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Biomedical subjects

A Efstratiadis

Publications and source records attributed to A Efstratiadis.

At least 19 recordsLinked to original sources
PubMed2001

Development of mammary adenocarcinomas by tissue-specific knockout of Brca2 in mice.

Cre-mediated deletion of exons 3 and 4 of the mouse Brca2 gene occurring specifically in mammary epithelial cells of conditional female mutants carrying a combination of loxP-modified and null Brca2 alleles resulted in a high incidence (77%) of breast tumors that were often palpable and developed in one or more glands after long latency (time for median tumor-free survival of approximately 1.4 years; total of 40 tumors in 20 animals). These invasive adenocarcinomas were histologically quite uniform, exhibiting predominantly a solid, nodular tumor pattern with very few variants, in striking contrast to the morphological heterogeneity of analogous Brca1-associated tumors. The karyotypes of tumor cells lacking Brca2 had various chromosomal aberrations and ranged from diploid to hypertetraploid, but this wide variability was incongruous with the histological appearance of carcinomas that was comparable between specimens. The implications of these observations in the context of models positing that Brca2 is involved in the maintenance of genomic stability are discussed.

Adenocarcinoma↗
PubMed2001

Tumorigenesis in mice carrying a truncating Brca1 mutation.

We generated mouse mutants carrying in the Brca1 locus a modification (Brca1(tr)) that eliminates the C-terminal half of the protein product and obtained results indicating that, depending on genetic background, the missing BRCT and/or other domains are dispensable for survival, but essential for tumor suppression. Most of the apparently hypomorphic Brca1(tr/tr) mutants developed various tumors. Lymphomas were detected at all ages, whereas sarcomas and carcinomas, including breast cancer, appeared after a long latency. The mammary tumors showed striking variability in histopathological patterns suggesting stochastic engagement of tumorigenic pathways in their progression, to which the Brca1(tr/tr) mutation was apparently a late participant.

Animals↗
PubMed2001

Nucleotide sequence, transcription map, and mutation analysis of the 13q14 chromosomal region deleted in B-cell chronic lymphocytic leukemia.

Deletions of the 13q14 chromosome region are associated with B-cell chronic lymphocytic leukemia (B-CLL) and several other types of cancer, suggesting the presence of a tumor suppressor gene. In previous studies the minimal region of deletion (MDR) was mapped to a less than 300-kilobase (kb) interval bordered by the markers 173a12-82 and 138G4/1.3R. For the identification of the putative tumor suppressor gene, the entire MDR (approximately 347 kb) has been sequenced, and transcribed regions have been identified by exon trapping, EST-based full-length complementary DNA cloning, database homology searches, and computer-assisted gene prediction analyses. The MDR contains 2 pseudogenes and 3 transcribed genes: CAR, encoding a putative RING-finger containing protein; 1B4/Leu2, generating noncoding transcripts; and EST70/Leu1, probably representing another noncoding gene (longest open reading frame of 78 codons). These genes have been sequenced in 20 B-CLL cases with 13q14 hemizygous deletion, and no mutations were found. Moreover, no somatic variants were found in the entire MDR analyzed for nucleotide substitutions by a combination of direct sequencing and fluorescence-assisted mismatch analysis in 5 B-CLL cases displaying 13q14-monoallelic deletion. The nondeleted allele of the CAR and EST70/Leu1 genes was expressed in B-CLL specimens, including those with monoallelic loss, whereas no expression of 1B4/Leu2 was detectable in B-CLL, regardless of the 13q14 status. These results indicate that allelic loss and mutation of a gene within the MDR is an unlikely pathogenetic mechanism for B-CLL. However, haplo-insufficiency of one of the identified genes may contribute to tumorigenesis. (Blood. 2001;97:2098-2104)

Animals↗
PubMed2001

Roles of growth hormone and insulin-like growth factor 1 in mouse postnatal growth.

