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Biomedical subjects

M I Núñez

Publications and source records attributed to M I Núñez.

At least 19 recordsLinked to original sources
PubMed2005

PARP-1-dependent 3-nitrotyrosine protein modification after DNA damage.

3-nitrotyrosine (NO2-Tyr) is thought to be a specific marker of cell injury during oxidative damage. We have evaluated the role of poly(ADP-ribose)polymerase-1 (PARP-1) in protein nitration after treatment of immortalized fibroblasts parp-1+/+ and parp-1-/- with the alkylating agent 2'-methyl-2'-nitroso-urea (MNU). Both cell lines showed increased iNOS expression following MNU treatment in parallel with a selective induction of tyrosine nitration of different proteins. PARP-1 deficient cells displayed a delayed iNOS accumulation, reduced number of nitrated proteins, and a lower global nitrotyrosine "footprint." We have identified the mitochondrial compartment as the major site of oxidative stress during DNA damage, being MnSOD one of the NO2-Tyr-modified proteins, but not in parp-1-/- cells. These results suggest that NO-derived injury can be modulated by proteins involved in the response to genotoxic damage, such as PARP-1, and may account for the limited oxidative injury in parp-1 knockout mice during carcinogenesis and inflammation.

Animals↗
PubMed2002

[Cytosolic pS2 levels in 154 non-small-cell lung carcinomas. Correlation with other clinical and biological parameters].

INTRODUCTION: pS2 was first identified as an estrogen induced molecule in a breast cancer cell line, but its gene responds to several growth factors and oncogenic proteins. Its main clinical application lies in breast carcinomas, where it is a molecular marker to identify breast cancer patients who will respond to hormone therapy. This study was performed to evaluate the significance of the pS2 cytosolic levels in non small cell lung carcinomas classified according to different clinical and biological parameters. Likewise, the results obtained were correlated with those observed in normal tissues. MATERIAL AND METHODS: The study group included 154 non small cell lung carcinomas (59 adenocarcinomas and 95 squamous carcinomas) and 54 normal lung samples. Cathepsin D, pS2, neuron specific enolase (NSE) and CA125 cytosolic concentrations were determined, as well as those of epidermal growth factor receptor (EGFR), erbB2 protein, CD44s, CD44v5 and CD44v6 on cell surface membranes. Likewise, clinical stage, histological grade, ploidy and cellular S-phase fraction were also considered as variables of the study. RESULTS: We observed cytosolic pS2 values greater than 1 ng/mg protein (positive cutoff) in 18/59 adenocarcinomas and in 10/95 squamous carcinomas (p:0.00177). In adenocarcinomas, pS2 positivity was not correlated with any clinical and biological parameters, but positive cases had lower EGFR (p:0.03770) concentrations and higher CA125 (p:0.01902) levels. In squamous carcinomas, pS2 positivity was associated with lower EGFR (p:0.02270) levels and higher erbB2 protein (p:0,00563) concentrations. CONCLUSIONS: Our results led us to suggest the following: 1) cytosolic pS2 concentrations are higher in tumoral than in normal samples; 2) adenoarcinomas had higher levels than squamous lung carcinomas; 3) in adenocarcinomas, positivity for pS2 is associated with lower EGFR levels and higher CA125 concentrations suggesting a worse outcome, and 4) in squamous lung tumors, positivity for pS2 was associated with lower EGFR and higher erbB2 oncoprotein concentrations, it not being possible to establish its clinical significance, although it may be correlated with a poor prognosis.

Adenocarcinoma↗
PubMed2002

Assessing the use of p16(INK4a) promoter gene methylation in serum for detection of bladder cancer.

OBJECTIVE: This study was undertaken to investigate whether hypermethylation in p16(INK4a) gene promoter could serve as plasma biomarker of bladder cancer. METHODS AND PATIENTS: We examined the p16(INK4a) status using methylation-specific PCR in 86 cancer patients and 49 controls (31 healthy people and 18 patients with benign urological diseases). RESULTS: The p16(INK4a) methylation was found in 22% of the serum samples and in 26% of the bladder cancer biopsies; one of them with carcinoma in situ. The presence of hypermethylated p16(INK4a) in serum seems to be a product from tumour cells because a strong statistical association was found between both matched DNA signals (p<0.0001). Using the control group, the presence of methylated p16(INK4a) in the serum of individuals with suspicion of bladder cancer was found to be associated with the tumour presence (p=0.0009). Aberrant p16(INK4a) methylation was also observed in one non-cancer patient, which is undergoing further assessment. CONCLUSIONS: According with our results, methylation of p16(INK4a) promoter may be involved in the bladder cancer genesis and the presence of p16(INK4a) methylated in serum of these patients could be useful in the cancer diagnosis with values of sensitivity, specificity and positive predictive value of 0.226, 0.950 and 0.98, respectively. These figures support the use of methylated p16(INK4a) as a new class of tumour marker in bladder cancer.

