Inhibition of telomerase limits the growth of human cancer cells.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In most human somatic cells, telomerase expression is repressed, and telomeres shorten progressively with each cell division. In contrast, most human tumors express telomerase, resulting in stabilized telomere length. These observations indicate that telomere maintenance is essential to the proliferation of tumor cells. We show here that expression of a mutant catalytic subunit of human telomerase results in complete inhibition of telomerase activity, reduction in telomere length and death of tumor cells. Moreover, expression of this mutant telomerase eliminated tumorigenicity in vivo. These observations demonstrate that disruption of telomere maintenance limits cellular lifespan in human cancer cells, thus validating human telomerase reverse transcriptase as an important target for the development of anti-neoplastic therapies.

E2F-5, a new E2F family member that interacts with p130 in vivo.

E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb. E2f-4 interacts with pRb-related p107 and not with pRb itself. Recently, a cDNA encoding a third member of the retinoblastoma gene family, p130, was isolated. p130 also interacts with E2F DNA binding activity, primarily in the G0 phase of the cell cycle. We report here the cloning of a fifth member of the E2F gene family. The human E2F-5 cDNA encodes a 346-amino-acid protein with a predicted molecular mass of 38 kDa. E2F-5 is more closely related to E2F-4 (78% similarity) than to E2F-1 (57% similarity). E2F-5 resembles the other E2Fs in that it binds to a consensus E2F site in a cooperative fashion with DP-1. By using a specific E2F-5 antiserum, we found that under physiological conditions, E2F-5 interacts preferentially with p130.

Methylation of the human telomerase gene CpG island.

The acquisition of expression of hTERT, the catalytic subunit of the telomerase enzyme, seems to be an essential step in the development of a majority of human tumors. However, little is known about the mechanisms preventing telomerase gene expression in normal and transformed cells that do not express hTERT. Using a methylation-specific PCR-based assay, we have found that the CpG island associated with the hTERT gene is unmethylated in telomerase-negative primary tissues and nonimmortalized cultured cells, indicating that mechanisms independent of DNA methylation are sufficient to prevent hTERT expression. The hTERT CpG island is methylated in many telomerase-negative and telomerase-positive cultured cells and tumors, but the extent of methylation did not correlate with expression of hTERT. Demethylation of DNA with 5-azacytidine in two cell lines induced expression of hTERT, suggesting that DNA methylation can contribute to hTERT repression in some cells. Together, these data show that the hTERT CpG island can undergo cytosine methylation in cultured cells and tumors and that DNA methylation may contribute to the regulation of the hTERT gene, but that CpG island methylation is not responsible for repressing hTERT expression in most telomerase-negative cells.

Cell cycle regulation by the retinoblastoma family of growth inhibitory proteins.

The retinoblastoma family of growth-inhibitory proteins act by binding and inhibiting several proteins with growth-stimulatory activity, the most prominent of which is the cellular transcription factor E2F. In higher organisms, progression through the cell division cycle is accompanied by the cyclical activation of a number of protein kinases, the cyclin-dependent kinases. Phosphorylation of retinoblastoma family proteins by these cyclin-dependent kinases leads to release of the associated growth-stimulatory proteins which in turn mediate progression through the cell division cycle.

Effects of monomethylfumarate on human granulocytes.

Monomethylfumarate (MMF) is the most active metabolite of the new antipsoriasis drug Fumaderm. Because granulocytes play an important role in the pathophysiology of psoriasis, the effects of this drug on the functional activities of these cells were investigated. MMF stimulated polarization and elastase release, and enhanced the intracellular killing of bacteria by granulocytes. This compound suppressed the formyl-Met-Nle-Phe (FMLP)-stimulated respiratory burst in these cells. MMF and dimethylfumarate but not its stereoisomer dimethylmaleate, fumaric acid, or dimethylmalate stimulated polarization of and elastase release by granulocytes, indicating that methylated fumarate derivatives interact with granulocytes in a specific fashion. MMF did not affect the binding of formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein isothiocyanate to the FMLP receptor on granulocytes. This compound induced an increase in the intracellular Ca++ ([Ca++]i) and cyclic adenosine monophsphate concentration. The agonistic effects of MMF on granulocytes are thought to be mediated by the rise in the [Ca++]i and the antagonistic effects by the increase in the cyclic adenosine monophosphate concentration. These effects of MMF on granulocytes may in part explain the beneficial action of methylated fumarate derivatives on psoriatic skin lesions.

