John R. W. Masters

Short tandem repeat profiling provides an international reference standard for human cell lines

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

HeLa cells 50 years on: the good, the bad and the ugly

HeLa cells — the first continuous cancer cell line — have been a mainstay of cancer research ever since their isolation from the aggressive glandular cervical cancer of a young woman more than 50 years ago. Knowledge of almost every process that occurs in human cells has been obtained using HeLa cells and the many other cell lines that have since been isolated. So why does fraud an ignorance surround the use of these and other human cancer cell lines?

Defective repair of cisplatin-induced DNA damage caused by reduced XPA protein in testicular germ cell tumours

Metastatic cancer in adults usually has a fatal outcome. In contrast, advanced testicular germ cell tumours are cured in over 80% of patients using cisplatin-based combination chemotherapy [1]. An understanding of why these cells are sensitive to chemotherapeutic drugs is likely to have implications for the treatment of other types of cancer. Earlier measurements indicate that testis tumour cells are hypersensitive to cisplatin and have a low capacity to remove cisplatin-induced DNA damage from the genome [2] [3]. We have investigated the nucleotide excision repair (NER) capacity of extracts from the well-defined 833K and GCT27 human testis tumour cell lines. Both had a reduced ability to carry out the incision steps of NER in comparison with extracts from known repair-proficient cells. Immunoblotting revealed that the testis tumour cells had normal amounts of most NER proteins, but low levels of the xeroderma pigmentosum group A protein (XPA) and the ERCC1-XPF endonuclease complex. Addition of XPA specifically conferred full NER capacity on the testis tumour extracts. These results show that a low XPA level in the testis tumour cell lines is sufficient to explain their poor ability to remove cisplatin adducts from DNA and might be a major reason for the high cisplatin sensitivity of testis tumours. Targeted inhibition of XPA could sensitise other types of cells and tumours to cisplatin and broaden the usefulness of this chemotherapeutic agent.

Human cancer cell lines: fact and fantasy.

Cancer cell lines are used in many biomedical research laboratories. Why, then, are they often described as unrepresentative of the cells from which they were derived? Here, I argue that they have been unjustly accused. Under the right conditions, and with appropriate controls, properly authenticated cancer cell lines retain the properties of the cancers of origin.

Check your cultures! A list of cross-contaminated or misidentified cell lines

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well‐recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handlers knowledge. Cross‐contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross‐contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response—how can a researcher know if their own cell lines are cross‐contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross‐contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross‐contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross‐contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

Curing metastatic cancer: lessons from testicular germ-cell tumours

Most metastatic cancers are fatal. More than 80% of patients with metastatic testicular germ-cell tumours (TGCTs), however, can be cured using cisplatin-based combination chemotherapy. Why are TGCTs more sensitive to chemotherapeutics than most other tumour types? Answers to this question could lead to new treatments for metastatic cancers.

Proliferative Heterogeneity in the Human Prostate: Evidence for Epithelial Stem Cells

Clonal analysis of human prostate epithelial cells was undertaken in order to identify stem cells. Two types of colony were distinguished, termed type I and type II. Type I colonies were relatively small and irregular and contained a loose mixture of differentiated and undifferentiated cells. In contrast, type II colonies were large, round, and homogeneous, consisting almost exclusively of small undifferentiated and dividing cells. The colony-forming efficiency was 5.8% ± 1.8 for freshly isolated epithelial cells. There were approximately 10 times as many type I as type II colonies and about 1 in 200 of the plated cells was capable of forming a type II colony. In three-dimensional culture on Matrigel, the type II colonies produced structures reminiscent of prostate epithelium, with luminal cells expressing markers of prostate epithelial differentiation, including the androgen receptor. On the basis of their proliferative characteristics and pluripotency, the type II colonies may be the progeny of stem cells and the type I colonies of a more differentiated transit-amplifying population.

