Targeting anti-apoptotic Bcl-2 by AT-101 to increase radiation efficacy: data from in vitro and clinical pharmacokinetic studies in head and neck cancer.

BACKGROUND: Pro-survival Bcl-2 family members can promote cancer development and contribute to treatment resistance. Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by overexpression of anti-apoptotic Bcl-2 family members. Increased levels of these anti-apoptotic proteins have been associated with radio- and chemoresistance and poor clinical outcome. Inhibition of anti-apoptotic Bcl-2 family members therefore represents an appealing strategy to overcome resistance to anti-cancer therapies. The aim of this study was to evaluate combined effects of radiation and the pan-Bcl-2 inhibitor AT-101 in HNSCC in vitro. In addition, we determined human plasma levels of AT-101 obtained from a phase I/II trial, and compared these with the effective in vitro concentrations to substantiate therapeutic opportunities. METHODS: We examined the effect of AT-101, radiation and the combination on apoptosis induction and clonogenic survival in two HNSCC cell lines that express the target proteins. Apoptosis was assessed by bis-benzimide staining to detect morphological nuclear changes and/or by propidium iodide staining and flow-cytometry analysis to quantify sub-diploid apoptotic nuclei. The type of interaction between AT-101 and radiation was evaluated by calculating the Combination Index (CI) and by performing isobolographic analysis. For the pharmacokinetic analysis, plasma AT-101 levels were measured by HPLC in blood samples collected from patients enrolled in our clinical phase I/II study. These patients with locally advanced HNSCC were treated with standard cisplatin-based chemoradiotherapy and received dose-escalating oral AT-101 in a 2-weeks daily schedule every 3 weeks. RESULTS: In vitro results showed that AT-101 enhances radiation-induced apoptosis with CI's below 1.0, indicating synergy. This effect was sequence-dependent. Clonogenic survival assays demonstrated a radiosensitizing effect with a DEF37 of 1.3 at sub-apoptotic concentrations of AT-101. Pharmacokinetic analysis of patient blood samples taken between 30 min and 24 h after intake of AT-101 showed a dose-dependent increase in plasma concentration with peak levels up to 300-700 ng/ml between 1.5 and 2.5 h after intake. CONCLUSION: AT-101 is a competent enhancer of radiation-induced apoptosis in HNSCC in vitro. In addition, in vitro radiosensitization was observed at clinically attainable plasma levels. These finding support further evaluation of the combination of AT-101 with radiation in Bcl-2-overexpressing tumors.

Activated ClpP kills persisters and eradicates a chronic biofilm infection.

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells-activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

Valproic acid sensitizes human glioma cells for temozolomide and γ-radiation.

Temozolomide (TMZ) is given in addition to radiotherapy in glioma patients, but its interaction with the commonly prescribed antiepileptic drug valproic acid (VPA) is largely unknown. Induction of DNA demethylation by VPA could potentially induce expression of the O(6)-methylguanine-DNA-methyltransferase (MGMT) protein, causing resistance to TMZ and thereby antagonizing its effect. Therefore, this study investigates the interaction between VPA, TMZ, and γ-radiation. Two glioma cell lines were used that differ in TMZ sensitivity caused by the absence (D384) or presence (T98) of the MGMT protein. VPA was administered before (24/48 h) or after (24 h) single doses of γ-radiation; or, after 24 h, VPA treatment was accompanied by a single dose of TMZ for another 24 h. For trimodal treatment the combination of VPA and TMZ was followed by single doses of γ-radiation. In both cell lines VPA caused enhancement of the radiation response after preincubation (DMF(0.2) 1.4 and 1.5) but not after postirradiation (DMF(0.2) 1.1 and 1.0). The combination of VPA and TMZ caused enhanced cytotoxicity (DMF(0.2) 1.7) in both the TMZ-sensitive cell line (D384) and the TMZ-resistant cell line (T98). The combination of VPA and TMZ caused a significant radiation enhancement (DMF(0.2) 1.9 and 1.6) that was slightly more effective than that of VPA alone. VPA does not antagonize the cytotoxic effects of TMZ. Preincubation with VPA enhances the effect of both γ-radiation and TMZ, in both a TMZ-sensitive and a TMZ-resistant human glioma cell line. VPA combined with TMZ may lead to further enhancement of the radiation response.

Prophylactic feeding tubes for patients with locally advanced head-and-neck cancer undergoing combined chemotherapy and radiotherapy-systematic review and recommendations for clinical practice.

