Most organisms coordinate their activities with the day-night cycle caused by the Earth’s rotation. Rather than simply responding to the daily light-dark transitions, it would appear that organisms have evolved real endogenous clocks to anticipate daily changes in the environment and to time biological processes that can maintain circadian behavior even in the absence of environmental cues. The overt manifestations of these clocks are circadian rhythms. Important examples of circadian rhythms in mammals, including humans, are sleep-wake cycle, daily fluctuations in body temperature, hormone secretion, blood pressure, liver metabolism etc.

It is well established now that circadian rhythmicity is a genetically controlled function, which is based on rhythmic transcriptional activation and repression of the set of evolutionary conserved genes that form a "core" clock mechanism. They are considered now to be an important aspect of normal human physiology as well as human pathology. Thus, the chances of asthmatic attacks are increased during the nighttime in patients with chronic asthma while strokes are more frequent in the few hours after waking.

Organism sensitivity to a variety of treatments is also known to be a function of the time of day. All these findings led to a concept of "chronotherapy", when medication used to treat the disease is delivered in the different doses at different times of the day. There is an increasing evidence that administration of chemotherapeutic agents on a programmed circadian pattern can reduce toxicity and significantly enhance efficacy of anticancer treatment. It is therefore essential to determine the mechanics of circadian control of drug activity to be able to optimize the conditions of therapy. It is especially important to have a reliable assay that would allow a physician to estimate the circadian status of the patient taking into account both individual variations in rhythmicity (often recognized as "morning larks" and "night owls") and possible changes induced by the disease.

In this work we plan to perform a comprehensive and systematic search for circadianly controlled genes using hybridization with high density oligonucleotide microarrays, the technology that allows one to simultaneously monitor the activity of virtually all genes. We will specifically focus on those tissues that can secrete proteins in easily accessible body fluids (salivary gland and kidney). Our experimental plan consists of four steps: (i) identification of all oscillating genes in these tissues, (ii) confirmation that they are indeed under a clock control mechanism, (iii) selection of those of them which encode secreted proteins to be further characterized as prospective targets for the development of a diagnostic assay, and (iv) investigating the molecular mechanisms regulating clock controlled genes.

DENIS C GUTTRIDGE PhD
THE OHIO STATE UNIVERSITY

There are many stages in life when cells in a human body are growing. One of these stages is when a baby is developing inside their mother before they are born. Another stage is after birth when babies grow up to be children and then grow up to be adults. And still another stage is when an injury occurs and the body needs to repair the damage tissue. In all of these stages, there has to be a way to tell cells when to grow so that tissues can be formed or injury can be repaired, and other ways to tell cells when to stop growing after the body is mature or after the injury has been healed. The body is very efficient at knowing when to grow cells and when to stop growing them, but occasionally, the human body makes mistakes because something has gone wrong with its molecular programming. When this occurs, cells may not receive the proper signals to tell them to stop growing. When cells continually grow out of control, it is referred to as cancer and this usually leads to death.

There are many molecules that are affected that allow cells to continually grow without stopping. One of these molecules is what we study in our laboratory called nuclear factor kappa B. This molecule is a protein made by cells which normally help these cells perform specialized tasks. But because mistakes are occasionally made in the body, nuclear factor kappa B can start functioning in other ways that it is not meant to. One of these ways is by making cells grow faster, and the research performed on this molecule indicates that its activity is required for cells to become cancerous. In our laboratory we are trying to understand how this molecule makes cells grow. Our hypothesis is nuclear factor kappa B controls the production of other molecules that actually make the decisions about whether a cell is suppose to grow or not grow. This is a process called cell cycle and in our research we are aiming to understanding how nuclear factor B is controlling cell cycle. Our hope is that by understanding how nuclear factor kappa B controls cell cycle we will better understand how it functions in cancer, and more importantly, we can begin to design drugs to block nuclear factor kappa B that may be useful in anti-cancer therapy.

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SUBRATA HALDAR PhD
METROHEALTH MEDICAL CENTER

Apoptosis is a naturally occurring process of cell death by suicide. This programmed cell death usually takes care of damaged cells. Cancer cells, however, have forgotten how to commit suicide and so continue to grow out of control. This ability of cancer cells to evade apoptosis is one of the keys for their fight against attack from chemotherapy.

