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Biomedical subjects

W A Schulz

Publications and source records attributed to W A Schulz.

At least 55 records · Page 3Linked to original sources
PubMed1997

p53 and MDM2 in the development and progression of bladder cancer.

OBJECTIVE: Earlier investigations have demonstrated that inactivation of the p53 tumor suppressor gene might play a role in the development and progression of bladder cancer. Complex formation with the MDM2 oncogene product is one mechanism inactivating the p53 protein. Therefore, the MDM2 and the p53 protein were investigated to study potential interactions in bladder cancer. METHOD: 200 archival bladder tissue specimens from 92 patients were studied by immunohistochemistry using monoclonal antibodies DO-1 against p53 and IF2 against MDM2. RESULTS: No staining was observed for p53 or MDM2 in normal urothelium. Alterations of both genes were rare in dysplasia. p53 accumulation was observed in 27-44% of the tumor stages examined. MDM2 overexpression increased from 18% in carcinoma in situ to 49% in T1 tumors, but was present in only 22% of the advanced tumors. Alterations of both genes were more frequent in high-grade lesions. To investigate the prognostic impact of these alterations 61 patients with superficial bladder tumors were followed for at least 2 years (mean 51 months). Multivariate analysis demonstrated that multifocal disease and p53 accumulation were significantly correlated with tumor progression (p = 0.0099 and 0.0135). MDM2 overexpression alone had no prognostic significance. Patients with alterations of both genes had a very high risk of tumor progression (p = 0.0064). CONCLUSION: These results demonstrate a positive correlation between p53 accumulation and MDM2 overexpression in the progression of bladder cancer which may have prognostic value.

Carcinoma in Situ↗
PubMed1996

Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma.

Alterations of DNA methylation were investigated in 6 urothelial carcinoma cell lines and 13 tumor tissues. The methylation of L1 LINE sequences was diminished in all cell lines (by 26 +/- 5%; range, 11-49%) and in most tumors (by 21 +/- 5%; range, 0-60%) compared to normal bladder mucosa. Hypermethylation of the calcitonin gene CpG island was restricted to cell lines and was not found in primary tumors, suggesting it had arisen during culture. In single-cell clones of a urothelial carcinoma cell line, both hypomethylation of L1 sequences and hypermethylation of the calcitonin gene persisted, indicating that they coexist within one cell. DNA methyltransferase expression did not correlate with the methylation status of the cell lines, but rather with histone H3 expression. Accordingly, it was down-regulated in quiescent cells. Aberrant expression of DNA methyltransferase is therefore not likely the cause for altered methylation patterns in urothelial carcinoma. L1 LINE hypomethylation seems to prevail in urothelial carcinoma and in this tumor might be useful for diagnostic or prognostic purposes.

Blotting, Northern↗
PubMed1996

Adjacent guanines as preferred sites for strand breaks in plasmid DNA irradiated with 193 nm and 248 nm UV laser light.

193 nm UV laser light induces single strand breaks as well as double strand breaks in plasmid DNA. The frequency of strand breaks is increased at sites where at least two guanine or, less frequently, a guanine and an adenine are adjacent to each other. 248 nm UV laser light induces predominantly single strand breaks with a less pronounced preference for guanine clusters. At both wavelengths, the presence of oxygen does not change the pattern of strand breaks, but in the presence of nitrous oxide, selectivity is lost; this is attributable to the production of the hydroxyl radical. These findings can be explained by a model in which the propagation of a radical or an electron hole along the DNA helix competes kinetically with the strand cleavage reaction. The difference in selectivity at the two different wavelengths is ascribed to the preferential light absorption by the purine bases at 193 nm.

Base Sequence↗
PubMed1996

Positive and negative regulatory regions in promoters of human glutathione transferase alpha genes.

