Junko Horiguchi-Yamada

Changes of G1 cyclins, cdk2, and cyclin A during the differentiation of HL60 cells induced by TPA.

Differentiation induction by 12-o-tetradecanoyl 13-acetate (TPA) results in the growth arrest of HL60 cells in the G1 phase. However, little is known about the changes of cell cycle-regulating genes during this differentiation process. We investigated the changes of mRNA for various cyclins (A, C, D1, D2, D3 and E) and cdk2. Synchronized HL60 cells began to proliferate immediately after release from cell cycle block and cell cycle synchrony was obvious until the second S phase. TPA-treated cells accumulated in G1 phase within 24 h and most of the cells were arrested in this phase at 36 h. The expression of cyclins and cdk2 was studied by Northern blot hybridization or the reverse-transcription polymerase chain reaction (RT-PCR). TPA treatment altered the expression of all genes studied. The expression of cdk2 and cyclin A mRNA was markedly down-regulated. Cyclin E mRNA expression was also prominently down-regulated from 12 h to 36 h, at which time a second increase of its expression was observed in control cells. In contrast, the expression of cyclin D1 mRNA was induced by TPA, while its expression in control cells was undetectable by Northern blot hybridization throughout the cell cycle. Cyclin C expression was faint and fluctuated irrelevant of cell cycle, but its expression in both control and TPA-treated cells was higher than at baseline. Cyclin D2 expression remained stable in control cells and TPA treatment resulted in slight down-regulation at 12 h, but no difference was observed after 24 h. Cyclin D3 mRNA expression was slightly induced at 6 h, a time when its expression was down-regulated in control cells. At 48 h, these cyclins (C, D2, and D3) showed almost same level of expression as the control. These findings suggest that the down-regulation of cyclin A and cdk2 expression contributes to the G1 arrest of HL60 cells during monocytic differentiation induced by TPA and that cyclin D1 plays an additional role other than the regulation of cell cycle progression.

Cytostatic concentrations of anticancer agents do not affect telomerase activity of leukaemic cells in vitro

Telomerase, the enzyme that maintains the ends of linear eukaryotic chromosomes, is more active in the majority of malignant tumours than in normal somatic cells. Telomerase plays a key role in the maintenance of chromosomal stability in tumours, but it still remains unknown whether anticancer agents can inhibit telomerase activity. In this study, we evaluated the effect of various anticancer agents (etoposide, cisplatin, irinotecan, mitomycin C and daunorubicin) on the telomerase activity of three human haematopoietic cancer cell lines (Daudi, K562 and U937). A decrease of telomerase activity was not observed in cells treated with IC50 doses of the drugs, except for irinotecan-treated Daudi cells and daunorubicin- and irinotecan-treated U937 cells. Propidium iodide staining disclosed that the cells with decreased telomerase activity were severely damaged. U937 cells exposed to 5 microM (IC90) etoposide showed three different stages of cell viability during treatment. Apoptotic cells with an intact plasma membrane still maintained high telomerase activity, while cells with plasma membrane damage lost telomerase activity. The mRNA of the telomerase catalytic subunit (hTERT) showed a decrease in expression along with the decline of telomerase activity. These results indicate that the concentrations of drugs resulting in cytostatic effects on cells do not affect telomerase activity.

