Biomedical subjects

N de Groot

Publications and source records attributed to N de Groot.

At least 37 records · Page 2Linked to original sources

PubMed1999

Collection of mRNA-like non-coding RNAs.

In last few years much data has accumulated which shows that in different cells various RNA transcripts are synthesized. They lack protein coding capacity and do not produce mature protein. It seems that they work mainly or exclusively on the RNA level. Their function and mechanism of action is poorly understood. In this paper we have collected all known RNA transcript and prepared a database for further structural and functional studies. This is the first collection of the nucleotide sequences of RNAs of this kind. The data can be accessed via WWW at: http://www.man.poznan.pl/5SData/ncRNA/inde x.html

Animals↗

PubMed1999

Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice.

The polymeric immunoglobulin receptor (pIgR), a transmembrane protein, transports dimeric IgA (dIgA) across the epithelial cells of the mucosal surfaces into the external secretions, for example milk from the mammary glands. The pIgR is consumed during the transcytosis of dIgA and is cleaved at the apical side of the epithelial cells, regardless of the binding to its ligand (dIgA), to form secretory component (SC). We hypothesize that the expression level of the endogenous murine pIgR gene in the epithelial cells is rate-limiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice over-expressing the pIgR gene in their mammary glands in order to examine the effect on dIgA levels in the milk. Here we report on the generation of transgenic mice and analysis of the expression level of pIgR in their mammary glands. We cloned and characterized the murine pIgR gene and constructed an expression cassette bearing the pIgR gene under the control of the regulatory sequences of the bovine alpha s1-casein gene. Four transgenic lines were made, expressing the pIgR construct at RNA and protein level only in their mammary glands. The levels of the SC protein in the milk ranged from 0.1 to 2.7 mg/ml during mid-lactation. These levels are 10-270 times higher than wild-type SC levels (0.01 mg/ml).

Animals↗

PubMed1998

The effect of retinoic acid on the activation of the human H19 promoter by a 3' downstream region.

The human H19 is paternally imprinted (maternally expressed). It is transcribed by RNA pol II, but has no protein product. Its function is unknown. We showed that the transcription of the human H19 gene is under the simultaneous control of both a 5' upstream (promoter) region and a 3' downstream region in cell lines derived from human choriocarcinomas. Moreover, the activation of the H19 promoter by retinoic acid in cells derived from human testicular germ cell tumors is dependent upon the 3' downstream region. The possibility that the action of retinoic acid on the H19 promoter is an indirect one and involves a member of the AP2 transcription factor family is discussed.

Antineoplastic Agents↗

PubMed1998

The dynamics of the imprinted H19 gene expression in the mouse model of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine.

The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is expressed in fetal bladder, down-regulated postnatally and is re-expressed in human bladder carcinoma. This study was designed to investigate the dynamics of the expression of H19 in the mouse bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of neoplastic transformation. BBN was administered to mice in the drinking water for 26-28 weeks. The bladders were removed at 5-10 week intervals for histopathological examination and for in situ hybridization for H19 RNA, using a 35S-labeled probe. Following BBN administration expression of H19 first appeared after 5 weeks in the lamina propria adjacent to the basement membrane, concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in epithelial cells. Invasive carcinomas, of the transitional and squamous sub-types, were seen after 20 weeks and more of BBN administration. At this stage H19 expression was observed in scattered tumor cells, in the connective tissue stroma of the tumor and in the lamina propria underlying the remaining hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal bladder but was absent in normal adult bladder. We conclude that, similar to humans, the H19 gene product is an oncofetal RNA molecule in the experimental mouse model of bladder carcinoma. In this model H19 is expressed in the connective tissue of the lamina propria prior to its expression in epithelial cells, concurrent with preneoplastic changes in the transitional epithelium of the bladder.

Animals↗

PubMed1998

Imprinted H19 oncofetal RNA is a candidate tumour marker for hepatocellular carcinoma.

AIMS/BACKGROUND: To study the expression of the H19 gene in hepatocellular carcinoma. H19 is an imprinted, maternally expressed gene, which is tightly linked, both physically and functionally, to the paternally expressed insulin-like growth factor 2 (IGF II). IGF II is known to be involved in liver carcinogenesis. H19 was first discovered in the fetal mouse liver to be under the same regulatory genes as alpha fetoprotein (alpha FP), a widely used tumour marker for hepatocellular carcinoma. METHODS: Using both radioactive and non-radioactive in situ hybridisation, the expression of the H19 gene was compared with the presence of alpha FP, as demonstrated by immunohistochemistry, in 18 cases of hepatocellular carcinoma. RESULTS: H19 expression was present in 13 of 18 cases, whereas staining for alpha FP was positive in only nine of 18 cases. Concordance was found in 12 of 18 tumours (66.7%). In general, the staining pattern for H19 was more diffuse than the immunohistochemical staining for alpha FP. CONCLUSIONS: The addition of a non-radioactive in situ hybridisation assay for H19 RNA to the panel of tumour markers used for the histopathological and cytological diagnosis of hepatocellular carcinoma might be useful.

