Scorpion venom heat-resistant peptide alleviates mitochondrial dynamics imbalance induced by PM2.5 exposure by downregulating the PGC-1α/SIRT3 signaling pathway.

Background: Epidemiological inquiry reveals that neuroinflammation and mitochondrial dysfunction caused by PM2.5 exposure are associated with Alzheimer's disease. Nevertheless, the molecular mechanisms of mitochondrial dynamics and neuroinflammation induced by PM2.5 exposure remain elusive. In this study, our objective was to explore the impact of PM2.5 on mitochondrial dynamics and neuroinflammation, while also examining the reparative potential of scorpion venom heat-resistant synthetic peptide (SVHRSP). Methods: Western blot and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were employed to ascertain the protein and gene levels of IL-1ß, IL-6, and TNF-α in BV2 cells. The concentration of IL-6 in the supernatant of the BV2 cell culture was measured by enzyme-linked immunosorbent assay. For the assessment of mitochondrial homeostasis, western blot, RT-qPCR, and cellular immunohistochemistry methods were utilized to investigate the protein and gene levels of DRP1 and MFN-2 in HT22 cells. In the context of signal pathway analyses, western blot, RT-qPCR, and immunofluorescence techniques were employed to detect the protein and gene expressions of PGC-1α and SIRT3 in HT22 cells, respectively. Following the transfection with siPGC-1αRNA, downstream proteins of PGC-1α/SIRT3 pathway in HT22 cells were investigated by Western blot and RT-qPCR. Results: The experimental findings demonstrated that exposure to PM2.5 exacerbated neuroinflammation, resulting in elevated levels of IL-1ß, IL-6, and TNF-α. Furthermore, it perturbed mitochondrial dynamics, as evidenced by increased DRP1 expression and decreased MFN-2 expression. Additionally, dysfunction was observed in the PGC-1α/SIRT3 signal pathway. However, intervention with SVHRSP ameliorated the cellular damage induced by PM2.5 exposure. Conclusions: SVHRSP alleviated neuroinflammation and mitochondrial dynamics imbalance induced by PM2.5 exposure by downregulating the PGC-1α/SIRT3 signaling pathway.

The tumor suppressor OVCA1 is a short half-life protein degraded by the ubiquitin-proteasome pathway.

Ovarian cancer gene 1 (OVCA1) is a tumor suppressor associated with ovarian cancer, which is involved in cell proliferation regulation, embryonic development and tumorigenesis. Loss of heterozygosity in the OVCA1 gene occurs in 50-86% of cases of ovarian cancer; however, the physiological and biochemical functions of OVCA1 are not yet clear. In the present study, the stability and degradation of OVCA1 were investigated in A2780, Hela and 293 cells. The results revealed that the OVCA1 protein was unstable by MG132 inhibiting proteasome mediated degradation, co-immunoprecipitation and half-life measurement experiments. The cellular protein levels of endogenous OVCA1 were too low to be detected by western blotting. In addition, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal inhibited the degradation of OVCA1 in cells. The co-immunoprecipitation assay revealed that the OVCA1 protein interacted with ubiquitin to form a poly-ubiquitinated complex in cells. The half-life of OVCA1, measured by inhibiting protein synthesis with cycloheximide, was <2 h. The present study demonstrated that OVCA1 may be degraded by the ubiquitin-mediated proteasome pathway and may be considered a short half-life protein. In conclusion, the regulation of OVCA1 protein degradation via the ubiquitin-proteasome pathway may represent a novel direction in the development of ovarian cancer therapy.

Cell Recognition Molecule L1 Regulates Cell Surface Glycosylation to Modulate Cell Survival and Migration.

Background: Cell recognition molecule L1 (L1) plays an important role in cancer cell differentiation, proliferation, migration and survival, but its mechanism remains unclear. Methodology/Principal: Our previous study has demonstrated that L1 enhanced cell survival and migration in neural cells by regulating cell surface glycosylation. In the present study, we show that L1 affected cell migration and survival in CHO (Chinese hamster ovary) cell line by modulation of sialylation and fucosylation at the cell surface via the PI3K (phosphoinositide 3-kinase) and Erk (extracellularsignal-regulated kinase) signaling pathways. Flow cytometry analysis indicated that L1 modulated cell surface sialylation and fucosylation in CHO cells. Activated L1 upregulated the protein expressions of ST6Gal1 (ß-galactoside α-2,6-sialyltransferase 1) and FUT9 (Fucosyltransferase 9) in CHO cells. Furthermore, activated L1 promoted CHO cells migration and survival as shown by transwell assay and MTT assay. Inhibitors of sialylation and fucosylation blocked L1-induced cell migration and survival, while decreasing FUT9 and ST6Gal1 expressions via the PI3K-dependent and Erk-dependent signaling pathways. Conclusion : L1 modulated cell migration and survival by regulation of cell surface sialylation and fucosylation via the PI3K-dependent and Erk-dependent signaling pathways.

Development and validation of an UPLC-Q/TOF-MS assay for the quantitation of neopanaxadiol in beagle dog plasma: Application to a pharmacokinetic study.

Neopanaxadiol (NPD), the main panaxadiol constituent of Panax ginseng C. A. Meyer (Araliaceae), has been regarded as the active component for the treatment of Alzheimer's disease. However, few references are available about pharmacokinetic evaluation for NPD. Accordingly, a rapid and sensitive method for quantitative analysis of NPD in beagle dog plasma based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry was developed and validated. Analytes were extracted from plasma by liquid-liquid extraction and chromatographic separation was achieved on an Agilent Zorbax Stable Bond C18 column. Detection was performed in the positive ion mode using multiple reaction monitoring of the transitions both at m/z 461.4 → 425.4 for NPD and internal standard of panaxadiol. All validation parameters, such as lower limit of quantitation, linearity, specificity, precision, accuracy, extraction recovery, matrix effect and stability, were within acceptable ranges and the method was appropriate for multitude sample determination. After oral intake, NPD was slowly absorbed and eliminated from circulatory blood system and corresponding plasma exposure was low. Application of this quantitative method will yield the first pharmacokinetic profile after oral administration of NPD to beagle dog. The information obtained here will be useful to understand the pharmacological effects of NPD.

Fucosyltransferase IV (FUT4) as an effective biomarker for the diagnosis of breast cancer.

Specific enzymes are involved in altered glycosylation of cancer. Fucosyltransferase IV (FUT4) is associated with the proliferation and metastasis of breast cancer. The application of FUT4 assay in the serum has not been reported yet. Here, the expression level of FUT4 in the breast cancer patient's tissues (n=60) was analyzed by immunohistochemistry (IHC) and the secreted FUT4 in blood serum samples (n=225) was detected by enzyme-linked immunosorbent assay (ELISA). Using low metastatic MCF-7 and high metastatic MDA-MB-231 breast cancer cell lines, FUT4 expression was also detected by reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunofluorescent staining. The conventional cancer biomarkers cancer antigen (CA15.3) and carcinoembryonic antigen (CEA) was analyzed by Elecsys-electrochemical immune assay (ECLIA) to compare specificity and sensitivity with that of FUT4. We have observed a significant high expression of FUT4 in breast cancer tissues and serums as compared to the normal tissues (P<0.01) and control serums (P<0.05). FUT4 expression was increased in MDA-MB-231 cells vs. that in MCF-7 cells. Furthermore, the results of receiver operating characteristic (ROC) analysis was shown, area under curve of FUT4 (AUC=0.784) was higher than that of CA15.3 (AUC=0.468) and CEA (AUC=0.563). The relation analysis is indicated FUT4 is significantly correlated with CA15.3 (r=0.234, P<0.05) and there is no significant correlation with CEA. In conclusion, this study suggests that FUT4 can serve as novel biomarker in the diagnosis and prognosis of breast cancer.

Pim1 serine/threonine kinase regulates the number and functions of murine hematopoietic stem cells.

The genes and pathways that govern the functions and expansion of hematopoietic stem cells (HSC) are not completely understood. In this study, we investigated the roles of serine/threonine Pim kinases in hematopoiesis in mice. We generated PIM1 transgenic mice (Pim1-Tx) overexpressing human PIM1 driven by vav hematopoietic promoter/regulatory elements. Compared to wild-type littermates, Pim1-Tx mice showed enhanced hematopoiesis as demonstrated by increased numbers of Lin(-) Sca-1 (+) c-Kit (+) (LSK) hematopoietic stem/progenitor cells and cobblestone area forming cells, higher BrdU incorporation in long-term HSC population, and a better ability to reconstitute lethally irradiated mice. We then extended our study using Pim1(-/-), Pim2(-/-), Pim3(-/-) single knockout (KO) mice. HSCs from Pim1(-/-) KO mice showed impaired long-term hematopoietic repopulating capacity in secondary and competitive transplantations. Interestingly, these defects were not observed in HSCs from Pim2(-/-) or Pim3(-/-) KO mice. Limiting dilution competitive transplantation assay estimated that the frequency of LSKCD34(-) HSCs was reduced by approximately 28-fold in Pim1(-/-) KO mice compared to wild-type littermates. Mechanistic studies demonstrated an important role of Pim1 kinase in regulating HSC cell proliferation and survival. Finally, our polymerase chain reaction (PCR) array and confirmatory real-time PCR (RT-PCR) studies identified several genes including Lef-1, Pax5, and Gata1 in HSCs that were affected by Pim1 deletion. Our data provide the first direct evidence for the important role of Pim1 kinase in the regulation of HSCs. Our study also dissects out the relative role of individual Pim kinase in HSC functions and regulation.

The BH3 mimetic ABT-737 induces cancer cell senescence.

ABT-737, a small molecule cell-permeable Bcl-2 antagonist that acts by mimicking BH3 proteins, induces apoptotic cell death in multiple cancer types. However, when incubated with this agent many solid tumor cell lines do not undergo apoptosis. The current study reveals a novel mechanism whereby ABT-737 when added to apoptosis-resistant cancer cells has profound biologic effects. In PV-10 cells, a renal cell carcinoma that does not die after ABT-737 treatment, this agent induces a two-fold change in the transcription of nearly 430 genes. Many of these induced mRNA changes are in secreted proteins, IL-6, IL-8, and IL-11 and chemokines CXCL2 and CXCL5, or genes associated with an "inflammatory" phenotype. Strikingly, these gene changes are highly similar to those changes previously identified in cellular senescence. Brief exposure of apoptosis-resistant renal, lung and prostate cancer cell lines to ABT-737, although not capable of inducing cell death, causes the induction of senescence-associated ß-galactosidase and inhibition of cell growth consistent with the induction of cellular senescence. Evidence indicates that the induction of senescence occurs as a result of reactive oxygen species elevation followed by low-level activation of the caspase cascade, insufficient to induce apoptosis, but sufficient to lead to minor DNA damage and increases in p53, p21, IL-6 and 8 proteins. By overexpression of a dominant-negative p53 protein, we show that ABT-737-induced cellular senescence is p53-dependent. Thus, in multiple cancer types in which ABT-737 is incapable of causing cell death, ABT-737 may have additional cellular activities that make its use as an anticancer agent highly attractive.

The Pim protein kinases regulate energy metabolism and cell growth.

The serine/threonine Pim kinases are overexpressed in solid cancers and hematologic malignancies and promote cell growth and survival. Here, we find that a novel Pim kinase inhibitor, SMI-4a, or Pim-1 siRNA blocked the rapamycin-sensitive mammalian target of rapamycin (mTORC1) activity by stimulating the phosphorylation and thus activating the mTORC1 negative regulator AMP-dependent protein kinase (AMPK). Mouse embryonic fibroblasts (MEFs) deficient for all three Pim kinases [triple knockout (TKO) MEFs] demonstrated activated AMPK driven by elevated ratios of AMPATP relative to wild-type MEFs. Consistent with these findings, TKO MEFs were found to grow slowly in culture and have decreased rates of protein synthesis secondary to a diminished amount of 5'-cap-dependent translation. Pim-3 expression alone in TKO MEFs was sufficient to reverse AMPK activation, increase protein synthesis, and drive MEF growth similar to wild type. Pim-3 expression was found to markedly increase the protein levels of both c-Myc and the peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), enzymes capable of regulating glycolysis and mitochondrial biogenesis, which were diminished in TKO MEFs. Overexpression of PGC-1α in TKO MEFs elevated ATP levels and inhibited the activation of AMPK. These results demonstrate the Pim kinase-mediated control of energy metabolism and thus regulation of AMPK activity. We identify an important role for Pim-3 in modulating c-Myc and PGC-1α protein levels and cell growth.

Regulation of Skp2 levels by the Pim-1 protein kinase.

The Pim-1 protein kinase plays an important role in regulating both cell growth and survival and enhancing transformation by multiple oncogenes. The ability of Pim-1 to regulate cell growth is mediated, in part, by the capacity of this protein kinase to control the levels of the p27, a protein that is a critical regulator of cyclin-dependent kinases that mediate cell cycle progression. To understand how Pim-1 is capable of regulating p27 protein levels, we focused our attention on the SCF(Skp2) ubiquitin ligase complex that controls the rate of degradation of this protein. We found that expression of Pim-1 increases the level of Skp2 through direct binding and phosphorylation of multiple sites on this protein. Along with known Skp2 phosphorylation sites including Ser(64) and Ser(72), we have identified Thr(417) as a unique Pim-1 phosphorylation target. Phosphorylation of Thr(417) controls the stability of Skp2 and its ability to degrade p27. Additionally, we found that Pim-1 regulates the anaphase-promoting complex or cyclosome (APC/C complex) that mediates the ubiquitination of Skp2. Pim-1 phosphorylates Cdh1 and impairs binding of this protein to another APC/C complex member, CDC27. These modifications inhibit Skp2 from degradation. Marked increases in Skp2 caused by these mechanisms lower cellular p27 levels. Consistent with these observations, we show that Pim-1 is able to cooperate with Skp2 to signal S phase entry. Our data reveal a novel Pim-1 kinase-dependent signaling pathway that plays a crucial role in cell cycle regulation.

p53-dependent induction of prostate cancer cell senescence by the PIM1 protein kinase.

The PIM family of serine threonine protein kinases plays an important role in regulating both the growth and transformation of malignant cells. However, in a cell line-dependent manner, overexpression of PIM1 can inhibit cell and tumor growth. In 22Rv1 human prostate cells, but not in Du145 or RWPE-2, PIM1 overexpression was associated with marked increases in cellular senescence, as shown by changes in the levels of beta-galactosidase (SA-beta-Gal), p21, interleukin (IL)-6 and IL-8 mRNA and protein. During early cell passages, PIM1 induced cellular polyploidy. As the passage number increased, markers of DNA damage, including the level of gammaH2AX and CHK2 phosphorylation, were seen. Coincident with these DNA damage markers, the level of p53 protein and genes transcriptionally activated by p53, such as p21, TP53INP1, and DDIT4, increased. In these 22Rv1 cells, the induction of p53 protein was associated not only with senescence but also with a significant level of apoptosis. The importance of the p53 pathway to PIM1-driven cellular senescence was further shown by the observation that expression of dominant-negative p53 or shRNA targeting p21 blocked the PIM1-induced changes in the DNA damage response and increases in SA-beta-Gal activity. Likewise, in a subcutaneous tumor model, PIM1-induced senescence was rescued when the p53-p21 pathways are inactivated. Based on these results, PIM1 will have its most profound effects on tumorigenesis in situations where the senescence response is inactivated.

A small molecule inhibitor of Pim protein kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma.

The serine/threonine Pim kinases are up-regulated in specific hematologic neoplasms, and play an important role in key signal transduction pathways, including those regulated by MYC, MYCN, FLT3-ITD, BCR-ABL, HOXA9, and EWS fusions. We demonstrate that SMI-4a, a novel benzylidene-thiazolidine-2, 4-dione small molecule inhibitor of the Pim kinases, kills a wide range of both myeloid and lymphoid cell lines with precursor T-cell lymphoblastic leukemia/lymphoma (pre-T-LBL/T-ALL) being highly sensitive. Incubation of pre-T-LBL cells with SMI-4a induced G1 phase cell-cycle arrest secondary to a dose-dependent induction of p27(Kip1), apoptosis through the mitochondrial pathway, and inhibition of the mammalian target of rapamycin C1 (mTORC1) pathway based on decreases in phospho-p70 S6K and phospho-4E-BP1, 2 substrates of this enzyme. In addition, treatment of these cells with SMI-4a was found to induce phosphorylation of extracellular signal-related kinase1/2 (ERK1/2), and the combination of SMI-4a and a mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor was highly synergistic in killing pre-T-LBL cells. In immunodeficient mice carrying subcutaneous pre-T-LBL tumors, treatment twice daily with SMI-4a caused a significant delay in the tumor growth without any change in the weight, blood counts, or chemistries. Our data suggest that inhibition of the Pim protein kinases may be developed as a therapeutic strategy for the treatment of pre-T-LBL.

The high-mobility group A1a/signal transducer and activator of transcription-3 axis: an achilles heel for hematopoietic malignancies?

Although HMGA1 (high-mobility group A1; formerly HMG-I/Y) is an oncogene that is widely overexpressed in aggressive cancers, the molecular mechanisms underlying transformation by HMGA1 are only beginning to emerge. HMGA1 encodes the HMGA1a and HMGA1b protein isoforms, which function in regulating gene expression. To determine how HMGA1 leads to neoplastic transformation, we looked for genes regulated by HMGA1 using gene expression profile analysis. Here, we show that the STAT3 gene, which encodes the signaling molecule signal transducer and activator of transcription 3 (STAT3), is a critical downstream target of HMGA1a. STAT3 mRNA and protein are up-regulated in fibroblasts overexpressing HMGA1a and activated STAT3 recapitulates the transforming activity of HMGA1a in fibroblasts. HMGA1a also binds directly to a conserved region of the STAT3 promoter in vivo in human leukemia cells by chromatin immunoprecipitation and activates transcription of the STAT3 promoter in transfection experiments. To determine if this pathway contributes to HMGA1-mediated transformation, we investigated STAT3 expression in our HMGA1a transgenic mice, all of which developed aggressive lymphoid malignancy. STAT3 expression was increased in the leukemia cells from our transgenics but not in control cells. Blocking STAT3 function induced apoptosis in the transgenic leukemia cells but not in controls. In primary human leukemia samples, there was a positive correlation between HMGA1a and STAT3 mRNA. Moreover, blocking STAT3 function in human leukemia or lymphoma cells led to decreased cellular motility and foci formation. Our results show that the HMGA1a-STAT3 axis is a potential Achilles heel that could be exploited therapeutically in hematopoietic and other malignancies overexpressing HMGA1a.

[Dynamic changes of microtubule in parthenogenetic and in vitro fertilized preimplantation embryos in mouse.].

In this study we detected dynamic changes and function of beta-tubulin, a subtype of microtubule, during the first cleavage period in mouse parthenogenetic and in vitro fertilized embryos. Firstly, we compared the developmental potential of in vitro fertilized, parthenogenetic, and in vivo fertilized embryos in culture. Then, the dynamic changes of beta-tubulin and nucleus in parthenogenetic and in vitro fertilized preimplantation embryos were detected by immunofluorescence and confocal microscopy to analyze the role of microtubules in meiotic division and embryonic development. The results indicated that the development rate of in vivo fertilized embryos was significantly higher than that of in vitro fertilized or parthenogenetic embryos (P<0.05). However, there was no significant difference in developmental potential between in vitro fertilized and parthenogenetic embryos. During in vitro fertilization, oocyte was activated when sperm entered it. Oocyte resumed the second meiotic division. Condensed maternal chromosomes aligning at the equator of the spindle were pulled to the spindle poles by kinetochore microtubules in anaphase. Furthermore, in telophase, there were microtubules between the two sets of decondensed maternal chromosomes. One set formed the second polar body (Pb(2)), which was extruded to the perivitelline space. The other set formed female pronucleus. Meanwhile, 5-8 h after fertilization, sperm chromatin condensed and decondensed to form male pronucleus. Microtubule composed mesosome and cytaster remodeling around male and female pronuclei to form long microtubules, which pull the pronuclei to get close. During 4-6 h parthenogenetic activation, SrCl(2) activated oocytes to resume meiosis. As a consequence, sister chromatids were pulled to spindle poles. Cytochalasin B, which was applied in the medium, inhibited the extrusion of Pb(2). Two haploid pronuclei in the cytoplasm were connected by microtubules. Compared with that in in vitro fertilization, oocyte is easier to be activated in parthenogenetic activation. Chemical activation is more efficient than sperm penetration in in vitro fertilization as indicated by earlier and better remodeling of the microtubules.

Retroviral insertional mutagenesis identifies genes that collaborate with NUP98-HOXD13 during leukemic transformation.

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

Notch1 mutations are important for leukemic transformation in murine models of precursor-T leukemia/lymphoma.

NOTCH1 is frequently mutated in human precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). In the current study, we found that 13 of 19 cell lines and 29 of 49 primary tumors from SCL/LMO1, OLIG2/LMO1, OLIG2, LMO1, NUP98/HOXD13, and p27(-/-)/SMAD3(+/-) mice had Notch1 mutations in either the heterodimerization (HD) or the glutamic acid/serine/threonine (PEST) domain but not both. Thymocytes from clinically healthy SCL/LMO1 mice aged 5 weeks did not have Notch1 mutations, whereas thymocytes from clinically healthy SCL/LMO1 mice aged 8 to 12 weeks did have Notch1 mutations and formed tumors upon transplantation into nude mice. Remarkably, all of the HD domain mutations that we identified were single-base substitutions, whereas all of the PEST domain mutations were insertions or deletions, half of which mapped to 1 of 2 mutational "hot spots." Taken together, these findings indicate that Notch1 mutations are very frequent events that are acquired relatively early in the process of leukemic transformation and are important for leukemic cell growth.

OLIG2 (BHLHB1), a bHLH transcription factor, contributes to leukemogenesis in concert with LMO1.

OLIG2 (originally designated BHLHB1) encodes a transcription factor that contains the basic helix-loop-helix motif. Although expression of OLIG2 is normally restricted to neural tissues, overexpression of OLIG2 has been shown in patients with precursor T-cell lymphoblastic lymphoma/leukemia (pre-T LBL). In the current study, we found that overexpression of OLIG2 was not only found in oligodendroglioma samples and normal neural tissue but also in a wide spectrum of malignant cell lines including leukemia, non-small cell lung carcinoma, melanoma, and breast cancer cell lines. To investigate whether enforced expression of OLIG2 is oncogenic, we generated transgenic mice that overexpressed OLIG2 in the thymus. Ectopic OLIG2 expression in the thymus was only weakly oncogenic as only 2 of 85 mice developed pre-T LBL. However, almost 60% of transgenic mice that overexpressed both OLIG2 and LMO1 developed pre-T LBL with large thymic tumor masses. Gene expression profiling of thymic tumors that developed in OLIG2/LMO1 mice revealed up-regulation of Notch1 as well as Deltex1 (Dtx1) and pre T-cell antigen receptor alpha (Ptcra), two genes that are considered to be downstream of Notch1. Of note, we found mutations in the Notch1 heterodimerization or proline-, glutamic acid-, serine-, and threonine-rich domain in three of six primary thymic tumors. In addition, growth of leukemic cell lines established from OLIG2/LMO1 transgenic mice was suppressed by a gamma-secretase inhibitor, suggesting that Notch1 up-regulation is important for the proliferation of OLIG2-LMO1 leukemic cells.

NUP98-HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia.

The myelodysplastic syndromes (MDSs) are a group of clonal hematopoietic stem-cell disorders characterized by ineffective hematopoiesis and dysplasia. A wide spectrum of genetic aberrations has been associated with MDS, including chromosomal translocations involving the NUP98 gene. Using a NUP98-HOXD13 fusion gene, we have developed a mouse model that faithfully recapitulates all of the key features of MDS, including peripheral blood cytopenias, bone marrow dysplasia, and apoptosis, and transformation to acute leukemia. The MDS that develops in NUP98-HOXD13 transgenic mice is uniformly fatal. Within 14 months, all of the mice died of either leukemic transformation or severe anemia and leucopenia as a result of progressive MDS. The NUP98-HOXD13 fusion gene inhibits megakaryocytic differentiation and increases apoptosis in the bone marrow, suggesting a mechanism leading to ineffective hematopoiesis in the presence of a hypercellular bone marrow. These mice provide an accurate preclinical model that can be used for the evaluation of MDS therapy and biology.

Distinct mechanisms lead to HPRT gene mutations in leukemic cells.

Leukemias are considered malignant clonal disorders arising from the accumulation of mutations in hematopoietic cells; the majority of these mutations are thought to be acquired somatically. Measurement of mutation frequency (Mf) at the hypoxanthine phosphoribosyltransferase (HPRT) locus has been developed as a method for estimating genomic instability. We investigated the Mf in 16 leukemic cell lines to determine whether these cell lines showed evidence of genomic instability. Although some leukemic cell lines had markedly elevated Mfs, the Mfs at the HPRT locus in leukemic cell lines were not always higher than those of B-lymphoblastoid cell lines and T lymphocytes from normal individuals. We were able to identify the HPRT mutation for 159 of 160 individual HPRT mutants. The HPRT mutations were characterized at a molecular level and classified as either gross chromosomal rearrangements (GCRs) or point mutations, such as single-nucleotide substitutions, insertions, or deletions. With rare exceptions, individual leukemic cell lines showed either point mutations or GCR, but not both. Of note, all the cell lines that primarily showed point mutations are known to be defective in mismatch repair machinery.

Leukemic transformation.

Leukemia results from the uncontrolled accumulation of primitive, poorly differentiated blood cells, and is a consequence of the accumulation of mutations in hematopoietic precursor cells. These mutations include point mutations (single base pair insertions, deletions, or substitutions), gross chromosomal rearrangements such as deletions, insertions, amplifications, and translocations, and epigenetic changes. It seems likely that mutations affecting at least two pathways are required for the development of leukemia. One of these pathways regulates cell accumulation; the second regulates hematopoietic differentiation. Molecularly targeted therapy, which interrupts functions of the leukemogenic proteins generated by mutations, has been developed and shown to be effective for several forms of malignancy. Therefore, it is our hope and belief that a clearer understanding of the mechanism(s) that underlie leukemic transformation will lead to effective new therapies for this dreaded disease.