Effects of azidothymidine on p33ING1b expression, apoptosis and senescence of TJ905 human glioblastoma cell line / 中华病理学杂志

<p><b>OBJECTIVES</b>To investigate the pharmacological effects of azidothymidine (AZT) on p33ING1b expression, senescence and apoptosis of TJ905 glioblastoma cells.</p><p><b>METHODS</b>TJ905 cells were treated with AZT at a serial concentrations of 50, 100 and 200 µmol/L. Semi-quantitative RT-PCR and cytochemical staining of senescence related-galactosidase (sβ-Gal) were used to evaluate the expression of p33ING1b mRNA and to label the senescent cells at the 1st, 3rd and 6th generations, respectively. In situ cell death detection and single cell gel electrophoresis were used to detect the apoptosis at the 3rd and 6th generations.</p><p><b>RESULTS</b>AZT induced the expression of p33ING1b mRNA and senescence of the tumor cells of the 1st generation in a dosage and time dependent manner. At the 6th generation, the relative amount of p33ING1b RT-PCR product (1.44±0.23) and sβ-Gal labeling index of 200 µmol/L group (45.62±6.74) were significantly higher than those of the 1st (0.95±0.13 and 7.82±2.40) and the 3rd generation cells (1.35±0.23, 26.27±7.17) of the same group, and cells of the same generation in the 50 µmol/L (0.85±0.24, 27.37±6.41) and 100 µmol/L groups (1.23±0.34, 35.49±5.12, P<0.01). There was a significant positive correlation between the p33ING1b mRNA expression and the labeling index of sβ-Gal. Pro-apoptotic effects of AZT became obvious at the 6th generation.</p><p><b>CONCLUSION</b>AZT upregulates the expression of p33ING1b, a possible mechanism in regulating senescence and apoptosis of the TJ905 cells.</p>

Azidothymidine inhibition of telomerase activity and proliferation of TJ905 human glioblastoma cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the pharmacological effects and underlying mechanism of azidothymidine (AZT) on human glioblastoma cells in vitro.</p><p><b>METHODS</b>The telomerase activity of human glioblastoma TJ905 cells was determined by TRAP assay after 24 hrs' incubation with 50, 100, 200 micromol/L AZT and control vehicle solution. Colony formation efficiencies of the cells were recorded. Cells of the 1st, 3rd and 6th generations were harvested, followed by evaluations of cyclin A protein expression by Western blot, cell cycle distribution by flow cytometry, apoptotic level by single cell gel electrophoresis and proliferation index by Ki-67 immunocytochemical staining.</p><p><b>RESULTS</b>AZT inhibited telomerase activity of TJ905 cells. Cyclin A expression levels in the cells treated with 50 and 100 micromol/L AZT were significantly lower than controls (P < 0.01), and down-regulation of the expression was in a dose- and time-dependent manner. Compared with controls, G(0)/G(1) phase cells were obviously decreased (P < 0.05 approximately 0.01) and S phase cells significantly increased (P < 0.05 approximately 0.01) after treatment with 50, 100 and 200 micromol/L AZT. The cell numbers of G(0)/G(1) and S phases at the 1st generation of above three treated groups changed in a dose-dependent manner, whereas S phase cells increases in all AZT treatment groups and G(0)/G(1) phase cell decrease in group treated with 50 micromol/L AZT were also in a time-dependent manner. Both the apoptotic cells of the 1st and 6th generations of all AZT treatment groups were significantly more than controls (P < 0.05 approximately 0.01), their numbers of the 6th generations of the three groups increased with AZT concentration (P < 0.05 approximately 0.01), and all of them were more than the 1st and 3rd generations of the same dosage group (P < 0.05 approximately 0.01). Colony formation efficiencies and Ki-67 labeling indexes of the three AZT treatment groups were distinctly lower than controls (P < 0.01), and they were also decreased with the elevation of AZT concentration and/or the elongation of the incubating time. The difference of any above parameter had no significance among the 1st, 3rd and 6th generations of control group (P > 0.05).</p><p><b>CONCLUSION</b>AZT blocks S/G(2) conversion of TJ905 cells by inhibition of telomerase activity and cyclin A expression, leading to an enhancement of apoptosis and suppression of cell proliferation.</p>

Expression of motilin and its precursor mRNA in normal parenchyma, benign and malignant tumors of human thyroid / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of motilin and its precursor mRNA in normal human thyroid. To compare the expression differences of motilin and it precursor mRNA between normal thyroid and intestines. To study the expression of motilin and its precursor mRNA in human thyroid tumors and their clinical implications.</p><p><b>METHODS</b>RT-PCR, Southern blot and molecular cloning were used to detect motilin transcript expression in human thyroid and mucous membrane of small intestine. Real-time PCR and immunohistochemical techniques were used to quantify motilin precursor mRNA and motilin peptide in thyroid tissue samples including adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter.</p><p><b>RESULTS</b>(1) The expression of motilin and its precursor mRNA in normal human thyroid was primarily in the thyroid C cells. (2) RT-PCR and Southern blot showed that motilin mRNA expressed in human thyroid was identical to that expressed in duodenum with identical sequence deposited in NCBI Genbank of America. (3) Immunohistochemistry, Western blot research and real-time PCR studies showed that motilin and its precursor mRNA were expressed in normal and tumor tissues of human thyroid. Thyroid tumors (acidophilic adenoma, medullary carcinoma, follicular carcinoma, papillary carcinoma and nodular goiter) showed intense and diffuse immunostaining for motilin peptide. Moreover, the expression of motilin and its precursor mRNA in thyroid medullar carcinoma and acidophilic adenoma were significantly higher than those of normal thyroid tissue (P < 0.05). The expression in thyroid follicular and papillary carcinomas were significantly lower than those of normal thyroid tissue (P < 0.05). There was no difference of the expression between nodular goiter and normal thyroid tissue (P > 0.05).</p><p><b>CONCLUSIONS</b>Motilin peptide and its precursor mRNA are expressed in C cells of human thyroid. The sequence of motilin is identical to that expressed in duodenum from NCBI Genbank of America. The expressions of both motilin and its precursor mRNA in thyroid medullary carcinoma and acidophilic adenoma are significantly increased. In contrast, their expressions in thyroid follicular and papillary carcinomas are significantly decreased. Motilin may regulate physiological functions of the thyroid through parafollicular cells. Motilin may be involved in the pathogenesis of medullary carcinoma and acidophilic adenoma of the thyroid.</p>