HER2 in a subset of breast cancers (HER2+) is associated with high histological grade and aggressive clinical course. Despite these distinctive features, the differences in response of HER2+ patients to both adjuvant cytotoxic chemotherapy and targeted therapy (e.g. trastuzumab) suggests that unrecognized biologic and clinical diversity is confounding treatment strategies. Furthermore, the small but established risk of cardiac morbidity with trastuzumab therapy compels efforts towards the identification of biomarkers that might help stratify patients.Over-expression of HER2+ patients. Staining with antibody directed at TRMT2A was found to be strongly associated with outcome in HER2+ patients. This association with outcome was tested in two independent validation cohorts; an existing staining dataset derived from tissue assembled at the Cleveland Clinic Foundation (CCF), and in a new retrospective study performed by staining archived paraffin blocks available at the Roswell Park Cancer Institute (RPCI).A single institution tissue array cohort assembled at the Clearview Cancer Institute of Huntsville (CCIH) was screened by immunohistochemistry staining using a large number of novel and commercially available antibodies to identify those with a univariate association with clinical outcome in HER2+ patients from the CCIH discovery cohort . This association with outcome was confirmed using 75 HER2+ patients from the CCF cohort  and 64 patients from the RPCI cohort . In bivariable analysis the association with outcome was independent of grade, tumor size, nodal status and the administration of conventional adjuvant chemotherapy in the CCIH and RPCI cohorts.TRMT2A staining showed a strong correlation with likelihood of recurrence at five years in 67 HER2+ breast cancer patients. These results suggest that TRMT2A expression should be further studied in the clinical trial setting to explore its predictive power for response to adjuvant cytotoxic chemotherapy in combination with HER2 targeted therapy.Studies from three independent single institution cohorts support TRMT2A protein expression as a biomarker of increased risk of recurrence in  A major challenge in the treatment of breast cancer is to accurately identify those patients who are more likely to develop recurrence so that appropriate therapy can be selected. Significant advances have been made in the development of combination chemotherapy regimens along with the development of effective targeted therapeutics. HoweverHER2 targeted monoclonal antibody, Trastuzumab, has been evaluated in four large randomized clinical trials demonstrating significant benefits in disease free survival (DFS) with the addition of one year of Trastuzumab to adjuvant chemotherapy[The HER2 gene is amplified in 15-20% of breast cancers and this molecular alteration carries with it a more aggressive clinical course . A HER2 motherapy. Howevermotherapy,7. In admotherapy,9. GivenHER2 positive tumors is in part reflected in biologic heterogeneity as assessed by gene expression profiling [HER2 positive tumors identified with immunohistochemistry and/or fluorescence in situ hybridization exhibit variation in global gene expression patterns related in part to expression of hormone receptor related genes and/or signatures that distinguish the aggressive luminal B tumor subtype[HER2 positive patients but has shown inconsistent results[HER2 positive tumors[The clinical heterogeneity of rofiling -12. HER2r subtype. Estroget results. Howeverve tumors,15.HER2 biologic diversity and or stratify HER2+ breast cancer into significantly different prognostic categories. We previously screened a large number of novel commercially available antisera, targeted by gene expression data, to identify panels of antibodies useful for breast tumor classification[HER2 expressing tumors and identified TRMT2A (previously known as HTF9C), a novel cell cycle regulated protein, as associated with aggressive clinical course [HER2 positive breast cancer patients into groups with different clinical outcomes may aid clinical decision-making. Furthermore, TRMT2A is a strong candidate biomarker worthy of further investigation as a predictive marker of response to adjuvant chemotherapy and HER2 targeted therapy.We have endeavored to translate gene expression based classification of carcinoma into IHC reagents that can be used to discover and validate the relationship between tumor classification and clinically significant phenotypes. In the ification. In thisl course ,17. We cHER2+ and 75 HER2+ cases were identified by IHC staining in the CCIH and CCF tissue arrays. The RPCI cohort was assembled by retrospective review of the pathology archives from January 1996 to January, 2006 to identify HER2+ breast cancer patients who where 3+ positive by immunohistochemistry using the Herceptest™ test kit . HER2 IHC status for all sixty-four cases identified was confirmed by re-staining. The clinicopathologic characteristics for the three patient cohorts are summarized in table Single institutional breast cancer cohorts from the Clearview Cancer Institute of Huntsville (CCIH), Cleveland Clinic Foundation (CCF), and Roswell Park Cancer Institute (RPCI) were used in this study. In all cohorts, patient tumor paraffin blocks were assigned an anonymous unique identifier linked to a clinical data base that contained treatment and outcome data blinded to investigators in this study. IRB approval was obtained for the use of patient blocks at each respective institute. Patients were excluded for study if they were male, had metastatic or multicentric disease at time of diagnosis, or had received trastuzumab in an adjuvant setting. Some study patients were treated variably with adjuvant cytotoxic chemotherapy. 67 Each tissue microarray (TMA) block contained a single 0.6 mm core sampled from representative paraffin blocks from each patient. Tissue array sections were de-paraffinized and dehydrated by submerging in xylene three times for 10 minutes each, followed by rinsing three times in 100% ethanol, two times in 95% ethanol and then treated by microwaving boiling for 11 minutes in 10 uM buffered citrate pH 6.0. Slides were allowed to cool to room temperature, and rinsed in distilled water followed by PBS. Slides were dipped in 0.03% hydrogen peroxide followed by rinsing with PBS and then stained using dilutions of antibodies in DAKO Diluent (DAKO Cytomation Inc.) for one hour at room temperature. Initial studies were performed with rabbit polyclonal affinity purified antibodies targeting a peptide located in the amino terminal third of the protein (s0545). A novel rabbit monoclonal antibody (S0722) directed to the same peptide was generated which showed very similar staining pattern to the polyclonal antibody as judged by comparison across a large breast cancer tissue array cohort (data not shown). The RPCI staining was performed using the rabbit monoclonal antibody. For each antibody, dilutions were first tested on a small 'titer' tissue array that had breast cancer cases with positive and negative cases in addition to a set of tumor derived cell lines suspended in paraffin. Secondary antibody was applied for one hour, and staining visualized using the DAKO Cytomation Envision staining kit according to the manufacturer's instructions. Cases were considered TRMT2A expressing if greater than 10% of cells showed cytoplasmic staining. Most positive cases show uniform staining across tumor cells within the sample. Nuclear staining in the absence of cytoplasmic staining was not scored as positive (16).Equal concentration of total cell line extracts from UACC62, MOLT4, LOXIMVI, RXF393, and OVCAR5 or recombinant, e.coli produced protein encoding four different human proteins (AbNova Inc) were run on a denaturing polyacrylamide gel, transferred to blotting paper. Blots were exposed to either a monoclonal antibody (s0722) directed at a synthetic peptide encoded within TRMT2A amino terminal third of the protein, a polyclonal antibody directed at the same peptide (s0545), or an independent polyclonal affinity-purified antibody (s0728) directed at a distinct, non-overlapping peptide in the middle third of the protein.HER2+ patients. The hypothesis that TRMT2A was related to outcome in HER2+ patients was subsequently evaluated in the existing CCF staining dataset as a prospectively designated hypothesis. The RPCI cohort was assembled explicitly to test the association identified and confirmed in the CCIH and CCF cohorts respectively. Staining was completed and scores were attached to the patient code number by the pathology group before the clinical records were linked to the immunohistochemistry dataset by the clinical investigator.We previously reported the screening of 126 novel and commercially available antibodies on the CCIH tissue array. Prior tHER2+ patients was assessed by a Cox proportional hazards model. Independence of TRMT2A from other clinical and pathologic prognostic parameters was also tested via Cox proportional hazards with each variable in a bi- or multivariable model. All univariate and multivariable Cox proportional hazard ratios and associated p-values were calculated using S-plus software . All reported p values are two sided.The relationship between all recurrence within five years of diagnosis and TRMT2A staining in HER2 expressing cases. As part of a large project aimed at translating the emerging gene expression based classification of carcinoma into an IHC-based classification, we had screened a large number of novel polyclonal affinity-purified antibodies for association with clinical outcome and selected thirty-seven for further study in an independent cohort assembled at the Cleveland Clinic Foundation (CCF)[HER2 positive patients  in the CCIH cohort  that included 69 ion (CCF). Of thest figure . TRMT2A in silico in the existing CCF cohort dataset which confirmed the association between TRMT2A staining and poor outcome   Figure . We subsHER2 expressing patients while estrogen receptor, progesterone receptor, nodal status, stage, and treatment with cytotoxic chemotherapy were not. We used a new rabbit monoclonal antibody directed at TRMT2A (S0722) which stained in a pattern very similar to the original polyclonal reagent across the CCIH cohort (data not shown). Western blot analysis with the rabbit monoclonal antibody showed an identical 38KD bands on multiple cell lines as the original polyclonal antibody and the same molecular weight band as a second polyclonal affinity purified antibody (s0728) directed at an independent epitope within the TRMT2A predicted protein. It also recognized only its cognate 68 kd in vitro transcribed and translated fusion protein in a western blot of four different fusion proteins loaded in equimolar amounts .In these three institutional cohorts from CCIH, CCF, and RPCI, TRMT2A stained 29%, 37% and 23% respectively. However, these subsets of patients contained 69%, 59% and 47% of the total number of To assess whether TRMT2A was providing information in HER2 expressing patients that was independent of standard clinical and pathologic factors in addition to ER and PR staining, we performed univariate and bivariate analysis with each prognostic factor and outcome Table .HER2+ patients with a high likelihood of recurrence. In these cohorts in which patients were treated with various cytotoxic chemotherapy regimens without Traztuzumab, estrogen and progesterone receptor expression showed an inconsistent relationship with outcome similar to inconsistent finding in the published literature [In the current report, we have demonstrated in three independent institutional cohorts that IHC-based detection of cytoplasmic TRMT2A identifies a subset of terature -15. TRMTTRMT2A was chosen for study based upon its identification as a novel open reading frame whose expression varies during the cell cycle similar to the oscillation of MCM, tubulin, CDC2, and other genes whose expression peak in S-phase and decrease during mitosis. Genes with similar expression patterns have been noted to be involved in different aspects of transiting the cell cycle and certain members have been noted to be elevated in tumor cells,18. The HER2+/TRMT2A+ breast cancers are more likely to recur treated with traditional cytotoxic therapy alone and therefore more aggressive treatment in the adjuvant setting may need to be considered for these patients, regardless of the other conventional risk factors. Whether or not HER2+/TRMT2A+ tumors are more likely to show benefit from trastuzumab cannot be determined from the current data but these data suggest that it should be studied in the clinical trial setting to further explore its relationship to response to adjuvant cytotoxic chemotherapy and HER2 targeted therapy.A greater understanding of the biologic heterogeneity within the HER2 positive cohort of breast cancers identified clinically would aid efforts to assess and predict outcome for this important subset of patients. Furthermore, prudent selection of treatment regimen and length of treatment could be helped by the identification of biomarkers that identify patients with a more aggressive clinical course as well as those most likely to respond to adjuvant therapy. Our data suggests that David G. Hicks, Bagi R. Janarthanan, R Vardarajan, Swati Kulkarni, Thaer Khoury, Daniel Dim, G. Thomas Budd, Brian J. Yoder, Raymond Tubbs, report no conflict of interest.  Rodney A. Beck, Brian Z. Ring, Robert S. Seitz, Douglas T. Ross are employees of, and stockholders in Clarient Inc which funded studies reported in this manuscript.  Marshall T. Schreeder, Noel C. Estopinal are stock holder of Clarient Inc which funded studies reported in this manuscript.DGH, BRJ, RV, SK, TK, DD, GTB, BJY, RT, MTS and NCE assembled paraffin blocks and clinical follow-up information for the cohorts reported in this manuscript. RAB performed wet work and IHC evaluation, BZR, RSS, performed the statistical analysis. DTR, BZR, RSS and DGH conceived the study and wrote the manuscript. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/108/prepub


Polyadenylated, mRNA-like transcripts with no coding potential are abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs) are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks (DSBs) that initiate homologous recombination. Most meiotic recombination is positioned at hotspots, but knowledge of the mechanisms is nebulous. In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs.prl class. DSB peaks map to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and intergenic regions, or most often within intergenic regions. Unconditional statistical tests revealed that this colocalization is non-random and robust (P≤5.5×10−8). Furthermore, we tested and rejected the hypothesis that the ncRNA loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes.We compared the genome-wide distribution of DSB peaks to that of polyadenylated ncRNA molecules of the Meiotic DSB hotspots are directed to loci that express polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism for what directs meiotic recombination to hotspots. It also reveals a likely biological function for enigmatic ncRNAs. We propose specific mechanisms by which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA loci, help to position recombination protein complexes at DSB hotspots within chromosomes. Small (∼20–30 nt) ncRNAs such as siRNA and miRNA can trigger the formation of euchromatin or heterochromatin, affect positively or negatively transcription, induce the deadenylation of mRNA, trigger the targeted degradation of mRNA, and regulate positively or negatively the translation of functional mRNAs  for hotspots across the genome In meiosis, a combination of crossover recombination structures (chiasmata) and sister chromatid cohesion distal to chiasmata help to align homologous chromosome pairs and ensure their proper segregation in the first meiotic division Figure 1i.e., genes) and in intergenic regions (IGRs), but they localize preferentially to large IGRs. Other than this general bias towards IGRs, no discrete features which might be responsible for DSB hotspot clustering were identified The meiotic recombination protein Spo11 (Rec12 in fission yeast) is a conserved, topoisomerase II-like enzyme which introduces dsDNA breaks (DSBs) that initiate recombination rec7 gene encompasses three polyadenylated, transcript from opposite strand RNA molecules, tos1, tos2, and tos3  (prl class of ncRNA molecules and the positions of prominent DSB clusters that initiate meiotic recombination.The 194 prominent DSB peaks encompass about 700 kbp (5.6%) of the genome sequence and those peaks are separated by regions essentially devoid of DSBs prl data set . The complete set of all detectable DSB peaks (weak and prominent) map to about 10.6% of the genome sequence prl molecules to fall within a peak by chance, and have 97.4% confidence that the number falling within a peak would be 12 or less. The probability of observing 24 or more occurrences out of 68 is low (P = 5.5×10−8)  across the genome.The preceding analyses considered the 194 prominent DSB peaks that collectively contain most of the meiotic DSBs catalyzed by Rec12 (Spo11). However, there are an additional 159 “weak” DSB peaks that encompass less than 5% of the genome and whose aggregate area integrals contribute 12% of the total DSBs measured across the genome prl loci are within IGRs prl loci and DSB peaks localizing preferentially, but independently, to IGRs. To explore this possibility further, we restricted the analysis to the 40% of the fission yeast genome comprised of IGRs, and we computed the corresponding conditional probabilities under the binomial distribution and null hypothesis of no association. DSB peaks (prominent and weak) cover 22% of DNA within IGRs, prominent peaks cover 14% of DNA within IGRs, and 59 prl loci are specific to IGRs. Twenty four of the 59 IGR-specific prl loci (41%) occur within a DSB peak (P = 9.6×10−4), 18 of which (31%) occur within a prominent peak (P = 8.6×10−4). We conclude that the intimate association between DSB peaks and prl loci does not arise from independently preferential localization of each to an IGR. Rather, the DSB peaks colocalize specifically with ncRNA loci that may be situated within ORFs, near ORF-IGR boundaries, or most often within IGRs  prl class help to activate DSB hotspots. This process may be conserved, because in mice one ncRNA has been shown to