Activation of clones producing self-reactive antibodies by foreign antigen and antiidiotype antibody carrying the internal image of the antigen.

Because we found in previous work that a high fraction of antibodies exhibiting various specificities bound to glutamic acid 50-tyrosine50 homopolymer (GT) and expressed pGAT cross-reactive idiotype (IdX), we studied the activation of clones producing multireactive antibodies in 1-mo-old MRL/lpr and C3H/HeJ mice bearing VHJ haplotype. The activation of such clones was studied after mice were immunized with GT in CFA, HP20 (an anti-Id MAb carrying the internal image of GT in the D region), and a synthetic peptide corresponding to the D segment of HP20. Our results indicate that immunized mice produced both GT- and self-reactive antibodies. Study of the immunochemical properties of MAb showed that they exhibit multispecific properties and bind with similar-affinity constants to GT or self-antigens such as DNA, Smith antigen (Sm), and IgG2a. An important fraction of antibodies obtained from MRL/lpr mice immunized with HP20 expressed pGAT IdX and some of these antibodies share IdX expressed on anti-DNA, Sm, and rheumatoid factor (RFs) antibodies. The hybridomas producing multispecific autoantibodies use heavy-chain- (VH) and light-chain-variable region (VK) genes from various V gene families, suggesting that they do not derive from the pool of GAT precursors. Sequencing of VH and VK genes of two antibodies show that they can use closely related VHJ558, unmutated VK1, or different VK genes than those used by anti-GT antibodies. Our data demonstrate that clones producing antibodies binding to GT and self-antigens with similar-affinity constants can be activated by foreign or anti-Id antibodies carrying the internal image of the antigen or even by a synthetic peptide corresponding to the D segment of anti-Id antibodies.

Critical role of the HMGI(Y) proteins in adipocytic cell growth and differentiation.

The high-mobility group I (HMGI) nonhistone chromosomal proteins HMGI(Y) and HMGI-C have been implicated in defining chromatin structure and in regulating the transcription of several genes. These proteins have been implicated in adipocyte homeostasis: a severe deficiency of fat tissue is found in mice with targeted disruption of the HMGI-C locus, and lipomagenesis in humans is frequently associated with somatic mutations of HMGI genes. The aim of this study was to examine the role of HMGI(Y) proteins in adipocytic cell growth and differentiation. First, we found that differentiation of the preadipocytic 3T3-L1 cell line caused early induction of HMGI(Y) gene expression. Suppression of HMGI(Y) expression by antisense technology dramatically increased the growth rate and impaired adipocytic differentiation in these cells. The process of adipogenic differentiation involves the interplay of several transcription factors, among which is the CCAAT/enhancer-binding protein (C/EBP) family of proteins. These factors are required for the transcriptional activation of adipocyte-specific genes. We also tested the hypothesis that HMGI(Y) might participate in transcriptional control of adipocyte-specific promoters. We found that HMGI(Y) proteins bind C/EBPbeta in vivo and in vitro. Furthermore, we show that HMGI(Y) strongly potentiates the capacity of C/EBPbeta to transactivate the leptin promoter, an adipose-specific promoter. Taken together, these results indicate that the HMGI(Y) proteins play a critical role in adipocytic cell growth and differentiation.

Double knockout of the ALL-1 gene blocks hematopoietic differentiation in vitro.

The ALL-1 gene is involved in translocations with many partner genes in different types of the acute leukemias, but it is not clear whether it acts as an oncogene or whether the fusion proteins resulting from the translocations have dominant negative effects. To distinguish between these two possibilities, we analyzed the ability of wild-type AB2.1 embryonal stem (ES) cells and of single or double ALL-1 gene knockout cells derived from them to differentiate along hematopoietic lineages after withdrawal of leukemia inhibitory factor, using in vitro colony formation assays. All-1 double knockout ES cells formed a significantly greater number of colonies with faster kinetics than wild-type and ALL-1 single knockout ES cells. Parental ES cells formed lineage-restricted colonies, whereas single and double knockout ES cells developed, at high frequency, immature and/or "biphenotypic" colonies, mimicking the aberrant hematopoiesis typical of leukemic patients. These data are consistent with the possibility that loss of function of the ALL-1 gene is important in leukemogenesis.

The expression of a truncated HMGI-C gene induces gigantism associated with lipomatosis.

Rearrangements of the HMGI-C gene have frequently been detected in human benign tumors of mesenchymal origin, including lipomas. The HMGI-C protein has three AT-hook domains and an acidic COOH-terminal tail. The HMGI-C modifications consist in the loss of the C-tail and the fusion with ectopic sequences. Recent results show that the loss of the COOH-terminal region, rather than the acquisition of new sequences, is sufficient to confer to HMGI-C the ability to transform NIH3T3 cells. Therefore, transgenic mice carrying a HMGI-C construct (HMGI-C/T), containing only the three AT-hook domains, were generated. The HMGI-C/T mice showed a giant phenotype, together with a predominantly abdominal/pelvic lipomatosis, suggesting a pivotal role of the HMGI-C truncation in the generation of human lipomas.

High level expression of the HMGI (Y) gene during embryonic development.

The HMGI protein family includes three proteins, named HMG-I, HMG-Y and HMGI-C. The first two proteins are coded for by the same gene, HMGI (Y), through an alternative splicing mechanism. Their expression is elevated in neoplastic tissues and cells and this overexpression has a causal role in the process of cellular neoplastic transformation. We demonstrate that the HMGI (Y) gene is expressed at very low levels in normal adult tissues, whereas in embryonic tissues it is expressed at high levels comparable to those detected in neoplastic tissues. Specifically, a very high expression of the HMGI (Y) gene was detected in all embryonic tissues at 8.5 dpc. Then in the following days, even though the gene is expressed essentially in all tissues, an abundant gene expression was restricted to some tissues. These results indicate an important role of the HMGI (Y) gene in development.

Altered gene expression in immunogenic poorly differentiated thyroid carcinomas from RET/PTC3p53-/- mice.

Cancers develop and progress via activation of oncogenes and loss of tumor suppressor genes, a progression that can be recapitulated through cross breeding mouse strains harboring genetic mutations. To define the role of RET/PTC3, p53 and Fhit in thyroid carcinogenesis, we intercrossed RET/PTC3 transgenics with p53-/- mice. This new strain, RET/PTC3p53-/-, succumb to rapidly growing and strikingly large multilobed thyroid tumors containing mixtures of both well and poorly differentiated, highly proliferative follicular epithelial cells. Interestingly, transplanted tumors from RET/PTC3p53-/- mice grew in SCID but not syngeneic immunocompetent mice indicating that these advanced tumors were immunogenic. RET/PTC3 protein expression was reduced to undetectable levels in tumors of older mice suggesting that the continued elevated expression of RET/PTC3 may not be necessary for tumor progression. Similarly, expression of Fhit protein was reduced in early tumors and undetected in older tumors irrespective of tumor histopathology. In contrast to RET/PTC3p53-/- mice, RET/PTC3Fhit-/- mice did not develop advanced thyroid carcinomas. These studies support a model of human thyroid cancer whereby thyroid epithelium expresses RET/PTC3 protein at early stages of tumor development, followed by the reduction of RET/PTC3 and loss of p53 function with progressive reduction of Fhit protein expression coincident with malignant progression.

Molecular analysis of the heterogeneity region of the human ribosomal spacer.

The human ribosomal non-transcribed spacers are 30 X 10(3) base-pairs (or 30 kb) in length with a limited length heterogeneity localized in a specific region downstream from the 3' end of the transcribed region. Total DNA digested with EcoRI and BamHI and hybridized with a probe containing the 3' end of the 28 S ribosomal RNA coding region shows four major bands of 3.9 kb, 4.6 kb, 5.4 kb and 6.2 kb. The 5.4 kb band is the most abundant in every individual, followed by the 4.6 kb band. The longest and the shortest size classes are less well-represented and may even be absent. Every individual shows his own pattern of relative abundance of non-transcribed spacer length classes that can be followed through generations. We decided to investigate the molecular structure of the heterogeneity region, in order to cast light onto the mechanisms underlying the origin and maintenance of this length heterogeneity. Pertinent spacer regions of eight ribosomal clones from two human genomic libraries were subcloned and analyzed by restriction mapping and nucleotide sequencing. In the minimal length class, there is a sequence of 700 base-pairs that appears to be tandemly duplicated once, twice or three times in the other length classes. This repeated DNA module contains a region consisting of repetitions of simple pyrimidine groups like C-T, C-T-T-T or C-C-C-T. DNA module repeats may differ by the length of this pyrimidine-rich region. However, these length variations are not continuous, as revealed by Southern transfer analysis of several individuals and different cloned gene units: instead, the repeated modules fall into two discrete length classes of about 700 base-pairs and 800 base-pairs. An imperfect duplication of a short sequence of 86/89 base-pairs is present at the boundary between the heterogeneity region and the upstream flanking region, representing a very ancient duplication event.

A genomic clone containing the promoter for the gene encoding the human lysosomal enzyme, alpha-galactosidase A.

We have isolated and characterized a human genomic clone for a lysosomal enzyme gene. The start point of transcription was identified using primer extension of poly(A)+ mRNA. This genomic clone is specific for human alpha-galactosidase A, and it includes sequences for the promoter, complete signal peptide, first exon, and part of the first intron. Direct and inverted repeat elements of 10, 11, 16, 19, and 22 nucleotides (nt) flank the promoter site. A (GA)n repeat element of approx. 60 nt with strong homology to similar elements identified in several species is located upstream from the promoter. A GGGCGG site specific for DNA-binding protein Sp1 is located near a CAAT box, and the CCGCCC inverted repeat of the Sp1 binding sequence is located by the TATA box. The sequence immediately flanking the ATG start codon of the human alpha-galactosidase A is highly homologous to sequences flanking the ATG start codons of the other human lysosomal hydrolases for which sequence information is available (beta-glucocerebrosidase, cathepsin B, cathepsin D, and beta-hexosaminidase alpha chain), but not for any of the other 133 human signal peptides examined. Our analysis also reveals that conversion of the propeptide to the mature enzyme involves cleavage of a C-terminal rather than an N-terminal fragment. This information about the normal alpha-galactosidase A gene will be useful for comparison to data obtained from patients with Fabry disease, who are characterized by a deficiency of this enzyme. This is the first genomic clone described to date for any lysosomal enzyme, and it establishes a reference for future analyses of the molecular events that mediate the expression of this important class of enzymes.

The complete nucleotide sequence of the ilvGMEDA cluster of Escherichia coli K-12.

The ilvGMEDA gene cluster of Escherichia coli K-12 has been the focus of intensive genetic and biochemical analysis for the past 30 years. Genetic regulation of the ilvGMEDA cluster involves attenuation, internal promoters, internal Rho-dependent termination sites, a site of polarity in the ilvG pseudogene of the wild-type organism, and autoregulation by the ilvA gene product, the biosynthetic L-threonine deaminase. We have now completed the nucleotide sequence of the 6600-bp cluster and have analyzed it, along with the ilvYC, ilvBN, and ilvIH genes, for codon frequencies and possible evolutionary relationships. The isoleucine content of each of the gene products of the ilvGMEDA cluster is quite similar (less than a two-fold variation), thus excluding one possible interpretation of the isoleucine-specific downstream amplification phenomenon. There is no evidence for retrograde evolution in the cluster since no significant homologies are detectable among genes that catalyze sequential reactions of the pathway. A highly significant homology does exist, however, between the threonine deaminases of yeast mitochondria and E. coli. The sequence at the boundary of the ilvA and ilvD genes is TAATAATG, so that the second TAA stop codon of ilvD overlaps the ATG initiation codon of ilvA.

The significance of idiotype-anti-idiotype interactions in the activation of self-reactive clones.