Adult↗

PubMed1997

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

Animals↗

PubMed1997

Unique expression patterns of H19 in human testicular cancers of different etiology.

The expression pattern of the imprinted human H19 gene was investigated in testicular cancers of different etiology, as well as in normal testicular parenchyma, parenchyma without germ cells, and adjacent to testicular germ cell tumors of adolescents and adults (TGCTs), using RNase protection analysis, mRNA in situ hybridization and reverse-transcription polymerase chain reaction. While different total expression levels were detected in spermatocytic seminomas, lymphomas, a Sertoli cell tumor and Leydig cell tumors, none showed a disturbance of monoallelic expression. Strikingly, the majority of invasive TGCTs revealed expression of both parental alleles. The total level of expression highly correlated with differentiation lineage and stage of maturation, similar to that as reported during early normal embryogenesis. Biallelic expression could also be determined specifically in testis parenchyma containing the preinvasive lesion of this cancer. We therefore conclude that within the adult testis, biallelic H19 expression is specific for TGCTs, and that the level of expression is dependent on differentiation lineage and maturation stage. This is in agreement with the proposed primordial germ cell-origin of this cancer, and might be related to retention of embryonic characteristics in TGCTs. In addition, our data argue against H19 being a tumor suppressor gene.

Adolescent↗

PubMed1997

Genomic imprinting and the endometrial cycle. The expression of the imprinted gene H19 in the human female reproductive organs.

H19 is an imprinted maternally expressed gene, which is not translated to protein and functions as an RNA molecule. It is closely related to the oppositely imprinted paternally expressed insulin-like growth factor 2 (IGF-2). While the biological function of H19 is not understood IGF-2 is a growth factor that plays a role in human follicular and endometrial differentiation. We examined the expression of H19 in the endometrium and ovary during the menstrual cycle by in situ hybridization applied to paraffin sections of human endometrium and ovaries at different stages of differentiation. In the endometrium, H19 expression was confined to the stroma and fluctuated with endometrial dating to reach its peak in the late secretory stage. IGF-2 was also prominently expressed in late secretory endometrium, but its expression was evident both in the stroma and glandular epithelium. Expression of H19 was not found in primordial, primary, and preantral follicles of the ovary, but prominent expression was evident in the theca of antral and cystic atretic follicles, and focal expression was noted in the granulosa of corpora lutea. An association between H19 expression during the menstrual cycle and the differentiation state of the human female reproductive tract, which is under hormonal control, is suggested.

Cell Differentiation↗

PubMed1997

The product of the imprinted H19 gene is an oncofetal RNA.

AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated.

Biomarkers, Tumor↗

PubMed1996

Characterization of the imprinted IPW gene: allelic expression in normal and tumorigenic human tissues.

IPW (Imprinted gene in the Prader-Willi syndrome region) is a recently identified paternally expressed gene. Previous work has demonstrated IPW expression in the human fetus and adult, with monoallelic expression in adult lymphoblasts and fibroblasts, and in fetal tissues. To further examine the expression of IPW, a series of experiments were carried out using RT-PCR to measure IPW expression in placentae and various fetal and tumor tissues. Biallelic expression of IPW was found in testicular germ cell tumor and bladder cancer cells, suggesting loss of imprinting in the latter case. Both H19 and Insulin-like growth Factor 2 (IGF2), two additional imprinted genes, also showed biallelic expression in those same tumors that demonstrated IPW biallelic expression. Of note, the naturally occurring parthenogenetic-derived mature teratoma unexpectedly expressed large amounts of IPW. Lastly, the pluripotent embryonal cancer cell line Tera-2 expressed IPW at the same level before and after differentiation induced by retinoic acid, suggesting that this gene functions in a 'housekeeping' capacity throughout cell growth. This was in contradistinction to H19 and IGF2, both of which showed significant transcriptional upregulation after Tera-2 cell differentiation.

Adult↗

PubMed1996

Relaxation of imprinting in carcinogenesis.

The study of genes at the heat of tumorigenesis has helped unveil an elaborate biochemical circuitry that governs the proliferation and differentiation of cells. Genomic imprinting is rapidly being recognized as a fundamental process in tumor biology. Aberrant relaxation of imprinted genes have been detected in a wide variety of cancers, of both embryonal and nonembryonal origin. However, despite a vast array of experimental observation, both the purpose and pathogenic mechanism of relaxation of imprinting remain an enigma. Hypotheses are examined in this regard along with speculation for future research.

Chromosome Aberrations↗

PubMed1996

Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts.

