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Monica Hollstein

Publications and source records attributed to Monica Hollstein.

12 recordsLinked to original sources
PubMed2007

An MVA vaccine overcomes tolerance to human p53 in mice and humans.

BACKGROUND: The cellular regulatory protein p53 is overexpressed by almost 50% of all malignancies making it an attractive target for a vaccine approach to cancer. A number of immunotherapy approaches targeting p53 have been evaluated successfully in murine models, but translation of these preclinical findings to the clinic has been unsuccessful. Prior studies in our laboratory employing murine models demonstrated that a modified vaccinia virus Ankara (MVA) vaccine expressing murine p53 could stimulate p53 specific immunity. Systemic administration of the MVA vaccine was able to effect the rejection of established tumors. To better understand the immunologic mechanisms that underlie the vaccine function of human p53, we utilized a murine model in which the murine germ line copy of p53 was replaced with a modified human one. These mice, referred to as Hupki, were evaluated as a tolerant model to explore the capacity of MVA expressing human p53 to overcome tolerance and reject human p53-expressing tumors. RESULTS: MVAp53 immunization of Hupki mice resulted in the generation of p53-specific CD8(+) T cells and the rejection of a highly aggressive murine mammary carcinoma cell line 4T1(H-2d) transfected with human p53 (4T1p53). An immunologic correlate of tumor protection was evaluated utilizing an overlapping peptide library spanning the full length of human p53. This reagent was also used in combination with MVAp53 to stimulate p53-specific CD8(+) T cell responses in cancer patients. CONCLUSION: These studies demonstrate the potential of MVAp53 to overcome tolerance to p53 for cancer immunotherapy.

Animals↗
PubMed2006

Ser46 phosphorylation regulates p53-dependent apoptosis and replicative senescence.

Posttranslational modification such as phosphorylation of p53 plays important roles in activating p53 responses to various cellular and genotoxic stresses. Cell line studies have shown that phosphorylation of Ser46 is correlated with the activation of p53 apoptotic activity. To address the physiological roles of Ser46 phosphorylation, we employed homologous recombination and LoxP/Cre-mediated deletion to introduce Ser46 to Ala missense mutation into the human p53 knock-in (HUPKI) allele in mice (p53hki(S46A)). p53 stabilization in response to various types of DNA damage is modestly reduced in p53hki(S46A) embryonic stem (ES) cells, mouse embryonic fibroblasts (MEFs) and thymocytes. In addition, p53-dependent apoptosis is partially impaired in p53hki(S46A) thymocytes and E1A/Ras-expressing mouse embryonic fibroblasts (MEFs) after DNA damage. Consistent with this finding, transcription of p53 target apoptotic genes is preferentially affected by S46A mutation after DNA damage. p53hki(S46A) MEFs proliferate and reach senescence normally but can be spontaneously immortalized more easily than wild type MEFs. In addition, p53hki(S46A) MEFs more readily escapes from Ras-induced senescence. Therefore, Ser46 phosphorylation activates p53-dependent apoptosis induced by DNA damage and cellular senescence induced by oncogenic stress.

Alanine↗
PubMed2006

MEF immortalization to investigate the ins and outs of mutagenesis.

The importance of tumor suppressor/oncogene mutations in tumor development is clear, but the causes of the DNA sequence changes in human cancers are not. Although elegant experiments with transgenic mice harboring lacZ or cII target sequences show that exposure to mutagenic human carcinogens can cause base substitutions in vivo, it does not follow from this that the mutations found in human cancers have to be the direct result of damage by external mutagens. They could be due to endogenously generated reactive oxygen species, or polymerase infidelity, for example. Specific patterns of mutations in the defined sequence of a test system set up to address this question can provide information on the molecular events leading to DNA sequence changes in humans if the experimentally induced mutations and patient tumor mutations are compared in the same gene. Fortuitously, inactivating point mutations in the p53 gene are driving events in the immortalization of murine embryonic fibroblasts (MEFs) in vitro. This discovery offers a natural biological strategy for selecting p53 mutants. Immortalized cell lines arising from primary MEFs harboring human p53 sequences (Hupki, human p53 knock-in) have p53 mutations that match p53 mutations in human tumors.