The human glutathione transferase (GST, EC 2.5.1.18) alpha class locus comprises several genes and pseudogenes. Genomic DNA encoding several human alpha-class-related genes and pseudogenes was cloned and characterized. Three distinct but highly similar 5'-flanking regions of GST alpha genes as well as a series of 5'-deletions were investigated for promoter activity by fusion to the luciferase reporter gene. Transient transfection of these luciferase constructs into human hepatoblastoma, kidney carcinoma, nephroblastoma or bladder carcinoma cells revealed that the promoters are active and contain both positive and negative regulatory regions that behave in a cell-type specific fashion. The 150 bp proximal promoter regions of the three sequences retained the same relative activities as the full length promoters. Two of them were equally active, whereas the third one showed only 20% of the activity of the two stronger promoters. Site-directed mutagenesis indicated that a conspicuous insertion of three nucleotides (TTT) in the weak promoter is not responsible for the different activities.

Cloning, Molecular↗
PubMed1996

Induction of mouse embryonal carcinoma cell differentiation and activation of the retinoic acid receptor beta 2 promoter by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (calcitriol) at 100 nmol/l elicited morphological differentiation and expression of collagen IV in mouse F9 embryonal carcinoma cells, and its effect was enhanced and accelerated by dibutyryl-cAMP (db-cAMP). The RAR beta 2 promoter was also activated, as evidenced by an increase in beta-galactosidase activity in an F9 reporter cell line with a stably integrated RAR beta 2-lacZ construct. All three effects were slower and less extensive with calcitriol than with retinoic acid, even in the presence of db-cAMP. Activation of the RAR beta 2 promoter by calcitriol required its TRE sequence, whereas db-cAMP required the CRE. TPA also activated the RAR beta 2 promoter, requiring a functional TRE. Thus, in the RAR beta 2 promoter the TRE sequence, whose function has so far been unidentified, mediates the effects of calcitriol and TPA. RAR beta 2 promoter activation by calcitriol was blocked by inhibitors of protein kinase C indicating that calcitriol elicits its effect via protein kinase C. Therefore, calcitriol induces differentiation of F9 mouse embryonal carcinoma cells at least in part by a pathway different from the classical one operative with retinoic acids.

Animals↗
PubMed1995

Turnover of glutathione S-transferase alpha mRNAs is accelerated by 12-O-tetradecanoyl phorbol-13-acetate in human hepatoma and colon carcinoma cell lines.

The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), known to induce murine glutathione S-transferase (GST) Ya, was examined for its effect on the expression of human GST alpha. Unexpectedly, 24-h treatment of the human hepatoma cell line HepG2 with 100 nmol/l TPA caused a decrease of the GST alpha mRNA level to below 5% of controls, i.e. opposite to the known response in the mouse. The level of mRNA for GST Mu was also decreased, but the mRNAs of c-jun and jun-B were elevated after 2 h. The decrease of GST alpha mRNAs was inhibited by staurosporine, suggesting an involvement of protein kinase C. Inhibition of transcription and translation by actinomycin D and cycloheximide also partially inhibited the effect of TPA on the expression of GST alpha. In the presence of actinomycin D, GST alpha mRNA halflife was 14.5 h, compared to 3.5 h in the presence of TPA. The calcium ionophore A23187 caused a loss of GST alpha mRNAs to levels almost as low as those obtained with TPA. The effects of TPA and the calcium ionophore were also observed in CaCo2 colon carcinoma cells. As a consequence of the decrease of mRNA levels, GST alpha protein levels and total GST enzyme activity were also diminished. Also, the morphology of the cells was changed after 3 h exposure to TPA. These data suggest that human GST alpha expression can be regulated at the level of mRNA stability by a pathway involving protein kinase C.

Animals↗
PubMed1995

Induction of gap junctional communication by 4-oxoretinoic acid generated from its precursor canthaxanthin.

The activity of 4-oxoretinoic acid as an inducer of gap junctional communication was investigated in C3H/10T1/2 murine fibroblasts. Two isomers of this retinoid, all-trans- and 13-cis-4-oxoretinoic acid, enhance gap junctional communication. This is accompanied by increased expression of connexin43 mRNA. Decomposition fractions of canthaxanthin were isolated by preparative high-performance liquid chromatography and shown to be active in the cell-cell communication assay. Two of the decomposition compounds were identified as all-trans- and 13-cis-4-oxoretinoic acid. Therefore, it is concluded that the biological activity of canthaxanthin regarding cell-cell communication is at least in part due to the formation of active decomposition products such as 4-oxoretinoic acid.

