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Biomedical subjects

J R Masters

Publications and source records attributed to J R Masters.

At least 19 recordsLinked to original sources
PubMed2005

Genome-wide screening for genetic changes in a matched pair of benign and prostate cancer cell lines using array CGH.

Copy number alterations in a matched pair of benign epithelial and prostate cancer cell lines derived from the same patient were assessed using array-based comparative genomic hybridisation (aCGH). The cancer cell line showed a gain of chromosome 7, deletion of chromosome 8, gains (including high level) and losses on chromosome 11, loss of 18p and gain of 20q. Deletions on chromosome 8 were confirmed with microsatellite markers. The aCGH results were compared to gene expression data obtained using DNA microarrays and suggested the involvement of caspases and ICEBERG on 11q and E2F1 on chromosome 20q.

Cell Line, Tumor↗
PubMed2003

Cancer stem cells.

Explore the source record for details and available documents.

Cell Division↗
PubMed2001

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.

Cell Line↗
PubMed2001

Epithelial cell differentiation pathways in the human prostate: identification of intermediate phenotypes by keratin expression.

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)

Aged↗
PubMed2001

The immortalized UROtsa cell line as a potential cell culture model of human urothelium.

The UROtsa cell line was isolated from a primary culture of normal human urothelium through immortalization with a construct containing the SV40 large T antigen. It proliferates in serum-containing growth medium as a cell monolayer with little evidence of uroepithelial differentiation. The working hypothesis in the present study was that this cell line could be induced to differentiate and express known features of in situ urothelium if the original serum-containing growth medium was changed to a serum-free formulation. We demonstrated that the UROtsa cells could be successfully placed into a serum-free growth medium consisting of a 1:1 mixture of Dulbeco's modified Eagle's medium and Ham's F-12 supplemented with selenium (5 ng/mL), insulin (5 microg/mL), transferrin (5 microg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). Under serum-free growth conditions, confluent UROtsa cells were shown by light microscopy to produce raised, three-dimensional structures. Routine ultrastructural examination disclosed these three-dimensional areas to consist of a stratified layer of cells that strongly resembled in situ urothelium. The cells displayed numerous desmosomal connections, complex interactions of the lateral membranes, and abundant intermediate filaments within the cytoplasm. Freeze fracture analysis demonstrated that the cells possessed tight-junction sealing strands and gap junctions. The overall morphology was most consistent with that found in the intermediate layers of in situ urothelium. The basal expression patterns of the metallothionein (MT) and heat shock proteins 27, 60, and 70 were determined in these cells, and expression was in agreement with that known to occur for in situ urothelium. The cells were also successfully tested for their ability to be stably transfected using expression vectors containing the MT-3 or MT-2A genes. The findings suggest that the UROtsa cells grown with a serum-free medium could be a valuable adjunct for studying environmental insult to the human urothelium in general and for the stress response in particular.

Cell Culture Techniques↗
PubMed2000

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.

Animals↗
PubMed2000

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.

Aged↗
PubMed2000

Comparison of marker protein expression in benign prostatic hyperplasia in vivo and in vitro.

OBJECTIVE: To use multiple immunofluorescence to compare the in vivo and in vitro expression of tissue-specific proteins in BPH. Materials and methods Pure populations of prostate epithelial and stromal cells were produced using standard methods. Serum-free media for epithelial cells were compared. Co-localization of proteins was compared in frozen-tissue sections and cultured cells by simultaneous multiple immunofluorescence, and recorded using a high-resolution charge-coupled device camera. RESULTS: In contrast to the other serum-free media tested, epithelial cells grew without squamous differentiation or vacuolation in prostate epithelial growth medium (PrEGM, Clonetics, BioWhittaker UK Ltd., Berks, UK). These cells were predominantly of a basal phenotype, with some cells showing a luminal phenotype. Most of the stromal cells had features of myofibroblasts, but smooth muscle cells and fibroblasts also were present. CONCLUSION: PrEGM is a commercially available serum-free medium in which primary cultures of prostate epithelial cells can be propagated reproducibly. This study provides a comprehensive description of tissue-specific protein expression in BPH in vivo and in vitro. The use of simultaneous multiple immunofluorescence to study co-localization has resulted in a more precise definition of phenotype than has previously been possible, thereby establishing the relevance of the in vitro model system BPH.

