QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 May 2006
doi: 10.1242/jcs.02966

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S. W. Articles by Zetter, B. R. Search for Related Content PubMed PubMed Citation Articles by Kim, S. W. Articles by Zetter, B. R. Social Bookmarking                         What's this?

Regulation of cell proliferation by the antizyme inhibitor: evidence for an antizyme-independent mechanism

¹ Program in Vascular Biology and Department of Surgery, Children's Hospital, Boston, MA 02115, USA
² Department of Psychiatry, Düsseldorf University, 40225 Düsseldorf, Germany
³ Department of Cellular and Molecular Physiology, Pennsylvania State College of Medicine, Hershey, PA 17033, USA

View larger version (36K): [in a new window]   Fig. 1. Effect of AZI on proliferation and anchorage-independent growth in AT2.1 prostate cancer cells. (A) 50 µg total cell lysate from AT2.1 cells stably transfected with pcDNA3 or pcDNA3-AZI constructs were analyzed for AZI and actin expression by western blotting. The stable AZI cell line used in these studies showed a fivefold increase in AZI expression relative to endogenous levels. (B) Proliferation of control () and AZI-overexpressing AT2.1 cells () in low serum (1% FBS). Cells were plated at a density of 1x104 cells in 12-well plates and the total number of cells per well were counted. Error bars indicate standard error of the mean (s.e.m.). (C) Proliferation of control () and AZI-overexpressing AT2.1 cells () grown in high serum (10% FBS) was assessed as described above. (D) Phase-contrast micrographs of control and AZI-overexpressing AT2.1 cells comparing colony formation in 0.45% noble agar, as described in the Materials and Methods. Colonies were visualized after 2 weeks using 0.02% Giemsa stain. AZI-overexpressing cells formed more colonies with diameters greater than 0.5 mm. Similar results were seen with a second clone that expressed AZI at twice the wild-type level (data not shown).

View larger version (15K): [in a new window]   Fig. 2. Effect of AZI on proliferation of non-transformed NIH-3T3 fibroblasts. (A) Western blot analysis of 50 µg of total cell lysate from NIH-3T3 cells stably infected with retroviral vectors pWZL or pWZL-AZI. (B) Proliferation of control () and AZI-overexpressing NIH-3T3 cells () grown in low serum (0.5% BCS). Cells were plated at a density of 5x104 cells in 12-well plates and the total number of cells per well were counted. Error bars indicate s.e.m. (C) To assess saturation density, control () and AZI-overexpressing NIH-3T3 cells () were plated at 1x104 cells in six-well plates in media containing 10% BCS and cell number assessed over 11 days.

View larger version (12K): [in a new window]   Fig. 3. AZI expression increases ODC activity in AT2.1 and NIH-3T3 cells. (A) ODC activity (pmol 14CO2/mg protein/hour) was measured in control and AZI-overexpressing AT2.1 cells, using [1-14C]-L-ornithine as the substrate. Total cell lysates were collected 24 hours after plating 5x105 cells in medium containing 10% serum. Error bars indicate s.e.m. (B) Effect of AZI overexpression on cellular ODC activity in NIH-3T3 fibroblasts. ODC activity was measured as above. (C) Polyamine uptake by AZI-overexpressing cells was measured following a 15 minute incubation with 0.25-10 µM [14C]spermine, as described in the Materials and Methods. No significant difference was observed in spermine uptake between AT2.1 vector control () and AZI-overexpressing cells (). Error bars indicate s.e.m.

View larger version (18K): [in a new window]   Fig. 4. Deletion of the putative antizyme-binding site and effect on antizyme binding. (A) Schematic of wild-type AZI and mutant AZI117-140. The putative antizyme(AZ)-binding site, designated residues 117-140, was deleted using overlap extension PCR. (B) Western blot analysis of wild-type AZI and AZI117-140 using AZI antibody. Proteins were synthesized using in vitro transcription and translation from pTriEx-Hygro-AZI and TriEx-Hygro-AZI117-140 constructs as S-tagged fusion proteins. (C) Loss of antizyme-binding by AZI117-140. The ability of S-tagged AZI or AZI117-140 to associate with radiolabeled antizyme was examined by affinity purification using S-protein agarose beads, followed by SDS-PAGE analysis and autoradiography. The mutant AZI117-140 was unable to bind antizyme.

View larger version (15K): [in a new window]   Fig. 5. Overexpression of AZI117-140 increases proliferation of AT2.1 cells without increasing ODC activity in AT2.1 cells. (A) Proliferation of AT2.1 cells stably transfected with pTriEx-Hygro, pTriEx-Hygro-AZI, or pTriEx-Hygro-AZI117-140, was measured as described in Materials and Methods, in media containing 1% FBS. Error bars indicate s.e.m. , Vector control AT2.1 cells; , AZI-overexpressing AT2.1 cells; , AZI117-140 overexpressing AT2.1 cells. The proliferation rate of AZI117-140 overexpressing cells decreased compared with wild-type AZI-overexpressing cells, but still demonstrated increased proliferation relative to vector controls. An average doubling time of 22.8±1.6 (s.d.) for control cells; 15.6±1.7 for AZI-overexpressing cells; and 18±1.7 for AZI117-140 overexpressing cells was determined from two separate experiments. (B) ODC activity was measured in control, AZI and AZI117-140-overexpressing AT2.1 cells. 5x105 cells were plated in RPMI supplemented with 10% FBS, and ODC activity assessed as described in the Materials and Methods. Error bars indicate s.e.m.

View larger version (90K): [in a new window]   Fig. 6. Effect of AZI RNAi on cell proliferation and cell-cycle protein levels. (A) Phase-contrast micrographs of AT2.1 cells transfected with control or AZI siRNA, 48 hours post transfection. (B) Quantification of cell number after scramble- or AZI-siRNA transfection. At 48 hours post transfection, cells were trypsinized and the number of cells per condition was quantified using a Coulter counter. (C) Western blot analysis of cell lysates from control or AZI siRNA-transfected AT2.1 cells, collected 48 hours following transfection. AZI siRNA-tranfected AT2.1 cells exhibited decreased levels of AZI (62±6% decrease) and cyclin D1 (56±4%, both mean ± s.e.m. from three independent experiments), but not of cyclin A or actin.

View larger version (26K): [in a new window]   Fig. 7. AZI shows ability to attenuate cyclin D1 degradation. Cyclin D1, wild-type AZI and AZI117-140 were synthesized by in vitro transcription and translation in separate reactions. All three proteins were radiolabeled with [35S]methionine. Translated proteins were combined and incubated in an ATP-regenerating buffer for 0, 30, 60, 120 and 180 minutes. The amount of cyclin D1 remaining at the indicated time points was assessed by SDS-PAGE and PhosphorImager analysis (lower panel). Results are representative of two independent experiments. , cyclin D1 alone; , Cyclin D1 + wild-type AZI; , % Cyclin D1 + AZI117-140.

View larger version (28K): [in a new window]   Fig. 8. Interaction of AZI and cyclin D1 in vitro and effect of AZI117-140 mutation. (A) 293 cells were transfected with combinations of plasmids expressing S-tagged AZI, FLAG-tagged antizyme and HA-tagged cyclin D1. After 48 hours, lysates were collected and incubated with S-protein agarose. AZI-interacting proteins were identified by western blot using anti-HA, anti-FLAG or anti-AZI antibodies. (B) Immunoprecipitation of transfected S-tagged AZI117-140, FLAG-tagged antizyme and HA-tagged cyclin D1 as described above. AZI117-140 interacted with cyclin D1 but not antizyme.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter