Isolation of tumour stem-like cells from benign tumours.

BACKGROUND: Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours. METHODS: Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice. RESULTS: In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments. CONCLUSION: This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.

Novel uses of insulin syringes to reduce dosing errors: a retrospective chart review of enoxaparin whole milligram dosing.

UNLABELLED: Enoxaparin is a low molecular weight heparin (LMWH) commonly used for thromboprophylaxis children. Enoxaparin dosing is based on patients' weight and results in decimal dosing. Due to the high concentration of enoxaparin the resultant decimal dose makes precise measurement difficult. Dilution is necessary and often results in ten-fold medication administration errors [Ghaleb MA, Barber N, Franklin BD, Yeung VWS, Khaki ZF, Wong ICK. Systematic review of medication errors in pediatric patients. Ann Pharmacother Oct 2006;40(10):1766-76, Raju TN, Kecskes S, Thornton JP, Perry M, Feldman S. Medication errors in neonatal and paediatric intensive-care units. Lancet Aug 12 1989;2(8659):374-6]. Enoxaparin may be administered in whole milligram doses via insulin syringe, where one milligram of enoxaparin equals one unit on the 100 unit graduated insulin syringe. STUDY DESIGN: A retrospective chart review of 514 children. Data was collected on underlying diagnosis, reason for anticoagulation, anti-Xa levels, hemorrhagic events, and medication errors identified. OUTCOME: to determine the occurrence rate of supra-therapeutic anticoagulation as indicated by anti-Xa levels >1.0 u/ml, when enoxaparin doses are rounded up to the whole milligram, and are administered using insulin syringes. The secondary objectives were to determine if the supra-therapeutic anti-Xa levels were associated with hemorrhagic events. To determine if children achieved and maintained therapeutic anti-Xa range using whole milligram dosing and to evaluate the impact of utilizing insulin syringes for administration on reducing dose measurement errors. RESULTS: All 514 patients were prescribed whole milligram enoxaparin dosing, and achieved therapeutic anti-Xa within a mean time of 2 days. No infant or child required decimal doses to achieve therapeutic levels. Five children achieved an initial supra-therapeutic anti-Xa level (1.04 -1.36 U/ml), requiring a single whole milligram dose decrease. There were no associated hemorrhagic events. CONCLUSION: Whole milligram enoxaparin dosing administered via an insulin syringe safely and effectively, achieved therapeutic levels in infants and children. The reduced incidence of enoxaparin dosing errors suggests that whole milligram enoxaparin dosing via an insulin syringe is a method that should be considered for standard of care.

DLK1: increased expression in gliomas and associated with oncogenic activities.

DLK1 (delta-like) is a transmembrane and secreted protein in the epidermal growth factor-like homeotic family. Although expressed widely during embryonic development, only a few tissues retain the expression in adults. Neuroendocrine tumors often highly express this protein; therefore, we hypothesized that brain tumors might also express it. This study found that the expression of DLK1 in gliomas was higher than that in normal brain (P < 0.05). After stable transfection of a DLK1 cDNA expression vector into GBM cell lines, their proliferation was increased. Furthermore, they lost contact inhibition, had enhanced anchorage-independent growth in soft agar, and had significantly greater capacity to migrate. Western blot studies showed that expression of cyclin D1, CDK2, and E2F4 were increased, and Rb levels were decreased in these cells. DLK1 was found on the cell surface and secreted in the medium from the transfected GBM cells. DLK1-enriched condition medium stimulated the growth of glioblastoma multiforme cell lines and explants. DLK1 antibody blocked cell growth stimulated by DLK1. In summary, these results suggest that DLK1 may play a role in the formation or progression of gliomas.

Cyclic GMP-specific phosphodiesterase inhibition and intracarotid bradykinin infusion enhances permeability into brain tumors.

