Biomedical subjects

J R Masters

Publications and source records attributed to J R Masters.

At least 37 records · Page 2Linked to original sources

PubMed1997

Complementation analysis of testis tumor cells.

Testis tumors are cured using cisplatin-based chemotherapy in over 80% of patients, and sensitivity to cisplatin is retained by testis tumor cells in vitro. The aim of this study was to use complementation analysis to determine how many genes control sensitivity to cisplatin in testis tumor cells. Four testis tumor cell lines were transfected with pSV2NEO and pBABE plasmids, conferring G418 and puromycin resistance, respectively. Self-crosses were generated to control for gene dosage, and the parentage of the hybrids was confirmed by PCR amplification of VNTR regions. Karyotyping confirmed that all the hybrids retained at least 88% of the combined number of chromosomes of the two parental cell lines. Cisplatin sensitivity was measured by clonogenic assay and complementation was not observed. This finding provides evidence that there is a single common mechanism controlling cisplatin sensitivity in testis tumor cells.

Antineoplastic Agents↗

PubMed1997

DNA repair capacity and cisplatin sensitivity of human testis tumour cells.

Metastatic testicular germ cell tumours can be cured using cisplatin-based combination chemotherapy. To investigate the role of DNA repair in cisplatin sensitivity, we measured the formation and removal of cisplatin adducts in the whole genome and in specific genomic regions in 3 testis and 3 bladder cancer cell lines. Following a 1 hr exposure to 50 microM cisplatin, the mean level of DNA platination was lower in the testis tumour cell lines. During a 72 hr post-treatment incubation period, the 3 bladder cell lines removed platinum from the overall genome, whereas 2 of the testis tumour cell lines showed relatively little reduction of DNA platination. The third testis tumour cell line, SuSa, showed an intermediate capacity to remove cisplatin. Cisplatin-induced damage and repair in selected regions of the actively transcribed N-ras gene and the inactive CD3delta gene were measured using quantitative PCR. The data were in agreement with those obtained with atomic absorption spectroscopy for the whole genome, showing that the bladder lines were repair-proficient: 2 of the testis tumour cell lines showed no repair, and the third testis line, SuSa, showed an intermediate level of repair in these 2 genes. Our findings confirm that reduced capacity to repair cisplatin-damaged DNA may contribute to the hypersensitivity of testis tumour cells to DNA-damaging agents.

Antineoplastic Agents↗

PubMed1997

KW-2149 (7-N-[2-[gamma-L-glutamylamino]ethyldithioethyl]mitomycin C). A new mitomycin C analogue activated by serum.

KW-2149 (7-N-[2-[gamma-L-glutamylamino]ethyldithioethyl]mitomycin C) is a new mitomycin-C analogue in clinical trial. This study demonstrates that KW-2149, unlike mitomycin C, is activated to a cytotoxic species by extracellular metabolism in serum. The metabolising activity differs between batches of serum and species of origin. Human serum had high activity (which resulted in a 150-fold enhancement of cytotoxicity), whereas mouse serum had low activity. In the presence of serum, the rate of uptake of 3H-KW-2149 into cells increased by 8-fold and drug binding to DNA by 32-fold. The metabolising activity of serum can partially be replaced by glutathione. No anticancer drug has previously been described whose toxicity is mediated by metabolism in serum.

Animals↗

PubMed1996

DNA repair in cisplatin-sensitive and resistant human cell lines measured in specific genes by quantitative polymerase chain reaction.

More than 80% of patients with testicular germ cell tumours (TGCT) are cured using cisplatin-based combination chemotherapy, and resistance to cisplatin is the final barrier to the cure of nearly all patients with this disease. In this study, we used quantitative polymerase chain reaction (Q-PCR) to investigate the role of DNA repair in cisplatin resistance in two genes, one transcribed and one not transcribed. Three pairs of cisplatin-sensitive and resistant cell lines were used, two derived from TGCT and one from a bladder cancer. In these pairs of sublines, we observed no major differences between the repair of cisplatin-induced damage in the transcribed and nontranscribed genes, nor did there appear to be any relationship between DNA repair capacity and the development of cisplatin resistance. Despite the strong indication that the sensitivity of testis tumour cells to cisplatin is related to their reduced ability to repair cisplatin-damaged DNA, these cells apparently do not become resistant to cisplatin by acquiring DNA repair capacity.