map to a well-defined meiotic recombination hotspot Transcripts of the The vast majority of fission yeast ncRNAs remain undiscovered prl loci are embedded entirely within DSB peaks , then the fraction of prl loci associated with DSB peaks also increases dramatically ? We propose two, not mutually exclusive, hypotheses on mechanism.prl-dependent chromatin remodeling, due either to some aspect of transcription at prl loci or mediated by the ncRNA molecules themselves, creates a preferential site for the nucleation of recombination protein complexes. This hypothesis fits comfortably within the prevailing orthodoxy, which posits that the relative accessibility of DNA in regions of “open” chromatin has a role in hotspot activity  base pair with their targets (RNA or DNA) and thereby deliver protein complexes to those targets e.g., as is implicated for the siRNA-containing RITS complex). Alternatively, it may involve the recognition of R-loop structures by protein complexes . In either case, the base pairing between ncRNA molecules and homologous chromosomal DNA would guide recombination to hotspots.Small ncRNAs . Second, they reveal a likely biological function for many of the enigmatic, polyadenylated ncRNA molecules that are so abundant in eukaryotes.One of our two alternative hypotheses is that the polyadenylated ncRNA molecules help to position meiotic recombination. For context, this hypothesis is consistent with, and might explain mechanistically, some of the many connections between transcription and recombination documented previously see . A few aprl class were identified among 987 random cDNA clones tosprlprl locations annotated in the Agilent microarray spreadsheet (Agilent Technologies). Seventeen prl loci were not annotated in that spreadsheet . These were added manually. One locus (prl52) was annotated twice (on chromosomes I and III). The position for chromosome I was used, as it is congruent with the location listed in the genome database. The distances between each prl locus and its two neighboring DSB peaks (left and right) were determined and tabulated in Excel 2005 . Ends of prl molecules (loci) that map within DSB peaks were assigned distance values of zero. Because 88% of the observed DSBs  were classified previously as falling within prominent DSB peaks Sixty-eight polyadenylated, ncRNA molecules of the P value for the observed number of mappings to DSB peaks was calculated using the exact binomial test of a single proportion. Exact binomial one-sided upper confidence limits were also calculated for the expected number of successes under the null hypothesis. Calculations were performed using the “BINOMDIST” function in a Microsoft Excel spreadsheet . The binomial-proportion estimates, sample sizes, observed success rates, null-hypothesis confidence limits, and calculated P values for each test scenario are given in the main text.Each locus expressing an ncRNA molecule can map either within or outside of a DSB peak. Thus, under random genome-wide placement with constant per-locus “success” probability of mapping within a peak (the null hypothesis), the set of ncRNA loci should approximate the binomial distribution with respect to DSB peaks. We therefore modeled the data using the Binomial distribution. Binomial proportion parameters (success probabilities per locus) were estimated as equal to the fraction of the genome encompassed by “all” and “prominent” DSB peaks. Conditional binomial proportion parameters  were similarly set equal to the fraction of IGRs encompassed by “all” and “prominent” DSB peaks. For every parameter estimate, the


Poecilia mexicana males show weaker expression of mating preferences when being observed by a rival. This may be an adaptation to reduce sperm competition risk, which arises because commonly preferred female phenotypes will receive attention also from surrounding males, and/or because other males can copy the focal male's mate choice. Do P. mexicana males indeed respond to perceived sperm competition risk? We gave males a choice between two females and repeated the tests under one of the following conditions: (1) an empty transparent cylinder was presented (control); (2) another ("audience") male inside the cylinder observed the focal male throughout the 2nd part, or (3) the audience male was presented only before the tests, but could not eavesdrop during the actual choice tests (non-specific sperm competition risk treatments); (4) the focal male could see a rival male interact sexually with the previously preferred, or (5) with the non-preferred female before the 2nd part of the tests (specific sperm competition risk treatments).Multidirectional interactions in social networks can have a profound effect on mate choice behavior; e.g., The strength of individual male preferences declined slightly also during the control treatment (1). However, this decrease was more than two-fold stronger in audience treatment (2), i.e., with non-specific sperm competition risk including the possibility for visual eavesdropping by the audience male. No audience effect was found in treatments (3) and (5), but a weak effect was also observed when the focal male had seen the previously preferred female sexually interact with a rival male .P. mexicana males tend to share their mating effort more equally among females when the resource value of their previously preferred mate decreases after mating with a rival male (perceived specific sperm competition risk), but this effect is comparatively weak.When comparing the two 'non-specific sperm competition risk' treatments, a very strong effect was found only when the audience male could actually observe the focal male during mate choice [treatment (2)]. This suggests that focal males indeed attempt to conceal their mating preferences so as to prevent surrounding males from copying their mate choice. When there is no potential for eavesdropping [treatment (3)], non-specific specific sperm competition risk seems to play a minor or no role. Our results also show that  An increasing body of literature considers the question of how mate choice is influenced by the social environment of the choosing individual (non-independent mate choice -7). TradXiphophorus hellerii) males are less likely to initiate a fight against observed winners , while in another half of trials it interacted with the previously non-preferred female [treatment (5)]. We scored sexual behaviors shown by the rival male  to make sure that the focal male would actually perceive sperm competition risk. All rival males exhibited sexual behavior .Finally, in treatments (4) and (5) we created an experimental situation where the focal male could observe the "audience" male  interact sexually with one of the two stimulus females , N = 30; treatment (2), N = 39; treatment (3), N = 27; treatment (4), N = 25; treatment (5), N = 24]. All statistical analyses were conducted using SPSS 12.0. Data are generally presented as means ± SE and were tested with Kolmogorov-Smirnov-tests for normality. All relative data were arcsine (square root)-transformed prior to statistical analysis.We scored a total of t-tests. Our major question was whether focal males would alter their individual choice decisions between the two parts of a trial, i.e., before and after presentation of an audience. We therefore compared the relative time males spent near the preferred female during the 1st part of a trial . The effect during treatment (4) was intermediate to treatments (1) and (2) . This effect could be due, primarily, to two reasons: neighboring males are likely to share intrinsic preferences for certain female phenotypes, such as large body size [Sperm competition in natural poeciliid populations can be intense ,58. Maleody size ,62. MatiP. mexicana males may even attempt to deceive rivals about their mate choice [So, why was the strongest effect seen in treatment (2)? We propose that males cease expressing mating preferences in the visual presence of a competitor to avoid being copied ,34. There choice . Using ae choice . This wae choice ,42. Againd testing period in treatment (2), where the strongest effect was seen. It needs to be stressed though that the audience male was presented during both 5-minute acclimation phases of the 2nd part in treatment (3)–still the effect was  weaker than in treatment (4), where the rival male was presented only during one (the first) 5-minute acclimation phase.One might argue that any difference among treatments in the decrease of male preferences was driven solely by different times of presentation of the audience/rival males. Indeed, the audience male was presented throughout the entire 2Non-specific sperm competition risk appears to play a vital role when the audience male can actually eavesdrop on the focal male's mate choice. Hence, audience effects, as reported in previous studies ,11 are bThe authors declare that they have no competing interests.MZ, KM, CHS and CK carried out the behavioral tests. MP and MZ conducted the statistical analyses and wrote the first draft of the manuscript. RT, BS, MZ and MP were equally involved in implementing the project, and developing later drafts of the manuscript. All authors read and approved the final manuscript.