To examine the relationship between growth hormone (GH) and insulin-like growth factor 1 (IGF1) in controlling postnatal growth, we performed a comparative analysis of dwarfing phenotypes manifested in mouse mutants lacking GH receptor, IGF1, or both. This genetic study has provided conclusive evidence demonstrating that GH and IGF1 promote postnatal growth by both independent and common functions, as the growth retardation of double Ghr/Igf1 nullizygotes is more severe than that observed with either class of single mutant. In fact, the body weight of these double-mutant mice is only approximately 17% of normal and, in absolute magnitude ( approximately 5 g), only twice that of the smallest known mammal. Thus, the growth control pathway in which the components of the GH/IGF1 signaling systems participate constitutes the major determinant of body size. To complement this conclusion mainly based on extensive growth curve analyses, we also present details concerning the involvement of the GH/IGF1 axis in linear growth derived by a developmental study of long bone ossification in the mutants.

Animals↗
PubMed2000

Conditional mutagenesis in mice with heat shock promoter-driven cre transgenes.

To explore the potential of a simple and rapid approach for ubiquitous conditional gene disruption, we have generated Cre-producer mouse transgenic lines (Hs-cre1, 6 and 7) expressing a recombinase transgene (cre) from a heat shock gene promoter and tested their performance in Cre-mediated excision of target DNA in crosses with Cre-responder strains carrying loxP-modified alleles of the genes encoding the Huntington's disease gene homolog (Hdh), the epidermal growth factor receptor (Egfr), and the type 1 insulin-like growth factor receptor (Igf1r). Analyses of progeny possessing various transgene/reporter combinations showed that cre expression can occur without heat shock in early embryos, but this constitutive transcription is stochastic and transgene dependent. Thus, Hs-cre1 behaves predominantly as a "deleter" strain, since the majority of progeny (approximately 70-85%) exhibit complete recombination, regardless of reporter locus. Lines Hs-cre6 and Hs-cre7, however, function successfully as "mosaicking" strains because, in addition to two extreme classes of progeny with 0% or 100% recombination, they generate an intermediate class of mosaics exhibiting various degrees of partial DNA excision. Notably, the frequency of offspring in each class varies between reporters, but mosaic embryos are consistently obtained in adequate numbers (approximately 30-60%). The Hs-cre6 transgene is also inducible and can be used to introduce mosaicism into adult tissues at preselected developmental times by heat shock treatment of mice with 0% recombination in tail DNA. By bypassing the lethality resulting from some gene knockouts, mosaic embryos and mice make particular mutational analyses possible and are also very useful for the identification of cell lineage-specific gene functions.

Animals↗
PubMed1999

Enhanced sensitivity to N-methyl-D-aspartate receptor activation in transgenic and knockin mouse models of Huntington's disease.

We used two mouse models of Huntington's disease (HD) to examine changes in glutamate receptor sensitivity and striatal electrophysiology. One model, a transgenic, consisted of mice expressing exon 1 of the human HD gene and carrying 141-157 CAG repeat sequences (R6/2 line). The second model, a CAG repeat "knockin," consisted of mice with different lengths of CAG repeats (CAG71 and CAG94 repeats). The effects of glutamate receptor activation were examined by visualizing neurons in brain slices with infrared videomicroscopy and differential interference contrast optics to determine changes in somatic area (cell swelling). Striatal and cortical neurons in both models (R6/2 and CAG94) displayed more rapid and increased swelling to N-methyl-D-aspartate (NMDA) than those in controls. This effect was specific as there were no consistent group differences after exposure to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) or kainate (KA). Intracellular recordings revealed that resting membrane potentials (RMPs) in the R6/2 transgenics were significantly more depolarized than those in their respective controls. RMPs in CAG94 mice also were more depolarized than those in CAG71 mice or their controls in a subset of striatal neurons. Confirming previous results, R6/2 mice expressed behavioral abnormalities and nuclear inclusions. However, CAG71 and CAG94 knockins did not, suggesting that increased sensitivity to NMDA may occur early in the disease process. These findings imply that NMDA antagonists or compounds that alter sensitivity of NMDA receptors may be useful in the treatment of HD.

