Biomedical subjects

M Vermey

Publications and source records attributed to M Vermey.

At least 19 recordsLinked to original sources

PubMed2001

Influence of the microenvironment on invasiveness of human bladder carcinoma cell lines.

To investigate the importance of the microenvironment in bladder cancer invasion, a panel of six bladder carcinoma cell lines (SD, RT112, JON, 1207, T24, and J82) was tested in both in vitro and in vivo invasion assays. Furthermore, invasiveness was correlated with the expression of components of the E-cadherin-catenin complex. The E-cadherin-negative cell lines, T24 and J82, displayed a high in vitro invasive capacity, whereas the E-cadherin-positive cell lines, SD and JON, completely lacked in vitro invasive capacity. In contrast, in vivo invasion was noted for all cell lines, with the exception of cell line JON. Most notably, SD formed highly invasive tumors in vivo. The in vivo invasiveness of the E-cadherin-positive bladder carcinoma cell lines was associated with a heterogeneous expression of the E-cadherin-catenin complex. The discrepancy between in vitro and in vivo invasive behavior implies that, in vivo, the microenvironment plays an important role in the establishment of the invasive phenotype. In addition, it was found that orthotopic xenografting of 1207 and T24 bladder carcinoma cells resulted in site-specific tumor take and an enhanced tumor outgrowth and invasiveness, respectively, compared with heterotopic (i.e., subcutaneous) inoculation. We conclude that the site-specific growth and invasion of the bladder carcinoma cell lines in vivo and the observed assay specific invasion (in vitro vs in vivo) points to an effect of the local (bladder) microenvironment on tumor cell behavior.

Animals↗

PubMed2000

E-cadherin promotes intraepithelial expansion of bladder carcinoma cells in an in vitro model of carcinoma in situ.

High-grade transitional cell carcinomas (TCCs) of the urinary bladder are frequently associated with carcinoma in situ, which may replace large areas of the mucosa of the urinary tract. The invasive component of TCCs often reveals a loss of expression of the cell-cell adhesion molecule E-cadherin, but the role of E-cadherin in the development and expansion of intraepithelial neoplasia is unknown. To study the underlying mechanism of intraepithelial expansion (IEE), we have developed an IEE assay. Human TCC cell lines were investigated in this IEE assay for their capacity to replace the surrounding normal murine urothelial cells. In vitro IEE appeared to be prominent in three (SD, RT112, and 1207) of the four E-cadherin-positive cell lines. Although the two E-cadherin-negative cell lines (T24 and J82) were able to penetrate surrounding normal urothelium as single cells, they largely lacked the capacity of IEE. These results prompted us to investigate whether the cell-cell adhesion molecule E-cadherin is an important determinant for IEE. T24 cells that were transfected with full-length mouse E-cadherin cDNA displayed an enhanced IEE rate. Transfection did not influence their proliferative capacity, their pattern and level of integrin expression, or their ability to expand in the absence of surrounding urothelium. The data suggest that E-cadherin-mediated cohesiveness is an important factor in the IEE of bladder carcinoma cells. These observations argue for a dual, paradoxical role of E-cadherin in bladder tumorigenesis. On the one hand, E-cadherin promotes the expansion of intraepithelial neoplasia; on the other hand, its loss correlates with invasive behavior.

Animals↗

PubMed1999

Pathological features of glycogen storage disease type II highlighted in the knockout mouse model.

Glycogen storage disease type II (GSDII; Pompe's disease) is an autosomal recessive disease caused by lysosomal alpha-glucosidase deficiency. Skeletal muscle weakness is the most conspicuous clinical symptom of patients suffering from GSDII and skeletal muscle also is prominently involved in the knockout mouse model of this disease. Thus far, however, little detailed information has been published on the pathological changes in other mouse tissues. This paper aims to provide these data and gives a record of the clinical course of the mouse model over a 2-year period. Four-month-old affected mice perform worse in a running wheel than their unaffected littermates, but do not yet display other clear signs of disease. The lysosomal glycogen storage, already evident at birth, becomes more severe in time, leading to muscle wasting by 9-10 months of age and then limb girdle weakness and kyphosis. The disease does not markedly shorten the animal's life span despite the serious tissue pathology, which is not limited to heart and skeletal muscle, but is also seen in the smooth muscle of blood vessels and of the respiratory, digestive, and urogenital tracts. In addition, the mice have lysosomal glycogen storage in the liver, kidney, spleen, and salivary gland; in Schwann cells of the peripheral nerves, and in a subset of neurons in the central nervous system. By pathological criteria, the knockout mouse model parallels the human infantile form of GSDII and is attractive for studying the possible reversal of tissue pathology and symptomatology under different therapeutic regimes.

Animals↗

PubMed1999

A comparative evaluation of various invasion assays testing colon carcinoma cell lines.