Intracarotid infusion of bradykinin selectively increases the delivery of compounds into brain tumors. This study sought to determine the role of cyclic GMP in increased permeability across the blood-tumor barrier (BTB) after infusion of bradykinin. In permeability studies, 186 Wistar rats with RG2 gliomas and C6 gliomas were used. Transport across the BTB was quantified by autoradiography and reported as a unidirectional transport, Ki, for [14C]dextran (Mr 70,000) and [14C]aminoisobutyric acid (Mr 103,000), with or without inhibition of cyclic GMP-specific phosphodiesterase or soluble guanylate cyclase. We also determined cyclic GMP levels in tumors and normal brain, with or without intracarotid bradykinin infusion, using RIA. Intracarotid infusion of bradykinin selectively increased permeability in RG2 tumors and C6 tumors for both tracers. Simultaneous infusion of bradykinin and a cyclic GMP-specific phosphodiesterase inhibitor, zaprinast (20 mg/kg), resulted in significantly increased permeability across the BTB, compared to intracarotid bradykinin infusion alone. Zaprinast also significantly prolonged the permeability effects of bradykinin. Pretreatment using i.v. infusion of the soluble guanylate cyclase inhibitor, LY-83583 (125 microg/kg), significantly attenuated the bradykinin effect of opening the BTB. Cyclic GMP levels in RG2 and C6 tumors were significantly increased after intracarotid bradykinin infusion (2.8- and 2.2-fold, respectively). Cyclic GMP levels in normal brain were not increased by bradykinin infusion. These results show that increasing cyclic GMP in tumor microvessels can increase permeability in response to bradykinin.

Increased brain tumor microvessel permeability after intracarotid bradykinin infusion is mediated by nitric oxide.

Nitric oxide (NO), a free radical gas implicated in a wide variety of biological reactions, is a novel signaling molecule that may regulate vasodilation, cerebral blood flow, and vascular permeability. This study was performed to determine whether NO mediates the selective increase in brain tumor microvessel permeability after intracarotid infusion of bradykinin in the RG2 rat glioma model. Intracarotid infusion of bradykinin selectively increased the transport of radiolabeled alpha-aminoisobutyric acid and dextran into brain tumors. Transport into normal brain was not increased. The administration of an NO synthase inhibitor, NG-nitro-L-arginine methyl ester, significantly inhibited the increased transport into tumors for both tracers. The inhibitory effect of NG-nitro-L-arginine methyl ester on the response to bradykinin was reversed by L-arginine. The expression of two NO synthase (NOS) isoforms in cultured RG2 glioma cell lines and intracerebral RG2 glioma was examined by immunohistochemistry and Western blot analysis. High levels of expression of neuronal NOS were detected in cultured and intracerebral RG2 cells but not in normal brain tissue, except in rare neuronal cells. The endothelial form of NOS was also expressed in cultured RG2 cells, but not as strongly as neuronal NOS expression. In intracerebral RG2 gliomas, expression of endothelial NOS in the tumor was detected at higher levels than in normal brain. These findings indicate that RG2 rat gliomas express high levels of NOS, which regulate the production of NO, compared with normal brain. We suggest that the selective permeability increase in brain tumor microvessels after bradykinin infusion is mediated by NO. Furthermore, the absence of high levels of NOS in normal brain may account for the attenuated permeability response to bradykinin in normal brain microvessels.

Overexpression of alpha4 chain-containing laminins in human glial tumors identified by gene microarray analysis.

