PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in indi... more PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103Q Exon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose-and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-B activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.
Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium ... more Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to r...
Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and... more Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and theaflavin-3,3'-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 microM) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC50s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 microM. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Amon...
Biochemical and Biophysical Research Communications, 1976
Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there o... more Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there occurs a transient increase in ornithine decarboxylase (EC4.1.1.17) (ODC) activity. The addition of 10 to 20 mM Na+, K+ or Mg++ completely inhibits this induction of ODC activity with no effect on cell growth. These cations also inhibit the increase of ODC activity in neuroblastoma cells and in H-35 cells which is induced by prostaglandin E1 plus 3-isobutyl-1-methyl-xanthine and by 15% fetal calf serum respectively. This inhibitory effect of low levels of cations on the induction of ODC activity in different cell lines suggests that the intracellular function of ODC, and of the products of the reaction it catalyzes (putrescine, spermidine and spermine), may be intimately involved with changes in cation pools.
Frontiers in bioscience : a journal and virtual library, Sep 1, 1997
Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent pr... more Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent protein serine/threonine kinase that phosphorylates many different nuclear proteins, including high mobility group (HMG) proteins, heterogeneous nuclear ribonuleoprotein (hnRNP) fractions, and nuclear matrix proteins, but not histones. The enzyme appears to be essential in growth regulation. However, it is not clear how the enzyme is regulated in vivo. To understand the regulation of this enzyme, we have searched for possible effectors for this enzyme. Spermine, at physiological concentrations, significantly stimlulates nuclear protein phosphorylation catalyzed by nuclear kinase II (NII kinase). Using various subnuclear fractions as substrates, we showed that the stimulatory effect of spermine was confined only to nuclear matrix proteins. Thus, spermine at 1 mM stimulated a >5-fold increase in nuclear matrix phosphorylation, but had little or no effect on the phosphorylation of HMG and ...
Cellular and Molecular Life Sciences Cmls, Dec 1, 2001
Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce ra... more Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system. The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects. Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action. On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered. Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT.
Biochimica Et Biophysica Acta General Subjects, Nov 9, 1990
Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lys... more Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.
The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in ... more The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in N-18 neuroblastoma cells in tissue culture was studied by the covalent incorporation of 8-azido-cyclic adenosine 3':5'-[32P]monophosphate, together with the techniques of sodium dodecyl sulfate:polyacrylamide gel electrophoresis and autoradiography. Greater than 95% of the total cAMP binding activity of N-18 neuroblastoma cells was identified as being regulatory subunits of the type I (RI) and type II (RII) species, with RI being the predominant form of the two (RI:RII = 3:1). The specific activity of RI but not of RII increased 3-fold when cells were grown in medium containing 1% rather than 10% fetal calf serum. Under the same conditions, the specific activity of acetylcholinesterase increased 3- to 5-fold. The increase in RI was inversely related to the serum concentration in the medium and was specific for cells at the stationary phase of growth. An increase in intracellular cAMP, concomitant with the increase in RI, was also observed. Morphological examination of stationary-phase neuroblastoma cells maintained in medium containing 1% fetal calf serum suggested the presence of a high proportion of highly-differentiated cells. It is proposed that the regulatory control of RI cAMP-binding protein by serum may involve modulation of intracellular cAMP and that the expression RI may be used as a biochemical index of differentiation in mouse neuroblastoma cells.
Page 1. Identification and characterization of a surface-associated, subtilisin-like serine prote... more Page 1. Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis PABLO HERNA´ NDEZ-ROMANO1, ROBERTO HERNA´ NDEZ1, ROSSANA ARROYO2, JOHN F. ALDERETE3 ...
Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3-monogallate mixture (TF-2), and thea... more Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3-monogallate mixture (TF-2), and theaflavin-3,3-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 M) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC 50 s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 M. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Among the tea polyphenols tested, TF-2 and, to a lesser degree, (؊)-epigallocatechin gallate inhibited cyclooxygenase (Cox)-2 gene expression. TF-2 at 50 M completely blocked the serum-induced Cox-2 gene expression at both mRNA and protein level. Other genes, including c-fos, c-myc, thymidine kinase, proliferating cell nuclear antigen, BRCA1, BRCA2, and Cox-1, were not significantly affected by TF-2. These findings suggest that TF-2 may be responsible, at least in part, for the chemopreventive activity in black tea extracts.
