Difference between genomic actions of estrogen versus raloxifene in human ovarian cancer cell lines.

Although there is growing evidence that estrogens promote tumor progression in epithelial ovarian cancer, the molecular mechanisms accounting for this are still unclear. Selective estrogen receptor modulators (SERMs) mimic estrogen action in certain tissues while opposing it in others. The molecular mechanisms of the effects of SERMs such as raloxifene on the tumor progression of epithelial ovarian cancer are also still unclear. Here, we show that various genomic actions of estrogen differ from those of raloxifene in human ovarian cancer cell lines expressing estrogen receptor alpha (ERalpha). 17beta-Estradiol (E2) induced the gene expression of c-Myc and IGF-1 and increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1. ERalpha silencing abolished the E2-stimulated c-Myc expression. E2 induced the recruitment of co-activators such as SRC-1, SRC-3 and CBP to the promoters of c-Myc and IGF-1, and SRC-1 silencing abolished both the E2-stimulated c-Myc expression and cell-cycle progression. In contrast, although raloxifene increased the binding of ERalpha to the AP1 site of the promoters of c-Myc and IGF-1, raloxifene had no effect on the gene expression of c-Myc or IGF-1. Raloxifene induced the recruitment of co-repressors such as HDAC2, N-CoR and SMRT to the promoter of IGF-1. Thus, the difference between the genomic actions exerted by estrogen and raloxifene in human ovarian cancer cell lines expressing ERalpha appear to be dependent on the recruitment of co-regulators.

Involvement of Sp-1 in the regulation of the Id-1 gene during trophoblast cell differentiation.

Id-1, a member of the helix-loop-helix transcription factor family, inhibits the differentiation of Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells. Id-1 is expressed at a high level in undifferentiated trophoblast stem cells and then down-regulated during early differentiation, and is thought to be a key regulator in the trophoblast giant-cell differentiation pathway. In this study, we analyzed the signaling mechanism regulating the high expression levels of Id-1 in undifferentiated Rcho-1 cells. Promoter deletion analysis revealed that a 31-bp sequence (Box-2 region), located between -200 and -169bp in the Id-1 promoter is necessary for the promoter activity. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that Box-2-binding activity was decreased during differentiation and that Sp-1 protein bound to this sequence. The protein level of Sp-1 was decreased during the differentiation. These results suggest that the Sp-1 protein level may regulate the Box-2-binding activity and the trophoblast giant-cell differentiation.

Prevalence of premenstrual syndrome and premenstrual dysphoric disorder in Japanese women.

To investigate the prevalence and impact of premenstrual symptoms in Japanese women, we developed the PSQ "The Premenstrual Symptoms Questionnaire" for the screening of premenstrual symptoms. The PSQ translates DSM-IV criteria into a rating scale with degrees of severity. One thousand one hundred and eighty-seven Japanese women between the ages of 20 and 49 yrs, who were seen at a clinic for uterine cancer screening, were assessed regarding their premenstrual symptoms using the PSQ. As many as 95% of these women were found to suffer from premenstrual symptoms. The rates of prevalence of moderate to severe PMS and PMDD in Japanese women were 5.3 and 1.2%, respectively, which are lower than those in Western women. Only 5.3% of women with moderate to severe PMS and PMDD were treated. The results of this study suggest that race and ethnicity influence the expression of premenstrual symptoms and that the current state of medical care for Japanese women with moderate to severe PMS and PMDD is not satisfactory.

STAT3-mediated constitutive expression of SOCS3 in an undifferentiated rat trophoblast-like cell line.

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.

Both estrogen and raloxifene cause G1 arrest of vascular smooth muscle cells.

The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17beta-estradiol (E(2)) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF)-stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E(2) or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E(2) and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E(2) and raloxifene may be mainly mediated by ERalpha.

A mechanism of resistance to TRAIL/Apo2L-induced apoptosis of newly established glioma cell line and sensitisation to TRAIL by genotoxic agents.