Various colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines Colon-26 and -38. None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro.

Animals↗

PubMed1997

Clonal growth of colorectal-carcinoma cell lines transplanted to nude mice.

It is generally assumed that tumor progression is a microevolutionary process in which increasingly aggressive clones, generated through genetic instability, emerge in an initially monoclonal lesion. The present study was undertaken to determine how rapidly a dominant clone will emerge from an initial polyclonal situation, and whether dominance of these clones is a prerequisite for the onset of metastasis. To this end, colon-carcinoma cells were infected in culture with an amphotropic retroviral vector containing the neomycin-phosphotransferase gene, which makes cells resistant to neomycin. A heterogeneous population of neomycin-resistant cells carrying random retroviral integrations was xenografted to the subcutis and to the cecum of nude mice. The xenografts obtained, as well as the available metastases, were analyzed as to viral integrations by Southern blotting. The results show that, (i) clonal selection already takes place during growth of the primary tumor; (ii) dominant clones also generate metastases. The retroviral integration pattern of metastases turned out to be identical to that found in the primary xenografts. This pattern remained unchanged in tumors obtained after serial transplantations of cells cultured from metastases.

Adenocarcinoma↗

PubMed1997

In vitro modulation of implantation and intraepithelial expansion of bladder tumor cells by epidermal growth factor.

A major problem in the management of bladder cancer is the high risk for recurrence of bladder tumors after transurethral resection. This has generally been attributed to the attachment and subsequent expansion of exfoliated tumor cells to the traumatized bladder wall. An in vitro cocultivation model was used to study the implantation and growth of human tumor cells in traumatized murine urothelium. Furthermore, we investigated in a time-course experiment whether stimulation of the regenerative activity of the normal urothelium by a growth factor could affect implantation and subsequent growth of bladder tumor cells. After inoculation on injured confluent cultures of murine urothelium, human T24 and SD bladder carcinoma cells preferentially attached to the denuded areas. SD cells expanded into the normal urothelium as a sharply demarcated tumor, while T24 cells infiltrated as single cells. Treatment of the primary urothelium with epidermal growth factor (EGF) stimulated the proliferation of the primary urothelium and reduced the implantation and growth of T24 considerably. EGF reduced the implantation of the SD tumor cells but could not prevent the further expansion at the expense of surrounding normal urothelium. Since EGF had no effect on migration or proliferation of SD or T24 cells, its modulation of expansive growth is most probably due to an increase in the regeneration of normal urothelium. This study suggests that recurrence of transitional cell carcinomas might in some instances be inhibited by stimulation of the regeneration of traumatized urothelium. The reported in vitro cocultivation model may be useful for studying additional factors involved in intraepithelial expansion of carcinoma cells.

Animals↗

PubMed1996

Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in mice causes male sterility associated with chromatin modification.

The ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis. The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination. We report the phenotype of the first animal mutant in the ubiquitin pathway: inactivation of the hHR6B-homologous gene in mice causes male infertility. Derailment of spermatogenesis becomes overt during the postmeiotic condensation of chromatin in spermatids. These findings provide a parallel between yeast sporulation and mammalian spermatogenesis and strongly implicate hHR6-dependent ubiquitination in chromatin remodeling. Since heterozygous male mice and even knockout female mice are completely normal and fertile and thus able to transmit the defect, similar hHR6B mutations may cause male infertility in man.

Amino Acid Sequence↗

PubMed1996

LacZ staining in paraffin-embedded tissue sections.

Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.

Animals↗

PubMed1995

Modulation of intra-epithelial expansion of human T24 bladder-carcinoma cells in murine urothelium by growth factors and extracellular-matrix components.

The high recurrence rate of bladder cancer is probably due to an efficient repopulation of the bladder by residual transformed cells after resection of the tumour. However, the regenerating capacity of the normal urothelial cells is very high. To study the balance between regenerating normal urothelium and out-growth of transformed urothelial cells, we recently developed an in vitro co-cultivation model. With this model system we studied the effects of growth factors and extracellular-matrix components on the intra-epithelial expansion of human T24 bladder-carcinoma cells in primary mouse-bladder explants. Exposure of the cultures to acidic fibroblast growth factor (aFGF) and laminin led to a dramatic increase in the number of invasive T24 cells into the primary urothelium. Epidermal growth factor (EGF) and collagen types I and IV counteracted the infiltration of individual T24 cells. EGF, aFGF, laminin and collagen types I and IV did not directly affect the migration and proliferation of T24 cells. Apparently, the efficacy of invasion of transformed urothelial cells into primary urothelium is not only dependent on the intrinsic characteristics of the transformed cells, but can be influenced to a considerable extent by exogenous components exerting their influence on the normal urothelium. The clinical relevance of this observation needs to be studied further.

