Showing papers by "Allan Bradley published in 1998"

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Abstract: Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome) We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53 We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis

Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis

Abstract: The Mdm2 proto-oncogene is amplified to high copy numbers in human sarcomas and is overexpressed in a wide variety of other human cancers. Because Mdm2 protein forms a complex with the p53 tumor suppressor protein and down-regulates p53 function, the oncogenic potential of Mdm2 is presumed to be p53-dependent. To model these conditions in mice, we have used the entire Mdm2 gene, under transcriptional control of its native promoter region, as a transgene to create mice that overexpress Mdm2. The transgenic mice are predisposed to spontaneous tumor formation, and the incidence of sarcomas observed in the Mdm2-transgenic mice in the presence or absence of functional p53 demonstrates that, in addition to Mdm2-mediated inactivation of p53, there exists a p53-independent role for Mdm2 in tumorigenesis.

Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair

Abstract: Germline mutations in the human MSH2, MLH1, PMS2 and PMS1 DNA mismatch repair (MMR) gene homologues appear to be responsible for most cases of hereditary non-polyposis colorectal cancer (HNPCC; refs 1-5). An important role for DNA replication errors in colorectal tumorigenesis has been suggested by the finding of frequent alterations in the length of specific mononucleotide tracts within genes controlling cell growth, including TGF-beta receptor type II (ref. 6), BAX (ref. 7) and APC (ref. 8). A broader role for MMR deficiency in human tumorigenesis is implicated by microsatellite instability in a fraction of sporadic tumours, including gastric, endometrial and colorectal malignancies. To better define the role of individual MMR genes in cancer susceptibility and MMR functions, we have generated mice deficient for the murine homologues of the human genes MLH1, PMS1 and PMS2. Surprisingly, we find that these mice show different tumour susceptibilities, most notably, to intestinal adenomas and adenocarcinomas, and different mutational spectra. Our results suggest that a general increase in replication errors may not be sufficient for intestinal tumour formation and that these genes share overlapping, but not identical functions.

Chromosomal Transposition of a Tc1/Mariner-Like Element in Mouse Embryonic Stem Cells

Abstract: Mouse has become an increasingly important organism for modeling human diseases and for determining gene function in a mammalian context. Unfortunately, transposon-tagged mutagenesis, one of the most valuable tools for functional genomics, still is not available in this organism. On the other hand, it has long been speculated that members of the Tc1/mariner-like elements may be less dependent on host factors and, hence, can be introduced into heterologous organisms. However, this prediction has not been realized in mice. We report here the chromosomal transposition of the Sleeping Beauty (SB) element in mouse embryonic stem cells, providing evidence that it can be used as an in vivo mutagen in mice.

Embryonic lethality and tumorigenesis caused by segmental aneuploidy on mouse chromosome 11.

Abstract: Chromosome engineering in mice enables the construction of models of human chromosomal diseases and provides key reagents for genetic studies. To begin to define functional information for a small portion of chromosome 11, deficiencies, duplications, and inversions were constructed in embryonic stem cells with sizes ranging from 1 Mb to 22 cM. Two deficiencies and three duplications were established in the mouse germline. Mice with a 1-Mb duplication developed corneal hyperplasia and thymic tumors, while two different 3- to 4-cM deficiencies were embryonically lethal in heterozygous mice. A duplication corresponding to one of these two deficiencies was able to rescue its haplolethality.

Thirteen years of manipulating the mouse genome: a personal history.

Abstract: In 1974, Dr. Ralph Brinster published a paper describing the consequences of injecting embryonal carcinoma cells, the predecessors of embryonic stem cells, into mouse blastocysts. Despite their early promise, embryonal carcinoma cells would not efficiently populate the germ line of mice. A decade later Elizabeth Robertson and I described the efficient generation of germline chimaeras from cultured embryonic stem cells and shortly afterwards the genetic manipulation of the mouse germline using ES cells. Our demonstration of the potency of Embryonic Stem cells gave birth to a new era in manipulative mouse genetics, one in which endogenous genes can now be mutated at will using gene targeting of retroviral mutagenesis. This review focuses on the development and testing of concepts and techniques during the thirteen years after we knew germline modification of endogenous genes in the mouse would be possible. This period is one in which more and more sophisticated tools for manipulating the mouse germline were developed and implemented. In this review I have taken the rare opportunity to reveal some of my thought processes, frustrations, successes and failures as we moved through this exciting period of rapid technological change. As I look forward to the next thirteen years, I feel that this will be an equally exciting period for manipulative genetics as we struggle to formulate concepts and design experiments that enable us to understand gene function in an era when the sequence of all genes will be known.

Functional characterization of BRCA1 and BRCA2: clues from their interacting proteins.

Abstract: The familial breast and ovarian cancersusceptibility genes, BRCA1 and BRCA2 have been thesubject of extensive functional analysis studies sincetheir cloning. Clues to their biological role inmaintaining the genomic integrity were provided by studiesthat revealed their interaction with the recombinationrepair protein HsRad51. The first clue of an interactionbetween HsRad51 and BRCA1 came from the colocalization of the characteristic nuclear foci formed bythese two proteins during S phase of the cell cycle. Aninteraction between murine Brca2 and MmRad51 wasdetected by the yeast two hybrid system. Utilizing the yeast two hybrid system and other techniquesseveral other Brca1 and Brca2 interacting proteins havebeen identified like, BARD1, importin-α, BIPs, RNApolymerase II holoenzyme, BRAP2 etc. Recently, mutations suggesting a role as a tumorsuppressor have been identified in the BARD1 gene inprimary human tumors. The identification of moleculesthat interact with Brca1 and Brca2 has greatly enhanced our knowledge of how BRCA1 and BRCA2 mayfunction as tumor suppressors.

A step toward genotype-based therapeutic regimens for breast cancer in patients with BRCA2 mutations?

Abstract: The positional cloning of a familial tumor suppressor gene is a monumental task often involving the coordinated efforts of international consortia of many laboratories. The euphoria that accompanies the successful cloning of a cancer-causing gene signals the initiation of many new avenues of study that attempt to define the activity of that gene’s product. These goals are laudatory, because by defining such a function and understanding how it is subverted by mutations, the hope is that eventually this information can be used to develop therapeutic approaches aimed at patients and their family members who participated selflessly in the genetic studies that ultimately led to the identification of the gene. The biologic functions of gene products are hugely diverse, and our knowledge base of gene function is currently so poor that the experimental pathways that lead to the elucidation of a gene’s function are not necessarily easy to discern. Usually, many years elapse between the cloning of a familial tumor suppressor gene and the identification of its function. Moreover, although valuable information will be garnered about a gene’s function, translating this knowledge into effective therapeutic regimens, based on the knowledge of an individual’s genetic lesion, is extremely difficult. However, an article appearing in this issue of the Journal (1), as well as two other articles (2,3), suggests that genotype-based therapeutics for familial breast cancer caused by lesions in the BRCA2 gene may be possible. All of these studies show that cell lines lacking functional BRCA2 are hypersensitive to agents that

The Ptentative nature of mouse knockouts.

Abstract: 322 nature genetics volume 20 december 1998 If you want a mutant mouse, of course, you can get it if you really want, although the extent to which you must try, try and try (again) will vary. Over the last decade, embryonic stem cell technology has moved from the domain of a few laboratories to that of the scientific community at large. Mutant mice afford unprecedented opportunities to experimentally address gene function in vivo in a malleable experimental context rarely provided by humans with analogous mutations. Consequently, announcement of a ‘disease’ gene often spawns multiple new research projects in many laboratories that will then compete to be the first to generate, analyse and report on a mouse strain missing the disease-gene homologue. Where a ‘hot’ gene is concerned, the number of groups at the starting line may be more than a dozen. While this duplication of effort might seem wasteful, the variety of alleles generated by parallel efforts is frequently beneficial in elucidating a gene’s function. There are some excellent examples of this; for instance, hypomorphic alleles of tumour suppressors Brca2 (refs 1,2) and Brca1 (ref. 3) have been as important as null alleles in offering insight into gene function. Competing groups often examine the effect of a null allele in different genetic backgrounds, which can give dramatically different phenotypes4,5, illustrating the effects of modifier alleles and pointing to genetic interactions which might go undetected if only one group had generated a targeted mutation. The relative ease with which mutant mice can now be made contrasts with the difficulty of making sense of the results. Phenotypic studies can take a long time, and it is not unusual to be left with the unsatisfying conclusion that a given mutation does not alter the phenotype6. This often reflects experimental limitations in knowing what assays to perform on a highly complex multicellular organism. Analysis of embryonic lethal phenotypes is also challenging, and it is not unusual for competing groups to disagree on the embryological cause of a developmental defect. Recently, independent knockout studies of the tumour suppressor Pten reached different conclusions on the embryonic function of this gene. While both groups agreed that Pten is essential for embryonic development, Antonio Di Cristofano and colleagues7 concluded that Pten is required for the differentiation of endoderm, mesoderm and ectoderm while Akira Suzuki and co-workers8 reported that Pten is required for patterning of the cephalic and caudal regions of the embryo and for placental development. What is the basis for these different conclusions? Failing to observe mutant embryos after embryonic day (E) 7.5, Di Cristofano et al. performed detailed examination of the difThe PTENtative nature of mouse knockouts

Introduction: BRCA1 and BRCA2 in mammary gland development and tumorigenesis.

Abstract: women and the second leading cause of cancer mortalfew years have provided clues about the function of ity. The previous two decades have increased our these gene products in cell growth and DNA damage understanding of basic cancer mechanisms and revolurepair. In the following set of articles, different aspects tionized our concepts about causality, molecular mechof BRCA1 and BRCA2 genetics, structure and funcanisms, diagnosis, treatment and prevention of many tion are reviewed. One common conclusion is that the cancers, including that of the breast. Credit is due to understanding of BRCA function is emerging remarka broad attack on the study of the fundamental aspects ably quickly and future studies will clarify the mechaof the molecular and genetic control of biological organism by which mutations in the genes result in nization and cell growth. Despite these intensive enhanced susceptibility to breast cancer. efforts, decreased mortality from this disease has been The discovery of the two breast cancer susceptievident only in this decade. New strategies are still bility genes raises multiple questions about the genetics needed for a complete understanding of breast cancer of the disease, some of which are reviewed in the in order to effect prevention and eventual cures. article by Gayther et al. For instance, in considering One important factor that alters the risk of breast the proportion of families whose breast cancer is due cancer is the inheritance of susceptibility genes. Breast to BRCA1 or BRCA2, the somewhat surprising answer cancer, as in other cancers, may be caused by mutations is that as yet unidentified susceptibility genes are likely which are acquired sporadically or by inheritance. The