[Establishment and characterization of lung adenocarcinoma cell line XLA-07].

OBJECTIVE: To establish and characterize a lung adenocarcinoma cell line from a female patient in Xuanwei, Yunnan province. METHODS: Surgical specimen of the lung adenocarcinoma was obtained and cultured immediately in RPMI 1640 medium with 10% fetal bovine serum and 10(5) U/L penicillin and 100 mg/L streptomycin. When stable proliferation of the cells was achieved after over 40 passages in culture, the biological features of the cell line were investigated by cell morphology, karyotyping, protein marker expression [cytokeratins (CKs), epithelial membrane antigen (EMA) and CD proteins], growth kinetics, cell cycle phase distribution, mitotic index, colony formation in soft agar, cell invasion and tumorigenicity in Balb/c nude mice. RESULTS: The established cell line was stably cultured for over 80 passages during a one-year period as an anchorage-dependent monolayer of short spindle, polygonal to epithelioid cells under phase contrast microscope. Microglandular cavities and disordered microfilaments were observed under transmission electron microscope. The growth curve presented in an "S" shape with the cell population doubled every 46.7 hours. The mitotic index was 1.5% and the colony formation rate was 8.3%. The cell cycle distribution included 76.9% in G(0)/G(1), 15.1% in S and 8.0% in G(2)/M. The cell line displayed a hypotriploid karyotype with a mode of 66 chromosomes and a median of 64 chromosomes. The cells expressed CK7, CK8, CK (Pan) and EMA by immunohistochemistry. A high level of cell surface expression of CD13 and CD59 was evident by flow cytometry. The cells were able to penetrate Matrigel in vitro but failed to form a stable xenograft in nude mice. CONCLUSION: A new human lung adenocarcinoma cell line, designated as XLA-07, is successfully established from a Xuanwei lung cancer patient.

Induction of lung epithelial cell transformation and fibroblast activation by Yunnan tin mine dust and their interaction.

Tumor-stroma interactions play a significant role in tumor development and progression. Our study employed an in vitro co-culture model of epithelial cells and fibroblasts to investigate the mechanism of and interaction between lung epithelial cell transformation and fibroblast activation induced by Yunnan tin mine dust. Epithelial cell transformation was evaluated using concanavalin A agglutination and anchorage-independent growth assays, and fibroblast activation was assessed via immunohistochemistry. The TGF-ß1/Smad pathway was monitored by Western blot analysis and ELISA. We found concanavalin A agglutination and anchorage-independent growth assays of dust-exposed epithelial cells were positive, dust-exposed fibroblasts expressed α-SMA, and during the mine dust-induced tumorigenesis, TGF-ß1/Smad signaling pathway changed. In conclusion, Yunnan tin mine dust is able to induce the malignant transformation of bronchial epithelial cells and fibroblast activation. Epithelial cells are the main target of mine dust. Bronchial epithelial cell transformation and fibroblast activation are correlated and synergistic. Their interdependence is related to the TGF-ß1/Smad signaling pathway.

[Role of protein kinase C-delta in hyperthermia-induced apoptosis in tongue squamous cell carcinoma Tca8113 cells].

OBJECTIVE: To study the role of protein kinase C-delta (PKC-delta) in hyperthermia-induced apoptosis in human tongue squamous cell carcinoma Tca8113 cells. METHODS: Tca8113 cells were treated at 43 degrees C in a heating water bath for 0, 40, 80, 120 min after pretreatment with Rottlerin, a specific inhibitor of PKC-delta, and equal volume dimethyl sulfoxide (DMSO) for 30 min, respectively. The cells were stained by propidium iodide (PI) and Rhodamine 123 to analysis apoptotic rate and the changes of mitochondrial transmembrane potential by flow cytometry (FCM). The total proteins were extracted for Western blotting analysis of activation and proteolysis of PKC-delta, and for colorimetric assay of relative activity of Caspase-3. RESULTS: Hyperthermia could induce proteolysis and activation of PKC-delta, and this was attenuated by Rottlerin. Apoptotic rate, decreasing of mitochondrial transmembrane potential and activity of Caspase-3 which being induced by hyperthermia in Tca8113 cells were inhibited by PKC-delta specific inhibitor Rottlerin. There were significantly statistical differences in apoptosis rates, mitochondrial transmembrane potential and activity of Caspase-3 between Rottlerin- and non-Rottlerin-pretreated cells after hyperthermia for 40, 80, 120 min (P < 0.01). CONCLUSION: Activated PKC-delta may facilitate hyperthermia-induced apoptosis in Tca8113 cells, and may be one of the mechanisms of apoptosis induced by hyperthermia.