Aged↗
PubMed2002

Interactions between radiotherapy and endocrine therapy in breast cancer.

Whenever radiation therapy is given with curative intent there is the risk of serious damage to normal tissue. This risk increases with the dose of radiation, as does the probability of local tumour control. In the attempt to cure, the doses reach a level that inevitably causes some undesirable adverse effects, ranging from undetectable, or minimal, to unacceptably severe. Over the last few years, a number of reports have suggested that the prediction of normal tissue response after radiotherapy may be achieved by assays on samples withdrawn from the patients prior to treatment, although recent reports have described mixed results. The ability to predict tumour response to anti-hormones in patients with breast cancer has important implications with regard to treatment. Recent discoveries promise to provide individualized treatment options. However, there are no data to support that, used jointly, the combination of radiotherapy and hormone therapy may achieve an enhancement of breast cancer tumour response. Nowadays, development in cancer therapy is increasingly arising out of studies in basic science; its implementation in the hands of clinicians is improving the management of patients with cancer. In addition, as the biological aspects of irradiation and hormonal therapy offer an explanation, at least in part, for the outcome observed in patients with breast cancer after therapy, we have focused this review on trying to analyse the most relevant experimental research about the relative roles of radiotherapy and hormonal therapy, the corresponding side-effects and, taking into account recent advances, future areas of research that we consider of major importance in the field.

Breast Neoplasms↗
PubMed2000

Capillary electrophoresis of DNA damage after irradiation: apoptosis and necrosis.

Apoptosis is a type of cellular death but also directly regulates tumorigenesis through different gene expression. This phenomenon is often used as end-point in studies of radio- and chemosensitivity of cancer cells. Restriction DNA fragments have been separated quickly, efficiently and successfully by capillary gel electrophoresis (CGE). In this study CGE has been applied to distinguish between the discrete pattern of degraded DNA produced by apoptosis and randomized DNA breaks produced by ionizing radiation. The influence of different variables has been discussed and an example of fast separation by CGE of the apoptotic fragments produced by UV light treatment is shown.

Apoptosis↗
PubMed2000

[Expression of the adhesion molecule CD44v6 in infiltrating ductal carcinomas of the breast is associated with hormone dependence. Our experience with 168 cases].

In order to investigate the possible hormone-dependence of CD44v6 in human breast cancer, we assayed the concentrations of this isoform in the membrane fraction of 168 invasive ductal carcinomas (IDC) and in 26 normal breast tissue samples, 18 fibradenomas (FAD), 3 fibrocystic disease specimens (FD), 7 mucinous carcinomas and 4 medullary carcinomas using the ELISA method. The results were compared with those of the estrogen (ER) and progesterone (PR) receptors, pS2, tissue type plasminogen activator (t-PA), cathepsin D, epidermal growth factor receptor (EGFR) and c-erbB2/neu oncoprotein concentrations. Menopausal status, size of the tumor in the cases of cancers, axillary lymph node involvement, histologic grade, ploidy, cellular synthesis phase, multifocality and multicentricity were also considered as variables. The cut-off value for CD44v6-positivity was set at 5 ng/mg prt. membrane protein content. 64/138 (38.1%) infiltrating ductal carcinomas scored positive. This was significantly higher than for the normal breast tissue (0/26; p: 0.0001), similar to that seen in the FAD (3/18), fibrocystic disease (0/3), infiltrating mucinous carcinomas (4/7) and lobular (3/15) and significantly lower than for the infiltrating medullary carcinomas (4/4; p: 0.027). There were no significant differences with the other groups of tissues studied. Furthermore, CD44v6-positive IDC showed significantly higher concentrations of ER, PR and cathepsin D and lower (p: 0.051) concentrations of EGFR when compared to their CD44v6-negative counterparts. The significant coexpression of ER, PR and cathepsin D seems to indicate a possible role for hormonal regulation of CD44v6 expression while the role of pS2 and t-PA, estrogen related proteins, was very reduced.