Animals↗

PubMed1995

Immunocytochemical characterization of explant cultures of human prostatic stromal cells.

The study of stromal-epithelial interactions greatly depends on the ability to culture both cell types separately, in order to permit analysis of their interactions under defined conditions in reconstitution experiments. Here we report the establishment of explant cultures of human prostatic stromal cells and their immunocytochemical characterization. As determined by antibodies to keratin and prostate specific acid phosphatase, only small numbers (< 5%) of epithelial cells were present in primary cultures; subsequent passaging further reduced epithelial cell contamination. Antibodies against intermediate filament proteins (keratins, vimentin, and desmin) and smooth muscle actin microfilaments demonstrated that stromal cells from benign prostatic hyperplasia and prostate carcinoma differed in regard to their differentiation markers. Two contrasting phenotypes were identified in cultures derived from these two different lesions: One exhibiting fibroblastic features, was predominant in cultures derived from benign lesions and a second, showing varying degrees of smooth muscle differentiation, was more abundant in carcinoma-derived cultures. These findings are indicative of a remarkable divergence in the stromal-epithelial relationships associated with these pathological conditions and may provide us with a potential tool for studying these processes.

Acid Phosphatase↗

PubMed1994

E-cadherin expression determines the mode of replacement of normal urothelium by human bladder carcinoma cells.

The high recurrence rate of human bladder cancer can be attributed to intraepithelial expansion of tumor cells or shedding and subsequent implantation of tumor cells elsewhere in the bladder. E-Cadherin is a calcium-dependent cell-cell adhesion molecule, and loss of E-cadherin by tumor cells is associated with increased tumor aggressiveness. Here we demonstrate that E-cadherin is also an important determinant of the mechanisms which are involved in the recurrence rate of bladder cancer. In a recently developed in vitro cocultivation model, we studied the effect of E-cadherin expression on the intraepithelial expansion pattern of six different human bladder carcinoma cell lines into primary murine urothelium. Bladder carcinoma cells lacking E-cadherin infiltrate into the primary urothelium as individual cells (pagetoid pattern). In contrast, a sharp demarcation is observed between E-cadherin-positive bladder cancer cells and the primary urothelium (carcinoma in situ pattern). With the same model, we demonstrate that only E-cadherin-positive bladder carcinoma cell lines could attach to and colonize the intact primary urothelium. We hypothesize that it is the latter process that plays an important role in the high recurrence rate that is observed in some of the patients.

Animals↗

PubMed1994

An in vitro model of urothelial regeneration: effects of growth factors and extracellular matrix proteins.

Although the cellular turnover of resting urothelium is very low, its regenerative capacity is known to be outstanding. In organotypic mouse urothelial cultures closely mimicking the differentiation and multilayering of normal urothelium, we examined the cell biological mechanisms underlying urothelial regeneration and the specific role of growth factors and several extracellular matrix (ECM) components. Exposure to epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) and culture on laminin resulted in enhanced expansion of the urothelium. Microscopy and assessment of proliferative activity revealed that enhanced urothelial expansion due to EGF could be attributed to increased proliferative activity and an increase in cell numbers, whereas aFGF-stimulated expansion must be considered the consequence of increased cellularity and migration. Laminin-enhanced urothelial expansion was shown to be the result of spreading of the entire urothelial organotypic culture. This was associated with a considerable decrease in the number of cell layers. A synergistic effect of growth factors and laminin was not found. This organotypic urothelial cell culture model seems to be very useful in studying strategies to improve urothelial regeneration.

Animals↗

PubMed1994

Characterization of distinct functions for growth factors in murine transitional epithelial cells in primary organotypic culture.

Although previous studies indicate that growth factors can affect several physiological processes in epithelia, their role in the biological dynamics of transitional epithelium of the bladder is not yet established. This study investigates the functional consequences of a direct action of EGF, TGF beta, FGF-1, FGF-2, PDGF-AA, and PDGF-BB on mouse urothelium in organoid-like primary cultures. Confluent and nonconfluent cultures served as a model for intact and regenerating urothelium, respectively. EGF and FGF-1 stimulated in both models under serum-free conditions the BrdU and [3H]-thymidine incorporation. This resulted in an increase in the number of cell layers, but the cultures assumed a less organoid-like morphology. In addition, EGF and FGF-1 stimulated the expansion of nonconfluent cultures. TGF beta inhibited proliferation, caused a decrease in the number of cell layers, and blocked expansion. Moreover, TGF beta induced the terminal differentiation and apoptosis of urothelial cells. In nonconfluent cultures PDGF-BB slightly stimulated the increase in the outgrowth area, but no other effect on the parameters for proliferation and differentiation was observed. FGF-2 and PDGF-AA did not affect any of the studied parameters. These data are consistent with the hypothesis that EGF and FGF-1 can promote wound healing and/or hyperplasia through direct action on the epithelial cells, while TGF beta promotes the development of a normal, differentiated transitional epithelium.