Autoantibodies against a wide range of self antigens have been found in the serum of normal mice and healthy humans. It is now accepted that the production of self reactive clones is not an aberration and that the immune system does not purge itself of the previously called "horror" clones. Recently it has been shown that the majority of autoantibodies are polyspecific and able to react with both self and foreign antigens. Following these observations, it was speculated that the activation of self reactive clones may result from common shared epitopes between self and foreign (bacterial, viral and parasitic) antigens. In this report, we show on the one hand that autoantibodies specific for Sm antigen express an idiotype originally found on antibodies specific for bacterial levan. On the other hand, using an anti-idiotypic antibody bearing the internal image of the glutamic-tyrosine polymer, we succeed in inducing polyspecific antibodies reacting with GT and self antigens in both normal and autoimmune disease prone mice. These findings may reflect one side of the mechanism(s) behind the activation of self reactive clones. The significance of idiotype-anti-idiotype interactions in the activation of self reactive clones which may be responsible for autoimmune diseases is discussed.

An agarose gel resolving a wide range of DNA fragment lengths.

To resolve DNA fragments ranging from several kilobases to some tens of base pairs in length, an agarose slab gel of steadily increasing thickness has been designed. During electrophoresis a gradient of decreasing electric-field strength is generated throughout the gel from the cathode end to the anode end. Shorter fragments which migrate further are decelerated, resulting in an increased linearity of the relationship between mobility and molecular weight.

Autoantibodies, LY-1, and immunoglobulin V gene expression in hybridomas obtained from young and from old New Zealand black mice.

We obtained a large number of hybridomas from 1-month-old and 16-month-old New Zealand black mice to study the fine specificities of the autoantibodies produced, the expression of Ly-1, and the expression of the immunoglobulin V gene families in this autoimmune strain. Analysis of the autoantibody specificities yielded 2 major classifications: those specific for a single autoantigen and those that exhibited multispecific binding. Among the multispecific antibodies, 2 categories were found: an antigen-inhibitable group and an antigen-noninhibitable group. A large proportion of VHJ558 and VH7183 gene families was observed in hybridomas obtained from 1-month-old mice, and in hybridomas obtained from 16-month-old mice, there was a large proportion of VHJ558 and VH36-60 gene families. Among the autoantibody kappa chains secreted by the hybridomas, there was a higher frequency of the V kappa 1, V kappa 8, and V kappa 9 gene families. Autoantibodies were produced by both the Ly-1+ and the Ly-1- B cell subsets.

Ly1 and V-gene expression among hybridomas secreting natural autoantibody.

This report presents evidence that multispecific natural autoantibodies (NAAb) arise from both Ly-1+ and Ly-1- B lymphocyte subsets based upon detection of mRNA transcript of Ly1 gene by Northern blotting among hybridomas from several mouse strains. Newborn Xid mice also possess B cells transcribing the Ly-1 gene that secrete multispecific NAAbs. Different VH and VK gene families were expressed at random in both Ly-1+ and Ly-1- hybridoma clones secreting NAAbs. Certain VK gene families VK1, -8, -9, -10 and -19, were preferentially used by NAAbs. No particular VH:VK pairings were observed among NAAbs multispecifically reactive with DNA and cytoskeleton proteins. The NAAbs reactive with BrMRBC exhibited predominant pairing between VH11:VK9 gene families, although, for the first time, we noted the participation of VK1, -10 and -19 gene families in encoding this autoantibody specificity.

Expression of Fhit protein during mouse development.

Fhit protein has a putative tumor suppressor function in several types of human and experimental cancers. To assess whether Fhit is involved in fetal development we have examined the distribution of Fhit protein in the 12- through 16-day postcoitum mouse fetus and in postnatal day 0 mouse pups by immunocytochemistry. High levels of Fhit protein were observed in the endodermal derivatives, namely, bronchi, trachea, esophagus, stomach, and intestine, in the 12- to 16-day postcoitum mouse fetus and in the postnatal day 0 pup. Other tissues showed a more restricted pattern of Fhit protein expression. These results suggest that Fhit may play a role in the development of specific tissues during mouse development.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors.

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

Identification and characterization of the ARP1 gene, a target for the human acute leukemia ALL1 gene.

