PubMed Health⌕ Search

Biomedical subjects

J F López-Sáez

Publications and source records attributed to J F López-Sáez.

At least 19 recordsLinked to original sources
PubMed2004

HSP90 and checkpoint-dependent lengthening of the G2 phase observed in plant cells under hypoxia and cold.

Proliferating cells of Allium cepa L. roots became adapted to hypoxia (5% oxygen) and cold (10 degrees C) by acquiring new steady-state kinetics of growth. The cell cycle time increased from the 17.6 h in control meristems up to 29.7 and 69.0 h under hypoxia and cold conditions, respectively. Acclimation of the proliferating cells was stress specific. No acclimation took place after 24 h of heat treatment (40 degrees C). Under cold treatment, all cycle phases enlarged uniformly. However, under hypoxia, while the G(1) and S cycle phases roughly doubled in their timing, the expected checkpoint-dependent lengthening of G(2) did not take place. This failure in lengthening G(2) in response to hypoxia correlated with a failure in the overinduction of a single peptide with a molecular mass of about 134 kDa which is among those recognised by an HSP90 antibody. Moreover, the presence of this large peptide of the HSP90 family proved to be a marker for cell proliferation. It was always absent from the contiguous differentiated cells of the root. Lastly, the mitochondrial chaperonin recognized by an HSP60 antibody in these roots not involved in photosynthesis was always higher in the proliferating than in the nonproliferating cells.

Cell Hypoxia↗
PubMed2003

G2 checkpoint targets late replicating DNA.

In the multinucleate cells induced in Allium cepa L. meristems, the nuclei surrounded by the largest cytoplasm environment complete replication earlier (advanced nuclei), but have a longer G2, than the others (delayed nuclei). Thus, all nuclei break down the nuclear envelope and start metaphase simultaneously. The present report shows that this synchronization relies on a checkpoint mechanism. When completion of replication was prevented in the delayed nuclei (due to in vivo 5-aminouracil feeding initiated when the advanced nuclei were already in G2), the metaphase was also further delayed in the advanced ones. In turn, some of the delayed nuclei overrode the G2 checkpoint (adaptation) and entered into mitosis with broken chromatids (Del Campo et al., 1997). Anoxic UVA (313 nm) irradiation apparently prevents the binding of regulatory proteins to Br-DNA. The present report shows that late replicating sequences are the targets of the checkpoint signal produced by the still replicating nuclei. This signal delays metaphase in the advanced nuclei, whose DNA is already fully replicated. Thus, when the already replicated sequences of late replicating DNA was modified in the advanced nuclei by bromosubstitution followed by anoxic UVA irradiation, they entered into mitosis without any delay, ignoring the inhibitory signals produced by the still replicating nuclei.

Animals↗
PubMed2003

Effect of caffeine on in vivo processing of alkylated bases in proliferating plant cells.

DNA damage was induced by either 2 mM ethylmethanesulfonate or 1 Gy of gamma-irradiation in Allium cepa L. root meristems. The percentage of DNA that migrated towards the anode during microelectrophoresis after alkali denaturation (pH approximately 13.5) of the isolated nuclei (comet assay) reflects the amount of single strand breaks present in them. There was some DNA migration (12.8+/-2.4%) in untreated roots. This percentage doubled at the end of 1.5 h treatment with the mono-functional alkylating agent 2 mM ethylmethanesulfonate, and trebled after a single exposure to 1 Gy of gamma-rays. A proportion of the DNA migration caused by these two treatments was reversed (repaired) by a 2 h long period of in vivo recovery. However, when 5 mM caffeine was applied after removal of the alkylating agent, the amount of DNA migrating to the comet tail over the same 2 h period was almost double that at the onset of recovery. In both control and irradiated nuclei, caffeine also increased the initial level of DNA migration in the comet assay, but to a lesser extent. These results indicate that caffeine increases the DNA damage that accumulates during the processing of alkylated bases and, to a lesser extent, of the DNA bases damaged by gamma-irradiation. Thus, the potentiation effect of caffeine on induced chromosomal damage may not just be due to caffeine-induced cancellation of the G2 checkpoint, but also to a direct effect this methylxantine has on the processing of DNA damage.

