Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity.

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP185₋206. However, ex-vivo T cell proliferation to PLP185₋206 peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.

Genomic instability in induced stem cells.

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.

B cell recruitment and selection in mouse GALT germinal centers.

In conventionally reared mice germinal centers (GCs) are chronically induced in Peyer's patches (PP), mesenteric lymph node (MLN), and isolated lymphoid follicles (ILF) of gut-associated lymphoid tissues (GALT), as a result of continuous B cell stimulation by commensal bacteria. It is generally thought that BCR-mediated antigen recognition controls the recruitment and thus selection of B cells within GALT GCs. However, recent results challenge this view and suggest that engagement of innate immune receptors by microbial antigens promotes B cell recruitment to, and maintenance within, the GC, irrespective of BCR specificity. We propose a scenario in which microbial determinants presented by follicular dendritic cells (FDCs) to innate receptors on B cells within the GC support the survival and concomitant expansion of somatically mutated, IgA-class-switched B cell clones expressing a variety of BCR specificities. From this pool, B cell mutants recognizing gut-derived antigens through their BCR are either, in GCs, drawn into the process of affinity maturation, or, in the lamina propria (LP) of the gut, locally selected to differentiate into plasmablasts, thus contributing to the continuous production of IgA antibodies required for an efficient protection against commensal and pathogenic microorganisms.

Relaxation of insulin-like growth factor 2 imprinting and discordant methylation at KvDMR1 in two first cousins affected by Beckwith-Wiedemann and Klippel-Trenaunay-Weber syndromes.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

The H19 endodermal enhancer is required for Igf2 activation and tumor formation in experimental liver carcinogenesis.

The expression of the linked but reciprocally imprinted Igf2 and H19 genes is activated in adult liver in the course of tumor development. By in situ hybridization analysis we have shown that both the Igf2 and H19 RNAs are expressed in the majority of the neoplastic nodules, and that hepatocellular carcinomas are developed in an experimental model of liver carcinogenesis. H19 is also highly activated in smaller and less distinct hyperplastic regions. The few neoplastic areas showing Igf2 but no H19 RNA display loss of the maternally inherited allele at the Igf2/H19 locus. These data are compatible with the existence of a common activation mechanism of these two genes during liver carcinogenesis and with a stronger H19 induction in the pre-neoplastic lesions. By using mice carrying a deletion of the H19 endodermal enhancer, we show that this regulatory element is necessary for the activation of the Igf2 and H19 genes upon induction of liver carcinogenesis. Furthermore, multiple sites of the H19 endodermal enhancer region become hypersensitive to DNase I when the carcinogenesis process is induced. Lastly, liver tumors developed in mice paternally inheriting the H19 enhancer deletion are found to have marked growth delays, increased frequency of apoptotic nuclei, and lack of Igf2 mRNA expression, thus indicating that this regulatory element plays a major role in the progression of liver carcinogenesis, since it is required for the activation of the anti-apoptotic Igf2 gene.

Expression and parental imprinting of the H19 gene in human rhabdomyosarcoma.

The expression of Insulin-like Growth Factor 2 (IGF-2) and H19, two genes located on human chromosome 11p15 and provided with cell growth modulating activity, is regulated by parental imprinting, in that the activity of their alleles is dependent on the parental origin. Parental bias in the genetic alterations of chromosome 11p15 observed in several pediatric cancers suggests the involvement of imprinted genes in tumor development. We have previously reported that the number of functional IGF-2 alleles is frequently increased in rhabdomyosarcoma (RMS), as a consequence of either relaxation of imprinting (LOI) or gene duplication. Here we show that the expression of the H19 gene is significantly suppressed with respect to normal muscle tissue in 13 out of 15 rhabdomyosarcomas with embryonal histology (ERMS) and in three out of 11 rhabdomyosarcomas classified as alveolar subtype (ARMS). Since a growth-inhibitory activity has been found associated with the H19 gene, the extinction of its expression can contribute to RMS development. Parental imprinting of the H19 gene was found conserved in all informative RMSs, including those whose ICF-2 imprinting was relaxed, indicating that LOI is a gene-specific event. Seven ERMSs and one ARMS displaying low H19 RNA levels showed an underrepresentation of the expressed allele in their genotype. This result is consistent with the paternal imprinting of the H19 gene and with the preferential loss of the maternal 11p15 alleles in these neoplasms. Low H19 expression was also found in four out of eight RMSs retaining the heterozygosity at 11p15, but showing IGF-2 LOI. These findings suggest that the genetic and epigenetic alterations affecting chromosome 11p15 in a high number of RMSs cause deregulation of more than one imprinted gene, possibly affecting tumor growth, including the extinction of H19 expression and an increase in the number of active IGF-2 alleles.

Relaxation of insulin-like growth factor-2 imprinting in rat cultured cells.

The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed state of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived 'post-confluent' cultures. The heritable changes in the expression, and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

Constitutive insulin-like growth factor-II expression interferes with the enterocyte-like differentiation of CaCo-2 cells.

In this study we have examined the role of insulin-like growth factor-II (IGF-II) in the differentiation of the CaCo-2 human colon carcinoma cell line. We have shown previously that IGF-II is an autocrine growth factor for CaCo-2 cells. IGF-II expression is high in proliferating, undifferentiated CaCo-2 cells and markedly decreases when cells become confluent and start to differentiate. To evaluate whether differentiation of CaCo-2 cells depends on an IGF-II related pathway, we treated cells with a blocking antibody to the IGF-I receptor that mediates most IGF-II biological effects. Treatment of preconfluent CaCo-2 cells with this antibody decreased by 40% autonomous cell proliferation and induced differentiation as shown by an increase in sucrase isomaltase activity and apolipoprotein A-I (apoA-I) mRNA levels. To examine the significance of autocrine IGF-II production in CaCo-2 cell differentiation, we generated stable CaCo-2 cell lines that constitutively express rat IGF-II under the control of a Rous sarcoma virus promoter. Sustained expression of IGF-II resulted in: (a) increased proliferative rate; (b) high IGF-I receptor number, even after reaching confluence; (c) increased capability of anchorage-independent growth; (d) inhibition of the expression of apoA-I and SI mRNAs. Analysis of several independent IGF-II-transfected clones showed an inverse correlation between IGF-II mRNA levels and expression of the differentiation markers, the cells expressing the higher levels of the transfected IGF-II being the less differentiated ones. Our data suggest that perturbation of IGF-II-mediated cell proliferation interferes with the enterocyte-like differentiation pathway of CaCo-2 cells.

Loss of heterozygosity of imprinted genes in SV40 t/T antigen-induced hepatocellular carcinomas.

Expression of the chromosomally linked Insulin-like Growth Factor II (IGF-II) and H19 genes is regulated by parental imprinting during development, since the maternally inherited IGF-II and the paternally inherited H19 alleles are inactive in fetal tissues. Here we show that expression of IGF-II and H19 genes is activated in transgenic mice during SV40 Tag-induced hepatocarcinogenesis and that imprinting of both genes is conserved in the liver tumors. Allelic imbalances of IGF-II and H19 genes and other chromosome 7 markers were detected in one third (13/39) of the hepatocellular carcinomas analysed. A strong bias on the allele retained in the neoplasms was observed, since underrepresentation or complete loss of maternal chromosome 7 was recognised in 12/13 cases. High levels of IGF-II mRNA were expressed by all carcinomas with relative excess of paternal chromosome 7 alleles and suppressed H19 expression was found in the neoplasms lacking the maternal alleles. Overall the results indicate that expression of imprinted genes is involved in progression of experimental liver tumors and suggest that the murine chromosome 7, whose loss may possibly cause the inactivation of a growth-inhibitory gene, is preferentially retained as paternal copy in the liver tumors because of parental imprinting of IGF-II gene.

Expression of insulin-like growth factor (IGF)-II and IGF-I receptor during proliferation and differentiation of CaCo-2 human colon carcinoma cells.