Differential gene expression in tumors often involves growth factors and extracellular matrix/basement membrane components. Here, 11,000- gene microarray was used to identify gene expression profiles in brain tumors including high-grade gliomas [glioblastoma multiforme (GBM) and anaplastic astrocytoma], low-grade astrocytomas, or benign extra-axial brain tumors (meningioma) in comparison with normal brain tissue. Histologically normal tissues adjacent to GBMs were also studied. All GBMs studied overexpressed 14 known genes compared with normal human brain tissue. Overexpressed genes belonged to two broad groups: (a) growth factor-related genes; and (b) structural/extracellular matrix-related genes. For most of these 14 genes, expression levels were lower in low-grade astrocytoma than in GBM and were barely detectable in normal brain. Despite normal-appearing histology, gene expression patterns of tissues immediately adjacent to GBM were similar to those of their respective primary GBMs. Two genes were consistently up-regulated in both high-grade and low-grade gliomas, as well as in histologically normal tissues adjacent to GBMs. These genes coded for the epidermal growth factor receptor (previously reported to be overexpressed in gliomas) and for the alpha4 chain of laminin, a major blood vessel basement membrane component. Changes in expression of this laminin chain have not been previously associated with malignant tumors. Overexpression of laminin alpha4 chain in GBM and astrocytoma grade II by gene microarray analysis was confirmed by semiquantitive reverse transcription-PCR and immunohistochemistry. Importantly, an alpha4 chain-containing laminin isoform, laminin-8 (alpha4beta1gamma1), was expressed mainly in blood vessel walls of GBMs and histologically normal tissues adjacent to GBMs, whereas another alpha4 chain-containing laminin isoform, laminin-9 (alpha4beta2gamma1), was expressed mainly in blood vessel walls of low-grade tumors and normal brain. GBMs that overexpressed laminin-8 had a shorter mean time to tumor recurrence (4.3 months) than GBMs with overexpression of laminin-9 (9.7 months, P = 0.0007). Up-regulation of alpha4 chain-containing laminins could be important for the development of glioma-induced neovascularization and glial tumor progression. Overexpression of laminin-8 may be predictive of glioma recurrence.

Novel human malignancy-associated gene (MAG) expressed in various tumors and in some tumor preexisting conditions.

We have identified a novel human malignancy-associated gene (MAG) expressed in various malignant tumors including glioblastomas and hepatocellular carcinomas (HCCs) and in tumor preexisting conditions such as hepatitis C virus- and hepatitis B virus-induced liver cirrhosis. The expression of MAG was characterized using reverse transcription-PCR (RT-PCR), rapid amplification of cDNA ends PCR, RNA dot blotting, RNase protection assay, and Northern blot analysis. Rapid amplification of cDNA ends PCR yielded a 536-bp MAG fragment in HCC, macroregenerative liver nodules with dysplasia, and liver cirrhosis but not in normal liver or placenta. By RT-PCR, MAG expression was not found in 12 different normal tissues but found in 46 of 51 (90%) premalignant and malignant tissues of various sites. Embryonic liver and brain were positive for MAG expression together with tumors from the same organs, but the corresponding normal adult tissues were negative. By RNase protection assay, MAG mRNA was expressed in the HepG2 liver tumor cell line and in an ovarian carcinoma but not in normal liver. The estimated transcript size from Northern blot analysis was 8.8 kb. This novel gene may play a role in the progression of premalignant conditions and in the development of HCC and other cancers.

Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration.

In this Phase I trial, patients' peripheral blood dendritic cells were pulsed with peptides eluted from the surface of autologous glioma cells. Three biweekly intradermal vaccinations of peptide-pulsed dendritic cells were administered to seven patients with glioblastoma multiforme and two patients with anaplastic astrocytoma. Dendritic cell vaccination elicited systemic cytotoxicity in four of seven tested patients. Robust intratumoral cytotoxic and memory T-cell infiltration was detected in two of four patients who underwent reoperation after vaccination. This Phase I study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed dendritic cell vaccine for patients with malignant glioma.

Previous Midlife Oestradiol Treatment Results in Long-Term Maintenance of Hippocampal Oestrogen Receptor α Levels in Ovariectomised Rats: Mechanisms and Implications for Memory.

