Novel role of Giα2 in cell migration: Downstream of PI3-kinase-AKT and Rac1 in prostate cancer cells.

Tumor cell motility is the essential step in cancer metastasis. Previously, we showed that oxytocin and epidermal growth factor (EGF) effects on cell migration in prostate cancer cells require Giα2 protein. In the current study, we investigated the interactions among G-protein coupled receptor (GPCR), Giα2, PI3-kinase, and Rac1 activation in the induction of migratory and invasive behavior by diverse stimuli. Knockdown and knockout of endogenous Giα2 in PC3 cells resulted in attenuation of transforming growth factor ß1 (TGFß1), oxytocin, SDF-1α, and EGF effects on cell migration and invasion. In addition, knockdown of Giα2 in E006AA cells attenuated cell migration and overexpression of Giα2 in LNCaP cells caused significant increase in basal and EGF-stimulated cell migration. Pretreatment of PC3 cells with Pertussis toxin resulted in attenuation of TGFß1- and oxytocin-induced migratory behavior and PI3-kinase activation without affecting EGF-induced PI3-kinase activation and cell migration. Basal- and EGF-induced activation of Rac1 in PC3 and DU145 cells were not affected in cells after Giα2 knockdown. On the other hand, Giα2 knockdown abolished the migratory capability of PC3 cells overexpressing constitutively active Rac1. The knockdown or knockout of Giα2 resulted in impaired formation of lamellipodia at the leading edge of the migrating cells. We conclude that Giα2 protein acts at two different levels which are both dependent and independent of GPCR signaling to induce cell migration and invasion in prostate cancer cells and its action is downstream of PI3-kinase-AKT-Rac1 axis.

A Leu to Ile but not Leu to Val change at HIV-1 reverse transcriptase codon 74 in the background of K65R mutation leads to an increased processivity of K65R+L74I enzyme and a replication competent virus.

BACKGROUND: The major hurdle in the treatment of Human Immunodeficiency virus type 1 (HIV-1) includes the development of drug resistance-associated mutations in the target regions of the virus. Since reverse transcriptase (RT) is essential for HIV-1 replication, several nucleoside analogues have been developed to target RT of the virus. Clinical studies have shown that mutations at RT codon 65 and 74 which are located in ß3-ß4 linkage group of finger sub-domain of RT are selected during treatment with several RT inhibitors, including didanosine, deoxycytidine, abacavir and tenofovir. Interestingly, the co-selection of K65R and L74V is rare in clinical settings. We have previously shown that K65R and L74V are incompatible and a R→K reversion occurs at codon 65 during replication of the virus. Analysis of the HIV resistance database has revealed that similar to K65R+L74V, the double mutant K65R+L74I is also rare. We sought to compare the impact of L→V versus L→I change at codon 74 in the background of K65R mutation, on the replication of doubly mutant viruses. METHODS: Proviral clones containing K65R, L74V, L74I, K65R+L74V and K65R+L74I RT mutations were created in pNL4-3 backbone and viruses were produced in 293T cells. Replication efficiencies of all the viruses were compared in peripheral blood mononuclear (PBM) cells in the absence of selection pressure. Replication capacity (RC) of mutant viruses in relation to wild type was calculated on the basis of antigen p24 production and RT activity, and paired analysis by student t-test was performed among RCs of doubly mutant viruses. Reversion at RT codons 65 and 74 was monitored during replication in PBM cells. In vitro processivity of mutant RTs was measured to analyze the impact of amino acid changes at RT codon 74. RESULTS: Replication kinetics plot showed that all of the mutant viruses were attenuated as compared to wild type (WT) virus. Although attenuated in comparison to WT virus and single point mutants K65R, L74V and L74I; the double mutant K65R+L74I replicated efficiently in comparison to K65R+L74V mutant. The increased replication capacity of K65R+L74I viruses in comparison to K65R+L74V viruses was significant at multiplicity of infection 0.01 (p = 0.0004). Direct sequencing and sequencing after population cloning showed a more pronounced reversion at codon 65 in viruses containing K65R+L74V mutations in comparison to viruses with K65R+L74I mutations. In vitro processivity assays showed increased processivity of RT containing K65R+L74I in comparison to K65R+L74V RT. CONCLUSIONS: The improved replication kinetics of K65R+L74I virus in comparison to K65R+L74V viruses was due to an increase in the processivity of RT containing K65R+L74I mutations. These observations support the rationale behind structural functional analysis to understand the interactions among unique RT mutations that may emerge during the treatment with specific drug regimens.

Replication-independent expression of anti-apoptosis marker genes in human peripheral blood mononuclear cells infected with the wild-type HIV-1 and reverse transcriptase variants.

Clinical trials with highly-active antiretroviral therapy (HAART) have shown that a substantial number of patients continue to show a decrease in viral load and/or increase or stable CD4(+) T-cell numbers even in the presence of multidrug resistant (MDR) viruses. We compared replication capacity (RC) and expression of anti-apoptosis marker genes (AAMGs) in human peripheral blood mononuclear (PBM) cells infected with NL4-3 (wild-type; WT) and mutant viruses. Replication kinetics assays showed a significant decrease in RC of all mutant viruses in comparison to the WT virus. The viruses containing patient-derived MDR RT without the K65R mutation (PSD5.2) replicated efficiently in comparison to the viruses with MDR RT containing the K65R mutation (PSD5.1), or the single mutations K65R and M184V. Compared with WT, a significant decrease in RCs of viruses: K65R (RC=0.39±0.02; p≤0.0001), M184V (RC=0.72±0.04; p≤0.0001), PSD5.1 (RC=0.32±0.04; p≤0.0001), and PSD5.2 (RC=0.90±0.04; p=0.002) was observed on day 10. RT-PCR-based apoptosis array was performed on total cellular RNA. Recombinant virus PSD5.2 showed a 1.5- to 6-fold upregulation in 8 AAMGs (AKT1, BAG3, BCL2A1, BFAR, BIRC2, BNIP1, BNIP3, and CFLAR) on day 1 and day 7 post-infection with respect to WT virus. PSD5.1 showed upregulation of only one gene (BAG1) on day 1 (1.75-fold) and day 7 (1.97-fold). Point mutant K65R showed a 1.5- to 4-fold upregulation of six AAMGs on day 7. Viruses with the M184V mutation showed upregulation of only one gene (BAG1). These observations indicate that the upregulation of specific AAMGs may not be dependent on the RCs of HIV-I variants, and that the possible interaction among mutated RT residues and viral and/or host proteins may induce CD4(+) T-cell-protective anti-apoptosis proteins.

Reverse transcriptase mutation K65N confers a decreased replication capacity to HIV-1 in comparison to K65R due to a decreased RT processivity.

In addition to K65R, the other mutation observed at HIV-1 RT codon 65 is K65N. While K65N appears to have a phenotypic effect similar to K65R, it is less frequent during clinical trials. We compared the relative impact of K→N with respect to K→R change on viral replication capacity (RC). Mutant viruses were created and replication kinetics assays were performed in PBM cells. Analysis of RCs revealed a significant loss in replication (p=0.004) for viruses containing K65N mutation in comparison to those with K65R mutation. RT processivity assays showed a significant decrease in the processivity of K65N RT in comparison to K65R RT. We demonstrated that the significant decrease in RC of K65N viruses is related to the impaired RT processivity of K65N RT in comparison to K65R, and that the selection of the K65R mutation may be favored in clinical use of antiretroviral drugs compared to K65N.