Susana Velasco - Academia.edu (original) (raw)
Papers by Susana Velasco
Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has... more Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has been extensively reported as a critical cancer related property that improves tumor cell adaptation and survival. CIN and its immediate consequence, aneuploidy, provoke adverse effects on cellular homeostasis that need to be overcome by developing efficient anti-stress mechanisms. Perturbations in these safeguard responses might be detrimental for cancer cells and represent an important tumor specific Achilles heel since CIN and aneuploidy are very rare events in normal cells. On the other hand, epitranscriptomic marks catalyzed by different RNA modifying enzymes have been found to change under several stress insults. Although CIN and aneuploidy are important intracellular stressors, their biological connection with RNA modifications is pending to be determined. In an in silico search for new cancer biomarkers, we have identified TRMT61B, a mitochondrial RNA methyltransferase enzyme, to...
Journal of Biological Chemistry, 1988
In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-... more In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.
Nature, 1995
Transcriptional activation of target genes represents an important component of the tumour-suppre... more Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.
Proceedings of the National Academy of Sciences, 2005
Chromosome 3p and 1p deletions are among the most frequent genetic changes in human lung cancer a... more Chromosome 3p and 1p deletions are among the most frequent genetic changes in human lung cancer and although candidate tumor suppressor genes have been identified in these regions, no causative correlations have been drawn between deletion or mutation of these and lung carcinogenesis. We identifyXPCandGadd45aas genes within each of these regions involved in lung tumor initiation and progression, respectively. One hundred percent ofXPC-/- mice develop multiple spontaneous lung tumors with a minority progressing to non-small cell lung adenocarcinoma, occasionally with metastasis to adjacent lymph nodes. Deletion ofGadd45aalone does not lead to increased lung tumors in mice, but coupled with anXPCdeletion, it results in lung tumor progression. Analysis of published data indicated allelic loss ofXPCin most human lung tumors and allelic loss ofGadd45ain some human lung and other cancer types. Because DNA repair capacity is compromised inXPC+/- cells, it is possible that the loss of a sin...
PLoS Biology, 2006
Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is ... more Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb m/m /Xpa À/À mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb m/m / Xpa À/À mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb m/m /Xpa À/À and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.
Oncogene, 2002
A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122... more A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122?L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc 7/7) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122?L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122?L allele in p53 7/7 mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122?L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122?L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122?L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122?L mutant allele in the Xpc 7/7 background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.
European Journal of Immunology, 2002
Cancer Research, 2007
Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple ... more Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple stresses, including currently used chemotherapeutic drugs. Nutlin-3a is a recently discovered small-molecule antagonist of the p53-destabilizing protein murine double minute-2 (MDM2) that induces cell cycle arrest and apoptosis in cancer cells with functional p53. Here, we report that nutlin-3a induces cellular senescence in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. No evidence of drug-induced apoptosis was observed in any case. Nutlin-induced senescence was strictly dependent on the presence of functional p53 as revealed by the fact that cells lacking p53 were completely insensitive to the drug, whereas cells lacking the tumor suppressor alternative reading frame product of the CDKN2A locus underwent irreversible cell cycle arrest. Interestingly, irreversibility was achieved in neoplastic cells faster than in their corresponding pare...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1985
Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein k... more Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of Mg-ATP stimulated 2-3-fold lyso-platelet-activating factor:acetyltransferase activity. This activation was due to an increase in the Vm~_~ of the acetylation reaction, whereas the K m for acetyI-CoA was not affected. The ATP derivative, AMPPNP, could not replace ATP and preincubation of the microsomes with the heat-stable inhibitor of protein kinase prevented the activation by Mg-ATP obtained in the presence of the protein kinase. Activation of the acetylation reaction by the protein kinase was reversible. Evidence is provided that the reversal of activation is due to dephosphorylation of the enzyme. These data provide evidence that in vitro lyso-platelet-activating factor:acetyltransferase from splenic microsomes is regulated by phosphorylation.
Journal of Clinical Investigation, 1991
![Research paper thumbnail of Quantitative Analysis of Translesion DNA Synthesis across a Benzo[a]pyrene-Guanine Adduct in Mammalian Cells](https://attachments.academia-assets.com/92749090/thumbnails/1.jpg)
](Journal of Biological Chemistry, 2004
Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized... more Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol. Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol, suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.
DNA Repair, 2006
Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of D... more Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of DNA polymerase kappa (PolÄ −/−) deficient mice. The spontaneous mutation rate in homozygous PolÄ −/− males was significantly higher than in isogenic wild-type mice (PolÄ +/+), but the ESTR mutation spectrum in PolÄ −/− animals did not differ from that in PolÄ +/+ males. We suggest that compromised translesion synthesis in PolÄ −/− mice may result in replication fork pausing which, in turn, may affect ESTR mutation rate.
The Journal of Cell …, 2010
Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has... more Chromosomal instability (CIN) is an important source of genetic and phenotypic variation that has been extensively reported as a critical cancer related property that improves tumor cell adaptation and survival. CIN and its immediate consequence, aneuploidy, provoke adverse effects on cellular homeostasis that need to be overcome by developing efficient anti-stress mechanisms. Perturbations in these safeguard responses might be detrimental for cancer cells and represent an important tumor specific Achilles heel since CIN and aneuploidy are very rare events in normal cells. On the other hand, epitranscriptomic marks catalyzed by different RNA modifying enzymes have been found to change under several stress insults. Although CIN and aneuploidy are important intracellular stressors, their biological connection with RNA modifications is pending to be determined. In an in silico search for new cancer biomarkers, we have identified TRMT61B, a mitochondrial RNA methyltransferase enzyme, to...
Journal of Biological Chemistry, 1988
In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-... more In order to characterize the mechanism of activation of the enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase (EC 2.3.1.67) which is the limiting step in the regulation of the synthesis of the potent inflammatory mediator 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; homogenates from human polymorphonuclear leukocytes were incubated in the presence of the catalytic subunit of cyclic AMP-dependent protein kinase and in the presence of a partially purified phospholipid sensitive, calcium-dependent protein kinase (PrKC). The first kinase was found to enhance up to 3-fold acetyltransferase activity in a dose- and time-dependent manner. In homogenates from PMN previously stimulated with complement-coated zymosan particles, the decay of acetyltransferase activity was partially prevented by the addition of soybean trypsin inhibitor and almost completely inhibited when the homogenates were supplemented with inhibitors of alkaline phosphatase such as 50 mM KF and 100 microM paranitrophenylphosphate. Under these conditions it was possible to initiate the decay of acetyltransferase activity by adding an excess of alkaline phosphatase. Preincubation of PMN with 12-O-tetradecanoylphorbol-13-acetate previous or simultaneously to the addition of ionophore A23187 reduced the increase in acetyltransferase produced by ionophore A23187, whereas the generation of superoxide anions was enhanced. Addition of partially purified PrKC to homogenates from ionophore A23187-stimulated PMN, reduced acetyltransferase activity by 63%, whereas only a 16% inhibition was observed on homogenates from resting PMN. These data indicate the modulation of acetyltransferase activity in human polymorphonuclear leukocytes by a phosphorylation-dephosphorylation mechanism linked to cyclic AMP-dependent protein kinase. Phospholipid sensitive, calcium-dependent protein kinase seems not to be involved in the mechanism of activation, but, most probably, in the generation of negative activation signals.
Nature, 1995
Transcriptional activation of target genes represents an important component of the tumour-suppre... more Transcriptional activation of target genes represents an important component of the tumour-suppressor function of p53 and provides a functional link between p53 and various growth-regulatory processes, including cell cycle progression (p21/WAF1), DNA repair (GADD45) and apoptosis (bax). Here we use a differential cloning approach to identify the gene encoding insulin-like growth factor binding protein 3 (IGF-BP3) as a novel p53-regulated target gene. Induction of IGF-BP3 gene expression by wild-type but not mutant p53 is associated with enhanced secretion of an active form of IGF-BP3 capable of inhibiting mitogenic signalling by the insulin-like growth factor IGF-1. Our results indicate that IGF-BP3 may link p53 to potential novel autocrine/paracrine signalling pathways and to processes regulated by or dependent on IGF(s), such as cellular growth, transformation and survival.
Proceedings of the National Academy of Sciences, 2005
Chromosome 3p and 1p deletions are among the most frequent genetic changes in human lung cancer a... more Chromosome 3p and 1p deletions are among the most frequent genetic changes in human lung cancer and although candidate tumor suppressor genes have been identified in these regions, no causative correlations have been drawn between deletion or mutation of these and lung carcinogenesis. We identifyXPCandGadd45aas genes within each of these regions involved in lung tumor initiation and progression, respectively. One hundred percent ofXPC-/- mice develop multiple spontaneous lung tumors with a minority progressing to non-small cell lung adenocarcinoma, occasionally with metastasis to adjacent lymph nodes. Deletion ofGadd45aalone does not lead to increased lung tumors in mice, but coupled with anXPCdeletion, it results in lung tumor progression. Analysis of published data indicated allelic loss ofXPCin most human lung tumors and allelic loss ofGadd45ain some human lung and other cancer types. Because DNA repair capacity is compromised inXPC+/- cells, it is possible that the loss of a sin...
PLoS Biology, 2006
Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is ... more Cockayne syndrome (CS) is a photosensitive, DNA repair disorder associated with progeria that is caused by a defect in the transcription-coupled repair subpathway of nucleotide excision repair (NER). Here, complete inactivation of NER in Csb m/m /Xpa À/À mutants causes a phenotype that reliably mimics the human progeroid CS syndrome. Newborn Csb m/m / Xpa À/À mice display attenuated growth, progressive neurological dysfunction, retinal degeneration, cachexia, kyphosis, and die before weaning. Mouse liver transcriptome analysis and several physiological endpoints revealed systemic suppression of the growth hormone/insulin-like growth factor 1 (GH/IGF1) somatotroph axis and oxidative metabolism, increased antioxidant responses, and hypoglycemia together with hepatic glycogen and fat accumulation. Broad genome-wide parallels between Csb m/m /Xpa À/À and naturally aged mouse liver transcriptomes suggested that these changes are intrinsic to natural ageing and the DNA repair-deficient mice. Importantly, wild-type mice exposed to a low dose of chronic genotoxic stress recapitulated this response, thereby pointing to a novel link between genome instability and the age-related decline of the somatotroph axis.
Oncogene, 2002
A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122... more A mutation in codon 122 of the mouse p53 gene resulting in a T to L amino acid substitution (T122?L) is frequently associated with skin cancer in UV-irradiated mice that are both homozygous mutant for the nucleotide excision repair (NER) gene Xpc (Xpc 7/7) and hemizygous mutant for the p53 gene. We investigated the functional consequences of the mouse T122?L mutation when expressed either in mammalian cells or in the yeast Saccharomyces cerevisiae. Similar to a non-functional allele, high expression of the T122?L allele in p53 7/7 mouse embryo fibroblasts and human Saos-2 cells failed to suppress growth. However, the T122?L mutant p53 showed wild-type transactivation levels with Bax and MDM2 promoters when expressed in either cell type and retained transactivation of the p21 and the c-Fos promoters in one cell line. Using a recently developed rheostatable p53 induction system in yeast we assessed the T122?L transactivation capacity at low levels of protein expression using 12 different p53 response elements (REs). Compared to wild-type p53 the T122?L protein manifested an unusual transactivation pattern comprising reduced and enhanced activity with specific REs. The high incidence of the T122?L mutant allele in the Xpc 7/7 background suggests that both genetic and epigenetic conditions may facilitate the emergence of particular functional p53 mutations. Furthermore, the approach that we have taken also provides for the dissection of functions that may be retained in many p53 tumor alleles.
European Journal of Immunology, 2002
Cancer Research, 2007
Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple ... more Cellular senescence is emerging as an important in vivo anticancer response elicited by multiple stresses, including currently used chemotherapeutic drugs. Nutlin-3a is a recently discovered small-molecule antagonist of the p53-destabilizing protein murine double minute-2 (MDM2) that induces cell cycle arrest and apoptosis in cancer cells with functional p53. Here, we report that nutlin-3a induces cellular senescence in murine primary fibroblasts, oncogenically transformed fibroblasts, and fibrosarcoma cell lines. No evidence of drug-induced apoptosis was observed in any case. Nutlin-induced senescence was strictly dependent on the presence of functional p53 as revealed by the fact that cells lacking p53 were completely insensitive to the drug, whereas cells lacking the tumor suppressor alternative reading frame product of the CDKN2A locus underwent irreversible cell cycle arrest. Interestingly, irreversibility was achieved in neoplastic cells faster than in their corresponding pare...
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1985
Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein k... more Incubation of rat splenic microsomes with the catalytic subunit of cyclic AMP-dependent protein kinase in the presence of Mg-ATP stimulated 2-3-fold lyso-platelet-activating factor:acetyltransferase activity. This activation was due to an increase in the Vm~_~ of the acetylation reaction, whereas the K m for acetyI-CoA was not affected. The ATP derivative, AMPPNP, could not replace ATP and preincubation of the microsomes with the heat-stable inhibitor of protein kinase prevented the activation by Mg-ATP obtained in the presence of the protein kinase. Activation of the acetylation reaction by the protein kinase was reversible. Evidence is provided that the reversal of activation is due to dephosphorylation of the enzyme. These data provide evidence that in vitro lyso-platelet-activating factor:acetyltransferase from splenic microsomes is regulated by phosphorylation.
Journal of Clinical Investigation, 1991
Journal of Biological Chemistry, 2004
Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized... more Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK ؉/؉ mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 ؎ 4%, which is an order of magnitude higher than in Escherichia coli. In PolK ؊/؊ MEFs, bypass was 16 ؎ 1%, suggesting that at least twothirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase (pol). In contrast, pol was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking pol. Expression of pol from a plasmid introduced into PolK ؊/؊ MEFs restored both the extent and fidelity of bypass across BP-G. Pol was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both pol and pol, suggesting that the biological role of pol in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that pol bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.
DNA Repair, 2006
Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of D... more Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of DNA polymerase kappa (PolÄ −/−) deficient mice. The spontaneous mutation rate in homozygous PolÄ −/− males was significantly higher than in isogenic wild-type mice (PolÄ +/+), but the ESTR mutation spectrum in PolÄ −/− animals did not differ from that in PolÄ +/+ males. We suggest that compromised translesion synthesis in PolÄ −/− mice may result in replication fork pausing which, in turn, may affect ESTR mutation rate.
The Journal of Cell …, 2010