Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed in vitro to carbon ions and argon ions at the HIRFL

Human hepatoma (SMMC-7721) and normal liver (L02) cells were irradiated with c-rays, 12C6+ and 36Ar18+ ion beams at the Heavy Ion Research Facility in Lanzhou (HIRFL). By using the Calyculin-A induced premature chromosome condensation technique, chromatid-type breaks and isochromatid-type breaks were scored separately. Tumor cells irradiated with heavy ions produced a majority of isochromatid break, while chromatid breaks were dominant when cells were exposed to c-rays. The relative biological effectiveness (RBE) for irradiation-induced chromatid breaks were 3.6 for L02 and 3.5 for SMMC-7721 cell lines at the LET peak of 96 keVlm 1 12C6+ ions, and 2.9 for both of the two cell lines of 512 keVlm 1 36Ar18+ ions. It suggested that the RBE of isochromatid-type breaks was pretty high when high-LET radiations were induced. Thus we concluded that the high production of isochromatid-type breaks, induced by the densely ionizing track structure, could be regarded as a signature of high-LET radiation exposure.

Effect of 80.55 MeV/u(12)C(6+) ions on radiosensitivity and cell cycle of human hepatoma cell lines

In this paper, the relationship between radiosensitivity, cell cycle alteration and the change of apoptosis in different human hepatoma cell lines irradiated by heavy ions were studied with the aim of building up the base data for clinical therapy. Exponentially growing hepatoma cell lines were irradiated by 80.55 MeV/u(12)C(6+) ions at a dose of 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy and 8 Gy. The radiosensitivity was assessed by means of the colony-forming assay. The DNA content, the percentage of each cell-cycle phase and the apoptosis rate were obtained with flow cytometry methods. After the irradiation, the SF2 (survival fraction at 2 gray) of SMMC-7721 cells were evidently lower than that of HepG2 cells. The S phase arrest, G2/M phase arrest delay and the apoptosis in the two hepatoma cell lines varied with the increase of the dose and repair time. The heavy ions could obviously kill the human hepatoma cell lines. Compared to HepG2 cells, SMMC-7721 cells were more radiosensitive to C-12(6+) ions.

A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with Co-60 gamma-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G(2) PCC was about five times more than G(1) PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G(1) and G(2) phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.

Correlation between initial chromatid damage and survival of various cell lines exposed to heavy charged particles

The biophysical characteristics of heavy ions make them a rational source of radiation for use in radiotherapy of malignant tumours. Prior to radiotherapy treatment, a therapeutic regimen must be precisely defined, and during this stage information on individual patient radiosensitivity would be of very great medical value. There are various methods to predict radiosensitivity, but some shortfalls are difficult to avoid. The present study investigated the induction of chromatid breaks in five different cell lines, including one normal liver cell line (L02), exposed to carbon ions accelerated by the heavy ion research facility in Lanzhou (HIRFL), using chemically induced premature chromosome condensation (PCC). Previous studies have reported the number of chromatid breaks to be linearly related to the radiation dose, but the relationship between cell survival and chromatid breaks is not clear. The major result of the present study is that cellular radiosensitivity, as measured by D-0, is linearly correlated with the frequency of chromatid breaks per Gy in these five cell lines. We propose that PCC may be applied to predict radiosensitivity of tumour cells exposed to heavy ions.

Analysis of three marine fish cell lines by RAPD assay

We tested the applicability of the random amplified polymorphic deoxyribonucleic acid (RAPD) analysis for identification of three marine fish cell lines FG. SPH, and RSBK and as a possible tool to detect cross-contamination. Sixth commercial 10-mer RAPD primers were tested on the cell lines and on samples collected from individual fish. The results obtained showed that the cell lines could be identified to the correspondent species on the basis of identical Patterns produced by 35-48% of the primers tested. the total mean similarity indices for cell lines versus correspondent species of individual fish ranged from 0.825 to 0.851. indicating the existence of genetic variation in these cell lines in relation to the species of their origin. Also, four primers, which gave a monomorphic hand pattern within species/line, but different among the species/line, were obtained. These primers can be useful for identification of these cell lines and for characterization of the genetic variation of these cell lines in relation to the species of their origin. This supported the use of RAPD analysis as an effective tool in species identification and cross-contamination test among different cell lines.

