Hetty M van Beerendonk

Leiden University Medical Centre, Leiden, South Holland, Netherlands

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Publications (8)33.84 Total impact

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    ABSTRACT: Chondrosarcoma is a malignant cartilaginous matrix-producing tumor that can be lethal in 10% to 50% of the patients. Surgery is the only effective treatment known as these tumors are notorious refractory to all types of conventional chemotherapy or radiotherapy. To identify a target for therapy, we want to determine whether estrogen signaling is active in chondrosarcoma because estrogen is important in the regulation of longitudinal growth that is initiated by chondrocyte proliferation and differentiation in the epiphyseal growth plate of long bones. We studied protein expression of the estrogen receptor in 35 cartilaginous tumors as well as mRNA levels for the estrogen receptor and for aromatase, an enzyme for estrogen synthesis and another potential therapeutic target. Furthermore, the activity of aromatase was determined in vitro by the tritiated water release assay. Dose-response experiments with chondrosarcoma cultured cells were done with estrogen, androstenedione, and exemestane. All chondrosarcomas tested showed mRNA and nuclear protein expression of the estrogen receptor. Also, aromatase mRNA was detected. The aromatase activity assay showed a functional aromatase enzyme in primary chondrosarcoma cultures and in a cell line. Growth of chondrosarcoma cell cultures can be stimulated by adding estrogen or androstenedione, which can be inhibited by exemestane. These results show, on the RNA, protein, and cell biological levels, that the ligand and the receptor are active in estrogen-mediated signal transduction. This observation implicates potential use of targeted drugs that interfere with estrogen signaling, such as those applied for treating breast cancer.
    Clinical Cancer Research 12/2005; 11(22):8028-35. · 7.84 Impact Factor
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    ABSTRACT: Loss of heterozygosity (LOH) at chromosomal band 9p21 is one of the few consistent genetic aberrations found in conventional chondrosarcoma. This locus harbours two cell-cycle regulators, CDKN2A/p16/INK4A and INK4A-p14ARF, which are inactivated in various human malignancies. It was therefore hypothesized that this locus also plays a role in the development of chondrosarcoma and this locus was investigated at protein, genetic, and epigenetic levels. Loss of p16 protein expression was detected by immunohistochemistry in 12 of 73 central chondrosarcomas and it correlated with increasing histological grade (p = 0.001). Loss of p16 protein expression was not found in 51 enchondromas, which are presumed to be potential precursors of conventional central chondrosarcoma. LOH at 9p21 was found in 15 of 39 chondrosarcomas (38%) but it did not correlate with loss of p16 protein expression. SSCP analysis of p16 did not reveal any mutations in 47 cases. Also, p14 was not the target of LOH, since it gave no aberrant bands on SSCP. To investigate whether an epigenetic mechanism was operating, methylation-specific PCR was used to look at p16 promotor methylation, which was identified in 5 of 30 tumours. However, this did not correlate with protein expression, or with LOH at 9p21. Cytogenetic data were available in a subset of cases. All tumours that showed chromosome 9 alterations also showed LOH and loss of INK4A/p16 protein expression. It is concluded that although some alterations were found at the DNA level and at the promoter expression level, the lack of correlation between LOH, promotor methylation, and protein expression indicates that a locus other than CDKN2A/p16 must be the target of LOH at 9p21. The correlation between INK4A/p16 protein expression and tumour grade, and the retention of expression in enchondromas, indicates that loss of INK4A/p16 protein expression may be an important event during tumour progression from enchondroma to conventional central chondrosarcoma, and in the progression in grade after recurrence of chondrosarcoma.
    The Journal of Pathology 04/2004; 202(3):359-66. · 7.59 Impact Factor
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    Journal of Pathology, 202, 359 - 366 (2004). 01/2004;
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    ABSTRACT: High quality RNA isolation from cartilaginous tissue is considered difficult because of relatively low cellularity and the abundance of extracellular matrix rich in glycosaminoglycans and collagens. Given the growing interest and technical possibilities to study RNA expression at a high throughput level, research on tissue with these characteristics is hampered by the lack of an efficient method for obtaining sufficient amounts of high quality RNA. This paper presents a robust protocol combining two RNA isolation procedures, based on a combination of Trizol and RNA specific columns, which has been developed to obtain high molecular weight RNA from fresh frozen and stored tissue of normal cartilage and cartilaginous tumours. Using this method, RNA was isolated from normal cartilage, peripheral chondrosarcoma, and central chondrosarcoma. The yields ranged from 0.1 to 0.5 microg RNA/mg tissue. RNA isolated with this method was stable and of high molecular weight. RNA samples from normal cartilage and from two chondrosarcomas isolated using this method were applied successfully in cDNA microarray experiments. The number of genes that give interpretable results was in the range of what would be expected from microarray results obtained on chondrosarcoma cell line RNA. Signal to noise ratios were good and differential expression between tumour and normal cartilage was detectable for a large number of genes. With this newly developed isolation method, high quality RNA can be obtained from low cellular tissue with a high extracellular matrix component. These procedures can also be applied to other tumour material.
