3, cDNA from 1 μg of total RNA; lane 4, cDNA from 0.5 μg of total RNA; lane 5, MW, X174DNA/HaeIII digest.
QRTPCR Determination of Cyclophilin A and CYP Expression
Samples of PBSPC cDNA described above were diluted 10 times with Milli-Q ultrapure water and analyzed by QRTPCR. Assay of cyclophilin A expression was used as internal control of gene expression in various clinical samples . In the cells, cyclophilin A expression was very high, suggesting that during handling and processing of samples, no significant RNA degradation occurred (Table 1). Thus, expression levels of CYPs were determined in subsequent experiments. Results revealed very high expression of CYP1B1 (more than 750,000 copies/μg of RNA), which was in stem cells not reported so far (Table 1). CYP2E1 expression was moderate (less than 10,000 copies/μg of RNA), and expression levels of CYP2C9 and CYP3A4 were below the limit of quantification (Table 1). Reaction efficiency was consistent in all assays, and calibration curves showed very good linearity (Table 1). Also, performance of all tests in samples representing 0.5 and 1 μg of total RNA was satisfactory, including zero signals in negative cDNA controls. Control experiment showed no expression of CYP1B1 in breast cancer cell line MDA-MB-435, high expression in resistant breast cancer cell line NCI/ADR-RES, and moderate expression in intestinal Caco-2 cell line (results not shown). The expression of CYP2C9 and 2E1 varied from low to high in these cells (up to 20 times difference, results not shown). CYP3A4 expression in the three cell lines used was negligible.
Table 1. Gene expressions of CYP1B1, 2C9, 2E1, 3A4, and cyclophilin A in CD34+ cells
Results of Immunoblotting Experiments
Western blotting was performed with cells washed and lysed as stated under Materials and Methods to liberate the P450 enzymes from membranes of endoplasmic reticulum. The procedure was sufficiently reliable to get samples yielding positive traces for P450 1B1 (Fig. 2A) in all samples studied as well as for the P450 2E1 (Fig. 2B). Under the same conditions, no expression of P450 2E1 was observed in human keratinocytes derived from HaCaT cell line serving as negative control (results not shown). On the other hand, no bands for P450 3A4/7 were observed. Also, no traces indicating the presence of the P450 2C9 protein were found, which is in line with the preliminary results showing absence of the specific activity of P450 2C9 in the PBSPC microsomes. Hence, the Western blotting confirmed the expression of two P450 forms, P450 1B1 and P450 2E1, in the CD34+ PBSPCs. In parallel, the Northern blotting experiments were performed also, confirming the same results obtained with Western blotting as well as with the QRTPCR (presence of the CYP2E1 and CYP1B1 mRNA and absence of the CYP3A4, CYP3A7, and CYP2C9 mRNA in the sample of the PBPSC total RNA, results not shown).
Figure 2. Immunoblotting of CD34+ cell lysate stained with antibodies against P450 1B1 (A) and P450 2E1 (B). (A): From left to right: lane 1, P450 1B1 standard (positive control); lanes 2–4, human liver microsomes (negative controls); lanes 5, 6, CD34+ cell lysate (two different protein concentrations, corresponding to 3 and 7 μg of protein/lane). (B): From left to right: lane 1, P450 2E1 standard (positive control); lane 2, human liver microsomes (positive control); lanes 3, 4, empty lanes; lanes 5–7, CD34+ lysate (5 μg of protein/lane in triplicate).
DISCUSSION
Our results show expression of two environmentally important P450s but not of other pharmacologically active ones (P450 3A4/7, P450 2C9) in CD34+ hematopoietic stem and progenitor cells. The presence of some P450 forms in the earliest phase of hematopoiesis may also suggest that besides the known protective role of P450 enzymes, both expressed forms may have so far unknown essential roles in development and maturation of hematopoietic cells.
ACKNOWLEDGMENTS
Anzenbacher P, Anzenbacherová E. Cytochromes P450 and metabolism of xenobiotics. Cell Mol Life Sci 2001;58:737–747.