GOALS: This work aimed to determine the benefits and risks of prophylactic feeding tubes for adult patients with squamous cell carcinoma of the head and neck who receive combined chemotherapy and radiotherapy with curative intent and to make recommendations on the use of prophylactic feeding tubes and the provision of adequate nutrition to this patient population. METHODS: A national multidisciplinary panel conducted a systematic review of the evidence and formulated recommendations to guide clinical decision-making. The draft evidence summary and recommendations were distributed to clinicians across Canada for their input. MAIN RESULTS: No randomized controlled trials have directly addressed this question. Evidence from studies in the target population was limited to seven descriptive studies: two with control groups (one prospective, one retrospective) and five without control groups. Results from ten controlled studies in patients treated with radiotherapy alone were also reviewed. CONCLUSIONS: The available evidence was insufficient to draw definitive conclusions about the effectiveness of prophylactic feeding tubes in the target patient population or to support an evidence-based practice guideline. After review of the evidence, of guidelines from other groups, and of current clinical practice in Canada, the multidisciplinary panel made consensus-based recommendations regarding comprehensive interdisciplinary clinical care before, during, and after cancer treatment. The recommendations are based on the expert opinion of the panel members and on their understanding of best clinical practice.

Absence of the MGMT protein as well as methylation of the MGMT promoter predict the sensitivity for temozolomide.

BACKGROUND: The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) can cause resistance to the alkylating drug temozolomide (TMZ). The purpose of this study was to determine the relationship between the MGMT status, determined by means of several techniques and methods, and the cytotoxic response to TMZ in 11 glioblastoma multiforme (GBM) cell lines and 5 human tumour cell lines of other origins. METHODS: Cell survival was analysed by clonogenic assay. The MGMT protein levels were assessed by western blot analysis. The MGMT promoter methylation levels were determined using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and quantitative real-time methylation-specific PCR (qMSP). On the basis of the results of these techniques, six GBM cell lines were selected and subjected to bisulphite sequencing. RESULTS: The MGMT protein was detected in all TMZ-resistant cell lines, whereas no MGMT protein could be detected in cell lines that were TMZ sensitive. The MS-MLPA results were able to predict TMZ sensitivity in 9 out of 16 cell lines (56%). The qMSP results matched well with TMZ sensitivity in 11 out of 12 (92%) glioma cell lines. In addition, methylation as detected by bisulphite sequencing seemed to be predictive of TMZ sensitivity in all six cell lines analysed (100%). CONCLUSION: The MGMT protein expression more than MGMT promoter methylation status predicts the response to TMZ in human tumour cell lines.

Strategies for the analysis of in vitro radiation sensitivity and prediction of interaction with potential radiation modifying agents.

PURPOSE: To better predict radiation-drug interactions in in vitro model systems, thorough assessment of the effects of in vitro exposure is required. The aim of this article is to show that both clonogenic capacity and cellular proliferation, which represent important different elements of tumour conduct, can be considered when assessing in vitro radio sensitisation. METHODS: A model was designed that can predict radiation-drug interactions based on changes in clonogenic capacity and cell proliferation by radiation modifying agents. RESULTS: Using this mechanistical model, the effect of combined exposure to radiation and potential drugs can be tested on both established cell lines and primary cells. In addition, we could obtain more information about the mechanisms underlying the radiation-drug interaction by assessing the results of in vitro exposure on tumour cell proliferation and clonogenic capacity according to our model. CONCLUSIONS: The significance of our model is not to replace the clonogenic gold standard but to give additional information about the radiation-drug combination by determining cell proliferation. Moreover, the advantage is that the interaction can also be predicted in cases where a clonogenic assay is not possible. Additional research into the biological effect of potential radio-sensitisers is warranted for future (pre)clinical studies.

Feeding outside the forest: the importance of crop raiding and an invasive weed in the diet of gallery forest ring-tailed lemurs (Lemur catta) following a cyclone at the Beza Mahafaly special reserve, Madagascar.

