Genomic mapping in outbred mice reveals overlap in genetic susceptibility for HZE ion- and γ-ray-induced tumors.

Cancer risk from galactic cosmic radiation exposure is considered a potential "showstopper" for a manned mission to Mars. Calculating the actual risks confronted by spaceflight crews is complicated by our limited understanding of the carcinogenic effects of high-charge, high-energy (HZE) ions, a radiation type for which no human exposure data exist. Using a mouse model of genetic diversity, we find that the histotype spectrum of HZE ion-induced tumors is similar to the spectra of spontaneous and γ-ray-induced tumors and that the genomic loci controlling susceptibilities overlap between groups for some tumor types. Where it occurs, this overlap indicates shared tumorigenesis mechanisms regardless of the type of radiation exposure and supports the use of human epidemiological data from γ-ray exposures to predict cancer risks from galactic cosmic rays.

Overexpression of Hp95 induces G1 phase arrest in confluent HeLa cells.

Xp95, a protein recently identified in Xenopus laevis, is potentially involved in progesterone-induced Xenopus oocyte maturation. In this study, we cloned a human homologue of Xp95, designated Hp95, and examined the effect of its overexpression on the growth properties of human malignant HeLa cells which have lost the contact inhibition of cell proliferation. We observed that although HeLa cells did not undergo G1 phase arrest at any stage after confluence, they were able to downregulate their G1 phase CDK activities in response to confluence. When Hp95 was overexpressed in HeLa cells by transfection with a constitutive or an inducible expression vector containing a full-length Hp95 transgene, HeLa cells became able to undergo G1 phase arrest and form a monolayer culture after confluence. However, the G1 phase CDK activities in these Hp95 overexpressing cells were not inhibited further as compared to control cells after confluence. These results indicate that the defects in HeLa cells that cause the loss of contact inhibition of cell proliferation are in components downstream of the G1 phase CDKs and that overexpression of Hp95 counteracts some of these defects.

Targeted disruption of the transition protein 2 gene affects sperm chromatin structure and reduces fertility in mice.

During mammalian spermiogenesis, major restructuring of chromatin takes place. In the mouse, the histones are replaced by the transition proteins, TP1 and TP2, which are in turn replaced by the protamines, P1 and P2. To investigate the role of TP2, we generated mice with a targeted deletion of its gene, Tnp2. Spermatogenesis in Tnp2 null mice was almost normal, with testis weights and epididymal sperm counts being unaffected. The only abnormality in testicular histology was a slight increase of sperm retention in stage IX to XI tubules. Epididymal sperm from Tnp2-null mice showed an increase in abnormal tail, but not head, morphology. The mice were fertile but produced small litters. In step 12 to 16 spermatid nuclei from Tnp2-null mice, there was normal displacement of histones, a compensatory translationally regulated increase in TP1 levels, and elevated levels of precursor and partially processed forms of P2. Electron microscopy revealed abnormal focal condensations of chromatin in step 11 to 13 spermatids and progressive chromatin condensation in later spermatids, but condensation was still incomplete in epididymal sperm. Compared to that of the wild type, the sperm chromatin of these mutants was more accessible to intercalating dyes and more susceptible to acid denaturation, which is believed to indicate DNA strand breaks. We conclude that TP2 is not a critical factor for shaping of the sperm nucleus, histone displacement, initiation of chromatin condensation, binding of protamines to DNA, or fertility but that it is necessary for maintaining the normal processing of P2 and, consequently, the completion of chromatin condensation.

Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice.

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.

Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene.

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

A chromosome 15 quantitative trait locus controls levels of radiation-induced jejunal crypt cell apoptosis in mice.