Chronic cocaine use is associated with disrupted dopaminergic neurotransmission but how this disruption affects overall brain function (other than reward/motivation) is yet to be fully investigated. Here we test the hypothesis that cocaine addicted subjects will have disrupted functional connectivity between the midbrain (where dopamine neurons are located) and cortical and subcortical brain regions during the performance of a sustained attention task.We measured brain activation and functional connectivity with fMRI in 20 cocaine abusers and 20 matched controls. When compared to controls, cocaine abusers had lower positive functional connectivity of midbrain with thalamus, cerebellum, and rostral cingulate, and this was associated with decreased activation in thalamus and cerebellum and enhanced deactivation in rostral cingulate.These findings suggest that decreased functional connectivity of the midbrain interferes with the activation and deactivation signals associated with sustained attention in cocaine addicts. With repeated use, cocaine leads to neuroadaptations in dopaminergic function (as well as neuroadaptations in other catecholamines and glutamatergic and gabaergic systems) The fluctuations of neural activity that mediate neuroadaptations Twenty healthy chronic cocaine abusers and 20 age-, gender-, and education-matched healthy control subjects participated in this study . ParticiSubjects were included in the study if they were (1) able to understand and give informed consent; and (2) had at least 12 years of education  and verbal intelligence ≥85 (estimated with the Wide Range Achievement Test reading subtest standard score). Subjects were excluded if they had: (3) history of head trauma or loss of consciousness (greater than 30 minutes) or other neurological disease of central origin (including seizures); (4) abnormal vital signs at time of screening or history of major medical conditions that may alter cerebral function, such as cardiovascular (including high blood pressure), endocrinological (including metabolic), oncological or autoimmune diseases for which the participant is required to take medications; (5) present or past history of psychiatric disorders other than substance abuse in cocaine abusers and nicotine dependence in all subjects; (6) positive urine screens for psychoactive drugs (or metabolites) other than cocaine ; (7) pregnancy, as confirmed with a urine test in all female subjects of childbearing age on study day; and (8) metal implants or other contraindications for MRI.p = 0.27; Fisher's exact test). There were also no significant differences in symptoms of depression, as assessed with the Beck Depression Inventory II edition p = 0.004; Fisher's exact test). Subjects were allowed to smoke regularly to minimize withdrawal symptoms. Specifically, seven cocaine subjects smoked within 4 hours prior the study and 4 additional cocaine subjects smoked within 6 hours prior the study. There was no difference in time since last cigarette  or in smoking frequency between the groups (P>0.54 t-test). Nineteen cocaine subjects met DSM-IV diagnosis for cocaine dependence or abuse, and had at least a 12-month  history of cocaine use (predominantly by the smoked route). Two cocaine dependent subjects also met diagnosis for cannabis or alcohol abuse. Two subjects had histories of past alcohol dependence. Seventeen of the cocaine subjects had a positive urine toxicology screen for cocaine on the day of the study, indicating that they used cocaine during the prior 72 hours. The urine toxicology screen was negative for the remaining three cocaine subjects and for all control subjects.Differences in age, education, and verbal IQ between the groups were not statistically significant . Similarly, the gender difference between groups was not statistically significant (We used a drug-word (DW) paradigm that has been previously described A 4-Tesla whole-body Varian/Siemens MRI scanner and a T2*-weighted single-shot gradient-echo planar imaging (EPI) pulse sequence  were used to map the blood oxygenation level dependent (BOLD) fMRI responses in the whole brain. Padding was used to minimize motion. Subject motion was monitored immediately after each fMRI run using a k-space motion detection algorithm 3 spatial resolution, axial orientation, 256 readout and 192×96 phase-encoding steps, 16 minutes scan time) and a modified T2-weigthed Hyperecho sequence 2 in-plane resolution, 5-mm thickness, no gap, 2 min scan time), and reviewed to rule out gross brain morphological abnormalities.Anatomical images were collected using a T1-weighted 3D-MDEFT sequence 3), and smoothed . Note that spatial normalization to the stereotactic space was carried out using the standard SPM2 EPI template.Image reconstruction was performed using an iterative phase correction method in IDL that minimizes signal-loss artifacts in EPI 3) and corrp <0.05, corrected for multiple comparisons at the cluster level, were considered significant in group analyses of brain activation.The general linear model used to calculate the individual BOLD contrast maps  for each fMRI time series consisted of a box-car design convolved with the canonical hemodynamic response function (HRF) and a high-pass filter (cut-off frequency: 1/520 Hz). The BOLD signal strength was estimated without the removal of global effects  to minimize false deactivation signals xyz =  mm; corrp <0.05 (corrected for multiple comparisons) were considered significant in group analysis of CM signals.A method recently proposed for studies based on blocked fMRI datasets 3 voxels fit into ROI that are symmetric around a peak voxel and that 3-mm isotropic voxels were used. We did not use n  = 0 ROIs because do not take full advantage of the 13-mm isotropic resels (include only one voxel). Similarly, n = 2 ROIs were not used because they include voxels that do not belong to the average functional cluster defined by the resels. Thus, n = 1 ROIs were used to maximize the number of voxels within the smoothness of the data. Thus, the average BOLD and CM signals in the left and right medial dorsal nuclei of the thalamus , cerebellum , and the rostral anterior cingulate cortex  were extracted from the individual BOLD and CM maps in 9×9×9 mm3 ROI volumes containing 27 voxels to using IDL. We selected these ROIs based on our previous fMRI studies http://www.talairach.org/) The relevant clusters were further evaluated with region-of-interest (ROI) analyses to identify potential outliers that might influence statistical analyses, and to report average values in a volume comparable to the image smoothness  during the DW task were not statistically significant, either between groups  or between words  , inferior (BA 40) and superior (BA 7) parietal cortices, thalamus, dorsal striatum, midbrain, and cerebellum and deactivated the parahippocampal (BA 30) and rostral anterior cingulate  gyri, precuneus (BA 31), amygdala, and insula (BA 13) , temporal (BA 21), and cingulate  cortex, ventral precuneus (BA 7), thalamus, parahippocampus, amygdala, putamen, caudate, cerebellum, and pons had positive CM .CM and BOLD signals in the thalamus were significantly correlated for control subjects , and rACC was significantly lower for cocaine subjects than for controls. In parallel and in absence of performance differences, cocaine subjects had lower activation in MDTHA and cerebellum and higher deactivation in rACC than controls, which is consistent with our previous findings in cocaine subjects tested with working memory xyz =  mm) with the rest of the brain using a different sample and a simplified version of the stimulation paradigm. Since the midbrain is relatively small and the imaging smoothing was larger than 12-mm is all directions, the functional responses in the selected seed region are representative of those in the entire midbrain. The midbrain  is the origin of the main dopamine (DA) projections to the forebrain, and cocaine addicts have lower dopaminergic function than controls Using the DW task we recently showed that drug words, but not neutral words, activated the midbrain in cocaine subjects but not in controls The MDTHA had lower CM and lower BOLD signals for cocaine abusers than for control subjects. These results are consistent with previous studies that documented reduced DA release This study also shows an association between hypo-activation and lower CM in the cerebellum for cocaine subjects compared to controls. The locus coeruleus, a homeostatic gray matter nucleus in the brainstem (lower midbrain/pons), is the main source of norepinephrinergic innervation to the thalamus and cerebellum The negative BOLD responses in the rACC were higher and the CM signals in rACC were lower for cocaine abusers than for control subjects. The rACC, which is innervated by DA, noradrenergic We did not exclude smokers as from our experience around 75% of cocaine subjects smoke cigarettes (vs. around 25% of controls). Fifteen cocaine subjects and five control subjects were smokers, and the group difference was statistically significant. Subjects were allowed to smoke regularly to minimize withdrawal symptoms. Previous studies have shown that nicotine can induce a dose-dependent increase in neuronal activity in a distributed system of brain regions, including the nucleus accumbens, amygdala, cingulate, and frontal lobes Summary: Using high-field (4 Tesla) fMRI, here we show that during processing of drug and matched neutral words cocaine abusers have similar accuracy and reaction times to matched controls, but lower BOLD and CM signals in the dorsal medial nucleus of the thalamus, cerebellum, and rostral anterior cingulate cortex. These findings suggest that lower recruitment of subcortical resources and impaired inhibition of cortical resources may be mediated by abnormal functional connectivity of catecholamine  pathways in cocaine abusers. The negative correlation of the thalamic CM  with years of cocaine use suggests that functional connectivity might be more sensitive than standard fMRI activation techniques for the detection of subtle functional neuroadaptations associated with drug addiction.