We have studied the expression of insulin-like growth factor type II (IGF-II) and its autocrine role during the proliferation and differentiation of the CaCo-2 colon carcinoma cell line. IGF-II RNA levels were high in proliferating cells and decreased by more than 10-fold when cells ceased to proliferate and differentiated. Immunoreactive IGF-II protein was high in the conditioned media of proliferating cells and decreased 20-fold in the media of differentiated cells. Reduced IGF-II expression was associated with a decrease in IGF-I receptor number that was high in proliferating cells (approximately 80,000 binding sites/cell) and reduced by 4-fold in differentiated cells. Exogenously added IGF-II was able to stimulate proliferation of serum-deprived cells in a dose-dependent fashion. IGF-II acted through the IGF-I receptor, since both basal and IGF-II-stimulated cell proliferation was inhibited by the monoclonal antibody alpha-IR3, which blocks the binding sites of the IGF-I receptor. The inhibition of CaCo-2 basal cell growth by the alpha-IR3 antibody suggests that IGF-II may act as an autocrine growth factor for these cells.

Three clustered Sp1 sites are required for efficient transcription of the TATA-less promoter of the gene for insulin-like growth factor-binding protein-2 from the rat.

Insulin-like growth factor-binding protein-2 (IGF-BP-2) transcription in rat liver varies with developmental age and fasting. To define the DNA elements required for efficient expression of the TATA-less rat IGFBP-2 gene, the native or mutated promoter was fused to a promoterless luciferase reporter gene and transfected into BRL-3A rat liver and 293 human embryonic kidney cell lines. Luciferase activity decreased approximately 25-fold with progressive 5' promoter deletions from nucleotide (nt) -581 to nt -189 (relative to ATG, +1). The smallest construct, however, still had > 21-fold greater luciferase activity than the promoterless construct. In DNase I foot-printing assays using native nt -276 to -36 promoter fragments or fragments containing block substitution mutations, BRL-3A nuclear extract and purified human transcription factor Sp1 protected a region from nt -234 to -215 containing one GC box and a broad region from nt -189 to -125 that contained three clustered but independent GC boxes. In gel retardation assays using an Sp1 oligonucleotide probe, BRL-3A extract formed two closely migrating complexes that were immunologically related to Sp1; Sp1 gave a single complex that co-migrated with the more retarded BRL-3A complex. Binding was competitively inhibited by oligonucleotides corresponding to each of the four GC boxes. The proximal three GC boxes were sufficient to allow trans-activation of the IGFBP-2 promoter by Sp1 in Drosophila SL2 cells. Independent block mutations indicated that all three of the GC boxes are required for promoter activity and are equally important. Thus, binding of Sp1 or Sp1-related proteins to three clustered GC boxes in the proximal IGFBP-2 promoter is essential for promoter activity. Multiple upstream regions also contribute to the full expression of the IGFBP-2 gene.

Extinction of insulin-like growth factor II gene expression in intratypic hybrids of rat liver cells.

The expression of the developmentally regulated insulin-like growth factor II (IGF-II) gene has been studied in somatic cell hybrids derived from rat liver cells. BRL3A cells, dedifferentiated variants of rat hepatocytes, producing high levels of IGF-II, were fused to BRL30E or FAO cells of the same embryonic lineage but not expressing detectable levels of IGF-II mRNA. We report here that the IGF-II gene is subject to extinction, since its specific RNA levels are decreased both in heterokaryons and stable cell hybrids. Transcriptional analysis in isolated nuclei from parental and hybrid cells showed that the IGF-II gene is transcribed at a similar rate in all cell types. Likewise, the stability of IGF-II cytoplasmic mRNA was equivalent in the high-expressing parental cells and in the hybrids. In contrast, the distribution of IGF-II mRNA between the nuclear and the cytoplasmic compartments differed markedly in parental and hybrid cell lines. The data presented show that the expression of the IGF-II gene is subject to a dominant negative control and suggest that the phenomenon involves mechanisms that operate at the posttranscriptional level.