Reduced levels of XPA, ERCC1 and XPF DNA repair proteins in testis tumor cell lines

Over 80% of patients with advanced metastatic testis tumors can be cured using cisplatin‐based combination chemotherapy. This is unusual as metastatic cancer in adults is usually incurable. Cell lines derived from testis tumors retain sensitivity to cisplatin in vitro. We previously investigated 2 testis tumor cell lines with a low capacity to remove cisplatin‐induced DNA damage and found that they had low levels of the DNA nucleotide excision repair proteins XPA, ERCC1 and XPF. To determine whether low levels of XPA, ERCC1 and XPF proteins are characteristic of testis tumor cell lines, we investigated 35 cell lines derived from cancers to determine whether groups of cell lines from diverse tissue origins differ from one another in constitutive levels of these NER proteins. Quantitative immunoblotting was used to compare groups of cell lines representing prostate, bladder, breast, lung, cervical, ovarian and testis cancers. Only the 6 testis tumor cell lines showed significantly lower mean levels of XPA (p = 0.001), XPF (p = 0.001) and ERCC1 (p = 0.004) proteins from the other groups. Our results encourage further investigation of the possibility that low levels of these nucleotide excision repair proteins could be related to the favorable response of testis tumors to cisplatin‐based chemotherapy.

Epithelial Cell Differentiation Pathways in the Human Prostate: Identification of Intermediate Phenotypes by Keratin Expression

SUMMARY The prostate grows slowly throughout adult life, leading to benign prostatic hyperplasia (BPH), which often results in urethral obstruction in later years. The symptoms of BPH are the second most common reason for surgery in men over 65. The aim of this study was to determine the relationship between cell proliferation and cell differentiation in BPH tissue. Using multiple antibodies, simultaneously detected with different fluorophore-conjugated secondary antibodies, several subpopulations of epithelial cells were detected. In addition to K14, basal cells also expressed keratins 15, 17, and 19 in various combinations, and some of the luminal cells also expressed K19 together with K8 and K18. Co-staining for cytokeratins and Ki-67 indicated that 44% of proliferative cells expressed K14 and 36% K19, although the difference was not statistically significant. This report provides a detailed description of the relationship between keratin expression and cell proliferation in the prostate and indicates that K19-positive cells form the link between the basal and luminal layers of the epithelium. (J Histochem Cytochem 49:271–278, 2001)

Hypomethylation of WNT5A, CRIP1 and S100P in prostate cancer

Oligoarray analysis of a matched pair of prostate cancer and normal cell lines derived from the same radical prostatectomy specimen identified 113 candidate hypomethylated genes that were overexpressed in the cancer cells and contained CpG islands. Hypomethylation of wingless-related MMTV integration site 5A (WNT5A), S100 calcium-binding protein P (S100P) and cysteine-rich protein 1(CRIP1) was confirmed in the cancer cells by bisulfite sequencing. Treatment of the corresponding normal prostate epithelial cells 1542-NPTX with the DNA methyltransferase inhibitor 5-Aza-2′-deoxycytidine (5-aza-CdR) induced higher levels of mRNA expression and partial loss of methylation on these genes. Primary prostate cancers were tested using methylation-specific polymerase chain reaction. WNT5A was hypomethylated in 11/17 (65%) tumors, S100P in 8/16 (50%) and CRIP1 in 13/20 (65%). Bisulfite sequencing of a section of the 5′ untranslated region (UTR) of WNT5A revealed that three CpG sites (15, 24 and 35) were consistently methylated (93%) in the normal cell line and normal tissues, but not in the prostate cancer cell line and eight primary prostate cancers. Multiple putative binding sites for the transcription factors SP1 and AP-2 were found adjacent to CpG sites 15 and 24. A putative c-Myb binding site was located within the CpG site 35. Anti-c-Myb antibody co-precipitation with WNT5A was methylation-sensitive in 1542-NPTX cells. It is likely that an epigenetic mechanism regulates WNT5A expression in prostate cancer.