Limitations in the measurement of c-myc oncoprotein and other nuclear antigens by flow cytometry

Recent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation-associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c-myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa-S3 cells. The cells were stained with monoclonal antibodies either to the c-myc oncoprotein or to p105, a prolifera-tion-associated nuclear antigen. Protein-levels were measured on the basis of anti-c-myc or anti-p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c-myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.     

Quantitative proteomic analysis of Myc oncoprotein function

This study applies a new quantitative proteomics technology to the analysis of the function of the Myc oncoprotein in mammalian cells. Employing isotope-coded affinity tag (ICAT) reagent labeling and tandem mass spectrometry, the global pattern of protein expression in rat myc-null cells was compared with that of myc-plus cells (myc-null cells in which myc has been introduced) to generate a differential protein expression catalog. Expression differences among many functionally related proteins were identified, including reduction of proteases, induction of protein synthesis pathways and upregulation of anabolic enzymes in myc-plus cells, which are predicted to lead to increased cell mass (cell growth). In addition, reduction in the levels of adhesion molecules, actin network proteins and Rho pathway proteins were observed in myc-plus cells, leading to reduced focal adhesions and actin stress fibers as well as altered morphology. These effects are dependent on the highly conserved Myc Box II region. Our results reveal a novel cytoskeletal function for Myc and indicate the feasibility of quantitative whole-proteome analysis in mammalian cells.     

Simultaneous quantification of c-myc oncoprotein, total cellular protein, and DNA content using multiparameter flow cytometry

Variations in total cellular protein content can confound interpretation of the significance of modulations of specific cellular proteins. In an effort to overcome this problem, a technique is described for the simultaneous measurement of a specific cellular protein, total cellular protein, and DNA content. The method utilizes dual-laser (uv and 488 nm) excitation and three fluorescent dyes: FITC, SR101, and DAPI. FITC-labelled antibody coupled with indirect immunofluorescence was used to quantify the c-myc oncoprotein, whereas SR101 and DAPI were used to measure total cellular protein and cellular DNA, respectively. Flow cytometric measurements of c-myc oncoprotein were compared to densitometric readings of p64c-myc. SR101 protein determinations were compared to those obtained by the Lowry technique. Results indicated that flow cytometric measurements correlated well with those obtained by the biochemical methods. The usefulness of the technique was further examined following treatment of exponentially growing HL-60 cells with 2.5 micrograms/ml cycloheximide for 0 to 12 h. Cycloheximide treatment was found to cause a significant decrease in c-myc oncoprotein content within 2 h (P less than 0.05), a relative increase in the proportion of G0/G1 cells and a modest decrease in total cellular protein. This technique appears to provide a rapid, quantitative approach, useful for investigating alterations in cellular growth balance occurring with cell differentiation, neoplastic transformation, or cell treatment with radiation or cytostatic drugs.     

Promotion of growth and apoptosis in c-myc nullizygous fibroblasts by other members of the myc oncoprotein family

c-myc nullizygous fibroblasts (KO cells) were used to compare the abilities of c-myc, N-myc and L-myc oncoproteins to accelerate growth, promote apoptosis, revert morphology, and regulate the expression of previously described c-myc target genes. All three myc oncoproteins were expressed following retroviral transduction of KO cells. The proteins all enhanced the growth rate of KO cells and significantly shortened the cell cycle transition time. They also accelerated apoptosis following serum deprivation, reverted the abnormal KO cell morphology, and modulated the expression of previously described c-myc target genes. In most cases, L-myc was equivalent to c-myc and N-myc in restoring all of the c-myc-dependent activities. These findings contrast with the previously reported weak transforming and transactivating properties of L-myc. Myc oncoproteins may thus impart both highly similar as well as dissimilar signals to the cells in which they are expressed.     

Possible function of the c-myc product: promotion of cellular DNA replication.

We have recently cloned a plasmid, pARS65, containing the sequences derived from mouse liver DNA which can autonomously replicate in mouse and human cells (Ariga et al., 1987). In this report, we show that replication of pARS65 in HL-60 cells can be inhibited by co-transfection with anti-c-myc antibody. In an in-vitro replication system using HL-60 nuclear extract, pARS65 functioned as a template. This in-vitro replication was also blocked by addition of anti-c-myc antibody. Specific binding activity of the c-myc product to pARS65 was detected by an immunobinding assay, suggesting that the c-myc protein promotes DNA replication through binding to the initiation site of replication. This has been substantiated using the antibody to help isolate a human DNA segment that can autonomously replicate in the cells.     

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity.     

An actin-binding function contributes to transformation by the Bcr-Abl oncoprotein of Philadelphia chromosome-positive human leukemias.

In Philadelphia chromosome-positive human leukemias, which include chronic myelogenous leukemia and some acute lymphocytic leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and Bcr-Abl fusion proteins are produced. The Bcr sequences activate the Abl tyrosine kinase which is required for the transforming function of Bcr-Abl. The Bcr sequences also enhance an F-actin-binding activity associated with c-Abl. Here, we show that binding of c-Abl and Bcr-Abl proteins to actin filaments in vivo and in vitro is mediated by an evolutionarily conserved domain at the C-terminal end of c-Abl. The c-Abl F-actin-binding domain contains a consensus motif found in several other actin-crosslinking proteins. Mutations in the consensus motif are shown to abolish binding to F-actin. Bcr-Abl proteins unable to associate with F-actin have a reduced ability to transform Rat-1 fibroblasts and to abrogate the requirement for interleukin-3 in the lymphoblastoid cell line Ba/F3. In transformed cells, Bcr-Abl induces a redistribution of F-actin into punctate, juxtanuclear aggregates. The binding to actin filaments has important implications for the pathogenic and physiological functions of the Bcr-Abl and c-Abl proteins.     