Most tumour cells are sensitive to TRAIL-induced apoptosis, but not normal cells; thus, cancer therapy using TRAIL is expected clinically. Several tumour cells are resistant to TRAIL-induced apoptosis, and various mechanisms of such resistance were reported in individual cases. In this study, we established a TRAIL-resistant glioma cell line, which completely lacked TRAIL receptors. In addition, this tumour cell line had wild-type p53 tumour-suppressive gene, suggesting new mechanisms for tumour cells to expand and escape from immune surveillance. The present study further explored the mechanisms that determine the sensitivity to TRAIL. We show that genotoxic agents such as cisplatin, doxorubicin and camptothecin, in addition to UV radiation, can induce TRAIL-R2 on the cell surface of TRAIL receptor-negative tumour cells. Newly synthesised TRAIL-R2 is functional, so apoptosis is effectively induced by TRAIL, but it is significantly inhibited by constitutive expression of dominant-negative p53. In addition, apoptosis induced by pretreatment of genotoxic agents and additional stimulation of TRAIL is efficiently inhibited by either antagonistic anti-TRAIL-R2 antibody or pan-caspase inhibitor z-VAD-FMK. Taken together, these findings suggest that resistance to TRAIL by lack of TRAIL receptors on glioma is restored by genotoxic agents, which support the new strategies for tumour killing by TRAIL-bearing cytotoxic cells in combination with genotoxic treatment.

Clinical features affecting the results of estrogen replacement therapy on bone density in Japanese postmenopausal women.

Although estrogen replacement therapy (ERT) has been established as an effective treatment for postmenopausal bone loss, the clinical features which predict the effects of ERT have not been well investigated in Japanese postmenopausal women. We analyzed the role of physical factors influencing the effect of ERT on vertebral bone mineral density (BMD) in 94 Japanese postmenopausal women treated for 2 years or longer. The increase in BMD with ERT is 17.6 +/- 27.6 mg/cm(2)/year (mean +/- SD) during the first 2 years. Rates of BMD change were negatively correlated with the estimated initial BMD, and positively correlated with age and years since menopause, while no correlation was noted with the body mass index by a simple correlation analysis. The relationships between BMD change and estimated initial BMD or age also held in a multiple regression analysis. The estimated initial BMD and age together accounted for 34.4% of the BMD change during ERT. Furthermore, there were very few (2.4%) nonresponders with a negative linear regression slope of BMD in the osteoporosis and osteopenia group, although 32.7% of the normal initial BMD group were nonresponders. These results suggest that the initial BMD and age are potent predictive factors of the ERT effect on BMD change in Japanese postmenopausal women.

Inhibitory effect of egualen sodium: a new stable derivative of azulene on histamine release from mast cell-like cells in the stomach.

We studied the inhibitory effect of egualen sodium (ES) (sodium 3-ethyl-7-isopropyl-1-azulenesulfonate 1/3 hydrate, KT1-32), a new derivative and more stable compound than azulene, on histamine release from the mucosal histaminocytes and elucidated the mechanism for this action. ES prevented the histamine release from isolated mast cell-like cells of the guinea pig stomach induced by A23187 in a dose-dependent fashion. ES dose-dependently inhibited the histamine release from lung pieces of sensitized guinea pigs induced by an antigen-antibody reaction. ES also inhibited histamine release from rat peritoneal mast cells induced by compound 48/80 or antigen-antibody reaction. ES exhibited the membrane stabilizing activity on DPPC liposomes. These findings suggest that ES may prevent histamine release from histaminocytes induced by various stimuli and the stabilizing action of the cell membrane may be responsible for the inhibition of histamine release.

Involvement of nuclear transcription factor Sp1 in regulating glucose transporter-1 gene expression during rat trophoblast differentiation.

Glucose transporter-1 (GLUT1) is important in placental glucose transport. However, the mechanism of regulation of placental GLUT1 expression remains to be elucidated. We show here that the level of GLUT1 protein in rat choriocarcinoma cells (Rcho-1) decreased during differentiation. To analyze the regulatory mechanism of rat GLUT1 (rGLUT1) gene expression, we transfected rGLUT1 promoter-chloramphenicol acetyltransferase constructs into Rcho-1 cells. Deletion analysis of the rGLUT1 promoter suggested that the region -76/-53 bp was essential for basal transcriptional activity. Electrophoretic mobility shift assays showed that transcription factors Sp1 and Sp3 bound two GC boxes in the region -99/-33 bp of the rGLUT1 promoter. Mutation analysis of the Sp1 binding sites revealed that the promoter-proximal site located between -76 and -53 bp was essential for basal rGLUT1 promoter activity. Furthermore, the decreased level of GLUT1 may result from a decreased level of Sp1 during differentiation. These findings suggest that Sp1 is involved in the regulation of rGLUT1 gene expression during rat trophoblast differentiation.

A case of undifferentiated carcinoma of the gallbladder with anomalous arrangement of the pancreaticobiliary ductal system.

