tretinoin and safingol

Cellular retinoic acid-binding protein-I (CRABP-I) gene expression is induced in mouse embryonal carcinoma P19 cells specifically by retinoic acid (RA) and the induction is enhanced by sphinganine. The effects of retinoic acid and sphinganine on CRABP-I gene expression can be accounted for by a stimulation of its transcription rate. Using a lacZ reporter system, it was determined that a DNA fragment containing a putative AP-1 binding site in the promoter region of CRABP-I gene is required for the up-regulation of CRABP-I gene transcription.

Topics: Animals; beta-Galactosidase; Carcinoma, Embryonal; DNA; Gene Expression; Gene Transfer Techniques; Mice; Promoter Regions, Genetic; Receptors, Retinoic Acid; Sphingosine; Transcription Factor AP-1; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

We have tested the effect of several bile acids on the proliferation and differentiation of the HL60 human promyelocytic leukemia cell line in vitro. Deoxycholate, chenodeoxycholate and lithocholic acid caused dose-dependent inhibition of cell proliferation and induction of differentiation along the monocyte/macrophage pathway as determined by morphology, NBT test, non-specific esterase, and staining by monoclonal antibodies against specific cell-surface antigens. Optimal effects were obtained at 100, 75, and 60 microM of the 3 bile acids respectively. Cell-cycle flow-cytometric analysis showed that a substantial fraction of HL60 cells accumulated at the G0/G1 transition. Protein-kinase-C inhibitors such as sphinganine and H-7 inhibited the differentiation-inducing effect of bile acids, suggesting a possible role for PKC in this regulation. When bile acids were combined with non-effective concentrations of all-trans retinoic acid, enhancement of the monocytic differentiation of THP-1 human leukemia cells was observed. Our findings demonstrate induction of tumor-cell differentiation by bile acids, compounds that present minimal undesirable effects in humans.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Bile Acids and Salts; Cell Cycle; Cell Differentiation; Cell Division; Chenodeoxycholic Acid; Deoxycholic Acid; Flow Cytometry; Humans; Isoquinolines; Leukemia, Promyelocytic, Acute; Lithocholic Acid; Monocytes; Piperazines; Protein Kinase C; Sphingosine; Tretinoin; Tumor Cells, Cultured

The efficiency of retinoic acid (RA)-induced differentiation was dependent on the position of HL-60 cells in the cell cycle. Our results demonstrated that cells at the G1/S border were more efficiently induced to differentiate by short exposure to RA than cells at other phases of the cell cycle. Synchronization of cells in G1/S phase by aphidicolin (APH) or mimosine (MIMO) increased the sensitivity of cells to RA short exposure treatment. Pretreatment with sphinganine (SP), a protein kinase C (PKC) inhibitor, potentiated RA-induced cell differentiation. By cell cycle analysis, SP was found to block the cell progression through the G1/S phase. Consequently, cells accumulated in the G1/S phase of the cell cycle. The present data therefore suggest a possible mechanism of action of SP to enhance RA-induced differentiation.

Topics: Aphidicolin; Cell Cycle; Cell Differentiation; DNA; Granulocytes; Humans; In Vitro Techniques; Leukemia, Myeloid; Mimosine; RNA; Sphingosine; Tretinoin; Tumor Cells, Cultured

Differential changes in the expression of PKC isoenzymes in the RA-induced differentiation were noted. As measured by Western blot analysis, our results indicated the expressions of PKC-alpha, and -beta isoenzymes decreased in the cell membrane but increased in the cytosol during the RA-induced granulocytic differentiation. The amounts of PKC-gamma, on the other hand, decreased in the cell membrane while there was no significant changes in the cytosol. Similarly, the expression of PKC-delta was not altered in the cytosol, but was slightly reduced during the SP enhancement of RA-induced differentiation. In contrast, there were virtually little changes in the expression of PKC-epsilon and -zeta in the cell membrane or in the cytosol during the RA-induced differentiation in the absence or presence of SP. Concomitant with the decreased total PKC activity, there was a decline in the generation of sn-1,2-diacylglycerol (DAG) during the RA-induced differentiation. SP, enhancing the RA-induced differentiation, also potentiated the decrease of DAG content.

Topics: Blotting, Western; Cell Differentiation; Cell Line; Diglycerides; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Granulocytes; Humans; Isoenzymes; Leukemia, Promyelocytic, Acute; Protein Kinase C; Sphingosine; Tretinoin; Tumor Cells, Cultured

Sphinganine (SP) pre-treatment potentiated the retinoic acid (RA)-induced (4-96h exposures) differentiation and increase of alkaline phosphatase (ALP) activity. A higher percentage of SP pre-treated cells in RA exposures resembled mature myelocytes or granulocytes; greater increase in ALP activity was observed. In cells exposed to RA alone for only a period of 24h, the ALP activity could still increase and reach a similar maximum ALP activity (8.5-10.0 units/mg protein) at 48h as it was under continuous RA treatment. In all cells with longer exposures (24-96h) to RA, SP pre-treatment increased ALP activity to more or less the same higher maximum (14.0-15.5 units/mg protein). SP, added 24h before or concomitantly, but not 24 nor 48h after the addition of RA, could potentiate the RA-induced differentiation and increase of ALP activity.

