The effect of cantharidins on leukemic stem cells.

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4-11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c-myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara-C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose-limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations.

Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope.

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.

Structural polarity and dynamics of male germline stem cells in an insect (milkweed bug Oncopeltus fasciatus).

Knowing the structure opens a door for a better understanding of function because there is no function without structure. Male germline stem cells (GSCs) of the milkweed bug (Oncopeltus fasciatus) exhibit a very extraordinary structure and a very special relationship with their niche, the apical cells. This structural relationship is strikingly different from that known in the fruit fly (Drosophila melanogaster) -- the most successful model system, which allowed deep insights into the signaling interactions between GSCs and niche. The complex structural polarity of male GSCs in the milkweed bug combined with their astonishing dynamics suggest that cell morphology and dynamics are causally related with the most important regulatory processes that take place between GSCs and niche and ensure maintenance, proliferation, and differentiation of GSCs in accordance with the temporal need of mature sperm. The intricate structure of the GSCs of the milkweed bug (and probably of some other insects, i.e., moths) is only accessible by electron microscopy. But, studying singular sections through the apical complex (i.e., GSCs and apical cells) is not sufficient to obtain a full picture of the GSCs; especially, the segregation of projection terminals is not tangible. Only serial sections and their overlay can establish whether membrane ingrowths merely constrict projections or whether a projection terminal is completely cut off. To sequence the GSC dynamics, it is necessary to include juvenile stages, when the processes start and the GSCs occur in small numbers. The fine structural analysis of segregating projection terminals suggests that these terminals undergo autophagocytosis. Autophagosomes can be labeled by markers. We demonstrated acid phosphatase and thiamine pyrophosphatase (TPPase). Both together are thought to identify autophagosomes. Using the appropriate substrate of the enzymes and cerium chloride, the precipitation of electron-dense cerium phosphate granules indicates the presence of enzymes and their location. Because the granules are very fine, they can be easily assigned to distinct cell organelles as the autophagosomes. Two methods, electron microscopy and immunocytochemistry, have pointed out a structural polarity and dynamics that are unprecedented for stem cells. We propose that these dynamics indicate a novel type of signal exchange and transduction between stem cells and their niche.

Quantification of the CD8+ T cell response against a mucin epitope in patients with breast cancer.

INTRODUCTION: Mucin 1, encoded by the MUC1 gene, is a tumor-associated antigen expressed on the surface of breast cancer cells. It would be of interest to see whether there is a naturally existing T cell immune response against mucin epitopes in cancer patients. MATERIALS AND METHODS: Using tetramer and interferon-gamma assays, the immune response to one MUC1 peptide epitope in the peripheral blood of breast cancer patients was quantified. The data were compared with the clinical course of the patients. RESULTS: CD8(+) T cells capable of recognizing the HLA-A*0201-restricted STAPPVHNV epitope were detected in 9 of 19 patients with a frequency ranging 0.01-0.082%. No significant difference was found between the occurrence of epitope-specific CD8(+) T cells of patients with progressive disease and disease-free patients. However, all patients with stable disease showed a specific immune response, including both patients with the highest frequency. CONCLUSIONS: The results of this study provide further evidence that a natural specific cellular immune response against this mucin epitope exists in breast cancer patients.

Constitutive activation of Flt3 and STAT5A enhances self-renewal and alters differentiation of hematopoietic stem cells.

OBJECTIVE: To model human leukemogenesis by transduction of human hematopoietic stem cells (HSC) with genes associated with leukemia and expressed in leukemic stem cells. METHODS: Constitutive activation of Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute myeloid leukemia (AML). Retroviral vectors expressing constitutively activated Flt3 and STAT5A were used to transduce human cord blood CD34(+) cells and HSC cell self-renewal and differentiation were evaluated. RESULTS: We have demonstrated that retroviral transduction of Flt3 mutations into CD34(+) cells enhanced HSC self-renewal as measured in vitro in competitive stromal coculture and limiting-dilution week-2 cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased myelopoiesis were noted together with strong activation of STAT5A. Consequently, transduction studies were undertaken with a constitutively active mutant of STAT5A (STAT5A[1( *)6]) and here also a marked, selective expansion of transduced CD34(+) cells was noted, with a massive increase in self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture. Differentiation was biased to erythropoiesis, including erythropoietin independence, with myeloid maturation inhibition. The observed phenotypic changes correlated with differential gene expression, with a number of genes differentially regulated by both the Flt3 and STAT5A mutants. These included upregulation of genes involved in erythropoiesis and downregulation of genes involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also characterized primary Flt3-ITD(+) AML bone marrow samples. Isolation of leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR(-) phenotype was undertaken with Flt3-ITD(+) AML samples resulting in co-purification of early CAFC. Gene expression of LSC relative to the bulk leukemic population revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell proliferation. CONCLUSION: Myeloid leukemogenesis is a multi-stage process that can involve constitutively activated receptors and downstream pathways involving STAT5, HOX genes, and HLF.

Stem cell autotomy and niche interaction in different systems.

The best known cases of cell autotomy are the formation of erythrocytes and thrombocytes (platelets) from progenitor cells that reside in special niches. Recently, autotomy of stem cells and its enigmatic interaction with the niche has been reported from male germline stem cells (GSCs) in several insect species. First described in lepidopterans, the silkmoth, followed by the gipsy moth and consecutively in hemipterans, foremost the milkweed bug. In both, moths and the milkweed bug, GSCs form finger-like projections toward the niche, the apical cells (homologs of the hub cells in Drosophila). Whereas in the milkweed bug the projection terminals remain at the surface of the niche cells, in the gipsy moth they protrude deeply into the singular niche cell. In both cases, the projections undergo serial retrograde fragmentation with progressing signs of autophagy. In the gipsy moth, the autotomized vesicles are phagocytized and digested by the niche cell. In the milkweed bug the autotomized vesicles accumulate at the niche surface and disintegrate. Autotomy and sprouting of new projections appears to occur continuously. The significance of the GSC-niche interactions, however, remains enigmatic. Our concept on the signaling relationship between stem cell-niche in general and GSC and niche (hub cells and cyst stem cells) in particular has been greatly shaped by Drosophila melanogaster. In comparing the interactions of GSCs with their niche in Drosophila with those in species exhibiting GSC autotomy it is obvious that additional or alternative modes of stem cell-niche communication exist. Thus, essential signaling pathways, including niche-stem cell adhesion (E-cadherin) and the direction of asymmetrical GSC division - as they were found in Drosophila - can hardly be translated into the systems where GSC autotomy was reported. It is shown here that the serial autotomy of GSC projections shows remarkable similarities with Wallerian axonal destruction, developmental axon pruning and dying-back degeneration in neurodegenerative diseases. Especially the hypothesis of an existing evolutionary conserved "autodestruction program" in axons that might also be active in GSC projections appears attractive. Investigations on the underlying signaling pathways have to be carried out. There are two other well known cases of programmed cell autotomy: the enucleation of erythroblasts in the process of erythrocyte maturation and the segregation of thousands of thrombocytes (platelets) from one megakaryocyte. Both progenitor cell types - erythroblasts and megakaryocytes - are associated with a niche in the bone marrow, erythroblasts with a macrophage, which they surround, and the megakaryocytes with the endothelial cells of sinusoids and their extracellular matrix. Although the regulatory mechanisms may be specific in each case, there is one aspect that connects all described processes of programmed cell autotomy and neuronal autodestruction: apoptotic pathways play always a prominent role. Studies on the role of male GSC autotomy in stem cell-niche interaction have just started but are expected to reveal hitherto unknown ways of signal exchange. Spermatogenesis in mammals advance our understanding of insect spermatogenesis. Mammal and insect spermatogenesis share some broad principles, but a comparison of the signaling pathways is difficult. We have intimate knowledge from Drosophila, but of almost no other insect, and we have only limited knowledge from mammals. The discovery of stem cell autotomy as part of the interaction with the niche promises new general insights into the complicated stem cell-niche interdependence.

