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Wolfgang A Schulz

Publications and source records attributed to Wolfgang A Schulz.

At least 19 recordsLinked to original sources
PubMed2006

In situ detection of global DNA hypomethylation in exfoliative urine cytology of patients with suspected bladder cancer.

Global DNA hypomethylation is a common phenomenon in bladder cancer. Therefore we investigated whether it is possible to detect and assess global DNA hypomethylation in bladder cancer using a specific monoclonal antibody for 5-methyl-cytosine. Cytospins from exfoliative urine cytology specimens of patients with bladder cancer or a history of bladder cancer, control patients with benign urological diseases and of young healthy volunteers were analyzed. Urothelial carcinoma (UC) cells showed various degrees of nuclear destaining indicating global DNA hypomethylation whereas all specimens from healthy volunteers showed granular nuclear staining indicating regular methylation of repeated DNA sequences. Lowest 5-methylcytosine immunostaining scores were observed in carcinoma cells and a statistically significant difference was observed between urothelial cells of healthy controls or patients with benign disease compared to bladder cancer patients (p<0.01, p<0.05, respectively). In UC cases even morphologically normal urothelial cells often displayed evident hypomethylation. Likewise, in patients with a history of UC, but no cystoscopic evidence of recurrence, morphologically non-malignant urothelial cells presented with some degree of demethylation. Our results strongly support the hypothesis of early global demethylation in bladder cancer. Immunocytochemical staining with the 5-methylcytosine antibody allows simultaneous individual assessment of nuclear morphology and methylation status of a given sample.

5-Methylcytosine↗
PubMed2006

Understanding urothelial carcinoma through cancer pathways.

Urothelial carcinoma (UC), the common histological subtype of bladder cancer, presents as a papillary tumor or as an invasive, often lethal form. To study UC molecular biology, candidate gene and genome-wide approaches have been followed. Here, it is argued that a 'cancer pathway' perspective is useful to integrate findings from both approaches. According to this view, papillary cancers typically exhibit activation of the MAPK pathway, as a consequence of oncogenic mutations in FGFR3 or HRAS, with increased Cyclin D1 expression. In contrast, invasive UC are characterized by severe disturbances in proximate cell cycle regulators, e.g. RB1 and CDKN2A/p16(INK4A), which decrease dependency on mitogenic signaling. In addition, these disturbances permit, promote and are in turn exacerbated by chromosomal instability, which is further enhanced by loss of TP53 function. In another vicious cycle, defective cell cycle regulation interacts with DNA methylation alterations. The transition toward invasive UC may require concomitant and interacting defects in cell cycle regulation and the control of genomic stability. Intriguingly, neither canonical WNT/beta-Catenin nor hedgehog signaling appear to play major roles in UC. This may reflect its origin from more differentiated urothelial cells possessing a high regenerative potential rather than a stem cell population.

Biomarkers, Tumor↗
PubMed2006

DNA methylation alterations in urothelial carcinoma.

In urothelial cancer, hypermethylation of specific genes and genome-wide hypomethylation, reflected in decreased methylation of LINE-1 retrotransposons, have both been reported, but were never investigated in the same specimens. We analyzed hypermethylation of six genes by methylation-specific PCR and LINE-1 hypomethylation by Southern blotting in 96 carcinoma tissues. Hypermethylation frequencies were: SFRP1 (55%), APC (45%), RASSF1A (35%), DAPK1 (29%), RARB2 (19%), and CDKN2A (2%). Three groups of cancers could be discerned, with escalating hypermethylation. Hypermethylation increased with tumor stage, particularly at the transition to invasive cancers, and RARB2 hypermethylation was indicative of lymph node involvement. A comparison to a previous study on prostate cancer using the same techniques suggests that hypermethylation in urothelial carcinoma occurs in a random rather than coordinated manner. LINE-1 hypomethylation was present in 90% of specimens, largely independent of hypermethylation. Lack of hypomethylation indicated a significantly better clinical prognosis. Bisulfite sequencing of SFRP1 demonstrated dense or patchy hypermethylation in tumor tissues that likely accounts for discrepant reported frequencies. In urothelial carcinoma cell lines, the same genes as in tissues were frequently hypermethylated. SFRP1 hypermethylation was concordant with lack of expression. 5-Aza-deoxycytidine induced its reexpression in some lines, whereas additional treatment with a histone deacetylase inhibitor was required in others. Thus, epigenetic SFRP1 inactivation occurs in a graduated manner. In conclusion, markers of genome-wide hypomethylation seem optimally suited for urothelial carcinoma detection, whereas combinations of hypermethylation and hypomethylation assays hold promise for classification.