Biomarkers, Tumor↗
PubMed1999

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.

Cell Extracts↗
PubMed1999

Gene-specific repair of Pt/DNA lesions and induction of apoptosis by the oral platinum drug JM216 in three human ovarian carcinoma cell lines sensitive and resistant to cisplatin.

JM216, an oral platinum drug entering into phase III clinical trial, exhibited comparable cytotoxicity to cisplatin in three human ovarian carcinoma cell lines: the sensitive (CH1), acquired resistant (CH1cisR) and intrinsically resistant (SKOV-3). Platinum accumulation and binding to DNA were similar in each of the three cell lines at equimolar doses, indicating that the resistant cell lines could tolerate higher intracellular platinum levels and platinum bound to DNA at IC50 concentrations of drug. Comparison with cisplatin demonstrated that intracellular platinum levels were marginally higher with JM216, but that platinum binding to DNA was similar for the two drugs in each of the cell lines. Each of the cell lines exhibited an ability to repair JM216 induced platinum/DNA lesions in the N-ras gene (gene-specific repair) at equitoxic concentrations of drug. However, this occurred to a greater extent in the two resistant cell lines such that by 24 h the CH1cisR and SKOV-3 had removed 72% and 67% respectively compared with approximately 32% for the CH1. Reduced gene-specific repair capacity in CH1 cells was also seen following incubation with 25 microM (or 5 microM - 2 x IC50) cisplatin, whereas the CH1cisR and SKOV-3 cell lines were repair proficient. JM216 induced apoptosis in the three cell lines following a 2h incubation with 2 x the IC50 of drug. Fluorescent microscopy of cells stained with propidium iodide showed that the detached cell population displayed typical apoptotic nuclei. Furthermore, field inversion gel electrophoresis demonstrated the presence of DNA fragments approximately 23-50 kb in size, indicative of apoptosis, in the detached cells. JM216 induced an S phase slow down in each of the three cell lines accompanied by a G2 block in the CH1 pair. Incubation with this concentration of JM216 also resulted in the induction of p53 in the CH1 and CH1cisR. These studies suggest that the relative sensitivity of the CH1 cell line to cisplatin and JM216 is at least partly attributable to a deficiency in gene-specific repair. The oral platinum drug, JM216, exerts its cytotoxic effects through the induction of apoptosis following a slow-down in S phase in both the sensitive and resistant lines.

Antineoplastic Agents↗
PubMed1999

Prognostic markers in clinically localised prostate cancer.

The prognostic value of immunohistochemical staining of P53, BCL-2, p27kip1, PSA, AR and MIB-1 was compared with that of established prognostic variables (Gleason score, surgical margins, tumour volume) following radical prostatectomy. Five groups were selected: negative margins with stable serum PSA (n=11), negative margins with rising serum PSA (n=7), positive margins with stable serum PSA (n=7), positive margins with rising serum PSA (6) and patients with micrometastatic disease diagnosed in lymph nodes removed during radical prostatectomy (n=8). Gleason score and tumour volume were of prognostic significance and immunohistochemical staining for MIB-1 and BCL-2 showed added independent prognostic significance in multivariate analysis.

Antigens, Nuclear↗
PubMed1999

Intravesical chemotherapy with epirubicin: a dose response study.

PURPOSE: We determined the difference in response to high and standard doses of intravesical epirubicin for treatment of superficial bladder cancer. MATERIALS AND METHODS: A total of 122 patients were entered into a randomized trial to compare the response of a marker tumor at 3 months, time to first recurrence and recurrence rates for 2 years after intravesical chemotherapy for superficial (pTa/pT1) bladder cancer. Patients were randomized to receive treatment for 1 hour with 1 (standard dose) or 2 mg./ml. (high dose) epirubicin (50 or 100 mg./50 ml. solution). RESULTS: There was no difference in the marker tumor response rate in 24 of 52 patients treated with the standard dose compared with 21 of 50 treated with the higher dose of epirubicin (p = 0.67). Similarly, the higher dose was not superior in regard to time to first recurrence, with a hazard ratio of 1.46 (p = 0.14, 95% confidence intervals 0.88 to 2.42). Considering the upper end of the confidence interval, we can reliably exclude an absolute difference of greater than 4% at 1 year for time to first recurrence in favor of higher dose chemotherapy. CONCLUSIONS: Epirubicin at double the standard dose for intravesical chemotherapy of superficial bladder cancer is not superior in regard to marker tumor response, time to first recurrence or recurrence rate.

