RESUMEN
The spindle assembly checkpoint (SAC) acts as a guardian against cellular threats that may lead to chromosomal missegregation and aneuploidy. Mad2, an anaphase-promoting complex/cyclosome-Cdc20 (APC/C(Cdc20)) inhibitor, has an additional homolog in mammals known as Mad2B, Mad2L2 or Rev7. Apart from its role in Polζ-mediated translesion DNA synthesis and double-strand break repair, Rev7 is also believed to inhibit APC/C by negatively regulating Cdh1. Here we report yet another function of Rev7 in cultured human cells. Rev7, as predicted earlier, is involved in the formation of a functional spindle and maintenance of chromosome segregation. In the absence of Rev7, cells tend to arrest in G2/M-phase and display increased monoastral and abnormal spindles with misaligned chromosomes. Furthermore, Rev7-depleted cells show Mad2 localization at the kinetochores of metaphase cells, an indicator of activated SAC, coupled with increased levels of Cyclin B1, an APC(Cdc20) substrate. Surprisingly unlike Mad2, depletion of Rev7 in several cultured human cell lines did not compromise SAC activity. Our data therefore suggest that besides its role in APC/C(Cdh1) inhibition, Rev7 is also required for mitotic spindle organization and faithful chromosome segregation most probably through its physical interaction with RAN.
Asunto(s)
Proteínas Mad2/metabolismo , Huso Acromático/metabolismo , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Aneuploidia , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , Citometría de Flujo , Células HCT116 , Células HeLa , Humanos , Inmunohistoquímica , Proteínas Mad2/genética , Mitosis/genética , Mitosis/fisiología , Huso Acromático/genéticaRESUMEN
We developed a telomerase-positive, infinite life span human fibroblast cell strain (MSU-1.0) by transfection of a v-MYC oncogene and spontaneous over-expression of transcription factors SP1/SP3. Loss of expression of p14(ALT) and enhanced expression of SPRY2 gave rise to the MSU-1.1 cell strain. Unlike MSU-1.0 cells, the MSU-1.1 cells can be malignantly transformed by expression of N-RAS(LYS61) or H-Ras(v12) oncoproteins (driven by their original promoters) and expression of a SRC-family protein, v-FES. MSU-1.1 cells can also be malignantly transformed by high expression of these RAS oncogenes or the v-K-RAS oncogene. PDGF-B transformed MSU-1.1 cells give rise to benign tumors (fibromas) in athymic mice. A second route to malignant transformation of the MSU-1.1 cells involves loss of functional TP53 protein by carcinogen treatment and loss of expression of wild type p16(INK). These studies indicate 6-8 "hits" are required to activate the oncogenes and inactivate the suppressor genes we identified.
Asunto(s)
Transformación Celular Neoplásica , Fibroblastos/patología , Animales , Células Cultivadas , Humanos , Transducción de Señal , Piel/patología , Proteína p14ARF Supresora de Tumor/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas ras/fisiología , Familia-src Quinasas/fisiologíaRESUMEN
BACKGROUND: DNA polymerase zeta (Polζ) is a specialized DNA polymerase that, unlike classical replicative polymerases, is capable of replicating past DNA lesions, i.e. of performing translesion synthesis (TLS). The catalytic subunit of hPolζ, hRev3, has been shown to play a critical role in DNA damage-induced mutagenesis in human cells, but less is known about the role of hRev7, the accessory subunit of hPolζ, in such mutagenesis. To address this question, we recently generated human fibroblasts with very significantly reduced levels of hRev7 protein and demonstrated that hRev7 is required to protect cells from ultraviolet(254 nm) (UV) radiation-induced cytotoxicity and mutagenesis (McNally et al., DNA Repair 7 (2008) 597-604). The goal of the present study was to determine whether hRev7 is similarly involved in the tolerance of DNA damage induced by benzo[a]pyrene diol epoxide (BPDE), the reactive form of the widespread environmental carcinogen benzo[a]pyrene. METHODS: To determine whether hRev7 also plays a role in protecting human cells from the cytotoxicity and mutagenesis induced by benzo[a]pyrene diol epoxide (BPDE), cell strains with reduced hRev7 were compared to their parental strain and a vector control strain for the effect of BPDE on cell survival, induction of mutations, and the ability to progress through the cell cycle. RESULTS: The results show that cell strains with reduced hRev7 are more sensitive to the cytotoxic effect of BPDE than the control strains, and progress through S-phase at a slower rate than the control cells following BPDE treatment, indicating that hRev7, and likely hPolζ, is required for efficient bypass of BPDE-induced DNA lesions. However, neither the frequency nor kinds of mutations induced by BPDE in cells with reduced hRev7 differ significantly from those induced in the control strains, suggesting that hPolζ is not essential for inserting nucleotides opposite BPDE-induced DNA damage. CONCLUSIONS: Taken together, our results which show that hRev7 is required for TLS past BPDE-induced DNA lesions but that it is not essential for inserting nucleotides opposite such lesions suggest a role for hPolζ in the extension step of translesion synthesis.
Asunto(s)
7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Daño del ADN , Mutágenos/toxicidad , Proteínas/fisiología , Línea Celular , Supervivencia Celular , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/fisiología , Humanos , Proteínas Mad2 , Mutación , Subunidades de Proteína/metabolismo , Subunidades de Proteína/fisiología , Proteínas/metabolismoRESUMEN
BACKGROUND: The activities of Rac1 and Cdc42 are essential for HRas-induced transformation of rodent fibroblasts. What is more, expression of constitutively activated mutants of Rac1 and/or Cdc42 is sufficient for their malignant transformation. The role for these two Rho GTPases in HRas-mediated transformation of human fibroblasts has not been studied. Here we evaluated the contribution of Rac1 and Cdc42 to maintaining HRas-induced transformation of human fibroblasts, and determined the ability of constitutively activated mutants of Rac1 or Cdc42 to induce malignant transformation of a human fibroblast cell strain. METHODS: Under the control of a tetracycline regulatable promoter, dominant negative mutants of Rac1 and Cdc42 were expressed in a human HRas-transformed, tumor derived fibroblast cell line. These cells were used to determine the roles of Rac1 and/or Cdc42 proteins in maintaining HRas-induced transformed phenotypes. Similarly, constitutively active mutants were expressed in a non-transformed human fibroblast cell strain to evaluate their potential to induce malignant transformation. Affymetrix GeneChip arrays were used for transcriptome analyses, and observed expression differences were subsequently validated using protein assays. RESULTS: Expression of dominant negative Rac1 and/or Cdc42 significantly altered transformed phenotypes of HRas malignantly transformed human fibroblasts. In contrast, expression of constitutively active mutants of Rac1 or Cdc42 was not sufficient to induce malignant transformation. Microarray analysis revealed that the expression of 29 genes was dependent on Rac1 and Cdc42, many of which are known to play a role in cancer. The dependence of two such genes, uPA and VEGF was further validated in both normoxic and hypoxic conditions. CONCLUSION(S): The results presented here indicate that expression of both Rac1 and Cdc42 is necessary for maintaining several transformed phenotypes in oncogenic HRas transformed human cells, including their ability to form tumors in athymic mice. Our data also indicate that expression of either activated Rac1 or Cdc42 alone is not sufficient for malignant transformation of human fibroblasts, although each is required for specific transformed phenotypes. Furthermore, our study elucidates that the expression of several highly significant cancer related genes require the activities of Rac1 and/or Cdc42 which may also play a critical role in cellular transformation.
