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Peter J Stambrook

Publications and source records attributed to Peter J Stambrook.

At least 19 recordsLinked to original sources
PubMed2006

The breast cancer susceptibility allele CHEK2*1100delC promotes genomic instability in a knock-in mouse model.

Allelic variants of CHEK2 contribute to an elevated risk for human breast cancer and possibly other cancer types. In particular, the CHEK2*1100delC polymorphic variant has been identified as a low-penetrance breast cancer susceptibility allele in breast cancer families with wild type BRCA1 and BRCA2. To better understand the molecular basis by which this allele increases risk for disease, we have generated a mouse in which the wild type CHEK2 (Chk2 in mouse) allele has been replaced with the 1100delC variant. Mouse embryo fibroblasts (MEFs) derived from these mice have an altered cell cycle profile in which a far greater proportion of cells are in S-phase and in G2 (4N) compared with wild type cells. The mutant cells show signs of spontaneous genomic instability as indicated by polyploidy and an increase in DNA double strand breaks.

Alleles↗
PubMed2006

An ageing question: do embryonic stem cells protect their genomes?

Many physiological and cellular processes contribute to the ageing of individuals. One hypothesis argues that the genomes of somatic cells accumulate mutations, which, in turn, alter the metabolism of the cells and contribute to the ageing process. The frequency of somatic mutation approaches 10(-4) and the majority of mutagenic events at heterozygous loci is due to loss of heterozygosity as a consequence of mitotic recombination. A corollary to the argument that somatic cells accumulate mutations is that cells of the germ line and ES cells have a greater requirement for maintaining the integrity of their genomes. In the former case, a high somatic mutation frequency predicts an increase in somatic disease, which limits our lifespan. The corollary is that cells of the germline and ES cells must minimize the mutational burden to limit the frequency of congenital disease and to ensure the proper transmission of undamaged DNA to the gene pool. This report describes two mechanisms utilized by murine ES cells to minimize DNA damage within the proliferative pool. In the first case, murine ES cells display a frequency of mutation and mitotic recombination that is about 100-fold lower than that observed in somatic cells. Second, ES cells lack a G1 checkpoint following DNA damage. When subjected to ionizing radiation, the fraction of apoptotic cells increases to about 40%. Ectopic expression of Chk2 is sufficient to establish a G1 arrest and the concomitant protection from cell death.

Aging↗
PubMed2006

Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage.

The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.

Ataxia Telangiectasia↗
PubMed2006

Exposure of mice to arsenic and/or benzo[a]pyrene does not increase the frequency of Aprt-deficient cells recovered from explanted skin of Aprt heterozygous mice.

Exposure to inorganic arsenic in drinking water is linked to cancer in humans, but the mechanism of arsenic-induced cancer is not clear. Arsenic is not a powerful point mutagen, but can cause chromosome malsegregation and mitotic recombination, two events that can cause loss of tumor suppressor alleles and thereby contribute to the evolution of cancerous cells. To determine whether arsenic increases the frequency of allele loss due to either malsegregation or mitotic recombination in vivo, Aprt(+/-) hybrid mice were exposed to sodium arsenite (10 mg/L) in their drinking water for 10 weeks. To determine whether arsenic enhances the action of a known mutagen, half of the arsenic-treated mice were exposed to benzo[a]pyrene (BaP) for 8 weeks by skin painting (500 nmoles/week). Cells were taken from painted dorsal skin and cultured in the presence of 2,6-diaminopurine (DAP), to select colonies lacking adenosine phosphoribosyl transferase (Aprt) activity. The frequency of DAP-resistant (DAP(r)) colonies varied substantially within the treatment groups, but there was no significant difference between the groups. Analysis of DNA from DAP(r) colonies suggested that mitotic recombination contributed to the loss of wild-type Aprt allele. Whether arsenic or BaP enhanced or diminished the frequency of this process could not be deduced from these data.

2-Aminopurine↗
PubMed2006

DNA damage response and mutagenesis in mouse embryonic stem cells.

Mutation in embryonic stem (ES) cells can potentially compromise multiple cell lineages and affect the well-being of subsequent generations. Thus, ES cells require sensitive mechanisms to maintain genomic integrity. One mechanism involves suppression of mutation. A complementary mechanism is to regulate the cell cycle checkpoint and facilitate cell death. Here, we describe the detailed protocols we have used to investigate DNA damage response and mutagenesis in mouse ES cells.

Adenine Phosphoribosyltransferase↗
PubMed2005

Co-mutagenic activity of arsenic and benzo[a]pyrene in mouse skin.

