Differential response to retinoic acid of Syrian hamster embryo fibroblasts expressing v-src or v-Ha-ras oncogenes.

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.     

Induction of cell differentiation in melanoma cells by inhibitors of IMP dehydrogenase: altered patterns of IMP dehydrogenase expression and activity

To study the induction of differentiation in human melanoma cells, we treated 12 melanoma cell lines with mycophenolic acid and tiazofurin, inhibitors of IMP dehydrogenase (IMPDH). In all cell lines studied, both agents inhibited cell growth and increased melanin content. However, the degree of growth inhibition did not necessarily correspond to the increase in melanin content. A detailed analysis of the HO and SK-MEL-131 cell lines indicated that mycophenolic acid and tiazofurin caused a time- and dose-dependent increase in the expression of a series of other maturation markers, including formation of dendrite-like structures, tyrosinase activity, and reactivity with the CF21 monoclonal antibody. The growth inhibition and melanogenesis induced by the IMPDH inhibitors was abrogated by the addition of exogenous guanosine. No such effect was observed after treatment of the cells with phorbol 12-myristate 13-acetate or retinoic acid, two other inducers of differentiation in these cells. The mycophenolic acid- and tiazofurin-treated cells also showed an increased level of IMPDH mRNA and protein, perhaps because of compensation for the inhibitor-mediated decrease in IMPDH activity. In contrast, treatment with phorbol 12-myristate 13-acetate or retinoic acid resulted in decreased levels of IMPDH mRNA and protein. The lack of a consistent pattern of IMPDH expression in the cells treated with IMPDH inhibitors and phorbol 12-myristate 13-acetate or retinoic acid suggests that the altered expression of IMPDH is not a general requirement for the induction of cell differentiation in these cells. Our results also suggest that IMPDH inhibitors may provide a useful approach to circumvent the differentiation block in melanoma.     

Cytotoxicity of tiazofurin and its arabinose and xylose analogues in K562 cells.

2-beta-D-Arabinofuranosylthiazole-4-carboxamide and 2-beta-D-xylofuranosyl-thiazole-4-carboxamide are sugar modified analogues of tiazofurin, a C-glycosyl nucleoside which after anabolism to the dinucleotide, TAD (thiazole-4-carboxamide adenine dinucleotide), exhibits antitumor activity. However, ara-T and xylo-T did not exhibit cytotoxicity. Compared to tiazofurin, only 12.5% of the ara-T and 8.8% of the xylo-T were metabolized to TAD derivatives by human myelogenous leukemia K562 cells. This was reflected in the finding that guanylate pools were not depressed after treatment with either tiazofurin derivative. These results provide evidence that the ribose moiety is essential for the metabolism and cytotoxicity of tiazofurin. This investigation should be helpful in the design of new analogues of tiazofurin for future clinical trials.     

Signaling pathways in retinoid chemoprevention and treatment of cancer

The Vitamin A metabolite, retinoic acid, has been shown to have chemopreventive and therapeutic activity for certain cancers such as head and neck, cervical, neuroblastoma and promyelocytic leukemia. Retinoic acid achieves these activities by inducing differentiation and/or growth arrest. A large number of studies have investigated the mechanism(s) by which retinoic acid alters the behavior of premalignant and tumor cells. Although much important data has been obtained, the exact signaling pathways required for retinoic acid to exert its biological effects remains elusive. In this review, we outline the role and function of retinoid nuclear receptors, followed by a discussion of how major signaling pathways are affected in different tumor types by retinoids. We conclude by examining the effect of retinoic acid on G1 cell cycle regulatory proteins in various tumors.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Action of tiazofurin and 8-Cl-cAMP in human colon and pancreatic cancer cells.

Tiazofurin and 8-Cl-cAMP are novel chemotherapeutic agents shown to be effective against various cancer cells in vitro and in vivo. They act through distinct mechanisms that might modulate the signal transduction pathway, which causes growth inhibition, differentiation and down-regulation of c-ras and c-myc oncogene expression. We examined the effects of tiazofurin and 8-Cl-cAMP on colony formation of HT-29 human colon cancer and BxPC-3 and PANC-1 human pancreatic cancer cell lines. The IC50 of 8-Cl-cAMP was 0.1 and 0.2 microM in the pancreatic and colon cancer cell lines, respectively, and tiazofurin yielded IC50s from 4 (PANC-1) to 18 microM (HT-29). Simultaneous incubation with 8-Cl-cAMP and tiazofurin had additive effects on the inhibition of colony formation in the three examined cell lines. These results indicate possible clinical usefulness of a combination of tiazofurin and 8-Cl-cAMP in the treatment of colon and pancreatic carcinomas.     

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.     

