A temporary compendium of thyroid hormone target genes in brain.

BACKGROUND: Thyroid hormone controls a number of developmental and physiological processes in the brain by directly acting on gene expression. Transcriptome analyses in rodent identified a number of thyroid hormone regulated genes in several brain areas at different stages. Genome wide analysis of chromatin occupancy in a neural cell line also identified a subset of genes which transcription is likely to be directly regulated by thyroid hormone receptors in neurons. However, the abundance of these data and apparent discrepancies between studies brought some confusion. RESULTS: We present here a meta-analysis of available data to identify recurrent themes in thyroid hormone action in brain cells. This provides a curated list of 734 regulated genes in rodent brain, and highlights a small number of likely direct target genes. Some of these genes are also regulated in amphibians during metamorphosis. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

An embryonic DNA-binding protein specific for the promoter of the retrovirus long terminal repeat.

Retrovirus expression is restricted in embryonal carcinoma (EC) cells but not in many differentiated cell lines. We used a very sensitive gel retardation assay to detect sequence-specific DNA-binding proteins in crude nuclear extracts obtained from EC and differentiated cells. Four binding sites were mapped in the noncoding sequences of the amphotropic murine leukemia virus. Strong binding to the CCAAT consensus sequence located in the promoter was specifically observed with EC nuclear extract. The binding protein is called EPBF (embryonal promoter-binding factor), and it is a candidate for the repressor of retrovirus transcription.

The transcription factor RFX3 directs nodal cilium development and left-right asymmetry specification.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Neuronal expression of a thyroid hormone receptor α mutation alters mouse behaviour.

In humans, alterations in thyroid hormone signalling are associated with mood and anxiety disorders, but the neural mechanisms underlying such association are poorly understood. The present study investigates the involvement of neuronal thyroid hormone receptor α (TRα) in anxiety, using mouse genetics and Cre/loxP technology to specifically alter TRα signalling in neurons. We evaluated the behaviour of mice expressing a dominant negative, neuron-specific mutation of TRα (TRαAMI/Cre3 mice), using the elevated-plus maze, light-dark box and open-field tests. In a first experiment, mice were housed individually, and the behaviour of TRαAMI/Cre3 mice differed significantly from that of control littermates in these 3 tests, suggesting heightened anxiety. In a second experiment, designed to evaluate the robustness of the results with the same 3 tests, mice were housed in groups. In these conditions, the behaviour of TRαAMI/Cre3 mice differed from that of control littermates only in the light-dark box. Thus, TRαAMI/Cre3 mice appear to be more likely to develop anxiety under stressful housing conditions than control mice. These results suggest that in adult mice, thyroid hormone signalling in neurons, via TRα, is involved in the control of anxiety behaviour.

Screening for germ line p53 mutations in children with malignant tumors and a family history of cancer.

We have undertaken a routine investigation of the p53 status for all the children treated at our institution either affected by multiple tumors or whose family displays at least one second degree relative or less, affected by cancer before the age of 45 years. We report here on the first set of ten such families, eight of which were identified through a proband with sarcoma. p53 exons 5 to 8 have been sequenced following polymerase chain reaction amplification performed on DNA isolated from total blood. A missense mutation affecting codons 248, 273, and 282 was identified in three families. The mutation was inherited in these three families and was detected in unaffected members. In seven families no mutation was detected in exons 5 to 8.

Primary chemotherapy in rhabdomyosarcomas and other malignant mesenchymal tumors of the orbit: results of the International Society of Pediatric Oncology MMT 84 Study.

PURPOSE: The MMT 84 multicentric prospective trial of the International Society of Pediatric Oncology (SIOP) was designed to (1) test the effectiveness of ifosfamide 3 g/m2 on days 1 and 2, vincristine 1.5 mg/m2 on days 1 and 14, and dactinomycin 0.9 mg/m2 on days 1 and 2 (IVA) repeated every 21 days; and (2) reduce late effects of treatment by reserving radiation therapy to the primary site for patients not achieving a complete response (CR) to primary chemotherapy. MATERIALS AND METHODS: Between 1984 and 1989, the MMT 84 study registered 34 children with nonmetastatic rhabdomyosarcomas (RMSs) and other malignant mesenchymal tumors (MMTs) of the orbit in this trial. RESULTS: The 4-year event-free survival rate is 62% +/- 9% (SD) and the 4-year survival rate 86% +/- 7% (SD). A total of 11 local recurrences occurred, 10 among 22 patients treated without initial radiation. Salvage of local failure was achieved in nine of 11 patients with the use of radiation and additional chemotherapy, but three later developed distant metastases and two have died. One isolated regional lymph node failure has occurred, while no patient relapsed with isolated distant metastases. Six of 12 patients who failed are alive with no evidence of disease from 16 to 50 months after relapse. The treatment was well tolerated in all patients, except for one with renal tubular acidosis and one who died of cardiotoxicity. Twelve patients remain in first remission without the use of radiation to the primary tumor from 27 to 84 months. CONCLUSION: Despite a higher incidence of local recurrence when treated by primary chemotherapy, early survival rates were not compromised and a significant number of patients avoided the late effects of radiation. However, longer follow-up is required to assess the ultimate outcome of patients treated in this manner.

Long-term follow-up of ifosfamide renal toxicity in children treated for malignant mesenchymal tumors: an International Society of Pediatric Oncology report.

The renal function of 74 children with malignant mesenchymal tumors in complete remission and who have received the same ifosfamide chemotherapy protocol (International Society of Pediatric Oncology Malignant Mesenchymal Tumor Study 84 [SIOP MMT 84]) were studied 1 year after the completion of treatment. Total cumulative doses were 36 or 60 g/m2 of ifosfamide (six or 10 cycles of ifosfamide, vincristine, and dactinomycin [IVA]). None of them had received cisplatin chemotherapy. Ages ranged from 4 months to 17 years; 58 patients were males and 42 females. The most common primary tumor site was the head and neck. Renal function was investigated by measuring plasma and urinary electrolytes, glucosuria, proteinuria, aminoaciduria, urinary pH, osmolarity, creatinine clearance, phosphate tubular reabsorption, beta 2 microglobulinuria, and lysozymuria. Fifty-eight patients (78%) had normal renal tests, whereas 16 patients (22%) had renal abnormalities. Two subsets of patients were identified from this latter group: the first included four patients (5% of the total population) who developed major toxicity resulting in Fanconi's syndrome (TDFS); and the second group included five patients with elevated beta 2 microglobulinuria and low phosphate reabsorption. The remaining seven patients had isolated beta 2 microglobulinuria. Severe toxicity was correlated with the higher cumulative dose of 60 g/m2 of ifosfamide, a younger age (less than 2 1/2 years old), and a predominance of vesicoprostatic tumor involvement. This low percentage (5%) of TDFS must be evaluated with respect to the efficacy of ifosfamide in the treatment of mesenchymal tumors in children.

High-dose busulfan and cyclophosphamide with autologous bone marrow transplantation support in advanced malignancies in children: a phase II study.

Twenty children with advanced, nonleukemic malignancies entered a phase II study of high-dose busulfan-cyclophosphamide followed by bone marrow transplantation (BMT). All had disease refractory to conventional and/or high-dose chemotherapy (HDC). There were ten neuroblastoma patients, six non-Hodgkin's lymphoma, three Ewing's sarcoma, and one rhabdomyosarcoma. Eight had primarily resistant disease, ten were in second progressive relapse, and two in third progressive relapse. One patient was not evaluable for response. Among the 19 evaluable patients the responses observed were complete response (CR), seven; partial response (PR), three; objective effect, five; and failure, four. However, survival was poor: 15 patients died, two are alive with disease, and three are alive with no evidence of disease (NED) at 8+, 11+, 14+ months post-BMT. Toxicity was high but considered as acceptable, taking into account the terminal state of these patients. Seven treatment-related deaths were observed. This combination therapy proved to be highly effective, with a response rate of 50%, and its value for eradication of residual disease in less advanced patients should be investigated.

Male gonadal function after chemotherapy for solid tumors in childhood.

The testicular function of 30 adolescent or adult males having undergone polychemotherapy in childhood was assessed by means of a spermogram or testicular biopsy. At the time of examination, the patients were pubertal and had completed chemotherapy between 1 and 20 years previously (mean, 9 years). All patients who were prepubertal or intrapubertal at the time of treatment achieved normal puberty with normal growth. Twenty patients presented with azoospermia and/or severe disturbances in the germinal line on biopsy. This series confirms the toxicity of alkylating agents, in particular that of the mechlorethamine, vincristine, procarbazine, and prednisone combination (MOPP) and that of cyclophosphamide (CPM). However, dactinomycin, vinblastine, and vincristine did not appear to have a toxic effect on spermatogenesis. The prepubertal state did not protect the gonads of 19 patients who were prepubertal at diagnosis: 12 are now sterile as a result of the treatment. An increase in basal follicle-stimulating hormone (FSH) levels gives a good indication of testicular damage, although normal levels do not rule out the possibility of azoospermia.

Juvenile granulosa cell tumor of the ovary in children: a clinical study of 15 cases.

Juvenile granulosa cell tumor (JGCT) in children accounted for 12% of all ovarian tumors treated in the Institut Gustave-Roussy (IGR) Pediatric Department from 1967 to 1985. The median age of the 15 girls was 8 years 7 months (range, 22 months to 15 years 7 months). Precocious pseudopuberty was present in six of the seven girls under 8 years. Of the other seven girls, one developed virilization symptoms. Surgery was the first treatment in each case. According to the Wollner classification, there were six stage I, one stage II, six stage III (including four ruptured tumors), and one stage IV JGCT cases. One patient was not available for staging. An adjuvant treatment (five chemotherapy and one radiotherapy combined with chemotherapy) was administered to six patients. Eleven girls are alive and free of disease, with a median follow-up of 6 years (range, 2 to 18 years). Four girls relapsed 6 to 17 months after surgery and died. Two of these relapses occurred in bone. The prognosis for JGCT in children is favorable for the lower stages when treated with surgery, but the best treatment for extensive and recurrent disease has yet to be determined.

Long-term sequelae of conservative treatment by surgery, brachytherapy, and chemotherapy for vulval and vaginal rhabdomyosarcoma in children.

Between 1970 and 1978, 17 girls with rhabdomyosarcoma (RMS) of the vulva or vagina were treated at the Institut Gustave-Roussy (IGR) by conservative treatment including surgery, brachytherapy, and chemotherapy. Twelve pubescent or postpubescent girls were studied for long-term sequelae. Eleven of 12 patients have had a normal puberty, two have a total of three healthy children, 11 have normal menses, and 10 normal menarche (one after hormonal replacement). Only one patient underwent hysterectomy following low-dose brachytherapy. Five girls have no vaginal complications, and three are sexually active. Three have had minimal vaginal sequelae, which required surgical correction to permit sexual intercourse. Four girls sustained serious sequelae (colorectal, vaginal, urethral, and ureteral stenosis). These sequelae, secondary to irradiation, are potentially avoidable in the future given the current advances in brachytherapy and improvements in dosimetry. This conservative treatment is useful and appropriate for girls with RMS of vulvar or vaginal origin when complete remission cannot be obtained with chemotherapy and partial colpectomy.

Repeated high-dose chemotherapy followed by purged autologous bone marrow transplantation as consolidation therapy in metastatic neuroblastoma.

Among 62 children over 1 year of age at diagnosis, who were treated for stage IV neuroblastoma, 33 entered complete remission (CR) or good partial remission (GPR) after conventional therapy and received high-dose chemotherapy (HDC) with in vitro purged autologous bone marrow transplantation (ABMT) as consolidation therapy. The HDC was a combination of carmustine (BCNU), teniposide (VM-26), and melphalan. Thirty-three patients received one course of this regimen, and 18 received two courses. At present, 16 of the 33 grafted patients are alive in continuous CR, with a median follow-up of 28 months. Toxicity of this regimen was tolerable, principally marked by bone marrow depression and gastrointestinal (GI) tract complications. Four complication-related deaths were observed. Relapse post-ABMT occurred most often in the bone marrow. Under this treatment, actuarial disease-free survival is improved compared with that observed under conventional therapy.

Hemorrhagic cystitis following high-dose chemotherapy and bone marrow transplantation in children with malignancies: incidence, clinical course, and outcome.

Two hundred ninety-one courses of high-dose chemotherapy (HDC) with bone marrow transplantation (BMT) in children with malignancies were reviewed in order to assess the incidence, clinical course, outcome, and predisposing factors of hemorrhagic cystitis. Hemorrhagic cystitis occurred in 19 HDC courses (6.5%). Three patients had grade I hematuria linked to thrombopenia, nine had grade II hematuria despite platelet levels greater than 50 x 10(9)/L, and seven had grade III hematuria with clots and bladder obstruction. Severe complications occurred in grade III patients, but no deaths were directly linked to the cystitis. Fourteen patients recovered within two to 120 days of onset. The other patients died before the cystitis resolved, either of a relapse of the malignancy or of infection. Predisposing factors were age (increased incidence in older children), conditioning regimen containing cyclophosphamide, previous vesical irradiation, association with prolonged aplasia, and hepatic complications. The role of busulfan was also probable. No viral agent was found.

Conservative treatment for girls with nonmetastatic rhabdomyosarcoma of the genital tract: A report from the Study Committee of the International Society of Pediatric Oncology.

