Biomedical subjects

F C S Ramaekers

Publications and source records attributed to F C S Ramaekers.

16 recordsLinked to original sources

PubMed2006

Role of nuclear lamina-cytoskeleton interactions in the maintenance of cellular strength.

The response of individual cells to cellular stress is vital for cellular functioning. A large network of physically interconnected cellular components, starting from the structural components of the cells' nucleus, via cytoskeleton filaments to adhesion molecules and the extracellular matrix, constitutes an integrated matrix that functions as a scaffold allowing the cell to cope with mechanical stress. Next to a role in mechanical properties, this network also has a mechanotransductional function in the response to mechanical stress. This signaling route does not only regulate a rapid reorganization of structural components such as actin filaments, but also stimulates for example gene activation via NFkappaB and other transcription factors. The importance of an intact mechano-signaling network is illustrated by the physiological consequences of several genetic defects of cellular network components e.g. actin, dystrophin, desmin and lamins. These give rise to an impaired response of the affected cells to mechanical stress and often result in dystrophy of the affected tissue. Recently, the importance of the cell nucleus in cellular strength has been established. Several new interconnecting proteins, such as the nesprins that link the nuclear lamina to the cytoskeleton, have been identified. Furthermore, the function of nuclear lamins in determining cellular strength and nuclear stability was illustrated in lamin-knock-out cells. Absence of the A-type lamins or mutations in these structural components of the nuclear lamina lead to an impaired cellular response to mechanical stress and disturbances in cytoskeletal organization. In addition, laminopathies show clinical phenotypes comparable to those seen for diseases resulting from genetic defects in cytoskeletal components, further indicating that lamins play a central role in maintaining the mechanical properties of the cell.

Animals↗

PubMed2006

Genomic integration of oncogenic HPV and gain of the human telomerase gene TERC at 3q26 are strongly associated events in the progression of uterine cervical dysplasia to invasive cancer.

Recently proposed events associated with the progression of cervical intraepithelial neoplasia (CIN) 2/3 to cervical carcinoma include integration of human papillomavirus (HPV) into the host genome, genomic instability, and an increase in chromosome 3q copy number. In particular, the gene coding for the RNA component of telomerase (TERC) at 3q26 has been implicated as a possible candidate gene. Since it is not known to date how these events are temporally related during cervical carcinogenesis, the aim of the present study was to assess the correlation between TERC gene copy number and the physical status of HPV during progression in cervical neoplasia. Solitary precursor lesions of the uterine cervix (CIN 2/3, n = 17), lesions associated with a micro-invasive carcinoma (CIN 3&mCA, n = 13), and advanced invasive carcinomas (invCA, n = 7) were analysed by fluorescence in situ hybridization (FISH) to determine the physical status of the virus and TERC gene copy number. The TERC gene was increasingly gained with progression of CIN 2/3 (3 of 17) through CIN 3&mCA (7 of 13) to invCA (5 of 7). In the lesions exhibiting gain of TERC, the virus was predominantly integrated. This was seen in eight of ten diploid lesions, indicating that these events can occur prior to aneuploidization and are strongly associated with the progression of CIN 3 to mCA and invCA (p < 0.001). With progression to carcinoma, a number of these lesions show polyploidization, resulting in aneuploidy and high TERC gene copy numbers. In conclusion, genomic integration of oncogenic HPV and gain of the human telomerase gene TERC appear to be important associated genetic events in the progression of uterine cervical dysplasia to invasive cancer.

Cell Transformation, Neoplastic↗

PubMed2006

Novel candidate tumour suppressor gene loci on chromosomes 11q23-24 and 22q13 involved in human insulinoma tumourigenesis.

Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumours. Previous molecular studies have shown that gain of chromosome 9q rather than MEN1 gene mutation is an important early event in tumour development and that chromosomal instability is associated with metastatic disease. In order to identify new gene loci and to define further the critical genetic events in insulinoma tumourigenesis, 27 insulinomas were investigated by array-based comparative genomic hybridization (array CGH) on 3.7 k genomic BAC arrays (resolution < or =1 Mb). Fluorescence in situ hybridization was used to validate alterations in a subset of tumours. Array CGH most frequently detected loss of chromosomes 11q and 22q and gains of chromosome 9q. The chromosomal regions of interest (CRI) included 11q24.1 (56%), 22q13.1 (67%), 22q13.31 (56%), and 9q32 (63%). Evaluation of the simultaneous occurrence of these aberrations in the individual tumours revealed that gain of 9q32 and loss of 22q13.1 are early genetic events in insulinomas, occurring independently of the other alterations. In tumours with increased genomic complexity, these alterations were often detected simultaneously, occurring in the same tumour cells. Losses of 11q24.1 and 22q13.31 were also associated with these more advanced tumour cases. The CRIs identified most likely harbour crucial candidate genes important in insulinoma tumourigenesis.

