Markku Kurkinen

Genome based cell population heterogeneity promotes tumorigenicity: The evolutionary mechanism of cancer

Cancer progression represents an evolutionary process where overall genome level changes reflect system instability and serve as a driving force for evolving new systems. To illustrate this principle it must be demonstrated that karyotypic heterogeneity (population diversity) directly contributes to tumorigenicity. Five well characterized in vitro tumor progression models representing various types of cancers were selected for such an analysis. The tumorigenicity of each model has been linked to different molecular pathways, and there is no common molecular mechanism shared among them. According to our hypothesis that genome level heterogeneity is a key to cancer evolution, we expect to reveal that the common link of tumorigenicity between these diverse models is elevated genome diversity. Spectral karyotyping (SKY) was used to compare the degree of karyotypic heterogeneity displayed in various sublines of these five models. The cell population diversity was determined by scoring type and frequencies of clonal and non‐clonal chromosome aberrations (CCAs and NCCAs). The tumorigenicity of these models has been separately analyzed. As expected, the highest level of NCCAs was detected coupled with the strongest tumorigenicity among all models analyzed. The karyotypic heterogeneity of both benign hyperplastic lesions and premalignant dysplastic tissues were further analyzed to support this conclusion. This common link between elevated NCCAs and increased tumorigenicity suggests an evolutionary causative relationship between system instability, population diversity, and cancer evolution. This study reconciles the difference between evolutionary and molecular mechanisms of cancer and suggests that NCCAs can serve as a biomarker to monitor the probability of cancer progression. J. Cell. Physiol. 219: 288–300, 2009.

Cloning and Characterization of a Novel Matrix Metalloproteinase (MMP), CMMP, from Chicken Embryo Fibroblasts CMMP, XENOPUS XMMP, AND HUMAN MMP19 HAVE A CONSERVED UNIQUE CYSTEINE IN THE CATALYTIC DOMAIN

We cloned a novel matrix metalloproteinase (MMP) called CMMP from cultured primary chicken embryo fibroblasts. The cDNA-derived CMMP sequence contains 472 amino acids including a putative 19-residue signal peptide and a unique cysteine in the catalytic domain, an insertion in a sequence motif that binds the structural (noncatalytic) zinc of MMPs. Strikingly, a homologously inserted cysteine is also found in Xenopus XMMP and human MMP19, two recently cloned novel members of the MMP family. Phylogenetic analysis suggest that XMMP and MMP19 represent founding members of the MMP family, whereas CMMP is related to collagenase MMPs. Bacterially produced recombinant CMMP (without the amino-terminal inhibition domain), which was autoproteolyzed at the carboxyl-terminal domain, digested casein and gelatin. As shown by Northern blotting, CMMP mRNA of 1.8 kilobase pairs was constitutively expressed in cultured primary chicken embryo fibroblasts and up-regulated by tumor necrosis factor-α and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, but it was not regulated by interleukin-1, basic fibroblast growth factor, or retinoic acid. CMMP mRNA of 1.8 kb was also detected in the head and body of 8-day-old chicken embryos and dramatically up-regulated in 9-day-old embryos.

Heterogeneity of Cell Death

Cell death constitutes a number of heterogeneous processes. Despite the dynamic nature of cell death, studies of cell death have primarily focused on apoptosis, and cell death has often been viewed as static events occurring in linear pathways. In this article we review cell death heterogeneity with specific focus on 4 aspects of cell death: the type of cell death; how it is induced; its mechanism(s); the results of cell death, and the implications of cell death heterogeneity for both basic and clinical research. This specifically reveals that cell death occurs in multiple overlapping forms that simultaneously occur within a population. Network and pathway heterogeneity in cell death is also discussed. Failure to integrate cell death heterogeneity within analyses can lead to inaccurate predictions of the amount of cell death that takes place in a tumor. Similarly, many molecular methods employed in cell death studies homogenize a population removing heterogeneity between individual cells and can be deceiving. Finally, and most importantly, cell death heterogeneity is linked to the formation of new genome systems through induction of aneuploidy and genome chaos (rapid genome reorganization).