PURPOSE: To report the results of a conservative multimodal approach in girls with nonmetastatic rhabdomyosarcoma (RMS) of the genital tract, treated in International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumors 84 and 89 protocols. PATIENTS AND METHODS: From 1984 to 1994, 38 girls with RMS of the genital tract (vulva, vagina, uterus) were treated in SIOP protocols. With the exception of patients with rare small tumors, which were resected at the start of the studies, all patients received initial chemotherapy (CHT) (ifosfamide, vincristine, and actinomycin D). Local treatment including surgery, brachytherapy (BT), and external-beam radiotherapy (ERT) was given only to girls who did not achieve complete remission (CR) with CHT or who subsequently relapsed. RESULTS: The primary tumor originated in the vulva or vagina in 27 girls and in the uterus in 11. The overall survival rate (+/- SE) was 91% +/- 6% at 5 years, and the event-free survival rate was 78% +/- 7%. At a median follow-up of 5 years, 30 girls were alive and in first CR and five were alive and in second CR. Four patients treated with complete resection of the tumor at diagnosis received less CHT. Thirteen patients were treated with CHT alone. In 17 patients, local treatment was necessary to achieve complete local control, for a residual mass after initial CHT (10 patients), for viable tumor on biopsy (three patients), or for local relapse (four patients). The local treatment used was radiotherapy (RT) (ERT in three patients, BT in seven), radical surgery with uterine ablation (three patients), RT and radical surgery (three patients), and conservative surgery with RT (one patient). CONCLUSION: Girls with nonmetastatic RMS of the genital tract have an excellent prognosis. We found no difference in outcome between uterine and vulvovaginal RMS. Local treatment does not seem necessary in patients who have a complete response to CHT. When a local treatment is needed, BT may be an alternative to radical surgery or ERT.

Identification of transcripts initiated from an internal promoter in the c-erbA alpha locus that encode inhibitors of retinoic acid receptor-alpha and triiodothyronine receptor activities.

The thyroid hormone receptor-coding locus, c-erbA alpha, generates several mRNAs originating from a single primary transcript that undergoes alternative splicing. We have identified for the first time two new transcripts, called TRdelta alpha1 and TRdelta alpha2 [mRNA for isoform alpha1 and alpha2 of the T3 receptor (TR), respectively], whose transcription is initiated from an internal promoter located within intron 7 of the c-erbA alpha gene. These two new transcripts exhibit tissue-specific patterns of expression in the mouse. These two patterns are in sharp contrast with the expression patterns of the full-length transcripts generated from the c-erbA alpha locus. TR alpha1 and TRdelta alpha2 mRNAs encode N-terminally truncated isoforms of T3R alpha1 and T3R alpha2, respectively. The protein product of TRdelta alpha1 antagonizes the transcriptional activation elicited by T3 and retinoic acid. This protein inhibits the ligand-induced activating functions of T3R alpha1 and 9-cis-retinoic acid receptor-alpha but does not affect the retinoic acid-dependent activating function of retinoic acid receptor-alpha. We predict that these truncated proteins may work as down-regulators of transcriptional activity of nuclear hormone receptors in vivo.

A novel growth-factor-dependent myeloid cell line derived from mouse bone marrow cells contains progenitors endowed with high proliferative potential.

Constitutive expression of human colony-stimulating factor-1 receptor (CSF-1R) confers long-lasting CSF-1-dependent proliferation to mouse myeloid cell lines. We developed mice transgenic for human CSF-1R because mouse CSF-1 cannot activate human CSF-1R. Then bone marrow cells from transgenic mice were plated onto MS-5 stromal cells expressing the membrane form of human CSF-1 (2M-1 cells) in order to combine the hematopoietic supporting properties of stromal cells and the proliferative effects of CSF-1. Thus, we were able to derive a hematopoietic cell line, called 47.10, that grew indefinitely under these conditions, whereas no cell line could be developed from nontransgenic mice. Proliferation of 47.10 cells is severely affected by neutralizing anti-CSF-1R monoclonal antibodies. Morphologic and cytofluorometry analysis established that most 47.10 cells are immature myelomonocytic cells. Consistent with this phenotype, the myeloid transcription factor PU.1, but not the erythroid transcription factor GATA-1, is expressed in 47.10 cells. A few 47.10 cells (3-5%) do not express lineage specific markers; they differentiate spontaneously to lineage-positive cells after replating on 2M-1 cells. In agar cultures, 47.10 cells form 7- and 14-day colonies in response to a cocktail of granulocyte/macrophage colony-stimulating factor (2.5 ng/mL), interleukin-3 (1 ng/mL), and mouse CSF-1 (10 ng/mL). Under these conditions, about 0.5% of 47.10 cells formed large 14-day colonies (>1 mm) composed of mature monocytes and granulocytes, reflecting the presence of progenitors endowed with high proliferative potential (HPP-47.10 cells). In conclusion, we have characterized a novel continuous myeloid cell line presenting a hierarchical structure similar to that of the bone marrow progenitor cell compartment.

