Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain

BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach (AU)

Defects in mTR stability and telomerase activity produced by the Dkc1 A353V mutation in dyskeratosis congenita are rescued by a peptide from the dyskerin TruB domain.

BACKGROUND: The predominant X-linked form of dyskeratosis congenita results from mutations in dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. MATERIALS AND METHODS: Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. RESULTS: Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. DISCUSSION: These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.

AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) acute myeloid leukemia-M2.

AML1-ETO fusion gene is generated from chromosomal translocation t(8;21) mainly in acute myeloid leukemia M2 subtype (AML-M2). Its spliced variant transcript, AML1-ETO9a, rapidly induces leukemia in murine model. To evaluate its clinical significance, AML1-ETO9a expression was assessed in 118 patients with t(8;21) AML-M2, using qualitative and nested quantitative reverse transcriptase (RT)-PCR methods. These cases were accordingly divided into the AML1-ETO9a-H group (n=86, positive for qualitative RT-PCR, with higher level of AML1-ETO9a by quantitative RT-PCR) and the AML1-ETO9a-L group (n=32, negative for qualitative RT-PCR, with lower but still detectable level of AML1-ETO9a by quantitative RT-PCR). C-KIT expression was significantly increased in the AML1-ETO9a-H group, as compared with the AML1-ETO9a-L group. Of the 36 patients harboring C-KIT mutations, 32 patients overexpressed AML1-ETO9a (P=0.0209). Clinically, AML1-ETO9a-H patients exhibited significantly elevated white blood cells count, less bone marrow aberrant myelocytes, increased CD56 but decreased CD19 expression (P=0.0451, P=0.0479, P=0.0149 and P=0.0298, respectively). Moreover, AML1-ETO9a overexpression was related to short event-free and overall survival time (P=0.0072 and P=0.0076, respectively). Taken together, these data suggest that AML1-ETO9a is correlated with C-KIT overexpression/mutations and indicates poor disease outcome in t(8;21) AML-M2.

A new fusion gene NUP98-IQCG identified in an acute T-lymphoid/myeloid leukemia with a t(3;11)(q29q13;p15)del(3)(q29) translocation.

NUP98 has been involved in multiple recurrent chromosome rearrangements in leukemia. We identified a novel fusion between NUP98 and IQ motif containing G (IQCG) gene from a de novo acute T-lymphoid/myeloid leukemia harboring t(3;11)(q29q13;p15)del(3)(q29). IQCG has two putative coiled-coil domains and one IQ domain. The FG repeat from NUP98 and the coiled-coil domain from IQCG were retained in the fusion protein. We demonstrated that NUP98-IQCG could form homodimer, heterodimerize with NUP98 or IQCG, bind co-activators and/or co-repressors, and show transcriptional activity in vitro. Expression of NUP98-IQCG inhibited 32Dcl3 cell apoptosis induced by Ara-C, and partially blocked granulocyte differentiation induced by G-CSF. Colony-forming assay and serial replating assays indicated that NUP98-IQCG was able to stimulate proliferation, partially block differentiation of hematopoietic stem/progenitor cells but was unable to confer transformation alone. Taken together, our data indicate that newly identified NUP98-IQCG fusion protein may play an essential role in leukemogenesis, but by itself may not be sufficient to induce leukemia.

Major form of NUP98/HOXC11 fusion in adult AML with t(11;12)(p15;q13) translocation exhibits aberrant trans-regulatory activity.

Three adult patients with de novo acute myeloid leukemia of distinct subtypes harboring t(11;12)(p15;q13) have been investigated to characterize the genes involved in that translocation. Through molecular cytogenetics, a chromosome break was detected at the 3' part of nucleoporin 98 (NUP98) gene at 11p15. Using rapid amplification of cDNA end, we identified the partner gene at 12q13, HOXC11. Molecular analysis showed that exon 12 of NUP98 was fused in-frame to exon 2 of HOXC11 in all three cases with t(11;12)(p15;q13). Therefore, this type of fusion may represent the major form of the NUP98-HOXC11 chimera so far reported. Moreover, two out of three cases had a confirmed deletion of the 3' part of NUP98 gene and more telomeric region of 11p harboring a group of tumor-suppressor genes. Interestingly, the NUP98-HOXC11 protein when assayed in a GAL4 reporter system, showed an aberrant trans-regulatory activity as compared to the wild-type HOXC11 in both COS-7 and HL-60 cells. Therefore, NUP98-HOXC11 may contribute to the leukemogenesis by interfering with the cellular mechanism of transcriptional regulation.

