pRb is an obesity suppressor in hypothalamus and high-fat diet inhibits pRb in this location.

pRb is frequently inactivated in tumours by mutations or phosphorylation. Here, we investigated whether pRb plays a role in obesity. The Arcuate nucleus (ARC) in hypothalamus contains antagonizing POMC and AGRP/NPY neurons for negative and positive energy balance, respectively. Various aspects of ARC neurons are affected in high-fat diet (HFD)-induced obesity mouse model. Using this model, we show that HFD, as well as pharmacological activation of AMPK, induces pRb phosphorylation and E2F target gene de-repression in ARC neurons. Some affected neurons express POMC; and deleting Rb1 in POMC neurons induces E2F target gene de-repression, cell-cycle re-entry, apoptosis, and a hyperphagia-obesity-diabetes syndrome. These defects can be corrected by combined deletion of E2f1. In contrast, deleting Rb1 in the antagonizing AGRP/NPY neurons shows no effects. Thus, pRb-E2F1 is an obesity suppression mechanism in ARC POMC neurons and HFD-AMPK inhibits this mechanism by phosphorylating pRb in this location.

Ethanol induces apoptotic death of beta-endorphin neurons in the rat hypothalamus by a TGF-beta 1-dependent mechanism.

BACKGROUND: We have previously shown that developing beta-endorphin neurons, in the arcuate nucleus of the hypothalamus become increasingly apoptotic when exposed to ethanol. As in the previous study we have observed an involvement in transforming growth factor beta 1 (TGF-beta1) in mediation of the apoptotic process, the present study was conducted to determine the ethanol-induced changes in this apoptotic regulatory peptide signaling in the arcuate nucleus of the hypothalamus of neonatal rats. METHODS: Pups were exposed to 11.34% ethanol in a milk-based diet or control diet on postnatal day (PND) 3 to PND7. Two hours after the last daily feeding, brains were collected and frozen in liquid nitrogen for analysis of various apoptosis regulatory proteins in the arcuate tissue by Western blots. Some animals were fixed in 4% paraformaldehyde and analyzed immunohistochemically. RESULTS: Ethanol exposure increased apoptotic death of beta-endorphin neurons in the arcuate nucleus of the hypothalamus. The cell death was associated with an increase in the tissue levels of TGF-beta1 in the mediobasal hypothalamus. This was correlated with a reduction in the arcuate level of retinoblastoma protein (Rb) phosphorylation. The reduced level of Rb phosphorylation was associated with an increased protein level of the cyclin dependent kinase inhibitor p27/kip but with a decreased protein level of cyclin dependent kinase 4 and cyclin D3. In addition, the apoptotic cell death was positively correlated with the level of Bclxs but negatively correlated with the level of the Bcl2. CONCLUSIONS: These results suggest that ethanol exposure increases TGF-beta1 signaling involving Bcl2 and Rb repression that may lead to apoptotic death of cells including beta-endorphin neurons in the arcuate nucleus of the hypothalamus.

Sesamin, a lignan of sesame, down-regulates cyclin D1 protein expression in human tumor cells.

Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol-lowering, lipid-lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF-7. Furthermore, sesamin dephosphorylates tumor-suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF-7 cell proliferation by sesamin is correlated with down-regulated cyclin D1 protein expression, a proto-oncogene that is overexpressed in many human cancer cells. It was found that sesamin-induced down-regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down-regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF-7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down-regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent.

High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells.

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

Brain-derived gangliosides regulate the cytokine production and proliferation of activated T cells.

Gangliosides may regulate the activity of the immune system in vivo, particularly within tissues such as neoplasms or the central nervous system, where they are most abundant. However, the specific mechanisms by which gangliosides modulate immune function remain incompletely understood. We have characterized the effects that brain-derived gangliosides have on specific steps of the T cell activation process in vitro. Gangliosides inhibit T cell proliferation downstream from the early activation events that are bypassed pharmacologically using the combination of a phorbol ester plus a calcium ionophore. These lipids block IL-2 and IFN-gamma gene transcription without inhibiting the production of IL-4 and IL-10 mRNA. This may be accounted for by the ability of gangliosides to prevent the activation of NF-kappaB in mitogen-stimulated T cells. Despite inhibiting IL-2 production, the antiproliferative effects of gangliosides are not reversed by adding supplemental IL-2 to the culture media. This defect persists because gangliosides also block the entry of activated T cells into the cell cycle. In this setting, phosphorylation of the retinoblastoma gene product, a protein whose phosphorylation state is an important regulator of normal cell cycle progression, is prevented. These studies help to define how gangliosides modulate T cell effector function in vitro. They also highlight the fact that certain T cell responses, namely the production of Th2-associated cytokines, are not inhibited by their actions.

Suppression of tumorigenesis by transcription units expressing the antisense E6 and E7 messenger RNA (mRNA) for the transforming proteins of the human papilloma virus and the sense mRNA for the retinoblastoma gene in cervical carcinoma cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.