Chromosomal Instability↗

PubMed2006

Nuclear lamins: laminopathies and their role in premature ageing.

It has been demonstrated that nuclear lamins are important proteins in maintaining cellular as well as nuclear integrity, and in maintaining chromatin organization in the nucleus. Moreover, there is growing evidence that lamins play a prominent role in transcriptional control. The family of laminopathies is a fast-growing group of diseases caused by abnormalities in the structure or processing of the lamin A/C (LMNA) gene. Mutations or incorrect processing cause more than a dozen different inherited diseases, ranging from striated muscular diseases, via fat- and peripheral nerve cell diseases, to progeria. This broad spectrum of diseases can only be explained if the responsible A-type lamin proteins perform multiple functions in normal cells. This review gives an overview of current knowledge on lamin structure and function and all known diseases associated with LMNA abnormalities. Based on the knowledge of the different functions of A-type lamins and associated proteins, explanations for the observed phenotypes are postulated. It is concluded that lamins seem to be key players in, among others, controlling the process of cellular ageing, since disturbance in lamin protein structure gives rise to several forms of premature ageing.

Aging, Premature↗

PubMed2006

Molecular parameters associated with insulinoma progression: chromosomal instability versus p53 and CK19 status.

Insulinomas represent the predominant syndromic subtype of endocrine pancreatic tumors (EPTs). Their metastatic potential cannot be predicted reliably using histopathological criteria. In the past few years, several attempts have been made to identify prognostic markers, among them TP53 mutations and immunostaining of p53 and recently cytokeratin 19 (CK19). In a previous study using conventional comparative genomic hybridization (CGH) we have shown that chromosomal instability (CIN) is associated with metastatic disease in insulinomas. It was our aim to evaluate these potential parameters in a single study. For the determination of CIN, we applied CGH to microarrays because it allows a high-resolution detection of DNA copy number changes in comparison with conventional CGH as well as the analysis of chromosomal regions close to the centromeres and telomeres, and at 1pter-->p32, 16p, 19 and 22. These regions are usually excluded from conventional CGH analysis, because they may show DNA gains in negative control hybridizations. Array CGH analysis of 30 insulinomas (15 tumors of benign, eight tumors of uncertain and seven tumors of malignant behavior) revealed that >or=20 chromosomal alterations and >or=6 telomeric losses were the best predictors of malignant progression. A subset of 22 insulinomas was further investigated for TP53 exon 5-8 gene mutations, and p53 and CK19 expression. Only one malignant tumor was shown to harbor an arginine 273 serine mutation and immunopositivity for p53. CK19 immunopositivity was detected in three malignant tumors and one tumor with uncertain behavior. In conclusion, our results indicate that CIN as well as telomeric loss are very powerful indicators for malignant progression in sporadic insulinomas. Our data do not support a critical role for p53 and CK19 as molecular parameters for this purpose.

Chromosomal Instability↗

PubMed2005

A-type lamins are essential for TGF-beta1 induced PP2A to dephosphorylate transcription factors.

Diseases caused by mutations in lamins A and C (laminopathies) suggest a crucial role for A-type lamins in different cellular processes. Laminopathies mostly affect tissues of mesenchymal origin. As transforming growth factor-beta1 (TGF-beta1) signalling impinges on the retinoblastoma protein (pRB) and SMADs, we tested the hypothesis that lamins modulate cellular responses to TGF-beta1 signalling, via the regulation of these transcription factors in mesenchymal cells. Here, we report that A-type lamins are essential for the inhibition of fibroblast proliferation by TGF-beta1. TGF-beta1 dephosphorylated pRB through PP2A, both of which, we show, are associated with lamin A/C. In addition, lamin A/C modulates the effect of TGF-beta1 on collagen production, a marker of mesenchymal differentiation. Our findings implicate lamin A/C in control of gene activity downstream of TGF-beta1, via nuclear phosphatases such as PP2A. This biological function provides a novel explanation for the observed mesenchymal dysfunction in laminopathies.

Animals↗

PubMed2005

Molecular aetiology and pathogenesis of basal cell carcinoma.

