Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML.

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

C/EBPα and MYB regulate FLT3 expression in AML.

The interaction between the receptor FLT3 (FMS-like tyrosine kinase-3) and its ligand FL leads to crucial signalling during the early stages of the commitment of haematopoietic stem cells. Mutation or over-expression of the FLT3 gene, leading to constitutive signalling, enhances the survival and expansion of a variety of leukaemias and is associated with an unfavourable clinical outcome for acute myeloid leukaemia (AML) patients. In this study, we used a murine cellular model for AML and primary leukaemic cells from AML patients to investigate the molecular mechanisms underlying the regulation of FLT3 gene expression and identify its key cis- and trans-regulators. By assessing DNA accessibility and epigenetic markings, we defined regulatory domains in the FLT3 promoter and first intron. These elements permit in vivo binding of several AML-related transcription factors, including the proto-oncogene MYB and the CCAAT/enhancer binding protein C/EBPα, which are recruited to the FLT3 promoter and intronic module, respectively. Substantiating their relevance to the human disease, our analysis of gene expression profiling arrays from AML patients uncovered significant correlations between FLT3 expression level and that of MYB and CEBPA. The latter relationship permits discrimination between patients with CEBPA mono- and bi-allelic mutations, and thus connects two major prognostic factors for AML.

Distinct regulation of c-myb gene expression by HoxA9, Meis1 and Pbx proteins in normal hematopoietic progenitors and transformed myeloid cells.

The proto-oncogenic protein c-Myb is an essential regulator of hematopoiesis and is frequently deregulated in hematological diseases such as lymphoma and leukemia. To gain insight into the mechanisms underlying the aberrant expression of c-Myb in myeloid leukemia, we analyzed and compared c-myb gene transcriptional regulation using two cell lines modeling normal hematopoietic progenitor cells (HPCs) and transformed myelomonocytic blasts. We report that the transcription factors HoxA9, Meis1, Pbx1 and Pbx2 bind in vivo to the c-myb locus and maintain its expression through different mechanisms in HPCs and leukemic cells. Our analysis also points to a critical role for Pbx2 in deregulating c-myb expression in murine myeloid cells cotransformed by the cooperative activity of HoxA9 and Meis1. This effect is associated with an intronic positioning of epigenetic marks and RNA polymerase II binding in the orthologous region of a previously described alternative promoter for c-myb. Taken together, our results could provide a first hint to explain the abnormal expression of c-myb in leukemic cells.

Bilateral ectopia lentis as a presenting feature of medulloepithelioma.

Ectopia lentis may be a feature of numerous systemic and ocular disorders. Kivela and Tarkkanen described an 8-year-old girl with medulloepithelioma who presented with ectopia lentis and a mass behind the temporal iris. Shields reported 2 children with medulloepithelioma who had ectopia lentis associated with neovascular glaucoma. To date, there has been no report of a child with ectopia lentis as the only presentation of an intraocular tumor. We present 2 children with malignant medulloepitheliomas who presented in this fashion.

Prevention of vomiting after general anesthesia for pediatric ophthalmic surgery.

We evaluated the effectiveness of a multifaceted general anesthesia protocol designed to minimize postoperative vomiting after pediatric eye surgery. A convenience sample of 150 consecutive children, aged 2 weeks to 18 years, who received general anesthesia for pediatric ophthalmic surgery was studied. General anesthesia was administered with induction by mask for 82.7% of the children and intravenously using propofol in 17.3% of the children. Anesthesia was maintained using halothane or isoflurane, oxygen, and air mixture for all patients. Morphine sulfate was used for additional pain relief, up to 0.1 mg/kg. Gastric aspiration was performed after intubation for each child. Metoclopramide, 0.15 mg/kg, and 0.1 mg/kg of ondansetron were administered before the end of each operation. Postoperatively, patients were monitored for vomiting for 24 hours. Postoperative vomiting occurred in 11 (7.3%) of 150 cases. Acute elevation of intraocular pressure was found in 5 of the 11 children who vomited. This vomiting was unresponsive to intravenous rescue ondansetron, but responded to lowering the intraocular pressure. The incidence of postoperative vomiting after general anesthesia for pediatric eye surgery can be substantially decreased by adopting a protocol designed to lessen the emetic effects of general anesthesia. Limited use of nitrous oxide for mask induction only, gastric emptying, and administration of metoclopramide and ondansetron intravenously in combination proved effective in reducing the incidence of postoperative vomiting.

