Romidepsin and tamoxifen cooperatively induce senescence of pancreatic cancer cells through downregulation of FOXM1 expression and induction of reactive oxygen species/lipid peroxidation.

BACKGROUND: Although improvement has been made in therapeutic strategies against pancreatic carcinoma, overall survival has not significantly enhanced over the past decade. Thus, the establishment of better therapeutic regimens remains a high priority. METHODS: Pancreatic cancer cell lines were incubated with romidepsin, an inhibitor of histone deacetylase, and tamoxifen, and their effects on cell growth, signaling and gene expression were analyzed. Xenografts of human pancreatic cancer CFPAC1 cells were medicated with romidepsin and tamoxifen to evaluate their effects on tumor growth. RESULTS: The inhibition of the growth of pancreatic cancer cells induced by romidepsin and tamoxifen was effectively reduced by N-acetyl cysteine and α-tocopherol, respectively. The combined treatment greatly induced reactive oxygen species production and mitochondrial lipid peroxidation, and these effects were prevented by N-acetyl cysteine and α-tocopherol. Tamoxifen enhanced romidepsin-induced cell senescence. FOXM1 expression was markedly downregulated in pancreatic cancer cells treated with romidepsin, and tamoxifen further reduced FOXM1 expression in cells treated with romidepsin. Siomycin A, an inhibitor of FOXM1, induced senescence in pancreatic cancer cells. Similar results were obtained in knockdown of FOXM1 expression by siRNA. CONCLUSION: Since FOXM1 is used as a prognostic marker and therapeutic target for pancreatic cancer, a combination of the clinically available drugs romidepsin and tamoxifen might be considered for the treatment of patients with pancreatic cancer.

Semisynthesis and biological evaluation of a cotylenin A mimic derived from fusicoccin A.

In an effort to overcome the unavailability of cotylenin A (CN A), an anticancer agent and a stabilizer of protein-protein interactions (PPIs) mediated by 14-3-3 proteins, ISIR-050 was designed as a CN A mimic. The synthesis was accomplished via a semisynthetic approach starting from fusicoccin A. ISIR-050 showed interferon-α (IFNα)-dependent growth inhibitory activity and a PPI stabilization effect similar to those of CN A. The biochemical analysis suggested that ISIR-050 and CN A induce the same pharmacological response to IFNα-treated cancer cells and that 14-3-3 proteins play a role in the mode of action.

Tamoxifen enhances romidepsin-induced apoptosis in T-cell malignant cells via activation of FOXO1 signaling pathway.

Although romidepsin as monotherapy appears to be useful for treating T-cell lymphoma, combined chemotherapy with other therapeutic agents is required for improvement of the treatment outcome. To establish safer and more effective regimens, systematic screening was conducted to identify suitable drugs to be used in combination with romidepsin for T-cell malignancies, and the underlying molecular mechanisms were examined. The most effective agent was tamoxifen. The combination of romidepsin and tamoxifen had a significant synergistic effect in inducing apoptosis. The growth-inhibitory effects of the combined treatment were reversed by α-tocopherol. FOXO1 expression was greatly upregulated in MOLT-4 cells treated with romidepsin plus tamoxifen. Knockdown of FOXO1 expression by siRNA significantly reduced the cell death induced by romidepsin plus tamoxifen. The combination of romidepsin and tamoxifen might be considered for the treatment of T-cell lymphoma patients.

Extracellular NM23 protein promotes the growth and survival of primary cultured human acute myelogenous leukemia cells.

An elevated serum level of NM23-H1 protein is found in acute myelogenous leukemia (AML), and predicts a poor treatment outcome in AML patients. To investigate the potential pathological link between the elevated serum level of this protein and poor prognosis, we examined the extracellular effects of recombinant NM23-H1 protein on the in vitro growth and survival of primary cultured AML cells at concentrations equivalent to the levels found in the serum of AML patients. Extracellular NM23-H1 protein promoted the in vitro growth and survival of AML cells and this activity was associated with the cytokine production and activation of the MAPK and signal transducers and activators of transcription signaling pathways. Inhibitors specific to MAPK signaling pathways inhibited the growth- and survival-promoting activity of NM23-H1. These findings indicate the novel biological action of extracellular NM23-H1 and its association with poor prognosis, and suggest an important role for extracellular NM23-H1 in the malignant progression of leukemia and a potential therapeutic target for these malignancies.