Ovariectomised rats that have received previous administration of oestradiol in midlife display enhanced cognition and increased hippocampal levels of oestrogen receptor (ER)α months after oestradiol treatment ended compared to ovariectomised controls. The present study aimed to investigate the mechanisms by which ERα levels are maintained following midlife oestradiol exposure and the role of ERα in memory in ageing females in the absence of circulating oestrogens. Unliganded ERα has increased interaction with the ubiquitin ligase, C-terminus of Hsc-70 interacting protein (CHIP), leading to increased degradation of the receptor. In our first experiment, we tested the hypothesis that midlife oestradiol exposure in ovariectomised rats results in decreased interaction between CHIP and hippocampal ERα, leading to increased levels of ERα. Middle-aged rats were ovariectomised and received oestradiol or vehicle implants. After 40 days, implants were removed. One month later, rats were killed and hippocampi were processed for whole protein western blotting and co-immunoprecipitation, in which ERα was immunoprecipitated from lysate. As expected, ERα protein expression was increased in rats previously treated with oestradiol compared to vehicle-treated rats. In rats treated with oestradiol, there was a decrease in CHIP-ERα interaction, suggesting that previous oestradiol treatment reduces interaction, slowing the degradation of ERα. In a second experiment, we determined the impact on memory of antagonism of ER in the absence of circulating oestrogens. Rats were ovariectomised and implanted with oestradiol capsules. Capsules were removed after 40 days. Rats received chronic i.c.v. infusion of ER antagonist, ICI 182 780, or artificial cerebrospinal fluid vehicle and were tested on a spatial memory radial-maze task. Rats treated with ICI 182 780 had significantly worse performance (more errors). These experiments provide evidence that previous midlife oestradiol treatment maintains hippocampal ERα by decreasing its interaction with CHIP and that activation of these receptors provides cognitive benefits in the absence of circulating oestrogens.

Clinical trial of Serratia marcescens extract and radiation therapy in patients with malignant astrocytoma.

PURPOSE: A clinical trial was undertaken to determine the safety and efficacy of combining a biologic response modifier derived from the bacterium Serratia marcescens (ImuVert) and radiation therapy (RT) in patients with newly diagnosed anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM). PATIENTS AND METHODS: Fifteen patients who had undergone either a gross total resection, a partial resection, or a biopsy were treated concurrently with ImuVert and RT. Safety and tolerance were examined by assessment of symptomatic reactions recorded at each ImuVert treatment. Efficacy of treatment was examined in terms of time to progression of tumor and survival. RESULTS: All patients experienced local reactions at the injection sites that consisted of erythema and induration. The majority of patients experienced flu-like symptoms. Hypotension was responsible for the most significant morbidity (which required fluid resuscitation and extended observation) and dose deescalation. No patients were removed from the study because of toxicity. There were no on-study deaths related to ImuVert treatment. Median time to progression was 33.4 weeks, and median survival was 78 weeks. CONCLUSION: These results compare favorably with those of recent studies in patients with malignant astrocytomas who received multimodality therapy.

High expression of the Glut1 glucose transporter in human brain hemangioblastoma endothelium.

The principal glucose transporter at the blood-brain barrier is Glut1, and GLUT1 expression is downregulated in high grade gliomas. In the present study, glucose transporter expression was studied in surgically resected hemangioblastoma tissue. Light microscopic immunochemistry indicated the high expression of the Glut1 glucose transporter isoform throughout the central vascular endothelium of this tissue. Glial fibrillary acidic protein (GFAP) was observed only at the tumor border, with no GFAP immunoreactivity in stromal cells, pericytes or endothelia in the central tumor regions. It is generally believed that more Glut1 is found in erythrocytes than any other cell, but quantitative electron microscopic immunogold analyses of Glut1-immunoreactive sites per micron of capillary membrane showed the Glut1 density in tumor endothelial membranes glucose transporter was 2-3-fold higher than in human red cells. In the same tissue samples, qualitative immunogold electron microscopy of human serum albumin indicated that this protein (MW 65,000) moved freely from the vascular space into pericapillary regions, confirming the leaky barrier characteristics of the hemangioblastoma. These studies show that Glut1 expression may be high in endothelia that are highly permeable and devoid of astroglial contacts. Thus, human cerebral hemangioblastomas may provide a novel system for studying the induction of Glut1 in the blood-brain barrier.

