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Biomedical subjects

Alfonso Bellacosa

Publications and source records attributed to Alfonso Bellacosa.

At least 19 recordsLinked to original sources
PubMed2006

The DNA N-glycosylase MED1 exhibits preference for halogenated pyrimidines and is involved in the cytotoxicity of 5-iododeoxyuridine.

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k(st) values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20- to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity.

Animals↗
PubMed2005

Epithelial-mesenchymal transition in development and cancer: role of phosphatidylinositol 3' kinase/AKT pathways.

Epithelial-mesenchymal transition (EMT) is an important process during development by which epithelial cells acquire mesenchymal, fibroblast-like properties and show reduced intercellular adhesion and increased motility. Accumulating evidence points to a critical role of EMT-like events during tumor progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. Several oncogenic pathways (peptide growth factors, Src, Ras, Ets, integrin, Wnt/beta-catenin and Notch) induce EMT and a critical molecular event is the downregulation of the cell adhesion molecule E-cadherin. Recently, activation of the phosphatidylinositol 3' kinase (PI3K)/AKT axis is emerging as a central feature of EMT. In this review, we discuss the role of PI3K/AKT pathways in EMT during development and cancer with a focus on E-cadherin regulation. Interactions between PI3K/AKT and other EMT-inducing pathways are presented, along with a discussion of the therapeutic implications of modulating EMT in order to achieve cancer control.

Cadherins↗
PubMed2005

Activation of AKT kinases in cancer: implications for therapeutic targeting.

The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.

Animals↗
PubMed2004

Altered gene expression in phenotypically normal renal cells from carriers of tumor suppressor gene mutations.

BACKGROUND: The inherently complex signaling networks of tumors result from genetic and epigenetic alterations that occur during cancer initiation and progression. METHODS: In an attempt to identify early molecular changes associated with dominantly inherited predisposition to "two-hit" renal tumors, the expression profiles of primary cultures of phenotypically normal renal epithelial cells from individuals bearing a germline mutation in either the von Hippel-Lindau (VHL) or the tuberous sclerosis complex (TSC) gene were compared to that of renal epithelial cells from control nonmutation carriers by microarray analysis. RESULTS: Reliability of the microarray data from pooled samples was confirmed by real-time RT-PCR. Principal Component Analysis revealed substantial differences in the gene expression profiles of the renal epithelial cells from VHL and TSC mutation carriers. In several instances, the microarray data confirm our present knowledge of the cellular pathways affected by biallelic VHL and TSC mutations. CONCLUSIONS: These findings demonstrate that heterozygosity for a mutant tumor suppressor gene may alter the expression profiles of phenotypically normal epithelial cells in a gene-specific manner. Detectable effects of "one-hit" represent early molecular changes in tumorigenesis that may serve as targets for chemopreventive intervention.

Biomarkers, Tumor↗
PubMed2004

A portrait of AKT kinases: human cancer and animal models depict a family with strong individualities.

More than a decade after their discovery, the three AKT kinase family members have emerged as central players in the signaling cascades that regulate cell growth, proliferation, survival and various aspects of intermediary metabolism. The mechanisms of activation of AKT kinases have been defined in relatively precise terms and new substrates are currently being validated in vivo. However, it is presently unclear whether AKT1, AKT2 and AKT3 are functionally redundant or whether each one performs specific functional role(s). In this review, we will summarize the signaling properties and highlight the specificities of AKT kinases that have emerged from the study of human cancer and animal models. While AKT kinases are an attractive target for pharmacological intervention, knowledge of the precise individual roles of AKT family members will improve the design of highly specific AKT-based therapeutics having reduced toxicity and improved efficacy.

Cell Enlargement↗
PubMed2004

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection.

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.

Cell Line, Tumor↗
PubMed2004

Use of RNA amplification in the optimal characterization of global gene expression using cDNA microarrays.

Microarray analysis of human tissue is frequently hindered by the limited amount of RNA available. Although amplification protocols can be utilized, the relative representation of transcripts present in the starting material must remain unaltered. In this study, 200 ng of total RNA derived from cultured renal epithelial cells from tuberous sclerosis complex (TSC) carriers and control individuals was amplified by in vitro transcription with T7 RNA polymerase. The resulting Cy-labeled cDNAs (from total or amplified RNA (aRNA)) were analyzed as direct replicates and dye-flips on slides containing 10,000 human cDNAs. The Pearson correlation coefficients for the direct replicate experiments were 0.80 (20 microg total RNA), 0.85 (40 microg total RNA), and 0.93 (2 microg of aRNA). Comparisons between the array data revealed that the majority of genes expressed in total RNA (97% for 20 microg and 85% for 40 microg) were also detected in aRNA. The correlation coefficient of the expression ratios for genes detected in both total RNA (40 microg) and aRNA was 0.63. Further, Student's t-test indicated no significant difference (P = 0.83) between these ratios. These results indicate that the number of expressed genes detected with total RNA is proportional to the amount of RNA used and underscore the requirement of large amounts of total RNA for a comprehensive characterization of gene expression profiles. RNA amplification allows the detection of a large number of genes expressed in the starting RNA population without altering their relative intensities significantly. Thus, an RNA amplification step improves the quality of gene expression results obtained by microarray analysis. This study indicates that high quality microarray data can be generated from small amounts of RNA, including those extracted from limiting clinical samples and microdissected histological specimens.

Gene Expression Profiling↗
PubMed2004

[Regulation of E-cadherin by the Akt protein kinase B].

E-cadherin is a cell adhesion molecule that is present in most normal epithelial cells. During malignant transformation, the expression of E-cadherin is frequently repressed. This protein is considered as a tumor-suppressor. The oncogenic form of Akt, a serine-threonine kinase, is multifunctionnal. One of its function is to induce the transcriptional repression of E-cadherin. This transcriptional repression induces cellular responses, epithelial cells becoming mesenchymal and invasive. At the molecular level, cells expressing a constitutive active form of Akt express also a transcription factor, Snail, known to repress the expression of E-cadherin.

