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Gerd P Pfeifer

Publications and source records attributed to Gerd P Pfeifer.

65 records · Page 4Linked to original sources
PubMed2002

Frequent hypermethylation of the RASSF1A gene in prostate cancer.

Recently, we have cloned and characterized the Ras association domain family 1A gene (RASSF1A) at 3p21.3, from which loss of genetic material is one of the most frequent events in several types of human solid tumors. The CpG island promoter region of this gene is highly methylated in several human cancers, most notably in small cell lung cancer, breast cancer, and renal cell carcinoma. In this study, we have analysed the methylation status of RASSF1A in primary prostate tumors and in the prostate cancer cell line LNCaP. In total, 37 out of 52 tumors (71%) were methylated at the promoter region of RASSF1A. The relative frequency of methylation was higher in more aggressive tumors compared with less malignant tumors. For instance, tumors with a Gleason score of 7-10 (25 out of 30, 83%) were significantly more methylated compared with Gleason 4-6 tumors (11 out of 20, 55%, P=0.032, Fisher's exact test). Coincident with a hypermethylated promoter, transcripts of RASSF1A were missing in LNCaP cells. Expression of RASSF1A was restored with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor. In conclusion, our data suggest that epigenetic inactivation of RASSF1A by methylation is a very common event in prostate cancer and might be involved in the progression of the disease. Testing for RASSF1A methylation should become useful in prostate cancer early detection and diagnosis and might aid prognosis by gauging the potential status of progression.

Chromosome Aberrations↗
PubMed2002

Reduced expression and increased CpG dinucleotide methylation of the rat APOBEC-1 promoter in transgenic rabbits.

Editing of apolipoprotein (apo) B mRNA in liver limits the plasma LDL levels in horses, dogs, rats or mice. Species such as man or rabbit do not edit the hepatic apo B mRNA and are therefore susceptible to atherosclerosis and coronary artery disease due to elevated plasma LDL levels. The catalytic subunit APOBEC-1 is the only missing component of the apo B mRNA editing enzyme complex in the human or rabbit liver. Here we describe the generation of transgenic rabbits in which APOBEC-1 expression is mediated by the proximal promoter of the rat APOBEC-1 gene. These transgenic rabbits are healthy and fertile, and rat APOBEC-1 mRNA is expressed in liver, intestine, kidney, lung, brain and muscle. The transgenic APOBEC-1 expression is low and not sufficient to induce editing in rabbit liver. In rat, the proximal APOBEC-1 promoter demonstrates a progressive loss of CpG dinucleotide methylation towards the core promoter region that is entirely unmethylated. In the transgenic rabbits, this distinct pattern of CpG methylation is lost, and throughout the entire rat APOBEC-1 promoter, >90% of the CpGs are methylated. Thus, the weak proximal rat APOBEC-1 promoter appears to be down-regulated in the rabbit and may be species-specific.

APOBEC-1 Deaminase↗
PubMed2002

Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG-->TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells.

Oxidative DNA damage may play an important role in human disease including cancer. Previously, mutational spectra have been determined using systems that include transition metal ions and hydrogen peroxide (H2O2). G-->T transversions and C-->T transitions were the most common mutations observed including some CC-->TT tandem mutations. C-->T transition mutations at methylated CpG dinucleotides are the most common mutations in human genetic diseases. It has been hypothesized that oxidative stress may increase the frequency of mutations at methylated CpG sequences. Here we have used a CpG-methylated shuttle vector to derive mutational spectra of copper/H2O2-induced DNA damage upon passage of the shuttle vector through human fibroblasts. We find that copper/H2O2 treatment produces higher numbers of CpG transition mutations when the CpGs are methylated but does not create clear C-->T hotspots at these sites. More strikingly, we observed that this treatment produces a substantial frequency of mutations that were mCG-->TT tandem mutations. Six of seven tandem mutations were of this type. mCG-->TT mutations (6/63 = 10% of all mutations) were observed only in nucleotide excision repair-deficient (XP-A) cells but were not found in repair-proficient cells. The data suggest that this novel type of mutation may be produced by vicinal or cross-linked base damage involving 5-methylcytosine and a neighboring guanine, which is repaired by nucleotide excision repair. We suggest that the underlying oxidative lesions could be responsible for the progressive neurodegeneration seen in XP-A individuals.

Ascorbic Acid↗
PubMed2002

Mutational signature of the proximate bladder carcinogen N-hydroxy-4-acetylaminobiphenyl: inconsistency with the p53 mutational spectrum in bladder cancer.

