Genetic engineering of carbohydrate biosynthetic pathways in transgenic mice demonstrates cell cycle-associated regulation of glycoconjugate production in small intestinal epithelial cells.

Proliferation, migration-associated differentiation, and cell death occur continuously and in a spatially well-organized fashion along the crypt-villus axis of the mouse small intestine, making it an attractive system for studying how these processes are regulated and interrelated. A pathway for producing glycoconjugates was engineered in adult FVB/N transgenic mice by expressing a human alpha 1,3/4-fucosyltransferase (alpha 1,3/4-FT; EC 2.4.1.65) along the length of this crypt-villus axis. The alpha 1,3/4-FT can use lacto-N-tetraose or lacto-neo-N-tetraose core chains to generate Lewis (Le) blood group antigens Le(a) or Le(x), respectively, and H type 1 or H type 2 core chains to produce Leb and Le(y). Single- and multilabel immunohistochemical studies revealed that expression of the alpha 1,3/4-FT results in production of Le(a) and Leb antigens in both undifferentiated proliferated crypt cells and in differentiated postmitotic villus-associated epithelial cells. In contrast, Le(x) antigens were restricted to crypt cells. Villus enterocytes can be induced to reenter the cell cycle by expression of simian virus 40 tumor antigen under the control of a promoter that only functions in differentiated members of this lineage. Bitransgenic animals, generated from a cross of FVB/N alpha 1,3/4-FT with FVB/N simian virus 40 tumor antigen mice, expand the range of Le(x) expression to include villus-associated enterocytes that have reentered the cell cycle. Thus, the fucosylations unveil a proliferation-dependent switch in oligosaccharide production, as defined by a monoclonal antibody specific for the Le(x) epitope. These findings show that genetic engineering of oligosaccharide biosynthetic pathways can be used to define markers for entry into, or progression through, the cell cycle and to identify changes in endogenous carbohydrate metabolism that occur when proliferative status is altered in a manner that is not deleterious to the system under study.

Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23).

The observation that overt type I diabetes is often preceded by the appearance of insulin autoantibodies and the reports that prophylactic administration of insulin to biobreeding diabetes-prone (BB-DP) rats, nonobese diabetic (NOD) mice, and human subjects results in protection from diabetes suggest that an immune response to insulin is involved in the process of beta cell destruction. We have recently reported that islet-infiltrating cells isolated from NOD mice are enriched for insulin-specific T cells, that insulin-specific T cell clones are capable of adoptive transfer of diabetes, and that epitopes present on residues 9-23 of the B chain appear to be dominant in this spontaneous response. In the experiments described in this report, the epitope specificity of 312 independently isolated insulin-specific T cell clones was determined and B-(9-23) was found to be dominant, with 93% of the clones exhibiting specificity toward this peptide and the remainder to an epitope on residues 7-21 of the A chain. On the basis of these observations, the effect of either subcutaneous or intranasal administration of B-(9-23) on the incidence of diabetes in NOD mice was determined. The results presented here indicate that both subcutaneous and intranasal administration of B-(9-23) resulted in a marked delay in the onset and a decrease in the incidence of diabetes relative to mice given the control peptide, tetanus toxin-(830-843). This protective effect is associated with reduced T-cell proliferative response to B-(9-23) in B-(9-23)-treated mice.

Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.

Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.

Insulin-like growth factor II mediates epidermal growth factor-induced mitogenesis in cervical cancer cells.

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Molecular heterogeneity of progenitors and radial migration in the developing cerebral cortex revealed by transgene expression.

We have analyzed the developmental pattern of beta-galactosidase (beta-gal) expression in the cerebral cortex of the beta 2nZ3'1 transgenic mouse line, which was generated using regulatory elements of the beta 2-microglobulin gene and shows ectopic expression in nervous tissue. From embryonic day 10 onward, beta-gal was expressed in the medial and dorsal cortices, including the hippocampal region, whereas lateral cortical areas were devoid of labeling. During the period of cortical neurogenesis (embryonic days 11-17), beta-gal was expressed by selective precursors in the proliferative ventricular zone of the neocortex and hippocampus, as well as by a number of migrating and postmigratory neurons arranged into narrow radial stripes above the labeled progenitors. Thus, the transgene labels a subset of cortical progenitors and their progeny. Postnatally, radial clusters of beta-gal-positive neurons were discernible until postpartum day 10. At this age, the clusters were 250 to 500 microns wide, composed of neurons spanning all the cortical layers and exhibiting several neuronal phenotypes. These data suggest molecular heterogeneity of cortical progenitors and of the cohorts of postmitotic neurons originating from them, which implies intrinsic molecular mosaicism in both cortical progenitors and developing neurons. Furthermore, the data show that neurons committed to the expression of the transgene migrate along very narrow, radial stripes.

Anti-nuclear antibody production and immune-complex glomerulonephritis in BALB/c mice treated with pristane.

The pathogenesis of systemic lupus erythematosus is thought to be primarily under genetic control, with environmental factors playing a secondary role. However, it has been shown recently that intraperitoneal injection of pristane (2,6,10,14-tetramethylpentadecane) induces autoantibodies typical of lupus in BALB/c mice, a strain not usually considered to be genetically susceptible to the disease. In this study, the induction of autoimmune disease by pristane was investigated. BALB/c mice receiving pristane were tested for autoantibody production and histopathological evidence of glomerulonephritis. Six of 11 mice developed IgM anti-single-stranded DNA antibodies shortly after receiving pristane and 4 developed IgM anti-histone antibodies, but anti-double-stranded DNA antibodies were absent. IgG anti-DNA and anti-histone antibodies were absent. In contrast, the lupus-associated anti-nuclear ribonucleoprotein/Sm and anti-Su autoantibodies produced by these mice were predominantly IgG. In addition to autoantibodies, most of the mice developed significant proteinuria. Light microscopy of the kidney showed segmental or diffuse proliferative glomerulonephritis. Electron microscopy showed subepithelial and mesangial immune-complex deposits and epithelial foot process effacement. Immunofluorescence revealed striking glomerular deposition of IgM, IgG, and C3 with a mesangial or mesangiocapillary distribution. Thus, pristane induces immune-complex glomerulonephritis in association with autoantibodies typical of lupus in BALB/c mice. These data support the idea that lupus is produced by an interplay of genetic and environmental factors and that unlike the MRL or (NZB x W)F1 mouse models, in which genetic susceptibility factors are of primary importance, environmental factors are of considerable importance in the autoimmune disease of pristane-treated BALB/c mice.

Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers.

c-Src is a nontransforming tyrosine kinase that participates in signaling events mediated by a variety of polypeptide growth factor receptors, including the epidermal growth factor receptor (EGFR). Overexpression and continual ligand stimulation of the EGFR results in morphological transformation of cells in vitro and tumor development in vivo. Elevated levels of c-Src and the EGFR are found in a variety of human malignancies, raising the question of whether c-Src can functionally cooperate with the EGFR during tumorigenesis. To address this issue, we generated c-Src/EGFR double overexpressors and compared their proliferative and biochemical characteristics to those of single overexpressors and control cells. We found that in cells expressing high levels of receptor, c-Src potentiated DNA synthesis, growth in soft agar, and tumor formation in nude mice. Growth potentiation was associated with the formation of a heterocomplex between c-Src and activated EGFR, the appearance of a distinct tyrosyl phosphorylation on the receptor, and an enhancement of receptor substrate phosphorylation. These findings indicate that c-Src is capable of potentiating receptor-mediated tumorigenesis and suggest that synergism between c-Src and the EGFR may contribute to a more aggressive phenotype in multiple human tumors.

Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.

Although the gene defect responsible for Huntington disease (HD) has recently been identified, the pathogenesis of the disease remains obscure. One potential mechanism is that the gene defect may lead to an impairment of energy metabolism followed by slow excitotoxic neuronal injury. In the present study we examined whether chronic administration of 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, can replicate the neuropathologic and clinical features of HD in nonhuman primates. After 3-6 weeks of 3-NP administration, apomorphine treatment induced a significant increase in motor activity as compared with saline-treated controls. Animals showed both choreiform movements, as well as foot and limb dystonia, which are characteristic of HD. More prolonged 3-NP treatment in two additional primates resulted in spontaneous dystonia and dyskinesia accompanied by lesions in the caudate and putamen seen by magnetic resonance imaging. Histologic evaluation showed that there was a depletion of calbindin neurons, astrogliosis, sparing of NADPH-diaphorase neurons, and growth-related proliferative changes in dendrites of spiny neurons similar to changes in HD. The striosomal organization of the striatum and the nucleus accumbens were spared. These findings show that chronic administration of 3-NP to nonhuman primates can replicate many of the characteristic motor and histologic features of HD, further strengthening the possibility that a subtle impairment of energy metabolism may play a role in its pathogenesis.

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

DNA synthesis and topoisomerase inhibitors increase transduction by adeno-associated virus vectors.

Viral vectors based on adeno-associated virus (AAV) preferentially transduce cells in S phase of the cell cycle. We recently found that DNA-damaging agents increased the transduction of nondividing cells. However, the optimal concentrations were toxic to cells. Here we show that the transduction of normal human fibroblasts by AAV vectors is increased by prior exposure to DNA synthesis inhibitors, such as aphidicolin or hydroxyurea, and topoisomerase inhibitors, such as etoposide or camptothecin. Transduction efficiencies could be increased > 300-fold in stationary cultures at concentrations that did not affect cell viability or proliferative potential. Both S-phase and non-S-phase cells were affected, suggesting that cellular functions other than replicative DNA synthesis may be involved. Applying these methods to gene transfer protocols should improve prospects for gene therapy by AAV vectors.