Reference genes for quantitative RT-PCR data in gastric tissues and cell lines

AIM: To evaluate the suitability of reference genes in gastric tissue samples and cell lines.METHODS: the suitability of genes ACTB, B2M, GAPDH, RPL29, and 18S rRNA was assessed in 21 matched pairs of neoplastic and adjacent nonneoplastic gastric tissues from patients with gastric adenocarcinoma, 27 normal gastric tissues from patients without cancer, and 4 cell lines using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). the ranking of the best single and combination of reference genes was determined by NormFinder, geNorm (TM), BestKeeper, and DataAssist (TM). in addition, GenEx software was used to determine the optimal number of reference genes. To validate the results, the mRNA expression of a target gene, DNMT1, was quantified using the different reference gene combinations suggested by the various software packages for normalization.RESULTS: ACTB was the best reference gene for all gastric tissues, cell lines and all gastric tissues plus cell lines. GAPDH + B2M or ACTB + B2M was the best combination of reference genes for all the gastric tissues. On the other hand, ACTB + B2M was the best combination for all the cell lines tested and was also the best combination for analyses involving all the gastric tissues plus cell lines. According to the GenEx software, 2 or 3 genes were the optimal number of references genes for all the gastric tissues. the relative quantification of DNMT1 showed similar patterns when normalized by each combination of reference genes. the level of expression of DNMT1 in neoplastic, adjacent non-neoplastic and normal gastric tissues did not differ when these samples were normalized using GAPDH + B2M (P = 0.32), ACTB + B2M (P = 0.61), or GAPDH + B2M + ACTB (P = 0.44).CONCLUSION: GAPDH + B2M or ACTB + B2M is the best combination of reference gene for all the gastric tissues, and ACTB + B2M is the best combination for the cell lines tested. (C) 2013 Baishideng Publishing Group Co., Limited. All rights reserved.

Molecular characterization of chordoma xenografts generated from a novel primary chordoma cell source and two chordoma cell lines.

OBJECT: Chordoma cells can generate solid-like tumors in xenograft models that express some molecular characteristics of the parent tumor, including positivity for brachyury and cytokeratins. However, there is a dearth of molecular markers that relate to chordoma tumor growth, as well as the cell lines needed to advance treatment. The objective in this study was to isolate a novel primary chordoma cell source and analyze the characteristics of tumor growth in a mouse xenograft model for comparison with the established U-CH1 and U-CH2b cell lines. METHODS: Primary cells from a sacral chordoma, called "DVC-4," were cultured alongside U-CH1 and U-CH2b cells for more than 20 passages and characterized for expression of CD24 and brachyury. While brachyury is believed essential for driving tumor formation, CD24 is associated with healthy nucleus pulposus cells. Each cell type was subcutaneously implanted in NOD/SCID/IL2Rγ(null) mice. The percentage of solid tumors formed, time to maximum tumor size, and immunostaining scores for CD24 and brachyury (intensity scores of 0-3, heterogeneity scores of 0-1) were reported and evaluated to test differences across groups. RESULTS: The DVC-4 cells retained chordoma-like morphology in culture and exhibited CD24 and brachyury expression profiles in vitro that were similar to those for U-CH1 and U-CH2b. Both U-CH1 and DVC-4 cells grew tumors at rates that were faster than those for U-CH2b cells. Gross tumor developed at nearly every site (95%) injected with U-CH1 and at most sites (75%) injected with DVC-4. In contrast, U-CH2b cells produced grossly visible tumors in less than 50% of injected sites. Brachyury staining was similar among tumors derived from all 3 cell types and was intensely positive (scores of 2-3) in a majority of tissue sections. In contrast, differences in the pattern and intensity of staining for CD24 were noted among the 3 types of cell-derived tumors (p < 0.05, chi-square test), with evidence of intense and uniform staining in a majority of U-CH1 tumor sections (score of 3) and more than half of the DVC-4 tumor sections (scores of 2-3). In contrast, a majority of sections from U-CH2b cells stained modestly for CD24 (scores of 1-2) with a predominantly heterogeneous staining pattern. CONCLUSIONS: This is the first report on xenografts generated from U-CH2b cells in which a low tumorigenicity was discovered despite evidence of chordoma-like characteristics in vitro. For tumors derived from a primary chordoma cell and U-CH1 cell line, similarly intense staining for CD24 was observed, which may correspond to their similar potential to grow tumors. In contrast, U-CH2b tumors stained less intensely for CD24. These results emphasize that many markers, including CD24, may be useful in distinguishing among chordoma cell types and their tumorigenicity in vivo.