Animals↗
PubMed1998

High-resolution YAC-cosmid-STS map of human chromosome 13.

We have assembled a high-resolution physical map of human chromosome 13 DNA (approximately 114 Mb) from hybridization, PCR, and FISH mapping data using a specifically designed set of computer programs. Although the mapping of 13p is limited, 13q (approximately 98 Mb) is covered by an almost continuous contig of 736 YACs aligned to 597 contigs of cosmids. Of a total of 10,789 cosmids initially selected from a chromosome 13-specific cosmid library (16,896 colonies) using inter-Alu PCR probes from the YACs and probes for markers mapped to chromosome 13, 511 were assembled in contigs that were established from cross-hybridization relationships between the cosmids. The 13q YAC-cosmid map was annotated with 655 sequence tagged sites (STSs) with an average spacing of 1 STS per 150 kb. This set of STSs, each identified by a D number and cytogenetic location, includes database markers (198), expressed sequence tags (93), and STSs generated by sequencing of the ends of cosmid inserts (364). Additional annotation has been provided by positioning 197 cosmids mapped by FISH on 13q. The final (comprehensive) map, a list of STS primers, and raw data used in map assembly are available at our Web site (genome1.ccc.columbia.edu/ approximately genome/) and can serve as a resource to facilitate accurate localization of additional markers, provide substrates for sequencing, and assist in the discovery of chromosome 13 genes associated with hereditary diseases.

Animals↗
PubMed1998

Mouse mutant embryos lacking huntingtin are rescued from lethality by wild-type extraembryonic tissues.

Mouse embryos nullizygous for a targeted disruption of the Huntington's disease gene homologue (Hdh), which encodes a protein (huntingtin) of unknown biochemical function, become developmentally retarded and disorganized, and die early in development. Using chimeric analysis, we demonstrate that extensively chimeric embryos derived by injection of Hdh null ES cells into wild-type host blastocysts are rescued from lethality. In contrast, when wild-type ES cells are injected into Hdh null blastocysts, the chimeric embryos are morphologically indistinguishable from Hdh null mutants derived from natural matings, and die shortly after gastrulation. Therefore, the primary defect in the absence of huntingtin lies in extraembryonic tissues, whereas the epiblast and its derivatives are affected secondarily. It is likely that the mutation results in impairment of the nutritive functions of the visceral endoderm, which otherwise appears to differentiate normally, as evidenced by the expression of several specific marker genes. Consistent with preliminary histochemical analysis indicating that at least the transport of ferric ions is defective in Hdh mutants and in conjunction with the known localization of huntingtin in the membranes of vesicles associated with microtubules, we hypothesize that this protein is involved in the intracellular trafficking of nutrients in early embryos.

Animals↗
PubMed1998

Genetics of mouse growth.

During development, mammalian organisms increase in size until a limit is reached that is mainly determined by the rate and duration of occurrence of cellular divisions increasing total cell number. This process is mostly regulated by an orchestration of the actions of genes participating in pathways that promote or inhibit growth through systemic or local effects. This view of growth control genes and of their effects on the cell cycle has begun emerging from the results of transgenic and gene knockout experiments, which have also re-emphasized the central involvement of some growth factors and hormones in growth signaling, although mechanistic relationships and details about the coordination of growth with patterning, differentiation and morphogenesis continue to remain largely elusive.

Animals↗
PubMed1997

Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith-Wiedemann and Simpson-Golabi-Behmel syndromes.