Cotylenin A, a new differentiation inducer, and rapamycin cooperatively inhibit growth of cancer cells through induction of cyclin G2.

Cotylenin A, a plant growth regulator, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), are potent inducers of differentiation of myeloid leukemia cells. Recently, we found that cotylenin A and rapamycin effectively inhibited the proliferation of several human breast cancer cell lines including MCF-7. Herein, we demonstrate that cotylenin A and rapamycin rapidly and markedly induced the cyclin G2 gene expression in several cancer cells including MCF-7 cells. The growth arrest of the MCF-7 cells at the G1 phase, induced by the treatment with cotylenin A and rapamycin or the culture in low serum medium, markedly induced the cyclin G2 gene expression. Anticancer drugs including doxorubicin, etoposide and 5-fluorouracil also induced cyclin G2 expression during induction of growth arrest of the MCF-7 cell at the G1 phase or G2/M phase. Ectopically inducible cyclin G2 expression potently inhibited the proliferation of MCF-7 cells. Furthermore, cyclin G2 knockdown induced by cyclin G2 small interfering RNA markedly reduced the potency of cotylenin A plus rapamycin to induce growth inhibition. Taken together, our results suggest that cotylenin A and rapamycin induce inhibition of cancer cell growth through the induction of cyclin G2.

Induction of differentiation of myeloid leukemia cells in primary culture in response to lithocholic acid acetate, a bile acid derivative, and cooperative effects with another differentiation inducer, cotylenin A.

Lithocholic acid (LCA) acetate induced the differentiation of human leukemia cells. Treatment with a combination of LCA acetate and cotylenin A, an inducer of the differentiation of leukemia cells, was more effective than that with LCA acetate or cotylenin A alone at inducing monocytic differentiation. LCA acetate activated mitogen-activated protein kinase (MAPK) before inducing differentiation. Cotylenin A did not activate MAPK, suggesting that cotylenin A has a different mode of action. The cooperative effects of LCA acetate and cotylenin A on inducing differentiation were, at least partly, due to the enhancement of LCA acetate-induced MAPK activation by cotylenin A.

TH588, an MTH1 inhibitor, enhances phenethyl isothiocyanate-induced growth inhibition in pancreatic cancer cells.

Chemotherapy and radiotherapy are the most common approaches in cancer therapy. They may kill cancer cells through the generation of high levels of reactive oxygen species (ROS), which leads to oxidative DNA damage. However, tumor resistance to ROS is a problem in cancer therapy. MTH1 sanitizes oxidized dNTP pools to prevent the incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, cancer cells require MTH1 activity to avoid the incorporation of oxidized dNTPs, which would result in DNA damage and cell death. By targeting a redox-adaptation mechanism, MTH1 inhibition represents a novel therapeutic strategy against cancer. However, recent reports have indicated that growth inhibition by MTH1 inhibitors may be due to off-target cytotoxic effects. TH588, one of the first-in-class MTH1 inhibitors, kills cancer cells by an off-target effect. However, a low concentration of TH588 may effectively inhibit MTH1 activity without inhibiting cell proliferation. Phenethyl isothiocyanate (PEITC) is a dietary anticarcinogenic compound and an inducer of ROS. In the present study, it has been demonstrated that combined treatment with PEITC and TH588 effectively inhibited the growth of pancreatic cancer MIAPaCa-2 and Panc-1 cells. The antioxidant N-acetylcysteine negated this synergistic growth inhibition. PEITC and TH588 cooperatively induced the formation of 8-oxo-deoxyguanine in nuclei and pH2AX foci, a marker of DNA damage. However, the combined effects are not associated with MTH1 mRNA expression in several cancer cell lines, suggesting that the possibility of an off-target effect of TH588 cannot be eliminated. These results suggest that the combination of PEITC and TH588 has potential as a novel therapeutic strategy against pancreatic cancer.

