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Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL., (© 2023 by The American Society of Hematology.)

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Adult neurogenesis occurs throughout life in the dentate gyrus (DG) and the subventricular zone (SVZ), where glia-like stem cells generate new neurons. Voluntary running is a powerful neurogenic stimulus triggering the proliferation of progenitor cells in the DG but, apparently, not in the SVZ. The antiproliferative gene Btg1 maintains the quiescence of DG and SVZ stem cells. Its ablation causes intense proliferation of DG and SVZ stem/progenitor cells in young mice, followed, during adulthood, by progressive decrease of the proliferative capacity. We have previously observed that running can rescue the deficit of DG Btg1-null neurogenesis. Here, we show that in adult Btg1-null SVZ stem and neuroblast cells, the reduction of proliferation is associated with a longer cell cycle and a more frequent entry into quiescence. Notably, running increases proliferation in Btg1-null SVZ stem cells highly above the levels of sedentary wild-type mice and restores normal values of cell cycle length and quiescence in stem and neuroblast cells, without affecting wild-type cells. Btg1-null SVZ neuroblasts show also increased migration throughout the rostral migratory stream and a deficiency of differentiated neurons in the olfactory bulb, possibly a consequence of premature exit from the cycle; running, however, normalizes migration and differentiation, increasing newborn neurons recruited to the olfactory circuitry. Furthermore, running increases the self-renewal of Btg1-null SVZ-derived neurospheres and, remarkably, in aged Btg1-null mice almost doubles the proliferating SVZ stem cells. Altogether, this reveals that SVZ stem cells are endowed with a hidden supply of self-renewal capacity, coupled to cell cycle acceleration and emerging after ablation of the quiescence-maintaining Btg1 gene and following exercise.

Physical exercise increases the generation of new neurons in adult neurogenesis. However, only few studies have investigated the beneficial effects of physical exercise in paradigms of impaired neurogenesis. Here, we demonstrate that running fully reverses the deficient adult neurogenesis within the hippocampus and subventricular zone of the lateral ventricle, observed in mice lacking the antiproliferative gene Btg1. We also evaluated for the first time how running influences the cell cycle kinetics of stem and precursor subpopulations of wild-type and Btg1-null mice, using a new method to determine the cell cycle length. Our data show that in wild-type mice running leads to a cell cycle shortening only of NeuroD1-positive progenitor cells. In contrast, in Btg1-null mice, physical exercise fully reactivates the defective hippocampal neurogenesis, by shortening the S-phase length and the overall cell cycle duration of both neural stem (glial fibrillary acidic protein(+) and Sox2(+)) and progenitor (NeuroD1(+)) cells. These events are sufficient and necessary to reactivate the hyperproliferation observed in Btg1-null early-postnatal mice and to expand the pool of adult neural stem and progenitor cells. Such a sustained increase of cell proliferation in Btg1-null mice after running provides a long-lasting increment of proliferation, differentiation, and production of newborn neurons, which rescues the impaired pattern separation previously identified in Btg1-null mice. This study shows that running positively affects the cell cycle kinetics of specific subpopulations of newly generated neurons and suggests that the plasticity of neural stem cells without cell cycle inhibitory control is reactivated by running, with implications for the long-term modulation of neurogenesis., (© 2014 AlphaMed Press.)

The four related mammalian MEX-3 RNA-binding proteins are evolutionarily conserved molecules for which the in vivo functions have not yet been fully characterized. Here, we report that male mice deficient for the gene encoding Mex3b are subfertile. Seminiferous tubules of Mex3b-deficient mice are obstructed as a consequence of the disrupted phagocytic capacity of somatic Sertoli cells. In addition, both the formation and the integrity of the blood-testis barrier are compromised owing to mislocalization of N-cadherin and connexin 43 at the surface of Sertoli cells. We further establish that Mex3b acts to regulate the cortical level of activated Rap1, a small G protein controlling phagocytosis and cell-cell interaction, through the activation and transport of Rap1GAP. The active form of Rap1 (Rap1-GTP) is abnormally increased at the membrane cortex and chemically restoring Rap1-GTP to physiological levels rescues the phagocytic and adhesion abilities of Sertoli cells. Overall, these findings implicate Mex3b in the spatial organization of the Rap1 pathway that orchestrates Sertoli cell functions.

The homeobox transcription factor CDX2 plays a crucial role in intestinal cell fate specification, both during normal development and in tumorigenic processes involving intestinal reprogramming. The CDX2 regulatory network is intricate, but it has not yet been fully uncovered. Through genome-wide screening of a 3D culture system, the RNA-binding protein MEX3A was identified as putatively involved in CDX2 regulation; therefore, its biological relevance was addressed by setting up cell-based assays together with expression studies in murine intestine. We demonstrate here that MEX3A has a repressive function by controlling CDX2 levels in gastric and colorectal cellular models. This is dependent on the interaction with a specific binding determinant present in CDX2 mRNA 3'untranslated region. We have further determined that MEX3A impairs intestinal differentiation and cellular polarization, affects cell cycle progression and promotes increased expression of intestinal stem cell markers, namely LGR5, BMI1 and MSI1. Finally, we show that MEX3A is expressed in mouse intestine, supporting an in vivo context for interaction with CDX2 and modulation of stem cell properties. Therefore, we describe a novel CDX2 post-transcriptional regulatory mechanism, through the RNA-binding protein MEX3A, with a major impact in intestinal differentiation, polarity and stemness, likely contributing to intestinal homeostasis and carcinogenesis.