Jejunal crypt cells undergo apoptosis in response to ionizing radiation exposure. In mice the number of cells deleted by apoptosis is determined by several factors including the dose of radiation, the time of day the apoptosis level is quantified, and the strain of mouse irradiated. We previously found that the difference in radiation-induced apoptosis levels between C57BL/6J (B6) and C3Hf/Kam (C3H) mice is controlled by multiple genes, and this set of genes is distinct from that controlling thymocyte apoptosis levels in the same strain combination. Here, we report that a new quantitative trait locus on chromosome 15, Rapop5, partly accounts for the murine strain difference in susceptibility to radiation-induced jejunal crypt cell apoptosis. In addition, we show sexual dimorphism in the extent of radiation-induced jejunal crypt cell apoptosis, with female mice having higher levels.

Haplotypes at ATM identify coding-sequence variation and indicate a region of extensive linkage disequilibrium.

Genetic variation in the human population may lead to functional variants of genes that contribute to risk for common chronic diseases such as cancer. In an effort to detect such possible predisposing variants, we constructed haplotypes for a candidate gene and tested their efficacy in association studies. We developed haplotypes consisting of 14 biallelic neutral-sequence variants that span 142 kb of the ATM locus. ATM is the gene responsible for the autosomal recessive disease ataxia-telangiectasia (AT). These ATM noncoding single-nucleotide polymorphisms (SNPs) were genotyped in nine CEPH families (89 individuals) and in 260 DNA samples from four different ethnic origins. Analysis of these data with an expectation-maximization algorithm revealed 22 haplotypes at this locus, with three major haplotypes having frequencies > or = .10. Tests for recombination and linkage disequilibrium (LD) show reduced recombination and extensive LD at the ATM locus, in all four ethnic groups studied. The most striking example was found in the study population of European ancestry, in which no evidence for recombination could be discerned. The potential of ATM haplotypes for detection of genetic variants through association studies was tested by analysis of 84 individuals carrying one of three ATM coding SNPs. Each coding SNP was detected by association with an ATM haplotype. We demonstrate that association studies with haplotypes for candidate genes have significant potential for the detection of genetic backgrounds that contribute to disease.

Abnormal spermatogenesis and reduced fertility in transition nuclear protein 1-deficient mice.

Transition nuclear proteins (TPs), the major proteins found in chromatin of condensing spermatids, are believed to be important for histone displacement and chromatin condensation during mammalian spermatogenesis. We generated mice lacking the major TP, TP1, by targeted deletion of the Tnp1 gene in mouse embryonic stem cells. Surprisingly, testis weights and sperm production were normal in the mutant mice, and only subtle abnormalities were observed in sperm morphology. Electron microscopy revealed large rod-like structures in the chromatin of mutant step 13 spermatids, in contrast to the fine chromatin fibrils observed in wild type. Steps 12-13 spermatid nuclei from the testis of Tnp1-null mice contained, in place of TP1, elevated levels of TP2 and some protamine 2 (P2) precursor. Most of the precursor was processed to mature P2, but high levels of incompletely processed forms remained in epididymal spermatozoa. Sperm motility was reduced severely, and approximately 60% of Tnp1-null males were infertile. We concluded that TP1 is not essential for histone displacement or chromatin condensation. The absence of TP1 may partially be compensated for by TP2 and P2 precursor, but this dysregulation of nucleoprotein replacement results in an abnormal pattern of chromatin condensation and in reduced fertility.

Tumor suppressor genes and breast cancer.

The genetic determinants for most breast cancer cases remain elusive. However, a mutation in a tumor suppressor gene, such as p53, BRCA1, BRCA2, or ATM, has been determined to be one mechanism of breast carcinogenesis. It has been established that inherited mutations in p53, BRCA1, and BRCA2 significantly contribute to breast cancer risk, although the importance of an inherited ATM mutation is controversial. Sporadic mutations in p53 are also common in breast cancer cells. The precise deficiencies that result from these genetic mutations have yet to be fully described. Although the functions of these genes are different, they are all involved in the maintenance of genomic stability after DNA damage. Mutations that impair the function of these four genes may adversely affect the manner in which DNA damage is processed. It is likely that the risk of breast cancer development is increased through this mechanism. In this article, we review the relevancy of p53, BRCA1, BRCA2, and ATM mutations to breast cancer development, and review the in vitro, in vivo, and clinical data exploring the mechanisms by which these mutations affect genomic integrity and DNA damage repair.

