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1.
Br J Cancer ; 90(2): 476-82, 2004 Jan 26.
Article in English | MEDLINE | ID: mdl-14735196

ABSTRACT

Specific chromosomal abnormalities are increasingly recognised to be associated with particular tumour subtypes. These cytogenetic abnormalities define the sites of specific genes, the alteration of which is implicated in the neoplastic process. We used comparative genomic hybridisation (CGH) to examine DNA from different breast and ovarian cancer cell lines for variations in DNA sequence copy number compared with the same normal control. We also compared different sources of the MCF7 breast line by both CGH and cDNA expression arrays. Some of the differences between the subcultures were extensive and involved large regions of the chromosome. Differences between the four subcultures were observed for gains of 2q, 5p, 5q, 6q, 7p, 7q, 9q, 10p, 11q, 13q, 14q, 16q, 18p and 20p, and losses of 4q, 5p, 5q, 6q, 7q, 8p, 11p, 11q, 12q, 13q, 15q, 19p, 19q, 20p, 21q, 22q and Xp. However, few variations were found between two subcultures examined, 5 months apart, from the same initial source. The RNA arrays also demonstrated considerable variation between the three different subcultures, with only 43% of genes expressed at the same levels in all three. Moreover, the patterns of the expressed genes did not always reflect our observed CGH aberrations. These results demonstrate extensive genomic instability and variation in RNA expression during subculture and provide supportive data for evidence that cell lines do evolve in culture, thereby weakening the direct relevance of such cultures as models of human cancer. This work also reinforces the concern that comparisons of published analyses of cultures of the same name may be dangerous.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA, Neoplasm/analysis , Genomic Instability , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Tumor Cells, Cultured , Cell Culture Techniques , Chromosome Aberrations , Female , Humans , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Reproducibility of Results
2.
J Med Chem ; 45(17): 3692-702, 2002 Aug 15.
Article in English | MEDLINE | ID: mdl-12166942

ABSTRACT

4-[N-[7-Bromo-2-methyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl]-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide (CB30865) is a quinazolin-4-one antitumor agent whose high growth-inhibitory activity (W1L2 IC(50) = 2.8 +/- 0.50 nM) is believed to have a folate-independent locus of action. In addition, CB30865 represents a class of compounds with unique biochemical characteristics such as a delayed, non-phase specific, cell-cycle arrest. The low aqueous solubility of CB30865 prompted a search for more water-soluble analogues for in vivo evaluation of this class of compounds. It was thought that aqueous solubility could be increased by the introduction of amino functionalities at the 2-position of the quinazolin-4-one ring. A variety of compounds (5a-j, 31a-c, 32, and 33) were synthesized in a linear fashion starting from 3-chloro-4-methylaniline. Most of these compounds (e.g., 5a, 5b, 5g) were significantly more water-soluble than CB30865 (636 microM for 5a at pH 6 and 992 microM for 5g at pH 6). In addition, some of them were up to 6-fold more cytotoxic than CB30865 (e.g., for 5a, W1L2 IC(50) = 0.49 +/- 0.24 nM) and retained its novel biochemical characteristics.


Subject(s)
Antineoplastic Agents/chemical synthesis , Quinazolines/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Quinazolines/chemistry , Quinazolines/pharmacology , Solubility , Structure-Activity Relationship , Tumor Cells, Cultured
3.
Br J Cancer ; 79(11-12): 1692-701, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10206279