We have studied the regulation of ATAD2 gene expression by androgens in prostate cells. ATAD2 is a coactivator of the androgen receptor (AR) and the MYC protein. We showed that ATAD2 expression is directly regulated by AR via an AR binding sequence (ARBS) located in the distal enhancer of its regulatory region. The gene is also regulated by the E2F1 transcription factor. Using knockdown and chromatin immunoprecipitation technique approaches, we could demonstrate that AR and E2F1 functionally collaborate and physically interact between each other. From the analysis of chromatin conformation, we conclude that this cooperation results from a chromatin looping over the ATAD2 promoter region between the ARBS and E2F1 binding site in an androgen-dependent manner. Furthermore, we could show that several genes overexpressed in prostate cancer and potentially involved in several aspects of tumor development have an ARBS and an E2F1 binding site in their regulatory regions and exhibit the same mechanism of regulation by both transcription factors as ATAD2.

Btg1 belongs to a family of cell cycle inhibitory genes. We observed that Btg1 is highly expressed in adult neurogenic niches, i.e., the dentate gyrus and subventricular zone (SVZ). Thus, we generated Btg1 knockout mice to analyze the role of Btg1 in the process of generation of adult new neurons. Ablation of Btg1 causes a transient increase of the proliferating dentate gyrus stem and progenitor cells at post-natal day 7; however, at 2 months of age the number of these proliferating cells, as well as of mature neurons, greatly decreases compared to wild-type controls. Remarkably, adult dentate gyrus stem and progenitor cells of Btg1-null mice exit the cell cycle after completing the S phase, express p53 and p21 at high levels and undergo apoptosis within 5 days. In the SVZ of adult (two-month-old) Btg1-null mice we observed an equivalent decrease, associated to apoptosis, of stem cells, neuroblasts, and neurons; furthermore, neurospheres derived from SVZ stem cells showed an age-dependent decrease of the self-renewal and expansion capacity. We conclude that ablation of Btg1 reduces the pool of dividing adult stem and progenitor cells in the dentate gyrus and SVZ by decreasing their proliferative capacity and inducing apoptosis, probably reflecting impairment of the control of the cell cycle transition from G1 to S phase. As a result, the ability of Btg1-null mice to discriminate among overlapping contextual memories was affected. Btg1 appears, therefore, to be required for maintaining adult stem and progenitor cells quiescence and self-renewal.

Spermatogenesis is a cyclic process in which diploid spermatogonia differentiate into haploid spermatozoa. This process is highly regulated, notably at the post-transcriptional level. MicroRNAs (miRNAs), single-stranded noncoding RNA molecules of about 20-25 nucleotides, are implicated in the regulation of many important biological pathways such as proliferation, apoptosis, and differentiation. We wondered whether miRNAs could play a role during spermatogenesis. The miRNA expression repertoire was tested in germ cells, and we present data showing that miR-34c was highly expressed only in these cells. Furthermore, our findings indicate that in male gonads, miR-34c expression is largely p53 independent in contrast to previous results showing a direct link in somatic cells between the miR-34 family and this tumor suppressor protein. In order to identify target genes involved in germinal lineage differentiation, we overexpressed miR-34c in HeLa cells, analyzed the transcriptome of these modified cells, and noticed a shift of the expression profile toward the germinal lineage. Recently, it has been shown that exogenous expression of Ddx4/Vasa in embryonic chicken stem cells (cESC) induces cESC reprogramming toward a germ cell fate. When we simultaneously expressed miR-34c in such cells, we could detect an up-regulation of germ cell-specific genes whereas the expression of other lineage specific markers remained unchanged. These data suggest that miR-34c could play a role by enhancing the germinal phenotype of cells already committed to this lineage.

Telomerase is essential for telomere maintenance, and its activation is thought to be a critical step in cellular immortalization and tumorigenesis. Human telomerase reverse transcriptase (hTERT) is a major component of telomerase activity. We show here that hTERT is expressed soon after lymphocyte activation and that its expression is inhibited by rapamycin, wortmannin, and FK506, which was the most potent inhibitor. These results suggest a potential role for the transcription factor nuclear factor of activated T cells (NFAT) in the regulation of hTERT expression. Five putative NFAT-binding sites were identified in the hTERT promoter. In luciferase assays, the hTERT promoter was activated by overexpressed NFAT1. Moreover, serial deletions revealed that the promoter activation was mainly due to a -40 NFAT1-binding site flanked by two SP1-binding sites. Mutation of the -40 NFAT-binding site caused a 53% reduction in the transcriptional activity of hTERT promoter. Simultaneous mutations of the -40 NFAT-responsive element together with one or both SP1-binding sites led to a more dramatic decrease in luciferase activity than single mutations, suggesting a functional synergy between NFAT1 and SP1 in hTERT transcriptional regulation. NFAT1 overexpression in MCF7 and Jurkat cell lines induced an increase in endogenous hTERT mRNA expression. Inversely, its down-regulation was induced by NFAT1 silencing. Furthermore, chromatin immunoprecipitation assay demonstrated that NFAT1 directly binds to two sites (-40 and -775) in the endogenous hTERT promoter. Thus, we show for the first time the direct involvement of NFAT1 in the transcriptional regulation of hTERT.

miR-16, a miRNA involved in cell proliferation and apoptosis regulation, may interfere with either oncogenic or tumor-suppressor pathways and is implicated in leukemogenesis. We then explored its expression in 93 childhood acute lymphoblastic leukemia (ALL) cases. A high miR-16 expression was associated with hyperleukocytosis and poor cytogenetic groups. In the whole group and in B-cell ALLs, disease-free survival (DFS) was significantly shorter for miR-16 above quartile 75. In T-cell ALLs, for both DFS and overall survival, a significant trend was found with a survival shortening from the lowest to the highest miR-16 levels. miR-16 expression neither significantly correlated with normal and malignant lymphocyte proliferation nor varied according to lymphocyte differentiation. The prognostic value of miR-16 in childhood ALL highlighted the complexity of miR-16 functions.

Background Information: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16., Results: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels., Conclusions: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.

Although the correlation between beta3-tubulin expression level in tumors has been well correlated with clinical outcome in patients receiving microtubule-targeted agents, the regulation of this protein remains poorly understood. Recently, new insight of regulatory processes was offered with the cloning of the gene promoter. In this study beta3-tubulin gene expression was induced in response to exposure to various antimicrotubule agents such as vinorelbine and paclitaxel. The exploration of the beta3-tubulin gene promoter by successive deletions followed by site-directed mutagenesis led to the localization of a vinorelbine-responsive element containing an activator-protein 1 (AP-1) site. Among the various antimicrotubule agents tested, it appeared that the implicated AP-1 site was activated only after exposure to vinorelbine. This study confirms the inducible nature of the beta3-tubulin gene promoter.

The btg1 (B-cell translocation gene 1) gene coding sequence was isolated from a translocation break point in a case of B-cell chronic lymphocytic leukaemia. We have already shown that BTG1, considered as an antiproliferative protein, strongly stimulates myoblast differentiation. However, the mechanisms involved in this influence remained unknown. In cultured myoblasts, we found that BTG1 stimulates the transcriptional activity of nuclear receptors (T3 and all-trans retinoic acid receptors but not RXRalpha and PPARgamma), c-Jun and myogenic factors (CMD1, Myf5, myogenin). Immunoprecipitation experiments performed in cells or using in vitro-synthesized proteins and GST pull-down assays established that BTG1 directly interacts with T3 and all-trans retinoic acid receptors and with avian MyoD (CMD1). These interactions are mediated by the transactivation domain of each transcription factor and the A box and C-terminal part of BTG1. NCoR presence induces the ligand dependency of the interaction with nuclear receptors. Lastly, deletion of BTG1 interacting domains abrogates its ability to stimulate nuclear receptors and CMD1 activity, and its myogenic influence. In conclusion, BTG1 is a novel important coactivator involved in the regulation of myoblast differentiation. It not only stimulates the activity of myogenic factors, but also of nuclear receptors already known as positive myogenic regulators.

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.

The CCR4-associated protein CAF1 has been demonstrated to play several roles in the control of transcription and of mRNA decay. To gain further insight into its physiological function, we generated CAF1-deficient mice. They are viable, healthy, and normal in appearance; however, mCAF1(-/-) male mice are sterile. The crossing of mCAF1(+/-) mice gave a Mendelian ratio of mCAF1(+/+), mCAF1(+/-), and mCAF1(-/-) pups, indicating that haploid mCAF1-deficient germ cells differentiate normally. The onset of the defect occurs during the first wave of spermatogenesis at 19 to 20 days after birth, during progression of pachytene spermatocytes to haploid spermatids and spermatozoa. Early disruption of spermatogenesis was evidenced by Sertoli cell vacuolization and tubular disorganization. The most mature germ cells were the most severely depleted, but progressively all germ cells were affected, giving Sertoli cell-only tubes, large interstitial spaces, and small testes. This phenotype could be linked to a defect(s) in germ cells and/or to inadequate Sertoli cell function, leading to seminiferous tubule disorganization and finally to a total disappearance of germ cells. The mCAF1-deficient mouse provides a new model of failed spermatogenesis in the adult that may be relevant to some cases of human male sterility.