Asunto(s)
Transformación Celular Neoplásica , Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Proteínas ras/metabolismo , Inductores de la Angiogénesis/metabolismo , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Tetraciclina/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Unión al GTP rho/metabolismoRESUMEN
We have reported that expression of Sprouty 2 (Spry2) is necessary for tumor formation by HRas(V12)-transformed fibroblasts. We now report on the role of Spry2 in the inhibition of UV(254 nm) radiation-induced apoptosis in HRas(V12)-transformed human fibroblasts. Silencing Spry2 in this context resulted in increased apoptosis, associated with decreased Akt activation and decreased phosphorylation of HDM2 at Ser-166, which has been shown to stabilize HDM2. As a consequence, when cells with silenced Spry2 were UV-irradiated, they exhibited diminished levels of HDM2 and elevated levels of p53. In agreement with these findings, overexpression of Spry2 in the parental non-transformed fibroblasts led to increased Akt activation and to the stabilization of HDM2. It also led to diminished expression of p53 and decreased apoptosis following UV irradiation. Silencing Spry2 in HRas-transformed cells decreased Rac1 activation, but independent expression of Spry2 in the non-transformed parental cells had no effect on Rac1, suggesting a specific involvement in the activation of Rac1 by Ras. Silencing Spry2 in HRas(V12)-transformed cells resulted in diminished interaction between HRas and Tiam1, a Rac1-specific nucleotide exchange factor. Expression of constitutively active Rac1 in cells with silenced Spry2 partly reversed the effect of Spry2 down-regulation. Furthermore, loss of Spry2 expression in HRas(V12)-transformed cells augmented the cytotoxicity of the DNA-damaging, chemotherapeutic agent cisplatin, a process that was also reversed by active Rac1. Together, these data show that Spry2 inhibits apoptosis in response to DNA damage by regulating Akt, HDM2, and p53, by a process mediated partly by Rac1.
Asunto(s)
Apoptosis , Daño del ADN/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Transgenes/genética , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular , Cisplatino/toxicidad , Regulación hacia Abajo , Activación Enzimática , Fibroblastos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Proteína Oncogénica p21(ras)/genética , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Translesion synthesis (TLS) refers to mechanisms by which specialized DNA polymerases incorporate nucleotides opposite fork-blocking lesions and extend replication until standard replicative polymerases take over. The first eukaryotic TLS polymerase discovered, S. cerevisiae Polzeta, consists of catalytic subunit Rev3 and non-catalytic subunit Rev7. Human homologs of these two proteins have been identified. Studies by Lawrence, Maher, and colleagues comparing UV((254nm))-irradiated human fibroblast cell strains expressing high levels of hRev3 antisense to their normal parental strains demonstrated that there was no difference in cell survival, but that the frequency of UV-induced mutations in the derivative strains was 10-fold lower than that of the parental strains, indicating that hRev3 plays a critical role in such mutagenesis. To examine the role of hRev7 in TLS, we generated human fibroblasts expressing hRev7 siRNA, identified two derivative cell strains with significantly reduced levels of hRev7, and compared them to their parental strain and a vector control for cell survival, induction of mutations, and ability to traverse the cell cycle following exposure to UV radiation. Cells with reduced hRev7 were approximately 2-times more sensitive to UV-induced cytotoxicity than the controls, indicating that unlike hRev3, hRev7 plays a protective role for cells exposed to UV radiation. When these cell strains were assayed for the frequency of mutations induced by UV in their HPRT gene, cell stains with reduced hRev7 were 5-times less sensitive to UV-induced mutagenesis than control strains. In addition, when these four strains were synchronized at the G1/S border, released from the block, UV-irradiated, and allowed to traverse the cell cycle, the rate of progression through S-phase of the cell strains with reduced hRev7 was significantly slower than that of the control strains. These data strongly support the hypothesis that hRev7 is required for TLS past UV-photoproducts, and together with hRev3, comprise hPolzeta.
