Epigenetic status of argininosuccinate synthetase and argininosuccinate lyase modulates autophagy and cell death in glioblastoma.

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Design of double-walled carbon nanotubes for biomedical applications.

Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT-siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom-designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells.

Paclitaxel resistance is associated with switch from apoptotic to autophagic cell death in MCF-7 breast cancer cells.

Taxanes remain first line chemotherapy in management of metastatic breast cancer and have a key role in epithelial ovarian cancer, with increasingly common use of weekly paclitaxel dosing regimens. However, their clinical utility is limited by the development of chemoresistance. To address this, we modelled in vitro paclitaxel resistance in MCF-7 cells. We show that at clinically relevant drug doses, emerging paclitaxel resistance is associated with profound changes in cell death responses and a switch from apoptosis to autophagy as the principal mechanism of drug-induced cytotoxicity. This was characterised by a complete absence of caspase-mediated apoptotic cell death (using the pan-caspase-inhibitor Z-VAD) in paclitaxel-resistant MCF-7TaxR cells, compared with parent MCF-7 or MDA-MB-231 cell lines on paclitaxel challenge, downregulation of caspase-7, caspase-9 and BCl2-interacting mediator of cell death (BIM) expression. Silencing with small interfering RNA to BIM in MCF-7 parental cells was sufficient to confer paclitaxel resistance, inferring the significance in downregulation of this protein in contributing to the resistant phenotype of the MCF-7TaxR cell line. Conversely, there was an increased autophagic response in the MCF-7TaxR cell line with reduced phospho-mTOR and relative resistance to the mTOR inhibitors rapamycin and RAD001. In conclusion, we show for the first time that paclitaxel resistance is associated with profound changes in cell death response with deletion of multiple apoptotic factors balanced by upregulation of the autophagic pathway and collateral sensitivity to platinum.

The cyclin-dependent kinase inhibitor p57(Kip2) is epigenetically regulated in carboplatin resistance and results in collateral sensitivity to the CDK inhibitor seliciclib in ovarian cancer.

BACKGROUND: Carboplatin remains a first-line agent in the management of epithelial ovarian cancer (EOC). Unfortunately, platinum-resistant disease ultimately occurs in most patients. Using a novel EOC cell line with acquired resistance to carboplatin: PEO1CarbR, genome-wide micro-array profiling identified the cyclin-dependent kinase inhibitor p57(Kip2) as specifically downregulated in carboplatin resistance. Presently, we describe confirmation of these preliminary data with a variety of approaches. METHODS: Cytotoxicity testing (MTT) and cell cycle blockade assessed drug responsiveness. Methylation specific PCR and pyrosequencing identified sites of promoter methylation in p57(Kip2). siRNA to p57(Kip2) was used to look at the changes in apoptosis of carboplatin treated EOC cells. EOC tissues (20 cases) were assessed for mRNA levels of p57(Kip2). RESULTS: Carboplatin resistance was reversed using 5-aza-cytidine in vitro. Promoter methylation sites and preferential sensitivity to seliciclib were seen in PEO1CarbR cells. Silencing p57(Kip)2 decreased the apoptotic response to the effects of platinum but produced sensitisation to seliciclib. EOC biopsies indicated an association of high levels of p57(Kip2)mRNA with complete responses to chemotherapy and improved outcome. CONCLUSION: We conclude that p57(Kip2) is a candidate biomarker of platinum sensitivity/resistance in EOC and such cases may show preferential response to the cyclin-dependent kinase inhibitor seliciclib.

AFM imaging of functionalized carbon nanotubes on biological membranes.

