Biomedical subjects

M Schutte

Publications and source records attributed to M Schutte.

At least 19 recordsLinked to original sources

PubMed2006

Association between the CHEK2*1100delC germ line mutation and estrogen receptor status.

Already published data were further analyzed regarding the association between the CHEK2*1100delC germ line mutation and estrogen receptor (ER) status in patients with breast cancer. The CHEK2*1100delC mutation was more prevalent among the patients with a positive ER status (4.2% versus 1.0%). An ER-negative status was beside CHEK2*1100delC mutation and independently associated with an earlier of age onset of breast cancer. There was a trend that an ER-negative status, beside the presence of a CHEK2*1100delC mutation, was associated with a worse disease-free survival. There might be an association between ER status and a CHEK2*1100delC mutation. More studies with larger number of patients are needed to further investigate the relation between CHEK2*1100delC and ER status.

Breast Neoplasms↗

PubMed2004

Effect of memantine on the alpha 7 neuronal nicotinic receptors, synaptophysin- and low molecular weight MAP-2 levels in the brain of transgenic mice over-expressing human acetylcholinesterase.

Transgenic mice over-expressing human acetylcholinesterase (hAChE-Tg) display memory impairments, cholinergic deficits and reduced dendritic branching. In this study, we found a reduced number of N-Methyl-D-Aspartate (NMDA) binding sites and reduced levels of low molecular weight (LMW) microtubule associated protein 2 (MAP-2), in addition to an increased number of alpha4 and alpha7 nicotinic receptor (nAChR) binding sites in the brain of hAChE-Tg mice. Treatment with memantine, 20 mg/kg/day during 14 days, significantly increased the number of [(125)I]alphabungarotoxin (alpha7 nAChR) binding sites in the frontal- and retrosplenial cortex of hAChE-Tg mice and synaptophysin- and LMW MAP-2 levels in the cortex of both hAChE-Tg and FVB/N controls. The findings reveal an alteration of the glutamatergic system in hAChE-Tg mice. Whether the effect of memantine on alpha7 nAChRs, synaptophysin- and LMW MAP-2 levels is a direct effect, or an indirect effect via the NMDA receptors, has to be further evaluated.

Acetylcholinesterase↗

PubMed2004

The CHEK2(*)1100delC mutation has no major contribution in oesophageal carcinogenesis.

In response to DNA damage, the cell cycle checkpoint kinase 2 (CHEK2) may phosphorylate p53, Cdc25A and Cdc25C, and regulate BRCA1 function, leading to cell cycle arrest and DNA repair. The truncating germline mutation CHEK2(*)1100delC abrogates kinase activity and confers low-penetrance susceptibility to breast cancer. We found CHEK2(*)1100delC in 0.5% of 190 oesophageal squamous cell carcinomas and in 1.5% of 196 oesophageal adenocarcinomas. In addition, we observed the mutation in 3.0% of 99 Barrett's metaplasias and 1.5% of 66 dysplastic Barrett's epithelia, both known precursor lesions of oesophageal adenocarcinoma. Since CHEK2(*)1100delC mutation frequencies did not significantly differ among oesophageal squamous cell carcinomas, adenocarcinomas and (dysplastic) Barrett's epithelia, as compared to healthy individuals, we conclude that the CHEK2(*)1100delC mutation has no major contribution in oesophageal carcinogenesis.

Adenocarcinoma↗

PubMed2004

Tumour characteristics and prognosis of breast cancer patients carrying the germline CHEK2*1100delC variant.