Animals↗
PubMed2005

Human p53 knock-in (hupki) mice do not differ in liver tumor response from their counterparts with murine p53.

Mouse models are important tools in toxicologic research. Differences between species in pathways contributing to tumor development, however, raise the question in how far mouse models are valid for human risk assessment. One striking difference relates to the frequency of spontaneous liver cancer which is high in certain mouse strains but rather low in humans. Similarly, mutation frequencies in cancer genes are characteristically different, i.e. P53 mutations are frequent in human but very rare in murine liver tumors, whereas Ras genes are often mutated in mouse liver tumors but hardly ever in human liver cancers. Since P53 has been shown to control oncogenic RAS in human cells, we hypothesized that this function of the tumor suppressor could differ in mouse hepatocytes. To test this hypothesis, we used hupki (human p53 knock-in) mice which carry a partly humanized P53 sequence (P53KI). In this study, we report the results of the first hepatocarcinogenesis experiment with this strain of mice. Mice of the genotypes P53KI/KI, P53WT/KI and P53WT/WT were treated with N-nitrosodiethylamine at 2 weeks of age and killed 35 weeks later. The frequency of liver tumors and glucose-6-phosphatase-altered liver lesions was almost identical in all three P53 genotypes and approximately 40-50% of liver tumors showed activating mutations in codon 61 of the Ha-Ras gene independent of genotype. Moreover, only very few P53-positive lesions were observed but without nuclear localization of the protein, suggesting the absence of P53 mutations. These data suggest that the hupki allele behaves like its murine ortholog in mouse hepatocarcinogenesis.

Animals↗
PubMed2004

p53 designer genes for the modern mouse.

Major efforts are underway to develop molecular strategies that target the p53 pathway for the treatment of cancer. Mouse strains with humanized p53 sequences that present the precise human DNA-binding domain as mutation target could be informative models to test p53 rescue drugs, and to explore experimentally the causes of human tumor mutations.

Animals↗
PubMed2004

Human tumor p53 mutations are selected for in mouse embryonic fibroblasts harboring a humanized p53 gene.

To date, there has been no way to examine induced human p53 gene mutations in cell cultures exposed to mutagenic factors, other than by restriction site analysis. Here, we used embryonic cells from our Hupki (human p53 knock-in) mouse strain to generate human p53 DNA-binding domain (DBD) mutations experimentally. Twenty cultures of untreated primary mouse Hupki fibroblasts and 20 short-wavelength UV light (UVC)-treated cultures (20J/m(2)) were passaged >20 times. Established Hupki embryonic fibroblast cell lines (HUFs) were genotyped by dideoxy DNA sequencing of p53 exons 4-9. Seven of the HUFs harbored point mutations in the humanized p53 DBD. Of the 9 mutations (6 single- and 1 triple-site mutation), 2 were at the most frequently mutated codons in human cancers (c.248 and c.273). The Affymetrix p53 GeneChip assay also readily identified the 6 single-base substitutions. All mutations in HUFs from UV-treated cultures were at dipyrimidine sites, including 3 nontranscribed strand C -->T transitions. The mutant HUFs were deficient in p53 transactivation function, and missense mutants had high levels of nuclear p53 protein. In a second experiment, primary Hupki cells were exposed to the carcinogen aristolochic acid I (AAI). Five of 10 cultures that became established within 2 months harbored p53 DBD mutations. All were transversions, including 4 A --> T substitutions on the nontranscribed strand, a hallmark of DNA mutation by AAI. We conclude that establishment of Hupki mouse fibroblasts in culture readily selects for p53 DBD mutations found in human tumors, providing a basis for generating experimental mutation patterns in human p53.

