fenretinide and Leukemia--Promyelocytic--Acute

Topics: Child; Fenretinide; Humans; Leukemia, Promyelocytic, Acute; Neurotoxicity Syndromes; Tretinoin; Vitamin A

Retinoids, the natural and synthetic derivatives of vitamin A, have been shown to regulate the growth and differentiation of a wide variety of cell types and consequently have enormous potential as chemotherapeutic agents. We have previously identified 2 genes, termed OVCA1 and OVCA2, which are located in a small region showing a high frequency of allelic loss in breast and ovarian tumors and share a common exon. Recent studies have suggested that expression of OVCA1 may be influenced by retinoids. Therefore, we analyzed the expression of OVCA1 and OVCA2 in cells in response to treatment with all-trans retinoic acid (RA) and N-(4-hydroxyphenyl)retinamide (4HPR), or under conditions of low serum and confluence, to determine further the roles of OVCA1 and OVCA2 in cell growth, apoptosis and differentiation. We show that OVCA2 mRNA and protein are ubiquitously expressed and that they are downregulated in the lung cancer cell line Calu-6 after treatment with RA and 4HPR. In addition, we observed that OVCA2 protein is proteolytically degraded in response to RA and 4HPR treatment in a time- and dose-dependent manner in the promyelocytic leukemia cell line HL60. In contrast, expression of the candidate tumor suppressor OVCA1 was not downregulated by these treatments. Furthermore, we demonstrate that OVCA2 is evolutionarily conserved and shows regional homology with dihydrofolate reductases (DHFRs), specifically with hydrolase folds found in alpha-beta hydrolases. Our results are in contrast to a previous report and show that OVCA2, not OVCA1 mRNA and protein, is downregulated in response to RA and 4HPR.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cell Division; COS Cells; Down-Regulation; Evolution, Molecular; Fenretinide; Genes, Tumor Suppressor; HeLa Cells; Humans; Leukemia, Promyelocytic, Acute; Lung Neoplasms; Minor Histocompatibility Antigens; Models, Molecular; Molecular Sequence Data; Proteins; Retinoids; RNA, Messenger; Sequence Homology; Tissue Distribution; Tretinoin; Tumor Cells, Cultured; Tumor Suppressor Proteins

All-trans-retinoic acid (RA) induces differentiation of acute promyelocytic leukemia cells both in vitro and in vivo and is an alternative to cytotoxic chemotherapy in the treatment of acute promyelocytic leukemia. However, despite a complete remission rate of about 90%, most patients relapse and are resistant to further treatment with RA. This resistance primarily is due to an increased systemic catabolism of RA. In this study we examined the catabolism of RA by the human acute promyelocytic leukemia cell line NB4 and the human myeloid leukemia cell line HL60. NB4 cells converted RA to 4-hydroxy-RA, 4-oxo-RA and more polar unidentified retinoids at a much greater rate than HL60 cells. Exposure of NB4 cells to RA induced RA catabolism. We found that 4-hydroxyphenylretinamide (4-HPR) inhibited the catabolism of RA. This inhibition was dosedependent and greater, on a molar basis, than the inhibition seen with the cytochrome P450 inhibitor, ketoconazole, or two synthetic retinoids, Ch55 and Am80. 4-HPR alone was a poor inducer of differentiation of NB4 cells. However, it markedly enhanced RA-induced differentiation and increased the level of retinoylation, the covalent binding of RA to proteins. These results suggest some retinoid analogs, including 4-HPR, may have clinical utility because of their ability to increase the biological half-life of RA.

Topics: Cell Differentiation; Drug Combinations; Fenretinide; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Tretinoin

All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t-RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells.

Topics: Apoptosis; Benzoates; CD18 Antigens; Cell Differentiation; Chromosomes, Human, Pair 15; Chromosomes, Human, Pair 17; Cytosol; Drug Resistance, Neoplasm; Enzyme Induction; Fenretinide; Gene Expression Regulation, Leukemic; Humans; Isoenzymes; Leukemia, Promyelocytic, Acute; Neoplasm Proteins; Neoplastic Stem Cells; Oncogene Proteins, Fusion; Protein Multimerization; Receptors, Retinoic Acid; Retinoids; Signal Transduction; Tetrahydronaphthalenes; Transglutaminases; Translocation, Genetic; Tretinoin; Tumor Cells, Cultured

Incubation of the HL-60 cells with 3 microM of RA and 4-HPR resulted in suppression of cell growth and decrease in cell viability. A significant percentage of the RA-treated cells also displayed differentiation towards neutrophils, as assayed by changes in nitroblue tetrazolium reduction (NBT) and alpha-naphthyl-acetate esterase (ANAE) activities, whereas the 4-HPR treated cells remained essentially undifferentiated. Flow cytometric analysis showed 4-HPR to cause partial cell arrest in the G2/M phase after a 3-day treatment and an additional G1 phase arrest after a 7-day treatment. With RA-treated cells, a reduction in the percentage of cells in the G1 phase was observed after 7 days of treatment. In 4-HPR-treated cells an extra peak, characteristic of cells undergoing apoptosis, was found in the cell cycle phase distribution analysis. Determination of specific protein expression changes by Western blot analysis showed that the p34cdc2 was down-regulated by both chemicals. Furthermore, RA induced bcl-2 but prevented the processing of actin, whereas 4-HPR had little effect on bcl-2 but increased the specific processing of actin. These results suggest that RA promotes neutrophil differentiation and the establishment of a semi apoptosis-resistant state, possibly through the overexpression of the bcl-2 gene. By contrast, 4-HPR may trigger apoptosis by inducing overall cyto-architectural changes and specific DNA fragmentation subsequent to increased turnover of the protein actin.

