Regulation of telomerase by human papillomaviruses.

The E6 oncoprotein of human papillomaviruses (HPVs) induces telomerase activity in primary human epithelial cells. This activity is dependent on association of E6 with E6AP, a cellular ubiquitin ligase. E6 activates the transcription of hTERT, the catalytic subunit of telomerase. E boxes near the start of hTERT transcription are required for E6; however, acetylated histones are only present in the E6 cells. We identified two isoforms of NFX1, a new binding partner of E6/E6AP. The NFX1- 91 isoform binds to an X-box motif located adjacent to the proximal E box, binds Sin3A and HDACs, repressing hTERT transcription. It preferentially binds E6/E6AP and is targeted for ubiquitin-mediated degradation. The NFX1-123 isoform has the opposite activity, increasing hTERT transcription or translation. This is the first example of viral oncoproteins disrupting regulation of telomerase, a critical event in tumorigenesis.

Differential activity of conditional MYC and its variant MYC-S in human mortal fibroblasts.

We have explored the effects of the conditional MYC-estrogen receptor fusion protein, MYC-ERTM, in human mortal fibroblasts, WI38, on cell-cycle entry, apoptosis and gene expression. The results indicate that activation of MYC-ERTM in WI38 cells is sufficient to cause S phase entry of quiescent cells, which is preceded by phosphorylation of Rb and activation of the Cdk2-associated kinase. We also analysed the MYC protein variant, MYC-S, which lacks part of the transcriptional activation domain but includes the conserved MYC box II and 26 amino acids N-terminal to it. MYC-S was previously shown to promote proliferation and apoptosis of immortalized rodent cell lines. The results indicate that MYC-S has undetectable activity as an inducer of S phase or apoptosis of quiescent WI38 cells. However, Myc-S stimulates proliferation of WI38 cells in the presence of 10% fetal calf serum. Surprisingly, we found that MYC-S, previously considered solely a repressor of specific reporter genes, is instead a weak transactivator of endogenous target genes both in mortal and immortalized cells. In addition, MYC-S exhibit a weak repressor activity upon an endogenous target gene only in immortalized cells. MYC-S transcriptional properties suggest that MYC box II and the adjacent N-terminal amino acids, while not sufficient for full repression function, participate in transactivation of endogenous target genes.

The Myc/Max/Mad network and the transcriptional control of cell behavior.

The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion.

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.

Direct activation of TERT transcription by c-MYC.

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

Myc target genes.

The myc family of proto-oncogenes belongs to the basic helix-loop-helix leucine-zipper (bHLHZ) class of transcription factors. Myc proteins function as transcriptional activators through heterodimerization with Max, but might also act as negative regulators of transcription. Identification of genes directly controlled by Myc-Max has proved difficult, but recent work is producing a growing list of candidates. Results to date suggest that Myc-Max influences cell growth and proliferation through direct activation of genes involved in DNA synthesis, RNA metabolism and cell-cycle progression.

Myc-Max heterodimers activate a DEAD box gene and interact with multiple E box-related sites in vivo.

The c-Myc protein is involved in cell proliferation, differentiation and apoptosis though heterodimerization with Max to form a transcriptionally active sequence-specific DNA binding complex. By means of sequential immunoprecipitation of chromatin using anti-Max and anti-Myc antibodies, we have identified a Myc-regulated gene and genomic sites occupied by Myc-Max in vivo. Four of 27 sites recovered by this procedure corresponded to the highest affinity 'canonical' CACGTG sequence. However, the most common in vivo binding sites belonged to the group of 'non-canonical' E box-related binding sites previously identified by in vitro selection. Several of the genomic fragments isolated contained transcribed sequences, including one, MrDb, encoding an evolutionarily conserved RNA helicase of the DEAD box family. The corresponding mRNA was induced following activation of a Myc-estrogen receptor fusion protein (Myc-ER) in the presence of a protein synthesis inhibitor, consistent with this helicase gene being a direct target of Myc-Max. In addition, as for c-Myc, the expression of MrDb is induced upon proliferative stimulation of primary human fibroblasts as well as B cells and down-regulated during terminal differentiation of HL60 leukemia cells. Our results indicate that Myc-Max heterodimers interact in vivo with a specific set of E box-related DNA sequences and that Myc is likely to activate multiple target genes including a highly conserved DEAD box protein. Therefore, Myc may exert its effects on cell behavior through proteins that affect RNA structure and metabolism.

The Max transcription factor network: involvement of Mad in differentiation and an approach to identification of target genes.

The small bHLHZip protein, Max, was originally identified through its interaction with Myc family proteins and appears to be an obligate partner for Myc function. Max has now been found to interact with at least two other proteins, Mad and Mxi1. These also belong to the bHLHZip class but are otherwise unrelated to Myc. Mad has been shown to abrogate the positive transcriptional activity of Myc and to inhibit Myc in co-transformation assays. This suggests that Mad may antagonize Myc function. Mad is rapidly induced upon differentiation, a time when Myc is frequently down-regulated. We show here evidence for Mad expression upon differentiation of myeloblasts, monoblasts, and keratinocytes. Mad:Max complexes are detected during differentiation and appear to replace the Myc:Max complexes present in proliferating cell populations. Since these complexes appear to form even in the presence of Myc, there may exist mechanisms that act to inhibit Myc:Max, or to promote Mad:Max, complex formation. We speculate that Max complex switching causes a change in the transcriptional activity of groups of target genes. Mad is not induced in all differentiating cell types, suggesting that other, possibly tissue-restricted, proteins might act in similar switch mechanisms to effect changes in transcriptional programs. We have also developed an approach to identification of the gene targets for Myc:Max complexes. By employing an immunoisolation procedure, we have begun characterization of several clones whose expression levels correlate with those of c-myc. Further identification of Myc-regulated genes may allow us to determine the molecular mechanism by which Myc governs cell proliferation and differentiation.