Adult↗
PubMed2006

Epigenetic control of CTCFL/BORIS and OCT4 expression in urogenital malignancies.

Aberrant hypomethylation in many cancers reactivates retrotransposons and selected single-copy genes such as cancer-testis antigens. Genes reactivated in this manner have recently been postulated to include CTCFL/BORIS, a presumptive testis-specific chromatin regulator, and OCT4/POU5F1, a transcriptional activator in pluripotent cells. We found both genes expressed at high levels in testis and at much lower levels in normal prostate tissue. In prostate and bladder carcinoma cell lines and cancer tissues expression remained largely unchanged, but individual prostate carcinomas showed modestly increased CTCFL expression compared to normal tissues. OCT4 expression was significantly decreased in cancer tissues. Promoter methylation in both genes paralleled expression levels. CTCFL, but not OCT4 was dramatically induced in cancer cell lines by 5-aza-2'-deoxycytidine, but neither gene by the histone deacetylase inhibitor suberoylanilide hydroxamic acid. Thus, CTCFL and OCT4 resemble cancer-testis antigens in being selectively hypomethylated and expressed in male germ cells but differ in lacking significant reexpression and hypomethylation in prostate carcinomas. DNA methylation appears the crucial mechanism in the control of CTCFL transcription, but less decisive in that of OCT4. These findings imply that inhibitors of DNA methylation used for cancer treatment may induce CTCFL expression. Immunohistochemistry demonstrated nuclear localization of CTCFL in developing spermatocytes, and cytoplasmatic localization in spermatogonia, Leydig cells, and epithelial prostate cells. Teratocarcinoma cell lines showed nuclear, and 5-aza-2'-deoxycytidine-treated prostate cancer lines nuclear or cytoplasmatic localization. These different localizations might indicate additional control of CTCFL function via intracellular compartmentation.

Adenocarcinoma↗
PubMed2006

Expression of death-associated protein kinase during tumour progression of human renal cell carcinomas: hypermethylation-independent mechanisms of inactivation.

Death-associated protein kinase (DAPK) is a pro-apoptotic Ca(2+)/calmodulin-dependent serine/threonine kinase that is widely expressed in tissues but kept silent in growing cells. Downregulation of DAPK transcription by CpG methylation has been demonstrated in a variety of tumours, providing a selective growth advantage during tumour progression. As the in vivo expression of DAPK in human renal cell carcinomas (RCCs) has not previously been analysed, 72 RCCs were investigated using semi-quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). We found that almost 92% (66/72) of all primary RCCs express DAPK mRNA and results obtained from methylation-specific PCR analyses suggest that aberrant CpG methylation of the DAPK promoter is absent even in DAPK non-expressing tumours. Comparison of early/intermediate with advanced tumour stages of clear cell RCCs showed that no significant changes in the expression levels of DAPK were evident. Chromophilic/papillary RCCs display no significantly different expression patterns of DAPK compared with stage-adjusted clear cell RCCs. Furthermore, on analysing the DAPK enzyme activity in RCC cell lines with DAPK mRNA and protein expression, only 1 out of 11 cell lines showed basal DAPK activity in kinase activity assays, suggesting that DAPK, although expressed in RCC, remains largely inactive. Our study demonstrates the in vivo expression of DAPK in RCCs and reveals that, in contrast to other tumour types, RCCs may not downregulate DAPK mRNA expression during tumour progression. Despite persistent DAPK transcription and translation, however, the markedly reduced DAPK enzyme activity in our RCC cell lines suggested a post-translational inactivation of DAPK in RCCs.

