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First published online 11 October 2005
doi: 10.1242/jcs.02616

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CREB-binding protein regulates apoptosis and growth of HMECs grown in reconstituted ECM via laminin-5

¹ Division of Medical Oncology, Duke University, Durham, NC 27710, USA
² Division of Hematology and Oncology, Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA
³ Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
⁴ RIKEN, Tsukuba Institute, Ibaraki 305-0074, Japan
⁵ Department of Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA

View larger version (32K): [in a new window]   Fig. 1. Partial karyotype and CBP expression of late passage HMEC-E6 cells (passage 20). (A) Partial karyotype of a single mitotic cell demonstrating two copies of an unbalanced translocation between chromosomes 13 and 16 involving 16p13 (arrows). Spectral karyotyping in classification colors (red, chromosome 13 material; orange, chromosome 16 material). (B) Partial karyotypes of two cells that demonstrate either loss of a more distal region of 16p or rearrangement at the CBP locus, 16p13. (C) CBP protein expression is decreased in late passage HMEC-E6 cells. Early and late passage HMEC-LXSN vector controls (LXSN) (passages 11 and 16) and HMEC-E6 cells (E6) (passages 11 and 18) were analyzed for CBP protein expression as described in Materials and Methods. Equal amounts of protein lysate were loaded per lane. Actin was used as a loading control.

View larger version (40K): [in a new window]   Fig. 2. Laminin-5 3-chain expression is decreased in rECM-resistant, CBP-poor, late passage HMEC-E6 cells. (A) Analysis of differential gene expression in early (E6E) and late (E6L) passage HMEC-E6 cells (passage 10 and 18) relative to early passage HMEC-LXSN controls (passage 10) (LXSN). Cells were grown in contact with rECM and harvested for differential gene expression. All RNA combinations used for array analysis were obtained from cells that were matched for passage number, cultured under the identical growth conditions, and harvested at identical confluency. Data were collected in triplicate using independent biological replicates. Color-coding: green, downregulation of gene expression; red, induction; black, no significant change; grey, no data available. (B,C) Semi-quantitative RT-PCR analysis of integrin and laminin-5 mRNA expression in early (E) and late (L) passage HMEC-E6 cells (E6) (passage 10 and 18) and HMEC-LXSN controls (LXSN) (passage 10 and 16). Expression was normalized to ß-actin. These data are representative of three separate experiments. *Significantly different from LXSN-E (P<0.05); #significantly different from E6-E (P<0.05). (D) Laminin-5 3-chain protein expression is decreased in rECM-resistant, late passage HMEC-E6 cells (E6) (passage 18) relative to early passage HMEC-E6 cells (E6) (passage 11) and early and late passage HMEC-LXSN controls (LX) (passage 10 and 18). Western analysis was performed as described in Materials and Methods. Equal amounts of protein lysate were loaded per lane. Actin serves as a loading control.

