Notch signaling is essential for vascular morphogenesis in mice.

The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.

Absence of human T-lymphotropic virus type I tax sequences in a population of normal blood donors in the Baltimore, MD/Washington, DC, area: results from a multicenter study.

BACKGROUND: It was reported recently that sequences corresponding to the human T-lymphotropic virus type I (HTLV-I) tax gene were detected in peripheral blood mononuclear cells from 8 to 11 percent of healthy blood donors without detectable antibodies to HTLV-I. A multicenter blind study was conducted to determine if these results could be independently confirmed. STUDY DESIGN AND METHODS: Specimens were collected from 100 anti-HTLV-I-negative healthy blood donors and from 11 anti-HTLV-I- or anti-HTLV-II-positive individuals. All samples were coded and distributed to each of four independent testing laboratories for polymerase chain reaction analysis to detect sequences of the HTLV-I or HTLV-II tax gene, using detailed procedures specified by the laboratory reporting the original observation. Each laboratory also tested a dilution panel of a plasmid containing HTLV-I tax to determine the analytical sensitivity of the procedure. RESULTS: The analytical sensitivity of the screening methods permitted detection of as few as 1 to 10 copies of the tax gene. However, HTLV-I tax sequences could not be detected in any of the anti-HTLV-I-negative blood donors at more than one test site. CONCLUSION: HTLV-I tax sequences appear not to be present in this population of 100 blood donors negative for anti-HTLV-I.

False-positive HIV-1 test results in a low-risk screening setting of voluntary blood donation. Retrovirus Epidemiology Donor Study.

CONTEXT: Persons at risk of human immunodeficiency virus 1 (HIV-1) infection, have been classified incorrectly as HIV infected because of Western blot results, but the frequency of false-positive Western blot results is unknown. OBJECTIVES: To determine the frequency of false-positive HIV-1 Western blot results in US blood donors and to make projections to other screened populations. Secondarily, to validate an algorithm for evaluating possible false-positive cases. DESIGN: A retrospective cohort study of HIV-1 enzyme immunoassay (EIA) and Western blot results from large blood donor screening programs in which donors with suspected false-positive Western blot results underwent HIV-1 RNA polymerase chain reaction (PCR) testing and follow-up HIV-1 serology. SETTING: Five US blood centers participating in the Retrovirus Epidemiology Donor Study. PARTICIPANTS: More than 5 million allogeneic and autologous blood donors who successfully donated blood at 1 of the 5 participating centers from 1991 through 1995. MAIN OUTCOME MEASURES: Rate of false positivity by Western blot and true HIV-1 infection status as determined by HIV-1 RNA PCR and by serologic follow-up of blood donors more than 5 weeks after donation. RESULTS: Of 421 donors who were positive for HIV-1 by Western blot, 39 (9.3%) met the criteria of possible false positivity because they lacked reactivity to p31. Of these, 20 (51.3%) were proven by PCR not to be infected with HIV-1. The false-positive prevalence was 4.8% of Western blot-positive donors and 0.0004% (1 in 251000) of all donors (95% confidence interval, 1 in 173000 to 1 in 379000 donors). CONCLUSIONS: A false diagnosis of HIV-1 infection can result from the combination of EIA and Western blot testing in blood donor and other HIV-1 screening programs. Individuals with a positive Western blot result lacking the p31 band should be counseled that, although they may be HIV infected, there is uncertainty about this conclusion. These individuals should be further evaluated by RNA PCR testing (if feasible) and HIV serologic analysis on a follow-up sample.

The chromosome location of the human homolog of the mouse mammary tumor-associated gene INT6 and its status in human breast carcinomas.

The INT6 gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog of INT6 is located on chromosome region 8q22-q23. A processed INT6 pseudogene is located on chromosome 6q. INT6 is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affecting INT6. Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 of INT6. Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked to INT6.

Capillary electrophoresis methodology for identification of cancer related gene expression patterns of fluorescent differential display polymerase chain reaction.

The mRNA differential display technique is a method for molecular survey and analysis of differential gene expression in eukaryotic cells and tissues. We have previously described the use of ABI Prism fluorescent technology to specifically amplify expressed sequence tags (ESTs) from several different biological paradigms. High throughput, fluorescent differential display performed on an automated sequencer (ABI 377) has proven to have significant cost cutting and time saving attributes compared to that of the radioactive differential display. Additionally, fluorescent tagged mRNA specific reverse transcription and PCR decreases the number of the inherent artifacts associated with radioactive differential display. We report here the application of a capillary electrophoresis system (ABI 310) to the identification of fluorescent differential display generated EST patterns. RNA samples from human and animal breast cancer paradigms were exposed to this technique and analyzed by the ABI 310 and the ABI 377. GeneScan and Genotyper software applications were used for rapid and semi-automated characterization of fluorescently labeled ESTs. Automated sample loading and uniform sample electrophoresis are among the main advantages of this system which significantly increase the precision and reproducibility of fluorescent differential display.

The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4).

The INT3 gene is frequently rearranged in mouse mammary tumor virus (MMTV)-induced mammary tumors of the CzechII mouse strain. We have completed the nucleotide sequence of the normal 6.5 Kb INT3 RNA and defined the intron/exon boundaries of the gene. The open reading frame of INT3 RNA should encode a 200 kd protein which shares 60% homology with the mouse homologue of Drosophila NOTCH. INT3 is unique among other members of the NOTCH family by containing 29 instead of 36 EGF-like repeats in the extracellular domain of the gene product. Five novel EGF-like repeats have been created as consequence of apparent small deletions which have occurred within the coding region for the extracellular domain during evolution. Nucleotide sequence analysis of host-viral junction fragments from nine independent MMTV-induced mammary tumors containing a rearranged INT3 gene reveals that all of the integration events occur within a 174 bp region 3' of the sequences encoding the LIN12 repeats in the INT3 extracellular domain and 5' of the sequences encoding the transmembrane domain. Therefore, the only tumorigenic INT3 mutations resulting from MMTV proviral insertions are those which results in the expression of the intracellular domain. This strongly suggests that MMTV-induced activation of INT3 is manifest in the absence of the regulatory action of the extracellular domain, including the LIN12 repeat sequences, leaving the expressed intracellular domain constitutively free to function in its role in mammary tumorigenesis.

Understanding mammary gland development through the imbalanced expression of growth regulators.

Functional differentiation of mammary tissue progresses in distinct phases spanning puberty and pregnancy. Here we have analyzed and compared the effects of transforming growth factor beta 1 (TGF beta 1), TGF alpha, and whey acidic protein (WAP), the Notch-related cell fate protein Int3, and p53 and pRb on mammary development. We chose transgene expression from the WAP gene promoter which is only active in mammary alveolar cells. The imbalanced expression of these molecules specifically altered development and differentiation of the gland. While TGF alpha did not disturb alveolar outgrowth, little or no alveolar structures developed in the presence of Int3. TGF beta 1, WAP, and the expression of SV40 T-antigen-which inactivates p53 and PRb-reduced overall alveolar development. The expression of individual milk protein genes was affected differentially by the transgenes. A WAP-lacZ transgene served as an additional indicator of terminal differentiation of alveolar cells, Homogeneous expression of lacZ was seen in mice transgenic for lacZ, or for TGF alpha and lacZ. In contrast, only a few differentiated cells were observed in the presence of TGF beta 1 and Tag. Thus, the expression of growth regulators in the same defined subset of mammary cells results in distinct developmental changes and a specific pattern of alveolar differentiation.

Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis.

Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4-kb tumor specific Int3 RNA transcript that encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development were curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene, which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter, the activity of which, unlike that of the MMTV long terminal repeat, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In nonbreeding WAP/Int3 females, mammary tumor incidence also reached 100%, but only after 70 weeks. The WAP/Int3 mammary tumors were highly malignant, and most tumor-bearing females, irrespective of breeding history, developed metastatic lung lesions. These results suggest that WAP promotor-targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP-driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into nontransgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated.