Adenocarcinoma, Mucinous↗
PubMed1999

[Clinical-biological differences between invasive ductal carcinomas and breast lobular carcinomas. Preliminary results].

In order to know the biological differences between both histological types, we have analyzed some clinical and biological parameters in tissues of women having infiltrating carcinomas (247 ductal (IDCs) 17 and lobular (ILCs) of the breast. Our preliminary results led us to suggest the following. 1) In negative axillary lymph node involvement (N-) patients, ILCs were more frequently associated with diploidy and the existence of multiple foci; likewise, they had higher pS2 cytosolic levels than IDC; 2) in N+ patients, ILC were associated with multicentricity and had higher concentrations of progesterone receptors and 3) these findings could help to explain us the clinical and biological peculiarities, as well as, the clinical outcome of both histological types.

Adult↗
PubMed1999

[Absence of correlation between the concentrations of carcinoembryonic antigen (CEA) and the tumor growth factor beta 2 (TGFB2) in the two main types of breast macrocysts].

In order to study the possible correlation between carcinoembryonic antigen (CEA) and cellular proliferation, we assayed the concentrations of this substance in the fluid of 77 bening macrocysts of the breast classified according to their Na+/K+ ratio and compared them with those of transforming growth factor beta 2. CEA levels correlated positively and significantly with the cationic ratio, the concentrations of albumin, glucose, Cl- and pH and were higher (range: 2.5-81.5, median 12.8 vs range: 0.4-41.5, median 3.2 ng/ml (p: 0.00000) in type 2 (Na+/K+ > 3) than in type 1 (Na+/K+ < 3) cysts. There was no correlation between CEA and TGFb2, nor between the former and dehydroepiandrosterone sulphate levels. These results led us to suggest that the high CEA concentrations in type 2 cysts seem to be the consequence of loos of cellular differentiation and disruption of the cyst wall lining as well as the acquisition of embryonary properties by the latter as a consequence of a reduced hormonal microenvironment.

Adult↗
PubMed1998

DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells.

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed-field gel electrophoresis (PFGE), we have quantified the initial "apparent" number of DNA double-strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter-individual variation in the measured dsb (1-5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation.

Aged↗
PubMed1998

Apoptosis after gamma irradiation. Is it an important cell death modality?

Apoptosis and necrosis are two different forms of cell death that can be induced by cytotoxic stress, such as ionizing radiation. We have studied the importance of apoptotic death induced after treatment with 6 Gy of gamma-irradiation in a panel of eight human tumour cell lines of different radiosensitivities. Three different techniques based on the detection of DNA fragmentation have been used, a qualitative one--DNA ladder formation --and two quantitative approaches--in situ tailing and comet assay. No statistically significant relationship between the two quantitative assays was found (r= 0.327, P = 0.159) so these methods seem to show different aspects of the process of cell death. The presence of the DNA ladder related well to the end-labelling method in that the least amount of end labelling was seen in samples in which necrotic degradation rather than apoptotic ladders were seen. However, as the results obtained by the comet assay are not in agreement with the DNA ladder experiments, we suggest that the distinction between the degraded DNA produced by apoptosis and necrosis may be difficult by this technique. Finally, although apoptosis has been proposed to be dependent on p53 functionality, and this may explain differences in cellular radiosensitivity, no statistically significant relationship was found between these parameters and apoptosis in the eight cell lines studied.

Apoptosis↗
PubMed1997

A comparison of p53 and p16 expression in human tumor cells treated with hyperthermia or ionizing radiation.

To assess the potential relationship between p53 and p16 proteins in the cellular response to stress, we have examined the levels of these proteins in a series of human tumor cell lines after treatment with either ionizing radiation or hyperthermia. We found that cells with abnormal radiation-induced G1 arrest (non-functional p53) had significantly higher constitutive levels of p16 than cells showing a normal G1 arrest (functional p53). Time-course experiments were done to test the effect of gamma-irradiation on intracellular levels of p16. The pattern of changes in p16 response was similar in all cell lines studied, and p16 expression was not related to cellular sensitivity to radiation or to the level of p53 induction after treatment. We also provide evidence that short-term exposure to high temperature causes p53 accumulation. Hyperthermia-induced p53 accumulation was greatest in those cells exhibiting the highest radiation-induced p53 accumulation, suggesting a possible relationship between p53 induction after these 2 different stresses. p16 synthesis was also induced in different cell lines after heat treatment, and this response was independent of p53 functionality. When we compared the level of p16 expression with the extent of G0/G1 arrest induced by heat, a linear correlation was found, raising the possibility that p16 may be involved in the control of cell cycle progression in response to heat treatment.

