技术资料

Zoledronic acid,a third-generation bisphosphonate,has been shown to reduce cell migration,invasion,and metastasis. However,the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT),a cellular process essential to the metastatic cascade,remain unclear. Therefore,the effects of zoledronic acid on EMT,using triple-negative breast cancer (TNBC) cells as a model system,were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers,N-cadherin,Twist,and Snail,and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability,induced cell-cycle arrest,and decreased the proliferative capacity of TNBC,suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype,the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins,BMI-1 and Oct-4,and both prevented and eliminated mammosphere formation. To understand the mechanism of these results,the effect of zoledronic acid on established EMT regulator NF-$$B was investigated. Zoledronic acid inhibited phosphorylation of RelA,the active subunit of NF-$$B,at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter,providing a direct link between inactivation of NF-$$B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased,correlating modulation of EMT to decreased self-renewal. On the basis of these results,it is proposed that through inactivation of NF-$$B,zoledronic acid reverses EMT,which leads to a decrease in self-renewal. View Publication

We investigated the expression status of periostin in breast cancer stem cells and its clinical implications in order to lay a foundation for managing breast cancer. CD44+/CD24-/line- tumor cells (CSC) from clinical specimens were sorted using flow cytometry. Periostin expression status was detected in CSC cells and 1,086 breast cancer specimens by Western blot and immunohistochemistry staining,with the CSC ratio determined by immunofluorescence double staining. The relationship between the periostin protein and clinico-pathological parameters and prognosis was subsequently determined. As a result,CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. Periostin protein was expressed higher in CSC cells compared to the control cells and was found to be related to CSC chemotherapy resistance. Moreover,periostin expression was found to be related to the CSC ratio in 1,086 breast cancer specimens (P = 0.001). In total,334 (30.76%) of the 1,086 breast cases showed high periostin expression. After universal and Spearman regression correlation analysis,periostin was observed to be related to histological grade,CSC ratio,lymph node metastasis,tumor size,and triple-negative breast cancer (all Ptextless0.05). Furthermore,periostin was shown to attain a significantly more distant bone metastasis and worse disease-specific survival than those with none or low-expressed periostin protein (P = 0.001). In the Cox regression test,periostin protein was detected as an independent prognostic factor (P = 0.001). In conclusion,periostin was found to be related to the CSC and an independent prognostic factor for breast cancer. It is also perhaps a potential target to breast cancer. View Publication

INTRODUCTION: Retinoic acid signaling plays key roles in embryonic development and in maintaining the differentiated status of adult tissues. Recently,the nuclear retinoic acid receptor (RAR) isotypes α,β and γ were found to play specific functions in the expansion and differentiation of the stem compartments of various tissues. For instance,RARγ appears to be involved in stem cell compartment expansion,while RARα and RARβ are implicated in the subsequent cell differentiation. We found that over-expressing c-Myc in normal mouse mammary epithelium and in a c-Myc-driven transgenic model of mammary cancer,disrupts the balance between RARγ and RARα/β in favor of RARγ. METHODS: The effects of c-Myc on RAR isotype expression were evaluated in normal mouse mammary epithelium,mammary tumor cells obtained from the MMTV-Myc transgenic mouse model as well as human normal immortalized breast epithelial and breast cancer cell lines. The in vivo effect of the RARα-selective agonist 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carboxamido]benzoic acid (Am580) was examined in the MMTV-Myc mouse model of mammary tumorigenesis. RESULTS: Modulation of the RARα/β to RARγ expression in mammary glands of normal mice,oncomice,and human mammary cell lines through the alteration of RAR-target gene expression affected cell proliferation,survival and tumor growth. Treatment of MMTV-Myc mice with the RARα-selective agonist Am580 led to significant inhibition of mammary tumor growth (˜90%,Ptextless0.001),lung metastasis (Ptextless0.01) and extended tumor latency in 63% of mice. Immunocytochemical analysis showed that in these mice,RARα responsive genes such as Cyp26A1,E-cadherin,cellular retinol-binding protein 1 (CRBP1) and p27,were up-regulated. In contrast,the mammary gland tumors of mice that responded poorly to Am580 treatment (37%) expressed significantly higher levels of RARγ. In vitro experiments indicated that the rise in RARγ was functionally linked to promotion of tumor growth and inhibition of differentiation. Thus,activation of the RARα pathway is linked to tumor growth inhibition,differentiation and cell death. CONCLUSIONS: The functional consequence of the interplay between c-Myc oncogene expression and the RARγ to RARα/β balance suggests that prevalence of RARγ over-RARα/β expression levels in breast cancer accompanied by c-Myc amplification or over-expression in breast cancer should be predictive of response to treatment with RARα-isotype-specific agonists and warrant monitoring during clinical trials. View Publication

BACKGROUND Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. DESIGN AND METHODS OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. RESULTS Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold,respectively,without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D,natural cytotoxicity receptors,and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred,expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion,persisted up to 4 weeks,were readily detectable in the human bone,inhibited myeloma growth and protected bone from myeloma-induced osteolysis. CONCLUSIONS These studies provide the rationale for testing expanded natural killer cells in humans. View Publication

