Clinical implications of quantitative real-time RT-PCR analysis of hTERT gene expression in human gliomas.

The presence of telomerase activity in a glioma may be a predictor of its malignant potential. Activation of telomerase is regulated at the transcriptional level of the human telomerase reverse transcriptase (hTERT). Here, we evaluated whether the amount of hTERT mRNA provides a molecular marker of glioma malignancy that would have clinical utility. We used a real-time RT-PCR to assess the number of hTERT transcripts in primary tumour samples derived from 70 glioma patients. Results were standardised by quantifying the number of ABL transcripts as internal control and expressed as hTERT/ABL ratio. The percentage of patients with detectable hTERT mRNA markedly increased with enhanced malignancy: low-grade gliomas expressed hTERT in one out of 14 cases (7.1%), anaplastic gliomas in four out of 13 cases (30.8%) and glioblastoma multiforme (GBM) tumours in 30 out of 43 cases (69.8%). The mean hTERT/ABL ratio was significantly higher in GBMs than in non-GBMs. Subdividing hTERT/ABL ratios as low (< pr = 25%) and high (>25%), we found that the overall survival among hTERT-positive GBMs was significantly worse in high hTERT expressors than in low hTERT expressors (P=0.0082). We conclude that the amount of hTERT mRNA may represent a diagnostic and prognostic indicator for GBM patients.

Inhibition of O6-alkylguanine-DNA alkyltransferase by O6-benzyl-N2-acetylguanosine increases chloroethylnitrosourea-induced apoptosis in Mer+ human melanoma cells.

The exposure of cells to -benzyl- 2-acetylguanosine (BNAG) and several guanine derivatives is known to reduce -alkylguanine-DNA alkyltransferase (AGAT) activity and to decrease the resistance of methyl enzyme repair positive (Mer ) cells to chloroethylnitrosoureas (CENUs) and. We evaluated the influence of AGAT activity inhibition by BNAG on the ability of two CENUs, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 3-(2-chloroethyl)-1-(2-methylsulphonyl)ethyl-3-nitrosourea (cystemustine), to induce an apoptotic response in two melanoma cell lines, M3 Dau (Mer ) and IPC 227F (Mer ). The apoptotic morphology of cells was assessed by microscopy after Wright-Giemsa or Hoechst 33342 staining of cells, and DNA internucleosomal cleavage was demonstrated by the ladder-like pattern of DNA separated by agarose gel electrophoresis. The concentration-dependent number of apoptotic cells assessed using a terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling (TUNEL) assay 72 h after BCNU or cystemustine treatment (0-400 microM for 2 h) was increased by prior AGAT depletion with BNAG pretreatment (300 microM for 4 h) in Mer cells. These results suggest that the DNA lesions on the position of guanine are a key event in inducing an apoptotic response in melanoma cells. We also observed that cystemustine was a more potent inducer of apoptosis than BCNU, and that the synergism with BNAG was more potent with cystemustine than with BCNU. These results suggest that the nature of the CENUs associated with an AGAT inhibitor is a determinant factor in forecasting the clinical efficacy of the association, especially in melanoma.

IL-6 gene amplification and expression in human glioblastomas.

The aggressiveness of human gliomas appears to be correlated with the upregulation of interleukin 6 (IL-6) gene. Using quantitative PCR methods, we detected amplification and expression of the IL-6 gene in 5 of 5 primary glioblastoma samples and in 4 of 5 glioblastoma cell lines. This finding suggests that the amplification of IL-6 gene may be a common feature in glioblastomas and may contribute to the IL-6 over-expression.

Circadian variation in O6-alkylguanine-DNA alkyltransferase activity in circulating blood mononuclear cells of healthy human subjects.