Cotylenin A and tyrosine kinase inhibitors synergistically inhibit the growth of chronic myeloid leukemia cells.

The treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs do not effectively eliminate CML stem cells. In fact, CML stem cells persist and cause relapse in the majority of patients upon discontinuation of the drug treatment. Transcriptomic and proteomic analyses have revealed that p53 and c-Myc play defining roles in CML stem cell survival, suggesting that the dual targeting of p53 and c-Myc may selectively eliminate stem cells in patients with CML. Since the downregulation of c-Myc and then upregulation of p21 (a target gene of p53) are commonly observed during the differentiation of acute myeloid leukemia cells induced by differentiation inducers, we hypothesized that differentiation-inducing agents may be useful in regulating c-Myc and p53 expression in CML cells. In the present study, we demonstrate that some differentiation-inducing agents effectively suppress the self-renewal ability of CML cells, and that the combination of these inducers with TKIs results in significantly greater inhibitory effects on CML cell growth compared to the use of TKIs or the inducer alone. The KU812 cells were treated with various concentrations of the inducers in the presence or absence of 30 nM imatinib for 4 days. Among the differentiation inducers we tested, cotylenin A (CN-A) was the most potent at inhibiting the self-renewal ability of the CML cells. CN-A induced the robust expression of CD38, a marker of committed progenitor and more differentiated myelomonocytic cells, and rapidly suppressed c-Myc expression and upregulated p21 expression in CML cells. Thus, these results suggest that CN-A may have potential to promote the elimination of stem cells in CML.

Therapeutic strategy using phenotypic modulation of cancer cells by differentiation-inducing agents.

A low concentration of differentiation inducers greatly enhances the in vitro and in vivo antiproliferative effects of interferon (IFN)alpha in several human cancer cells. Among the differentiation inducers tested, the sensitivity of cancer cells to IFNalpha was most strongly affected by cotylenin A. Cotylenin A, which is a novel fusicoccane diterpene glycoside with a complex sugar moiety, affected the differentiation of leukemia cells that were freshly isolated from acute myelogenous leukemia patients in primary culture. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5 were early genes induced by the combination of cotylenin A and IFNalpha in carcinoma cells. Neutralizing antibody to TRAIL inhibited apoptosis, suggesting that cotylenin A and IFNalpha cooperatively induced apoptosis through the TRAIL signaling system. Combined treatment preferentially induced apoptosis in human lung cancer cells while sparing normal lung epithelial cells. In an analysis of various cancer cell lines, ovarian cancer cells were highly sensitive to combined treatment with cotylenin A and IFNalpha in terms of the inhibition of cell growth. This treatment was also effective toward ovarian cancer cells that were refractory to cisplatin, and significantly inhibited the growth of ovarian cancer cells as xenografts without apparent adverse effects. Ovarian cancer cells from patients were also sensitive to the combined treatment in primary cultures. Combined treatment with cotylenin A and IFNalpha may have therapeutic value in treating human cancers including ovarian cancer.

Japanese apricot extract (MK615) potentiates bendamustine-induced apoptosis via impairment of the DNA damage response in lymphoma cells.

Bendamustine, a hybrid molecule of a purine analog and alkylator, induces cell death by the activation of apoptosis and the DNA damage response. The agent MK615 is produced from Japanese apricot and contains a number of cyclic triterpenes that exhibit antitumor activities. In the present study, the combined effects of bendamustine and MK615 on lymphoma cells were investigated. The combined compounds synergistically induced apoptosis in all lymphoid cell lines examined. MK615 inhibited the bendamustine-induced phosphorylation of checkpoint kinase 1 and 2. As ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) kinases are key mediators of the DNA damage response, the effects of the combination of bendamustine and ATM/ATR inhibitors (KU-60019 and VE-821) on lymphoma cells were investigated. KU-60019 and/or VE-821 potentiated bendamustine activity in all cell lines tested, but did not affect MK615 activity, suggesting that these inhibitors have the same underlying mechanism of action as that of MK615. The results of the present study suggest that it may be feasible to use ATM/ATR inhibitors in combination with bendamustine for treating malignant lymphoma.