Bradykinin selectively opens blood-tumor barrier in experimental brain tumors.

Bradykinin, infused in low doses (10 micrograms/kg/min) through the carotid artery ipsilateral to RG2 glioma in rats, significantly increased the permeability in tumor capillaries to six different tracers of varying molecular weights compared with intracarotid infusion of saline alone. Permeability in normal brain capillaries was not significantly increased by intracarotid bradykinin infusion. Tracers used to examined permeability included radiolabeled alpha-aminoisobutyric acid (AIB; MW 103), sucrose (MW 342.3), inulin (MW 5000), and dextran (MW 70,000), horseradish peroxidase (HRP) and Evans blue (EB). Permeability was expressed as the unidirectional transfer constant K(i) (microliter/g/min). The permeabilities (K(i)) of tumors in the bradykinin group versus the control saline group for AIB, sucrose, inulin, and dextran were 25.91 +/- 6.78 vs. 13.95 +/- 4.29 (p < 0.01), 17.90 +/- 2.65 vs. 10.75 +/- 4.55 (p < 0.01), 23.92 +/- 6.99 vs. 6.20 +/- 4.37 (p < 0.01), and 17.84 +/- 1.00 vs. 1.47 +/- 1.24 (p < 0.001), respectively (mean +/- SD). Permeability of RG2 gliomas to high molecular weight dextran (70,000) was 12-fold higher in the bradykinin group than in the saline infusion group. Intracarotid infusion of bradykinin did not significantly increase the blood volume in tumor or brain tissue despite its known vasodilative effect. The permeability of normal brain capillaries was unaffected by intracarotid bradykinin infusion. The increased permeability was reversed 20 min after stopping the intracarotid infusion. Electron microscopic and gross qualitative analysis was performed using HRP and EB. Intracarotid bradykinin infusion increased HRP and EB within tumor tissue but not normal tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracarotid infusion of leukotriene C4 selectively increases blood-brain barrier permeability after focal ischemia in rats.

Intracarotid infusions of leukotriene C4 (LTC4) were used to open selectively the blood-brain barrier (BBB) in ischemic tissue after middle cerebral artery (MCA) occlusion in rats. BBB permeability was determined by quantitative autoradiography using [14C]aminoisobutyric acid. Seventy-two hours after MCA occlusion, LTC4 (4 micrograms total dose) infused into the carotid artery ipsilateral to the MCA occlusion selectively increased the unidirectional transfer constant for permeability Ki approximately threefold within core ischemic tissue and tissue adjacent ot the ischemic core. No effect on BBB permeability was seen within nonischemic brain tissue or in ischemic tissue after only 24 h after MCA occlusion. gamma-Glutamyl transpeptidase (gamma-GTP) activity was decreased in capillaries in ischemic tissue at 48 and 72 h after infarction, compared to high gamma-GTP in normal brain capillaries and moderate gamma-GTP in capillaries in the ischemic tissue at 24 h after infarction. These findings suggest that normal brain capillaries resist the vasogenic effects of LTC4. In contrast, LTC4 increases permeability in capillaries of ischemic tissue, where gamma-GTP is decreased. gamma-Glutamyl transpeptidase, an enzyme that inactivates LTC4 to LTD4 and LTE4 to LTF4, may act as an "enzymatic barrier" in normal brain capillaries to leukotrienes.

Imaging peripheral benzodiazepine receptors in brain tumors in rats: in vitro binding characteristics.