Cytotoxic agents such as chloroethylnitrosoureas (CENUs) mostly alkylate DNA on the O6-guanine position. This highly mutagenic lesion can be repaired by O6-alkylguanine-DNA alkyltransferase (AGT), which removes the alkyl group by accepting it to the cysteine residue of its active site. AGT activity displayed a circadian rhythm in mouse liver, coincident with that of CENU tolerability. We investigated whether AGT activity displayed a circadian rhythm in human circulating mononuclear cells (MNCs). The study was performed in 12 healthy volunteers aged 19 to 31 years. Circadian synchronization was verified with rest/activity cycle as assessed with wrist actigraphy and plasma cortisol and melatonin rhythms. Subjects were hospitalized for 24 hr and blood samples were obtained at 08:00, 12:00, 16:00, 20:00, 22:00, 00:00, 02:00, 04:00 and 08:00 overnight. MNCs were isolated on Ficoll immediately after blood sampling and frozen at -196 degrees C until AGT activity determination by HPLC. Mean AGT activity (+/- SEM) varied from 821 +/- 67 fmol/mg of total proteins at noon (trough), up to 1,055 +/- 80 fmol/mg at midnight (peak), i.e., by approximately 30%. A circadian rhythm was statistically validated with both analysis of variance (p < 0.009) and cosinor (p < 0.02) for AGT activity in MNCs (acrophase +/- SD at 00:30 +/- 210 min) as well as for MNC circulating count and for plasma cortisol and melatonin concentrations. Despite individual variations in the extent of AGT activity rhythm (more or less pronounced according to subject), AGT activity displayed a circadian rhythm in human MNCs of our healthy study group subjects. The results warrant to further investigate AGT rhythmicity both in circulating MNCs and in target tissues of cancer patients, as a prerequisite for clinical testing of chronotherapy with alkylating agents.

Interleukin-6 overexpression as a marker of malignancy in human gliomas.

OBJECT: Glioblastomas multiforme (GBMs) grow rapidly and are highly resistant to treatment compared with other glioma types and grades. Consequently, it is of major interest to identify markers of aggressiveness in these tumors that could represent new therapeutic targets. Interleukin (IL)-6 is frequently produced in gliomas and, given its manifold properties, could be considered as a candidate marker. Expression of IL-6 may be involved in cell growth, resistance to chemotherapy and radiotherapy (via an antiapoptotic pathway), and angiogenesis. This study was conducted to test this hypotheses and to evaluate the suitability of IL-6 as a target in the treatment of GBMs. METHODS: The authors studied the relationship between the level of IL-6 gene expression as assessed using semiquantitative reverse transcription-polymerase chain reaction and by determining various histological types and grades in a series of 59 gliomas. It was found that GBMs displayed a significantly higher level of IL-6 expression than other types of glioma (p < 0.001). Immunohistochemical analysis revealed that IL-6 was produced mainly by malignant cells and a few vascular endothelial cells. CONCLUSIONS: It can be inferred from these findings that IL-6 gene expression is related to glioma aggressiveness and that IL-6 may play a central role in GBM behavior. Interleukin-6, therefore, could be considered as a new potential target in the treatment of GBMs.

O(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in human glioblastomas in relation to patient characteristics and p53 accumulation.

Repair of cytotoxic DNA damage by O(6)-methylguanine-DNA methyltransferase (MGMT) is a potentially important factor of chemoresistance to chloroethylnitrosoureas (CENUs), commonly used in the treatment of glioblastoma multiforme (GBM). The value of p53 as a prognostic factor in GBMs remains unclear, but a possible relationship between MGMT gene expression and p53 has been suggested. To further examine these GBM characteristics in vivo, we assessed MGMT gene expression using semi-quantitative RT-PCR and p53 alteration by immuno-histochemistry on a series of 39 GBMs. MGMT gene expression was inversely correlated with age (p < 0.03), consistent with the results of others. Interestingly, tumors from male patients had higher MGMT mRNA amounts than tumors from female patients (p < 0.03). No prognostic implication was observed either for MGMT gene expression or for p53 accumulation. However, MGMT gene expression was significantly lower in p53-altered GBM, regardless of the percentage of positive cells (p < 0.01). Our observation suggests that in human glial tumors, a low level of MGMT gene expression might promote p53 alteration, probably via mutation of its gene. Int. J. Cancer (Pred. Oncol.) 84:416-420, 1999.

