Biomedical subjects

R Grümmer

Publications and source records attributed to R Grümmer.

At least 19 recordsLinked to original sources

PubMed2005

Vascular development in endometriosis.

Endometriosis, defined as the presence of endometrial tissue outside the uterus, is an estrogen-dependent disease which causes pelvic pain and subfertility in women of reproductive age. The condition has a dramatic impact on the professional, social and marital life of sufferers. Direct and indirect evidence suggests that angiogenesis is required for the development and persistence of endometriosis. In this review the state-of-the-art with regard to our understanding of the role of angiogenesis in the ectopic implantation and survival of menstrual endometrial tissue will be discussed.

Blood Vessels↗

PubMed2005

Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4.

Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.

Adenovirus E1A Proteins↗

PubMed2004

Different regulatory pathways of endometrial connexin expression: preimplantation hormonal-mediated pathway versus embryo implantation-initiated pathway.

Transformation of the endometrium into the receptive phase is under the control of ovarian steroid hormones and is modulated by embryonic signals during implantation. We have previously shown that this differentiation process is accompanied by a suppression of gap junction connexins (Cx) 26 and 43 before implantation followed by a local induction of both connexins in the implantation chamber. In the present study, we demonstrate that connexin gene expression in the rodent endometrium is regulated via two distinct signaling pathways during these different stages of early pregnancy. During preimplantation, transcription of connexins can be induced by estrogen via an estrogen receptor (ER)-dependent pathway. Additionally, Cx26 and Cx43 are induced by embryonic signals during implantation and delayed implantation as well as during artificially induced decidualization. In contrast to the estrogen-induced expression, this embryonic/decidual-associated induction of Cx26 and Cx43 could not be blocked by antiestrogen, thus pointing to another regulatory pathway independent of the ER. Studies in ERalpha and ERbeta knockout mice confirmed these different pathways, demonstrating that in the endometrium, estrogen-mediated Cx26 gene induction, but not induction during decidualization, is dependent on functional ERalpha. To evaluate potential embryonic signals regulating Cx26 expression, uteri of pseudopregnant animals were incubated with different mediators in an organ-culture model, showing that catechol estrogen and mediators of the inflammatory cascade such as prostaglandin F(2alpha) and interleukin-1beta are able to induce Cx26 expression through the ER-independent pathway. Thus, the present study demonstrates that endometrial expression of Cx26 and Cx43 is induced via estrogen and ERalpha during preimplantation but then utilizes an ER-independent signaling pathway during embryo implantation and decidualization.

Animals↗

PubMed2002

Functional significance of gap junctional coupling in preimplantation development.

Gap junctional intercellular coupling allows cells to share low molecular weight metabolites and second messengers, thus facilitating homeostatic and developmental processes. Gap junctions make their appearance very early in rodent development, during compaction in the eight-cell stage. Surprisingly, preimplantation mouse embryos lacking the gap junction protein connexin 43 develop normally and establish full-term pregnancies despite severely reduced gap junctional coupling. It was suggested that this might be explained by the presence of at least five additional connexins known to be expressed in blastocysts. In the present study, we set out to clarify the number of connexins present in preimplantation rodent embryos and the role of gap junctional coupling, if any, in blastocyst development. We provide evidence from reverse transcription-polymerase chain reaction analysis that the genes encoding 3 additional connexins (connexin 30 or beta6, connexin 36 or alpha9, and connexin 57 or alpha10) are also transcribed in preimplantation mouse embryos. Furthermore, we show that multiple connexins are expressed in rat preimplantation embryos, indicating that multiplicity of connexin expression may be a common feature of early mammalian embryogenesis. We could detect no up-regulation of any of 3 coexpressed connexins examined in mouse embryos lacking connexin 43. Impaired intercellular coupling caused either by the loss of connexin 43 or by treatment of cultured embryos with the gap junctional coupling blocker 18alpha-glycyrrhetinic acid (AGA) had no discernable effect on either apoptosis or glucose utilization, parameters known to be affected by gap junctional coupling in other contexts. These results, taken together with the reported inability of AGA to perturb blastocyst formation, imply that gap junctional coupling is not essential during this developmental period. We propose that connexin expression and the assembly of multiple types of gap junction channels in preimplantation embryos facilitates the diversification of communication pathways that will appear during postimplantation development. New evidence of this diversification is presented using rat blastocyst outgrowths.

