Diamond Blackfan anemia is mediated by hyperactive Nemo-like kinase.

Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure syndrome associated with ribosomal gene mutations that lead to ribosomal insufficiency. DBA is characterized by anemia, congenital anomalies, and cancer predisposition. Treatment for DBA is associated with significant morbidity. Here, we report the identification of Nemo-like kinase (NLK) as a potential target for DBA therapy. To identify new DBA targets, we screen for small molecules that increase erythroid expansion in mouse models of DBA. This screen identified a compound that inhibits NLK. Chemical and genetic inhibition of NLK increases erythroid expansion in mouse and human progenitors, including bone marrow cells from DBA patients. In DBA models and patient samples, aberrant NLK activation is initiated at the Megakaryocyte/Erythroid Progenitor (MEP) stage of differentiation and is not observed in non-erythroid hematopoietic lineages or healthy erythroblasts. We propose that NLK mediates aberrant erythropoiesis in DBA and is a potential target for therapy.

Disruption of the 5S RNP-Mdm2 interaction significantly improves the erythroid defect in a mouse model for Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by haploinsufficiency of genes encoding ribosomal proteins (RPs). Perturbed ribosome biogenesis in DBA has been shown to induce a p53-mediated ribosomal stress response. However, the mechanisms of p53 activation and its relevance for the erythroid defect remain elusive. Previous studies have indicated that activation of p53 is caused by the inhibition of mouse double minute 2 (Mdm2), the main negative regulator of p53, by the 5S ribonucleoprotein particle (RNP). Meanwhile, it is not clear whether this mechanism solely mediates the p53-dependent component found in DBA. To approach this question, we crossed our mouse model for RPS19-deficient DBA with Mdm2(C305F) knock-in mice that have a disrupted 5S RNP-Mdm2 interaction. Upon induction of the Rps19 deficiency, Mdm2(C305F) reversed the p53 response and improved expansion of hematopoietic progenitors in vitro, and ameliorated the anemia in vivo. Unexpectedly, disruption of the 5S RNP-Mdm2 interaction also led to selective defect in erythropoiesis. Our findings highlight the sensitivity of erythroid progenitor cells to aberrations in p53 homeostasis mediated by the 5S RNP-Mdm2 interaction. Finally, we provide evidence indicating that physiological activation of the 5S RNP-Mdm2-p53 pathway may contribute to functional decline of the hematopoietic system in a cell-autonomous manner over time.

Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite.

High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancer cells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in the hepatocellular carcinoma cell line Huh7. Treatment with 20 µM of selenite reduced the viability of Huh7 cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold. Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxic effect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutral sphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 µM of SK1-II prior to treatment with 10 µM of selenite caused induction of apoptosis and gave rise to a 2.5-fold increase in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 µM of SK1-II combined with 10 µM of selenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides. The formation of reactive oxygen species (ROS) after treatment with 10 µM of selenite was maximally enhanced by 1 µM of SK1-II. Moreover, combined treatment with SK1-II and 10 µM of selenite synergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell line MIHA remained unaffected by the same treatment. These results raise the possibility that a combination of selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant, at doses that are non-toxic to normal liver cells.

Discovery of a novel series of inhibitors of human cytomegalovirus primase.

Infection by human cytomegalovirus (hCMV) remains a potent threat to susceptible people throughout the world. We have discovered a series of imidazolyl-pyrimidine compounds, which were found to be irreversible inhibitors of the hCMV UL70 primase based on results from radiolabeling and SAR studies. Two promising analogs are described that rival ganciclovir and cidofovir in antiviral potency and possess improved cytotoxicity profiles.

Erythropoiesis in the Rps19 disrupted mouse: Analysis of erythropoietin response and biochemical markers for Diamond-Blackfan anemia.

The human ribosomal protein S19 gene (RPS19) is mutated in approximately 20% of patients with Diamond-Blackfan anemia (DBA), a congenital disease with a specific defect in erythropoiesis. The clinical expression of DBA is highly variable, and subclinical phenotypes may be revealed by elevated erythrocyte deaminase (eADA) activity only. In mice, complete loss of Rps19 results in early embryonic lethality whereas Rps19+/- mice are viable and without major abnormalities including the hematopoietic system. We have performed a detailed analysis of the Rps19+/- mice. We estimated the Rps19 levels in hematopoietic tissues and we analyzed erythrocyte deaminase activity and globin isoforms which are used as markers for DBA. The effect of a disrupted Rps19 allele on a different genetic background was investigated as well as the response to erythropoietin (EPO). From our results, we argue that the loss of one Rps19 allele in mice is fully compensated for at the transcriptional level with preservation of erythropoiesis.

Effects of HsRad51 overexpression on cell proliferation, cell cycle progression, and apoptosis.

Expression of the DNA repair and recombination protein human Rad51 (HsRad51) is increased in transformed cells and in cancer cell lines. In order to study the effects of acute HsRad51 ectopic overexpression on cell proliferation, cell cycle progression, and apoptosis, we generated clones of the human fibrosarcoma cell line HT1080 carrying a HsRad51 transgene under a repressible promoter. The HsRad51-overexpressing cells showed decreased plating efficiency and growth rate in a dose-dependent manner with regard to the degree of overexpression. An accumulation of HsRad51-overexpressing cells in G(2) was observed following release of cells after synchronization with double thymidine block. Moreover, the fraction of apoptotic cells measured by annexin V-FACS increased with the time of HsRad51 overexpression. In the light of these observations, sustained increased levels of HsRad51 may contribute to tumor progression by causing a selection for cells tolerant to the growth-suppressive and apoptosis-inducing effects of acute HsRad51 overexpression.

Caspase-3 mediated cleavage of HsRad51 at an unconventional site.

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41-kDa full-length protein into a 33-kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S-100 extract. In this study, site-directed mutagenesis was used for mapping of the cleavage site to AQVD274 downward arrow G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine-treated apoptotic MCF-7 cells, which lack caspase-3, indicates that caspase-3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33-kDa fragment was generated by recombinant caspase-3 and -7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 downward arrow G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33-kDa product by recombinant caspase-3, whereas caspase-7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 downward arrow G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 downward arrow N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 downward arrow G and DVLD187 downward arrow N cleavage products of HsRad51 appeared at equal concentrations of caspase-3. Moreover both fragments were generated by induction of apoptosis in MDA-MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.

p53 splice acceptor site mutation and increased HsRAD51 protein expression in Bloom's syndrome GM1492 fibroblasts.

GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.

DNA double-strand break repair, DNA-PK, and DNA ligases in two human squamous carcinoma cell lines with different radiosensitivity.

PURPOSE: Variation in sensitivity to radiotherapy among tumors has been related to the capacity of cells to repair radiation-induced DNA double-strand breaks (DSBs). DNA-dependent protein kinase (DNA-PK) and DNA ligases may affect DNA dsb rejoining. This study was performed to compare rate of rejoining of radiation-induced DSBs, DNA-PK, and DNA ligase activities in two human squamous carcinoma cell lines with different sensitivity to ionizing radiation. METHODS AND MATERIALS: Cell survival of two human squamous carcinoma cell lines, UM-SCC-1 and UM-SCC-14A, was determined by an in vitro clonogenic assay. DSB rejoining was studied using pulsed field gel electrophoresis (PFGE). DNA-PK activity was determined using BIOTRAK DNA-PK enzyme assay system (Amersham). DNA ligase activity in crude cell extracts was measured using [5'-33P] Poly (dA) x (oligo (dT) as a substrate. Proteolytic degradation of proteins was analyzed by means of Western blotting. RESULTS: Applying the commonly used linear-quadratic equation to describe cell survival, S = e-alphaD-betaD2, the two cell lines roughly have the same alpha value (approximately 0.40 Gy(-1)) whereas the beta value was considerably higher in UM-SCC-14A (0.067 Gy(-2)+/-0.007 Gy(-2) [SEM]) as compared to UM-SCC-1 (0.013 Gy(-2)+/-0.004 Gy(-2) [SEM]). Furthermore, UM-SCC-1 was more proficient in rejoining of X-ray-induced DSBs as compared to UM-SCC-14A as quantified by PFGE. The constitutive level of DNA-PK activity was 1.6 times higher in UM-SCC-1 as compared to UM-SCC-14A ( < 0.05). The constitutive level of DNA ligase activity was similar in the two cell lines. CONCLUSIONS: The results suggest that the proficiency in rejoining of DSBs is associated with DNA-PK activity but not with total DNA ligase activity.

Proteolytic cleavage of HsRad51 during apoptosis.

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T-lymphocyte cell lines, Jurkat and PFI-285. Apoptosis was induced by camptothecin or anti-Fas monoclonal antibody (anti-Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP-ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S-100 extract in a cell-free system. In each case, cleavage of HsRad51 was abolished by acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase-2, -3 or -6 to -10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA-dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.

P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro.

To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.

Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes.

The S. cerevisiae RAD51 gene product exerts important functions in meiotic and mitotic recombination, as well as in the repair of DNA double-strand breaks. We have studied the expression of the human RAD51 (HsRAD51) gene in primary human peripheral blood lymphocytes (PBLs). The HsRAD51 mRNA level increased three fold in mitogen stimulated PBLs, with a peak in the late S phase. A five fold increase of HsRAD51 protein levels was observed in late G2. Specific inhibition of DNA synthesis with aphidicolin did not block the induction of the HsRAD51 protein, indicating that HsRAD51 expression is independent of DNA replication. In contrast, after inhibition of RNA synthesis with actinomycin D and protein synthesis with cycloheximide, the HsRAD51 protein level decreased rapidly. Taken together, these results indicate that the HsRAD51 gene is transcriptionally regulated in human PBLs, and exerts its function during the late S and G2 phases of the cell cycle.