Molecular cytogenetic characterization and clinical relevance of additional, complex and/or variant chromosome abnormalities in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by typical morphological manifestation, t(15;17) translocation and active response to all-trans retinoic acid (ATRA) in the great majority of patients. However, a subset of APL cases may present atypical phenotypic, cytogenetic or molecular features at different stages of the disease. The biological and clinical significance of these features sometimes remains obscure. In this study, 284 APL patients were cytogenetically analyzed and precise diagnosis was performed according to the molecular cytogenetic results. Twenty-six APL patients were identified as having additional, complex and/or variant chromosomal abnormalities at diagnosis or at relapse, 16 of them being further analyzed using fluorescence in situ hybridization (FISH) or chromosome painting (CP). Interestingly, some of these chromosomal aberrations were found to be associated with atypical morphology and/or drug response, indicating a genotype-phenotype correlation. Analysis of the complex karyotype may also allow a better understanding of the levels of cellular origin of the leukemogenesis. Examination of the remission induction and survival data showed that the presence of the additional/complex chromosome abnormalities was related to the prognosis in both primarily diagnosed and relapsed patients in this series.

Feasibility and clinical significance of real-time quantitative RT-PCR assay of PML-RARalpha fusion transcript in patients with acute promyelocytic leukemia.

INTRODUCTION: To study the relationship between the expression level of the PML-RARalpha fusion transcripts and the clinical status and efficiency of the therapy in acute promyelocytic leukemia (APL) patients, we applied a very sensitive and specific real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) system to quantify the dose of PML-RARalpha fusion transcripts in a series of APL patients at distinct disease stages. MATERIALS AND METHODS: A total of 31 APL patients (19 males and 12 females; aged from 8 to 74 years) from eight hospitals in Shanghai were analysed. Real-time Quantitative RT-PCR was used to measure the normalized dose (DoseN) of PML-RARalpha fusion transcripts. RESULTS: A wide range of PML-RARalpha DoseN above 1 x 10(3) was noted in 25 newly diagnosed patients. PML-RARalpha DoseN was significantly decreased after remission induction with ATRA, ATRA/chemotherapy or As2O3 and further reduced after consolidation. The fact that all patients with long disease free survival had a constantly low PML-RARalpha DoseN below 2 x 10(2) and a higher level predicted impending relapse suggests that this value could serve as a 'threshold' for molecular remission. PML-RARalpha DoseN was also of prognostic value in a group of relapsed patients, since good response to As2O3 reinduction was accompanied by a remarkable reduction of fusion transcript level, whereas patients with high PML-RARalpha Dose(N) after the second CR tended to relapse again rapidly. CONCLUSION: These results confirm that real-time RT-PCR assay for PML-RARalpha transcripts in APL patients is useful in reflecting leukemic burden, assessing response to treatment and indicating the ultimate clinical outcome or curability of disease.

Gene expression profiling in the human hypothalamus-pituitary-adrenal axis and full-length cDNA cloning.

The primary neuroendocrine interface, hypothalamus and pituitary, together with adrenals, constitute the major axis responsible for the maintenance of homeostasis and the response to the perturbations in the environment. The gene expression profiling in the human hypothalamus-pituitary-adrenal axis was catalogued by generating a large amount of expressed sequence tags (ESTs), followed by bioinformatics analysis (http://www.chgc.sh.cn/ database). Totally, 25,973 sequences of good quality were obtained from 31,130 clones (83.4%) from cDNA libraries of the hypothalamus, pituitary, and adrenal glands. After eliminating 5,347 sequences corresponding to repetitive elements and mtDNA, 20,626 ESTs could be assembled into 9, 175 clusters (3,979, 3,074, and 4,116 clusters in hypothalamus, pituitary, and adrenal glands, respectively) when overlapping ESTs were integrated. Of these clusters, 2,777 (30.3%) corresponded to known genes, 4,165 (44.8%) to dbESTs, and 2,233 (24.3%) to novel ESTs. The gene expression profiles reflected well the functional characteristics of the three levels in the hypothalamus-pituitary-adrenal axis, because most of the 20 genes with highest expression showed statistical difference in terms of tissue distribution, including a group of tissue-specific functional markers. Meanwhile, some findings were made with regard to the physiology of the axis, and 200 full-length cDNAs of novel genes were cloned and sequenced. All of these data may contribute to the understanding of the neuroendocrine regulation of human life.