Ovarian cancer evolution through stochastic genome alterations: defining the genomic role in ovarian cancer

Abstract Ovarian cancer is the fifth leading cause of death among women worldwide. Characterized by complex etiology and multi-level heterogeneity, its origins are not well understood. Intense research efforts over the last decade have furthered our knowledge by identifying multiple risk factors that are associated with the disease. However, it is still unclear how genetic heterogeneity contributes to tumor formation, and more specifically, how genome-level heterogeneity acts as the key driving force of cancer evolution. Most current genomic approaches are based on ‘average molecular profiling.’ While effective for data generation, they often fail to effectively address the issue of high level heterogeneity because they mask variation that exists in a cell population. In this synthesis, we hypothesize that genome-mediated cancer evolution can effectively explain diverse factors that contribute to ovarian cancer. In particular, the key contribution of genome replacement can be observed during major transitions of ovarian cancer evolution including cellular immortalization, transformation, and malignancy. First, we briefly review major updates in the literature, and illustrate how current gene-mediated research will offer limited insight into cellular heterogeneity and ovarian cancer evolution. We next explain a holistic framework for genome-based ovarian cancer evolution and apply it to understand the genomic dynamics of a syngeneic ovarian cancer mouse model. Finally, we employ single cell assays to further test our hypothesis, discuss some predictions, and report some recent findings.

CLONING AND DEVELOPMENTAL REGULATION OF TISSUE INHIBITOR OF METALLOPROTEINASES-3 (TIMP3) IN XENOPUS LAEVIS EARLY EMBRYOS

We cloned a cDNA encoding tissue inhibitor of metalloproteinases-3 (TIMP3) from the frog Xenopus laevis. Similar to TIMP3 from other species, Xenopus TIMP3 has 188 residues including 12 conserved cysteines and Asn184, a putative site for N-linked sugars. Xenopus TIMP3 is 84% identical with human TIMP3. As shown by Northern blotting and RT-PCR, Xenopus TIMP3 mRNA is maternally inherited in eggs and midblastula (stage 8) embryos, downregulated in gastrula and then upregulated in neurula and pretailbud embryos. In select adult tissues, TIMP3 mRNA is present in heart, muscle, liver, skin, intestine and ovaries. These results suggest that TIMP3 is involved in the regulation of expression of matrix metalloproteinases in Xenopus early development and adult tissue remodeling.

Matrix Metalloproteinase 21

Publisher Summary This chapter examines the structural chemistry and the biological aspects of Matrix metalloproteinase 21 (XMMP). The sequence of XMMP contains 604 amino acids including a putative signal peptide of 22 residues with a characteristic hydropathy profile. There are no other significant hydrophobic segments, suggesting that XMMP is a secretory protein. At the end of the propeptide, after the cysteine switch PRCGVPD motif, XMMP has a 37 amino acid insertion domain followed by a putative cleavage site for intracellular activation by furin proteinases. The catalytic domain has a unique cysteine; an insertion in a sequence motif that binds the structural zinc in MMPs. XMMP lacks a proline-rich linker peptide or a hinge region that is typically found in other MMPs between the catalytic domain and C-terminal hemopexin/vitronectin like domain. The linker peptide is the most variable region of MMPs, and plays a role in MMP activity and substrate specificity. XMMP has a calculated molecular weight of 70115 and a catalytically active form 47072. The sequence of XMMP is significantly different from other MMPs. It is found that in a phylogenetic tree of MMPs, XMMP is at the very bottom, providing an outlier from which the branching and MMP evolution begins.

Is Alzheimer's dementia a systemic disease?

P2-176 ARE CORTICAL SOURCES OFAUDITORY Figure 1. Individual values of the cortical (LORETA) sources of auditory P3a and P3b peak in the Ab-negative and Ab-positive aMCI subjects. For P3a peak, the individual values for Brodmann area (BA) 8, BA 9, BA 10, ODDBALL EVENT-RELATED POTENTIALS AN EARLY DIAGNOSTIC MARKEROFALZHEIMER’S DISEASE? BA 11, BA 44, BA 45, BA 46, and BA 47 are reported. For P3b, the individual values for BA 5, BA7, BA 39, BA 40, BA 22, BA 42, BA 23, and BA 31 are reported. Claudio Babiloni, Claudio Del Percio, Nicola Marzano, Susanna Cordone, Giuseppe Noce, Christina Bagnoli, Paolo Maria Rossini, Andrea Soricelli, Flavio Nobili, David Bartres, Olivier Blin, Pierre Payoux, Regis Bordet, Bernhard W. Muller, Magda Tsolaki, Lucilla Parnetti, Ulrich Hegerl, Tilman Hensch, Juergen Dukart, Alessandro Bartolino, Gianluigi Forloni, Jill Richardson, Giovanni Battista Frisoni, Sapienza University of Rome, Rome, Italy; IRCCS San Raffaele Pisana, Rome, Italy; University of Foggia, Foggia, Italy; IRCCS SDN, Naples, Italy; IRCCS Istituto Centro SanGiovanni di Dio Fatebenefratelli, Brescia, Italy; Catholic University of Rome, Rome, Italy; University of Genoa, Genoa, Italy; Universitat de Barcelona and IDIBAPS, Barcelona, Spain; Mediterranean Institute of Cognitive Neurosciences, Marseille, France; Universit e de Toulouse, Toulouse, France; Lille 2 University, Lille, France; University of Duisburg-Essen, Essen, Germany; Aristotle University of Thessaloniki, Thessaloniki, Greece; University of Perugia, Perugia, Italy; University of Leipzig, Leipzig, Germany; Roche, Basel, Switzerland; IRCCS Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy; Neurosciences Therapeutic Area, U.K., United Kingdom. Contact e-mail: claudio.babiloni@uniroma1.it

INSULIN RECEPTORS IN INTACT HIPPOCAMPUS AND HIPPOCAMPAL CULTURED CELLS FUNCTION DOWNSTREAM OF SRC-FAMILY KINASES TO PROTECT NEURONS AGAINST EXCITOTOXIC GLUTAMATE-INDUCED CELL DEATH

to evaluate 5-HT and 5-HIAA breakdown metabolite were performed. Results: 5-HT1B, p11, and SERT protein and gene expression were downregulated in APPswe Sh-SY5Y (P<0.001 and <0, 0001 respectively), and 5-HT production was reduced and 5-HIAA production increased. Similarly, in the Tg2576 mice, hippocampus analysis showed lower levels of 5HT1B, SERT, and p11 (P<0.001). Conclusions: These data collectively show that the 5HT1B receptor and related systems are downregulated in AD. Exploring the behavioral consequences of these changes is warranted.

WHY "AMYLOID CASCADE" IS A MISGUIDED HYPOTHESIS OF ALZHEIMER'S DEMENTIA ETIOLOGY

Background: Alzheimer’s dementia(AD) refers to mind disorders characterized by a progressive and irreversible memory loss and mental decline. Causes for AD are not know, and there is no cure or treatment for AD Methods: We argue, research and development of treatments for AD, the way it has been done,is too limited and takes too long time to have any impact on the disease. We revisit recent clinical drug trials to understand why the trials failed and,indeed, why they could not have succeeded in the first place. Results: Did the trials fail simply because our ideas about AD etiology are not correct.Did the trials fail because what was done was too little or too late, at a point of no return for AD.We review alternative on-going drug trials and therapies for preclinical AD, that is,on asymptomatic patients and people at risk, designed to prevent or slow the progression of ADConclusions:We predict these trials are going to fail too because amyloid plaques and neurofibrillary tangles are not causing AD but are the outcome of brain cells dying.

Disturbances of innate immunity in Alzheimer's disease: Possible therapeutic approach

Background: Alzheimer’s disease (AD) is the leading cause of dementia in the elderly, characterized by neurofibrillary tangles, senile plaques and a progressive loss of neuronal cells in selective brain regions. Mitochondrial dysfunction is a prominent and early feature of the disease, although the underlying mechanism is still not clear. Mitochondria are dynamics organelles that undergo continual fission and fusion events which serve crucial physiological function. Our recent studies demonstrated that an altered balance in mitochondrial fission and fusion was likely an important mechanism leading to mitochondrial and synaptic/neuronal dysfunction in AD brain. Mutations in presenilins (PS) cause early-onset familial form of AD (fAD). PS1 is found in mitochondria and mutant PS1 affects mitochondrial function and transport, suggesting that PS1 mutants may cause mitochondrial/neuronal dysfunction through regulation of mitochondrial dynamics. A detailed investigation into the potential role of PS1 in mitochondrial dynamics is warranted. Methods: In this study, we investigated the effect of PS1 fAD mutations on mitochondrial dynamics in neuroblastoma M17 cells and mouse primary neurons by confocal microscopy analysis.Results:Compared with control M17 cells, M17 cells overexpressing PS1 fAD mutants displayed fragmented mitochondria and abnormal and uneven mitochondrial distribution with mitochondria accumulating around the perinuclear area while more remote cytoplasmic areas were not covered by mitochondria. Importantly, fibroblasts from fAD patients with PS1 mutations also demonstrated abnormal mitochondrial dynamics. Our further studies in primary neurons revealed that PS1 knockout (KO) neurons demonstrated significant changes in mitochondria morphology, distribution and movement which could be prevented by coexpression of wild-type PS1, but not fAD-causing PS1 mutant, suggesting that PS1 is involved in the regulation of mitochondrial dynamics which may be impaired by PS1 fAD mutations. Most importantly we found that PS1 physically interacted with DLP1, a key regulator of both mitochondrial fission and distribution. Conclusions: These studies suggest that fADassociated PS1 mutants may cause impaired regulation of mitochondrial dynamics through specific interaction with DLP1 which causes mitochondrial dysfunction and redistribution which adversely affects neuronal functions in AD.