Topics: Alkaline Phosphatase; Cell Differentiation; Cell Line; Drug Synergism; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Sphingosine; Time Factors; Tretinoin; Tumor Cells, Cultured

Induction of differentiation, inhibition of cell growth, and localization of nucleophosmin in HL-60 cells under the treatment of retinoic acid (RA) were studied. Bright nucleolar fluorescence was observed in control promyelocytic growing cells. The addition of RA in the culture system resulted in time- and dose-dependent induction of differentiation, cell growth inhibition, and nucleophosmin translocation from nucleoli to nucleoplasm. Unlike the control cells, many fewer nucleophosmin-associated preribosomal ribonucleoprotein particles (pre-rRNPs) could be obtained from nucleoli of RA-treated cells. Addition of sphinganine, an inhibitor of protein kinase C, facilitated the RA-induced differentiation, nucleophosmin translocation, and cell growth inhibition. Cells treated with sphinganine were more responsive to RA. Differentiation, translocation of nucleophosmin, and inhibition of cell growth occurred with lesser doses of RA or in shorter incubation times in the presence of sphinganine. Significant numbers of HL-60 cells could be rescued from the effects of RA upon the removal of RA after 2-h drug exposure. Pretreatment but not posttreatment of HL-60 cells with sphinganine, however, modulated the reversibility of the effects induced by short-exposure RA treatment. These results indicated that RA therapy can be improved by the pretreatment or the concurrent use of a modulator of protein kinase C activity. Nucleophosmin translocation as observed by immunofluorescence may be a simple and rapid method for assessing inhibition of cellular growth in response to differentiation inducers such as RA in cancer chemotherapy.

Topics: Cell Compartmentation; Cell Differentiation; Cell Division; Drug Administration Schedule; Drug Synergism; Growth Inhibitors; In Vitro Techniques; Leukemia, Myeloid; Nuclear Proteins; Nucleophosmin; Ribonucleoproteins; Sphingosine; Tretinoin; Tumor Cells, Cultured

The differentiation of HL-60 promyelocytic cells toward mature granulocytic cells induced by retinoic acid (RA) was accompanied by a decrease in protein kinase C (PKC) activity. The enhancement of RA-induced differentiation and the potentiation of the decrease of PKC activity by sphinganine (SP) seemed to correlate with each other. Kinetically, PKC activity during RA-induced differentiation without SP decreased to its lowest (75% of the control) after 48h; about 50% of the reduction was observed at 24h. In the presence of SP, PKC activity decreased more rapidly to its lowest (60% of the control) within 24h of incubation of RA. SP, added 24h before or concomitantly with the addition of RA, could potentiate the RA-induced differentiation and the reduction of PKC activity. Our results indicate that the effect of SP and the role of PKC during RA-induced differentiation may be critical at the early stages of induction of differentiation (within 24h of RA exposure).

Topics: Cell Differentiation; Cell Division; Cell Line; Drug Synergism; Granulocytes; Humans; Leukemia, Promyelocytic, Acute; Protein Kinase C; Sphingosine; Tretinoin; Tumor Cells, Cultured

The differentiation of HL-60 promyelocytic cells toward mature myelocytic cells induced by retinoic acid (RA) was accompanied by a quantitative similar increase in alkaline phosphatase (ALP) activity. The potentiation of RA-induced differentiation and the enhancement of ALP activity by sphinganine seemed to correlate with each other. The combination of RA and sphinganine increased in parallel the percentage of mature cells and the ALP activities. Short exposures (4-8h) of HL-60 cells to RA promoted differentiation and ALP activity to a fraction (about 50%) of their maximums which were achieved in cells after 24h or longer RA exposure. Our results indicate that the key step for the induction of ALP activity and the differentiation by RA probably takes place within 24h of incubation.

Topics: Alkaline Phosphatase; Cell Differentiation; Cell Line; Enzyme Induction; Granulocytes; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Sphingosine; Tretinoin

The human cell line HL-60 was used to investigate the role of protein kinase C in the regulation of retinoic acid-induced maturation of promyelocytic leukemia cells by growth and differentiation factors found in serum. Cells grown in serum-containing medium differentiated less than cells in serum-free medium due to several factors, including albumin binding of retinoic acid. Addition of an inhibitor (sphinganine) of protein kinase C, an enzyme that participates in cellular responses to many serum factors, facilitated the retinoic acid-induced differentiation. Cells treated with both retinoic acid and sphinganine produced more superoxide when stimulated by formylmethionylleucylphenylalanine; hence, this combination generated a more functional population of cells. The ability of sphinganine to promote retinoic acid-induced differentiation suggests that retinoic acid therapy might be improved by the concurrent use of a modulator of protein kinase C activity.

Topics: Blood Physiological Phenomena; Cell Differentiation; Humans; Leukemia, Promyelocytic, Acute; N-Formylmethionine Leucyl-Phenylalanine; Protein Kinase C; Sphingosine; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin