Loss of p53 induces leukemic transformation in a murine model of Jak2 V617F-driven polycythemia vera.

As leukemic transformation of myeloproliferative neoplasms (MPNs) worsens the clinical outcome, reducing the inherent risk of the critical event in MPN cases could be beneficial. Among genetic alterations concerning the transformation, the frequent one is TP53 mutation. Here we show that retroviral overexpression of Jak2 V617F mutant into wild-type p53 murine bone marrow cells induced polycythemia vera (PV) in the recipient mice, whereas Jak2 V617F-transduced p53-null mice developed lethal leukemia after the preceding PV phase. The leukemic mice had severe anemia, hepatosplenomegaly, pulmonary hemorrhage and expansion of dysplastic erythroid progenitors. Primitive leukemia cells (c-kit+Sca1+Lin- (KSL) and CD34-CD16/32-c-kit+Sca1-Lin- (megakaryocyte-erythroid progenitor; MEP)) and erythroid progenitors (CD71+) from Jak2 V617F-transduced p53-null leukemic mice had leukemia-initiating capacity, however, myeloid differentiated populations (Mac-1+) could not recapitulate the disease. Interestingly, recipients transplanted with CD71+ cells rapidly developed erythroid leukemia, which was in sharp contrast to leukemic KSL cells to cause lethal leukemia after the polycythemic state. The leukemic CD71+ cells were more sensitive to INCB18424, a potent JAK inhibitor, than KSL cells. p53 restoration could ameliorate Jak2 V617F-transduced p53-null erythroleukemia. Taken together, our results show that p53 loss is sufficient for inducing leukemic transformation in Jak2 V617F-positive MPN.

Evi1 defines leukemia-initiating capacity and tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Relapse of chronic myeloid leukemia (CML) is triggered by stem cells with a reconstituting capacity similar to that of hematopoietic stem cells (HSCs) and CML stem cells are a source of resistance in drug therapy with tyrosine kinase inhibitors (TKIs). Ecotropic viral integration site 1 (EVI1), a key transcription factor in HSC regulation, is known to predict poor outcomes in myeloid malignancies, however, incapability of prospective isolation of EVI1-high leukemic cells precludes the functional evaluation of intraindividual EVI1-high cells. Introduction of CML into Evi1-internal ribosomal entry site (IRES)-green fluorescent protein (GFP) knock-in mice, a versatile HSC-reporter strain, enables us to separate Evi1-high CML cells from the individual. Evi1-IRES-GFP allele models of CML in chronic phase (CML-CP), by retroviral overexpression of BCR-ABL and by crossing BCR-ABL transgenic mice, revealed that Evi1 is predominantly enriched in the stem cell fraction and associated with an enhanced proliferative as well as a leukemia-initiating capacity and that Evi1-high CML-CP cells exhibit resistance to TKIs. Overexpressing BCR-ABL and NUP98-HOXA9 in Evi1-IRES-GFP knock-in mice to model CML in blast crisis (CML-BC), in which Evi1-high cells turned to be a major population as opposed to a minor population in CML-CP models, showed that Evi1-high CML-BC cells have a greater potential to recapitulate the disease and appear resistant to TKIs. Furthermore, given that Evi1 heterozygosity ameliorates CML-CP and CML-BC development and that the combination of Evi1 and BCR-ABL causes acute myeloid leukemia resembling CML-BC, Evi1 could regulate CML development as a potent driver. In addition, in human CML-CP cases, we show that EVI1 is highly expressed in stem cell-enriched CD34+CD38-CD90+ fraction at single-cell level. This is the first report to clarify directly that Evi1-high leukemic cells themselves possess the superior potential to Evi1-low cells in oncogenic self-renewal, which highlights the role of Evi1 as a valuable and a functional marker of CML stem cells.

Imprinting in neurons.

Although most imprinted genes display parent-origin-specific gene expression in tissues where they are transcribed, some genes are imprinted in a tissue-specific manner. Genes that show brain-specific imprinting or brain-specific lack of imprinting present a unique opportunity to study the process of imprinting during tissue differentiation. In this review, I introduce the systematic study of brain-cell-lineage-specific imprinting using a primary brain cell culture system, where neurons or glial cells are cultured separately. Two reports using the primary brain cell culture revealed brain-cell-lineage-specific imprinting in Ube3a and Igf2r, which had previously been described to show brain-specific imprinting and brain-specific lack of imprinting, respectively. Such brain-cell-lineage-specific imprinting was associated with cell-specific epigenetic modifications, especially with their reciprocally imprinted antisense non-coding RNAs, Ube3a-ATS and Air. These results emphasize the necessity of imprinting analysis at the cell level rather than in whole brain tissue during brain differentiation. The brain cell culture system provides us with a new powerful tool to understand the molecular mechanism of brain-specific imprinting.

Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a.

The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted Ube3a expression is detected predominantly in the hippocampus, cerebellar Purkinje cells and the olfactory bulb. Therefore, imprinting of mouse Ube3a is thought to be region-specific with different levels of silencing of the paternal Ube3a allele in different brain regions. To determine cell types of imprinted Ube3a expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT-PCR and immunofluorescence were performed to determine the allelic expression of the gene. The Ube3a gene encodes two RNA transcripts in the brain, sense and antisense. The sense transcript was expressed maternally in neurons but biallelically in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and only from the paternal allele. Our data present evidence of brain cell type-specific imprinting, i.e. neuron-specific imprinting of Ube3a in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts present only in neurons suggests that the neuron-specific imprinting mechanism is related to the lineage determination of neural stem cells.

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.

Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

A 30-base-pair element in the first intron of SOX9 acts as an enhancer in ATDC5.

SOX9 is a transcription factor that is essential for chondrogenesis and testis differentiation, and haploinsufficiency of SOX9 causes campomelic dysplasia, severe skeletal malformation syndrome with variably penetrant XY sex reversal. Here we demonstrate that in several cell lines that express SOX9, 30-bp element in the first intron of human SOX9 gene act as a potential enhancer in the ATDC5 chondroprogenitor cell line, despite the apparent absence of cell-specific regulatory elements within a 5.5-kb promoter region. Deletion and site-specific mutational analyses reveal that the last 12 bp of the 30-bp element are critical for transcriptional activity, while 5'-half sequences are necessary for full transactivation. Gel retardation assays indicate the possible involvement of several binding factors along the length of this element. These results suggest that functionally interdependent elements in the 30-bp enhancer region of the first intron account for basal expression levels of Sox9 in ATDC5.

[Long-term outcome of monotherapy by Cohen ureterocystoneostomy for vesicoureteral reflux in spina bifida patients].

BACKGROUND: The conventional surgical treatment for vesioureteral reflux (VUR) is spina bifida patients is ureterocystoneostomy. Various newer therapies, including augmentation enterocystoplasty and minimally invasive subureteral collagen injection, have been introduced. However, all of these procedures have specific advantages and disadvantages, and no guidelines for deciding on the surgical treatment of VUR in spina bifida patients have yet been established. In this study, the long-term outcome of the Cohen procedure, a method of ureterocystoneostomy, was examined. PATIENTS AND METHODS: Among spina bifida patients in whom VUR was treated by the Cohen procedure alone from 1984 to 1989, 27 patients who could be followed up for 5 years or longer were enrolled in the study (11 males and 16 females, with a mean age of 13.4 years at surgery). In principle, they were followed up using annual cystography, excretory urography, and blood and urine tests. At the final assessment, they were examined for the presence at VUR and for morphological abnormalities of the urinary tract. Their renal function was also assessed. They were followed for 6 to 13 years (mean: 8.9 years), and the mean age at final assessment was 22.2 years. RESULTS: Among 42 ureters in the 27 patients examined, 38 ureters (90.5%) in 22 patients (81.5%) did not have VUR postoperatively. Four ureters in 4 patients had the recurrence of VUR, and in another patient new occurrence was detected postoperatively. Augmentation ileocystoplasty was performed to treat the postoperative decrease of bladder compliance in 4 patients. Among 22 patients who had hydronephrosis preoperatively, 9 (40.9%) showed improvement and none suffered from aggravation of this condition. None of the patients showed a decline of renal function, except for 1 who had a serum creatinine of 2.5 mg/dl preoperatively and developed end-stage renal failure at 7 years postoperatively. CONCLUSIONS: The Cohen procedure has an excellent anti-reflux effect. It is one of the therapeutic options for VUR in patients with good bladder compliance.

Two Thai families with Norrie disease (ND): association of two novel missense mutations with severe ND phenotype, seizures, and a manifesting carrier.

We describe two Thai families with Norrie disease (ND) in three generations, including 10 affected males and one manifesting female. All affected males in each family had severely defective eye development with complete loss of vision. In addition, three male patients (one from family 1 and two from family 2) suffered from epilepsy, and one female carrier from one family manifested blindness with phthisis bulbi in her right eye. Mutation analysis of the ND gene (NDP) revealed two different novel missense mutations (L16P and S75P) that co-segregated with ND in each family, suggesting that the newly appearing proline at codon 16 or codon 75 alters the conformation of the ND protein and contributes to the severe phenotype of ND in each family. Other studies suggest that epileptic seizures or growth retardation that is associated with ND is the consequence of loss of contiguous genes, because most such patients had deletions extending beyond the Norrie locus. Our finding that the three affected males in the two families with the missense mutations had epilepsy does not support a contiguous gene effect, but favors the pleiotropism of NDP, at least as far as the epileptic manifestation is concerned. The unilateral blindness in the female carrier may have been due to non-random X-inactivation.

[A case of polyarteritis nodosa presenting as a mass in scrotum].

A 16-year-old boy with a painful tumor in the left scrotum was referred to our department. CT scans showed a low density area in the left testis, so we diagnosed a left testicular tumor and performed left inguinal orchiectomy. Histological examination revealed polyarteritis nodosa (PN) of the testis and epididymis. Systemic examination revealed no other evidence of PN. Although induration developed in the right epididymis after the operation, it resolved with steroid therapy. The patient is currently asymptomatic and is being followed at our clinic. The pathogenesis and management of this rare condition are discussed.

The novel gene, gamma2-COP (COPG2), in the 7q32 imprinted domain escapes genomic imprinting.

The gene MEST (or PEG1) on chromosome 7q32 is paternally expressed in human fetal tissues as a result of genomic imprinting. Since some imprinted genes are clustered, we speculated that an imprinted gene cluster might exist at 7q32. We have sought to isolate additional human genes close to MEST and to characterize their allelic expression patterns. Here, we report the biallelic expression of the gene, gamma2-COP (coatomer protein complex, subunit gamma 2, HUGO-approved symbol COPG2), and monoallelic expression of the transcript, CIT1, which is located in intron 20 of gamma2-COP. Recently, gamma2-COP was reported to be a novel imprinted gene that overlaps the 3'-untranslated region (3'-UTR) of MEST in a tail-to-tail orientation. However, our results revealed biallelic expression in all fetal tissues and adult blood lymphocytes. On the other hand, CIT1 was an antisense transcript of gamma2-COP intron 20 and was expressed from the paternal allele in all fetal tissues examined. Adult blood lymphocytes showed biallelic expression. We identified additional MEST 3'-UTR sequence, which overlaps the last four exons and introns of gamma2-COP. This additional MEST 3'-UTR may complicate analysis of gamma2-COP imprinting. Our data indicate that the region containing MEST at 7q32 is an imprinted domain, but gamma2-COP adjacent to MEST escapes genomic imprinting.

Changes in liver fatty acid unsaturation after partial hepatectomy in the rat.

The purpose of this study was to assess changes in the degree of fatty acid unsaturation in rat liver after partial hepatectomy. This is the first study in which liver fatty acid unsaturation has been analyzed over a long period of regeneration until day 28 after operation. The relationship between changes in unsaturation and fatty acid composition in the regenerating liver were also investigated in this study. Proton nuclear magnetic resonance spectroscopy revealed significantly elevated levels of unsaturation with a maximum on day 5 after partial hepatectomy, compared with untreated controls (11.72+/-0.55 vs. 11.05+/-0.26%, P < 0.05). No significant changes in unsaturation were found in day 1 regenerating liver, which is rich in absolute amounts of fatty acids. Based on gas-liquid chromatography, the relative amounts of oleic acid (18:1n-9) and linoleic acid (LA; 18:2n-6) were increased, while polyunsaturated fatty acids such as arachidonic acid (20:4n-6) and docosahexaenoic acid (DHA; 22:6n-3) were decreased on day 1. On the other hand, on day 5 of regeneration, while most fatty acids were returning to their preoperative control levels, only DHA was higher than the control value (7.69+/-0.58 vs. 5.57+/-0.37%, P < 0.001). The high levels of unsaturation on day 5 were found to be partly due to the increase in DHA. The findings suggest that some significant signals are transmitted during the regeneration process owing to alterations in the membrane structure by the high levels of fatty acid unsaturation and the increase in DHA levels on day 5 after partial hepatectomy.

Construction of a physical and transcript map flanking the imprinted MEST/PEG1 region at 7q32.

MEST/PEG1, a gene expressed paternally in mesodermal derivatives in early embryonic stages, is the first imprinted gene mapped to chromosome 7q32. Since imprinted genes are clustered in general at a chromosomal region, we speculated that a similar imprinted-gene cluster may exist at chromosome region 7q32 and that the functions of some such genes may contribute to the phenotype of Silver-Russell syndrome including maternal uniparental disomy for chromosome 7 (maternal UPD7). As an initial step toward the isolation of imprinted genes at 7q32, we adopted an integrated approach involving the construction of a PAC contig and ESTs mapping in the vicinity of MEST. Here, we have constructed a complete contig of PAC and BAC clones and a transcript map spanning the entire approximately 1-Mb region between D7S530 and D7S649. We developed 60 novel STSs and precisely mapped 47 genes/ESTs. This map contains a putative autistic disorder locus that has been suggested to be localized near markers D7S530 and D7S684. This integrated physical and transcript map provides a valuable resource for identification of an imprinted gene(s) in this region as well as a candidate gene(s) for autistic disorder.

Submucosal tumor (SMT)-like esophageal squamous cell carcinoma with gastric metastasis.

We present here a case of submucosal tumor-like esophageal cancer with metastasis to the stomach. A 60-year-old man, whose ability to swallow was impaired, was admitted to Kyorin University School of Medicine Hospital. Gastrointestinal endoscopy demonstrated a small bulging mass in the lower esophagus and a large submucosal mass in the gastric cardia. The gastric lesion was growing rapidly and becoming easily hemorrhagic. It appeared rich in blood flow by angiography. Surgical treatment was adopted after a diagnosis of esophageal cancer and a gastric submucosal tumor was made. However, in the end, the gastric mass was identified as a metastasis from the esophageal squamous cell carcinoma. Moreover, the primary esophageal lesion displayed quite a special type of histology, a so-called submucosal tumor-like pattern, which was covered by normal epithelium and grew mainly in the submucosal layer of the esophagus. Gastric metastasis from esophageal cancer is relatively rare, and it is quite rare that an esophageal squamous cell carcinoma grows as a submucosal tumor. Finally, the patient died of pneumonia and metastasis to the liver on the 110th day of hospitalization. Intramural metastasis to the stomach from esophageal cancer should be treated in its advanced stage, and poor prognosis can be expected from aggressive treatment. It is necessary to recognize this complication so that appropriate therapy can be carried out on patients with esophageal cancer, and the situation needs to be carefully evaluated, including the stomach, both before and after treatment.

ASC, a novel 22-kDa protein, aggregates during apoptosis of human promyelocytic leukemia HL-60 cells.

The cytoskeletal and/or nuclear matrix molecules responsible for morphological changes associated with apoptosis were identified using monoclonal antibodies (mAbs). We developed mAbs against Triton X-100-insoluble components of HL-60 cells pretreated with all-trans retinoic acid. In particular, one mAb recognized a 22-kDa protein that exhibited intriguing behavior by forming an aggregate and appearing as a speck during apoptosis induced by retinoic acid and other anti-tumor drugs. Cloning and sequencing of its cDNA revealed that this protein comprises 195 amino acids and that its C-terminal half has a caspase recruitment domain (CARD) motif, characteristic of numerous proteins involved in apoptotic signaling. We referred to this protein as ASC (apoptosis-associated speck-like protein containing a CARD). The ASC gene was mapped on chromosome 16p11.2-12. The antisense oligonucleotides of ASC were found to reduce the expression of ASC, and consequently, etoposide-mediated apoptosis of HL-60 cells was suppressed. Our results indicate that ASC is a novel member of the CARD-containing adaptor protein family.