Iris Shahar

Neuroprotection by NGF in the PC12 In Vitro OGD Model

Abstract: Neurodegenerative disorders and chronic disability due to stroke in the brain or spinal cord afflict a large sector of the population. To investigate the mechanism involved in ischemic stroke and to develop neuroprotective drugs/therapies, in vivo and in vitro, pharmacological models are needed. To investigate the cellular and molecular neuroprotective mechanisms of nerve growth factor (NGF), a member of the nervous system neurotrophin family of growth factors, under ischemia, we used an oxygen‐glucose‐deprivation (OGD) device and pheochromocytoma PC12 cells exposed to a paradigm of ischemic insult. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, conferred 30% of neuroprotection. Time‐course experiments showed marked activation of the ERK, JNK, and p‐38 MAPK isoforms during the OGD phase, but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD‐induced activation of JNK 1, and 20% and 50% attenuation of OGD‐induced activation of p‐38 α and β, respectively. The effect of NGF on gene expression in the PC12 ischemic model using Affymatrix Rat DNA‐Microarray technology indicates that only 6% of the genes are differentially regulated (induced/suppressed) by OGD insult and/or NGF. These findings support the notion that pretreatment with NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms and differential gene expression. This ischemic model may be useful to investigate molecular mechanisms of OGD‐induced neurotoxicity and NGF‐induced neuroprotection, and to generate novel therapeutic concepts for stroke treatment.

Sil overexpression in lung cancer characterizes tumors with increased mitotic activity

Sil (SCL interrupting locus) was cloned from the most common chromosomal rearrangement in T-cell acute lymphoblastic leukemia. It is an immediate early gene whose expression is associated with cell proliferation. Sil protein levels are tightly regulated during the cell cycle, reaching peak levels in mitosis and disappearing on transition to G1. A recent study found Sil to be one of 17 genes whose overexpression in primary adenocarcinomas predicts metastatic spread. We hypothesized that Sil might have a role in carcinogenesis. To address this question, we utilized several approaches. Using a multitumor tissue array, we found that Sil protein expression was increased mostly in lung cancer, but also at lower levels, in a subset of other tumors. Microarray gene expression analysis and immunohistochemistry of lung cancer samples verified these observations. Sil gene expression in lung cancer correlated with the expression of several kinetochore check-point genes and with the histopathologic mitotic index. These observations suggest that overexpression of the Sil gene characterizes tumors with increased mitotic activity.

Gene expression profiling of in vivo UVB-irradiated human epidermis.

Background: Several recent studies have employed microarray profiling to study UVB‐regulated gene expression in human skin. These studies are all based on UV‐irradiated cultured cells that differ substantially from the intact tissues they are supposed to imitate. The purpose of the present study was to analyze the differential expression of UVB‐regulated genes in intact human epidermis following in vivo UV irradiation.

Expression and chromosomal organization of mouse meiotic genes

Microarray technology which enables large scale analysis of gene expression and thus comparison between transcriptomes of different cell types, cells undergoing different treatments or cells at different developmental stages has also been used to study the transcriptome involved with spermatogenesis. Many new germ cell‐specific genes were determined, and the resulting genes were classified according to different criteria. However, the biological significance of these classifications and their clustering according to developmental transcriptional patterns during spermatogenesis have not yet been addressed. In this study we utilized mouse testicular transcriptome analysis at five distinct post‐natal ages (Days 7, 10, 12, 14, and 17), representing distinct meiotic stages, in an attempt to better understand the biological significance of genes clustered into similar expression patterns during this process. Among 790 sequences that showed an expression level change of twofold or more in any of the five key stages that were monitored, relative to the geometric average of all stages, about 40% peaked and about 30% were specifically suppressed at post‐natal day 14 (representing the early pachytene stage of spermatocytes), reflecting tight transcriptional regulation at this stage. We also found that each of the six main transcription clusters that were determined was characterized by statistically significant representation of genes related to specific biological processes. Finally, our results indicated that genes important for meiosis are not randomly distributed along the mouse genome but rather preferentially located on specific chromosomes, suggesting for the first time that chromosomal location might be a regulating factor of meiotic gene expression. Mol. Reprod. Dev. 77: 241–248, 2010.

Exercise training alters the molecular response to myocardial infarction.

PURPOSE We and others have shown that swimming exercise training performed before irreversible coronary occlusion improves the outcome of the heart injury and alters gene expression at the remodeling phase. The purpose of the current study was to identify temporal changes in the molecular response to myocardial infraction of prior exercise trained rats during the acute, the subacute, and the chronic phases postinfarction. METHODS Rats underwent a 7-wk swimming or sedentary protocol and were subjected to surgical induction of acute myocardial infarction (MI). Hearts were removed before and at 4 h, 2 d, and 4 wk after surgery. RNA extracted from the surviving myocardium of the MI hearts or from corresponding tissues in the non-MI hearts was subjected to multitranscript profiling. Results for representative transcripts were validated by reverse transcription and quantitative polymerase chain reaction amplification. RESULTS Global analysis of the 3686 detected transcripts generated a two-branch dendrogram that distinguished the pre-MI and the 4-h groups from the 2-d and the 4-wk groups and indicated that early after MI, the impact of infarction on the genes expressed overrides the training effect, whereas at 4 wk, the exercised hearts differ markedly from the nonexercised. Clustering the 1500 genes that showed the highest variance over time indicated differential expression of transcription regulators and proapoptotic genes 4 h and 2 d after MI and of stress-related and profibrotic genes 4 wk later in the exercised compared with sedentary hearts. CONCLUSION Swimming exercise training conducted before acute MI reprograms the surviving myocardium for altered molecular response to MI that explains, in part, the protected cardiac phenotype of the exercised animals.

Malignant phenotype correlating with drug resistance in two human neuroblastoma cell lines

SummaryThe main cause for the failure of chemotherapeutic treatment of advanced cancer probably lies in the emergence of drug resistant clones. In the present study we compared the sensitivity to adriamycin (ADR) and the capacity of ADR uptake in two human neuroblastoma cell lines differing in properties relevant to metastatic potential, the GP2 and MB, of low- and high-malignancy phenotype, respectively.Examination of the ADR effect onin vitro proliferative capacity of the two cell lines revealed a higher sensitivity of GP2 as compared to the MB variant.Intracellular ADR accumulation was determined by fluorocytometry, spectrofluorometry and fluorescence microscopy. According to the three methods, the GP2 line cells, representing a low-malignancy phenotype, had a higher uptake ability than the MB cells, possessing a phenotype of higher aggressiveness.The quantitative determination revealed that over a broad range of ADR concentrations, the GP2 cells accumulated 2–3.5 folds the amount of cytotoxic agent penetrating the MB cells. The FACS analysis showed that the cell population of each of the variants consisted of two subpopulations varying in their ability to accumulate ADR. In the GP2 line the high permeability subpopulation represented nearly half of the total cell population, whereas in the MB line this subpopulation represented a minority.The correlation observed between ADR uptake capacity and sensitivity to the cytotoxic agent, as evidenced by its effect on proliferative capacity, suggests that the resistance of the MB cells is due to a P-G-P modification-related mechanism.