Exposure to inorganic arsenic in drinking water is linked to skin, lung and bladder cancer in humans. The mechanism of arsenic-induced cancer is not clear, but exposure to arsenic and polycyclic arylhydrocarbons (PAH) is more carcinogenic than exposure to either type of carcinogen alone. Arsenic can also generate reactive oxygen species, suggesting that oxidation of DNA may play a role in carcinogenesis. Oxidization of guanosines in polyG tracts is known to cause frameshift mutations, and such events can be detected in situ using the G11 placental alkaline phosphatase (PLAP) transgenic mouse model, which reports frameshift mutations in a run of 11 G:C basepairs by generating cells containing heat-resistant alkaline phosphatase activity. PAH can also induce frameshift mutations. In the study described here, FVB/N mice carrying the G11 PLAP transgene were crossed to C57Bl/6 mice. Half of the hybrid mice were given drinking water with sodium arsenite (10 mg/L) for 10 weeks. Half of the arsenic treated mice were also exposed to benzo[a]pyrene (BaP) by skin painting (500 nmol/week) for 8 weeks. Another group of mice was exposed to BaP but not arsenic. The effect on frameshift mutation was assessed by staining sections of skin tissue to detect cells with PLAP activity. Arsenic alone had no significant effect. On average, mice given BaP alone had approximately three times more PLAP-positive (PLAP+) cells. By contrast, mice exposed to both arsenic and BaP exhibited 10-fold more PLAP+ cells in the skin, and these cells were often arranged in large clusters, suggesting derivation from stem cells. Whereas combined treatment produced more PLAP+ cells, stable BaP adduct levels and arsenic burdens were not higher in mice exposed to both agents compared to mice exposed to either one agent or the other.

Alkaline Phosphatase↗
PubMed2005

The Plk3-Cdc25 circuit.

Polo-like kinases (Plks) are key regulators of the cell cycle, especially in the G2 phase and mitosis. They are incorporated into signaling networks that regulate many aspects of the cell cycle, including but not limited to centrosome maturation and separation, mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. The Plks have well conserved 30-amino-acid elements, designated polo boxes (PBs), located in their carboxyl-termini, which with their flanking regions constitute a functional Polo-box domain (PBD). Members of the Plk family exist in a variety of organisms including Polo in Drosophila melanogaster; Cdc5 in Saccharomyces cerevisiae; Plo1 in Schizosaccharomyces pombe; Plx1 in Xenopus laevis; and Plk1, Snk/Plk2, Fnk/Prk/Plk3, and Sak in mammals. Polo, Cdc5, and Plo1 are essential for viability. The Plks can be separated into two groups according to their functions. The first group (Polo, Cdc5, plo1, Plx1, and Plk1) primarily performs mitotic functions, whereas the second group (Plk2 and Plk3) appears to have additional functions during the G1, S, and G2 phases of the cell cycle. Several contributions to this issue will discuss different aspects of Plk involvement in cell-cycle regulation. This review, therefore, will focus on the role of Plk3 in regulating Cdc25 phosphatase function and its effect on the cell cycle.

Animals↗
PubMed2004

Mlh1 mediates tissue-specific regulation of mitotic recombination.

Mitotic recombination (MR) between chromosome homologs in somatic cells is a major pathway to the loss of heterozygosity (LOH), which may cause cancer if tumor suppressor genes are involved. MR can be suppressed by DNA sequence heterology (homeology) in hybrid mice from matings between species or between subspecies. We now report that MR is relatively suppressed in F1 hybrids between inbred strains C57BL/6 and 129S2. The frequency of MR in fibroblasts is lower in F1 hybrid mice than in either of the two parental strains. However, MR in T cells is not affected by strain background. Thus, relatively small genetic differences are capable of restricting MR in a tissue-specific manner. Using Mlh1-deficient mice, we tested the role of mismatch repair in MR in two isogenic cell types. In fibroblasts of C57BL/6 x 129S2 F1 mice, the suppression of MR is alleviated in the absence of MLH1. In contrast, MR is not affected by Mlh1 status in T cells. The frequency of point mutations at the reporter gene loci Aprt and Hprt, on the other hand, is significantly increased in both T cells and fibroblasts of Mlh1(-/-) mice. Thus, different cell types respond differently to MLH1 deficiency, and the contribution of MR to tumorigenesis may be tissue-dependent in the absence of mismatch repair.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Restoration of an absent G1 arrest and protection from apoptosis in embryonic stem cells after ionizing radiation.