View larger version (36K): [in a new window]   Fig. 3. Loss or suppression of CBP expression inhibits CBP occupancy of the LAMA3A promoter and suppresses LAMA3A promoter activity and laminin-5 expression. (A) Late passage HMEC-E6 cells do not exhibit CBP occupancy of the 277 bp AP-1-rich region of the LAMA3A promoter. ChIP was performed in HMEC-LXSN controls (LXSN) (passage 11 and 16), and early passage HMEC-E6 cells (E6) (passage 11) and compared with CBP-poor, late passage HMEC-E6 cells (E6) (passage 18). Input controls test the integrity of the DNA samples. (B) LAMA3A promoter activity was measured in early and late passage HMEC-LXSN controls (LXSN) (passage 11 and 16), and early passage HMEC-E6 cells (E6) (passage 10) and compared with CBP-poor, late passage HMEC-E6 cells (E6) (passage 18). Data represent two independent experiments performed in triplicate. (C) CBP protein expression is suppressed by antisense ODNs. HMEC-LXSN vector controls (LXSN) (passage 12) and early passage HMEC-E6 cells (E6) (passage 12) were cultured in the presence of (1) no treatment, (2) active CBP-specific ODN (A3342V), and (3) inactive CBP ODN (scrA3342V). Resultant cells were analyzed for CBP protein expression as described in Materials and Methods. Equal amounts of protein lysate were added per lane. Actin was used as a loading control. (D) CBP protein expression is suppressed in rECM culture on days 1-11. Early passage HMEC-E6 cells (passage 12) were cultured in rECM and treated daily with active CBP-specific ODN (A3342V) (AS) or inactive CBP ODN (scrA3342) (S). Resultant cells were tested for CBP protein expression as described in Materials and Methods. Equal amounts of protein lysate were added per lane and actin was used as a loading control. (E) Early passage HMEC-E6 cells (passage 10) were treated with CBP-specific ODN (A3342A; As) and inactive CBP ODN (scrA3342V; Scr), grown in contact with rECM, and tested by ChIP to determine whether suppression of CBP expression resulted in a loss of CBP binding to the AP-1-rich site of the LAMA3A promoter. Input controls test the integrity of the DNA samples. (F) Suppression of CBP expression results in decreased LAMA3A promoter activity. Early passage HMEC-LXSN controls (passage 11) and HMEC-E6 cells (passage 11) grown in rECM and treated with CBP-specific antisense ODNs (A3342V) (AS-CBP) exhibit decreased LAMA3A promoter activity relative to cells treated with inactive ODNs (scrA3342V) (scr-CBP). Data represent two independent experiments performed in triplicate. Error bars show standard error. (G) Laminin-5 3-chain mRNA expression is suppressed by antisense ODNs. Early passage HMEC-E6 cells (passage 12) were cultured in the presence of no treatment (C), inactive CBP ODN (scrA3342V; Scr), and active CBP-specific ODN (A3342V; As). Resultant cells were analyzed for laminin-5 3-chain mRNA expression by semi-quantitative RT-PCR as described in Materials and Methods. Actin was used as a loading control.

View larger version (35K): [in a new window]   Fig. 4. Expression of CBP in late passage HMEC-E6 cells promotes CBP binding to the LAMA3A promoter and increases LAMA3A promoter activity and laminin-5 expression. (A) CBP protein expression in (1) early passage HMEC-E6 cells (passage 10), (2) late passage HMEC-E6 cells (passage 18; ), (3) late passage HMEC-E6-CBP– vector controls (passage 18; (–)CBP), and (4) late passage HMEC-E6-CBP+ cells (passage 18; (+)CBP). Equal amounts of protein lysate were added per lane. Actin was used as a loading control. (B) Exogenous expression of CBP in late passage HMEC-E6-CBP+ cells promotes CBP occupancy of the 277 bp AP-1-rich region of the LAMA3A promoter. ChIP was performed in (1) early passage HMEC-E6 cells, (2) late passage HMEC-E6 cells (passage 17; ) and (3) late passage HMEC-E6-CBP– vector controls (passage 17; CBP(–)) and compared with late passage HMEC-E6-CBP+ cells (passage 17; CBP(+)). Input controls test the integrity of the DNA samples. (C) LAMA3A promoter activity was measured in (1) early passage HMEC-E6 cells (E6E; passage 10), (2) late passage HMEC-E6 cells (E6L no tx; passage 18), and (3) late passage HMEC-E6-CBP– vector controls (E6L(–)CBP; passage 18) and compared with late passage HMEC-E6-CBP+ cells (E6L(+)CBP; passage 18). Data represent two independent experiments performed in triplicate. (D) Laminin-5 protein expression in (1) early passage HMEC-E6 cells (passage 10), (2) late passage HMEC-E6 cells (passage 18; ), (3) late passage HMEC-E6-CBP– vector controls (passage 18; (–)CBP), and (4) late passage HMEC-E6-CBP+ cells (passage 18; (+)CBP). Equal amounts of protein lysate were added per lane. Actin was used as a loading control.