Identification and cloning of xp95, a putative signal transduction protein in Xenopus oocytes.

A 95-kDa protein in Xenopus oocytes, Xp95, was shown to be phosphorylated from the first through the second meiotic divisions during progesterone-induced oocyte maturation. Xp95 was purified and cloned. The Xp95 protein sequence exhibited homology to mouse Rhophilin, budding yeast Bro1, and Aspergillus PalA, all of which are implicated in signal transduction. It also contained three conserved features including seven conserved tyrosines, a phosphorylation consensus sequence for the Src family of tyrosine kinases, and a proline-rich domain near the C terminus that contains multiple SH3 domain-binding motifs. We showed the following: 1) that both Xp95 isolated from Xenopus oocytes and a synthetic peptide containing the Src phosphorylation consensus sequence of Xp95 were phosphorylated in vitro by Src kinase and to a lesser extent by Fyn kinase; 2) Xp95 from Xenopus oocytes or eggs was recognized by an anti-phosphotyrosine antibody, and the relative abundance of tyrosine-phosphorylated Xp95 increased during oocyte maturation; and 3) microinjection of deregulated Src mRNA into Xenopus oocytes increased the abundance of tyrosine-phosphorylated Xp95. These results suggest that Xp95 is an element in a tyrosine kinase signaling pathway that may be involved in progesterone-induced Xenopus oocyte maturation.

Induction of transforming growth factor alpha in irradiated mouse jejunum.

PURPOSE: To determine the involvement of the mitogenic growth factors transforming growth factor alpha (TGF alpha), epidermal growth factor (EGF), and the EGF receptor (EGF-R) in the proliferative response after irradiation of the mouse jejunum. METHODS AND MATERIALS: C3Hf/Kam mice were whole-body irradiated with 5 and 11 Gy 250 kV X rays. Mice were killed 1-10 days after irradiation, and immunohistochemistry, in situ hybridization (ISH), and RNase protection assays were performed. RESULTS: Damage to the jejunal crypts caused by irradiation resulted in a strong proliferative response 1-5 days after 5 Gy and 3-6 days after 11 Gy. Expression of TGF alpha, EGF, and EGF-R increased at 1-2 days and decreased at 4-8 days after 5- or 11-Gy irradiation. Also, TGF alpha mRNA increased during the early phase of the proliferative response (1-2 days after 5 or 11 Gy) followed by a decrease at 4 days after 5 Gy and 8 days after 11 Gy. CONCLUSION: These data indicate that, at the beginning of the proliferative response after irradiation, the transcription of TGF alpha mRNA is increased, and that it is inhibited just before compensatory proliferation decreases. Thus, active regulation of TGF alpha expression takes place at least at the transcriptional level, resulting in upregulation of TGF alpha production and increased TGF alpha levels in the crypts during the proliferative response.

Diurnal variations in the expression of radiation-induced apoptosis.

Experiments were performed to determine whether diurnal variations in apoptosis in the mouse small intestine after irradiation with 2.5 Gy gamma rays depended on the time of day that the mice were irradiated, the time of day that the mice were sacrificed or the interval between irradiation and sacrifice. Experiments were performed with a 12-h light:dark regimen with the light period from 6:00 to 18:00 h. With fixed intervals of 6 h and 24 h between irradiation and sacrifice, a peak in induced apoptosis (16%) was observed in mice sacrificed at 8:00 h, two times higher than the nadir of response at 23:00 h (8%). When variable intervals were used between irradiation and measurement of apoptosis, i.e. sacrifice, at 8:00 h or 23:00 h, the induced apoptosis was dependent on the interval, with a peak for 18-h intervals. However, the level of apoptosis was always about twofold higher when measured at 8:00 h than at 23:00 h. No correlation was observed between diurnal variations in apoptosis and survival of mouse intestinal crypts. The diurnal variations in apoptosis after irradiation can be interpreted either in terms of expression of apoptosis during the G2/M phase of the cell cycle in partially synchronized cells, or in terms of a systemic mechanism such as diurnal variation in the neurohormone melatonin.