Molecular cloning of six novel Krüppel-like zinc finger genes from hematopoietic cells and identification of a novel transregulatory domain KRNB.

To clone zinc finger genes expressed in hematopoietic system, we designed primers based on conserved Cys(2)/His(2) zinc finger sequences to amplify corresponding domains from mRNA of normal bone marrow and leukemia cell line NB4. DNA fragments of novel zinc finger genes were chosen and used as probe pool to screen cDNA libraries or subject to rapid amplification of cDNA ends in order to obtain full-length cDNA. Six cDNAs including whole open reading frame of zinc finger proteins, named as ZNF191, ZNF253 (BMZF-1), ZNF255 (BMZF-2), ZNF256 (BMZF-3), ZNF257 (BMZF-4), and ZNF254 (BMZF-5) were obtained. All six belong to the Krüppel-like zinc finger gene family, and typical transcriptional regulatory motifs exist in the N-terminal moiety, such as the SCAN box in ZNF191, and the KRAB domains in ZNF253, ZNF254, ZNF256, and ZNF257. A previously undefined sequence nominated as Krüppel-related novel box, which may represent a new transregulatory motif, was revealed at the N terminus of ZNF255. The transregulatory function of non-zinc finger regions of ZNF191, ZNF253, and ZNF255 were addressed in yeast and mammalian cells. The results indicated that ZNF255 might be a conditional transactivator, whereas ZNF253 and ZNF191 displayed a suppressive effect on the transcription in yeast and/or mammalian systems.

Studies on treatment of acute promyelocytic leukemia with arsenic trioxide: remission induction, follow-up, and molecular monitoring in 11 newly diagnosed and 47 relapsed acute promyelocytic leukemia patients.

Fifty-eight acute promyelocytic leukemia (APL) patients (11 newly diagnosed and 47 relapsed) were studied for arsenic trioxide (As2O3) treatment. Clinical complete remission (CR) was obtained in 8 of 11 (72.7%) newly diagnosed cases. However, As2O3 treatment resulted in hepatic toxicity in 7 cases including 2 deaths, in contrast to the mild liver dysfunction in one third of the relapsed patients. Forty of forty-seven (85.1%) relapsed patients achieved CR. Two of three nonresponders showed clonal evolution at relapse, with disappearance of t(15;17) and PML-RARalpha fusion gene in 1 and shift to a dominant AML-1-ETO population in another, suggesting a correlation between PML-RARalpha expression and therapeutic response. In a follow-up of 33 relapsed cases over 7 to 48 months, the estimated disease-free survival (DFS) rates for 1 and 2 years were 63.6% and 41.6%, respectively, and the actual median DFS was 17 months. Patients with white blood cell (WBC) count below 10 x 10(9)/L at relapse had better survival than those with WBC count over 10 x 10(9)/L (P =.038). The duration of As2O3-induced CR was related to postremission therapy, because there was only 2 of 11 relapses in patients treated with As2O3 combined with chemotherapy, compared with 12 of 18 relapses with As2O3 alone (P =.01). Reverse transcription polymerase chain reaction (RT-PCR) analysis in both newly diagnosed and relapsed groups showed long-term use of As2O3 could lead to a molecular remission in some patients. We thus recommend that ATRA be used as first choice for remission induction in newly diagnosed APL cases, whereas As2O3 can be either used as a rescue for relapsed cases or included into multidrug consolidation/maintenance clinical trials.

Genomic sequence, structural organization, molecular evolution, and aberrant rearrangement of promyelocytic leukemia zinc finger gene.