Multifunctional carbon nanotubes are promising for biomedical applications as their nano-size, together with their physical stability, gives access into the cell and various cellular compartments including the nucleus. However, the direct and label-free detection of carbon nanotube uptake into cells is a challenging task. The atomic force microscope (AFM) is capable of resolving details of cellular surfaces at the nanometer scale and thus allows following of the docking of carbon nanotubes to biological membranes. Here we present topographical AFM images of non-covalently functionalized single walled (SWNT) and double walled carbon nanotubes (DWNT) immobilized on different biological membranes, such as plasma membranes and nuclear envelopes, as well as on a monolayer of avidin molecules. We were able to visualize DWNT on the nuclear membrane while at the same time resolving individual nuclear pore complexes. Furthermore, we succeeded in localizing individual SWNT at the border of incubated cells and in identifying bundles of DWNT on cell surfaces by AFM imaging.

A gene expression profiling approach assessing celecoxib in a randomized controlled trial in prostate cancer.

BACKGROUND: We performed a pilot study, looking at the COX-2 inhibitor celecoxib, on newly diagnosed prostate cancer patients in the neo-adjuvant setting using DNA microarray analysis. PATIENTS AND METHODS: This was a single-blinded, randomized controlled phase II presurgical (radical prostatectomy) 28-day trial of celecoxib versus no drug in patients with localized T1-2 N0 M0 prostate cancer. cDNA microarray analysis was carried out on prostate cancer biopsies taken from freshly obtained radical prostatectomy samples. Results were confirmed by qPCR analysis of a selection of genes. RESULTS: Multiple genes were differentially expressed in response to celecoxib treatment. Statistical analysis of microarray data indicated 24 genes were up-regulated and 4 genes down-regulated as a consequence of celecoxib treatment. Gene changes e.g. survivin, SRP72kDa, were associated with promoting apoptotic cell death, enhancement of antioxidant processes and tumour suppressor function (p73 and cyclin B1 up-regulation). CONCLUSION: Celecoxib at 400 mg b.i.d. for 4 weeks perioperatively gave rise to changes in gene expression in prostate cancer tissue consistent with enhancement of apoptosis and tumour suppressor function. Given the short time interval for the duration of this study, the data are encouraging and provide a good rationale for conducting further trials of celecoxib in prostate cancer.

The context and potential of epigenetics in oncology.

Cancer has long been known to be a disease caused by alterations in the genetic blueprint of cells. In the past decade it has become evident that epigenetic processes have a function, at least equally important, in neoplasia. Epigenetics describes the mechanisms that result in heritable alterations in gene expression profiles without an accompanying change in DNA sequence. Genetics and epigenetics intricately interact in the pathogenesis of cancer (Esteller, 2007). In this review, we paint a broad picture of current understanding of epigenetic changes in cancer cells and reflect on the immense clinical potential of emerging knowledge of epigenetics in the diagnosis, prognostic assessment, treatment, and screening of cancer.

Dielectrophoretic analysis of changes in cytoplasmic ion levels due to ion channel blocker action reveals underlying differences between drug-sensitive and multidrug-resistant leukaemic cells.

Dielectrophoresis (DEP)--the motion of particles in non-uniform AC fields-has been used in the investigation of cell electrophysiology. The technique offers the advantages of rapid determination of the conductance and capacitance of membrane and cytoplasm. However, it is unable to directly determine the ionic strengths of individual cytoplasmic ions, which has potentially limited its application in assessing cell composition. In this paper, we demonstrate how dielectrophoresis can be used to investigate the cytoplasmic ion composition by using ion channel blocking agents. By blocking key ion transporters individually, it is possible to determine their overall contribution to the free ions in the cytoplasm. We use this technique to evaluate the relative contributions of chloride, potassium and calcium ions to the cytoplasmic conductivities of drug sensitive and resistant myelogenous leukaemic (K562) cells in order to determine the contributions of individual ion channel activity in mediating multi-drug resistance in cancer. Results indicate that whilst K(+) and Ca(2+) levels were extremely similar between sensitive and resistant lines, levels of Cl(-) were elevated by three times to that in the resistant line, implying increased chloride channel activity. This result is in line with current theories of MDR, and validates the use of ion channel blockers with DEP to investigate ion channel function.

COX-2 inhibition: a possible role in the management of prostate cancer?

