Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

The transcriptional program in the response of human fibroblasts to serum.

The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

Identification of B94 (TNFAIP2) as a potential retinoic acid target gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by a block to myeloid differentiation caused by expression of the fusion oncoprotein promyelocytic leukemia-retinoic acid receptor alpha (PML-RARalpha). The purpose of this study was to identify genes that are regulated in a PML-RARalpha-dependent fashion by retinoic acid (RA), because such genes may be integrally involved in APL pathogenesis and/or myeloid differentiation. A cDNA microarray approach was used to identify genes induced in response to RA in TF1 myeloid leukemia cells expressing PML-RARalpha (TF1-PR cells). The B94 gene (TNFAIP2; Unigene Hs.101382), originally identified as a tumor necrosis factor alpha-inducible gene in endothelial cells, was one of several genes found to be induced by RA specifically in TF1-PR cells, but not in TF1-neo (control) cells. The induction of B94 was most pronounced in cells expressing the PML-RARalpha short isoform and was negligible in cells that expressed a mutant PML-RARalpha protein containing a deletion of the PML coiled-coil domain. B94 induction by RA occurred within 1 h, did not require new protein synthesis, and was inhibited by actinomycin D, suggesting rapid transcriptional activation. B94 was also induced by RA in NB4, UF1, and HL-60 cells, but not in other hematopoietic cell lines tested, suggesting that its up-regulation by RA may be specific to cells that express PML-RARalpha or are at the late myeloblast or promyelocyte stage of myeloid development. A screen of bone marrow cells from normal donors or patients with acute myelogenous leukemia showed that B94 was highly expressed in normal marrow and in marrow from patients with acute myelogenous leukemia French-American-British subtypes M0-M2, but was repressed in marrow cells from APL patients. Treatment of APL blasts in vitro with all-trans-RA resulted in up-regulation of B94 mRNA. These results suggest that B94 plays a role in myeloid development and support the hypothesis that B94 is a target gene of PML-RARalpha in APL.

Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation.

We used a new approach, termed dynamic cortical deformation (DCD), to study the neuronal, vascular, and glial responses that occur in focal cerebral contusions. DCD produces experimental contusion by rapidly deforming the cerebral cortex with a transient, nonablative vacuum pulse of short duration (25 milliseconds) to mimic the circumstances of traumatic injury. A neuropathological evaluation was performed on brain tissue from adult rats sacrificed 3 days following induction of either moderate (4 psi, n = 6) or high (8 psi, n = 6) severity DCD. In all animals, DCD produced focal hemorrhagic lesions at the vacuum site without overt damage to other regions. Examination of histological sections showed localized gross tissue and neuronal loss in the cortex at the injury site, with the volume of cell loss dependent upon the mechanical loading (p < 0.001). Axonal pathology shown with neurofilament immunostaining (SMI-31 and SMI-32) was observed in the subcortical white matter inferior to the injury site and in the ipsilateral internal capsule. No axonal injury was observed in the contralateral hemisphere or in any remote regions. Glial fibrillary acidic protein (GFAP) immunostaining revealed widespread reactive astrocytosis surrounding the necrotic region in the ipsilateral cortex. This analysis confirms that rapid mechanical deformation of the cortex induces focal contusions in the absence of primary damage to remote areas 3 days following injury. Although it is suggested that massive release of neurotoxic substances from a contusion may cause damage throughout the brain, these data emphasize the importance of combined injury mechanisms, e.g. mechanical distortion and excitatory amino acid mediated damage, that underlie the complex pathology patterns observed in traumatic brain injury.

Selective loss and selective sparing of neurons in the thalamic reticular nucleus following human cardiac arrest.

Neurons in the portion of the human thalamic reticular nucleus (RT) associated with the prefrontal cortex and mediodorsal thalamic nuclei were found to be selectively vulnerable to ischemic neuronal damage following relatively short (< or = 5-min) duration cardiac arrest. In contrast, selective sparing of these RT neurons occurred in cases with longer (> 10-min) duration of arrest that was sufficient to produce extensive ischemic neuronal damage throughout the cerebral cortex and thalamic relay nuclei. The selective degeneration of RT neurons appears to require the sustained activity of corticothalamic or thalamocortical projections to the RT following the ischemic insult. Loss of RT neurons associated with the frontal cortex and mediodorsal thalamus may be the biological basis of some types of persisting cognitive deficits in attentional processing experienced by patients following cardiac arrest, open heart surgery, or other forms of brief global cerebral ischemia.