Structure and function of the c-myc DNA-unwinding element-binding protein DUE-B

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.     

The human oncoprotein MDM2 arrests the cell cycle: elimination of its cell-cycle-inhibitory function induces tumorigenesis.

The human oncoprotein MDM2 (hMDM2) overexpresses in various human tumors. If amplified, the mdm2 gene can enhance the tumorigenic potential of murine cells. Here, we present evidence to show that the full-length human or mouse MDM2 expressed from their respective cDNA can inhibit the G0/G1-S phase transition of NIH 3T3 and normal human diploid cells. The protein harbors more than one cell-cycle-inhibitory domain that does not overlap with the p53-interaction domain. Deletion mutants of hMDM2 that lack the cell-cycle-inhibitory domains can be stably expressed in NIH 3T3 cells, enhancing their tumorigenic potential. The tumorigenic domain of hMDM2 overlaps with the p53-interaction domain. Some tumor-derived cells, such as Saos-2, H1299 or U-2OS, are relatively insensitive to the growth-inhibitory effects of hMDM2. These observations suggest that hMDM2 overexpression in response to oncogenic stimuli would induce growth arrest in normal cells. Elimination or inactivation of the hMDM2-induced G0/G1 arrest may contribute to one of the steps of tumorigenesis.     

Structure of the Bcr-Abl oncoprotein oligomerization domain.

The Bcr-Abl oncoprotein is responsible for a wide range of human leukemias, including most cases of Philadelphia chromosome-positive chronic myelogenous leukemia. Oligomerization of Bcr-Abl is essential for oncogenicity. We determined the crystal structure of the N-terminal oligomerization domain of Bcr-Abl (residues 1-72 or Bcr1-72) and found a novel mode of oligomer formation. Two N-shaped monomers dimerize by swapping N-terminal helices and by forming an antiparallel coiled coil between C-terminal helices. Two dimers then stack onto each other to form a tetramer. The Bcr1-72 structure provides a basis for the design of inhibitors of Bcr-Abl transforming activity by disrupting Bcr-Abl oligomerization.     

A role for isoprenoid lipids in the localization and function of an oncoprotein

Intermediates of the cholesterol biosynthetic pathway are covalently attached to a number of eukaryotic proteins, including the Ras oncoprotein. Ras protein is post-translationally processed at its carboxyl terminus in three steps, resulting in a COOH-terminal cysteine residue to which a polyisoprenoid moiety, probably farnesyl, is attached in a thioether linkage. Polyisoprenylation of Ras protein is required for its membrane association and for the oncogenicity of mutant forms of the protein. Inhibition of polyisoprenylation may offer a route by which Ras-mediated tumors can be pharmacologically suppressed. Other proteins that are polyisoprenylated include nuclear lamin B, fungal mating factors, and subunits of trimeric guanine nucleotide-binding proteins. A consensus sequence for polyisoprenylation (Cys-aliphatic-aliphatic-X) has been identified at the COOH-terminus of modified proteins. Recent evidence indicates that proteins can be modified by several different polyisoprenoids.     

Gibbon and marmoset c-myc nucleotide sequences.

The nucleotide sequences of the gibbon and marmoset myc loci have been determined by the dideoxy ribomethod. The number of mutations which occurred during evolution and the branches affected were deduced according to the principle of maximum parsimony, from a comparison with known mammal sequences. As previously observed for the human and chimpanzee myc genes, an Alu repeat belonging to subclass III was observed in the second intron of the gibbon myc gene. In contrast, no such element was found in the marmoset gene. Alignment of the Myc amino acid (aa) sequences provided clues for detecting which aa or which protein regions have been more heavily mutated. Conversely, some regions remained free of mutations and remained unchanged from mouse to human, most probably in connection with some important embedded property(ies). An intriguing feature of the human Myc protein is duplication of 50 aa out of 439. Strikingly, most of these aa remain unchanged in mouse, rat, cat, marmoset, gibbon, chimpanzee and human.     

Presence and role of c-myc proto-oncogene product in mammalian sperm cell function

The presence and role of c-myc protein was investigated in mature sperm cells of the human, mouse, and rabbit. The monoclonal antibodies against c-myc protein (c-myc) reacted with the acrosomal region of the sperm of these mammalian species in the indirect immunofluorescence technique. The c-myc monoclonal antibody (MCA) recognized c-myc protein of 62 and 64 kDa on Western blots of lithium diiodosalicylate-solubilized sperm preparations of these species. The c-myc MCA showed a dose-dependent inhibition of human sperm penetration of zona-free hamster eggs, inhibition of murine in vitro fertilization, and reduced in vivo fertilization in rabbits. There was no effect of the antibody on percent sperm motility, though the antibody significantly affected various motility characteristics such as mean and maximum amplitude of lateral head displacement and curvilinear velocity involved in hyperactivation phenomenon of human sperm cells. These results suggest that c-myc or c-myc-like protein is present in mature sperm cells and may have a role in sperm cell function especially in capacitation and/or acrosome reaction.