The 14-3-3 protein was shown to be present into the parasitophorous vacuole of Toxoplasma gondii-infected human monocyte cells and in the excreted/secreted antigens (ESA). The ESA 14-3-3 protein migrates electrophoretically as the cytosol and the main membranous 14-3-3 isoforms. The excretion/secretion of 14-3-3 was not sensitive to cycloheximide, a protein synthesis inhibitor, even at a concentration which inhibited the production of 14-3-3 inside the tachyzoites. Recombinant 14-3-3/GST protein was used to test the presence of 14-3-3 antibodies in different human sera. A positive immunoreactivity was observed with sera corresponding to acute toxoplasmosis and a possible involvement of 14-3-3 in host immunity is discussed.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by the presence of multiple gastrointestinal polyps and an increased risk for various types of cancers. Inactivating germline mutations of the LKB1 gene, which encodes a serine/threonine kinase, are responsible for the majority of PJS cases. Here, we show that the heteromeric complex containing the molecular chaperones Hsp90 and Cdc37/p50 interacts with the kinase domain of LKB1. Treatment of cells with either geldanamycin or novobiocin, two pharmacological inhibitors of Hsp90 causes the destabilization of LKB1. Furthermore, geldanamycin treatment leads to the ubiquitination and the rapid degradation of LKB1 by the proteasome-dependent pathway. In addition, we found that a LKB1 point mutation identified in a sporadic testicular cancer, weakens the interaction of LKB1 with both Hsp90 and Cdc37/p50 and enhances its sensitivity to the destabilizing effect of geldanamycin. Collectively, our results demonstrate that the Hsp90/Cdc37 complex is a major regulator of the stability of the LKB1 tumor suppressor. Furthermore, these data draw attention to the possible adverse consequences of antitumor drugs that target Hsp90, such as antibiotics related to geldanamycin, which could disrupt LKB1 function and promote the development of polyps and carcinomatous lesions.

A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.

Background: Several recent reports have connected protein methylation with differentiation. Furthermore, the BTG/TOB proteins have also been implicated in such control. BTG1 and 2 have been shown to interact with PRMT1 (predominant cellular arginine N-methyltransferase of type I)., Results: First, we have studied the interaction between PRMT1 and the proteins of the BTG/TOB family. We show that boxC, a sequence present only in BTG1 and BTG2, is essential for this association. Using boxC peptide, we have investigated the importance of PRMT1/BTG protein association during type I protein methylation reactions. Finally, we show that the addition of boxC fused to penetratin interferes with the neuronal differentiation of PC12 cells and ES cell-derived neurones., Conclusions: Taken together, these results indicate that PRMT1/BTG proteins could play a key role in the arginine methylation-mediated signalling pathway as well as in neuronal differentiation.

Here we report the specific regulation of gadd45 expression in human melanoma cell lines following UVB radiation. This solar wavelength is likely to be involved in melanoma aetiology. We have previously shown that gadd45 expression is strongly enhanced in a p53-independent manner following UVB irradiation, unlike the other p53 target genes studied. Furthermore, gadd45 is specifically activated in melanocytes since its induction in response to UVB, is not observed in other skin cells such as keratinocytes or fibroblasts. To investigate this particular regulation of gadd45, we analysed the UVB-induced response of different gadd45 promoter regions. Thus, a minimal promoter region of 50 bp length, responsible for gadd45 activation in melanoma cell lines following UVB irradiation, was determined. In electrophoretic mobility shift assays (EMSAs), we showed that this region (-106/-56) of the gadd45 promoter which contains two identical octamers, binds the POU family gene products oct-1 and N-oct3. Given the specific expression pattern of N-oct3 in melanocyte, we invalidated the expression of this transcription factor in melanoma cells: such an abrogation of N-oct3 protein expression in melanoma cells impeded gadd45 UVB-response. Thus the response of melanocyte to UVB may use an original and previously undescribed pathway.

We have previously shown that BTG1 stimulates myoblast differentiation. In addition, this protein displays a major nuclear localization in confluent myoblasts, decreasing during the early steps of differentiation, and is essentially detected in the cytoplasm of mature myotubes. To identify the domains involved in the cellular trafficking of BTG1, we observed the localization of several BTG1 sequences fused to betaGalactosidase. The highly conserved B box among all members of the BTG family induces a significant nuclear localization of the betaGal moiety, enhanced by presence of the BTG1 carboxy-terminal sequence. In addition, a functional Nuclear Export Signal (NES) overlaps the B box. Moreover, presence of the first 43 NH(2)-terminal amino acids reduced the nuclear localization of each chimeric protein tested. Last, the BTG1 amino-terminal domain bears an LxxLL motif favouring nuclear accumulation, and another region encompassing the A box inhibiting nuclear localization. In contrast to a BTG1 mutant exclusively localized in the cytoplasm, transient expression of a mutant displaying a nuclear localization enhanced myoblasts withdrawal from the cell cycle and terminal differentiation, thus mimicking the myogenic influence of BTG1. In conclusion, several regions of BTG1 are implicated in its cellular localization, and BTG1 myogenic activity is induced at the nuclear level.

The product of the B-cell translocation gene 1 (BTG1), a member of an antiproliferative protein family including Tis-21/PC3 and Tob, is thought to play an important role in the regulation of cell cycle progression. We have shown in a previous work that triiodothyronine (T3) stimulates quail myoblast differentiation, partly through a cAMP-dependent mechanism involved in the stimulation of cell cycle withdrawal. Furthermore, we found that T3 or 8-Br-cAMP increases BTG1 nuclear accumulation in confluent myoblast cultures. In this study, we report that BTG1 is essentially expressed at cell confluence and in differentiated myotubes. Whereas neither T3 nor cAMP exerted a direct transcriptional control upon BTG1 expression, we found that AP-1 activity, a crucial target involved in the triiodothyronine myogenic influence, repressed BTG1 expression, thus probably explaining the low BTG1 expression level in proliferating myoblasts. In transient transfection studies, we demonstrated that an AP-1-like sequence located in the BTG1 promoter was involved in this negative regulation. Our present data also bring evidence that the stimulation of BTG1 nuclear accumulation by T3 or 8-Br-cAMP probably results from an increased nuclear import or retention in the nucleus. Lastly, BTG1 overexpression in quail myoblasts mimicked the T3 or 8-Br-cAMP myogenic influence: (i) inhibition of myoblast proliferation due to an increased rate of myoblast withdrawal from the cell cycle; and (ii) stimulation of terminal differentiation. These data suggest that BTG1 is probably involved in T3 and cAMP myogenic influences. In conclusion, BTG1 is a T3 target involved in the regulation of myoblast differentiation., (Copyright 1999 Academic Press.)

The tumour suppressor gene PTEN (also called MMAC1 or TEP1) is somatically mutated in a variety of cancer types [1] [2] [3] [4]. In addition, germline mutation of PTEN is responsible for two dominantly inherited, related cancer syndromes called Cowden disease and Bannayan-Ruvalcaba-Riley syndrome [4]. PTEN encodes a dual-specificity phosphatase that inhibits cell spreading and migration partly by inhibiting integrin-mediated signalling [5] [6] [7]. Furthermore, PTEN regulates the levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3) by specifically dephosphorylating position 3 on the inositol ring [8]. We report here that the dauer formation gene daf-18 is the Caenorhabditis elegans homologue of PTEN. DAF-18 is a component of the insulin-like signalling pathway controlling entry into diapause and adult longevity that is regulated by the DAF-2 receptor tyrosine kinase and the AGE-1 PI 3-kinase [9]. Others have shown that mutation of daf-18 suppresses the life extension and constitutive dauer formation associated with daf-2 or age-1 mutants. Similarly, we show that inactivation of daf-18 by RNA-mediated interference mimics this suppression, and that a wild-type daf-18 transgene rescues the dauer defect. These results indicate that PTEN/daf-18 antagonizes the DAF-2-AGE-1 pathway, perhaps by catalyzing dephosphorylation of the PIP3 generated by AGE-1. These data further support the notion that mutations of PTEN contribute to the development of human neoplasia through an aberrant activation of the PI 3-kinase signalling cascade.

The neuropeptide bombesin stimulates tumour cell proliferation in vitro. Through pharmacological testing, 20-40% of human colorectal tumours have been shown to be equipped with bombesin/gastrin releasing peptide receptor (GRP-R). The aim of the present study was to test whether GRP-R expression is correlated with tumour characteristics and usual prognostic factors in colorectal adenocarcinomas. A sensitive reverse transcription (RT)-competitive polymerase chain reaction (PCR) method was validated by studying GRP-R mRNA in separated layers of normal colonic wall, and GRP-R mRNA levels (in parallel with binding studies) in colon cancer cell lines LoVo and Caco-2. GRP-R mRNA levels were then determined in 29 surgical tumour specimens and the results compared with tumour histology and, using histochemistry, with the accumulation of p53 protein and a Ki-67 cell proliferation index. The mRNA was not detected in normal colonic epithelium, whereas a distinct signal was observed after amplification in 27/29 (93%) tumour specimens. Estimates of mRNA levels in the 27 positive tumours ranged from 52 to 8000 amol/0.25 microgram total RNA, and were significantly higher in poorly/moderately differentiated tumours (P < 0.05) and in tumours with lymphatic vessel invasion (P < 0.01). There was no relationship with p53 accumulation or to the proliferation index. Our results show that GRP-R mRNA can be detected in most colorectal tumour specimens, and suggest a link between high mRNA levels and both tumour dedifferentiation and lymph vessel invasion, but not proliferation.

Both BTG1 and BTG2 are involved in cell-growth control. BTG2 expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest. In order to investigate the mechanism underlying Btg-mediated functions, we looked for possible functional partners of Btg1 and Btg2. Using yeast two-hybrid screening, protein-binding assays, and transient transfection assays in HeLa cells, we demonstrated the physical in vitro and in vivo interaction of both Btg1 and Btg2 with the mouse protein mCaf1 (i.e. mouse CCR4-associated factor 1). mCaf1 was identified through its interaction with the CCR4 protein, a component of a general transcription multisubunit complex, which, in yeast, regulates the expression of different genes involved in cell-cycle regulation and progression. These data suggest that Btg proteins, through their association with mCaf1, may participate, either directly or indirectly, in the transcriptional regulation of the genes involved in the control of the cell cycle. Finally, we found that box B, one of two conserved domains which define the Btg family, plays a functional role, namely that it is essential to the Btg-mCaf1 interaction.