MPM-2 epitope sequence is not sufficient for recognition and phosphorylation by ME kinase-H.

Monoclonal antibody MPM-2 recognizes a large family of mitotic phosphoproteins in a phosphorylation-dependent manner. The antigenic phosphoepitope, designated the MPM-2 epitope, putatively consists of hydrophobic residue-Thr/Ser-Pro-hydrophobic residue-uncharged/basic residue. In this study, we addressed whether this sequence motif contains all the information necessary for recognition and phosphorylation by the kinase that phosphorylates most MPM-2 antigens. A fusion protein between glutathione S-transferase and a 19-residue peptide that contained two representative MPM-2 epitope sequences overlapping with two potential MAP kinase phosphorylation sites was constructed. Both the MPM-2 epitope sequences in the fusion protein (GST-MPM2) were phosphorylated by Xenopus egg extract, making the fusion protein MPM-2 reactive. However, while MAP kinase phosphorylated both the MPM-2 epitope sequences, neither ME kinase-H, a good candidate for a major MPM-2 epitope kinase, nor mitotic cdc2 kinase, which is known to phosphorylate certain MPM-2 antigens in vitro, phosphorylated GST-MPM2 to any significant extent. Furthermore, depletion of MAP kinase activity removed most, if not all, of the GST-MPM2 phosphorylating activity from crude Xenopus egg extracts. These results suggest that additional or different structural information than that provided by the deduced MPM-2 epitope sequence is required for recognition and phosphorylation by ME kinase-H or other major MPM-2 epitope kinases. They also offer a valid explanation for selective phosphorylation of certain MPM-2 antigens by MAP kinase as well as selective recognition of certain phosphorylated MAP kinase substrates by MPM-2.

Genotype selection to rapidly breed congenic strains.

Congenic strains can now be constructed guided by the transmission of DNA markers. This allows not only selection for transmission of a desired, donor-derived differential region but also selection against the transmission of unwanted donor origin genomic material. The additional selection capacity should allow congenic strains to be produced in fewer generations than is possible with random backcrosses. Here, we consider modifications of a standard backcross breeding scheme to produce congenic mice by the inclusion of genotype-based selective breeding strategies. Simulation is used to evaluate the consequences of each strategy on the number of chromosomes that contain unwanted, donor-derived genetic material and the average length of this unwanted donor DNA for each backcross generation. Our prototypic strategy was to choose a single mouse to sire each generation using criteria designed to select against the transmission of chromosomes, other than the one containing the replacement genomic region, that contain any donor origin sequence at all. This chromosome elimination strategy resulted in an average of 16.4 chromosomes free of donor DNA in mice of the third backcross (N3) generation. A strategy based solely on positive selection for the replacement region required six backcross generations to achieve the same results.

Identification of quantitative trait loci controlling levels of radiation-induced thymocyte apoptosis in mice.

Thymocyte apoptosis levels are higher in C57BL/6J mice than in C3Hf/Kam mice. Low-dose irradiation increases the numbers of thymocytes undergoing apoptosis, but the strain difference persists. We mapped three loci controlling radiation-induced thymocyte apoptosis levels in F2 intercross progeny of these strains. The strongest association of a genomic region with an apoptosis level occurred in a region of chromosome 11 known to harbor a locus (or loci) important in the pathogenesis of several rodent models of autoimmune disease. Additional loci influencing radiation-induced thymocyte apoptosis were identified on chromosomes 9 and 16. The genetic polymorphisms underlying these loci may have an evolutionary role in fine-tuning the apoptotic response in T cells and may be important in the etiology of lymphoproliferative disorders and autoimmunity.