Antineoplastic Agents↗

PubMed1996

Hypersensitivity of human testicular tumors to etoposide-induced apoptosis is associated with functional p53 and a high Bax:Bcl-2 ratio.

Metastatic testicular cancers are curable, whereas bladder cancers and most other solid tumors are not. Cell lines derived from human testicular (GH, GCT27, and 833K) and bladder (RT4, RT112, and HT1376) tumors retain this differential chemosensitivity in vitro. We have investigated the hypothesis that differential sensitivity to chemotherapy is related to differences in the threshold of susceptibility to undergoing apoptosis. Sensitivity to etoposide was not directly related to the frequency of DNA strand breaks. DNA damage was on average 2-fold greater in the testicular than the bladder tumor cell lines; in contrast, the testicular tumor lines were 15-fold more sensitive to etoposide cytotoxicity than the bladder tumor lines (IC90 values of 19 +/- 6 versus 293 +/- 180 microM, respectively). Using equidamaging (550 rad equivalents) etoposide treatments, the percentage of cells that underwent drug-induced apoptosis was on average higher in the testicular tumor cell lines than the bladder tumor cell lines. The testicular tumor lines have two characteristics that could confer sensitivity to drug-induced apoptosis. First, they have functional p53: the product of the p53-dependent gene waf-1 was increased after etoposide treatment. Second, the testicular tumor lines expressed relatively high levels of the apoptosis-promoting protein Bax, but there was no expression of the suppressor of apoptosis Bcl-2. In contrast, only one of the three bladder cell lines (RT4) had functional p53, and all of the bladder lines had readily detectable levels of Bcl-2 and low levels of Bax. In the testicular cell lines, increases in p53 and p53-transactivated genes were associated with apoptosis but not arrest in G1. In contrast, in the bladder cell line (RT4), increases in p53 and Waf-1 were associated with both arrest in G1 and apoptosis. The differences in the ratio of Bax:Bcl-2 could contribute to the differential sensitivity of the two tumor types. However, in contrast to earlier reports, the ratio of Bax and Bel-2 was not perturbed by DNA damage.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Genetic basis of drug sensitivity in human testis tumour cells.

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98orC1). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning.

Antineoplastic Agents↗

PubMed1996

Response to epirubicin in patients with superficial bladder cancer and expression of the topoisomerase II alpha and beta genes.

Biopsies of superficial bladder cancer were analysed to study the relationship between response to epirubicin and the expression of the human topoisomerase II alpha and beta genes. Tissue samples were obtained prior to treatment and a marker tumour was left in the bladder. Transcript levels of both genes were generally lower in biopsies taken following treatment failure. Levels of topoisomerase II mRNA were uniformly lower in tumour tissue than in biopsies of normal tissue.

Aged↗

PubMed1996

ThioTEPA pharmacokinetics during intravesical chemotherapy: the influence of dose and volume of instillate on systemic uptake and dose rate to the tumour.

ThioTEPA is given intravesically in a variety of schedules to treat superficial bladder cancer. In this study, the influence of the dose of ThioTEPA and the volume of instillate on the dose rate to the tumour and the systemic uptake of ThioTEPA was investigated in eight patients with pTa or pTl disease. Each patient received four courses of ThioTEPA consisting of 30 mg of drug/30 ml of distilled water, 30 mg/60 ml, 60 mg/30 ml and 60 mg/60 ml. Blood and urine samples were obtained for 8 h following instillation, and ThioTEPA and TEPA levels were measured. The AUC(infinity) values (areas under the concentration-time curve, extrapolated to infinity) in plasma were approximately 2 factors higher at the two 60-mg doses. However, the AUC value in the bladder was nearly 70% higher when 60 mg of drug was instilled in 30 ml of distilled water as compared with 60 mg in 60 ml. Thus, by decreasing the volume of instillate it is possible to increase the dose rate to the tumour without increasing the systemic toxicity.