Asunto(s)
Fibroblastos/efectos de la radiación , Proteínas Mad2/metabolismo , Mutagénesis , Fase S/genética , Rayos Ultravioleta , Supervivencia Celular , Fibroblastos/metabolismo , Humanos , Proteínas Mad2/genética , MutaciónRESUMEN
Sprouty 2 (Spry2) acts as an inhibitor of receptor tyrosine kinase signaling in various cellular contexts. Interestingly, Spry2 also prevents the c-Cbl-induced degradation of epidermal growth factor receptor (EGFR). We compared human fibroblasts malignantly transformed by overexpression of H-Ras(V12) oncogene to their nontransformed parental cells and found that the malignant cells express a high level of Spry2. These cells also exhibited an increase in the level of EGFR compared with their precursor cells. We found that intact EGFR was required if H-Ras-transformed cells were to grow in the absence of exogenous growth factors or form large colonies in agarose. When we decreased expression of Spry2, using a Spry2-specific shRNA, the H-Ras(V12)-transformed fibroblasts could no longer form large colonies in agarose, grow in reduced levels of serum, or form tumors in athymic mice. The level of active H-Ras in these cells remained unaltered. A similar, but less pronounced, effect in tumor formation was observed when Spry2 was down-regulated in human patient-derived fibrosarcoma cell lines. In H-Ras-transformed cells Spry2 sustained the level and the downstream signaling activity of EGFR. In the parental, non-H-Ras-transformed fibroblasts, expression of Spry2 resulted in the inhibition of H-Ras and ERK activation, suggesting that the positive effect of Spry2 in tumor formation is specific to H-Ras transformation. Co-immunoprecipitation studies with H-Ras-transformed cells revealed that Spry2 and H-Ras interact and that H-Ras interacts with Spry2-binding partners, c-Cbl and CIN85, in a Spry2-dependent manner. These data show that Spry2 plays a critical role in the ability of H-Ras-transformed cells to form tumors in athymic mice.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Sarcoma/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular Transformada , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fibroblastos/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Ratones , Ratones Desnudos , Proteína Oncogénica p21(ras)/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Sarcoma/genética , Sarcoma/patologíaRESUMEN
Xeroderma pigmentosum variant (XPV) patients have normal DNA excision repair, yet are predisposed to develop sunlight-induced cancer. They exhibit a 25-fold higher than normal frequency of UV-induced mutations and very unusual kinds (spectrum), mainly transversions. The primary defect in XPV cells is the lack of functional DNA polymerase (Pol) eta, the translesion synthesis DNA polymerase that readily inserts adenine nucleotides opposite photoproducts involving thymine. The high frequency and striking difference in kinds of UV-induced mutations in XPV cells strongly suggest that, in the absence of Pol eta, an abnormally error-prone polymerase substitutes. In vitro replication studies of Pol iota show that it replicates past 5'T-T3' and 5'T-U3' cyclobutane pyrimidine dimers, incorporating G or T nucleotides opposite the 3' nucleotide. To test the hypothesis that Pol iota causes the high frequency and abnormal spectrum of UV-induced mutations in XPV cells, we identified an unlimited lifespan XPV cell line expressing two forms of Pol iota, whose frequency of UV-induced mutations is twice that of XPV cells expressing one form. We eliminated expression of one form and compared the parental cells and derivatives for the frequency and kinds of UV-induced mutations. All exhibited similar sensitivity to the cytotoxicity of UV((254 nm)), and the kinds of mutations induced were identical, but the frequency of mutations induced in the derivatives was reduced to
Asunto(s)
ADN Polimerasa Dirigida por ADN/deficiencia , Mutación/efectos de la radiación , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Secuencia de Aminoácidos , Línea Celular , ADN Polimerasa Dirigida por ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Fibroblastos , Humanos , Datos de Secuencia Molecular , Transfección , Rayos Ultravioleta , Xerodermia Pigmentosa/patología , ADN Polimerasa iotaRESUMEN
In a study of the role of transcription factor Sp1 in the formation of tumors by human fibrosarcoma cell lines that overexpress it [Cancer Res., 65 (2005) 1007], we found that expression of an Sp1-specific ribozyme, not only reduced the level of Sp1 protein, but also that of Sp3 protein, and that when the protein levels of these two transcription factors in the fibrosarcoma cell lines were reduced to near that found in normal human fibroblasts, the cell lines could no longer form tumors. An Sp1-specific ribozyme could reduce the level of expression of both Sp1 protein and Sp3 protein if the promoter of the Sp1 gene and that of the Sp3 gene both have Sp1/Sp3 transcription factor binding sites and if such sites are critically responsible for the level of expression of both Sp1 and Sp3 protein in the cells. The Sp1 minimal promoter has been identified and it has two Sp1/Sp3 sites [J. Biol. Chem. 276 (2001) 22126]. To characterize the Sp3 promoter, we isolated 2.1 kb of the 5'-flanking region of the Sp3 gene, which contains Sp1/Sp3 binding sites, and using an expression reporter assay, showed that it has promoter activity. We then systematically reduced the size of the 5' flanking region, and determined that the nt-339 to nt-39 fragment, which contains an Sp1/Sp3 binding site at nt-181 and another at nt-168, retained the same promoter activity as the 2.1 kb region. Electrophoretic mobility shift assays indicated that both Sp3 protein and Sp1protein bind to these two sites. By mutating either or both of these binding sites, we showed using the reporter assay that each site is required for full promoter activity. We then designed an Sp3-specific ribozyme, expressed it in a human fibrosarcoma cell line in which Sp1 protein and Sp3 protein are expressed at high levels, and found that, indeed, the level of expression of both proteins was significantly reduced.