Guidelines for the use of cell lines in biomedical research.

Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.

Allelic imbalance and biochemical outcome after radical prostatectomy.

OBJECTIVE: To compare the incidence of allelic imbalance (AI) in men with rapid disease progression with those who remained disease free after radical prostatectomy, with the aim of identifying genetic markers to predict prognosis and guide further treatment. PATIENTS AND METHODS: Tumour and normal DNA were extracted from two matched groups of 31 men with extracapsular node-negative (pT3N0) prostate cancer who had undergone radical prostatectomy. One group comprised men who developed biochemical recurrence within 2 years of surgery and one group were prostate-specific antigen (PSA) free for at least 3 years. Men were matched for Gleason grade, preoperative PSA and pathological stage. Analysis was performed by genotyping. RESULTS: Allelic imbalance was analysed using 30 markers, and was seen in at least one marker in 57 (92%) of the cases. Deletion at marker D10S211 (10p12.1) was significantly more common in the relapse group than the non-relapse group (35 vs 5%, P=0.03). CONCLUSIONS: This study demonstrates significant association between AI on chromosome 10 and biochemical progression after radical prostatectomy.

Rho family GTPases are activated during HGF-stimulated prostate cancer-cell scattering.

An important process in embryogenesis and cancer-cell metastasis is the conversion of epithelial cells to a migratory phenotype, a phenomenon known as epithelial-mesenchymal transition (E-MT). To achieve E-MT, cells dissociate from neighbouring cells and adopt a migratory morphology. This transition requires remodelling of their cell shape and substratum adhesions; activities that require extensive reorganisation of the actin cytoskeleton. Hepatocyte growth factor (HGF)-induced scattering of Madin Darby canine kidney (MDCK) cells is a routinely used model of E-MT, in which actin cytoskeletal rearrangement is known to be dependent on Rho family GTPases. We have developed a novel model of HGF-induced E-MT using the human prostate cancer cell line, DU145. This model overcomes the limitation of using a canine cell line and facilitates the study of E-MT in human cancer. We demonstrate for the first time the scattering response of individual DU145 cells to HGF in real time and have characterised changes in actin cytoskeletal organisation and cell adhesions as these cells respond to HGF. HGF-induced scattering of DU145 cells is dependent on the activity of Rho family GTPases, and using this model, we are able to demonstrate for the first time that endogenous Cdc42 is activated downstream of HGF. Furthermore we have also shown that the response of DU145 cells to HGF is dependent on a phosphatidylinositide 3-kinase pathway.

The comparative values of bone marrow aspirate and trephine for obtaining bone scan-targeted metastases from hormone-refractory prostate cancer.

Samples of metastatic prostate cancer to bone are difficult to obtain. The aim of this study was to compare the results of bone marrow aspirate and trephine biopsy for obtaining metastatic hormone-refractory prostate cancer (HRPC) samples using previous diagnostic planar 99(m)Tc-HDP bone scans to guide the procedure. All samples taken were for the purposes of research and molecular studies on HRPC. Twenty patients with HRPC had bone marrow aspirate and trephines taken from lesions in the posterior superior iliac spine or sacro-iliac region when shown on diagnostic 99(m)Tc-HDP bone scans. Three patients also underwent plain X-ray, 18F-positron emission tomography bone scan, pelvic MRI scan and 99(m)Tc nanocolloid bone marrow scans. These images were used to assess if the extra imaging information provided, such as three-dimensional localisation of the bone metastases, was of value for target bone metastases. Cancer cells were obtained in 15/20 (75%) cases in which a trephine biopsy was attempted and 0/20 of cases in which a bone marrow aspiration was attempted. The additional information provided by the range of other imaging investigations was of little benefit in obtaining tumour samples, but did suggest why negative biopsies were obtained in some cases after targeting with planar bone scans. We recommend the use of bone marrow trephine biopsy alone, guided by previous diagnostic 99(m)Tc planar bone scan as a practical method to obtain prostate cancer cells from bone metastases.

Amplification of the androgen receptor gene in bone metastases from hormone-refractory prostate cancer.

The aim of this study was to examine the prevalence of androgen receptor (AR) amplification in metastases to bone and other sites in patients with hormone-refractory prostate cancer (HRPC) and to compare these findings with those in pretreatment primary tumour samples from the same patients. Tissue from 24 patients with HRPC was available for study, together with 13 primary tumour specimens. AR gene amplification and copy number for X-chromosome were assessed by fluorescence in situ hybridization (FISH) using a SpectrumOrange-labelled probe at locus Xq11-13 for the AR gene and a SpectrumGreen-labelled alpha-satellite probe for the X-chromosome (Vysis, UK, Ltd.). A minimum of 20 nuclei were scored in each of three tumour areas by two independent observers. Samples from 18/24 patients with HRPC (12 bone marrow biopsies, three local tumour recurrences, and three lymph nodes) and nine primary tumour specimens were adequate for FISH analysis. Results were expressed as a mean ratio of AR gene copy number : mean X-chromosome number, with a ratio of greater than 1.5 defined as amplification. AR gene amplification was seen in 9/18 (50%) cases of HRPC and in none of the primary (untreated) tumour specimens (p = 0.0048, Fisher's exact test). For the 12 bone marrow samples, AR gene amplification occurred in 5/12 (38%) cases. Elevated copy number for chromosome X occurred in 3/18 (17%) HRPC and 4/9 (44%) matched primary tumours. This study shows for the first time that AR gene amplification can be demonstrated by FISH in bone metastases from HRPC patients. Because bone marrow biopsies can be obtained from most patients with HRPC, the findings provide a rational basis for the routine selection of patients who may respond more favourably to second-line anti-androgen therapy.