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Andrei Thomas-Tikhonenko

Publications and source records attributed to Andrei Thomas-Tikhonenko.

18 recordsLinked to original sources
PubMed2006

Epigenetic histone modifications do not control Igkappa locus contraction and intranuclear localization in cells with dual B cell-macrophage potential.

Somatic rearrangement of the Ig genes during B cell development is believed to be controlled, at least in part, by accessibility of the loci to the recombinational machinery. Accessibility is poorly understood, but appears to be controlled by a combination of histone posttranslational modifications, large scale Ig locus contractions, and changes in intranuclear localization of the loci. These changes are regulated by developmental stage-specific as well as tissue-specific mechanisms. We previously isolated a murine B cell lymphoma line, Myc5, that can oscillate between the B cell and macrophage lineages depending upon growth conditions. This line provides an opportunity to study tissue-specific regulation of epigenetic mechanisms operating on the Ig loci. We found that when Myc5 cells are induced to differentiate from B cells into macrophages, expression of macrophage-specific transcripts was induced (M-CSFR, F4/80, and CD14), whereas B cell-specific transcripts decreased dramatically (mb-1, E47, IRF4, Pax5, and Igkappa). Loss of Igkappa transcription was associated with reduced Igkappa locus contraction, as well as increased association with heterochromatin protein-1 and association of the Igkappa locus with the nuclear periphery. Surprisingly, however, we found that histone modifications at the Igkappa locus remained largely unchanged whether the cells were grown in vivo as B cells, or in vitro as macrophages. These results mechanistically uncouple histone modifications at the Igkappa locus from changes in locus contraction and intranuclear localization.

Alleles↗
PubMed2006

B-Lymphoma cells with epigenetic silencing of Pax5 trans-differentiate into macrophages, but not other hematopoietic lineages.

In mice, zygotic or pro-B-cell-specific knock-out of the Pax5 gene allows differentiation of pro-B-cells into all hematopoietic lineages. We previously generated and characterized a murine B-cell lymphoma, dubbed Myc5, whose cells spontaneously lose Pax5 expression when cultured in vitro, but regain it when re-injected into syngeneic mice. In cultured Myc5 cells, the loss of Pax5 correlates with the acquisition of myeloid markers, such as CD11b and F4/80. Here, we sought to determine whether these cells are truly B-macrophage-restricted or, like Pax5-null progenitors, can give rise to additional hematopoietic lineages. In vitro differentiation assays with various cytokines showed that Myc5 cells do not differentiate into NK cells, dendritic cells, neutrophils, or osteoclasts. At the same time, in the presence of macrophage colony-stimulating factor (M-CSF), they readily phagocytose latex beads and provide T-cell help. Both phenomena are indicative of the bona fide macrophage phenotype. Conversely, enforced Pax5 re-expression in macrophage-like Myc5 cells led to down-regulation of the M-CSF receptor and re-acquisition of some B-cell surface markers (e.g., CD79a) and lineage-specific transcription factors (e.g., IRF4 and Blimp). Retrovirally encoded Pax5 also restored expression of several master B-cell differentiation proteins, such as the IL-7 receptor and transcription factor E2A. In contrast, levels of EBF were unaffected by Pax5 suggesting that EBF acts exclusively upstream of Pax5 and might contribute to Pax5 expression. Indeed, transduction with an EBF-encoding retrovirus partly reactivated endogenous Pax5. Our data reveal the complex relationship between B-cell-specific transcription factors and suggest the existence of numerous feedback mechanisms.

Animals↗
PubMed2006

Augmentation of tumor angiogenesis by a Myc-activated microRNA cluster.

Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2'-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92-transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.

Animals↗
PubMed2006

Kit-activating mutations in AML: lessons from PU.1-induced murine erythroleukemia.