Immediate up-regulation of the calcium-binding protein S100P and its involvement in the cytokinin-induced differentiation of human myeloid leukemia cells.

Cytokinins are important purine derivatives that act as redifferentiation-inducing hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA) and kinetin are very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to IPA using cDNA microarrays and compared the results with those obtained with other inducers of differentiation, such as all-trans retinoic acid (ATRA), 1 alpha,25-dihydroxyvitamin D3 (VD3) and cotylenin A (CN-A). Many genes were up-regulated, and only a small fraction were down-regulated, upon exposure to the inducers. IPA and CN-A, but not ATRA or VD3, immediately induced the expression of mRNA for the calcium-binding protein S100P. The up-regulation of S100P was confirmed at the protein expression level. We also examined the expression of other S100 proteins, including S100A8, S100A9 and S100A12, and found that IPA preferentially up-regulated S100P at the early stages of differentiation. IPA-induced differentiation of HL-60 cells was suppressed by treatment with antisense oligonucleotides against S100P, suggesting that S100P plays an important role in cell differentiation.

Cotylenin A-induced differentiation is independent of the transforming growth factor-beta signaling system in human myeloid leukemia HL-60 cells.

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-beta and 1alpha, 25-dihydroxyvitamin D(3) (VD3) resulted in increased differentiation compared to separate treatments, but TGF-beta did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-beta. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-beta antibody or an inhibitor of TGF-beta signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-beta signaling system, although TGF-beta acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.

Cotylenin A, a differentiation-inducing agent, and IFN-alpha cooperatively induce apoptosis and have an antitumor effect on human non-small cell lung carcinoma cells in nude mice.

Cotylenin A, a novel inducer of the differentiation of leukemia cells, and IFN-alpha synergistically inhibited the growth of and induced apoptosis in several human non-small cell lung carcinoma cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor DR5 were the early genes induced by the combination of cotylenin A and IFN alpha in lung carcinoma cells. Neutralizing antibody to TRAIL inhibited apoptosis, suggesting that cotylenin A and IFN alpha cooperatively induced apoptosis through the TRAIL signaling system. This combined treatment preferentially induced apoptosis in human lung cancer cells while sparing normal lung epithelial cells and significantly inhibited the growth of human lung cancer cells as xenografts without apparent adverse effects, suggesting that this combination may have therapeutic value in treating lung cancer.

Tamoxifen enhances the differentiation-inducing and growth-inhibitory effects of all-trans retinoic acid in acute promyelocytic leukemia cells.

All-trans retinoic acid (ATRA) is valuable in differentiation therapy for acute promyelocytic leukemia (APL). However, ATRA has had limited success as a single agent, due to the development of resistance. We found that tamoxifen effectively enhanced the differentiation-inducing effect of ATRA. Tamoxifen alone inhibited the proliferation of myeloid leukemia cell lines while only slightly increasing morphologic differentiation. Tamoxifen effectively enhanced the growth-inhibiting actions of various differentiation-inducing agents. ATRA in the presence of tamoxifen increased NBT reduction and the expression of CD11b in HL-60 cells more effectively than ATRA alone. Tamoxifen also enhanced the differentiation induced by the other inducers tested. ATRA induced the differentiation of APL cell lines NB4 and HT93 and APL cells in primary culture, and this differentiation was also enhanced by tamoxifen. Tamoxifen is one of the most widely used drugs for the treatment of cancer and has few side effects. The combination of ATRA and tamoxifen might be considered for the treatment of APL patients in whom it can be difficult to apply arsenic trioxide or anthracyclines.

Combined treatment with cotylenin A and phenethyl isothiocyanate induces strong antitumor activity mainly through the induction of ferroptotic cell death in human pancreatic cancer cells.