Apoptosis Regulatory Proteins↗
PubMed2006

Imbalances of chromosome arm 1p in pediatric and adult germ cell tumors are caused by true allelic loss: a combined comparative genomic hybridization and microsatellite analysis.

Previous studies on childhood germ cell tumors (GCTs) report highly variable frequencies of losses at chromosome arm 1p. Since deletions at 1p portend a poor prognosis in other embryonal tumors, this study aims to clarify the question of the frequency of true allelic loss at 1p and whether it constitutes a prognostic parameter. We analyzed 13 GCTs from different gonadal and extragonadal sites of children (4 teratomas, 9 malignant GCTs) and 18 GCTs of adolescents and adults (3 teratomas; 15 malignant GCTs) using automated microsatellite analysis with 23 polymorphic markers and chromosomal "high resolution" comparative genomic hybridization (HR-CGH). With this combined approach, we detected loss of heterozygosity (LOH) at 1p in 8/9 childhood malignant GCTs with concordant data from HR-CGH and microsatellite analyses. In contrast, LOH at 1p was not detected in childhood teratomas (0/4) and constituted a rare event in GCTs of adolescence and adulthood (3/18). The commonly deleted region was located at distal 1p36-pter, with a proximal boundary between the markers D1S450 and D1S2870. These data unequivocally demonstrate that deletion at 1p is common in childhood GCTs and results in allelic loss. This observation argues for the presence of a classical tumor suppressor at distal 1p. Considering the high frequency of LOH at 1p and the overall favorable prognosis of childhood GCTs, a prognostic impact of LOH at 1p in childhood GCTs appears unlikely. However, since two postpubertal tumors with LOH at 1p progressed, a prognostic relevance in this age group seems possible, warranting a prospective evaluation.

Adolescent↗
PubMed2006

L1 retrotransposons in human cancers.

Retrotransposons like L1 are silenced in somatic cells by a variety of mechanisms acting at different levels. Protective mechanisms include DNA methylation and packaging into inactive chromatin to suppress transcription and prevent recombination, potentially supported by cytidine deaminase editing of RNA. Furthermore, DNA strand breaks arising during attempted retrotranspositions ought to activate cellular checkpoints, and L1 activation outside immunoprivileged sites may elicit immune responses. A number of observations indicate that L1 sequences nevertheless become reactivated in human cancer. Prominently, methylation of L1 sequences is diminished in many cancer types and full-length L1 RNAs become detectable, although strong expression is restricted to germ cell cancers. L1 elements have been found to be enriched at sites of illegitimate recombination in many cancers. In theory, lack of L1 repression in cancer might cause transcriptional deregulation, insertional mutations, DNA breaks, and an increased frequency of recombinations, contributing to genome disorganization, expression changes, and chromosomal instability. There is however little evidence that such effects occur at a gross scale in human cancers. Rather, as a rule, L1 repression is only partly alleviated. Unfortunately, many techniques commonly used to investigate genetic and epigenetic alterations in cancer cells are not well suited to detect subtle effects elicited by partial reactivation of retroelements like L1 which are present as abundant, but heterogeneous copies. Therefore, effects of L1 sequences exerted on the local chromatin structure, on the transcriptional regulation of individual genes, and on chromosome fragility need to be more closely investigated in normal and cancer cells.

Journal Article↗
PubMed2006

Genomic and expression analysis of the 3q25-q26 amplification unit reveals TLOC1/SEC62 as a probable target gene in prostate cancer.

Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.

Cell Line, Tumor↗
PubMed2005

Relationship of NKX3.1 and MYC gene copy number ratio and DNA hypomethylation to prostate carcinoma stage.