We studied the mutagenicity of the proximate bladder carcinogen, N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) in embryonic fibroblasts of the Big Blue mouse. Treatment of these cells with increasing concentrations of N-OH-AABP for 24 h resulted in a dose-dependent increase in mutation frequency of the cII transgene up to 12.8-fold over the background. Single base substitutions comprised 86% of the N-OH-AABP-induced mutations and 74% of the spontaneous cII mutations (sequenced number of mutant plaques, 141 and 145, respectively). Of these, 63 and 36%, respectively, occurred at guanine residues along the cII gene. Whereas G to T transversions predominated in the induced cII mutations (47%), insertion was the most spontaneously derived cII mutation (19%). Mapping of N-OH-AABP-induced DNA adducts along the cII gene by terminal transferase-dependent PCR showed the formation of DNA adducts at specific nucleotide positions. Five preferential DNA adduction sites were established, of which four were major mutation sites for N-OH-AABP, especially for G to T transversions. This unique mutational signature of N-OH-AABP in the cII gene was, however, in sharp contrast with the mutational spectrum of the p53 gene in human bladder cancer. G to A transitions are the dominant type of p53 mutations (53%), being also prevalent in almost all of its five mutational hotspots (codons 175, 248, 273, 280, and 285). In addition, the majority of mutations in three of these hotspots (codons 175, 248, and 273) are at a methylated CpG site, whereas in the cII gene neither the preferential N-OH-AABP DNA adduction sites nor the induced mutational hotspots are biased toward methylated CpG dinucleotides. We conclude that N-OH-AABP leaves a characteristic mutational signature in the cII transgene, which is consistent with its preferential DNA adduction profile. However, the pattern of mutation induced by N-OH-AABP in the cII gene is largely at odds with the mutational spectrum of the p53 gene in human bladder cancer.

Aminobiphenyl Compounds↗
PubMed2002

Frequent epigenetic silencing of the CpG island promoter of RASSF1A in thyroid carcinoma.

Loss of heterozygosity of chromosome 3p21 is one of the most frequent alterations in solid tumors, including thyroid carcinomas. Recently, we have characterized the novel tumor suppressor gene RASSF1 located in this locus. The RASSF1A isoform is epigenetically inactivated in a variety of human primary tumors. In this study, we investigated the expression and methylation status of the RASSF1 gene in thyroid carcinoma. In nine thyroid cancer cell lines, the RASSF1A promoter CpG island was methylated completely, and expression was absent. Treatment of these cell lines with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the transcription of RASSF1A. The methylation status of the RASSF1A promoter was analyzed in 38 primary thyroid tumors, including 1 poorly differentiated thyroid carcinoma, 5 medullary thyroid carcinoma (MTC), 10 follicular thyroid carcinoma (FTC), 9 undifferentiated thyroid carcinoma (UTC), and 13 papillary thyroid carcinoma (PTC). In 71% of thyroid carcinomas, the RASSF1A CpG island was hypermethylated. Methylation frequency was higher in the aggressive forms of thyroid carcinoma and was found in 80% of MTC, in 78% of UTC, and in 70% of FTC, compared with 62% in the more benign PTC. RASSF1A inactivation was detected in all stages of thyroid carcinoma scored by Tumor-Node-Metastasis classification. Additionally, we analyzed the methylation frequency of the CpG island of cell cycle inhibitor p16(INK4a) in the same thyroid tumors. The p16 gene was inactivated in 56 and 25% of cell lines and primary tumors, respectively. p16 methylation was detected in 56% of UTC, 10% of FTC, and 25% of PTC but not in MTC. In UTC, which belongs to the most aggressive carcinomas in humans, the most common combined inactivation of RASSF1A and p16 was detected. In general, 90% of tumors with p16 inactivation were also silenced for RASSF1A expression. However, RASSF1A hypermethylation was detected three times more frequently in thyroid cancers. Thus, RASSF1A inactivation may play a crucial role in the malignancy of thyroid carcinoma.

Carcinoma↗
PubMed2002

RASSF3 and NORE1: identification and cloning of two human homologues of the putative tumor suppressor gene RASSF1.

RASSF1A, one of the two major isoforms of the putative tumor suppressor gene RASSF1, located at 3p21.3, is inactivated in a variety of human cancers including lung, breast, bladder and renal cell carcinomas. We have isolated and cloned two human homologues of this gene, RASSF3 and NORE1, located at 12q14.1 and 1q32.1, respectively. Both RASSF3 and NORE1 share almost 60% homology, at the amino acid level, with RASSF1. The RASSF3 gene contains five exons and encodes a 247 amino acid protein (MW of 28.6 kDa) with a highly conserved Ras association (RalGDS/AF-6) (RA) domain at the C-terminus. RASSF3 is ubiquitously expressed in all normal tissues and cancer cell lines analysed. NORE1, which is homologous to the previously described mouse Nore1 gene, exists in at least two spliced isoforms, A and B. Transcript A encodes a protein of 418 amino acids (MW or 47 kDa) while transcript B contains an ORF of 265 aa (MW of 30.5 kDa). Both share a RA domain, encoded by exons 3 through 6. NORE1A and NORE1B are expressed in most of the normal tissues analysed but they appear to be down-regulated in several cancer cell lines. However, contrary to RASSF1A, gene silencing by methylation of the CpG islands at which the two NORE1 transcripts initiate is not a common event in human primary tumors. RASSF3 and NORE1B are very similar, at the N-terminus, to the splice variant C of RASSF1 (RASSF1C), which does not seem to be involved in tumorigenesis. NORE1A is most closely related to RASSF1A, for sequence homology and genomic organization. However, aberrations in tumors have so far not been found. The presence of a Ras association domain common to NORE1, RASSF1, and RASSF3 suggests their possible involvement in Ras-like signaling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1.

