BIN1 is a novel MYC-interacting protein with features of a tumour suppressor.

BIN1 is a novel protein that interacts with the functionally critical Myc box regions at the N terminus of the MYC oncoprotein. BIN1 is structurally related to amphiphysin, a breast cancer-associated autoimmune antigen, and RVS167, a negative regulator of the yeast cell cycle, suggesting roles in malignancy and cell cycle control. Consistent with this likelihood, BIN1 inhibited malignant cell transformation by MYC. Although BIN1 is expressed in many normal cells, its levels were greatly reduced or undetectable in 14/27 carcinoma cell lines and 3/6 primary breast tumours. Deficits were functionally significant because ectopic expression of BIN1 inhibited the growth of tumour cells lacking endogenous message. We conclude that BIN1 is an MYC-interacting protein with features of a tumour suppressor.

Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment.

Farnesyl transferase inhibitors (FTIs) are a novel class of antitumor drugs that block the oncogenic activity of Ras. Because FTIs lack significant cell toxicity in vitro and in vivo, a significant question is how they cause tumor regression. We now report that FTIs are in fact potent activators of apoptosis in Ras-transformed cells if attachment to substratum is prevented. When cultured at high density or on polyHEMA, a nonadherent substrate, Ras-transformed cells exhibited massive DNA degradation and cell death within 24 h of treatment with the FTI L-739,749. Death was p53-independent and was inhibited by the apoptosis suppressor BCL-XL. Furthermore, apoptosis was significantly attenuated by ectopic expression of a farnesyl-independent form of RhoB, a Rho protein previously implicated as a critical target for inhibition by FTIs. The findings suggest a link between FTIs and Rho-dependent adhesion signaling. Furthermore, our work indicates that FTIs revert cells to a state in which cell-substratum attachment is necessary for viability and suggests that apoptosis forms the basis for drug-induced tumor regression.

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.

New Myc-interacting proteins: a second Myc network emerges.

Despite its intensive investigation for almost two decades, c-Myc remains a fascinating and enigmatic subject. A large and compelling body of evidence indicates that c-Myc is a transcription factor with central roles in the regulation of cell proliferation, differentiation, and apoptosis, but its exact function has remained elusive. In this review we survey recent advances in the identification and analysis of c-Myc-binding proteins, which suggest insights into the transcriptional roles of c-Myc but which also extend the existing functional paradigms. The C-terminal domain (CTD) of c-Myc mediates interaction with Max and physiological recognition of DNA target sequences, events needed for all biological actions. Recently described interactions between the CTD and other cellular proteins, including YY-1, AP-2, BRCA-1, TFII-I, and Miz-1, suggest levels of regulatory complexity beyond Max in controlling DNA recognition by c-Myc. The N-terminal domain (NTD), which includes the evolutionarily conserved and functionally crucial Myc Box sequences (MB1 and MB2), contains the transcription activation domain (TAD) of c-Myc as well as regions required for transcriptional repression, cell cycle regulation, transformation, and apoptosis. In addition to interaction with the retinoblastoma family protein p107, the NTD has been shown to interact with alpha-tubulin and the novel adaptor proteins Binl, MM-1, Pam, TRRAP, and AMY-1. The structure of these proteins and their effects on c-Myc actions suggest links to the transcriptional regulatory machinery as well as to cell cycle regulation, chromatin modeling, and apoptosis. Investigations of this emerging NTD-based network may reveal how c-Myc is regulated and how it affects cell fate, as well as providing tools to distinguish the physiological roles of various Myc target genes.

The polyproline region of p53 is required to activate apoptosis but not growth arrest.

p53 is a pivotal regulator of apoptosis but its mechanism of action is obscure. We report that the polyproline (PP) region located between p53's transactivation and DNA binding domains is necessary to induce apoptosis but not cell growth arrest. The PP region was dispensable for DNA binding, inhibition of SAOS-2 tumor cell growth, suppression of E1A + RAS cell transformation, and cell cycle inhibition. A temperature-sensitive dominant inhibitory p53 mutant lacking PP (p53ts deltaPP) retained its ability to cooperate with adenovirus E1A in transformation of primary BRK cells. However, while activation of wt p53 induced apoptosis in E1A + p53ts-transformed cells, activation of p53 deltaPP induced cell cycle arrest but not apoptosis in E1A + p53ts deltaPP-transformed cells. Similarly, PP deletion abolished apoptosis in LoVo colon carcinoma cells, which are killed by wt p53 overexpression. Transactivation was largely unaffected by PP deletion. Significantly, BAX induction was intact, indicating that additional events are required for p53 to induce apoptosis. As a recently described site for familial mutation in at least one breast cancer family, the PP region represents a domain that may be altered in human tumors. We concluded that p53's ability to induce apoptosis is dispensable for inhibiting cell growth and transformation and that the PP region plays a crucial role in apoptotic signaling.

c-Myc induces apoptosis in epithelial cells by both p53-dependent and p53-independent mechanisms.