Animals↗
PubMed2003

The base excision repair enzyme MED1 mediates DNA damage response to antitumor drugs and is associated with mismatch repair system integrity.

Cytotoxicity of methylating agents is caused mostly by methylation of the O6 position of guanine in DNA to form O6-methylguanine (O6-meG). O6-meG can direct misincorporation of thymine during replication, generating O6-meG:T mismatches. Recognition of these mispairs by the mismatch repair (MMR) system leads to cell cycle arrest and apoptosis. MMR also modulates sensitivity to other antitumor drugs. The base excision repair (BER) enzyme MED1 (also known as MBD4) interacts with the MMR protein MLH1. MED1 was found to exhibit thymine glycosylase activity on O6-meG:T mismatches. To examine the biological significance of this activity, we generated mice with targeted inactivation of the Med1 gene and prepared mouse embryonic fibroblasts (MEF) with different Med1 genotype. Unlike wild-type and heterozygous cultures, Med1-/- MEF failed to undergo G2-M cell cycle arrest and apoptosis upon treatment with the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Similar results were obtained with platinum compounds' 5-fluorouracil and irinotecan. As is the case with MMR-defective cells, resistance of Med1-/- MEF to MNNG was due to a tolerance mechanism because DNA damage accumulated but did not elicit checkpoint activation. Interestingly, steady state amounts of several MMR proteins are reduced in Med1-/- MEF, in comparison with Med1+/+ and Med1+/- MEF. We conclude that MED1 has an additional role in DNA damage response to antitumor agents and is associated with integrity of the MMR system. MED1 defects (much like MMR defects) may impair cell cycle arrest and apoptosis induced by DNA damage.

Animals↗
PubMed2003

RNA repair: damage control.

RNA in a cell is subject to many of the same insults as DNA. RNA damage can induce apoptosis and may be exploited for anti-cancer chemotherapy. It is a surprise, however, to learn that cells may repair RNA damage, suggesting a far greater significance of RNA in genotoxic stress.

Alkylation↗
PubMed2003

The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines.

Epithelial-mesenchymal transition (EMT) is an important process during development and oncogenesis by which epithelial cells acquire fibroblast-like properties and show reduced intercellular adhesion and increased motility. Squamous cell carcinoma lines engineered to express constitutively active Akt underwent EMT, characterized by down-regulation of the epithelial markers desmoplakin, E-cadherin, and beta-catenin and up-regulation of the mesenchymal marker vimentin. The cells lost epithelial cell morphology and acquired fibroblast-like properties. Additionally, E-cadherin was down-regulated transcriptionally. The cells expressing constitutively active Akt exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. AKT is activated in many human carcinomas, and the AKT-driven EMT may confer the motility required for tissue invasion and metastasis. These findings suggest that future therapies based on AKT inhibition may complement conventional treatments by controlling tumor cell invasion and metastasis.

Cadherins↗
PubMed2003

Genetic hits and mutation rate in colorectal tumorigenesis: versatility of Knudson's theory and implications for cancer prevention.

The multistep model of carcinogenesis is now widely accepted for colorectal cancer and other common epithelial tumors of the adult. The challenges ahead are to define the number of genetic hits necessary for conversion of a normal cell into a cancer cell and to determine whether the observed increase in the mutation rate (mutator phenotype) is required. The beauty of Knudson's two-hit theory is its ability to explain diverse experimental situations and guide specific predictions, including some directly relevant to cancer prevention.

Adenoma↗
PubMed2003

Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.

We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A.

Animals↗
PubMed2003

The phosphoinositide 3-kinase/AKT1 pathway involvement in drug and all-trans-retinoic acid resistance of leukemia cells.

Disruption of the apoptotic pathways may account for resistance to chemotherapy and treatment failures in human neoplastic disease. To further evaluate this issue, we isolated a HL-60 cell clone highly resistant to several drugs inducing apoptosis and to the differentiating chemical all-trans-retinoic acid (ATRA). The resistant clone displayed an activated phosphoinositide 3-kinase (PI3K)/AKT1 pathway, with levels of phosphatidylinositol (3,4,5) trisphosphate higher than the parental cells and increased levels of both Thr 308 and Ser 473 phosphorylated AKT1. In vitro AKT1 activity was elevated in resistant cells, whereas treatment of the resistant cell clone with two inhibitors of PI3K, wortmannin or Ly294002, strongly reduced phosphatidylinositol (3,4,5) trisphosphate levels and AKT1 activity. The inhibitors reversed resistance to drugs. Resistant cells overexpressing either dominant negative PI3K or dominant negative AKT1 became sensitive to drugs and ATRA. Conversely, if parental HL-60 cells were forced to overexpress an activated AKT1, they became resistant to apoptotic inducers and ATRA. There was a tight relationship between the activation of the PI3K/AKT1 axis and the expression of c-IAP1 and c-IAP2 proteins. Activation of the PI3K/AKT1 axis in resistant cells was dependent on enhanced tyrosine phosphorylation of the p85 regulatory subunit of PI3K, conceivably due to an autocrine insulin-like growth factor-I production. Our findings suggest that an up-regulation of the PI3K/AKT1 pathway might be one of the survival mechanisms responsible for the onset of resistance to chemotherapeutic and differentiating therapy in patients with acute leukemia.

Antineoplastic Agents↗
PubMed2002

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice.

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of beta-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat alpha-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt(max)) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt(min), was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, beta-adrenergic receptor (beta-AR) density, and beta-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-beta is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-beta/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting beta-AR signaling capacity.

Animals↗