3' deletions cause aniridia by preventing PAX6 gene expression.

Aniridia is a panocular human eye malformation caused by heterozygous null mutations within PAX6, a paired-box transcription factor, or cytogenetic deletions of chromosome 11p13 that encompass PAX6. Chromosomal rearrangements also have been described that disrupt 11p13 but spare the PAX6 transcription unit in two families with aniridia. These presumably cause a loss of gene expression, by removing positive cis regulatory elements or juxtaposing negative DNA sequences. We report two submicroscopic de novo deletions of 11p13 that cause aniridia but are located >11 kb from the 3' end of PAX6. The clinical manifestations are indistinguishable from cases with chain-terminating mutations in the coding region. Using human x mouse retinoblastoma somatic cell hybrids, we show that PAX6 is transcribed only from the normal allele but not from the deleted chromosome 11 homolog. Our findings suggest that remote 3' regulatory elements are required for initiation of PAX6 expression.

Identification of wild-type and exon 5 deletion variants of estrogen receptor beta in normal human mammary gland.

We have examined messenger RNA (mRNA) expression of estrogen receptor (ER) alpha, wild-type ERbeta (mRNA and protein), and ERbeta exon 5 deletion variants (ERbeta delta5) in samples of normal human mammary gland obtained from 37 premenopausal subjects undergoing reduction mammoplasty. Comparing individual expression, ERbetaP mRNA predominated, expressed in 34 of 37 samples (91%), whereas ERalpha was found in 21 of 37 cases (57%). Receptor combinations were then analyzed and compared. Most samples either coexpressed ERalpha with ERbeta (54%) or expressed just ERbeta (38%). Immunohistochemical analysis revealed that ERbeta mRNA expression mirrored that of protein. Immunoreactivity was observed in the nucleus with additional evidence of cytoplasmic staining in those epithelial cells lining the breast ducts. Sporadic immunoreactivity was also detected in stromal cells. Expression of wild type and ERbeta delta5 was analyzed, and their association with ERalpha was compared. Most samples coexpressed wild-type ERbeta and the splice variant (62%; P = 0.05), with 30% exclusively expressing wild-type ERbeta. Although samples coexpressing wild type and variant ERbeta showed no statistical association with ERalpha, those samples expressing only wild-type ERP, showed a trend toward associations with ERalpha (P = 0.07). In conclusion, our data would support a role for ERbeta in the normal human mammary gland, where we propose it may be the dominant receptor.

Direct activation of oestrogen receptor-alpha by interleukin-6 in primary cultures of breast cancer epithelial cells.

Interleukin 6 (IL-6) is secreted by breast tumours and shows synergistic activity with 17beta-oestradiol (E2), leading to increases in reductive 17beta-hydroxysteroid dehydrogenase activity in breast cancer epithelial cells. However, the mechanisms involved are poorly understood. Using short-term epithelial cultures established from primary breast tumours, we have examined whether IL-6 could directly affect transcriptional activity of oestrogen reception alpha (ERalpha). Tumour epithelial cultures were established from 15 breast tumours, grown to 70% confluence and transiently transfected with a plasmid reporter containing the vitellogenin oestrogen response element and the luciferase coding sequence (ERE-TK-LUC). Following transfection, cells were incubated with E2, IL-6, the pure anti-oestrogen ZM 182780 or combinations of these substances for 48 h. Luciferase activity was then measured in cell lysates. E2 caused a dose-dependent increase in luciferase expression, causing a maximum threefold stimulation at 100 pM. In the presence of IL-6, transcriptional activity was increased by up to 2.5-fold in ERalpha+ cultures (11/15). In combination with E2, synergistic effects were observed with increases in luciferase activity of up to sixfold over controls. This effect could be blocked by treatment with ZM 182780. Pre-incubation of cells with an antibody directed against the signalling component of IL-6, gp130, was ineffective in blocking the E2 response. This antibody reduced, but did not completely block the effect of IL-6 either alone or in combination with E2, suggesting cross-talk between the two signalling pathways. In conclusion, these results provide evidence for direct transcriptional activation of ERalpha by IL-6.