The role of different extracellular matrix (ECM)-degrading enzymes in the normal functioning of the placenta is well documented. Heparan sulphate proteoglycan (HSPG) is an integral constituent of the placental and decidual ECM. Because this proteoglycan specifically interacts with various macromolecules in the ECM, its degradation may disassemble the matrix. Hence, in the case of the placenta, this may facilitate normal placentation and trophoblast invasion. Crude placental specimens were collected from first and third trimester placentas. Heparanase (endo-beta-glucuronidase) was isolated and purified by ammonium sulphate precipitation followed by sequential chromatographies on carboxymethyl-, heparin- and ConA-Sepharose columns. The placental enzyme was further characterized for its molecular weight and specific inhibition by heparin, and was shown to resemble heparanase expressed by highly metastatic tumor cells and activated cells of the immune system. In order to locate the source of heparanase activity in the placenta, primary cytotrophoblast cultures were established. Intact cells, as well as conditioned medium and cell lysates, were analysed for heparanase activity using metabolically sulphate-labelled ECM as a natural substrate. Heparanase was highly active in lysates of cytotrophoblasts. This activity was also expressed by intact cytotrophoblasts seeded on ECM, but no activity could be detected in the culture medium. Incubation of the cytotrophoblasts in contact with ECM resulted in release of ECM-bound basic fibroblast growth factor (bFGF). We propose that the cytotrophoblastic heparanase facilitates placentation, through cytotrophoblast extravasation and localized neovascularization.

Cells, Cultured↗

PubMed1996

Hyaluronan, CD44 and its variant exons in human trophoblast invasion and placental angiogenesis.

Both hyaluronan and one of its receptors, CD44, can be demonstrated in the early human conceptus and in placental stroma. The variants of CD44 resulting from variable exon splicing are found in metastasizing human malignancies and are also involved in hyaluronan uptake and degradation. The resulting hyaluronan fragments are known to be highly angiogenic. We postulated that the self-limited process of trophoblast invasion of the uterine decidua results in part from the strategy of alternative splicing of CD44, similar to that used by invasive cancer cells in the course of metastatic spread and possibly angiogenesis. Monoclonal antibodies specific for CD44s and for an exon expressed during metastatic tumour progression, CD44v7, were used to examine this hypothesis. In this study we found human trophoblasts, for the first time, to express CD44. Intermediate trophoblasts of first and second trimester exhibited the standard form of CD44 while extravillous trophoblasts, which are responsible for the invading characteristics of the placenta, were positive for the alternatively spliced form, the CD44v7-8. Moreover, in the case of placenta accreta there was a prominent membrane staining of the trophoblasts that were embedded in the fibrin layer over the myometrium. The highly metastatic choriocarcinoma cells also expressed CD44v7-8. We propose, therefore, that the invading trophoblasts utilize the alternatively splicing machinery. These cells retain their invasive capabilities through the permissive ECM by carrying the CD44v7-8 isoform, which binds weakly to hyaluronan and thus prevents it from being degraded by intracellular hyaluronidase.

Alternative Splicing↗

PubMed1996

Double mutants of Saccharomyces cerevisiae with alterations in global genome and transcription-coupled repair.

The nucleotide excision repair (NER) pathway is thought to consist of two subpathways: transcription-coupled repair, limited to the transcribed strand of active genes, and global genome repair for nontranscribed DNA strands. Recently we cloned the RAD26 gene, the Saccharomyces cerevisiae homolog of human CSB/ERCC6, a gene involved in transcription-coupled repair and the disorder Cockayne syndrome. This paper describes the analysis of yeast double mutants selectively affected in each NER subpathway. Although rad26 disruption mutants are defective in transcription-coupled repair, they are not UV sensitive. However, double mutants of RAD26 with the global genome repair determinants RAD7 and RAD16 appeared more UV sensitive than the single rad7 or rad16 mutants but not as sensitive as completely NER-deficient mutants. These findings unmask a role of RAD26 and transcription-coupled repair in UV survival, indicate that transcription-coupled repair and global genome repair are partially overlapping, and provide evidence for a residual NER modality in the double mutants. Analysis of dimer removal from the active RPB2 gene in the rad7/16 rad26 double mutants revealed (i) a contribution of the global genome repair factors Rad7p and Rad16p to repair of the transcribed strand, confirming the partial overlap between both NER subpathways, and (ii) residual repair specifically of the transcribed strand. To investigate the transcription dependence of this repair activity, strand-specific repair of the inducible GAL7 gene was investigated. The template strand of this gene was repaired only under induced conditions, pointing to a role for transcription in the residual repair in the double mutants and suggesting that transcription-coupled repair can to some extent operate independently from Rad26p. Our findings also indicate locus heterogeneity for the dependence of transcription-coupled repair on RAD26.

Adenosine Triphosphatases↗

PubMed1996

Developmentally imprinted genes as markers for bladder tumor progression.

