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H te Riele

Publications and source records attributed to H te Riele.

At least 19 recordsLinked to original sources
PubMed2006

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3.

We have previously demonstrated that site-specific insertion, deletion or substitution of one or two nucleotides in mouse embryonic stem cells (ES cells) by single-stranded deoxyribo-oligonucleotides is several hundred-fold suppressed by DNA mismatch repair (MMR) activity. Here, we have investigated whether compound mismatches and larger insertions escape detection by the MMR machinery and can be effectively introduced in MMR-proficient cells. We identified several compound mismatches that escaped detection by the MMR machinery to some extent, but could not define general rules predicting the efficacy of complex base-pair substitutions. In contrast, we found that four-nucleotide insertions were largely subject to suppression by the MSH2/MSH3 branch of MMR and could be effectively introduced in Msh3-deficient cells. As these cells have no overt mutator phenotype and Msh3-deficient mice do not develop cancer, Msh3-deficient ES cells can be used for oligonucleotide-mediated gene disruption. As an example, we present disruption of the Fanconi anemia gene Fancf.

Animals↗
PubMed2001

Escape from premature senescence is not sufficient for oncogenic transformation by Ras.

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.

Animals↗
PubMed2001

Overlapping functions of the pRb family in the regulation of rRNA synthesis.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

3T3 Cells↗
PubMed2000

Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions.

The retinoblastoma suppressor pRB belongs to the family of so-called pocket proteins, which also includes p107 and p130. These proteins may functionally overlap in cell cycle control and tumor suppression. We have generated an isogenic set of embryonic stem (ES) cell lines carrying single or compound loss-of-function mutations in the Rb gene family, including a cell line completely devoid of all three pocket proteins. None of the knockout combinations affected the growth characteristics of ES cells; however, concomitant ablation of all three pocket proteins strongly impaired their differentiation capacity. For the generated genotypes, primary mouse embryonic fibroblasts (MEFs) also were obtained. While inactivation of Rb alone did not alleviate the senescence response of MEFs, pRB/p107-deficient MEFs, after having adapted to in vitro culturing, continued to proliferate at modest rate. Additional ablation of p130 rendered MEFs completely insensitive to senescence-inducing signals and strongly increased their proliferation rate. Although triple-knockout MEFs retained anchorage dependence, they lacked proper G(1) control and showed increased cell turnover under growth-inhibiting conditions.

Animals↗
PubMed2000

p27kip1-independent cell cycle regulation by MYC.

MYC transcription factors are potent stimulators of cell proliferation. It has been suggested that the CDK-inhibitor p27kip1 is a critical G1 phase cell cycle target of c-MYC. We show here that mouse embryo fibroblasts deficient for both p27kip1 and the related p21cip1 are still responsive to stimulation by c-MYC and can be arrested in G1 by a dominant negative mutant of c-MYC. This growth arrest can be overruled by ectopic expression of E2F or adenovirus E1A, but not by a mutant of E1A defective for binding to retinoblastoma family proteins. We show that fibroblasts with a genetic disruption of all three retinoblastoma family members (pRb, p107 and p130) are unresponsive to a dominant negative c-MYC mutant. These data indicate that p27kip1 is not the only rate limiting cell cycle target of c-MYC and suggest that regulation of E2F is also essential for c-MYC's mitogenic activity.

Adenovirus E1A Proteins↗
PubMed2000

Acceleration of lymphomagenesis in mismatch-repair deficient mice by exposure to genotoxic agents.

Hemizygosity for genes that are essential for DNA mismatch repair (MMR) was found to underlie cancer predisposition in hereditary nonpolypsis colorectal cancer (HNPCC). Loss of the wild-type allele generates a MMR-deficient cell compartment with a high propensity to oncogenic transformation. MMR deficiency not only accelerates spontaneous mutagenesis resulting from DNA replication errors, but also affects the cellular response to genotoxic agents. To study the consequences of MMR deficiency in vitro and to provide experimental access to HNPCC we have generated MMR-deficient cell lines and mice. The combination of MMR deficiency and exposure to genotoxic agents strongly accelerated lymphomagenesis.

Animals↗
PubMed1999

Msh2 status modulates both apoptosis and mutation frequency in the murine small intestine.

Deficiency in genes involved in DNA mismatch repair increases susceptibility to cancer, particularly of the colorectal epithelium. Using Msh2 null mice, we demonstrate that this genetic defect renders normal intestinal epithelial cells susceptible to mutation in vivo at the Dlb-1 locus. Compared with wild-type mice, Msh2-deficient animals had higher basal levels of mutation and were more sensitive to the mutagenic effects of temozolomide. Experiments using Msh2-deficient cells in vitro suggest that an element of this effect is attributable to increased clonogenicity. Indeed, we show that Msh2 plays a role in the in vivo initiation of apoptosis after treatment with temozolomide, N-methyl-N'-nitro-N-nitrosoguanidine, and cisplatin. This was not influenced by the in vivo depletion of O6-alkylguanine-DNA-alkyltransferase after administration of O6-benzylguanine. By analyzing mice mutant for both Msh2 and p53, we found that the Msh2-dependent apoptotic response was primarily mediated through a p53-dependent pathway. Msh2 also was required to signal delayed p53-independent death. Taken together, these studies characterize an in vivo Msh2-dependent apoptotic response to methylating agents and raise the possibility that Msh2 deficiency may predispose to malignancy not only through failed repair of mismatch DNA lesions but also through the failure to engage apoptosis.

Animals↗
PubMed1999

Mouse MutS-like protein Msh5 is required for proper chromosome synapsis in male and female meiosis.

Members of the mammalian mismatch repair protein family of MutS and MutL homologs have been implicated in postreplicative mismatch correction and chromosome interactions during meiotic recombination. Here we demonstrate that mice carrying a disruption in MutS homolog Msh5 show a meiotic defect, leading to male and female sterility. Histological and cytological examination of prophase I stages in both sexes revealed an extended zygotene stage, characterized by impaired and aberrant chromosome synapsis, that was followed by apoptotic cell death. Thus, murine Msh5 promotes synapsis of homologous chromosomes in meiotic prophase I.

Animals↗
PubMed1999

Microsatellite instability in human cancer: a prognostic marker for chemotherapy?

The majority of tumors associated with the nonpolyposis form of familial colorectal cancer (HNPCC) shows a specific form of genetic instability which is manifested by length alterations of mono- or dinucleotide repeat sequences [e.g., (A)n or (CA)n]. This phenomenon was termed the RER+ (replication error-positive) phenotype, MSI or MIN (microsatellite instability), and found to result from defects in the cells' DNA mismatch repair system. This system recognizes and restores misincorporated bases or slippage errors which frequently occur during DNA replication. Loss of DNA mismatch repair therefore strongly accelerates the evolutionary process of mutagenesis and selection which underlies the development of cancer. In addition to mutation avoidance, DNA mismatch repair also plays a crucial role in the toxicity of a number of DNA-damaging drugs that are used in cancer chemotherapy. In experimental systems, mismatch-repair-deficient cells are highly tolerant to the methylating chemotherapeutic drugs streptozocin and temozolomide and, albeit to a lesser extent, to cisplatin and doxorubicin. These drugs are therefore expected to be less effective on mismatch-repair-deficient tumors in humans. MIN was also found in a substantial portion of sporadic (nonfamilial) human tumors. However, in many cases the extent of microsatellite instability was not as dramatic as found in HNPCC-related tumors and the underlying genetic defect is unclear. Therefore, while the mismatch repair status of tumors may become an important determinant in the choice of chemotherapeutic intervention, the significance of MIN in sporadic cancer remains elusive.

Base Pair Mismatch↗
PubMed1999

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

Alleles↗
PubMed1998

Mouse embryonic stem cells carrying one or two defective Msh2 alleles respond abnormally to oxidative stress inflicted by low-level radiation.

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted Msh2 alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from Msh2(+/-) and Msh2(-/-) mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from Msh2(+/+) mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.

Alleles↗
PubMed1998

Flp-mediated tissue-specific inactivation of the retinoblastoma tumor suppressor gene in the mouse.