Hepatocyte growth factor (HGF) protects c-met-expressing Burkitt''s lymphoma cell lines from apoptotic death induced by DNA damaging agents

The relative sensitivity of neoplastic cells to DNA damaging agents is a key factor in cancer therapy. In this paper, we show that pretreatment of Burkitt's lymphoma cell lines expressing the c-met protooncogene with hepatocyte growth factor (HGF) protects them from death induced by DNA damaging agents commonly used in tumour therapy. This protection was observed in assays based on morphological assessment of apoptotic cells and DNA fragmentation assays. The protection was dose- and time-dependent — maximal protection requiring pre-incubation with 100 ng/ml HGF for 48 h. Western blotting analysis and flow cytometric studies revealed that HGF inhibited doxorubicin- and etoposide-induced decreases in the levels of the anti-apoptotic proteins Bcl-XL, and to a lesser extent Bcl-2, without inducing changes in the pro-apoptotic Bax protein. Overall, these studies suggest that the accumulation of HGF within the microenvironment of neoplastic cells may contribute to the development of a chemoresistant phenotype.

Staurosporine-induced apoptosis and hydrogen peroxide-induced necrosis in two human breast cell lines.

The use of apoptosis-inducing agents in the treatment of malignant cancer is increasingly being considered as a therapeutic approach. In this study, the induction of apoptosis and necrosis was examined in terms of temporal dose responses, comparing a malignant and nonmalignant breast cell line. Staurosporine (SSP)-induced apoptosis and H2O2-induced necrosis were evaluated by two cytotoxicity assays, neutral red (NR) and methyl-thiazolyl tertrazolium (MTT), in comparison with a differential dye uptake assay, using Hoechst33342/propidium iodide (Hoechst/PI). Confirmatory morphological assessment was also performed by routine resin histology and transmission electron microscopy. Cell viability was assessed over a 0.5-48 h time course. In nonmalignant HBL-100 cells, 50 nM SSP induced 100% apoptosis after a 48 h exposure, while the same exposure to SSP caused only 4% apoptosis in metastatic T47D cells. Although complete apoptosis of both cell lines was induced by 50 M SSP, this effect was delayed in T47D (24 h) compared with HBL-100 (4 h). Results also showed that neither MTT or NR can distinguish between the modes of cell death, nor detect the early onset of apoptosis revealed by Hoechst/PI.

Methylation status of oestrogen receptor-a-gene promoter sequences in human ovarian epithelial cell lines.

We have determined the methylation status of the CpG island of the oestrogen receptor gene in seven human ovarian cell lines. Cell lines expressing oestrogen receptor showed no evidence of hypermethylation. In three of four cell lines that produced no detectable oestrogen receptor protein, hypermethylation was observed at the NotI site of the CpG island. These results indicate that aberrant hypermethylation may be responsible for a significant proportion of epithelial ovarian tumours in which oestrogen receptor expression is lost.