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Factor de Transcripción Sp1/metabolismo , Factores de Transcripción/metabolismo , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Clonación Molecular , ADN/química , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Femenino , Vectores Genéticos/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Unión Proteica , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/fisiología , Factor de Transcripción Sp3 , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Sitio de Iniciación de la Transcripción , TransfecciónRESUMEN
Sp1 is a transcription factor for many genes, including genes involved in tumorigenesis. We found that human fibroblast cells malignantly transformed in culture by a carcinogen or by stable transfection of an oncogene express Sp1 at 8-fold to 18-fold higher levels than their parental cells. These cell lines form fibrosarcomas in athymic mice with a very short latency, and the cells from the tumors express the same high levels of Sp1. Similar high levels of Sp1 were found in the patient-derived fibrosarcoma cell lines tested, and in the tumors formed in athymic mice by these cell lines. To investigate the role of overexpression of Sp1 in malignant transformation of human fibroblasts, we transfected an Sp1 U1snRNA/Ribozyme into two human cell lines, malignantly transformed in culture by a carcinogen or overexpression of an oncogene, and into a patient-derived fibrosarcoma cell line. The level of expression of Sp1 in these transfected cell lines was reduced to near normal. The cells regained the spindle-shaped morphology and exhibited increased apoptosis and decreased expression of several genes linked to cancer, i.e., epithelial growth factor receptor, urokinase plasminogen activator, urokinase plasminogen activator receptor, and vascular endothelial growth factor. When injected into athymic mice, these cell lines with near normal levels of Sp1 failed to form tumors or did so only at a greatly reduced frequency and with a much longer latency. These data indicate that overexpression of Sp1 plays a causal role in malignant transformation of human fibroblasts and suggest that for cancers in which it is overexpressed, Sp1 constitutes a target for therapy.
Asunto(s)
Transformación Celular Neoplásica/genética , Fibrosarcoma/genética , Factor de Transcripción Sp1/genética , Animales , Apoptosis/genética , Secuencia de Bases , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Vectores Genéticos/genética , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN sin Sentido/genética , ARN Catalítico/genética , ARN Nuclear Pequeño/genética , Factor de Transcripción Sp1/biosíntesis , Factor de Transcripción Sp3 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , TransfecciónRESUMEN
PURPOSE: Modulation of platinating agent cytotoxicity has important clinical implications as a result of their widespread use in the treatment of many different cancers. O6-Benzylguanine (BG) enhances the cytotoxicity of cisplatin against several human tumor lines. The purpose of our work was to elucidate whether BG affects pathways prior to DNA damage (i.e., glutathione, GSH) or following DNA damage (i.e., nucleotide excision repair, NER). METHODS: In efforts to determine the mechanism of enhancement we: (1) evaluated whether different sequences of BG plus cisplatin treatment differed in their ability to enhance cisplatin-induced cytotoxicity and DNA platination; (2) determined the effect of BG on GSH and glutathione S-transferase (GST) activity and; (3) determined whether BG enhanced cisplatin-induced cytotoxicity in cells lacking specific enzymes in the NER pathway. Colony-forming assay, atomic absorption spectroscopy and HPLC were employed to measure tumor cell growth inhibition, quantitate the amount of platinum on DNA, and determine intracellular GSH concentrations, respectively. RESULTS: Increased cytotoxicity and platination of DNA was observed when cells were exposed to BG prior to and/or during cisplatin treatment and not when BG followed cisplatin treatment. BG did not significantly alter GST activity with minimal depletion of GSH. In contrast, buthionine sulfoximine (BSO) caused a much more dramatic decrease in GSH than BG that was not accompanied by a dramatic increase in sensitivity to cisplatin. Furthermore, BG enhanced the cytotoxicity of cisplatin in a series of cell lines deficient in NER. CONCLUSIONS: Overall, our results suggest that the mechanism of enhancement involves neither the GSH nor the NER pathways, but triggers an event prior to DNA platination damage that ultimately results in increased cytotoxicity, apoptosis and increased platination levels.