Guengerich FP, Chun YJ, Kim D et al. Cytochrome P450 1B1: a target for inhibition in anticarcinogenesis strategies. Mutat Res 2003;523-524:173–182.
Shimada T, Hayes CL, Yamazaki H et al. Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res 1996;56:2979–2984.
Guengerich FP, Kim DH, Iwasaki M. Role of human cytochrome P450 in the oxidation of many low molecular weight cancer suspects. Chem Res Toxicol 1991;4:168–179.
Johnsrud EK, Koukouritaki SB, Divakaran K et al. Human hepatic CYP2E expression during development. J Pharm Exp Ther 2003;307:402–407.
Lieber CS. Cytochrome P450 2E1: its physiological and pathological role. Physiol Rev 1997;77:517–544.
Upadhya SC, Tirumalai PS, Boyd MR et al. Cytochrome P4502E (CYP2E) in brain: constitutive expression, induction by ethanol and localization by fluorescence in situ hybridization. Arch Biochem Biophys 2000;373:23–34.
Kirchheiner J, Brockmoller J. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther 2005;77:1–16.
Enayetallah AE, French RA, Thibodeau MS et al. Distribution of soluble epoxide hydrolase and of cytochrome P450 2C8, 2C9, and 2J2 in human tissues. J Histochem Cytochem 2004;52:447–454.
Keshava C, McCanlies EC, Weston A. CYP3A4 polymorphisms-potential risk factors for breast and prostate cancer: a HuGE review. Am J Epidemiol 2004;160:825–841.
Kitada M, Kato T, Ohmori S et al. Immunochemical characterization and toxicological significance of P-450HFLb purified from human fetal livers. Biochim Biophys Acta 1992;1117:301–305.
Vollweiler JL, Zielske SP, Reese JS et al. Hematopoietic stem cell gene therapy: progress toward therapeutic targets. Bone Marrow Transplant 2003;32:1–7.
Hodges VM, Molloy GY, Wickramasinghe SN. Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow- derived macrophages and human haemopoietic cell lines. Br J Haematol 2000;108:151–156.
Nguyen LT, Ramanathan M, Weinstock-Guttman B et al. Detection of cytochrome P450 and other drug-metabolizing enzyme mRNAs in peripheral blood mononuclear cells using DNA arrays. Drug Metab Dispos 2000;28:987–993.
Bernauer U, Vieth B, Ellrich R et al. CYP2E1 expression in bone marrow and its intra- and interspecies variability: approaches for a more reliable extrapolation from one species to another in the risk assessment of chemicals. Arch Toxicol 2000;73:618–624.
Haufroid V, Toubeau F, Clippe A et al. Real-time quantification of cytochrome P4502E1 mRNA in human peripheral blood lymphocytes by reverse transcription-PCR: method and practical application. Clin Chem 2001;47:1126–1129.
Carlsson E, Fredriksson J, Groop L et al. Variation in the calpain-10 gene is associated with elevated triglyceride levels and reduced adipose tissue messenger ribonucleic acid expression in obese Swedish subjects. J Clin Endocrinol Metab 2004;89:3601–3605.
Schmid H, Cohen CD, Henger A et al. Validation of endogenous controls for gene expression analysis in microdissected human renal biopsies. Kidney Int 2003;64:356–360.
Kousalová L, Anzenbacherová E, Baranová J et al. Presence of cytochrome P450 enzymes in human CD34+ hematopoietic progenitor cells. Gen Physiol Biophys 2004;23:251–257.
Guillén MI, Donato MT, Jover R et al. Oncostatin M down-regulates basal and induced cytochromes P450 in human hepatocytes. J Pharmacol Exper Ther 1998;285:127–134.
Hukkanen J, Hakkola J, Antilla S et al. Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in humanbronchoalveolar macrophages and peripheral blood lymphocytes. Mol Carcinog 1997;20:224–230.
Nagai F, Hiyoshi Y, Sugimachi K et al. Cytochrome P450 (CYP) expression in human myeloblastic and lymphoid cell lines. Biol Pharm Bull 2002;25:383–385.(Pavel Soueka, Pavel Anzen)