TP53 mutations have shown that loss of heterozygosity (LOH) occurs during passaging and is associated with genomic instability, such as chromosomal aberrations and aneuploidy. to investigate the genomic changes associated with LOH in Li-Fraumeni (LF) fibroblasts, we have analysed cell strains at increasing population doublings (PD) using Comparative Genomic Hybridization (CGH). We have looked at three groups of cell strains: LF mutation-carrying strains which showed LOH for TP53, LF mutation-carrying strains which did not show LOH, and strains from normal individuals. Using CGH, we have detected loss of distinct chromosomal regions associated with LOH in 4 out of 5 mutation-carrying strains. In particular we have found loss of chromosomal regions containing genes involved in cell cycle control or senescence, including loss of 9p and 17p in these strains. Other recurrent changes included loss of chromosomes 4q and 6q, regions shown to contain one or more tumour suppressor genes. No genomic alterations were detected at cumulative PD in the normal strains or in the LF mutation-carrying strains which did not show LOH for TP53. We have also analysed the three groups of strains for microsatellite instability and somatic TP53 mutations, and have found genetic alterations in only one strain. © 2000 Cancer Research CampaignStudies of Li-Fraumeni syndrome fibroblasts heterozygous for germline


Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated  In addition such approaches do not allow for simultaneous detection of key stromal cell genes or for genes that may be modulated as a consequence of crucial epithelial-stromal interactions.Prostatic growth, differentiation and adult function are dependent upon the presence of androgens. It is well established that androgens control growth and differentiation via mesenchymal-epithelial interactions. In the adult prostate, androgens are believed to act through the stromal androgen receptor (AR) to maintain a growth-quiescent gland IGF1, IGF2, TGFB3, BMP5, MMP2, COX2, and GSTM5. However, these studies used tissues enriched for epithelial cells. As a result, the analyses could have missed potentially crucial genes expressed predominantly in stromal cells. These studies did not address whether any of the patients had undergone prior androgen-ablation therapy. Approaches based on RNA extracted from tissues also do not allow for direct manipulation of hormonal stimulation, limiting the ability to more directly detect genes regulated by androgens, which might serve as predictors of response to therapies targeting androgenic stimulation or as novel targets for alternate drug therapy in this androgen driven disease process.More biologically relevant studies utilizing cDNA microarray analysis of BPH tissues have been reported. Luo, et al. analyzed the expression of 6,500 human genes in prostate cancer and BPH tissues from patients undergoing radical prostatectomy or transurethral resection of the prostate (TURP) in vitro, which would likely result in altered gene expression patterns from those which would be seen in vivo.In another relevant study, gene expression profiles in prostatic stromal cells from different prostate zones were analysed, comparing normal and diseased tissues in vivo and allows for direct manipulation of androgens, either by castration or hormonal supplementation of the host. The resulting data set was then analyzed to identify relatively highly expressed genes which were androgen regulated. In an effort to identify potential biomarkers amenable to future blood-based testing, emphasis was placed on genes whose products were known or predicted to be extracellular. Expression of these genes was then analyzed using qRT-PCR with cDNA pools derived from tissues from patients with minimal, mild, moderate and severe BPH pathology changes, or with CaP, to determine whether expression correlated with disease status.In the present study we used oligonucleotide microarrays to examine the expression of androgen regulated genes in benign human prostate tissue growing as xenografts in severe combined immunodeficient (SCID) mice De-identified human prostate tissue samples were obtained from the Vanderbilt Tissue Acquisition Core via the Department of Pathology in accordance with Vanderbilt IRB protocols. All patients signed informed consent approving the use of their tissues for unspecified research purposes. All experiments involving animals were conducted according to the Animal Welfare Act and approved by the Vanderbilt Institutional Animal Care and Use Committee. Animal care/welfare and veterinary oversight was provided by the Vanderbilt Divison of Animal Care.3, prostate wt <34 g, No BPH nodules; Mild BPH: TZ volume 4.5–8.99 cm3 or prostate wt 34–44.99 g or BPH nodules; Moderate BPH: TZ volume 9–16.99 cm3 or prostate wt 45–59.99 g and BPH nodules; Severe BPH: TZ volume ≥17 cm3 or prostate wt ≥60 g and BPH nodules. Prostate cancer tissues were classified as moderately differentiated (Gleason scores 5–6) or poorly differentiated (Gleason scores 8–9). Snap frozen fresh tissue cores procured as described below were processed for RNA and histology To determine the severity of BPH pathology changes for cases utilized for RNA extraction, TZ areas affected by glandular and stromal hyperplasia were outlined in whole mount sections from radical prostatectomy (RP) samples obtained via the Department of Pathology in accordance with Vanderbilt IRB protocols. TZ volumes were determined by planimetry with a digitized graphics tablet, in a manner identical to our routine determination of total tumor volume in RPs De-identified human prostate tissue samples were obtained from the Vanderbilt Tissue Acquisition Core via the Department of Pathology in accordance with Vanderbilt IRB protocols. 6 mm diameter cores obtained intraoperatively from fresh RP specimens were procured from the right and left TZ and PZ from mid to base and from mid to apex as described RNA extraction of snap-frozen TZ tissues and harvested xenografts was performed using a modification of previously described methods P-value cut-off of 0.05, indicating 284 expected false positive genes. A Welch t-test was then used to identify genes with significant differences  in expression between days 0 and 14 across all tissue samples. This list was then filtered by signal expression value, retaining the top 95% of signal dynamic range, yielding 784 genes/probes. This method of analysis was independently performed for both the castrate and testosterone supplemented data sets. All statistical analyses of microarray data were performed by the VMSR.Due to the considerable variation inherent in individual patients, the time 0 for each tissue sample was used as the control or reference sample rather than a standard reference sample across the entire experiment. Data were normalized by Lowess using GeneTraffic software  and imported into GeneSpring  for subsequent analysis. Initially, only day 0 and day 14 time points were considered, with genes that had at least a 1.5-fold upregulation in the day 14 sample vs. day 0 selected, which yielded 5,679 genes/probes. An ANOVA analysis was used to identify genes with significant differences in expression, with a P≤0.05 within our microarray data), 2) expressed at significantly higher levels in the prostate relative to other tissues (P≤0.05 by hypergeometric test), and 3) extracellular space or cell surface (Gene Ontogeny categories). These criteria yielded a set of genes that included previously validated biomarkers, including KLK3, ACPP, and MSMB. Several additional genes known or suspected to be involved in BPH based upon prior studies were “manually” included for further study: IGF1, IGF1R, TGFB1, TGFB3, TGFBR1, and TGFBR2. A total of 84 gene targets (and the 18S rRNA housekeeping control gene) were chosen for confirmation of expression in patient samples, several assessed with redundant probes.Potential candidate genes were systematically selected with overlapping bioinformatics criteria, employing WebGestalt: 1) androgen-regulated  was designed to assay candidate genes from the microarray analysis and control genes, for a total of 96 targets. 