Transcription Factors: Normal and Malignant Development of Blood Cells

Transcription Factors: Normal and Malignant Development of Blood Cells, edited by Katya Ravid and Jonathan Licht. New York, Wiley-Liss, Inc, 2001; 622 pages. Nobody doubts that hematopoietic differentiation stems from the transcription of genes involved in lineage specificity. Protein-DNA interaction played out by transcription factors and their cognate DNA sequences must be coordinately regulated throughout the process of blood cell development. Vigorous efforts have been made over the last 15 years to elucidate the precise role of individual transcription factors. So much has been learned that it has become overwhelming for clinicians, and even for researchers, to catch up on new discoveries about transcription in hematopoiesis. Transcription Factors: Normal and Malignant Development of Blood Cells, edited by Katya Ravid and Jonathan Licht, is a comprehensive manual that provides information about the major transcription factors for blood cell development and aberrant pathways by translocation. This volume (622 pages and 111 figures), which weighs about one kilogram, has 32 chapters and is divided into six parts. The first part deals with transcription factors relating to the normal differentiation of megakaryocytic and erythroid cells. The topics addressed in Part One are GATA-1 and friend of GATA (FOG), nuclear factor erythroid 2 (NF-E2), TAL1/SCL, erythroid Kruppel-like factor (EKLF), and an overview of transcription factors implicated in megakaryocytic differentiation. Part Two, which focuses on myeloid differentiation, consists of the topics RUNX1(AML1)/CBFB ( -subunit–encoding core binding factor), PU.1, CCAAT/ enhancer-binding proteins, homeobox genes, retinoic acid receptors, and vitamin D3 receptors. Part Three, which addresses lymphoid differentiation, contains eight chapters, and includes information about Ikaros, PU.1, Pax5(BSAP), Janus kinases and STAT families, E2A, Bcl-6, octamer factors, and early B-cell factor (EBF). Each chapter (15-20 pages) is written by researchers in the field, most in the United States and a few in Europe, and can be read easily. Authors have intended to describe their works using culture systems and, to a greater extent, gene-targeted animal models. Knowledge obtained by knockout and knockin methods is reviewed in the chapters and makes this book an up-todate guide for hematologists. Part Four describes transcription factors involved in leukemia due to chromosomal translocation and targets nine factors: retinoic acid receptor (RAR) , inv(16), EVI1, t(8;21), TEL/ETV6, mixed lineage leukemia (MLL), coactivators, LMO2, and acetyltransferases CREB-binding protein (CBP)/p300. Each chapter includes information based on experiments by the authors and other researchers and discusses the structure and function of the gene, its role in differentiation, fusion partner genes, and the function of chimera genes in leukemogenesis. Part Five, “Oncogenesis and Hematopoiesis,” is relatively short, with two chapters, one for myc and myb and another for NFB. Readers will encounter broad descriptions, derived from numerous references, about the structure of these oncogenes and their roles in hematopoiesis and transformation. The last section contains a list of transcription factors implicated in hematopoiesis and a table of chromosomal translocations associated with disruption of transcriptional regulators in leukemia and lymphoma. This section is useful for locating information about the genes in chromosomal translocations that are relevant in the clinical settings of hematological malignancies. The editors summarize their conclusions in the introduction, presenting the general characteristics of transcription factors in hematopoiesis. Their main points are extracted as follows:

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.

Interferon-α repressed telomerase along with G1-accumulation of Daudi cells

The implications of telomerase on senescence and human carcinogenesis are widely accepted, but the changes of telomerase activity along with cell cycle modulation by anticancer treatment still remain obscure. In this paper, we issued whether the telomerase activity fluctuated along with cell cycle of cultured cancer cells using the antiproliferative effect of interferon-α (IFN-α). Daudi Burkitt lymphoma cells, treated with IFN-α, showed proliferation inhibition and cell cycle arrest at G1. The telomerase activity at 72 h was repressed to about 20% of control cells. Furthermore, after 72 h IFN-α treatment, the cells in G1 phase showed the marked decrease of telomerase activity, while cells in S and G2/M still possessed it. Among expressions of telomerase-related genes, only the catalytic subunit of telomerase (hTERT) decreased from 48 h, while the template RNA component (hTERC) and telomerase-associated protein 1 (TEP-1) were not affected. The downregulation of c-Myc preceded the change of hTERT. Moreover, the analysis of cells treated with IFN-α for 24 h revealed that cells in G1-to-S transition mainly expressed high hTERT, while S and G2/M cells had higher level of telomerase activity than that of G1 cells. These results indicate that (i) the expression of hTERT precedes the telomerase activity which is higher in S and G2/M phases than G1 phase, (ii) IFN-α repressed the telomerase activity in a cell cycle-dependent manner with the downregulation of hTERT.