Animals↗

PubMed1994

Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.

The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes.

Animals↗

PubMed1994

Expression of growth factors and receptors during specific phases in regenerating urothelium after acute injury in vivo.

We investigated the spatio-temporal changes in RNA and protein expression of growth factors and their receptors by in situ hybridization and immunocytochemistry during regeneration after acute injury of mouse urothelium in vivo. These data were correlated with changes in morphology and proliferation during regeneration. Except for an enhanced muscular transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta type II receptor expression, changes in expression patterns of growth factors or receptors were confined to the urothelium. Increased mucosal RNA expression of insulin-like growth factor-II (IGF-II) and particularly of type I IGF receptor, as well as fibroblast growth factor-1 (FGF-1) but not of FGF-2, coincided with re-epithelialization and urothelial proliferation. Both high levels of urothelial TGF-beta 1 RNA and protein expression were associated with re-epithelialization and differentiation. In addition, TGF beta type II receptor protein expression was similarly enhanced in the same urothelial cells. Platelet-derived growth factor-A (PDGF-A) RNA was expressed constitutively in the mucosa but decreased in the reepithelialization phase. The data are consistent with the notion that urothelial regeneration can be achieved by paracrine or autocrine acting, urothelium-derived growth factors. Since analogous growth factor RNA expression patterns in regenerating skin epidermis have been found, a more general growth factor-regulated mechanism for epithelial regeneration may be suggested.

Animals↗

PubMed1993

Characterization of mouse urothelial cell lines in different phases of transitional-cell carcinogenesis.

Altered cellular responsiveness to growth factors is one of the factors involved in carcinogenesis. In order to study the role of growth factors in transitional-cell carcinogenesis, we established 3 urothelial cell lines from normal mouse urothelium, designated g/G, NUC-5 and NUC-I. These cell lines were studied by light and transmission electron microscopy, karyotyping, grafting in syngeneic mice, growth-factor response in vitro under serum-free conditions, and EGF receptor expression. In the presence of insulin or insulin-like growth factors I and II, proliferation of the non-tumorigenic DNA-tetraploid g/G and DNA aneuploid NUC-5 cells is stimulated by EGF, TGF alpha, bFGF and aFGF. This stimulation can be abolished in g/G but not in NUC-5 cells by simultaneous addition of TGF beta. Proliferation of g/G and NUC-5 cells can also be stimulated by PDGF-AA. The spindle-cell-like NUC-I cells are DNA aneuploid and tumorigenic in syngeneic mice; they express low levels of EGF receptors and their autonomous proliferation is only affected by insulin or insulin-like growth factors. Each of these cell lines seems to reflect a different phase in transitional-cell carcinogenesis: g/G cells have gained immortality, have become tetraploid, but are non-tumorigenic and growth-factor-dependent. NUC-5 cells have become aneuploid, have a growth-factor responsiveness different from that of normal epithelial cells, but are still non-tumorigenic. NUC-I cells are aneuploid, tumorigenic, and growth-factor-independent. These urothelial cell lines provide a suitable tool for further studies in transitional-cell carcinogenesis.

Animals↗

PubMed1993

An in vitro model of intra-epithelial expansion of transformed urothelial cells.

Replacement of normal urothelium by pre-cancerous epithelium may explain the high recurrence rate of human bladder cancer. An in vitro model was designed in order to study the mechanisms of expansion of transformed urothelial cells at the expense of normal urothelium. For this purpose, mouse bladder explants were allowed to expand on a transparent porous membrane. Subsequently, cell sheets of the non-tumorigenic mouse urothelial cell line NUC-5, the tumorigenic mouse urothelial cell line NUC-5 Py and the human bladder-carcinoma cell line T24 were inoculated adjacent to the primary explant. Daily measurements of the outgrowth of the bladder explant were performed, and all cultures were terminated on day 24. At this time the post-confluent primary urothelial cell outgrowths still showed proliferative activity, as demonstrated by bromodeoxyuridine incorporation. In due course the non-tumorigenic NUC-5 cells were replaced by the bladder outgrowth. T24 and NUC-5 Py cells were able to inhibit the bladder outgrowth, or even infiltrate or replace the explant. This was confirmed by immunohistochemistry with (species-specific) anti-cytokeratin antibodies and by microscopic evaluation of cross-sections of the porous membrane. This co-cultivation model appears to be suitable for the in vitro study of the mechanisms of intra-epithelial expansion of transformed urothelial cell lines.

Animals↗

PubMed1991

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.

Antibodies, Monoclonal↗