Cloning, expression and chromosomal localization of a new putative receptor-like protein tyrosine phosphatase.

We have isolated a mouse cDNA of 5.7 kb, encoding a new member of the family of receptor-like protein tyrosine phosphatases, termed mRPTP mu. The cDNA predicts a protein of 1432 amino acids (not including signal peptide) with a calculated Mr of 161,636. In addition, we have cloned the human homologue, hRPTP mu, which shows 98.7% amino acid identity to mRPTP mu. The predicted mRPTP mu protein consists of a 722 amino acid extracellular region, containing 13 potential N-glycosylation sites, a single transmembrane domain and a 688 amino acid intracellular part containing 2 tandem repeats homologous to the catalytic domains of other tyrosine phosphatases. The N-terminal extracellular part contains a region of about 170 amino acids with no sequence similarities to known proteins, followed by one Ig-like domain and 4 fibronectin type III-like domains. The intracellular part is unique in that the region between the transmembrane domain and the first catalytic domain is about twice as large as in other receptor-like protein tyrosine phosphatases. RNA blot analysis reveals a single transcript, that is most abundant in lung and present in much lower amounts in brain and heart. Transfection of the mRPTP mu cDNA into COS cells results in the synthesis of a protein with an apparent Mr of 195,000, as detected in immunoblots using an antipeptide antibody. The human RPTP mu gene is localized on chromosome 18pter-q11, a region with frequent abnormalities implicated in human cancer.

Biological reprogramming in acquired resistance to endocrine therapy of breast cancer.

Endocrine therapies targeting the proliferative effect of 17ß-estradiol through estrogen receptor α (ERα) are the most effective systemic treatment of ERα-positive breast cancer. However, most breast tumors initially responsive to these therapies develop resistance through molecular mechanisms that are not yet fully understood. The long-term estrogen-deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibitors in postmenopausal women. To elucidate this resistance, genomic, transcriptomic and molecular data were integrated into the time course of MCF7-LTED adaptation. Dynamic and widespread genomic changes were observed, including amplification of the ESR1 locus consequently linked to an increase in ERα. Dynamic transcriptomic profiles were also observed that correlated significantly with genomic changes and were predicted to be influenced by transcription factors known to be involved in acquired resistance or cell proliferation (for example, interferon regulatory transcription factor 1 and E2F1, respectively) but, notably, not by canonical ERα transcriptional function. Consistently, at the molecular level, activation of growth factor signaling pathways by EGFR/ERBB/AKT and a switch from phospho-Ser118 (pS118)- to pS167-ERα were observed during MCF7-LTED adaptation. Evaluation of relevant clinical settings identified significant associations between MCF7-LTED and breast tumor transcriptome profiles that characterize ERα-negative status, early response to letrozole and tamoxifen, and recurrence after tamoxifen treatment. In accordance with these profiles, MCF7-LTED cells showed increased sensitivity to inhibition of FGFR-mediated signaling with PD173074. This study provides mechanistic insight into acquired resistance to endocrine therapies of breast cancer and highlights a potential therapeutic strategy.

Interaction of c-Myc with the pRb-related protein p107 results in inhibition of c-Myc-mediated transactivation.

The product of the c-myc proto-oncogene, c-Myc, is a sequence-specific DNA binding protein with an N-terminal transactivation domain and a C-terminal DNA binding domain. Several lines of evidence indicate that c-Myc activity is essential for normal cell cycle progression. Since the abundance of c-Myc during the cell cycle is constant, c-Myc's activity may be regulated at a post-translational level. We have shown previously that the N-terminus of c-Myc can form a specific complex with the product of the retinoblastoma gene, pRb, in vitro. These data suggested a model in which pRb, or pRb-related proteins, regulate c-Myc activity through direct binding. We show here that the pRb-related protein p107, but not pRb itself, forms a specific complex with the N-terminal transactivation domain of c-Myc in vivo. Binding of p107 to c-Myc causes a significant inhibition of c-Myc transactivation. Expression of c-Myc releases cells from a p107-induced growth arrest, but not from pRb-induced growth arrest. Our data suggest that p107 can control c-Myc activity through direct binding to the transactivation domain and that c-Myc is a target for p107-mediated growth suppression.

Intracellular signalling by binding sites for the antipsoriatic agent monomethylfumarate on human granulocytes.