Anomalous junction of the pancreaticobiliary duct (AJPBD) is a congenital anomaly associated with gallbladder carcinoma. Especially patients with noncystic dilatation and without dilatation of the biliary tract are at risk of gallbladder carcinoma. A 56-year-old woman with advanced gallbladder cancer associated with AJPBD but without dilatation of the biliary tract was treated at our hospital. Although histologically cancer cells remained in the layer of the proprial mucosa, extensive metastases to lymph nodes including the paraaorta and peripancreas were detected. According to the TNM classification this case was of Stage IVB. The cancer consisted of medullary round cells, and was diagnosed as undifferentiated carcinoma. After surgery poor prognosis was expected, but three years have elapsed with no recurrence. The case is of interest because of two points of discrepancy: the primary cancer did not show deep invasion but demonstrated extensive lymph node metastases; the cancer was histologically malignant but prognosis was relatively good.

[Case report: a recurrent gastric cancer in the terminal stage, associated with obstructive jaundice which responded significantly to oral administration of TS-1].

TS-1, a novel oral formation of 5-fluorouracil that consists of 1M tegafur (5-FU), 0.4M CDHP and 1M Oxo, is reported to achieve a higher response rate of 49% in patients with advanced gastric cancer in a late phase II study. We report a case of recurrent gastric cancer that responded significantly to the short-term administration of TS-1. A 73-year-old man, who had undergone a curative distal gastrectomy with D2 lymphadenectomy 2 years earlier, had presented with obstructive jaundice resulting from cancerous lymphadenopathy. PTCD was performed for drainage, but cholestasis disappeared completely through the two courses of oral administration of TS-1. The serum level of transaminase and bilirubin remained within normal limits, even with PTCD unequipped, until the patient died of the original disease. The adverse effects observed with the drug were anemia (grade 1) and skin pigmentation (grade 2), both of which improved soon after discontinuing the medication. In conclusion, TS-1 may be well-tolerable and effective in some cases of terminal-stage and/or recurrent gastric cancer, especially those associated with obstructive jaundice arising from the cancerous lymphadenopathy, in that patient QOL can be maintained to a much greater extent.

Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells.

Raloxifene is a tissue-selective estrogen receptor modulator. The effect of estrogen on cardiovascular disease is mainly dependent on direct actions on the vascular wall involving activation of endothelial nitric oxide synthase (eNOS) via Akt and extracellular signal-regulated protein kinase (ERK) cascades. Although raloxifene is also known to activate eNOS in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells and simian virus 40-transformed rat lung vascular endothelial cells (TRLECs), the raloxifene analog LY117018 caused acute phosphorylation of eNOS that was unaffected by actinomycin D and was blocked by the pure estrogen receptor antagonist ICI182,780. Activation of Akt by raloxifene reached a plateau at 15-30 min and declined thereafter, a similar time frame to that of Akt activation by 17beta-estradiol. On the other hand, both activation and phosphorylation of ERK by raloxifene showed a biphasic pattern (peaks at 5 min and 1 h), whereas ERK activation and phosphorylation by 17beta-estradiol reached a plateau at 5 min and declined thereafter. A MEK inhibitor, PD98059, had no effect on the raloxifene-induced Akt activity, suggesting an absence of cross-talk between the ERK and Akt cascades. Either exogenous expression of a dominant-negative Akt or pretreatment of TRLECs with PD98059 decreased the raloxifene-induced eNOS phosphorylation. Moreover, raloxifene stimulated the activation of Akt, ERK, and eNOS in Chinese hamster ovary cells expressing estrogen receptor alpha but not Chinese hamster ovary cells expressing estrogen receptor beta. Our findings suggest that raloxifene-induced eNOS phosphorylation is mediated by estrogen receptor alpha via a nongenomic mechanism and is differentially mediated by Akt- and ERK-dependent cascades.

IFN-gamma induces the apoptosis of WEHI 279 and normal pre-B cell lines by expressing direct inhibitor of apoptosis protein binding protein with low pI.

Interferon-gamma plays a crucial role in induction of Th1 response but is predominantly a negative regulator of B cell differentiation and Th2 response, so it is a key molecule in determining cellular or humoral immunity. In this study, we demonstrate that IFN-gamma induces apoptosis in WEHI 279 mouse B cells and IL-7-dependent mouse pre-B cells by disrupting mitochondrial membrane potential and cytochrome c release via down-regulation of Bcl-2 and Bcl-x(L). Furthermore, this apoptotic signal is promoted by the de novo synthesis of endogenous direct inhibitor of apoptosis protein binding protein with low pI (DIABLO) by IFN-gamma and its release from mitochondria into the cytosol. Inhibition of DIABLO expression by antisense oligonucleotide is sufficient to decrease caspase activities and DNA fragmentation, but not cytochrome c release from mitochondria, suggesting that DIABLO plays a critical role in promoting apoptotic signals downstream of mitochondrial events. Thus, these findings demonstrate a signaling pathway during B cell apoptosis induced by IFN-gamma and possible mechanisms by which B cell differentiation is negatively regulated by Th1-type cytokines.