Thalamic retrograde degeneration following cortical injury: an excitotoxic process?

Traumatic or stroke-like injuries of the cerebral cortex result in the rapid retrograde degeneration of thalamic relay neurons that project to the damaged area. Although this phenomenon has been well documented, neither the basis for the relay neuron's extreme sensitivity to axotomy nor the mechanisms involved in the degenerative process have been clearly identified. Physiological and biochemical studies of the thalamic response to cortical ablation indicate that pathological overexcitation might contribute to the degenerative process. The responses of thalamic projection neurons, protoplasmic astrocytes, and inhibitory thalamic reticular neurons in adult mice were examined from one to 120 days following ablation of the somatosensory cortex as part of an investigation of the role of excitotoxicity in thalamic retrograde degeneration. The responses of thalamic neurons to cortical ablation were compared with those produced by intracortical injection of the convulsant excitotoxin kainic acid, since the degeneration of neurons in connected brain structures distant to the site of kainic acid injection is also thought to occur via an excitotoxic mechanism. Within two days after either type of cortical injury, protoplasmic astrocytes in affected regions of the thalamic ventrobasal complex and the medial division of the posterior thalamic nuclei became reactive and expressed increased levels of immunohistochemically detectable glial fibrillary acidic protein. Within the affected regions of the ventrobasal complex an increased intensity of puncta positive for glutamate decarboxylase immunoreactivity, presumably due to an increase in its content within the terminals of the reciprocally interconnected thalamic reticular neurons, was also evident. These immunohistochemically detectable alterations in the milieu of the damaged thalamic neurons preceded the disappearance of the affected relay neurons by at least two days following cortical ablation and by seven to 10 days following intracortical kainic acid injection. Regions of the thalamus containing reactive astrocytes corresponded very closely to the regions undergoing retrograde degeneration. Protoplasmic astrocytes in these areas remained intensely reactive up to 60 days after cortical injury. Levels of glutamate decarboxylase were only transiently elevated in the degenerating regions of the ventrobasal complex following cortical ablation and returned to normal by 14 days. Increased glutamate decarboxylase immunoreactivity was transiently seen through the entire ventrobasal complex following intracortical kainic acid injection but was markedly more intense in degenerating regions. These patterns of labeling did not return to normal until 50 days after intracortical kainic acid injection, well after the death of the relay neurons. Cortical ablation and intracortical kainic acid injection produce similar alterations in thalamic neuronal and glial populations.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of gene expression signatures from breast tumors and breast tissue derived cell lines.

Cell lines derived from human tumors have historically served as the primary experimental model system for exploration of tumor cell biology and pharmacology. Cell line studies, however, must be interpreted in the context of artifacts introduced by selection and establishment of cell lines in vitro. This complication has led to difficulty in the extrapolation of biology observed in cell lines to tumor biology in vivo. Modern genomic analysis tool like DNA microarrays and gene expression profiling now provide a platform for the systematic characterization and classification of both cell lines and tumor samples. Studies using clinical samples have begun to identify classes of tumors that appear both biologically and clinically unique as inferred from their distinctive patterns of expressed genes. In this review, we explore the relationships between patterns of gene expression in breast tumor derived cell lines to those from clinical tumor specimens. This analysis demonstrates that cell lines and tumor samples have distinctive gene expression patterns in common and underscores the need for careful assessment of the appropriateness of any given cell line as a model for a given tumor subtype.

Selective loss of neurons from the thalamic reticular nucleus following severe human head injury.