View larger version (54K): [in a new window]   Fig. 5. Expression and distribution of 3-integrin and laminin-5 3-chain in HMECs that express altered levels of CBP. (A) Immunofluorescence characterization of 3-integrin and laminin-5 3-chain expression in cells sensitive and resistant to rECM-mediated growth arrest and apoptosis. Frozen section of early and late passage HMEC-LXSN controls (LXSN; passage 10 and 16) and HMEC-E6 cells (E6; passage 10 and 18) were grown in rECM for 6 days, cryosectioned, and immunostained for localization of 3-integrin and laminin-5 3-chain expression. 3-integrin and laminin-5 3-chain expression was primarily localized at the basal surface of early and late passage HMEC-LXSN and early passage HMEC-E6 cells (arrowheads). By contrast, CBP-poor late passage HMEC-E6 cells showed dispersed membrane and intracellular staining of 3-integrin (arrow) and qualitatively decreased laminin-5 3-chain expression. (B) Immunofluorescent characterization of 3ß1-integrin and laminin-5 3-chain expression in HMECs treated with CBP antisense ODNs. Frozen section of early passage HMEC-LXSN vector controls (LXSN) (passage 11) and HMEC-E6 cells (E6) (passage 11) treated either with CBP antisense ODN (A3342V; CBP-as) or inactive CBP ODN (scrA3342V; CBP-scr). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for 3-integrin or laminin-5 3-chain. 3-integrin and laminin-5 3-chain expression was primarily localized at the basolateral surface in HMEC-LXSN and HMEC-E6 cells treated with inactive CBP ODNs (arrowheads). By contrast, HMEC-LXSN and HMEC-E6 cells treated with antisense CBP ODNs demonstrated disorganized membrane and cytosolic staining of 3-integrin (arrows) and markedly reduced laminin-5 3-chain expression. (C) Immunofluorescent characterization of 3ß1-integrin and laminin-5 3-chain expression in late passage HMEC-E6 cells expressing exogenous CBP. Frozen sections of immunostained late passage HMEC-E6-CBP– vector controls (CBP(–); passage 17) and late passage HMEC-E6-CBP+ cells (CBP(+); passage 17). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for 3-integrin or laminin-5 3-chain. HMEC-E6-CBP– cells demonstrated disorganized membrane and cytosolic staining of 3-integrin and markedly reduced levels of laminin-5 3-chain expression. By contrast, HMEC-E6-CBP+ cells exhibited a qualitative increase in laminin-5 3-chain expression. Although there was persistent membrane and cytosolic staining of 3-integrin and laminin-5 3-chain, there was also increased localization at the basolateral surface (arrowheads).

View larger version (33K): [in a new window]   Fig. 6. Modulation of CBP expression alters proliferation in rECM culture. (A-D) Inhibition of CBP expression in HMECs by antisense ODNs results in enhanced proliferation in rECM. Ki-67 staining indices (A,C) in early passage HMEC-LXSN cells (passage 11) (A) and early passage HMEC-E6 cells (passage 11) (C). The mean number of nuclei per cell cluster (B,D) in early passage HMEC-LXSN vector controls (passage 10) (B) and early passage HMEC-E6 cells (passage 11) (D) treated with either CBP antisense ODN (A3342V; CBP AS) or inactive CBP ODN (scrA3342V; CBP scr). Two hundred cells were surveyed per time point and indices were calculated from an average of three independent experiments. Error bars show standard error.

View larger version (85K): [in a new window]   Fig. 7. Inhibition of CBP in early passage HMECs by antisense ODNs blocks apoptosis in rECM. Electron micrographs of early passage HMEC-LXSN control cells (passage 11) (A) and early passage HMEC-E6 cells (passage 11) (B) treated with CBP antisense (A3342V) ODN and grown in rECM for 9 days. Cells formed large, dense, irregularly shaped, multicellular colonies that have no central lumen (A,B). By contrast, early passage HMEC-E6 cells (passage 10) treated with inactive CBP ODN (scrA3342V) (C) underwent apoptosis when grown in rECM for 7 days as shown by (1) nuclear condensation (n), (2) cell shrinkage and separation, and (3) margination of chromatin (mr). Percentage of apoptotic cells in early passage HMEC-E6 cells (passage 11) (D) and early passage HMEC-LXSN controls (passage 11) (E) treated either with active (A3342V; CBP-as) or inactive (scrA3342V; CBP-scr) CBP-specific ODNs. Apoptosis was measured by TUNEL-staining. Apoptotic index was measured by calculating the percentage of TUNEL-stained cells relative to the total number of cells surveyed. Data represents an average of three independent experiments. Error bars show standard error.