OBJECTIVE: High stage prostate cancers have been reported to frequently harbor chromosome 8 alterations and hypomethylation of LINE-1 retrotransposons. The potential of these parameters for molecular staging of prostate carcinoma was investigated. METHODS: High molecular weight DNA was extracted from 63 carcinoma tissues (22 pT2, 38 pT3, 3 pT4). Chromosome 8 alterations were followed by determining the ratio of NKX3.1 (at 8p21) to MYC (at 8q24) gene copy numbers (NKX3.1:MYC ratio) using a new real-time PCR technique. LINE-1 hypomethylation was quantified by Southern blot analysis. RESULTS: In 42 carcinomas NKX3.1 copy numbers were altered, with decreases in 32 cases. Copy numbers of MYC were increased in 38 cases and diminished in four. The NKX3.1:MYC ratio was altered in 45 specimens, with a decrease in all but two. NKX3.1 loss was associated with tumor stage (p<0.03) and MYC gain with Gleason score (p<0.03). The NKX3.1:MYC ratio was highly significantly associated with tumor stage (p<0.002), displaying 66% sensitivity and 87% specificity. LINE-1 hypomethylation was related (p<0.004) to tumor stage, but exhibited lower sensitivity (59%) and specificity (77%). CONCLUSION: A straightforward PCR technique detecting chromosome 8 alterations might be useful to predict which prostate cancers are organ-confined while determination of hypomethylation appears to be somewhat less well suited.

Aged↗
PubMed2005

Amplification and overexpression of the ID4 gene at 6p22.3 in bladder cancer.

BACKGROUND: Amplifications at 6p22.3 are prevalent in advanced stage bladder cancer (TCC). Previous studies have identified SOX4, CDKAL, and E2F3 as targets of this amplification and therefore potential oncogenes, but the more telomeric DEK gene too has been reported as overexpressed and amplified. We have therefore investigated whether the intermediate region harboring the oncogene candidate ID4 is also part of the amplicon. RESULTS: Expression of E2F3, DEK, and ID4 was investigated by real-time RT-PCR in 28 TCC compared to 6 normal bladder tissues and in 15 TCC cell lines compared to cultured normal urothelial cells. Expression of E2F3 as well as DEK increased on average in tumor vs. normal tissues (3-fold and 2.5-fold, resp.), but only the increase for E2F3 was statistically significant (p = 0.039). ID4 overexpression was observed in selected specimens. Each of the three genes was overexpressed in several cell lines, up to 150-fold (ID4), 30-fold (E2F3), and 9-fold (DEK), but these increases were not correlated to each other. Instead, moderate (DEK) to excellent (ID4) correlations were observed with copy number increases of microsatellites near each gene. Microsatellite copy number increases were highly heterogeneous across the investigated several Mb region revealing at least three subregions of amplification. CONCLUSION: Extending previous reports, our data indicate that the 6p22.3 amplicon in TCC is highly heterogeneous and targets several genes in a variable fashion. Among these, expression of E2F3 and DEK appear to be generally increased in TCC, with additional increases caused by amplifications. In contrast, over-expression of ID4, which is normally predominantly expressed in testes and brain, appears to depend more strictly on gene amplification. Accordingly, the effect of amplifications at 6p22.3 in bladder cancer is expected to be non-uniform, thereby contributing to the highly variable biological and clinical behavior of advanced stage tumors. ID4 is a potential oncogene in a small subset of bladder cancers.

Aged↗
PubMed2005

Multiple mechanisms downregulate CDKN1C in human bladder cancer.