The GABAergic neurons of the thalamic reticular nucleus, or nucleus reticularis thalami (RT), have been implicated as important components in attentional processing systems. Neurons in the RT are exquisitely sensitive to degeneration following kainic and domoic acid toxicity, experimental global ischemia, human cardiac arrest, and experimental closed head injury in nonhuman primates. The present study was performed to establish whether the selective loss of human RT neurons occurred following severe head injury. Brains from 37 human nonsurvivors of head injury were examined for evidence of RT neuronal loss. RT lesions in were found in 36 of 37 cases, representing 65 of 73 (89%) of the reticular nuclei examined. The incidence of RT lesions was similar in all age groups: 13 of 14 cases (92.9%) in the pediatric (< or = 16 years) group, 33 of 37 (89.2%) in the young adult (18-45 years) group, and 19 of 22 (86.4%) in the older adult (> 45 years) group. RT lesions were characterized by loss of one fourth to three fourths of the neurons from the region of the nucleus associated with the frontal cortex and thalamic mediodorsal (MD) and ventrolateral (VL) nuclei. Sparing of RT neurons correlated highly with the presence of extensive frontal cortical lesions, suggesting that an intact corticothalamic projection was necessary for RT degeneration following head injury. A pathologic cascade with a prominent excitotoxic component is proposed. The loss of these inhibitory thalamic reticular neurons and the resultant thalamic and neocortical neuronal dysfunctions may underlie some forms of attentional deficits that persist following head injury.

Selective vulnerability of hippocampal neurons in acceleration-induced experimental head injury.

Traumatically induced subtotal hippocampal neuronal loss traditionally has been considered a consequence of intracranial hypertension and impaired cerebral perfusion. We have examined the frequency and distribution of hippocampal lesions in an acceleration model of brain injury in 54 anesthetized nonhuman primates undergoing physiologic monitoring and subjected postinjury to comprehensive neuropathologic examination. Hippocampal lesions occurred in 32/54 animals (59%). These lesions always involved the CA-1 hippocampal subfield and were bilateral in 24 animals. Hippocampal involvement was not associated with marked elevation of intracranial pressure or depression of cerebral perfusion pressure. These lesions occurred in the absence of involvement of other brain regions considered selectively vulnerable to hypoxic insults. Hippocampal damage occurred in 46% of animals with mild injury characterized by brief periods of unconsciousness and no residual neurologic deficit. Ninety-four percent of animals with severe injuries and prolonged posttraumatic coma had hippocampal involvement. Traumatically induced selective neuronal necrosis of the hippocampus is a specific lesion not explained by the conventional mechanistic theories of head injury. An alternative hypothesis, such as excitotoxicity involving glutamate or other neurotransmitters, may account for the lesions demonstrated in this study.

Modification of the cortical impact model to produce axonal injury in the rat cerebral cortex.

Diffuse axonal injury (DAI) is a form of brain injury that is characterized by morphologic changes to axons throughout the brain and brainstem. Previous biomechanical studies have shown that primary axonal dysfunction, ranging from minor electrophysiologic disturbances to immediate axotomy, can be related to the rate and level of axonal deformation. Some existing rodent head injury models display varying degrees of axonal injury in the forebrain and brainstem, but the extent of axonal damage in the forebrain has been limited to the contused hemisphere. This study examined whether opening the dura mater over the contralateral hemisphere could direct mechanical deformation across the sagittal midline and produce levels of strain sufficient to cause a more widespread, bilateral forebrain axonal injury following cortical impact. Intracranial deformation patterns produced by this modified cortical impact technique were examined using surrogate skull-brain models. Modeling results revealed that the presence of a contralateral craniotomy significantly reduced surrogate tissue herniation through the foramen magnum, allowed surrogate tissue movement across the sagittal midline, and resulted in an appreciable increase in the shear strain in the contralateral cortex during the impact. To evaluate the injury pattern produced using this novel technique, rat brains were subjected to rigid indentor impact injury of their left somatosensory motor cortex (1.5 mm indentation, 4.5-4.9 m/sec velocity, and 22 msec dwell time) and examined after a 2-7 day survival period. Neurofilament immunohistochemistry revealed numerous axonal retraction balls in the subcortical white matter and overlying deep cortical layers in the right hemisphere beneath the contralateral craniotomy. Retraction balls were not seen at these positions in normals, sham controls, or animals that received cortical impact without contralateral craniotomy and dural opening. The results from these physical modeling and animal experiments indicate that opening of the contralateral dura mater permits translation of sufficient mechanical deformation across the midline to produce a more widespread pattern of axonal injury in the forebrain, a pattern that is distinct from those produced by existing fluid percussion and cortical impact techniques.

Biomechanical analysis of experimental diffuse axonal injury.