We tested the hypothesis that wild-type p53 activity is required for c-Myc-dependent apoptosis in epithelial cells. Primary baby rat kidney epithelial cell lines were generated by immortalization through the concerted action of c-Myc and a temperature-sensitive (ts) dominant inhibitory mutant allele of p53 (BRK myc/p53ts cells). When shifted to the permissive temperature for wild-type p53 activity, the BRK myc/p53ts cells underwent growth arrest and apoptosis. However, apoptosis also could be induced by serum deprivation at the nonpermissive temperature, when p53 was in the mutant state. Bcl-2 suppressed both modes of cell death. Apoptosis induced by wild-type p53 but not by serum deprivation was accompanied by G1 cell cycle arrest and increased expression of the Bcl-2 antagonist Bax. We concluded that c-Myc could induce apoptosis in epithelial cells by at least two mechanisms that could be distinguished by their p53 requirement. Our results support the possibility that c-Myc-dependent cell death might be exploited for therapeutic ends during carcinoma development, without regard to p53 status of the target cell.

Bin1 functionally interacts with Myc and inhibits cell proliferation via multiple mechanisms.

The tumor suppressor Bin1 was identified through its interaction with the N-terminal region of Myc which harbors its transcriptional activation domain. Here we show that Bin1 and Myc physically and functionally associate in cells and that Bin1 inhibits cell proliferation through both Myc-dependent and Myc-independent mechanisms. Bin1 specifically inhibited transactivation by Myc as assayed from artificial promoters or from the Myc target genes ornithine decarboxylase (ODC) and alpha prothymosin (pT). Inhibition of ODC but not pT required the presence of the Myc binding domain (MBD) of Bin1 suggesting two mechanisms of action. Consistent with this possibility, a non-MBD region of Bin1 was sufficient to recruit a repression function to DNA that was unrelated to histone deacetylase. Regions outside the MBD required for growth inhibition were mapped in Ras cotransformation or HepG2 hepatoma cell growth assays. Bin1 required the N-terminal BAR domain to suppress focus formation by Myc whereas the C-terminal U1 and SH3 domains were required to inhibit adenovirus E1A or mutant p53, respectively. All three domains contributed to Bin1 suppression of tumor cell growth but BAR-C was most crucial. These findings supported functional interaction between Myc and Bin1 in cells and indicated that Bin1 could inhibit malignant cell growth through multiple mechanisms.

Regulation of E2F1-induced apoptosis by poly(ADP-ribosyl)ation.

The transcription factor adenovirus E2 promoter-binding factor (E2F)-1 normally enhances cell-cycle progression, but it also induces apoptosis under certain conditions, including DNA damage and serum deprivation. Although DNA damage facilitates the phosphorylation and stabilization of E2F1 to trigger apoptosis, how serum starvation renders cells vulnerable to E2F1-induced apoptosis remains unclear. Because poly(ADP-ribose) polymerase 1 (PARP1), a nuclear enzyme essential for genomic stability and chromatin remodeling, interacts directly with E2F1, we investigated the effects of PARP1 on E2F1-mediated functions in the presence and absence of serum. PARP1 attenuation, which increased E2F1 transactivation, induced G2/M cell-cycle arrest under normal growth conditions, but enhanced E2F1-induced apoptosis in serum-starved cells. Interestingly, basal PARP1 activity was sufficient to modify E2F1 by poly(ADP-ribosyl)ation, which stabilized the interaction between E2F1 and the BIN1 tumor suppressor in the nucleus. Accordingly, BIN1 acted as an RB1-independent E2F1 corepressor. Because E2F1 directly activates the BIN1 gene promoter, BIN1 curbed E2F1 activity through a negative-feedback mechanism. Conversely, when the BIN1-E2F1 interaction was abolished by PARP1 suppression, E2F1 continuously increased BIN1 levels. This is functionally germane, as PARP1-depletion-associated G2/M arrest was reversed by the transfection of BIN1 siRNA. Moreover, PARP-inhibitor-associated anti-transformation activity was compromised by the coexpression of dominant-negative BIN1. Because serum starvation massively reduced the E2F1 poly(ADP-ribosyl)ation, we conclude that the release of BIN1 from hypo-poly(ADP-ribosyl)ated E2F1 is a mechanism by which serum starvation promotes E2F1-induced apoptosis.

The primary structure of human gamma-glutamyl transpeptidase.

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.

Synthesis of human gamma-glutamyl transpeptidase (GGT) during the fetal development of liver.

We have determined expression of human GGT gene encoding gamma-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.

Elevated cytokine levels in synovial fluid of rheumatoid arthritis correlates with the presence of cytomegalovirus genome.

Synovial fluid aspirated from 34 patients with symptomatic rheumatoid arthritis (RA) was evaluated for the presence of human cytomegalovirus (CMV) genomic material using polymerase chain reaction (PCR), and for levels of interleukin 8 (IL-8) and IL-6 using enzyme-linked immunoadsorbence assay. IL-8 and IL-6 levels were significantly higher in CMV DNA-positive RA patients than CMV DNA-negative RA patients and at least 10-fold higher than in both corresponding control groups of patients with osteoarthritis (OA). These findings suggest an association between elevated IL-8 and IL-6 levels and the presence of the CMV genome in RA patients.

Cytomegalovirus genomes demonstrated by polymerase chain reaction in synovial fluid from rheumatoid arthritis patients.

Synovial fluid from 20 patients with rheumatoid arthritis was analyzed to detect human cytomegalovirus (CMV) genomic material using polymerase chain reaction. Of 20 samples tested, 9 were positive for CMV by either ethidium bromide staining or Southern blotting. In contrast, CMV was detected in only 2 of 18 control patients with osteoarthritis. These results suggest an etiologic relationship between CMV and rheumatoid arthritis.

The c-MYC-interacting proapoptotic tumor suppressor BIN1 is a transcriptional target for E2F1 in response to DNA damage.

The E2F1 transcription factor, which was originally identified as a cell-cycle initiator, mediates apoptosis in response to DNA damage. As E2F1-induced apoptosis is an attractive mechanism for cancer therapy, it is critical to fully elucidate its effector pathways. Here, we show that the c-MYC-interacting proapoptotic tumor suppressor, BIN1, is transcriptionally activated by E2F1 and mediates E2F1-induced apoptosis in response to DNA damage. Acting through the DNA-binding and transactivation domains, ectopically expressed E2F1 activated the human BIN1 promoter, which contains canonical E2F-recognition sites. Conversely, depletion of E2F1 by small interfering RNA or germline deletion led to BIN1 deficiency. DNA-damaging agents (which included etoposide) increased BIN1 levels, unless E2F1 was deficient. Moreover, endogenous E2F1 protein interacted directly with the BIN1 gene promoter in chromatin, particularly after etoposide treatment. Notably, suppression of BIN1 expression using an antisense (AS) technique attenuated the cell death mediated by E2F1 and etoposide. Although the p53 tumor suppressor, its sibling protein p73, and caspases are well-known E2F1 effectors for DNA damage-induced apoptosis, AS-BIN1 did not compromise their apoptotic functions. Our results collectively suggest that BIN1 is a novel transcriptional target of E2F1 that triggers a unique mode of cell death in response to DNA damage.

Japanese encephalitis virus nonstructural protein NS3 has RNA binding and ATPase activities.

Sequence data suggest that Japanese encephalitis virus (JEV) protein NS3 is a multifunctional protein with sequence motifs characteristic of a protease and a helicase. To examine the functions of JEV-NS3, a fusion protein of NS3 in Escherichia coli was generated. Analysis by Western blot using monospecific rabbit antisera generated against the fusion protein (anti-MBJEN3) showed that NS3 was localized in the membrane fraction of JEV-infected cells and the particulate fraction of bacteria extracts. The addition of anti-MBJEN3 sera reduced JEV-specific RNA synthesis activity in a in vitro system. In addition, NS3 was shown to exhibit RNA binding and ATPase activities, suggesting this protein has an important role in viral RNA replication in virus-infected cells.

Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells.

Clinical evidence suggests that hepatitis C virus (HCV) is etiologically involved in hepatic cancer and liver cirrhosis. To investigate whether the HCV nonstructural protein NS3 has oncogenic activity, NIH 3T3 cells were transfected with an expression vector containing cDNA for the 5'- or 3'-half sequence of the HCV genome segment encoding NS3. Only cells transfected with the 5'-half cDNA rapidly proliferated, lost contact inhibition, grew anchorage independently in soft agar, and formed tumors in nude mice. PCR analysis confirmed the presence of the 5'-half DNA in the transfectants. These results suggest that the 5' region of the HCV genome segment encoding NS3 is involved in cell transformation.

Detection of human cytomegalovirus genome in uterus tissue.

To analyze persistent infection by human cytomegalovirus (HCMV) in vivo, specimens obtained from various sources and autopsied organs were examined for the presence of HCMV DNA, mRNA transcripts and antigens by polymerase chain reaction, in situ hybridization and immunostaining. The HCMV genome was detected in lung, liver, kidney, and blood vessels at an average positive rate of 15%. The highest PCR-positive rate was observed with cervical smears. Subsequent examination of uterus tissues from patients with myoma revealed HCMV transcripts and antigens in glandular epithelial cells, leukocytes, endothelial cells, and others, indicating productive HCMV infection of cervical tissue.

Different antibody response to a neutralizing epitope of human cytomegalovirus glycoprotein B among seropositive individuals.

The amino-terminal portion of human cytomegalovirus glycoprotein B (HCMV-gB) was expressed as a fusion protein to analyze the neutralizing epitope recognized by human monoclonal antibody C23 and the humoral immune response to this epitope. The linear neutralizing epitope was further localized to the peptide within 17 amino acids (position 68-84) which were conserved between two HCMV laboratory strains. Ten out of 17 HCMV-seropositive human sera contained the antibody against this epitope. Although seven sera were negative for reacting with the fusion protein, the viruses isolated from the same patients retained the epitope. The immunogenicity of the epitope and the possible application of C23 human monoclonal antibody for passive immunization against HCMV infections are discussed.

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.

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.