ALL1, the human homologue of Drosophila trithorax, is directly involved in human acute leukemias associated with abnormalities at 11q23. Using the differential display method, we isolated a gene that is down-regulated in All1 double-knockout mouse embryonic stem (ES) cells. The gene, designated ARP1 (also termed RIEG, Ptx2, or Otlx2), is a member of a family of homeotic genes containing a short motif shared with several homeobox genes. Using a bacterially synthesized All1 polypeptide encompassing the AT-hook motifs, we identified a 0.5-kb ARP1 DNA fragment that preferentially bound to the polypeptide. Within this DNA, a region of approximately 100 bp was protected by the polypeptide from digestion with ExoIII and DNase I. Whole-mount in situ hybridization to early mouse embryos of 9.5-10.5 days indicated a complex pattern of Arp1 expression spatially overlapping with the expression of All1. Although the ARP1 gene is expressed strongly in bone marrow cells, no transcripts were detected in six leukemia cell lines with 11q23 translocations. These results suggest that ARP1 is up-regulated by the All1 protein, possibly through direct interaction with an upstream DNA sequence of the former. The results are also consistent with the suggestion that ALL1 chimeric proteins resulting from 11q23 abnormalities act in a dominant negative fashion.

The tumor spectrum in FHIT-deficient mice.

Mice carrying one inactivated Fhit allele (Fhit +/- mice) are highly susceptible to tumor induction by N-nitrosomethylbenzylamine, with 100% of Fhit +/- mice exhibiting tumors of the forestomach/squamocolumnar junction vs. 25% of Fhit +/+ controls. In the current study a single N-nitrosomethylbenzylamine dose was administered to Fhit +/+, +/-, and -/- mice to compare carcinogen susceptibility in +/- and -/- Fhit-deficient mice. At 29 weeks after treatment, 7.7% of wild-type mice had tumors. Of the Fhit -/- mice 89.5% exhibited tumors (average 3.3 tumors/mouse) of the forestomach and squamocolumnar junction; half of the -/- mice had medium (2 mm diameter) to large (>2 mm) tumors. Of the Fhit +/- mice 78% exhibited tumors (average 2.4 tumors/mouse) and 22% showed medium to large tumors. Untreated Fhit-deficient mice have been observed for up to 2 years for spontaneous tumors. Fhit +/- mice (average age 21 mo) exhibit an average of 0.94 tumors of different types; Fhit -/- mice (average age 16 mo) also showed an array of tumors (average 0.76 tumor/mouse). The similar spontaneous and induced tumor spectra observed in mice with one or both Fhit alleles inactivated suggests that Fhit may be a one-hit tumor suppressor gene in some tissues.

Muir-Torre-like syndrome in Fhit-deficient mice.

To investigate the role of the Fhit gene in carcinogen induction of neoplasia, we have inactivated one Fhit allele in mouse embryonic stem cells and produced (129/SvJ x C57BL/6J) F(1) mice with a Fhit allele inactivated (+/-). Fhit +/+ and +/- mice were treated intragastrically with nitrosomethylbenzylamine and observed for 10 wk posttreatment. A total of 25% of the +/+ mice developed adenoma or papilloma of the forestomach, whereas 100% of the +/- mice developed multiple tumors that were a mixture of adenomas, squamous papillomas, invasive carcinomas of the forestomach, as well as tumors of sebaceous glands. The visceral and sebaceous tumors, which lacked Fhit protein, were similar to those characteristic of Muir-Torre familial cancer syndrome.

FHIT gene therapy prevents tumor development in Fhit-deficient mice.

The tumor suppressor gene FHIT spans a common fragile site and is highly susceptible to environmental carcinogens. FHIT inactivation and loss of expression is found in a large fraction of premaligant and malignant lesions. In this study, we were able to inhibit tumor development by oral gene transfer, using adenoviral or adenoassociated viral vectors expressing the human FHIT gene, in heterozygous Fhit(+/-) knockout mice, that are prone to tumor development after carcinogen exposure. We therefore suggest that FHIT gene therapy could be a novel clinical approach not only in treatment of early stages of cancer, but also in prevention of human cancer.