ABSTRACT
In order to investigate the role of calcium pathway in myeloid differentiation, the expression level of genes related to calcium pathway in all-trans retinoic acid (ATRA)-induced NB4 cell differentiation was detected by cDNA microarray, some of which were further confirmed by quantitative real time RT-PCR. At the same time, the expressions of these genes in NB4-R1 cells treated with ATRA and 8-CPT-cAM P alone or in combination, and in differentiation of primary cells from ATRA-induced newly diagnosed APL patients were detected by real time RT-PCR. The results showed that during differentiation of ATRA-induced NB4 cells, the expressions of genes related to calcium concentration had changed, the expression of downstream effectors in calcium pathway was up-regulated and confirmed by real time RT-PCR assay. The expression of genes related to calcium concentration did not change significantly when NB4-R1 cells were treated by ATRA or 8-CPT-cAMP alone, but expression changes of those genes were similar to the changes in ATRA-induced NB4 cell differentiation when NB4-R1 cells were treated by ATRA combined with 8-CPT-cAMP. In addition, the expression changes of those genes in ATRA-induced primary cells of patients with APL were also similar to changes in ATRA-induced NB4 cell differentiation. It is concluded that calcium pathway may be involved in ATRA-induced differentiation in APL cell.
Subject(s)
Humans , Calcium , Metabolism , Cell Differentiation , Gene Expression Regulation, Leukemic , Leukemia, Promyelocytic, Acute , Genetics , Metabolism , Signal Transduction , Tretinoin , Pharmacology , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the role of inhibitor of differentiation 1 (ID1) in ATRA-induced acute promyelocytic leukemia (APL) cells differentiation.</p><p><b>METHODS</b>The expression of ID1 was detected by cDNA microarray, cycloheximide inhibition test, real-time RT-PCR and western blot.</p><p><b>RESULTS</b>The expression of ID1 gene was up-regulated in ATRA-induced NB4 cells and APL cells from two patients and was independent on other proteins synthesis. ID1 expression level reached the peak at 2 h in NB4 cells induced by ATRA, its relative expression level was (359.4 +/- 48.7)-fold greater than control. ID1 expression level reached the peak at 2 h in bone marrow cells from APL patents treated with ATRA, and its level detected 3 times in one of the patient was (311.1 +/- 48.7) fold of control. The expression of ID1 protein was not up-regulated in ATRA resistant NB4-R2 cells after ATRA treatment.</p><p><b>CONCLUSION</b>ID1 may be involved in ATRA-induced granulocytic differentiation as an ATRA-targeted gene.</p>
Subject(s)
Humans , Antineoplastic Agents , Therapeutic Uses , Cell Differentiation , Inhibitor of Differentiation Protein 1 , Genetics , Metabolism , Leukemia, Promyelocytic, Acute , Drug Therapy , Metabolism , Pathology , Oligonucleotide Array Sequence Analysis , Tretinoin , Therapeutic UsesABSTRACT
The retinoid N-4-hydroxyphenyl retinamide (4-HPR also known as fenretinide), a synthetic derivative of all trans retinoic acid (ATRA), has shown as an efficient chemopreventive, chemotherapeutic agent and a potent inducer of apoptosis in various cancer cell types in vitro, including leukemic cells. However the mechanisms by which 4-HPR has the apoptotic effects is not completely elucidated. This study was aimed to investigate the effect of 4-HPR on several leukemic cells and explore its mechanisms of effect on U937 cells. The cell growth and proliferation experiments were performed [corrected] cell apoptosis was detected by annexin V; reactive oxygen species (ROS) and mitochondrial transmembrane potential (DeltaPsim) were determined; protein [corrected] expression was detected by Western blot. The results showed that 4-HPR inhibited the proliferation of U937 cells in a dose- and time-dependent manner. 4-HPR markedly [corrected] induced apoptosis in U937 cells, triggered the generation of ROS, induced the loss of mitochondrial transmembrane potential, decreased the expression of procaspase-8 and procaspase-3. Pretreatment of L-ascorbic acid suppressed the generation of ROS, disruption of mitochondrial potential, activation of caspases and apoptosis. It is concluded that the generation of ROS followed by the disruption of mitochondrial transmembrane potential plays an important role on 4-HPR-induced apoptosis in leukemic cells, suggesting that 4-HPR may be one of mitochondrial-targeted agents with clinical potential in treating cancer.