Animals↗

PubMed2002

Responsiveness of endometrial genes Connexin26, Connexin43, C3 and clusterin to primary estrogen, selective estrogen receptor modulators, phyto- and xenoestrogens.

Phytohormones and chemical compounds revealing estrogenic effects are of increasing interest for their possible influence on the physiology of the reproductive tract. The gap junction connexin (Cx) genes Cx26 and Cx43, the plasma glycoprotein clusterin gene and the complement C3 gene are highly regulated by estrogen in rat endometrium. To test the value of these genes as markers for estrogenic responsiveness we analyzed the effects of estradiol, diethylstilbestrol, the selective estrogen receptor modulators (SERMs) raloxifene and tamoxifen, the phytoestrogens genistein and daidzein, and the industrial compounds DDT (1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl) ethane) and polychlorinated biphenyl (PCB) on the transcription of these genes in rat endometrium in vivo. Enhancement of Cx26 and decrease of clusterin transcripts expression by estradiol was observed at 0.03 micro g/250 g body weight (BW), and induction of C3 expression was observed at 0.05 micro g/250 g BW. A comparable effect was obtained by a tenfold higher concentration of diethylstilbestrol. Tamoxifen had a regulatory effect on this set of genes at about a 300-fold higher concentration, while raloxifen revealed much weaker estrogenic activity. No effect on Cx43 transcripts was observed with any of the compounds at the concentrations used. An effect of genistein was observed only on Cx26 expression, while PCB decreased clusterin transcripts. These results show that Cx26, C3 and clusterin reveal a comparable sensitivity to estrogens and SERMs. With respect to the phytoestrogen genistein, however, Cx26 seems to be the most sensitive gene. The analysis of clusters of estrogen-sensitive endometrial genes could help to identify estrogenic substances, assess their potency, and elucidate their mechanism of action.

Animals↗

PubMed2001

Enhancement of connexin 43 expression increases proliferation and differentiation of an osteoblast-like cell line.

Bone cells form a functional syncytium as they are coupled by gap junctions composed mainly of connexin 43 (Cx43). To further understand the role of Cx43 in bone cell growth and differentiation, we stably transfected Cx45-expressing UMR 106-01 cells with Cx43 using an expression vector containing rat Cx43 cDNA. Three stably transfected clones were analyzed, all of which showed altered expression of Cx43 and/or Cx45 as was obvious from immunocytochemistry and Northern blotting. Double whole-cell patch clamping revealed single-channel conductances of 20 (Cx45) and 60 pS (Cx43). The overexpression of Cx43 led to an increase in dye coupling concomitant with elevated gap-junctional conductance. The phenotype of the transfected clones was characterized by an increased proliferation (4- to 7-fold) compared to controls. Moreover, a transfectant clone with 10- to 12-fold enhanced Cx43 expression showed a significantly increased calcium content of the extracellular matrix and enlarged mineralization nodules, while alkaline phosphatase was moderately increased. We conclude that enhanced gap-junctional coupling via Cx43 significantly promotes proliferation and differentiation of UMR cells.

Alkaline Phosphatase↗

PubMed2001

Expression of the gap junction connexins Cx43, Cx45 and Cx26 in human uterine leiomyomata.

Uterine leiomyomata of 34 premenopausal women undergoing leiomyomectomy or hysterectomy, and in four cases the corresponding myometrium, were collected at laparotomy or laparoscopy to investigate the ability of these benign smooth muscle cell tumors to express different connexins. Immunohistochemical and Northern blot analyses were performed for the characterization of the expression of connexins Cx43, Cx45, Cx26 and Cx32. Immunofluorescence revealed the presence of Cx43 in most leiomyomata. Only seven leiomyomata lacked Cx43 expression. Cx45 was expressed in 13, a weak Cx26 immunostaining was found in seven cases, whereas Cx32 could not be detected. No correlations between the 17 beta-estradiol or progesterone serum levels and the expression patterns of the connexins Cx43, Cx45 and Cx26 could be observed. Gonadotropin-releasing hormone (GnRH)-agonist or progestin treatment did not influence the connexin expression pattern. Northern blot analyses confirmed these results; however, transcripts of Cx26 were not detectable. Connexin transcripts between myomata and the corresponding myometrium showed no obvious differences. Our data show that uterine leiomyomata are capable of expressing different connexins comparable to the corresponding myometrium, but do not respond to different hormonal conditions. The ability to express the appropriate connexins could explain why these tumors, though developing independently of hormonal levels, are still differentiated benign smooth muscle tumors.