Nore and RASSF1A are noncatalytic proteins that share 50% identity over their carboxyterminal 300 AA, a segment that encompasses a putative Ras-Rap association (RA) domain. RASSF1 is expressed as several splice variants, each of which contain an RA domain, however the 340 AA RASSF1A, but not the shorter RASSF1C variant, is a putative tumor suppressor. Nore binds to Ras and several Ras-like GTPases in a GTP dependent fashion however neither RASSF1 (A or C) or the C. elegans Nore/RASSF1 homolog, T24F1.3 exhibit any interaction with Ras or six other Ras-like GTPases in a yeast two-hybrid expression assay. A low recovery of RASSF1A (but not RASSF1C) in association with RasG12V is observed however on transient expression in COS cells. Nore and RASSF1A can each efficiently homodimerize and heterodimerize with each other through their nonhomologous aminoterminal segments. Recombinant RASSF1C exhibits a much weaker ability to homodimerize or heterodimerize; thus the binding of RASSF1C to Nore is very much less than the binding of RASSF1A to Nore. The association of RASSF1A with RasG12V in COS cells appears to reflect the heterodimerization of RASSF1A with Nore, inasmuch the recovery of RASSF1A with RasG12V is increased by concurrent expression of full length Nore, and abolished by expression of Nore deleted of its RA domain. The preferential ability of RASSF1A to heterodimerize with Nore and thereby associate with Ras-like GTPases may be relevant to its putative tumor suppressor function.

Animals↗
PubMed2002

MBD3L1 and MBD3L2, two new proteins homologous to the methyl-CpG-binding proteins MBD2 and MBD3: characterization of MBD3L1 as a testis-specific transcriptional repressor.

Methylation of cytosines at CpG dinucleotides is essential for mammalian development. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of proteins that contain a methyl-CpG binding domain (MBD). Except for MBD4, these proteins are involved in gene silencing imposed by methylated DNA. We have identified a human gene that codes for a protein that is 42% identical to MBD3 and 38% identical to MBD2 but lacks the methyl-CpG binding domain. The recombinant protein does not bind to methylated DNA in vitro. The corresponding mouse Mbd3L1 gene was also cloned. The MBD3L1 gene is expressed specifically in testis. During spermatogenesis, expression of MBD3L1 is observed only in round spermatids, suggesting a role for the gene product in the postmeiotic stages of male germ cell development. The MBD3L1 protein is localized to discrete areas in the nucleus and contains an N-terminal transcriptional repression domain. This repression is independent of histone deacetylase inhibition. A homologue of MBD3L1, MBD3L2, was also identified and cloned. Expression of MBD3L2 was found in germ cell tumors and some somatic tissues. These novel proteins may function as counterparts of MBD2 and/or MBD3 in developmental stage-specific transcriptional repression.

Amino Acid Sequence↗
PubMed2002

Mapping polycyclic aromatic hydrocarbon and aromatic amine-induced DNA damage in cancer-related genes at the sequence level.

Genomic injury induced by environmental carcinogens, such as polycyclic aromatic hydrocarbons and aromatic amines, is the initial step that can trigger mutagenesis and carcinogenesis. In addition to the physico-chemical property of DNA damaging agents, several important factors such as primary sequence, chromatin structure, methylation, protein association, and transcriptional activity can affect not only the initial level and distribution of DNA damage but also the efficiency of repair. Therefore, mapping the DNA damage induced by environmental agents in cancer-related genes such as p53 and ras at the sequence level provides essential information for assessing their carcinogenic potential. Recently, using the E. coli nucleotide excision enzyme complex, UvrABC nucleases in combination with ligation-mediated polymerase chain reaction, we developed a method to map DNA damage in the p53 and ras genes. These studies led us to conclude that targeted DNA damage, in combination with growth selection, contributes greatly in shaping the mutation spectrum in these genes in human cancer. Here we present the rationale and details of this approach, typical experimental results and necessary precautions.

Amino Acids, Aromatic↗
PubMed2002

Methylation of the RASSF1A gene in human cancers.

Loss of genetic material from chromosome 3p21.3 is one of the most common and earliest events in the pathogenesis of lung cancer and many other solid tumors. The chromosomal area 3p21.3 is thought to harbor at least one important tumor suppressor gene, which, despite many years of investigation, has remained elusive. In our previous studies, we have identified and cloned a gene from the common homozygous deletion area at 3p21.3. The gene, named RASSF1A (Ras ASSociation domain Family 1A), has homology to a mammalian Ras effector. The RASSF1A gene is epigenetically inactivated in a large percentage of human lung cancers, in particular small cell carcinomas. A high frequency of methylation of RASSF1A is found also in breast cancers, renal cell carcinomas, ovarian, gastric and bladder cancers, and in neuroblastomas. The RASSF1A gene is a candidate for a tumor suppressor gene in 3p21.3.

Animals↗