Response to DNA damage and cell-cycle regulation differ markedly between embryonic stem (ES) cells and somatic cells. ES cells require exquisitely sensitive mechanisms to maintain genomic integrity and do so, in part, by suppressing spontaneous mutation. Spontaneous mutation frequency in somatic cells is approximately 10(-4) compared with 10(-6) for ES cells. ES cells also lack a G(1) checkpoint and are hypersensitive to IR and other DNA-damaging agents. These characteristics facilitate apoptosis and the removal of cells with a mutational burden from the population, thereby keeping the population free of damaged cells. Here, we identify signaling pathways that are compromised and lead to a natural absence of aG(1) arrest in ES cells after DNA damage. The affected pathways are those mediated by p53 and p21 and by ATM, Chk2, Cdc25A, and Cdk2. In ES cells, Chk2 kinase is not intranuclear as in somatic cells but is sequestered at centrosomes and is unavailable to phosphorylate Cdc25A phosphatase and cause its degradation. Although ectopic expression of Chk2 does not rescue the p53/p21 pathway, its expression is sufficient to allow it to phosphorylate Cdc25A, activate downstream targets, restore a G(1) arrest, and protect the cell from apoptosis.

Animals↗
PubMed2004

Cdc25C phosphorylation on serine 191 by Plk3 promotes its nuclear translocation.

Mitosis in human cells is initiated at the end of G2 by activation of the Cdc2/cyclin B complex. Activation occurs by dephosphorylation of the inhibitory residues, threonine 14 (T14) and tyrosine 15 (Y15), on Cdc2 by the Cdc25C phosphatase. Entry into mitosis is regulated by the subcellular relocalization of Cdc2/cyclin B, which is rapidly imported into the nucleus at the end of G2. Here, we show that polo-like kinase 3 (Plk3) is able to phosphorylate Cdc25C primarily on S191, and to a lesser extent on S198 in vitro, both of which are within a nuclear exclusion motif. Following transfection, the S191D Cdc25C mutant leads to an enhanced accumulation of Cdc25C in the nucleus, while the S191A mutant facilitated the Cdc25C nuclear exclusion. Furthermore, translocation of Cdc25C to the nucleus was accompanied by a decrease in Cdc2 phosphorylation on Y15. Plk3-WT overexpression led to a sharp increase in Cdc25C nuclear accumulation, while Plk3-KD overexpression failed to do so. The effect of Plk3 overexpression on Cdc25C was reversed by coexpression of a Plk3 siRNA. These results support a role for the polo kinases in coordinating the translocation and perhaps the timing of both Cdc25C and its target Cdc2/cyclin B to the nucleus upon entry into mitosis.

Active Transport, Cell Nucleus↗
PubMed2004

Major histocompatibility gene therapy: the importance of haplotype and beta 2-microglobulin.

OBJECTIVES/HYPOTHESIS: Alloantigen gene therapy with the genes for the Class I major histocompatibility complex (MHC) HLA-B7 and beta 2-microglobulin in HLA-B7-negative patients has potential efficacy in the treatment of head and neck cancer, although the mechanism of response is unclear. Whether tumor regression is due to a response to HLA-B7 in HLA-B7-negative patients (i.e., due to "foreign" antigen) or simply to MHC overexpression is unknown. Therefore, a mouse model was used to compare tumor growth following syngeneic MHC transfection to alloantigenic MHC transfection. The importance of the beta 2-microglobulin gene was also evaluated. STUDY DESIGN: Prospective animal study. METHODS: The head and neck cancer cell line SCC-VII that grows in immunocompetent C3H mice, which are MHC haplotype H2-K, was used. Stable transfections were made with H2-K, H2-K, and beta 2-microglobulin in the SCC-VII cells. To test the importance of MHC "foreignness," mice were injected with SCC-VII cells, SCC-VII plus H2-K plus beta 2-microglobulin transfected cells, and SCC-VII plus H2-K plus beta 2-microglobulin transfected cells. To evaluate beta 2-microglobulin, mice were injected with SCC-VII cells, SCC-VII plus H2-K plus beta 2-microglobulin transfected cells, SCC-VII plusH2-K transfected cells, and SCC-VII plus beta 2-microglobulin transfected cells. Tumor growth in all groups was compared statistically. RESULTS: Major histocompatibility complex foreignness was a part of the antitumor response. Foreign MHC routinely abrogated tumor growth, whereas syngeneic MHC only slowed tumor growth. beta 2-microglobulin aided the MHC tumor inhibition but did not inhibit tumor without the MHC. CONCLUSION: The antitumor response was greater when the MHC gene used was foreign. beta 2-microglobulin increased the efficacy of MHC gene therapy. Both of these findings are important when designing clinical trials of immunologically based gene therapies for head and neck cancer.