A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.

In a mouse mammary tumor model system in which carcinogenic progression can be investigated, we have found a unique mutation of Int-3 associated with progression from premalignant lobular hyperplasia to tumor. Sequence analysis of the rearranged fragment revealed an insertion of an intracisternal type A particle (IAP) within the Int-3 gene. Int-3 is mutated frequently in mouse mammary tumor virus (MMTV)-induced mammary tumors by insertion of MMTV proviral DNA into this intragenic region. In these mutations, the insertion produces a chimeric Int-3 transcript encoding the cytoplasmic portion of the Int-3 protein driven by the MMTV long terminal repeat promoter. In this case, the IAP DNA was inserted in the opposite transcriptional orientation relative to Int-3; nevertheless, a similar chimeric RNA transcript driven by a cryptic promoter in the oppositely oriented 5' IAP long terminal repeat was generated. This is the first demonstration that an insertional mutation unrelated to MMTV activates an Int gene commonly associated with mammary tumorigenesis.

The chromosomal location of the mouse mammary tumor gene Int6 and related pseudogenes in the mouse genome.

The Int6 gene is a common insertion site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors. We have determined that this gene is located centromeric of the Myc protooncogene on mouse chromosome 15. In the mouse genome there are several other Int6-reactive restriction fragments that are located on mouse chromosomes 6, 11, 14, 17, and 18. Nucleotide sequence analysis of four of six of these additional Int6 fragments showed that they contain processed Int6 pseudogenes. Comparisons between the Int6 genes of the inbred mouse laboratory strains and the wild mouse species Mus spretus and Mus mus musculus indicate that some pseudogenes were present before divergence of these species and others were acquired since their separation.

Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development.

INT3 is interrupted by retroviral DNA insertion in approximately 18% of primary Czech mouse mammary tumors induced by mouse mammary tumor virus. One consequence of these insertions is the production of a 2.4-kilobase, tumor-specific RNA transcript encoding the entire intracellular domain of the Int3 protein which is initiated from the 3' long terminal repeat promoter of the inserted viral genome. Female mice (FVB-3) transgenic for a genomic fragment comprised of this truncated region of INT3 express the 2.4-kilobase truncated INT3 transcript and exhibit focal mammary tumors at 100% penetrance. INT3 is a member of a family of genes, highly conserved through evolution and characterized by Drosophila melanogaster Notch and Caenorhabditis elegans lin-12, the function of which relates to cell fate determination. Upon transfection into the appropriate hosts, expression vectors of truncated Notch and lin-12, representing their respective cytoplasmic domains, have been demonstrated to effect their complete gene function with respect to cell fate determination. This suggests that the extracellular portion of these proteins function only to regulate activity. Reciprocal transplantation of transgenic FVB-3 and normal mammary tissue to the epithelium-divested fat pads of the respective donor females demonstrates that FVB-3 mammary epithelium is unable to grow and/or to functionally differentiate. However, normal epithelium grows and fully differentiates in transgenic FVB-3 fat pads, indicating that the dysfunction of FVB-3 mammary glands is due to a deficiency inherent in their epithelium. Electron microscopy reveals that transgenic INT3 epithelial cells do not form intercellular junctional complexes in the developing subadult mammary gland. The hormonal stimulation of pregnancy overcomes the deficiency for ductal growth so apparent in the virgin gland such that pregnant FVB-3 glands produce complete ductal systems. Nevertheless, during pregnancy, FVB-3 mammary cells fail to form secretory lobules and to produce milk. Examination of INT3 expression by immunocytochemistry and reverse transcriptase-PCR show that INT3 is expressed constitutively in mammary stroma and epithelia at all stages of postpubertal mammary evolution. These results indicate that deregulated expression of a truncated Int3 in mammary epithelial cells limits their capacity to perform the cell fate decisions required for morphogenesis and functional differentiation.

Int-6, a highly conserved, widely expressed gene, is mutated by mouse mammary tumor virus in mammary preneoplasia.

With a unique mouse mammary tumor model system in which mouse mammary tumor virus (MMTV) insertional mutations can be detected during progression from preneoplasia to frank malignancy, including metastasis, we have discovered a new common integration site (designated Int-6) for MMTV in mouse mammary tumors. MMTV was integrated into Int-6 in a mammary hyperplastic outgrowth line, its tumors and metastases, and two independent mammary tumors arising in unrelated mice. The Int-6 gene is ubiquitously expressed as a 1.4-kb RNA species in adult tissues and is detected beginning at day 8 of embryonic development. The nucleotide sequence of Int-6 is unrelated to any of the known genes in the GenBank database. MMTV integrates within introns of the gene in the opposite transcriptional orientation. In each tumor tested, this results in the expression of a truncated Int-6/long terminal repeat (LTR) chimeric RNA species which is terminated at a cryptic termination signal in the MMTV LTR. Since the nonrearranged Int-6 alleles in these tumors contain no mutations, we favor the conclusion that truncation of the Int-6 gene product either biologically activates its function or represents a dominant-negative mutation.

Cloning and characterization of the mouse gene encoding mammary-derived growth inhibitor/heart-fatty acid-binding protein.

From a mouse genomic library we isolated and characterized a gene, Fabph1, encoding mammary-derived growth inhibitor (MDGI)/heart fatty-acid-binding protein (H-FABP). Exon sequences were identical with a MDGI-encoding cDNA isolated previously from the mammary gland of pregnant mice. The product of this gene has also been detected in heart, where it had been termed H-FABP. It has an intron/exon structure similar to other FABP-encoding genes. In addition to this expressed gene, we isolated a related intronless pseudogene, Fabph-ps, with an open reading frame which was highly conserved when compared with Fabph1. Fabph1 was positioned on chromosome (Chr) 4 using interrelated sequence locus, Fabph-rs1, to Chr 8. A Mus spretus-specific related sequence, Fabph-rs2, was identified on Chr 17 by analysis of interspecies crosses. The 5'-flanking region of Fabph1 contains putative transcription factor-binding elements which could account for its constitutive expression in muscle tissue, as well as for its developmental stage-dependent expression in mammary epithelium.

Mouse mammary tumor gene int-3: a member of the notch gene family transforms mammary epithelial cells.

Expression of a 2.3-kb RNA species is induced in mammary tumors as a consequence of insertional mutagenesis at the int-3 locus by the mouse mammary tumor virus. The nucleotide sequence and biological activity of this mammary tumor-specific int-3 RNA species were determined. It contains an open reading frame which encodes a 57-kDa protein. The translated protein possesses six nearly contiguous 32-amino-acid repeats which are related to a similar motif in the Saccharomyces cerevisiae cdc-10-encoded cell cycle protein. In addition, the int-3 cdc-10 repeats are bounded by the PEST amino acid sequence motif which is commonly found in proteins having a rapid turnover and may represent sites for phosphorylation. The int-3 cdc-10 repeat sequences are 50% identical to a portion of the intracellular domain of the neurogenic Drosophila notch gene product. Activation of expression of a recombinant int-3 genomic DNA fragment encoding the 2.3-kb RNA species in HC11 mouse mammary epithelial cells in vitro induces anchorage-independent growth in soft agar.

Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.

Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV). Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6. To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences. All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed. Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age. Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice. The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated. In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.

Long-term in vivo expression of genes introduced by retrovirus-mediated transfer into mammary epithelial cells.

Nonimmortalized mouse mammary epithelial cells expressing Escherichia coli beta-galactosidase from a murine amphotropic packaged retroviral vector were injected into the epithelium-divested mammary fat pads of syngeneic mice. Mammary glands formed from the injected mammary epithelial cells contained ductal and lobular cells, both of which expressed beta-galactosidase when examined in situ more than 12 months later. These results indicate that stable recombinant gene expression can be achieved in vivo in the mammary gland without altering the growth properties of normal mammary epithelium.