Absorption↗

PubMed1996

Sensitivity of testis tumour cells to chemotherapeutic drugs: role of detoxifying pathways.

In contrast to most other types of cancer, metastatic testicular germ cell tumours (TGCT) are cured in most patients using cisplatin-based combination chemotherapy. The biochemical mechanisms underlying this sensitivity have not been defined. Drug detoxification can modulate response to chemotherapy in vivo and in vitro, and therefore we measured levels of glutathione (GSH), glutathione-S-transferase (GST) and both constitutive and cisplatin- and dexamethasone-induced levels of metallothionein (MT) in five human testis tumour cell lines. The levels were compared with those in five human bladder cancer cell lines and two cell lines with cisplatin resistance acquired in vitro. GSH levels were relatively low in the testis tumour cell lines, as might be expected in drug-sensitive cells, and there was a 77-fold increase in GSH levels in the cisplatin-resistant testis tumour cell line. GST levels were similar in the two cell types, while metallothionein levels were relatively high in the testis tumour cell lines. These data indicate that GSH may contribute to the sensitivity of TGCT to chemotherapy, and that GSH expression may be involved in the acquisition of cisplatin resistance in these tumours.

Antineoplastic Agents↗

PubMed1996

5 alpha-reductase expression by prostate cancer cell lines and benign prostatic hyperplasia in vitro.

5 alpha-reductase (5 alpha R) activity in two human prostate cancer cell lines was compared to that in benign prostatic hyperplasia (BPH) tissue and COS cells transfected with and expressing the human genes for 5 alpha-reductase type 1 (5 alpha R1) and type 2 (5 alpha R2). Comparisons were based on pH profiles and sensitivities to selective inhibitors of 5 alpha-reductase In the cancer lines, activity was greatest over the pH range 7-8, compared to a sharp peak of activity between pH 5-5.5 in BPH tissue and COS cells expressing 5 alpha R2. Finasteride and SKF105,657 were potent inhibitors of 5 alpha-reductase activity in BPH tissue and COS cells expressing 5 alpha R2, but weak inhibitors in the cancer lines and in COS cells expressing 5 alpha R1. In contrast, UK117,026 was a more potent inhibitor of 5 alpha-reductase activity in the prostate cancer cell lines and in COS cells expressing 5 alpha R1. These data indicate that human prostate cancer cell lines express 5 alpha-reductase activity similar to that in COS cells transfected with 5 alpha R1, but different from that in BPH tissue. This may be a consequence of in vitro culture. Alternatively, it may reflect a change occurring as a result of neoplastic transformation in which case it will be important to select appropriate inhibitors in the clinic.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1995

Relative cytotoxicities of adriamycin and epirubicin in combination with lonidamine against human bladder cancer cell lines.

We have used a panel of bladder cancer cell lines to compare the toxicities of Adriamycin and epirubicin, two drugs used intravesically to treat superficial transitional cell cancer (TCC) of the bladder, alone and in combination with lonidamine, an agent known to be active against anthracycline-resistant disease. Comparing concentrations reducing colony-forming ability by 50%, epirubicin and Adriamycin were similar in their cytotoxicities, although epirubicin was more potent against every line except an Adriamycin-resistant subline. Combinations of the two drugs with a non-cytotoxic concentration (1 microgram/ml) of lonidamine were tested using the Adriamycin-resistant subline MGH-U1R and its sensitive parental line MGH-U1. The addition of lonidamine caused a two-fold increase in the sensitivity of the resistant subline to both drugs, while having no effect on the sensitivity of the parental line. The data indicate that this combination might be of value in anthracycline-resistant disease.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1995

Immortalisation of human urothelial cells.

A cell line derived from the urothelium lining the ureter of a 12-year-old girl was immortalised using a temperature-sensitive SV40 large T-antigen gene construct, and designated UROtsa. Following immortalisation, UROtsa cells expressed SV40 large T-antigen, but did not acquire characteristics of neoplastic transformation, including growth in soft agar or the development of tumours in nude mice. Metaphase spreads had a normal chromosomal appearance and number. UROtsa cells remained permissive for cell growth at 39 degrees C, indicating that they did not retain temperature sensitivity. UROtsa provides an in vitro model of "normal" urothelium.