Malignant melanoma is a particularly spiteful form in this regard. Not only is this form of malignancy chemoresistant, but it also is highly metastatic. The melanoma cells have mastered the art of avoiding apoptosis by switching off a downstream player in p53 mediated cell suicide pathway. Unlike other cancers, melanoma cells often carry functional or wild form of p53. Wild form of p53 usually cooperates to kill tumor cells during chemotherapy. But in the case of malignant melanoma, cancer cells do not respond to therapy despite the presence of functional p53. The reason is due to silencing of a downstream pro-apoptotic effector gene Apaf-1 by methylation. Interestingly, the gene can be turned on by methylation inhibitors and melanoma cells become sensitive to destruction by chemotherapeutics. Our research program proposed here will be directed towards the development of a preclinical model in athymic mice.

One of the scientific priorities in melanoma research is to identify checkpoint gene in apoptotic signaling. Our studies all together might lead us to a novel therapeutic intervention target such as Apaf-1 for a subpopulation of metastatic melanoma.

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Cancer can be induced by environmental stress or genetic mutations. Acute Myelocytic Leukemia (AML) is a form of leukemia (cancer of blood cells), which is caused by a chromosomal translocation resulting in mutant fusion proteins. Aberrant recruitment of these mutant proteins with co-factors, SMRT and N-CoR, have been linked to AML. In addition, SMRT also serve as a cofactor for several molecules that have been implicated in T-cell leukemias and B-cell lymphomas.

In this proposal, we focus on one of the SMRT-interaction proteins, GPS2/AMF-1, which also associates with viral oncoproteins and the tumor suppressor protein, p53. We will examine how SMRT activity is regulated by GPS2/AMF-1. Our studies will shed light on our current knowledge of the function of SMRT and the molecular mechanism underlying these leukemias and lymphomas and may have therapeutic implications.

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HUA LOU PHD
CASE WESTERN RESERVE UNIVERSITY

Cancer development is a complex process involving many changes at the cellular level. During cancer development, certain proteins can be turned on or off by changes at the level of transcription. Proteins may also change their composition by adding or deleting a few amino acids through a process called "alternative splicing", which often leads to dramatic changes of the protein function.

Production of these alternative forms of a protein is tightly controlled by a large number of cellular enzymes in normal cells, whereas this tight control is often lost in cancer cells. For example, the human calcitonin/calcitonin gene-related peptide (CGRP) gene can generate two peptide hormones, the calcium-regulating hormone calcitonin and the neurotransmitter CGRP, through alternative splicing. In normal human body, calcitonin is predominantly produced in thyroid cells and CGRP is produced in a number of neuron cells. However, when the thyroid cells become malignant, a large amount of CGRP is produced while the level of calcitonin is reduced. In fact, CGRP has been used as a cancer marker for this type of cancer.

In this proposal, we propose initial experiments of a long-term study to understand what regulates the change from calcitonin to CGRP production in medullary thyroid carcinomas (MTC). We will use a cell line derived from a rat MTC. The major advantage of using this cell line is that it can be induced to behave more like normal thyroid cells, i.e., predominantly producing calcitonin, by inclusion of a hormone called dexamethasone in the cell culture medium. We will establish this inducible system to study factors that control production of either calcitonin or CGRP. These studies will provide evidence for the hypothesis that cellular enzymes regulating alternative splicing are changed in cancer cells. Future studies using this system will reveal factors that are responsible for the changes in alternative splicing in cancer cells. As a result, therapies may be developed that are targeted at these factors instead of the various downstream proteins.

http://genetics.cwru.edu/lou.html

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NARENDRA NARAYANA PHD
CASE WESTERN RESERVE UNIVERSITY

Approximately 10,000 new cases of childhood cancer are diagnosed each year in the United States. Cancer is a disease caused by uncontrolled cellular growth. A tumor is an uncontrolled growth of cells.

Leukemias and solid tumors represent two major types of childhood cancers. Gonadoblastoma, a solid tumor in children, is associated with abnormal functioning of gonads. Modifications or deletions in human DNA (genes) required for testicular differentiation in the formation of an embryo confer a high risk of gonadoblastoma. The Doublesex (DSX) protein important in fly’s sex-determination has a new DNA-binding domain (DM) highly conserved among metazoans. Its function in gene regulation and conservation in mammals suggests a direct relevance to human organogenesis and genetic cancer susceptibility.

Structure determination of the DSX protein will shed light on the molecular mechanisms that underlie the sex determination and has an immediate relevance to the mechanisms of organogenesis and genetic cancer.