Asunto(s)
Antineoplásicos/farmacología , Cisplatino/farmacología , Reparación del ADN/efectos de los fármacos , Glutatión/metabolismo , Guanina/análogos & derivados , Guanina/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Sinergismo Farmacológico , Humanos , Platino (Metal)/metabolismo , Células Tumorales CultivadasRESUMEN
Changes in expression of hepatocyte growth factor (HGF) and its receptor, MET, are associated with formation and malignant progression of human tumors. In the present study, 10 of 11 human fibrosarcoma cell lines tested expressed significantly higher levels of MET than were found in a series of normal human fibroblast lines. Still more significant, MET was constitutively phosphorylated in all 11 fibrosarcoma lines, whereas the normal fibroblasts exhibited very low levels of the phosphorylated form. All the cell lines expressed HGF mRNA. To determine the role of MET and/or HGF in tumorigenesis, a fibrosarcoma line expressing high levels of MET protein and low levels of HGF/NK2 mRNA was stably transfected with a hammerhead ribozyme targeting MET. In addition, a fibrosarcoma line expressing high levels of both MET protein and HGF/NK2 mRNA was transfected with a ribozyme targeting MET, or with a ribozyme targeting MET and another targeting HGF. The transfectant cell lines no longer formed tumors, or did so at a greatly reduced frequency and/or longer latency. Because Sp1 is a transcription factor for MET, we assayed the cell lines for their level of Sp1 protein. Sp1 was markedly overexpressed in 7 of the 11 fibrosarcoma lines compared to normal fibroblast lines. Deletion analysis and site-directed mutagenesis of the MET promoter revealed that tandem Sp1 sites in the proximal promoter are critical for transcription of MET. Increased expression of Sp1 in a normal human fibroblast line containing a MET promoter-luciferase construct resulted in a dose-dependent increase in luciferase. Conversely, inhibition of Sp1 binding to DNA in a fibrosarcoma cell line, using an Sp1 decoy, dramatically reduced MET expression. Taken together, these results indicate that in human fibrosarcoma cells, high levels of the phosphorylated form of MET are required for tumor formation and that Sp1 can function to control the level of MET.
Asunto(s)
Fibrosarcoma/patología , Regulación Neoplásica de la Expresión Génica , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-met/genética , ARN Catalítico/farmacología , Factor de Transcripción Sp1/genética , Animales , Northern Blotting , Regulación hacia Abajo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosarcoma/metabolismo , Fibrosarcoma/terapia , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Luciferasas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mutagénesis Sitio-Dirigida , Mutación/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Eliminación de Secuencia , Factor de Transcripción Sp1/metabolismo , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
In 1997, McCormick and co-workers identified a novel putative tumor suppressor gene, designated ST7, encoding a unique protein with transmembrane receptor characteristics [Qing et al. (1999) Oncogene 18, 335-342]. Using degenerate primers corresponding to the highly conserved region of the ligand-binding domains of members of the low-density lipoprotein receptor (LDLR) superfamily, Ishii et al. [Genomics (1998) 51, 132-135] discovered a low-density lipoprotein receptor-related protein (LRP) that closely resembles ST7. Later, another LRP closely resembling ST7 and LRP3 was found (murine LRP9) [Sugiyama et al. (2000) Biochemistry 39, 15817-15825]. These results strongly suggested that ST7 was also a novel member of the low-density lipoprotein receptor superfamily. Proteins of this superfamily have been shown to function in endocytosis and/or signal transduction. To evaluate the relationship of ST7 to the LDLR superfamily proteins and to determine whether ST7 may function in endocytosis and/or signal transduction, we used proteomic tools to analyze the functional motifs present in the protein. Our results indicate that ST7 is a member of a subfamily of the LDLR superfamily and that its cytoplasmic domain contains several motifs implicated in endocytosis and signal transduction. Use of the yeast two-hybrid system to identify proteins that associate with ST7's cytoplasmic domain revealed that this domain interacts with three proteins involved in signal transduction and/or endocytosis, viz., receptor for activated protein C kinase 1 (RACK1), muscle integrin binding protein (MIBP), and SMAD anchor for receptor activation (SARA), suggesting that ST7, like other proteins in the LDLR superfamily, functions in these two pathways. Clearly, ST7 is an LRP, and therefore, it should now be referred to as LRP12.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina Endopeptidasas , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Endocitosis/fisiología , Humanos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Factores Reguladores Miogénicos/metabolismo , Péptidos/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol) , Estructura Terciaria de Proteína , Receptores de Cinasa C Activada , Receptores de LDL/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (+/-)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not possible to demonstrate that the decrease in mutagenesis reflected decreased hRev3 protein. However, the conclusion is supported by the fact that in a similar study with a strain expressing a high level of antisense hREV1, a very similar result was obtained, i.e. UV or BPDE mutagenesis was virtually eliminated.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/biosíntesis , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , ADN/efectos de los fármacos , ADN/efectos de la radiación , Proteínas de Unión al ADN , ADN Polimerasa Dirigida por ADN/genética , Hipoxantina Fosforribosiltransferasa/genética , Mutagénesis , Mutágenos/toxicidad , ARN sin Sentido/genética , Rayos Ultravioleta/efectos adversosRESUMEN
When DNA replication stalls at a fork-blocking lesion, cells use damage tolerance pathways to continue replication. One pathway, "translesion synthesis," involves specialized DNA polymerases that can use damaged DNA as a template. Translesion synthesis can result in mutations (i.e., can be error-prone), but it can also be error-free. An alternative pathway has been hypothesized (sometimes called "damage avoidance"), by which cells make temporary use of an undamaged copy of the blocked sequence as a template, i.e., the newly synthesized daughter strand of the sister duplex or the allelic copy. This pathway is error-free. Evidence of the use of the daughter strand of the sister duplex as a template in intact mammalian cells has not been available heretofore. To determine whether hMms2, a ubiquitin-conjugating enzyme-like protein, plays a critical role in such damage avoidance, a human fibroblast cell strain in which both error-prone translesion synthesis and error-free damage avoidance can be detected and quantified simultaneously, and several derivative strains in which expression of hMms2 protein had been eliminated or greatly decreased, were compared for their ability to avoid translesion synthesis past UV(254nm)-induced DNA photoproducts. Loss of hMms2 protein eliminated the ability of the latter strains to use an allelic copy of a target gene for damage avoidance, i.e., to produce a wild-type gene from two nonfunctional allelic copies of that gene. Molecular analysis of the wild-type gene showed that this process involves gene conversion unassociated with crossing-over. That the loss of hMms2 also eliminated use of the daughter strand of the sister duplex as a template for damage avoidance could be inferred from the fact that the frequency of mutations induced by UV in the single copy HPRT gene of the derivative strains was significantly higher than that observed in the parental strain. These data indicate that hMMS2 is essential for human cells to carry out damage avoidance by using either type of homolog, and that damage avoidance and translesion synthesis are alternative pathways for tolerating fork-blocking photoproducts.