There is mounting evidence to support a role for cyclooxygenase-2 (COX-2) inhibitors (coxibs) in the management of prostate cancer. This review considers the current evidence base for the use of coxibs in prostate cancer as well as their adverse event profile. A systematic literature review using the search terms 'cyclooxygenase', 'COX-2', 'coxibs', 'cardiovascular risk', and 'prostate cancer' was performed using Medline. Celecoxib appears safer in terms of cardiovascular toxicity than other coxibs, and this may relate to its lower selectivity for the COX-2 enzyme. This lower selectivity also provides rationale for its putative broader anti-cancer effects, via non-COX-2-dependent pathways that affect cell cycle regulation, angiogenesis, and hypoxic modulation. There are also interacting relationships between COX-2, chronic inflammation, and prostate cancer. There is much promise for the coxibs as anti-cancer agents. The future might be to pharmacologically adapt these agents to exert their COX-2 independent mechanisms of action while minimizing their COX-2-dependent adverse cardiovascular effects.

Copper bis(diphosphine) complexes: radiopharmaceuticals for the detection of multi-drug resistance in tumours by PET.

Experience with imaging of the multi-drug resistance (MDR) phenotype in tumours using technetium-99m sestamibi, a substrate of the P-glycoprotein (Pgp) transporter, suggests that better quantification of images and separation of MDR from other variables affecting tracer uptake in tumours are required. One approach to these problems is the development of short half-life positron-emitting tracers which are substrates of Pgp. Several lipophilic cationic copper(I) bis(diphosphine) complexes labelled with copper-64 have been synthesised and evaluated in vitro as substrates for Pgp. The synthesis is rapid and efficient with no need for purification steps. The chemistry is suitable for use with very short half-life radionuclides such as copper-62 (9.7 min) and copper-60 (23.7 min). Incubation of the complexes with human serum in vitro showed that they are sufficiently stable in serum to support clinical imaging, and the more lipophilic members of the series are taken up rapidly by cells (Chinese hamster ovary and human ovarian carcinoma) in vitro with great avidity. Uptake in human ovarian carcinoma cells is significantly reduced after several months of conditioning in the presence of doxorubicin, which induces increased Pgp expression. Uptake in hooded rat sarcoma (HSN) cells, which express Pgp, is significantly increased in the presence of the MDR modulator cyclosporin A. Biodistribution studies in hooded rats show rapid blood clearance, excretion through both kidneys and liver, and low uptake in other tissues. The one complex investigated in HSN tumour-bearing rats showed uptake in tumour increasing up to 30 min p.i. while it was decreasing in other tissues. We conclude that diphosphine ligands offer a good basis for development of radiopharmaceuticals containing copper radionuclides, and that this series of complexes should undergo further evaluation in vivo as positron emission tomography imaging agents for MDR.

Incidence of P-glycoprotein overexpression and multidrug resistance (MDR) reversal in adult soft tissue sarcoma.

Multidrug resistance (MDR) is a widespread problem in the treatment of neoplastic diseases and may limit the effectiveness of treatment of adult soft tissue sarcomas (STS). We examined the levels of expression of the MDR marker P-glycoprotein (Pgp) in fresh, surgical material and matched paraffin-embedded tissue using MRK-16 and JSB-1 monoclonal antibodies. Using fresh tumour material in short-term culture an assessment of doxorubicin sensitivity (MTT assay) and MDR modulation using PSC-833 in daunorubicin (DNR) accumulation experiments (FACS analysis) was carried out. 44 patients were studied at various disease stages with a mean follow-up duration of 487 days (range: 45-1095 days). Immunocytochemistry and immunohistochemistry showed 62% and 58%, respectively, of STS samples were positive for Pgp. Patients showing negative Pgp expression had a median survival of 544 days versus 431 days for Pgp-positive patients (P=0.311), with disease-free survival medians of 508 and 355 days, respectively (P=0.203). In vitro doxorubicin sensitivity was not informative in this respect and there was no apparent relationship between this and Pgp expression. Eleven out of 29 samples evaluated for MDR modulation showed enhanced tumour cell DNR accumulation. However, the effects of PSC-833 on drug accumulation in clinical material were modest compared with those seen for MDR cell lines, with a maximum of only 20% enhancement. Moreover, there was no relationship between the extent of PSC-833 effects on accumulation and the levels of Pgp seen in the STS samples. Nevertheless, we show evidence that a proportion of cases of STS express moderate to high levels of Pgp. There may be a role for MDR modulating agents in association with doxorubicin in the treatment of these tumours, either in the adjuvant setting or at first relapse.

