Mutation of the MXI1 gene in prostate cancer.

The Mxi1 protein negatively regulates Myc oncoprotein activity and thus potentially serves a tumour suppressor function. MXI1 maps to chromosome 10q24-q25, a region that is deleted in some cases of prostate cancer. We have detected mutations in the retained MXI1 alleles in four primary prostate tumours with 10q24-q25 deletions. Two tumours contained inactivating mutations, whereas two others contained the identical missense mutation. Fluorescence in situ hybridization also demonstrated loss of one MXI1 allele in an additional tumour lacking chromosome 10 abnormalities. MXI1 thus displays allelic loss and mutation in some cases of prostate cancer that may contribute to the pathogenesis or neoplastic evolution of this common malignancy.

Expression and analysis of COOH-terminal deletions of the human thrombospondin molecule.

Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.

Isolation of type C virions from a normal human fibroblast strain.

Type C virions were spontaneously released from cultures of a diploid human cell strain. The varions have properties of known type C RNA tumor viruses and share antigenic determinants with the major interspecies-specific antigen (p30) of simian sarcoma virus. Antiserum to reverse transcriptase of gibbon ape leukemia virus inhibits the reverse transcriptase of the putative human virions and that of simian sarcoma virus, but has no effect on the corresponding enzymes of avian or murine RNA tumor viruses.

Antisense-mediated reduction in thrombospondin reverses the malignant phenotype of a human squamous carcinoma.

Thrombospondin (TSP) is a trimeric glycoprotein which is synthesized and incorporated into the extracellular matrix by a wide variety of cells. TSP is involved in a number of cellular processes which govern tumor cell behavior including mitogenesis, attachment, migration, and differentiation. To directly assess the role of TSP in tumor cell growth and spread, a human squamous carcinoma cell line, with high TSP production and an invasive phenotype, was transfected with a TSP cDNA antisense expression vector. Five unique transfected clones were obtained with reduced TSP production. Expression of the transfected antisense sequence in these clones was verified by a ribonuclease protection assay. These clones demonstrated reduced growth rates in vitro when compared with a vector transfected control. After subcutaneous inoculation into athymic mice, the antisense clones formed either no tumors or tumors that were slow growing and highly differentiated. This contrasted with the vector-transfected clone which produced poorly differentiated, rapidly growing, invasive tumors. Our results argue in favor of a direct role for TSP in determining the malignant phenotype of certain human tumors.

Rhabdomyosarcomas do not contain mutations in the DNA binding domains of myogenic transcription factors.

Skeletal myogenesis is regulated by a group of transcription factors (MyoD, myogenin, myf5, and myf6) that are "basic helix-loop-helix" proteins that bind to the promoters of muscle-specific genes and promote their expression. We have previously shown that after a mutation of Leu122 to Arg the DNA binding basic domain of MyoD confers c-myc-like functional characteristics to the protein. In this study we used single-strand conformation polymorphism analysis to determine whether such mutations occur naturally in rhabdomyosarcomas. We have found that the basic domains of all the myogenic factors remain unaltered in rhabdomyosarcomas. Selection against such mutations may be the result of functional redundancy of these myogenic transcription factors.

Regulation of megakaryocyte phenotype in human erythroleukemia cells.

Induction of human erythroleukemia (HEL) cells with nanomolar tumor-promoting phorbol myristate acetate (PMA) diesters results in the synchronous acquisition of multiple markers of the megakaryocyte phenotype. Induced cells markedly increase their content of cytoplasm and show features of morphological maturation. At the ultrastructural level, PMA-treated cells show increases in cytoplasm, nuclear lobulation and nucleolar content, and free ribosomes. Limited numbers of cells also express alpha-granules and nascent demarcation membrane systems. Functionally, PMA-stimulated HEL cells express increased amounts of the megakaryocyte/platelet proteins: glycoprotein IIb/IIIa, platelet factor 4, von Willebrand factor, glycoprotein Ib, and thrombospondin. No changes are observed in antigenic markers of the erythroid (glycophorin A) or macrophage lineages (MO-1 or MO-2). The increases in antigenic expression are rapid, reaching maximum levels within 3-4 d under serum-free conditions. Treatment with PMA also abruptly (within 1-2 d) inhibits cellular division in these cells. Washout studies indicate that phorbols exert their effect within 18-24 h, the approximate cell cycle time for these cells. Consistent with proliferative arrest, c-myc proto-oncogene transcripts begin to decline within 8 h of PMA treatment, although transcripts of c-myb are unaffected. Importantly, megakaryocyte differentiation is associated with endomitotic DNA synthesis (i.e., continued DNA synthesis in the absence of mitosis and cytokinesis), with HEL cells reaching a DNA content of 3-12 times that of unstimulated cells. Endomitosis is coordinately regulated with changes in antigenic expression and cell size such that those cells having the highest DNA content are the largest and also express the greatest levels of antigen.