Increased expression of estrogen receptor beta mRNA in tamoxifen-resistant breast cancer patients.

Tamoxifen is currently the first-line therapy for treatment of hormone-dependent breast cancer. However, despite initial benefits, most patients eventually relapse. Two groups of patients were identified: (a) a tamoxifen-sensitive group (n = 8); and (b) a tamoxifen-resistant group (n = 9). Using reverse transcription-PCR, the relative expression of mRNA for both estrogen receptor (ER) beta and transforming growth factor beta1 was determined in each patient group and quantified against a known reference standard. ER-beta mRNA was significantly up-regulated in the tamoxifen-resistant group as compared with the tamoxifen-sensitive group (P = 0.001 by Fisher's exact test), and, consistent with previous findings, transforming growth factor beta1 was also up-regulated in the tamoxifen-resistant cohort (P = 0.02). The importance of ER-beta in tamoxifen resistance was validated using tamoxifen-sensitive and -resistant cell lines, in which it was demonstrated that ER-beta mRNA was significantly up-regulated in the resistant cells. These results lend further support to a role for ER-beta as a poor prognostic factor in breast cancer.

In vivo and in vitro expression of steroid-converting enzymes in human breast tumours: associations with interleukin-6.

Enzymes modulating local steroid availability play an important role in the progression of human breast cancer. These include isoforms of 17beta-hydroxysteroid dehydrogenase (17-HSD), aromatase and steroid sulphatase (STS). The aim of this study was to investigate the expression, by reverse transcription polymerase chain reaction, of 17-HSD types I-IV, aromatase and steroid STS in a series of 51 human breast tumour biopsies and 22 primary cultures of epithelial and stromal cells derived from these tumours, giving a profile of the steroid-regulating network for individual tumours. Correlations between enzyme expression profiles and expression of the interleukin (IL)-6 gene were also sought. All except one tumour expressed at least one isoform of 17-HSD, either alone or in combination with aromatase and STS. Expression of 17-HSD isoforms I-IV were observed in nine tumours. Of the 15 tumours which expressed three isoforms, a combination of 17-HSD II, III and IV was most common (6/15 samples). The majority of tumours (n = 17) expressed two isoforms of 17-HSD with combinations of 17-HSD II and IV predominant (7/17 samples). Eight tumours expressed a single isoform and of these, 17-HSD I was in the majority (5/8 samples). In primary epithelial cultures, enzyme expression was ranked: HSD I (86%) > STS (77%) > HSD II (59%) > HSD IV (50%) = aromatase (50%) > HSD III (32%). Incidence of enzyme expression was generally reduced in stromal cultures which were ranked: HSD I (68%) > STS (67%) > aromatase (48%) > HSD II (43%) > HSD IV (28%) > HSD III (19%). Expression of IL-6 was associated with tumours that expressed > or = 3 steroid-converting enzymes. These tumours were of higher grade and tended to come from patients with family history of breast cancer. In conclusion, we propose that these enzymes work in tandem with cytokines thereby providing sufficient quantities of bioactive oestrogen from less active precursors which stimulates tumour growth.

Evidence for transcriptional activation of ERalpha by IL-1beta in breast cancer cells.

Estrogen is mitogenic in breast cancer where IL-1beta also fulfils a role. The aim of this study was to determine any relationship between IL-1beta and ERalpha in breast cancer. By RT-PCR, 26/77 tumours expressed IL-1beta, and 57/77 expressed ERalpha. Samples which were IL-1beta positive were categorised against those which expressed ERalpha. Of the 26 tumours which expressed IL-1beta, all were ERalpha positive. We next examined whether IL-1beta could directly activate ERalpha. MCF-7 cells stably transfected with a plasmid reporter (ERE-TK-LUC) were incubated with either 17beta-estradiol (E2, 10-9-10-13 M), IL-1beta (10 ng/ml), the pure antiestrogen ZM 182780 (10 nM) or combinations of these substances. Transcriptional activity was measured in cell lysates 48 h later. E2 caused a dose-dependent increase in luciferase activity. With IL-1beta, transcriptional activity was typically half of the E2 response. To determine the role of the IL-1 receptor, parallel cultures were incubated with IL-1 receptor antagonist. This reduced, but did not completely block the effect of IL-1beta, suggesting that IL-1beta was affecting transcriptional activity via another pathway. Confirmation that the effect was via ERalpha was verified using the pure antiestrogen, ZM 182370, which completely abrogated the effects of E2, when added alone or in combination with IL-1beta. These results provide compelling evidence for direct transcriptional activation of ERalpha by IL-1beta. Interactions of these factors may thus modulate hormonal activity in human breast tumours.