The treatment of pancreatic cancer, one of the most aggressive gastrointestinal tract malignancies, with current chemotherapeutic drugs has had limited success due to its chemoresistance and poor prognosis. Therefore, the development of new drugs or effective combination therapies is urgently needed. Cotylenin A (CN-A) (a plant growth regulator) is a potent inducer of differentiation in myeloid leukemia cells and exhibits potent antitumor activities in several cancer cell lines. In the present study, we demonstrated that CN-A and phenethyl isothiocyanate (PEITC), an inducer of reactive oxygen species (ROS) and a dietary anticarcinogenic compound, synergistically inhibited the proliferation of MIAPaCa-2, PANC-1 and gemcitabine-resistant PANC-1 cells. A combined treatment with CN-A and PEITC also effectively inhibited the anchorage-independent growth of these cancer cells. The combined treatment with CN-A and PEITC strongly induced cell death within 1 day at concentrations at which CN-A or PEITC alone did not affect cell viability. A combined treatment with synthetic CN-A derivatives (ISIR-005 and ISIR-042) or fusicoccin J (CN-A-related natural product) and PEITC did not have synergistic effects on cell death. The combined treatment with CN-A and PEITC synergistically induced the generation of ROS. Antioxidants (N-acetylcysteine and trolox), ferroptosis inhibitors (ferrostatin-1 and liproxstatin), and the lysosomal iron chelator deferoxamine canceled the synergistic cell death. Apoptosis inhibitors (Z-VAD-FMK and Q-VD-OPH) and the necrosis inhibitor necrostatin-1s did not inhibit synergistic cell death. Autophagy inhibitors (3-metyladenine and chloroquine) partially prevented cell death. These results show that synergistic cell death induced by the combined treatment with CN-A and PEITC is mainly due to the induction of ferroptosis. Therefore, the combination of CN-A and PEITC has potential as a novel therapeutic strategy against pancreatic cancer.

Cytokinin-induced differentiation of human myeloid leukemia HL-60 cells is associated with the formation of nucleotides, but not with incorporation into DNA or RNA.

Cytokinins are important purine derivatives that act as hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA), kinetin and benzyladenine were very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. The metabolism of cytokinins to their nucleotides was closely associated with cytokinin-induced differentiation and growth inhibition. When the cells were incubated with [14C]-benzyladenine, radioactivity was significantly incorporated into RNA and DNA. However, the radioactive nucleotides in RNA or DNA were adenine nucleotides, not benzyladenine nucleotides, suggesting that cytokinins were not incorporated into RNA and DNA. The benzyladenine nucleotides were not stably released into the medium in intact form. Cytokinins effectively induced a phosphorylated (active) form of mitogen-activated protein kinase (MAPK). MAPK activation was necessary for cytokinin-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by cytokinins. These results suggest that cytokinin nucleotides themselves play an important role in inducing the differentiation of HL-60 cells.

Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts.

INTRODUCTION: Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G1 arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. METHOD: We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. RESULTS: We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G1 phase, rather than apoptosis, and induced senescence-associated beta-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G2, transforming growth factor-beta-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. CONCLUSION: Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A.

Clinical significance of intracytoplasmic nm23-H1 expression in diffuse large B-cell lymphoma.

PURPOSE: Recently, we established an ELISA technique for measuring nm23-H1 protein in serum and found that the serum nm23-H1 level is a potential prognostic factor for patients with non-Hodgkin's lymphoma. EXPERIMENTAL DESIGN: We used immunohistochemistry to examine the expression of nm23-H1 by the lymphoma cells in patients with diffuse large B-cell lymphoma (DLBCL). RESULTS: By analyzing a consecutive series of 172 untreated DLBCL patients, we found that 100 (58.1%) were strongly positive. The cytoplasmic nm23 expression in lymphoma cells correlated significantly with the serum nm23-H1 level. There was a significant correlation between patients with cytoplasmic nm23-positive lymphoma and those with performance status 2-4, stage III/IV, bulky mass, B symptoms, elevated serum level of soluble interleukin 2 receptor, and elevated serum level of C-reactive protein. Overall and progression-free survival rates were significantly lower in patients with nm23-H1-positive lymphomas than in those with nm23-H1-negative lymphomas. Similar difference was seen between patients with high and low serum levels of nm23-H1. Thus, the correlation between presence or absence of cytoplasmic nm23-H1 expression and serum nm23-H1 levels suggests that serum nm23-H1 is produced directly by lymphoma cells. CONCLUSION: We suggest that nm23-H1 expression is a prognostic factor for DLBCL, and that it is as important as serum nm23-H1, both of which are useful for planning a treatment strategy.