Animals↗
PubMed2004

DNA adducts and p53 mutations in a patient with aristolochic acid-associated nephropathy.

BACKGROUND: Aristolochic acid-associated nephropathy (AAN) is a specific type of renal disease that predisposes patients to a high risk of urothelial carcinoma. The authors have analyzed DNA from a patient who had urothelial malignancy 6 years after presenting with AAN and later had a breast carcinoma that metastasized to the liver. METHODS AND RESULTS: DNA was isolated from the primary breast tumor, the liver tumor, and the original urothelial malignancy and assayed for aristolochic acid (AA)-DNA adducts and mutations in the p53 gene. The adduct detected was the adenosine adduct of aristolochic acid I 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI). In DNA from the breast and liver tumors the authors showed the same missense mutation in codon 245 (GGC-->GAC; Gly-->Asp) of exon 7 of p53. In contrast, DNA extracted from the urothelial tumor showed an AAG to TAG mutation in codon 139 (Lys-->Stop) of exon 5. CONCLUSION: A to T transversions, as observed here, are the typical mutations observed in the H-ras gene of tumors induced when rodents are treated with AA and correspond with DNA adduct formation at adenosine residues. These data indicate the probable molecular mechanism whereby AA causes urothelial malignancy.

Aristolochia↗
PubMed2003

Cell type- and promoter-specific roles of Ser18 phosphorylation in regulating p53 responses.

Phosphorylation of mouse p53 at Ser18 occurs after DNA damage. To determine the physiological roles of this phosphorylation event in p53-dependent DNA damage responses, a Ser18 to Ala missense mutation was introduced into the germline of mice. Thymocytes and fibroblasts from the knock-in mice show reduced transactivation of many p53 target genes following DNA damage. p53 protein stabilization and DNA binding are similar in knock-in and wild type mice, but C-terminal acetylation was defective, consistent with a role for Ser18 in the recruitment of transcriptional co-activators. The apoptotic response of knock-in thymocytes to ionizing radiation is intermediate between that of wild type and p53 null thymocytes. Despite impaired transcriptional and apoptotic responses, the knock-in mice are not prone to spontaneous tumorigenesis. This indicates that neither phosphorylation of p53 on Ser18 by ATM nor a full transcriptional response is essential to prevent spontaneous tumor formation in mice.

Animals↗
PubMed2002

Mouse models for generating P53 gene mutation spectra.

The p53 tumor suppressor gene lends itself to mutation spectra analysis, because the frequency of point mutations in human tumors is high, the locations of inactivating tumor mutations are numerous and dispersed, and all possible base substitutions are observed in human cancer. P53 tumor mutations induced experimentally in mice exposed to carcinogens have been described, but have not yet contributed significantly to our understanding of mutagenic mechanisms or of the origins of mutations in human cancers. Recently, gene-targeting technology has allowed development of a new mouse model, which explores experimentally the endogenous and environmental factors that may contribute to neoplastic disease in humans.

Animals↗
PubMed2002

The IARC TP53 database: new online mutation analysis and recommendations to users.

Mutations in the tumor suppressor gene TP53 are frequent in most human cancers. Comparison of the mutation patterns in different cancers may reveal clues on the natural history of the disease. Over the past 10 years, several databases of TP53 mutations have been developed. The most extensive of these databases is maintained and developed at the International Agency for Research on Cancer. The database compiles all mutations (somatic and inherited), as well as polymorphisms, that have been reported in the published literature since 1989. The IARC TP53 mutation dataset is the largest dataset available on the variations of any human gene. The database is available at www.iarc.fr/P53/. In this paper, we describe recent developments of the database. These developments include restructuring of the database, which is now patient-centered, with more detailed annotations on the patient (carcinogen exposure, virus infection, genetic background). In addition, a new on-line application to retrieve somatic mutation data and analyze mutation patterns is now available. We also discuss limitations on the use of the database and provide recommendations to users.

Computational Biology↗
PubMed2002

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.

Adenocarcinoma↗