Alkylation↗
PubMed2003

Human syndromes with genomic instability and multiprotein machines that repair DNA double-strand breaks.

The present report deals with the functional relationships among protein complexes which, when mutated, are responsible for four human syndromes displaying cancer proneness, and whose cells are deficient in DNA double-strand break (DSB) repair. In some of them, the cells are also unable to activate the proper checkpoint, while in the others an unduly override of the checkpoint-induced arrest occurs. As a consequence, all these patients display genome instability. In ataxia-telangiectasia, the mutated protein (ATM) is a kinase, which acts as a transducer of DNA damage signalling. The defective protein in the ataxia-telangiectasia-like disorder is a DNase (the Mre11 nuclease) that in vivo produces single-strand tails at both sides of DSBs. Mre11 is always present with the Rad50 ATPase in a protein machine: the nuclease complex. In mammals, this complex also contains nibrin, the protein mutated in the Nijmegen syndrome. Nibrin confers new abilities to the nuclease complex, and can also bind to BRCA1 (one of the two proteins mutated in familial breast cancer). BRCA1 has a central motif that binds with high affinity to cruciform DNA, a structure present in places where the DNA loops are anchored to the chromosomal axis or scaffold. The BRCA1 x cruciform DNA complex should be released to allow the nuclease complex to work in DNA recombinational repair of DSBs. BRCA1 also acts as a scaffold for the assembly of ATPases such as Rad51, responsible for the somatic homologous recombination. Loss of the BRCA1 gene prevents cell survival after exposure to cross-linkers. The BRCA1-RING domain is an E3-ubiquitin ligase. It can mono-ubiquitinate the FANCD2 protein, mutated in one of the Fanconi anemia complementation groups, to regulate it. Finally, during DNA replication, the nuclease complex and its activating ATM kinase are integrated in the BRCA1-associated surveillance complex (BASC) that contains, among others, enzymes required for mismatch excision repair. In short, the proteins missing in these syndromes have in common their BRCA1-mediated assembly into multimeric machines responsible for the surveillance of DNA replication, DSB recombinational repair, and the removal of DNA cross-links.

Apoptosis↗
PubMed2001

Fanconi anemia lymphocytes: effect of DL-alpha-tocopherol (Vitamin E) on chromatid breaks and on G2 repair efficiency.

The high frequency of chromosomal breaks in Fanconi anemia (FA) lymphocytes has been related to the increased oxidative damage shown by these cells. The effect of 100 microM DL-alpha-tocopherol (Vitamin E) on the level of chromosomal damage in mitosis was studied in lymphocytes from five FA patients and from age matched controls, both under basal conditions and when G2 repair was prevented by 2.5 mM caffeine (G2 unrepaired damage). In addition, the effect of this antioxidant on G2 duration and the efficiency of G2 repair was also evaluated in the sample. alpha-Tocopherol (AT) decreased the frequency of chromosomal damage (under basal and inhibited G2 repair conditions) and the duration of G2 in FA cells. This antioxidant protective effect, expressed as the decrease in chromatid breaks, was greater in FA cells (50.8%) than in controls (25%). The efficiency of the G2 repair process (G2 R rate) defined as the ratio between the percentage of chromatid breaks repaired in G2 and the duration of this cell cycle phase was lesser in FA cells (10.6) than in controls (22.6). AT treatment slightly increased this G2 R rate, both in FA cells and controls. These results suggest that an increased oxidative damage and a lower G2 repair rate may be simultaneously involved in the high frequency of chromatid damage detected in FA cells.

Adolescent↗
PubMed2001

Ataxia telangiectasia: G2 checkpoint and chromosomal damage in proliferating lymphocytes.

