技术资料

Microtubules are among the most strategic subcellular targets of anticancer chemotherapeutics. Despite this fact,new antimicrotubule agents that possess unique mechanisms of cytotoxic action and have broader antineoplastic spectra than the vinca alkaloids have not been introduced over the last several decades--until the recent development of taxol. Unlike classical antimicrotubule agents like colchicine and the vinca alkaloids,which induce depolymerization of microtubules,taxol induces tubulin polymerization and forms extremely stable and nonfunctional microtubules. Taxol has demonstrated broad activity in preclinical screening studies,and antineoplastic activity has been observed in several classically refractory tumors. These tumors include cisplatin-resistant ovarian carcinoma in phase II trials and malignant melanoma and non-small cell lung carcinoma in phase I studies. Taxol's structural complexity has hampered the development of feasible processes for synthesis,and its extreme scarcity has limited the use of a conventional,broad-scale screening approach for evaluation of clinical antitumor activity. However,taxol's unique mechanism of action,its spectrum of preclinical antitumor activity,and tumor responses in early clinical trials have generated renewed interest in pursuing its development. View Publication

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance,hypoxic resistance,high tumorigenicity,high cell invasion,and metastatic abilities are characteristics of these cells,which are responsible for breast cancer recurrence. Therefore,the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique,technologies that depend on cell surface markers,ALDEFLUOR assays,and in situ detection. Identification technologies include mammosphere cultures,limited dilution in vitro,and in-vivo animal models. This review provides an important reference for breast cancer stem cell research,which will explore new methods for the treatment of patients with breast cancer. View Publication

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality yet identified in any pediatric leukemia and gives rise to the TEL-AML1 fusion product. To investigate the effects of TEL-AML1 on hematopoiesis,fetal liver hematopoietic progenitor cells (HPCs) were transduced with retroviral vectors expressing this fusion protein. We show that TEL-AML1 dramatically alters differentiation of HPCs in vitro,preferentially promoting B-lymphocyte development,enhancing self-renewal of B-cell precursors,and leading to the establishment of long-term growth factor-dependent pre-B-cell lines. However,it had no effect on myeloid development in vitro. Further experiments were performed to determine whether TEL-AML1 also demonstrates lineage-specific activity in vivo. TEL-AML1-expressing HPCs displayed a competitive advantage in reconstituting both B-cell and myeloid lineages in vivo but had no effect on reconstitution of the T-cell lineage. Despite promoting these alterations in hematopoiesis,TEL-AML1 did not induce leukemia in transplanted mice. Our study provides a unique insight into the role of TEL-AML1 in leukemia predisposition and a potential model to study the mechanism of leukemogenesis associated with this fusion. View Publication

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program,by reprogramming of a more differentiated cell type by oncogenic insults,or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5,K14,and vimentin),luminal (E-cadherin,K8,K18,or K19),and stem/progenitor (CD49f,CD29,CD44,and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways,such as Notch,Wnt,Hedgehog,and LIF,in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers,these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer. View Publication

In somatic cells,eroded telomeres can induce DNA double-strand break signaling,leading to a form of replicative senescence or apoptosis,both of which are barriers to tumorigenesis. However,cancer cells might display telomere dysfunctions which in conjunction with defects in DNA repair and apoptosis,enables them to circumvent these pathways. Chronic lymphocytic leukemia (CLL) cells exhibit telomere dysfunction,and a subset of these cells are resistant to DNA damage-induced apoptosis and display short telomeres. We show here that these cells exhibit significant resection of their protective telomeric 3' single-stranded overhangs and an increased number of telomere-induced foci containing gammaH2AX and 53BP1. Chromatin immunoprecipitation and immunofluorescence experiments demonstrated increased levels of telomeric Ku70 and phospho-S2056-DNA-PKcs,2 essential components of the mammalian nonhomologous end-joining DNA repair system. Notably,these CLL cells display deletions of telomeric signals on one or 2 chromatids in parallel with 11q22 deletions,or with 13q14 deletions associated with another chromosomal aberration or with a complex karyotype. Taken together,our results indicate that a subset of CLL cells from patients with an unfavorable clinical outcome harbor a novel type of chromosomal aberration resulting from telomere dysfunction. View Publication

Chronic myeloid leukaemia (CML) is caused by a defined genetic abnormality that generates BCR-ABL,a constitutively active tyrosine kinase. It is widely believed that BCR-ABL activates Akt signalling that suppresses the forkhead O transcription factors (FOXO),supporting the proliferation or inhibiting the apoptosis of CML cells. Although the use of the tyrosine kinase inhibitor imatinib is a breakthrough for CML therapy,imatinib does not deplete the leukaemia-initiating cells (LICs) that drive the recurrence of CML. Here,using a syngeneic transplantation system and a CML-like myeloproliferative disease mouse model,we show that Foxo3a has an essential role in the maintenance of CML LICs. We find that cells with nuclear localization of Foxo3a and decreased Akt phosphorylation are enriched in the LIC population. Serial transplantation of LICs generated from Foxo3a(+/+) and Foxo3a(-/-) mice shows that the ability of LICs to cause disease is significantly decreased by Foxo3a deficiency. Furthermore,we find that TGF-beta is a critical regulator of Akt activation in LICs and controls Foxo3a localization. A combination of TGF-beta inhibition,Foxo3a deficiency and imatinib treatment led to efficient depletion of CML in vivo. Furthermore,the treatment of human CML LICs with a TGF-beta inhibitor impaired their colony-forming ability in vitro. Our results demonstrate a critical role for the TGF-beta-FOXO pathway in the maintenance of LICs,and strengthen our understanding of the mechanisms that specifically maintain CML LICs in vivo. View Publication

Reversine (RV) is the synthetic purine identified from a protein kinase-based screen of purine mimetics and it has been shown to induce muscle myoblast differentiation into progenitor cells that can be further converted into other cell lineages. Since protein kinases play a pivotal role in cell cycle control,we hypothesize that RV might affect the proliferation of cancer cells. Herein we report that RV inhibited growth of cultured human tumor cells,respectively,PC-3,HeLa,CWR22Rv1,and DU-145 cells,and induced accumulation of polyploidal cells with textgreater or =4N DNA content. However,RV was without effect on growth of normal prostate epithelial cells. RV-treated PC-3 cells showed enlarged nuclei and an estimated 100-fold increase in cell size. Moreover,PC-3 cells treated with RV for 2-4 days were accompanied by a marked increase in the expression of p21(WAF1),a modest elevation in the levels of cyclin D3 and CDK6 and concomitantly,also a substantial reduction in cyclin B and CDK1. These results suggest that RV may induce polyploidy and increase in cell size by up-regulating p21(WAF1) and cyclin D3/CDK6,while simultaneously suppressing the expression of cyclin B and CDK1. View Publication

Therapies that target signaling pathways critical to the pathogenesis and progression of squamous cell carcinoma of the head and neck (HNSCC) are needed. One such target,phosphatidylinositol 3-kinase,and its downstream target serine/threonine kinase,Akt,are up-regulated in HNSCC. Targeted therapy could consist of inhibitors of these kinases or,alternatively,of inhibitors of the pathways that they regulate. To explore the effect of Akt inhibition on the growth and survival of HNSCC tumors,we evaluated the effect of a novel Akt inhibitor,KP372-1,on the growth,survival,and sensitivity to anoikis of HNSCC cell lines in culture. Using Western blotting of head and neck cancer cell lines and squamous mucosa and carcinoma specimens,we found that Akt was highly phosphorylated in head and neck cancer cell lines and human head and neck squamous carcinoma specimens. Treatment of HNSCC cell lines with KP372-1 blocked the activation of Akt,inhibited head and neck cancer cell proliferation,and induced apoptosis and anoikis in several HNSCC cell lines. Furthermore,KP372-1 decreased the phosphorylation of the S6 ribosomal (Ser240/244) protein,which is a downstream target of Akt. Taken together,these findings indicate that KP372-1 may be a useful therapeutic agent for HNSCC and should be further evaluated in preclinical models of HNSCC. View Publication

The phosphatidylinositol 3' kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway,which is a critical regulator of cell proliferation and survival,is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens,and hypothesised that KP372-1,an Akt inhibitor,would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells,leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling,further preclinical evaluation of this compound for treatment of thyroid cancer is warranted. View Publication

Bone is the most common site for metastasis in patients with solid tumours. Bisphosphonates are an effective treatment for preventing skeletal related events and preserving quality of life in these patients. Zoledronic acid (ZA) is the most potent osteoclast inhibitor and is licensed for the treatment of bone metastases. Clodronate and pamidronate are also licensed for this indication. In addition,ZA has been demonstrated to exhibit antitumour effect. Direct and indirect mechanisms of anti-tumour effect have been postulated and at many times proven. Evidence exists that ZA antitumour effect is mediated through inhibition of tumour cells proliferation,induction of apoptosis,synergistic/additive to inhibitory effect of cytotoxic agents,inhibition of angiogenesis,decrease tumour cells adhesion to bone,decrease tumour cells invasion and migration,disorganization of cell cytoskeleton and activation of specific cellular antitumour immune response. There is also clinical evidence from clinical trials that ZA improved long term survival outcome in cancer patients with and without bone metastases. In this review we highlight the preclinical and clinical studies investigating the antitumour effect of bisphosphonates with particular reference to ZA. View Publication

The role of autophagy,a lysosomal degradation pathway which prevents cellular damage,in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis,malignant transformation of HSCs leads to leukemia. Therefore,mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study,we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions,a severe myeloproliferation,and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species,as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded,Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions,the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively,these data show that Atg7 is an essential regulator of adult HSC maintenance. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication