153 results on '"Reddy, E. P."'
Search Results
2. Effect of feeding higher concentrations of limiting amino acids on performance, slaughter variables and nitrogen retention in broiler chicken fed graded levels of toasted guar (Cyamopsis tetragonoloba) meal.
- Author
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Rao SVR, Raju MVLN, Prakash B, Rajkumar U, Srilatha T, and Reddy EPK
- Subjects
- Amino Acids metabolism, Animal Feed analysis, Animal Nutritional Physiological Phenomena, Animals, Body Weight, Digestion, Food Additives analysis, Male, Nitrogen pharmacology, Weight Gain drug effects, Amino Acids pharmacology, Chickens metabolism, Cyamopsis chemistry, Food Additives pharmacology, Nitrogen metabolism
- Abstract
1. An experiment was conducted to study the effect of supplementing higher concentrations (100% vs. 110%) of critical amino acids (CAA) on performance (body weight gain - BWG, feed efficiency - FE), slaughter variables and nitrogen retention in broiler chicken (1-6 weeks of age) fed graded levels of toasted guar meal (TGM) as a protein source in diets. 2. The TGM was included at five graded concentrations (0, 50, 100, 150 and 200 g/kg) in iso-caloric and iso-protein diets with either the recommended concentration (100%) of CAA (lysine, total sulphur amino acids, threonine, tryptophan and valine) or at 10% higher (110%) concentration. A metabolism trial of 3-day duration was conducted during 6th week of age to study nitrogen retention. 3. The TGM levels and CAA concentration at 21 or 42 d of age did not influence BWG, FI and FE. BWG was not affected with inclusion of TGM up to 100 g/kg in starter and overall production (1-42 d of age) phases. The FE improved with TGM supplementation during starter phase, while at the end of experiment (42 d), FE was depressed by inclusion of TGM in dose dependant manner. All performance variables improved with increase in concentration of CAA from 100% to 110%. 4. Breast meat weight improved and abdominal fat weight reduced with higher levels of CAA in diet. Retention of nitrogen reduced with increase in level of TGM in broiler diet. Increasing concentrations of CAA in diet improved nitrogen retention. 5. It was concluded that TGM could be incorporated up to 100 g/kg with 100% CAA and up to 150 g/kg with 110% CAA without affecting performance. Increasing CAA concentration (110%) in diets significantly improved BWG and FE (21 and 42 d), breast meat weight and nitrogen retention in broiler chicken.
- Published
- 2018
- Full Text
- View/download PDF
3. Effect of supplementing germinated sprouts of pulses on performance, carcass variables, immune and oxidative stress indicators in broiler chickens reared during tropical summer season.
- Author
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Rama Rao SV, Prakash B, Rajkumar U, Raju MVLN, Srilatha T, and Reddy EPK
- Subjects
- Animal Feed analysis, Animals, Chickens immunology, Dietary Supplements, Lipid Peroxidation, Male, Random Allocation, Seasons, Tropical Climate, Chickens growth & development, Diet veterinary, Oxidative Stress, Seedlings, Vigna
- Abstract
An experiment was conducted to study the effects of supplementing sprouts of pulses on performance, carcass variables, immune responses, and anti-oxidant variables in broiler chicken (day 1 to 6 weeks of age) reared during summer season in tropical region. Sprouts of black gram (BG, Vigna mungo), green gram (GG, Vigna radiata), and wild gram (WG, Vigna trilobata) were produced by soaking the pulses in water for 16 h and incubating at 37 °C for 24 h. Total phenolic content in sprouts of WG, BG, and GG was 102, 96.1, and 79.2 mg GAE/g, respectively, while the anti-radical activity in the sprouts was 61, 58, and 52%, respectively. A total of 200-day-old broiler male chicks were equally and randomly distributed in to 4 groups, each having 10 replicates of 5 chicks and housed in battery brooders in open-sided poultry house. Each of these groups was fed sprouts of BG, GG, or WG at 5% of feed intake, while the control group without feeding sprouts was kept for comparison. The trial was conducted during mid summer season (April and May, 2017). Feed conversion ratio (FCR) was reduced (P < 0.05) in broilers fed sprouted pulses compared to the control group at day 21. However, the body weight gain and FCR at 42 days of age, slaughter variables, and immune responses were not affected due to feeding of sprouted pulses. Feeding of sprouts significantly (P < 0.05) reduced lipid peroxidation and increased (P < 0.05) the activities of glutathione peroxidase, glutathione reductase, and superoxide dismutase in liver and spleen of broilers compared to the control group. Based on the results, it is concluded that oxidative stress in broiler chicken reared in tropical summer could be reduced by supplementing sprouted pulses without affecting performance, carcass variables, and immune responses.
- Published
- 2018
- Full Text
- View/download PDF
4. Lipid abnormalities, lipoprotein (a) and apoprotein pattern in non-dialyzed patients with chronic kidney disease.
- Author
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Rao AM, Bitla AR, Reddy EP, Sivakumar V, and Srinivasa Rao PV
- Abstract
The present study was carried out to explore the altered lipid, lipoprotein and apoprotein abnormalities along with lipoprotein (a) in chronic kidney disease patients with stage I to V which were further divided into group 1 (stage I and II), group 2 (stage III and IV) and group 3 (stage V). 50 chronic kidney disease patients with stage I to V and 20 healthy normal subjects as controls were recruited for this study. Among the various parameters tested triglyceride levels were high in group 1 and 2, whereas VLDL cholesterol, Lp (a) and apo B levels were significantly high in all the groups when compared to controls (P<0.05). However, LDL cholesterol level was significantly low in group 3 only as compared to control group (P<0.05). Apoprotein AI values also showed significant decrease in all groups as compared to controls (P<0.05). Though total cholesterol levels in group 1 and LDL levels in group 1 and 2 were higher than controls, but the values attained not statistically significant (P>0.05). In conclusion high levels of VLDL cholesterol, Lp (a), apo B and low levels of apoprotein AI as reported in this study are the major lipid disorders in the development of cardiovascular complications at all the stages in these patients.
- Published
- 2010
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5. New Therapeutics Targeting Colon Cancer Stem Cells.
- Author
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Thenappan A, Li Y, Shetty K, Johnson L, Reddy EP, and Mishra L
- Abstract
The recent identification of tumor-initiating colorectal cancer (CRC) stem cells in the pathogenesis of CRC has provided a potential target for novel therapeutics. Many details about CRC stem cells, however, remain poorly understood. Several potential markers of CRC stem cells have been proposed, including CD133, CD44, and, recently, Lgr5. Attention also has been drawn to control of stem cell self-renewal, proliferation, and differentiation by the Wnt and transforming growth factor (TGF)-β pathways. Disruption of Wnt signaling, via loss of APC (adenomatous polyposis coli), is among the earliest events in the multistage progression of CRC and likely occurs in basal crypt stem cells, generating a neoplastic cell population that then expands upward to occupy the rest of the crypt. TGF-β signaling is a key tumor suppressor pathway, and mutations in the type II receptor and Smad4 are observed in CRC specimens and are associated with more aggressive disease in tumors with disrupted Wnt signaling. Loss of the TGF-β adaptor protein β(2)-spectrin is associated with loss of colonic cell polarity and architecture, and its expression parallels that of Smad4. This review suggests rational approaches to target CRC stem cells as a novel and effective way to treat advanced and difficult-to-treat CRC.
- Published
- 2009
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6. Styryl sulfonyl compounds inhibit translation of cyclin D1 in mantle cell lymphoma cells.
- Author
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Prasad A, Park IW, Allen H, Zhang X, Reddy MV, Boominathan R, Reddy EP, and Groopman JE
- Subjects
- Apoptosis drug effects, Cell Line, Tumor, Eukaryotic Initiation Factor-4E physiology, Extracellular Signal-Regulated MAP Kinases physiology, Forkhead Box Protein O1, Forkhead Transcription Factors metabolism, Glycine pharmacology, Humans, Lymphoma, Mantle-Cell genetics, Lymphoma, Mantle-Cell pathology, Mitogen-Activated Protein Kinase Kinases physiology, Phosphoinositide-3 Kinase Inhibitors, Phosphorylation, Protein Kinases physiology, Proto-Oncogene Proteins c-akt physiology, Proto-Oncogene Proteins c-myc antagonists & inhibitors, Signal Transduction drug effects, TOR Serine-Threonine Kinases, p38 Mitogen-Activated Protein Kinases physiology, raf Kinases physiology, Antineoplastic Agents pharmacology, Cyclin D1 genetics, Glycine analogs & derivatives, Lymphoma, Mantle-Cell drug therapy, Protein Biosynthesis drug effects, Sulfones pharmacology
- Abstract
Mantle cell lymphoma (MCL) is characterized by the uncontrolled overexpression of cyclin D1. Styryl sulfonyl compounds have shown potent antitumor activity against MCL by inducing cell-cycle arrest and apoptosis. However, the exact molecular mechanism by which these compounds function is yet to be elucidated. Here, we show that the prototypical styryl sulfonyl compound ON 01910.Na decreased cyclin D1 and c-Myc protein levels in MCL cells, whereas mRNA levels of cyclin D1 were minimally affected. Notably, ON 01910.Na suppressed eukaryotic translation initiation factor 4E (eIF4E)-mediated cyclin D1 mRNA translation, decreased levels of phosphorylated Akt, mammalian target of Rapamycin (mTOR) and eIF4E-binding protein (eIF4E-BP), lowered the cap site binding activity of eIF4E and directly inhibited activity of phosphatidylinositol-3 kinase (PI-3K). Analysis of apoptotic signaling pathways revealed that ON 01910.Na induced the release of cytochrome c from mitochondria, altered expression of Bcl-2 family of proteins and stimulated activation of caspases. Taken together, styryl sulfonyls can cause a rapid decrease of cyclin D1 by blocking cyclin D1 mRNA translation through inhibition of the PI-3K/Akt/mTOR/eIF4E-BP signaling pathway and triggering a cytochrome c-dependent apoptotic pathway in MCL cells.
- Published
- 2009
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7. Evaluation of the novel mitotic modulator ON 01910.Na in pancreatic cancer and preclinical development of an ex vivo predictive assay.