The purpose of this paper is to present results from methodologies used in our laboratory that are targeted toward identifying specific brain injury thresholds. Results from studying one form of brain injury, diffuse axonal injury, are presented in this report. Physical models, or surrogates, of the skull-brain complex are used to estimate the relationship between inertial loading and brain deformation. A porcine model of diffuse axonal injury, developed with information from these physical models and earlier in vitro tissue modeling studies, is used to correlate histologic and radiologic evidence of axonal injury to predicted regions of injury from the experimental and theoretical analysis. These results form the basis for developing improved diffuse brain injury tolerance levels, as well as identifying new means of diagnostic and treatment techniques for diffuse axonal injury.

Degeneration of neurons in the thalamic reticular nucleus following transient ischemia due to raised intracranial pressure: excitotoxic degeneration mediated via non-NMDA receptors?

Transient global ischemia was produced in rats by cisternal fluid infusion, producing a negative cerebral perfusion pressure by elevating the intracranial pressure (ICP) 25-50 mm Hg above mean arterial pressure (MAP). Animals were allowed to survive for 2-7 days following a transient ischemic episode of 5-30 min. The brains were examined for signs of ischemic degeneration in Nissl-stained sections and adjacent sections reacted with antisera against glial fibrillary acidic protein (GFAP) or aspartate aminotransferase (AAT). Neurons in the thalamic reticular nucleus (RT), a pure population of gamma-aminobutyric acid (GABA)ergic neurons which project their axons to thalamic relay nuclei, were found to have the lowest threshold for degeneration in this model, consistently undergoing degeneration under conditions which completely spared the hippocampal CA1 from degeneration. Whereas it took up to 30 min of complete ischemia to produce degeneration of CA1 neurons when ICP was raised using room temperature infusion fluids, 15 min of ischemia under these conditions was sufficient to produce extensive degeneration of neurons in the entire ventral 3/4 of the RT. Prolonged (greater than 25 min) episodes of partial ischemia (ICP less than or equal to MAP) were also sufficient to produce massive degeneration of RT neurons. The lesion in the RT was most clearly evident in sections reacted with antisera to GFAP, labeling intensely reactive protoplasmic astrocytes within the regions of the RT where neuronal degeneration had occurred. Neuronal loss and accompanying proliferation of microglial cells were evident in Nissl-stained sections but the extent of the neuronal loss was most clearly obvious in sections reacted with an antisera to AAT, an enzyme present in detectable quantities in GABAergic neurons. Pretreatment with the non-competitive NMDA antagonist MK-801 at doses sufficient to completely prevent massive degeneration of the hippocampal CA1 failed to prevent the degeneration of RT neurons, suggesting that if RT degeneration involves an excitotoxic process it acts through non-NMDA receptors.

Degeneration of hippocampal CA1 neurons following transient ischemia due to raised intracranial pressure: evidence for a temperature-dependent excitotoxic process.

Degeneration of hippocampal CA1 neurons occurs following transient complete ischemia produced by raised intracranial pressure. Both systemic injection of MK-801 and profound cerebral hypothermia produced by cisternal infusion of room temperature (22-25 degrees C) fluids protect vulnerable CA1 neurons from degeneration. Hypothermia appears to decrease hippocampal extracellular levels of glutamate during and after ischemia but provides only relative protection from ischemia as CA1 degeneration does occur with prolonged (30 min) periods of ischemia. Elevated intracranial pressure appears to produce ischemic degeneration in the hippocampus via an NMDA receptor mediated excitotoxic process which is highly temperature dependent.

The AMPA antagonist NBQX protects thalamic reticular neurons from degeneration following cardiac arrest in rats.

Thalamic reticular (RT) neurons are selectively vulnerable to degeneration following global ischemia. The degenerative mechanism is thought to involve an excitotoxic component, mediated in part by sustained post-ischemic activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) type excitatory amino acid (EAA) receptors. In order to test this hypothesis, the selective competitive AMPA type EAA antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxalinedione) was administered at 30 mg/kg to rats 1, 3, and 6 h after resuscitation from 10 min cardiac arrest. NBQX treatment resulted in a 2-fold increase of spared RT neurons, from a mean density of 3.6 +/- 0.8 x 10(3) neurons/mm3 in cardiac arrest cases to 7.4 +/- 1.1 x 10(3) neurons/mm3 in the NBQX treated group, which represents sparing of 41.7% of the normal population of RT neurons, and protection of 26.9% of vulnerable RT neurons. Neurons within the central core of the RT manifest both a higher degree of vulnerability to ischemic degeneration, > 92% loss, and a higher sensitivity to sparing following NBQX administration, 460% increased sparing, than neuronal sub-populations in the medial or lateral 1/3 of the RT. Protection by post-arrest administration of NBQX suggests that sustained post-arrest stimulation of AMPA receptors is an important component in the process of ischemic degeneration of RT neurons.