Blotting, Northern↗

PubMed2001

Peritoneal endometriosis: validation of an in-vivo model.

BACKGROUND: The current medical treatment of endometriosis, a common gynaecological disease, is still associated with a high recurrence rate. To establish an appropriate in-vivo model to evaluate new therapeutic strategies we validated the nude mouse model for the intraperitoneal cultivation of human endometrial tissue. METHODS: Human endometrium of the proliferative phase was implanted into the peritoneal cavity of normal cycling and ovariectomized athymic mice and of cycling non-obese diabetic (NOD)-severe combined immuno-deficiency (SCID) mice. Morphology, proliferation, differentiation, and angiogenesis in the ectopic endometrium at different time points after implantation was investigated. RESULTS: Adhesion of endometrial fragments was observed from day 2 onwards. The lesions persisted for up to 28 days revealing a well preserved glandular morphology. The glandular epithelium maintained cytokeratin expression even after 14 days of culture. With progressing culture, glands exhibited vimentin staining in combination with a decrease of surrounding stromal cells. Proliferation of glandular epithelium could be demonstrated throughout the investigated period of 28 days, whereas expression of oestrogen and progesterone receptors was maintained only in endometriotic lesions grown in cycling but not in ovariectomized mice. Neoangiogenesis occurred from day 4 onwards, independent from the intraperitoneal localization of the ectopic lesions. CONCLUSIONS: This in-vivo model is a promising tool to test the effect of compounds such as different hormone agonists/antagonists or anti-angiogenic factors to develop new therapeutic concepts in endometriosis.

Adult↗

PubMed2000

Expression of the imprinted genes MEST/Mest in human and murine placenta suggests a role in angiogenesis.

In the mouse fetus, Mest is widely expressed in mesoderm derived tissues. In separate studies in mice and in humans, it has been shown to be maternally imprinted, that is, only the paternally inherited allele is active. Here, we show that starting with implantation, Mest is also expressed in maternal decidua of the mouse and in placenta of both humans and mice. Expression in murine decidua was restricted to endothelial cells. After Day 7, expression in the decidua gradually decreased. Mest-specific RT-PCR and restriction fragment length variant (RFLV) analysis of decidualized endometrium isolated from (M. musculus x M. spretus)F1 females showed that only the paternally derived Mest allele was activated in the decidual endothelium. In the mouse extraembryonic tissues, Mest transcripts were detected in derivatives of extraembryonic mesoderm only. Here, hemangioblast precursor cells and endothelial cells were positive. At all developmental stages of the mouse, trophoblast-derived cells were clearly devoid of Mest transcripts. In the human placenta MEST transcripts were also detected in hemangioblast precursor cells, however, MEST was also expressed in villous and invasive cytotrophoblast. In a human choriocarcinoma/trophoblastic tumour grown in a nude mouse, human MEST was expressed in the tumour cells, whereas murine Mest was expressed in endothelia of the murine capillaries. The expression pattern exhibited by both Mest and MEST in extraembryonic tissues during development and during formation of choriocarcinoma/trophoblast tumour suggests a functional role of the MEST proteins related to oncofetal angiogenesis. Dev Dyn 2000;217:1-10.

Animals↗

PubMed1999

Transfection with different connexin genes alters growth and differentiation of human choriocarcinoma cells.

To examine the role of cell-cell communication via gap junctions in controlling proliferation and differentiation we transfected the malignant trophoblast cell line Jeg-3, which exhibits extremely low cell-cell communication mediated by endogenously expressed connexin40, with connexin26, connexin40, and connexin43, respectively. In vitro growth of all cell clones transfected with connexin genes was significantly reduced compared to controls. This effect corresponded to a significant increase in total junctional conductance of all clones. Single-channel conductances for channels formed by the transfected connexins were in the range of the values published previously. Though total junctional conductance varied highly among clones and even within one clone, differentiation of the cells indicated by beta-hCG secretion was most prominent in the clones that revealed the largest amount of well-coupled cell pairs. Connexin26 channels enable cells of one clone to reduce drastically growth rate and produce significantly higher secretion of beta-hCG. Connexin43 had only moderate effects on the differentiation properties of Jeg-3 cells. These findings suggest that restoration of cell-cell communication plays a role in growth reduction and in differentiation of tumor cells and that different channel proteins might have different effects.