- Author
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Jimeno A, Chan A, Cusatis G, Zhang X, Wheelhouse J, Solomon A, Chan F, Zhao M, Cosenza SC, Ramana Reddy MV, Rudek MA, Kulesza P, Donehower RC, Reddy EP, and Hidalgo M
- Subjects
- Animals, Antimetabolites, Antineoplastic therapeutic use, Biopsy, Fine-Needle, Cell Line, Tumor, Cyclin B metabolism, Cyclin B1, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology, Deoxycytidine therapeutic use, Female, Glycine pharmacology, Glycine therapeutic use, Humans, Mice, Mice, Nude, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology, Predictive Value of Tests, Sulfones therapeutic use, cdc25 Phosphatases metabolism, Gemcitabine, Antimetabolites, Antineoplastic pharmacology, Drug Resistance, Neoplasm drug effects, Glycine analogs & derivatives, Pancreatic Neoplasms drug therapy, Sulfones pharmacology, Xenograft Model Antitumor Assays
- Abstract
The pupose of this study was to evaluate the activity of ON 01910.Na, a mitotic inhibitor, in in vitro and in vivo models of pancreatic cancer and to discover biomarkers predictive of efficacy. Successive in vitro and in vivo models were used; these included cell line-derived and patient-derived tumors from our PancXenoBank, a live collection of freshly generated pancreatic cancer xenografts. ON 01910.Na showed equivalent activity to gemcitabine against pancreatic cancer cell lines in vitro. The activity of the agent correlated with suppression of phospho-CDC25C and cyclin B1. These markers were optimized for a fine-needle aspirate ex vivo rapid assay. Cyclin B1 mRNA evaluation yielded the most optimal combination of accuracy and reproducibility. Next, nine patient-derived tumors from the PancXenoBank were profiled using the assay developed in cell lines and treated with ON01910.Na for 28 days. Two cases were cataloged as potential responders and seven as resistants. There was a correlation between the ex vivo assay and sensitivity to the tested agent, as the two cases prospectively identified as sensitive met prespecified criteria for response. Of the seven tumors of predictive resistant, only one was found to be sensitive to ON 01910.Na. In addition, there was a good correlation between cyclin B1 downregulation ex vivo and changes in cyclin B1 protein post-treatment. The novel mitotic inhibitor, ON 01910.Na, showed activity in preclinical model of pancreatic cancer. A rapid assay was rationally developed that not only identified cases sensitive to ON 01910.Na, but also anticipated the pharmacodynamic events occurring after in vivo exposure.
- Published
- 2009
- Full Text
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8. Liver stem cells and hepatocellular carcinoma.
- Author
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Mishra L, Banker T, Murray J, Byers S, Thenappan A, He AR, Shetty K, Johnson L, and Reddy EP
- Subjects
- Biomarkers analysis, Carcinoma, Hepatocellular pathology, Cell Lineage, Embryonic Stem Cells cytology, Hedgehog Proteins physiology, Humans, Liver cytology, Liver growth & development, Liver Regeneration physiology, Liver Transplantation, Living Donors, Male, Neoplastic Stem Cells physiology, Prognosis, Receptors, Notch physiology, Signal Transduction physiology, Transforming Growth Factor beta physiology, Wnt Proteins physiology, beta Catenin physiology, Carcinoma, Hepatocellular therapy, Neoplastic Stem Cells pathology
- Abstract
Although the existence of cancer stem cells (CSCs) was first proposed over 40 years ago, only in the past decade have these cells been identified in hematological malignancies, and more recently in solid tumors that include liver, breast, prostate, brain, and colon. Constant proliferation of stem cells is a vital component in liver tissues. In these renewing tissues, mutations will most likely result in expansion of the altered stem cells, perpetuating and increasing the chances of additional mutations and tumor progression. However, many details about hepatocellular cancer stem cells that are important for early detection remain poorly understood, including the precise cell(s) of origin, molecular genetics, and the mechanisms responsible for the highly aggressive clinical picture of hepatocellular carcinoma (HCC). Exploration of the difference between CSCs from normal stem cells is crucial not only for the understanding of tumor biology but also for the development of specific therapies that effectively target these cells in patients. These ideas have drawn attention to control of stem cell proliferation by the transforming growth factor beta (TGF-beta), Notch, Wnt, and Hedgehog pathways. Recent evidence also suggests a key role for the TGF-beta signaling pathway in both hepatocellular cancer suppression and endoderm formation, suggesting a dual role for this pathway in tumor suppression as well as progression of differentiation from a stem or progenitor stage. This review provides a rationale for detecting and analyzing tumor stem cells as one of the most effective ways to treat cancers such as HCC.
- Published
- 2009
- Full Text
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9. JNK signaling in apoptosis.
- Author
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Dhanasekaran DN and Reddy EP
- Subjects
- Cell Nucleus enzymology, Humans, Mitochondria enzymology, Phosphorylation, Apoptosis genetics, MAP Kinase Kinase 4 metabolism, Signal Transduction
- Abstract
Jun N-terminal kinases or JNKs play a critical role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways. JNKs activate apoptotic signaling by the upregulation of pro-apoptotic genes through the transactivation of specific transcription factors or by directly modulating the activities of mitochondrial pro- and antiapoptotic proteins through distinct phosphorylation events. This review analyses our present understanding of the role of JNK in apoptotic signaling and the various mechanisms by which JNK promotes apoptosis.
- Published
- 2008
- Full Text
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10. Celecoxib and a novel COX-2 inhibitor ON09310 upregulate death receptor 5 expression via GADD153/CHOP.
- Author
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He Q, Luo X, Jin W, Huang Y, Reddy MV, Reddy EP, and Sheikh MS
- Subjects
- Apoptosis drug effects, Celecoxib, Cell Line, Tumor, Cyclooxygenase 2 Inhibitors therapeutic use, Drug Screening Assays, Antitumor, Humans, Indoles therapeutic use, Pyrazoles therapeutic use, Sulfonamides therapeutic use, TNF-Related Apoptosis-Inducing Ligand metabolism, Up-Regulation drug effects, Cyclooxygenase 2 Inhibitors pharmacology, Gene Expression Regulation, Neoplastic drug effects, Indoles pharmacology, Neoplasm Proteins metabolism, Neoplasms prevention & control, Pyrazoles pharmacology, Receptors, TNF-Related Apoptosis-Inducing Ligand biosynthesis, Sulfonamides pharmacology, Transcription Factor CHOP metabolism
- Abstract
Cyclooxygenase-2 (COX-2) inhibitors are promising anticancer agents but their long-term use at high doses is associated with adverse cardiovascular events. The molecular mechanisms underlying the anticancer or toxic cardiovascular effects of COX-2 inhibitors remain unknown. Here we report that COX-2-selective celecoxib and a novel COX-2 inhibitor ON09310 upregulate death receptor 5 (DR5) and cooperate with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the ligand for DR5, to induce apoptosis in COX-2-positive and -negative cancer cells. We also show that both agents engage GADD153/CHOP to transcriptionally upregulate DR5 expression; GADD153/CHOP is a C/EBP homologous transcription factor implicated in cellular stress response and apoptosis. Based on our results, we propose that (1) these agents appear to mediate their effects, at least in part, by engaging GADD153/CHOP to activate DR5-dependent apoptotic pathway and (2) their regulation of GADD153/CHOP and DR5 expression appears to occur independent of their COX-2 inhibitory effects. Our results also indicate that ON09310 is generally more potent than celecoxib and, at lower concentration, strongly cooperates with TRAIL to induce apoptosis. Taken together, our findings form the basis for future in-depth studies to further explore the utility of TRAIL and/or agonistic anti-DR5 antibodies in combination with low-dose COX-2 inhibitors as a rational approach for cancer prevention and treatment.
- Published
- 2008
- Full Text
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11. Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling.
- Author
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Tang Y, Kitisin K, Jogunoori W, Li C, Deng CX, Mueller SC, Ressom HW, Rashid A, He AR, Mendelson JS, Jessup JM, Shetty K, Zasloff M, Mishra B, Reddy EP, Johnson L, and Mishra L
- Subjects
- Animals, Apoptosis, Calcium-Binding Proteins deficiency, Calcium-Binding Proteins genetics, Calcium-Binding Proteins metabolism, Cell Line, Cell Proliferation, Cell Separation, Down-Regulation, Gene Expression Profiling, Glycoproteins deficiency, Glycoproteins genetics, Glycoproteins metabolism, Humans, Liver cytology, Liver metabolism, Liver Neoplasms pathology, Mice, Mice, Knockout, Proteinase Inhibitory Proteins, Secretory, STAT3 Transcription Factor metabolism, Interleukin-6 metabolism, Liver Neoplasms metabolism, Signal Transduction, Stem Cells metabolism, Transforming Growth Factor beta metabolism
- Abstract
Cancer stem cells (CSCs) are critical for the initiation, propagation, and treatment resistance of multiple cancers. Yet functional interactions between specific signaling pathways in solid organ "cancer stem cells," such as those of the liver, remain elusive. We report that in regenerating human liver, two to four cells per 30,000-50,000 cells express stem cell proteins Stat3, Oct4, and Nanog, along with the prodifferentiation proteins TGF-beta-receptor type II (TBRII) and embryonic liver fodrin (ELF). Examination of human hepatocellular cancer (HCC) reveals cells that label with stem cell markers that have unexpectedly lost TBRII and ELF. elf(+/-) mice spontaneously develop HCC; expression analysis of these tumors highlighted the marked activation of the genes involved in the IL-6 signaling pathway, including IL-6 and Stat3, suggesting that HCC could arise from an IL-6-driven transformed stem cell with inactivated TGF-beta signaling. Similarly, suppression of IL-6 signaling, through the generation of mouse knockouts involving a positive regulator of IL-6, Inter-alpha-trypsin inhibitor-heavy chain-4 (ITIH4), resulted in reduction in HCC in elf(+/-) mice. This study reveals an unexpected functional link between IL-6, a major stem cell signaling pathway, and the TGF-beta signaling pathway in the modulation of mammalian HCC, a lethal cancer of the foregut. These experiments suggest an important therapeutic role for targeting IL-6 in HCCs lacking a functional TGF-beta pathway.
- Published
- 2008
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12. Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.
- Author
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Kitisin K, Ganesan N, Tang Y, Jogunoori W, Volpe EA, Kim SS, Katuri V, Kallakury B, Pishvaian M, Albanese C, Mendelson J, Zasloff M, Rashid A, Fishbein T, Evans SR, Sidawy A, Reddy EP, Mishra B, Johnson LB, Shetty K, and Mishra L
- Subjects
- Animals, Carrier Proteins genetics, Cell Line, Tumor, Cyclin D, Cyclins antagonists & inhibitors, Humans, Liver Neoplasms, Experimental metabolism, Mice, Mice, Knockout, Microfilament Proteins deficiency, Microfilament Proteins genetics, Phosphorylation, Receptors, Transforming Growth Factor beta metabolism, Retinoblastoma metabolism, Signal Transduction genetics, Spectrin deficiency, Spectrin genetics, Transforming Growth Factor beta2 metabolism, Transforming Growth Factor beta2 physiology, Tumor Suppressor Proteins genetics, Tumor Suppressor Proteins physiology, Carcinoma, Hepatocellular etiology, Carcinoma, Hepatocellular metabolism, Carrier Proteins physiology, Cyclins metabolism, Liver Neoplasms, Experimental etiology, Microfilament Proteins physiology, Signal Transduction physiology, Spectrin physiology, Transforming Growth Factor beta2 antagonists & inhibitors
- Abstract
Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.
- Published
- 2007
- Full Text
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13. Hematopoietic cytokine receptor signaling.
- Author
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Baker SJ, Rane SG, and Reddy EP
- Subjects
- Animals, Cytokines physiology, Hematopoietic Stem Cells enzymology, Hematopoietic Stem Cells metabolism, Humans, Cytokines metabolism, Hematopoiesis physiology, Hematopoietic Stem Cells physiology, Receptors, Cytokine physiology, Signal Transduction physiology
- Abstract
Hematopoiesis is the cumulative result of intricately regulated signaling pathways that are mediated by cytokines and their receptors. Proper culmination of these diverse pathways forms the basis for an orderly generation of different cell types. Recent studies conducted over the past 10-15 years have revealed that hematopoietic cytokine receptor signaling is largely mediated by a family of tyrosine kinases termed Janus kinases (JAKs) and their downstream transcription factors termed STATs (signal transducers and activators of transcription). Aberration in these pathways, such as that caused by the recently identified JAK2V617F mutation, is an underlying cause for diseases such as leukemias and other myeloproliferative disorders. This recent discovery, when coupled with the fact that STATs are activated by oncoproteins such as BCR-ABL, underscores the importance of the JAK-STAT pathway in both normal cellular development and disease states.
- Published
- 2007
- Full Text
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14. Evaluation of novel cell cycle inhibitors in mantle cell lymphoma.
- Author
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Park IW, Reddy MV, Reddy EP, and Groopman JE
- Subjects
- Antineoplastic Agents chemistry, Apoptosis drug effects, Blotting, Western, Caspase 3 metabolism, Caspase Inhibitors, Cell Cycle Proteins metabolism, Cell Division drug effects, Cell Line, Tumor, Cell Survival drug effects, Cysteine Proteinase Inhibitors pharmacology, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, Flow Cytometry, G2 Phase drug effects, Humans, In Situ Nick-End Labeling, Lymphoma, Mantle-Cell metabolism, Lymphoma, Mantle-Cell pathology, Molecular Structure, Styrenes chemistry, Styrenes pharmacology, Sulfones chemistry, Antineoplastic Agents pharmacology, Cell Cycle drug effects, Sulfones pharmacology
- Abstract
Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. In this study, we tested a novel class of kinase inhibitors, styryl sulfones, which differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including cyclin-dependent kinase 4, p53, mouse double minute 2 (MDM2), and cyclin D as well as increased cyclin B expression. Using both terminal deoxy transferase uridine triphosphate nick end-labelling and poly ADP-ribose polymerase assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of B-cell lymphoma family molecules. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus, styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.
- Published
- 2007
- Full Text
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15. Scaffold proteins of MAP-kinase modules.
- Author
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Dhanasekaran DN, Kashef K, Lee CM, Xu H, and Reddy EP
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing metabolism, Animals, Humans, Mitogen-Activated Protein Kinases chemistry, Mitogen-Activated Protein Kinases metabolism, Nuclear Matrix-Associated Proteins chemistry, Nuclear Matrix-Associated Proteins metabolism, Adaptor Proteins, Signal Transducing physiology, Mitogen-Activated Protein Kinases physiology, Nuclear Matrix-Associated Proteins physiology
- Abstract
Mitogen-activated protein kinases (MAPKs) regulate critical signaling pathways involved in cell proliferation, differentiation and apoptosis. Recent studies have shown that a novel class of scaffold proteins mediates the structural and functional organization of the three-tier MAPK module. By linking the MAP3K, MAP2K and MAPK into a multienzyme complex, these MAPK-specific scaffold proteins provide an insulated physical conduit through which signals from the respective MAPK can be transmitted to the appropriate spatiotemporal cellular loci. Scaffold proteins play a determinant role in modulating the signaling strength of their cognate MAPK module by regulating the signal amplitude and duration. The scaffold proteins themselves are finely regulated resulting in dynamic intra- and inter-molecular interactions that can modulate the signaling outputs of MAPK modules. This review focuses on defining the diverse mechanisms by which these scaffold proteins interact with their respective MAPK modules and the role of such interactions in the spatiotemporal organization as well as context-specific signaling of the different MAPK modules.
- Published
- 2007
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16. Granulocyte colony-stimulating factor-induced upregulation of Jak3 transcription during granulocytic differentiation is mediated by the cooperative action of Sp1 and Stat3.
- Author
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Mangan JK, Tantravahi RV, Rane SG, and Reddy EP
- Subjects
- Animals, Cells, Cultured, Electrophoretic Mobility Shift Assay, Janus Kinase 3, Mice, Protein-Tyrosine Kinases metabolism, Up-Regulation, Cell Differentiation, Gene Expression Regulation, Granulocyte Colony-Stimulating Factor pharmacology, Granulocytes cytology, Protein-Tyrosine Kinases genetics, STAT3 Transcription Factor metabolism, Sp1 Transcription Factor metabolism, Transcription, Genetic
- Abstract
We previously demonstrated that Jak3 is a primary response gene for G-CSF and ectopic overexpression of Jak3 can accelerate granulocytic differentiation of normal mouse bone marrow cells induced by G-CSF and GM-CSF. To gain insight into the regulation of G-CSF-induced transcription of Jak3, we constructed deletion and linker scanning mutants of the Jak3 promoter sequences and performed luciferase reporter assays in the murine myeloid cell line 32Dcl3, with and without G-CSF stimulation. These experiments showed that mutation of a -67 to -85 element, which contained a putative Sp1 binding site, or mutation of a -44 to -53 GAS element resulted in a marked reduction of Jak3 promoter activity. Electrophoretic mobility shift assays revealed that Sp1 and Stat3 present in nuclear lysates of 32Dcl3 cells stimulated with G-CSF can bind to the -67 to -85 element and -44 to -53 GAS element, respectively. In addition, cotransfection of a constitutively active mutant of Stat3 along with a Jak3 promoter/luciferase reporter resulted in enhanced Jak3 promoter activity. Together, these results demonstrate that activation of Jak3 transcription during G-CSF- induced granulocytic differentiation is mediated by the combined action of Sp1 and Stat3, a mechanism also shown to be important in IL-6-induced monocytic differentiation.
- Published
- 2006
- Full Text
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17. Critical interactions between TGF-beta signaling/ELF, and E-cadherin/beta-catenin mediated tumor suppression.
- Author
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Katuri V, Tang Y, Li C, Jogunoori W, Deng CX, Rashid A, Sidawy AN, Evans S, Reddy EP, Mishra B, and Mishra L
- Subjects
- Animals, Cadherins physiology, Carrier Proteins physiology, Cell Adhesion, Epithelial Cells physiology, Gastrointestinal Neoplasms physiopathology, Gene Expression Profiling, Mice, Microfilament Proteins physiology, Signal Transduction, Smad4 Protein biosynthesis, Smad4 Protein genetics, beta Catenin physiology, Carrier Proteins biosynthesis, DNA-Binding Proteins, Gastrointestinal Neoplasms genetics, Microfilament Proteins biosynthesis, Transforming Growth Factor beta physiology
- Abstract
Inactivation of the transforming growth factor-beta (TGF-beta) pathway occurs often in malignancies of the gastrointestinal (GI) system. However, only a fraction of sporadic GI tumors exhibit inactivating mutations in early stages of cancer formation, suggesting that other mechanisms play a critical role in the inactivation of this pathway. Here, we show a wide range of GI tumors, including those of the stomach, liver and colon in elf+/- and elf+/- / Smad4+/- mutant mice. We found that embryonic liver fodrin (ELF), a beta-Spectrin originally identified in endodermal stem/progenitor cells committed to foregut lineage, possesses potent antioncogenic activity and is frequently inactivated in GI cancers. Specifically, E-cadherin accumulation at cell-cell contacts and E-cadherin-beta-catenin-dependent epithelial cell-cell adhesion is disrupted in elf+/- / Smad4+/- mutant gastric epithelial cells, and could be rescued by ectopic expression of full-length elf, but not Smad3 or Smad4. Subcellular fractionation revealed that E-cadherin is expressed mainly at the cell membrane after TGF-beta stimulation. In contrast, elf+/- / Smad4+/- mutant tissues showed abnormal distribution of E-cadherin that could be rescued by overexpression of ELF but not Smad3 or Smad4. Our results identify a group of common lethal malignancies in which inactivation of TGF-beta signaling, which is essential for tumor suppression, is disrupted by inactivation of the ELF adaptor protein.
- Published
- 2006
- Full Text
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18. Human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer activity is inhibited by transcriptional factors, from the upstream stimulating factors family, via E-box and newly identified initiator element (Inr) in FSH-R non-expressing cells.
- Author
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Putowski LT, Schillings WJ, Lee CM, Reddy EP, and Jakowicki JA
- Subjects
- Animals, CHO Cells, Cloning, Molecular, Cricetinae, DNA-Binding Proteins genetics, Gene Expression, Humans, Luciferases genetics, Mutagenesis, Polymerase Chain Reaction, Recombinant Fusion Proteins, Regulatory Sequences, Nucleic Acid, Sequence Analysis, DNA, Transcription Factors genetics, Transfection, Upstream Stimulatory Factors, DNA-Binding Proteins physiology, E-Box Elements genetics, Enhancer Elements, Genetic genetics, Promoter Regions, Genetic genetics, Receptors, FSH genetics, Transcription Factors pharmacology, Transcription Factors physiology
- Abstract
To localize the regulatory elements in the human follicle-stimulating hormone receptor (FSH-R) promoter/enhancer and to determine the role of upstream stimulatingfactors (USFs) in these elements, we transiently transfected constructs of FSH-R promoter/enhancer in pGL3 luciferase reporter plasmids into Chinese hamster ovary cells and the activities were determined by measuring luciferase luminescence of the cell lysates. The 5'-flanking regions of the human FSH-R gene from nt -1485 to -1 with respect to the gene translation start site were amplified by polymerase chain reaction (PCR) and subcloned in pGL3. Deletion mutants were created using PCR or restriction enzyme digestion. Mutation in the E-box sequence from nt -124 to -119 (E-box 3), in the construct from -224 to nt -1 or in the Inr element, which encompasses the transcriptional start site at nt -99, resulted in a substantial reduction in the human FSH-R promoter/enhancer activity. Overexpression of upstream stimulating factor-1 (USF1) suppresses the activity of the human FSH-R promoter/enhancer via Inr and E-box elements. Upstream stimulating factor-2 (USF2) decreases FSH-R promoter/enhancer activity by acting on E-box 3. The results indicate that E-box 3 and the Inr element are important elements of the human FSH-R promoter/ enhancer. USF family members inhibit FSH-R gene activity by acting via these elements. USF1 and USF2 suppress human FSH-R promoter/enhancer activity by acting on E-box 3. USF1 also decreases activity by interacting with the Inr element.
- Published
- 2004
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19. Genomic organization and characterization of the promoter region of murine GSTM2 gene.
- Author
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Kumar A and Reddy EP
- Subjects
- Animals, Base Sequence, DNA chemistry, DNA genetics, DNA isolation & purification, Exons, Introns, Luciferases genetics, Luciferases metabolism, Mice, Mice, Inbred Strains, Molecular Sequence Data, Mutation, Proto-Oncogene Proteins c-myb genetics, Proto-Oncogene Proteins c-myb physiology, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Sequence Analysis, DNA, Sequence Deletion, Sequence Homology, Nucleic Acid, Transcriptional Activation, Tumor Cells, Cultured, Genes genetics, Glutathione Transferase genetics, Promoter Regions, Genetic genetics
- Abstract
In this study, we have isolated the genomic clone of the murine GSTM2 gene and determined its sequence. Consistent with the class mu genefamily, the mGSTM2 gene consists of eight exons. The exon-intron boundaries and the distribution of coding sequences within the exons of the known GST class mu family members were found to follow a similar pattern suggesting that various members of this family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. By primer extension, the start of transcription was determined to be 40 bp upstream of the initial AUG codon. To gain an understanding of the mGSTM2 regulation, we have also cloned and analyzed its promoter region. Maximal activity was observed in a 170 bp 5'-flanking region. The activity was decreased by 3-fold in a 402 bp 5'-flanking region suggesting the presence of repressor elements. While no TATA box was identified, the presence of an SP1 site at position -38 was noted. Deletion of this SP1 site completely abrogated promoter activity. The promoter contained eight putative Myb responsive elements and its transcriptional activity was upregulated by t-Myb but not by c-Myb.
