PMC

6BG and MeI block 17-β estradiol (E2)-stimulated growth of ER-positive cells. (A and D) Histograms from FC analysis of the populations of MCF7 and T47D cells, respectively, at G₁, S, and G₂ phases after culture in full medium alone (Ctr), Cdex medium, and full medium with 6BG for 15 h. (B and E) Summary of the percentages of cells at G₁, S, and G₂ phases for cells grown in Cdex medium alone (Cdex), treated with E2 for 15 h (E2), treated with MeI (1 mM) followed by E2 (E2+MeI), and treated with 6BG (50 μM) followed by E2 (E2+6BG). (C and F) R-MGMT analysis by protease V8 and Pab.MGMT in extracts from the cells analyzed in panels B and E after 2 h of E2 treatment.

Inhibition of ER response by 6BG and MeI is independent of p53. MCF7 cells grown in normal medium were treated with 2 Gy of γ irradiation (from a cobalt 65 source), 60 μM 6BG, or 1 mM MeI. Total cell extracts were prepared at 1, 3, and 6 h after treatment for Western blot analysis using Mab.DO1 for p53 and Mab.3B8 for MGMT as shown in panels A and B, respectively. The cell extracts were also treated with protease V8 to confirm the presence of R-MGMT as shown in panel C. Panel D shows the FC analysis of the cell population profiles 24 h after treatment with 6BG, Mel, and γ irradiation. Ctr, untreated control cells. The inserted table gives the summary of the average percentage of cells at G₁, S, and G₂ from three independent experiments.

6BG inhibits the growth of breast cells expressing ER and MGMT (mer⁺). (A) Flow cytometry analysis of breast (MCF7 [panel 1], T47D [panel 2], BT549 [panel 3], and MDA-231 [panel 4]) and virus-infected (HeLa CCL2 [panel 5] and MRC5.SV40 [panel 6]) cells after 6BG treatment (60 μM) for 15 h (). The histograms and tables represent the gated cell populations (events, y axis) and percentages, respectively, with different DNA contents stained by PI (FL2-A on the x axis defines the G₁, S, or G₂ cells). Ctr, control. (B) Immunoblot of MGMT and R-MGMT by Pab.MGMT in total cell extracts. Cell lines are numbered as in panel A. Control indicates untreated samples, whereas 6BG indicates 6BG-treated (2 h) samples. The 6BG+V8 panel shows the 6BG-treated samples digested with protease V8 for R-MGMT analysis (). Two hundred micrograms of cell extract/lane was used. (C) Immunoblot of ER as for panel B by Pab.HC20. (D) Immunoblot of SRC-1 as for panel B by the antibody Pab.N19.

The LXXLL motif of R-MGMT binds to ER in vitro and in vivo. (A) The scheme for binding of recombinant ER-α to immobilized GST-(K107L)MGMT. (B) Immunoblot of recombinant ER-α (by Mab.F10) bound to immobilized GST-(wt)MGMT and GST-(K107L)MGMT (visualized by Mab.3B8). (C) Peptide competition. The top panel is the sequence of the peptide used. Peptides (7 μg) were added to the ER-α before binding to the immobilized GST-(K107L)MGMT. The associated ER-α was analyzed by immunoblot using Mab.F10. (D) Mammalian two-hybrid assay. The top panel shows the region of SRC-1 (NR) used, similar to MGMT in size and in positioning of the LXXLL motif. Codons are numbered. The bottom panel shows the average luciferase activity obtained from three independent experiments in the ER⁻ MGMT⁺ HeLa CCL2 cells normalized to the control β-Gal activities obtained. V represents the VP-16-containing vector, and M represents the GAL4 DNA-binding domain-containing vector. The fusion constructs are V-K107L-MGMT, V-SRC-1 (NR), and M-ER. The luciferase activities were normalized to β-Gal activities from triplicate experiments. (E) Effect of 6BG on mammalian two-hybrid assay. The experiment is similar to that shown in panel D except that the ER⁻ MGMT MRC5.SV40 cells were used and 6BG (25 μM) was added together with E2.