In mice, the imprinted Igf2 gene (expressed from the paternal allele), which encodes a growth-promoting factor (IGF-II), is linked closely to the reciprocally imprinted H19 locus on chromosome 7. Also imprinted (expressed from the maternal allele) is the Igf2r gene on chromsome 17 encoding the type 2 IGF receptor that is involved in degradation of excess IGF-II. Double mutant embryos carrying a deletion around the H19 region and also a targeted Igf2r allele, both inherited maternally, have extremely high levels of IGF-II (7- and 11-fold higher than normal in tissues and serum, respectively) as a result of biallelic Igf2 expression (imprint relaxation by deletion of H19-associated sequence) in combination with lack of the IGF2R-mediated IGF-II turnover. This excess of IGF-II causes somatic overgrowth, visceromegaly, placentomegaly, omphalocele, and cardiac and adrenal defects, which are also features of the Beckwith-Wiedemann syndrome (BWS), a genetically complex human disorder associated with chromosomal abnormalities in the 11p15.5 region where the IGF2 gene resides. In addition, the double mutant mouse embryos exhibit skeletal defects and cleft palate, which are manifestations observed frequently in the Simpson-Golabi-Behmel syndrome, another overgrowth disorder overlapping phenotypically, but not genetically, with BWS.

Abnormalities, Multiple↗
PubMed1997

Growth-promoting interaction of IGF-II with the insulin receptor during mouse embryonic development.

Genetic analyses of dwarfing phenotypes resulting from targeted mutagenesis of the genes encoding the insulin-like growth factors (IGF-I and IGF-II) and their cognate type 1 IGF receptor (IGF1R) have demonstrated that this signaling system is a major determinant of mouse embryonic growth. Of the two IGF ligands, IGF-I interacts exclusively with IGF1R, whereas IGF-II recognizes an additional receptor (XR), because the growth retardation of embryos lacking both IGR1R and IGF-II (30% of normal birthweight) is more severe than that manifested in either class of single Igf1r or Igf2 null mutants (45 and 60% of normal, respectively). To determine whether XR is the insulin receptor (IR), we examined embryos nullizygous for both Igf1r and Insr. While the growth of embryos lacking solely IR is affected very mildly and only at the end of gestation, concomitant absence of IGF1R results in a severe growth-deficiency phenotype (30% of normal size at birth) that is first detected at Embryonic Day 13.5 and is also characterized by transient edema, curly tail, generalized organ hypoplasia, including the muscles, developmental delays in ossification, and thin epidermis. The Igf1r/Insr double nullizygotes are phenotypically indistinguishable from double mutants lacking IGF1R and IGF-II and from other double and triple mutants in which all of the IGF ligand/receptor interactions have been eliminated. Therefore, these results provide genetic evidence that the growth-promoting function of IGF-II during mouse embryogenesis is mediated in part by signaling through the insulin receptor.

Animals↗
PubMed1997

Cloning and gene mapping of the chromosome 13q14 region deleted in chronic lymphocytic leukemia.

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.

Alleles↗
PubMed1997

Targeted mutations of breast cancer susceptibility gene homologs in mice: lethal phenotypes of Brca1, Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizygous embryos.

Mutations of the human BRCA1 and BRCA2 genes encoding tumor suppressors have been implicated in inherited predisposition to breast and other cancers. Disruption of the homologous mouse genes Brca1 and Brca2 by targeting showed that they both have indispensable roles during embryogenesis, because nullizygous embryos become developmentally retarded and disorganized, and die early in development. In Brca1 mutants, the onset of abnormalities is earlier by one day and their phenotypic features and time of death are highly variable, whereas the phenotype of Brca2 null embryos is more uniform, and they all survive for at least 8.5 embryonic days. Observations with Brca1/Brca2 double nullizygotes raise the possibility that the two developmental pathways could be linked. Interestingly, the impact of the Brca1 or Brca2 null mutation is less severe in a p53 null background.

Animals↗
PubMed1997

Insulin-like growth factor II stimulates cell proliferation through the insulin receptor.