In concert with its ligand, the stem cell factor (SCF), the receptor tyrosine kinase c-Kit acts as a key signaling molecule for a number of cell types, including hematopoietic stem cells, mast cells, melanocytes and germ cells. Gain-of-function mutations in c-Kit have been described in a number of human cancers, including testicular germinomas, acute myeloid leukemia and gastrointestinal stromal tumors. Yet their contribution to neoplastic growth is incompletely understood. Now Kosmider et al report the acquisition of Kit mutations in 86% of late-stage eryhtroleukemias in Spi-1/PU.1 transgenic mice. Without Kit mutations, these mice suffer from a benign disease whose hallmark is erythropoietin-dependent expansion of undifferentiated red blood cell precursors. Newly acquired Kit mutations affect codon 814 or 818, and ectopic expression of these mutants in nonmalignant pro-erythroblasts confers erythropoietin independence and tumorigenicity. Using tyrosine kinase inhibitors PP1, PP2, and imatinib mesylate (a.k.a. Gleevac), the authors demonstrate that Kit mutations are important for the autonomous expansion of malignant cells via the MEK/Erk1/2 and PI3K/Akt pathways. These findings validate the notion that one differentiation-blocking (e.g., PU.1 activation) and one proliferative (e.g., c-Kit mutations) event are required for the development of frank leukemia.

Animals↗
PubMed2006

Activation of transferrin receptor 1 by c-Myc enhances cellular proliferation and tumorigenesis.

Overexpression of transferrin receptor 1 (TFRC1), a major mediator of iron uptake in mammalian cells, is a common feature of human malignancies. Therapeutic strategies designed to interfere with tumor iron metabolism have targeted TFRC1. The c-Myc oncogenic transcription factor stimulates proliferation and growth by activating thousands of target genes. Here we demonstrate that TFRC1 is a critical downstream target of c-Myc. Using in vitro and in vivo models of B-cell lymphoma, we show that TFRC1 expression is activated by c-Myc. Chromatin immunoprecipitation experiments reveal that c-Myc directly binds a conserved region of TFRC1. In light of these findings, we sought to determine whether TFRC1 is required for c-Myc-mediated cellular proliferation and cell size control. TFRC1 inhibition decreases cellular proliferation and results in G1 arrest without affecting cell size. Consistent with these findings, expression profiling reveals that TFRC1 depletion alters expression of genes that regulate the cell cycle. Furthermore, enforced TFRC1 expression confers a growth advantage to cells and significantly enhances the rate of c-Myc-mediated tumor formation in vivo. These findings provide a molecular basis for increased TFRC1 expression in human tumors, illuminate the role of TFRC1 in the c-Myc target gene network, and support strategies that target TFRC1 for cancer therapy.

Animals↗
PubMed2006

Infection & neoplastic growth 101: the required reading for microbial pathogens aspiring to cause cancer.

The role of infectious agents in the development of cancer is well-established. For example, numerous RNA and DNA viruses express dedicated oncoproteins that are able to transform cells in vitro and induce rapid tumor formation in experimental animals. Curiously, these acutely-transforming viruses are seldom associated with naturally occurring neoplasms of humans or animals. Conversely, the microbial pathogens that are linked to cancers in natural hosts rarely encode acutely transforming proteins. Moreover, they tend to down-regulate most of their genes except for genes affecting cell survival, often via the NF-kappaB pathway. This allows the pre-neoplastic cell to avoid triggering cytotoxic immunity and the ensuing acute inflammation. These host responses not only kill tumor cells directly, but also strongly suppress the development of new blood vessels (neovascularization), which is absolutely essential for neoplastic growth. Chronic inflammation, on the other hand, promotes neoplastic cell growth and may even be conducive to neovascularization. Thus, cancer-associated microbial pathogens must follow the "less is more" principle. According to this principle, only minor perturbations in cell proliferation and death are tolerated by the host, but in the long run, they are all that is necessary for tumorigenesis.

Bacterial Infections↗
PubMed2005

Metastasis-associated protein 1 (MTA1) is an essential downstream effector of the c-MYC oncoprotein.

The c-myc oncogene is among the most commonly overexpressed genes in human cancer. c-myc encodes a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor (c-MYC) that activates a cascade of downstream targets that ultimately mediate cellular transformation. Although a large number of genes are regulated by c-MYC, only a few have been functionally linked to c-MYC-mediated transformation. By expression profiling, the metastasis-associated protein 1 (MTA1) gene was identified here as a target of the c-MYC oncoprotein in primary human cells, a result confirmed in human cancer cells. MTA1 itself has been previously implicated in cellular transformation, in part through its ability to regulate the epithelial-to-mesenchymal transition and metastasis. MTA1 is a component of the Mi-2/nucleosome remodeling and deacetylating (NURD) complex that contains both histone deacetylase and nucleosome remodeling activity. The data reported here demonstrate that endogenous c-MYC binds to the genomic MTA1 locus and recruits transcriptional coactivators. Most importantly, short hairpin RNA (shRNA)-mediated knockdown of MTA1 blocks the ability of c-MYC to transform mammalian cells. These data implicate MTA1 and the Mi-2/NURD complex as one of the first downstream targets of c-MYC function that are essential for the transformation potential of c-MYC.

Adenosine Triphosphatases↗
PubMed2005

Inactivation of Myc in murine two-hit B lymphomas causes dormancy with elevated levels of interleukin 10 receptor and CD20: implications for adjuvant therapies.

Overexpression of c-Myc and inactivation of p53 are hallmarks of human Burkitt's lymphomas. We had previously showed that transduction of murine p53-null bone marrow cells with a Myc-encoding retrovirus is sufficient for B lymphomagenesis. To address the role of Myc in tumor sustenance, we generated lymphomas induced by the Myc-estrogen receptor fusion protein (MycER). Engrafted hosts were continuously treated with the ER ligand 4-hydroxytamoxifen (4-OHT) to allow tumor formation. Subsequent inactivation of MycER via 4-OHT deprivation resulted in tumor stasis but only partial regression. At the cellular level, dormant neoplastic lymphocytes withdrew from mitosis and underwent further B-cell differentiation. Concomitantly, they up-regulated genes involved in lymphocyte proliferation and survival, most notably interleukin 10 receptor alpha (IL10Ralpha) and CD20, the target for antibody therapy with Rituxan. We found that overexpression of IL10Ralpha affords significant proliferative advantages and in 4-OHT-deprived animals correlates with eventual tumor relapse. Both dormant and relapsing tumors maintain IL10Ralpha expression suggesting that they might be sensitive to emerging drugs targeting the IL-10 pathway. Up-regulation of CD20 following Myc inactivation was also observed in immortalized human lymphocytes. Importantly, in this system, Myc(OFF)CD20(HIGH) cells were more prone to Rituxan-induced apoptosis than Myc(ON)CD20(MED). Thus, targeting Myc, while moderately effective on its own, shapes the phenotype of dormant neoplastic cells and sensitizes them to adjuvant molecular therapies.

Alleles↗
PubMed2005

Targeting beta-transducin repeat-containing protein E3 ubiquitin ligase augments the effects of antitumor drugs on breast cancer cells.

beta-Transducin repeat-containing proteins (beta-TrCP) serve as substrate recognition component of E3 ubiquitin ligases that control stability of important regulators of cell cycle and signal transduction. beta-TrCP function is essential for the induction of nuclear factor kappaB transcriptional activities, which play a key role in proliferation and survival of cancer cells and are often constitutively up-regulated in human breast cancers. Here we show that inhibition of beta-TrCP either by RNAi approach or by forced expression of a dominant-negative beta-TrCP mutant suppresses growth and survival of human breast cancer cells. In addition, inhibition of beta-TrCP augments the antiproliferative effects of anticancer drugs such as doxorubicin, tamoxifen, and paclitaxel on human mammary tumor cells. These data provide the proof of principle that targeting beta-TrCP might be beneficial for anticancer therapies.

Antineoplastic Agents↗
PubMed2005

Functional validation of genes implicated in lymphomagenesis: an in vivo selection assay using a Myc-induced B-cell tumor.

The involvement of the c-Myc transcription factor in neoplastic transformation is well documented. However, which of its numerous target genes are crucial for tumorigenesis remains a frequently contested issue. We have recently established a non-transgenic murine model for B-cell lymphoma based on neoplastic conversion of p53-null bone marrow cells by conditionally active Myc. Using this model, we have identified a number of genes whose expression levels are affected by Myc during B-lymphomagenesis. Here we discuss their possible roles in neoplastic processes and describe an experimental approach allowing in vivo validation of these roles. We demonstrate that lymphoma cells overexpressing one of the Myc targets, the interleukin-10 receptor gene, have a very strong selective advantage over low IL10R expressors. Furthermore, Mcl1, a presumptive IL10R effector, also confers selective advantages when overexpressed in Myc-transformed hematopoietic cells. Thus, both IL10R and Mcl1 might be amenable to therapeutic interventions, and new targets can be identified and validated using the selection approach.

Animals↗
PubMed2004

B cell-specific loss of histone 3 lysine 9 methylation in the V(H) locus depends on Pax5.

Immunoglobulin heavy chain rearrangement (V(H)-to-DJ(H)) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse V(H) locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non-B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous V(H)-to-DJ(H) recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the V(H) locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell-specific V(H)-to-DJ(H) recombination.

Animals↗
PubMed2004

Myc-transformed epithelial cells down-regulate clusterin, which inhibits their growth in vitro and carcinogenesis in vivo.

Effective treatment of malignant carcinomas requires identification of proteins regulating epithelial cell proliferation. To this end, we compared gene expression profiles in murine colonocytes and their c-Myc-transformed counterparts, which possess enhanced proliferative potential. A surprisingly short list of deregulated genes included the cDNA for clusterin, an extracellular glycoprotein without a firmly established function. We had previously demonstrated that in organs such as skin, clusterin expression is restricted to differentiating but not proliferating cell layers, suggesting a possible negative role in cell division. Indeed, its transient overexpression in Myc-transduced colonocytes decreased cell accumulation. Furthermore, clusterin was down-regulated in rapidly dividing human keratinocytes infected with a Myc-encoding adenovirus. Its knockdown via antisense RNA in neoplastic epidermoid cells enhanced proliferation. Finally, recombinant human clusterin suppressed, in a dose-dependent manner, DNA replication in keratinocytes and other cells of epithelial origin. Thus, clusterin appears to be an inhibitor of epithelial cell proliferation in vitro. To determine whether it also affects neoplastic growth in vivo, we compared wild-type and clusterin-null mice with respect to their sensitivity to 7, 12-dimethylbenz(a)anthracene /12-Otetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis. We observed that the mean number of papillomas/mouse was higher in clusterin-null animals. Moreover, these papillomas did not regress as readily as in wild-type mice and persisted beyond week 35. The rate of progression toward squamous cell carcinoma was not altered, although those developing in clusterin-null mice were on average better differentiated. These data suggest that clusterin not only suppresses epithelial cell proliferation in vitro but also interferes with the promotion stage of skin carcinogenesis.

Animals↗
PubMed2004

Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity.

Tumor necrosis factor alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha family of death receptor ligands and holds great therapeutic potential as a tumor cell-specific cytotoxic agent. Using a panel of established tumor cell lines and normal cells, we found a significant difference between the number of TRAIL-sensitive cells expressing high levels of c-myc and TRAIL-resistant cells expressing low levels of c-myc (P < 0.05, n = 19). We also found a direct linear correlation between c-myc levels and TRAIL sensitivity in TRAIL-sensitive cell lines (r = 0.94, n = 6). Overexpression of c-myc or activation of a myc-estrogen receptor (ER) fusion sensitized TRAIL-resistant cells to TRAIL. Conversely, small interfering RNA (siRNA)-mediated knockdown of c-myc significantly reduced both c-myc expression and TRAIL-induced apoptosis. The gene encoding the inhibitor of caspase activation, FLICE inhibitory protein (FLIP), appears to be a direct target of c-myc-mediated transcriptional repression. Overexpression of c-myc or activation of myc-estrogen receptor (ER) decreased FLIP levels both in cell culture and in mouse models of c-myc-induced tumorigenesis, while knocking down c-myc using siRNA increased FLIP expression. Chromatin immunoprecipitation and luciferase reporter analyses showed that c-myc binds and represses the human FLIP promoter. c-myc expression enhanced TRAIL-induced caspase 8 cleavage and FLIP cleavage at the death-inducing signaling complex. Combined siRNA-mediated knockdown of FLIP and c-myc resensitized cells to TRAIL. Therefore, c-myc down-regulation of FLIP expression provides a universal mechanism to explain the ability of c-myc to sensitize cells to death receptor stimuli. In addition, identification of c-myc as a major determinant of TRAIL sensitivity provides a potentially important screening tool for identification of TRAIL-sensitive tumors.

Animals↗
PubMed2003

Infection and cancer: the common vein.

The role of infectious agents in the development of cancer is well documented. The pathogenesis of various human neoplasms ranging from non-Hodgkin lymphoma (NHL) to cervical carcinoma frequently involves a chronic, most often viral, infection. At the same time, there is compelling evidence that certain acute infections result in the inhibition of neoplastic growth. The basis for this phenomenon is often thought to be concomitant anti-tumor immunity. Yet, experimental data supporting this hypothesis are scarce, and other non-immune anti-tumor factors could be involved. For instance, since virtually all aggressive tumors outstrip their blood supply, development of new vessels, or angiogenesis, is a limiting factor during neoplastic growth. In this review, we will discuss recent studies that implicate anti-angiogenesis in infection-mediated tumor suppression and suggest that this mechanism could also complement cytotoxic immunity arising from the use of cancer vaccines.

Angiogenesis Inhibitors↗
PubMed2002

Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/reactivation of the EBF/Pax5 pathway.

B lymphomagenesis is an uncontrolled expansion of immature precursors that fail to complete their differentiation program. This failure could be at least partly explained by inappropriate expression of several oncogenic transcription factors, such as Pax5 and Myc. Both Pax5 and c-Myc are implicated in the pathogenesis of non-Hodgkin lymphomas. To address their role in lymphomagenesis, we analyzed B-cell lymphomas derived from p53-null bone marrow progenitors infected in vivo by a Myc-encoding retrovirus. All Myc-induced lymphomas invariably maintained expression of Pax5, which is thought to be incompatible with terminal differentiation. However, upon culturing in vitro, several cell lines spontaneously down-regulated Pax5 and its target genes CD19, N-Myc, and MB1. Unexpectedly, other B-cell markers (eg, CD45R) were also down-regulated, and markers of myeloid lineage (CD11b and F4/80 antigen) were acquired instead. Moreover, cells assumed the morphology reminiscent of myeloid cells. A pool of F4/80-positive cells as well as several single-cell clones were obtained and reinjected into syngeneic mice. Remarkably, pooled cells rapidly re-expressed Pax5 and formed tumors of relatively mature lymphoid phenotype, with surface immunoglobulins being abundantly expressed. Approximately half of tumorigenic single-cell clones also abandoned myeloid differentiation and gave rise to B lymphomas. However, when secondary lymphoma cells were returned to in vitro conditions, they once again switched to myeloid differentiation. This process could be curbed via enforced expression of retrovirally encoded Pax5. Our data demonstrate that some Myc target cells are bipotent B-lymphoid/myeloid progenitors with the astonishing capacity to undergo successive rounds of lineage switching.

Animals↗
PubMed2002

A non-transgenic mouse model for B-cell lymphoma: in vivo infection of p53-null bone marrow progenitors by a Myc retrovirus is sufficient for tumorigenesis.

The c-Myc oncoprotein is strongly implicated in B-cell neoplasms such as human Burkitt lymphomas and mouse plasmocytomas. Transgenic mice in which the myc gene is juxtaposed to an immunoglobulin enhancer (E(mu)-myc) also develop B-cell lymphomas, but relatively late in life. In addition, these neoplasms are invariably clonal, suggesting the involvement of additional mutations. Such mutations frequently affect the p53 tumour suppressor gene or its positive regulator Arf, hinting that inactivation of the p53 pathway might be the second hit required for the progression towards malignancy. However, even tumours arising in E(mu)-myc/Arf-null animals are thought to be clonal. This observation raised doubts whether overexpression of Myc in p53-null B-cell precursors is sufficient for tumorigenesis. To address this question, we have established a new, non-transgenic mouse model of B-lymphoma. This model is based on isolation of primary bone marrow (BM) cells, admixing them with packaging cells producing a Myc-encoding retrovirus (LMycSN), and subcutaneous injection into a host with which BM cells are syngeneic. Predictably, wild type BM cells infected in vivo by LMycSN were not tumorigenic. However, LMycSN-infected p53-null BM cells readily gave rise to B-cell lymphomas composed predominantly of late pro-B/small pre-B-cells. In these tumours, heavy chain gene rearrangements were analysed using two independent PCR-based assays. All neoplasms with DJ-rearrangements were found to be polyclonal. This result suggests that inactivation of p53 and overexpression of Myc is all that is necessary for the development of full-fledged B-lymphomas. Our model would also be instrumental in assessing the transforming potential of Myc mutants and in studying cooperation between Myc and other oncogenes.

Animals↗
PubMedDate not supplied

An essential role of Th1 responses and interferon gamma in infection-mediated suppression of neoplastic growth.

We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth. Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFN-gamma), in this phenomenon. Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV). In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection. To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses. When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth. However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts. This enhanced resistance correlated with dramatically elevated levels of circulating IFN-gamma, a principal Th1 cytokine. Furthermore, while interleukin-12 and tumor necrosis factor a were dispensable for tumor suppression, in animals deficient in IFN-gamma production or signaling, tumor growth and neovascularization were markedly enhanced. Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFN-gamma and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance.

Acute Disease↗