BACKGROUND: The germline CHEK2*1100delC variant has been associated with breast cancer in multiple case families where involvement of BRCA1 and BRCA2 has been excluded. METHODS: We have investigated the tumour characteristics and prognosis of carriers of this germline variant by means of a prospective cohort study in an unselected cohort of 1084 consecutive patients with primary breast cancer. Data were collected for 34 patients with a germline CHEK2*1100delC mutation and for 102 patients without this mutation, stratified by age and date of diagnosis of the first primary breast cancer (within 1 year). RESULTS: Carriers developed steroid receptor positive tumours (oestrogen receptor (ER): 91%; progesterone receptor (PR): 81%) more frequently than non-carriers (ER: 69%; PR: 53%; p = 0.04). Mutation carriers more frequently had a female first or second degree relative with breast cancer (p = 0.03), or had any first or second degree relative with breast or ovarian cancer (p = 0.04). Patients with the CHEK2 variant had a more unfavourable prognosis regarding the occurrence of contralateral breast cancer (relative risk (RR) = 5.74; 95% confidence interval (CI) 1.67 to 19.65), distant metastasis-free survival (RR = 2.81; 95% CI 1.20 to 6.58), and disease-free survival (RR = 3.86; 95% CI 1.91 to 7.78). As yet, no difference with respect to overall survival has been found at a median follow up of 3.8 years. CONCLUSION: We conclude that carrying the CHEK2*1100delC mutation is an adverse prognostic indicator for breast cancer. If independently confirmed by others, intensive surveillance, and possibly preventive measures, should be considered for newly diagnosed breast cancer cases carrying the CHEK2*1100delC variant.

Adult↗

PubMed2003

Allelotype of 28 human breast cancer cell lines and xenografts.

Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (<25%) and 17 arms had moderate allelic loss frequencies (25-50%). Five chromosomal arms were deleted in more than half of the breast cancer samples (8p, 10q, 13q, 17p, and 17q). Three of these frequently lost chromosomal arms had not been identified as such by comparative genome hybridisation, illustrating the higher sensitivity of microsatellite analysis for the detection of allelic losses. As we present allelic loss data of individual samples, our allelotype should not only aid the identification of new breast cancer genes but also provides a baseline for myriad studies involving these breast cancer cell lines.

Alleles↗

PubMed2001

Mutant E-cadherin breast cancer cells do not display constitutive Wnt signaling.

Participation of E-cadherin in the Wnt signaling pathway was suggested because of the dual role of beta-catenin in cell adhesion and the Wnt signaling cascade. Whereas beta-catenin interacts at the cell membrane with the cell adhesion protein E-cadherin, in the nucleus it activates Wnt target genes through formation of transcriptionally active complexes with members of the Tcf/Lef family of transcription factors. Here, we analyzed by PCR and direct cycle sequencing 26 human breast cancer cell lines for alterations in the E-cadherin gene. Genetic alterations were identified in eight cell lines. Five cell lines had truncating mutations, whereas three cell lines had in-frame deletions in the gene transcript and expressed mutant E-cadherin proteins at the cell membrane. Involvement of E-cadherin in the Wnt pathway was evaluated through determination of the activity of a Tcf reporter gene, which had been transiently transfected into 15 breast cancer cell lines. None of six E-cadherin mutant cell lines and four cell lines that exhibit transcriptional silencing of the E-cadherin gene showed Tcf-mediated transcriptional activation. E-cadherin wild-type cell line DU4475 exhibited constitutive Tcf-beta-catenin signaling activity and was found to express truncated APC proteins. These results indicate that if cellular transformation occurred through mutation of E-cadherin, it is not mediated via constitutive activation of the Wnt signaling pathway.

Breast Neoplasms↗

PubMed2000

Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors.

BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.

Breast Neoplasms↗

PubMed2000

Immunohistochemical p16INK4a analysis of archival tumors with deletion, hypermethylation, or mutation of the CDKN2/MTS1 gene. A comparison of four commercial antibodies.

The MTS1/CDKN2/p16 gene encoding the p16INK4a tumor-suppressor protein is commonly inactivated by homozygous deletion or hypermethylation of the promoter in a wide range of human malignancies. In select tumor types, including pancreatic adenocarcinomas, intragenic mutations are found in a significant percentage of cases. The immunoreactivity of mutant p16 proteins has not been comprehensively studied. Moreover, the immunohistochemical properties of commercially available antibodies have not been described in detail. We studied 35 pancreatic adenocarcinomas with a molecularly defined p16 status (16 homozygous deletions, 3 hypermethylated cases, and 16 tumors with an intragenic mutation in one allele associated with loss of the second allele). In addition, we studied nine cell lines (three homozygous deletions, three hypermethylated lines, and three intragenic mutations). Paraffin sections of the tumors and cell blocks were reacted with four different anti-p16 antibodies: polyclonal and monoclonal (clone G175-405) antibodies from PharMingen, monoclonal antibody DCS-50 from Oncogene Science, and monoclonal antibody ZJ11 from Neo-Markers. Optimal staining conditions were established for each antibody. The pancreatic carcinomas with homozygous p16 deletions were largely devoid of nuclear staining (admixed nonneoplastic cells served as internal positive controls); only one adenocarcinoma each reacted with DCS-50 and the polyclonal antibody, and five were positive with ZJ11, suggesting that nonspecific nuclear staining can occur under certain conditions. Antibody DCS-50 produced nuclear staining in all three hypermethylated carcinomas, whereas G175-405 stained none of them. Three of the four antibodies produced nuclear immunoreactivity in 7 to 14 of the 16 carcinomas carrying p16 mutations; G175-405 showed only weak reactivity in one case. Cytoplasmic staining was present in all carcinomas and cell lines and with all antibodies and therefore cannot be considered specific; it was strongest with G175-405. Thus, we found antibody G175-405 to be the most specific, and monoclonals DCS-50 and ZJ11 the least specific for wild-type p16. However, the former tends to give stronger cytoplasmic background staining. For tumor types in which p16 mutations are uncommon, the PharMingen polyclonal antibody may be a suitable alternative.

