Antiproliferative effects of gefitinib are associated with suppression of E2F-1 expression and telomerase activity.

BACKGROUND: Gefitinib (Iressa, ZD1839) is a selective epidermal growth factor receptor tyrosine kinase inhibitor. E2F-1 is a critical determinant in cell cycle. Growth signals up-regulate telomerase activity. The effects of gefitinib on E2F-1 and telomerase in A549, H23 and A431 cells were examined. MATERIALS AND METHODS: Cell proliferation and cell cycle progression were measured by the WST-1 assay and flow cytometry. The expression of E2F-1 and cyclin-dependent kinase inhibitors was evaluated, and hTERT mRNA expression and telomerase activity were analyzed. RESULTS: In the A431 and A549 cells, treatment with gefitinib inhibited cell proliferation and was associated with an increase in G1-phase. In both cell types, gefitinib decreased the expression of E2F-1 mRNA and protein, followed by the suppression of hTERT mRNA and telomerase activity. In the H23 cells, gefitinib did not affect cell proliferation. CONCLUSION: The antiproliferative effects of gefitinib may be, at least in part, due to the inhibition of E2F-1 expression and telomerase activity.

HTLV-I Tax protein inhibits apoptosis induction but not G1 arrest by pyrrolidinedithiocarbamate, an anti-oxidant, in adult T cell leukemia cells.

OBJECTIVE: We examined the anti-tumor effect of pyrrolidinedithiocarbamate (PDTC) on HTLV-1-infected T clones and the mechanism of HTLV-1 Tax protein inhibition of PDTC-induced apoptosis. MATERIALS AND METHODS: Tax-nonproducing clones S1T and Su9T01, Tax-producing clones K3T and F6T, and Tax cDNA stably transfected S1TcTax clones S1TcTax04 and S1TcTax05 were examined for PDTC inhibition of thymidine incorporation and apoptosis induction by ISEL method. In addition, S1TcTax clones were analyzed by DNA histography and DNA fragmentation and also examined for p53, p21, or Bax protein expression by Western blot. RESULTS: PDTC inhibited thymidine incorporation of all four HTLV-1-infected T cells in a similar dose-dependent manner, but K3T and F6T were more resistant than S1T and Su9T01 in apoptosis induction. S1TcTax clones also showed resistance to PDTC-induced apoptosis as compared to Tax-nonproducing S1T and S1Tneo. DNA histography demonstrated that PDTC induces G1 arrest and apoptosis in S1T and S1Tneo, and that S1TcTax clones are also sensitive to PDTC in G1 arrest but resistant in apoptosis induction. DNA fragmentation also demonstrated ladder formation only in S1Tneo but not in S1TcTax04. Western blots demonstrated higher expression of p53 and p21 proteins in S1Tneo than in S1TcTax04 during whole phase after PDTC stimulation with moderate enhancement in S1Tneo but small in S1TcTax04. Bax protein expression was detected only at early phase in S1Tneo but was not detected in S1TcTax04. CONCLUSION: These findings suggest that PDTC-induced apoptosis is related with Bax, and that G1 arrest is possibly related with p21. Tax might inhibit apoptosis induction mainly via inhibition of Bax expression preceded at least in part by p53 inhibition.

Human T-cell lymphotropic virus type I Tax activates lung resistance-related protein expression in leukemic clones established from an adult T-cell leukemia patient.

OBJECTIVE: We examined the significance of human T-cell lymphotropic virus type I (HTLV-I) Tax protein-induced resistance to anticancer drugs and the relationship between Tax and multidrug resistance proteins. MATERIALS AND METHODS: S1T cell, a leukemic non-Tax-producing T-cell clone established from an adult T-cell leukemia (ATL) patient, S1TcTax05 and S1TcTax10 clones, transfected with Tax stably expressing cDNA, and S1Tneo, transfected with a neomycin-resistant gene, were examined for Tax-related anticancer drug resistance. Resistance of those cells to the anticancer drugs doxorubicin, etoposide, cisplatin, and vindesine was tested with the MTT method. Expression of multidrug resistance protein mRNAs (MDR1, MRP1, cMOAT/MRP2, and LRP) was analyzed with reverse transcriptase polymerase chain reaction (RT-PCR). Doxorubicin subcellular distribution in those cells was examined by fluorescence microscopy. RESULTS: S1TcTax05 and S1TcTax10 showed resistance to doxorubicin, etoposide, and vindesine, but not to cisplatin as compared with S1T or S1Tneo. RT-PCR demonstrated that MRP1 mRNA was expressed, but MDR1, cMOAT, and LRP mRNAs were not in S1T or S1Tneo. Marked expression of LRP mRNA was detected, but no change of MDR1, MRP1, or cMOAT mRNA expression in Tax-expressing S1TcTax05 and S1TcTax10. Fluorescence microscopy demonstrated that doxorubicin was distributed mainly in the cytoplasm of S1TcTax05 and S1TcTax10, and in the nucleus of S1T and S1Tneo. CONCLUSIONS: These findings suggest that Tax-related drug resistance of ATL cells is due to LRP and not MDR1, as reported previously. These findings in cells derived from an ATL patient suggest a novel mechanism for drug resistance in Tax-expressing ATL cells.

Repeated waon therapy improves pulmonary hypertension during exercise in patients with severe chronic obstructive pulmonary disease.

OBJECTIVES: Repeated Waon therapy, which uses a far infrared-ray dry sauna system, improved the vascular endothelial function and the cardiac function in patients with chronic heart failure. In patients with chronic obstructive pulmonary disease (COPD), pulmonary hypertension (PH) is associated with a poor prognosis. We investigated whether repeated Waon therapy improves PH, cardiac function, exercise tolerance, and the quality of life (QOL) in patients with COPD. METHODS: Consecutive 13 patients with COPD, who met the Global Initiative for Chronic Obstructive Lung Disease criteria and had breathlessness despite receiving conventional treatments, were recruited for this study. They underwent Waon therapy at 60 degrees C in sauna for 15 min following 30 min warmth with blankets outside of the sauna room. This therapy was performed once a day, for 4 weeks. Cardiac function, exercise tolerance, and St. George's Respiratory Questionnaire (SGRQ) were assessed before and 4 weeks after Waon therapy. RESULTS: Right ventricular positive dP/dt at rest elevated significantly from 397 +/- 266 to 512 +/- 320 mmHg/s (p = 0.024) after the therapy. While the PH at rest did not significantly decrease, the PH during exercise decreased significantly from 64 +/- 18 to 51 +/- 13 mmHg (p = 0.028) after Waon therapy. Furthermore, the therapy prolonged the mean exercise time of the constant load of cycle ergometer exercise test from 360 +/- 107 to 392 +/- 97 s (p = 0.032). The total scores of SGRQ improved from 59.7 +/- 16.9 to 55.3 +/- 17.2 (p = 0.002). In addition, no adverse effects were observed related to Waon therapy. CONCLUSIONS: Repeated Waon therapy improved right ventricular positive dP/dt, PH during exercise, exercise tolerance and the QOL in patients with severe COPD.

Autocrine and/or paracrine growth of adult T-cell leukaemia tumour cells by interleukin 15.

We previously demonstrated that interleukin 2 (IL-2) autocrine/paracrine growth in adult T-cell leukaemia (ATL) cells was closely correlated with clinical aggressiveness. In the present study, we compared the significance of IL-15 and IL-2 in growth of ATL cells and clinical aggressiveness. Thirty-seven patients with ATL were examined: 19 acute and 18 chronic. Autonomous growth and IL-2- or IL-15-responsive growth activities of ATL cells were measured by [3H]-thymidine incorporation after 24 h cultures in vitro. All of the autonomous, IL-15- and IL-2-responsive growth activities of acute-type cells were higher than those of chronic type (P = 0.04, P = 0.03 and P = 0.02 respectively). IL-15- and IL-2-responsive growth activities were highly correlated (P = 0.0001, R2 = 0.837). Enzyme-linked immunosorbent assay (ELISA) showed detectable serum levels of IL-15 and IL-2 in 18 out of 19 and 14 out of 17 patients respectively. Reverse transcription polymerase chain reaction (RT-PCR) revealed IL-15 and IL-2 mRNA expression in 8 out of 11 patients' cells. Anti-IL-2 antibody partially inhibited autonomous growth of ATL cells; anti-IL-15 antibody was less effective. In situ immunochemistry detected IL-15 in cells of three patients and was consistent with the results of RT-PCR. These results suggest that ATL cells grow in an IL-15 autocrine/paracrine manner and that this growth is related to disease aggressiveness in a manner similar to IL-2.