The yeast-derived Flp-frt site-specific DNA recombination system was used to achieve pituitary-specific inactivation of the retinoblastoma (Rb) tumor suppressor gene. Whereas mice carrying only frt sites in both alleles of Rb remain tumor free, tumorigenesis ensues when the Flp recombinase is expressed. The rate of tumorigenesis in these mice depends both on the expression level of the Flp recombinase and on the presence of frt sites in one or both Rb alleles. This permitted a more accurate definition of the consecutive steps in pituitary tumorigenesis. Our study illustrates the potential of this approach for studying sporadic cancer in a defined mouse model.

Alleles↗
PubMed1998

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Animals↗
PubMed1998

p107 is a suppressor of retinoblastoma development in pRb-deficient mice.

Hemizygosity for the retinoblastoma gene RB in man strongly predisposes to retinoblastoma. In the mouse, however, Rb hemizygosity leaves the retina normal, whereas in Rb-/- chimeras pRb-deficient retinoblasts undergo apoptosis. To test whether concomitant inactivation of the Rb-related gene p107 is required to unleash the oncogenic potential of pRb deficiency in the mouse retina, we inactivated both Rb and p107 by homologous recombination in embryonic stem cells and generated chimeric mice. Retinoblastomas were found in five out of seven adult pRb/p107-deficient chimeras. The retinal tumors showed amacrine cell differentiation, and therefore originated from cells committed to the inner but not the outer nuclear layer. Retinal lesions were already observed at embryonic day 17.5. At this stage, the primitive nuclear layer exhibited severe dysplasia, including rosette-like arrangements, and apoptosis. These findings provide formal proof for the role of loss of Rb in retinoblastoma development in the mouse and the first in vivo evidence that p107 can exert a tumor suppressor function.

Animals↗
PubMed1998

Mouse models for hereditary nonpolyposis colorectal cancer.

Hemizygous germ-line defects in mismatch repair (MMR) genes underlie hereditary nonpolyposis colorectal cancer (HNPCC). Loss of the wild-type allele results in a mutator phenotype, accelerating tumorigenesis. Tumorigenesis specifically occurs in the gastrointestinal and genitourinary tracts; the cause of this tissue specificity is elusive. To understand the etiology and tissue distribution of tumors in HNPCC, we have developed mouse models carrying a deficiency in the MMR gene Msh2. Most of the completely Msh2-deficient mice succumbed to lymphomas at an early age; lymphomagenesis was synergistically enhanced by exposure to ethylnitrosourea. Lymphomas were absent in immunocompromised Tap1-/-;Msh2-/- mice; these mice generally succumbed to HNPCC-like tumors. Together, these data suggest that the HNPCC tumor spectrum is determined by exposure of MMR-deficient cells to exogenous mutagens, rather than by tissue-specific loss of the wild-type MMR allele or by immune surveillance. Msh2 hemizygous mice had an elevated tumor incidence that, surprisingly, was rarely correlated with loss of the Msh2+ allele. To develop a model for intestinal tumorigenesis in HNPCC, we introduced the Min allele of the Apc tumor suppressor gene. We observed loss of the wild-type Msh2 allele in a significant fraction of intestinal tumors in Apc+/Min;Msh2+/- mice. In some of the latter tumors, one area of the tumor displayed loss of the Msh2+ allele, but not of the Apc+ allele, whereas another area displayed the inverse genotype. This apparent biclonality might indicate a requirement for collaboration between independent tumor clones during intestinal tumorigenesis.

Adenomatous Polyposis Coli Protein↗
PubMed1995

Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.

To investigate the role of the presumed DNA mismatch repair (MMR) gene Msh2 in genome stability and tumorigenesis, we have generated cells and mice that are deficient for the gene. Msh2-deficient cells have lost mismatch binding and have acquired microsatellite instability, a mutator phenotype, and tolerance to methylating agents. Moreover, in these cells, homologous recombination has lost dependence on complete identity between interacting DNA sequences, suggesting that Msh2 is involved in safeguarding the genome from promiscuous recombination. Msh2-deficient mice display no major abnormalities, but a significant fraction develops lymphomas at an early age. Thus, Msh2 is involved in MMR, controlling several aspects of genome stability; loss of MMR-controlled genome stability predisposes to cancer.

Animals↗
PubMed1995

An HMG-box-containing T-cell factor required for thymocyte differentiation.

Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.

Animals↗