Changes of cell cycle-regulating genes in interferon-treated Daudi cells

Interferon (IFN) modulates the expression of several genes and some of themare considered to be responsible for the inhibition of cellular growth. However, the alterations of cell cycle-regulating genes produced by IFN still remain unclear. Accordingly, we studied the expression of cell cycle-regulating genes during IFN-induced growth arrest. Cell cycle synchronized and unsynchronized Daudi Burkitt lymphoma cells were treated with IFN. Both the cell cycle distribution and the expression of cell cycle-regulating genes (cdk2, cdc2, cyclins A, B, C, D3 cdc25, and well) were studied by flow cytometry and by Northern blot hybridization or the reverse-transcription polymerase chain reaction, respectively. Treated cells passed through the first G1 phase and gradually accumulated in the following G1 phase. Expression of cyclins A, B, and D3 oscillated along with the cell cycle progression in control cells, and the alterations of cyclin B expression were especially prominent. Both cdc2 and cdk2 also showed changes, but these were not so distinct as observed with cyclin B. Expression of cdc25 and weel was little affected by cell cycle progression. In IFN-treated cells, expression of cyclins A and B were down-regulated, while that of cyclin C was not. Cyclin D3 expression was also down-regulated at 48 h, followed by an increase at 72 h. Expression of both cdc2 and cdk2 was down-regulated, especially that of the later. Weel expression was down-regulated by IFN but, the expression of cdc25 remained stable. These findings suggest that the modulation of cell cycle-regulating genes, particular by cyclin A and cdk2, plays an important role in IFN-induced cellular growth arrest.

Inactivation of ERK accelerates erythroid differentiation of K562 cells induced by herbimycin A and STI571 while activation of MEK1 interferes with it.

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ERK pathway.

Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells

Interferon (IFN) is one of the potent antiproliferative cytokines and is used to treat some selected cancers. IFN arrests the growth of Burkitt Iymphoma derived cell line Daudi cells in the G1 phase. G1-to-S progression is controlled by positive and negative regulatory genes. Therefore, we investigated the effects of IFN on G1-controlling genes. Expression of cyclin-dependent kinases (Cdks 2, 3, 4, 5, 6), MO 15/Cdk7, and cyclins E and H was studied to assess positive regulators, while p15Ink4B, p16Ink4, p18, p21CipI, and p27Kip1 were assessed as negative regulators. Cdks 2, 4, 6 and cyclin E were markedly down-regulated. MO15/Cdk7 expression showed little change, but its regulatory subunit (cyclin H) was down-regulated like cyclin E. Expression of p15Ink4B and p16Ink4 was not observed. p18 was induced until 48 h and its expression returned to the initial level at 72 h. In contrast, p21Cip1 mRNA expression remained at the baseline level throughout IFN treatment, while the expression of p27Kip1 increased at 48 and 72 h. Taken together, these data indicate that IFN changes the messenger RNA of G1-controlling genes towards the suppression of G1-to-S transition.

Both NUP98/TOP1 and TOP1/NUP98 transcripts are detected in a de novo AML with t(11;20)(p15;q11).

The NUP98 gene is involved in several chromosomal abnormalities associated with acute leukemia. The recurrent t(11;20)(p15;q11) chromosomal translocation results in generation of the NUP98/TOP1 chimeric gene. This abnormality has been observed primarily in therapy‐related leukemias, and TOP1/NUP98 transcripts have not been demonstrated. We describe a case of de novo acute myeloid leukemia with t(11;20)(p15;q11), with no known history of exposure to chemicals. The translocation occurred in intron 13 of NUP98 and intron 7 of TOP1, as in the three previously reported cases. The breakpoint in NUP98 was exactly the same as that found in a previously reported case. In addition, a reciprocal TOP1/NUP98 transcript was detected for the first time in our patient.

Differential responses of Bcl-2 family genes to etoposide in chronic myeloid leukemia K562 cells

Etoposide is a potent anticancer agent that is used to treat various tumors. We have investigated the dose-dependent effect of etoposide on apoptosis using chronic myeloid leukemia K562 cells treated with low (5 μM) or high (100 μM) concentrations of the drug. At a low concentration, etoposide induced little apoptosis at 24 h, while about 20% of the cells showed apoptosis morphologically at a high concentration. Processing of caspase-3 was slightly detected from 12 h and became obvious at 24 h with 100 μM etoposide. Caspase-3-like protease activity was detected at 24 h with a high concentration. Moreover, these changes were accompanied by cleavage of poly ADP ribose polymerase (PARP). Changes of the mRNA levels of most apoptosis-regulating genes were not prominent at both concentrations, except for the rapid induction of c-IAP-2/HIAP-1 and the down-regulation of Bcl-XL by 100 μM etoposide. The downregulation of Bcl-XL protein occurred from 6 h, while Bax protein conversely showed a slight increase from 6 h. Taken together, the present findings show that the dose-dependent apoptotic effect of etoposide is based on a change in the balance between Bcl-XL and Bax, which precedes the activation of caspase-3.