- Published
- 2001
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20. Characterization of an alternatively spliced AATYK mRNA: expression pattern of AATYK in the brain and neuronal cells.
- Author
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Baker SJ, Sumerson R, Reddy CD, Berrebi AS, Flynn DC, and Reddy EP
- Subjects
- Amino Acid Sequence, Animals, Apoptosis Regulatory Proteins, Base Sequence, Blotting, Western, Brain cytology, Carcinoma genetics, Carcinoma metabolism, Cell Differentiation, Cloning, Molecular, DNA Primers chemistry, DNA, Complementary genetics, Embryonal Carcinoma Stem Cells, Gene Expression, Humans, Immunoenzyme Techniques, Molecular Sequence Data, Neoplastic Stem Cells metabolism, Polymerase Chain Reaction, Protein Biosynthesis, Protein-Tyrosine Kinases metabolism, Rats, Reverse Transcriptase Polymerase Chain Reaction, Transcription, Genetic, Tumor Cells, Cultured, Alternative Splicing genetics, Brain enzymology, Neurons enzymology, Protein-Tyrosine Kinases genetics, RNA, Messenger biosynthesis
- Abstract
The AATYK gene encodes a tyrosine kinase whose expression is up-regulated during the apoptosis and differentiation of 32Dcl3 myeloblastic cells. Because high levels of AATYK mRNA have also been detected in the brain, and because these transcripts differ in size from that observed in the 32Dcl3 cell line, it was of interest to determine whether this gene encodes mRNAs that are alternatively spliced and whether these mRNAs are expressed in a tissue-specific manner. We have isolated a novel, alternatively spliced AATYK mRNA using cDNA library screening and RT-PCR, whose expression is readily detected in the brain but not myeloid cells. Western blot analysis revealed that the AATYK protein was expressed in virtually all regions of the adult rat brain in which neurons are present, including olfactory bulb, forebrain, cortex, midbrain, cerebellum and pons. Immunohistochemical labeling of adult brain sections showed the highest levels of AATYK expression in the cerebellum and olfactory bulb. Expression of AATYK was also up-regulated as a function of RA-induced neuronal differentiation of p19 embryonal carcinoma cells, supporting a role for this protein in mature neurons and neuronal differentiation.
- Published
- 2001
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21. The T cell-dependent B cell immune response and germinal center reaction are intact in A-myb-deficient mice.
- Author
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Vora KA, Lentz VM, Monsell W, Rao SP, Mettus R, Toscani A, Reddy EP, and Manser T
- Subjects
- Animals, Antibodies blood, Antibody Formation genetics, B-Lymphocytes metabolism, B-Lymphocytes pathology, Cell Differentiation genetics, Cell Differentiation immunology, Chickens, Female, Germinal Center metabolism, Germinal Center pathology, Immunoglobulin Class Switching genetics, Injections, Intraperitoneal, Lymphocyte Activation genetics, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Proto-Oncogene Proteins c-myb, Spleen abnormalities, Spleen growth & development, Spleen immunology, T-Lymphocytes metabolism, gamma-Globulins administration & dosage, gamma-Globulins immunology, Avian Proteins, B-Lymphocytes immunology, Germinal Center immunology, Proto-Oncogene Proteins deficiency, Proto-Oncogene Proteins genetics, T-Lymphocytes immunology, Trans-Activators deficiency, Trans-Activators genetics
- Abstract
Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.
- Published
- 2001
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22. Janus kinases: components of multiple signaling pathways.
- Author
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Rane SG and Reddy EP
- Subjects
- Animals, Cytokines physiology, Enzyme Activation, Humans, Janus Kinase 1, Janus Kinase 2, Protein-Tyrosine Kinases chemistry, Structure-Activity Relationship, Substrate Specificity, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins, Signal Transduction
- Abstract
Cytoplasmic Janus protein tyrosine kinases (JAKs) are crucial components of diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. Evidence to date, indicates that JAK kinase function may integrate components of diverse signaling cascades. While it is likely that activation of STAT proteins may be an important function attributed to the JAK kinases, it is certainly not the only function performed by this key family of cytoplasmic tyrosine kinases. Emerging evidence indicates that phosphorylation of cytokine and growth factor receptors may be the primary functional attribute of JAK kinases. The JAK-triggered receptor phosphorylation can potentially be a rate-limiting event for a successful culmination of downstream signaling events. In support of this hypothesis, it has been found that JAK kinase function is required for optimal activation of the Src-kinase cascade, the Ras-MAP kinase pathway, the PI3K-AKT pathway and STAT signaling following the interaction of cytokine/interferon receptors with their ligands. Aberrations in JAK kinase activity, that may lead to derailment of one or more of the above mentioned pathways could disrupt normal cellular responses and result in disease states. Thus, over-activation of JAK kinases has been implicated in tumorigenesis. In contrast, loss of JAK kinase function has been found to result in disease states such as severe-combined immunodeficiency. In summary, optimal JAK kinase activity is a critical determinant of normal transmission of cytokine and growth factor signals.
- Published
- 2000
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23. IL-3 signaling and the role of Src kinases, JAKs and STATs: a covert liaison unveiled.
- Author
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Reddy EP, Korapati A, Chaturvedi P, and Rane S
- Subjects
- Animals, Apoptosis, Cytokines metabolism, Humans, Janus Kinase 1, Phosphatidylinositol 3-Kinases metabolism, Receptors, Interleukin-3 metabolism, STAT1 Transcription Factor, Transcriptional Activation, DNA-Binding Proteins metabolism, Interleukin-3 metabolism, Protein-Tyrosine Kinases metabolism, Signal Transduction, Trans-Activators metabolism, src-Family Kinases metabolism
- Abstract
Hematopoiesis is the cumulative result of intricately regulated signal transduction cascades that are mediated by cytokines and their cognate receptors. Proper culmination of these diverse signaling pathways forms the basis for an orderly generation of different cell types and aberrations in these pathways is an underlying cause for diseases such as cancer. Over the past several years, downstream events initiated upon cytokine/growth factor stimulation have been a major focus of biomedical research. As a result, several key concepts have emerged allowing a better understanding of the complex signaling processes. A group of novel transcription factors, termed signal transducers and activators of transcription (STATs) appear to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Until recently, the JAK proteins were considered to be the tyrosine kinases, which dictated the levels of phosphorylation and activation of STAT proteins, forming the basis of the JAK-STAT model. However, over the past few years, increasing evidence has accumulated which indicates that at least some of the STAT protein activation may be mediated by members of the Src gene family following cytokine/growth factor stimulation. Studies have demonstrated that the Src-family of tyrosine kinases can phosphorylate and activate certain STAT proteins, in lieu of JAK kinases. In such a scenario, JAK kinases may be more crucial to phosphorylation of the cytokine/growth factor receptors and in the process create docking sites on the receptors for binding of SH2-containing proteins such as STATs, Src-kinases and other signaling intermediates. Tyrosine phosphorylation and activation of STAT proteins can be achieved either by JAKs or Src-kinases depending on the nature of STAT that is being activated. This forms the basis for the JAK-Src-STAT model proposed in this review. The concerted action of JAK kinases, members of the Src-kinase family and STAT proteins, leads to cell proliferation and cell survival, the end-point of the cytokine/growth factor stimulus. Oncogene (2000).
- Published
- 2000
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24. Cloning of murine G1RP, a novel gene related to Drosophila melanogaster g1.
- Author
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Baker SJ and Reddy EP
- Subjects
- Amino Acid Sequence, Animals, Apoptosis drug effects, Base Sequence, Blotting, Northern, Cell Line, Cloning, Molecular, DNA, Complementary chemistry, DNA, Complementary genetics, DNA, Complementary isolation & purification, Drosophila Proteins, Drosophila melanogaster genetics, Gene Expression Regulation drug effects, Interleukin-3 pharmacology, Male, Mice, Molecular Sequence Data, RNA drug effects, RNA genetics, RNA metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Tissue Distribution, Up-Regulation, DNA-Binding Proteins genetics, Zinc Fingers genetics
- Abstract
To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we performed representational difference analysis (RDA) using 32Dcl3 myeloblastic cells that were deprived of IL-3 for 24h. We have isolated a novel cDNA (g1-related protein, G1RP) that is homologous to g1, a Drosophila melanogaster zinc-finger protein that is expressed in the mesoderm. Northern blot analysis using RNAs derived from 32Dcl3 cells that have been grown in the absence of IL-3 demonstrates that the G1RP message is upregulated in these cells following the removal of IL-3, suggesting that this gene may regulate growth factor withdrawal-induced apoptosis of myeloid precursor cells.
- Published
- 2000
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- View/download PDF
25. Cell cycle control of pancreatic beta cell proliferation.
- Author
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Rane SG and Reddy EP
- Subjects
- Animals, Cell Division, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinases metabolism, Cyclins metabolism, Humans, Insulin physiology, Signal Transduction, Cell Cycle, Diabetes Mellitus pathology, Islets of Langerhans cytology, Proto-Oncogene Proteins
- Abstract
Diabetes mellitus ensues as a consequence of the body's inability to respond normally to high blood glucose levels. The onset of diabetes is due to several pathological changes, which are a reflection of either the inability of the pancreatic beta cells to secrete sufficient insulin to combat the hyperglycemia or a state of insulin resistance in target tissues. However, the significance of changes in beta cell mass and decreased beta cell proliferation or growth in progression of diabetes has been under-appreciated. Beta cells, like all other cells of our body are under the regulatory checks and balances enforced by changes in cell cycle progression. However, very little is known regarding the key components of the cell cycle machinery regulating cell cycle control of beta cells. Knowledge of key elements involved in cell cycle regulation of beta cells will go a long way in improving our understanding of the replication capacity and developmental biology of beta cells. This information is essential for us to design new approaches that can be used to correct beta cell deficiency in diabetes. This review focuses on the current knowledge of factors important for proliferation of beta cells and proposes a cell cycle model for regeneration of the beta cell population lost or reduced in diabetes.
- Published
- 2000
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26. Oncogenic mutant of Galpha12 stimulates cell proliferation through cycloxygenase-2 signaling pathway.
- Author
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Dermott JM, Reddy MR, Onesime D, Reddy EP, and Dhanasekaran N
- Subjects
- 3T3 Cells, Animals, Cell Division genetics, Cyclooxygenase 2, Fibroblasts physiology, Mice, Mutation, Cell Transformation, Neoplastic genetics, Fibroblasts pathology, GTP-Binding Proteins genetics, Isoenzymes physiology, Prostaglandin-Endoperoxide Synthases physiology, Signal Transduction genetics
- Abstract
Expression of the GTPase-deficient, activated mutant alpha-subunit of the heterotrimeric G protein G12 (Galpha12QL) leads to the neoplastic transformation of fibroblast cell lines. The mitogenic pathway regulated by Galpha12QL includes an extensive signaling network involving several small GTPases and various kinases. In addition, Galpha12QL has been shown to potentiate the serum-induced phospholipase-A2 activity in NIH3T3 cells. In the present study, we demonstrate that cycloxygenase-2 (COX-2) pathway is involved in the mitogenic pathway activated by Galpha12QL. Expression of Galpha12QL and not Galpha13QL, stimulates the serum-induced release of arachidonic acid in NIH3T3 cells. Furthermore, expression of Galpha12QL or the stimulation of wild-type Galpha12 induces the expression of COX-2. Our results also indicate that the COX-2 inhibitor acutely disrupts the DNA-synthesis stimulated by Galpha12QL in NIH3T3 cells. These studies, for the first time, identify the crucial role of COX-2 in Galpha12-mediated regulation of cell proliferation and suggest a role for prostaglandin-derived autocrine loop in Galpha12-mediated signaling pathways.
- Published
- 1999
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27. The myb gene family in cell growth, differentiation and apoptosis.
- Author
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Oh IH and Reddy EP
- Subjects
- Gene Expression, Humans, Apoptosis, Cell Differentiation, Cell Division, Oncogenes genetics
- Abstract
The myb gene family consists of three members, named A, B and c-myb which encode nuclear proteins that function as transcriptional transactivators. Proteins encoded by these three genes exhibit a tripartate structure with an N-terminal DNA-binding domain, a central transactivation domain and a C-terminal regulatory domain. These proteins exhibit highest homology in their DNA binding domains and appear to bind DNA with overlapping sequence specificities. Transactivation by myb gene family varies considerably depending on cell type and promoter context suggesting a dependence on interaction with other cell type specific co-factors. While the C-terminal domains of A-Myb and c-Myb proteins exert a negative regulatory effect on their transcriptional transactivation function, the C-terminal domain of B-Myb appears to function as a positive regulator of this activity. One or more of these proteins interact with other transcription factors such as Ets-2, CEBP and NF-M. In addition, expression of these genes is cell cycle-regulated and inhibition of their expression with antisense oligonucleotides has been found to affect cell cycle-progression, cell division and/or differentiation. Members of the myb gene family exhibit different temporal and spatial expression patterns suggesting a distinctive function for each of these genes. Gene knockout experiments show that these genes play an essential role in development. Loss of c-myb function results in embryonic lethality due to failure of fetal hepatic hematopoiesis. A-myb null mutant mice, on the other hand are viable but exhibit growth abnormalities, and defects in spermatogenesis and female breast development. While the role of c-myb in oncogenesis is well established, future experiments are likely to provide further clues regarding the role of A-myb and B-myb in tumorigenesis.
- Published
- 1999
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28. The v-myc oncogene.
- Author
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Lee CM and Reddy EP
- Subjects
- Animals, Apoptosis, Cell Differentiation, Cell Division, Cell Transformation, Neoplastic genetics, Gene Expression Regulation, Neoplastic, Humans, Macrophage Colony-Stimulating Factor genetics, Genes, myc physiology
- Abstract
v-myc is the viral homolog of c-myc transduced by several acute transforming retroviruses, many of which encode this gene as a Gag-Myc fusion protein. The v-myc oncogene can transform several lineages of mammalian and avian cells either alone or in cooperation with other oncogenes. While the Gag portion of the Gag-Myc fusion protein and the nuclear localization signal each appear to be dispensable for transformation, the N- and C-termini of the Myc sequence have been found to be essential for transformation. All v-myc genes contain point mutations which seem to confer a greater potency to v-myc in the process of transformation, proliferation, and apoptosis. In v-myc-transformed myelomonocytic cells, secondary events occur, such as the expression of colony stimulating factor-1 (CSF-1) which play a critical role in immortalization and subsequent tumor progression. Inhibition of the autocrine loop of CSF-1 was found to induce apoptosis in the immortalized cells. While overexpression of v-Myc blocks terminal differentiation of hematopoietic cells, this is not sufficient to block the differentiation of certain neural and skeletal muscle cells. Recent developments on the effects of v-myc on cell growth, transformation, differentiation and apoptosis are discussed in this review.
- Published
- 1999
- Full Text
- View/download PDF
29. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.
- Author
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Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, and Barbacid M
- Subjects
- Animals, Cell Line, Cyclin-Dependent Kinase 4, Cyclin-Dependent Kinases metabolism, Diabetes Mellitus enzymology, Diabetes Mellitus metabolism, Enzyme Activation, Female, Gene Expression Regulation, Hyperplasia, Infertility, Female genetics, Infertility, Male genetics, Islets of Langerhans cytology, Islets of Langerhans enzymology, Male, Mice, Mice, Inbred Strains, Mutagenesis, Site-Directed, Spermatogenesis genetics, Cyclin-Dependent Kinases genetics, Diabetes Mellitus genetics, Insulin deficiency, Islets of Langerhans pathology, Proto-Oncogene Proteins
- Abstract
To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.
- Published
- 1999
- Full Text
- View/download PDF
30. Modulation of life and death by the TNF receptor superfamily.
- Author
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Baker SJ and Reddy EP
- Subjects
- Animals, Antigens, CD physiology, CD40 Antigens metabolism, Calcium-Calmodulin-Dependent Protein Kinases physiology, Carrier Proteins physiology, Co-Repressor Proteins, Fas-Associated Death Domain Protein, Humans, JNK Mitogen-Activated Protein Kinases, Molecular Chaperones, Multigene Family, Proteins metabolism, Proteins physiology, Receptor-Interacting Protein Serine-Threonine Kinases, Receptors, Tumor Necrosis Factor, Type I, TNF Receptor-Associated Factor 1, TNF Receptor-Associated Factor 3, Tumor Necrosis Factor-alpha physiology, fas Receptor physiology, Adaptor Proteins, Signal Transducing, Apoptosis genetics, Intracellular Signaling Peptides and Proteins, Mitogen-Activated Protein Kinases, Nuclear Proteins, Receptors, Tumor Necrosis Factor physiology, Signal Transduction
- Abstract
The tumor necrosis factor receptor (TNFR) superfamily represents a growing family, with over 20 members having been identified thus far in mammalian cells. These proteins share significant homologies in their extracellular ligand binding domains and intracellular effector (death) domains. These receptors appear to transmit their signals via protein-protein interactions, which convey either a death or survival signal. Isolation and characterization of death domain containing proteins (TRADD, FADD/MORT-1, RIP), TRAF domain containing proteins (TRAF1-6) as well as new members and adaptor proteins such as DAXX have provided new insights to our understanding of signaling mechanisms associated with this family of receptors. While the death signals seem to be associated with the activation of both the caspase and JUN kinase pathways, the survival signals are mediated via the activation of the NF-kappaB pathway.
- Published
- 1998
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- View/download PDF
31. Effect of Semicarpus anacardium on the cell cycle of DU-145 cells.
- Author
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Tripathi YB, Tripathi P, Ramana Reddy MV, Dutt S, Tewari DS, and Reddy EP
- Abstract
The nuts of Semicarpus anacardium (Anacardiaceae) are one of the most favoured medicine in the Indian System of Medicine for the management of arthritis and several other free radical mediated diseases. It is also recommended for the management of the breast cancer. In this report we have investigated its role on the cell cycle and cell viability on the DU-145 cells (transformed prostate cells) by flow cytometric technique. It was observed that the plant extract significantly arrests the cell cycle at G-1 stage, and induced apoptosis. On higher concentrations, it affects the cell viability. The response was dose dependent., (Copyright © 1998 Gustav Fischer Verlag. Published by Elsevier GmbH.. All rights reserved.)
- Published
- 1998
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32. The role of v-Fgr myristoylation and the Gag domain in membrane binding and cellular transformation.
- Author
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Baker SJ, Cosenza SC, and Reddy EP
- Subjects
- 3T3 Cells, Animals, Binding Sites genetics, Cats, Mice, Mutation, Protein Binding, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, Sarcoma Viruses, Feline genetics, Cell Transformation, Viral genetics, Gene Products, gag genetics, Gene Products, gag metabolism, Retroviridae Proteins, Oncogenic genetics, Retroviridae Proteins, Oncogenic metabolism
- Abstract
The v-fgr oncogene encodes a chimeric oncoprotein composed of feline sarcoma virus (FeSV)-derived gag and cellular-derived actin and c-Fgr sequences. v-Fgr is myristoylated and membrane bound, two criteria which must be met for src kinases to induce cellular transformation. Although inhibition of myristoylation resulted in a decreased ability of v-Fgr to sediment with membranes from an NIH-3T3 P100 fraction, deletion of the gag domain caused nearly all of the protein to remain unbound and cytosolic. Systematic deletions within gag indicate that while amino acids 3 through 9 are critical determinants of myristoylation and/or define a domain which directs membrane localization, these residues cooperate with additional gag sequences when anchoring the protein to the plasma membrane. Furthermore, nonmyristoylated and/or cytoplasmic variants of v-Fgr failed to induce anchorage-independent growth of NIH-3T3 cells, indicating that proper subcellular localization of v-Fgr is a key factor in its ability to induce transformation., (Copyright 1998 Academic Press.)
- Published
- 1998
- Full Text
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33. Differential regulation of the retinoblastoma family of proteins during cell proliferation and differentiation.
- Author
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Garriga J, Limón A, Mayol X, Rane SG, Albrecht JH, Reddy EP, Andrés V, and Graña X
- Subjects
- 3T3 Cells metabolism, Animals, Cell Differentiation physiology, Cell Division physiology, Glioblastoma metabolism, Granulocyte Colony-Stimulating Factor pharmacology, Granulocytes cytology, Granulocytes drug effects, Humans, Liver Regeneration physiology, Male, Mice, Mice, Inbred BALB C, Muscle, Skeletal cytology, Muscle, Skeletal metabolism, Nuclear Proteins biosynthesis, Nuclear Proteins metabolism, Phosphoproteins biosynthesis, Phosphoproteins metabolism, Phosphorylation, Protein Processing, Post-Translational, Rats, Rats, Sprague-Dawley, Retinoblastoma Protein biosynthesis, Retinoblastoma Protein metabolism, Retinoblastoma-Like Protein p107, Retinoblastoma-Like Protein p130, Tumor Cells, Cultured, Nuclear Proteins physiology, Phosphoproteins physiology, Proteins, Retinoblastoma Protein physiology
- Abstract
In the present study we have analysed the regulation of pocket protein expression and post-transcriptional modifications on cell proliferation and differentiation, both in vivo and in vitro. There are marked changes in pocket protein levels during these transitions, the most striking differences being observed between p130 and p107. The mechanisms responsible for regulating pocket protein levels seem to be dependent on both cell type and pocket protein, in addition to their dependence on the cell growth status. Changes in retinoblastoma protein and p107 levels are independent of their state of phosphorylation. However, whereas p130 phosphorylation to forms characteristic of quiescent/differentiated cells results in the accumulation of p130 protein, phosphorylation of p130 to one or more forms characteristic of cycling cells is accompanied by down-regulation of its protein levels. We also show here that the phosphorylation status and protein levels of p130 and p107 are regulated in vivo as in cultured cells. In vivo, changes in p130 forms are correlated with changes in E2F complexes. Moreover, the modulation of p130 and p107 status during cell differentiation in vitro is consistent with the patterns of protein expression and phosphorylation status found in mouse tissues. Thus in addition to the direct disruption of pocket protein/E2F complexes induced by cyclin/cyclin-dependent kinase, the results we report here indicate that the differential modulation of pocket protein levels constitutes a major mechanism that regulates the pool of each pocket protein that is accessible to E2F and/or other transcription factors.