Expression of the imprinted CDKN1C gene at chromosome 11p15.5 encoding the cell cycle inhibitor p57(KIP2) is disturbed in Beckwith-Wiedemann syndrome and in several human cancers by different mechanisms. Many advanced urothelial cancers (TCC) display downregulation of CDKN1C expression. The responsible mechanisms were investigated in TCC cell lines, with cultured normal urothelial cells (UEC) as controls. CDKN1C mRNA expression was diminished in 12/15 TCC lines and p57(KIP2) protein was decreased accordingly. Because CDKN1C is expressed from the maternal allele only, LOH at 11p15.5 represents one mechanism of downregulation. In 3 cell lines, several polymorphic markers flanking CDKN1C were homozygous compatible with this mechanism. Hypermethylation of the CDKN1C promoter, a reported cause of downregulation in other cancers, was detected by bisulfite sequencing in several cell lines and appeared associated with downregulation in at least one cell line. The methylation inhibitor 5-aza-2'deoxycytidine induced CDKN1C expression in this cell line and others. A third reported mechanism involves a switch of both alleles toward a paternal imprinting pattern, indicated by hypomethylation of a differentially methylated region (DMR) in the imprinting center (IC2). This hypomethylation was detected in most TCC lines, and was associated with re-expression of the non-coding LIT1 RNA and with downregulation of CDKN1C in several. Thus, CDKN1C downregulation in TCC seems to occur by several different mechanisms. This finding and the ability of p57(KIP2) to induce senescence in urothelial cells make CDKN1C a good candidate for a tumor suppressor at 11p in TCC.

Carcinoma, Transitional Cell↗
PubMed2005

Estimating cancer survival and clinical outcome based on genetic tumor progression scores.

MOTIVATION: In cancer research, prediction of time to death or relapse is important for a meaningful tumor classification and selecting appropriate therapies. Survival prognosis is typically based on clinical and histological parameters. There is increasing interest in identifying genetic markers that better capture the status of a tumor in order to improve on existing predictions. The accumulation of genetic alterations during tumor progression can be used for the assessment of the genetic status of the tumor. For modeling dependences between the genetic events, evolutionary tree models have been applied. RESULTS: Mixture models of oncogenetic trees provide a probabilistic framework for the estimation of typical pathogenetic routes. From these models we derive a genetic progression score (GPS) that estimates the genetic status of a tumor. GPS is calculated for glioblastoma patients from loss of heterozygosity measurements and for prostate cancer patients from comparative genomic hybridization measurements. Cox proportional hazard models are then fitted to observed survival times of glioblastoma patients and to times until PSA relapse following radical prostatectomy of prostate cancer patients. It turns out that the genetically defined GPS is predictive even after adjustment for classical clinical markers and thus can be considered a medically relevant prognostic factor. AVAILABILITY: Mtreemix, a software package for estimating tree mixture models, is freely available for non-commercial users at http://mtreemix.bioinf.mpi-sb.mpg.de. The raw cancer datasets and R code for the analysis with Cox models are available upon request from the corresponding author.

Biomarkers, Tumor↗
PubMed2005

Homozygous deletions of CDKN2A caused by alternative mechanisms in various human cancer cell lines.

The CDKN2A tumor-suppressor locus on chromosome band 9p21, which encodes p16(INK4A), a negative regulator of cyclin-dependent kinases, and p14(ARF1), an activator of TP53, is inactivated in many human cancers by point mutation, promoter hypermethylation, and, often, deletion. Homozygous deletions are unusually prevalent at this locus in very different human cancers. In the present study, we compared deletions in squamous cell carcinoma of the head and neck (SCCHN) cell lines to those in T-cell acute lymphatic leukemia (T-ALL), glioma, and bladder carcinoma (TCC) cell lines. Of 14 SCCHN lines, 10 showed homozygous deletions of CDKN2A, one displayed promoter hypermethylation with gene silencing, and one had a frameshift deletion in exon 2. Many deletion ends were in or proximal to the repetitive sequence clusters flanking the locus. Breakpoint junctions displayed variable microhomologies or insertions characteristic of DNA repair by nonhomologous end-joining. In general, deletions were much smaller in SCCHN than in TCC and glioma. In T-ALL, breakpoints were near consensus sites for recombination mediated by RAG (recombination activating genes) enzymes, and the structure of the junctions was consistent with this mechanism. We suggest that different mechanisms of CDKN2A deletion prevail in different human cancers. Aberrant RAG-mediated recombination may be responsible in T-ALL, and exuberant DNA repair by nonhomologous end-joining is the likely prevailing mechanism in SCCHN, but a distinct mechanism in TCC and glioma remains to be elucidated.