    Journal of Clinical Pathology 11/2001; 54(10):778-82. · 2.44 Impact Factor
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    ABSTRACT: Loss of heterozygosity (LOH) at the long arm of chromosome 16 occurs in at least half of all breast tumors and is considered to target one or more tumor suppressor genes. Despite extensive studies by us and by others, a clear consensus of the boundaries of the smallest region of overlap (SRO) could not be identified. To find more solid evidence for SROs, we tested a large series of 712 breast tumors for LOH at 16q using a dense map of polymorphic markers. Strict criteria for LOH and retention were applied, and results that did not meet these criteria were excluded from the analysis. We compared LOH results obtained from samples with different DNA isolation methods, ie., from microdissected tissue versus total tissue blocks. In the latter group, 16% of the cases were excluded because of noninterpretable LOH results. The selection of polymorphic markers is clearly influencing the LOH pattern because a chromosomal region seems more frequently involved in LOH when many markers from this region are used. The LOH detection method, i.e., radioactive versus fluorescence detection, has no marked effect on the results. Increasing the threshold window for retention of heterozygosity resulted in significantly more cases with complex LOH, i.e., several alternating regions of loss and retention, than seen in tumors with a small window for retention. Tumors with complex LOH do not provide evidence for clear-cut SROs that are repeatedly found in other samples. On disregarding these complex cases, we could identify three different SROs, two at band 16q24.3 and one at 16q22.1. In all three tumor series, we found cases with single LOH regions that designated the distal region at 16q24.3 and the region at 16q22.1. Comparing histological data on these tumors did not result in the identification of a particular subtype with LOH at 16q or a specific region involved in LOH. Only the rare mucinous tumors had no 16q LOH at all. Furthermore, a positive estrogen content is prevalent in tumors with 16q LOH, but not in tumors with LOH at 16q24.3 only.
    Cancer Research 03/2001; 61(3):1171-7. · 8.65 Impact Factor
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    ABSTRACT: Canavan disease is a severe progressive autosomal recessive disorder, which is characterised by spongy degeneration of the brain. The disease is caused by mutations in the aspartoacylase gene. Two different mutations were reported on 98% of the alleles of Ashkenazi Jewish patients, in which population the disease is highly prevalent. In non-Jewish patients of European origin, one mutation (914C > A) is found in 50% of the alleles, the other alleles representing all kinds of different mutations. We here describe the results of the mutation analysis in 17 European, non-Jewish patients. Ten different mutations were found, of which four had not been described before (H21P, A57T, R168H, P181T). A deletion of exon4, which until now had only been described once, was revealed in all five alleles of Turkish origin tested, indicating that this is a founder effect in the Turkish population.
    European Journal of HumanGenetics 08/2000; 8(7):557-60. · 4.32 Impact Factor
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    ABSTRACT: In a search for candidate tumor suppressor genes within a 650-kb common region of loss of heterozygosity (LOH) at 16q24.3 in breast cancer tissues, a 2.6-kb cDNA, named copine VII (CPNE7), was characterized. The gene is 2654 bp and codes for a 633-residue protein with high homology to the other members of the copine family, such as copine I, copine III, and N-copine. The predicted amino acid sequence contains two copies of a C2 domain in the N-terminus. Since these domains have been found in several membrane-binding proteins involved in different intracellular processes, copine VII was viewed as a potential tumor suppressor gene. Mutation analysis was carried out by single-strand conformation polymorphism analysis of 18 breast tumor tissue samples with ascertained LOH on chromosome 16q24.3. Since only two polymorphisms were identified, no evidence was found to indicate that copine VII is the tumor suppressor gene at 16q24.3 involved in breast cancer.
    Genomics 11/1999; 61(2):219-26. · 3.01 Impact Factor
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    Journal of Clinical Pathology, 52, 778 - 782 (2001).