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1.
Mol Cell Biol ; 30(22): 5306-17, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20855525

ABSTRACT

Src family kinase (SFK) activity is elevated in many cancers, and this activity correlates with aggressive tumor behavior. The α6ß4 integrin, which is also associated with a poor prognosis in many tumor types, can stimulate SFK activation; however, the mechanism by which it does so is not known. In the current study, we provide novel mechanistic insight into how the α6ß4 integrin selectively activates the Src family member Fyn in response to receptor engagement. Both catalytic and noncatalytic functions of SHP2 are required for Fyn activation by α6ß4. Specifically, the tyrosine phosphatase SHP2 is recruited to α6ß4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the ß4 subunit. Fyn is recruited to the α6ß4/SHP2 complex through an interaction with phospho-Y580 in the C terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6ß4-dependent invasion. Of significance for tumor progression, phosphorylation of Y580-SHP2 and SFK activation are increased in orthotopic human breast tumors that express α6ß4 and activation of this pathway is dependent upon Y1494.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Integrin alpha6beta4/metabolism , Neoplasm Invasiveness , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins c-fyn/metabolism , Amino Acid Sequence , Cell Line, Tumor , Enzyme Activation , Female , Humans , Integrin alpha6beta4/genetics , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Proto-Oncogene Proteins c-fyn/genetics , Signal Transduction/physiology , src Homology Domains
2.
Cancer Res ; 68(21): 8779-87, 2008 Nov 01.
Article in English | MEDLINE | ID: mdl-18974120

ABSTRACT

Expression of the alpha6beta4 integrin is associated with poor patient prognosis and reduced survival in a variety of human cancers. In recent years, a limited number of in vivo studies have examined the contribution of this integrin receptor to cancer progression and they have revealed that the alpha6beta4 integrin plays a multifaceted role in regulating tumor development and progression. In the current study, we investigated the mechanism by which one tyrosine residue in the beta4 subunit cytoplasmic domain, Y1494, contributes to the tumor-promoting functions of the alpha6beta4 integrin in vivo. We show that Y1494 participates in the stimulation of diverse signaling pathways that promote alpha6beta4-dependent tumor growth and invasion. Mutation of Y1494 inhibits the ability of the alpha6beta4 integrin to support anchorage-independent growth in vitro and tumor development and angiogenesis in vivo, a result that mimics the loss of total expression of the beta4 subunit. Our results support the hypothesis that Y1494 regulates alpha6beta4-dependent anchorage-independent growth through activation of the extracellular signal-regulated kinase 1/2 signaling pathway, and invasion through the combined activation of phosphatidylinositol 3-kinase and Src. Collectively, our results identify Y1494 as a major regulatory site for signaling from the alpha6beta4 integrin to promote tumor development and progression.


Subject(s)
Integrin alpha6beta4/physiology , Signal Transduction/physiology , Tyrosine/physiology , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Immunohistochemistry , Integrin alpha6beta4/chemistry , Neovascularization, Pathologic , Phosphatidylinositol 3-Kinases/metabolism , src-Family Kinases/metabolism
3.
Anal Bioanal Chem ; 392(6): 1189-96, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18791706

ABSTRACT

Dihydroxyacetone phosphate (DHAP) is a glycolytic intermediate that has been found to be significantly elevated in the erythrocytes of diabetic patients and patients with triosephosphate isomerase deficiency. DHAP spontaneously breaks down to methylglyoxal, a potent glycating agent that reacts with proteins and nucleic acids in vivo to form advanced glycation endproducts (AGEs). Like methylglyoxal, DHAP itself is also a glycating metabolite, capable of condensing with proteins and altering their structure or function. The objective of this investigation was to evaluate the susceptibility of nucleotides to nonenzymatic attack by DHAP, and to determine the factors influencing the rate and extent of nucleotide glycation by this sugar. Of the four nucleotide triphosphates (ATP, CTP, GTP and UTP) that were studied, only GTP was reactive, forming a wide range of UV and fluorescent products with DHAP. Increases in temperature and nucleotide concentration enhanced the rate and extent of GTP glycation by DHAP and promoted the heterogeneity of AGEs. Capillary electrophoresis, HPLC, and mass spectrometry allowed for a thorough analysis of the glycated products and demonstrated that the reaction of DHAP with GTP occurred via the classical Amadori pathway.