1 µg of total RNA was reverse transcribed into single-stranded cDNA using High-Capacity cDNA Archive kit (Applied Biosystems). Following cDNA synthesis, RNA was degraded by alkaline hydrolysis, adjusted to neutral pH, and cDNA purified by adsorption to silica gel  and eluted in 64 µl of 10 mmol/L Tris HCl (pH 8.5). cDNA quantities were measured spectrophotometrically . cDNA was diluted to 0.25 ng/µl in 1X TaqMan Universal PCR Master Mix (Applied Biosystems), loaded into the microfluidic card, sealed and centrifuged. Cards were then cycled on an ABI Prism 7900HT sequence detection system, and data analyzed with SDS 2.1 software (Applied Biosystems). After normalization to the endogenous control, 18S rRNA, levels were expressed relative to the control RNA pool calibrator (fold change). RNAs prepared from each category of BPH tissues were pooled from 5–10 patients. Control RNA was pooled from patients with no appreciable TZ expansion. The analysis included four replicates for the mild and severe BPH pools, as well as the moderately differentiated and poorly differentiated prostate cancer pools, and eight replicates for the control and moderate BPH pools.Human prostate tissue samples from the paraffin embedded whole mount blocks from the 43 patients originally characterized for BPH pathology severity and utilized for corresponding TZ derived RNA were obtained from the Vanderbilt Tissue Acquisition Core via the Department of Pathology and tissue microarrays were generated by one of the authors (MPR). The microarrays contained three 0.6 mm core samples, two from the TZ and one from PZ away from the cancer involved area. Staining of tissue sections was performed using a previously described protocol The immunostained slides were reviewed by a pathologist (MPR) blinded to the BPH pathology severity of the case and results expressed in a semi-quantitative manner. Percentage staining was scored on a scale of 0 to 4, where 0 = no staining, 1 = less than 25%, 2 = 25% to 50%, 3 = 50% to 75%, and 4 = 75% to 100%. The intensity of staining was scored on a scale of 1 to 3, where 1 = mild, 2 = moderate, and 3 = marked. For a sample yielding no staining, the intensity score was also 0. When both stromal and epithelial staining was present, scoring was done separately. To calculate a protein expression index, the percentage score was summed with the intensity score. The combined scores were plotted for each BPH pathology category , and Kruskal-Wallis tests were used to compare the combined scores across different disease severity status.KLK3 (PSA). In order to allow for this expected biologic variability between individual patient tissues, each patient's tissue derived RNA samples were re-centered to that patient's time 0 controls. For the primary analysis, two time points were considered, days 0 and 14, yielding 5,679 genes with a 1.5 fold or greater increase in expression. An ANOVA analysis was performed on these 5,679 genes, with a P-value cut-off of 0.05, which predicts 284 false positives. Genes that had at least a 1.5-fold increase or decrease in expression in the day 14 sample vs. day 0 were then filtered by signal expression value, retaining the top 95% of signal dynamic range, yielding 784 genes. The microarray data have been deposited in NCBI's Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE17862).We profiled gene expression patterns from human prostate TZ xenografts, following the addition or subtraction of testosterone. KLK3 (PSA), MSMB (beta-microseminoprotein), and TMEPAI (transmembrane prostate androgen-induced protein) were all previously well known to be regulated by androgens in human prostate TRPM8 , a putative prognostic marker and therapeutic target in prostate cancer ACPP (prostatic acid phosphatase) SCGB1A1 (uteroglobin) NDRG1 (N-myc downstream regulated gene 1) CREB3L4 (cAMP responsive element binding protein 3-like 4) RNASEL has been reported as a candidate hereditary prostate cancer gene PTGDS (prostaglandin D2 synthase), FGFBP1 (fibroblast growth factor binding protein 1), KLK11 , CXCL5 (chemokine (C-X-C motif) ligand 5), FKBP11 (FK506-binding protein 11) and TIMP1 .TMEPAI (transmembrane prostate androgen-induced protein) was the most highly expressed androgen-regulated gene identified. Additional highly expressed, androgen-regulated genes included KLK3 (PSA), MSMB (beta-microseminoprotein), ACPP (prostatic acid phosphatase) and SCGB1A1 (uteroglobin).GLI1 is up-regulated in mouse prostate following castration ANNAT1 (annexin 1), which has been reported to decrease in androgen stimulated prostate cancer compared with benign prostatic epithelium KLK3, ACPP, and MSMB, further validating our experimental approach. Other genes of interest added “manually” for RT-PCR analysis were IGF1, IGF1R, TGFB1, TGFB3, TGFBR1, and TGFBR2. The expression levels of these target genes were measured in RNA pools prepared from TZ tissues designated as having mild, moderate, and severe BPH changes, as well as moderately and poorly differentiated prostate cancer. RNA for each category of BPH or prostate cancer tissues was pooled from 5–10 patient samples. Control RNA was pooled from TZ samples from patients with no appreciable TZ expansion from glandular and stromal hyperplasia.To further investigate the expression levels of androgen regulated genes identified by microarray analysis, quantitative RT-PCR analysis was performed on selected targets using RNA pools derived from human prostate tissues. A major clinical need is to identify biomarkers that provide prognostic information regarding BPH progression or that may predict response to treatments, including 5 alpha reductase inhibitors. Secreted proteins that can be readily measured in blood are particularly attractive clinical targets. From the gene expression profiles in hormone manipulated TZ xenografts, potential biomarkers were systematically selected with overlapping bioinformatics criteria, employing WebGestalt. Gene targets selected were 1) androgen-regulated within our microarray data, 2) expressed at significantly higher levels in the prostate relative to other tissues, and 3) known or predicted to express secreted or cell surface proteins. These criteria yielded a set of candidate genes  that incThe results of the qRT-PCR analyses are shown in CDH13, CHRNB1, COL4A5, EFEMP2, EMP3, F10 (16-fold), FGF2, FGFBP1 (10-fold), IGF1, IGF2 (18-fold), LIPG, RARB, SGCA, SGCD, TGFB1, TGFB3 (14-fold), TGFBR2 and TIMP2 of the “high in BPH, moderate in prostate cancer” group; SMOC1 (26-fold) in the “high in BPH, low in prostate cancer” group; and SCGB3A1 (15-fold) and SCGB1A1 (14-fold) in the “moderate in BPH, low in prostate cancer” group.A number of genes had expression levels in the BPH pools that increased with severity of disease. Some of the genes had expression levels that were particularly elevated in BPH, but relatively lower in the prostate cancer pools. These genes would be expected to have the best potential as candidate BPH biomarkers, including specificity compared to prostate carcinoma. Those expressed extracellularly and with an average expression level at least three-fold greater in BPH RNA pools than in the prostate cancer RNA pools included: FGF2, SMOC1 and TIMP2 were chosen for IHC testing, as these genes had markedly increased RNA expression levels in BPH relative to controls, and antibodies were available that produced specific and satisfactory staining of human TZ tissues. Representative immunostaining for each antibody is shown in To determine if the differences in the RNA expression levels observed in BPH tissues were also present at the protein level and to further characterize the epithelial and/or stromal localization of potential increased expression, we performed immunohistochemistry (IHC) on a BPH tissue microarray (TMA). The array contained 129 tissue sections from 43 unique patients, containing BPH tissues with pathologies ranging from minimal to severe as defined in P-values ranging from 0.10 to 0.96.To more objectively analyze immunostaining results and allow for statistical analysis, immunoreactivity on TMA sections was scored semiquantitatively as described in the in vivo. The expression of a subset of these genes was correlated at the RNA level with disease status in BPH tissues.There is a need for BPH-specific markers that correlate with disease progression, which could be used for the early identification of patients likely to progress to more severely symptomatic disease. The timely use of existing or novel non-surgical therapies in these patients may reduce the significant cost and morbidity of surgical intervention. Hyperplastic growth of the prostate transition zone associated with clinical BPH may be the result of the abnormal expression of key androgen responsive genes, including those involved in prostatic development, and which lead to an imbalance between cell division and cell death. Although several candidate mediators have been suggested to play a role in BPH, the androgen regulated genes that are important for both normal and abnormal prostate growth remain to be completely defined. We have identified a panel of genes regulated by androgens in human transition zone prostate tissue A subset of these genes was investigated at the protein level by IHC. Expression in epithelium and/or stroma was confirmed in tissues of varying severity BPH pathology. However, no obvious correlation was seen between IHC intensity and BPH pathology severity. This panel of genes provides a valuable dataset for androgen regulated genes that could be etiologically involved in the abnormal epithelial and stromal proliferation characteristic of BPH. Future investigation will be necessary to establish the biologic significance of specific genes in the evolution of BPH and the possible suitability as a target for pharmacologic intervention. It is desirable to have biomarkers of clinical utility in BPH prior to acquisition of tissue as a consequence of therapeutic intervention . As such, tissue based quantitation of mRNA levels or IHC intensity is unlikely to be a clinically useful test. Our studies provide a candidate list for further investigation using more suitable test substrates, such as urine for mRNA or protein or serum for protein. The highly relevant model of androgen regulated expression in intact TZ tissues analogous to actual BPH tissues increases the likelihood that such candidate targets could have prognostic or therapeutic relevance.We used oligonucleotide microarrays to examine the expression of androgen regulated genes in normal human TZ prostate tissue growing as xenografts in male SCID mice. Among genes with androgen regulated expression that we identified, those with higher expression in prostate relative to other tissues and those of the cell surface or extracellular compartment have greatest potential to serve as useful clinical biomarkers. The most promising candidates were culled with WebGestalt bioinformatics tools. The expression of these genes was screened using qRT-PCR analysis of cDNA pools derived from BPH or prostate cancer tissues, to determine whether expression was correlated with disease status.When generating the androgen regulated gene lists, we used a 14 day time point in order to look for genes chronically up-regulated in response to androgens. Genes identified as up-regulated by androgens at this longest time point investigated were also generally found to gradually increase levels at earlier time points. Genes transiently up-regulated at earlier time points may reflect genes indirectly induced during growth and differentiation, and would likely be poor choices for markers of prostatic disease.in vivo. However, since the exact ratios of stroma to epithelia are variable, genes identified as altered at the RNA level in intact tissues could be related to changes in epithelium, stroma or both. A weakness of the method is that the tissue used for RNA extraction is destroyed, and even adjacent tissues from the same patient may have markedly differing stromal-epithelial ratios. Therefore, this approach requires subsequent validation by in situ hybridization or immunohistochemistry to determine cell type specificity.Tissue obtained as a surgical specimen from prostatectomy can be subjected to prolonged blood supply interruption during the surgical procedure, compromising RNA quality. The use of xenografts allows the tissue to recover from damage induced during surgery. Xenografts can be harvested rapidly with less severe ischemia, thus allowing for the isolation of high quality RNA. In addition to producing high quality tissue, this method vs use of cell lines also allows us to study tissues with intact stromal-epithelial interactions occurring ACPP, CXCL5, FGFBP1, FKBP11, KLK11, PTGDS, and TIMP1. CXCL5 was recently shown to be elevated in serum from patients with BPH and may potentially distinguish between BPH and prostate cancer among patients presenting with low serum PSA FKBP11 expression was up-regulated in mouse prostate by androgen Several genes identified in our microarray analysis were previously known to be regulated by androgens or involved in prostatic disease. These included the up-regulated genes GLI1, ANNAT1 (annexin 1), BCL2, and CLIC4. Compatible with our observations, expression of GLI1 was reported to increase in mouse prostate after the withdrawal of androgen BCL2 and CLIC4, have been reported to have anti-apoptotic, and proapoptotic activities, respectively. Cardillo et al. found elevated BCL2 expression in apoptosis resistant BPH following androgen deprivation CLIC4 is involved in p53 mediated apoptosis We also identified genes in our microarray analysis that were down-regulated by androgens. These included EMP3, FGF2, FGFBP1, IGF1, IGF2, TGFB3, TGFBR2, and TIMP2. FGF2 (fibroblast growth factor 2), IGF1 (insulin-like growth factor 1), and IGF2 (insulin-like growth factor 2), have altered expression levels in BPH and prostate cancer Serum PSA commonly used for prostate cancer screening is clearly related to prostate volume, which is increased as a consequence of TZ expansion due to glandular and stromal hyperplasia seen in BPH. Biomarkers of greater specificity for either CaP or BPH are greatly needed. Fourteen genes were identified as being highly elevated in BPH and had BPH/PCa expression ratios of at least 5-fold . Of thesF10 (upregulated 16-fold), LIPG, SGCA and SMOC1 (upregulated 26-fold). Coagulation Factor X (F10) is a serine protease that can be activated by cancer procoagulant (CP), a cysteine protease produced by malignant and embryonic tissues LIPG) is involved in lipoprotein metabolism and is elevated in inflammation SGCA  encodes a component of the dystrophin-glycoprotein complex (DGC) in striated muscle, which is critical to the stability of muscle fiber membranes and to the linking of the actin cytoskeleton to the extracellular matrix. SMOC1) belongs to the BM-40 family, which has been implicated in tissue remodeling, angiogenesis and bone mineralization Other genes identified from the microarray analysis as androgen regulated and that were also highly expressed in pathologic BPH tissues had not been previously implicated in BPH or prostate cancer. These genes included Analysis of gene expression differences in RNA samples extracted from intact tissues does not specifically allow for localization of the increased or decreased altered genes to the glandular and/or stromal compartments in TZ tissues, both of which likely contribute fundamentally to evolution of BPH, nor does it necessarily translate to altered levels of more biologically relevant proteins. We examined the expression of a subset of genes of interest in BPH tissues at the protein level by immunohistochemistry (IHC), using a tissue microarray containing sections with BPH pathologies ranging from minimal to severe. IHC staining patterns were assessed semi-quantitatively for FGF2, SMOC1, and TIMP2. The expression of FGF2 in BPH has been previously reported by several groups As all three of these genes have been found to have altered expression in prostate cancer tissues and in much larger volumes of BPH tissue (by qRT-PCR), they may very well play a role in BPH progression, but based on results of IHC, it does not appear to be simply based on uniform over-expression throughout the transition zone.The fact that expression arrays on intact human TZ tissues xenografted into hormonally manipulated host mice identified many genes well known to be androgen regulated validates our approach. However, this method may still miss genes with low expression levels that could be important in pathogenesis, although such genes would seem less likely to make good biomarkers. When we examined the expression of a subset of these highly expressed genes from the microarray data by RT-PCR in BPH tissues, we found several with elevated levels in BPH tissues relative to normal controls. Our study confirms the known or postulated role of many of these in prostate pathology, and identifies additional genes that appear worthy of further investigation. The detailed analysis of each of these genes will require substantial further investigation, but may provide further insight into the molecular etiology of BPH pathogenesis.In summary, our results demonstrate the feasibility of using a mouse xenograft model to characterize the gene expression profiles of intact human prostate tissues in response to androgens. The model has the unique advantage of maintaining intact epithelial-stromal interactions during hormone manipulation, cellular interactions which are likely crucial in the progressive evolution of this androgen dependent pathologic condition. A number of genes were identified that were regulated by androgens, some that were previously well characterized, and many more whose possible roles in prostate development and pathology remain largely unknown. Additionally, a subset of these androgen regulated genes were found to be over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease severity. Such knowledge may aid the discovery of new diagnostic and therapeutic targets for the early detection and treatment of BPH and prostate cancer.


The challenge in acute stroke is still to reperfuse as early as possible the ischemic territory. Since fibrinolytic therapies have a limited window with potential risk of bleeding, having a nonpharmacologic mean to recruit vessels in area surrounding necrosis might be useful. We propose here to use antigravity suit inflated at “venous” pressure levels to shift blood towards thoracic and brain territories. We report two cases of spectacular clinical recovery after acute carotid occlusion. We report two cases where an anti-gravity suit  was A 28-year-old woman was seen 2 hours after the sudden onset of total right brachial monoplegia, right facial palsy, and mutism  due to a left middle cerebral artery (MCA) infarct. She received intravenous recombinant tissue plasminogen activator (rt-PA) 2.5 hoursafter stroke onset, without subsequent improvement. Cerebral MRI diffusion-weighted images and T2 flair-weighted images performed 24 hours later showed increased signal in the whole superficial left MCA territory Figures . The MR Two days after stroke onset, there was no clinical improvement and the patient developed bradycardia (40 pulses/min) and hypotension (100/70 mmHg). Lower body positive pressure (LBPP) was applied using an anti-gravity suit  for 150 minutes. The anti-gravity suit included three independent bladders connected to three pneumatic extremities with manometers providing instantaneous pressure values. The garment was applied from ankle to costal margin and the bladder pressures were set at 20 mmHg on the lower limbs and 10 mmHg on the abdominal area, to keep a positive pressure gradient , 4. DuriDuring LBPP application and up to 48 hours later, there was a 50% increase in systolic and diastolic MCA velocities . The RI Three days after LBPP application the patient started to recover her right hand mobility and her facial palsy started to improve. One week after LBPP application she had completely recovered her right arm and facial mobility. She finally went back home 3 weeks after hospitalisation.On MRI performed 5 days after stroke onset, diffusion-weighted images showed reduced diffusion abnormalities and reduced infarct size on T2 flair-weighted images Figures . The MRAA 61-year-old. man was admitted in our stroke unit after 2 reversible episodes of left monocular blindness associated with a total right distal upper limb paralysis. His medical past history was hypertension, hypercholesterolemia, and tobacco use. At the admission the clinical examination showed a total right wrist extension paresis, the so-called “pseudoradial paresis” related to a small left frontal infarction visible on MRI (DWI). The MR angiography (MRA) and the duplex scanning showed an occlusion of the left internal carotid artery due to atherosclerosis and a negative flow in the ophthalmic artery downstream. The intracranial MRA showed poor collateral supply by anterior communicating artery and an apparent nonfunctional posterior communicating artery. We decided to apply LBPP process for 2 reasons: (1) the worsening of the right hand deficit (see movie), (2) this worsening was not related to a fall in systemic blood pressure (167/84 mmHg). The LBPP was applied once during 90 minutes following the same procedure than in case one. The repeated measurements of blood pressure and heart rate did not show any significant changes along the procedure. The transcranial Doppler showed a decreased of the left MCA resistance to flow index  while there was slight changes on the MCA velocities (64/32 to 75/45 cm/sec). The left ophthalmic artery flow remained negative during and after LBPP application. Heart rate and blood pressure remained stable during LBPP application. The clinical improvement was sustained after anti-G suit deflation (as presented on the movie) consisting in the recovery of the pseudoradial palsy with persisting ataxia lasting few days. The total recovery was obtained before discharge.We report two patients with symptomatic carotid artery occlusion in whom LBPP was applied with an anti-gravity suit during the acute phase of stroke. The patients made a remarkable recovery in parallel with significant cerebral hemodynamic improvement not related to global systemic hemodynamic changes.LBPP is known to shift blood from the lower body compartment towards the upper part of the body, which improves cardiac preload and output , 6, 7 an Several mechanisms might be discussed for neurological improvement: (1) recanalization of carotid occlusion, (2) development of collaterals, and (3) recruitment of smaller vessels in watershed fields. For the first patient, it is unlikely that rt-PA administration played a major role in this dramatic improvement since there was no improvement 24 hours after treatment, which decreases the likelihood of a favourable outcome to 20% , 10, andThe reopening of the carotid artery, which was unusually rapid for the spontaneous evolution of a dissection , may havAnother possible beneficial effect could be the recruitment of smaller vessels in watershed fields especially in case 2. The clinical improvement during the LBPP application could be related to the new partition in the frontal region of blood which could play a role in the recruitment of small vessels by an efficient loading or even a reopening. Two recent reports showed the impact of a mechanical external counter-pulsation on cerebral velocities in healthy volunteers  and on fEven if this paper is limited to two patients, without control group, the remarkable recovery observed in these two patients together with the improvement in hemodynamic parameters on TCD might be related to LBPP application through improvement of cerebral perfusion. We need definitively, further investigation during the acute phase of ischemic stroke to conclude on the beneficial effect of this noninvasive tool.


ARNT) and cathepsin K (CTSK) genes lie in a tandem head-to-tail arrangement on human chromosome 1. The twogenes are in extremely close proximity; the usual CTSK transcription start site isless than 1.4 kb downstream of the end of the longest reported ARNT transcript.By generating an RT-PCR product that overlaps both the 3′ end of ARNT andthe 5′ end of CTSK, we show that ARNT transcripts may extend through theARNT–CTSK intergenic region and progress into the CTSK gene. Furthermore, by using quantitative RT-PCR from several tissues to detect the ARNT expressionsignature in CTSK introns, we show that ARNT transcripts can read through intoCTSK as far as CTSK intron 3, extending approximately 3.7 kb downstream of theend of the longest previously described ARNT mRNA. Given that ARNT and CTSK are expressed in an overlapping range of tissues, ARNT read-through may have anegative impact on CTSK transcript levels by interfering with CTSK expression.We also present evidence for novel CTSK transcripts following sequence analysisof CTSK-derived ESTs and RT-PCR products. These transcripts show alternate 5′splicing and or 5′ extension and are sometimes initiated from a cryptic alternativepromoter which is upstream of the known CTSK promoter and possibly in the 3′UTR of ARNT.The aryl hydrocarbon receptor nuclear translocator (