Melanoma-cell toxicity of cystemustine combined with O6-benzyl-N2-acetylguanosine.

Cystemustine (N'-(2-chloroethyl)-N-(2-(methylsulphonyl)ethyl)-N'-nitrosourea) is a new chloroethylnitrosourea (CENU) being used in phase II clinical trials of disseminated melanoma. Clinical results show that tumour regression has only been observed in 25% of melanomas treated by CENUs. Tumour resistance to CENU is known to be mainly due to a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT). The poor remission rate of melanoma with CENUs is attributed to the fact that metastases contain high MGMT levels. Previously, we have shown that O6-benzyl-N2-acetylguanosine (BNAG), an MGMT inhibitor, can be combined with cystemustine by intravenous administration, and increases the antitumour effect of cystemustine in resistant human melanoma. In the work presented here, we investigated the in vitro pharmacological effect of this combination on the DNA of human melanoma cells (M3Dau cells). A quantitative polymerase chain reaction (QPCR) assay was used to measure DNA damage in a fragment (2.7 kb) of the hprt gene. The results show that treatment with BNAG enhances the number of lesions in the DNA of cystemustine-treated resistant malignant melanocytes, which may account for the high tumour-cell toxicity of the combination of cystemustine and BNAG.

Circadian rhythm in toxic effects of cystemustine in mice: relevance for chronomodulated delivery.

Cystemustine is a new nitrosourea with high anti-tumor activity and a short plasma half-life in mice. The influence of circadian dosing time upon its toxicities was first investigated in a total of 368 synchronized male B6D2F1 mice. Late survival rate varied from 4% in mice receiving a single dose of cystemustine (conventional lethal dose 50%) at 7 hours after light onset (HALO) up to 88% in mice treated at 15 or at 19 HALO. Target organ toxicities (bone marrow, circulating blood cells, spleen, colon and duodenum) were studied following a single slightly lower dose of cystemustine. Leukopenia was the major hematologic effect encountered. Leukocyte count nadir occurred 7 days after injection and was lowest following cystemustine at 7 HALO as compared to 13 or 19 HALO. Recovery was faster after cystemustine at 19 HALO as compared to other dosing times. Bone-marrow necrotic lesions were more pronounced 1 day after cystemustine at 7 HALO than after cystemustine at 19 HALO. Thus, a large-amplitude circadian rhythm characterized the toxicity of this nitrosourea in mice. The lowest cystemustine toxicity was found near the middle of the active span of the rest-activity circadian cycle of mice.

Circadian variation in O6-methylguanine-DNA methyltransferase activity in mouse liver.

Bifunctional chloroethylating cytostatic agents produce lethal DNA lesions, as a result of the formation of O6-alkylguanines. These lesions can be repaired by O6-methylguanine-DNA methyltransferase (MGMT). This ubiquitous nuclear and cytosolic enzyme removes the alkyl group by accepting it to the cysteine residue of its active site, thus preventing the formation of DNA interstrand cross-links. The role of the circadian organization in cellular protection against such DNA insults was examined in male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (LD12:12). MGMT activity was determined in liver of mice obtained at eight different circadian times, located 3 h apart. MGMT activity varied 5-fold along the 24 h time-scale, from 7 +/- 1 pmol/g of tissue at 7 h after light onset (HALO), during the rest span, up to 32 +/- 9 pmol/g at 19 HALO (second mid to late activity span). This large amplitude circadian rhythm in MGMT activity may be an important determinant of the susceptibility rhythms to alkylating agents. The greatest DNA repair activity occurred at night when mice were active, eat and drink, and thus are at a higher risk of being exposed to chemical insults.