In January 2005, a cyclone hit southern Madagascar, including the Beza Mahafaly Special Reserve, disrupting the flowering/fruiting cycle of Tamarindus indica, leaving Lemur catta without its major food resource during reproductive periods. We studied two adjacent groups of L. catta during the late gestation period, and both groups ventured outside the reserve to feed. The Red group (RG) fed daily on cultivated sweet potato (Ipomoea batatas) leaves in a nearby field, and both groups consumed leaves and stems of the invasive terrestrial flowering herb Mexican prickly poppy (Argemone mexicana), growing outside the reserve. The Green group (GG) spent significantly more time feeding than did RG, and more time feeding inside the forest compared to outside. The members of RG spent half of their time feeding in the crops, and nearly half of their diet consisted of easy-to-process sweet potato leaves. Additionally, RG defended and restricted GG's access to the crop territory. Of the two non-forest foods, A. mexicana leaves were higher in protein and most minerals (P, Mg, K and Na, but not Ca) and lower in fiber than sweet potato leaves, but sweet potato leaves were preferred by RG. L. catta is a markedly flexible primate with respect to diet, and switches to fallback foods from outside the forest during periods of low food availability. In the highly seasonal and unpredictable climate of southern Madagascar, such behavioral adaptations are important to the survival of this species.

Differential radiosensitizing potential of temozolomide in MGMT promoter methylated glioblastoma multiforme cell lines.

PURPOSE: To investigate the radiosensitizing potential of temozolomide (TMZ) for human glioblastoma multiforme (GBM) cell lines using single-dose and fractionated gamma-irradiation. METHODS AND MATERIALS: Three genetically characterized human GBM cell lines (AMC-3046, VU-109, and VU-122) were exposed to various single (0-6 Gy) and daily fractionated doses (2 Gy per fraction) of gamma-irradiation. Repeated TMZ doses were given before and concurrent with irradiation treatment. Immediately plated clonogenic cell-survival curves were determined for both the single-dose and the fractionated irradiation experiments. To establish the net effect of clonogenic cell survival and cell proliferation, growth curves were determined, expressed as the number of surviving cells. RESULTS: All three cell lines showed MGMT promoter methylation, lacked MGMT protein expression, and were sensitive to TMZ. The isotoxic TMZ concentrations used were in a clinically feasible range of 10 micromol/L (AMC-3046), 3 micromol/L (VU-109), and 2.5 micromol/L (VU-122). Temozolomide was able to radiosensitize two cell lines (AMC 3046 and VU-122) using single-dose irradiation. A reduction in the number of surviving cells after treatment with the combination of TMZ and fractionated irradiation was seen in all three cell lines, but only AMC 3046 showed a radiosensitizing effect. CONCLUSIONS: This study on TMZ-sensitive GBM cell lines shows that TMZ can act as a radiosensitizer and is at least additive to gamma-irradiation. Enhancement of the radiation response by TMZ seems to be independent of the epigenetically silenced MGMT gene.

Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration.

PURPOSE: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines. MATERIALS AND METHODS: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 - 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis. RESULTS: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 microM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 microM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 microM) or Celecoxib (50 microM) and U87 with NS-398 (200 microM) after radiation resulted even in radioprotection. CONCLUSIONS: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0-6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24-72 h exposure to 250-750 microM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 microM meloxicam for 24 h increased the fraction of cells in the radiosensitive G(2)/M cell cycle phase in D384 (18-27%) and U251 (17-41%) cells. 750 microM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.

Genetic profiling of a distant second glioblastoma multiforme after radiotherapy: Recurrence or second primary tumor?

OBJECT: In nearly all patients with glioblastoma multiforme (GBM) a local recurrence develops within a short period of time. In this paper the authors describe two patients in whom a second GBM developed after a relatively long time interval at a site remote from the primary tumor. The genetic profiles of the tumors were compared to discriminate between distant recurrence and a second primary tumor. METHODS: Both patients harboring a supratentorial GBM were treated with surgery and local high-dose radiotherapy. Local control of the disease at the primary tumor site was achieved. Within 2 years, a second GBM developed in both patients, not only outside the previously irradiated target areas but infratentorially in one patient and in the opposite hemisphere in the other. The tumors were examined for the presence of several genetic alterations that are frequently found in GBMs--a loss of heterozygosity at chromosome regions 1p36, 10pl5, 19q13, and 22q13, and at the CDKN2A, PTEN, DMBT1, and TP53 gene regions; a TP53 mutation; and EGFR amplification. In the first patient, genetic profiling revealed that the primary tumor had an allelic imbalance for markers in several chromosome regions for which the second tumor displayed a complete loss. In the second patient, genetic profiling demonstrated the presence of genetic changes in the second tumor that were identical with and additional to those found in the primary tumor. CONCLUSIONS: Based on the similarities between the genetic profiles of the primary and the second tumors in these patients, the authors decided that in each case the second distant GBM was a distant recurrence rather than a second independent primary tumor.