Animals↗

PubMed1995

Exceptional sensitivity of testicular germ cell tumour cell lines to the new anti-cancer agent, temozolomide.

Metastatic testicular germ cell tumours are cured in approximately 85% of patients using cisplatin-based combination chemotherapy. Patients who fail to respond have a poor prognosis, and there is a need for more effective treatments for cisplatin-resistant disease. In this study, it is shown that two of four cell lines derived from human non-seminomatous testicular germ cell tumours are exceptionally sensitive to temozolomide, a new imidazotetrazine which can cross the blood-brain barrier in mice. In addition, three pairs of cisplatin-resistant sublines show little cross-resistance to temozolomide. These data suggest that temozolomide might have activity against non-seminomatous testicular germ cell tumours which have relapsed following cisplatin-containing chemotherapy, and could have a role in the treatment of patients with metastatic lesions in the brain.

Antineoplastic Agents↗

PubMed1995

Heat shock protein expression in testis and bladder cancer cell lines exhibiting differential sensitivity to heat.

Testis cancer cells are more sensitive than bladder and most other cancer cells to chemotherapeutic drugs both in the clinic and in vitro. In this study we show that they are also more sensitive than bladder cancer cells to heat. Since heat and drug sensitivity may be related to the ability of a cell to mount a stress response, constitutive and induced levels of heat shock proteins (HSPs) in three testis and three bladder human cancer cell lines were measured using Western blotting and scanning densitometry. No correlation between constitutive levels of HSP 90 or HSP 73/72 and cellular heat sensitivity was found. However, HSP 27 levels were much lower in the testis tumour cells, suggesting that low HSP 27 expression might contribute to heat sensitivity. Protein synthesis studies using [35S]methionine indicated that, for the same heat shocks, the kinetics of synthesis and decay of HSP 90 and HSP 73/72 in 833K (the most heat sensitive testis cells) was similar to or greater than that in HT1376 (the most heat-resistant bladder cells). Both 833K and HT1376 developed thermotolerance, and this followed an increase in synthesis of HSPs. These results indicate that, although there are differences in the constitutive levels of HSPs between testis and bladder cancer cells, both cell types are capable of mounting an induced heat shock response and can develop a similar degree of thermotolerance.

Cell Survival↗

PubMed1994

Culture and characterisation of human urothelium in vivo and in vitro.

The aim of this study was to culture human urothelium and generate enough cells for subsequent reconstructive surgery. Using a modification of the Rheinwald-Green method for the routine culture of keratinocytes from patients with burns, we successfully cultured 91% of 57 biopsies from the renal pelvis, ureter, bladder and urethra of paediatric patients. The cells could be split one to three up to 9 times at 7-10 day intervals, giving a surface area of 1000 cm2 after a 2 month culture period. Primary cultures could not be initiated in defined medium MCDB153, although cells initiated using the Rheinwald-Green method could subsequently be propagated in this medium. Cytokeratin patterns in vitro were similar to those in vivo in the expression of keratins 7, 18 and 19 (characteristic of simple epithelia) and keratin 13 (characteristic of non-cornified stratified epithelia). Cultured urothelium also expressed keratin 14 (characteristic of cornified stratified epithelium) in about 25% of cells and keratin 16 (characteristic of fast-growing cells). These findings indicate that urothelial cells can be propagated in vitro for autologous grafting, and the next step is to identify substrates suitable for urothelial cell growth and differentiation and surgical manipulation.

Cells, Cultured↗

PubMed1994

Comparison of testosterone metabolism in benign prostatic hyperplasia and human prostate cancer cell lines in vitro.

