Differentiation of Monocytic Cells Is Accompanied by a Change in the Expression of the Set of Oct-1 Isoforms

Changes in the expression level of Oct-1A, Oct-1L, Oct-1X, and Oct-1Z isoforms and CD14 surface antigen during differentiation of HL-60 monocytic cells induced in vitro by dimethyl sulfoxide were studied, and the expression level of the four Oct-1 isoforms in vivo in human monocytes was determined.     

Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.     

Protein kinase C beta1 is implicated in the regulation of neuroblastoma cell growth and proliferation.

To investigate a putative involvement of protein kinase C (PKC) isoforms in supporting neuroblastoma cell proliferation, SK-N-BE(2) neuroblastoma cells were transfected with expression vectors coding for the C2 and V5 regions from different PKC isoforms. These structures have been suggested to inhibit the activity of their corresponding PKC isoform. The PKC fragments were fused to enhanced green fluorescent protein to facilitate the detection of transfected cells. Expression of the C2 domain from a classical PKC isoform (PKCalpha), but not of C2 domains from novel PKCdelta or PKCepsilon, suppressed the number of neuroblastoma cells positive for cyclin A and bromodeoxyuridine incorporation. This indicates a role for a classical isoform in regulating proliferation of these cells. Among the V5 fragments from PKCalpha, PKCbetaI, and PKCbetaII, the PKCbetaI V5 had the most suppressive effect on proliferation markers, and this fragment also displaced PKCbetaI from the nucleus. Furthermore, a PKCbeta-specific inhibitor, LY379196, suppressed the phorbol ester- and serum-supported growth of neuroblastoma cells. There was a marked enhancement by LY379196 of the growth-suppressive and/or cytotoxic effects of paclitaxel and vincristine. These results indicate that PKCbetaI has a positive effect on the growth and proliferation of neuroblastoma cells and demonstrate that inhibition of PKCbeta may be used to enhance the effect of microtubule-interacting anticancer agents on neuroblastoma cells.     

Induction of serum amyloid A genes is associated with growth and apoptosis of HC11 mammary epithelial cells

In this study, we examined the expression and functions of serum amyloid A (SAA) isoforms during apoptosis of HC11 mammary gland epithelial cells. Expression of SAA mRNAs and apoptosis were increased in HC11 cells by serum withdrawal and gradually decreased upon the addition of serum, or epidermal growth factor (EGF). TNFalpha treatment of HC11 cells also induced expression of SAA genes, and the effect on SAA1 and SAA2 expression was suppressed by treatment with MG132, and in cells transfected with a dominant negative mutant form of IkappaBalpha. Similar results were observed in response to interleukin-1 (IL-1), IL-6 and interferon gamma (IFNgamma). Furthermore, overexpression of the SAA1 and SAA2 isoforms suppressed growth and accelerated apoptosis of HC11 cells by increasing caspase 3/7 and caspase 8 activities, but the apoptotic effect of tumor necrosis factor alpha (TNFalpha) on HC11 cells was not enhanced. We found that expression of SAA1 and SAA2, but not SAA3, was regulated by an NFkappaB-dependent pathway, and that overexpression of SAA isoforms accelerated the apoptosis of HC11 cells.     

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.     

Expression of a G protein subunit, alpha i-1, in Balb/c 3T3 cells leads to agonist-specific changes in growth regulation

Cellular receptors for many hormones, neurotransmitters, and growth factors are coupled to intracellular effector enzymes or ion channels through a set of heterotrimeric G proteins. In order to determine whether isoforms of G protein alpha subunits contribute differentially to mitogenic responses, we introduced an alpha subunit isoform, alpha i-1, into Balb/c 3T3 cells that normally lack this subtype. Balb/c 3T3 cells transfected with a plasmid containing cDNA encoding alpha i-1 expressed the alpha i-1 protein as judged both by the appearance of immunoreactive alpha i-1 protein on Western blots and by two-dimensional analysis of the proteins [32P]ADP-ribosylated by pertussis toxin. The amount of alpha i-1 expressed is less than the amount of alpha subunits endogenously present in these cells. Expression of alpha i-1 in the transfected cells slightly blunts stimulation of adenylylcyclase by GTP, guanosine 5'-3-O-(thio)triphosphate, or forskolin, but has no major effect on the ability of thrombin to inhibit the enzyme. In contrast, the expression of alpha i-1 has significant effects on cell growth and on the mitogenic response to thrombin. The alpha i-1-transfected cells have a doubling time that is twice as long as control cells transfected with the same plasmid without a cDNA insert. Despite their slower growth, thymidine incorporation in response to thrombin is greater in transfected than in control cells. Thrombin-stimulated DNA synthesis is sensitive to inhibition by pertussis toxin and is 5-fold more sensitive to inhibition by pertussis toxin in transfected cells than in control cells. The changes are receptor-specific since the mitogenic response to platelet-derived growth factor is indistinguishable between control and transfected cells. These studies suggest that the alpha i subunit composition of the cell may have profound effects on its growth and its response to stimulation through a specific cell surface receptor.