View larger version (58K): [in a new window]   Fig. 8. Expression of CBP in late passage HMEC-E6 cells promotes growth regulation and apoptosis in rECM culture. (A,B) Exogenous expression of CBP in apoptosis-resistant late passage HMEC-E6 cells results in decreased growth in rECM culture. (A) Ki-67 staining indices in late passage HMEC-E6 cells (passage 17) expressing exogenous CBP (CBP(+)) or transduced with the LXSN control vector (CBP(–)). (B) The number of nuclei per cell cluster formed by late passage HMEC-E6 cells (passage 17) expressing exogenous CBP (CBP(+)) or transduced with the LXSN control vector (CBP(–)). Two hundred cells were surveyed per time point and indices were calculated from an average of three independent experiments. Error bars show standard error. (C,D) Electron micrographs of late passage HMEC-E6 cells (passage 17) expressing exogenous CBP ((+)CBP) or transduced with the LXSN control vector ((–)CBP) grown in rECM for 7 days. Late passage HMEC-E6-CBP(–) control cells formed large, dense, irregularly shaped, multicellular colonies that have no central lumen (C). By contrast, late passage HMEC-E6-CBP+ cells underwent apoptosis when grown in rECM for 7 days (D) as shown by (1) nuclear condensation (n), (2) cell shrinkage and separation, and (3) margination of chromatin (mr). (E) Percentage of apoptotic cells in late passage HMEC-E6 cells (passage 16) expressing exogenous CBP (CBP(+)) or transduced with the LXSN control vector (CBP(–)). Apoptosis was measured by TUNEL-staining. Apoptotic index was measured by calculating the percentage of TUNEL-stained cells relative to the total number of cells surveyed. Data represents an average of three independent experiments. Error bars show standard error.

View larger version (33K): [in a new window]   Fig. 9. Suppression of laminin-5 3-chain in early passage HMEC-E6 cells blocks apoptosis in rECM. (A) Laminin-5 3-chain mRNA expression is compared by semi-quantitative RT-PCR in three early passage HMEC-E6 cells (passage 10) clones expressing siRNA sequences directed against laminin-5 3-chain (si#1, si#2, si#3) or control siRNA (si-cont), and in HMEC-E6 control cells (no tx). Actin was used as a loading control. (B,C) Suppression of laminin-5 3-chain expression in early passage HMEC-E6 cells results in increased proliferation in rECM culture. (B) Ki-67 staining indices in early passage HMEC-E6 cells (passage 11) expressing siRNA sequence #2 directed against laminin-5 3-chain or siRNA control. (C) DAPI staining of cell nuclei demonstrates the number of nuclei per cell cluster formed by early passage HMEC-E6 cells (passage 11) expressing either siRNA sequence #2 directed against laminin-5 3-chain or siRNA control. Two hundred cells were surveyed per time point and indices were calculated from an average of three independent experiments. Error bars show standard error. (D) Electron micrographs of early passage HMEC-E6 cells (passage 11) expressing siRNA sequence #2 directed against laminin-5 3-chain (si-laminin-5) or control siRNA (si-control). Early passage HMEC-E6 cells expressing control siRNA underwent apoptosis when grown in rECM for 7 days as shown by (1) nuclear condensation (n), (2) cell shrinkage and separation, and (3) margination of chromatin (mr). By contrast, HMEC-E6 cells expressing siRNA directed against laminin-5 3-chain did not undergo apoptosis and instead formed large, dense, irregularly shaped multicellular colonies that have no central lumen. (E) Relative levels of TdT-positive cells in early passage HMEC-E6 cells (passage 10) treated either with siRNA sequence #2 directed against laminin-5 3-chain (laminin-5 si-RNA) or control siRNA (si-control). Apoptosis was measured by TUNEL-staining. Data represents an average of two independent experiments in triplicate. Error bars show standard error. (F) Immunofluorescent characterization of laminin-5 3-chain expression in early passage HMEC-E6 cells (passage 12) expressing si-RNA directed against laminin-5 3-chain (si-laminin-5) or control siRNA (si-control). Cells were grown in rECM for 6 days, cryosectioned, and immunostained for laminin-5 3-chain. Laminin-5 3-chain expression was primarily localized at the basolateral surface in early passage HMEC-E6 cells expressing control siRNA. By contrast, early passage HMEC-E6 cells expressing si-RNA directed against laminin-5 3-chain exhibited markedly reduced laminin-5 3-chain expression.