Antibodies↗

PubMed1999

Transforming growth factor-beta responsiveness in DPC4/SMAD4-null cancer cells.

DPC4/SMAD4 is a candidate tumor suppressor gene with a strikingly high frequency of gene alterations in pancreatic cancer that suggests a discrete role for DPC4 in these tumors. DPC4 tumor-suppressive function has been implicated to mediate the transforming growth factor-beta (TGFbeta)-suppressive pathway; however, in a DPC4-null pancreatic cancer cell line, TGFbeta growth-inhibitory and transcriptional responses were found to be DPC4-independent. This was observed within native cells having a natural homozygous deletion and in clones engineered for stable expression of wild-type DPC4 integrated into the genome. This observation contrasted with the absolute DPC4 dependence of TGFbeta responses in a breast cancer cell line studied in parallel. This growth-inhibitory response to TGFbeta in DPC4-null cells relied on an intact ras effector pathway. These data further suggest a major categorization of TGFbeta responses into DPC4-dependent and -independent signaling pathways and specifically suggest that disruption of the TGFbeta-independent signal might be a basis of selection for the emergence of DPC4 alterations during tumorigenesis in the pancreas and other sites.

Alleles↗

PubMed1999

DPC4/SMAD4 gene alterations in human cancer, and their functional implications.

The vast majority of pancreatic carcinomas have allelic loss of chromosome 18. Detailed analysis revealed a consensus region of homozygous deletion at 18q21.1 in one third of pancreatic carcinomas. The DPC4 gene (also known as SMAD4) was located in this region, and was found to be inactivated by intragenic mutations in another 20% of pancreatic carcinomas. The Dpc4 protein was shown to mediate TGF beta-stimulated gene transcription through sequence-specific binding to DNA. Eleven mutant Dpc4 proteins, identified in human carcinomas, were all found to be impaired in their ability to regulate gene transcription. A functional grouping of the mutant proteins could be made in those that were deficient in DNA binding, those that had impaired nuclear translocation, and those that had affected their transcription activation domain. The results imply gene transcription mediated by Dpc4 as a critical tumour suppressive pathway in pancreatic carcinoma.

Activin Receptors, Type I↗

PubMed1998

Identification of 12p as a region of frequent deletion in advanced prostate cancer.

The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.

Adenocarcinoma↗

PubMed1998

Dpc4 transcriptional activation and dysfunction in cancer cells.

Dpc4 (Smad4) is implicated in mediation of signals from transforming growth factor (TGF) beta and related ligands, and wild-type Dpc4 mediates TGF-beta-stimulated gene transcription at specific DNA sequences bound by Dpc4 [Smad binding element (SBE)]. We characterized panels of DPC4 tumor mutations and cancer cell lines. Amino acid substitutions within the NH2-terminal third of Dpc4 weakened or ablated SBE-mediated gene regulation by a disruption of DNA binding. An interaction of the COOH-terminal end with the DNA-binding domain of Dpc4 was evident but was not required to explain the functional impairment produced by NH2-terminal DPC4 mutations. Both substitution and truncation mutations of the COOH-terminal half of DPC4 lacked the ability to regulate transcription while retaining the sequence-specific DNA-binding function, but through differing mechanisms. A modular domain to redistribute Dpc4 to the nuclear compartment allowed SBE-mediated transcriptional activation in a cell line having a TGF-1 receptor defect and was sufficient to restore SBE-mediated transactivation ability to COOH-terminal DPC4 missense mutants. Cells harboring DPC4 alterations had a universal impairment of the TGF-beta-stimulated SBE transcriptional response. These studies identify the loss of SBE-mediated gene regulation as a uniform outcome of the selection for DPC4 alterations during tumorigenesis. They raise the possibility of restoration of some Dpc4-associated transcriptional events in cancer cells through the targeted redistribution of wild-type and some missense mutant forms of Dpc4 to the nucleus.