Animals↗
PubMed1995

Efficacy of all-trans-beta-carotene, canthaxanthin, and all-trans-, 9-cis-, and 4-oxoretinoic acids in inducing differentiation of an F9 embryonal carcinoma RAR beta-lacZ reporter cell line.

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

Animals↗
PubMed1995

Regulation of DNA methyltransferase during differentiation of F9 mouse embryonal carcinoma cells.

DNA becomes demethylated when F9 mouse embryonal carcinoma cells differentiate into parietal endoderm. DNA methyltransferase (DNA-MTase) activity decreased by 50% during 1 week of differentiation. The level of DNA-MTase mRNA was also diminished accordingly, but the transcription rate of the DNA-MTase gene measured by run-on transcription was essentially unchanged, indicating regulation of DNA-MTase expression at a posttranscriptional step. The decline of DNA-MTase mRNA paralleled that of histone H3 mRNA in accord with the notion that DNA-MTase is preferentially expressed in the S phase of the cell cycle. Since DNA-MTase expression decreases in parallel with DNA synthesis, DNA demethylation during differentiation of F9 cells appears not to be due to limited expression of DNA-MTase. However, the plasmid pAFP7000CAT, alpha-fetoprotein (AFP), which is strongly de novo methylated when transfected into F9 stem cells became only weakly methylated after transfection into the F9 parietal endoderm derivative P1, indicating that the activity of DNA-MTase within parietal endoderm cells is more strongly diminished than is apparent from measurements of mRNA amounts and of overall DNA-MTase activity in vitro. The discrepancy between DNA-MTase expression and its actual activity within the cell indicates the existence of a novel mechanism controlling the activity of DNA-MTase.

Animals↗
PubMed1995

Inactivation of tumor suppressor genes and deregulation of the c-myc gene in urothelial cancer cell lines.

Recent investigations have demonstrated p53 and Rb alterations in a subset of transitional cell carcinoma (TCC). Further genetic changes during tumor progression include overexpression of the c-myc gene in a significant number of mainly invasive bladder tumors. To study the possible interactions between these genes in TCC, urothelial cancer cell lines were chosen as an in vitro model. Expression and mutation of p53 was studied in 15 bladder cancer cell lines by immunocytochemistry, Western blot, polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing of double stranded PCR products of exons 4, 5, 7 and 8 of genomic DNA. C-myc expression and gene structure were studied using Northern and Southern blot techniques Rb protein expression was analyzed by Western blot. Twelve of 15 cell lines showed either p53 mutations or abnormal protein expression. Consistent with previous studies, five cell lines did not express Rb protein. None of the cell lines studied retained both tumor suppressor genes in a functional form. The c-myc gene appeared to be intact in all cell lines and copy numbers were close to normal. Northern analysis demonstrated that all cell lines expressed c-myc mRNA but evidence for altered regulation was found in at least two cell lines. Our data suggest that amplification or translocation are not the underlying mechanism for c-myc overexpression in urothelial tumors. No correlation between loss of Rb protein and c-myc expression was observed. The results presented here for the cell lines match well those obtained in vivo. Thus, these cell lines may provide a suitable model for further analysis of molecular alterations in urothelial cancer.

Base Sequence↗
PubMed1994

Lowered amounts of the tissue-specific transcription factor LFB1 (HNF1) correlate with decreased levels of glutathione S-transferase alpha messenger RNA in human renal cell carcinoma.

Human renal cell carcinoma is characterized by the loss of differentiation markers such as glutathione S-transferase alpha (GST-alpha). In this paper we show that the promoter of a GST-alpha gene contains a functional binding site for the cell-specific transcription factor LFB1 (HNF1). To investigate the potential role of LFB1 in the down-regulation of GST-alpha expression, we have compared the amount and the binding activity of the LFB1 protein between normal kidney and tumor tissue. By Western analysis and gel retardation assay using a monoclonal antibody specific for LFB1 we show that in 11 of 14 carcinomas the amount of LFB1 is clearly reduced compared to the corresponding normal tissue and that in all 14 renal carcinomas LFB1 binding activity is diminished. As in the same samples the abundance of GST-alpha mRNA is lower than in the normal tissue, we postulate that the loss of LFB1 binding activity might be responsible for the decreased expression of the GST-alpha gene in renal cell carcinoma.

Antibodies, Monoclonal↗
PubMed1994

Enhancement of reporter gene de novo methylation by DNA fragments from the alpha-fetoprotein control region.

The 5'-upstream region of the rat alpha-fetoprotein (AFP) gene strongly increased de novo methylation of an adjacent chloramphenicol acetyltransferase (CAT) gene upon transfection into F9 mouse embryonal carcinoma cells. The same effect was exerted by a distal 775-base pair (bp) fragment and by 300- and 1-kb fragments preceding the transcriptional start site, but not by other parts of the control region. Further division of the larger, strongly active fragments resulted in a gradual decrease of methylation and clonal variation in the methylation patterns. The effect of the 775-bp fragment did not depend on its orientation. It was ablated by insertion of the mouse metallothionein I promoter between the AFP gene fragment and the CAT gene, but not by its insertion upstream of the AFP gene fragment. Two fragments from the AFP control region increasing methylation contained B1 and B2 small interspersed repetitive elements, respectively. B1 and B2 sequences of different origin also acted strongly to increase methylation. These findings support the idea that mammalian genes contain specific sequences involved in regulating their methylation. The effects of these sequences appear to be exerted in cis, to be dependent on proximity, but not on orientation, and to require an optimal size of 500-700 bp. Small retrotransposon sequences within such elements may be particularly effective in attracting de novo methylation.

Animals↗
PubMed1994

Exemption of satellite DNA from demethylation in immortalized differentiated derivatives of F9 mouse embryonal carcinoma cells.

DNA methylation in F9 embryonal carcinoma cells at various stages of retinoic acid-induced differentiation was compared to that in immortalized differentiated derivatives of this cell line. Different repetitive sequences, such as L1 LINE, GAPDH pseudogenes, and major and minor satellite DNA, lost their methylation to similar extents during F9 differentiation into parietal endoderm cells. In the immortalized derivatives 8a and P1, which phenotypically resemble F9 cells at intermediate stages of differentiation, methylation patterns diverged: Methylation of L1 and GAPDH sequences was strongly and moderately diminished, respectively, whereas methylation of satellite DNA was almost as high as that in stem cells. P19 embryonal carcinoma cells and D3 embryonic stem cells possessed methylation patterns similar to those of F9 stem cells. In immortalized cell lines with differentiated phenotypes derived from P19, methylation was diminished uniformly throughout the genome, but the overall level of methylation remained higher than that in terminally differentiated F9 cells. Treatment with the methylation inhibitor deoxyazacytidine halted the proliferation of 8a, P1, and F9 cells and elicited marked changes in morphology. However, markers of differentiation were not induced in F9 cells. The finding that all immortalized differentiated derivatives of embryonal carcinoma cells retained a higher level of DNA methylation--at least in parts of their genomes--than did terminally differentiated cells may indicate a function of demethylation of DNA and of satellite sequences in particular in terminal differentiation of extraembryonic tissues.

Animals↗
PubMed1994

P53 accumulation in precursor lesions and early stages of bladder cancer.