Virofection: a new procedure to achieve stable expression of genes transferred into early embryos.

A new procedure, virofection, designed to stabilize the expression of transfected DNA has been developed. It exploits the capacity of retroviruses to integrate their genome into the chromosomes of host cells. The co-transfection of two plasmids, one carrying the genome of a defective retrovirus vector, the other one encoding all the retroviral proteins, results in a transient production of infectious virus particles. These particles can infect the neighboring cells and this leads to the stable integration of the vector genome. This procedure is time-saving and appears to be quite efficient. When applied to chicken embryonic fibroblasts cultured in vitro, it resulted in the stable expression of the lacZ gene in more than 30% of the cells, and did not induce chronic viremia. Stable lacZ expression was also achieved in chicken embryos in ovo. Virofection appears to be a promising and generally applicable method for implementing stable, safe and efficient gene transfer in vitro and in vivo.

Phase II study of rapid-scheduled etoposide in paediatric soft tissue sarcomas.

Twenty three patients with paediatric soft tissue sarcomas who had relapsed or refractory disease were treated with a rapid schedule of intravenous etoposide (100 mg/m2 daily on three consecutive days, weekly over 3 weeks). The regimen was well tolerated with predictable myelotoxicity. In 19 patients with rhabdomyosarcoma, there was a response rate of 42%. This appears to be better than previously reported with conventional three weekly schedules. These data indicate that for rhabdomyosarcoma, as for some other tumours, a divided dose regimen may be the optimal schedule and is worthy of further evaluation.

Treatment of non-metastatic rhabdomyosarcomas in childhood and adolescence. Results of the second study of the International Society of Paediatric Oncology: MMT84.

The second International Society of Paediatric Oncology (SIOP) study for rhabdomyosarcoma (MMT84) had several goals. The two principal aims were: (1) to improve the survival of children with rhabdomyosarcoma; and (2) to reduce the late effects from therapy by restricting the indications for surgery and/or radiotherapy after good response to initial chemotherapy. A further aim was to investigate the role of high-dose chemotherapy in young patients with parameningeal primary tumours. 186 previously untreated eligible patients entered the study. Patients with completely resected primary tumour received three courses of IVA (ifosfamide, vincristine and actinomycin D). Patients with incompletely resected tumour received six to 10 courses of IVA according to stage. Patients achieving complete remission with chemotherapy alone did not usually receive radiotherapy or undergo extensive surgery, but patients remaining in partial remission received local therapy with surgery and/or radiotherapy. Only patients over 5 years of age with parameningeal disease and patients over 12 years with tumours at any site were given systematic irradiation. Complete remission was achieved in 91% (170/186) of all patients. With a median follow-up of 8 years, the 5-year overall survival was 68% (+/- 3% standard error of the mean (SEM) and the 5-year event-free survival 53% (+/- 4% SEM). These results show an improvement over previous SIOP study (RMS75) in which survival was 52% and event-free survival was 47%. Among the 54 patients who exhibited isolated local relapse, 35% (19/54) survived in further remission longer than 2 years after retreatment, including local therapy (surgery +/- radiotherapy). Analysis of the overall burden of therapy received by all surviving children (including primary treatment and treatment for relapse if required) showed that 24% (28/116) were treated by limited surgery followed by three courses of IVA, 29% (34/116) were treated by chemotherapy alone (after initial biopsy) and 13% (15/116) received chemotherapy plus conservative local treatment (limited surgery or radiotherapy for residual disease). Only 34% (39/116) received intensive local therapy defined as radical wide field radiotherapy or radical surgery or both. Compared with the results obtained in the previous SIOP study, treatment in MMT84 was based on response to initial chemotherapy and, despite an overall reduction of the use of local therapy, significantly improved survival for patients with non-metastatic disease. This trial, also for the first time, provides evidence that retreatment after local relapse can achieve long-term second remissions.

Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development.

We have generated transgenic reporter mice to analyze the spatio-temporal distribution of thyroid hormone signaling during mouse brain development. The reporter system, utilizing a chimeric yeast Gal4 DNA-binding domain-thyroid hormone alpha ligand-binding domain fusion protein to drive lacZ expression, revealed that thyroid hormone signaling starts in the midbrain roof several days before the onset of thyroid gland function, and that it remains highly heterogeneous in the central nervous system throughout pre- and postnatal development. We speculate that this heterogeneity might provide neural cells with positional information during development.