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity.

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 triggered apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the proapoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high-dose Ara-C therapy.

Down-regulation of the human tumor antigen mucin by gemcitabine on the pancreatic cancer cell line capan-2.

The nucleoside analogue gemcitabine displays therapeutic effects mainly against breast, ovarian and pancreatic cancer. Mucin, encoded by the gene MUC1, is a well-established tumor antigen expressed on these tumors. Knowledge of possible effects of chemotherapeutic drugs on the level of mucin epitope expression is important for the design of clinical protocols combining chemo- and immunotherapeutic approaches. In this study, we determined the influence of gemcitabine on the mucin expression of the human pancreatic carcinoma cell line Capan-2. The cells were treated with three different concentrations (0.01 microg/ml, 0.1 microg/ml and 0.25 microg/ml) of gemcitabine or were left untreated and were analyzed after 24 hours, 3 and 5 days. Flow cytometric analysis showed a dose-dependent decrease of mucin expression on the cell surface which remained over 5 days. The strongest reduction of mucin expression was detectable 24 hours after application of the drug. The down-regulation of the tumor antigen mucin by gemcitabine might weaken an immune response against mucin-expressing tumors, which are under treatment with this chemotherapeutic drug.

Structural characterization and primary in vitro cell culture of locust male germline stem cells and their niche.

The establishment of in vitro culture systems to expand stem cells and to elucidate the niche/stem cell interaction is among the most sought-after culture systems of our time. To further investigate niche/stem cell interactions, we evaluated in vitro cultures of isolated intact male germline-niche complexes (i.e., apical complexes), complexes with empty niche spaces, and completely empty niches (i.e., isolated apical cells) from the testes of Locusta migratoria and the interaction of these complexes with isolated germline stem cells, spermatogonia (of transit-amplifying stages), cyst progenitor cells, cyst progenitor cell-like cells, cyst cells, and follicle envelope cells. The structural characteristics of these cell types allow the identification of the different cell types in primary cultures, which we studied in detail by light and electron microscopy. In intact testes germline stem cells strongly adhere to their niche (the apical cell), but emigrate from their niche and form filopodia if the apical complex is put into culture with "standard media." The lively movements of the long filopodia of isolated germline stem cells and spermatogonia may be indicative of their search for specific signals to home to their niche. All other incubated cell types (except for follicle envelope cells) expressed rhizopodia and lobopodia. Nevertheless isolated germline stem cells in culture do not migrate to empty niche spaces of nearby apical cells. This could indicate that apical cells lose their germline stem cell attracting ability in vitro, although apical cells devoid of germline stem cells either by emigration of germline stem cells or by mechanical removal of germline stem cells are capable of surviving in vitro up to 56 days, forming many small lobopodia and performing amoeboid movements. We hypothesize that the breakdown of the apical complex in vitro with standard media interrupts the signaling between the germline stem cells and the niche (and conceivably the cyst progenitor cells) which directs the typical behavior of the male regenerative center. Previously we demonstrated the necessity of the apical cell for the survival of the germline stem cell. From these studies we are now able to culture viable isolated germline stem cells and all cells of its niche complex, although DNA synthesis stops after Day 1 in culture. This enables us to examine the effects of supplements to our standard medium on the interaction of the germline stem cell with its niche, the apical cell. The supplements we evaluated included conditioned medium, tissues, organs, and hemolymph of male locusts, insect hormones, mammalian growth factors, Ca(2+) ion, and a Ca(2+) ionophore. Although biological effects on the germline stem cell and apical cell could be detected with the additives, none of these supplements restored the in vivo behavior of the incubated cell types. We conclude that the strong adhesion between germline stem cells and apical cells in vivo is actively maintained by peripheral factors that reach the apical complex via hemolymph, since a hemolymph-testis barrier does not exist. The in vitro culture model introduced in this study provides a platform to scan for possible regulatory factors that play a key role in a feedback loop that keeps germline stem cell division and sperm disposal in equilibrium.

Apoptosis of male germ-line stem cells after laser ablation of their niche.

Male germ-line stem cells (GSCs) and their niche-the apical cells or hub cells-display a unique feature at the apices of insect testicular follicles. In the locust, Locusta migratoria, the niche consists of only one large apical cell surrounded by about 60 GSCs. The apical cell can be readily identified in the intact follicle. Using laser ablation it is feasible to destroy the apical cell exclusively without injuring neighboring GSCs or any other cells. The most immediate effect on GSCs is the loss of their structural polarity. Beginning about 3 h after laser treatment chromatin starts to clump and condense in individual GSCs, and some show the first signs of cellular breakdown. These symptoms intensify during the 96-h observation period after laser ablation of the apical cell. TUNEL staining and electron microscopic observations confirm an apoptotic cell death of the GSCs. Laser ablation of individual GSCs had no effect on neighboring GSCs or the apical cell. Destroyed apical cells were not replaced during the observation period. Mitotic divisions of GSCs ceased after about 24 h after apical cell ablation. It is speculated that it might be a general principle in stem cell-niche relationships that stem cells undergo apoptosis when the niche is dysfunctional. This could be a control mechanism to prevent tumor growth of orphaned GSCs.

Tumor cell specific toxicity of Inula helenium extracts.

The aim of the research program was to identify botanical extracts with antineoplastic activity. In this respect extracts prepared from Inula helenium roots showed a remarkable activity. As evidenced by the MTT assay, the Inula helenium extract revealed a highly selective toxicity toward four different tumor cell lines (HT-29, MCF-7, Capan-2 and G1), but a much lower toxicity against healthy human peripheral blood lymphocytes (PBLs) from two donors. The extract-induced death of tumor cells was studied extensively by electron microscopy. There was a remarkable similarity of morphological alterations observed in the four cell lines: patchy chromatin condensations, cytoplasmic vesiculation, swelling and rupture of mitochondria. The morphology of cellular breakdown bore more resemblance to necrotic than to apoptotic cell death, which was supported by the failure to mark early apoptotic events by Annexin V. It has been pointed out recently that compounds inducing cell death with necrotic-like morphology could be very beneficial in cases where cancerous cells have gained resistance to apoptosis. In this context, the remarkable difference in cytotoxicity exerted by the Inula helenium extract, which was over 100-times higher in the tumor cell lines than in the PBLs, makes the extract an excellent candidate for further anticancerous investigations, especially since the Inula helenium extract was not mutagenic in the Ames test.

Hematopoiesis controlled by distinct TIF1gamma and Smad4 branches of the TGFbeta pathway.

Tissue homeostasis in mammals relies on powerful cytostatic and differentiation signals delivered by the cytokine TGFbeta and relayed within the cell via the activation of Smad transcription factors. Formation of transcription regulatory complexes by the association of Smad4 with receptor-phosphorylated Smads 2 and 3 is a central event in the canonical TGFbeta pathway. Here we provide evidence for a branching of this pathway. The ubiquitious nuclear protein Transcriptional Intermediary Factor 1gamma (TIF1gamma) selectively binds receptor-phosphorylated Smad2/3 in competition with Smad4. Rapid and robust binding of TIF1gamma to Smad2/3 occurs in hematopoietic, mesenchymal, and epithelial cell types in response to TGFbeta. In human hematopoietic stem/progenitor cells, where TGFbeta inhibits proliferation and stimulates erythroid differentiation, TIF1gamma mediates the differentiation response while Smad4 mediates the antiproliferative response with Smad2/3 participating in both responses. Thus, Smad2/3-TIF1gamma and Smad2/3-Smad4 function as complementary effector arms in the control of hematopoietic cell fate by the TGFbeta/Smad pathway.

Virus-like particles derived from major capsid protein VP1 of different polyomaviruses differ in their ability to induce maturation in human dendritic cells.

As polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) have been demonstrated to be highly immunogenic, we studied their interaction with human dendritic cells (hDCs). Exposure of hDCs to VLPs originating from murine (MPyV) or hamster polyomavirus (HaPyV) induced hDC maturation. In contrast, exposure of hDCs to VLPs derived from human polyomaviruses (BK and JC) and simian virus 40 (SV40) only marginally induced DC maturation. The hDCs stimulated by HaPyV- or MPyV-derived VLPs readily produced interleukin-12 and stimulated CD8-positive T-cell responses in vitro. The highest frequencies of activated T cells were again observed after pulsing with HaPyV- and MPyV-derived VLPs. Monocyte-derived hDCs both bound and internalized the various tested polyomavirus VP1-derived VLPs with different levels of efficiency, partially explaining their individual maturation potentials. In conclusion, our data suggest a high variability in uptake of polyomavirus-derived VLPs and potency to induce hDC maturation.

Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis.

To investigate the role of constitutively active internal tandem duplication (ITD) mutants of the Fms-like tyrosine kinase 3 (Flt3) receptor in leukemogenesis, we introduced the Flt3-ITD, W51, into human cord blood CD34+ cells and evaluated their phenotype in diverse hematopoietic assays. W51 expression resulted in a strong proliferative advantage and enhanced erythropoiesis as determined by immunophenotyping, colony assays, and molecular analyses. In MS-5 stromal cocultures, numerous early cobblestone areas (CAs) were generated within 10 to 14 days. Such W51-associated early CAs disappeared by 4 weeks, yet retained self-renewal properties as demonstrated by generation of secondary and tertiary CAs upon replating. This phenotype appears related to the expression of W51 since it was abolished by exposure to the FLT3 inhibitor, AG1295, but not to the c-kit inhibitor PD16. Wild-type Flt3-overexpressing CD34+ cells exposed to high levels of its physiologic ligand did not produce early CAs, highlighting differences in intracellular signaling between wild-type Flt3 and W51. W51-associated signal transducer and activator of transcription 5 (Stat5) activation plays a major role in this phenotype, although additional downstream targets of W51 may be relevant. Flt3-ITD+ acute myeloid leukemia (AML) blasts from patients invariably generated early AG1295-sensitive CAs in MS-5 cocultures, further validating the phenotype observed in transduced CD34+ cells.

The migrastatin family: discovery of potent cell migration inhibitors by chemical synthesis.

The first asymmetric total synthesis of (+)-migrastatin (1), a macrolide natural product with anti-metastatic properties, has been accomplished. Our concise and flexible approach utilized a Lewis acid-catalyzed diene aldehyde condensation (LACDAC) to install the three contiguous stereocenters and the trisubstituted (Z)-alkene of migrastatin (2 + 3 --> 21). Construction of the two remaining stereocenters and incorporation of the glutarimide-containing side chain was achieved by an anti-selective aldol addition of propionyl oxazolidinone 28 to angelic aldehyde 27, followed by a Horner-Wadsworth-Emmons (HWE) coupling of 32 with glutarimide aldehyde 5. Finally, the assembly of the macrocycle was realized by a highly (E)-selective ring-closing metathesis (35 --> 37). Utilizing the power of diverted total synthesis (DTS), a series of otherwise inaccessible analogues was prepared and evaluated for their potential as tumor cell migration inhibitors in several in vitro assays. These studies revealed a dramatic increase in activity when the natural motif was considerably simplified, presenting macrolactones 45 and 48, as well as macrolactam 55, macroketone 60, and CF(3)-alcohol 71 as promising anti-metastatic agents.