ABSTRACT

Three lipophilic quinazoline-based aminomethyl pyridine compounds, which differ only in the position of the nitrogen in their pyridine ring, are described. CB300179 (2-pyridine), CB300189 (4-pyridine) and CB30865 (3-pyridine) all inhibited isolated mammalian TS with IC50 values of 508, 250 and 156 nM respectively. CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1-100 nM for several mouse and human cell types). CB300179 and CB300189 were active in the micromolar range. Against W1L2 cells, CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. Furthermore, combinations of precursors and end products of folate metabolism, e.g. dThd/hypoxanthine (HX) or leucovorin (LV), did not prevent activity. CB30865 did not interfere with the incorporation of tritiated dThd, uridine or leucine after 4 h. A cell line was raised with acquired resistance to CB30865 (W1L2:R865; > 200-fold), which was not cross-resistant to CB300179 or CB300189. In addition, W1L2:R865 cells were as sensitive as parental cells to agents from all the major chemotherapeutic drug classes. CB300179 and CB300189 induced an S phase accumulation (preventable by co-administration of dThd). No cell cycle redistribution was observed following exposure (4-48 h) to an equitoxic concentration of CB30865. In the NCI anticancer drug-discovery screen, CB30865 displayed a pattern of activity which was not consistent with known anti-tumour agents. These data suggest that CB30865 represents a class of potent potential anti-tumour agents with a novel mechanism of action.


Subject(s)
Antineoplastic Agents/therapeutic use , Folic Acid/pharmacology , Lipid Metabolism , Quinazolines/therapeutic use , Animals , Cell Cycle/drug effects , Cell Division/drug effects , Cell Survival/drug effects , Chemical Precipitation , Colony-Forming Units Assay , Cytoprotection , DNA, Neoplasm/biosynthesis , Humans , Leukemia L1210/drug therapy , Leukemia L1210/pathology , Mice , Solubility , Tumor Cells, Cultured
4.
J Inorg Biochem ; 77(1-2): 95-104, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10626360

ABSTRACT

The technique of comparative genomic hybridisation (CGH) has until recently been used to screen for common genomic abnormalities in fresh tumour material; it has identified previously unrecognised regions of amplification associated with poor prognosis subtypes of breast cancer and lymphoma. Our group has applied this technique to resistant cell lines and their sensitive counterparts in order to define chromosomal abnormalities associated with acquired drug resistance. We have demonstrated the applicability of this technique to the study of drug resistance using cell lines with known mechanisms of resistance. The ability to detect novel genomic alterations in cell lines with novel mechanisms of resistance was also demonstrated. We subsequently examined the CGH profiles of seven different cell lines made resistant to three platinum analogues and showed the most consistent abnormalities to involve over-representation of regions 4q and 6q. More recently, we have applied the CGH technique to a series of testicular germ cell tumours (TGCTs) collected as formalin-fixed paraffin-embedded biopsy specimens from patients, both pre- and post-therapy using a platinum-based regimen (POMB/ACE). Previous reports have shown over-representation of X, 7q, 8q and 12p and loss of 13q to occur in 25% of primary TGCTs. Over-representation of 12p was confirmed in the majority of these biopsy samples; deletion of 13q was noted in the initial biopsies of several patients. We also demonstrated alterations of 4p, 4q, 5q and 6q in this series of patients. Newly acquired deletions of 2q and 18q and amplifications of 8q were frequently observed in post-chemotherapy samples from resistant tumours. The CGH studies on these patients with TGCT will not only enable us to correlate our observations on clinical material with those from long-term cell lines, but should also identify sites of key genes involved in clinical platinum resistance.


Subject(s)
Antineoplastic Agents/pharmacology , Chromosome Aberrations , Drug Resistance, Neoplasm/genetics , In Situ Hybridization/methods , Platinum Compounds/pharmacology , Chromosomes, Human , Cisplatin/pharmacology , Doxorubicin/pharmacology , Humans , In Situ Hybridization, Fluorescence , Male , Organoplatinum Compounds/pharmacology , Quinazolines/pharmacology , Testicular Neoplasms/drug therapy , Testicular Neoplasms/genetics , Tumor Cells, Cultured
5.
Cytometry ; 33(1): 56-66, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9725559

ABSTRACT

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound.


Subject(s)
Growth Inhibitors/pharmacology , Pyridines/pharmacology , Thymidylate Synthase/antagonists & inhibitors , Animals , Bisbenzimidazole , Bromodeoxyuridine , Cell Cycle , Cell Division/drug effects , DNA, Neoplasm/analysis , Flow Cytometry , Folic Acid/analogs & derivatives , Folic Acid Antagonists , Humans , Mice , Propidium , Quinazolines , Staining and Labeling , Tumor Cells, Cultured
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