Effect of O6-(4-bromothenyl)guanine on different temozolomide schedules in a human melanoma xenograft model

The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (ATase) is a major component of resistance to treatment with methylating agents and nitrosoureas. Inactivation of the protein, via the administration of pseudosubstrates, prior to chemotherapy has been shown to improve the latter's therapeutic index in animal models of human tumours. We have also shown that rational scheduling of temozolomide, so that drug is administered at the ATase nadir after the preceding dose, increases tumour growth delay in these models. We now report the results of combining these two approaches. Nude mice bearing A375M human melanoma xenografts were treated with vehicle or 100 mg/kg temozolomide ip for 5 doses spaced 4, 12 or 24 hr apart. Each dose was preceded by the injection of vehicle or 20 mg/kg 4BTG. All treatments resulted in significant delays in tumour quintupling time compared with controls: by 6.2, 5.9 and 16.8 days, respectively, for 24-, 12- and 4-hourly temozolomide alone and by 22.3, 21.3 and 22.1 days, respectively, in combination with 4BTG. Weight loss due to TMZ was unaffected by the presence of 4BTG. This was of the order of 6.2-10.6% with 24- and 12-hourly administration and 17.4-20.1% (p < 0.0001) with 4-hourly treatment. In our model, combining daily temozolomide with 4-BTG confers increased antitumour activity equivalent to that achieved by compressing the temozolomide schedule but with less toxicity. Using temozolomide schedule compression with 4-BTG does not improve on this result, suggesting that ATase inactivation with pseudosubstrates is a more promising means of enhancing the activity of temozolomide than compressed scheduling.     

Primary osteosarcoma of the skull

Primary osteogenic sarcoma of the skull is an exceedingly rare condition. An adult male patient is described, who had a painless swelling in the right forehead that had rapidly enlarged in the previous 6 months. Radiological investigations showed a large destructive mass lesion involving the right side of the frontal bone with extension into the frontal sinus, causing marked extradural compression of brain parenchyma. Histopathological examination confirmed the lesion to be primary osteogenic sarcoma.     

Expression of differential nitric oxide synthase isoforms in human normal gastric mucosa and gastric cancer tissue

The present study investigated the expression and distribution of three isoforms of nitric oxide synthase (NOS) in different anatomical regions of the human stomach and in gastric neoplastic tissues by immunohistochemistry using specific antibodies. Intracellular localization of individual isoenzymes of NOS was detected in normal gastric mucosa. Gastric cancer tissues had a marked reduction of all three NOS isoforms expression. The expression of the endothelial NOS, neuronal NOS and inducible NOS in the tumor tissue was significantly lower than in normal gastric mucosa (P = 0.01, P = 0.02, P < 0.01, respectively). In the tumor tissue the expression of inducible NOS was significantly lower than the expression of both constitutive forms of NOS (P < 0.01). There was a tendency to higher expression of both constitutive forms of NOS in earlier stages T2 of the tumor compared to advanced T4 tumor. In contrast, the expression of inducible NOS was higher than in the advanced T4 tumor than in the earlier stages T2 of the tumor. The mapping of the expression of endothelial NOS, neuronal NOS and inducible NOS in human stomach showed higher expression of NOS isoforms in the distal third than in the proximal third of the stomach (P = 0.03, P = 0.04, P = 0.01, respectively). We conclude that there is greater expression of NOS in the stomach corpus and in antrum than in the proximal third of the normal human stomach mirroring the anatomical predilection of common pathological changes in this part of the human stomach. Furthermore, there was loss of the expression of individual isoenzymes in gastric neoplasms.      

O6-(4-bromothenyl)guanine reverses temozolomide resistance in human breast tumour MCF-7 cells and xenografts

Tumour resistance to chemotherapy involving methylating agents such as DTIC (dacarbazine) and temozolomide is linked to expression of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (MGMT). There is considerable interest in improving the efficacy of such O(6)-alkylating chemotherapy by the prior inactivation of MGMT. We have examined the effect of the modified guanine base, O(6)-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib) on MGMT activity and cell or xenograft tumour growth inhibition by temozolomide in the human breast carcinosarcoma cell line, MCF-7. PaTrin-2 effectively inactivated MGMT in MCF-7 cells (IC(50) approximately 6 nM) and in xenografts there was complete inactivation of MGMT within 2 h of dosing (20 mg kg(-1) i.p.) and only slight recovery by 24 h. MGMT inactivation in a range of murine host tissues varied between complete and approximately 60%, with extensive recovery by 24 h. PaTrin-2 (10 microM) substantially increased the growth inhibitory effects of temozolomide in MCF-7 cells (D(60)=10 microM with PaTrin-2 vs 400 microM without). In MCF-7 xenografts, neither temozolomide (100 mg kg(-1) day(-1) for 5 days) nor PaTrin-2 (20 mg kg(-1) day(-1) for 5 days) had any significant effect on tumour growth. In contrast, the PaTrin-2-temozolomide combination produced a substantial tumour growth delay: median tumour quintupling time was increase by 22 days (P<0.005) without any significant increase in toxicity as assessed from animal weight. A PaTrin-2-temozolomide combination may therefore be beneficial in the treatment of human breast cancers.     