Lack of prognostic significance of ploidy and S-phase measurements in advanced ovarian cancer.

DNA ploidy and S-phase measurements were made in fresh tumour samples obtained from 27 patients with either untreated or previously treated ovarian cancer. In addition, an assessment of in vitro chemosensitivity to post-operative chemotherapy was carried out. Whilst we found that patients with aneuploid tumours tended to survive longer (median survival 441 days versus 268 days for diploid tumours; p = 0.308) and this was associated with clinical response (p = 0.137) this was not statistically significant. Tumours with a high S-phase fraction (i.e. > 9% median) showed a median survival of 362 days compared with 484 days for those with a low S-phase fraction (< 9%; p = 0.79). Whilst a high level of predictability for chemosensitive patients (81%) was achieved, chemosensitivity per se was not related to the respective tumour DNA ploidy or S-phase fraction. In addition, when considering disease stage i.e. FIGO stage III or IV, or extent of residual disease between diploid and aneuploid groups, there were no statistically significant differences. However, there was a tendency for stage III tumours to be aneuploid (p = 0.189). Aneuploid tumours also showed the highest S-phase fractions (p = 0.03), indicative of a high proliferation rate. In conclusion, we have shown that DNA content and ploidy status do not appear to be major prognostic indicators for the management of ovarian cancer.

Susceptibility of fibromatosis cells in short-term culture to Ifosfamide: a possible experimental treatment in clinically aggressive cases.

Purpose. Deep fibromatoses are large, often rapidly growing but benign soft tissue tumours. Although surgery is the mainstay of treatment, in unremitting and aggressive cases the use of cytotoxic chemotherapy may produce objective tumour responses. Fresh tumour samples from four patients with fibromatosis were investigated as part of a study of drug resistance in soft tissue tumours.Methods. Following short-term culture of fibromatosis cells in vitro , chemosensitivity to 4-hydroperoxy-ifosfamide, the active form of ifosfamide and doxorubicin was tested. Following 72-h continuous exposure to each drug, surviving cell fraction was assessed using the lactate dehydrogenase assay.Results. Mean IC(50) values for ifosfamide and doxorubicin were 6.2 and 0.35 micromol/l, respectively. In samples of soft tissue sarcoma (STS) from the same study the mean IC(50) values for ifosfamide and doxorubicin were 14.8 and 1.69 micromol. The difference in mean ifosfamide IC(50) values for fibromatosis and STS samples was statistically significant.Discussion. We are not aware of any other report suggesting the use of ifosfamide in this condition. These observations suggest that, for patients with inoperable or progressive lesions of fibromatosis causing significant morbidity, it may be valuable to include ifosfamide in experimental treatment regimens.

Drug resistance studies using fresh human ovarian carcinoma and soft tissue sarcoma samples.

The relevance of continuous cell line cultures to the problem of clinical anticancer drug resistance is unclear. There is also mounting scepticism regarding the use of tumour cell lines with in vitro acquired drug resistance, possessing high levels of resistance unlikely to be seen in the clinical setting. To overcome some of these problems we have initiated a study of drug resistance using fresh tumour material obtained from patients suffering from ovarian cancer and soft tissue sarcoma (STS). Studies involving ovarian cancer have involved over 30 specimens of stage III-IV disease. For these samples we have specifically focused on the multidrug-resistant (MDR) phenotype, examining the role of proteins P-glycoprotein (Pgp), multidrug resistance-protein (MRP) and lung-resistance-associated protein (LRP). Techniques have involved chemosensitivity testing, immunocytochemistry and flow cytometry, to measure Pgp function (drug efflux capacity with modulator reversal). Pgp was the most commonly expressed marker and its expression correlated with survival. MDR modulation using cyclosporins was shown to chemosensitise a proportion of the samples. Hence, in vitro screening can help to identify patients likely to benefit from resistance reversal strategies. Studies involving STS have looked at a combination of MDR and p53 disruption (commonly seen in this disease). Data have been examined alongside clinical data and the course of disease has been closely monitored. Although our studies are ongoing, we have identified a group of patients with aggressive disease showing marked drug resistance in vitro. All patients have relapsed with persistent disease following chemotherapy or radiotherapy. A number of chemoresistant patients showed a combination of p53 disruption in the presence of an MDR phenotype. Feedback from these translational studies should be used to guide the selection of patients for clinical trials using resistance reversal strategies and may suggest new targets for drug development.

N-(Hydroxymethyl) melamines.

1. The N-(hydroxymethyl) melamines are analogs of the antitumor agent hexamethylmelamine (HMM) which do not require bioactivation to exert their antitumor effects. 2. Trimelamol (N2,N4,N6-trihydroxymethyl-N2,N4,N6-trimethylmelamine; TM) was developed as a water-soluble antitumor agent for intravenous administration. 3. Phase I and II trials of TM showed promising activity versus platinum-refractory ovarian cancer, but unfortunately further clinical development was halted due to formulation difficulties. 4. Stable analogs of TM were synthesized in an effort to overcome this shortcoming and these were evaluated in a number of in vitro and in vivo studies. 5. While the stable analogs showed good in vitro cytotoxicity in tumor cell lines, only one analog, CB7646 [bis-N-(hydroxymethyl)trimethylmelamine], showed comparable in vivo antitumor activity to that seen for TM. 6. Both TM and CB7646 were curative in human ovarian and breast cancer xenograft models, including the HX110P ovarian cancer xenograft with acquired resistance to carboplatin. 7. As CB7646 possesses favorable formulation characteristics, relating to its superior stability over that for TM, it is currently being developed for phase I clinical trial. 8. The N-(hydroxymethyl) melamines are capable of overcoming many forms of drug resistance, based on data obtained in in vitro and in vivo studies, and thus show promise as agents in the treatment of heavily pretreated, refractive tumors.

Chemosensitivity testing of fresh and continuous tumor cell cultures using lactate dehydrogenase.

We have examined the use of the LDH (lactate dehydrogenase) assay for chemosensitivity testing in established and primary cultures of sarcoma, leukaemia and ovarian cancer in parallel with the MTT assay. The method we describe is rapid, sensitive and ideal for 96-well plate assays using adherent or suspension cultures. Excellent agreement between the two methods was observed (r = 0.936) using a variety of antitumour agents, with some notable exceptions. In the Bax (human synovial sarcoma) cell line MTT colour production by control cells was very low, thus MTT-->formazan production could not be relied upon as a definitive end point equating with cell number. In contrast, colour production of control cells using the LDH assay was significantly greater and all cultures tested were suitable for titration of chemosensitivity. There was a discrepancy between IC50 values obtained either by cell counting or MTT in the HTB88 (human leiomyosarcoma) line treated with 5-FU (59.9 microM vs > 200 microM, respectively). However, cell counting agreed well with the LDH assay (IC50 47.3 microM). Whilst the MTT assay remains a reliable method for chemosensitivity testing, the LDH assay may prove more appropriate in certain experimental settings.

Pre-clinical development of the anti-tumour agent CB 7646, bis N-(hydroxymethyl) trimethylmelamine, a stable analogue of trimelamol.

