ABSTRACT
BACKGROUND: Lissencephaly is a severe brain malformation in part caused by mutations in the LIS1 gene. LIS1 interacts with microtubule-associated proteins, and enhances transport of microtubule fragments. Previously we showed that LIS1 interacts with HIV-1 Tat protein and that this interaction was mediated by WD40 domains of LIS1. In the present study, we analyze the effect of LIS1 on Tat-mediated transcription of HIV-1 LTR. RESULTS: Tat-mediated HIV-1 transcription was upregulated in 293 cells transfected with LIS1 expression vector. The WD5 but not the N-terminal domain of LIS1 increases Tat-dependent HIV-1 transcription. The effect of LIS1 was similar to the effect of okadaic acid, an inhibitor of protein phosphatase 2A (PP2A). We then analyzed the effect of LIS1 on the activity of PP2A in vitro. We show that LIS1 and its isolated WD5 domain but not the N-terminal domain of LIS1 blocks PP2A activity. CONCLUSION: Our results show that inhibition of PP2A by LIS1 induces HIV-1 transcription. Our results also point to a possibility that LIS1 might function in the cells as a yet unrecognized regulatory subunit of PP2A.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/pharmacology , HIV-1/metabolism , Microtubule-Associated Proteins/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Transcription, Genetic/drug effects , Up-Regulation , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Cell Line , Gene Expression Regulation, Viral , Gene Products, tat/genetics , Gene Products, tat/metabolism , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Phosphoprotein Phosphatases/genetics , Protein Phosphatase 2 , tat Gene Products, Human Immunodeficiency VirusABSTRACT
BACKGROUND: Increasing evidence suggests that adjuvant systemic chemotherapy is necessary for the survival of breast cancer patients. Antitumor agents are more effective when used in combination with drugs exhibiting different mechanisms of action than when used alone. Previous studies from this laboratory have shown that raloxifene (RAL) attenuation of 5-fluorouracil/methotrexate (5-FU/MTX) cytotoxicity to breast cancer cells was sequence-dependent. The aim was to evaluate the same combination of RAL, 5-FU and MTX to determine the most effective regimes and cellular mechanisms of action to mitigate MTX cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: The sequence-dependent interaction among MTX, 5-FU and RAL on the proliferation and viability of human bone marrow HS-5 cells was determined by the MTT assay and the Trypan blue dye exclusion assay by exposing the cells to MTX, 5-FU and RAL alone, RAL 24 h prior to 5-FU followed 2 h later by MTX, and 5-FU 2 h prior to MTX followed 24 h later by RAL. The control cells were untreated. RESULTS: The growth rate in MCF-7 in early RAL was 68 +/- 3.07% and late RAL 37 +/- 2.05% of the control rate, whereas in bone marrow the same drug combinations exhibit a significant protection against MTX cytotoxity, with the early RAL combination yielding 81 +/- 3.77% and late 54 +/- 2.74% of the control. The finding was further supported by cell flow cytometry and Western blot analysis. CONCLUSION: Sequence-dependent administration of RAL in combination with 5-FU/MTX may have maximum antineoplastic activity in breast cancer while at the same time provide protection to human bone marrow.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Bone Marrow Diseases/prevention & control , Raloxifene Hydrochloride/pharmacology , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Blotting, Western , Bone Marrow/drug effects , Bone Marrow Diseases/chemically induced , Breast Neoplasms/drug therapy , Cell Cycle/drug effects , Cell Growth Processes/drug effects , Drug Interactions , Flow Cytometry , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Methotrexate/administration & dosage , Methotrexate/adverse effects , Raloxifene Hydrochloride/administration & dosageABSTRACT
BACKGROUND: HIV-1 Tat activates transcription of HIV-1 viral genes by inducing phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Tat can also disturb cellular metabolism by inhibiting proliferation of antigen-specific T lymphocytes and by inducing cellular apoptosis. Tat-induced apoptosis of T-cells is attributed, in part, to the distortion of microtubules polymerization. LIS1 is a microtubule-associated protein that facilitates microtubule polymerization. RESULTS: We identified here LIS1 as a Tat-interacting protein during extensive biochemical fractionation of T-cell extracts. We found several proteins to co-purify with a Tat-associated RNAPII CTD kinase activity including LIS1, CDK7, cyclin H, and MAT1. Tat interacted with LIS1 but not with CDK7, cyclin H or MAT1 in vitro. LIS1 also co-immunoprecipitated with Tat expressed in HeLa cells. Further, LIS1 interacted with Tat in a yeast two-hybrid system. CONCLUSION: Our results indicate that Tat interacts with LIS1 in vitro and in vivo and that this interaction might contribute to the effect of Tat on microtubule formation.