c-myc antisense transcripts accelerate differentiation and inhibit G1 progression in murine erythroleukemia cells.

Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units. Sixteen clones were isolated, each containing unrearranged c-myc sequences and expressing high levels of antisense transcripts. All antisense clones examined contained reduced amounts of cytoplasmic endogenous c-myc transcripts. The kinetics of reaccumulation of endogenous c-myc mRNA during a 24-h exposure to dimethyl sulfoxide (DMSO) were also retarded and the ultimate transcript levels attained were less than in control cells. Antisense clones grew as well as control F-MEL cells in medium containing 10% fetal calf serum but at only a half and a quarter of the control rates in media containing 5 and 2% serum, respectively. Antisense clones differentiated faster and to a greater degree than control cells following DMSO exposure. myc antisense transcript expression was increased by growing cells in methotrexate, which resulted in an enhanced response to DMSO. Fluorescence-activated cell sorter (FACS) analysis of cellular DNA content indicated that a greater fraction of antisense nuclei contained a G0/G1 2n DNA content following a 24-h exposure to DMSO. When density-arrested antisense clones were diluted into fresh medium to allow reentry into the cell cycle, they incorporated less [3H]thymidine than control cells. FACS analysis showed that this was because only a portion of the cell population was entering S phase. Whereas control cells did not increase in size following release from density arrested antisense cells contained a subpopulation which were initially smaller and which eventually attained the same size as control cells. Quiescent antisense cells thus comprise two populations, each arrested at a different point in G1. Dilutional replating allowed both populations to reenter the cell cycle. We propose a model which postulates that certain minimal myc levels are necessary for cells to traverse G1. Those with insufficient levels, due, for example, to antisense inhibition, are unable to completely traverse G1 during density arrest and synchronize at an earlier point than do control cells. This earlier point may be along the differentiation pathway and may account for the greater responsiveness of antisense cells to DMSO induction. This model postulates that F-MEL cells overexpressing myc fail to differentiate because myc levels are never sufficiently low enough to allow cells to enter the differentiation pathway.

Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways.

Immediate early genes involved in controlling cell proliferation are rapidly and transiently induced following stimulation of susceptible cells with serum. To study how oncoproteins regulate immediate early genes, we examined serum inducibility of these genes in cells transformed by various oncoproteins. We found that induction of the immediate early gene, c-fos, by serum stimulation was markedly attenuated in four independent cell lines stably transformed by the v-Src tyrosine kinase. Cells chronically transformed by other oncoproteins implicated in tyrosine kinase signaling pathways, including v-Sis, v-Ras, and v-Raf, showed the same pattern of attenuation. In contrast, serum inducibility of c-fos was not attenuated in cells transformed by simian virus 40, which is thought to transform cells through a different pathway. Cell cycle analyses showed that proliferation of these transformed cell lines could be arrested effectively in 0.1% serum, demonstrating that the attenuation was not simply due to continuous cycling of transformed cells after serum deprivation. Moreover, serum inducibility of other immediate early genes, including c-jun, junB, egr-1, and NGFI-B, also was strikingly attenuated by these same oncoproteins. Nuclear run-on transcription assays established that this attenuation of serum inducibility occurred at the transcriptional level. Finally, flow cytometric analysis demonstrated that serum-starved v-Src-transformed cells were viable and able to progress into S phase of the cell cycle after serum stimulation, even though the induction of immediate early genes was greatly attenuated in these cells. Our results suggest that activation of immediate early genes is repressed by chronic stimulation of tyrosine kinase signaling pathways in transformed cells.

The leucine zipper of c-Myc is required for full inhibition of erythroleukemia differentiation.

The leucine zipper motif has been observed in a number of proteins thought to function as eucaryotic transcription factors. Mutation of the leucine zipper interferes with protein dimerization and DNA binding. We examined the effect of point mutations in the leucine zipper of c-Myc on its ability to dimerize in vitro and to inhibit Friend murine erythroleukemia (F-MEL) differentiation. Glutaraldehyde cross-linking studies failed to provide evidence for homodimerization of in vitro-synthesized c-Myc protein, although it was readily demonstrated for c-Jun. Nevertheless, whereas transfected wild-type c-myc sequences strongly inhibited F-MEL differentiation, those with single or multiple mutations in the leucine zipper were only partially effective in this regard. Since the leucine zipper domain of c-Myc is essential for its cooperative effect in ras oncogene-mediated transformation, this study emphasizes the close relationship that exists between transformation and hematopoietic commitment and differentiation. c-Myc may produce its effects on F-MEL differentiation through leucine zipper-mediated heterodimeric associations rather than homodimeric ones.

Regulation of the junB gene by v-src.

The proteins encoded by cellular and viral src genes are believed to be involved in the transmission of mitogenic signals, the nuclear recipients of which are largely unknown. In this work, we report that four different v-src-transformed cell lines from three different species possess elevated levels of junB transcripts. Transient expression of junB promoter-chloramphenicol acetyltransferase constructs in NIH 3T3 cells was used to demonstrate that the increase in junB transcripts was specifically associated with v-src expression and could not be recapitulated with a c-src, v-H-ras, or v-raf expression vector. Deletion mutants were used to localize the v-src-responsive region in the junB promoter to a 121-nucleotide region encompassing the CCAAT and TATAA elements. This region is distinct from one in the 5' untranslated region of the junB gene which is required to maintain its high-level basal expression. Point mutagenesis of the junB TATAA box completely abolished v-src responsiveness, suggesting that proteins which bind to this element are modified by src transformation. Several v-src and c-src mutants were used to demonstrate that elevated tyrosine kinase activity of src proteins is required for the observed effects on junB expression. Finally, homology between the TATAA box regions of junB and the unrelated but src-responsive gene 9E3/CEF-4 suggests that modulation of gene activity through proteins which bind to this region may be a recurrent, although not exclusive, theme in src transforming action. Our results suggest that src proteins may modulate some nuclear effectors through pathways not involving cellular ras or raf gene products.

Constitutive expression of a c-myb cDNA blocks Friend murine erythroleukemia cell differentiation.

A full-length human c-myb cDNA clone has been isolated from a CCRF-CEM leukemia cell cDNA library. The plasmid vector contains simian virus 40-derived promotor, splice, and polyadenylation sequences as well as a transcription unit for a dihydrofolate reductase cDNA. We have introduced this construct into Friend erythroleukemia (F-MEL) cells and have isolated a number of clones which contain intact and transcriptionally active human c-myb sequences. F-MEL clones expressing the highest levels of the human c-myb mRNA differentiate poorly in response to dimethyl sulfoxide. Two clones which initially expressed low levels of human c-myb transcripts and which differentiated normally were subsequently inhibited in their ability to differentiate when grown in successively higher concentrations of methotrexate, due to amplification and enhanced expression of plasmid sequences. The inhibitory effect on F-MEL differentiation appeared to be independent of the early decline in c-myc transcripts which were normally regulated in all cases examined. Our results indicate that constitutive expression of a nontruncated human c-myb cDNA can exert profound effects on erythroid differentiation and argue for a causal role of c-myb in the F-MEL differentiation process.

Depletion of c-myc with specific antisense sequences reverses the transformed phenotype in ras oncogene-transformed NIH 3T3 cells.

ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.

OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 mitogen-activated protein kinase. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were used to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through PPI.

Modulation of cis-platinum resistance in Friend erythroleukemia cells by c-myc.

The molecular genetic basis for cancer cell resistance to the important family of platinum coordination complex drugs is poorly understood. The observation that malignant cells commonly become resistant to therapy, while normal cells rarely do, suggests that certain molecular processes involved in malignancy, e.g., oncogene activation, might also play a role in drug resistance. Since aberrant expression and amplification of various myc oncogenes have been implicated in the prognosis of human cancers, the poor therapeutic response of some of these tumors might be explained if high levels of myc gene products rendered cells more refractory to therapy. We tested this hypothesis by determining the effect of varying the level of c-myc expression on resistance to cis-platinum and ionizing radiation in Friend murine erythroleukemia cells expressing varying levels of c-myc gene product. We found that a) the degree of cis-platinum resistance correlated directly with the level of c-myc expression, b) glucocorticoid induction of murine mammary tumor virus promoter-driven c-myc sequences significantly increased cis-platinum resistance, c) restoring c-myc transcript levels to normal restored the original cis-platinum sensitivity at a rate which paralleled that of induced c-myc transcript depletion, and d) c-myc transcript level had no effect on ionizing radiation response. These findings suggest that c-myc levels may influence therapeutic success in some tumors and may regulate specific processes by which cells cope with DNA damage caused by DNA cross-linking agents such as the platinum analogues, but not ionizing radiation.

Inverse regulation of cyclin B1 by c-Myc and p53 and induction of tetraploidy by cyclin B1 overexpression.

We have shown previously that mitotic spindle inhibitors allow the c-Myconcoprotein to uncouple mitosis from DNA synthesis, resulting in the acquisition of tetraploidy. This can also occur in the absence of spindle inhibition if c-Myc deregulation is combined with inactivation of the p53 tumor suppressor. Under these conditions, cyclin B1 protein is induced but retains its normal cell cycle regulation. We now show that the cyclin B1 promoter is directly but oppositely regulated by c-Myc and p53. Enforced expression of cyclin B1 also induces tetraploidy, either after mitotic spindle inhibition or in the absence of such inhibition if cyclin B1 is coexpressed with c-Myc. Cyclin B1 represents a new class of c-Myc target genes that is also regulated by p53. It is also the first identified downstream effector of c-Myc able to produce the chromosomal instability that characterizes virtually all tumor cells.

Lack of transcriptional repression by max homodimers.

Max, a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) protein, plays a central role in the transcriptional regulation of myc oncoprotein-responsive genes. Myc-max heterodimers bind to consensus E-box motifs near or within the promoters of these genes and activate gene expression, whereas heterodimers between max and members of the mad family of bHLH-ZIP proteins promote transcriptional repression. In contrast to all other members of the myc network, max readily homodimerizes and binds to identical E-box sites in vitro. However, the role for max homodimers in transcriptional repression in vivo is unclear. Upstream stimulatory factor (USF) is a bHLH-ZIP protein which does not interact with members of the myc-max-mad family. By replacing the HLH-ZIP domain of max with that from USF, we created a chimeric protein, max(USF), which was indistinguishable from max with respect to its ability to homodimerize and bind DNA. As expected, however, max(USF) was unable to heterodimerize with any of the tested max partner proteins and was incapable of suppressing c-myc target genes. Thus, transcriptional repression is an exclusive property of max-mad heterodimers and cannot be achieved by max homodimers alone.

MYC oncogenes and human neoplastic disease.

c-myc, N-myc and L-myc are the three members of the myc oncoprotein family whose role in the pathogenesis of many human neoplastic diseases has received wide empirical support. In this review, we first summarize data, derived mainly from non-clinical studies, indicating that these oncoproteins actually serve quite different roles in vivo. This concept necessarily lies at the heart of the basis for the observation that the deregulated expression of each MYC gene is reproducibly associated with only certain naturally occurring malignancies in humans and that these genes are not interchangeable with respect to their aberrant functional consequences. We also review evidence implicating each of the above MYC genes in specific neoplastic diseases and have attempted to identify unresolved questions which deserve further basic or clinical investigation. We have made every attempt to review those diseases for which significant and confirmatory evidence, based on studies with primary tumor material, exists to implicate MYC members in their causation and/or progression.

Mmip1: a novel leucine zipper protein that reverses the suppressive effects of Mad family members on c-myc.

C-myc, a member of the basic helix-loop-helix-leucine zipper (bHLH-ZIP) protein family activates target genes in heterodimeric association with another bHLH-ZIP protein, Max. Max readily homodimerizes, competes with C-myc-Max heterodimers, and represses transcription. Four additional bHLH-ZIP proteins, Mad1, Mxi1, Mad3 and Mad4, heterodimerize with Max and also repress transcription of c-myc-responsive genes. We employed a yeast two-hybid approach to identify proteins which interact with Mxi. We identified a novel ZIP-containing protein, Mmip1 (Mad member-interacting protein 1) that strongly dimerizes with all four Mad members, but not with c-myc, Max, or with unrelated HLH proteins. The Mmip1-Mxi association is mediated by the ZIP domain of each polypeptide and is as strong or stronger than the associations between c-myc and Max or Max and Mxi1. In vitro, Mmip1 can inhibit DNA binding by Max-Mad heterodimers and, in vivo, can reverse the suppressive effects of Mad proteins on c-myc functions. Mmipl is found in a variety of cells types, is induced by serum stimulation, and can be co-immunoprecipitated from fibroblasts in association with Mxi1. By interfering with the dimerization between Max and Mad family member proteins, Mmip1 can indirectly up-regulate the transcriptional activity of c-myc and suppress the antiproliferative actions of Mad proteins.

Mmip-2/Rnf-17 enhances c-Myc function and regulates some target genes in common with glucocorticoid hormones.

Members of the Mad family of basic-helix-loop-helix-leucine zipper proteins inhibit the transcriptional activity of the c-Myc oncoprotein. Mmip-2/Rnf-17 is a RING-finger protein that interacts with all four known Mad proteins, redistributes them to the cytoplasm, and thus enhances c-Myc function. We generated cell lines in which Mmip-2/Rnf-17 was rendered glucocorticoid (GC)-inducible. Stable expression of Mmip-/Rnf-17 resulted in the expected transport of the most abundant endogenous mad protein, Mxi1, to the cytoplasm. Compensatory increases in Mxi1 and Mad3 transcripts, similar to those previously described in Mad1 null hematopoietic cells, were also seen. Mmip-2/Rnf-17 also sensitized cells to several different pro-apoptotic stimuli and regulated a subset of c-Myc target genes. Unexpectedly, some of these genes were also found to be modulated solely by GCs. Thus, the inhibition of Mad proteins by Mmip-2/Rnf-17 modulates c-Myc function by enhancing its ability to regulate a subset of its potential target genes. Our results also identify a previously unrecognized overlap between genes regulated by c-Myc- and GCs and provide a potential molecular basis for their regulation of common cellular functions.

Genetic dissection of c-myc apoptotic pathways.

All biological functions mediated by the c-myc oncoprotein require an intact transactivation domain (TAD). We compared TAD mutants for their ability to promote apoptosis of 32D myeloid cells in response to interleukin-3 (IL-3) deprivation and exposure to chemotherapeutic drugs, and to activate ornithine decarboxylase, an endogenous c-myc target. Different sub-regions of the TAD were required to mediate each function. cDNA microarrays were then used to identify multiple c-myc-regulated transcripts, some of which were also modulated by IL-3 or cytotoxic drugs, as well as by specific sub-regions of the TAD. Several of the c-myc-regulated transcripts had also been previously identified as targets for IFN-gamma. The functional consequences of their deregulation were manifested by a marked sensitivity of c-myc-overexpressing cells to IFN-gamma-mediated apoptosis. Our results establish that several well-characterized functions of c-myc are separable and correlate with the expression of a novel group of target genes, some of which also mediate the apoptotic action of IFN-gamma.