Progesterone inhibits transcriptional activation of human chorionic gonadotropin-alpha gene through protein kinase A pathway in trophoblast cells.

The purpose of this study was to analyze the mechanism of transcriptional inhibition of human chorionic gonadotropin-alpha (hCGalpha) gene by progesterone in trophoblast cells. We stably transfected -290 bp hCGalpha promoter-CAT constructs (-290halphaCAT) into Rcho-1 cells and monitored the promoter activities. Differentiation-dependent activation of -290 bp hCGalpha promoter containing a tandem repeat of cAMP response element (CRE) was inhibited by progesterone in a dose-dependent manner. To further analyze the mechanism of the progesterone action, Rcho-1 cells stably transfected with -290halphaCAT were treated with forskolin in the presence of progesterone. Progesterone inhibited forskolin-induced transcriptional activation of hCGalpha gene. Moreover, progesterone inhibited forskolin-induced transcriptional activation of CRE-CRE-tk-CAT. These results suggest that progesterone may inhibit cAMP-induced transcriptional activation of hCGalpha gene through CRE. Although progesterone did not alter the amount of CRE-binding protein (CREB), which is a main transcriptional factor bound to CRE(s) on hCGalpha promoter, progesterone abolished forskolin-induced CREB phosphorylation. In addition, pretreatment with progesterone abolished forskolin-induced activation of nuclear protein kinase A (PKA). In conclusion, progesterone inhibits hCGalpha gene transcription, at least in part, via the CRE region by inhibiting CREB phosphorylation through PKA pathway in trophoblast cells.

Effects of bezafibrate and simvastatin on plasma lipoproteins in hypercholesterolemia resistant to hormone replacement therapy.

OBJECTIVES: Estrogen replacement therapy has favorable effects on serum lipoprotein levels in postmenopausal women with hypercholesterolemia. However, there are some patients who fail to respond to hormone replacement therapy (HRT) to lower the serum cholesterol level. In these cases, a conventional lipid-lowering therapy will be applied in addition to HRT, while the effects of these drugs are not well understood. In this study, we studied the effects of simvastatin and bezafibrate administered in addition to HRT. METHODS: Patients who were hypercholesterolemic even after HRT were randomly assigned to three treatment groups: HRT only (control group, n=10), HRT+simvastatin (10 mg/day, n=10), or HRT+bezafibrate (400 mg/day, n=10). Serum lipids and lipoprotein levels were measured throughout 12 weeks. RESULTS: The serum triglyceride levels were decreased by 24+/-28 and 38+/-13% in the HRT+simvastatin and HRT+bezafibrate groups, respectively. HRT+simvastatin decreased the total cholesterol (21+/-10%) and low-density lipoprotein cholesterol (28+/-12%) levels without affecting the high-density lipoprotein cholesterol (HDL-C) level, while HRT+bezafibrate increased the HDL-C level (12+/-11%). CONCLUSIONS: Treatment with simvastatin or bezafibrate in addition to HRT should be considered in cases of postmenopausal hypercholesterolemia in which HRT alone fails to lower the serum lipoprotein levels.

Identification of a novel T-cell epitope in soluble egg antigen of Schistosoma japonicum.

Identification of T-cell epitopes harbored in soluble egg antigen (SEA) of Schistosoma japonicum and study of the immunological properties are essential for understanding the immunopathology and the control of schistosomiasis. As a follow-up to our previous work, the 66- to 80-kDa fragment from SEA was partially digested with protease, fractionated by reverse-phase high-pressure liquid chromatography, and found to be carrying a peptide which stimulated proliferation and gamma interferon (IFN-gamma) production of Th1 clones specific to SEA. Sequence analysis showed that the peptide was composed of 12 amino acids lined up as DLAVELAYLGNL. A synthetic homologue induced proliferation and IFN-gamma and interleukin-2 (IL-2) production, but not IL-4 or IL-6 production, by the Th1 clones as well as by the spleen cells from SEA-immunized mice, thus indicating that the peptide carries a Th1 epitope of SEA.

Involvement of mitochondrial permeability transition and caspase-9 activation in dimethyl sulfoxide-induced apoptosis of EL-4 lymphoma cells.

