Overexpression of amplified genes is often associated with the acquisition of resistance to cancer therapeutic agents in vitro. We have identified a similar molecular mechanism in vivo for endocrine treatment failure in human prostate cancer which involves amplification of the androgen receptor (AR) gene. Comparative genomic hybridization shows that amplification of the Xq11-q13 region (the location), is common in tumours recurring during androgen deprivation therapy. We found high-level AR amplification in seven of 23 (30%) recurrent tumours, but in none of the specimens taken from the same patients prior to therapy. Our results suggest that AR amplification emerges during androgen deprivation therapy by facilitating tumour cell growth in low androgen concentrations.

The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 ؎ 22% when the treatment is started 12 h before ischemia and by 63 ؎ 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1␤-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E2 production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

Ischemic stroke is the most common lifethreatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inf lammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinf lammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre-and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemiainduced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1␤-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inf lammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.

Minocycline, a semisynthetic tetracycline derivative, protects brain against global and focal ischemia in rodents. We examined whether minocycline reduces excitotoxicity in primary neuronal cultures. Minocycline (0.02 M) significantly increased neuronal survival in mixed spinal cord (SC) cultures treated with 500 M glutamate or 100 M kainate for 24 hr. Treatment with these excitotoxins induced a dose-dependent proliferation of microglia that was associated with increased release of interleukin-1␤ (IL-1␤) and was followed by increased lactate dehydrogenase (LDH) release. The excitotoxicity was enhanced when microglial cells were cultured on top of SC cultures. Minocycline prevented excitotoxin-induced microglial proliferation and the increased release of nitric oxide (NO) metabolites and IL-1␤. Excitotoxins induced microglial proliferation and increased the release of NO metabolites and IL-1␤ also in pure microglia cultures, and these responses were inhibited by minocycline. In both SC and pure microglia cultures, excitotoxins activated p38 mitogen-activated protein kinase (p38 MAPK) exclusively in microglia. Minocycline inhibited p38 MAPK activation in SC cultures, and treatment with SB203580, a p38 MAPK inhibitor, but not with PD98059, a p44/42 MAPK inhibitor, increased neuronal survival. In pure microglia cultures, glutamate induced transient activation of p38 MAPK, and this was inhibited by minocycline. These findings indicate that the proliferation and activation of microglia contributes to excitotoxicity, which is inhibited by minocycline, an antibiotic used in severe human infections.

Repetitive spreading depression (SD) waves, involving depolarization of neurons and astrocytes and upregulation of glucose consumption, is thought to lower the threshold of neuronal death during and immediately after ischemia. Using rat models for SD and focal ischemia we investigated the expression of cyclooxygenase-1 (COX-1), the constitutive form, and cyclooxygenase-2 (COX-2), the inducible form of a key enzyme in prostaglandin biosynthesis and the target enzymes for nonsteroidal anti-inf lammatory drugs. Whereas COX-1 mRNA levels were undetectable and uninducible, COX-2 mRNA and protein levels were rapidly increased in the cortex, especially in layers 2 and 3 after SD and transient focal ischemia. The cortical induction was reduced by MK-801, an N-methyl-D-aspartic acid-receptor antagonist, and by dexamethasone and quinacrine, phospholipase A 2 (PLA 2 ) inhibiting compounds. MK-801 acted by blocking SD whereas treatment with PLA 2 inhibitors preserved the wave propagation. NBQX, an ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid͞kainate-receptor antagonist, did not affect the SD-induced COX-2 expression, whereas COXinhibitors indomethacin and diclofenac, as well as a NO synthase-inhibitor, N G -nitro-L-arginine methyl ester, tended to enhance the COX-2 mRNA expression. In addition, ischemia induced COX-2 expression in the hippocampal and perifocal striatal neurons and in endothelial cells. Thus, COX-2 is transiently induced after SD and focal ischemia by activation of N-methyl-D-aspartic acid-receptors and PLA 2 , most prominently in cortical neurons that are at a high risk to die after focal brain ischemia.