Molecular cloning of a splice variant of Caenorhabditis elegans YNK1, a putative element in signal transduction.

YNK1 is a 98.3-kDa protein whose sequence was originally deduced from a genomic sequence in Caenorhabditis elegans. It was recently found that YNK1 is homologous to three different proteins implicated in signal transduction, suggesting that YNK1 is a signal transduction protein. In this report we describe the isolation of a full-length cDNA that encodes a splice variant of YNK1, designated YNK1a. We also present evidence that both YNK1 and YNK1a transcripts exist in vivo. Furthermore, using an antibody raised against a YNK1a recombinant protein, we demonstrate that the YNK1 protein is expressed in vivo throughout development.

Genetic basis of strain variation in levels of radiation-induced apoptosis of thymocytes.

Levels of radiation-induced apoptosis of thymocytes were compared in C57BL/6J and C3Hf/Kam mice. For up to 8 h after irradiation, levels of apoptosis were higher in the C57BL/6J strain for all doses assayed. The heritability of the strain difference in the extent of apoptosis resulting from irradiation was investigated using a breeding study. Analysis of the F1 and F2 intercross progeny of these two strains provided strong evidence for a maternal effect but little evidence of sex linkage. The results indicate that in this strain combination the level of apoptosis of thymocytes after irradiation is a heritable trait that is likely to be controlled by few genes. These genes should be detectable in a mapping study.

Strain difference in jejunal crypt cell susceptibility to radiation-induced apoptosis.

Levels of radiation-induced jejunal crypt cell apoptosis were compared in C57BL/6J, C3Hf/Kam and C3H/HeJ mice. Apoptosis levels were consistently lower in the C3H strains than in C57BL/6J. Although other explanations are possible, the strain difference is most likely to have a genetic basis, and in fact a preliminary analysis of the F2 progeny of C3H/HeJ and C57BL/6J mice indicates that more than one gene is involved. Both C3H strains also had lower levels of radiation-induced thymocyte apoptosis than C57BL/6J mice. Jejunal crypt cell apoptosis levels did not co-segregate with thymocyte apoptosis levels in the F2 progeny of C57BL/6J and C3H/HeJ mice. These results imply that the genes responsible for the difference in radiation-induced thymocyte apoptosis levels between these two strains are not the same as those responsible for the strain difference in radiation-induced jejunal crypt cell apoptosis levels. The experiments reported here identify strain-specific differences in levels of radiation-induced crypt cell apoptosis and are a first step towards identifying genetic polymorphisms that influence sensitivity of the small intestine to damage from ionizing radiation.

Ex vivo gene delivery of platelet-derived growth factor increases 0-2A progenitors in adult rat spinal cord.

The O-2A progenitor cell, which serves as a stem cell for the myelinating oligodendrocyte, has been implicated as a major target for radiation-induced spinal cord injury. In an attempt to increase the number of O-2A cells in the spinal cord, we applied an ex vivo gene therapy procedure for delivering platelet derived growth factor (PDGF). Recombinant fibroblasts expressing PDGF A chain were injected into the cisterna magna of adult rats, which resulted in cell seeding of the subarachnoid space of the cervical spinal cord. The number of O-2A progenitors in the cervical spinal cord was then assessed with an in vitro clonogenic assay. O-2A cells were found to be increased 8 days after recombinant cell injection, and they remained elevated up to at least 14 days. Analysis of O-2A colonies indicated that the implantation of PDGF-expressing cells increased the number of O-2A progenitors without affecting their in vitro proliferation potential or differentiation capacity. These data suggest that implantation of PDGF-expressing cells in the subarachnoid space of the cervical spinal cord may influence a stem cell population critical to the repair of demyelinated lesions.

Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3.

It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.