Jae Goo Seol

Induction of apoptosis by vitamin D3 analogue EB1089 in NCI-H929 myeloma cells via activation of caspase 3 and p38 MAP kinase.

EB1089, a novel 1,25‐dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI‐H929 human myeloma cells. EB1089 inhibited cell growth of NCI‐H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose‐dependent manner. We could also detect apoptosis in NCI‐H929 cells exposed to EB1089 (1 × 10−7 m for 72 h) using the sub‐G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down‐regulation of the Bcl‐2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089‐treated NCI‐H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose‐ and time‐dependent manner. In addition, EB1089 caused the down‐regulation of p44 extracellular signal‐related kinase (ERK) activity and up‐regulation of the p38 kinase activity during apoptosis of NCI‐H929 cells. These results suggest that EB1089 inhibits growth of NCI‐H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.

Monensin-mediated growth inhibition in human lymphoma cells through cell cycle arrest and apoptosis

Summary. Monensin, a Na+ ionophore, regulates many cellular functions, including apoptosis. We investigated the in vitro antiproliferative effect of monensin on nine human lymphoma cell lines. Monensin significantly inhibited the proliferation of all the lymphoma cell lines examined with a 50% inhibition concentration of about 0·5 μmol/l, and induced a G1 and/or a G2‐M phase arrest in these cell lines. To address the antiproliferative mechanism of monensin, we examined the effect of this drug on cell‐cycle‐related proteins in CA46 cells (both G1 and G2 arrest) and Molt‐4 cells (G2 arrest). Treatment with monensin for 72 h decreased CDK4 and cyclin A levels in CA46 cells, and cdc2 levels in Molt‐4 cells. In monensin‐treated CA46 cells, increased p21‐CDK2, p27‐CDK2 and p27‐CDK4 complex forms were observed. And, in monensin‐treated Molt‐4 cells, increased p21–cdc2 complex form was detected. Furthermore, the activities of CDK2‐ and CDK4‐associated kinases were reduced in association with Rb hypophosphorylation in monensin‐treated CA46 cells. The activity of cdc2‐associated kinase was decreased in both cell lines, which was accompanied by induction of Wee1. Also, monensin induced apoptosis in these cell lines, as evidenced by annexin V binding assay and flow cytometric detection of sub‐G1 DNA content. This apoptotic process was associated with loss of mitochondria transmembrane potential (Δψm). Taken together, these results demonstrated for the first time that monensin potently inhibits the proliferation of human lymphoma cell lines via cell cycle arrest and apoptosis.

Antibacterial properties of a pre-formulated recombinant phage endolysin, SAL-1

To evaluate the phage endolysin SAL-1 as a therapeutic agent for Staphylococcus aureus infections, the in vitro and in vivo antibacterial properties of a pre-formulation containing recombinant SAL-1 as an active pharmaceutical ingredient were investigated. The stable pre-formulation (designated SAL200) uniquely included calcium ions and Poloxamer 188 as enhancing and stabilising ingredients, respectively. SAL-1 was successfully produced with no extraneous amino acids by decreasing the culture temperature and was highly purified using a two-step chromatography procedure consisting of ion exchange and hydrophobic interaction chromatography. SAL200 exhibited rapid and effective bactericidal activity against encapsulated and biofilm-forming S. aureus as well as against planktonic S. aureus cells. In addition, SAL200 demonstrated increased effectiveness in the serum environment, with a significantly reduced minimum bactericidal concentration compared with that determined in culture medium. In in vitro antibacterial tests performed against 425 clinical isolates [including 336 meticillin-resistant S. aureus (MRSA) isolates and 1 vancomycin-intermediate S. aureus isolate], collected from 421 patients and four animals, SAL200 exhibited obvious antibacterial activity against all S. aureus isolates tested. Intravenous injection of SAL200 in a mouse model of MRSA infection prolonged the viability of mice and significantly reduced bacterial counts in the bloodstream and splenic tissue. The results presented in this article strongly support SAL200 as a highly potent bactericidal agent against MRSA with an adequate pharmaceutical formulation.

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)2D3, and vitamin D3 analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D3‐sensitive HL‐60 cells, whereas vitamin D3 insensitive K562 cells did not change its activity. This down‐regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34− cells > leukaemic CD34+ cells > normal CD34− cells > normal CD34+ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S‐phase, cytotoxicity to cytosine arabinoside and percentage of CD34+ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.

Ontogeny of natural killer cells and T cells by analysis of BCR-ABL rearrangement from patients with chronic myelogenous leukaemia.

Chronic myelogenous leukaemia (CML) is a haematological malignant disorder characterized by the Philadelphia chromosome (Ph) and BCR–ABL gene rearrangement. This abnormal fusion gene can be considered to serve as a marker for the transformed cell clone in CML and is found in all cells arising from the same malignant precursor cell. It has been detected in CML cells of the myeloid, monocytic, erythroid and B‐lymphocytic lineages. However, it is still arguable as to whether T lymphocytes or natural killer (NK) cells carry this marker. Answering this question would clarify the ontogenic relationship between NK cells and T cells. We examined 12 CML patients and studied the expression of BCR–ABL rearrangement by fluorescence in situ hybridization (FISH) in both NK cells and T cells sorted by flow cytometry. The purity of T cells was 95·6–99·8% and that of NK cells was 95·3–99·3% after sorting. Neither NK cells nor T cells showed any positive BCR–ABL signal with the exception of one patient who recovered from a lymphoid blastic crisis. We speculate that T cells and NK cells originate from BCR–ABL‐negative stem cells.