Recent insights into the cell biology of the epidermis and its appendages are transforming our understanding of the pathogenesis of basal cell carcinoma (BCC). The significant progress that has been made warrants a comprehensive review of the molecular and cellular pathology of BCC. The items addressed include environmental and genetic risk factors, the biology of the putative precursor cell(s), and the contribution of aberrations in processes such as apoptosis, cell proliferation, differentiation and signalling to carcinogenesis. Furthermore, established and novel treatment modalities are discussed with particular attention to future biological approaches.

Apoptosis↗

PubMed2005

Chromosomal instability predicts metastatic disease in patients with insulinomas.

Endocrine pancreatic tumors (EPTs) comprise a highly heterogeneous group of tumors with different clinical behavior and genetic makeup. Insulinomas represent the predominant syndromic subtype of EPTs. The metastatic potential of insulinomas can frequently not be predicted using histopathological criteria, and also molecular markers indicating malignant progression are unreliable because of the small number of cases per subtype studied so far. For the identification of reliable indicators of metastatic disease, we investigated 62 sporadic insulinomas (44 benign and 18 tumors with metastases) by means of comparative genomic hybridization (CGH). In addition, the role of MEN1 (multiple endocrine neoplasia type 1) gene mutations was determined to assess specific chromosomal alterations associated with dysfunction of this endocrine tumor-related tumor suppressor gene. Only one case with a somatic MEN1 mutation was identified (1527del7bp), indicating that the MEN1 gene plays a minor pathogenic role in sporadic insulinomas. CGH analysis revealed that the total number of aberrations per tumor differs strongly between the benign and the malignant group (4.2 vs 14.1; P<0.0001). Furthermore, chromosome 9q gain was found to be the most frequent aberration in both benign and malignant insulinomas, whereas chromosome 6q losses and 12q, 14q and 17pq gains are strongly associated with metastatic disease. Our study shows that chromosomal instability, as defined by > or =5 gains together with > or =5 losses, or total number of gains and losses > or =8, rather than parameters such as tumor size and proliferation index, is the most powerful indicator for the development of metastatic disease in patients with sporadic insulinoma.

Adult↗

PubMed2004

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization.

We have applied the fluorescence loss of intensity after photobleaching (FLIP) technique to study the molecular dynamics and organization of nuclear lamin proteins in cell lines stably transfected with green fluorescent protein (GFP)-tagged A-type lamin cDNA. Normal lamin A and C proteins show abundant decoration of the inner layer of the nuclear membrane, the nuclear lamina, and a generally diffuse localization in the nuclear interior. Bleaching studies revealed that, while the GFP-tagged lamins in the lamina were virtually immobile, the intranuclear fraction of these molecules was partially mobile. Intranuclear lamin C was significantly more mobile than intranuclear lamina A. In search of a structural cause for the variety of inherited diseases caused by A-type lamin mutations, we have studied the molecular organization of GFP-tagged lamin A and lamin C mutants R453W and R386K, found in Emery-Dreifuss muscular dystrophy (EDMD), and lamin A and lamin C mutant R482W, found in patients with Dunnigan-type familial partial lipodystrophy (FPLD). In all mutants, a prominent increase in lamin mobility was observed, indicating loss of structural stability of lamin polymers, both at the perinuclear lamina and in the intranuclear lamin organization. While the lamin rod domain mutant showed overall increased mobility, the tail domain mutants showed mainly intranuclear destabilization, possibly as a result of loss of interaction with chromatin. Decreased stability of lamin mutant polymers was confirmed by flow cytometric analyses and immunoblotting of nuclear extracts. Our findings suggest a loss of function of A-type lamin mutant proteins in the organization of intranuclear chromatin and predict the loss of gene regulatory function in laminopathies.

Animals↗

PubMed2004

Temporal and spatial variations in structural protein expression during the progression from stunned to hibernating myocardium.

BACKGROUND: Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. METHODS AND RESULTS: Expression of structural protein (desmin, desmoplakin, titin, cardiotin, alpha-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed alpha-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33+/-3 mm Hg. CONCLUSIONS: Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

Actinin↗

PubMed2004

Electric discharge plasmas influence attachment of cultured CHO K1 cells.

