In vitro testing of platinum-based drugs on a panel of human ovarian tumour cell lines.

Much improvement in the treatment of ovarian cancer has been achieved since the introduction of platinum compounds in the 1980s, with the result that single-agent platinum-based therapy following primary surgery is now the standard treatment for advanced ovarian cancer. The main therapeutic effect of chemotherapy is based on the sensitivity of the patient's tumour to the drug. However, testing a new chemical compound on humans requires much care, time and resources, whereas prior testing of drugs on cancer cell lines may indicate those drugs particularly suited to treatment of a specific disease. This study investigates the actions of two established platinum-based chemotherapeutic agents (cisplatin and carboplatin) on a panel of 10 human ovarian cancer cell lines. Each cell line was plated onto 96-well tissue culture plates, incubated for 72 hours with the drug, formalin-fixed and then assessed using the methylene blue colorimetric microassay to detect viable cells. The IC50 values for each cell line were calculated in order to assess the toxicity of each drug, and a wide range of responses were observed across the 10 cell lines investigated. This suggests that the panel reflected the heterogeneous nature of ovarian cancer, a malignancy in which a huge range of drug sensitivities can be seen even among tumours of the same histological type. The results indicate that the panel could be of use either as a primary screen to test new drugs against ovarian cancer or to investigate the drug resistance that is so common in this disease.

Mycoplasma detection in cell cultures: a comparison of four methods.

Mycoplasma is a common contaminant of tissue culture samples. Infection is persistent, difficult to detect and diagnose, and very difficult to cure. The concentration of mycoplasma in infected cultures can be as high as 10(7) colony-forming units per mL, and their presence can change many of the cell reactions, including altering cell growth rate, inducing morphological changes or cell transformation, and mimicking virus infection. Therefore, it should be assumed that a mycoplasma-contaminated cell line may be significantly influenced in every respect, and, thus, experimental data derived from such a cell line is likely to be invalid. Contamination is not obvious, either macroscopically or microscopically; thus, routine mycoplasma testing is essential for any cell culture laboratory. Many of the testing procedures developed so far are time-consuming, expensive, inconclusive and unsuitable for screening large numbers of test specimens. This study compares DNA staining, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and PCR ELISA, to determine which is the best procedure for routine assessment of cell cultures. All four methods gave reproducible results with both infected and non-infected cell lines. Both ELISA methods were easy to perform, reproducible and easily interpreted.

Characterisation of seven human ovarian tumour cell lines.

Characteristics of a panel of seven human ovarian tumour cell lines are presented. Positive staining with HMFG2 and ultrastructural identification of desmosomes confirmed the epithelial nature of the cell lines. The lines showed wide variations in ploidy, doubling times and clonogenicity in soft agar. Both vimentin and keratin were equally expressed in five lines, one line showed strong preferential expression of keratin and one line showed preferential expression of vimentin. Karyotypic changes associated with ovarian cancer were identified in all the lines. Four of the seven cell lines showed loss of chromosome material distal to 11p13-15. These cell lines offer considerable potential for research into the biology and genetics of ovarian cancer.

Use of XTT for quantitating clonogenic growth in soft agar.

The purpose of this study was to investigate the use of the tetrazolium salt. XTT, in a microassay for quantitation of anchorage-independent growth of cells in soft agar, using two human ovarian tumour cell lines (OAW 42 and OAW 28). The response of OAW 42 to Cis-platinum, EGF, TGF ß and insulin, and of OAW 28 to EGF, TGF ß and insulin were determined both by visual colony counting and use of the XTT assay. Drug inhibition and growth factor inhibition and stimulation were clearly demonstrated, with increases or decreases in visually counted colony numbers being paralleled by increases or decreases in optical density values obtained by using XTT. The XTT colorimetric assay was simple to perform and reproducible with low variability, and was found to be an acceptable alternative to visual colony counting for the quantitation of cell line response to drugs and growth factors under anchorage-independent growth conditions.

The methylene blue colorimetric microassay for determining cell line response to growth factors.

The validity of the methylene blue colorimetric microassay for determining the response of monolayers of human ovarian tumour cell lines to different growth factors was investigated. Linearity of the relationship between cell density and optical density was confirmed for each cell line (r=0.989-0.999,p<0.001), and when initial cell density was optimised to give exponential growth over the assay period, differences in response to medium supplements were obvious. The response of target cells to growth factors, obtained using the methylene blue assay, were compared with, and found to parallel, previously documented responses obtained non-colorimetrically. Thus Mink lung epithelial cells (MLEC) were inhibited by TGß (Holleyet al., 1983), EGF had an inhibitory effect on A431 cells (Gill & Lazar, 1981; Barnes, 1982), and the mesothelial cell line showed a proliferative response to EGF and hydrocortisone (Connell and Rheinwald, 1983).The methylene blue colorimetric microssay was found to be a simple, reliable, sensitive method with low variability, for determining the response of cultured cells to growth factors.