- Published
- 1998
- Full Text
- View/download PDF
34. Src kinases and not JAKs activate STATs during IL-3 induced myeloid cell proliferation.
- Author
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Chaturvedi P, Reddy MV, and Reddy EP
- Subjects
- Animals, Apoptosis, CSK Tyrosine-Protein Kinase, Cell Division, Cell Line, Cell Line, Transformed, DNA metabolism, Enzyme Activation, Janus Kinase 2, Mice, Mitogen-Activated Protein Kinase 1 metabolism, Mutagenesis, Protein-Tyrosine Kinases genetics, STAT3 Transcription Factor, src-Family Kinases, DNA-Binding Proteins metabolism, Interleukin-3 pharmacology, Mitogens pharmacology, Protein-Tyrosine Kinases metabolism, Proto-Oncogene Proteins, Trans-Activators metabolism
- Abstract
Interaction of IL-3 with its receptor is known to activate STAT-3 via phosphorylation of Tyrosine 701, which facilitates its dimerization and translocation to the nucleus, leading to the transcription of its target genes. In this communication, we have investigated the nature of tyrosine kinases that mediate STAT-3 phosphorylation during IL-3-mediated activation of myeloid cell proliferation. Our results show that interaction of IL-3 with its receptor leads to the activation of c-Src kinase activity, which in turn facilitates the binding of c-Src to STAT-3. This association leads to the phosphorylation of STAT-3, allowing this transcription factor to translocate to the nucleus. Expression of a dominant negative mutant of src (AMSrc) in these cells results in a block to IL-3 mediated phosphorylation of STAT-3, and its ability to bind to DNA. On the other hand, expression of a dominant negative mutant of JAK2 (JAK2KE) had no effect on IL-3-mediated activation of STAT-3. Our results also show that AMSrc does not affect the phosphorylation of JAK2, suggesting that JAK and STAT phosphorylation events are mediated by two independent pathways. Inhibition of c-Src activation by AMSrc, which leads to a block to STAT-3 activation, results in a dramatic inhibition of cell proliferation mediated by IL-3. However, expression of AMSrc does not activate apoptotic pathways. In contrast, expression of JAK2KE results in accelerated apoptosis of 32Dcl3 cells grown in the absence of IL-3 with concomitant down-regulation of Erk-2 kinase activity. These results suggest that Src family kinases mediate the phosphorylation of STATs and play a critical role in signal transduction pathways associated with myeloid cell proliferation while JAK kinases mediate the activation of Erk-2 pathway which appears to provide antiapoptotic signals. Thus the activation of JAKs and STATs appear to be two independent but related events, which dictate two separate biological outcomes, the combination of which results in proliferation and survival of myeloid precursor cells.
- Published
- 1998
- Full Text
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35. The C-terminal domain of B-Myb acts as a positive regulator of transcription and modulates its biological functions.
- Author
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Oh IH and Reddy EP
- Subjects
- Animals, Gene Expression Regulation, Mice, Proto-Oncogene Proteins c-myb, Cell Cycle Proteins, DNA-Binding Proteins genetics, Oncogenes, Proto-Oncogene Proteins genetics, Trans-Activators genetics, Transcription Factors genetics, Transcription, Genetic
- Abstract
The myb gene family consists of three members, named A-, B-, and c-myb. All three members of this family encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In this report, we have examined the biochemical and biological activities of murine B-myb and compared these properties with those of murine c-myb. In transient transactivation assays, murine B-myb exhibited transactivation potential comparable to that of c-myb. An analysis of deletion mutants of B-myb and c-myb showed that while the C-terminal domain of c-Myb acts as a negative regulator of transcriptional transactivation, the C-terminal domain of B-Myb functions as a positive enhancer of transactivation. To compare the biological activities of c-myb and B-myb, the two genes were overexpressed in 32Dcl3 cells, which are known to undergo terminal differentiation into granulocytes in the presence of granulocyte colony-stimulating factor (G-CSF). We observed that c-myb blocked the G-CSF-induced terminal differentiation of 32Dcl3 cells, resulting in their continued proliferation in the presence of G-CSF. In contrast, ectopic overexpression of B-myb blocked the ability of 32D cells to proliferate in the presence of G-CSF and accelerated the G-CSF-induced granulocytic differentiation of these cells. Similar studies with B-myb-c-myb chimeras showed that only chimeras that contained the C-terminal domain of B-Myb were able to accelerate the G-CSF-induced terminal differentiation of 32Dcl3 cells. These studies show that c-myb and B-myb do not exhibit identical biological activities and that the carboxyl-terminal regulatory domain of B-Myb plays a critical role in its biological function.
- Published
- 1998
- Full Text
- View/download PDF
36. AATYK: a novel tyrosine kinase induced during growth arrest and apoptosis of myeloid cells.
- Author
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Gaozza E, Baker SJ, Vora RK, and Reddy EP
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Differentiation, Cells, Cultured, Cloning, Molecular, Cytokines physiology, Enzyme Induction, Granulocyte Colony-Stimulating Factor physiology, In Vitro Techniques, Mice, Molecular Sequence Data, Protein Biosynthesis, Protein Structure, Tertiary, Protein-Tyrosine Kinases isolation & purification, Transcription, Genetic, Up-Regulation, src Homology Domains, Apoptosis genetics, Bone Marrow Cells enzymology, Protein-Tyrosine Kinases genetics
- Abstract
Apoptosis, or programmed cell death, is a process where developmental or environmental stimuli activate a genetic program to implement a series of events that culminate in cell death. To study the nature of genes that are induced during the apoptotic death of myeloid precursor cells, we utilized the 32Dcl3 cell line, which is derived from normal mouse bone marrow, is non-tumorigenic and diploid. These cells are strictly dependent on IL-3 for growth and apoptose when deprived of IL-3. However, when these cells are transferred to medium containing G-CSF, the cell number increases 4-5-fold and after 12 days the entire population is differentiated into granulocytes followed by apoptotic death. In our search for genes that are induced during apoptosis and/or terminal differentiation of 32Dcl3 cells, we identified a novel gene termed AATYK (Apoptosis Associated Tyrosine Kinase), whose expression is dramatically upregulated during IL-3 deprivation as well as G-CSF-induced terminal differentiation. In this report, we describe the sequence of the cDNA clone, derived from the mRNA transcript of this gene. These studies show that this gene encodes a protein with a tyrosine kinase domain at the N-terminal end and a proline-rich domain at the C-terminal end. We also report that the expression of this gene is blocked in v-abl or bcr-abl transformed myeloid cells which are unable to apoptose when grown in the absence of IL-3. However, AATYK expression is induced in 32D cells transformed by the v-abl gene when these cells are incubated in the presence of DMSO, which induces growth arrest and apoptotic death of the cells. On the other hand, DMSO fails to induce apoptosis or AATYK expression in 32D cells transformed by the bcr-abl oncogene, suggesting that AATYK expression may be a necessary pre-requisite for the induction of growth arrest and/or apoptosis of myeloid precursor cells.
- Published
- 1997
- Full Text
- View/download PDF
37. cAMP-induced NF-kappaB (p50/relB) binding to a c-myb intronic enhancer correlates with c-myb up-regulation and inhibition of erythroleukemia cell differentiation.
- Author
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Suhasini M, Reddy CD, Reddy EP, DiDonato JA, and Pilz RB
- Subjects
- Animals, Colforsin pharmacology, Cyclic AMP analogs & derivatives, Genes, Reporter, Leukemia, Erythroblastic, Acute metabolism, Leukemia, Erythroblastic, Acute pathology, Mice, Protein Binding, RNA, Messenger genetics, RNA, Messenger metabolism, Subcellular Fractions metabolism, Transcription Factors metabolism, Tumor Cells, Cultured, Cell Differentiation genetics, Cyclic AMP pharmacology, Enhancer Elements, Genetic, Introns, Leukemia, Erythroblastic, Acute genetics, NF-kappa B metabolism, Oncogenes
- Abstract
During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogues and the adenyl cyclase-stimulating agent forskolin. We found that these drugs prevented the late down-regulation of c-myb which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels was due to increased mRNA transcription, we examined the transcription factors NF-kappaB and AP-1 which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contained p50 and RelB; in co-transfection assays, p50 and RelB transactivated a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells which could cooperate with NF-kappaB. We conclude that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers.
- Published
- 1997
- Full Text
- View/download PDF
38. Murine A-myb gene encodes a transcription factor, which cooperates with Ets-2 and exhibits distinctive biochemical and biological activities from c-myb.
- Author
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Oh IH and Reddy EP
- Subjects
- 3T3 Cells, Animals, Cell Differentiation, Gene Expression Regulation, Developmental, Granulocyte Colony-Stimulating Factor pharmacology, Granulocytes cytology, Hematopoiesis, Macromolecular Substances, Mice, Promoter Regions, Genetic, Proto-Oncogene Protein c-ets-2, Proto-Oncogene Proteins c-myb, Sequence Deletion, Structure-Activity Relationship, Transcription, Genetic, Transcriptional Activation, DNA-Binding Proteins, Proto-Oncogene Proteins physiology, Repressor Proteins, Trans-Activators physiology, Transcription Factors
- Abstract
The myb gene family consists of three members, named A-, B-, and c-myb, which encode nuclear proteins that bind to DNA and function as regulators of transcription. Our results show that murine A-myb is a poor transactivator of transcription compared with murine c-myb. Deletion of the COOH-terminal domain of A-Myb, or co-expression with Ets-2 resulted in increased transactivation potential. While ectopic overexpression of c-myb in 32Dcl3 cells results in a block to the ability of these cells to undergo terminal differentiation resulting in indefinite growth in granulocyte-colony-stimulating factor (G-CSF), similar overexpression of A-myb results in growth arrest and concomitant terminal differentiation of 32D cells into granulocytes. Co-expression of A-myb and ets-2 in these cells results in the restoration of the proliferative activity of the cells in G-CSF, but fails to induce a block to G-CSF-induced terminal differentiation. However, overexpression of the COOH-terminal deletion mutant of A-myb results in a block to G-CSF-induced differentiation of 32D cells, suggesting that the distinctive biological phenotypes produced by A-myb and c-myb genes are mediated by their COOH-terminal domains.
- Published
- 1997
- Full Text
- View/download PDF
39. Abrogation of interleukin-3 dependence of myeloid cells by the v-src oncogene requires SH2 and SH3 domains which specify activation of STATs.
- Author
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Chaturvedi P, Sharma S, and Reddy EP
- Subjects
- Acute-Phase Proteins metabolism, Animals, Cell Line, Electrophoresis, Polyacrylamide Gel, Hematopoietic Stem Cells drug effects, Mice, Protein-Tyrosine Kinases metabolism, Recombinant Fusion Proteins pharmacology, Retroviridae Proteins, Oncogenic pharmacology, STAT1 Transcription Factor, STAT3 Transcription Factor, STAT5 Transcription Factor, Signal Transduction, Structure-Activity Relationship, DNA-Binding Proteins metabolism, Hematopoietic Stem Cells metabolism, Interleukin-3 metabolism, Milk Proteins, Oncogene Protein pp60(v-src) pharmacology, Trans-Activators metabolism, src Homology Domains
- Abstract
The v-src oncogene encodes a nonreceptor tyrosine kinase. When this gene was expressed in the myeloblastic cell line 32Dcl3, it was found to abrogate interleukin-3 (IL-3) dependence of this cell line and to block its ability to terminally differentiate into granulocytes in response to granulocyte colony-stimulating factor (GCSF). In contrast, a highly related tyrosine kinase gene, v-fgr, fails to render this cell line IL-3 independent for growth or to block its ability to undergo terminal differentiation in the presence of GCSF. The active structural domains of v-src that are responsible for the abrogation of IL-3 dependence of myeloid cells and the mechanisms by which v-src transforms these cells are at present unclear. To identify the domains in v-src which are responsible for this activity, we constructed several chimeric recombinants between the v-src and the related Src family member v-fgr by replacing portions of v-src with corresponding domains of v-fgr. These chimeric DNAs were transfected into 32Dcl3 cells and examined for their abilities to render this cell line IL-3 independent. Our results show that only chimeras containing both the SH3 and the SH2 domains of v-src were capable of rendering the 32Dcl3 cell line IL-3 independent. To understand the possible mechanisms underlying the IL-3-independent growth of v-src-transformed 32Dcl3 cells, we examined the phosphorylation status of JAK-1, JAK-2, and JAK-3 kinases in the v-src- and v-fgr-transformed 32Dcl3 cells. Our results show that none of the JAK kinases are constitutively phosphorylated by v-src or v-fgr. We then examined the phosphorylation status of the STAT (signal transducers and activators of transcription) family of transcription factors. Our results show that STAT1, STAT3, and STAT5 exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, while such constitutive phosphorylation is not seen in v-fgr-transformed cell lines. Our results also show that STAT3 coimmunoprecipitates with v-Src, suggesting that the activation of STAT3 occurs due to direct association with v-Src. However, STAT1 and STAT5, which also exist in a constitutively phosphorylated state in v-src-transformed 32Dcl3 cells, do not coimmunoprecipitate with v-Src, suggesting that these proteins either interact weakly with v-Src or are phosphorylated by a mechanism distinctive from that of STAT3.
- Published
- 1997
- Full Text
- View/download PDF
40. Arrest of spermatogenesis and defective breast development in mice lacking A-myb.
- Author
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Toscani A, Mettus RV, Coupland R, Simpkins H, Litvin J, Orth J, Hatton KS, and Reddy EP
- Subjects
- Animals, Female, Gene Expression, Gene Targeting, Germ-Line Mutation, Infertility, Male genetics, Male, Mammary Glands, Animal anatomy & histology, Mammary Glands, Animal embryology, Mammary Glands, Animal growth & development, Meiosis, Mice, Molecular Sequence Data, Pregnancy, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-myb, Spermatogenesis genetics, Stem Cells, Testis anatomy & histology, Testis embryology, Testis growth & development, Trans-Activators genetics, Mammary Glands, Animal physiology, Proto-Oncogene Proteins physiology, Spermatogenesis physiology, Testis physiology, Trans-Activators physiology
- Abstract
The Myb gene family currently consists of three members, named A-, B- and c-myb. These genes encode nuclear proteins that bind DNA in a sequence-specific manner and function as regulators of transcription. In adult male mice, A-myb is expressed predominantly in male germ cells. In female mice, A-myb is expressed in breast ductal epithelium, mainly during pregnancy-induced ductal branching and alveolar development. We report here that mice homozygous for a germline mutation in A-myb develop to term but show defects in growth after birth and male infertility due to a block in spermatogenesis. Morphological examination of the testes of A-myb-/- males revealed that the germ cells enter meiotic prophase and arrest at pachytene. In adult homozygous null A-myb female mice, the breast epithelial compartment showed underdevelopment of breast tissue following pregnancy and the female mice were unable to nurse their newborn pups. These results demonstrate that A-myb plays a critical role in spermatogenesis and mammary gland development.
- Published
- 1997
- Full Text
- View/download PDF
41. Nuclear oncology and the Imagene concept.
- Author
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Urbain JL, Vekemans MC, Lemieux SK, Cosenza SC, Senadhi VK, Milestone BN, and Reddy EP
- Subjects
- Humans, Molecular Probes, Peptides, Radioimmunodetection trends, Radiopharmaceuticals, Tomography, Emission-Computed trends, Genome, Human, Medical Oncology trends, Nuclear Medicine trends
- Abstract
Over the past 2 decades we have witnessed an explosion of new radioisotopic tracers aimed at detecting, staging and eventually treating tumors. In fact, nuclear oncology has evolved into a field on its own. Aside from aspecific radioisotopic tracers such as thallium 201 or gallium 67, clinicians and oncologists can now use specific radiolabeled monoclonal antibodies and metabolic tracers. In the near future, molecular probes based on the sequencing of the human genome with an exquisite specificity should also become available. In this article, we shall review the most recent developments in this new field.
- Published
- 1997
42. Structural organization and chromosomal mapping of JAK3 locus.
- Author
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Kumar A, Toscani A, Rane S, and Reddy EP
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromosome Mapping, Cloning, Molecular, Conserved Sequence, Humans, In Situ Hybridization, Fluorescence, Janus Kinase 1, Janus Kinase 2, Janus Kinase 3, Mice, Molecular Sequence Data, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Transcription, Genetic, Chromosomes, Human, Pair 19, Protein-Tyrosine Kinases genetics, Proto-Oncogene Proteins
- Abstract
In this study, we have isolated the genomic DNA clone encoding the murine JAK3 gene and determined its sequence. Partial genomic clones of the JAK1 and JAK2 genes encoding the tyrosine kinase domain were also isolated and compared with JAK3. The genomic structure of JAK3 consists of 23 exons. The exon/intron boundaries and the distribution of coding sequences within the exons of each of the three JAK genes were found to follow a similar pattern suggesting that various members of the JAK family have originated from a single primordial gene by duplication and the structure has been closely maintained through evolution. Using in situ hybridization and FISH analysis, we have mapped the JAK3 gene to human chromosome 19p13.1-p13.2.
- Published
- 1996
43. Transcriptional activation potential of normal and tumor-associated myb isoforms does not correlate with their ability to block GCSF-induced terminal differentiation of murine myeloid precursor cells.
- Author
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Patel G, Tantravahi R, Oh IH, and Reddy EP
- Subjects
- Animals, Cell Differentiation drug effects, Cell Differentiation physiology, Cell Line, Genetic Vectors, Granulocyte Colony-Stimulating Factor pharmacology, Hematopoietic Stem Cells drug effects, Hematopoietic Stem Cells physiology, Isomerism, Mice, Neoplasm Proteins genetics, Phenotype, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-myb, Retroviridae genetics, Trans-Activators genetics, Transformation, Genetic, Granulocyte Colony-Stimulating Factor antagonists & inhibitors, Hematopoietic Stem Cells cytology, Neoplasm Proteins physiology, Proto-Oncogene Proteins physiology, Trans-Activators physiology, Transcriptional Activation
- Abstract
The myb gene has been shown to be an important regulator of hematopoietic cell proliferation, differentiation and apoptosis. Activation of the myb gene into an oncogenic form has involved structural alterations to the coding sequences. Thus, the v-myb gene encoded by the Avian Myeloblastosis Virus, is truncated at both the 5' and 3' ends. Additionally, tumor cells containing rearrangements in the myb locus, such as the ABPL tumors or NFS60 tumor cell line have recently been shown to display a heterogeneity of structure. In this study, we examined the growth and differentiation properties of clonal cell lines derived from 32Dcl3 which harbor myb transgenes; derived from v-myb, and the ABPL-1, ABPL-2, ABPL-4 and NFS-60 cell lines. Retroviral vectors containing the appropriate myb cDNAs were produced, transfected into packaging cell lines, and the viruses were used to generate the 32D derivative cell clones. Abrogation of IL-3 dependence was never observed in any cell line. Expression of c-myb, ABPL-1-myb and ABPL-2-myb isoforms in 32D cells resulted in a block to their ability to terminally differentiate into granulocytes at the pro-myelocytic stage. However, expression of ABPL-4-myb or NFS60-myb in these cells failed to result in a similar effect. These cells differentiated into granulocytes in the presence of G-CSF, albeit more slowly than control 32Dcl3 cells. We also examined the ability of various Myb-isoforms to transactivate transcription of reporter genes containing Myb-binding elements in their promoter/enhancer sequences, to determine whether the phenotypic effects produced by these various isoforms correlate with their ability to transactivate transcription. Our results show that while v-myb and c-myb transactivated transcription equally well, the NFS60-myb exhibited the highest levels of transcriptional transactivation. The ABPL-1, ABPL-2 and ABPL-4-myb isoforms showed very low levels of transcriptional transactivation potential with the same reporter genes. These results suggest that the ability of various Myb-isoforms to transactivate transcription does not by itself correlate with their ability to induce a block to G-CSF-induced terminal differentiation of myeloid precursor cells.
- Published
- 1996
44. Murine myeloid leukemic cells with disrupted myb loci show splicing anomalies that account for heterogeneous sizes in myb proteins.
- Author
-
Tantravahi R, Dudek H, Patel G, and Reddy EP
- Subjects
- Amino Acid Sequence, Base Sequence, Chromosome Mapping, Gene Rearrangement, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Protein Biosynthesis, Proto-Oncogene Proteins c-myb, RNA, Messenger genetics, RNA, Messenger metabolism, Ribonucleases metabolism, Transcription, Genetic, Transcriptional Activation, Transformation, Genetic, Tumor Cells, Cultured, Leukemia, Myeloid genetics, Leukemia, Myeloid metabolism, Proto-Oncogene Proteins biosynthesis, Proto-Oncogene Proteins genetics, RNA Splicing, Trans-Activators biosynthesis, Trans-Activators genetics
- Abstract
The ABPL tumor cell lines represent a group of myeloid cell lines which contain an altered myb locus due to viral insertional mutagenesis within the third exon of c-myb. Immunoprecipitation analysis of the proteins produced in three ABPL lines revealed an interesting anomaly. Despite the invariant position of the virus integration event, the three ABPL tumor cell lines we examined (ABPL-1, ABPL-2 and ABPL-4) produced three different sized proteins. In this report, we examined the molecular basis for this protein size heterogeneity. Molecular cloning and sequence analysis of the cDNAs derived from the myb transcripts show that ABPL-1 tumor produces a tripartate mRNA containing sequences derived from the viral gag and env genes fused to the myb coding region. This results in the synthesis of a 74 kd protein. In the ABPL-2 tumor line, a gag-myb fusion protein is produced which is of 68 kd. In ABPL-4 cell line a gag-myb fusion protein is produced which contains an internal deletion of coding sequences derived from exons 13 and 14. This deletion results in the synthesis of a 59 kd protein in ABPL-4 tumor cell line. These observations were further confirmed by RNase protection assays which demonstrate the presence of aberrantly spliced mRNAs in ABPL-1 and ABPL-4 tumor cells but not in cells containing an undisrupted c-myb locus. In vitro translation and immuno-precipitation analysis of the cRNAs derived from the ABPL-1, ABPL-2 and ABPL-4 cDNAs show the synthesis of protein products that were identical to Myb proteins produced by these tumors in vivo. These results suggest that integration of Mo-MuLV within the c-myb locus not only results in deletions of the 5' end of the transcript but splicing aberrations within the encoded mRNA, which results in the synthesis of a heterogeneous array of proteins, not seen in normal hematopoietic cells.
- Published
- 1996
45. Temporal patterns of A-myb and B-myb gene expression during testis development.
- Author
-
Latham KE, Litvin J, Orth JM, Patel B, Mettus R, and Reddy EP
- Subjects
- Animals, Cell Division physiology, DNA-Binding Proteins genetics, Down-Regulation, Female, Gene Expression, Male, Meiosis physiology, Mice, Mice, Inbred C57BL, Prophase physiology, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-myb, RNA, Messenger genetics, RNA, Messenger metabolism, Spermatocytes cytology, Spermatocytes metabolism, Spermatogonia cytology, Spermatogonia metabolism, Testis embryology, Trans-Activators genetics, Transcription Factors genetics, Cell Cycle Proteins, DNA-Binding Proteins biosynthesis, Proto-Oncogene Proteins biosynthesis, Spermatogenesis physiology, Testis growth & development, Testis metabolism, Trans-Activators biosynthesis, Transcription Factors biosynthesis
- Abstract
We recently reported the cloning and sequencing of the mouse A-myb proto-oncogene cDNA and the abundant expression of this mRNA primarily in the testis of adult mice. The A-myb mRNA is detectable by in situ hybridization specifically in the spermatogenic cells, and is downregulated during terminal differentiation. A low level of expression is observed in a few other tissues, including ovary, spleen and brain. We have extended those studies by examining A-myb and B-myb expression during testis development in the mouse. The A-myb and B-myb genes are both expressed in a cell- and stage-specific manner during testis development. The B-myb mRNA is expressed most highly in gonocytes of the fetal testis and in spermatogonia and early spermatocytes in the adult. B-myb expression decreases at day 18 post partum, coincident with the initial appearance of late pachytene spermatocytes. B-myb expression was also detectable in some interstitial cells of the fetal and adult testis. The A-myb mRNA was not detectable by in situ hybridization in fetal day 15.5 gonocytes but was detectable at a low abundance by RT-PCR in fetal and newborn mice. A-myb mRNA expression increased at post-natal day 10, when primary spermatocytes first appear. In the adult, the A-myb mRNA was expressed highly in a sub-population of spermatogonia and in primary spermatocytes, but was not detectable in spermatids. This expression of A-myb is consistent with the meiotic arrest that is observed in A-myb-deficient male mice. We conclude that B-myb may play a critical role in controlling the proliferation or differentiation of gonocytes and spermatogonia and possibly the somatic lineages as well, whereas A-myb is required for progression through the first meiotic prophase. These distinct roles for B-myb and A-myb during spermatogenesis may reflect distinct transactivation potentials of the two proteins. Further studies to determine the functions of A-myb and B-myb in the developing testis should improve our understanding of the molecular events associated with spermatogenesis and differentiation of the Sertoli and other somatic cell types of the testis.
- Published
- 1996
46. Multi-unit anti-BCR-ABL ribozyme therapy in chronic myelogenous leukemia.
- Author
-
Leopold LH, Shore SK, and Reddy EP
- Subjects
- Animals, Base Sequence, Fusion Proteins, bcr-abl biosynthesis, Fusion Proteins, bcr-abl genetics, Humans, Kinetics, Molecular Sequence Data, RNA, Catalytic chemistry, RNA, Catalytic metabolism, RNA, Messenger metabolism, Substrate Specificity, Transfection, Tumor Cells, Cultured, Fusion Proteins, bcr-abl antagonists & inhibitors, Genetic Therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive therapy, RNA, Catalytic therapeutic use
- Abstract
In this review, we summarize and update our data on the development of a multi-unit anti-BCR/ABL ribozyme. In vitro studies comparing several anti-BCR/ABL ribozymes demonstrated that a triple-unit ribozyme is the most efficient. Detailed kinetic analysis revealed this ribozyme to have a lower Kcat, most likely due to non homologous bases at restriction enzyme sites used in ribozyme construction. Delivery of this ribozyme to a BCR/ABL transformed cell line by a novel vehicle targeting the folate receptor resulted in a 3 log reduction in BCR/ABL mRNA when analyzed by RT-PCR. This delivery strategy reversed the IL-3 independence of this cell line. Retroviral vectors containing genes coding for the multi-unit ribozyme have been constructed and their use to effect BCR/ABL transformed cell biology is discussed.
- Published
- 1996
- Full Text
- View/download PDF
47. Identification of a novel Bcl-2 related gene, BRAG-1, in human glioma.
- Author
-
Das R, Reddy EP, Chatterjee D, and Andrews DW
- Subjects
- Amino Acid Sequence, Base Sequence, Gene Expression, Humans, Molecular Sequence Data, Sequence Analysis, DNA, Apoptosis genetics, Brain Neoplasms genetics, Genes genetics, Glioma genetics, Open Reading Frames genetics
- Abstract
Programmed cell death (apoptosis) plays a major role in embryogenesis, in mature organ homeostasis and in many disease states including cancer. Apoptosis occurs as an orderly cell-intrinsic suicide program regulated by a family of genes related to Bcl-2. Here, we describe the cloning and molecular characterization of a gene homologous to Bcl-2 from a human glioma. This gene named BRAG-1 (for brain-related apoptosis gene) has an open reading frame that encodes for a protein of 31 kDa sharing significant sequence homology with the Bcl-2 family of genes in the BH1 and BH2 regions. Northern blot analyses revealed that BRAG-1 is expressed in human gliomas as a 1.8 kb message. This gene, interestingly, was found to be expressed predominantly in normal human brain as a 4.5 kb transcript which is different in size from the message found in tumor tissues. These results suggest that BRAG-1 may be rearranged in human gliomas leading to its over-expression as a truncated transcript. Utilizing a bacterial expression vector, we produced BRAG-1 protein which was found to cross-react with a Bcl-2 monoclonal antibody, further suggesting structural and immunological similarity to Bcl-2.
- Published
- 1996
48. Transducers of life and death: TNF receptor superfamily and associated proteins.
- Author
-
Baker SJ and Reddy EP
- Subjects
- Animals, Apoptosis, Humans, Proteins physiology, TNF Receptor-Associated Factor 1, TNF Receptor-Associated Factor 2, fas Receptor physiology, Receptors, Tumor Necrosis Factor physiology, Signal Transduction
- Abstract
Signal transduction pathways which are initiated by members of the TNF superfamily utilize receptors which are devoid of intrinsic catalytic activity. Isolation and characterization of death domain (TNF-RI, Fas, TRADD, FADD/MORT-1, RIP) and TRAF domain-containing proteins (TRAF-1, TRAF-2, TRAF-3) have partially bridged a large molecular gap within one of several signaling pathways which originate at the plasma membrane and terminate in the nucleus. The ability of these two protein families to selectively dimerize and bind to related receptors allows them to govern diverse cellular responses which culminate in cellular proliferation, differentiation, effector functions, and apoptosis.
- Published
- 1996
49. Cellular factors binding to a novel cis-acting element mediate steroid hormone responsiveness of mouse mammary tumor virus promoter.
- Author
-
Lee KI, Reddy EP, and Reddy CD
- Subjects
- 3T3 Cells, Animals, Base Sequence, Breast Neoplasms, Cell Line, Chloramphenicol O-Acetyltransferase biosynthesis, Female, Gene Expression, Genes, Viral, Humans, Mammary Tumor Virus, Mouse metabolism, Mice, Molecular Sequence Data, Mutagenesis, Mutagenesis, Site-Directed, Plasmids, Polymerase Chain Reaction, Receptors, Progesterone metabolism, Repetitive Sequences, Nucleic Acid, Sequence Deletion, Transcription, Genetic drug effects, Transfection, Tumor Cells, Cultured, DNA-Binding Proteins metabolism, Dexamethasone pharmacology, Mammary Tumor Virus, Mouse genetics, Progesterone pharmacology, Promoter Regions, Genetic, Receptors, Glucocorticoid metabolism, Regulatory Sequences, Nucleic Acid, Transcriptional Activation
- Abstract
Steroid hormone receptors regulate mouse mammary tumor virus (MMTV) gene expression by binding to hormone response DNA elements present in the long terminal repeat. Tissue-specific expression of MMTV is unlikely to be regulated by steroid hormone-receptor complex alone, and mammary cell-specific factors might play a role in the hormone-induced transcriptional activation. In this report we have investigated the function of a novel cis-acting element designated Kil (-204 to -188) which is located adjacent to the distal glucocorticoid response element, in steroid hormone-induced transcription of MMTV. Electrophoretic mobility shift assays indicate that cellular factors bind to the Kil element, and dexamethasone stimulation results in alterations in the binding pattern of proteins in this region. By transient transfection assays using wild type and deletion mutants of the Kil element, we show that this novel cis-acting element is necessary for hormone-induced transcription of MMTV and functions in mammary tumor cells but not in NIH/3T3 cells. Mutagenesis of the Kil sequence suggests that the entire Kil element functioning as one unit is necessary for hormone-induced transcription of MMTV. When placed in the context of heterologous promoters, neither Kil element nor glucocorticoid response element is able to induce significant hormone-induced transcription of MMTV. The presence of both the DNA elements in tandem results in optimal induction of transcription in the presence of steroid hormones. Our results also indicate that the Kil element functions in human breast carcinoma cell lines such as T47D and MCF-7. These results suggest that Kil element in combination with distal glucocorticoid response element functions as a mammary cell-specific enhancer to regulate MMTV transcription.
- Published
- 1995
- Full Text
- View/download PDF
50. B cell differentiation: role of E2A and Pax5/BSAP transcription factors.
- Author
-
Baker SJ and Reddy EP
- Subjects
- Animals, B-Lymphocytes cytology, Base Sequence, Cell Differentiation, Consensus Sequence, Enhancer Elements, Genetic, Gene Expression Regulation, Genes, Immunoglobulin, Genes, Switch, Helix-Loop-Helix Motifs, Hematopoiesis, Mice, Mice, Transgenic, Molecular Sequence Data, PAX5 Transcription Factor, Promoter Regions, Genetic, RNA, Messenger genetics, TCF Transcription Factors, Transcription Factor 7-Like 1 Protein, B-Lymphocytes physiology, DNA-Binding Proteins physiology, Nuclear Proteins physiology, Transcription Factors
- Abstract
Transcriptional regulation of lineage specific genes has the ability to dictate both the proliferative and differentiative potentials of a pluipotent precursor cell. The E2A and Pax5/BSAP genes encode transcription factors which bind to B cell specific promoters and enhancers and guide the development of immature, but committed cells into mature B lymphocytes which express and secrete immunoglobulins. In vitro analysis has consistently suggested that these proteins regulate distinct classes of genes during B cell differentiation; however, recent targeted gene disruption and transgene expression in mice has indicated that these genes may actually be components of a single regulatory mechanism which is essential for both B lymphocyte differentiation and proliferation.
- Published
- 1995
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