Pathways of testosterone metabolism in tissue slices and cell suspensions of human benign hyperplastic prostate (BPH) tissue and human prostate cancer cell lines (DU145, HPC-36M, PC-3/MA2 and LNCaP) were investigated. Thin layer chromatography analysis was used to identify the following tritiated metabolites: testosterone, 5 alpha-dihydrostestosterone (DHT), 5 alpha-androstane-3 alpha/3 beta-17 beta-diol (androstanediols), 4-androstene-3,17-dione (androstenedione) and 5 alpha-androstanedione. The predominant pathway for testosterone metabolism in BPH was via 5 alpha-reductase producing 5 alpha-dihydrotestosterone (71% and 75% total metabolites in slices and suspensions incubated for 24 h, respectively). The cancer cell lines DU145 and HPC-36M resembled BPH by metabolizing testosterone predominantly to DHT (68% and 82% total metabolites, respectively), although the rate of metabolism was much lower in the cell lines (0.099 and 0.05 pmol testosterone/mg protein/h in DU145 and HPC-36M) compared to the BPH cell suspensions (6.4 pmol testosterone/mg protein/h). In contrast, PC-3/MA2 contained high 17 beta-HSD activity forming large amounts of 4-androstene-3,17-dione (84% total metabolites), converting testosterone at a rate faster (12.8 pmol testosterone/mg protein/h) than the BPH cell suspensions. LNCaP rapidly converted testosterone exclusively to a glucuronide conjugate (7.4 pmol testosterone/mg protein/h), although after incubation with [3H]-4-androstene-3,17-dione, 5 alpha-reductase activity was demonstrated. LNCaP was the only cell line whose growth and colony-forming ability was stimulated by testosterone and DHT. BPH and all the cell lines tested had 5 alpha-reductase activity, but only the prostate tissue and the cell lines DU145 and HPC-36M converted testosterone predominantly to DHT.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1994

Superficial bladder cancer: the response of a marker tumour to a single intravesical instillation of epirubicin.

OBJECTIVE: To determine the response of a marker tumour to a single instillation of intravesical epirubicin. PATIENTS AND METHODS: Eighty-one patients (54 men, 27 women) with a mean age of 69.3 years (range 36-92) with superficial bladder cancer were randomized to receive a single instillation of intravesical epirubicin. At the initial cystoscopy all but one papillary marker tumour was resected. Subsequently the patients were randomized to receive either intravesical epirubicin at a concentration of 1 mg/ml (n = 40) or 2 mg/ml (n = 41) in 50 ml of saline for 1 h. The response of the marker tumour was determined at 3 months (first check cystoscopy). The toxicity associated with both treatments was also recorded. RESULTS: A complete response (no visible or microscopic bladder carcinoma) was observed in 46% (95% confidence interval (CI) 35-57%) of patients. No patient experienced systemic side-effects. Chemical cystitis and bladder irritability were the most frequent local side-effects, occurring in 15% (95% CI 8-24%) of the patients. CONCLUSIONS: A single instillation of intravesical epirubicin has a demonstrable effect in superficial bladder cancer. The results compare favourably with more onerous regimes. Side-effects were minimal at 1 mg/ml and acceptable at 2 mg/ml.

Administration, Intravesical↗

PubMed1993

The differential induction of collateral resistance to 62.5 MeV (p-->Be+) neutrons and 4 MeV photons by exposure to cis-platinum.

PURPOSE: To determine the relative sensitivity to cis-platinum, 4 MeV photons and 62.5 MeV (p-->Be+) neutrons in five human tumor cell lines, and their cis-platinum resistant variants. METHODS AND MATERIALS: The degree of cross-resistance of five human in-vitro cell lines to photons or fast neutrons was analysed for both cisplatinum-sensitive and resistant variants. RESULTS: The development of acquired cis-platinum resistance conferred collateral resistance to 62.5 MeV (p--Be+) neutrons in all five cell lines, but did not consistently decrease the photon sensitivity of these same cells. CONCLUSION: The reduction in photon and neutron sensitivity following the development of acquired cis-platinum resistance may possibly be regulated by different mechanisms. The reduction in neutron sensitivity was primarily due to a 1.3-1.7 fold reduction in the magnitude of the initial slope (alpha), which was independent of the degree of platinum resistance induced, suggesting a non-stochiometric relationship between the mechanisms responsible for acquired cis-platinum, and 62.5 MeV (p-->Be+) neutron resistance.

Cell Survival↗