Administration, Intravesical↗
PubMed1998

KW-2149 (7-N-[2-[gamma-L-glutamylamino]ethyldithioethyl] mitomycin C): DNA interactions and drug uptake following serum activation.

7-N-[2-[gamma-L-glutamylamino]ethyldithio-ethyl] mitomycin C (KW-2149) is a mitomycin-C analogue currently being evaluated in clinical trials. It has been shown that KW-2149 is unusual in that it is activated by serum, resulting in an increase in potency of up to 200-fold. To investigate the mechanism by which KW-2149 is activated, the abilities of mitomycin-C, KW-2149 and its metabolites M-18 (symmetrical disulphide dimer) and M-16 (methyl sulphide form) to interact with DNA were compared, and the influence of serum and glutathione on the sequence-specificity of KW-2149-DNA interactions was determined. Following reduction by glutathione both KW-2149 and M-18 are more efficient crosslinking agents of naked DNA, with the metabolite M-18 showing superior activity. The efficiency of DNA interstrand crosslinking in cells by KW-2149 was also increased by the addition of serum. Using the potassium/SDS precipitation method it was found that KW-2149 and M-18 crosslink protein to DNA whilst mitomycin C and M-16 do not. All four compounds produced almost identical patterns of adducts. Serum and glutathione did not alter the pattern of DNA adducts, but did increase the efficiency of adduct formation. Our earlier studies had indicated that the mechanism of activation of KW-2149 by serum is related to cellular uptake, and we therefore studied the effects of certain metabolic inhibitors, temperature and competitive inhibition on drug uptake. The results suggest that uptake is passive, and this indicates that a component in serum modifies KW-2149 to a form that passively enters cells more rapidly.

Antibiotics, Antineoplastic↗
PubMed1998

Mouse-human somatic cell hybrids: loss of mouse and human chromosomes.

Testicular germ cell tumors are unusual because they can be cured in over 80% of patients with combination chemotherapy. In order to identify chromosomes carrying genes controlling drug sensitivity, fusions were made between a mouse embryonal carcinoma (EC) cell line, F9, and a human bladder cancer cell line, MGH-UI. In contrast to some previous reports, interspecies hybrids of mouse EC cells with human cells were easy to produce. Six independent hybrids were cloned and grown for 10 further passages and karyotyped. Surprisingly, all the independent hybrids retained approximately 80% of the 40 mouse chromosomes and approximately 80% of the 83 human chromosomes. Despite the positive selection for mouse chromosomes and the absence of selection for human chromosomes, it appears that some of both sets of chromosomes are essential for these hybrids.

Animals↗
PubMed1998

In vitro assessment of a collagen sponge for engineering urothelial grafts.

OBJECTIVE: To investigate the deposition of urinary crystals and the growth characteristics of urothelial cells on a collagen sponge, as a preliminary step in engineering urothelial autologous grafts. MATERIALS AND METHODS: Collagen sponges were exposed to a continuous flow of urine at pH 5.3 and 6.3 for 1 week. The sponges were examined microscopically for crystal deposition and analysed for their calcium content. Two cell lines, RT112, derived from a well-differentiated transitional cell carcinoma, and UROtsa, an immortalized urothelial cell line, were seeded on the collagen sponges. Cells were cultured for 6, 12 and 21 days. The pattern of growth was analysed by histology and immunostaining with a pan-cytokeratin antibody. Growth was assayed to quantify cell proliferation on the sponges. RESULTS: No crystals were evident on any of the collagen sponges. Calcium deposition was negligible at pH 5.3. Although calcium levels were measurable at pH 6.3, the levels were very low. Both cell lines attached and grew in a stratified manner on the collagen sponge, RT112 forming a layer 6-8 cells thick, and UROtsa a layer 4-6 cells thick; cell proliferation was maximal at 5-10 days. The sponge remained easy to handle after 3 weeks in culture. CONCLUSION: These findings show that collagen sponges support the growth and stratification of urothelial cells, and indicate that the collagen sponge is a suitable substrate for developing urothelial autologous grafts.

Calcium↗