Pim1 cooperates with E2a-Pbx1 to facilitate the progression of thymic lymphomas in transgenic mice

Mice transgenic for the leukemia oncogene E2A-PBX1 invariably develop lethal, high-grade T-cell lymphomas by 5 months of age. In this study, retroviral insertional mutagenesis was employed to identify oncogenes that cooperate with the E2A-PBX1 transgene in lymphomagenesis. Neonatal retroviral infection substantially reduced length of survival due to accelerated development of lymphomas (81 versus 130 days). The Pim1 gene was targeted by retroviral insertions in 48% of accelerated lymphomas whereas less than 5% contained activated c-Myc and none contained activated Pim2. However, Pim1 DNA rearrangements were frequently sub-stoichiometric and not present at all sites of involvement in an otherwise monoclonal lymphoma indicating that Pim1 activation occurred late in the course of lymphomagenesis. Tumor subpopulations containing activated Pim1 alleles displayed a substantial growth advantage over Pim1 negative cells following serial transfer to secondary, syngeneic recipients. Cooperative interactions were observed in intercrossed Pim1 and E2A-PBX1 transgenic mice in which all double transgenic progeny developed lethal, diffuse T lineage lymphomas by 3 months of age, whereas only 13% of E2A-PBX1 and none of Pim1 single transgenic intercross progeny developed lymphomas by 1 year. Tumors from double transgenic mice were monoclonal providing evidence that additional genetic events were required for transformation. Therefore, Pim1 and E2a-Pbx1 cooperate in T lineage lymphomagenesis but they are not sufficient and the role of Pim1 is more likely to be associated with tumor progression.     

Tat-induced lesions in transgenic mice do not correlate with the HIV-1 LTR transactivation

A pilot study was conducted in 7 normal volunteers to demonstrate the feasibility of employing pharmacokinetic tailoring to achieve matching plasma opioid concentration-time curves after epidural (e.p.) and intravenous (i.v.) alfentanil administration. Each subject participated in 1 pretest and 2 test sessions. Our pain model was cutaneous electrical stimulation of the finger and toe, adjusted to produce a baseline pain report of 5 (strong pain on a 0-5 scale). On test day 1, subjects received e.p. alfentanil (750 micrograms) and an i.v. saline infusion. Serial measurements of analgesia, end tidal CO2, pupil size, subjective side effects, and plasma alfentanil concentrations were conducted before and at various time intervals over a 4-h period after alfentanil administration. On test day 2, subjects received e.p. saline and a pharmacokinetically tailored i.v. infusion (using individual pharmacokinetics determined on the pretest day) designed to achieve a plasma concentration-time profile identical to that observed on the epidural day. The same battery of effect measurements was administered as on the 1st test day. Plasma alfentanil was measured to verify the accuracy of the tailored infusion. Plasma alfentanil concentration profiles were nearly identical on both test days. Peak plasma alfentanil concentrations were near the reported minimum effective analgesic concentration (MEAC). Overall, analgesia was slightly greater with e.p. administration. Onset of pain relief was rapid, and duration was approximately 1.5 h with e.p. and 1 h with i.v. alfentanil. There were no differences in pupil size, ETCO2, or subjective side effects between e.p. versus i.v. administration. We conclude that systemic redistribution from the epidural space appears to account for most, but not all, of the analgesia.     

Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1

Mice completely deficient for Dvl1, one of three mouse homologs of the Drosophila segment polarity gene Dishevelled, were created by gene targeting. Dvl1-deficient mice are viable, fertile, and structurally normal. Surprisingly, these mice exhibited reduced social interaction, including differences in whisker trimming, deficits in nest-building, less huddling contact during home cage sleeping, and subordinate responses in a social dominance test. Sensorimotor gating was abnormal, as measured by deficits in prepulse inhibition of acoustic and tactile startle. Thus, Dvl1 mutants may provide a model for aspects of several human psychiatric disorders. These results are consistent with an interpretation that common genetic mechanisms underlie abnormal social behavior and sensorimotor gating deficits and implicate Dvl1 in processes underlying complex behaviors.     

Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene

PURPOSE: To characterize the morphological changes in the eyes of transgenic mice harboring different mutations in type II collagen gene to elucidate the function of this collagen in the eye, and to find out whether these animals could function as models for the human arthro-ophthalmopathies of the Kniest, Stickler and Wagner types. METHODS: Three genetically engineered mouse lines representing two types of mutations in the triple-helical domain of type II collagen and their nontransgenic littermates used as controls were analyzed on day 18.5 embryonic development. After genotyping by polymerase chain reaction (PCR) and Southern hybridization the embryos were prepared for routine histology. Polarization microscopy was done on hyaluronidase-treated sections. RESULTS: Histological analysis revealed several genotype-dependent abnormalities in the eyes of the transgenic mice. Most striking changes were observed in the vitreous architecture; in one line of mice the vitreous was tightly packed in the posterior region of the vitreous space with thick fibrils, empty cavities and dense membrane-like material. The other mutation resulted in reduced filament density of the vitreous. In the most severely affected phenotype the internal limiting membrane was detached from the retinal layers and was markedly thickened, and the posterior lens capsule was thickened. The anterior chamber was shallow or absent in all transgenic lines but was well formed in the normal animals. Changes were also observed in the lens, corneal and scleral structures. CONCLUSIONS: The ocular changes observed in transgenic mice harboring mutations in type II collagen gene show similarities to the human ocular findings in Kniest dysplasia, and in Stickler and Wagner syndromes. We therefore propose that these animals could serve as models for systematic analysis of vitreoretinal degeneration and other abnormalities, as seen in these syndromes.     

Chimeric homeobox gene E2A-PBX1 induces proliferation, apoptosis, and malignant lymphomas in transgenic mice

Expression of the homeobox fusion gene E2A-PBX1 under control of the immunoglobulin heavy chain enhancer efficiently induced malignancies in transgenic mice. All animals died before 5 months of age with lymphomas that demonstrated phenotypes consistent with transitional intermediate thymocytes (CD4+/CD8+/CD3med). E2A-PBX1 also markedly altered lymphoid development in pretumorous animals, reducing the number of thymocytes and bone marrow B lineage progenitors to 20% of normal levels. In spite of the observed reductions in lymphoid cells, premalignant animals contained significantly increased numbers of cycling thymocytes, but a higher proportion was also undergoing apoptosis, suggesting that increased cell death resulted in the marked lymphopenias. These data indicate that the chimeric homeodomain protein E2A-PBX1 paradoxically induces both proliferation and apoptosis in lymphoid cells, suggesting an in vivo association between nuclear oncogene-induced cell cycle progression and programed cell death.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

Altered tumor growth and metastasis of a T-cell lymphoma in Timp-1 transgenic mice

The concept of tumor suppression by tissue inhibitor of metalloproteinases (TIMPs) has evolved primarily from studies of genetically modulated tumor cells. The next step is to focus on the host and assess the protective potential of host TIMP-1 on primary tumor growth and metastasis. We generated two transgenic mouse lines with altered Timp-1 expression in skin and liver: one overexpressed Timp-1 (Timp-1(high)), and the other had antisense RNA-mediated Timp-1 reduction (Timp-1(low)). ESbL-lacZ T-lymphoma cells provided the tumor challenge, as they form primary tumors upon intradermal injection with spontaneous metastasis to liver. Metastases were examined in X-Gal-stained whole-organ mounts. Timp-1 overexpression inhibited intradermal tumor growth and spontaneous metastasis, leading to prolonged survival of the mice. The opposite effects occurred in Timp-1(low) mice, leading to shorter host survival. Experimental metastasis assays showed that Timp-1-compromised livers in Timp-1(low) mice showed at least a doubling of metastatic foci and numerous additional micrometastases, indicative of increased host susceptibility. However, Timp-1(high) mouse livers showed an unaltered metastatic load in the experimental metastasis assay. In conclusion, these data demonstrate that Timp-1 levels within a tissue predetermine the development and progression of T-cell lymphoma.     

BCR-ABL induces neurite-like structures and BCR lacking the SH2-binding domain induces cell rounding in PC12 cells

The activated tyrosine kinase oncoprotein BCR-ABL is responsible for pathogenesis of Philadelphia chromosome-positive human leukemias. Because BCR carries a GAP (GTPase-activating protein) activity toward cytoskeleton-related small GTP-binding proteins, we utilized a neuronal PC12 cell system to test morphogenic potentials of BCR-ABL or BCR. We report here unique morphological phenotypes of PC12 cells expressing either BCR-ABL or a BCR mutant which lacks the SH2-binding domain (BCR Delta162-413). Although MAP kinase was not activated in PC12 cells expressing BCR-ABL, they showed incomplete neurite extensions even in the absence of the nerve growth factor (NGF). Overproduction of BCR Delta162-413 in PC12 cells, on the other hand, induced cell rounding in the absence of NGF. Interestingly, those cells could hardly make terminal differentiation in the presence of NGF and continued to grow without changing their round shape, although NGF receptor as well as MAP kinase appeared to be activated. Interestingly, the botulinum C3 toxin induced neurite-like structures in PC12 cells overexpressing BCR Delta162-413 without NGF.     

Three-dimensional computed tomography in the head and neck diseases with bony abnormalities

The purpose of this study was to define the role of three-dimensional (3D) computed tomography (CT) in the head and neck diseases with bony abnormalities. Thirty-two patients were examined with a low dose radiation technique. Three-dimensional CT clearly delineated bony lesions in 27 of 32 patients. Three-dimensional CT could not demonstrate subtle bony erosions infiltrated by tumor, a temporal bone fracture, and a blow-out fracture, although two-dimensional (2D) images obtained before the 3D reconstructions clearly depicted those lesions. These two kinds of CT technique were thought to be complimentary.     

Partial loss of tolerance liability to morphine analgesia in mice lacking the nociceptin receptor gene

In mice lacking the nociceptin (or orphanin FQ) receptor gene, when 10 mg/kg of morphine was subcutaneously given, a potent analgesia in the tail pinch test was observed. The analgesic effect of morphine was equivalent among wild-type, heterozygous and homozygous mutant mice. When morphine was given to such mice in a dose of 10 mg/kg once per day for 5 days, wild-type and heterozygous mice showed marked tolerance or reduction in the morphine analgesia on the 5th day, while homozygous mice showed only 50% reduction in the peripheral analgesia of morphine. These findings suggest that nociceptin or its receptor plays important roles in the in vivo mechanism for the development of morphine tolerance.     

Evi-1 raises AP-1 activity and stimulates c-fos promoter transactivation with dependence on the second zinc finger domain

Evi-1 is a gene, encoding a zinc finger protein, associated with a common viral integration site in murine leukemias. It is suggested that Evi-1 plays important roles in embryogenesis and transformation of myeloid cells. To elucidate mechanisms by which Evi-1 induces such biological effects, we analyzed the relationship between Evi-1 and AP-1 which could regulate cellular proliferation and differentiation. When Evi-1 was expressed, transactivation through a 12-O-tetradecanoylphorbol 13-acetate-responsive element was observed in NIH3T3 and P19 cells. Evi-1-transfected P19 cells showed some differentiated phenotypes and increased expression of endogenous c-Jun and c-Fos. These results indicate that Evi-1 raises AP-1 activity. Evi-1 caused stimulation of the c-fos promoter transactivation, which seems to be a main mechanism of AP-1 activation, through at least two portions of the promoter. Evi-1 has the first zinc finger domain at the N terminus and the second zinc finger domain near the C-terminus. We constructed deletion mutants of Evi-1 and investigated the functions of these domains. It was shown that the second zinc finger domain is essential for the activation of AP-1 and transactivation of the c-fos promoter.     

Rapid methods for the analysis of immunoglobulin gene hypermutation: application to transgenic and gene targeted mice

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Little is known about the mechanism, but the availability of rapid facile assays for monitoring immunoglobulin hypermutation would greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the process. Here we describe two such assays. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PCR to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry immunoglobulin transgenes, we exploited the fact that hypermutation extends into the region flanking the 3'-side of the rearranged J segments. We show that PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germline counterpart is unequivocally known. This assay was particularly useful for analysing endogenous immunoglobulin gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, we show that one can exploit the fact that, following denaturation/renaturation, the PCR amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes which can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS -Wagner et al. (1995) Nucleic Acids Res. 23, 3944-3948-. Such assays enabled us, by example, to show that antibody hypermutation proceeds in the absence of the p53 tumour suppressor gene product.     

Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene

To elucidate the role of A1, a new member of the Bcl-2 family of apoptosis regulators active in hematopoietic cell apoptosis, we established mice lacking A1-a, a subtype of the A1 gene in mice (A1-a-/- mice). Spontaneous apoptosis of peripheral blood neutrophils of A1-a-/- mice was enhanced compared with that of either wild-type mice or heterozygous mutants (A1-a+/- mice). Neutrophil apoptosis inhibition induced by lipopolysaccharide treatment in vitro or transendothelial migration in vivo observed in wild-type mice was abolished in both A1-a-/- and A1-a+/- animals. On the other hand, the extent of tumor necrosis factor alpha-induced acceleration of neutrophil apoptosis did not differ among A1-a-/-, A1-a+/-, and wild-type mice. The descending order of A1 mRNA expression was wild-type, A1-a+/-, and A1-a-/-. Taken together, these results suggest that A1 is involved in inhibition of certain types of neutrophil apoptosis.     

Inner ear and maternal reproductive defects in mice lacking the Hmx3 homeobox gene

The Hmx homeobox gene family is of ancient origin, being present in species as diverse as Drosophila, sea urchin and mammals. The three members of the murine Hmx family, designated Hmx1, Hmx2 and Hmx3, are expressed in tissues that suggest a common functional role in sensory organ development and pregnancy. Hmx3 is one of the earliest markers for vestibular inner ear development during embryogenesis, and is also upregulated in the myometrium of the uterus during pregnancy. Targeted disruption of the Hmx3 gene results in mice with abnormal circling behavior and severe vestibular defects owing to a depletion of sensory cells in the saccule and utricle, and a complete loss of the horizontal semicircular canal crista, as well as a fusion of the utricle and saccule endolymphatic spaces into a common utriculosaccular cavity. Both the sensory and secretory epithelium of the cochlear duct appear normal in the Hmx3 null animals. The majority of Hmx3 null females have a reproductive defect. Hmx3 null females can be fertilized and their embryos undergo normal preimplantation development, but the embryos fail to implant successfully in the Hmx3 null uterus and subsequently die. Transfer of preimplantation embryos from mutant Hmx3 uterine horns to wild-type pseudopregnant females results in successful pregnancy, indicating a failure of the Hmx3 null uterus to support normal post-implantation pregnancy. Molecular analysis revealed the perturbation of Hmx, Wnt and LIF gene expression in the Hmx3 null uterus. Interestingly, expression of both Hmx1 and Hmx2 is downregulated in the Hmx3 null uterus, suggesting a hierarchical relationship among the three Hmx genes during pregnancy.     

Zinc finger protein GFI-1 has low oncogenic potential but cooperates strongly with pim and myc genes in T-cell lymphomagenesis

The gfi-1 gene encodes a zinc finger containing protein that is specifically expressed in T-lymphocytes and is a frequent target of proviral insertion in T-cell lymphoma provoked by infection with MoMuLV--a non acute transforming retrovirus. Expression of a gfi-1 transgene targeted to T-cells by the lck proximal promoter provokes a reduction of peripheral CD4 and CD8 positive T-cells but nevertheless weakly predisposes transgenic animals for the development of T-cell lymphoma. Forced coexpression of the serine/threonine kinase Pim-1 can partially restore normal T-cell numbers in double pim-1/gfi-1 transgenic mice. Moreover, the combinatorial expression of Pim-1 and Gfi-1 leads to accelerated development of T-cell lymphoma with a mean latency period of 114 days. A similar accelerated rate of lymphoma development was observed when lck-gfi-1 mice were crossed with mice that carry a L-myc gene targeted to be expressed at high levels in T-cells. The results show that gfi-1 can act with low activity as a dominant oncogene when overexpressed but also demonstrate that it is most efficient only in the presence of a cooperative partner protein as for example Pim-1 or L-Myc. In addition, the results suggest that Pim-1 and Gfi-1 are acting synergistically in both T-cell lymphomagenesis and T-cell development.     

Metastasis of mammary carcinomas in GRS/A hybrid mice transgenic for the mts1 gene

Transgenic mice, carrying the mts1 gene, one of the genes involved in the acquisition of the metastatic phenotype, were generated. The mts1 gene was placed under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter leading to overexpression in the lactating mammary gland of transgenic animals. Animals bearing the transgene appear phenotypically normal. Animals of two transgenic lines (Tg463 and Tg507) were crossed with the GRS/A mice. The GRS/A strain is characterized by high incidence of mammary tumors which rarely metastasize. 40% of the tumor bearing hybrid GRS/A mts1 females were found to develop secondary tumors in the lungs. The Mts1 protein was detected in the transgene primary tumor cells as well as in the corresponding metastases. Nontransgenic littermates expressed the Mts1 protein only in the stromal cells surrounding the tumor but not in the tumor cells by itself. Taken together these observations indicate that overexpression of the mts1 gene in the mouse mammary carcinoma cells gives rise to more aggressive tumors which are able to metastasize.     

Targeted expression of activated erbB-2 to the epidermis of transgenic mice elicits striking developmental abnormalities in the epidermis and hair follicles

The erbB-2 proto-oncogene belongs to a receptor tyrosine kinase family that includes the epidermal growth factor receptor, erbB-2, erbB-3, and erbB-4. erbB-2 is expressed in basal cells of the squamous epithelia and the outer root sheath of the hair follicles, but its function in epidermal development has not been well studied. To investigate its role in the skin, we created transgenic mice harboring an activated erbB-2 oncogene under the control of the human keratin 14 promoter. The keratin 14 promoter directed its expression to cells in which erbB-2 is normally expressed, whereas the activated receptor gene ensured increased signaling. All transgenic founder mice exhibited extensive and striking skin phenotype, including epidermal hyperplasia, preneoplasia, papilloma, hyperkeratosis, and dyskeratosis. The majority of the hair follicles were replaced by bizarre hyperproliferative intradermal squamous invaginations, whereas the rest of the follicles exhibited severe hyperplasia and disorganization. All but one of the transgenic mice died before or shortly after birth, probably as a consequence of defects in the skin and esophagus. These observations demonstrate that the skin is sensitive to erbB-2 signaling, suggesting an important role for this receptor tyrosine kinase in epidermal growth, differentiation, and hair follicle morphogenesis.