R-MGMT disrupts the ER–SRC-1 interaction and fails to associate with the CBP/p300 complex. (A) Quantification of MGMT and ER proteins. Lanes labeled MCF7 and T47D contain 200 μg of total cell extracts, whereas lanes with numbers contain the purified recombinant MGMT and ER proteins, with numbers indicating amounts in nanograms (ng). Western blotting was performed with Mab.3B8 for MGMT and with Pab.HC20 for ER. (B) Immunoblot analysis of SRC-1 and ER in SRC-1 immunoprecipitates from MCF7 nuclear extracts. The lanes labeled Ctr, Mel, and 6BG are untreated (control), Mel (1 mM)-treated, and 6BG (50 μM)-treated cells grown in full medium for 2 h, respectively. The top panel is the immunoblot of the SRC-1 protein visualized by Pab.N19 in the SRC-1 Mab.677-F11 immunoprecipitate, and the bottom panel is the coimmunoprecipitated ER shown by Pab.HC20. (C) Immunoblot analysis of ER (Mab.F10) and MGMT or R-MGMT (Mab.3B8) in ER immunoprecipitates (Pab.H184), similar to panel B. (D) Survey of breast cells. Immunoblots of ER and MGMT in reported breast cell lines are shown: note the absence of the dual ER-positive and MGMT-deficient phenotypes. (E) Immunoblot analysis of SRC-1 and ER in SRC-1 immunoprecipitates from SV-ER (ER⁺ MGMT⁻) nuclear extracts, similar to panel B: note that the levels of ER protein were not affected by MeI and 6BG treatments compared to the MCF7 (ER⁺ MGMT⁺) cells in panel B. (F) Immunoblot analysis of CBP/p300 (Pab.451) and MGMT (Pab.MGMT) and in CBP/p300 immunoprecipitates (Mab.CBP/p300), similar to panel B.

Effect of R-MGMT on the cell cycle and transcription. (A) Growth arrest. MCF7 cells were transfected with expression vector (Ctr) or vectors fused with wt or K107L mutant MGMT cDNAs for 24 h () before analysis by FC using dual channels to quantify the fluorescence in the cells from the stainings of MGMT by Mab.3B8 and of DNA by PI. Panels a, b, and c show the distributions of transfected cells stained by MGMT Mab.3B8 (FL1-A, y axis) versus their DNA contents (FL2-A, x axis). Cells with greater MGMT stainings than the control (Ctr), due to the expression of the MGMT cDNAs, are gated (the region R) to generate the histograms (d, e, and f) where the cell populations (events, y axis) were plotted against their DNA contents (x axis) representing G₁, S, and G₂ phases. The arrow in panel c indicates that a large population of cells expressing the K107L-MGMT proteins are at G₁ phase. (B) Inhibition of RNA synthesis. Cells were labeled for 6 h with [³H]uridine after being cultured under full medium (FM) (lane 2), FM with 6BG (lane 1, pretreatment with 60 μM 6BG for 2 h before labeling), Cdex medium (lane 3), Cdex with E2 (lane 4) (pretreatment with 100 nM E2 for 2 h before labeling), or Cdex with E2 and 6BG together (lane 5) (pretreatment with 6BG and E2 together for 2 h before labeling). The labeled total RNAs isolated (2 μg/lane) were resolved on a 1% agarose gel. The top panel is the autoradiograph of the membrane with the labeled RNAs resolved by the agarose gel. The histogram is a summary of the ³H counts from 2 μg of labeled RNAs (averaged from three independent experiments). The bottom panel is the description of the samples. (C) RT-PCR analysis of the levels of PR, pS2, PBGD, and β-actin mRNAs obtained from cells cultured for 24 h, similar to panel B without [³H]uridine labeling. (D) Abbreviations of the wt and Mu (K107L mutant) MGMT full-length (FL) cDNAs used for transfection into the SV-ER cells (). M is the first methionine. (E) Reporter assay. Top panel, immunoblot of the expressed ER (Pab.HC20) and MGMT (Pab.MGMT) proteins in SV-ER cells grown in Cdex medium 24 h after cotransfection with ERE-Luc and MGMT (Wt, Mu, Wt-2A, and Mu-2A) constructs followed by 6 h of treatment with 1 μM E2. SV indicates the parental ER- and MGMT-negative MRC5.SV40 cells. Lower panel, the corresponding luciferase activities in the cell extracts.