Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models.

Necrostatin-1 (Nec-1) is widely used in disease models to examine the contribution of receptor-interacting protein kinase (RIPK) 1 in cell death and inflammation. We studied three Nec-1 analogs: Nec-1, the active inhibitor of RIPK1, Nec-1 inactive (Nec-1i), its inactive variant, and Nec-1 stable (Nec-1s), its more stable variant. We report that Nec-1 is identical to methyl-thiohydantoin-tryptophan, an inhibitor of the potent immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Both Nec-1 and Nec-1i inhibited human IDO, but Nec-1s did not, as predicted by molecular modeling. Therefore, Nec-1s is a more specific RIPK1 inhibitor lacking the IDO-targeting effect. Next, although Nec-1i was ∼100 × less effective than Nec-1 in inhibiting human RIPK1 kinase activity in vitro, it was only 10 times less potent than Nec-1 and Nec-1s in a mouse necroptosis assay and became even equipotent at high concentrations. Along the same line, in vivo, high doses of Nec-1, Nec-1i and Nec-1s prevented tumor necrosis factor (TNF)-induced mortality equally well, excluding the use of Nec-1i as an inactive control. Paradoxically, low doses of Nec-1 or Nec-1i, but not Nec -1s, even sensitized mice to TNF-induced mortality. Importantly, Nec-1s did not exhibit this low dose toxicity, stressing again the preferred use of Nec-1s in vivo. Our findings have important implications for the interpretation of Nec-1-based data in experimental disease models.

A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase.

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.

Bin1 mediates apoptosis by c-Myc in transformed primary cells.

The Bin1 gene encodes a c-Myc-interacting adapter protein with tumor suppressor and cell death properties. In this study, we offer evidence that Bin1 participates in a mechanism through which c-Myc activates programmed cell death in transformed primary chick or rat cells. Antisense or dominant inhibitory Bin1 genes did not affect the ability of c-Myc to drive proliferation or transformation, but they did reduce the susceptibility of cells to c-Myc-induced apoptosis. Protein-protein interaction was implicated, suggesting that Bin1 mediates a death or death sensitization signal from c-Myc. Our findings offer direct support for the "dual signal" model of Myc apoptotic function, based on interactions with a binding protein. Loss of Bin1 in human tumors may promote malignant progression in part by helping to stanch the death penalty associated with c-Myc activation.

Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma.

The genetic events underlying the development of prostate cancer are poorly defined. c-Myc is often activated in tumors that have progressed to metastatic status, so events that promote this process may be important. Bin1 is a nucleocytoplasmic adaptor protein with features of a tumor suppressor that was identified through its ability to interact with and inhibit malignant transformation by c-Myc. We investigated a role for Bin1 loss or inactivation in prostate cancer because the human Bin1 gene is located at chromosome 2q14 within a region that is frequently deleted in metastatic prostate cancer but where no tumor suppressor candidate has been located. A novel polymorphic microsatellite marker located within intron 5 of the human Bin1 gene was used to demonstrate loss of heterozygosity and coding alteration in 40% of informative cases of prostate neoplasia examined. RNA and immunohistochemical analyses indicated that Bin1 was expressed in most primary tumors, even at slightly elevated levels relative to benign tissues, but that it was frequently missing or inactivated by aberrant splicing in metastatic tumors and androgen-independent tumor cell lines. Ectopic expression of Bin1 suppressed the growth of prostate cancer lines in vitro. Our findings support the candidacy of Bin1 as the chromosome 2q prostate tumor suppressor gene.

Losses of the tumor suppressor BIN1 in breast carcinoma are frequent and reflect deficits in programmed cell death capacity.

Oncogenic activation of MYC occurs often in breast carcinoma and is associated with poor prognosis. Loss or inactivation of mechanisms that restrain MYC may therefore be involved in tumor progression. In this study, we show that the MYC-interacting adaptor protein BIN1 is frequently missing in malignant breast cells and that this loss is functionally significant. BIN1 was expressed in normal and benign cells and tissues but was undetectable in 6/6 estrogen receptor-positive or estrogen receptor-negative carcinoma cell lines examined. Similarly, complete or partial losses of BIN1 were documented in 30/50 (60%) cases of malignant breast tissue analyzed by immuno-histochemistry or RT-PCR. Abnormalities in the organization of the BIN1 gene were apparent in only a minority of these cases, suggesting that most losses were due to epigenetic causes. Nevertheless, they were functionally significant because ectopic BIN1 induced programmed cell death in malignant cells lacking endogenous BIN1 but had no effect on the viability of benign cells. We propose that loss of BIN1 may contribute to breast cancer progression by eliminating a mechanism that restrains the ability of activated MYC to drive cell division inappropriately.

Mechanism for elimination of a tumor suppressor: aberrant splicing of a brain-specific exon causes loss of function of Bin1 in melanoma.

Loss of tumor suppressors that restrain important oncoproteins such as c-Myc may contribute to malignant progression. Bin1 is an adapter protein with features of a tumor suppressor that was identified through its interaction with and inhibition of the oncogenic properties of c-Myc. In this study, we analyzed the patterns of Bin1 expression in normal melanocytes and melanoma cells at different stages of tumor progression. Evidence is provided that Bin1 function is abrogated in melanoma cells by a mechanism based on aberrant splicing of a tissue-specific exon. Specifically, most melanoma cells inappropriately expressed exon 12A, which is spliced alternately into Bin1 isoforms found in brain but not into isoforms found in melanocytes and many other nonneuronal cells. Exon 12A sequences abolished the ability of Bin1 to inhibit malignant transformation by c-Myc or adenovirus E1A. Similarly, these sequences abolished the ability of Bin1 to induce programmed cell death in melanoma cells that endogenously expressed exon 12A. Our findings suggest that aberrant splicing of Bin1 may contribute to melanoma progression, and they define a mechanism by which the activity of a tumor suppressor can be eliminated in cells.

Bau, a splice form of Neurabin-I that interacts with the tumor suppressor Bin1, inhibits malignant cell transformation.

Bin1 is a nucleocytoplasmic adaptor protein and tumor suppressor. A novel protein termed Bau was identified through its ability to interact with a region of Bin1 required to inhibit malignant cell transformation by certain oncogenes. Bau is a splice form of Neurabin-I, one of two related F-actin-binding proteins that are proposed to link cadherin-based cell-cell adhesion sites with the growth regulatory kinase p70S6K. Bau lacks actin- and p70S6K-binding domains found in Neurabin-I but includes coiled-coil domains that are part of its central domain as well as additional sequences not found in Neurabin-I. Interaction with Bin1 requires the presence of the U3 region which is alternately spliced in muscle cells. Bau localizes to the nucleus and cytosol. Like Bin1, Bau can suppress oncogene-mediated transformation and inhibit tumor cell growth. We suggest that Bau may link Bin1 to the Neurabin-I/p70S6K system in muscle and other cells, perhaps providing a mechanism to influence adhesion-dependent signals which affect cell fate.

The murine Bin1 gene functions early in myogenesis and defines a new region of synteny between mouse chromosome 18 and human chromosome 2.

We cloned and functionally characterized the murine Bin1 gene as a first step to investigate its physiological roles in differentiation, apoptosis, and tumorigenesis. The exon-intron organization of the >/=55-kb gene is similar to that of the human gene. Consistent with a role for Bin1 in apoptosis, the promoter included a functional consensus motif for activation by NF-kappaB, an important regulator of cell death. A muscle regulatory module defined in the human promoter that includes a consensus recognition site for myoD family proteins was not conserved in the mouse promoter. However, Bin1 is upregulated in embryonic development by E10.5 in myotomes, the progenitors of skeletal muscle, supporting a role in myogenesis and suggesting that the mouse and human genes may be controlled somewhat differently during development. In C2C12 myoblasts antisense Bin1 prevents induction of the cell cycle kinase inhibitor p21WAF1, suggesting that it acts at an early time during the muscle differentiation program. Interspecific mouse backcross mapping located the Bin1 locus between Mep1b and Apc on chromosome 18. Since the human gene was mapped previously to chromosome 2q14, the location of Bin1 defines a previously unrecognized region of synteny between human chromosome 2 and mouse chromosome 18.

Inhibition of apoptosis by Marek's disease viruses.

Strains of Marek's disease virus (MDV), a herpesvirus, have been shown to augment the development of lymphoid leukosis induced by retroviruses, the avian leukosis virus (ALV) or the reticuloendotheliosis virus. In this study we explored the possibility that the ability to augment lymphoid leukosis may be correlated with the ability of different strains of MDV to block apoptosis. Subclones of the ALV-transformed B cell line, DT40, which was free of MDV DNA were infected with either R2/23 strain of MDV-1, SB1 strain of MDV-2, or turkey herpes virus (HVT), a MDV-3. We found that most of the normal DT40 cells and DT40 cells infected with the R2/23 became apoptotic when cultured in serum-reduced medium. By contrast, the frequency of apoptotic cells was greatly reduced in the DT40-SB1 and DT40-HVT subclones. These findings suggest that because the SB-1 strain persists in the ALV-infected cells, it may augment lymphoid leukosis by inhibiting apoptosis and providing a survival advantage to the B cells which have a deregulated myc proto-oncogene.

Regulation of Bcl-xL expression in human keratinocytes by cell-substratum adhesion and the epidermal growth factor receptor.

Cell-substratum adhesion is an essential requirement for survival of human neonatal keratinocytes in vitro. Similarly, activation of the epidermal growth factor receptor (EGF-R) has recently been implicated not only in cell cycle progression but also in survival of normal keratinocytes. The mechanisms by which either cell-substratum adhesion or EGF-R activation protect keratinocytes from programmed cell death are poorly understood. Here we describe that blockade of the EGF-R and inhibition of substratum adhesion share a common downstream event, the down-regulation of the cell death protector Bcl-xL. Expression of Bcl-xL protein was down-regulated during forced suspension culture of keratinocytes, concurrent with large-scale apoptosis. Similarly, EGF-R blockade was accompanied by down-regulation of Bcl-xL steady-state mRNA and protein levels to an extent comparable to that observed in forced suspension culture. However, down-regulation of Bcl-xL expression by EGF-R blockade was not accompanied by apoptosis; in this case, a second signal, generated by passaging, was required to induce rapid and large-scale apoptosis. These findings are consistent with the conclusions that (i) Bcl-xL represents a shared molecular target for signaling through cell-substrate adhesion receptors and the EGF-R, and (ii) reduced levels of Bcl-xL expression through EGF-R blockade lower the tolerance of keratinocytes for cell death signals generated by cellular stress.

Structural analysis of the human BIN1 gene. Evidence for tissue-specific transcriptional regulation and alternate RNA splicing.

BIN1 is a putative tumor suppressor that was identified through its interaction with the MYC oncoprotein. To begin to identify elements of BIN1 whose alteration may contribute to malignancy, we cloned and characterized the human BIN1 gene and promoter. Nineteen exons were identified in a region of >54 kilobases, six of which were alternately spliced in a cell type-specific manner. One alternately spliced exon encodes part of the MYC-binding domain, suggesting that splicing controls the MYC-binding capacity of BIN1 polypeptides. Four other alternately spliced exons encode amphiphysin-related sequences that were included in brain-specific BIN1 species, also termed amphiphysin isoforms or amphiphysin II. The 5'-flanking region of BIN1 is GC-rich and lacks a TATA box but directs transcriptional initiation from a single site. A approximately 0. 9-kilobase fragment from this region was sufficient for basal transcription and transactivation by MyoD, which may account for the high levels of BIN1 observed in skeletal muscle. This study lays the foundation for genetic and epigenetic investigations into the role of BIN1 in normal and neoplastic cell regulation.

Bcl-2 expressed using a retroviral vector is localized primarily in the nuclear membrane and the endoplasmic reticulum of chicken embryo fibroblasts.

A complementary DNA for human bcl-2 was cloned into the replication competent avian retrovirus vector RCASBP, and the resulting virus was used to express human Bcl-2 protein at high levels in chicken embryo fibroblasts. The expression of Bcl-2 did not transform or significantly alter the longevity of the chicken embryo fibroblasts in the presence of normal amounts of serum. However, the expression of Bcl-2 blocked c-Myc-induced apoptosis in these cells. Fractionation of the infected chicken embryo fibroblasts indicated that the protein was distributed equally between nuclear and high density cytoplasmic membranes. Immunofluorescence analysis by confocal microscopy and immunoelectron microscopy showed that the Bcl-2 protein was primarily associated with the nuclear membrane and with the endoplasmic reticulum. Reduced amounts of the protein were associated with other membranes in the cytoplasm. These data show that, in this system, the Bcl-2 protein associates with the nuclear membrane and intracytoplasmic membranes but is not preferentially associated with mitochondria.

Persistence of Marek's disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.

In ovo infection with the avian retrovirus RAV-1 leads to persistent infection of the central nervous system.

The ability of an avian retrovirus to cause central nervous system (CNS) disease was investigated in chickens infected in ovo with Rous associated virus-1. Viral envelope and core proteins and mature virions were found throughout CNS parenchyma, with the highest amounts localized in the granular layer of the cerebellum, in blood vessel endothelium, and the choroid plexus. This distribution was established by the time of hatching and persisted throughout the 14 weeks of observation. The highest levels of integrated proviral DNA and viral mRNA, were present in the cerebellum, consistent with the distribution of viral antigens. Mononuclear cell infiltrates were evident throughout the CNS, consistent with an inflammatory process. However, demyelination or vacuolar changes, as observed in other retroviral-induced CNS diseases, were not detected. Clinical symptoms of progressive neurologic dysfunction, i.e., weakness or paralysis of the hindlimbs, imbalance, and ataxia, were present in 7 of 38 infected chickens before termination of the experiment at 14 weeks posthatch. Viral antigens or lymphocyte infiltration were not detected in peripheral nerves. These findings suggest that the avian system may provide a valuable model to analyze the mechanisms governing retroviral induced CNS disease.