Goniosurgery for prevention of aniridic glaucoma.

OBJECTIVE: To report the long-term success and complications of modified goniosurgery to prevent aniridic glaucoma, an entity that typically is difficult to control medically or surgically. DESIGN: A retrospective review of the medical charts. RESULTS: Fifty-five eyes in 33 patients who had aniridia without glaucoma and who underwent goniosurgery were identified. Ninety-one procedures were performed on the 55 eyes by 1 surgeon (D.S.W.). Each eye had an average of 1.65 procedures and an average of 200 degrees of goniosurgery. Average age at time of initial goniosurgery was 36.6 months. There were no operative complications. No eye had a decrease in visual acuity at last follow-up. All eyes had a preoperative intraocular pressure (IOP) less than 21 mm Hg. At last follow-up (average, 9 years 6 months; range, 8 months to 24 years), 49 eyes (89%) had IOPs less than 22 mm Hg without medications. The remaining 6 eyes (11%) had IOPs of 22 mm Hg or less with up to 2 types of eyedrops. CONCLUSIONS: Without prophylactic goniotomy, aniridic glaucoma may be expected in half of patients, and when it occurs, it is extremely difficult to control. Prophylactic goniosurgery in selected eyes of young patients with aniridia may be effective in preventing aniridic glaucoma.

Coexpression of estrogen receptor alpha and beta: poor prognostic factors in human breast cancer?

The cloning of a second estrogen receptor (ER), ER beta, has prompted a reevaluation of the role of ERs in breast cancer. The aim of this study was to determine the expression of both ER isoforms in normal (n = 23) and malignant (n = 60) human breast tissue by reverse transcription-PCR and correlate this information with known prognostic factors including tumor grade and node status. In normal breast tissue, expression of ER beta predominated, with 22% of samples exclusively expressing ER beta; this was not observed in any of the breast tumor samples investigated. Most breast tumors expressed ER alpha, either alone or in combination with ER beta. Interestingly, those tumors that coexpressed ER alpha and ER beta were node positive (P = 0.02; Fisher's exact test) and tended to be of higher grade. Because antiestrogens are agonists when signaling through the AP1 element, overexpression of ER beta in tumors expressing both ER subtypes may explain the failure of antiestrogen therapy in some breast cancer patients. Thus, ER beta may be a useful prognostic factor in patients with breast cancer.

Short-term primary culture of epithelial cells derived from human breast tumours.

As experimental models for breast cancer, most studies rely on established human breast cancer cell lines. However, many of these lines were established over 20 years ago, many from pleural effusions rather than the primary tumour, so the validity of using them as representative models is questionable. This paper describes our experiences, over a 3-year period, in establishing short-term epithelial-cell-enriched preparations from primary breast tumours based on differential centrifugation followed by culture in selective media. Epithelial cells were successfully cultured from 55% of samples, but culture success did not appear to be correlated with tumour histology, stage, grade or node status. Epithelial cell-enriched cultures were immunopositive for broad-spectrum cytokeratin and epithelial membrane antigen (EMA). Positivity for keratin 19 confirmed that the cultures contained tumour-derived cells, which additionally showed significantly higher activity of the reductive pathway of the steroid-converting enzyme 17beta-hydroxysteroid dehydrogenase type I. That the cultures contained tumour and not normal epithelial cells was further substantiated by the complete absence of the calmodulin-like gene NB-1 in tumour-derived cultures; this is only associated with normal breast epithelia. Eighty-five per cent of cultures established from oestrogen receptor (ER)-positive tumours expressed ER in vitro; this was functional in 66% of cultures, although ER-positive phenotype was gradually lost over time. In conclusion, epithelial cells can be isolated and maintained as short-term cultures from primary breast tumours irrespective of histopathological or clinical details, providing a model system with a greater biological and clinical relevance than breast cancer cell lines.