Subject(s)
Dihydroxyacetone Phosphate/metabolism , Glycation End Products, Advanced/metabolism , Guanosine Triphosphate/metabolism , Buffers , Chromatography, High Pressure Liquid/methods , Dihydroxyacetone Phosphate/chemistry , Electrophoresis, Capillary/methods , Fluorescence , Glycation End Products, Advanced/chemistry , Glycosylation , Guanosine Triphosphate/chemistry , Kinetics , Spectrometry, Fluorescence/methods , Spectrophotometry, Ultraviolet/methods , Temperature , Time Factors
4.
Med Oncol ; 25(3): 294-8, 2008.
Article in English | MEDLINE | ID: mdl-18074245

ABSTRACT

Breast cancer is a rare disease in men representing nearly 1% of the total breast cancer cases worldwide. While treatments developed for women with breast cancer are often applied to treat men with breast cancer, however, lack of awareness of this disease leads to its detection at a later stage in men. This review discusses male breast cancer and draws comparisons with female breast cancer and discusses current treatments available to treat this disease. It is believed that this review shall provide concise and relevant information increasing the awareness of issues revolving around male breast cancer (MBC).


Subject(s)
Breast Neoplasms, Male/epidemiology , Breast Neoplasms, Male/therapy , Breast Neoplasms/therapy , Antineoplastic Agents, Hormonal/therapeutic use , Aromatase Inhibitors/therapeutic use , Breast Neoplasms/epidemiology , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Breast Neoplasms, Male/metabolism , Breast Neoplasms, Male/psychology , Chemotherapy, Adjuvant , Estrogens/metabolism , Female , Humans , Male , Neoplasm Staging , Sex Characteristics , Tamoxifen/therapeutic use
5.
Med Oncol ; 25(2): 113-24, 2008.
Article in English | MEDLINE | ID: mdl-17973095

ABSTRACT

Estrogen has been implicated in promoting breast cancer in a majority of women. Endocrine therapy controlling estrogen production has been the guiding principle in treating breast cancer for more than a century. A greater understanding of this disease at a molecular level has led to the development of molecules that inhibit estrogen production by inhibiting the aromatase enzyme, that is the primary source of estrogen in postmenopausal women. This review examines the evolution of aromatase inhibitor (AI) based therapies over the past three decades. The third generation aromatase inhibitors (anastrozole, letrozole and exemestane), which have been found to be extremely specific and effective in an adjuvant/neoadjuvant/extended adjuvant setting are discussed from a biochemical and clinical perspective. A comprehensive discussion of the past, present, and future of aromatase inhibitors is conducted in this review.


Subject(s)
Aromatase Inhibitors/therapeutic use , Breast Neoplasms/drug therapy , Aminoglutethimide/therapeutic use , Androstadienes/therapeutic use , Androstenedione/analogs & derivatives , Androstenedione/therapeutic use , Aromatase Inhibitors/pharmacology , Clinical Trials as Topic , Fadrozole/therapeutic use , Female , Humans , Letrozole , Nitriles/therapeutic use , Triazoles/therapeutic use
6.
Anal Bioanal Chem ; 390(2): 679-88, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17965853

ABSTRACT

Methylglyoxal and glyoxal are generated from the oxidation of carbohydrates and lipids, and like D-glucose have been shown to nonenzymatically react with proteins to form advanced glycation end products (AGEs). AGEs can occur both in vitro and in vivo, and these compounds have been shown to exacerbate many of the long-term complications of diabetes. Earlier studies in our laboratory reported D-glucose, D-galactose, and D/L-glyceraldehyde formed AGEs with nucleosides. The objective of this study was to focus on purines and pyrimidines and to analyze these DNA nucleoside derived AGE adducts with glyoxal or methylglyoxal using a combination of analytical techniques. Studies using UV and fluorescence spectroscopy along with mass spectrometry provided for a thorough analysis of the nucleoside AGEs and demonstrated that methylglyoxal and glyoxal reacted with 2'-deoxyguanosine via the classic Amadori pathway, and did not react appreciably with 2'-deoxyadenosine, 2'-deoxythymidine, and 2'-deoxycytidine. Additional findings revealed that methylglyoxal was more reactive than glyoxal.