Monomethylfumarate (MMF), the most active metabolite of the new antipsoriasis drug Fumaderm, stimulates an anti-inflammatory mediator profile in human leucocytes and inhibits the proliferation of keratinocytes. These effects of MMF on cells in vitro may in part explain the beneficial action of Fumaderm in patients. In addition, we have reported that MMF stimulates an increase in the intracellular free Ca2+ concentration ([Ca2+]i) and the cyclic adenosine monophosphate (cAMP) concentration in granulocytes and keratinocytes. Because Ca2+ and cAMP control many physiological cellular responses, including cell proliferation and inflammatory mediator production, the present study focused on the intracellular signal transduction pathway which links interaction between MMF and granulocytes with increases in [Ca2+]i and the cAMP concentration. The increase in [Ca2+]i in granulocytes after MMF depended both on extracellular Ca2+ and Ca2+ from intracellular stores. Ca2+ is essential for the increase in the cAMP concentration after stimulation with MMF. The results found for pharmacological inhibitors of various protein kinases suggest that a staurosporine-sensitive protein kinase different from protein kinase C (PKC) and protein kinase A is involved in the MMF-induced increase in [Ca2+]i in granulocytes. As MMF activated protein tyrosine kinase (PTK), and inhibition of this protein kinase partially reduced the increase in [Ca2+]i in granulocytes, PTK activity most likely is involved. In addition, activation of protein kinase histone 4 (PKH4) probably plays a part in the MMF-stimulated increase in [Ca2+]i in granulocytes as well. As MMF stimulated an increase in the GTP-ase activity of membranes and pertussis toxin (PTX) inhibited the increase in the [Ca2+]i and PKH4 activity of granulocytes stimulated by this compound, it is concluded that MMF activates PTX-sensitive G proteins. Competition binding studies with radiolabelled dimethylfumarate (DMF) and unlabelled DMF and MMF revealed the presence of specific binding sites for methylated fumarates on granulocytes. In summary, MMF binds to specific sites on the plasma membrane of cells. This interaction activates pertussis toxin-sensitive G proteins which then stimulate an increase in PTK and PKH4 activity. These protein kinases may regulate the rise in [Ca2+]i and the intracellular cAMP concentration. Elevated [Ca2+]i and intracellular cAMP concentration stimulate protein kinases that regulate transcription factors. Activation of these factors results in induction of downstream gene expression and thus controls cell functions, e.g. cell proliferation and production of inflammatory mediators, as has been found for cells incubated with MMF.

Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins.

E2F transcription factors are key regulators of transcription during the cell cycle. E2F activity is regulated at the level of transcription and DNA binding and by complex formation with the retinoblastoma pocket protein family. We show here that free E2F-1 and E2F-4 transcription factors are unstable and that their degradation is mediated by the ubiquitin-proteasome pathway. Both E2F-1 and E2F-4 are rendered unstable by an epitope in the carboxyl terminus of the proteins, in close proximity to their pocket protein interaction surface. We show that binding of E2F-1 to pRb or E2F-4 to p107 or p130 protects E2Fs from degradation, causing the complexes to be stable. The increased stability of E2F-4 pocket protein complexes may contribute to the maintenance of active transcriptional repression in quiescent cells. Surprisingly, adenovirus transforming proteins, which release pocket protein-E2F complexes, also inhibit breakdown of free E2F. These data reveal an additional level of regulation of E2F transcription factors by targeted proteolysis, which is inhibited by pocket protein binding and adenovirus early region 1 transforming proteins.

Functional analysis of Burkitt's lymphoma mutant c-Myc proteins.

The c-myc gene encodes a sequence-specific DNA binding protein that activates transcription of cellular genes. Transcription activation by Myc proteins is regulated by phosphorylation of serine and threonine residues within the transactivation domain and by complex formation with the retinoblastoma-related protein p107. In Burkitt's lymphoma, missense mutations within the c-Myc transactivation domain have been found with high frequency. It has been reported that mutant c-Myc proteins derived from Burkitt's lymphoma cell lines are resistant to inhibition by p107, thus providing a rationale for the increased oncogenic activity of these mutant c-Myc proteins. It has been suggested that these mutant c-Myc proteins resist down-modulation by p107 because they lack cyclin A-cdk2-dependent phosphorylation. Here, we have examined three different Burkitt's lymphoma mutant c-Myc proteins found in primary Burkitt's lymphomas and one mutant c-Myc protein detected in a Burkitt's lymphoma cell line. All four have an unaltered ability to activate transcription and are sensitive to inhibition of transactivation by p107. Furthermore, we provide evidence that down-modulation of c-Myc transactivation by p107 does not require phosphorylation of the c-Myc transactivation domain by cyclin A-cdk2. Our data indicate that escape from p107-induced suppression is not a general consequence of all Burkitt's lymphoma-associated c-Myc mutations, suggesting that other mechanisms exist to deregulate c-Myc function.

