Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer.

Overexpression of RhoA in cancer indicates a poor prognosis, because of increased tumor cell proliferation and invasion and tumor angiogenesis. We showed previously that anti-RhoA small interfering RNA (siRNA) inhibited aggressive breast cancer more effectively than conventional blockers of Rho-mediated signaling pathways. This study reports the efficacy and lack of toxicity of intravenously administered encapsulated anti-RhoA siRNA in chitosan-coated polyisohexylcyanoacrylate (PIHCA) nanoparticles in xenografted aggressive breast cancers (MDA-MB-231). The siRNA was administered every 3 days at a dose of 150 or 1500 microg/kg body weight in nude mice. This treatment inhibited the growth of tumors by 90% in the 150-microg group and by even more in the 1500-microg group. Necrotic areas were observed in tumors from animals treated with anti-RhoA siRNA at 1500 microg/kg, resulting from angiogenesis inhibition. In addition, this therapy was found to be devoid of toxic effects, as evidenced by similarities between control and treated animals for the following parameters: body weight gain; biochemical markers of hepatic, renal, and pancreatic function; and macroscopic appearance of organs after 30 days of treatment. Because of its efficacy and the absence of toxicity, it is suggested that this strategy of anti-RhoA siRNA holds significant promise for the treatment of aggressive cancers.

An LNA-based loss-of-function assay for micro-RNAs.

MicroRNAs (miRNAs) have recently emerged as being essential for development and for the control of cell proliferation/differentiation in various organisms. However, little is known about miRNA function and mode of action at the cellular level. We have designed a miRNA loss-of-function assay, based on chemically modified locked nucleic acids (LNA) antisense oligonucleotides and usable in tissue culture cells. We show that LNA/DNA mixed oligonucleotides form highly stable duplexes with miRNAs in vitro. Ex vivo, the target miRNA becomes undetectable in cells transfected with the antisense oligonucleotide. The effect is dose-dependent, long-lasting, and specific. Moreover, using a reporter assay, we show that antisense LNA/DNA oligonucleotides inhibit short non-coding RNAs at the functional level. Thus LNA/DNA mixmers represent powerful tools for functional analysis of miRNAs.

Anti-RhoA and anti-RhoC siRNAs inhibit the proliferation and invasiveness of MDA-MB-231 breast cancer cells in vitro and in vivo.

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

CBP/p300 and muscle differentiation: no HAT, no muscle.

Terminal differentiation of muscle cells follows a precisely orchestrated program of transcriptional regulatory events at the promoters of both muscle-specific and ubiquitous genes. Two distinct families of transcriptional co-activators, GCN5/PCAF and CREB-binding protein (CBP)/p300, are crucial to this process. While both possess histone acetyl-transferase (HAT) activity, previous studies have failed to identify a requirement for CBP/p300 HAT function in myogenic differentiation. We have addressed this issue directly using a chemical inhibitor of CBP/p300 in addition to a negative transdominant mutant. Our results clearly demonstrate that CBP/p300 HAT activity is critical for myogenic terminal differentiation. Furthermore, this requirement is restricted to a subset of events in the differentiation program: cell fusion and specific gene expression. These data help to define the requirements for enzymatic function of distinct coactivators at different stages of the muscle cell differentiation program.

Cell cycle-dependent recruitment of HDAC-1 correlates with deacetylation of histone H4 on an Rb-E2F target promoter.

The transcription factor E2F, which is a key element in the control of cell proliferation, is repressed by Rb and other pocket proteins in growth-arrested differentiating cells, as well as in proliferating cells when they progress through early G1. It is not known whether similar mechanisms are operative in the two situations. A body of data suggests that E2F repression by pocket proteins involves class I histone deacetylases (HDACs). It has been hypothesized that these enzymes are recruited to E2F target promoters where they deacetylate histones. Here we have tested this hypothesis directly by using formaldehyde cross-linked chromatin immunoprecipitation (XChIP) assays to evaluate HDAC association in living cells. Our data show that a histone deacetylase, HDAC-1, is stably bound to an E2F target promoter during early G1 in proliferating cells and released at the G1-S transition. In addition, our results reveal an inverse correlation between HDAC-1 recruitment and histone H4 acetylation on specific lysines.

The Rb/chromatin connection and epigenetic control: opinion.