Cell Communication↗

PubMed1999

Characteristic growth of human choriocarcinoma xenografts in nude mice.

In order to investigate the mechanisms and regulation of trophoblast vessel invasion, we established a model using malignant trophoblast cells grown in nude mice. The human choriocarcinoma cell line Jeg-3 established fast-growing tumours within 2 weeks after subcutaneous injection into nude mice. Interestingly, instead of neoangiogenesis the tumours were characterized by large blood-filled lacunae. Staining for hCG-beta and for mouse panendothelial antigen revealed that the majority of the lacunae were lined by trophoblast cells. The blood supply of the lacunae resulted from invasion of the choriocarcinoma cells into the host vessels at the junctional zone between tumour and mouse tissue. These large blood-filled lacunae led to a much faster expansion of tumours with less necrotic areas compared to tumours of non-trophoblastic origin (Hec-1A and HeLa), which revealed neovascularization. Jeg-3 choriocarcinoma cells eroded host vessels and replaced the endothelial lining in a similar way to trophoblast cells during the establishment of a haemochorial placenta. This model can be useful in investigating the cell biological mechanisms of trophoblast vessel invasion and replacement of the endothelium by trophoblast cells.

Animals↗

PubMed1999

Gap junction connexin genes cx26 and cx43 are differentially regulated by ovarian steroid hormones in rat endometrium.

In rat endometrium, expression of gap junction connexin-26 (cx26) in the epithelium and cx43 in the uterine stroma is suppressed by progesterone before implantation. For further study of connexin gene regulation we analyzed expression of cx26, cx43, and cx32 in the endometrium of ovariectomized rats treated with different ratios of 17beta-estradiol (E2) and progesterone (P). A hormonal ratio of E2 to P that mimics conditions during pregnancy (0.1 microg E2 and 4 mg P) suppressed expression of cx26 and cx43. By changing the ratio to higher E2 levels (1 microg E2), cx26, in contrast to cx43, was not suppressed even by application of a high P concentration (10 mg). Time-course experiments supplying E2 alone led to an early gene response of cx26 within 3 h, whereas induction of cx43 transcripts was not detected until 14 h after E2 treatment. Simultaneous application of the antiestrogen ICI 182780 abolished E2-mediated induction of both connexins. No hormonal regulation of cx32 could be detected. As already shown for cx43 gene induction in the myometrium, E2-mediated induction of cx26 expression in the endometrium also required newly synthesized transcription factors. It can be concluded that only a hormonal ratio resembling conditions during pregnancy is able to suppress the expression of both cx26 and cx43 and that cx26 gene expression is induced earlier by E2 and is likely to be more sensitive to a shift in the E2 to P ratio than cx43.

Animals↗

PubMed1998

Regulation of gap junction connexins in the endometrium during early pregnancy.

In mammalian species embryo implantation into uterine tissue is restricted to a limited time period, the receptive phase. For successful implantation appropriate differentiation of the receptive endometrium is under the control of ovarian steroid hormones. In addition, locally acting embryonic signals are needed to modulate the maternal environment before invasion of the trophoblast is permitted. The expression pattern of gap junction channel proteins, connexins (cx), is directly related to this process. In rodents as well as in rabbit and humans the receptive endometrium is characterized by a lack of such cell-to-cell communication channels. In the rat endometrium cx26 is suppressed in the epithelium and cx43 in the stromal compartment by maternal progesterone, a phenomenon that can be observed similarly in human endometrium. Experimental approaches revealed that both connexin genes react very sensitively to progesterone and estrogen treatment. In rat and rabbit connexin expression is induced locally in the endometrium in response to the implanting blastocyst. In both species this induction of connexins can be mimicked by a traumatic stimulus. In conclusion, suppression of connexin expression in the endometrium is a characteristic cell biological indication for receptivity in different species. The limited induction of direct cell-to-cell communication properties, probably due to locally acting blastocyst signals, seems to be a precondition for embryo implantation.