Peripheral benzodiazepine binding constants for transplanted RG-2 gliomas and HD and LK Walker 256 tumors (metastatic breast carcinoma) were determined in Wistar rats using autoradiography. In addition, Kd and Bmax parameters for peripheral benzodiazepine receptors on RG-2 tumors were directly visualized using digital image analysis of autoradiograms. High specific binding of [3H]PK11195, a selective peripheral benzodiazepine ligand, had excellent topographical correlation to areas of histologically verified tumor. Scatchard analysis suggested a single class of peripheral binding sites with similar binding affinities in RG-2 and LK Walker 256 tumors and normal cortex. Bmax was 20-fold greater in glial tumors and 11.6- and 10.6-fold greater in LK and HK Walker 256 tumors, respectively, compared to normal cortex. The location of metastatic tumors, either intracerebrally or subcutaneously, did not effect their Kd or Bmax values. Kd and Bmax values for RG-2 tumors were similar whether determined densitometrically or by direct visualization with image analysis. Binding parameters within normal brain were difficult to visualize by image analysis due to the low level of specific binding. The ability to label specifically intracerebral tumor cells and to characterize the binding parameters shown in this study suggest that peripheral benzodiazepine receptor ligands could be utilized by PET to analyze directly a variety of tumors in humans.

A herpes simplex virus type 1 mutant deleted for gamma34.5 and LAT kills glioma cells in vitro and is inhibited for in vivo reactivation.

To create an oncolytic herpes simplex virus type 1 (HSV-1) that is inhibited for reactivation, we constructed a novel herpes recombinant virus with deletions in the gamma34.5 and LAT genes. The LAT gene was replaced by the gene for green fluorescent protein, thereby allowing viral infection to be followed. This virus, designated DM33, is effective in killing primary and established human glioma cell lines in culture. DM33 is considerably less virulent following intracerebral inoculation of HSV-susceptible BALB/c mice than the wild-type HSV-1 strain McKrae. The safety of this virus is further supported by the retention of its sensitivity to ganciclovir and its relatively limited toxicity against cultured human neuronal cells, astrocytes, and endothelial cells. The ability of DM33 to spontaneously reactivate was tested in a rabbit ocular infection model that accurately depicts human herpes infection and reactivation. Following ocular infection of rabbits, spontaneous reactivation was detected in 83% (15/18) of the eyes infected with wild-type McKrae. In contrast, none of the eyes infected with DM33 had detectable reactivation. The efficacy of this virus in cultured human glioma cell lines, its safety, confirmed by its inability to reactivate, and its attenuated neurovirulence make DM33 a promising oncolytic agent for tumor therapy.

Thallium-201 SPECT imaging of brain tumors: methods and results.

Recent studies suggest that thallium-201 (201Tl) planar scans of brain tumors more accurately reflect viable tumor burden than CT, MRI, or radionuclide studies with other single-photon emitting compounds. We have previously reported the utility of 201Tl SPECT index in distinguishing low- from high-grade gliomas elsewhere. Here we describe the technical considerations of deriving a simple 201Tl index, based on uptake in the tumor normalized to homologous contralateral tissue, from SPECT images of brain tumors. We evaluated the importance of consistently correcting for tissue attenuation, as it may achieve better lesion discrimination on qualitative inspection, and the methodologic limitations imposed by partial volume effects at the limits of resolution.

Stimulation of cell growth and DNA synthesis by peripheral benzodiazepine.

The effects of peripheral benzodiazepine receptor ligands on cell proliferation were evaluated. PK11195 increased the growth rate of C6 glioma cells by 20-30% in the nanomolar range in serum free medium. [3H]thymidine incorporation into C6 glioma cells also were increased 22% and 25% after treatment by PK11195 and Ro5-4864, respectively. The effect of PK11195 as a mitogenic agent was estimated by mitogenic agent was estimated by [3H]thymidine incorporation using Swiss 3T3 cells. PK11195 increased DNA synthesis 170% over control at 10 nM. Higher concentrations of benzodiazepines showed inhibition of the DNA synthesis. Peripheral benzodiazepine binding sites underwent downregulation after exposure to serum free medium or to 10 nM PK11195. These findings suggest that peripheral benzodiazepines may be involved in the regulation of cell proliferation as a growth factor in lower concentration and as a antiproliferative agent in higher concentration.

