Derivation and basic characterization of colorectal carcinoma primary cell lines.

BACKGROUND: Colorectal carcinoma is one of the most common malignancies in western countries. Among different approaches to its research, primary cancer cell lines can play an important role. AIM: The main purposes of this study were: 1) to establish an effective and reproducible method of colorectal cancer cell isolation and cultivation from primary tumours and lymph node metastases and 2) to elucidate the biological features of the tumours favouring successful cell cultivation. MATERIALS AND METHODS: The tumour cells were obtained from colectomy specimens. Primary tumour and lymph node metastasis tissue was used for establishing the tissue cultures. Colectomy samples were further processed for routine histopathological assessment: tumour grade, stage, angioinvasion and perineural spread were evaluated. Features of tissue culture cells were assessed using phase contrast microscopy and immune-histochemical techniques. WST-1 assay and X-CELLigence real time analysis were carried out for viability and proliferation testing before and after treatment with irinotecan and oxaliplatin. Molecular features of the tumour including K-RAS/B-RAF/N-RAS mutations were tested using allele-specific PCR. Results of the cultivation process were compared to the histopathological and molecular features of the tumours. RESULTS: In total, we obtained 33 samples from the primary site of tumours and 20 samples from lymph node metastases; in total, 27 cell lines were successfully isolated. Morphologic features characteristic of tumour cells in primary cell lines and epithelial differentiation (positive for AE1/AE3 cytokeratin) were evaluated. Higher tumour stage, angioinvasion and presence of perineural spread in primary tumour correlated positively with successful cell isolation from lymph node metastasis. All samples tested were NRAS wild-type. No correlation was found between molecular phenotype and the cell culture features. A higher proliferation potential was observed in the primary tumour cells, whereas higher sensitivity to irinotecan was found in the lymph node metastatic cells. CONCLUSIONS: Using mechanical dissociation, we successfully derived and cultivated CRC cells from primary tumours and lymph node metastases with success rate 3 % and 70% respectively. Primary tumour features favouring successful establishment of cell cultures were identified.

The role of autophagic cell death and apoptosis in irinotecan-treated p53 null colon cancer cells.

The roles of autophagic cell death and apoptosis induced by topoisomerase inhibitor irinotecan in colon cancer cells with deleted p53 were investigated during 48 h. We report that irinotecan-dependent cytotoxicity and proapoptotic activity were reduced in the present model while autophagy levels significantly increased. Upon p53 transfection, cell demise rates increased, with cells bearing the features of apoptosis and autophagic cell death. The subsequent studies into mechanisms of cell death process revealed the important role of Bax in mediating mitochondrial and lysosomal leakage which might serve as leading signals for both apoptosis and autophagic cell death. These results suggest that different modes of cell death in p53 null colon cancer cells treated with cytostatics (irinotecan) may be activated simultaneously. Moreover, their interactions possibly occur at several stages and aren't mutually exclusive. This might thus lead to a potential synergism with interesting therapeutic ramifications.