Base Sequence↗
PubMed2005

Hedgehog signaling in normal urothelial cells and in urothelial carcinoma cell lines.

Constitutive activation of hedgehog signaling, often caused by PTCH1 inactivation and leading to inappropriate activation of GLI target genes, is crucial for the development of several human tumors including basal cell carcinoma of the skin and medulloblastoma. The PTCH1 gene at 9q22 is also considered as a candidate tumor suppressor in transitional cell carcinoma (TCC), of which >50% show LOH in this region. However, only rare mutations have been found in PTCH1. We have therefore investigated GLI-dependent promoter activity and expression of hedgehog pathway components in TCC cell lines and proliferating normal urothelial cells. Normal urothelial cells cultured in serum-free medium, but not TCC lines exhibited low, but significant promoter activity under standard growth conditions. Accordingly, GLI1-3 and PTCH1 mRNAs were expressed at moderate levels, and sonic hedgehog (SHH) mRNA expression was low to undetectable. In co-transfection experiments GLI1 increased promoter activity significantly in one TCC line and further in normal urothelial cells, but less strongly in other TCC lines. Expression patterns of GLI factor mRNAs did not correlate with inducibility. No significant effects of SHH or cyclopamine on proliferation were observed, ruling out autocrine effects. However, SHH induced GLI-dependent promoter activity in normal urothelial cells. Taken together, our data suggest that the hedgehog pathway is weakly active in normal adult urothelial cells and of limited importance in TCC.

Carcinoma, Transitional Cell↗
PubMed2005

Causes and consequences of DNA hypomethylation in human cancer.

While specific genes are hypermethylated in the genome of cancer cells, overall methylcytosine content is often decreased as a consequence of hypomethylation affecting many repetitive sequences. Hypomethylation is also observed at a number of single-copy genes. While global hypomethylation is highly prevalent across all cancer types, it often displays considerable specificity with regard to tumor type, tumor stage, and sequences affected. Following an overview of hypomethylation alterations in various cancers, this review focuses on 3 hypotheses. First, hypomethylation at a single-copy gene may occur as a 2-step process, in which selection for gene function follows upon random hypo methylation. In this fashion, hypomethylation facilitates the adaptation of cancer cells to the ever-changing tumor tissue microenvironment, particularly during metastasis. Second, the development of global hypomethylation is intimately linked to chromatin restructuring and nuclear disorganization in cancer cells, reflected in a large number of changes in histone-modifying enzymes and other chromatin regulators. Third, DNA hypomethylation may occur at least partly as a consequence of cell cycle deregulation disturbing the coordination between DNA replication and activity of DNA methyltransferases. Finally, because of their relation to tumor progression and metastasis, DNA hypomethylation markers may be particularly useful to classify cancer and predict their clinical course.

Chromatin↗
PubMed2005

Hypomethylation of the XIST gene promoter in prostate cancer.

In a process denoted "global hypomethylation" repetitive DNA sequences like LINE-1 retrotransposons become hypomethylated in human cancers, including a subset of prostate carcinomas. It is less well known to what extent single-copy sequences are affected by this phenomenon. Therefore, we have analyzed methylation and expression of the XIST gene by bisulfite sequencing and real-time RT-PCR. The promoter of this single-copy gene is strongly methylated in normal male cells, including leukocytes and normal prostate. In prostate cancer tissues and particularly in cell lines, partial hypomethylation was observed paralleling that of LINE-1 sequences. Weak XIST expression was found in normal prostate tissues, but none in leukocytes. Only slight increases in expression of this gene were found in cancer tissues and cell lines. Our data suggest that hypomethylation in prostate cancer is indeed "global," affecting repeat and unique sequences in parallel. Detection of partially hypomethylated XIST alleles in prostate cancer tissues might be useful for the identification of cases with pronounced hypomethylation, which tend to be more aggressive.

Aged↗