The promyelocytic leukemia zinc finger gene (PLZF) is involved in chromosomal translocation t(11;17) associated with acute promyelocytic leukemia. In this work, a 201-kilobase genomic DNA region containing the entire PLZF gene was sequenced. Repeated elements account for 19.83%, and no obvious coding information other than PLZF is present over this region. PLZF contains six exons and five introns, and the exon organization corresponds well with protein domains. There are at least four alternative splicings (AS-I, -II, -III, and -IV) within exon 1. AS-I could be detected in most tissues tested whereas AS-II, -III, and -IV were present in the stomach, testis, and heart, respectively. Although splicing donor and acceptor signals at exon-intron boundaries for AS-I and exons 1-6 were classical (gt-ag), AS-II, -III, and -IV had atypical splicing sites. These alternative splicings, nevertheless, maintained the ORF and may encode isoforms with absence of important functional domains. In mRNA species without AS-I, there is a relatively long 5' UTR of 6.0 kilobases. A TATA box and several transcription factor binding sites were found in the putative promoter region upstream of the transcription start site. PLZF is a well conserved gene from Caenorhabditis elegans to human. PLZF paralogous sequences are found in human genome. The presence of two MLL/PLZF-like alignments on human chromosome 11q23 and 19 suggests a syntenic replication during evolution. The chromosomal breakpoints and joining sites in the index acute promyelocytic leukemia case with t(11;17) also were characterized, which suggests the involvement of DNA damage-repair mechanism.

Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full-length cDNA cloning.

Hematopoietic stem/progenitor cells (HSPCs) possess the potentials of self-renewal, proliferation, and differentiation toward different lineages of blood cells. These cells not only play a primordial role in hematopoietic development but also have important clinical application. Characterization of the gene expression profile in CD34(+) HSPCs may lead to a better understanding of the regulation of normal and pathological hematopoiesis. In the present work, genes expressed in human umbilical cord blood CD34(+) cells were catalogued by partially sequencing a large amount of cDNA clones [or expressed sequence tags (ESTs)] and analyzing these sequences with the tools of bioinformatics. Among 9,866 ESTs thus obtained, 4,697 (47.6%) showed identity to known genes in the GenBank database, 2, 603 (26.4%) matched to the ESTs previously deposited in a public domain database, 1,415 (14.3%) were previously undescribed ESTs, and the remaining 1,151 (11.7%) were mitochondrial DNA, ribosomal RNA, or repetitive (Alu or L1) sequences. Integration of ESTs of known genes generated a profile including 855 genes that could be divided into different categories according to their functions. Some (8.2%) of the genes in this profile were considered related to early hematopoiesis. The possible function of ESTs corresponding to so far unknown genes were approached by means of homology and functional motif searches. Moreover, attempts were made to generate libraries enriched for full-length cDNAs, to better explore the genes in HSPCs. Nearly 60% of the cDNA clones of mRNA under 2 kb in our libraries had 5' ends upstream of the first ATG codon of the ORF. With this satisfactory result, we have developed an efficient working system that allowed fast sequencing of 32 full-length cDNAs, 16 of them being mapped to the chromosomes with radiation hybrid panels. This work may lay a basis for the further research on the molecular network of hematopoietic regulation.

[Alternative combination chemotherapy with mitomycin C, vincristine, methotrexate, 5-fluorouracil, cis-platinum and adriamycin for adenocarcinoma of the lung].

Efficacy of an alternative combination chemotherapy with MMC, VCR, MTX, 5-FU, CDDP and ADM for adenocarcinoma of the lung is reported. Forty-one advanced cases (stage III: 9; IV: 32) were chosen for the chemotherapy. Two combination chemotherapies MMC + VCR + MTX + 5-FU + CDDP: MVMFP; MMC + VCR + MTX + 5-FU + ADM: MVMFA were repeated alternatively for 8 consecutive weeks with 2 interposed rest weeks, and this regimen was completed in 34 cases. All 41 cases were evaluable. Three and 26 cases achieved complete and partial responses, respectively. The response rate was 70.7%, and the median survival time was 13 months. The adverse effects of the chemotherapy observed were tolerable: alopecia (63.4%), gastrointestinal symptoms (14.5%), bone marrow toxicity (12.2%) and liver dysfunction (4.8%). These results indicate that our MVMFP/MVMFA alternative chemotherapy is quite effective for adenocarcinoma of the lung, comparable or superior to conventional chemotherapies.