The catalytic DNA topoisomerase II inhibitor ICRF-193 and all-trans retinoic acid cooperatively induce granulocytic differentiation of acute promyelocytic leukemia cells: candidate drugs for chemo-differentiation therapy against acute promyelocytic leukemia.

OBJECTIVE: Although all-trans retinoic acid (ATRA) can bring about complete remission of acute promyelocytic leukemia (APL), the incidence of early recurrence is considerably high. Thus, chemotherapeutic agents, such as anthracycline agents or cytosine arabinoside (AraC), are generally co-administered with ATRA. The therapeutic outcome of APL patients has significantly improved by chemo-differentiation therapy. Late-phase toxicities, such as cardiotoxicity and secondary carcinogenesis, are becoming clinically important. Therefore, we must identify the most suitable chemotherapeutic agents for the treatment of APL. METHODS: We examined the effects of ICRF-193 and several other anticancer drugs on the growth and differentiation of APL cell lines (NB4 and HT-93) and other myeloid leukemia cell lines (HL-60 and U937). RESULTS: If anticancer agents were available that not only inhibited the proliferation of APL cells but also induced their differentiation, they would be very useful for the treatment of APL. DNR slightly induced the differentiation of APL cells. On the other hand, other DNA topoisomerase II inhibitors, such as ICRF-154 and ICRF-193, significantly induced the differentiation of APL cell lines and leukemia cells freshly isolated from APL patients. These drugs effectively cooperated with ATRA in inhibiting the growth and inducing the differentiation of APL cells, whereas DNR did not. The incidence of cardiotoxicity and secondary carcinogenesis associated with ICRF-193 are much lower than that with DNR. CONCLUSION: These results suggest that ICRF-193 may be useful in the treatment of patients with APL.

Role of MmTRA1b/phospholipid scramblase1 gene expression in the induction of differentiation of human myeloid leukemia cells into granulocytes.

OBJECTIVE: We previously cloned a human normal counterpart (MmTRA1b/phospholipid scramblase 1) of the mouse leukemogenesis-associated gene MmTRA1a. MmTRA1b gene expression was increased during differentiation of human monoblastic leukemia U937 cells using some differentiation inducers but not 1alpha,25-dihydroxyvitamin D(3) (a typical monocytic differentiation inducer). To further elucidate the role of human MmTRA1b gene expression in the differentiation of myelogenous leukemia cells, we measured MmTRA1b gene expression in several myeloid leukemia cell lines and primary leukemia cells. MATERIALS AND METHODS: The expression of MmTRA1b mRNA was determined by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS: Expression of the MmTRA1b gene was markedly induced during granulocytic differentiation of promyelocytic leukemia NB4 and HT93 cells induced by all-trans retinoic acid (ATRA). The level of MmTRA1b mRNA was significantly increased during differentiation toward granulocytes, but not monocytes/macrophages, in bipotential myeloid leukemia HL-60 cells. The level of MmTRA1 mRNA was not increased during erythroid differentiation induced by hemin in erythroid leukemia K562 and HEL cells or during megakaryocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate in K562 cells. Expression of the MmTRA1b gene also was not induced when apoptosis of NB4 cells was induced by antileukemic drugs. ATRA-induced differentiation of antisense MmTRA1b-transfected NB4 cells was significantly suppressed. On the other hand, ATRA induced the differentiation of MmTRA1b-transfected NB4 cells more efficiently than that of mock-transfected cells. MmTRA1b mRNA also was clearly induced in ATRA-treated primary acute promyelocytic leukemia cells during granulocytic differentiation. CONCLUSION: MmTRA1b mRNA was specifically induced during granulocytic differentiation of acute promyelocytic leukemia cells and was associated with induction of their differentiation.