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NYWANA SIZEMORE PHD
THE CLEVELAND CLINIC FOUNDATION

Knowledge of the mechanisms responsible for tumor formation and development is essential for cancer detection and treatment. Inactivation of tumor suppressor genes is one of the most frequent events in the development and progression of tumors.

PTEN is a tumor suppressor gene, which codes for an important protein in normal cells. PTEN, in its normal form, suppresses tumors, but when mutated, it is most often found in cancers that are difficult to treat and usually fatal, including breast cancer. Ordinarily this gene suppresses tumors by negatively regulating an enzyme called phosphatidylinositol 3 kinase (PI3K) that controls normal cellular growth and processes. Inactivation of PTEN renders it unable to carry out its negative regulation of PI3K. This aberrant continuous signal from PI3K activates the oncogene protein product AKT. The inappropriate activation of AKT then leads to activation of a set of kinases called the I Kappa B Kinases (IKKs). The IKKs control the activation of two transcription factors, Nuclear factor kappa B (NFkB) and b-catenin, which regulate the expression of many important genes necessary for a cell to function normally. However, when these same genes are inappropriately expressed they can contribute to tumor development.

We want to study the signal transduction pathways that lead to activation of these two transcription factors in breast carcinogenesis. A second goal is to identify and study the regulation of the genes controlled by these two transcription factors responsible for controlling breast cancer proliferation, survival, and metastasis. We also want to study the impact of inhibiting these signal transduction pathways on the malignant properties of breast cancer cells, so we might discover better ways to treat breast cancer in people.

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KARL ANDREW WERBOVETZ, Ph.D.
THE OHIO STATE UNIVERSITY

Many drugs that are now used to treat cancer affect a protein called tubulin. Although tubulin is a good target for cancer therapy, very little is known about how drugs attach to tubulin.

The purpose of our work is to learn more about how drugs attach to tubulin. To do this, we will make special tags for tubulin that resemble known cancer drugs. These tags will be specially designed to allow us find out where they attach to tubulin. We will then use special equipment to find these tags on tubulin. When we find these tags on the tubulin protein, we can use known tubulin computer maps to figure out what the surface of the protein looks like at the point where the tags are attached.

If we know what the protein looks like where the tags are attached, we can use this information to make new drugs that stick better to tubulin. We hope that our work leads to better drugs to fight cancer in the future.

Cancer is the No. 2 killer in the U.S. and it is estimated that over half a million people will die this year from cancer.

In the last decade, death rates from cancer declined significantly, attributed largely to improved effectiveness of anti-cancer treatment. For an anti-cancer drug to be effective, it has to be toxic to cancer cells, but less harmful to normal cells. For instance, paclitaxel, a widely utilized chemical in treating cancer, is very effective in triggering the cancer cells to commit suicide. Understanding how this drug achieves its toxicity to cancer will help to maximize drug efficacy and identify new target for anti-cancer treatment.

We believe that an enzyme protein inside the cell, called MEKK1, plays a key role as a mediator of paclitaxel toxicity. We will use cells with or without MEKK1 to study what drug-induced suicide programs are controlled by this enzyme and how this enzyme interacts with other proteins to make a cancer cell more vulnerable to drug-induced cell death.

We expect that results generated from this study will provide valuable information for the strategic design and the improvement of anti-cancer treatments.

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The goal of this project is to identify additional targets of the Raf-1 protein kinase. Raf-1 facilitates the transfer of the phosphate group form ATP, an energy currency of cell, to other proteins (substrates) in a process known as protein phosphorylation.

The attachment of phosphates to other proteins is a major on-off switch used by cells to regulate protein function. Protein phosphorylation has been shown to play an important role in regulating many cellular processes including growing and maturation. Of the over 100 cancer causing genes (oncogenes) that have been discovered, a significant majority of them are kinases. Deregulation of Raf-1 kinase activity in cells has been shown to promote the changing of normal cells to tumors.

Despite years of intensive research, only one biologically relevant substrate protein has been identified. However, there are several impressive lines of evidence suggesting that additional important substrates remain unidentified. Identification of additional Raf-1 substrates will illuminate the molecular mechanism of Raf-1-mediated transformation. A thorough understanding of the transformation functions of Raf-1 will expedite our overall understanding of cancer.

http://www.mco.edu/depts/biochem