Recent investigations have demonstrated alterations of the p53 tumor-suppressor gene in a considerable number of transitional-cell carcinoma (TCC) specimens. Thus far, these investigations have been restricted to either papillary TCC or invasive bladder cancer. To obtain further information on a possible involvement of p53 in bladder cancer development or tumor progression, investigations of precursor lesions and early stages of this disease are required. Immunohistochemical examination of 6 dysplasias and 24 carcinomas in situ (TIS) showed p53 accumulation, which is suggestive of p53 inactivation, in 2 (33%) and 9 (38%) of these specimens, respectively. This ratio was similar in 9 T1 lesions (33%) and in 14 cases of muscle-infiltrative disease (35%). In papillary tumors, p53 accumulation was observed exclusively in 3/10 moderately differentiated or high-grade lesions but not in 1 Ta G1 tumor. The expression of p53 accumulation was a consistent finding. The examination of tumor recurrences yielded either the presence or the absence of p53 overexpression in the primary and recurrent tumors of 7/8 patients. Similarly, in multifocal TCC, p53 accumulation was also either present or absent in 10/11 cases examined. These results suggest the existence of at least two different subgroups of TCC, with p53 accumulation being present in one of these groups. The observation of p53 accumulation in dysplasia and in TIS is a prerequisite for a possible involvement of p53 in bladder cancer carcinogenesis, although it does not prove this assumption.

Carcinoma in Situ↗
PubMed1994

Expression of NAD(P)H:quinone oxidoreductase and glutathione S-transferases alpha and pi in human renal cell carcinoma and in kidney cancer-derived cell lines.

NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GST) are enzymes of interest in cell defence and drug resistance. Relative levels of NQOR mRNA in renal cell carcinomas were 28 +/- 24% (n = 21) of those in non-neoplastic tissue and the enzyme activity decreased from 41 +/- 39 to 18 +/- 27 mU/mg protein (n = 23). In three of the cases, there was no measurable NQOR enzyme activity at all, indicating a polymorphism in the population for this gene. Relative GST-alpha mRNA levels in the tumours were on average 6 +/- 6% (n = 22) of the control value, whereas for GST-pi mRNA smaller decreases as well as increases were found in the tumours as compared to control tissue, but, on average, the level remained unchanged. Overall GST activity measured with CDNB as a substrate was 152 +/- 157 mU/mg protein in tumour tissue and 342 +/- 177 mU/mg protein in non-neoplastic tissue (n = 23). In all kidney tumour-derived cell lines NQOR mRNA was strongly expressed and on a per protein basis NQOR activity was about 10-fold higher than in the kidney tumour samples. GST-pi but not GST-alpha mRNA was also present. Total GST enzyme activities in these cell lines were similar to those in kidney tumour samples. HepG2 cells exhibited expression of NQOR and GST-alpha; GST-pi was not detectable. NQOR activity in HepG2 was about four-fold higher than in kidney-derived cell lines. Thus, NQOR and GST-alpha are both down-regulated in renal carcinoma, but their expression diverges in carcinoma cell lines.

Aged↗
PubMed1994

Loss of heterozygosity at the NAD(P)H: quinone oxidoreductase locus associated with increased resistance against mitomycin C in a human bladder carcinoma cell line.

The human bladder carcinoma cell line RT112 and the mitomycin C-resistant cell line RT112MMC, derived from RT112 cells, were examined for their expression of NAD(P)H:quinone oxidoreductase (NQOR) and glutathione S-transferases (GSTs). RT112 cells were 40-fold more sensitive towards mitomycin C than RT112MMC cells. The NQOR mRNA level in RT112MMC cells was decreased to 15% as compared to RT112 cells. NQOR enzyme activity was 391 +/- 140 mU/mg protein in RT112 cells, whereas NQOR activity in RT112MMC cells was not measurable. As shown by a fast PCR-based assay and DNA-sequencing, the cell line RT112 is heterozygous, whereas RT112MMC is homozygous for a null allele of the NQOR gene without enzymatic activity. Accordingly, both wild-type and null allele mRNAs were present in RT112 cells, whereas only null allele mRNA was found in RT112MMC. The lack of NQOR enzyme activity in RT112MMC cells was thus associated with loss of heterozygosity at the NQOR locus. By a PCR-RFLP assay, three kidney carcinoma patients without measurable NQOR enzyme activity were shown to be homozygous for the null allele. The PCR assay described here is useful for examination of large numbers of samples. The relative amount of GST-Pi mRNA was decreased by 30% in RT112MMC as compared to RT112, contributing to a diminished level of GST enzyme activity, using CDNB as a substrate, from 95 +/- 62 mU/mg protein in RT112 to 26 +/- 6 mU/mg protein in RT112MMC.

Base Sequence↗