Loss of p21 increases sensitivity to ionizing radiation and delays the onset of lymphoma in atm-deficient mice

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by growth retardation, cerebellar ataxia, oculocutaneous telangiectasias, and a high incidence of lymphomas and leukemias. In addition, AT patients are sensitive to ionizing radiation. Atm-deficient mice recapitulate most of the AT phenotype. p21cip1/waf1 (p21 hereafter), an inhibitor of cyclin-dependent kinases, has been implicated in cellular senescence and response to γ-radiation-induced DNA damage. To study the role of p21 in ATM-mediated signal transduction pathways, we examined the combined effect of the genetic loss of atm and p21 on growth control, radiation sensitivity, and tumorigenesis. As might have been expected, our data provide evidence that p21 modifies the in vitro senescent response seen in AT fibroblasts. Further, it is a downstream effector of ATM-mediated growth control. In addition, however, we find that loss of p21 in the context of an atm-deficient mouse leads to a delay in thymic lymphomagenesis and an increase in acute radiation sensitivity in vivo (the latter principally because of effects on the gut epithelium). Modification of these two crucial aspects of the ATM phenotype can be related to an apparent increase in spontaneous apoptosis seen in tumor cells and in the irradiated intestinal epithelium of mice doubly null for atm and p21. Thus, loss of p21 seems to contribute to tumor suppression by a mechanism that operates via a sensitized apoptotic response. These results have implications for cancer therapy in general and AT patients in particular.

α1 and α2 Integrins Mediate Invasive Activity of Mouse Mammary Carcinoma Cells through Regulation of Stromelysin-1 Expression

Tumor cell invasion relies on cell migration and extracellular matrix proteolysis. We investigated the contribution of different integrins to the invasive activity of mouse mammary carcinoma cells. Antibodies against integrin subunits α6 and β1, but not against α1 and α2, inhibited cell locomotion on a reconstituted basement membrane in two-dimensional cell migration assays, whereas antibodies against β1, but not against α6 or α2, interfered with cell adhesion to basement membrane constituents. Blocking antibodies against α1 integrins impaired only cell adhesion to type IV collagen. Antibodies against α1, α2, α6, and β1, but not α5, integrin subunits reduced invasion of a reconstituted basement membrane. Integrins α1 and α2, which contributed only marginally to motility and adhesion, regulated proteinase production. Antibodies against α1 and α2, but not α6 and β1, integrin subunits inhibited both transcription and protein expression of the matrix metalloproteinase stromelysin-1. Inhibition of tumor cell invasion by antibodies against α1 and α2 was reversed by addition of recombinant stromelysin-1. In contrast, stromelysin-1 could not rescue invasion inhibited by anti-α6 antibodies. Our data indicate that α1 and α2 integrins confer invasive behavior by regulating stromelysin-1 expression, whereas α6 integrins regulate cell motility. These results provide new insights into the specific functions of integrins during tumor cell invasion.

A fusion pore phenotype in mast cells of the ruby-eye mouse

Using patch-clamp capacitance and amperometric techniques, we have identified an exocytotic phenotype that affects the function of the fusion pore, the molecular structure that connects the lumen of a secretory vesicle with the extracellular environment during exocytosis. Direct observation of individual exocytotic events in mast cells from the ruby-eye mouse (ru/ru) showed a 3-fold increase in the fraction and duration of transient fusion events with respect to wild-type mice. The fraction of the total fusion events that were transient increased from 0.22 ± 0.02 (wild type) to 0.65 ± 0.02 (ru/ru), and the average duration of these events increased from 418 ± 32 ms (wild type) to 1207 ± 89 ms (ru/ru). We also show that this phenotype can reduce and delay an evoked secretory response by causing the fusion of vesicles that have been previously emptied by repeated cycles of transient fusion. The exocytotic phenotype that we describe here may be a cause of diseases like platelet storage pool deficiency and prolonged bleeding times for which the ruby-eye mouse serves as an animal model. Furthermore, the identification of the gene causing the fusion pore phenotype reported here will illuminate the molecular mechanisms regulating exocytotic fusion.

Receptors for oxidized low-density lipoprotein on elicited mouse peritoneal macrophages can recognize both the modified lipid moieties and the modified protein moieties: Implications with respect to macrophage recognition of apoptotic cells

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4°C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

p53-dependent and -independent responses to cisplatin in mouse testicular teratocarcinoma cells

Testicular cancers respond favorably to chemotherapy with the platinum-containing drug cis-diamminedichloroplatinum(II) (cisplatin). One factor that could explain the efficacy of cisplatin is the low frequency of p53 mutations observed in this tumor type. The present study examines the p53-mediated responses in murine testicular teratocarcinoma cells exposed to the drug. Cisplatin treatment of teratocarcinoma cells with a wild-type p53 gene resulted in accumulation of the p53 protein through posttranscriptional mechanisms; induction of p53-target genes was also observed. Drug treatment resulted in rapid apoptosis in p53-wild-type cells but not in p53−/− teratocarcinoma cells. In the latter cells, cisplatin exposure caused prolonged cell cycle arrest accompanied by induction of the p21 gene. Clonogenic assays demonstrated that the p53 mutation did not confer resistance to cisplatin. These experiments suggest that cisplatin inhibits cellular proliferation of testicular teratocarcinoma cells by two possible mechanisms, p53-dependent apoptosis and p53-independent cell cycle arrest.

Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells

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.

A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells

We describe a mouse model in which p27Kip1 transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18–26 h of transgene expression, the G1 phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27Kip1 and control of G1 phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G1 phase length to a putative physiological maximum despite ongoing p27Kip1 transgene expression.

Metabolism of Polyamines in Transgenic Cells of Carrot Expressing a Mouse Ornithine Decarboxylase cDNA1

The metabolisms of arginine (Arg), ornithine (Orn), and putrescine were compared in a nontransgenic and a transgenic cell line of carrot (Daucus carota L.) expressing a mouse Orn decarboxylase cDNA. [14C]Arg, [14C]Orn, and [14C]putrescine were fed to cells and their rates of decarboxylation, uptake, metabolism into polyamines, and incorporation into acid-insoluble material were determined. Transgenic cells showed higher decarboxylation rates for labeled Orn than the nontransgenic cells. This was correlated positively with higher amounts of labeled putrescine production from labeled Orn. With labeled Arg, both the transgenic and the nontransgenic cells exhibited similar rates of decarboxylation and conversion into labeled putrescine. When [14C]putrescine was fed, higher rates of degradation were observed in transgenic cells as compared with the nontransgenic cells. It is concluded that (a) increased production of putrescine via the Orn decarboxylase pathway has no compensatory effects on the Arg decarboxylase pathway, and (b) higher rates of putrescine production in the transgenic cells are accompanied by higher rates of putrescine conversion into spermidine and spermine as well as the catabolism of putrescine.

Prostaglandin E2 receptors of the EP2 and EP4 subtypes regulate activation and differentiation of mouse B lymphocytes to IgE-secreting cells.

Prostaglandin E2 (PGE2) is a potent lipid molecule with complex proinflammatory and immunoregulatory properties. PGE2 can shape the immune response by stimulating the production of IgE antibody by B lymphocytes and the synthesis of T-helper type 2 cytokines [e.g., interleukin (IL)-4, IL-10], while inhibiting production of Th1 cytokines (e.g., interferon-gamma, IL-12). It is unknown what type of receptor binds PGE2 and modulates these responses. Recent analyses in nonhematopoietic cells have identified six PGE2 receptors (EP1, EP2, EP3 alpha, EP3 beta, EP3 gamma, and EP4). This investigation examines quiescent B lymphocytes and reports that these cells express mRNA encoding EP1, EP2, EP3 beta, and EP4 receptors. The immunoregulatory functions of each receptor were investigated using small molecule agonists that preferentially bind EP receptor subtypes. Unlike agonists for EP1 and EP3, agonists that bound EP2 or EP2 and EP4 receptors strongly inhibited expression of class II major histocompatibility complex and CD23 and blocked enlargement of mouse B lymphocytes stimulated with IL-4 and/or lipopolysaccharide. PGE2 promotes differentiation and synergistically enhances IL-4 and lipopolysaccharide-driven B-cell immunoglobulin class switching to IgE. Agonists that bound EP2 or EP2 and EP4 receptors also strongly stimulated class switching to IgE. Experiments employing inhibitors of cAMP metabolism demonstrate that the mechanism by which EP2 and EP4 receptors regulate B lymphocyte activity requires elevation of cAMP. In conclusion, these data suggest that antagonists to EP2 and EP4 receptors will be important for diminishing allergic and IgE-mediated asthmatic responses.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.

High levels of the p53 protein are immunohistochemically detectable in a majority of human nonmelanoma skin cancers and UVB-induced murine skin tumors. These increased protein levels are often associated with mutations in the conserved domains of the p53 gene. To investigate the timing of the p53 alterations in the process of UVB carcinogenesis, we used a well defined murine model (SKH:HR1 hairless mice) in which the time that tumors appear is predictable from the UVB exposures. The mice were subjected to a series of daily UVB exposures, either for 17 days or for 30 days, which would cause skin tumors to appear around 80 or 30 weeks, respectively. In the epidermis of these mice, we detected clusters of cells showing a strong immunostaining of the p53 protein, as measured with the CM-5 polyclonal antiserum. This cannot be explained by transient accumulation of the normal p53 protein as a physiological response to UVB-induced DNA damage. In single exposure experiments the observed transient CM-5 immunoreactivity lasted for only 3 days and was not clustered, whereas these clusters were still detectable as long as 56 days after 17 days of UVB exposure. In addition, approximately 70% of these patches reacted with the mutant-specific monoclonal antibody PAb240, whereas transiently induced p53-positive cells did not. In line with indicative human data, these experimental results in the hairless mouse model unambiguously demonstrate that constitutive p53 alterations are causally related to chronic UVB exposure and that they are a very early event in the induction of skin cancer by UVB radiation.

Distribution of parthenogenetic cells in the mouse brain and their influence on brain development and behavior.

A systematic analysis of parthenogenetic (PG) cell fate within the central nervous system (CNS) was made throughout fetal development and neonatal and adult life. Chimeras were made between PG embryos carrying a ubiquitously expressed lacZ transgene and normal fertilized embryos. After detailed histological analysis, we find that the developmental potential of PG cells is spatially restricted to certain parts of the brain. PG cells are prevalent in telencephalic structures and are largely excluded from diencephalic structures, especially the hypothalamus. These spatial restrictions are established early in development. Behavioral studies with chimeras identified an increase in male aggression when the proportion of PG cells in the brain was high. These studies demonstrate that imprinted genes play key roles in development of the CNS and may be involved in behavior.