Clinical analysis of 82 cases of acute promyelocytic leukemia with PML-RARα short isoform in children and adults.

Background: Acute promyelocytic leukemia (APL) with PML/RARα fusion gene is a distinct variant of acute myeloid leukemia. According to the different break sites of the PML gene, there are three transcripts: Long (bcr1), Variant (bcr2) and Short (bcr3). Methods: We retrospectively analyzed 82 APL cases with PML-RARα short isoform. Results: A total of 384 patients with APL were seen, of which 85(22.14%) had PML/RARα short isoform (bcr3) and 82 met the inclusion criteria. The median age was 33.5 years (range, 2-72 years). The incidences of hemorrhage in the intermediate- and high-risk group were higher, but only the incidence between medium and low risk differed statistically (P=0.006), and the incidences of fever, fatigue, splenomegaly, and lymph node enlargement and differentiation syndrome (DS) in those groups were not statistically significant (P>0.05). FLT3 gene mutation rate and the mortality rate of the high-risk group were significantly higher than that of other groups (P=0.040 and P=0.004, P=0.041 and P=0.037, respectively). The mortality rate was lowest (4.26%) in the group treated with ATRA combined with arsenic and anthracycline. The 3-year OS and the 3-year DFS of the low and intermediate-risk group were better (P=0.019 and P=0.017, respectively). Conclusions: ATRA combined with arsenic and anthracycline had significant impact on outcomes in APL with PML-RARα short isoform.

Short PDE4 Isoforms as Drug Targets in Disease.

The second messenger, cyclic adenosine monophosphate (cAMP), is a master regulator of signal transduction that maintains cell homeostasis. A fine balance between cAMP synthesis by adenylyl cyclase and degradation by phosphodiesterases (PDEs) underpins receptor-specific responses. As multiple receptors rely on cAMP for signaling, PDEs shape three-dimensional, localized gradients of the cyclic nucleotide to drive appropriate signaling cascades. Of the 11 PDE families, PDE4, which comprises long, short, and supershort isoforms and a dead-short isoform, is of great interest due to its implication in disease. Aberrant PDE4 expression and post-translational modifications are hallmarks of several clinical indications for which curative treatment is not yet available. While some PDE4-specific small molecule inhibitors directed against the active site are approved for clinical use, they are limited by severe side effects owing to the high degree of conservation of the catalytic domain between over 20 unique isoforms. Some attempts to use the different modular structure that exists between long and shorter isoforms are now bearing success. However, these inhibitors are exclusively aimed at PDE4 long isoforms, which have been the focus of the majority of research in this area. Here, we have summarised literature on the lesser-studied short PDE4 isoforms and provide a record of the discovery, regulation, and disease relevance of this class of enzymes that represent an untapped target for specific inhibition in the future.

Short-Form Thymic Stromal Lymphopoietin (sfTSLP) Is the Predominant Isoform Expressed by Gynaecologic Cancers and Promotes Tumour Growth.

Thymic stromal lymphopoietin (TSLP) is an epithelial cell derived cytokine belonging to the IL-7 family and a key initiator of allergic inflammation. Two main isoforms of TSLP, classified as long- (lfTSLP) and short-form (sfTSLP), have been reported in human, but their expression patterns and role(s) in cancers are not yet clear. mRNA expression was examined by isoform-specific RT-PCR and RNA in situ hybridisation. Epigenetic regulation was investigated by chromatin immunoprecipitation-PCR and bisulfite sequencing. Tumour progression was investigated by gene overexpression, cell viability assay, cancer organoid culture and transwell invasion. Signals were investigated by proteome profiler protein array and RNA-sequencing. With the use of isoform-specific primers and probes, we uncovered that only sfTSLP was expressed in the cell lines and tumour tissues of human ovarian and endometrial cancers. We also showed the epigenetic regulation of sfTSLP: sfTSLP transcription was regulated by histone acetylation at promoters in ovarian cancer cells, whereas silencing of the sfTSLP transcripts was regulated by promoter DNA methylation in endometrial cancer cells. In vitro study showed that ectopically overexpressing sfTSLP promoted tumour growth but not invasion. Human phosphokinase array application demonstrated that the sfTSLP overexpression activated phosphorylation of multiple intracellular kinases (including GSK3α/ß, AMPKα1, p53, AKT1/2, ERK1/2 and Src) in ovarian cancer cells in a context-dependent manner. We further investigated the impact of sfTSLP overexpression on transcriptome by RNA-sequencing and found that EFNB2 and PBX1 were downregulated in ovarian and endometrial cancer cells, suggesting their role in sfTSLP-mediated tumour growth. In conclusion, sfTSLP is predominantly expressed in ovarian and endometrial cancers and promotes tumour growth.

NFATc1/αA and Blimp-1 Support the Follicular and Effector Phenotype of Tregs.

CD4+CXCR5+Foxp3+ T-follicular regulatory (TFR) cells control the germinal center responses. Like T-follicular helper cells, they express high levels of Nuclear Factor of Activated T-cells c1, predominantly its short isoform NFATc1/αA. Ablation of NFATc1 in Tregs prevents upregulation of CXCR5 and migration of TFR cells into B-cell follicles. By contrast, constitutive active NFATc1/αA defines the surface density of CXCR5, whose level determines how deep a TFR migrates into the GC and how effectively it controls antibody production. As one type of effector Treg, TFR cells express B lymphocyte-induced maturation protein-1 (Blimp-1). Blimp-1 can directly repress Cxcr5 and NFATc1/αA is necessary to overcome this Blimp-1-mediated repression. Interestingly, Blimp-1 even reinforces the recruitment of NFATc1 to Cxcr5 by protein-protein interaction and by those means cooperates with NFATc1 for Cxcr5 transactivation. On the contrary, Blimp-1 is necessary to counterbalance NFATc1/αA and preserve the Treg identity. This is because although NFATc1/αA strengthens the follicular development of Tregs, it bears the inherent risk of causing an ex-Treg phenotype.

De novo small deletion affecting transcription start site of short isoform of AUTS2 gene in a patient with syndromic neurodevelopmental defects.

Disruption of the autism susceptibility candidate 2 (AUTS2) gene through genomic rearrangements, copy number variations (CNVs), and intragenic deletions and mutations, has been recurrently involved in syndromic forms of developmental delay and intellectual disability, known as AUTS2 syndrome. The AUTS2 gene plays an important role in regulation of neuronal migration, and when altered, associates with a variable phenotype from severely to mildly affected patients. The more severe phenotypes significantly correlate with the presence of defects affecting the C-terminus part of the gene. This article reports a new patient with a syndromic neurodevelopmental disorder, who presents a deletion of 30 nucleotides in the exon 9 of the AUTS2 gene. Importantly, this deletion includes the transcription start site for the AUTS2 short transcript isoform, which has an important role in brain development. Gene expression analysis of AUTS2 full-length and short isoforms revealed that the deletion found in this patient causes a remarkable reduction in the expression level, not only of the short isoform, but also of the full AUTS2 transcripts. This report adds more evidence for the role of mutated AUTS2 short transcripts in the development of a severe phenotype in the AUTS2 syndrome.

MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase.

The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits. Many chaperones shared between yeast and humans are involved in COX assembly. Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors. Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process. Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A. By the same analysis, we identified the short isoform of the myofibrillogenesis regulator 1 (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes.

Epigenetic regulation of human DCLK-1 gene during colon-carcinogenesis: clinical and mechanistic implications.

Colorectal carcinogenesis is a multi-step process. While ~25% of colorectal cancers (CRCs) arise in patients with a family history (genetic predisposition), ~75% of CRCs are due to age-associated accumulation of epigenetic alterations which can result in the suppression of key tumor suppressor genes leading to mutations and activation of oncogenic pathways. Sporadic colon-carcinogenesis is facilitated by many molecular pathways of genomic instability which include chromosomal instability (CIN), micro-satellite instability (MSI) and CpG island methylator phenotype (CIMP), leading towards loss of homeostasis and onset of neoplastic transformation. The unopposed activation of Wnt/ß-catenin pathways, either due to loss of APC function or up-regulation of related stimulatory pathways, results in unopposed hyperproliferation of colonic crypts, considered the single most important risk factor for colon carcinogenesis. Hypermethylation of CpG islands within the promoters of specific genes can potentially inactivate DNA repair genes and/or critical tumor suppressor genes. Recently, CpG methylation of the 5' promoter of human (h) DCLK1 gene was reported in many human epithelial cancers, including colorectal cancers (CRCs), resulting in the loss of expression of the canonical long isoform of DCLK1 (DCLK1-L) in hCRCs. Instead, a shorter isoform of DCLK1 (DCLK1-S) was discovered to be expressed in hCRCs, from an alternate ß promoter of DCLKL1-gene; the clinical and biological implications of these novel findings, in relation to recent publications is discussed.

Altered expression of the tachykinins substance P/neurokinin A/hemokinin-1 and their preferred neurokinin 1/neurokinin 2 receptors in uterine leiomyomata.

OBJECTIVE: To study the expression levels of tachykinins and tachykinin receptors in uterine leiomyomas and matched myometrium. DESIGN: Laboratory study. SETTING: University research laboratories and academic hospital. PATIENT(S): Women undergoing hysterectomy for symptomatic leiomyomas. INTERVENTION(S): Quantitative polymerase chain reaction, immunohistochemistry and Western blot. MAIN OUTCOME MEASURE(S): Expression and tissue immunostaining of substance P, neurokinin A, hemokinin-1, neurokinin 1 receptor full-length (NK1R-Fl) and truncated (NK1R-Tr) isoforms, and neurokinin 2 receptor (NK2R) in paired samples of leiomyoma and adjacent normal myometrium. RESULT(S): TAC1 messenger RNA (mRNA) was significantly up-regulated in leiomyomas, whereas intense immunoreaction for the three peptides was particularly abundant in connective tissue cells. Differential regulation of TACR1 mRNA was observed, and at the protein level there was a significant increased expression of NK1R short isoform (NK1R-Tr). TACR2 mRNA was significantly up-regulated in leiomyomas, although levels of NK2R protein were similar in normal and tumor cells. CONCLUSION(S): These and our previous data demonstrate that the whole tachykinin system is differentially regulated in leiomyomas. The increased expression of NK1R-Tr might stimulate leiomyoma growth in a similar way to that observed in other steroid-dependent tumors.

SVCT2 transporter expression is post-natally induced in cortical neurons and its function is regulated by its short isoform.

Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium-vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post-natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1-15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed. The sodium-vitamin C cotransporter, SVCT2, is induced in neurons within the inner layer of the brain cortex during post-natal development, mainly in pyramidal cortex neurons. Two different isoforms, SVCT2wt and SVCT2sh, were detected. Using in vitro studies, we suggest a molecular interaction between SVCT2wt and SVCT2sh, which may regulate the affinity of vitamin C uptake.

Dopamine receptor D2S gene transfer improves the sensitivity of GH3 rat pituitary adenoma cells to bromocriptine.

Bromocriptine, a dopamine agonist (DA), has been used in the treatment of prolactinomas. Recent studies have indicated that dopamine 2 receptor short isoform (D2S) may play an important role in suppressing PRL synthesis and prolactinoma cell growth under DA treatment. In the current study, we investigated the role of D2S in the therapeutic action of bromocriptine in GH3 using both in vitro and in vivo approaches. Infection of adenovirus-D2S increased D2S expression in GH3 cells (P<0.05). D2S expression significantly decreased the GH3 cell viability subjected to bromocriptine treatment in vitro (P<0.05). In nude mice, adenovirus-D2S transfection sensitized GH3 xenograft to bromocriptine treatment evidenced by the significant inhibition of D2S expressed tumor growth as compared with vector control. Furthermore, decrease of Bcl-2 expression, increase of Bax, and active Caspase-3 were found in D2S expressed GH3 xenograft subjected to bromocriptine treatment. In summary, our study indicates that D2S expression plays a critical role in the therapeutic action of bromocriptine in pituitary adenomas and that adenovirus-mediated D2S gene transfer combined with bromocriptine may provide a novel treatment for DA-resistant prolactinomas.

Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes.

The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system.

Detection of Long and Short isoforms of PML-RARA mRNA by RT-PCR in Acute Promyelocytic Leukemia / 대한혈액학회지

BACKGROUND: Chromosomal translocation t (15 ; 17), the breakpoints of which are in the PML gene on chromosome 15 and RARA gene on chromosome 17, is specifically found in acute promyelocytic leukemia (APL). According to the site of breakpoint on PML gene, two major isoforms (Long or Short) of PML-RARA mRNA are produced. METHODS: To detect long (L) and short (S) isoforms, we extracted RNA and amplified PML-RARA mRNA by RT-PCR from leukemic cells of 20 cases of APL. We compared the result of cytogenetic study and the clinical response after chemotherapy or ATRA therapy for remission induction with the isoforms of PML-RARA mRNA. RESULTS: In 19 cases (94%) among 20 cases with APL, PML-RARA mRNA was positive, and negative in a case who showed only i (17q) without t (15;17). In 12 cases (63.2%), L isoform of PML-RARA mRNA was detected, and S isoform (36.8%) in 7 cases of APL. All the cases with t (15;17) were positive for PML-RARA mRNA. In a case of trisomy 8 without t (15;17), PML-RARA mRNA of L isoform was detected. There was no significant difference between L and S isoform in laboratory findings and clinical response after chemotherapy or ATRA treatment. Excluding 6 cases with death before or within 10 days of ATRA treatment or chemotherapy, among 13 patients of positive PML-RARA mRNA, 11 cases (84.6%) reached to complete remission, but a case of negative PML-RARA mRNAwas resistent to ATRA treatment. CONCLUSION: This study suggests that detection of PML-RARA mRNA with two major isofroms using RT-PCR is more sensitive to diagnose APL and to detect minimal residual disease than cytogenetic study and that further study with more cases may be substantiated the types of PML-RARA mRNA isoform as a prognostic marker.