The expression of a adenosine cyclic 3':5'-monophosphate (cAMP)-binding protein, regulatory subun... more The expression of a adenosine cyclic 3':5'-monophosphate (cAMP)-binding protein, regulatory subunit of the type I cAMPdependent protein kinase (R,), and its functional significance in the differentiation of N-18 mouse neuroblastoma cells were examined. 8-Azidoadenosine cyclic 3':5'-[32P]monophosphate, a photoaffinity-labeling analog of cAMP, and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to identify and quantitate cAMP-binding proteins in cell extracts. The induction of differentiation of N-18 mouse neuroblastoma cells, initiated either by adding dibutyryl aden osine cyclic 3':5'-monophosphate to the growth medium or by culturing cells in medium supplemented with 1% fetal calf serum, led to a 3-fold increase in the amount of 8-azidoadenosine cyclic 3':5'-[32P]monophosphate incorporated into R,, when assayed in vitro. This increased incorporation was attrib utable to an increase in the amount of RI rather than to an increase in the affinity of R, for 8-azidoadenosine cyclic 3':5'-[32P]monophosphate. The subunit molecular weight, isoelectric point, and immunoreactivity of R, were found to be identical to that of the regulatory subunit of the type I cAMP-dependent protein kinase purified from bovine skeletal muscle. The in crease in RI was not accompanied by an increase in the cAMPdependent protein kinase activity. DEAE-cellulose column chromatography confirmed the induction of R, as a free cAMPbinding protein in the differentiated neuroblastoma cells. The possibility of a growth-dependent regulation of R, was also examined. Addition of 2% dimethyl sulfoxide to cultures of N-18 mouse neuroblastoma cells inhibited cell growth without increasing the specific activity of RI. Dimethyl sulfoxide had no effect on neurite outgrowth or acetylcholinesterase activity, two parameters characteristic of differentiated cells. The fact that the induction of RI coincided with differentiation of the neuroblastoma cells suggests that the expression of RI may be used as a biochemical index of differentiation in these cells. The presence of a free cAMP-binding protein, not associated with cAMP-dependent protein kinase in neuroblastoma cells, raises important considerations concerning the action of cAMP in the regulation of growth and differentiation.
PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in indi... more PolyQ-expanded huntingtin (mHtt) variants form aggregates, termed inclusion bodies (IBs), in individuals with and models of Huntington's disease (HD). The role of IB versus diffusible mHtt in neurotoxicity remains unclear. Using a ponasterone (PA)inducible cell model of HD, here we evaluated the effects of heat shock on the appearance and functional outcome of Htt103Q Exon1-EGFP expression. Quantitative image analysis indicated that 80-90% of this mHtt protein initially appears as "diffuse" signals in the cytosol, with IBs forming at high mHtt expression. A 2-h heat shock during the PA induction reduced the diffuse signal, but greatly increased mHtt IB formation in both cytosol and nucleus. Dose-and time-dependent mHtt expression suggested that nucleated polymerization drives IB formation. RNA-mediated knockdown of heat shock protein 70 (HSP70) and heat shock cognate 70 protein (HSC70) provided evidence for their involvement in promoting diffuse mHtt to form IBs. Reporter gene assays assessing the impacts of diffuse versus IB mHtt showed concordance of diffuse mHtt expression with the repression of heat shock factor 1, cAMP-responsive element-binding protein (CREB), and NF-B activity. CREB repression was reversed by heat shock coinciding with mHtt IB formation. In an embryonic striatal neuron-derived HD model, the chemical chaperone sorbitol similarly promoted the structuring of diffuse mHtt into IBs and supported cell survival under stress. Our results provide evidence that mHtt IB formation is a chaperone-supported cellular coping mechanism that depletes diffusible mHtt conformers, alleviates transcription factor dysfunction, and promotes neuron survival.
Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium ... more Osmolytes are organic solutes that change the protein folding landscape shifting the equilibrium towards the folded state. Herein, we use osmolytes to probe the structuring and aggregation of the intrinsically disordered mutant Huntingtin (mHtt) vis-a-vis the pathogenicity of mHtt on transcription factor function and cell survival. Using an inducible PC12 cell model of Huntington’s disease (HD), we show that stabilizing polyol osmolytes drive the aggregation of Htt103QExon1-EGFP from a diffuse ensemble into inclusion bodies (IBs), whereas the destabilizing osmolyte urea does not. This effect of stabilizing osmolytes is innate, generic, countered by urea, and unaffected by HSP70 and HSC70 knockdown. A qualitatively similar result of osmolyte-induced mHtt IB formation is observed in a conditionally immortalized striatal neuron model of HD, and IB formation correlates with improved survival under stress. Increased expression of diffuse mHtt sequesters the CREB transcription factor to r...
Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and... more Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3'-monogallate mixture (TF-2), and theaflavin-3,3'-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 microM) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC50s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 microM. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Amon...
Biochemical and Biophysical Research Communications, 1976
Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there o... more Abstract L1210 cells reinitiate growth after dilution with fresh medium; during this time there occurs a transient increase in ornithine decarboxylase (EC4.1.1.17) (ODC) activity. The addition of 10 to 20 mM Na+, K+ or Mg++ completely inhibits this induction of ODC activity with no effect on cell growth. These cations also inhibit the increase of ODC activity in neuroblastoma cells and in H-35 cells which is induced by prostaglandin E1 plus 3-isobutyl-1-methyl-xanthine and by 15% fetal calf serum respectively. This inhibitory effect of low levels of cations on the induction of ODC activity in different cell lines suggests that the intracellular function of ODC, and of the products of the reaction it catalyzes (putrescine, spermidine and spermine), may be intimately involved with changes in cation pools.
Frontiers in bioscience : a journal and virtual library, Sep 1, 1997
Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent pr... more Nuclear kinase II (nuclear casein kinase 2) is a multifunctional, second messenger-independent protein serine/threonine kinase that phosphorylates many different nuclear proteins, including high mobility group (HMG) proteins, heterogeneous nuclear ribonuleoprotein (hnRNP) fractions, and nuclear matrix proteins, but not histones. The enzyme appears to be essential in growth regulation. However, it is not clear how the enzyme is regulated in vivo. To understand the regulation of this enzyme, we have searched for possible effectors for this enzyme. Spermine, at physiological concentrations, significantly stimlulates nuclear protein phosphorylation catalyzed by nuclear kinase II (NII kinase). Using various subnuclear fractions as substrates, we showed that the stimulatory effect of spermine was confined only to nuclear matrix proteins. Thus, spermine at 1 mM stimulated a >5-fold increase in nuclear matrix phosphorylation, but had little or no effect on the phosphorylation of HMG and ...
Cellular and Molecular Life Sciences Cmls, Dec 1, 2001
Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce ra... more Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system. The effects are unique to NAA: none ofthe NAA-related compounds examined (an NAD precursor/niacin, NAD analogs, and poly(ADP-ribose) polymerase inhibitors) exerted similar effects. Thus, NAD-related metabolism and poly(ADP-ribosyl)ation are unlikely related to the NAA action. On the other hand, histone acetyltransferase (HAT) activity was elevated in NAA-exposed cells, while in aged cells, HAT activity and histone H4 acetylation were lowered. Taken together, the results suggest that NAA may cause rejuvenation by restoring, at least in part, altered gene expression in aged cells through its activation of HAT.
Biochimica Et Biophysica Acta General Subjects, Nov 9, 1990
Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lys... more Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.
The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in ... more The regulation of cyclic adenosine 3':5'-monophosphate (cAMP)-binding protein in N-18 neuroblastoma cells in tissue culture was studied by the covalent incorporation of 8-azido-cyclic adenosine 3':5'-[32P]monophosphate, together with the techniques of sodium dodecyl sulfate:polyacrylamide gel electrophoresis and autoradiography. Greater than 95% of the total cAMP binding activity of N-18 neuroblastoma cells was identified as being regulatory subunits of the type I (RI) and type II (RII) species, with RI being the predominant form of the two (RI:RII = 3:1). The specific activity of RI but not of RII increased 3-fold when cells were grown in medium containing 1% rather than 10% fetal calf serum. Under the same conditions, the specific activity of acetylcholinesterase increased 3- to 5-fold. The increase in RI was inversely related to the serum concentration in the medium and was specific for cells at the stationary phase of growth. An increase in intracellular cAMP, concomitant with the increase in RI, was also observed. Morphological examination of stationary-phase neuroblastoma cells maintained in medium containing 1% fetal calf serum suggested the presence of a high proportion of highly-differentiated cells. It is proposed that the regulatory control of RI cAMP-binding protein by serum may involve modulation of intracellular cAMP and that the expression RI may be used as a biochemical index of differentiation in mouse neuroblastoma cells.
Page 1. Identification and characterization of a surface-associated, subtilisin-like serine prote... more Page 1. Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis PABLO HERNA´ NDEZ-ROMANO1, ROBERTO HERNA´ NDEZ1, ROSSANA ARROYO2, JOHN F. ALDERETE3 ...
Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3-monogallate mixture (TF-2), and thea... more Theaflavin (TF-1), theaflavin-3-monogallate and theaflavin-3-monogallate mixture (TF-2), and theaflavin-3,3-digallate (TF-3) are the major black tea polyphenols. Here we compared the effects of these polyphenols on cell growth, apoptosis, and gene expression in normal and cancerous cells. We showed that TF-2 (10-50 M) inhibited the growth of SV40 transformed WI38 human cells (WI38VA) and Caco-2 colon cancer cells but had little effect on the growth of their normal counterparts. The IC 50 s of TF-2 for the growth inhibition of WI38 and WI38VA cells were, respectively, 300 and 3 M. The other two black tea polyphenols, TF-1 and TF-3, did not exhibit such differential growth-inhibitory effect. TF-2, but not TF-1 or TF-3, induced apoptosis in transformed WI38VA cells but not in normal WI38 cells, suggesting that apoptosis was responsible, at least in part, for the differential growth-inhibitory effect of TF-2. Cox-2 has been implicated in intestinal carcinogenesis. Among the tea polyphenols tested, TF-2 and, to a lesser degree, (؊)-epigallocatechin gallate inhibited cyclooxygenase (Cox)-2 gene expression. TF-2 at 50 M completely blocked the serum-induced Cox-2 gene expression at both mRNA and protein level. Other genes, including c-fos, c-myc, thymidine kinase, proliferating cell nuclear antigen, BRCA1, BRCA2, and Cox-1, were not significantly affected by TF-2. These findings suggest that TF-2 may be responsible, at least in part, for the chemopreventive activity in black tea extracts.
The expression of a adenosine cyclic 3':5'-monophosphate (cAMP)-binding protein, regulatory subun... more The expression of a adenosine cyclic 3':5'-monophosphate (cAMP)-binding protein, regulatory subunit of the type I cAMPdependent protein kinase (R,), and its functional significance in the differentiation of N-18 mouse neuroblastoma cells were examined. 8-Azidoadenosine cyclic 3':5'-[32P]monophosphate, a photoaffinity-labeling analog of cAMP, and high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to identify and quantitate cAMP-binding proteins in cell extracts. The induction of differentiation of N-18 mouse neuroblastoma cells, initiated either by adding dibutyryl aden osine cyclic 3':5'-monophosphate to the growth medium or by culturing cells in medium supplemented with 1% fetal calf serum, led to a 3-fold increase in the amount of 8-azidoadenosine cyclic 3':5'-[32P]monophosphate incorporated into R,, when assayed in vitro. This increased incorporation was attrib utable to an increase in the amount of RI rather than to an increase in the affinity of R, for 8-azidoadenosine cyclic 3':5'-[32P]monophosphate. The subunit molecular weight, isoelectric point, and immunoreactivity of R, were found to be identical to that of the regulatory subunit of the type I cAMP-dependent protein kinase purified from bovine skeletal muscle. The in crease in RI was not accompanied by an increase in the cAMPdependent protein kinase activity. DEAE-cellulose column chromatography confirmed the induction of R, as a free cAMPbinding protein in the differentiated neuroblastoma cells. The possibility of a growth-dependent regulation of R, was also examined. Addition of 2% dimethyl sulfoxide to cultures of N-18 mouse neuroblastoma cells inhibited cell growth without increasing the specific activity of RI. Dimethyl sulfoxide had no effect on neurite outgrowth or acetylcholinesterase activity, two parameters characteristic of differentiated cells. The fact that the induction of RI coincided with differentiation of the neuroblastoma cells suggests that the expression of RI may be used as a biochemical index of differentiation in these cells. The presence of a free cAMP-binding protein, not associated with cAMP-dependent protein kinase in neuroblastoma cells, raises important considerations concerning the action of cAMP in the regulation of growth and differentiation.
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