Hyperthermia-induced apoptosis in Tca8113 cells is inhibited by heat shock protein 27 through blocking phospholipid scramblase 3 phosphorylation.

PURPOSE: Hyperthermia induces tumour cell apoptosis through the mitochondrial apoptotic pathway; however, the signal transduction mechanism underlying this process still needs to be fully elucidated. Phospholipid scramblase 3 (PLS3), a target of protein kinase C-delta (PKC-delta), resides in mitochondria and plays pivotal roles in regulating apoptotic response. Activated PLS3 facilitates cardiolipin (CL) translocation from the mitochondrial inner membrane to the outer leaflet of the mitochondrial outer membrane and triggers apoptosis. MATERIALS AND METHODS: The tongue squamous cell carcinoma Tca8113 cells were transfected or co-transfected using Lipofectamine 2000 with plasmids pCMV-6xHis-PLS3, pCMV-6xHis-PLS3 (T21A), pHA-PKC-delta, pHA-PKC-delta-KD (K376R), pHA-Hsp27, and empty control plasmid pcDNA3.1. The transfected cells were heated in water bath at 43 degrees C for 20 min, 40 min and 60 min. Assessments of apoptosis and redistribution of mitochondrial cardiolipin were performed by flow cytometry. PLS3, PKC-delta, Hsp27, phosphorylation of PLS3 and PLS3/PKC-delta interaction were detected by western blotting. RESULTS: In our study the results show that elevated levels of the wild-type PLS3, but not the PLS3 (T21A) mutant, is able to increase hyperthermia-induced CL translocation and apoptosis. Wild-type PKC-delta facilitates PLS3 phosphorylation, PKC-delta/PLS3 interaction, and CL translocation, which consequently promote apoptosis. In contrast, heat shock protein 27 (Hsp27) blocks PKC-delta-induced PLS3 phosphorylation, suppresses PKC-delta/PLS3 interaction and CL translocation, and inhibits apoptosis. CONCLUSIONS: Our findings suggest that phosphorylation of PLS3 by PKC-delta is involved in the hyperthermia-induced apoptotic signal transduction pathway in Tca8113 cells, and that Hsp27 blocks this pathway to suppress hyperthermia-induced apoptosis.

[Study on the distribution of vitamin D receptor gene start codon polymorphism in the Achangs and Hans].

OBJECTIVE: To detect the difference between the Chinese Achang and Han ethnic groups in Yunnan province in the distribution of vitamin D receptor (VDR) gene start codon polymorphism. METHODS: Polymerase chain reaction-restriction fragment length polymorphism, gene sequencing and genetic analysis methods were used. A restriction fragment length polymorphism in the start codon of VDR (Fok I) gene was tested in the Achangs (n=68) and the Hans (n=92). RESULTS: The frequencies of FF, Ff and ff genotypes were found to be 18%, 35% and 47% in the Achangs, and 22%, 52% and 26% in the Hans, respectively. A significant difference was seen in the frequency distribution of VDR genotype between the Achangs and the Hans(Chi2=7.716, P=0.021). CONCLUSION: The Achang and Han ethnic groups differ in the frequency distribution of VDR gene start codon polymorphism.