␤-amyloid (A␤), derived form the ␤-amyloid precursor protein (APP), is important for the pathogenesis of Alzheimer's disease (AD), which is characterized by progressive decline of cognitive functions, formation of A␤ plaques and neurofibrillary tangles, and loss of neurons. However, introducing a human wild-type or mutant APP gene to rodent models of AD does not result in clear neurodegeneration, suggesting that contributory factors lowering the threshold of neuronal death may be present in AD. Because brain ischemia has recently been recognized to contribute to the pathogenesis of AD, we studied the effect of focal brain ischemia in 8-and 20-month-old mice overexpressing the 751-amino acid isoform of human APP. We found that APP751 mice have higher activity of p38 mitogen-activated protein kinase (p38 MAPK) in microglia, the main immune effector cells within the brain, and increased vulnerability to brain ischemia when compared with age-matched wild-type mice. These characteristics are associated with enhanced microglial activation and inflammation but not with altered regulation of cerebral blood flow, as assessed by MRI and laser Doppler flowmetry. Suppression of inflammation with aspirin or inhibition of p38 MAPK with a selective inhibitor, SD-282, abolishes the increased neuronal vulnerability in APP751 transgenic mice. SD-282 also suppresses the expression of inducible nitric-oxide synthase and the binding activity of activator protein 1. These findings elucidate molecular mechanisms of neuronal injury in AD and suggest that antiinflammatory compounds preventing activation of p38 MAPK in microglia may reduce neuronal vulnerability in AD.T he ␤-amyloid precursor protein (APP) is a ubiquitous transmembrane glycoprotein and the source of ␤-amyloid peptides (A␤), which are the principal components of amyloid plaques in the brain of patients with Alzheimer's disease (AD), an age-related neurodegenerative disease associated with progressive decline of cognitive functions (1, 2). Other hallmarks of AD include the formation of neurofibrillary tangles in neurons, loss of synapses, and decreases in cell density in the distinct regions of the brain. These histopathological changes are observed in familial AD, which is caused by mutations in the APP or presenilin genes, in sporadic AD, and in individuals with Down's syndrome, who carry an extra copy of chromosome 21 and overexpress wild-type APP several fold in the brain (3-6).Substantial evidence indicates importance of APP for the pathogenesis of AD, but the mechanism how APP increases neuronal vulnerability in AD is unclear. A large number of epidemiological studies indicate that inflammatory events are involved because antiinflammatory drugs slow the progression of the disease, and reactive microglia and proinflammatory molecules that are secreted by microglia are present at sites of amyloid plaques (7-11). The inflammation hypothesis is further supported by the findings that secreted derivatives of APP (sAPP-␣) and A␤ activate microglial cells resulting in death ...

DNA damage induced by low doses of ionizing radiation causes apoptosis, which is partially mediated via the generation of free radicals. Both free radicals and apoptosis are involved in the majority of brain diseases, including stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Because previous studies have shown that tetracycline derivatives doxycycline and minocycline have anti-in¯amma-tory effects and are protective against brain ischemia, we studied whether minocycline and doxycycline or ceftriaxone, a cephalosporin antibiotic with the potential to inhibit excitotoxicity, protect neurons against ionizing radiation in primary cortical cultures. A single dose of 1 Gy signi®cantly increased lactate dehydrogenase release, induced DNA fragmentation in neurons and triggered microglial proliferation. Treatment with minocycline (20 nM), doxycycline (20 nM) and ceftriaxone (1 mM) signi®cantly reduced irradiation-induced lactate dehydrogenase release and DNA fragmentation. The most ef®cient protection was achieved by minocycline treatment, which also inhibited the irradiation-induced increase in microglial cell number. Our results suggest that some tetracycline derivatives, such as doxycycline and minocycline, and ceftriaxone, a cephalosporin derivative, protect neurons against apoptotic death.

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