New evidence for an extra-hepatic role of N-acetylglucosaminyltransferase III in the progression of diethylnitrosamine-induced liver tumors in mice.

N-acetylglucosaminyltransferase III (GlcNAc-TIII) is encoded by the Mgat3 gene and catalyzes the addition of the bisecting GlcNAc to the core of N-glycans. Mice lacking GlcNAc-TIII due to the insertion mutation Mgat3tmlPst (termed Mgat3neo), exhibit retarded progression of liver tumors induced by diethylnitrosamine (DEN; M. Bhaumik et al, Cancer Res., 58: 2881-2887, 1998). This phenotype seemed to be due to a reduction, in activity or amount, of a circulating glycoprotein(s) that enhances DEN-induced liver tumor progression. Here, we provide new evidence to support this hypothesis. First, we show that mice with a deletion mutation of the Mgat3 gene coding exon (Mgat3tmlJxm, termed Mgat3delta) also exhibit retarded progression of DEN-induced liver tumors. At 7 months there was a significant decrease in liver weight (approximately 27%; P < 0.01), reflecting reduced tumor burden in Mgat3delta/delta mice. In addition, tumors were generally fewer and smaller, and histological changes were less severe in Mgat3delta/delta livers. Therefore, tumor progression is retarded in mice with two different null mutations in the Mgat3 gene. Second, we show that the development of DEN-induced tumors is unaltered by high levels of GlcNAc-TIII in the liver of transgenic mice. The Mgat3 gene coding exon under the control of the major urinary protein (MUP) promoter was used to generate transgenic mice that express GlcNAc-TIII in liver. Following DEN injection and phenobarbitol treatment, however, no significant differences were observed between MUP/Mgat3 transgenic and control mice in either tumor numbers or liver weight. The combined data provide strong evidence that retarded progression of tumors in mice lacking GlcNAc-TIII is due to the absence of the bisecting GlcNAc residue on N-glycans of a circulating glycoprotein(s) from a tissue other than liver.

Progression of hepatic neoplasms is severely retarded in mice lacking the bisecting N-acetylglucosamine on N-glycans: evidence for a glycoprotein factor that facilitates hepatic tumor progression.

The glycosyltransferase termed GlcNAc-TIII is dedicated to the transfer of a single N-acetylglucosamine (GlcNAc) residue (the bisecting GlcNAc), to a subset of N-glycans in glycoproteins. The addition of this GlcNAc is differentially regulated during development and is induced in certain cancers, particularly in hepatic tumorigenesis. To investigate a functional role for the bisecting GlcNAc in the development of liver cancer, the Mgat3 gene that codes for GlcNAc-TIII, was inactivated by targeted gene disruption, and the susceptibility of Mgat3-/- mice to tumor induction was tested. After a single injection with diethylnitrosamine and subsequent treatment with phenobarbitol for 6 months, Mgat3+/+ and Mgat3+/- mice had grossly enlarged livers that contained numerous tumors. By stark contrast, Mgat3-/- mice had livers of normal size, and only 50% of mice had one to four small tumors. However, histological examination showed that Mgat3-/- livers had significant numbers of basophilic foci, and by 10-12 months after diethylnitrosamine injection, tumors had developed in Mgat3-/- mice. Therefore, initiation occurred in Mgat3-/- mice but progression was severely retarded. Assays for Mgat3 gene expression in tumor tissue gave an unexpected result. In contrast to the situation in the rat, hepatic tumor formation in the mouse was not accompanied by a dramatic increase of GlcNAc-TIII activity nor of glycoproteins with a bisecting GlcNAc, nor of Mgat3 gene expression in tumor tissue from wild-type mice. The data suggest that a glycoprotein factor with the bisecting GlcNAc facilitates tumor progression in liver. In the absence of the bisecting GlcNAc in Mgat3-/- mice, the factor is reduced in activity, and tumor progression is severely retarded.

Cloning and chromosomal mapping of the mouse Mgat3 gene encoding N-acetylglucosaminyltransferase III.

Complex and hybrid N-linked carbohydrates synthesized by mammalian cells may possess a N-acetylglucosamine residue known as the bisecting GlcNAc. The transfer of this residue is catalyzed by the enzyme UDP-N-acetylglucosamine:beta-D-mannoside beta 1,4-N-acetylglucosaminyltransferase III (GlcNAc-TIII; EC 2.4.1.144). To begin to investigate biological functions for carbohydrates with a bisected GlcNAc residue, we have cloned and partially characterized the mouse gene (Mgat3) encoding GlcNAc-TIII. A rat GlcNAc-TIII-encoding cDNA was used to isolate clones from a mouse strain 129 Sv liver genomic DNA library. An NsiI genomic DNA fragment containing an ORF with 96% identity to rat GlcNAc-TIII was subcloned into a mammalian expression vector and transfected into Chinese hamster ovary (CHO) cells. The transfectants expressed GlcNAc-TIII activity only when the ORF was in the sense orientation. Southern analysis showed that Mgat3 is present in a single copy in the mouse genome. Mapping by restriction-fragment length polymorphism analysis of backcross progeny located Mgat3 to mouse chromosome 15, at a position homologous with region 22q12.3-q13.1 in the human genome. Northern analyses of adult tissues showed that Mgat3 is expressed at high levels in kidney and brain, and at lower levels in many other tissues.

Pregnancy rates following fimbriectomy reversal via neosalpingostomy: a 10-year retrospective analysis.

OBJECTIVE: To establish parameters associated with successful fimbriectomy reversal and to estimate monthly fecundability and cumulative pregnancy rates through life-table analysis. DESIGN: Series report. SETTING: University-based infertility clinic. PATIENT(S): Forty-one women undergoing surgery for tubal sterilization reversal. INTERVENTION(S): Surgical fimbriectomy reversal. MAIN OUTCOME MEASURE(S): Time from sterilization to reversal, laparoscopy vs. laparotomy, uni- vs. bilateral fimbriectomy reversal, Bruhat vs. suture, tubal lengths, postsurgical hysterosalpingogram, ovulation induction, incidence of pregnancy and outcome, and life-table analysis to determine pregnancy rate. RESULT(S): The mean time from sterilization to reversal was 11.5 years. Of the 41 women who underwent fimbriectomy reversal, 6 (14.6%) conceived. Sixteen reversals were performed by laparotomy resulting in 4 (25%) pregnancies, whereas 25 were performed laparoscopically resulting in 2 (8%) pregnancies. Eight had unilateral salpingostomies and 33 bilateral, of which 1 of 8 (12.5%) and 5 of 33 (15.2%) conceived, respectively. Using the Bruhat technique, 1 of 11 (9%) conceived vs. 5 of 30 (16.7%) that underwent reversal using sutures. The mean postoperative tubal length for the 6 women who conceived was 8 cm vs. 6.7 cm in the 35 women who did not conceive. Postoperatively, 26 women received ovulation induction and 1 (3.8%) conceived whereas 5 (33.3%) conceptions occurred in 15 women who did not require ovulation induction. Using life-table analysis with 619 postsurgical cycles, the monthly fecundability was.0097. The cumulative conception rate after 5 years was 31.2%. CONCLUSION(S): Neosalpingostomy for the reversal of fimbriectomy sterilization represents a viable option for fertility restoration. The best candidates for this procedure are spontaneously ovulatory and have a tubal length of more than 7 cm.

Evidence for translational control of beta-tubulin synthesis during differentiation of Leishmania donovani.

Tubulin biosynthesis was rapidly induced during transformation of the mammalian (amastigote) stage of the kinetoplastid parasite Leishmania donovani to flagellated promastigotes. However, transcription of beta-tubulin genes occurred constitutively, as judged by nascent RNA synthesis in isolated nuclei and Northern blotting of steady-state mRNA. Two mRNA species of 2.2 and 2.4 kb were shared by the two cell-types, while a third 2.6 kb species, constituting about 20% of the total, was present in large amounts in promastigotes. RNase protection experiments demonstrated sequence microheterogeneity in the 5'-untranslated region, the pattern of which was identical in promastigotes and amastigotes. By primer extension assays, heterogeneity in the 5'-terminal cap structure of amastigote beta-tubulin mRNA and differential pausing of reverse transcriptase within the mini-exon leader region were detected. These differences correlated with enhanced translational efficiency of tubulin mRNA from promastigotes in a rabbit reticulocyte lysate system. The results indicate that translational control plays a major role in tubulin induction during L. donovani differentiation.

CHO cells provide access to novel N-glycans and developmentally regulated glycosyltransferases.

Chinese hamster ovary (CHO) cells express only a subset of the glycosyltransferases activities known to exist. They do not express several fucosyltransferases, galactosyltransferases, sialyltransferases or N-acetylglucosaminyltransferases. However, following mutagenesis or transfection with large amounts of DNA, rare mutants that express a transferase activity de novo have been obtained. The first CHO mutant of this type was LEC10, which expresses the N-acetylglucosaminyltransferase, GlcNAc-TIII, that adds the bisecting GlcNAc to complex N-glycans. Several analogous gain-of-function mutants have now been characterized and, all express a new glycosyltransferase activity. In several cases, expression is known to reflect gene activation at the transcriptional level. Thus, CHO cells contain quiescent glycosyltransferase genes that may be activated by mutational events. Several of these transferases have properties distinct from previously described enzymes. In fact, the most recently characterized dominant CHO mutants, LEC14 and LEC18, each express a GlcNAc-T activity that creates novel N-glycans never before observed in glycoproteins from any other source. In these and possibly other cases, it appears the CHO genome has provided access to new GlcNAc-Ts that may be difficult to identify by conventional methods.