Animals↗

PubMed1998

Expression of the gap-junction connexins 26 and 30 in the rat cochlea.

Gap junction channels which are responsible for direct intercellular communication are composed of connexin proteins. Different connexins are distributed in a tissue-specific manner. Up to now only connexin26 has been identified to be widely expressed in the inner ear. In order to investigate the role of additional gap junction proteins, the expression of connexin30 and 43 was investigated in the rat cochlea. Connexin26 and connexin30 were both expressed in the spiral limbus, the spiral ligament, the stria vascularis and between supporting cells of the organ of Corti. Double-labeling experiments suggest that both connexins are partly colocalized between cells. Weak staining of connexin43 could only be detected in the stria vascularis, the spiral ligament and between organ of Corti supporting cells. The corresponding transcripts for connexin26, 30 and 43 could be detected by Northern blot analysis. The expression of different gap junction channels in the cochlea suggests functional diversity. Gap junctions in the inner ear may control ion concentrations of cochlear fluids or act as conduits through which glucose and other metabolites diffuse.

Animals↗

PubMed1996

Regulation of connexin31 gene expression upon retinoic acid treatment in rat choriocarcinoma cells.

The controlled invasiveness of the trophoblast is based on the balance between invasive properties at implantation and the differentiation program of the developing placenta. During placental development in rats a switch of connexin gene expression has been observed in parallel to the switch from the invasive to the differentiated phenotype of trophoblast cells. To investigate the role of connexin expression for trophoblast invasion, proliferation, and differentiation, we studied one rat trophoblast (HRP-1) and one rat choriocarcinoma cell line (Rcho-1). The choriocarcinoma cells were characterized by expression of cx31 and a lack of E-cadherin, corresponding to the invasive trophoblast in vivo, whereas HRP-1 cells expressed cx43, normally found in the spongiotrophoblast and in late giant cells, and E-cadherin. Upon retinoic acid treatment, Rcho-1 cells irreversibly lost cx31 expression, accompanied by a loss of functional coupling. No changes in regard to connexin expression and cell-cell communication could be observed in HRP-1 cells. In addition, treatment of Rcho-1 cells with retinoic acid for 7 days upregulated expression of cx43 transcript, but no protein could be found. Proliferation was clearly reduced and the mean volume of cells doubled from Day 4 to Day 7 of retinoic acid treatment in Rcho-1 cells, while both parameters were not affected in HRP-1 cells. Both cell lines showed a similar invasion rate using a Matrigel invasion assay, and invasion was equally suppressed upon retinoic acid treatment. Thus the different connexin expression appears more likely to play a role in regulating proliferation and differentiation along the multilineage pathway than invasiveness of rat trophoblast cells.

Animals↗

PubMed1996

Connexins and E-cadherin are differentially expressed during trophoblast invasion and placenta differentiation in the rat.

We have characterized the spatial and temporal expression pattern of six different connexin genes and E-cadherin during trophectoderm development in the rat. During the initial phase of trophoblast invasion at 6 days postcoitum (dpc), the trophoblast expressed E-cadherin but no connexin expression could be observed. With progressing invasion of the polar trophoblast into the maternal decidua, from 7 dpc onwards E-cadherin expression in the ectoplacental cone cells was lost and was now restricted to the extraembryonic ectoderm. In the ectoplacental cone and extraembryonic ectoderm instead connexin31 mRNA and protein could be found. This pattern was maintained up to day 10 postcoitum. The start of labyrinthine trophoblast differentiation from day 11 postcoitum onwards was characterized by persisting expression of E-cadherin in the extraembryonic ectoderm and its derivative, the chorionic plate. In addition to E-cadherin, from 10 dpc onwards, connexin26 started to be expressed in the chorionic plate, and both molecules remained coexpressed in the labyrinthine trophoblast of the mature placenta. During this differentiation process connexin31 remained expressed mainly in the proliferating spongiotrophoblast. From day 14 postcoitum onwards, the expression of connexin31 in the spongiotrophoblastic cells decreased, and in parallel they started to express connexin43. The trophoblastic giant cells, first characterized by connexin31, lost all of the investigated connexins during midgestation on day 12 postcoitum but started to express connexin43 from day 18 postcoitum onwards. Our studies suggest that loss of E-cadherin and induction of connexin31 expression is correlated with the proliferative and invasive stages of the ectoplacental cone, whereas appearance of connexin26, E-cadherin and connexin43 reflects the switch to the differentiated phenotypes of the mature placenta.