R- cells are 3T3-like fibroblasts generated from mouse embryos nullizygous for a targeted disruption of the genes encoding the type 1 insulin-like growth factor (IGF) receptor (IGF1R). These cells fail to proliferate in serum-free medium supplemented with purified growth factors, in contrast to their wild-type counterparts. However, when R- cells overexpress the insulin receptor from a stably integrated plasmid, R-/IR cells, they become capable of growing in serum-free medium supplemented solely with insulin or IGF-II, but not with IGF-I. Moreover, the introduction into R-/IR cells of an additional plasmid expressing IGF-II causes these cells to proliferate in serum-free medium without growth factor supplementation. From these results, we conclude that IGF-II can stimulate cell proliferation not only through its cognate IGF1R but also through the insulin receptor.

3T3 Cells↗
PubMed1996

Mouse mutants lacking the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds.

The cation-dependent and cation-independent mannose 6-phosphate receptors (CD- and CI-MPRs) bind the phosphomannosyl recognition marker of lysosomal hydrolases, but in mammals the latter also interacts with insulin-like growth factor II (IGF-II). While IGF signaling is mediated by the type 1 IGF receptor (IGF1R), the type 2 receptor (IGF2R/CI-MPR) serves IGF-II turnover. Mouse mutants inheriting maternally a targeted disruption of the imprinted Igf2r gene, which is normally expressed only from the maternal allele, have increased serum and tissue levels of IGF-II and exhibit overgrowth (135% of normal birthweight) and generalized organomegaly, kinky tail, postaxial polydactyly, heart abnormalities, and edema. These mutants usually die perinatally, but a small minority can survive depending on genetic background and can occasionally reproduce, except for some females characterized by an imperforate vagina and hydrometrocolpos. Consistent with the hypothesis that lethality in the absence of IGF2R-mediated turnover is caused by excess of IGF-II overstimulating IGF1R, Igf2r mutants are completely rescued when they carry a second mutation eliminating either IGF-II or IGF1R. Normal embryonic development of the Igf1r/Igf2r double mutants, which differ from wild-type siblings only in the pattern of postnatal growth, appears to occur by signaling of IGF-II, being in excess, through a genetically identified unknown receptor, since triple mutants lacking IGF1R, IGF2R, and IGF-II are nonviable dwarfs (30% of normal size). In contrast with the Igf2r/Igf2 double mutants, mice lacking IGF2R/CI-MPR and CD-MPR survive in an IGF-II null background at a very low frequency and only for a few postnatal weeks, indicating that the mannose 6-phosphate-mediated lysosomal enzyme trafficking is essential for viability.

Animals↗
PubMed1996

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

BRCA2 Protein↗
PubMed1996

Effects of an Igf1 gene null mutation on mouse reproduction.

Both sexes of adult mice homozygous for a targeted mutation of the Igf1 gene, encoding insulin-like growth factor 1, are infertile dwarfs (approximately 30% of normal size). The testes are reduced in size less than expected from the degree of dwarfism but sustain spermatogenesis only at 18% of the normal level. The epididymides are overall nearly allometric to the reduced body weight, but the distal regions of the duct, vas deferens, seminal vesicles, and prostate are vestigial. Despite the mutational impact on the epididymis, capacitated sperm are able to fertilize wild type eggs in vitro. It is hypothesized that the infertility of male mutants is caused by failure of androgenization resulting in absence of mating behavior, due to drastically reduced levels of serum testosterone (18% of normal). This hormonal deficiency was correlated with an ultrastructural analysis of mutant Leydig cells revealing a significant developmental delay, while assays in organ culture showed that the basal and LH-stimulated production of testosterone by testicular parenchyma is reduced in comparison with wild type controls. The female mutants fail to ovulate even after administration of gonadotropins, which is apparently the primary cause of their infertility, and possess an infantile uterus that exhibits a dramatic hypoplasia especially in the myometrium. The phenotypic manifestations of the mutation were correlated with the localization of transcripts for insulin-like growth factor I and its cognate receptor in wild type reproductive tissues by in situ hybridization.

Animals↗