Maternal perinatal calorie restriction temporally regulates the hepatic autophagy and redox status in male rat.

Intrauterine growth restriction (IUGR) leads to adult obesity, cardiovascular disease, and non-alcoholic fatty liver disease/steatohepatitis. Animal models have shown that combined intrauterine and early postnatal calorie restriction (IPCR) ameliorates these sequelae in adult life. The mechanism by which IPCR protects against adult onset disease is not understood. Autophagy, a lysosomal degradative process, recycles cellular constituents and eliminates damaged organelles, proteins, and oxidants. In this study, we hypothesized that IPCR could regulate autophagy in the liver of male rat offspring. At birth (d1) of male IUGR rat offspring and on day 21 (p21) of life, IPCR male rat offspring had a profound decrease in hepatic autophagy in all three stages of development: initiation, elongation, and maturation. However, upon receiving a normal diet ad-lib throughout adulthood, aged IPCR rats (day 450 of life (p450)), had increased hepatic autophagy, in direct contrast to what was seen in early life. The decreased autophagy at d21 led to the accumulation of ubiquitinated proteins and lipid oxidative products, whereas the increased autophagy in late life had the opposite effect. Oxidized lipids were unchanged at d1 by IUGR treatment indicating that decreased autophagy precedes oxidative stress in early life. When cellular signaling pathways regulating autophagy were examined, the 5' adenosine monophosphate-activated protein kinase pathway (AMPK), and not endoplasmic stress pathways, was found to be altered, suggesting that autophagy is regulated through AMPK signaling pathway in IPCR rats. Taken together, this study reveals that the perinatal nutritional status establishes a nutritionally sensitive memory that enhances hepatic autophagy in late life, a process that perhaps acts as a protective mechanism to limited nutrition.

The hepatic transcriptome of young suckling and aging intrauterine growth restricted male rats.

Intrauterine growth restriction leads to the development of adult onset obesity/metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, stroke, dyslipidemia, and non-alcoholic fatty liver disease/steatohepatitis. Continued postnatal growth restriction has been shown to ameliorate many of these sequelae. To further our understanding of the mechanism of how intrauterine and early postnatal growth affects adult health we have employed Affymetrix microarray-based expression profiling to characterize hepatic gene expression of male offspring in a rat model of maternal nutrient restriction in early and late life. At day 21 of life (p21) combined intrauterine and postnatal calorie restriction treatment led to expression changes in circadian, metabolic, and insulin-like growth factor genes as part of a larger transcriptional response that encompasses 144 genes. Independent and controlled experiments at p21 confirm the early life circadian, metabolic, and growth factor perturbations. In contrast to the p21 transcriptional response, at day 450 of life (d450) only seven genes, largely uncharacterized, were differentially expressed. This lack of a transcriptional response identifies non-transcriptional mechanisms mediating the adult sequelae of intrauterine growth restriction. Independent experiments at d450 identify a circadian defect as well as validate expression changes to four of the genes identified by the microarray screen which have a novel association with growth restriction. Emerging from this rich dataset is a portrait of how the liver responds to growth restriction through circadian dysregulation, energy/substrate management, and growth factor modulation.

Expression profiling of attenuated mitochondrial function identifies retrograde signals in Drosophila.

Mitochondria are able to modulate cell state and fate during normal and pathophysiologic conditions through a nuclear-mediated mechanism collectively termed as a retrograde response. Our previous studies in Drosophila melanogaster have clearly established that progress through the cell cycle is precisely regulated by the intrinsic activity of the mitochondrion by specific signaling cascades mounted by the cell. As a means to further our understanding of how mitochondrial energy status affects nuclear control of basic cell decisions, we have employed Affymetrix microarray-based transcriptional profiling of Drosophila S2 cells knocked down for the gene encoding subunit Va of the complex IV of the mitochondrial electron transport chain. The profiling data identify transcriptional upregulation of glycolytic genes, and metabolic studies confirm this increase in glycolysis. The data provide a model of the shift of metabolism from a predominately oxidative state toward a predominately aerobic glycolytic state mediated through transcriptional control. The transcriptional changes alter many signaling systems, including p53, insulin, hypoxia-induced factor α, and conserved mitochondrial retrograde responses. This rich dataset provides many novel targets for further understanding the mechanism whereby the mitochondrion manages energy substrate disposition and directs cellular fate decisions.

Metabolic control of G1-S transition: cyclin E degradation by p53-induced activation of the ubiquitin-proteasome system.