There is a checkpoint pathway in eukaryotic cells that depends on ATM (ataxia telangiectasia mutated) kinase which activates the processes leading to the repair of DNA damage and also lengthens the G(2) stage of the cell cycle. In cells from ataxia telangiectasia patients, due to their lack of active ATM kinase, an increase in chromosomal aberrations and a failure to induce G(2) lengthening could be expected. However, the basal G(2) timing in ataxia telangiectasia cells was longer than in controls and was further extended after X-ray irradiation (0.4 Gy), although to a lesser extent than in controls. Moreover, in control cells caffeine shortened G(2) and increased chromosomal damage 7-fold, while in ataxia telangiectasia cells caffeine only trebled aberration yield without shortening G(2). As caffeine is an inhibitor of ATM kinase, these results suggest the existence of some redundant ATM-independent checkpoint in G(2) of ataxia telangiectasia cells. The differential response to caffeine of ataxia telangiectasia and control lymphocytes may be explained by the presence of two different subpathways in the G(2) checkpoint: one regulating the processing and repair of damaged DNA and the other controlling G(2) timing. While in controls both subpathways may be mediated by ATM kinase, in ataxia telangiectasia cells caffeine-sensitive ATR kinase and the caffeine-insensitive DNA-PK kinases might be responsible for DNA repair and the G(2) delay subpathways, respectively. Confirmation of this model in ataxia telangiectasia cells with another cell type in which both subpathways are mediated by DNA-PK should define whether a metylxanthine such as caffeine may also have an additional direct inhibitory effect on DNA repair.

Ataxia Telangiectasia↗
PubMed1999

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation.

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.

Adult↗
PubMed1998

Cell proliferation and cancer.

The discovery that phosphorylation of selected proteins by cyclin-dependent kinases is the engine which makes the cycle run provides a new image of the control of proliferation and of its deregulation. The high conservation of this machinery in the different eukaryotic organisms emphasizes its early origin and its importance for life. It also makes the extrapolation of findings between different species feasible. The control of proliferation relies basically on accelerating and braking mechanisms which act on the engine driving the cycle. This review particularly stresses the importance of checkpoint or tumor suppressor pathways as transduction systems of negative signals which may induce a cycle braking operation. They prevent any important cycle transition, as the initiation of proliferation, that of replication, mitosis, etc., until the DNA and other cellular conditions make such a progression safe. These checkpoint pathways are able to recognize and transduce signals about the adequacy of initiating or continuing proliferation for a cell at a particular time, under a particular set of external and internal conditions. Crucial components of these pathways are proteins encoded by some of the checkpoint genes that evaluate the final balance of mitogenic and antimitogenic pathways reaching them and, if the balance is negative, they prevent temporarily cycle inititation or its progression by inhibiting the corresponding cyclin-dependent kinases. On the other hand, when the balance becomes positive, they allow the activation of the cyclin-dependent kinases. Uncontrolled cell proliferation associated with cancer always depends on the functional abrogation of at least one of the checkpoint pathways. The checkpoint or tumor suppressor protein p53 is one of the proteins in them, and mutations in the gene encoding it are present in more than half of all human tumours. The review touches new pharmacological strategies which have been opened by the discovery of portions of some of the signal transduction cascades involved in the transient brake of cell proliferation. Restoration of checkpoint pathways either prevents further proliferation of cells with damaged genome until repair is over or, alternatively, the dismantling of these checkpoints induce those cells to commit suicide (apoptosis). The fact that both restoration and dismantling of checkpoint pathways sensitive to DNA damage have not disturbing effects on any other proliferating cell with undamaged DNA makes these selective strategies promissing.

Animals↗
PubMed1997

Competence for nuclear replication and the NOR-chromosomes of Allium cepa L.

Autotetraploid (4n = 32) cells were induced in Allium cepa L. root meristems by successively treating with a multipolarizing agent in anaphase (carbetamide) and an inhibitor of cell plate formation in telophase (caffeine). This treatment produced cells with their 32 chromosomes distributed in more than two nuclei. During G1, one of the nuclei in the resulting trinucleate cells had a DNA content which was equivalent to an average of 16 chromosomes, while the other 16 chromosomes were randomly distributed in two aneuploid nuclei. In the set of 16 chromosomes forming the onion diploid complement, there are 4 NOR-chromosomes and 5 chromosomes carrying DNA domains providing a nucleus with the competence to replicate, as previously shown. Expected probabilities derived from the different possible models for cosegregation of both kinds of chromosomes in the two aneuploid nuclei of the trinucleate cells were estimated by a computer simulation. These expected values were compared with the recorded frequencies of aneuploid nuclei which were able to organize a nucleolus and to respond to inducers of replication. The present data are compatible with the existence of sequences providing a nucleus with the competence to replicate in three out of the four NOR-bearing chromosomes, as well as in two other chromosomes of this diploid complement. The scarcity of chromosomes bearing early origins able to initiate replication in a nucleus is a striking feature of this huge onion genome.

Allium↗
PubMed1997

Frailty of two cell cycle checkpoints which prevent entry into mitosis and progression through early mitotic stages in higher plant cells.

Allium cepa L. root meristems were given two short caffeine treatments spaced by 15 hours, the time which roughly corresponds to the duration of one cell cycle. In this way two subsequent cytokineses were prevented, and multinucleate cells with their in complement distributed into two, three or four nuclei were formed. Though all nuclei started to replicate synchronously in these cells, some of them (fast nuclei) completed their replication earlier than others (slow nuclei). The present report shows that two successive checkpoints operate before prometaphase in these cells. The first one prevents the entry of the fast nuclei into prophase until the slow ones have completed their replication. The second checkpoint ensures the synchronous entry into prometaphase after all nuclei have reached and finished prophase. By treating the multinucleate cells with an inhibitor of DNA synthesis at that time when fast but not slow nuclei had finished their replication, it was observed that both checkpoint mechanisms became leaky with time. Under these conditions the fast nuclei entered prophase in the presence of nuclei which were prevented from finishing the replication of their DNA. Subsequently, even prometaphase was triggered after a prolonged prophase. Finally, as expected from the presence of mitotic stages in these cells, nuclei with incompletely replicated DNA endured premature chromosome condensation. The prematurely condensed chromosomes either remained in a prometaphase-like stage until reconstitution nuclei formed or they followed the progression of the fast nuclei into metaphase and anaphase leading to the appearance of acentric chromosomal segments which after reconstitution gave rise to aneuploid nuclei containing unstable and broken DNA.

Allium↗
PubMed1996

Nucleolar organizer expression in Allium cepa L. chromosomes.

Roots from Allium cepa L. (cv. Francesa) bulbs in which a maximum of two nucleoli per nucleus developed were selected for this study. Five rDNA clusters were detected by fluorescent in situ hybridization on chromosomal squashes (2n = 16) with a rhodamine-labelled wheat rDNA repeat. The rDNA clusters were located on four chromosomes: the largest cluster occurred on the small arm of a single homologue of the smallest pair 8. Its homologue showed two different small rDNA clusters, one near each telomere. The two homologues of the satellited chromosomes 6 also showed different rDNA contents, which were intermediate to those found in pair 8. The same five well-differentiated hybridization signals were observed in interphase cells that were inactive in transcription because they were in dormant roots, or in proliferating ones in which the synthesis of the large rRNA precursor was prevented. After multipolarizing agent was applied in anaphase followed by inhibition of cytokinesis, multinucleate autotetraploid cells were formed, which often contained more than four nucleoli. Thus, at least two of the three nucleolar organizer regions that consistently failed to develop a nucleolus in normal mononucleate cells were capable of developing nucleoli when segregated into different nuclei in multinucleate cells.

Chromosome Mapping↗
PubMed1992

A limited number of chromosomes makes a nucleus competent to respond to inducers of replication and mitosis in a plant.

Multinucleate tetraploid cells with unbalanced chromosomal distribution in aneuploid nuclei were obtained in Allium cepa L. root meristems. For this, their natural diploid cells were treated with a multipolarizing agent (1 h carbetamide) followed by an inhibitor of cytokinesis (1 h caffeine). Data from these multinucleate cells with aneuploid nuclei suggest that only four out of the thirty-two chromosomes of their autotetraploid complement possess DNA sequences making the nucleus competent to respond to inducers of replication and mitosis. Direct observation of cells where a single replicated chromosome had reached mitosis showed that this chromosome was the one bearing the nucleolar organizer. Six specific chromosomes would confer competence to the nucleus to respond to inducers of replication but not to those producing chromosome condensation. Another four different chromosomes would confer the nucleus with the ability to respond to mitotic inducers but not to replication inducers. The rest of the chromosomal complement seemed to lack any of the DNA sequences needed for these two important cycle transitions. In a nutshell, certain DNA sequences distributed in a few chromosomes of the onion complement are an intranuclear requirement to initiate replication and mitosis in these plant cells.

Allium↗
PubMed1991

Effects of caffeine and cycloheximide during G2 prophase in control and X-ray-irradiated human lymphocytes.

The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.

Antimutagenic Agents↗
PubMed1988

Fanconi's anemia lymphocytes: effect of caffeine, adenosine and niacinamide during G2 prophase.

In this investigation peripheral blood lymphocytes from 3 Fanconi's anemia (FA) patients, 2 FA heterozygotes and 4 normal subjects were treated with caffeine and/or adenosine, and/or niacinamide during G2 prophase. Caffeine dramatically increased breakage levels in homozygote and heterozygote cells. Niacinamide and adenosine decreased the amount of chromosomal aberrations detected in FA homozygote and heterozygote lymphocytes treated and untreated with caffeine during G2 prophase. Caffeine sensitivity of heterozygote lymphocytes is proposed as a new clinical test to explore heterozygosis in individuals of FA families.

Adenosine↗
PubMed1986

Relationship of chromosomal damage induced by caffeine to growth temperature and ATP level in proliferating cells.

Caffeine is known to induce chromosomal aberrations in proliferating cells when they are incubated during G2 and mitotic prophase. In the present paper, this caffeine effect has been analyzed in Allium cepa root meristems growing at different culture temperatures under steady-state kinetics. Caffeine (1-10 mM) induces chromosomal aberrations in a dose-dependent manner, and the treatment efficiency correlates linearly with the square of caffeine concentration. The efficiency of caffeine incubations, within the range 5-25 degrees C during equivalent cycle time periods has also been studied. It has been found that the lower the culture temperature, the higher the level of chromosomal aberrations. Moreover, at different temperatures, the level of chromosomal aberrations is a simple function of caffeine concentration and the ATP level. Therefore, the efficiency of caffeine treatment appears to be determined by some interaction between caffeine concentration and cellular ATP level. Our present results demonstrate that the influence of growth temperature on the chromosome-breaking effect of caffeine can be, at least partially, explained by the ATP levels during the incubation periods. In short, under different kinetics of plant cell proliferation, the ATP level, and/or something correlating with it, could explain the efficiency of caffeine in inducing chromosomal aberrations: the lower the ATP level, the higher the caffeine efficiency.

Adenosine Triphosphate↗
PubMed1985

Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage.

Proliferating plant cells treated during the late S period with 5-aminouracil (AU), give the typical response that DNA-damaging agents induce, characterized by: an important mitotic delay, and a potentiation of the chromosome damage by caffeine post-treatment. The study of labelled prophases, after a tritiated thymidine pulse, allowed evaluation of the mitotic delay induced by AU as well as its reversion by caffeine, while chromosome damage was estimated by the percentage of anaphases and telophases showing chromosomal aberrations. Post-treatment with adenosine alone has shown no effect on mitotic delay or chromosomal damage. However, when cells after AU were incubated in caffeine plus adenosine, the chromosome damage potentiation was abolished without affecting the caffeine action on mitotic delay. As a consequence, we postulate that caffeine could have two effects on G2 cells with damaged DNA: the first, to cancel their mitotic delay and the second to inhibit some DNA-repair pathway(s). Only this last effect could be reversed by adenosine.

Adenosine↗