Regulation of the retinoblastoma protein-related p107 by G1 cyclin complexes.

The orderly progression through the cell cycle is mediated by the sequential activation of several cyclin/cyclin-dependent kinase (cdk) complexes. These kinases phosphorylate a number of cellular substrates, among which is the product of the retinoblastoma gene, pRb. Phosphorylation of pRb in late G1 causes the release of the transcription factor E2F from pRb, resulting in the transcriptional activation of E2F-responsive genes. We show here that phosphorylation of the pRb-related p107 is also cell cycle regulated. p107 is first phosphorylated at 8 hr following serum stimulation of quiescent fibroblasts, which coincides with an increase in cyclin D1 protein levels. Consistent with this, we show that a cyclin D1/cdk4 complex, but not a cyclin E/cdk2 complex, can phosphorylate p107 in vivo. Furthermore, phosphorylation of p107 can be abolished by the overexpression of a dominant-negative form of cdk4. Phosphorylation of p107 results in the loss of the ability to associate with E2F-4, a transcription factor with growth-promoting and oncogenic activity. A p107-induced cell cycle block can be released by cyclin D1/cdk4 but not by cyclin E/cdk2. These data indicate that the activity of p107 is regulated by phosphorylation through D-type cyclins.

The pRB-related protein p107 contains two growth suppression domains: independent interactions with E2F and cyclin/cdk complexes.

Unregulated expression of either the retinoblastoma protein (pRB) or the related protein p107 can cause growth arrest of sensitive cells in the G1 phase of the cell cycle. However, growth arrests mediated by p107 and pRB are not identical. Through structure-function and co-expression analyses we have dissected the p107 molecule into two domains that independently are able to block cell cycle progression. One domain corresponds to the sequences needed for interaction with the transcription factor E2F, and the other corresponds to the interaction domain for cyclin A or cyclin E complexes. In cervical carcinoma cell line C33A, which was previously shown to be sensitive to p107 but resistant to pRB growth suppression, only the cyclin binding domain is active as a growth suppressor. Furthermore, we show that these two independent domains are functional in untransformed mouse fibroblasts. Together, these results provide experimental evidence for the presence of two functional domains in p107 and pinpoint an important functional difference between p107 and pRB.

N-myc suppresses major histocompatibility complex class I gene expression through down-regulation of the p50 subunit of NF-kappa B.

In neuroblastoma, N-myc suppresses the expression of major histocompatibility complex (MHC) Class I antigens by reducing the binding of a nuclear factor to the enhancer-A element in the MHC Class I gene promoter. We show here that the p50 subunit of NF-kappa B is part of this complex and that expression of p50 mRNA is suppressed by N-myc. Transfection of a p50 expression vector in neuroblastoma cells that express N-myc at a high level leads to restoration of factor binding to the MHC Class I gene enhancer, restores enhancer activity and leads to re-expression of MHC Class I antigens at the cell surface. These data indicate that the p50 subunit of NF-kappa B is involved in the regulation of MHC Class I antigen expression and that N-myc down-regulates MHC Class I gene expression primarily through suppression of p50 expression.

Cell-cell adhesion mediated by a receptor-like protein tyrosine phosphatase.

