The HPV E7 oncoprotein inhibits tumor necrosis factor alpha-mediated apoptosis in normal human fibroblasts.

Tumor necrosis factor-alpha (TNF) is a cytokine that induces programmed cell death, apoptosis, in a number of cell types and is employed by cytotoxic T cells to eliminate virus infected cells. Consequently, many viruses have acquired mechanisms to undermine these host cell defense mechanisms and cause resistance to TNF-mediated apoptosis. Here we show that normal human diploid fibroblasts that express the human papillomavirus type 16 E7 oncoprotein have a decreased propensity to undergo apoptosis in response to TNF treatment. The ability of E7 to undermine TNF-mediated apoptosis correlates with cellular transformation. While E7 does not generally subvert signaling by tumor necrosis factor receptor 1, pro-caspase 8 activation is decreased in E7-expressing cells. E7 also provides some protection from apoptosis caused by stimulation of the TNF receptor 1-related cytokine receptor Fas, where induction of apoptosis occurs much slower in this cell type. Hence, E7-expressing normal human fibroblasts exhibit a specific defect that obstructs cytokine-mediated activation of pro-caspase 8 and apoptosis.

p107 and p130: versatile proteins with interesting pockets.

p107 and p130 were originally identified as targets of the transforming domains of viral oncoproteins encoded by small DNA tumor viruses. Together with pRB, the protein product of the retinoblastoma gene (Rb), p107 and p130 represent a family of closely related proteins that play critical roles in the regulation of cell proliferation. p107, p130, and pRB are transcriptional regulators whose activities are coupled to the cell cycle. Each of these proteins associates with E2F and is directly regulated by phosphorylation by cyclin-dependent kinases. In vivo studies of p107 and p130 function have revealed that their roles overlap extensively with one another and with pRB. In addition, the analysis of mice (and cell lines derived from these animals) deficient in these proteins shows that the individual members of this family harbor distinct functions that, at present, are poorly understood. The characterization of tumor cells continues to emphasize the important and somewhat unique role of pRB in tumor suppression, and the evidence linking the specific inactivation of p107 or p130 to tumor development remains quite limited. In this review we summarize the biochemical and functional properties of p107 and p130, and we compare and contrast these properties to those of pRB.

Nuclear organization of DNA replication in primary mammalian cells.

Using methods that conserve nuclear architecture, we have reanalyzed the spatial organization of the initiation of mammalian DNA synthesis. Contrary to the commonly held view that replication begins at hundreds of dispersed nuclear sites, primary fibroblasts initiate synthesis in a limited number of foci that contain replication proteins, surround the nucleolus, and overlap with previously identified internal lamin A/C structures. These foci are established in early G(1)-phase and also contain members of the retinoblastoma protein family. Later, in S-phase, DNA replication sites distribute to regions located throughout the nucleus. As this progression occurs, association with the lamin structure and pRB family members is lost. A similar temporal progression is found in all the primary cells we have examined but not in most established cell lines, indicating that the immortalization process modifies spatial control of DNA replication. These findings indicate that in normal mammalian cells, the onset of DNA synthesis is coordinately regulated at a small number of previously unrecognized perinucleolar sites that are selected in early G(1)-phase.

Requirements for cell cycle arrest by p16INK4a.

Analysis of tumor-derived mutations has led to the suggestion that p16INK4a, cyclin D1, cdk4, and the retinoblastoma protein (pRB) are components of a regulatory pathway that is inactivated in most tumor cells. Cell cycle arrest induced by p16INK4a, an inhibitor of cyclin D-dependent kinases, requires pRB, and it has been proposed that this G1 arrest is mediated by pRB-E2F repressor complexes. By comparing the properties of primary mouse embryonic fibroblasts specifically lacking pRB-family members, we find that pRB is insufficient for a p16INK4a-induced arrest. In addition to pRB, a second function provided by either p107 or p130, two pRB-related proteins, is required for p16INK4a to block DNA synthesis. We infer that p16INK4a-induced arrest is not mediated exclusively by pRB, but depends on the nonredundant functions of at least two pRB-family members.

Combinatorial roles for pRB, p107, and p130 in E2F-mediated cell cycle control.