Non-thermal plasmas can be generated by electric discharges in gases. These plasmas are reactive media, capable of superficial treatment of various materials. A novel non-thermal atmospheric plasma source (plasma needle) has been developed and tested. Plasma appears at the end of a metal pin as a submillimetre glow. We investigate the possibility of applying the plasma needle directly to living tissues; the final goal is controlled cell treatment in microsurgery. To resolve plasma effects on cells, we study cultured Chinese hamster ovarian cells (CHO-K1) as a model system. When these are exposed to the plasma, instantaneous detachment of cells from the surface and loss of cell-cell interaction is observed. This occurs in the power range 0.1-0.2 W. Cell viability is assessed using propidium iodide (PI) and cell tracker green (CTG) fluorescent staining utilizing confocal laser scanning microscopy (CLSM). Detached cells remain alive. Use of higher doses (plasma power >0.2 W) results in cell necrosis. In all cases, plasma-influenced cells are strictly localized in submillimetre areas, while no reaction in surrounding cells is observed. Due to its extreme precision, plasma treatment may be applicable in refined tissue modification.

Animals↗

PubMed2004

Human follicular stem cells: their presence in plucked hair and follicular cell culture.

BACKGROUND AND OBJECTIVES: A considerable portion of the hair follicle remains attached to plucked hair and can be used for follicle cell culture. In this study we have phenotyped these cells in an attempt to identify the stem cell fraction. Reports in the literature have indicated that this cell population may be positive for cytokeratin (CK) 19. Because stem cells in general need to be protected from apoptosis, the presence of the apoptosis-suppressing Bcl-2 protein, together with the absence of the apoptosis-promoting Bax and the CK profile may be used as an indicator of the stem cell population in the hair follicle, and in cultures of hair follicle cells. METHODS: Hair follicles from skin biopsies and plucked hair were derived from the scalps of healthy volunteers. Follicular cells were cultured from the plucked hairs. These hair follicles, plucked hairs and cultured cells were examined for their CK profiles, which are indicative of the type of cell (basal/stem cells) and for their status with respect to the proliferation marker Ki-67, Bax and Bcl-2. RESULTS: We found coexpression for CK19 and Bcl-2, but not Bax in two distinct areas, localized in the upper and lower third of the follicle from both skin biopsies and plucked hairs, while proliferation markers were negative in these areas. CK19 and Bcl-2 were also coexpressed in combination in a fraction of the follicular cell culture. The skin basal cell marker CK14 could be found throughout the outer root sheath of the hair follicle from both skin biopsies and plucked hairs, as well as in the follicular cell culture. CONCLUSIONS: Thus, CK19/Bcl-2-positive and Bax-negative cells can be obtained from cells derived from plucked hair and are retained in cultures made from these cells. If this phenotype represents follicular stem cells, our finding endorses the assumption that stem cells are located in the bulge area of the hair follicle, as we did not find them in or near the dermal papilla.

Adult↗

PubMed2004

Proteasomes act in the pre-mitochondrial signal transduction route towards roscovitine-induced apoptosis.

The role of the ubiquitin-proteasome pathway during roscovitine induced apoptosis was evaluated in the non-small cell lung carcinoma cell line MR65. To this end specific inhibitors of proteasome activity, MG132 and lactacystin were used. Addition of MG132 or lactacystin, 1 h prior to the addition of the CDK-inhibitor roscovitine to the cell cultures inhibited apoptosis significantly, as measured by PS exposure, cytokeratin 18 cleavage and caspase-3 activation. Furthermore, we show that inhibition of proteasome activation prior to induction of apoptosis by roscovitine prevents loss of mitochondrial inner transmembrane potential (DeltaPsim). In addition we found that MG132 and lactacystin prevent release of cytochrome c from the mitochondrion. In contrast to the above findings we see no effect of proteasome inhibition in Fas-mediated apoptosis. Taken together our data suggest a specific role for proteasomes very early in roscovitine-induced apoptosis, upstream from the caspase cascade and mitochondrion.

Acetylcysteine↗

PubMed2003

Proliferation and aneusomy predict survival of young patients with astrocytoma grade II.