Topics: Actins; Blotting, Western; CDC2 Protein Kinase; Cell Cycle; Cell Differentiation; Cell Division; Fenretinide; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tretinoin

The cancer chemopreventive retinoid N-(4-hydroxyphenyl)-all-trans retinamide (HPR) was recently shown by us to have antiproliferative and apoptotic effects on human leukemic cell lines, including those unresponsive to all-trans retinoic acid (ATRA). We have now characterized further the process of HPR-induced cell death. We report that inhibitors of RNA transcription and of protein synthesis, activators of protein kinase C (PKC), inhibitors of tyrosine kinases, Zn++, and the antioxidants acetylcysteine, ascorbic acid, alpha-tocopherol, and deferoxamine suppressed HPR-induced apoptosis. HL60 cells induced toward monocytic differentiation by 1,25 dihydroxyvitamin-D3 [1,25(OH)2D3], but not those induced toward the granulocytic differentiation by ATRA, showed reduced responses to HPR. The transport of HPR by cells with different sensitivity to the retinoid, however, was similar, even after treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which induces unresponsiveness to HPR. The expression of the apoptosis-related genes bcl-2, p53, and c-myc was examined to determine their role in HPR-triggered cell death. The levels of bcl-2 mRNA were markedly diminished by 24 hours of HPR treatment in all cell lines except in the relatively HPR-insensitive line K422. However, probably because of its long half-life, bcl-2 protein levels were either unchanged or only slightly decreased. Downregulation of p53 mRNA was also observed within 24 hours of HPR exposure in NB4 but not K422 cells, but no changes in the amount of p53 protein were found. Suppression of c-myc transcription was observed in all cells except K422. The protective role of bcl-2 on cell death by HPR was investigated in HL60 as well as 697 pre-B leukemia and Jurkat T-acute lymphocytic leukemia (T-ALL) cells constitutively expressing high levels of bcl-2 proteins due to gene transfer manipulation. Compared with control cells, the onset of apoptosis in these cells with deregulated bcl-2 production was delayed by at least 24 hours. These findings establish that cell death by HPR requires RNA transcription and protein synthesis and is regulated by the activation of PKC. Although changes in bcl-2, p53, and c-myc expression are found in cells treated with HPR, the time-course of these events suggests that HPR-triggered apoptosis is not directly controlled by these genes. Finally, while ectopic overexpression of bcl-2 does not protect cells from death by HPR, it markedly delays its onset.(

Topics: Anticarcinogenic Agents; Antimetabolites; Antioxidants; Apoptosis; Cell Differentiation; DNA, Neoplasm; Enzyme Activation; Fenretinide; Gene Expression Regulation, Leukemic; Genes, myc; Genes, p53; Glucuronates; Humans; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Lymphoma, Follicular; Neoplasm Proteins; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Protein Kinase C; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of particular clinical interest in cancer chemoprevention. We have examined the in vitro effects of HPR on lymphoid and myeloid malignant cell lines and found that at concentrations between 10(-5) and 3 x 10(-7) M it induces a dose-dependent growth inhibition (the peak plasma concentration in patients treated with HPR is 1 to 2 x 10(-6) M). The antiproliferative effect of HPR was, in all cell lines except K422, more potent than that induced by an equimolar dose of all-trans retinoic acid (RA). Also, this effect was irreversible on HL60 and DoHH2 cells that had been exposed to HPR (3 x 10(-6) M) for 24 h, but reversible on Raji and DHL4 exposed to the retinoid for 48 and 72 h, respectively. Time-course growth analysis showed that HPR at 3 x 10(-6) M or below induces a rapid fall of thymidine uptake and viability (> 90%), whereas between 10(-6) and 3 x 10(-7) M exhibits cytostatic effects. Interestingly, the RA-resistant HL-60R and NB306 cells, characterized by a point mutation in the retinoic acid receptor (RAR) and by the loss of the pml/RAR protein, respectively, were, like the parental RA-inducible HL-60 and NB4 cell lines, fully responsive to HPR, thereby suggesting that HPR and RA could act through different receptors or pathways. DNA flow-cytofluorimetric analysis revealed that HPR does not block cells in a specific phase of the cell cycle but triggers programmed cell death or apoptosis. This phenomenon was evidenced both by the visualization, on gel electrophoresis, of fragmented DNA, and by the "in cell" enzymatic labeling of DNA breaks with fluorescent dUTP. With the latter method, apoptotic cells become detectable by 6 h following exposure to 3 x 10(-6) M HPR. Ultrastructural examination of HPR-treated samples showed cells with chromatin compaction and cytoplasm condensation, characteristic of apoptotic cells. In conclusion, our study demonstrates that HPR suppresses malignant cell growth and induces apoptosis at pharmacologically relevant doses. The differential responsiveness by a number of cell lines, especially HL-60R and NB306, to HPR and RA indicates that these compounds may act through different receptors. The clinical use of HPR, particularly in retinoic acid-unresponsive acute promyelocytic leukemia patients, is suggested.

Topics: Apoptosis; Cell Cycle; Cell Division; DNA; Drug Resistance; Fenretinide; Humans; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured