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Biomedical subjects

M S Soengas

Publications and source records attributed to M S Soengas.

16 recordsLinked to original sources
PubMed2006

Adenovirus-mediated TA-p73beta gene transfer increases chemosensitivity of human malignant melanomas.

Malignant melanoma is the most aggressive form of skin cancer and has proven to be highly resistant to conventional chemotherapy. Intriguingly, the p53 tumor suppressor, a main mediator of chemoresistance in other tumor types, is rarely mutated in melanoma. However, we have previously shown that anti-apoptotic isoforms of p73 (deltaTA-p73), another member of the p53 family, are overexpressed in metastatic melanomas. DeltaTA-p73 can oppose the pro-apoptotic functions of p53 and full length p73, and thus it could contribute to melanoma chemoresistance. In this study, we use an efficient adenoviral-based gene transfer approach to introduce a transcriptionally active form of p73 (TA-p73beta) in melanoma cells, with the objective of overcoming drug resistance. Interestingly, TA-p73beta significantly sensitized 5 out of 7 aggressive melanoma cell lines to the standard therapeutic agents adriamycin and cisplatin. More importantly, TA-p73beta displayed a synergistic effect in vivo allowing adriamycin or cisplatin to block melanoma cell growth in mouse xenograft models (p < 0.05). In summary, our data show that Ad-mediated TA-p73beta gene expression can markedly sensitize a subset of melanoma cell lines to adriamycin and cisplatin in vitro and in vivo, suggesting a new chemosensitization strategy for malignant melanomas.

Adenoviridae↗
PubMed2003

Recombinant mistletoe lectin induces p53-independent apoptosis in tumour cells and cooperates with ionising radiation.

Mistletoe extracts are used as alternative cancer treatment in addition to standard chemotherapy and radiation treatment and have an immunostimulatory and pain-relieving effect. A direct antitumour effect of mistletoe extracts against tumour cells of lymphoid origin has been linked to the D-galactoside-specific mistletoe lectin I. In this study, we investigated the cellular effect of bacterially expressed, recombinant mistletoe lectin alone or in combination with ionising radiation in a genetically defined p53-wild-type and p53-deficient E1A/ras-transformed murine tumour cells system. Downregulation of the proliferative activity and cell killing by recombinant mistletoe lectin occurred in a clear dose response (0.1-1 ng ml(-1)). Induction of apoptosis was p53-independent, but apoptosis-associated factor-1-dependent. Cellular treatment with lectin in combination with ionising radiation resulted in both p53-wild-type and p53-deficient tumour cells in an at least additive, antiproliferative effect and enhanced activation of caspase-3. Combined treatment with ionising radiation and lectin revealed a similar cytotoxic effect in human, p53-mutated adenocarcinoma cells. Thus, recombinant mistletoe lectin alone and in combination with ionising radiation bypasses often prevalent apoptotic deficiencies in treatment-resistant tumour cells.

Adenocarcinoma↗
PubMed2001

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma.

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

Antineoplastic Agents↗
PubMed2000

Protein kinase C inhibitor and irradiation-induced apoptosis: relevance of the cytochrome c-mediated caspase-9 death pathway.

Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.

Animals↗
PubMed1999

Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition.

The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.

Animals↗
PubMed1998

Differential requirement for caspase 9 in apoptotic pathways in vivo.

Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9-/- embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9-/- thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9-/- ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9-/- ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells.

Animals↗
PubMed1998

Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.

Caspases are fundamental components of the mammalian apoptotic machinery, but the precise contribution of individual caspases is controversial. CPP32 (caspase 3) is a prototypical caspase that becomes activated during apoptosis. In this study, we took a comprehensive approach to examining the role of CPP32 in apoptosis using mice, embryonic stem (ES) cells, and mouse embryonic fibroblasts (MEFs) deficient for CPP32. CPP32(ex3-/-) mice have reduced viability and, consistent with an earlier report, display defective neuronal apoptosis and neurological defects. Inactivation of CPP32 dramatically reduces apoptosis in diverse settings, including activation-induced cell death (AICD) of peripheral T cells, as well as chemotherapy-induced apoptosis of oncogenically transformed CPP32(-/-) MEFs. As well, the requirement for CPP32 can be remarkably stimulus-dependent: In ES cells, CPP32 is necessary for efficient apoptosis following UV- but not gamma-irradiation. Conversely, the same stimulus can show a tissue-specific dependence on CPP32: Hence, TNFalpha treatment induces normal levels of apoptosis in CPP32 deficient thymocytes, but defective apoptosis in oncogenically transformed MEFs. Finally, in some settings, CPP32 is required for certain apoptotic events but not others: Select CPP32(ex3-/-) cell types undergoing cell death are incapable of chromatin condensation and DNA degradation, but display other hallmarks of apoptosis. Together, these results indicate that CPP32 is an essential component in apoptotic events that is remarkably system- and stimulus-dependent. Consequently, drugs that inhibit CPP32 may preferentially disrupt specific forms of cell death.

Animals↗
PubMed1997

Structural features of phi29 single-stranded DNA-binding protein. I. Environment of tyrosines in terms of complex formation with DNA.

The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi29 is absolutely required for viral DNA replication in vivo. About approximately 95% of the intrinsic tyrosine fluorescence of phi29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi29 SSB fluorescence emission (phiF = 0.067 +/- 0.010), (ii) the fast motions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega (cooperativity) defining phi29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = K omega, being largely independent of temperature. Altogether, the fluorescent properties of phi29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.

Bacillus Phages↗
PubMed1997

Structural features of phi29 single-stranded DNA-binding protein. II. Global conformation of phi29 single-stranded DNA-binding protein and the effects of complex formation on the protein and the single-stranded DNA.

The strand-displacement mechanism of Bacillus subtilis phage phi29 DNA replication occurs through replicative intermediates with high amounts of single-stranded DNA (ssDNA). These ssDNA must be covered by the viral ssDNA-binding protein, phi29 SSB, to be replicated in vivo. To understand the characteristics of phi29 SSB-ssDNA complex that could explain the requirement of phi29 SSB, we have (i) determined the hydrodynamic behavior of phi29 SSB in solution and (ii) monitored the effect of complex formation on phi29 SSB and ssDNA secondary structure. Based on its translational frictional coefficient (3.5 +/- 0.1) x 10(8) gs(-1), and its rotational correlation time, 7.0 +/- 0.5 ns, phi29 SSB was modeled as a nearly spherical ellipsoid of revolution. The axial ratio (p = a/b) could range from 0.8 to 1.0 (oblate model, a < b) or 1.0 to 3.2 (prolate model, a > b). Far-UV CD spectra, indicated that phi29 SSB is highly organized within a wide range of temperatures (15 to 50 degrees C), being mainly constituted by beta-sheet elements (approximately 50%, at pH 7). Complex formation with ssDNA, although inducing minimal changes on the global conformation of phi29 SSB, had a clear stabilizing effect against pH and temperature increase of the solution samples. On the other hand, phi29 SSB binding leads to non-conservative changes of the near-UV CD spectra of ssDNA, which are consistent with different nearest-neighbor interactions of the nucleotide bases upon complex formation. The above results will be compared to those reported for other SSBs and discussed in terms of the functional roles of phi29 SSB.

Bacillus Phages↗
PubMed1995

Helix-destabilizing activity of phi 29 single-stranded DNA binding protein: effect on the elongation rate during strand displacement DNA replication.

The single-stranded DNA (ssDNA)-binding protein (SSB) of bacteriophage phi 29 is one of the virus-encoded proteins required for viral DNA replication. We have found that phi 29 SSB has helix-destabilizing activity since it removes secondary structure of the ssDNA in phi 29 replicative intermediates, as revealed by electron microscopy, and displaces oligonucleotides annealed to M13 ssDNA. To investigate the mechanism of the SSB-dependent stimulation of phi 29 DNA replication we have characterized the helix-destabilizing activity of phi 29 SSB and measured its effect on the DNA elongation rate by phi 29 DNA polymerase, which does not require an accessory helicase. The use of replication reactions where strand displacement is either required (phi 29 DNA replication) or not (conversion of primed M13 ssDNA into double-stranded DNA (dsDNA)) has allowed us to find that (1) strand displacement DNA replication was affected by lowering the temperature or by increasing the salt concentration, since the DNA elongation rate on the phi 29 template was three to fourfold slower than on primed M13 ssDNA, (2) under those conditions, addition of phi 29 SSB stimulated to different extents the DNA elongation rate during phi 29 DNA replication, whereas it had a marginal effect on primed M13 ssDNA replication, and (3) phi 29 SSB increased four to sixfold the phi 29 DNA elongation rate by phi 29 DNA polymerase strand displacement mutants, reaching approximately 50% the rate of the wild-type enzyme. The implications of the helix-destabilizing properties of the phi 29 SSB under conditions in which DNA opening is impaired are discussed.

Bacillus Phages↗
PubMed1995

Protein-nucleic acid interactions in bacteriophage phi 29 DNA replication.

phi 29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the phi 29 DNA ends. Structure-function studies have been carried out in the phi 29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of phi 29 DNA polymerase contains the 3'-5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of phi 29 DNA replication. The structure of the protein p6-DNA complex has been determined, as well as the main signals at the phi 29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an alpha-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The phi 29 protein p5 is the single-stranded DNA binding (SSB) protein involved in phi 29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the phi 29 SSB-ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19,285-bp phi 29 DNA molecule by a factor of 4000 after 1 h of incubation at 30 degrees C. The infectivity of the in vitro amplified DNA was identical to that of phi 29 DNA obtained from virions.

Bacillus Phages↗
PubMed1994

3'-->5' exonuclease active site of phi 29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism.

The residues forming the 3'-->5' exonuclease active site of phi 29 DNA polymerase, located at the N-terminal conserved motifs Exo I, Exo II and Exo III, have been defined by site-directed mutagenesis (Bernad, A., Blanco, L., Lázaro, J. M., Martin, G., and Salas, M. (1989) Cell 59, 219-228; Soengas, M. S., Esteban, J. A., Lázaro, J. M., Bernad, A., Blasco, M. A., Salas, M., and Blanco, L. (1992) EMBO J. 11, 4227-4237). To understand their catalytic role, the residual exonuclease activity of mutants at these active site residues has been kinetically studied. The critical function of residues Asp12, Glu14, Asp66, and Asp169 is supported by a 10(5)-fold reduction in the exonuclease catalytic rate upon single mutation. Residue Tyr165 seems to play a secondary role in the exonuclease reaction based on the 10(2)-10(3)-fold reduced catalytic rate of mutants Y165F and Y165C. Most of the mutants were specially active in the presence of Mn2+ ions, which could be indicative of a direct involvement of these residues in a metal ion-assisted exonucleolytic reaction. The data obtained strongly suggest that the 3'-->5' exonuclease active site of phi 29 DNA polymerase is structurally and functionally similar to that of the Escherichia coli DNA polymerase I. In addition, these residues were also very important for the strand displacement ability of phi 29 DNA polymerase, suggesting a structural overlapping of this activity with the 3'-->5' exonuclease.

Bacillus Phages↗
PubMed1994

Complex formation between phage phi 29 single-stranded DNA binding protein and DNA.

Bacteriophage phi 29 gene 5 encodes a single-stranded DNA (ssDNA) binding protein (SSB) which stimulates viral DNA replication. In the present study, a structural characterization of the complex between ssDNA and the phi 29 SSB was carried out using electron microscopy, band-shift assays and nuclease digestion as well as by monitoring changes in the intrinsic fluorescence of phi 29 SSB upon binding. Phage phi 29 SSB behaves as a monomer in solution and forms complexes with ssDNA which have a homogeneous structure, as if they consist of a continuous array of protein bound to DNA. Interaction of phi 29 SSB with ssDNA leads to a quenching of its tyrosine-dependent intrinsic fluorescence. This fluorescence quenching was directly proportional to the amount of phi 29 SSB bound to the ssDNA and the maximal quenching upon binding was very high (Qmax = 94.6 +/- 3.5%). Direct titration experiments have allowed us to estimate that the stoichiometry (n) of binding to ssDNA was 3.4(+/- 0.3) nucleotides per phi 29 SSB monomer. Both Qmax and n are independent of the salt concentration, suggesting the existence of only one major binding mode. At low salt concentrations, the effective binding constant (Keff = K omega) to poly(dT) was 2.2 x 10(5) M-1, the intrinsic binding constant (K) and the cooperativity parameter (omega) being 4.3 x 10(3) M-1 and 51, respectively. At increasing salt concentrations, the Keff exhibited a small, but significant, decrease. The possible functional significance of the binding parameters of phi 29 SSB during viral DNA replication is discussed.

Bacillus Phages↗
PubMed1992

Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities.

In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes. Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation. In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity. Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease. In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors. Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process. Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Amino Acid Sequence↗