The AMPA antagonist NBQX provides partial protection of rat cerebellar Purkinje cells after cardiac arrest and resuscitation.

Purkinje cell loss in adult rats resuscitated following cardiac arrest is analogous to that seen following human cardiac arrest. Administration of the competitive AMPA antagonist NBQX to rats resuscitated after 10 min duration cardiac arrest rescued 21.5% of the vulnerable Purkinje cell population. These results support the hypothesis that sustained postischemic overexcitation of AMPA receptors may be a driving force in the process of Purkinje cell degeneration.

Neural transplantation: autoradiographic analysis of histogenesis in neocortical transplants.

Neurohistogenesis in neocortical transplants obtained from 15-, 16-, 17-, 18-, 19-, 20- and 21-day-old embryos, was studied employing [3H]thymidine autoradiography. The neural tissues were transplanted in the midvermis of cerebellum of the host animals. Following transplantation the host animals in different groups were injected with the radiochemical at 6 hr, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 day intervals, to label the neurons forming on different days in the developing transplants. Analysis of autoradiograms showed that all the neocortical transplants did undergo histogenesis in the host cerebellum, and that it was similar to that seen in a normally developing neocortex. Transplants from the 15-day embryos showed histogenesis lasting for 9 days, and at the other extreme transplants from the 21-day embryos showed histogenesis lasting only for 1 day. Histogenesis in other transplants fell between these two extremes in a graded fashion in relation to the age of the donor embryos. The magnitude of histogenesis in transplants from different donor embryos was closely related to the final size of the transplants. Transplants from 15-day embryos were the largest in size, and they were followed by those from 16-, 17-, 18-, 19-, 20- and 21-day donor embryos in a graded fashion. All transplants were intraparenchymal, and histologically appeared normal. They contained fully differentiated neurons, and were anatomically integrated with the host cerebellum without any glial scar tissue or necrotic tissue intervening between them.

Degeneration of rat thalamic reticular neurons following intrathalamic domoic acid injection.

Domoic acid (DA), an analog of kainic acid, produces attentional deficits in humans who have ingested shell fish contaminated with this excitotoxin. The thalamic reticular nucleus (RT), by virtue of its location, connections and intrinsic properties, has been implicated in attentional processes. This study demonstrated the vulnerability of RT neurons following intrathalamic DA injections in rats. Lesions were characterized by almost total neuronal loss throughout the RT and sparing of adjacent populations of relay neurons in the VL and VPL. Los of RT neurons may underlie some types of attentional deficits observed in humans following DA poisoning.

The partial mu opiate agonist buprenorphine protects a sub-population of thalamic reticular neurons following cardiac arrest in rats.

Extensive loss of neurons occurs from the middle region of the thalamic reticular nucleus (RT) in rats resuscitated after 10 min of cardiac arrest. Administration of the partial mu opiate agonist buprenorphine 45 min after resuscitation produces a small but significant increase in spared neurons along the medial and lateral margins of the middle RT, regions where mu receptors have been localized. Systemic administration of mu agonists may augment endogenous opiate mechanisms that contribute to the relative ischemic resistance of subpopulations of RT neurons.

The small molecule FKBP ligand GPI 1046 induces partial striatal re-innervation after intranigral 6-hydroxydopamine lesion in rats.

Extensive unilateral striatal deafferentation was produced by intranigral 6-hydroxydopamine (6-OHDA) in rats. Beginning 60 days after 6-OHDA injection animals received a 14-day course of treatment with either the small molecule FKBP ligand GPI 1046 (10 mg/kg) or its vehicle alone. Striatal dopaminergic innervation density was determined from high power image analysis of striatal tyrosine hydroxylase (TH) immunohistochemistry. GPI 1046 treatment did not alter TH fiber density in the contralateral striatum but did produce significantly higher striatal TH fiber density in the ipsilateral caudate-putamen. This striatal re-innervation occurred in the absence of increased nigral sparing, and appears to reflect the GPI 1046 induced sprouting of residual TH+ fibers spared by the 6-OHDA lesion.