We observed that dimethyl sulfoxide (DMSO) induced apoptotic changes in the EL-4 murine lymphoma cell line and that effect was dependent on the concentration and time period. Incubating cells over a period of 18 h, 2.5% DMSO was found to induce sub-G1 peak in DNA histograms analyzed by flowcytometer and nucleosomal ladder formation in DNA gel electrophoresis. We also found down-regulation of Bcl-2, collapse of mitochondrial membrane potential (delta psi m) occurred following DMSO treatment, and release of cytochrome c from the mitochondria to cytosol. These observations suggest that DMSO converted its pro-apoptotic signal at the mitochondria. In the involvement of caspases, caspase-9 and -3, but not caspase-8, were found to be activated responding to DMSO treatment. Inhibitory experiments demonstrated that caspase cascade of mitochondrial apoptotic pathway was indispensable for DMSO-induced apoptosis. In the caspase cascade, caspase-9 was an upstream initiator and its primary signal could be transduced and amplified by caspase-3, -6 and -7. Kinetic study of these data showed mitochondrial dysfunction and caspase activation occurred at 12 h and apoptotic change of nuclear DNA at 18 h, providing another support for the transduction of DMSO pro-apoptotic signal via the mitochondrial pathway.

Wide use of skin-lightening soap may cause mercury poisoning in Kenya.

In a previous study, we speculated that some of the high mercury levels observed in head hair from a total of 14 subjects who resided around Lake Victoria, Tanzania, might be attributable to the habitual use of toilet soap containing considerable amounts of mercury (Harada et al. Sci Total Environ 1999;227:249-256). In August 1998, the current study was conducted to investigate if such mercury-containing soap was also available in the surroundings of Lake Victoria, Kenya, and if so, its toxic effects. A total of nine goldminers, 44 fishermen and their families, and 12 residents of Kisumu City, Kenya, volunteered for the study. Fourteen types of toilet soap were collected in Kisumu. Total mercury content was very significantly higher than in European-made soap (0.47-1.7%, as mercury iodide) compared with Kenya-made soap (0.41 x 10(-4)-6.2 x 10(-4)%). Indeed, all the subjects with a high hair mercury level (> 36.1 ppm) had made habitual use of European-made soap, accompanied by various symptoms, such as tremor, lassitude, vertigo, neurosthenia, and black and white blots, suggesting inorganic-mercury poisoning. On the other hand, any subject who had used soap other than the European-made soap, did not exceed a mercury level of 10 ppm in hair that is well within normal limits (Harada et al. Sci Total Environ 1999:227:249-256). The findings obtained suggest that the mercury-containing soap must be barred from circulation without delay, and that the residents' health in addition to the environmental pollution in Lake Victoria (Kenya as well as Tanzania) should be kept under close observation.

[All-trans retinoic acid combined with low-dose cytosine arabinoside treatment for acute myelogenous leukemia with trilineage myelodysplasia--a case report].

AML with trilineage myelodysplasia (AML/TMDS) is recognized as having of poor prognosis due to its resistance to chemotherapy in comparison with de novo AML. An AML/TMDS patient who failed to respond to ordinary induction therapy achieved complete remission with combination therapy consisting of all-trans retinoic acid (ATRA) and low-dose Ara-C. No serious toxicity was observed. ATRA combined with low-dose Ara-C could be an alternative treatment for this kind of patient.

Down-regulation of Pim-1 and Bcl-2 is accompanied with apoptosis of interleukin-6-depleted mouse B-cell hybridoma 7TD1 cells.

Here we report, interleukin-6 (IL-6) dependent mouse B-cell hybridoma, 7TD1 cells underwent apoptotic cell death with the starvation of IL-6. First, 7TD1 cells cultured without IL-6 arrested at G0/G1 phase (maximum accumulation at 24 h ) of the cell cycle. After that, the parameters of apoptosis namely, decreased mitochondrial transmembrane potential (DeltaPsi(m)), activation of caspases, DNA fragmentation and morphological changes (condensed nucleus and formation of apoptotic bodies) were observed. As evidents by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, down-regulation of Pim-1 (a serine/threonine kinase) and Bcl-2 was observed in the IL-6-depleted 7TD1 cells. There was no change in the expression of c-Myc, Bcl-xL and Mcl-1, even at 48 h of IL-6-depletion. Taken together, these results indicate that IL-6 withdrawn from the 7TD1 cells resulted in G0/G1 arrest and then caspase-dependent apoptosis via mitochondrial pathway by down-regulation of Pim-1 and Bcl-2, which may be essential for anti-apoptotic signals of IL-6.