Animals↗
PubMed2003

Impaired expression of an organic cation transporter, IMPT1, in a knockout mouse model for kidney stone disease.

The imprinted multimembrane-spanning polyspecific transporter-like gene 1 ( IMPT1) encodes a predicted protein with organic cation transport capabilities. As a first step in understanding the function of IMPT1, we identified the renal structures expressing this gene in knockout mice with adenine phosphoribosyltransferase (APRT) deficiency and 2,8-dihydroxyadenine (DHA) nephrolithiasis. IMPT1 mRNA was not detected using a standard in situ hybridization (ISH) protocol, but we observed intense staining in cortico-medullary tubules and glomeruli in wild-type mice using an improved reverse transcription-polymerase chain reaction (RT-PCR) ISH procedure. IMPT1 mRNA expression was significantly decreased in the cortical region in kidney sections from APRT-deficient male mice. APRT-deficient female mice are less severely affected by DHA-induced kidney stone disease, and we observed only a modest reduction in IMPT1 expression in kidneys from these mice. IMPT1 expression in APRT heterozygous mice was comparable to that in wild-type mice, suggesting imprinting of one of the parental alleles. These findings suggest that decreased IMPT1 mRNA expression may contribute to the impaired renal function in APRT-deficient male mice, and that RT-PCR ISH is a valuable tool for localizing the site of expression of transcripts that are not detectable using standard ISH procedures.

Adenine↗
PubMed2003

Alloantigen gene therapy for head and neck cancer: evaluation of animal models.

BACKGROUND: Human trials of alloantigen gene therapy, using the class I major histocompatibility complex (MHC) HLA-B7, have demonstrated the potential efficacy of this treatment for head and neck cancer. Its mechanism remains unclear. An immune-competent mouse model of MHC gene therapy to test factors potentially important to the tumor response is needed. METHODS: Two cell lines were used, B4B8 cells that grow in Balb/c mice and SCC-VII cells that grow in C3H mice. The mouse MHC H2-K(b) was used as the therapeutic gene, because it is an alloantigen to both mice strains. Plasmids that encode the H2-K(b) cDNA were prepared, and the cell lines were transfected. Mice were injected subcutaneously with naive cells to determine the tumor kinetics and serve as controls. Mice were injected with H2-K(b) transfected cells and tumor growth was compared with controls. Mice that did not grow tumor were rechallenged with naive cells to assess for tumor immunity. Mice were injected with transfected and naive cells admixed to determine whether the concentration of the alloantigen is important. RESULTS: B4B8 tumors grew slowly, whereas SCC-VII tumors grew rapidly. Transfection with H2-K(b) plasmid prevented or inhibited tumor growth of both the B4B8 and SCC-VII tumors. This growth inhibition was independent of the number of cells injected. In the mice that did not grow tumor, tumor immunity was demonstrated after challenge with naive cells in both models. There was no relationship between induction of immunity and the timing of the challenge or initial cell quantity. The mice injected with a mixture of naive and transfected cells grew tumor, although growth was delayed in the B4B8 model. CONCLUSIONS: The results demonstrate that the two mouse models can serve as a rapid and slow growing tumor model of alloantigen gene therapy. In addition, it was noted that initial tumor cell number is not a significant factor for predicting tumor response and demonstrated that in both of these models alloantigen gene therapy results in significant antitumor immunity.

Animals↗
PubMed2003

Antitumor effect of secreted Flt3-ligand can act at distant tumor sites in a murine model of head and neck cancer.

The Flt3 ligand (Flt3-L) manifests antitumor activity, presumably due to its capacity to recruit dendritic cells and cause their proliferation. To assess whether local production of Flt3-L can mediate a "distant bystander" effect, murine B4B8 squamous cell carcinoma cells were transfected with a plasmid encoding a secretory form of Flt3-L to produce B4B8FL cells. Similarly, B4B8FL and B4B8 cells were transfected with herpes simplex virus thymidine kinase (HSVTK) to produce B4B8TK and B4B8FL/TK cells, which should be sensitive to ganciclovir (GCV), to know whether the effects of Flt3-L and HSVTK/GCV would be synergistic. To test for a distant bystander effect in vivo, B4B8FL, B4B8TK, and B4B8FL/TK cells were injected subcutaneously into the left flank of syngeneic Balb/c mice, and naïve B4B8 cells were injected into the right flank. The formation of tumors derived from B4B8FL and B4B8FL/TK cells was significantly delayed in both flanks compared with naïve B4B8 and B4B8TK cells. Growth of B4B8TK tumors in the ipsilateral flank was retarded following GCV treatment, but in contrast to B4B8FL and B4B8FL/TK cells, no distant bystander effect in the contralateral flank was observed. Immunohistochemistry showed lymphocytic infiltrates in both flanks of the B4B8FL and B4B8FL/TK groups. The data indicate that in these cells, local secretion of Flt3-L is sufficient to evoke a distant bystander effect but that expression of HSVTK, even after GCV administration, is not. Furthermore, the combination of local Flt3-L and HSVTK production, together with GCV administration, does not enhance the distant bystander effect produced by Flt3-L alone.

