[Reversing effect of brassinolide on multidrug resistance of-CCRF-VCR1000 cells and a preliminary investigation on its mechanisms].

AIM: To investigate the effect of brassinolide, a plant growth modulator, on multidrug resistance (MDR) of human T lymphoblastoid cell line CCRF-VCR 1000 which was obtained by progressively addition of vincristine (VCR) to sensitive CCRF-CEM cells, and to explore preliminarily the mechanism of reversing action. METHODS: MTT method was used to detect the resistant factor of resistant cell line and the reversing fold after addition of brassinolide. The intracellular accumulation of rhodamine 123, a fluorescent dye transported by P-glycoprotein was detected by flow cytometry, the catalytic activity of topoisomerase II was assessed by Sulliven method to find the effect of brassinolide on resistance. The protein expression of p53 was measured using Western blotting in the sensitive cells and resistant cells to explore the effect of brassinolide. RESULTS: The resistant factors of CCRF-VCR cells on adriamycin, VP-16 and VCR are respectively as 153.1, 55.9 and 8123.1 folds comparing to the sensitive cell line CCRF-CEM. After treatment of brassinolide under the concentration of 0.001 - 10.0 microg x mL(-1), the resistance of CCRF-VCR was reversed partly with the reversing folds respectively as 4.4 - 11.6. The intracellular accumulation of rhodamine 123 was significantly reduced in the resistant cells. After treatment of brassinolide, the accumulation increased, the level of fluorescent dye was situated between resistant cells and sensitive cells. No alteration of the catalytic activity of topoisomerase II was found among three groups. The level of protein expression of p53 in resistant cells was higher than that of sensitive cells. After brassinolide treatment, the expression of p53 in CCRF-VCR cells restored to the level of sensitive cells. CONCLUSION: Brassinolide could effectively reverse the resistance of CCRF-VCR cells by inhibiting the effusion of drug transported by P-glucoprotein. To down regulate the abnormal expression of p53 maybe one of the mechanisms of reversing MDR for brassinolide.

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12h of light and 12h of darkness (LD12:12) for at least 3wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of approximately 2 x 2 mm2). Ten days later, a tumor sample (area of approximately 5 mm2) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G1, S or G2-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G2-M phase cells. The approximate average distribution patterns of G1 and G2-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G1, 21 HALO for S and 15 HALO for G2-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.

[Circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing human nasopharyngeal carcinoma].

BACKGROUND & OBJECTIVE: No report was found on research of circadian rhythms of nasopharyngeal carcinoma(NPC). This study was designed to investigate the circadian rhythms of DNA synthesis and apoptosis correlated gene expression in bone marrow cells of nude mice bearing NPC and to collect necessary data for making clinical chronochemotherapy schedule of NPC. METHODS: Sixty-nine BALB/C nude mice were synchronized with an alternative lighting regimen with 12 hours in light and 12 hours in dark (LD 12:12) for at least 3 weeks. Human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells were implanted into each mouse. Ten days after transplantation, the mice were sacrificed, bone marrow cells were collected in the 3, 7, 11, 15, 19, and 23 hours after light onset (HALO). Single cell suspension was obtained and stained with propidium iodide. The cellular DNA content was measured by flow cytometry. Data were analyzed with analysis of variance(ANOVA) and Cosinor analysis. Proteins were extracted from bone marrow cells and determined; Bcl-2, p53, and p21 expression were tested using Western blot analysis. RESULTS: The proportion of bone marrow cells in phase G1, S, and G2/M varied according to circadian sampling time with statistical significance (ANOVA). Moreover, such variation in G1 and G2/M was statistically valid by Cosinor analysis with an acrophase located at 10.8 HALO and 1.8 HALO, respectively. The distribution curves of phase G1 and G2/M fit to Cosinor changes but not for S phase cells. The expression of p53 and Bcl-2 protein level in bone marrow cells varied in 24 h time scale. No p21 protein expression was found in this experiment. CONCLUSION: DNA synthesis of bone marrow cells in nude mice bearing human nasopharyngeal poorly differentiated squamous cell carcinoma (CNE-2) cells varies according to circadian rhythm. The expression of p53 and Bcl-2 protein level in bone marrow cells varies in 24 h time scale.