The importance of immunohistochemical expression of EGFr in squamous cell carcinoma of the oral cavity treated with surgery and postoperative radiotherapy.

PURPOSE: The aim of this study was to investigate the prognostic significance of epidermal growth factor (EGFr) expression in oral cavity squamous cell carcinoma (OCSCC) treated with curative surgery and postoperative radiotherapy. METHODS AND MATERIALS: This retrospective study included 165 OCSCC patients. The expression of EGFr was assessed on paraffin-embedded tissue of the primary tumor by immunohistochemistry using a monoclonal antibody directed against EGFr. Intensity of the EGFr expression was scored by two authors blinded for the clinical outcome. RESULTS: In the univariate analysis, locoregional control at 3 years (LRC) in the EGFr-negative cases was 69% compared with 77% in the EGFr-positive cases (p = 0.22). In the multivariate analysis for local control, a significant interaction was found between EGFr and overall treatment time of radiation (OTT). After stratification for EGFr expression, the OTT was of no importance in the EGFr-negative cases, whereas a significant difference in LRC was found in the EGFr-positive cases, in which the LRC after 3 years was 69% and 94% in case of an OTT of 0-42 days and >42 days, respectively (p = 0.009; hazard ratio = 3.42; 95% confidence interval, 1.28-8.96). No significant association was found between EGFr expression and overall survival. CONCLUSIONS: In the present study, no association was found between EGFr expression and outcome regarding locoregional control and overall survival. However, the results of the present study suggest that patients with squamous cell carcinoma of the oral cavity with high EGFr expression benefit more from a reduction of the overall treatment time of postoperative radiation than those with low EGFr expression.

Expression of cyclooxygenase-2 and epidermal growth factor receptor in primary and recurrent glioblastoma multiforme.

PURPOSE: To investigate the pattern and level of cyclooxygenase-2 (COX-2) expression in a series of high grade primary and recurrent glioblastoma multiforme (GBM) and correlation with time to recurrence and patients' survival following therapy. The relationship between COX-2 and epidermal growth factor receptor (EGFR) immunoreactivities was evaluated. MATERIALS AND METHODS: Specimens of 14 primary and 14 recurrent GBMs (eight pairs) following surgery and full course radiation therapy were processed for immunostaining on COX-2 and EGFR. Tumor cell positivity was semi-quantitatively scored. COX-2 scores of the primary tumor and recurrence were correlated with the time to radiological tumor progression and patients' survival. RESULTS: COX-2 positive tumor cells were disseminated throughout the tumor parenchyma. The intensity and pattern of COX-2 expression were heterogeneous, with predominant expression in areas surrounding tumor necrosis. Scoring of COX-2 positivity revealed values between 1 and 80% of the cells. Primary GBMs with COX-2 expression levels between 25% and 70% of the tumor cells showed a shorter time to radiological recurrence than GBMs with <10% COX-2 positive tumor cells (respectively, 219 +/- 50 and 382 +/- 77 days). No correlation was found between the COX-2 expression in the primary tumor and patients' survival (r (s) = -0.073) following therapy. No correlation was found either between COX-2 and EGFR immunoreactivity. CONCLUSIONS: Immunohistochemical expression of COX-2 in GBM showed large variation. Hence, determination of COX-2 expression in tumor specimen for each individual might be relevant for selection of those patients, who could benefit from adjuvant therapy with selective COX-2 inhibitors.

Correlation of tumor- and stromal-derived MT1-MMP expression with progression of human ovarian tumors in SCID mice.

OBJECTIVE: Human ovarian carcinoma samples were orthotopically implanted into SCID mice to investigate the contribution of matrix metalloproteases (MMPs) to the spread of ovarian tumors. METHODS: Mice were inoculated with patient tumor samples, and developed ovarian tumors over a 16-week period with metastasis occurring in some mice. Species-specific quantitative RT-PCR was used to identify the source of tumor-associated MMPs. RESULTS: Membrane-type (MT)1-MMP mRNA was significantly increased in high-grade tumors, tumors with evidence of serosal involvement, and tumors in which distant metastases were detected. The increase in MT1-MMP expression was predominantly from the human tumor cells, with a minor contribution from the mouse ovarian stroma. Neither human nor mouse MT2-MMP were correlated with tumor progression and MT3-MMP levels were negligible. While tumor cells did not produce significant amounts of MMP-2 or MMP-9, the presence of tumor was associated with increased levels of MMP-2 expression by mouse ovarian stroma. Stromal-derived MT1-MMP was greater in large tumors and was associated with stromal MMP-2 expression but neither was significantly linked with metastasis. CONCLUSIONS: These studies indicate that tumor-derived MT1-MMP, more so than other gelatinolytic MMPs, is strongly linked to aggressive tumor behavior. This orthotopic model of human ovarian carcinoma is appropriate for studying ovarian tumor progression, and will be valuable in the further investigation of the metastatic process.

Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution.

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.

Direct observation of domains in model stratum corneum lipid mixtures by Raman microspectroscopy.

Several studies on intact and model stratum corneum (SC), the top layer of the epidermis, have suggested the presence of crystalline domains. In the present work, we used micro-Raman mapping to detect lipid domains in model lipid mixtures formed by an equimolar mixture of ceramides, cholesterol, and palmitic acid, the three main lipid species of SC. We were able to determine the spatial distribution of the three compounds individually based on the systematic analysis of band areas. As a control, we studied freeze-dried lipid mixtures, and the Raman microspectroscopy reported faithfully the homogeneous distribution of the three compounds. Spectral mapping was then performed on hydrated equimolar mixtures carefully annealed. In this case, clear phase separations were observed. Domains enriched in cholesterol, ceramides, or palmitic acid with a size of a few tens of square microns were detected. These findings constitute the first direct evidence of the formation of heterogeneous domains in the SC lipid models in a bulk phase. Raman microspectroscopy is an innovative approach to characterize the conditions leading to the formation of domains and provides new insights into the understanding of the skin barrier.

Potential role of mental practice using motor imagery in neurologic rehabilitation.

For many patients with damage to the central nervous system (CNS), execution of motor tasks is very difficult, sometimes impossible, even after early participation in an active rehabilitation program. Several investigators have recently proposed that mental practice could be used by these patients as a therapeutic tool to improve their performance of motor functions, yet very little empirical work addresses this issue directly. This article discusses the rationale for investigating mental practice as a means of promoting motor recovery in patients with a neurologic disorder. We first present evidence supporting the existence of a similarity between executed and imagined actions using data from psychophysical, neurophysiologic, and brain imaging studies. This parallel is then extended to the repetition of movements during physical and mental practice of a motor skill. Finally, a new model is proposed to emphasize the key role of motor imagery as an essential process of mental practice, and also to stimulate additional research on this type of training in the rehabilitation of patients with motor impairments of cerebral origin.

Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas.

Studies have suggested that an imbalance of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may contribute to the malignant phenotype of gliomas. In this study, we have undertaken a detailed analysis of expression of the TIMP family in normal human brain and malignant gliomas at both the mRNA and protein level. Reverse transcription-PCR (RT-PCR) analyses of total RNA from surgical tumour specimens revealed unique expression patterns for the 4 members of the TIMP family, with TIMP-1 and -4 showing positive and negative correlations, respectively, with glioma malignancy. By RT-PCR, TIMP-2 and TIMP-3 expression did not change with tumour grade. In situ hybridization localized TIMP-1 to glial tumour cells and also to the surrounding tumour vasculature. TIMP-4 transcripts were predominantly localized to tumour cells, though minor expression was found in vessels. Recombinant TIMP-4 reduced invasion of U251 glioma cells through Matrigel, and U87 clones overexpressing TIMP-4 showed reduced invasive capacity in vitro. TIMP-4, but not TIMP-1, blocked Membrane Type-1-MMP-mediated progelatinase-A (MMP-2) activation in human umbilical vein endothelial cells. The differential expression and localization of individual TIMPs may contribute to the pathophysiology of human malignant gliomas, particularly with regard to tumour vascularization.

The importance of using absolute mutant frequencies to compare mutation spectra.

Because damage to the cellular DNA is very hazardous for a cell, it is important to identify compounds, which can cause DNA damage. To investigate the mutagenic effect of a particular agent of interest, usually mutation spectra are determined in a selected target gene. The most commonly used method to compare different mutation spectra with each other, is the comparison of the percentages of each type of mutation. In this paper, it is emphasized that comparison of percentages can lead to incorrect conclusions and therefore another determinant, the absolute mutant frequency, should be used.