DNA↗

PubMed1997

Abrogation of the Rb/p16 tumor-suppressive pathway in virtually all pancreatic carcinomas.

The Rb/p16 tumor-suppressive pathway is abrogated frequently in human tumors, either through inactivation of the Rb or p16INK4a/CDKN2/MTS1 tumor-suppressor proteins, or through alteration or overexpression of the cyclin D1 or cyclin-dependent kinase 4 oncoproteins. We reported previously that the p16 gene was genetically inactivated in 82% of pancreatic carcinomas. Nearly half of these inactivations were by intragenic mutation of p16, and the remainder were by homozygous deletion of the gene. Here, we analyzed pancreatic carcinomas for additional mechanisms by which the Rb/p16 pathway might be inactivated. Transcriptional silencing of the p16 gene in association with methylation of its 5'-CpG island was examined by methylation-specific PCR in 18 pancreatic carcinomas. Nine of these were known to harbor an intragenic mutation in p16, and nine had a wild-type p16 coding sequence. Seven of the 18 tumors were hypermethylated, and all 7 were p16 wild-type (P = 0.001). Complete silencing of transcription from methylated wild-type gene sequences was demonstrated. Immunohistochemical analysis revealed normal expression levels of the Rb protein in all carcinomas studied. None of the carcinomas had genomic amplification of the cyclin D1 or CDK4 genes, and none had mutation of the p16-binding domain of CDK4. An additional p16 mutation was identified. In total, the Rb/p16 pathway was abrogated in 49 of the 50 carcinomas (98%) studied, all through inactivation of the p16 gene. Similar results were obtained in an independently analyzed series of 19 pancreatic carcinomas. These data demonstrate the central role of the Rb/p16 pathway in the development of pancreatic carcinoma.

Carrier Proteins↗

PubMed1997

Tumor-suppressive pathways in pancreatic carcinoma.

During tumorigenesis, positive selection is exerted upon those tumor cells that alter rate-limiting regulatory pathways. A corollary of this principle is that mutation of one gene abrogates the need for alteration of another gene in the same pathway and also that the coexistence in a single tumor of mutations in different genes implies their involvement in distinct tumor-suppressive pathways. We studied 42 pancreatic adenocarcinomas for genetic alterations in the K-ras oncogene and the p16, p53, and DPC4 tumor suppressor genes. All of them had the K-ras gene mutated. Thirty-eight % of the tumors had four altered genes, another 38% had three altered genes, 15% had two altered genes, and 8% of the tumors had one altered gene. Interestingly, we noted a high concordance of DPC4 and p16 inactivations (P = 0.007), suggesting that the genetic inactivation of p16 increases the selective advantage of subsequent mutation in DPC4. No statistically significant association was identified between the alteration of these cancer genes and pathological or clinical parameters. This type of multigenic analysis in human tumors may serve to substantiate experimental tumor models and thus increase our understanding of the truly physiologically relevant tumor-suppressive pathways that are abrogated during human tumorigenesis.

Adenocarcinoma↗

PubMed1997

Genomic sequencing of DPC4 in the analysis of familial pancreatic carcinoma.

A first-degree relative with pancreatic cancer is found in 5% to 10% of patients with pancreatic carcinomas, suggesting an inherited predisposition for this neoplasm. The recently identified DPC4 tumor suppressor gene is a strong candidate for the gene responsible for the familial form of pancreatic carcinoma. DPC4 was identified in a consensus area of homozygous deletion in pancreatic carcinomas, and it is biallelically inactivated in approximately 50% of sporadic pancreatic carcinomas. The coding sequence of this gene is 1660 nucleotides in length, covering 11 exons. We describe optimized primers and conditions used in polymerase chain reaction and cycle sequencing of the entire DPC4 coding sequence of 25 individuals (eight with pancreatic carcinoma) from 11 kindreds with a familial aggregation of pancreatic carcinoma. No mutations in the coding sequences of the DPC4 gene were found; hence, it appears that germline mutations in DPC4 cannot account for many of the familial aggregations of pancreatic carcinoma.