Gene expression abnormalities in human glial tumors identified by gene array.

Novel genes specific for human oligodendroglioma and glioblastoma multiforme (GBM) were detected using the gene array analysis [18,376 genes Gene Discovery Array (GDA) from Incyte Genomics, Inc.]. Eleven genes were chosen based on the highest ratios of differential expression identified by GDA between histologically normal adjacent tissue and brain tumor tissue. The differential expression of those 11 genes was verified by semiquantitative RT-PCR and Northern analysis on 22 samples of glial and other tumors of the brain, as well as of normal embryonic and adult brain tissue. Gene no. 5 (an EST) was more expressed by GDA analysis in histologically normal adjacent brain tissue than in the corresponding oligodendroglioma. By RT-PCR, this gene was expressed in a number of brain tumors but not in normal adult and embryonic brain. By GDA analysis, gene no. 7 (oligophrenin-1) gave the highest ratio compared to other genes in brain tissue adjacent to the GBM vs. GBM. By RT-PCR, oligophrenin-1 was expressed in tumors and tumor-adjacent tissue, whereas meningioma and corpus callosum were negative. Gene no. 11 (an EST) was expressed only in brain tumors but not in normal brain by Northern analysis (message size 1.5 kb) and RT-PCR. GDA analysis successfully identified genes preferentially expressed in brain tumors, which was confirmed by Northern analysis and semiquantitative RT-PCR. The validity of gene arrays for tumor-specific gene discovery is discussed. Study of differential gene expression in glial tumors should help identify the mechanism/s of transformation of normal glial cells to malignant.

Gene expression of GLUT3 and GLUT1 glucose transporters in human brain tumors.

GLUT3 glucose transporter gene expression is confined to neurons, while GLUT1 gene expression is limited to endothelial cells in normal brain. Thus far, neither of the GLUT genes has been shown to be consistently expressed in glial cells in adult brain in vivo under normal conditions. However, GLUT gene expression may be aberrant in human brain glial tumors. The present investigation shows that the GLUT1 and GLUT3 transcripts are differentially expressed in a series of 20 human brain tumors. The GLUT1/actin mRNA ratio increased in parallel to the astrocytoma grade, compared to a control human brain cortex, although no change in this ratio was seen in 5 meningiomas. Immunoreactive GLUT1 protein was not detectable in human brain tumors, including high-grade gliomas. Both 4.2 or 2.7 kb GLUT3/actin mRNA ratios showed a linear correlation with the glioma grade (P < 0.025), and the GLUT3-immunoreactive protein was also expressed in high grade gliomas. These studies provide evidence for induction of GLUT1 and GLUT3 gene expression in malignant glial cells, and the mRNA levels correlate with the biologic aggressiveness of the tumor. The detection of immunoreactive GLUT3, but not GLUT1, in the high grade gliomas suggest the GLUT3 isoform may be the predominant glucose transporter in highly malignant glial cells of human brain.

Topographical and temporal specificity of human intraoperative optical intrinsic signals.

The goal of this study was to determine the topographical and temporal specificity of neuronal and vascular responses using an intraoperative optical technique (iOIS). The face, thumb, index, and middle fingers were stimulated individually to obtain separate maps of cortical activation. Peak optical responses provided unique, non-overlapping cortical brain maps. Non-peak signals were more dispersed and produced overlapping responses from different digits. Peak iOIS responses colocalized with electrocortical stimulation mapping and evoked potentials. Temporally, we observed statistically significant specificity corresponding to sequential cortical activation during early optical signals (500-1750 ms), but later perfusion responses were non-specific. To our knowledge, this is the first report of either topographical specificity in overlapping spatial patterns, and/or temporal specificity in early perfusion profiles. These results therefore may have significant implications for other perfusion dependent functional imaging techniques.