Animals↗
PubMed2002

Mammalian Polo-like kinase 3 (Plk3) is a multifunctional protein involved in stress response pathways.

The Polo-like kinases (Plks) are a conserved family of kinases that contribute to cell cycle regulation, particularly in G2 and mitosis. In mammals, there are at least three members of the Plk family. Here we show that Plk3 is a stress response protein that becomes phosphorylated following DNA damage or mitotic spindle disruption. Phosphorylation enhances its kinase activity and is dependent upon ataxia telangiectasia-mutated (ATM) in the former case but not the latter. Plk3 associates with complexes of multiple sizes ranging from 150 to greater then 600 kDa. In its unphosphorylated form it elutes from a sizing column at about 400 kDa whereas it associates with complexes of 150 and 600 kDa when phosphorylated. Among the proteins with which it physically associates and utilizes, as substrates are Chk2 and P53. It phosphorylates Chk2 on a residue different from threonine 68 (Thr68), the principal target for ATM. While ATM is necessary for phosphorylation and activation of Chk2 in vivo, Plk3 seems to contribute to its full activation. In its phosphorylated form it also coelutes and forms a complex with unpolymerized tubulin. In aggregate, the data argue that Plk3 is a multifunctional protein that associates with multiple complexes and that contributes to response to stress incurred by DNA damage and mitotic spindle disruption, albeit via different pathways.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2002

Visualization of mosaicism in tissues of normal and mismatch-repair-deficient mice carrying a microsatellite-containing transgene.

To determine the frequency of mutation in different cell types of mammals, transgenic mice that allow mutant cells to be visualized in situ were used. These mice carry a defective allele of the human placental alkaline phosphatase (PLAP) gene. The allele does not produce enzyme because the reading frame is shifted by an insertion of 7 G:C basepairs. The insertion is adjacent to four existing G:C basepairs, so the allele has a tract of 11Gs. The G11 PLAP allele was studied in wildtype mice and in mice deficient in mismatch-repair (MMR) due to lack of either Pms2 or Mlh1. PLAP(+) cells were counted in brain, heart, kidney, and liver. In wildtype mice, there was an average of between 5 and 30 PLAP(+) events per million cells. No cells with alkaline phosphatase activity were detected in tissues from mice lacking the PLAP gene. In MMR-deficient mice, the number of PLAP(+) allele was increased by at least three-order of magnitude in brain, heart and kidney, but <10-fold in liver. These data show that MMR is vital to maintaining repeat stability in brain, heart and kidney cells. The reason for the different results in the liver is not clear. Cells in the liver were shown to be capable of expressing of PLAP enzyme and PLAP mRNA was present in this organ.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Radiation-induced genetic instability in vivo depends on p53 status.

In response to ionizing radiation and other agents that damage DNA, the p53 tumor suppressor protein activates multiple cellular processes including cell cycle checkpoints and programmed cell death. Although loss of p53 function is associated with radiation-induced genetic instability in cell lines, it is not clear if this relationship exists in vivo. To study the role of p53 in maintenance of genetic stability in normal tissues following irradiation, we have measured mutant frequencies at the adenine phosphoribosyltransferase (Aprt) and hypothanine-guanine phosphoribosyltransferase (Hprt) loci and examined mechanisms of loss of heterozygosity (LOH) in normal T cells of p53-deficient, Aprt heterozygous mice that were subjected to whole-body irradiation with a single dose of 4Gy X-rays. The radiation-induced mutant frequency at both the Aprt and Hprt loci was elevated in cells from mice with different p53 genotypes. The radiation-induced elevation of p53-/- mice was significantly greater than that of p53+/- or p53+/+ mice and was caused by several different kinds of mutational events at the both chromosomal and intragenic levels. Most significantly, interstitial deletion, which occurs rarely in unirradiated mice, became the most common mechanism leading to LOH in irradiated p53 null mice. These observations support the idea that absence or reduction of p53 expression enhances radiation-induced tumorigenesis by increasing genetic instability at various loci, such as those for tumor suppressor genes.

Animals↗