Biomedical subjects

Joan Gil

Publications and source records attributed to Joan Gil.

31 records · Page 2Linked to original sources

PubMed2003

Dopamine induces autophagic cell death and alpha-synuclein increase in human neuroblastoma SH-SY5Y cells.

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinson's disease and other such diseases. We used SH-SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress-induced pathways, and expression of alpha-synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 microM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N-acetylcysteine and ascorbic acid. Dopamine activates the stress-response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases alpha-synuclein expression. Both cell viability and the increase in alpha-synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3-methyladenine. This indicates that oxidative stress, stress-activated kinases, and factors involved in autophagy up-regulate alpha-synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration.

Autophagy↗

PubMed2003

5-Aminoimidazole-4-carboxamide riboside induces apoptosis in Jurkat cells, but the AMP-activated protein kinase is not involved.

5-Aminoimidazole-4-carboxamide (AICA) riboside, a precursor of purine nucleotide biosynthesis, induces apoptosis in Jurkat cells. Incorporation of AICAriboside into the cells is necessary for this effect since addition of nitrobenzylthioinosine, a nucleoside-transport inhibitor, completely protects Jurkat cells from apoptosis. Adenosine, but not other nucleosides, also protects Jurkat cells from AICAriboside-induced apoptosis. The apoptotic effect is caspase-dependent since caspases 9 and 3 are activated and the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) blocks apoptosis. Furthermore, AICAriboside induces mitochondrial cytochrome c release. AICAriboside, when phosphorylated to AICAribotide (ZMP), is a specific activator of the AMP-activated protein kinase (AMPK) in certain cell types. However, AICAriboside does not activate AMPK in Jurkat cells. Moreover, 5-iodotubercidin, an inhibitor of AICAriboside phosphorylation, does not inhibit apoptosis in Jurkat cells. These results indicate that AICAriboside induces apoptosis independently of ZMP synthesis and AMPK activation in Jurkat cells.

Adenosine↗

PubMed2003

Acadesine activates AMPK and induces apoptosis in B-cell chronic lymphocytic leukemia cells but not in T lymphocytes.

Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment.

AMP-Activated Protein Kinases↗

PubMed2003

Human papillomavirus-related cervical lesions in adolescents: a histologic and morphometric study.

OBJECTIVE: To evaluate the cytopathic effect of human papillomavirus (HPV) infection in adolescents. METHODS: Cervical biopsies from 100 patients, 50 from adolescents age 14 to 20 and 50 from mature women age 35 to 64, all diagnosed with HPV-related lesions (condylomas), were studied histologically and morphometrically. Fifty were associated with low-grade squamous intraepithelial lesions and 50 with high-grade squamous intraepithelial lesions. RESULTS: Epithelial cells with large hyperchromatic, often bizarre-shaped, nuclei, staining positive for HPV-16, were identified in most samples from adolescents. These nuclei were, on average, almost twice as large as those seen in biopsies from older women (P = 0.003), and they mostly occupied the lower half of the epithelium (P = 0.0008). These large cells were further analyzed for texture by a novel image-analysis approach, the autocorrelation factor beta, that revealed a markedly different, smoother nuclear structure, suggestive of a large viral load, different from the irregular chromatin pattern seen in dysplastic nuclei. CONCLUSIONS: These peculiar nuclei were ubiquitous in the adolescent cervical biopsies and probably represent a primary abundant productive viral infection. They should not necessarily be interpreted as dysplastic.

Adolescent↗

PubMed2003

Protein kinases in the regulation of apoptosis in B-cell chronic lymphocytic leukemia.

The involvement of several protein kinase pathways in the regulation of apoptosis and cell survival has been analyzed in a wide range of models. This article reviews current understanding of the protein kinases involved in the control of apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Protein kinase C (PKC), phosphatidylinositol 3-kinase (P13K) and nuclear factor-kappa B (NF-kappaB) play important roles in the survival of these leukemic cells. These survival pathways affect proteins involved in the control of apoptosis by altering their expression or function. The elucidation of the signal transduction network involved in the survival of B-CLL cells could provide novel pharmacological targets for the therapy of B-CLL.

Animals↗

PubMed2003

Applications of image analysis to anatomic pathology: realities and promises.

Image Analysis in Pathology is viewed as an ancillary method meant to provide objective support in the resolution of difficult problems. Its Achilles heel is the process of nuclear segmentation (delimitation of the nuclear membrane) which is extremely difficult in pathology materials. Although interactive segmentation procedures are available no reliable fully automatic method has been described. The only application of image analysis that has truly succeeded in Pathology is DNA ploidy measurement. A very desirable application is the quantitation of immunohistochemical markers, which is technically challenging, has been resolved only in certain cases and is unlikely to have a general solution. Nuclear quantitation has repeatedly proven to be helpful in reaching differential diagnoses, in particular when based on size distributions of nuclear profiles rather than its average, but is hampered by the segmentation problem discussed above. Texture analysis of chromatin is an exciting, mathematically complex application likely to succeed, for which many approaches have been described. Finally a diagnosis (classification) can be obtained based on algorithms applied to multiple descriptors of tumor cells (for instance nuclear sizes, chromatin texture, shape, etc). The best classificatory approaches are neural networks (a form of artificial intelligencee), multivariate analysis, and logistic regression (statistical).

Algorithms↗

PubMed2003

p63 and TTF-1 immunostaining. A useful marker panel for distinguishing small cell carcinoma of lung from poorly differentiated squamous cell carcinoma of lung.

We studied the usefulness of p63 and thyroid transcription factor-1 (TTF-1) immunostains for differentiating poorly differentiated squamous cell carcinoma (PDSCC) from small cell lung carcinoma (SCLC). We used monoclonal antibodies reactive to p63 or TTF-1 to stain 4-microns-thick sections from 30 formalin-fixed, paraffin-embedded lung biopsy and resection specimens and 7 alcohol-fixed, formalin-postfixed, paraffin-embedded cell blocks from lung fine-needle aspirations (FNAs). For p63, we used a streptavidin-biotin kit, diaminobenzidine as the chromogen, and a hematoxylin counterstain. We used automated immunostaining for TTF-1. The 37 cases included 23 SCLCs, 13 PDSCCs, and 1 carcinoma initially diagnosed as PDSCC. All 23 SCLCs were negative or, rarely, equivocal for p63; 20 (87%) of 23 were TTF-1+; nuclear staining ranged from strong and/or frequent to weak and/or uncommon. All 13 PDSCCs were TTF-1-/p63+ with intense staining of 50% to 100% of tumor cells. One case originally diagnosed as PDSCC was TTF-1+/p63-, suggestive of SCLC; after morphologic reexamination and immunostaining for neuroendocrine markers, it was reclassified as intermediate-type SCLC. TTF-1 immunostaining showed equal or increased sensitivity in alcohol-fixed cytologic cell block samples compared with formalin-fixed biopsy material; in 1 SCLC case, the biopsy specimen was TTF-1-; however, the FNA cell block stained positively. p63 and TTF-1 appear to be useful for differentiating SCLC from lung PDSCC in formalin-fixed and alcohol-fixed, formalin-postfixed material.

Antibodies, Monoclonal↗

PubMed2002

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia.

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Image analysis and morphometry in the diagnosis of breast cancer.

Image Analysis, a complicated field still in the early stages of application to Pathology, has the capability of rendering major contributions to the diagnosis, prognosis, and management of malignancies of the breast. The present review summarizes the main problems and the general approach to the use of this technique for quantitating immunohistochemical stain results, obtaining DNA histograms, and making de novo diagnoses in routine materials of the Pathology service. In the case of diagnosis, the main steps are sampling, segmentation, and measures of chromatin texture. Currently, the limiting factor for all routine applications of image analysis is probably the absence of a reliable automatic nuclear segmentation.

Breast Neoplasms↗

PubMed2002

Calmodulin prevents activation of Ras by PKC in 3T3 fibroblasts.

We have shown previously (Villalonga, P., López- Alcalá, C., Bosch, M., Chiloeches, A., Rocamora, N., Gil, J., Marais, R., Marshall, C. J., Bachs, O., and Agell, N. (2001) Mol. Cell. Biol. 21, 7345-7354) that calmodulin negatively regulates Ras activation in fibroblasts. Hence, anti-calmodulin drugs (such as W13, trifluoroperazine, or W7) are able to induce Ras/ERK pathway activation under low levels of growth factors. We show here that cell treatment with protein kinase C (PKC) inhibitors abolishes W13-induced activation of Ras, Raf-1, and ERK. Consequently, PKC activity is essential for achieving the synergism between calmodulin inhibition and growth factors to activate Ras. Furthermore, whereas the activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) does not induce Ras activation in 3T3 cells, activation is observed if calmodulin is simultaneously inhibited. This indicates that calmodulin is preventing Ras activation by PKC. Treatment of cells with epidermal growth factor receptor or platelet-derived growth factor receptor tyrosine kinase inhibitors does not abrogate the activation of Ras by calmodulin inhibition. This implies that epidermal growth factor receptor and platelet-derived growth factor receptor tyrosine kinase activities are dispensable for the activation of Ras by TPA plus W13, and, therefore, Ras activation is not a consequence of the transactivation of those receptors by the combination of the anti-calmodulin drug plus TPA. Furthermore, K-Ras, the isoform previously shown to bind to calmodulin, is the only one activated by TPA when calmodulin is inhibited. These data suggest that direct interaction between K-Ras and calmodulin may account for the inability of PKC to activate Ras in 3T3 fibroblasts. In vitro experiments showed that the phosphorylation of K-Ras by PKC was inhibited by calmodulin, suggesting that calmodulin-dependent modulation of K-Ras phosphorylation by PKC could be the mechanism underlying K-Ras activation in fibroblasts treated with TPA plus W13.

3T3 Cells↗

PubMed2002

Involvement of protein kinase C and phosphatidylinositol 3-kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3-kinase) to study their involvement in TPA- and IL-4-induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.

Aged↗

PubMed2002

P63 in pulmonary epithelium, pulmonary squamous neoplasms, and other pulmonary tumors.

p63 is a p53-homologous nuclear protein that appears to play a crucial role in regulation of stem cell commitment in squamous and other epithelia. In this study, p63 expression was examined in benign lung and in neoplasms of pulmonary origin. Eighty sections from routinely fixed and processed archival bronchoscopic biopsy or lobectomy specimens were pretreated with citric acid (pH 6.0) for antigen retrieval, then incubated overnight with anti-p63 monoclonal antibody 4A4. Slides were stained using a streptavidin-biotin kit and diaminobenzidine as chromagen, and were counterstained with hematoxylin. In normal lung, p63 intensely stained nuclei of bronchial reserve cells but did not stain ciliated cells, alveolar epithelial cells, or nonepithelial cells. The lower strata of squamous metaplastic bronchial epithelium stained positively. All squamous-cell carcinomas stained positively (n = 30). In some well-differentiated carcinomas, staining was found at the periphery of tumor nests but was negative in central zones showing squamous maturation. Poorly differentiated carcinomas showed very high proportions (80% to 100%) of p63-positive nuclei. All small-cell carcinomas were p63 negative (n = 9). Staining of bronchioloalveolar carcinomas (n = 7) and adenocarcinomas (n = 23) was variable: some tumors showed no detectable staining, others showed heterogeneously positive staining. Adenosquamous carcinomas (n = 5) displayed a unique basalar staining pattern. Carcinoid tumors were almost entirely negative (n = 5). We conclude that p63 is expressed in benign bronchial stem cells, in neoplastic cells with either squamous differentiation or squamous differentiating potential, and in a subpopulation of adenocarcinomas. p63 immunostaining may also aid in some histopathologic distinctions, such as in small biopsies where the differential diagnosis is poorly differentiated squamous carcinoma versus small-cell carcinoma. A stem cell biology-based classification system for squamous carcinomas is proposed.

Adenocarcinoma↗

PubMed2001

Light Regulation of Gibberellin Biosynthesis and Mode of Action.

Some phenotypic effects produced in plants by light are very similar to those induced by hormones. In this review, the light-gibberellin (GA) interaction in germination, de-etiolation, stem growth, and tuber formation (process regulated by GAs) are discussed. Germination of lettuce and Arabidopsis seeds depends on red irradiation (R), which enhances the expression of GA 3-oxidase genes (GA3ox) and leads to an increase in active GA content. De-etiolation of pea seedling alters the expression of GA20ox and GA3ox genes and induces a rapid decrease of GA1 content. Stem growth of green plants is also affected by diverse light irradiation characteristics. Low light intensity increases stem elongation and active GA content in pea and Brassica. Photoperiod controls active GA levels in long-day rosette (spinach and Silene) and in woody plants (Salix and hybrid aspen) by regulating different steps of GA biosynthesis, mainly through transcript levels of GA20ox and GA3ox genes. Light modulation of stem elongation in light-grown plants is controlled by phytochrome, which modifies GA biosynthesis and catabolism (tobacco, potato, cowpea, Arabidopsis) and GA-response (pea, cucumber, Arabidopsis). In Arabidopsis and tobacco, ATH1 (a gene encoding an homeotic transcription factor) is a positive mediator of a phyB-specific signal transduction cascade controlling GA levels by regulating the expression of GA20ox and GA3ox. Tuber formation in potato is controlled by photoperiod (through phyB) and GAs. Inductive short-day conditions alter the diurnal rhythm of GA20ox transcript abundance, and increases the expression of a new protein (PHOR1) that plays a role in the photoperiod-GA interaction.

Journal Article↗