Formulation difficulties prevented the otherwise promising clinical development of the anti-tumour agent trimelamol (TM; tris-[hydroxymethyl]trimethylmelamine]). A synthetic analogue programme resulted in the identification of CB 7646 (bis N-[hydroxymethyl]trimethylmelamine) as a compound with improved stability and favourable formulation characteristics. The in vitro and in vivo activity of CB 7646 was shown to be very similar to that of TM. In addition, curative activities were shown in the PXN/65 human ovarian cancer xenograft and the MX-1 and T-61 human breast cancer xenograft models. The effectiveness of the N-(hydroxymethyl)melamines against platinum-refractory disease was noted in the phase I clinical trial of TM. In line with this finding, the present study confirmed the effective activity of both TM and CB 7646 against various forms of platinum resistance in in vitro models. Curative activity for TM and CB 7646 was seen in the HX110P human ovarian cancer xenograft with acquired carboplatin resistance. Animal studies indicated less neurotoxicity for CB 7646 than for TM. The pharmacokinetic profile of CB 7646 indicated a decreased plasma elimination, indicative of slower in vivo degradation than for TM. CB 7646, therefore, represents a promising candidate for clinical development, designed to supersede TM.

The activity of N-(hydroxymethyl) melamines in fresh human ovarian tumour cells and xenografts.

Trimelamol (TM) was developed as a water soluble analogue of the oral chemotherapeutic agent hexamethyl-melamine (HMM), to be administered i.v., in an effort to avoid dose limiting emesis. Because of formulation difficulties due to its inherent instability the development of TM was halted. In vivo studies using a human ovarian cancer xenograft model PXN/65 showed TM to be curative in the dose range of 15-60 mg/kg i.p. daily x 5, for 4 weeks. Conversely, HMM given at the highest dose (60 mg/kg i.p., or 90 mg/kg p.o.) indicated only very modest tumour growth delays. In vitro chemosensitivity testing using primary ovarian tumour cultures showed that in 12/23 cases indicating reduced sensitivity to cisplatin or carboplatin, sensitivity to TM was increased. TM was curative in the carboplatin-resistant HX 110P human ovarian cancer xenograft and promising activity was seen in the MX-1 human breast cancer xenograft. In spite of enhanced stability in aqueous solution and good in vitro cytotoxicity, the TM analogues CB 7669 (triscyanomethyl) and CB 7639 (tristrifluoroethyl) showed disappointing in vivo antitumour activity which may be explained by the need for prolonged exposure. TM analogues with intermediate stability are currently under development in an effort to further the clinical development of this promising group of antitumour agents.

The role of the N-(hydroxymethyl)melamines as antitumour agents: mechanism of action studies.

The hexamethylmelamine analogue trimelamol (tris-hydroxymethyl[trimethyl]melamine) and its equicytotoxic stable analogues CB 7547, CB 7639 and CB 7669 have been used to clarify the mechanism of action for the N-(hydroxymethyl)melamines as antitumour agents. Two main mechanisms have been proposed and explored: (i) formation of a reactive iminium species forming covalent adducts with DNA; and (ii) local formaldehyde release leading to cytotoxic damage. 32P-postlabelling and thermal denaturation experiments showed these compounds to be interactive with cytosine and guanine. Trimelamol gave rise to DNA-interstrand crosslinks in naked plasmid DNA and in cultured cell lines, whereas the analogues failed to do so under a variety of experimental conditions. Along with our observations that cell lines with acquired resistance to the N-(hydroxymethyl)melamines showed no significant cross-resistance to classical bifunctional alkylating agents, DNA crosslinking may play only a minor role in their mechanism of action. In cultured cell lines treatment with formaldehyde, trimelamol and CB 7639 gave rise to high levels of DNA-protein crosslinks with a gradual disappearance over a 24 hr period. Along with our earlier observation that resistance to trimelamol coincides with cross-resistance to formaldehyde, we conclude that formaldehyde-release may be an important factor in their cytotoxicity. Further, the cytotoxicity of trimelamol or formaldehyde towards human ovarian cancer cells was not influenced by glutathione depletion. As the precise mechanism of action for the N-(hydroxymethyl)melamines is apparently not shared by many commonly used anticancer agents, this may confer their broad-spectrum activity versus heavily pretreated tumours.