Subject(s)
DNA/chemistry , Deoxyguanosine/chemistry , Glyoxal/chemistry , Nucleosides/chemistry , Chromatography, High Pressure Liquid , Enzymes/metabolism , Glycosylation , Hydrogen-Ion Concentration , Methylation , Molecular Structure , Nucleosides/chemical synthesis , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry , Temperature
7.
Bioorg Chem ; 35(6): 417-29, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17937966

ABSTRACT

Guanosine 5'-triphosphate (GTP) plays a significant role in the bioenergetics, metabolism, and signaling of cells; consequently, any modifications to the structure of the molecule can have profound effects on a cell's survival and function. Previous studies in our laboratory demonstrated that like proteins, purines, and pyrimidines can nonenzymatically react with sugars to generate advanced glycation endproducts (AGEs) and that these AGEs can form in vitro under physiological conditions. The objective of this investigation was twofold. First, it was to evaluate the susceptibility of ATP, GTP, CTP, and TTP to nonenzymatic modification by D-glucose and DL-glyceraldehyde, and second to assess the effect of various factors such as temperature, pH and incubation time, and sugar concentration on the rate and extent of nucleotide triphosphate AGE formation. Of the four nucleotide triphosphates that were studied, only GTP was significantly reactive forming a heterogeneous group of compounds with DL-glyceraldehyde. D-Glucose exhibited no significant reactivity with any of the nucleotide triphosphates, a finding that was supported by UV and fluorescence spectroscopy. Capillary electrophoresis, high-performance liquid chromatography and mass spectrometry allowed for a thorough analysis of the glycated GTP products and demonstrated that the modification of GTP by dl-glyceraldehyde occurred via the classical Amadori pathway.


Subject(s)
Glycation End Products, Advanced/chemistry , Glyceraldehyde/chemistry , Guanosine Triphosphate/chemistry , Buffers , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Glucose/chemistry , Hydrogen-Ion Concentration , Spectrometry, Fluorescence , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Temperature
8.
Anal Biochem ; 360(2): 235-43, 2007 Jan 15.
Article in English | MEDLINE | ID: mdl-17097593

ABSTRACT

Advanced glycation end products (AGEs) play a significant role in the pathophysiology of diabetes leading to such conditions as atherosclerosis, cataract formation, and renal dysfunction. While the formation of nucleoside AGEs was previously demonstrated, no extensive studies have been performed to assess the effect of AGEs on DNA structure and folding. The objective of this study was to investigate the nonenzymatic glycation of two DNA oligonucleotide duplexes with one duplex consisting of deoxy-poly(A)15 and deoxy-poly(T)15 and the other consisting of deoxy-poly(GA)15 and deoxy-poly(CT)15. With D-glucose, D-galactose, D/L-glyceraldehyde, and D-glucosamine serving as the model glycating carbohydrates, D-glucosamine was found to exhibit the greatest effect on the stability and structure of the oligonucleotide duplexes, a finding that was confirmed by circular dichroism. The nonenzymatic glycation of deoxy-poly(AT) by D-glucosamine destabilized the deoxy-poly(AT) structure and changed its conformation from A form to X form. D-glucosamine also altered the conformation of deoxy-poly(GA)15 and deoxy-poly(CT)15 from A form to B form. Capillary electrophoresis and ultraviolet and fluorescence spectroscopy revealed that, of the various purines and pyrimidines, 2'-deoxyguanosine and guanine were most reactive with D-glucosamine. The nonenzymatic modification of nucleic acids warrants further investigation because this phenomenon may occur in vivo, altering DNA structure and/or function.


Subject(s)
Circular Dichroism/methods , Nucleic Acid Conformation , Oligodeoxyribonucleotides/analysis , Electrophoresis, Capillary , Galactose/metabolism , Glucosamine/metabolism , Glucose/metabolism , Glyceraldehyde/metabolism , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/metabolism , Spectrophotometry, Ultraviolet
9.
Bioorg Chem ; 35(1): 11-24, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16970975

ABSTRACT

Glyoxylate is a 2 carbon aldo acid that is formed in hepatic tissue from glycolate. Once formed, the molecule can be converted to glycine by alanine-glyoxylate aminotransferase (AGAT). In defects of AGAT, glyoxylate is transformed to oxalate, resulting in high levels of oxalate in the body. The objective of this study was 2-fold. First, it was to determine, if akin to D-glucose, D-fructose or DL-glyceraldehyde, glyoxylate was susceptible to non-enzymatic attack by amino containing molecules such as lysine, arginine or glucosamine. Second, if by virtue of its molecular structure and size, glyoxylate was as reactive a reagent in non-enzymatic reactions as DL-glyceraldehyde; i.e., a glycose that we previously demonstrated to be a more effective glycating agent than D-glucose or D-fructose. Using capillary electrophoresis (CE), high performance liquid chromatography and UV and fluorescence spectroscopy, glyoxylate was found to be a highly reactive precursor of advanced glycation like end products (AGLEs) and a more effective promoter of non-enzymatic end products than D-glucose, D-fructose or DL-glyceraldehyde.


Subject(s)
Arginine/chemistry , Glucosamine/chemistry , Glycation End Products, Advanced/chemistry , Glyoxylates/chemistry , Lysine/chemistry , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Fructose/chemistry , Glucose/chemistry , Glyceraldehyde/chemistry , Lysine/analogs & derivatives , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet
10.
Anal Bioanal Chem ; 386(6): 1633-40, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16972054

ABSTRACT

The advanced glycation end products (AGEs) of DNA nucleobases have received little attention, perhaps due to the fact that adenine, guanine, cytosine and thymine do not dissolve under mild pH conditions. To maintain nucleobases in solution, alkaline pH conditions are typically required. The objectives of this investigation were twofold: to study the susceptibility of DNA nucleobases to nonenzymatic attack by different sugars, and to evaluate the factors that influence the formation of nucleobase AGEs at pH 12, i.e., in an alkaline environment that promotes the aldo-keto isomerization and epimerization of sugars. Varying concentrations of adenine, guanine, thymine and cytosine were incubated over time with constant concentrations of D-glucose, D-galactose or D/L-glyceraldehyde under different conditions of temperature and ionic strength. Incubation of the nucleobases with the sugars resulted in a heterogeneous assembly of AGEs whose formation was monitored by UV/fluorescence spectroscopy. Capillary electrophoresis and HPLC were used to resolve the AGEs of the DNA adducts and provided a powerful tool for following the extent of glycation in each of the DNA nucleobases. Mass spectrometry studies of DNA adducts of guanine established that glycation at pH 12 proceeded through an Amadori intermediate.


Subject(s)
Alkalies , DNA/analysis , DNA/chemistry , Purines/analysis , Purines/chemistry , Pyrimidines/analysis , Pyrimidines/chemistry , Chromatography, High Pressure Liquid , DNA/metabolism , Electrophoresis, Capillary , Glycosylation , Hydrogen-Ion Concentration , Molecular Structure , Purines/metabolism , Pyrimidines/metabolism , Spectrum Analysis
11.
Anal Biochem ; 345(2): 171-80, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-16143291

ABSTRACT

Reducing sugars can react with the free amino groups of proteins to form a heterogeneous group of compounds known as advanced glycation endproducts (AGEs) or Maillard reaction products. The objective of this investigation was to monitor the nonenzymatic glycation of DNA nucleosides and to characterize the formation of nucleoside AGEs using capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), UV fluorescence spectroscopy, and mass spectrometry. Deoxyguanosine, deoxyadenosine, deoxythymidine, and deoxycytidine were used as the model nucleosides and were incubated over time with glucose, galactose, or glyceraldehyde. Under increasing concentrations and time, deoxyguanosine exhibited the highest rate of glycation with glyceraldehyde. Deoxyadenosine and deoxycytidine exhibited comparable reactivity with glyceraldehyde and no appreciable reactivity with galactose or glucose. No reactivity was observed between deoxythymidine and the sugars. A combination of CE, HPLC, UV fluorescence spectroscopy, and mass spectrometry provided a convenient method for characterizing nucleoside AGEs and for monitoring the physical factors that influence the formation of sugar adducts of DNA nucleosides.


Subject(s)
DNA/chemistry , Glycation End Products, Advanced/analysis , Maillard Reaction , Nucleosides/metabolism , Chromatography, High Pressure Liquid , Electrophoresis, Capillary , Kinetics , Mass Spectrometry , Spectrometry, Fluorescence
12.
Anal Biochem ; 343(2): 237-43, 2005 Aug 15.
Article in English | MEDLINE | ID: mdl-15992760

ABSTRACT

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.


Subject(s)
Electrophoresis, Capillary/methods , Glucosamine/metabolism , Glycation End Products, Advanced/analysis , Buffers , Chromatography, High Pressure Liquid/methods , Glyceraldehyde/metabolism , Hexoses/metabolism , Hydrogen-Ion Concentration , Oxidation-Reduction , Spectrometry, Fluorescence , Time Factors
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