Exudative retinal detachment in X-linked retinoschisis.

X-linked retinoschisis is a vitreoretinal dystrophy characterized by foveal and peripheral retinoschisis in the nerve fiber layer. Although many associated peripheral retinal findings have been reported, few reports have described massive exudative retinal detachments in patients with X-linked retinoschisis. The authors report the unusual occurrence of Coats'-like exudative retinopathy in two patients with X-linked retinoschisis. Both patients had peripheral massive exudative retinal detachments.

Goniosurgery for prevention of aniridic glaucoma.

PURPOSE: We conducted a retrospective study to report the long-term success and complications of modified goniosurgery to prevent aniridic glaucoma, an entity that typically is difficult to control medically or surgically. METHODS: Fifty-five eyes in 33 patients who had aniridia without glaucoma and who had goniosurgery were identified. Ninety-one procedures were performed on 55 eyes by 1 surgeon (D.S.W.). Each eye had an average of 1.65 procedures and an average of 200 degrees of goniosurgery. Average patient age at time of initial goniosurgery was 37 months. There were no operative complications. RESULTS: No eye had a decrease in visual acuity at last follow-up. All eyes had a preoperative intraocular pressure (IOP) of less than 21 mm Hg. At last follow-up (average, 9 years 6 months; range, 8 months to 24 years), 49 eyes (89%) had IOP of less than 22 mm Hg without medications. The remaining 6 eyes (11%) had IOP of less than or equal to 22 mm Hg with up to 2 eye drops. Of 224 aniridic eyes of 112 patients that were seen for eye care by 1 of the authors (D.S.W.), 119 eyes (53%) demonstrated glaucoma, as defined by IOP of greater than 21 mm Hg. CONCLUSIONS: Without prophylactic goniotomy, aniridic glaucoma may be expected in half of patients, and when it occurs, it is extremely difficult to control. Prophylactic goniosurgery in selected eyes of young patients with aniridia is effective in preventing aniridic glaucoma.

Mitomycin C-augmented trabeculectomy in refractory congenital glaucoma.

OBJECTIVE: The purpose of the study is to determine the safety and efficacy of mitomycin C-augmented trabeculectomy in children with developmental glaucoma treated previously by conventional procedures. DESIGN: Retrospective review of all cases of developmental glaucoma with previously failed conventional procedures that underwent mitomycin C-augmented trabeculectomy between January 1992 and December 1994. PARTICIPANTS: A total of 19 eyes of 13 patients were included in the study. Nineteen eyes included primary congenital glaucoma (15 eyes) with documented failure of primary trabeculotomy, Axenfeld-Reiger syndrome (2 eyes) and aniridia (2 eyes). INTERVENTION: Mitomycin C-augmented (0.4 mg/ml for 3 minutes) trabeculectomy was the chosen intervention. MAIN OUTCOME MEASURES: Preoperative and postoperative intraocular pressures (IOPs), visual acuities, success rate, bleb characteristics, time of surgical failure, and complications were the main outcome measures. RESULTS: The mean IOP was reduced from a preoperative level of 33.74 +/- 10.70 mmHg to 11.89 +/- 1.33 mmHg (P < 0.0001) with the percentage reduction in IOP being 64.75. The mean follow-up was 19.52 +/- 2.65 months. Visual acuity was maintained in all the cases after surgery. Complete success as defined in the authors' study was achieved in 18 eyes (94.74%). Only one patient was classified as a qualified success. The bleb was characterized by its large elevated, avascular, transparent appearance in all the eyes. The only significant complication was retinal detachment in an eye with buphthalmos, aphakia, and large axial length. However, the retina was reattached successfully by retinal reattachment surgery, and visual acuity improved to the preoperative level of 20/200. It was not possible to determine the cause of retinal detachment or to assess the possible role of mitomycin C in this complication. CONCLUSIONS: Mitomycin C-augmented trabeculectomy is a viable option in eyes with failed conventional trabeculotomy surgery.