The balance between cell differentiation and proliferation is regulated at the transcriptional level. In the cell cycle, the transition from G1 to S phase (G1/S transition) is of paramount importance in this regard. Indeed, it is only before this point that cells can be oriented toward the differentiation pathway: beyond, cells progress into the cycle in an autonomous manner. The G1/S transition is orchestrated by the transcription factor E2F. E2F controls the expression of a group of checkpoint genes whose products are required either for the G1-to-S transition itself or for DNA replication (e.g. DNA polymerase alpha). E2F activity is repressed in growth-arrested cells and in early G1, and is activated at mid-to-late G1. E2F is controlled by the retinoblastoma tumor suppressor protein Rb. Rb represses E2F mainly by recruiting chromatin remodeling factors (histone deacetylases and SWI/SNF complexes), the DNA methyltransferase DNMT1, and a histone methyltransferase. This review will focus on the molecular mechanisms of E2F repression by Rb during the cell cycle and during cell-cycle exit by differentiating cells. A model in which Rb irreversibly represses E2F-regulated genes in differentiated cells by an epigenetic mechanism linked to heterochromatin, and involving histone H3 and promoter DNA methylation, is discussed.

Unambiguous demonstration of triple-helix-directed gene modification.

Triple-helix-forming oligonucleotides (TFOs), which can potentially modify target genes irreversibly, represent promising tools for antiviral therapies. However, their effectiveness on endogenous genes has yet to be unambiguously demonstrated. To monitor endogenous gene modification by TFOs in a yeast model, we inactivated an auxotrophic marker gene by inserting target sequences of interest into its coding region. The genetically engineered yeast cells then were treated with psoralen-linked TFOs followed by UV irradiation, thus generating highly mutagenic covalent crosslinks at the target site whose repair could restore gene function; the number of revertants and spectrum of mutations generated were quantified. Results showed that a phosphoramidate TFO indeed reaches its target sequence, forms crosslinks, and generates mutations at the expected site via a triplex-mediated mechanism: (i) under identical conditions, no mutations were generated by the same TFO at two other loci in the target strain, nor in an isogenic control strain carrying a modified target sequence incapable of supporting triple-helix formation; (ii) for a given target sequence, whether the triplex was formed in vivo on an endogenous gene or in vitro on an exogenous plasmid, the nature of the mutations generated was identical, and consistent with the repair of a psoralen crosslink at the target site. Although the mutation efficiency was probably too low for therapeutic applications, our results confirm the validity of the triple-helix approach and provide a means of evaluating the effectiveness of new chemically modified TFOs and analogs.

Autonomously binding protein detected on ets box of c-fos serum response element in proliferating cells.

The serum response element (SRE) in the c-fos promoter contains an ets box whose integrity is required for full activation of this proto-oncogene by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. Electrophoretic mobility shift assays (EMSA) detect a protein in nuclear extracts that binds to the wild-type SRE, but not to an SRE containing a mutated ets box. Competition studies using unlabeled probes, and supershift experiments using antibodies and in vitro translated core serum response factor (SRF) indicate that the protein in question is not YY1, SAP-1, nor Elk-1 and that it does not exhibit ternary complex factor (TCF) activity, so that it may correspond to an autonomously binding Ets family protein. The complete disappearance of this "Ets-like autonomous binding factor" upon terminal differentiation of both L6alpha2 myoblastic and PC12 pheochromocytoma cells points to a possible role in the proliferation/differentiation process.

Nitric oxide mediation of active immunosuppression associated with graft-versus-host reaction.

In the immunosuppression accompanying the lethal systemic graft-versus-host reaction (GVHR) directed against minor histocompatibility antigens in irradiated adult mice, we previously determined that non-T, non-B, L-leucine methyl ester (LME)-sensitive cells were implicated via two different mechanisms: one, which is interferon-gamma (IFN-gamma)-dependent and affects both T-cell proliferative responses and thymus-independent antibody production by CD5(+) B cells; and a second, which is IFN-gamma-independent and affects B-cell proliferative responses. Because IFN-gamma induces the production of nitric oxide (NO), a potent immunosuppressive molecule, we investigated the involvement of NO in the suppression mediated by the LME-sensitive cells. Inducible NO synthase (iNOS) mRNA, iNOS protein, and the stable end products of iNOS pathway, L-citrulline and nitrite, were detected early in GVHR in LME-sensitive spleen cells taken ex vivo and could be amplified in vitro by T and B mitogens. Inhibition of NO production with arginine analogs (aminoguanidine, N(G)-monomethyl-L-arginine [LMMA]), like anti-IFN-gamma antibodies, reversed suppression of both T-cell responses to concanavalin A and CD5(+) B-cell responses, but not of B-cell response to lipopolysaccharides (LPS). The GVHR-associated, IFN-gamma-dependent immunosuppression of T-cell proliferation and of antibody synthesis by CD5(+) B cells is the consequence of NO production by LME-sensitive cells. Immunohistochemical analyses indicate that these cells belong to the macrophage lineage.

Histone acetyltransferase activity of CBP is controlled by cycle-dependent kinases and oncoprotein E1A.

Transforming viral proteins such as E1A force cells through the restriction point of the cell cycle into S phase by forming complexes with two cellular proteins: the retinoblastoma protein (Rb), a transcriptional co-repressor, and CBP/p300, a transcriptional co-activator. These two proteins locally influence chromatin structure: Rb recruits a histone deacetylase, whereas CBP is a histone acetyltransferase. Progression through the restriction point is triggered by phosphorylation of Rb, leading to disruption of Rb-associated repressive complexes and allowing the activation of S-phase genes. Here we show that CBP, like Rb, is controlled by phosphorylation at the G1/S boundary, increasing its histone acetyltransferase activity. This enzymatic activation is mimicked by E1A.

Intracellular signaling events in CD77-mediated apoptosis of Burkitt's lymphoma cells.

In the hematopoietic system CD77, a glycolipid surface antigen, is restricted to group I Burkitt's lymphoma (BL) cell lines and a subset of germinal center B lymphocytes. Recently, we have reported that recombinant B subunits of Verotoxin, which specifically binds to CD77, induce programmed cell death of CD77+ BL cells. Here, we show that an anti-CD77 monoclonal antibody (38.13) immobilized on tissue culture dishes also induces apoptosis, and we have explored the signal transducing events leading to this cell death. We show that ligation of CD77 antigen causes an increase of the intracellular Ca2+ concentration owing to an influx of extracellular Ca2+ through calcium channels. Chelation of extracellular Ca2+ with EGTA partially prevents anti-CD77-induced apoptosis, indicating that this process is probably Ca2+ dependent. We show that the cross-linking of CD77 provokes an increase of intracellular cAMP levels followed by cAMP-dependent protein kinase activation. We report that BL cells produce ceramide when they are exposed to 38.13 but, unexpectedly, without a concomitant decrease in sphingomyelin or CD77 content. Finally, we provide evidence that C2-ceramide, calcium ionophore, and forskolin (which increases intracellular levels of cAMP) independently induce apoptosis of CD77+ BL cells and, moreover, that C2-ceramide and forskolin strongly synergize to cause cell death. The possible role of CD77-mediated apoptosis in the B cell selection that occurs in germinal centers is discussed.

Recruitment of transcription factors to the target site by triplex-forming oligonucleotides.

Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA target at physiological salt and pH conditions. The stability of the triplex formed under these conditions is very high: >90% of the triplex remains intact after 24 h of incubation. Bound to the double-stranded target DNA, the oligonucleotide retains its ability to interact specifically with transcription factors, recruiting them to the proximity of the target DNA. Our results suggest that this type of oligonucleotide may prove useful in the design of new tools for artificial modulation of gene expression.

Peptide nucleic acids directed to the promoter of the alpha-chain of the interleukin-2 receptor.

Two 10-mer oligopyrimidine peptide nucleic acids (PNAs) were designed to interfere with IL-2R alpha promoter expression by binding to the regulatory sequences overlapping SRF and NF-kappa B transcription factor sites. Specific complexes were formed on each target sequence, and clearly involved (1) Hoogsteen hydrogen bonds as shown by experiments in which the purine strand of a single or double-stranded target was substituted with 7-deazadeoxyguanosine, (2) P-loop formation on double-helical DNA as evidenced by susceptibility to a single-strand-specific nuclease. When formed on a single-stranded DNA target, these highly stable complexes were responsible for efficient physical blockage of T7 DNA polymerase elongation on the template DNA containing the target oligopurine sequence. On a double-stranded target, these complexes only formed at low ionic strength and were slowly dissociated at physiological ionic strength (pH 6.5) with a t1/2 of 6.5-7 h. The salt-dependent instability of preformed complexes on a plasmid target was probably the critical factor responsible for their lack of significant sequence-specific effect on IL-2R alpha promoter activity inside living cells.

Induction and suppression of delayed-type hypersensitivity to non-MHC antigens during lethal graft-versus-host reaction.