Regulation of N-linked glycosylation. Neuronal cell-specific expression of a 5' extended transcript from the gene encoding N-acetylglucosaminyltransferase I.

A key transferase in the generation of mature N-linked carbohydrates is the medial Golgi enzyme N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-TI) which is encoded by the Mgat-1 gene. Previous studies revealed two size classes of Mgat-1 mRNA that are differentially expressed in mouse tissues. Nearly all tissues possess a shorter form (approximately 2.9 kb), whereas brain has predominantly a longer Mgat-1 mRNA (approximately 3.3 kb). We now show, by in situ hybridization of horizontal sections of adult mouse brain, that Mgat-1 RNA levels vary markedly in different brain cell types with the greatest expression being in neuronal cells. The differential expression of the approximately 2.9 kb and approximately 3.3 kb Mgat-1 mRNAs is likely to be controlled by tissue-specific promoters since the size difference between the mRNAs was found to residue entirely in the 5' untranslated region of the approximately 3.3 kb mRNA. Evidence for this was obtained by an RNase H strategy, reverse transcription-polymerase chain reaction (RT-PCR) and 5' anchored PCR. All of the 0.4 kb difference in size was localized upstream of the previously isolated cDNA sequence. Sequence information from this region was obtained from a mouse brain cDNA library by a PCR amplification strategy and a probe specific for the 3.3 kb mRNA was generated. This probe hybridized uniquely to the approximately 3.3 kb Mgat-1 mRNA and Southern blot analysis showed that the new sequence is physically linked to the Mgat-1 gene. A tissue culture model which displays an increase in expression of the approximately 3.3 kb Mgat-1 mRNA transcript during differentiation to neuronal cells has been developed in P19 embryonal carcinoma (EC) cells. The combined data suggest that 5' exons in the Mgat-1 gene are differentially utilized by tissue-specific promoters and that transcription factor(s) which specify production of the approximately 3.3 kb Mgat-1 mRNA are induced by retinoic acid treatment of P19 EC cells.

WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Two critical hits for promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is associated with chromosomal translocations that always involve the RARalpha gene, which variably fuses to one of several distinct loci, including PML or PLZF (X genes). Due to the reciprocity of the translocation, X-RARalpha and RARalpha-X fusion proteins coexist in APL blasts. PLZF-RARalpha transgenic mice (TM) develop leukemia that lacks the differentiation block at the promyelocytic stage that characterizes APL. We generated TM expressing RARalpha-PLZF and PLZF-RARalpha in their promyelocytes. RARalpha-PLZF TM do not develop leukemia. However, PLZF-RARalpha/RARalpha-PLZF double TM develop leukemia with classic APL features. We demonstrate that RARalpha-PLZF can interfere with PLZF transcriptional repression and that this is critical for APL pathogenesis, since leukemias in PLZF(-/-)/PLZF-RARalpha mutants and in PLZF-RARalpha/RARalpha-PLZF TM are indistinguishable. Thus, both products of a cancer-associated translocation are crucial in determining the distinctive features of the disease.

UCI-VULV-1, a vulvar squamous carcinoma cell line.

Squamous carcinoma of the vulva (SCV) is an uncommon neoplasm of uncertain etiology. There is evidence that there are two subgroups of SCV, one associated with human papilloma virus (HPV) and a second HPV-negative group. The UCI-VULV-1 cell line, obtained from a lymph node metastasis of an SCV, grows with a population doubling time of approximately 60 hr. The saturation density is 10(5) cells/cm2. The cell line does not exhibit anchorage independence and is weakly tumorigenic. The cells range in appearance from an abundant spindle cell to a less common larger, flat cell. All of the cells are immunoreactive for high-molecular-weight keratin, but only the flat cells, which form squamous pearls in vivo, are immunoreactive for low-molecular-weight keratin. The cell line expresses epidermal growth factor (EGF), transforming growth factor-alpha, the EGF receptor, and p53 protein. Polymerase chain reaction revealed no HPV DNA within the cells. Early passage cells exhibited karyotypic heterogeneity with few similarities to previous described SCV karyotypes. The cells display sensitivity to cis-platinum in concentrations toxic to many ovarian and cervical carcinoma lines. UCI-VULV-1 may be helpful for studying the properties of the HPV-negative form of SCV.

A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome).

Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.