PURPOSE: Developmentally imprinted genes, such as H19 and insulin-like growth factor-II (IGF-II), play an important role during human embryogenesis and also have been implicated in the pathogenesis of embryonal tumors of childhood. Since H19 is expressed in human fetal bladder, we evaluated 35 bladder carcinomas for H19 expression by in situ hybridization analysis and correlated expression with tumor grade. As a prelude to gene transfer studies to determine if H19 is a bladder tumor oncogene, we also evaluated bladder cell lines for expression of H19, IGF-II, IGF-I and the type I IGF receptor. MATERIALS AND METHODS: H19 expression was evaluated by in situ hybridization analysis in bladder tumor specimens. Northern analysis was used to evaluate the expression of H19, IGF-II, IGF-I and the type I IGF receptor in bladder cell lines. RESULTS: H19 was expressed preferentially in advanced stage tumors: 2 of 12 grade I tumors were H19 positive, whereas 9 of 11 grade II and 7 of 10 grade III tumors expressed H19 (p = 0.004). Additionally, 6 of 6 carcinoma in situ tumors were H19 positive, whereas normal bladder mucosa cells were H19 negative. We found that 3 of 11 cell lines (HT-1376, HT-1197 and 5637) express high levels of H19 mRNA, and each of these cell lines and J82 also express IGF-II. All cell lines examined expressed the type I IGF receptor, whereas there was no detectable IGF-I mRNA. CONCLUSIONS: These data demonstrate that H19 is an oncodevelopmental marker of bladder tumor progression and raise the possibility that H19 may have oncogenic properties in bladder cancer.

Biomarkers, Tumor↗

PubMed1995

The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma.

The imprinted H19 gene is highly expressed in human embryos, fetal tissues and is nearly completely shut off in adults. However, it is reexpressed in a number of tumors including bladder carcinoma, demonstrating that H19 RNA is an oncofetal RNA. Tumors induced by injection of bladder carcinoma cell lines express H19 in contrast to the cells before injection. These observations support the notion of a positive correlation between H19 expression and bladder carcinoma. Loss of imprinting of H19 and IGF-2 was observed in samples of human bladder carcinoma.

Animals↗

PubMed1995

H19 expression and tumorigenicity of choriocarcinoma derived cell lines.

Certain embryonal tumors demonstrate a loss of heterozygosity at the parentally imprinted region of chromosome 11p15.5. It has been hypothesized that this implicates a tumor suppressor gene at this locus. The human H19 gene maps to 11p15.5, is expressed in fetal tissues including the placenta and is paternally imprinted. Here we show that the abundance of H19 transcripts in cells of two choriocarcinoma derived cell lines (JAr and JEG-3) differs greatly. While JAr cells express high levels of H19 RNA, the expression of H19 in JEG-3 cells is much lower than that of normal trophoblasts. Cells of these two cell lines were subcutaneously injected into nude mice with subsequent tumor formation. A fivefold increase in the H19 RNA level was measured in tumors derived from JEG-3 cell lines as compared to these cells before injection. However this increase in H19 RNA did not alter the clonogenicity in soft agar nor the growth rate of the cells derived from these tumors as compared to the original JEG-3 cells. Nevertheless, the cells retaining the elevated level of H19 transcripts were more tumorigenic than the original cells. We propose that there is a selection of cells expressing high levels of H19 from the total JEG-3 cell population during the microevolution of tumor formation. These observations, together with our previous publications on H19 expression in human cancers, do not support the notion of a tumor suppressor role for the H19 gene.

Animals↗

PubMed1995

The interaction between cytotrophoblasts and their derived tumor cells.

Previous experiments demonstrated that human cytotrophoblasts and cells of the choriocarcinoma cell line JAr interact in vitro. As a result of this interaction there is an increased synthesis of CG and hPL, probably as a result of the increased CG and hPL synthesis by the cytotrophoblasts. In the present investigation we studied this interaction in greater detail and found that both cytotrophoblasts and JAr cells undergo changes in their biological properties as a result of this interaction. JAr cells and cytotrophoblasts cocultured for 72 hr were fractionated according to their size by centrifugal elutriation. The number of cells in the fraction which contain the largest cells was very significantly increased as a result of the coculture. This increase was due to an increase in the number of cells of both cell types. This fraction was the most active one in the synthesis of CG and hPL. The synthesis of DNA by the JAr nuclei in this fraction of the cocultured cells was almost completely inhibited but in the parallel fraction of the JAr cells cultivated alone the level of DNA synthesis was equal to that of all other JAr cell fractions. Heterokaryons are formed in the coculture. In these heterokaryons a factor which inhibits DNA synthesis in the cytotrophoblasts may inhibit DNA synthesis in JAr nuclei and at least be partly responsible for the inhibition of DNA synthesis observed.

Animals↗