www.med.uoc.gr Department  of  Biochemistry - Division of Basic Sciences - University  of  Crete
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Kardassis Dimitris

Research Directions


Our lab is focusing on the following areas of research:

1) TGF? signaling and cancer

Transforming Growth Factor β (TGFβ) is a pleiotropic cytokine that controls several key biological processes including cell proliferation, differentiation, apoptosis and extracellular matrix production. TGFβ signals via heteromeric complexes of type I and type II serine/threonine kinase receptors and via intracellular effectors called Smads. Following their activation by TGFβ, type I receptors phosphorylate a subset of the Smad family called R-Smads (Smad2 and SMad3) causing their heterooligomerization with a common Smad (Smad4) and their translocation to the nucleus where they activate the transcription of target genes in cooperation with DNA binding cofactors and coactivators.

The basic TGF? signaling engine

Deregulation of TGFβ signaling either by mutations in key signaling components or by negative interference with other signaling pathways is a common characteristic in many types of cancer (pancreatic, colon, breast cancer).

We are concentrating our efforts on several aspects of TGFβ signaling. We are studying the structure-function relationship in Smad proteins and the characterization of tumorigenic mutations. By using a large panel of Smad mutants generated in our laboratory, we are investigating the structural requirements for Smad oligomerization, nuclear translocation and transcriptional activation. We are characterizing in detail a novel transactivation domain in the linker region of Smad3 identified recently in our laboratory (Prokova et al, NAR 2005).Using various in vitro and in vivo assays including a recently described protein-protein interaction assay based on biotinylation in vivo, we are analyzing inactivating mutations in Smad proteins found frequently in cancer patients. In collaboration with Dr George Mosialos (Fleming Institute, Athens) and Dr Christos Tsatsanis (U. of Crete Medical School) we are studying the molecular mechanisms of inhibition of TGFβ signaling and Smad functions by oncogenic proteins such as the Latent Membrane Protein 1 of the Epstein Barr Virus and the product of the Tpl2/Cot oncogene. Finally, we are characterizing the mechanisms of transcriptional activation by Smad proteins by analyzing the promoter regions of novel TGFβ target genes. We have initiated this effort by focusing on the promoter of the RhoB small GTPase gene which is involved in the TGFβ-induced cytoskeletion reorganization in cancer cells (collaboration with Dr Christos Stournaras, U. of Crete Medical School).

Structural detail in the region surrounding a tumorigenic mutation in Smad4 protein (E239).
Mutations introduced are shown in yellow.


2) Regulation and functions of the cdk inhibitor p21/WAF1

p21WAF1/Cip1 (p21) is a potent cell cycle inhibitor downstream of the p53 tumor suppressor protein. In the nucleus, p21 is an inhibitor of G1 and G2 cyclin-dependent kinases as well as of the Proliferating Nuclear Antigen (PCNA). In contrast, cytosolic sequestration of p21 which is a frequent case in cancer cells, alters the functional properties of p21 from growth inhibitory to tumor promoting and metastatic. Promotion of tumor growth and metastasis by cytosolic p21 is exerted via inhibition of the apoptotic pathway and the regulation of actin cytoskeleton organization.

Summary of the various stress factors that active the p53 tumor suppressor protein and the opposite functions played by the cdk inhibitor p21 in nucleus and the cytoplasm.

We are interested in understanding the molecular mechanisms that govern the response of the p21 gene to cytokines (TGFβ) and to chemotherapeutic drugs that activate the p53 tumor suppressor protein (5-Fluorouracil, Mithramycin A). We are studying the role played by members of the Sp1 family of ubiquitous transcription factors in the above p21 gene responses by characterizing the physical and functional interactions between Sp proteins and signaling mediators (p53, Smads, AP1).

The role of transcription factor Sp1 in the transcriptional activation of p21 gene by chemotherapeutic drugs.

As mentioned above, the intracellular localization of p21 determines the decision between cell cycle arrest and apoptosis We are planning to apply gene and protein transfer technologies in order to evaluate the therapeutic potential of nuclear p21 in breast cancer. This will be accomplished by delivering purified TAT-p21 fusion proteins or recombinant adenoviruses expressing p21, either wild type or mutant, into breast cancer cells or animals models of cancer and by following their growth and metastatic properties. Furthermore, we are planning to characterize the regulatory mechanisms involved in p21 nucleo-cytoplasmic shuttling in breast cancer cells expressing high or low levels of the her/neu oncogene. We propose to study the role of p21 in the apoptotic response of breast cancer cells undergoing chemotherapy using gene silencing technology.