Cell cycle progression is precisely regulated by diverse extrinsic and intrinsic cellular factors. Previous genetic analysis in Drosophila melanogaster has shown that disruption of the mitochondrial electron transport chain activates a G1-S checkpoint as a result of a control of cyclin E by p53. This regulation does not involve activation of the p27 homologue dacapo in flies. We demonstrate that regulation of cyclin E is not at the level of transcription or translation. Rather, attenuated mitochondrial activity leads to transcriptional upregulation of the F-box protein archipelago, the Fbxw7 homologue in flies. We establish that archipelago and the proteasomal machinery contribute to degradation of cyclin E in response to mitochondrial dysfunction. Our work provides in vivo genetic evidence for p53-mediated integration of metabolic stress signals, which modulate the activity of the ubiquitin-proteasome system to degrade cyclin E protein and thereby impose cell cycle arrest.

Maternal embryonic leucine zipper kinase is a key regulator of the proliferation of malignant brain tumors, including brain tumor stem cells.

Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.

Distinct transcription profiles of primary and secondary glioblastoma subgroups.

Glioblastomas are invasive and aggressive tumors of the brain, generally considered to arise from glial cells. A subset of these cancers develops from lower-grade gliomas and can thus be clinically classified as "secondary," whereas some glioblastomas occur with no prior evidence of a lower-grade tumor and can be clinically classified as "primary." Substantial genetic differences between these groups of glioblastomas have been identified previously. We used large-scale expression analyses to identify glioblastoma-associated genes (GAG) that are associated with a more malignant phenotype via comparison with lower-grade astrocytomas. We have further defined gene expression differences that distinguish primary and secondary glioblastomas. GAGs distinct to primary or secondary tumors provided information on the heterogeneous properties and apparently distinct oncogenic mechanisms of these tumors. Secondary GAGs primarily include mitotic cell cycle components, suggesting the loss of function in prominent cell cycle regulators, whereas primary GAGs highlight genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences. These data highlight that the development of gene pathway-targeted therapies may need to be specifically tailored to each subtype of glioblastoma.

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation.

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.

Gene expression profiling of gliomas strongly predicts survival.

In current clinical practice, histology-based grading of diffuse infiltrative gliomas is the best predictor of patient survival time. Yet histology provides little insight into the underlying biology of gliomas and is limited in its ability to identify and guide new molecularly targeted therapies. We have performed large-scale gene expression analysis using the Affymetrix HG U133 oligonucleotide arrays on 85 diffuse infiltrating gliomas of all histologic types to assess whether a gene expression-based, histology-independent classifier is predictive of survival and to determine whether gene expression signatures provide insight into the biology of gliomas. We found that gene expression-based grouping of tumors is a more powerful survival predictor than histologic grade or age. The poor prognosis samples could be grouped into three different poor prognosis groups, each with distinct molecular signatures. We further describe a list of 44 genes whose expression patterns reliably classify gliomas into previously unrecognized biological and prognostic groups: these genes are outstanding candidates for use in histology-independent classification of high-grade gliomas. The ability of the large scale and 44 gene set expression signatures to group tumors into strong survival groups was validated with an additional external and independent data set from another institution composed of 50 additional gliomas. This demonstrates that large-scale gene expression analysis and subset analysis of gliomas reveals unrecognized heterogeneity of tumors and is efficient at selecting prognosis-related gene expression differences which are able to be applied across institutions.

MKP-1-induced dephosphorylation of extracellular signal-regulated kinase is essential for triggering nitric oxide-induced apoptosis in human breast cancer cell lines: implications in breast cancer.

Apoptosis is regulated by a series of biochemical events that commits a cell to death. We are interested in understanding and have been investigating the mechanisms by which nitric oxide (NO) induces apoptosis in human breast cancer cell lines. In this study, we investigated the possible interplay of extracellular signal-regulated kinase (ERK) and Akt pathways in NO-induced apoptosis. MKP-1 transcripts were induced in these cells as early as 4 h, peaking at 8 h leading to inactivation of ERK1/2 at 16-24 h after exposure to NO. We also found 50% decrease in the levels pAkt at 24 h of DETA-NONOate treatment. The inactivation of ERK1/2 preceded the dephosphorylation of Akt and apoptosis. NO was not able to inactivate ERK1/2 or Akt or to induce apoptosis in the presence of a phosphatase inhibitor, sodium orthovanadate, or antisense oligonucleotides, suggesting a cross-talk between the two pathways. NO also up-regulated MKP-1 in another breast cancer cell line, ZR 75-30, which led to inactivation of ERK1/2 and induced apoptosis. In MDA-MB-231, NO did not induce MKP-1, and there was no ERK inactivation or apoptosis. Our results indicate that expression of MKP-1 by NO leading to dephosphorylation of ERK1/2 is the initial essential event that commits the cells to the apoptotic pathway in breast cancer cells.