c-yes protein kinase is associated with a 38 kD protein in cerebellum.

p62c-yes, the protein product of the yes proto-oncogene, was found in association with a cellular protein of 38 kD in chicken cerebella. The complex was detected by immunoprecipitation of cerebellar membranes with affinity purified anti-yes IgG followed by in vitro phosphorylation of the immunocomplex. Both proteins were found to be phosphorylated exclusively on tyrosine. The sedimentation profile of the yes kinase indicated that a fraction of p62c-yes was complexed with the 38 kD protein and comigrated in the gradient with a molecular mass of approximately 150 kD. We have previously described the association of p60c-src with a 38 kD protein, referred to as p38 [Grandori, C. and Hanafusa, H., J. Cell Biol. (1988), 107: 2125-2135]. Comparison of the src-associated p38 with the yes-associated 38 kD protein indicates that they are indistinguishable by one-dimensional peptide mapping. Association of p38 with more than one member of the src-family of tyrosine kinases makes this protein an attractive probe to study the structural and functional aspects of these enzymes.

p60c-src is complexed with a cellular protein in subcellular compartments involved in exocytosis.

We found high levels of the c-src gene product in neuroendocrine tissues from adult animals. To understand the role of this proto-oncogene product, the subcellular localization of p60c-src was studied in neuroendocrine tissue from adrenal medulla. The results indicate that p60c-src was highly enriched in chromaffin granule membranes, in stable association with a protein of 38 kD. The complex with the 38-kD protein was also detected in brain, a tissue known to carry high levels of p60c-src. The 38-kD protein is not calpactin I, II, or synaptophysin. Comparison of its peptide map showed a high degree of conservation among the different species and tissues examined. The interaction between p60c-src and the 38-kD protein involves disulphide bonds that are stable even when the cell fractionation is performed in the presence of a reducing agent. Since the presence of disulphide bonds among cytoplasmic proteins is very unlikely, the possibility of a noncovalent association between p60c-src and the 38-kD protein in vivo is discussed. The 38-kD protein may be involved in a function of p60c-src related to secretory organelles.

Phosphorylation of cellular proteins in Rous sarcoma virus-infected cells: analysis by use of anti-phosphotyrosine antibodies.

The protein substrates for the tyrosine protein kinases in cells transformed by avian sarcoma viruses were analyzed by gel electrophoresis in combination with immunoblotting or immunoprecipitation by antibodies against phosphotyrosine. We found that greater than 90% of phosphotyrosine-containing cellular proteins can be immunoprecipitated by these antibodies. The level of phosphotyrosine-containing cellular proteins detectable by this method markedly increased upon transformation with Rous sarcoma virus, and more than 20 distinct bands of such proteins were found in lysates of Rous sarcoma virus-transformed cells. Most of these phosphotyrosine-containing proteins had not been identified by other methods, and their presence appeared to correlate with morphological transformation in cells infected with various Rous sarcoma virus mutants and Y73, PRCII, and Fujinami sarcoma viruses. However, considerably different patterns were obtained with cells infected with nontransforming Rous sarcoma virus mutants that encode nonmyristylated src kinases, indicating that most substrates that correlate with transformation can only be recognized by p60v-src associated with the plasma membrane.

Amino acid substitutions sufficient to convert the nontransforming p60c-src protein to a transforming protein.

We have previously shown that Rous sarcoma virus variants that carry the cellular homolog (c-src) of the viral src gene (v-src) do not transform chicken embryo fibroblasts. We also have shown that replacement of sequences upstream or downstream from the BglI site of the cellular src gene with the corresponding regions of v-src restored transforming activity to the hybrid genes. Since there are only six amino acid changes between p60c-src and p60v-src within the sequences upstream from BglI, we constructed chimeric molecules involving v-src and c-src to determine the effect of each amino acid substitution on the biological activities of the gene product. We found that the change from Thr to Ile at position 338 or the replacement of a fragment of c-src containing Gly-63, Arg-95, and Thr-96 with a corresponding fragment of v-src containing Asp-63, Trp-95, and Ile-96 converted p60c-src into a transforming protein by the criteria of focus formation, anchorage-independent growth, and tumor formation in newborn chickens. These mutations also resulted in elevation of the protein kinase activity of p60c-src.

Decline of natural cytotoxicity of human lymphocytes following infection with human T-cell leukemia/lymphoma virus (HTLV).

Cell-mediated natural cytotoxicity (CMNC) of fresh or long-term cultured lymphocytes collected from HTLV-positive patients or infected in vitro with the virus, was tested against K562 target cells. Severe depression of reactivity was found in fresh lymphocytes of three patients with advanced disease, in 12 in vitro established T-cell malignant lines, and two HTLV-infected cord blood (C5/MJ and C91/PL) lines. Moreover, all (eight) HTLV-1 infected cell lines listed showed a significant inhibition of CMNC of peripheral blood lymphocytes of healthy donors. Whether virus infection promotes the outgrowth of pre-existing suppressor cells and/or produce changes of the T-lymphocyte function is unknown.