We have recently reported that transformation of murine NIH 3T3 cells by v-fos oncogene interfered with Hsp70 and Hsp25 accumulation after heat shock. Here, we have investigated the effect mediated by other oncogenes on the accumulation of these stress proteins. We report that T-antigen transformation of NIH 3T3 cells delayed and reduced the accumulation of Hsp25 after heat shock and decreased the heat-mediated phosphorylation of this protein. This decreased level of Hsp25 correlated with a reduced accumulation of the corresponding mRNA and was related to T-antigen level. In contrast, T-antigen had no effect on the expression of the major stress protein Hsp70 nor did it interfere with the level of Hsp90 or Hsp60. We report also that v-src or Ha-ras oncogenes delayed Hsp25 accumulation after heat shock but that only v-src reduced the heat-induced phosphorylation of this protein. v-src, but not Ha-ras, interfered with Hsp70 expression and none of these oncogenes had an effect on Hsp60 or Hsp90 levels. Taken together, these observations suggest that an altered accumulation of Hsp25 after heat shock is a common characteristic of NIH 3T3 fibroblasts transformed by different oncogenes.

Cell cycle regulation is critical for maintenance of genome integrity. A prominent factor that guarantees genomic stability of cells is p53 (ref. 1). The P53 gene encodes a transcription factor that has a role as a tumour suppressor. Identification of p53-target genes should provide greater insight into the molecular mechanisms that mediate the tumour suppressor activities of p53. The rodent Pc3/Tis21 gene was initially described as an immediate early gene induced by tumour promoters and growth factors in PC12 and Swiss 3T3 cells. It is expressed in a variety of cell and tissue types and encodes a remarkably labile protein. Pc3/Tis21 has a strong sequence similarity to the human antiproliferative BTG1 gene cloned from a chromosomal translocation of a B-cell chronic lymphocytic leukaemia. This similarity led us to speculate that BTG1 and the putative human homologue of Pc3/Tis21 (named BTG2) were members of a new family of genes involved in growth control and/or differentiation. This hypothesis was recently strengthened by the identification of a new antiproliferative protein, named TOB, which shares sequence similarity with BTG1 and PC3/TIS21 (ref. 7). Here, we cloned and localized the human BTG2 gene. We show that BTG2 expression is induced through a p53-dependent mechanism and that BTG2 function may be relevant to cell cycle control and cellular response to DNA damage.

In a previous work, we have shown that T3 induces a potent stimulation of avian myoblast differentiation. In this study, we demonstrated that this hormone did not affect MyoD and myogenin expression. As numerous data suggest that T3 could affect the cAMP pathway, we have studied its involvement in the myogenic activity of triiodothyronine on quail myoblast. In agreement with Zalin and Montagues (Cell 2, 103-108 (1974)), we observed a transient rise in myoblast intracellular cAMP level some hours before the onset of terminal differentiation. Interestingly, this rise occurred earlier in T3-treated than in control myoblasts, and cAMP production was significantly increased by the hormone. Moreover, T3 increased CREB transcriptional activity, thus suggesting that the entire cAMP signaling pathway was stimulated by this hormone. In addition, we observed that addition of an inhibitor of adenylate cyclase activity prior to the cAMP rise dramatically inhibited myoblast differentiation. Last, we showed that cAMP mimicked all T3 actions upon myoblast differentiation: (1) T3 and cAMP reduced myoblast proliferation by increasing the number of postmitotic myoblasts at cell confluence; (2) T3 and cAMP increased BTG1 nuclear accumulation; (3) T3 and cAMP stimulated terminal differentiation only when added during the proliferative phasis. These data strongly suggest that the transient rise in cAMP production could be essential for myoblast terminal differentiation. In addition, it appears that, at least in avian myoblasts, T3 stimulation of terminal differentiation involves the cAMP pathway.

Post-transplant lymphoproliferative disorders (PTLD) are well-known complications of iatrogenic immune deficiency and are thought to result from the proliferation of B cells infected by the Epstein-Barr virus (EBV). Some large cell lymphomas occurring in the general population carry a rearrangement of the Bcl6/Laz3 zinc-finger-encoding gene. 15 EBV-associated PTLD were tested for the presence of Bcl6/Laz3 rearrangements by Southern blot analysis using two specific probes (F370, F372). One out of 15 cases displayed a rearranged band independent of the germline one. In contrast, 10 lymphoblastoid cell lines and one lymphoblastoid cell line passaged in an SCID mouse carried only germline alleles of Bcl6/Laz3 after Southern blot hybridization. This indicates that genetic abnormalities may also play an important role in the development of some PTLD.