The clinical course of astrocytoma grade II (AII) is highly variable and not reflected by histological characteristics. As one of the best prognostic factors, higher age identifies rapid progressive A II. For patients over 35 years of age, an aggressive treatment is normally propagated. For patients under 35 years, there is no clear guidance for treatment choices, and therefore also the necessity of histopathological diagnosis is often questioned. We studied the additional prognostic value of the proliferation index and the detection of genetic aberrations for patients with A II. The tumour samples were obtained by stereotactic biopsy or tumour resection and divided into two age groups, that is 18-34 years (n=19) and > or =35 years (n=28). Factors tested included the proliferation (Ki-67) index, and numerical aberrations for chromosomes 1, 7, and 10, as detected by in situ hybridisation (ISH). The results show that age is a prognostic indicator when studied in the total patient group, with patients above 35 years showing a relatively poor prognosis. Increased proliferation index in the presence of aneusomy appears to identify a subgroup of patients with poor prognosis more accurately than predicted by proliferation index alone. We conclude that histologically classified cases of A II comprise a heterogeneous group of tumours with different biological and genetic constitution, which exhibit a highly variable clinical course. Immunostaining for Ki-67 in combination with the detection of aneusomy by ISH allows the identification of a subgroup of patients with rapidly progressive A II. This is an extra argument not to defer stereotactic biopsy in young patients with radiological suspicion of A II.

Adolescent↗

PubMed2003

The garlic-derived organosulfur component ajoene decreases basal cell carcinoma tumor size by inducing apoptosis.

Although the therapeutic role of ajoene, an organosulfur compound of garlic, in cardiovascular diseases and mycology has been established, its usefulness in cancer treatment has only recently been suggested. We applied ajoene topically to the tumors of 21 patients with either nodular or superficial basal cell carcinoma (BCC). A reduction in tumor size was seen in 17 patients. Immunohistochemical assays for Bcl-2 expression in a selection of these tumors before and after treatment showed a significant decrease in this apoptosis-suppressing protein. On average, the percentage of tumor cells expressing the proliferation marker Ki-67 was not decreased, which suggests that the action of ajoene is not explained by a cytostatic effect. To obtain further insight into the mode of action of ajoene, the BCC cell line TE354T and a short-term primary culture of BCC were analyzed for apoptosis induction after treatment with the drug. Apoptosis was detected by morphology of the cells and by flow cytometry. Ajoene induced apoptosis in a dose- and time-dependent manner in these cultures. Taking together the results of the in vivo and in vitro studies, we conclude that ajoene can reduce BCC tumor size, mainly by inducing the mitochondria-dependent route of apoptosis.

Aged↗

PubMed2003

Lamin expression in normal human skin, actinic keratosis, squamous cell carcinoma and basal cell carcinoma.

BACKGROUND: Aberrant expression patterns of nuclear lamins have been described in various types of cancer depending on the subtype of cancer, its aggressiveness, proliferative capacity and degree of differentiation. In general, the expression of A-type lamins (lamins A and C) has been correlated with a non-proliferating, differentiated state of cells and tissues. OBJECTIVES: To establish and compare the expression patterns of lamins in normal human skin, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). METHODS: Expression patterns of the individual lamin subtypes were studied immunohistochemically. The proliferation capacity of the tumour cells was detected using a specific antibody to Ki-67, and was related to the A-type lamin expression patterns. RESULTS: In normal skin, lamin A was expressed in the suprabasal cell compartment of the epidermis, whereas the basal cells were mostly unstained. BCCs and SCCs stained positive in most cells, while the epidermis overlying BCC and SCC and the epidermis in AK stained homogeneously and strongly in the basal cells in addition to the suprabasal cells. Lamin C was expressed in some basal cells of normal epidermis while the suprabasal cells stained strongly positive. Both BCCs and SCCs stained strongly positive for lamin C, with the difference that in BCC the staining was predominantly present in nucleolar structures with occasional staining of the nuclear envelope. The epidermis overlying SCC showed strong positivity in the lamina of virtually all cells. The expression of lamin C in the basal cells of AK resembled the expression pattern seen in the epidermis overlying BCC, i.e. a nucleolar staining next to nuclear envelope staining. Lamin B1 and B2 were found in virtually all cells in normal epidermis, AK, BCC, SCC and the epidermis overlying cancer. The percentage of Ki-67-expressing cells was highest in BCC (45%), and gradually decreased via epidermis overlying BCC, AK, SCC, and epidermis overlying SCC, to normal skin (11%). Simultaneous expression of A-type lamins and Ki-67 occurred in approximately 50% of the proliferating (Ki-67 positive) cells in BCC and SCC. CONCLUSIONS: Significant changes occur in the expression patterns of A-type lamins in both premalignant and malignant lesions of the skin. The profound overlap of lamin A and Ki-67 staining patterns indicates that the proliferating tumour cells may obtain a certain degree of differentiation. Finally, lamin A expression in the basal cell layer of the apparently normal epidermis overlying BCC may suggest its involvement in the primary process.

Basal Cell Carcinoma↗