A delayed-type hypersensitivity reaction (DTH) to non-MHC Ag was detected during a lethal graft-vs-host reaction (GVH) induced by incompatibility for non-MHC Ag alone. In this model, when appropriate doses of B10.D2 bone marrow and lymphoid cells are grafted to irradiated (DBA/2 x B10.D2)F1 recipients, the ensuing GVH, directed against those DBA/2 non-MHC Ag absent from the B10.D2 background, results in virtually 100% mortality in less than 3 mo; donor alloimmunization against the host histocompatibility Ag considerably reduces mortality, and survival rates of 80 to 100% are common. The experiments reported here show that: 1) the cell responsible for DTH induction expresses the CD4+ CD8- phenotype; 2) CD4+ cells likewise seem to play a predominant role in the pathology of lethal GVH in this genetic combination; 3) the alloimmunization protocol that abrogates mortality also abolishes GVH-associated DTH; and 4) this suppressive effect, as shown elsewhere for the protection against mortality, is mediated by CD4- CD8- "double negative" Thy-1+ CD3+ T suppressor cells. Thus, there is a good parallel between lethal GVH and its associated DTH as concerns both induction and suppression of the two phenomena, suggesting that mortality and DTH may represent different manifestations of a common underlying mechanism. Initiation of the effector phase of DTH in the adoptive transfer model seems to be dependent on the presence of a Thy-1+ double-negative cell in the transfer inoculum; the possible relationship of this double-negative cell to the Th-1-type CD4+ DTH-mediating cell recently shown to induce lethal GVH is discussed.

Lethal graft-versus-host reaction against non-H-2 antigens. I. Prevention of GVH-associated immunodeficiency by preimmunizing the donor against host-specific non-H-2 antigens.

Injection of B10.D2 cells into irradiated H-2d compatible (DBA/2xB10.D2)F1 recipients provokes a lethal GVH that can be abrogated by donor preimmunization against host-specific DBA/2 non-H-2 antigens. To study the possible relationship between the observed protection and restoration of immune responsiveness, we compared spleen cellularity, selected T and B cell functions, and NK activity in GVH and protected mice during the 1st month after grafting. Normal and isografted mice served as controls. GVH was found to be characterized by an early stimulation phase associated with splenomegaly and increased percentages (but not numbers) of Lyt-2+ and L3T4+ cells, followed by profound aplasia and abrogation of IL-2 production. Response to a B cell mitogen (LPS) is depressed, and cells from GVH mice exert a strong suppressive effect on the LPS and PHA responsiveness of normal cells. Suppression appears to be mediated by a radioresistant, nylon nonadherent, asialo GM1 negative cell expressing a low level of Thy-1 antigen. In contrast, protection correlates with progressive restoration of spleen cellularity and LPS responsiveness, with decreased but clearly detectable IL-2 production, and transient nonspecific suppressor activity. The immune status of protected mice resembles that of isografted controls. No correlation was found between mortality (or protection) and either PHA responsiveness, which remained depressed in all grafted mice throughout the observation period, or NK activity, which was strongly depressed in both GVH and protected mice. In conclusion, protection correlates with the disappearance of nonspecific suppressor cells and the restoration of cellularity and certain nonspecific immune functions. Donor immunization against host-specific non-H-2 antigens, which protects against mortality, also protects against GVH-associated immune deficiency.

Abrogation of the lethal graft-vs.-host reaction developed to non-H-2 antigens: involvement of T suppressor cells distinct from veto cells.

The mortality induced by graft-vs.-host reaction (GVHR) in (DBA/2 x B10.D2)F1 recipients transplanted with cells from H-2d-identical B10.D2 donors can be abrogated by preimmunizing the donors with parent-strain spleen cells from normal DBA/2 mice. The experiments described here were designed to explore the possibility that the observed protection might be mediated by veto cells contained in the immunizing cell inoculum; the reasoning was based on an analogy with the cytotoxic T lymphocyte response to non-H-2 antigens where suppression can be mediated by veto cells, present in the spleens of normal mice, which are radiosensitive and largely Lyt-2+. We show that the intensity of the protection against GVHR mortality is a function of the immunizing cell dose, and that protection remains effective when optimal doses of immunizing cells are (a) irradiated or (b) pretreated with anti-Thy-1 serum. GVHR suppression is abrogated when, before transfer to F1 recipients, suppressor cells from spleens of immunized donors are pretreated with antiserum directed against Lyt-1.2 (expressed by B10.D2 but not by DBA/2, which expresses Lyt-1.1); in contrast, it is not significantly affected when these same cells are pretreated with anti-Lyt-2.2 alloantiserum. We conclude that when the antigen load is great enough the immunizing cells play a largely passive role in the observed suppression. The protection against GVHR mortality seen in this H-2-compatible combination is transferable by Lyt-1+2- suppressor T cells originating in mice given high doses of alloantigen. These suppressor cells are therefore distinct from the splenic veto T cells effective against cytotoxic T lymphocyte responses to non-H-2 antigens. The mechanism of the observed suppression and its relationship to Mls product(s) are discussed.

Abrogation of lethal graft-versus-host reaction directed against non-H-2 antigens: role of Mlsa and K/I region antigens in the induction of unresponsiveness by alloimmunization.

A graft-versus-host reaction (GVHR) directed against DBA/2 non-H-2 antigens alone can be induced by grafting B10.D2 bone marrow and spleen cells intravenously to heavily irradiated, H-2d compatible (DBA/2 X B10.D2)F1 adult mice. Under the experimental conditions used, only 0-10% of recipients survive, but the survival is greatly increased by donor alloimmunization, a few days prior to grafting, against host-specific (DBA/2) non-H-2 antigens and non-specific (foreign) H-2 antigens. The increased survival is mediated by alloimmunization-activated suppressor cells which can decrease the intensity of the immune reaction developed by normal B10.D2 cells both in vivo (GVHR) and in vitro (proliferative response measured in mixed lymphocyte culture, MLC). The present experiments were designed to explore the antigenic requirements for inducing suppression. The results showed that in GVHR the protective effect induced by donor alloimmunization against the specific non-H-2 antigens, which leads to 70-80% survival, is due primarily, if not entirely, to immunization against Mlsa antigens. Results of MLC experiments confirmed this conclusion, showing that immunization against Mlsa antigens is sufficient to account for the suppressive effect induced by the specific immunization. In addition, they indicated that the non-specific protective effect induced by donor alloimmunization against foreign H-2 antigens, which leads to 20-30% survival, is due to immunization against antigens encoded by the K and/or I region(s) of the H-2 complex; immunization against D region encoded antigens alone has no effect.

Parameters involved in the induction and abrogation of the lethal graft-versus-host reaction directed against non-H-2 antigens.

The grafting of cells from donors incompatible for non-H-2 antigens alone can lead to GvHR mortality in up to 100% of lethally irradiated adult recipients. GvHR severity correlates with the number of mature immunocompetent cells present in the bone marrow inoculum. Histologic and clinical manifestations of GvHR observed in these mice differ from those seen when GvHR is induced across an H-2 barrier. The number of non-H-2 genes capable of influencing GvHR mortality is probably great, and their effects may vary as a function of sex. The non-H-2 genes influence GvHR mortality mainly via their interactions, the consequences of which are complex and can result in either cumulative or suppressive effects. GvHR mortality is considerably reduced by donor immunization, shortly before grafting, against host-specific non-H-2 antigens; and it is virtually abrogated by an additional immunization of the donors against nonspecific (foreign) H-2 antigens. Three weeks after grafting, these "protected" mice are easily distinguishable from those undergoing lethal GvHR, as assessed by both clinical appearance and histologic examination; in contrast, they are nearly indistinguishable from control mice grafted with syngeneic cells. However, depending upon the conditions used for the immunization, an additional immunization against nonspecific H-2 antigens can lead to acceleration rather than suppression of GvHR mortality; this phenomenon is not seen, under the same experimental conditions, after immunization against specific non-H-2 antigens alone. It is therefore suggested that a "second signal" provided by an additional nonspecific stimulus can potentiate either the establishment of specific suppression or the activation of a secondary ("positive") response. Suppressive effects of the specific and nonspecific immunizations are cumulative, and both treatments activate suppressor cells. The intensity of suppression induced by both specific and nonspecific immunizations is antigen dose-dependent. At equivalent antigen doses the specific immunization is considerably more effective than the nonspecific immunization, and is detectable after injection of as few as 2.5 X 10(5) cells. In both cases, irradiation of the immunizing cells abolishes the suppression induced by the lower cell doses tested, while it merely decreases the intensity of the suppression induced by the higher cell doses tested. The impairment of suppression after irradiation of the immunizing cells is not attributable to a modification of their homing pattern, but to the fact that proliferation of the immunizing cells, which leads to an augmentation of the antigen dose, is abolished by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)