Numerous studies have implicated the pRB family of nuclear proteins in the control of cell cycle progression. Although over-expression experiments have revealed that each of these proteins, pRB, p107, and p130, can induce a G(1) cell cycle arrest, mouse knockouts demonstrated distinct developmental requirements for these proteins, as well as partial functional redundancy between family members. To study the mechanism by which the closely related pRB family proteins contribute to cell cycle progression, we generated 3T3 fibroblasts derived from embryos that lack one or more of these proteins (pRB(-/-), p107(-/-), p130(-/-), pRB(-/-)/p107(-/-), pRB(-/-)/p130(-/-), and p107(-/-)/p130(-/-)). By comparing the growth and cell cycle characteristics of these cells, we have observed clear differences in the manner in which they transit through the G(1) and S phases as well as exit from the cell cycle. Deletion of Rb, or more than one of the family members, results in a shortening of G(1) and a lengthening of S phase, as well as a reduction in growth factor requirements. In addition, the individual cell lines showed differential regulation of a subset of E2F-dependent gene promoters, as well as differences in cell cycle-dependent kinase activity. Taken together, these observations suggest that the closely related pRB family proteins affect cell cycle progression through distinct biochemical mechanisms and that their coordinated action may contribute to their diverse functions in various physiological settings.

Opposing roles of pRB and p107 in adipocyte differentiation.

The retinoblastoma (RB) family of proteins, pRB, p107, and p130, have been postulated to be partially redundant in their ability to regulate progression through the G(1) phase of the cell cycle. However, pRB appears to be unique in its capacity as a classical tumor suppressor, possibly because of a specialized role in maintaining the balance between proliferation and differentiation. A variety of studies have in fact revealed an apparent role for pRB in cellular differentiation and development. However, roles for p107 and p130 in differentiation have not yet been established, and knockout mouse studies have indicated that they may be functionally redundant during development, and possibly perform a role in differentiation distinct from that of pRB. Using adipogenesis as a model, we have indeed found distinct roles for the pRB family proteins in regulating differentiation. 3T3 fibroblasts deficient in p107 and p130 differentiate with high efficiency, whereas pRB(-/-) 3T3 cells exhibit defects in their differentiation potential. Moreover, over-expression of pRB in wild-type cells promotes differentiation, whereas over-expression of p107 antagonizes differentiation. The seemingly opposing roles of pRB family members in adipocyte differentiation can be explained, at least in part, by a requirement for pRB in maintaining cell cycle exit as well as potentiating the activity of the differentiation-associated transcription factor, C/EBPalpha. p107 does not affect C/EBPalpha-driven transcription and is not required for cell cycle exit, but instead, loss of p107 lowers the requirement for the differentiation factor PPARgamma. These findings suggest contrasting biological roles for individual members of the pRB family of proteins that may explain why pRB, but not p107, is commonly mutated during human tumor development.

Timing of cyclin E gene expression depends on the regulated association of a bipartite repressor element with a novel E2F complex.

Transient induction of the cyclin E gene in late G1 gates progression into S. We show that this event is controlled via a cyclin E repressor module (CERM), a novel bipartite repressor element located near the cyclin E transcription start site. CERM consists of a variant E2F-binding site and a contiguous upstream AT-rich sequence which cooperate during G0/G1 to delay cyclin E expression until late G1. CERM binds the protein complex CERC, which disappears upon progression through G0-G1 and reappears upon entry into the following G1. CERC disappearance correlates kinetically with the liberation of the CERM module in vivo and cyclin E transcriptional induction. CERC contains E2F4/DP1 and a pocket protein, and sediments faster than classical E2F complexes in a glycerol gradient, suggesting the presence of additional components in a novel high molecular weight complex. Affinity purified CERC binds to CERM but not to canonical E2F sites, thus displaying behavior different from known E2F complexes. In cells nullizygous for members of the Rb family, CERC is still detectable and CERM-dependent repression is functional. Thus p130, p107 and pRb function interchangeably in CERC. Notably, the CERC-CERM complex dissociates prematurely in pRb-/- cells in correspondence with the premature expression of cyclin E. Thus, we identify a new regulatory module that controls repression of G1-specific genes in G0/G1.

Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1.