Receptor-like protein tyrosine phosphatases (receptor-PTPs) represent a novel family of transmembrane proteins that are thought to play important roles in cellular regulation. They consist of a cytoplasmic catalytic region, a single transmembrane segment and an extracellular, putative ligand-binding domain, but the nature of their physiological ligands is unknown. We have recently cloned a new receptor-PTP (RPTP mu), the ectodomain of which includes an Ig-like and four fibronectin type III-like domains, suggesting that RPTP mu may be involved in cell-cell or cell-matrix interactions. To test this hypothesis, we expressed RPTP mu in insect Sf9 cells using recombinant baculovirus. We demonstrate that RPTP mu dramatically promotes cell-to-cell adhesion in a homophilic, Ca(2+)-independent manner. No adhesion is observed in Sf9 cells expressing a chimeric RPTP mu molecule containing the extracellular domain of the epidermal growth factor receptor. Furthermore, cells expressing an enzymatically inactive, point-mutated RPTP mu or a truncated form of RPTP mu, lacking the entire catalytic region, show adhesive properties indistinguishable from those of wild-type RPTP mu, indicating that the catalytic domain is not essential for RPTP mu-mediated adhesion. These results assign a physiological role for RPTP mu in signaling cell-cell recognition.

E2F-4, a new member of the E2F gene family, has oncogenic activity and associates with p107 in vivo.

The E2F family of transcription factors controls the expression of genes that are involved in cell cycle regulation. E2F DNA-binding activity is found in complex with the retinoblastoma protein, pRb, and with the pRb-related p107 and p130. To date, cDNAs for three members of the E2F gene family have been isolated. However, all three E2Fs associate in vivo exclusively with pRb. We report here the cloning and functional analysis of a fourth E2F family member. E2F-4 encodes a 413-amino-acid protein with significant homology to E2F-1. E2F-4 antibodies recognize a 60-kD protein in anti-p107 immunoprecipitates, indicating that E2F-4 associates with p107 in vivo. Like the other E2Fs, E2F-4 requires DP-1 for efficient DNA binding and transcriptional activation of E2F site-containing promoters. Increased expression of E2F-4 and DP-1 in SaoS-2 osteosarcoma cells causes a shift from G1-phase cells to S and G2/M-phase cells, suggesting a role for E2F-4 in regulation of cell-cycle progression. We show that expression of E2F-4 and DP-1 together with an activated ras oncogene in rat embryo fibroblasts, causes transformation, indicating that E2F-4 has oncogenic activity.

Creation of human tumour cells with defined genetic elements.

During malignant transformation, cancer cells acquire genetic mutations that override the normal mechanisms controlling cellular proliferation. Primary rodent cells are efficiently converted into tumorigenic cells by the coexpression of cooperating oncogenes. However, similar experiments with human cells have consistently failed to yield tumorigenic transformants, indicating a fundamental difference in the biology of human and rodent cells. The few reported successes in the creation of human tumour cells have depended on the use of chemical or physical agents to achieve immortalization, the selection of rare, spontaneously arising immortalized cells, or the use of an entire viral genome. We show here that the ectopic expression of the telomerase catalytic subunit (hTERT) in combination with two oncogenes (the simian virus 40 large-T oncoprotein and an oncogenic allele of H-ras) results in direct tumorigenic conversion of normal human epithelial and fibroblast cells. These results demonstrate that disruption of the intracellular pathways regulated by large-T, oncogenic ras and telomerase suffices to create a human tumor cell.

Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization.

The immortalization of human cells is a critical step during tumorigenesis. In vitro, normal human somatic cells must overcome two proliferative blockades, senescence and crisis, to become immortal. Transformation with viral oncogenes extends the life span of human cells beyond senescence. Such transformed cells eventually succumb to crisis, a period of widespread cellular death that has been proposed to be the result of telomeric shortening. We now show that ectopic expression of the telomerase catalytic subunit (human telomerase reverse transcriptase or hTERT) and subsequent activation of telomerase can allow postsenescent cells to proliferate beyond crisis, the last known proliferative blockade to cellular immortality. Moreover, we demonstrate that alteration of the carboxyl terminus of human telomerase reverse transcriptase does not affect telomerase enzymatic activity but impedes the ability of this enzyme to maintain telomeres. Telomerase-positive cells expressing this mutant enzyme fail to undergo immortalization, further tightening the connection between telomere maintenance and immortalization.

hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.

Telomerase, the ribonucleoprotein enzyme that elongates telomeres, is repressed in normal human somatic cells but is reactivated during tumor progression. We report the cloning of a human gene, hEST2, that shares significant sequence similarity with the telomerase catalytic subunit genes of lower eukaryotes. hEST2 is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Moreover, the message is up-regulated concomitant with the activation of telomerase during the immortalization of cultured cells and down-regulated during in vitro cellular differentiation. Taken together, these observations suggest that the induction of hEST2 mRNA expression is required for the telomerase activation that occurs during cellular immortalization and tumor progression.