Carrier Proteins↗
PubMed1996

Relationship between DNA damage, rejoining and cell killing by radiation in mammalian cells.

The prevailing hypothesis on the mechanism of radiation-induced cell killing identifies the genetic material deoxyribonucleic acid (DNA) as the most important subcellular target at biologically relevant doses. In this review we present new data and summarize the role of the DNA double-strand breaks (dsb) induced by ionizing radiation and DNA dsb rejoining as determinants of cellular radiosensitivity. When cells were irradiated at high dose-rate, two molecular end-points were identified which often correlated with radiosensitivity: (1) the apparent number of DNA dsb induced per Gy per DNA unit and (2) the half-time of the fast component of the DNA dsb rejoining kinetics. These two molecular determinants, not mutually exclusive, may be linked through a common factor such as the conformation of DNA.

Cell Death↗
PubMed1996

Relationship between p53 status and radiosensitivity in human tumour cell lines.

We examined the relationship between p53 levels before and after irradiation, radiation-induced cell cycle delays, apoptotic cell death and radiosensitivity in a panel of eight human tumour cell lines. The cell lines differed widely in their clonogenic survival after radiation, (surviving fraction at 2 Gy: SF2=0.18-0.82). Constitutive p53 protein levels varied from 2.2 +/- 0.4 to 6.3 +/- 0.3 optical density units (OD) per 10(6) cells. p53 after irradiation (6 Gy) also varied between the cell lines, ranging from no induction to a 1.6-fold increase in p53 levels 4 h after treatment. p53 function was also assessed by G1 cell cycle arrest after irradiation. The cellular response to radiation, measured as G0/G1 arrest, and the induction of apoptosis were in good agreement. However, a trace amount of DNA ladder formation was found in two cell lines lacking G1 arrest. Overall cellular radiosensitivity correlated well with the level of radiation-induced G1 arrest (correlation coefficient r=0.856; P=0.0067), with p53 constitutive levels (r=0.874, P=0.0046), and with p53 protein fold induction (r=-0.882, P=0.0038). Our data suggest that (1) the constitutive p53 level, (2) G1 arrest after irradiation, or (3) the p53 protein response to radiation may be good predictive tests for radiosensitivity in some cell types.

Apoptosis↗
PubMed1996

Radiosensitivity of human breast cancer cell lines of different hormonal responsiveness. Modulatory effects of oestradiol.

Treatments which inhibit or retard progression of the cell through the cell cycle have been reported to reduce the effectiveness of ionizing radiation by increasing cellular radioresistance. We studied cellular radiosensitivity and radiation-induced DNA damage (double-strand break, dsb) in both hormone-sensitive and non-sensitive human breast cancer cell lines. After 72h of culture in an oestradiol-deprived medium, MCF-7 BUS and T47D B8 breast cancer cells showed a significant delay in growth, whereas no effect was seen in EVSA-T cell line. In oestradiol-free medium, MGF-7 BUS cells were arrested mainly in G(zero)/G1 phase (85-90% in G(zero)/G1, 5-7% in S, and 6-8% in G2/M). The growth-delayed MCF-7 BUS cells showed reduced radiosensitivity (survival fraction at 2 Gy, SF2 = 63%; initial DNA damage 1.00 dsb/Gy/DNA unit) in comparison with proliferating cells (SF2 = 33%, initial DNA damage 2.70 dsb/Gy/DNA unit). The radio-protective effect of oestrogen deprivation was abolished by rescuing MCF-7 cells with oestrogen-containing medium. At 24h after rescue, MCF-7 BUS cells reached a cell cycle distribution close to that found under standard culture conditions and their radiosensitivity was correspondingly increased (SF2 = 40%, DNA damage = 2.52 dsb/Gy/DNA unit). Our findings indicate that: (1) sensitivity to radiation and the proportion of proliferating cells are probably related, and (2) differences in radiosensitivity reflect differences in radiation-induced DNA damage.

Breast Neoplasms↗
PubMed1995

Radiation-induced DNA double-strand break rejoining in human tumour cells.

Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell.

Breast Neoplasms↗