Proteome analysis of NIH3T3 cells transformed by activated Galpha12: regulation of leukemia-associated protein SET

Galpha(12), the alpha-subunit of the G12 family of heterotrimeric G proteins is involved in the regulation of cell proliferation and neoplastic transformation. GTPase-deficient, constitutively activated mutant of Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) has been previously shown to induce oncogenic transformation of NIH3T3 cells promoting serum- and anchorage-independent growth. Reduced growth-factor dependent, autonomous cell growth forms a critical defining point at which a normal cell turns into an oncogenic one. To identify the underlying mechanism involved in such growth-factor/serum independent growth of Galpha(12)QL-transformed NIH3T3, we carried out a two-dimensional differential proteome analysis of Galpha(12)QL-transformed NIH3T3 cells and cells expressing vector control. This analysis revealed a total of 22 protein-spots whose expression was altered by more than 3-folds. Two of these spots were identified by MALDI-MS analysis as proliferating cell nuclear antigen (PCNA) and myeloid-leukemia-associated SET protein. The increased expressions of these proteins in Galpha(12)QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated Galpha(12) readily increased the expression of SET protein by 24 h. As SET has been previously reported to be an inhibitor of phosphatase PP2A, the nuclear phosphatase activity was monitored in cells expressing activated Galpha(12). Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in Galpha(12)QL cells compared to vector control cells. Thus, our results from differential proteome analysis presented here report for the first time a role for SET in Galpha(12)-mediated signaling pathways and a role for Galpha(12) in the regulation of the leukemia-associated SET-protein expression.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

The cellular responses to ras and nuclear oncogenes were investigated in purified populations of rat Schwann cells. v-Ha-ras and SV40 large T cooperate to transform Schwann cells, inducing growth in soft agar and allowing proliferation in the absence of added mitogens. Expression of large T alone reduces their growth factor requirements but is insufficient to induce full transformation. In contrast, expression of v-Ha-ras leads to proliferation arrest in Schwann cells expressing a temperature-sensitive mutant of large T at the restrictive temperature. Cells arrest in either the G1 or G2/M phases of the cell cycle, and can re-enter cell division at the permissive temperature even after prolonged periods at the restrictive conditions. Oncogenic ras proteins also inhibit DNA synthesis when microinjected into Schwann cells. Adenovirus E1a and c-myc oncogenes behave similarly to SV40 large T. They cooperate with Ha-ras oncogenes to transform Schwann cells, and prevent ras-induced growth arrest. Thus nuclear oncogenes fundamentally alter the response of Schwann cells to a ras oncogene from cell cycle arrest to transformation.     

Activation of the platelet-derived growth factor receptor by the bovine papillomavirus E5 transforming protein.

The bovine papillomavirus E5 gene encodes a 44 amino acid membrane-associated protein that can induce tumorigenic transformation of rodent fibroblast cell lines. Genetic studies suggest that the E5 protein may transform cells by influencing the activity of cellular proteins involved in growth regulation. We report here that the endogenous cellular beta type receptor for the platelet-derived growth factor (PDGF) is constitutively activated in C127 and FR3T3 cells stably transformed by the E5 protein, but not in these cell types transformed by a variety of other oncogenes. In C127 cells, a metabolic precursor as well as the mature form of the receptor is activated by E5 transformation. Activation of the receptor also occurs upon acute E5-mediated transformation of these cells and precedes mitogenic stimulation in this system. Moreover, activation of the receptor by addition of PDGF or the v-sis gene to untransformed cells is sufficient to induce DNA synthesis and stable growth transformation. We propose that the PDGF receptor is an important cellular intermediate in the transforming activity of the bovine papillomavirus E5 protein. There is a short region of sequence similarity between the fibropapillomavirus E5 proteins and PDGF, suggesting that the E5 proteins may activate the PDGF receptor by binding directly to it.     

Transformation-sensitive and growth-related changes of protein synthesis in REF52 cells. A two-dimensional gel analysis of SV40-, adenovirus-, and Kirsten murine sarcoma virus-transformed rat cells using the REF52 protein database

Two-dimensional gels of normal and virally transformed REF52 cells have been quantified and compared using the QUEST system for construction and analysis of protein databases. The REF52 protein map is based on more than 1600 high quality spots, and the relative amounts of these proteins are studied in 79 gels representing 12 major experiments. REF52 cells transformed by SV40, adenovirus, and Kirsten murine sarcoma virus (KiMSV) are compared to normal REF52 cells at several stages of growth from low density to confluence and after refeeding confluent cells. In addition, early (1-4 h) and late (21-24 h) responses to serum stimulation were measured in normal, SV40-and adenovirus-transformed cells. The database has been analyzed with respect to 1) known marker proteins and protein sets, 2) global comparison of protein patterns, and 3) selection of unknown spots which have interesting patterns of regulation. For the marker proteins, which include the tropomyosin family and the proliferation-sensitive nuclear antigen, new aspects of regulation by growth and transformation have been revealed. Proliferation-sensitive nuclear antigen, a protein known to be involved in DNA synthesis, is growth-regulated in normal cells and overexpressed in some SV40- and adenovirus-transformed cells. Global comparisons reveal no overall correlation between growth-regulated changes and transformation-induced changes; however, a set of 26 coregulated proteins, including proliferation-sensitive nuclear antigen, was found to be overexpressed in REF52 cells transformed by SV40 or adenovirus. These proteins are synthesized at rates that correlate with the rate of cell proliferation in REF52 and Kirsten murine sarcoma virus-transformed cells but, in SV40- and adenovirus-transformed cells, these proteins are synthesized at high levels independent of the rate of growth. These data suggest that the transforming proteins of SV40 and adenovirus share a function that results in deregulation of the genes coding for a class of cell cycle-regulated proteins.     

Abolition of cyclin-dependent kinase inhibitor p16Ink4a and p21Cip1/Waf1 functions permits Ras-induced anchorage-independent growth in telomerase-immortalized human fibroblasts

Human cells are more resistant to both immortalization and malignant transformation than rodent cells. Recent studies have established the basic genetic requirements for the transformation of human cells, but much of this work relied on the expression of transforming proteins derived from DNA tumor viruses. We constructed an isogenic panel of human fibroblast cell lines using a combination of gene targeting and ectopic expression of dominantly acting mutants of cellular genes. Abolition of p21(Cip1/Waf1) and p16(Ink4a) functions prevented oncogenically activated Ras from inducing growth arrest and was sufficient for limited anchorage-independent growth but not tumorigenesis. Deletion of the tumor suppressor p53 combined with abolition of p16(Ink4a) function failed to mimic the introduction of simian virus 40 large T antigen, indicating that large T antigen may target additional cellular functions. Ha-Ras and Myc cooperated only to a limited extent, but in the absence of Ras, Myc cooperated strongly with the simian virus 40 small t antigen to elicit aggressive anchorage-independent growth. The experiments reported here further define specific components of human transformation pathways.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Functional role of BK virus tumor antigens in transformation.

We have examined the role of the human papovavirus BK virus (BKV) tumor (T) antigen(s) in the maintenance of transformation and have identified the domain of T antigen essential for transformation. BKV-transformed BHK 21 and NIH 3T3 cells expressing antisense T-antigen RNA lose their ability to grow in soft agar, indicating the need for the continued expression of T antigen for the maintenance of the transformed phenotype. Experiments using translation termination linker insertion and deletion mutagenesis of BKV T antigen demonstrate that amino acids 356 to 384 are essential for transformation. Although BKV T antigen shares 100, 95, and 82% amino acid homology with that of simian virus 40 (SV40) for the nuclear localization signal, p53-binding domain, and DNA-binding domain, respectively, the transformation domains of BKV and SV40 T antigens share only 54% homology. Also, BKV T antigen lacks a substantial portion of the ATPase domain of SV40, and our results indicate the dispensability of the remaining portion for transformation by this protein. We suggest that the differences in the amino acids in the identified transformation domains together with the differences in the ATPase domains may account for the differences in the transformation potentials of the two proteins.     

The role of the IGF-I receptor in the growth and transformation of mammalian cells

Abstract. Recent developments in the molecular biology of the insulin-like growth factor I (IGF-I) receptor have clarified its role in cellular growth and transformation. Although cells homozygous for a targeted disruption of the IGF-I receptor genes can grow in serum-supplemented medium, the IGF-I receptor is required for optimal growth, and is required equally in all phases of the cell cycle. The receptor plays an even more stringent role in cellular transformation and tumorigenicity, which seem to be dependent on its normal expression in several cell types. The expression of both the IGF-I receptor and its ligands is regulated by other growth factors (especially PDGF and EGF), by oncogenes (like SV40 T antigen and c-myb) and by tumour suppressor genes (like WT1 and RB). The picture emerging from these studies is that several transforming agents may exert their growth promoting effects through the direct or indirect activation of the IGF autocrine loop.     

Lysosomal enzymes possess a common antigenic determinant in the cellular slime mold, Dictyostelium discoideum

Antisera have been prepared against two lysosomal enzymes of the cellular slime mold, Dictyostelium discoideum. The two purified enzyme preparations used for immunization, N-acetylglucosaminidase and beta-glucosidase-1, show no cross-contamination with each other and no significant contamination by other lysosomal enzymes. However, antisera raised against either enzyme bind equally well to seven different lysosomal enzymes and show no preference for the enzyme against which they were raised. A total of 10 different antisera have been examined and all show similar results. Preadsorption of antisera with either purified enzyme removes all antibody activity against the other enzyme. Evidence is presented which indicates that the same species of antibodies are responsible for the precipitation of seven lysosomal enzymes. These data are discussed in terms of the proposal that the antigen that is shared by the lysosomal enzymes is a post-translational modification of the enzyme proteins. We have sought to further characterize the distribution of this common antigen among cellular proteins. We show that N-acetylglucosaminidase and beta-glucosidase-1 represent less than 5% of the total common antigen containing proteins in the cell. Precipitation of 35S-labeled cellular proteins from vegetative cells indicates that as much as 15-30% of the total cell protein may possess the common antigen. Preadsorption experiments confirm that all of the proteins immunoprecipitated in these experiments are recognized by the same antibodies that precipitate the lysosomal enzyme activities. Most of the labeled proteins are secreted into the medium along with the lysosomal enzyme activities during axenic growth. During the developmental phase of the life cycle of Dictyostelium, the total amount of the common antigen decreases about 2-fold relative to total cell protein. However, the synthesis of antigenic proteins continues throughout most of development.     

Alteration in cyclic adenosine 3':5'-monophosphate binding proteins during the phenotypic change of mouse macrophages transformed by a temperature-sensitive mutant (tsA640) of SV40

By using a photoaffinity ligand, cell extracts from transformed macrophages that were established by infection with temperature-sensitive mutants (tsA640) of simian virus 40 (SV40) were examined for cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins. At the nonpermissive temperature for SV40 large T antigen, 39.0 degrees C, no significant cAMP-binding proteins could be detected, such as primary mouse macrophages. At the permissive temperature of 33.0 degrees C, cAMP-binding proteins appeared later than SV40 T antigen expression and cellular DNA synthesis. The profile of cAMP-binding proteins was similar to that of resting, but not proliferating, mouse clonal fibroblasts (BALB/c 3T3). These and previous results suggest that SV40 T antigen influences the expression of cAMP-binding proteins in tsA640-transformed macrophages; the large/small T antigen converts the profile of cAMP-binding proteins from macrophage to fibroblastic cells.     

The herpes simplex virus virulence factor ICP34.5 and the cellular protein MyD116 complex with proliferating cell nuclear antigen through the 63-amino-acid domain conserved in ICP34.5, MyD116, and GADD34.

The herpes simplex virus (HSV) virulence factor ICP34.5, the mouse myeloid differentiation protein MyD116, and the hamster growth arrest and DNA damage protein GADD34 share a 63-amino-acid carboxyl domain which has significant homologies to otherwise divergent proteins. Here we report that both ICP34.5 and its cellular homolog MyD116 complex through the conserved domain with proliferating cell nuclear antigen. In addition, HSV infection induces a novel 70-kDa cellular protein detectable by antisera to both ICP34.5 and GADD34, demonstrating that this novel protein possesses homology with the 63-amino-acid conserved domain.     

Grow-ING, Age-ING and Die-ING: ING proteins link cancer, senescence and apoptosis

The INhibitor of Growth (ING) family of plant homeodomain (PHD) proteins induce apoptosis and regulate gene expression through stress-inducible binding of phospholipids with subsequent nuclear and nucleolar localization. Relocalization occurs concomitantly with interaction with a subset of nuclear proteins, including PCNA, p53 and several regulators of acetylation such as the p300/CBP and PCAF histone acetyltransferases (HATs), as well as the histone deacetylases HDAC1 and hSir2. These interactions alter the localized state of chromatin compaction, subsequently affecting the expression of subsets of genes, including those associated with the stress response (Hsp70), apoptosis (Bax, MDM2) and cell cycle regulation (p21WAF1, cyclin B) in a cell- and tissue-specific manner. The expression levels and subcellular localization of ING proteins are altered in a significant number of human cancer types, while the expression of ING isoforms changes during cellular aging, suggesting that ING proteins may play a role in linking cellular transformation and replicative senescence. The variety of functions attributed to ING proteins suggest that this tumor suppressor serves to link the disparate processes of cell cycle regulation, cell suicide and cellular aging through epigenetic regulation of gene expression. This review examines recent findings in the ING field with a focus on the functions of protein-protein interactions involving ING family members and the mechanisms by which these interactions facilitate the various roles that ING proteins play in tumorigenesis, apoptosis and senescence.