Carcinoma↗

PubMed1997

DPC4 gene mutation in colitis associated neoplasia.

BACKGROUND: Colitis associated dysplasia and cancer often have deletions involving the long arm of chromosome 18q, suggesting the location of a tumour suppressor gene critical for their tumorigenesis. The DPC4 gene, which is genetically inactivated in pancreatic and other cancers, has recently been described. AIM: Because DPC4 is located at 18q21.1, the hypothesis that it could be a mutated tumour suppressor gene in colitis associated neoplasms was tested. PATIENTS: Advanced neoplastic lesions from six patients having chronic colitis were analysed for DPC4. METHODS: Individual exons of DPC4 were amplified by the polymerase chain reaction (PCR) and sequenced from genomic DNA of tissue specimens dissected by cryostat. RESULTS: DPC4 was found to have biallelic inactivation in one of three neoplasms shown to have allelic loss of 18q. The mutation had been acquired somatically in a plaque of high grade dysplasia. The mutation created a non-sense codon, which would cause premature termination of protein translation. CONCLUSION: The DPC4 gene is a target of 18q LOH events in colitis associated neoplasia.

Adult↗

PubMed1996

Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas.

Germline mutations in BRCA2 predispose carriers to the development of breast, ovarian, and a variety of other cancers. The original localization of the BRCA2 gene was aided by its homozygous deletion in a pancreatic carcinoma; indeed, an excess of pancreatic carcinoma has been seen in some BRCA2 cancer families. To determine the involvement of BRCA2 in pancreatic carcinomas, we screened for BRCA2 alterations in an unselected panel of 41 adenocarcinomas of the pancreas (30 pancreatic adenocarcinoma xenografts and 11 pancreatic cancer cell lines). Of the 15 (27%) that had allelic loss at the BRCA2 locus, 4 (9.8%) had abnormalities in the second allele upon screening of the entire BRCA2 gene by in vitro synthesized protein assay. Three of the four mutations were considered germline in origin (7.3% overall; two were confirmed in normal tissue, and one was the 6174delT mutation from the pancreatic cancer cell line CAPAN-1, for which normal tissue was unavailable). The identification of two 6174delT mutations in this series prompted us to evaluate the prevalence of this mutation in an overlapping consecutive series of 245 patients who underwent pancreatoduodenectomy for adenocarcinoma of the pancreas. Sequence analysis of this limited region of the gene identified two additional mutations: (a) one additional germline 6174delT mutation (2 of 245, 0.8% overall); and (b) a second nearby germline 6158insT mutation. One of the patients with a germline mutation had a single relative with breast cancer, and another had a single relative with prostate cancer. None had a family history of pancreatic cancer. The incidence of germline BRCA2 mutations in apparently sporadic pancreatic cancer may be at least as high as in breast or ovarian cancer. Our results suggest that some familial risks for carcinoma will be evident only through a population-based application of gene screening techniques because a low disease penetrance of the germline mutations in some families often evades clinical suspicion.

Adenocarcinoma↗

PubMed1996

Allelic loss and mutational analysis of the DPC4 gene in esophageal adenocarcinoma.

DPC4, a recently cloned gene located on 18q2l.l, is inactivated in almost one half of pancreatic adenocarcinomas. To determine whether DPC4 inactivation is involved in esophageal adenocarcinoma, we have analyzed aneuploid populations from biopsies of 35 patients with Barrett's esophagus who had premalignant epithelium, adenocarcinoma, or both. Sixteen of 35 patients (46%) had allelic loss at l8q21.1, including 7 patients who had only premalignant tissue present in their Barrett segment. In addition, three of four patients (75%) with l8q21.1 loss in their aneuploid populations had the allelic loss present in diploid cells. Mutational analysis of DPC4 did not reveal any inactivating alterations in the gene. These data indicate that allelic losses at l8q are selected during neoplastic progression in Barrett's esophagus, but the targeted gene remains to be identified.

Alleles↗