Studies of purine and tiazofurin metabolism in drug sensitive human chronic myelogenous leukemia K 562 cells

Antineoplastic activity of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is mediated by an anabolite of the drug thiazole-4-carboxamide adenine dinucleotide (TAD), an analog of NAD which inhibits IMP dehydrogenase activity resulting in the depletion of guanylate pools and cell death. Human chronic myelogenous leukemia K 562 cells were found to be sensitive to tiazofurin with an IC50 of 19.2 microM. TAD content in K 562 cells (1.3 nmol/10(9)/h) was in the range found in susceptible murine and human tumor cells. Studies were conducted to relate tiazofurin toxicity with biochemical effects by examining nucleotide pools. Among the nucleotides, only guanylate pools were significantly depleted by the drug. To further study the effect of the drug on the purine nucleotide de novo and salvage biosynthetic pathways, flux of radiolabelled formate and guanine was employed. The results showed that de novo synthesis of guanylates was curtailed primarily by the drug's action without influencing adenylate biosynthesis or salvage of guanine to guanylates. These studies show that K 562 cells are sensitive to selective inhibition of de novo guanylate pathway indicating that human chronic myelogenous leukemia in blast crisis might be a good candidate for Phase II clinical trials with tiazofurin.     

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RAR alpha, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.     

Cell cycle dependent regulation of IMP dehydrogenase activity and effect of tiazofurin.

The activity of IMP dehydrogenase (IMP DH), the rate-limiting enzyme of de novo GTP biosynthesis, was shown to be increased in cancer cells. Tiazofurin, an inhibitor of IMP dehydrogenase, proved to be an effective agent in the treatment of refractory granulocytic leukemia. To examine the cell cycle dependent alterations of GTP synthesis and sensitivities to tiazofurin, we measured IMP DH activities and GTP pools, as well as the effects of tiazofurin on cell cycle phase enriched HL-60 cells. We now show that IMP DH activities and GTP concentrations are increased in S-phase enriched fractions of HL-60 cells. Moreover, the depletion of GTP concentrations by tiazofurin is most effective in S-phase enriched HL-60 cells. These results may be utilized in cancer chemotherapy to combine tiazofurin with biologic response modifiers which recruit quiescent leukemic cells into the cell cycle.     

Retinoid metabolism and cancer

Retinoids play important roles in cell differentiation and apoptosis, notably in epithelial tissues. Their utility in cancer therapy has been demonstrated in specific cancer types. Use of retinoic acid (RA) in the treatment of acute promyelocytic leukemia was the first successful example of retinoid-based differentiation therapy. RA has since been evaluated for treatment of other cancers, revealing variable effectiveness. The observation that expression of enzymes involved in RA biosynthesis is suppressed during tumorigenesis suggests that intra-tumor depletion in RA levels may contribute to tumor development and argues for the use of retinoids in cancer treatment. However, the induction of RA-inactivating enzymes is one of the mechanisms that may limit the efficacy of retinoid therapy and contribute to acquired resistance to RA treatment, suggesting that retinoic acid metabolism blocking agents may be effective agents in differentiation therapy.     

Phosphorylation of translation factors in response to anoxia in turtles,Trachemys scripta elegans: role of the AMP-activated protein kinase and target of rapamycin signalling pathways

Molecular and Cellular Biochemistry - Arsenic trioxide (As2O3) has recently been successfully used to treat all trans retinoic acid (ATRA) resistant relapsing acute promyelocytic leukemia. However,...     

PRAM-1 is a novel adaptor protein regulated by retinoic acid (RA) and promyelocytic leukemia (PML)-RA receptor alpha in acute promyelocytic leukemia cells

The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.     

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.     

Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60

all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.     

Altered nuclear cofactor switching in retinoic-resistant variants of the PML-RARα oncoprotein of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) results from a reciprocal translocation that fuses the gene for the PML tumor suppressor to that encoding the retinoic acid receptor alpha (RARα). The resulting PML-RARα oncogene product interferes with multiple regulatory pathways associated with myeloid differentiation, including normal PML and RARα functions. The standard treatment for APL includes anthracycline-based chemotherapeutic agents plus the RARα agonist all-trans retinoic acid (ATRA). Relapse, which is often accompanied by ATRA resistance, occurs in an appreciable frequency of treated patients. One potential mechanism suggested by model experiments featuring the selection of ATRA-resistant APL cell lines involves ATRA-resistant versions of the PML-RARα oncogene, where the relevant mutations localize to the RARα ligand-binding domain (LBD). Such mutations may act by compromising agonist binding, but other mechanisms are possible. Here, we studied the molecular consequence of ATRA resistance by use of circular dichroism, protease resistance, and fluorescence anisotropy assays employing peptides derived from the NCOR nuclear corepressor and the ACTR nuclear coactivator. The consequences of the mutations on global structure and cofactor interaction functions were assessed quantitatively, providing insights into the basis of agonist resistance. Attenuated cofactor switching and increased protease resistance represent features of the LBDs of ATRA-resistant PML-RARα, and these properties may be recapitulated in the full-length oncoproteins.     

A new C-nucleoside analogue of tiazofurin: synthesis and biological evaluation of 2-beta-D-ribofuranosylimidazole-4-carboxamide (imidazofurin)

2-Beta-D-ribofuranosylimidazole-4-carboxamide, an imidazole analogue of the antitumor agent tiazofurin, was synthesized and evaluated for the growth inhibitory activity of human myelogenous leukemia K562 cells.     

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.