The effect of v-fos transformation on the cellular response to heat shock has been investigated. NIH 3T3 fibroblasts were transfected with the FBR p75gag-fos gene fusion under the control of the long terminal repeat (LTR) promoter of Finkel-Biskin-Reilly (FBR) murine sarcoma virus and with the gene encoding hygromycin resistance. Several hygromycin-resistant clone isolates, that expressed various levels of p75gag-fos oncoprotein, were analyzed as they displayed properties of transformed cells, such as altered morphology, shorter doubling time, serum-independent growth and foci formation in soft agar. The thermal response of these clones was compared to that of the control cells expressing the hygromycin-resistance gene only. Here, we report that the v-fos-transformed clones displayed an enhanced thermosensitivity which resulted in a reduced tolerance to thermal stress. Heat-treated v-fos-transformed cells displayed a decreased expression and accumulation of the major stress proteins Hsp68 (68-kDa heat-shock protein) and Hsp25 which probably resulted of a reduced accumulation of the corresponding mRNAs. This effect was particularly intense at the level of Hsp25. These alterations in cell survival and stress-protein expression appeared correlated to the level of p75gag-fos. At least for Hsp68, the transcription of this gene was not found altered by v-fos expression suggesting that this oncogene increases the turn-over of Hsp68 mRNA. After the heat-shock treatment, v-fos transformation also reduced the time period during which the constitutively expressed stress protein Hsc70 redistributes inside the nucleus. Since Hsp68 and Hsp25 are molecular chaperones that in vivo protect cells against the deleterious effects of heat shock, it is conceivable that their reduced accumulation and altered cellular distribution following heat shock may contribute, at least in part, to the thermosensitivity of v-fos-transformed NIH 3T3 fibroblasts.

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.

Transplant recipients are at increased risk for the development of post-transplant lymphoproliferative disorders (PTLDs). PTLDs harbor genomes of the Epstein-Barr virus, a herpesvirus that immortalizes B cells in vitro. At least five viral proteins are required for immortalization. Two of them are particularly important. Latent membrane protein (LMP) has transforming activity in fibroblasts, and Epstein-Barr antigen (EBNA)2 transactivates the expression of numerous cellular and viral genes. To determine whether the expression of EBNA2 and LMP is related to the histological and clinical presentation of PTLD, we tested their expression in 14 Epstein-Barr virus-positive cases. Using monoclonal antibodies to EBNA2 and LMP on paraffin sections, we found an expression of both proteins in 2 of 3 polymorphic PTLD and in 7 of 8 cases of monomorphic, large cell PTLD, without plasmacytic differentiation. One polymorphic and one large cell PTLD expressed LMP only. LMP and EBNA2 were found particularly in immunoblasts. The number of positive cells was extremely variable in the different cases as well as within the same biopsy. Three cases of PTLD had morphological and phenotypical features of plasmacytomas and did not stain for EBNA2 or LMP. This suggests that the expression of EBNA2 and LMP is related to the differentiation stage of the infected cells and that other viral or cellular proteins may contribute to tumor growth.

Group I and Epstein-Barr virus-negative Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface Ig receptors or after exposure to a calcium ionophore. We show here that tumor necrosis factor (TNF)-alpha protects these B cell lines against Ca(2+)-dependent apoptosis. Protection was associated with up-regulation of bcl-2 mRNA and protein expression. The increase of Bcl-2 expression induced by TNF-alpha was inhibited by chelerythrine, a specific inhibitor of protein kinase C (PKC), suggesting that Bcl-2 expression was dependent on PKC activation. Furthermore, we show that phorbol esters and cyclosporin A (CsA), which prevent Ca(2+)-dependent apoptosis, up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expression is controlled by calcineurin since we have shown that FK506 but not rapamycin had the same effect on Bcl-2 expression, whereas okadaic acid, an inhibitor of phosphatases 1, 2A and 2C, was ineffective. These data provide direct evidence that TNF-alpha prevents Ca(2+)-dependent apoptosis by a Bcl-2-dependent mechanism mediated by PKC.

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M(r) (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.

Post-transplant lymphoproliferative disorders (PTLD) are potentially fatal complications of organ transplants. Impairment of the immune system by immunosuppressive drugs is the assumed cause of PTLD. The Epstein-Barr virus (EBV) is detected in most of the PTLD studied and is considered as the main aetiological agent. The clinical course of PTLD patients remains unpredictable, some lymphoproliferations regress after discontinuation of the immunosuppressive treatment, others behave as true malignant tumours. The mechanism by which a viro-induced lymphoproliferation evolves to an autonomous tumour remains unclear, and little is known about the genetic changes that occur during this process. We report two cases of fatal EBV-associated PTLD in heart transplant recipients. Both tumours were monoclonal and carried numerous chromosomal abnormalities, including a classic t(8;14)(q24;q32) with rearrangement of the MYC proto-oncogene. One tumour demonstrated an amplification of the proto-oncogene N-MYC. The EBNA2 gene was not expressed in tumoral cells, suggesting that the chromosomal abnormalities contributed the function of EBNA2 in these cells. The morphology of the tumours indicated that the cases presented here were not Burkitt's lymphomas. These findings provide some clues with regard to the genetic changes which lead to a B-cell malignancy in some transplant patients.

The human BTG1 gene (expressing an anti-proliferative function) is an evolutionarily conserved gene homologous to the murine PC3/TIS21 genes. Here, we report the cloning and sequencing of the murine BTG1 coding region and chicken BTG1 cDNA. The putative human and mouse BTG1 proteins are 100% identical; the chicken BTG1 cDNA contains an open reading frame of 170 amino acids with a 91% identity to its human and murine counterparts. The 3'-untranslated region of BTG1 is also highly conserved (82% homology between human and chicken), suggesting that it plays a key role in the regulation of BTG1 expression. These data confirm that BTG1 is phylogenetically highly conserved and that BTG1 and PC3/TIS21 may constitute the first members of a new family of functionally related genes.