Fanconi anemia (FA) is a rare inherited genomic instability syndrome representing one of the best examples of hematopoietic stem cell deficiency. Although FA might be an excellent candidate for bone marrow (BM) genetic correction ex vivo,knockout animal models are not sufficient to guide preclinical steps,and gene therapy attempts have proven disappointing so far. Contributing to these poor results is a characteristic and dramatic early BM-cells die-off when placed in culture. We show here that human primary FA BM cell survival can be ameliorated by using specific culture conditions that limit oxidative stress. When coupled with retrovirus-mediated transfer of the main complementation group FANCA-cDNA,we could achieve long-term reconstitution of the stem cell compartment both in vitro and in vivo. Gene-corrected BM cultures grew for textgreater120 days,and after cultured cell transplantation into NOD/SCID mice,clonogenic human cells carrying the FANCA transgene could be detected 6 months after transduction. By comparison,untransduced cells died in culture by 15 days. Of necessity for ethical reasons,experiments were conducted on a very limited number of primary BM cells. By using low cytokine regimen and conditions matching regulatory requirements,a contingent of gene-corrected cells slowly emerges with an unmet potential for in vivo engraftment. Future therapeutic applications of stem cells might be expanding from these data. In addition,we provide a model of gene-corrected human primary cell growth that carries the potential to better delineate the combined role of both DNA damage and oxidative stress in the pathogenesis of FA. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23,MYB was a good candidate gene. Our molecular investigations,based on fluorescence in situ hybridization and rapid amplification of cDNA ends,revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together,these results establish,for the first time,a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia. View Publication

Oligomerization is an important regulatory mechanism for many proteins,including oncoproteins and other pathogenic proteins. The oncoprotein Bcr-Abl relies on oligomerization via its coiled coil domain for its kinase activity,suggesting that a designed coiled coil domain with enhanced binding to Bcr-Abl and reduced self-oligomerization would be therapeutically useful. Key mutations in the coiled coil domain of Bcr-Abl were identified that reduce homo-oligomerization through intermolecular charge-charge repulsion yet increase interaction with the Bcr-Abl coiled coil through additional salt bridges,resulting in an enhanced ability to disrupt the oligomeric state of Bcr-Abl. The mutations were modeled computationally to optimize the design. Assays performed in vitro confirmed the validity and functionality of the optimal mutations,which were found to exhibit reduced homo-oligomerization and increased binding to the Bcr-Abl coiled coil domain. Introduction of the mutant coiled coil into K562 cells resulted in decreased phosphorylation of Bcr-Abl,reduced cell proliferation,and increased caspase-3/7 activity and DNA segmentation. Importantly,the mutant coiled coil domain was more efficacious than the wild type in all experiments performed. The improved inhibition of Bcr-Abl through oligomeric disruption resulting from this modified coiled coil domain represents a viable alternative to small molecule inhibitors for therapeutic intervention. View Publication

Pim kinases are Ser/Thr kinases with multiple substrates that affect survival pathways. These proteins are overexpressed in acute myeloid leukemia (AML) blasts and we hypothesized that Pim kinase inhibition would affect AML cell survival. Imidazo[1,2-b]pyridazine compound,SGI-1776 inhibits Pim-1,Pim-2 and Pim-3,and was evaluated in AML-cell line,-xenograft model,and -primary blasts. Treatment of AML cells with SGI-1776 results in a concentration-dependent induction of apoptosis and we investigated its effect on Pim kinase functions. Phosphorylation of traditional Pim kinase targets,c-Myc(Ser62) and 4E-BP1 (Thr36/Thr47),were both decreased in actively cycling AML cell lines MV-4-11,MOLM-13 and OCI-AML-3. Levels of antiapoptotic proteins Bcl-2,Bcl-x(L),XIAP,and proapoptotic Bak and Bax were unchanged; however,a significant reduction in Mcl-1 was observed. This was correlated with inhibition of global RNA and protein synthesis and MCL-1 transcript decline after SGI-1776 treatment. These data suggest that SGI-1776 mechanism in AML involves Mcl-1 protein reduction. Consistent with cell line data,xenograft model studies with mice bearing MV-4-11 tumors showed efficacy with SGI-1776. Importantly,SGI-1776 was also cytotoxic in AML primary cells,irrespective of FLT3 mutation status and resulted in Mcl-1 protein decline. Pim kinase inhibition may be a new strategy for AML treatment. View Publication

Overexpression of wild-type MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We evaluated whether MN1 plays a functional role in leukemogenesis. We demonstrate using retroviral gene transfer and bone marrow (BM) transplantation that MN1 overexpression rapidly induces lethal AML in mice. Insertional mutagenesis and chromosomal instability were ruled out as secondary aberrations. MN1 increased resistance to all-trans retinoic acid (ATRA)-induced cell-cycle arrest and differentiation by more than 3000-fold in vitro. The differentiation block could be released by fusion of a transcriptional activator (VP16) to MN1 without affecting the ability to immortalize BM cells,suggesting that MN1 blocks differentiation by transcriptional repression. We then evaluated whether MN1 expression levels in patients with AML (excluding M3-AML) correlated with resistance to ATRA treatment in elderly patients uniformly treated within treatment protocol AMLHD98-B. Strikingly,patients with low MN1 expression who received ATRA had a significantly prolonged event-free (P = .008) and overall (P = .04) survival compared with patients with either low MN1 expression and no ATRA,or high MN1 expression with or without ATRA. MN1 is a unique oncogene in hematopoiesis that both promotes proliferation/self-renewal and blocks differentiation,and may become useful as a predictive marker in AML treatment. View Publication