Organochlorine pesticides and mercury in cottonmouths (Agkistrodon piscivorus) from northeastern Texas, USA

Despite their ecological importance and global decline, snakes remain poorly studied in ecotoxicology. In this study, we examined organochlorine (OC) pesticide and mercury accumulation in cottonmouths (Agkistrodon piscivorus) living on a contaminated site in northeastern Texas (USA). Mercury and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) were detected in all snakes examined. Other OCs, including p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), methoxychlor, aldrin, and heptachlor, also were detected, but less frequently. Concentrations of p,p'-DDE were higher in fat than in liver, while mercury concentrations were highest in liver, followed by kidney and tail clips. One animal contained the highest mercury concentration yet reported for a snake (8,610 ng/g). Mercury concentrations in liver and kidney were higher in males than females, while no intersex differences in p,p'-DDE concentrations were observed. Concentrations of p,p'-DDE in fat were correlated positively with body size in male cottonmouths but not females, suggesting a slower rate of accumulation in females. Body size strongly predicted mercury concentrations in liver, kidney, and tail clips of both sexes. Tail clips were strong predictors of mercury in liver and kidney in males but not females, suggesting possible sex-dependent differences in mercury toxicokinetics. Both long-term field studies and controlled laboratory investigations are needed to adequately assess the response of cottonmouths to chronic contaminant exposure.     

Analyzing Mycobacterium tuberculosis proteomes for candidate vaccine epitopes

Secreted antigens of Mycobacterium tuberculosis (Mtb) induce strong T cell responses and interferon-gamma (IFN-gamma) secretion, both of which are integral in the defense against Mtb. We used web-based tools (SignaIP and Prosite) to identify putative secreted proteins from Mtb genomes CDC 1551 and H37Rv. We then used EpiMatrix, a proprietary pattern-matching algorithm, to do a preliminary analysis of these proteins for regions that contained a high number of class II MHC binding motif matches. The use of bioinformatics tools reduced the number of potential epitopes to be screened to 5% of the 1.3 million overlapping peptides. Peripheral blood mononuclear cells (PBMC) were obtained from healthy, asymptomatic tuberculin skin test-positive donors. Of the 17 highest-ranking peptide candidates that could be synthesized for this preliminary in vitro evaluation, 15 (88%) stimulated IFN-gamma response, and eight (47%) stimulated lymphocyte proliferation in vitro. IFN-gamma ELISpot assays were therefore a more sensitive test for T cell response to these peptides than were proliferation assays. One highly promiscuous epitope (MT2281-26-J, WRRRPLSSALLSFGLLLGGLPL) induced IFN-gamma secretion in PBMC from 11 of 25 Mtb immune subjects (44%). Overall, 15 epitopes, and MT2281-26-J in particular, are candidates for inclusion in a multi-epitope TB vaccine. These findings support the systematic application of bioinformatics tools to whole genomes when used in combination with in vitro methods for screening and confirming epitopes.     

Sensitization of a human ovarian cancer cell line to temozolomide by simultaneous attenuation of the Bcl-2 antiapoptotic protein and DNA repair by O6-alkylguanine-DNA alkyltransferase

Temozolomide is an alkylating agent that mediates its cytotoxic effects via O(6)-methylguanine (O(6)-meG) adducts in DNA. O(6)-alkylguanine-DNA-alkyltransferase (MGMT) can repair such adducts and therefore constitutes a major resistance mechanism to the drug. MGMT activity can be attenuated in vitro and in vivo by the pseudosubstrate O(6)-(4-bromothenyl)guanine (PaTrin-2, Patrin, Lomeguatrib), which in clinical trials is in combination with temozolomide. Resistance to cytotoxic agents can also be mediated by the Bcl-2 protein, which inhibits apoptosis and is frequently up-regulated in tumor cells. Attenuation of Bcl-2 expression can be affected by treatment of cells with the antisense oligonucleotide, oblimersen sodium (Genasense), currently in phase III clinical trials in combination with the methylating agent dacarbazine. Using a human ovarian cancer cell line (A2780) that expresses both Bcl-2 and MGMT, we show that cells treated with active dose levels of either oblimersen (but not control reverse sequence or mismatch oligonucleotides) or PaTrin-2 are substantially sensitized to temozolomide. Furthermore, the exposure of oblimersen-pretreated cells to PaTrin-2 leads to an even greater sensitization of these cells to temozolomide. Thus, growth of cells treated only with temozolomide (5 microg/mL) was 91% of control growth, whereas additional exposure to PaTrin-2 alone (10 micromol/L) or oblimersen alone (33 nmol/L) reduced this to 81% and 66%, respectively, and the combination of PaTrin-2 (10 micromol/L) and oblimersen (33 nmol/L) reduced growth to 25% of control. These results suggest that targeting both Bcl-2 with oblimersen and MGMT with PaTrin-2 would markedly enhance the antitumor activity of temozolomide and merits testing in clinical trials.