The BTG1 gene locus has been shown to be involved in a t(8;12)(q24;q22) chromosomal translocation in a case of B-cell chronic lymphocytic leukemia. We report here the cloning and sequencing of the human BTG1 cDNA and establish the genomic organization of this gene. The full-length cDNA isolated from a lymphoblastoid cell line contains an open reading frame of 171 amino acids. BTG1 expression is maximal in the G0/G1 phases of the cell cycle and is down-regulated when cells progress throughout G1. Furthermore, transfection experiments of NIH3T3 cells indicate that BTG1 negatively regulates cell proliferation. The BTG1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG1 and PC3 are not cognate genes. We then postulate that these two genes are the first members of a new family of antiproliferative genes.

We performed molecular cloning and sequencing of the breakpoints of a new chromosomal translocation involving the MYC protooncogene locus. This secondary t(8;12)(q24;q22) was associated with a primary t(11;14)(q13;q32) translocation in a case of B-cell chronic lymphocytic leukemia (CLL) in blastic transformation. In this leukemia, Northern blot and nuclease analyses SI showed that MYC was strongly expressed with initiation of the transcription at both the 5' and 3' promoters as observed in Burkitt's lymphomas; no coding change was observed in MYC putative regulatory sequences. The breakpoint on chromosome 8 mapped to the 3' end of the MYC locus, in a region containing a potential Z-DNA tract, and where we identified two DNase 1 hypersensitive sites. A rearranged MYC gene fragment was cloned and shown to contain chromosome 12 information by Southern blot analysis and by in situ hybridization. A genomic probe subcloned from the isolated region of the chromosome 12 recognized a 1.8 kb transcript in virtually all the tissues tested but a preferential expression of this new gene, which we termed BTG1 (for B-cell translocation gene 1) was observed in the CLL cells and in tissues of lymphoid origin. This chromosome 12 coding sequence is conserved in evolution and a transcript of similar size is present in murine tissues.

The t(14;18) chromosomal translocation is widely recognized as a cytogenetic abnormality associated with follicular lymphomas, but estimates of its frequency in this type of lymphoma vary from less than 40% to almost 90% according to the geographic origin of the patients. Using two human genomic probes for major and minor breakpoint cluster regions mapping at chromosome 18q21, we have analysed 131 cases of B non-Hodgkin's lymphomas obtained from France, by the Southern blot technique. The genotypic study was complemented in most cases by immunophenotypic and cell kinetic analyses. The BCL2 gene located at 18q21 band was rearranged in 39 of 56 (70%) follicular lymphomas and in 9 of 74 (12%) diffuse lymphomas; probes for major and minor breakpoint regions detected two thirds and one third of the rearrangements respectively. Regarding the morphologic subtypes of follicular and diffuse lymphomas, no significant differences were observed irrespective of the probe used. Review of the literature showed that comparable results have been obtained previously using both cytogenetic and molecular approaches and our results support the view that the global incidence of the t(14;18)(q32;q21) translocation in follicular lymphomas is about 70% with wide geographic variations. The immunological study provides evidence for a significant correlation of BCL2 rearrangement with surface immunoglobulin gamma isotype expression and with the lack of reactivity of the malignant cells with an antibody against the CD5 cluster. In the cases where cell kinetics was analysed, we did not find any significant difference between the rate of proliferation and BCL2 rearrangement. These data should be compared with previously reported observations made in humans or in transgenic mice and enable us to propose a model accounting for the role of BCL2 in B cell tumorigenesis.

The t(11;14)(q13;q32) is a recurring translocation associated with some chronic B-cell lymphocytic malignancies; the putative protooncogene BCL1, located at the chromosome band 11q13, can be involved during the translocation process. In order to determine if BCL1 rearrangement is associated with a particular subtype of lymphoma, we analysed 131 B-cell non-Hodgkin's lymphoma samples by Southern blot analysis, using a BCL1 probe. The BCL1 locus was rearranged in 9 out of 25 (36%) cases of intermediate lymphocytic cell lymphomas (ILL), in 1 out of 8 cases of diffuse small cleaved cell lymphoma, in 1 out of 12 cases of diffuse mixed cell lymphoma, and in 1 out of 21 cases of diffuse large cell lymphoma. In contrast, BCL1 was never found rearranged in any of the 46 follicular lymphomas analysed. The BCL2 gene was in germ-line configuration in all ILL. Sequential hybridization of Southern blots with JH, C mu, and BCLI probes identified comigrating fragments in only one case of ILL, which suggests that, in all the other cases, either the rearrangement of BCL1 did not result from a t(11;14) translocation or the break on chromosome 14 occurred outside the JH or C mu regions. These results indicate that rearrangement of the BCL1 locus may be closely associated with ILL and could be considered as a genotypic marker of this lymphoma subtype.