Unregulated expression of the transcription factor E2F promotes the G1-to-S phase transition in cultured mammalian cells. However, there has been no direct evidence for an E2F requirement in this process. To demonstrate that E2F is obligatory for cell cycle progression, we attempted to inactivate E2F by overexpressing dominant-negative forms of one of its heterodimeric partners, DP-1. We dissected the functional domains of DP-1 and separated the region that facilitate heterodimer DNA binding from the E2F dimerization domain. Various DP-1 mutants were introduced into cells via transfection, and the cell cycle profile of the transfected cells was analyzed by flow cytometry. Expression of wild-type DP-1 or DP-1 mutants that bind to both DNA and E2F drove cells into S phase. In contrast, DP-1 mutants that retained E2F binding but lost DNA binding arrested cells in the G1 phase of the cell cycle. The DP-1 mutants that were unable to bind DNA resulted in transcriptionally inactive E2F complexes, suggesting that the G1 arrest is caused by formation of defective E2F heterodimers. Furthermore, the G1 arrest instigated by these DP-1 mutants could be rescued by coexpression of wild-type E2F or DP protein. These experiments define functional domains of DP and demonstrate a requirement for active E2F complexes in cell cycle progression.

Downregulation of c-myc expression after heat shock in human B-cell lines is independent of 5' mRNA sequences.

The effect of heat-shock on the expression of c-myc genes in different chromosomal contexts was investigated in a panel of human B-lymphoid cell lines. Burkitt's lymphoma cell lines with c-myc translocation breakpoints upstream of the first exon, within the exon itself, or in the first intron showed downregulation of c-myc levels as did a cell line without any translocation. The c-myc mRNA of cell lines with translocation breakpoints within the c-myc gene have previously been reported to have prolonged half-lives. After heat shock, the levels of these mRNA species were reduced with similar kinetics as the normal c-myc mRNA. An exception was an RNA species where the only c-myc sequences are derived from exon 1, showing that sequences from this part of the c-myc gene are not sufficient to mediate the rapid downregulation. Nuclear run-on analysis did not show reduced transcription of c-myc after heat shock and a comparison of cytoplasmic and total RNA did not indicate accumulation of longer, unspliced c-myc mRNA species. These observations suggest a posttranscriptional, cytoplasmic downregulation targeting exons 2 and/or 3. B-lymphoma lines transfected with a hsp70 promoter-linked c-myc gene were deficient in their ability to reinitiate proliferation after heat shock, providing a physiological rationale for the normal downregulation of c-myc after this type of physical stress.

Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.

Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBP delta. Likewise, MIX is a direct inducer of C/EBP beta gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBP delta and C/EBP beta accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP alpha. Conclusive evidence has already shown that C/EBP alpha regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBP delta and C/EBP beta play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP alpha. Conditions facilitating the precocious expression of either C/EBP delta or C/EBP beta were observed to accelerate adipogenesis and, in the case of C/EBP beta, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP beta effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBP beta in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat-laden adipocytes.

Analysis of c-Myc domains involved in stimulating SV40 replication.

We have demonstrated previously that overproduction of c-Myc, N-Myc and, to a lesser extent, L-Myc facilitates the replication of simian virus 40 (SV40)-based vectors in human lymphoid cells. Using a series of c-myc deletion mutants, we investigated which c-Myc regions are important in stimulating SV40 replication. The ability of c-Myc to promote SV40 replication was significantly reduced by deletions in the second exon domain, formerly shown to be crucial for c-Myc's transforming capacity. The c-myc mutants with a disrupted basic region (b) or leucine zipper (Zip) motif were also unable to stimulate SV40 replication. These regions are implicated in protein-DNA and protein-protein interactions, respectively, suggesting that the c-Myc protein might be associated with the DNA-protein replication complex. We present data obtained from gel mobility shift assays and from an immunocomplex-binding assay substantiating this hypothesis.

Nuclear colocalization of c-myc protein and hsp70 in cells transfected with human wild-type and mutant c-myc genes.

Using immunofluorescence and electron microscopy we have studied the localization of wild-type and mutant c-myc proteins transiently expressed in CV-1 cells. In agreement with our previous observations, wild-type c-myc protein accumulated in large amorphous globules in the nucleus. All mutant proteins tested accumulated in the nucleus as well, but gave rise to morphologically different inclusion bodies. Many small globules appeared in cells transfected with D145-262 (deletion of amino acids 145-262), while cells transfected with D371-412 or D414-433 generated structures looking like a fine network or like beads on a string. In addition, a particulate cytoplasmic staining appeared in some cells transfected with the wild-type gene and in cells transfected with mutants D145-262 or D414-433. Since the c-myc protein has been reported to stimulate expression of exogenous hsp70 protein, we also examined the intracellular distribution of hsp70 in the transfected cells. Double immunofluorescence microscopy revealed that hsp70 codistributed with the c-myc protein in distinct globules in the nucleus of many but not all myc-positive cells. However, the levels of hsp70 transcripts were not significantly raised compared to nontransfected and vector-transfected cells. Likewise, the levels of hsp70 protein did not vary significantly. These findings indicate that overexpression of c-myc stimulates translocation of preexisting hsp70 from the cytoplasm into the nucleus, rather than influencing hsp70 expression. Conceivably, this may represent one of several mechanisms whereby the cell deals with excessive amounts of c-myc protein.

Reintroduction of a normal retinoblastoma gene into retinoblastoma and osteosarcoma cells inhibits the replication associated function of SV40 large T antigen.

The product of the retinoblastoma (Rb) gene can form complexes with the transforming proteins of small DNA tumor viruses, including SV40 large T antigen (Tag), adenovirus E1A, and the human papilloma virus E7. The strong correlation between their ability to transform and their ability to bind Rb protein suggests that these oncoproteins exert their effect through blocking the Rb function. SV40 Tag causes oncogenic cell transformation of rodent cells, and it is also required for viral DNA replication. In this paper, we investigated the effect of the Rb protein on the SV40 replication associated function of Tag. We present evidence suggesting that the complex formation between Rb and Tag interferes with the viral DNA replication. In Y79 retinoblastoma and Saos-2 osteosarcoma cells, which lack functional Rb protein, a SV40 based plasmid vector, pSVEpR4, replicates well. In the same cells reconstituted for Rb expression with an intact Rb gene introduced by retroviral mediated gene transfer, pSVEpR4 replicates to a considerably lower level. The inhibitory effect of Rb protein was surmounted by increasing the intracellular level of Tag. Increasing amounts of Tag in wild-type Rb negative Y79 cells had virtually no effect on SV40 replication. Furthermore, the overexpression of Tag in Rb reconstituted Y79 cells did not alter the growth rate of the cells. These data suggest that Rb protein interacts with Tag and modulates its ability to promote SV40 DNA replication.

The effect of myc proteins on SV40 replication in human lymphoid cells.

We have previously demonstrated that over-expression of c-myc facilitates the replication of SV40 DNA in human Burkitt lymphoma cells (BL). In this paper we investigated the ability of N-myc and L-myc to substitute for c-myc in promoting SV40 DNA replication. Vectors expressing either a chimeric N-myc/c-myc, N-myc or L-myc gene were constructed and co-transfected with a plasmid containing the SV40 origin of replication and the early genes encoding SV40 T antigens (Tag). The chimeric N-myc/c-myc and the N-myc proteins enhance SV40 replication in the human lymphoma line BJAB at levels comparable to c-myc. The stimulative effect of N-myc is also evident in the BL cell line RAMOS. However, L-myc stimulated SV40 replication only in RAMOS cells.

Elevated expression of c-myc and N-myc produces distinct changes in nuclear fine structure and chromatin organization.

The proto-oncogenes c-myc and N-myc encode nuclear phosphoproteins with unknown function. Here, c-myc or N-myc, or hybrid constructs of the two, were transfected into fibroblastic cells (CV-1) using SV40-based high expression vectors. The cells were studied by indirect immunofluorescence microscopy and transmission electron microscopy to determine the localization of the two myc proteins within the nucleus and their influence on nuclear fine structure and chromatin organization. In c-myc transfected cells the overproduced protein product accumulated in large amorphous globules that displaced the normal chromatin and did not stain for DNA. In N-myc transfected cells condensed chromatin loops were formed. They were attached to the nuclear envelope and by traction in the latter they may have contributed to give the nucleus its irregular shape in these cells. During mitosis the chromatin loops persisted as clearly identifiable entities within the chromosomes, suggesting a rigid conformation that did not allow normal chromosome packaging. These findings suggest that the c-myc and N-myc proteins bind to different structures and may have different functions. Observations on cells transfected with hybrid constructs indicated that both the second and third exon of c-myc were required to yield a product that behaved like the c-myc protein. In contrast, domains encoded by the second exon of N-myc were sufficient to give rise to a product that morphologically behaved like the N-myc protein.

Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells.

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.