3) Transcriptional regulation of genes involved in lipoprotein metabolism

a) Transcriptional regulation of the human apolipoprotein genes by hormore nuclear receptors: For many years we have been studying, using in vitro and in vivo approaches, the mechanisms that govern the regulation of transcription of the genes coding for of all major human apolipoproteins in liver and intestinal cells. In recent years we have been focusing our efforts on the specific role of orphan and ligand dependent hormone nuclear receptors on the expression of these genes by identifying and characterizing Hormone Response Elements (HREs) present on the promoters and the enhancers of the genes of the two apolipoprotein gene clusters in humans: the apoA-I/C-III/A-IV gene cluster on chromosome 11 and the apoE/C-I/C-IV/C-II gene cluster on chromosome 19. We are also studying the effect of pro- and inflammatory cytokines on the transcriptional activity of huclear receptors and the expression of genes of the apoA-I/C-III/A-IV gene cluster,

Schematic representation of putative synergistic interactions between nuclear receptors bound to the IR-1 of the (214/226) HCR-1 and the HREs
CIIB and CIIC present in the proximal apoC-II promoter

Signal transduction cascades triggered by pro- and anti- inflammatory cytokines that affect positively or negatively the activity of hormone nuclear receptors which bind to the HREs of the genes of the apoA-I/C-III/A-IV gene cluster.

b) Transcriptional regulation of the human apoE gene in macrophages and in the brain: Apolipoprotein E (apoE) is a key protein component of plasma lipoproteins and promotes the clearance of excess cholesterol from the plasma by binding to several members of the LDL receptor family. ApoE deficiency in animal models and in human patients with type III hyperlipoproteinemia is associated with premature atherosclerosis. ApoE has lately emerged as an important molecule in several biological processes and diseases such as cognitive function, immunoregulation, Alzheimer's disease (AD), and even infectious diseases. ApoE expression is regulated by a complex interaction of developmental, hormonal, dietary and pathological factors.

We are concentrating our efforts on the identification andcharacteriztion of the regulatory elements responsible for the basal level of apoE expression in the healthy state and for the modulation of its expression in pathological states. The experiments are focused on two different cell types involved in two distinct major diseases in which inflammation takes an important counterpart: macrophages, one of the key cell types involved in atherogenesis and astrocytes, one of the major cell types implicated in Alzheimer’s Disease (AD).

The beneficial effect of apoE production by macrophages has been shown in numerous animal studies: transgenic mice expressing apoE only in macrophages are protected against atherosclerosis, even though the plasma levels of apoE are exceedingly low and the animals are hypercholesterolemic; transgenic mice with normal level of apoE expression except in macrophages are more susceptible to atherosclerosis. On the other hand, it was demonstrated that apoE plays a key role in neuronal repair after injury by redistributing lipids to regenerating axons and to Schwann cells; also it has a protective role for AD.

c) Transcriptional regulation of the lipid transporter ABCA1: Clinical and epidemiological studies have shown an inverse correlation between HDL cholesterol and the risk of coronary heart disease events. In fact, low HDL cholesterol were found with high frequency in patients with premature myocardial infarction. Formation of HDL requires functional interactions of lipid-free apoA-I with the lipid transporter ABCA1. Specific mutations in ABCA1 and/or deficiency in apoA-I prevent the formation of HDL. ABCA1 functions in the efflux of cellular cholesterol and phospholipids and these functions are severly reduced in patients with Tangier disease that have defective ABCA1 forms. In contrast, overexpression of ABCA1 is associated with elevated plasma HDL levels, decreased plasma LDL levels and protection from atherosclerosis. Selective increase in the expression of ABCA1 that will ultimately result in increase in HDL requires a thorough understanding of transcriptional regulatory mechanisms that control the expresison of the ABCA1 gene.

Schematic representation of the role of the ABCA1 lipid transporter in HDL biogenesis and the reverse cholesterol transport.

Our aim is to understand the molecular mechanisms that control the hepatic expression of the ABCA1 gene in vitro and in vivo. Specifically, we are characterizing important regulatory regions present in the upstream ABCA1 promoter (class1/2 transcripts) as well as in the first intron, which contains several transcription initiation sites for the class 3 transcripts of this gene. We are also studying the role of Sp1 in the regulation of ABCA1 gene expression by cholesterol via the Liver X Receptors (LXRs). Finally, we are studying the mechanisms of differentiation-induced transacription of the ABCA1 gene in macrophages.

Patterns of expression and organization of the 5’ region of the human ABCA1 gene showing the sites of intitation of transcription of the Class1-3 transcripts.


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