Animals↗

PubMed1996

Expression pattern of different gap junction connexins is related to embryo implantation.

Successful implantation in mammals requires a close interaction between the embryo and the uterus. Direct cell-cell communication via gap junctions seems to play an important role in the preparation of the uterus for embryo implantation and in the regulation of trophoblast invasion. During preimplantation in the rat the gap junctional proteins connexin (cx) 26 and cx43 are suppressed. This loss of cell-cell communication seems to be important for transformation of the endometrium into the receptive phase. The suppressive effect is mediated by progesterone as demonstrated by the application of antigestagens. At implantation, however, a spatial and temporal pattern of connexin expression is induced in response to embryo recognition. cx26 is locally expressed in the uterine epithelium of the implantation chamber, cx43 in the surrounding decidua prior to invasion. With progressing invasion, the decidual cells surrounding the invading trophoblast in addition to cx43 reveal cx26. In this phase, the invasive partner, the blastocyst, is characterized by coexpression of cx43 and cx31. During trophoblast invasion however, cx31 becomes restricted to the cells of the invasive ectoplacental cone, cx43 to the embryo proper. It seems that compartmentalization of the trophoblast and the inner cell mass is established by two different connexins. During placental differentiation connexin expression switches from cx31 to cx26 and cx43, indicating the end of the invasive phase. The highly regulated pattern of connexin expression in the endometrium as well as in the trophoblast suggests a key role of this different intercellular pathways in regulating the invasion process of the trophoblast into its host tissue, the endometrium.

Animals↗

PubMed1996

The role of matrix contact and of cell-cell interactions in choriocarcinoma cell differentiation.

Cell differentiation is supported much better by gels of extracellular matrix than by the same matrix provided as a rigid substrate. Many cell types including normal and malignant trophoblast cells, however, form multicellular multilayered aggregates on matrix gels with increased cell-to-cell contacts as compared to regular monolayers on rigid matrix substrates. In such cultures, it remained open, so far, whether stimulated expression of differentiation markers is caused by enhanced cell-to-cell communication or is displayed only by cells in direct contact to the gel. Therefore, choriocarcinoma cells (BeWo) were grown as aggregates: (a) on gels of the basement membrane-like Matrigel, (b) on plastic coated with poly-HEMA, or (c) as aggregates (spheroids) in suspension culture. Production of the differentiation marker chorionic gonadotropin was stimulated significantly in aggregates attached to gels of Matrigel or to the poly-HEMA substrate but not in suspended spheroids. With respect to cell-cell communications, however, expression of E-cadherin mRNA was not altered in any type of aggregates, as compared to control cultures on plastic. The expression of connexin43 mRNA (not of connexin26) was increased only in suspended spheroids, while microinjection of the fluorescent dye Lucifer Yellow suggested that cell communication via gap junctions was absent from cells grown as monolayers and was not induced in any type of aggregate. When cells were grown on gels of Matrigel, the relevance of direct cellular contact to the substrate for differentiation was analyzed by immunohistochemistry. Trophoblastic differentiation markers (chorionic gonadotropin, placental lactogen, placenta-type alkaline phosphatase, and pregnancy-specific glycoprotein beta 1) as well as the proliferation marker Ki-67 were not preferentially expressed in cells that were in contact with the gel. Similar random distributions of all these markers were also observed in spheroids cultured in suspension. The distributions of several matrix molecules and of different integrins were comparable between aggregates on matrix gels and those in suspension culture. According to these data, cell-cell communication appears to play a subordinate role for cytodifferentiation in cell aggregates on matrix gels, so that substrate anchorage and physical properties of the substrate may be the decisive factors. Interestingly, however, direct contact to the substrate does not seem to be essential for the stimulation of differentiation in cells on matrix gels. The results are discussed in the context of the "tensegrity"-model for cell-matrix interactions in which proper mechanical properties of the substrate are important for the regulation of cell differentiation by allowing a balanced integrity of external and cell-internal tensile forces.

Actins↗